role in immune evasion [276]. HIF-1α is also involved in decreasing the anti-tumor immune response via its control of the PD-1/PD-L1 in immune cells and tumor cells. The hypoxia-inducible element (HRE), where HIF-1α binds, was found in the PD-L1 proximal promoter [277]. Combined, the above information suggests that HIF-1α might serve as a therapeutic strategy to overcome both TNBC metabolic rewiring and immunosuppression.

#### **7. Conclusions**

Immune checkpoint inhibition has evolved significantly to reflect the immunogenic potential of TNBC, among other cancers. Despite some positive clinical outcomes with anti-PD-1/PD-L1 and anti-CTLA-4 antibodies, the monotherapy only provides benefit to a subset of the patients, warranting further studies. To date, a large number of clinical trials are looking at immune checkpoint inhibition as a treatment modality to complement chemotherapy. Since enrichment of CSCs and tumor reconstitution are often associated with chemotherapeutic agents, novel combination strategies include normalizing tumor-associated vasculature, modulating the TME, and targeting a multitude of receptors and transcription factors, which may lead to a more effective and durable response for TNBC treatment. In addition to active immunity, therapies that strengthen passive immunity or counter the metabolic reprogramming of tumors may be advantageous in future research. Although significant progress has been made, many challenges remain in the field when looking for a combinatorial immunotherapy to target TNBC.

**Author Contributions:** K.M., S.E.-S., V.D. and L.W. conceived of and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partially funded by operating grants from Cancer Research Society/University of Ottawa (24064) and Natural Sciences and Engineering Research Council RGPIN-2019-0522 to LW; New Frontiers in Research Fund NFRE-2019-00674 to V.D. and L.W.; Canadian Institutes of Health Research-The Canada Graduate Scholarship to SE.

**Conflicts of Interest:** All authors declare no conflict of interest.

#### **References**

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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

 Despite the significant benefit of the use of conventional chemotherapy and monoclonal antibodies in the prognosis of breast cancer patients and although the recent approval of the anti-PD-L1 antibody atezolizumab in combination with chemotherapy has been a milestone for the treatment of patients with metastatic triple-negative breast cancer, immunologic treatment of breast tumors remains a great challenge. In this review, we summarize current breast cancer classification and standard of care, the main obstacles that hinder the success of immunotherapies in breast cancer patients, as well as different approaches that could be useful to enhance the response of breast tumors to immunotherapies.

**Keywords:** immunotherapy; breast cancer; resistance; checkpoint; targeted treatment; personalized medicine

#### **1. Introduction**

#### *1.1. Breast Cancer*

According to the last Global Cancer Statistics (GLOBOCAN 2018), breast cancer represented 11.6% of all cancers, which places this disease as the second most commonly diagnosed cancer after lung cancer, and caused 6.6% of the total cancer deaths in 2018 [1]. Among women, incidence rates for breast cancer significantly exceeded those for other cancers in both transitioned and transitioning countries, remaining as the most frequently diagnosed cancer and the leading cause of cancer death in women worldwide [1].

Although for the majority of breast cancer patients it is not possible to identify a specific risk factor [2], these are diverse and well documented and include obesity, physical inactivity, alcohol consumption, use of hormone therapy, high breast density, and hereditary susceptibility due to mutations in autosomal dominant genes [3], which represents between 5–10% of all breast cancer cases in women [3]. Among these genetic alterations, mutations affecting *BRCA1* and *BRCA2* genes, which control DNA repair and transcriptional regulation in response to DNA damage, can lead to the accumulation of genetic alterations and greatly increase lifetime risk to develop different types of malignancies, including breast cancer [4]. Indeed, mutations in BRCA1 and BRCA2 genes are associated with an increased risk of inherited breast and ovarian cancer, representing the strongest

susceptibility markers that have been identified for breast cancer worldwide, with an estimated 45–80% lifetime risk of breast cancer for BRCA1-BRCA2 mutation carriers [4]. In a similar manner, mutations affecting *TP53* are also related to triple negative breast cancer [3].

As with other types of cancer, early diagnosis greatly increases the chances for successful treatment, allowing for a 20% reduction in overall mortality rates [5]. In this regard, despite reported handicaps of screening programs like high overdiagnosis rates and costs, risks that are derived from ionizing radiation, or false positive biopsy recommendations, both mammography, breast self-examinations, clinical breast examinations, digital breast tomosynthesis, ultrasonography, magnetic resonance imaging, and oncogene identification represent the main tools for early diagnosis, sorting out, and prevention of risk factors as well as timely treatment to lessen breast cancer morbidity [5].

Besides screening programs, adjuvant chemotherapy has also had a significant impact on the prognosis of breast cancer patients, having significantly improved their overall survival, disease-free survival [6], and death rates related to breast-cancer since the early 1990s [7]. In this respect, breast cancer has traditionally been classified into three subtypes with different prognoses and treatment responses [8,9] (Table 1).

**Table 1.** Breast cancer classification and standard of care.

HR+: Positive for Hormone Receptors. ER+: Expressing Estrogen Receptors. PR+: Expressing Progesterone Receptors. HER2: Positive for Human Epidermal Growth Factor Receptor 2 (Receptor tyrosine-protein kinase ERBB2, CD340). TNBC: Triple Negative Breast Cancer. CK5/6+: Expressing cytokeratin 5/6. EGFR+: Expressing Epidermal Growth Factor Receptor.

Even though most breast cancer patients are diagnosed early enough to be successfully treated with surgery, chemotherapy, radiotherapy, or a combination thereof [8], nearly 30% of women that are initially diagnosed with early-stage disease will eventually develop a metastatic disease [7], which ultimately leads to patient death. In this scenario, and given their high efficacy and selectivity, the development of cancer immunotherapies and other treatment strategies targeting tumor cells have positioned themselves as promising options to win the battle against breast cancer in opposition to conventional treatments that lack tumor selectivity and cause more side effects.

#### *1.2. Immunotherapy as an Option for Cancer Treatment*

According to the cancer immunoediting model [15], the relation between tumor cells and the immune system is a dynamic process which consists of three main phases (Figure 1):

	- Genetic abnormalities lead to the production of new antigens by tumor cells, which are processed and presented as antigen-derived peptides on the cell surface in association with Human Leukocyte Antigen class I (HLA-I).
	- Neoantigens that are present in tumor microenvironment are recognized, processed, and presented on the surface of Antigen Presenting Cells (APCs) as antigen-derived peptides in association with Human Leukocyte Antigen class-II (HLA-II), which can be recognized by helper T-cell receptors and leads to B-cell and cytotoxic T-cell stimulation and maturation.
	- After T-cell activation by co-stimulatory signals provided by APCs, T-cells recognize neoantigens presented by HLA-I and attack the targeted tumor cell by the secretion of cytotoxic granules and/or via Fas cell surface death receptor (FAS) and caspase activation.

One of the characteristics of advanced tumors is their capability to evade adaptive immune responses [17], which would explain the direct relationship between tumor growth and immune evasion [18]. Since mechanisms leading to tumor evasion are diverse [19] (Table 2), a significant effort has been made in recent years to develop new strategies to trigger tumor cell death by stimulating the patient's natural defenses to recognize and destroy tumor cells.

**Table 2.** Immune system mechanisms of tumor evasion.

**Table 2.** *Cont.*

CD95: Fas/APO-1. CD95L: CD95 ligand. APC: Antigen Presenting Cell/Dendritic Cell. HLA: Human Leukocyte Antigen. IL: Interleukin. JAK-3: Janus kinase 3. M-CSF: Macrophage Colony-Stimulating Factor. NF-kB: Nuclear Factor-kappa-B transcription complex. TGF: Transforming Growth Factor. VEGF: Vascular Endothelial Growth Factor.

These findings have been the basis for the development of different modalities of anticancer immunotherapy, including tumor-targeting immunotherapies, oncolytic viruses, anticancer vaccines, or adoptive cell immunotherapies [24] that are designed to target tumor cells and work with the immune system at different levels (Figure 2). As a result of the great success achieved in different studies and trials that demonstrate the efficacy of immunotherapies not only against primary tumors, but also preventing metastasis and recurrence [25], cancer immunotherapy has become the fourth pillar of cancer care, complementing surgery, cytotoxic therapy, and radiotherapy [26].

**Figure 1.** Cancer Immunoediting. ELIMINATION: (1) Neoantigen production by transformed cells. (2) Neoantigen presentation on the surface of transformed cells, associated with HLA-I. (3) Neoantigen recognition and processing by antigen presenting cells. (4) Neoantigen presentation on the surface of antigen presenting cells, associated with HLA-II. (5) T-cell activation in the presence of co-stimulatory signals. (6) Transformed cell recognition by activated T cells and elimination. EQUILIBRIUM: (7) Transformed cells with a resistant or non-immunogenic phenotype escape elimination and proliferate, although the immune system is still able to control the tumor growth. ESCAPE: (8) Uncontrolled proliferation of cells with a resistant or a non-immunogenic phenotype, leading to tumor progression and metastasis.

**Figure 2.** Modalities of cancer immunotherapy.

In this regard, the recent approval of treatments based on the use of checkpoint inhibitors has been a turning point for the treatment of patients with different tumors including melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, bladder cancer, head and neck squamous cell carcinoma, Merkel-cell carcinoma, microsatellite instability high, or mismatch repair deficient solid tumors [27], since they have demonstrated that they significantly increase survival rates when compared to standard therapy among different tumor types [23]. The impact of the basic studies allowed the development of checkpoint inhibitor therapies, which is the reason that James P. Allison and Tasuku Honjo won the Nobel Prize in Physiology and Medicine in 2018.

#### *1.3. Checkpoint Inhibitors*

Provided that activated CD8<sup>+</sup> (cytotoxic T lymphocytes or T-cells) recognize and destroy pathogen-infected or aberrant cells like cancer cells, they are considered the main effectors of cell-mediated immunity. On the other hand, since T cells also increase antibody responses through the action of CD4 (T-helper cells) and the enhancement of antibody production by B cells, their activation represents a critical step for the initiation and regulation of the immune response [28].

In accordance with the two-signal model of lymphocyte activation, both co-stimulatory signals and antigen-specific signals mediating the engagement of T-cell receptor (TCR) to HLA-II expressed on the surface of antigen-presenting cells participate in T-cell activation and maturation [29]. The subsequent response is regulated by a balance between co-stimulatory and inhibitory signals, or immune-checkpoints [30], at multiple steps during the immune response [31], which limits tissue damage and allows for the maintenance of self-tolerance. Given their immunosuppressive functions, dysregulated expression of inhibitory signals implies a major advantage in the tumor microenvironment, leading to immune evasion (Table 2).

Nowadays, and due to their association with the inhibition of lymphocyte activity and subsequent anergy, different immune checkpoint receptor-ligand combinations are the subject of intense study as tools for cancer treatment by restoring immune system function either as mono or in combination therapies [30,32]. Among these, and because of their central role during the immune response and peripheral tolerance, both Cytotoxic T-Lymphocyte-Associated Antigen 4 (CTLA4, CD152) and Programmed Cell Death Protein 1 (PD-1, CD279)/Programmed Cell Death Protein 1 Ligand (PD-L1, CD274) pathways have proved to be valid targets for the development of new cancer treatments and have allowed for the clinical approval of a number of CTLA and PD-1/PD-L1 checkpoint inhibitors (Table 3).

**Table 3.** Approved checkpoint inhibitors for cancer treatment.

**Table 3.** *Cont.*

CTLA4: Cytotoxic T-lymphocyte associated protein 4; PD-1: Programmed Cell Death Protein 1; PD-L1: Programmed Cell Death Protein 1-Ligand; MSI-H: Microsatellite instability high; dMMR: Mismatch repair deficient.

As previously mentioned, apart from T-cell receptor interaction with HLA-II, T-cell activation is controlled by further antigen-independent costimulatory signals such as CD28 (Cluster of Differentiation 28) and CTLA-4. In this respect, and contrary to CD28 signals, which are required for T-cell activation and cytokine secretion, CTLA-4 signaling inhibits T-cell activation, which is especially important in lymph nodes where CTLA4 neutralizes potentially autoreactive T-cells at the initial stage of naïve CD4 and CD8 cell activation [31]. Both CD28 and CTLA-4 can be stimulated by CD80 (B7-1) and CD86 (B7-2) ligands that are expressed on activated APCs, leading to T-cell proliferation and differentiation through the production of growth cytokines when there is an elevated CD28:CD80/CD86 ratio [42] or to dephosphorylation of T-cell receptor signaling proteins by tyrosine phosphatases [43], leading to T-cell inactivation and anergy, in the case of an increased CTLA-4:CD80/CD86 ratio [42].

Since CTLA-4 binds to CD80/86 with very high affinity, this receptor mediates immunosuppression by competing for CD28 and also by inducing CD80/86 removal from antigen presenting cells' surface [33]. For this reason, by blocking the interaction between CTLA-4 and CD80/86 ligands, CTLA-4 inhibitors can prevent T-cells exhaustion and boost the antitumor T-cell response [44]. Despite the demonstrated survival benefit of ipilimubab in patients with advanced melanoma, severe immune-mediated adverse effects, high cost, and modest response rates (ranging between 4% and 16% when used in monotherapy) [44] remain as the main impediments for its use.

On the other hand, PD-1 predominantly regulates previously activated T-cells at the later stages of an immune response [31], mainly within tissue and tumors [30,31]. The expression of this membrane receptor, which can be temporarily induced in activated CD8 T-cells, natural killer T-cells, or myeloid cells following the activation and stimulation of T-cell receptor by cytokines and interleukin, is constitutive in T-cells exhibiting the exhausted phenotype [23]. PD-1 binding to its ligand, PD-L1, promotes the dephosphorylation of T-cell receptor proximal signal components and leads to the inhibition of signaling pathways commanded by protein kinases including PI3K/AKT (phosphoinositide 3-kinase/Protein Kinase B), PTEN (phosphatase and tensin homolog), CK2 (casein kinase 2) [37], and RAS/MEK/ERK (mitogen-activated protein kinase MAPK/extracellular-signal-regulated-kinase), which decreases T-cell proliferation, survival, cytokine production, and other effector functions [23]. Thus, by interrupting the interaction between PD-1 and PD-L1, checkpoint inhibitors can restore antitumor immune responses and promote immune-mediated elimination of tumor cells [32]. Although nivolumab alone or combined with ipilimumab significantly improves the overall and complete response rates compared with ipilimumab alone in patients with metastatic melanoma [45], response rates to PD-1/PD-L1 blocking therapies only ranges between 20–38% among different tumor types [23], which implies that the majority of advanced stage patients cannot benefit from these therapies.

Despite the low response rates and immune-related adverse events in some cancer patients, both CTLA4 and PD-1/PD-L1 inhibitors have broadly demonstrated their value to boost potent and durable anti-tumor responses and to increase the average life expectancy for metastatic cancer patients [23].

#### **2. Breast Cancer Immunotherapy**

#### *2.1. First Approaches*

The first cancer immunotherapy treatments were based on the use of humanized monoclonal antibodies with the ability to bind and neutralize a targeted altered molecule expressed by cancer cells and on which their survival and proliferation depends. The approval in September 1998 of trastuzumab (Herceptin®, Genentech, Inc., South San Francisco, CA, United States) represented the release of the first antibody for the treatment of metastatic breast cancer patients with HER2 (Receptor tyrosine-protein kinase ERBB2, CD340) overexpression and/or gene amplification, which represented a milestone in the treatment of breast cancer. After trastuzumab, other different anti-HER2 monoclonal antibodies including lapatinib (Tykerb®, GlaxoSmithKline, Brentford, United Kingdom), neratinib (Nerlynx®, Puma Biotechnology, Los Angeles, CA, United States), gefitinib (Iressa®, AstraZeneca, Cambridge, United Kingdom), or afatinib (Giotrif®, Boehringer Ingelheim Pharmaceuticals, Inc., Ingelheim am Rhein, Germany) [8] as monotherapy or in combination with conventional treatments have contributed to increasing the number of therapeutic options for breast cancer patients.

Although the use of monoclonal antibodies targeting altered proteins has definitely improved the outcome of cancer patients, modest response rates (Table 4) and resistance development [46] remain as the major impediments for treatment success and require the search for new approaches apart from combined therapies, among which antibody-drug conjugates (ADC) such as the recently FDA approved ado-trastuzumab emtansine (Kadcyla®, Genentech, Inc., South San Francisco, CA, United States) [47] and T cell bispecific antibodies stand out among the most promising strategies for breast cancer patients [48].

**Table 4.** Approved humanized monoclonal antibodies for breast cancer treatment.

**Table 4.** *Cont.*

As a result of the latest studies in this field and in line with the encouraging long-term success of checkpoint inhibitors in the treatment of different tumors, distinct research groups have focused their efforts in developing analog treatments for breast cancer patients. In fact, as a result of the findings from the Phase III double-blind IMpassion130 trial (ClinicalTrials.gov ID NCT02425891), which reported a 40% reduced risk of disease progression or death in patients receiving atezolizumab plus nab-placlitaxel or placebo [58–60], in March 2019, the FDA approved the first checkpoint inhibitor immunotherapy drug, the anti-PD-L1 antibody atezolizumab (Tecentriq ®), in combination with chemotherapy (Abraxane®) for the treatment of triple-negative, metastatic breast cancer patients with positive PD-L1 protein expression [61]. However, despite this great milestone, modest complete response rates (7.1%, 95% CI, 4.9–9.9 and 10.3%, 95% CI, 6.3–15.6 in PD-L1 positive subgroup) and immune-mediated serious adverse events such as pneumonitis, hepatitis, colitis, and endocrinopathies that can cause treatment discontinuation [59] remain as notable impediments for the success of this treatment and justify the search of new therapeutic strategies.

#### *2.2. Mechanisms of Immune Evasion in Breast Cancer*

As stated above, tumor immune evasion can occur as a result of defective tumor-directed T-cell activation, deficient activated T-cell infiltration into the tumor microenvironment, or because of the tumor cell resistance to cytotoxic action of the immune cells [62].

#### 2.2.1. Breast Tumor Microenvironment

Immunogenicity is defined as the ability to induce a humoral and/or cell-mediated adaptive immune response. In fact, both the burden of tumor mutations and the load of neo-epitopes represent two of the factors that are linked to response to checkpoint inhibitors in different malignancies like melanoma or lung cancer [63]. However, although tumor neoantigens that are produced as a result of breast cancer cells' genomic instability can be recognized by the immune system and induce T-cell responses and antitumor immunity [62,64], the immunogenicity of breast cancer can be rather heterogeneous, depending to a large extent on the specific subtype of breast cancer [65].

In the particular case of HER2-positive breast tumors, gene profiling studies have shown that highly suspicious calcifications are associated with decreased immune system activity and *ERBB2* overexpression [66]. For this reason, breast calcifications would be useful not only in the radiological assessment of breast lesions [67], but also in the management of breast cancer patient candidates for immunotherapy. On the other hand, although estrogen receptor-negative and HER2-positive have shown evidence of immunogenicity [65], these types of inflammatory breast tumors are rare (1–5% of cases) [68] when compared to triple negative breast tumors, which are unique among breast cancer subtypes in having strong antigen expression [69] and high stromal and tumor-infiltrating lymphocytes, parameters with a strong prognostic and predictive significance to immunotherapy and chemotherapy [62,63,70,71]. Accordingly, triple negative breast tumors with high infiltration of tumor-associated macrophages have been found to have a higher risk of metastasis and lower rates of disease-free survival and overall survival, having been proposed as potentially useful prognostic markers for triple negative breast cancer patients [72,73].

Except for these immunogenic subtypes, breast tumors have historically been classified as immunologically silent [62] or "cold" tumors, characterized by the presence of low mutation and neoantigen burden and few effector tumor infiltrating lymphocytes, factors proposed as prognostic markers [62], and metastasis to lymph nodes correlation [74].

Since non-inflamed tumors represent a significant impediment to the success of T-cell-based immunotherapies, different studies have aimed their efforts towards developing new strategies to increase the presence of immune infiltrates and hence, to improve patient prognosis. Among these, in addition to directly causing cell damage [75], the use of local tumor hyperthermia has proven to be a valuable tool as an immunotherapy strategy for cancer [76] by boosting immune cell activation and increasing the sensitivity of tumor cells to anti-tumor immune responses by different mechanisms, including:

In this context, different studies are reporting promising results for hyperthermia as complementary treatment to surgery, chemo, radio, and immunotherapy in breast cancer patients [75,77–79]. However, convincing data about the benefit of the combination of hyperthermia with checkpoint inhibitors for breast cancer treatment should be provided by multicenter clinical trials in which related side-effects are also evaluated [79]. Likewise, radiation has also shown to increase mutational load of tumors, optimize antigen presentation, and decrease immune suppressors in the tumor microenvironment, priming the tumor for immunotherapy [71], which justifies additional studies in these fields.

Besides the presence of high tumor infiltrating lymphocytes, recognition of tumor cells is a critical step for the success of the immune response. In this regard, although estrogen has an immunoenhancing impact on the immune system [80] with an apparent effect in all major innate and adaptive immune cells [81], high levels of estrogens may interfere with HLA-II expression and IFN-γ signaling, with significant implications for tumor immune escape [82]. Estrogens are also well known to be a risk factor for breast cancer by enhancing the expression of genes involved in tumor cell survival and proliferation as well as growth factors including vascular endothelial growth factor (VEGF) [83], epidermal growth factor (EGF), insulin growth factor (IGF), fibroblast growth factor (FGF) [69,84], and their receptors [8]. Since estrogen presence in tumor microenvironment can also play a significant immunosuppressive

role by promoting tolerance of weakly immunogenic tumor cells [69], the use of antiestrogen therapies in combination with aromatase inhibitors could be a rational strategy to enhance the response to immunotherapies. However, although adjuvant hormonal therapy combined with HER2-targeted agents in hormone receptor-positive and HER2-positive breast cancer patients already represents a standard treatment, recent studies have shown that estrogen deprivation promotes transcriptional programs that favor immune evasion and increases PD-L1 expression in metastasis arising from breast cancer patients receiving adjuvant hormonal therapy for their local disease [85]. For this reason, the use of hormone-therapies in combination with PD-1/PD-L1 blocking immunotherapies should be thoroughly investigated. On the other hand, and for the reasons mentioned above, the application of conventional monoclonal antibodies targeting one or more growth factors would be a useful adjuvant to enhance the efficacy of breast cancer immunotherapy by improving APCs function [86,87].

#### 2.2.2. Changes in Breast Tumor Cells

Instead of loss of the targeted protein, resistance to cancer immunotherapies, such as monoclonal antibodies, is frequently due to the activation of alternate pathways [53] like immunosuppressive checkpoint pathways.

Among these, and largely due to the FDA approval of atezolizumab, blockade of the PD-1/PD-L1 pathway constitutes one of the most promising strategies for breast cancer immunotherapy. Despite this important addition to the number of therapeutic options available for metastatic breast cancer patients, it is important to note that the objective response rate achieved by atezolizumab was 53% versus 33% for the placebo group [88] and that to date, it has been approved for the treatment of the triple-negative subtype, which only constitutes 10–15% of breast carcinomas [89], with positive PD-L1 protein expression, which occurs in approximately 20% of breast cancers (mainly HER2-positive and triple negative) [62].

Similarly to the PD-L1 pathway, different randomized clinical trials are currently evaluating the effect of PD-1 inhibitors as monotherapy or in combination with conventional and non-conventional treatments [62,65] in breast cancer patients with results that although modest, are encouraging. In this respect, even though PD-L1 status remains the core predictor for anti-PD-1/PD-L1 therapies and patient selection [23], the validity of PD-L1 expression as a prognostic marker remains controversial [62] and justifies the need to develop new immunotherapy biomarker panels as well as new strategies to improve response rates.

Another major impediment to immunotherapy success in breast cancer patients is the selection of apoptosis-resistant cells, which constitutes one of the hallmarks of cancer [17]. Since both chemo- and immunotherapies directly or indirectly activate the cellular apoptosis machinery, tumor sensitivity to anti-cancer treatments will significantly depend on the level of expression of anti-apoptotic proteins [24] in general and, more specifically, on the existence of a pro-survival profile characterized by an increased ratio between anti-/pro-apoptotic proteins [24,90,91].

Provided that antiapoptotic proteins such as clusterin (APO-J) [91,92], BCL-2, BMF [24] as well as different pro-survival kinases [8] are frequently altered in metastatic breast cancer, the use of profiling techniques or systematic mapping of anti-apoptotic gene dependencies would be justified in order to effectively select those patients that could better benefit from combined treatments of protein inhibitors and immunotherapy. In this regard, different studies have already evidenced the need to use therapies with a combination of inhibitors targeting different anti-apoptotic proteins in order to achieve better clinical benefits and avoid the activation of alternate pro-survival pathways [8,23,24].

HLA-I expression on the surface of breast tumor cells, which is positively correlated with tumor-infiltrating lymphocytes, is essential for an effective cytotoxic response [93] and the subsequent success of T-cell mediated immunotherapies. For this reason, loss or changes in HLA-I expression, which is another of the hallmarks of cancer [17], also represent a significant impediment for breast cancer immunotherapy.

Total loss of HLA-I is found in 37% of in situ breast carcinomas, 43% of the primary tumors, and 70% of the lymph node metastases [94]. Since HLA-I expression in these tumors is related with a pro-death phenotype characterized by an increased proapoptotic *BAX*/antiapoptotic *BCL2* ratio [94], preliminary studies for patients' selection would be justified in order to ensure the success of immunotherapies in breast cancer patients.

In the case of triple negative breast tumors, HLA-I expression is variable, contributing when altered to the development of an immunosuppressive tumor microenvironment and immune escape [69]. However, since the activation of the HLA-II presentation pathway occurs in approximately 30% of triple negative breast cancer patients [95], associated with the presence of tumor infiltrating lymphocytes and improved prognosis [95,96], the expression of both receptors are factors that must be taken into consideration prior to the application of immunotherapy treatment.

With respect to HER2 overexpressing tumors, although this receptor-tyrosine kinase represents a valuable target for T-cell based immunotherapies, these tumors may escape cytotoxic T lymphocyte-mediated lysis by downregulating HLA-I, since the expression of both receptors is inversely correlated with breast cancer cells [69,97]. Similarly, in normal and cancerous breast tissues, HLA-I expression is inversely correlated with the expression of estrogen receptors, which may be related to the low level of tumor-infiltrating lymphocytes [93], and hence, with the failure of the T-cell cytotoxic response. It is worth emphasizing at this point that provided that agents targeting different protein kinases such as Mitogen Activated Protein Kinase (MAPK) or HER2 may increase HLA-I expression in breast cancer cells [97,98], the use of kinase inhibitors would be a valuable strategy to increase the antitumor effects of T-cell based immunotherapies. Similarly, strategies aimed at inducing HLA-II expression in tumor cells may be valuable tools to increase patient response and prognosis to such therapies [96].

#### **3. Conclusions**

#### *Where to go*

The improvement of the response rates for immunotherapies remains a great challenge for cancer treatment in general and for breast cancer in particular. Considering the relatively limited T-cell infiltration in most breast cancers, the development of novel strategies that are aimed to enable sufficient lymphocyte infiltration as well as to generate de novo T-cell responses that overlap the immunosuppressive tumor environment may be key to the success of this kind of therapy in breast cancer patients [63].

Among the different approaches that are currently being considered and despite their limited efficacy when delivered as a monotherapy [99], oncolytic viruses have demonstrated their safety [100] and ability in targeting and killing cancer cells as well as in stimulating immunotherapeutic effects in patients [101], positioning themselves as a promising strategy to increase treatment efficacy when used in combination therapy [99–101] and as a unique platform for personalized treatment of patients with advanced breast cancer [101].

Recent evidence on the role of tumor-associated macrophages in breast tumor growth, progression, treatment resistance, and metastasis has paved the way for the development of novel macrophage-targeted breast treatment strategies, such as the inhibition of macrophage recruitment, repolarization of tumor-associated macrophages to an antitumor phenotype, and the enhancement of macrophage-mediated tumor cell killing or phagocytosis, which are currently being evaluated in clinical trials [102]. Despite the promising results of preclinical studies, these therapies have proved limited clinical efficacy, therefore the development of new strategies that improve the effectiveness of such treatments is necessary.

On the other hand, results of adoptive cell immunotherapies, which includes Chimeric Antigen Receptor (CAR) T cell therapy and Tumor-Infiltrating Lymphocyte (TIL) therapy, based on the isolation of antitumor T cells from the primary tumor, further ex vivo expansion and activation, and subsequent reinfusion of such cells into the patient [65], are also proving valuable in both preclinical and clinical studies for the treatment of patients with breast cancer in general and HER2 positive tumors in particular [65,103]. In a similar line, next-generation sequencing and bioinformatic technologies have become a fundamental tool to facilitate neoantigen identification and the consequent improvement of personalized neoantigen-based translational immunotherapy studies [104], as well as to develop neoantigen vaccines to induce neoantigen-specific T-cell responses through the activation of antigen-presenting cells [105,106].

Results obtained with nanoparticles are no less important, having been postulated as the great asset to overcome the limitations of existing immunotherapy, being able to improve overall anti-cancer immune responses with minimal systemic side effects [107]. However, although nanoparticles in different in vitro and in vivo breast cancer models [108] have already proven their efficacy in defeating the immune-suppressive effect of tumor microenvironment [107] and drug resistance [108], as well as in delivering neoantigens and adjuvants to tumor cells [107], reducing the side effects of anticancer drugs [109], certain nanoparticles like titanium dioxide, silica, and gold complexes can lead to the formation of micrometer-size gaps in the blood vessel's endothelial walls and the intravasation of surviving cancer cells into the surrounding vasculature, which increases the risk of metastasis [110].

Another main drawback of immunotherapies, especially within a combined regimen, is the occurrence of immune-related side-effects affecting different organs such as the skin (rash, pruritus) or gastrointestinal tract (diarrhea, colitis) (Table 4). Although the severity of these immune-related adverse events are generally mild, life-threatening complications may also occur [111] and would end up, in many cases, in a reduction of the optimal treatment dose or medication discontinuation. For this reason, there is still a strong need for further research in order to develop biomarker panels that allow for patient selection and predict the response to immunotherapies and immune-related adverse events.

**Author Contributions:** M.G.-A. contributed to the conceptualization, writing and review and M.R. contributed to the supervision and review of the manuscript.

**Funding:** This research was partially supported by grants from Consejería de Salud de la Junta de Andalucía (PI 16/0298) and the European Regional Development Fund.

**Acknowledgments:** The authors wish to thank Adolfo Reque for his helpful advice during the preparation of this work.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Immunotherapy in Pediatric Solid Tumors—A Systematic Review**

#### **Raoud Marayati** †**, Colin H. Quinn** † **and Elizabeth A. Beierle \***

Division of Pediatric Surgery, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL 35294, USA<EMAIL_ADDRESS>(R.M<EMAIL_ADDRESS>(C.H.Q.)

**\*** Correspondence<EMAIL_ADDRESS>
† These authors contributed equally to this work.

Received: 16 November 2019; Accepted: 12 December 2019; Published: 14 December 2019

**Abstract:** Despite advances in the treatment of many pediatric solid tumors, children with aggressive and high-risk disease continue to have a dismal prognosis. For those presenting with metastatic or recurrent disease, multiple rounds of intensified chemotherapy and radiation are the typical course of action, but more often than not, this fails to control the progression of the disease. Thus, new therapeutics are desperately needed to improve the outcomes for these children. Recent advances in our understanding of both the immune system's biology and its interaction with tumors have led to the development of novel immunotherapeutics as alternative treatment options for these aggressive malignancies. Immunotherapeutic approaches have shown promising results for pediatric solid tumors in early clinical trials, but challenges remain concerning safety and anti-tumor efficacy. In this review, we aim to discuss and summarize the main classes of immunotherapeutics used to treat pediatric solid tumors.

**Keywords:** pediatric solid tumors; immunotherapy; chimeric antigen receptors; cancer vaccines; oncolytic viral therapy; immune checkpoint inhibitors; immunomodulation

#### **1. Introduction**

Immunotherapy is being popularized as an approach to target pediatric cancer. This treatment modality has proven effective in pediatric hematological malignancies such as acute lymphocytic leukemia (ALL), but there remains much to be learned before we can harness the potential of immunotherapy in the treatment of solid tumors. Here, we examine two broad immunotherapy approaches that may be utilized for the treatment of pediatric solid tumors: direct utilization of the immune system properties and immune system modulation. Within each of these categories, we discuss the benefits and challenges of each therapy for solid tumors and specifically highlight the effects on pediatric populations. The overarching objective of this review is to discuss immunotherapies that are currently in use as well as those with potential future use in the treatment of pediatric solid tumors.

#### **2. Direct Utilization of the Immune System**

#### *2.1. Oncolytic Virus-Based Therapy*

Oncolytic virus-based therapy is an emerging approach designed to target a variety of cancers. The concept for utilizing oncolytic virotherapy in cancer treatment originated from observations that patients with Hodgkin's lymphoma temporarily improved following a hepatitis infection [1]. Oncolytic viruses are constructed by altering the genetic profile of a viral vector to render the virus apathogenic while maintaining its ability to infect, replicate, and spread amongst host cells. Oncolytic viruses are also often engineered with specific receptors for cancer cells, rendering them target-specific and

potentially more efficacious [2]. The cancer cells will then behave as hosts and will be subjected to the oncolytic effects of the virus.

The benefit of oncolytic viral therapy is twofold: (1) it harnesses a virus's innate ability to lyse cancer cells and (2) it has the potential to trigger a cytotoxic immune response. In cancer cells, the upregulation of DNA replication assists in the production of viral progeny. The buildup of progeny results in lysis of the cells and infection of neighboring cancer cells [3]. This approach is effective for solid tumors, as viral delivery may be accomplished through direct intratumoral injections, resulting in direct killing of the malignant cells without producing severe systemic side effects or unwanted hepatic degradation of the virus, which may occur with systemic injection [4]. As a result of viral-mediated tumor cell lysis, pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and tumor-associated antigens (TAA) are released. These molecular signals initiate an immune response directed at the tumor even if this tumor has previously and successfully evaded the immune system [5]. These molecular signaling molecules allow for an intact immune system to utilize natural killer (NK) cells, dendritic cells (DCs), and other antigen-presenting cells (APCs) to directly target the cancer cells [6].

A variety of replicating viruses have been studied as cancer therapeutics, including adenoviruses, herpesviruses, paramyxoviruses, picornaviruses, poxviruses, reoviruses, rhabdoviruses, and togaviruses [7]. In pediatrics, variants of oncolytic Herpes simplex virus (oHSV) have been shown effective in a variety of solid tumors, such as glioblastoma, neuroblastoma, and sarcoma [8]. oHSVs have been genetically engineered to allow for selective uptake or replication of the virus by tumor cells but not healthy tissue [9,10]. Additionally, particular oHSVs have been engineered to produce chemokines or increased amounts of TAA, which stimulates and bolsters the immune system response directed toward the tumor [6,11].

There is great potential to use the immune response to target tumors through oHSV.  Following this innate immune response, an adaptive response may ensue [12,13]. Such a reaction could potentially lead to immune memory, negating the need for retreatment and theoretically, tumor relapse. This built-in defense mechanism could then take over for the destruction of most of the tumor. Barriers to this response, especially in solid tumors, include complete viral clearance, dense fibrosis surrounding the tumor, and the tumor microenvironment (TME) [5]. Combination therapy provides a means to overcome these barriers. In melanoma, combining T-VEC, a modified herpes simplex virus, with a MEK inhibitor (trametinib) produced an increased infiltration of CD8+ T cells into the tumor and a decreased tumor size in vivo [14]. A pre-clinical investigation of the TME in sarcoma showed that modulation of tumor-associated macrophages (TAMs) could potentiate an immune response. This study focused on Ewing sarcoma and oHSV. The investigators demonstrated that by targeting the TME with trabectedin, a currently approved chemotherapeutic, the M2 macrophage population was decreased, allowing for uninhibited viral infection by the rRp450 virus. They also showed that combining rRp450 with trabectedin in an in vivo xenograft model of Ewing sarcoma significantly decreased tumor volume and increased animal survival [15]. Such studies provide an avenue for future investigations in viral therapy.

Currently, there are no commercially available viral therapies that are routinely used for extracranial solid tumors in pediatric patients. T-VEC is a viral therapy available for the treatment of melanoma in adults, but studies have not specifically tested its use in a pediatric-only population [16]. There are only four clinical trials listed on www.clinicaltrials.gov that are actively recruiting for viral therapy in pediatric cancers: oHSV in cerebellar tumors (NCT03911388), oHSV in supratentorial brain tumors (NCT02457845), adenovirus in gliomas (NCT03178032), and an oncolytic poliovirus in gliomas (NCT03043391). Considering there are 28 actively recruiting clinical trials listed for adults, there is obvious room for expansion of this therapeutic approach for pediatric solid tumors.

#### *2.2. Antigen-Targeting Therapy*

Tumor antigen-targeting therapy, initially based on antibody–drug conjugates (ADC), utilizes specific monoclonal antibodies (mAbs) as an approach to target cancer cells and cause antibody-dependent cell-mediated toxicity (ADCC). Initially, these mAbs were used to assist in more direct drug delivery of chemotherapeutics [17]. In pediatrics, this model has been employed to target a tumor-specific antigen, GD2, which is a di-ganglioside expressed on neuroblastoma and osteosarcoma. In these tumors, a specific antibody targeting GD2 has been developed and is used in clinical treatment [18,19]. Additionally, a newer target for neuroblastoma treatment is anaplastic lymphoma kinase (ALK). An mAb targeting the ALK surface receptor, mAb30/49, led to decreased tumor cell proliferation and viability in vitro [20].

In order to increase the efficacy of these antibodies, researchers are examining techniques to evoke the activity of immune cells, such as NK cells, to involve them in tumor cell lysis. Investigators have demonstrated this concept using an anti-GD2 antibody, hu14.18K322A, combined with IL-15. This combination resulted in decreased tumor cell viability in vitro and growth in vivo, as well as in an increase in mature NK cells in the TME [21].

To take advantage of the immune system, antibodies have been developed in combination with proteins designed to elicit an immune-stimulating response. FDA-approved drugs, such as dinutuximab (Ch14.18, murine/human chimeric antibody to GD2) and naxitamab (hu3F8, a humanized mAB to GD2), are administered in combination with granulocyte-macrophage colony stimulating factor (GM-CSF) to boost the immune response [22]. A phase I clinical trial studied naxitamab with GM-CSF and its effects on resistant neuroblastoma (NCT012757626). A total of 31 children had evaluable disease. Of those, 14 (45%) had a complete or partial tumor response, 5 (16%) had stable disease, and 11 (35%) had progressive disease [23]. To study the role of the immune system in more depth, researchers have utilized dinutuximab in a patient-derived xenograft (PDX) model. Investigators have shown that following surgical resection of neuroblastoma in a PDX model, increased animal survival and decreased tumor invasiveness were achieved with the administration of dinutuximab and activated NK cells [24]. For the mAb to achieve such an immune reaction independently, a different technology must be applied.

Bispecific antibodies, unlike normal antibodies, elicit a cytotoxic T cell response against a specific tumor target. The Bispecific T Engager (BiTE) technology activates a T cell response by binding to CD3 on T cells [25]. The molecule combines the CD3 binding site with a second site that is tumor-specific. BiTE directly targets the cancer and limits damage to non-malignant tissue (Figure 1). The direct activation of cytotoxic T cells limits the need for other anti-cancer interventions. Currently, clinical trials with BiTE antibodies are limited to just two TAA: CD19 and EpCAM [26]. Of the two, only anti-CD19 (blinatumomab) has been investigated in children and it has been limited to hematologic malignancies. Blinatumomab was administered to children with relapsed/refractory ALL. In this phase I/II study, 39% of the children that received the determined dosage and treatment plan achieved a minimal residual disease response [27]. Elitzur and colleagues reported 11 pediatric patients with ALL who were treated with blinatumomab as a bridge to further therapy after suffering from severe chemotherapy toxicities. All 11 children went on to resume standard chemotherapy with an overall survival of 80% [28]. Further preclinical studies and clinical trials with other TAA for BiTE antibodies will be required before this promising technology may be translated for clinical use in the treatment of pediatric solid tumors.

**Figure 1.** A schematic representation of the Bispecific T-cell Engager (BiTE) technology. The BiTE antibody connects the CD3 binding site on T cells with a tumor-associated antigen (TAA) specific to tumor cells. This triggers T cell activation and cytokine release, ultimately resulting in an anti-tumor response. The anti-CD3 single-chain variable fragment (scFv, shown in purple) is shared by all BiTE antibodies. The target antigen-specific scFv (in light green) is different for each BiTE antibody and can recognize targets such as CD19 or EpCAM.

#### *2.3. Immune Checkpoint Inhibitors*

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), the first immune-checkpoint receptor to be targeted clinically, is expressed on the surface of activated T cells and transmits an inhibitory signal to T cells. Normal T cell activation requires the engagement of the T cell receptor (TCR)/CD3 complex and the CD28 co-stimulatory signal, which then leads to increased expression of the co-inhibitory signal, CTLA-4. CTLA-4 binds to B7 molecules (CD80 and CD86) with greater affinity, thus out-competes CD28 for their shared ligands, preventing T cell activation. CTLA-4 signaling is utilized by some tumor cells to evade T cell anti-tumor activity. Thus, CTLA-4 blockade potentiates effective immune responses against tumor cells [29]. CTLA-4 is also found in regulatory T cells (Tregs) and contributes to their inhibitory function. CTLA-4 blockade in Tregs results in their decreased immunosuppression [30].

Preclinical data suggest that pediatric solid tumors have high expression of CTLA-4. In a panel of 34 adult and pediatric tumor cell lines, including osteosarcoma, rhabdomyosarcoma, and neuroblastoma, CTLA-4 expression was found at different densities on 88% of the cell lines examined, with higher intensity of staining in osteosarcoma [31]. In addition, 20 pediatric patients, 11 with osteosarcoma and 9 with Ewing sarcoma, had significantly increased expression of CTLA-4 on both CD4+ and CD8+ T cells obtained from peripheral blood samples compared to healthy controls [32]. These findings indicate that targeting CTLA-4 may be useful in these pediatric tumor types.

Ipilimumab is a mAb directed toward CTLA-4 signaling. Ipilimumab is FDA-approved for the treatment of adults and children with unresectable or metastatic melanoma. Recently, a phase I clinical trial (NCT01445379) included a total of 33 patients aged 2–21 years with recurrent or refractory solid tumors treated with CTLA-4 blockade. In this study, ipilimumab was well tolerated and resulted in increased activation of cytotoxic T lymphocyte without increased infiltration of Tregs; however, no objective tumor regression was observed [33].

Programmed cell death receptor 1 (PD-1) and its ligands (PD-L1 and PD-L2) are also part of the immune checkpoint pathway. PD-1 plays a role in downregulating T cell activation, which leads to tumor tolerance, while PD-Ls inhibit cytokine production and anti-tumor lymphocytes in the TME [34]. PD-1 is also highly expressed on Tregs and, when engaged by its ligand, is thought to enhance the activity and proliferation of these cells [35].

Several preclinical studies examined the expression of PD-1 and PD-L1 in pediatric cancer subtypes, with conflicting results. Only 9% of 451 pediatric tumors expressed PD-L1 in at least 1% of tumor cells, with the highest expressors being Burkitt lymphoma (80%), glioblastoma multiforme (36%), and neuroblastoma (14%) [36]. Conversely, in another study of children with advanced melanoma, relapsed or refractory solid tumors, or lymphoma, 33% of 689 screened tumors were positive for PD-L1 expression [37]. Of note, PD-L1 staining was associated with inferior survival among neuroblastoma patients [36], and higher expression of PD-1 correlated with disease progression in patients with osteosarcoma [38].

Pembrolizumab, an anti-PD-1 antibody, is FDA-approved for the treatment of both adults and children with refractory Hodgkin's lymphoma. Nivolumab, another anti-PD-1 antibody, has shown responses in adult solid tumors [39,40]. In pediatric solid tumors, these therapies remain under investigation.  In a retrospective review of 10 children with recurrent or refractory brain tumors treated with nivolumab, 9 patients had radiographic disease progression. Three patients had partial response at the primary tumor site, of whom two had progression of metastatic disease [42]. In other small studies of nivolumab treatment in pediatric brain tumor patients, results were mixed. [43,44]. Currently, a phase I/II trial (NCT03585465) is assessing nivolumab in combination with chemotherapy in pediatric patients with refractory/relapsing solid tumors or lymphoma. Two other trials are evaluating nivolumab alone: NCT02992964 is a pilot study of nivolumab in pediatric patients with refractory/recurrent hypermutated malignancies, and NCT02901145 is evaluating nivolumab in progressive/relapsed pediatric solid tumors, including osteosarcoma, Ewing sarcoma, neuroblastoma, and rhabdomyosarcoma.

Dual checkpoint blockade is hypothesized to prevent immune escape and may be promising in the treatment of pediatric solid tumors. Combinations of CTLA-4 and PD-1 antibodies are currently being investigated [45]. In an implantable murine model of metastatic osteosarcoma, treatment with anti-PD-L1 antibody resulted in downregulation of PD-L1 expression and upregulation of CD80/CD86 expression on tumor cells, as well as upregulation of CTLA-4 on tumor infiltrating CD8+ T cells [46]. Furthermore, combination therapy of PD-1/CTLA-4 signaling blockade resulted in complete protection from metastasis in 50% of treated mice as well as in T cell memory protection against future tumor inoculation [46]. Currently, NCT02304458 is an ongoing phase I/II trial evaluating PD-1/CTLA-4 signaling blockade combination therapy in pediatric patients with relapsed/refractory solid tumors.

#### **3. Modulation of the Immune System**

#### *3.1. Tumor Microenvironment: Cancer-Associated Fibroblasts, Tumor-Associated Macrophages, and Myeloid-Derived Suppressor Cells*

Many non-tumor cells including macrophages and fibroblasts are present in the TME and affect the malignant potential of tumor cells. Cancer-associated fibroblasts (CAFs) and TAMs are two of the primary infiltrating stromal cells.

CAFs are activated fibroblasts that play an important role in promoting tumor growth, invasion, and angiogenesis [47]. In a study of 60 primary neuroblastoma tumors, increased CAFs were associated with significantly higher microvascular proliferation and Schwannian stroma-poor histopathology, both poor prognostic factors [48]. In addition, blocking CAF-derived prostaglandin E2 (PGE2) production with a small molecule inhibitor was shown to reduce neuroblastoma cell growth, impair angiogenesis, and reduce tumor growth in vivo [49]. Further, in a genetically modified murine lung carcinoma model, depletion of CAFs resulted in significant inhibition of tumor growth and enhanced anti-tumor

immunity [50]. While CAFs could be a potential therapeutic target in pediatric solid tumors, there are currently no methods suitable for clinical translation, and further studies are needed to guide the development of such stroma-directed therapy.

TAMs, which most closely resemble M2 macrophages, are major contributors to the TME. Whether TAMs promote or impede tumor growth is tissue-dependent. High infiltration of TAMs was first described in neuroblastoma and shown to be associated with worse prognosis [51,52]. On the contrary, TAMs play a beneficial role in medulloblastoma and induced tumor growth suppression in vitro as well as in various mouse models [53]. Furthermore, the presence of TAMs, detected by genome-wide mRNA profiling and immunohistochemistry, was shown to be associated with suppression of metastasis and improved overall survival in patients with high-grade osteosarcoma [54], thus providing a rationale for the use of macrophage-activating agents such as liposomal muramyl tripeptide phosphatidylethanolamine (L-MTP-PE). L-MTP-PE is a synthetic analog of a bacterial wall component that induces the activation of monocytes and macrophages in the TME, thereby promoting their anti-tumor activity [55]. Conflicting results exist regarding the utility of L-MTP-PE [56]. A report from the Children's Oncology Group (COG) analyzed whether the addition of L-MTP-PE would improve outcomes in patients with osteosarcoma and found a statistically significant improvement of the overall survival from 70 to 78% [57]. However, 91 patients with metastatic osteosarcoma were separately analyzed, with no significant survival difference seen with the administration of L-MTP-PE [58].

Cancer cells also recruit myeloid-derived suppressor cells (MDSCs) to the TME as a mechanism to successfully evade the immune system. MDSCs are a population of tumor-infiltrating cells with immune-suppressive and tumor-promoting activity. MDSCs suppress both adaptive and innate arms of immunity through direct inhibition of the cytotoxic functions of T cells and NK cells [59]. Inhibition of MDSCs in three different immunocompetent mouse models of neuroblastoma resulted in the inhibition of tumor growth [60]. Thus, immunotherapies aimed at eliminating this suppressor cell subset could be advantageous in targeting the TME by counteracting the tumor escape mechanism and resuscitating the immune system. There are currently no known such therapies for pediatric solid tumors.

#### *3.2. Cytokines and Growth Factors*

Cytokines and growth factors that influence immune cells' proliferation, phenotype, or function remain under investigation with respect to treatment of pediatric solid tumors.

An example of anti-tumor cytokine therapy involves interleukin 2 (IL-2). IL-2 is a gamma-c cytokine produced by T helper 1 cells that functions to activate T cell proliferation and facilitate the maintenance of NK cells [61]. Currently, IL-2 is FDA-approved for treating adults with renal cell carcinoma (RCC) and malignant melanoma [62]. In children with large refractory sarcoma or neuroblastoma, several phase I and II trials utilizing IL-2 as monotherapy have shown no measurable anti-tumor effects, and relapses occurred despite immune activation [63–65]. Of note, one of five children with RCC had a complete response which was consistent with the 10–20% response rate observed in adults [64]. However, IL-2 administered with alternating cycles of GM-CSF plus the mAb ch14.18 (dinutuximab) resulted in higher rates of event-free (66% versus 46%) and overall (86% versus 75%) survival after 2 years compared to standard therapy alone in children with high-risk neuroblastoma [18].

Alpha-interferon (IFN-α) is another cytokine known to activate cytotoxic T lymphocytes and NK cells [66]. IFN-α is FDA-approved for use in adults to treat malignant melanoma, chronic myelogenous leukemia (CML), hairy cell leukemia, and acquired immunodeficiency syndrome (AIDS)-related Kaposi sarcoma. A limited number of studies have evaluated the use of IFN therapy to treat pediatric solid tumors. High-dose IFN-α administered for 4 weeks followed by a lower maintenance dose for 48 weeks was feasible in children with resected stage III melanoma and was associated with less toxicity than in adults treated with the same regimen [67]. However, 2 out of 15 patients were taken off that study for recurrent disease during maintenance therapy [67]. A phase II study (NCT00041145) of pegylated

IFN-α in 32 children with diffuse intrinsic pontine glioma (DIPG) reported prolonged median time to progression without significant improvement of the two-year survival rate [68], concluding that monotherapy with pegylated IFN-α may not be adequate, and further evaluation for use in combination studies is needed. Recently, a phase II trial (NCT00678951) explored the effect of IFN-α in children with unresectable plexiform neurofibromas and found both clinical and radiographic improvements; weekly injections of IFN-α resulted in at least doubling of the time to progression [69]. Currently, a combination of pegylated IFN-α, chemotherapy, and surgery is being tested in a phase III COG trial to treat patients, including children over the age of 5, with high-grade osteosarcoma (NCT00134030).

The cytokine receptor activator of nuclear factor-κB ligand (RANKL) is a member of the tumor necrosis factor (TNF) family that, in addition to being expressed on the surface of osteoblasts, is released by activated T lymphocytes [70]. RANKL regulates bone metabolism and plays a role in the pathophysiology of bone metastasis. RANKL induces osteoclast activation, which then mediates bone resorption and release of growth factors, resulting in a cycle of bone destruction and tumor proliferation [71]. Denosumab, a RANKL antibody, inhibits this osteoclast-mediated bone destruction. It has been used in phase II clinical trials in adults with multiple myeloma and metastatic breast and prostate cancer where it suppressed bone resorption [72]. In a study of 40 patients including 14 children, RANKL was expressed in 75% of high-grade osteosarcomas, and its expression correlated with a more aggressive clinical course, poor response to neoadjuvant chemotherapy, and poor event-free survival [73]. Denosumab may thus have a utility in the treatment of osteosarcoma and is currently being evaluated in a phase II clinical trial (NCT02470091) in children with recurrent or refractory osteosarcoma.

TNF, a peptide produced by macrophages and lymphocytes, has cytostatic and cytolytic effects on tumor cells in vitro [74] as well as stimulates necrosis and tumor regression in vivo [75]. Therapies incorporating recombinant TNF have been limited by the development of systemic toxicities, including hypotension, hemorrhagic gastritis, hyperbilirubinemia, and elevated creatinine [76]. Recombinant TNF has been studied in combination with dactinomycin in a phase I trial in 21 patients with refractory malignancies, including sarcoma and Wilms tumor. Evidence of anti-tumor activity was observed in only three patients, including one with Wilms tumor. Based on the anti-tumor activity observed in that patient with Wilms tumor, a phase II trial evaluated the combination of TNF and dactinomycin in patients with relapsed or refractory Wilms tumor. The combination was well tolerated and resulted in complete response in 16% and stable disease in 26% of patients [77].

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is another member of the TNF superfamily. TRAIL activates death receptors expressed on tumor cells, such as TRAIL-R1 and TRAIL-R2, inducing apoptosis [78]. Osteosarcoma, Ewing sarcoma, and rhabdomyosarcoma cell lines that express TRAIL death receptor were found to be sensitive to TRAIL-mediated apoptosis [79,80]. Lexatumumab, an agonistic human mAB against TRAIL-R2, binds and activates TRAIL-R2. Lexatumumab was evaluated in a phase I trial (NCT00428272) in pediatric patients with recurrent or progressive solid tumors, including osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, soft tissue sarcoma, hepatoblastoma, and nephroblastoma [81]. While no patients experienced either a complete or a partial response, several showed evidence of anti-tumor activity. A patient with osteosarcoma demonstrated resolution of the clinical symptoms and positron emission tomography activity, ongoing for more than 1 year off therapy, while a patient with hepatoblastoma showed a dramatic biomarker response [81].

GM-CSF is a myeloid growth factor that stimulates hematopoietic stem cells to make granulocytes and monocytes. In acute myeloid leukemia (AML), GM-CSF led to sensitization of leukemic cells and enhanced the cytotoxicity effects of chemotherapy [82]. Inhaled GM-CSF was evaluated in three adolescents with pulmonary metastases from osteosarcoma and Ewing sarcoma (NCT00673179). There were virtually no toxicities, and a patient with Ewing sarcoma demonstrated a complete response [83].

Other cytokines studied include IFN-γ, which is produced by NK cells and T cells in response to viral and intracellular bacterial infections as well as during anti-tumor responses. IFN-γ is currently FDA-approved for the treatment of children with osteopetrosis and chronic granulomatous disease. In addition, INF-γ has shown activity against Ewing sarcoma in combination with a TRAIL agonist in preclinical models [84] and has potential for clinical translation.

#### *3.3. Chimeric Antigen Receptor T Cell Therapy*

Chimeric antigen receptor (CAR) T cell therapy has been rapidly expanding in pediatric cancer therapeutics. In this approach, autologous T cells are collected from the patient, expanded, and subsequently engineered to express CARs, which are designed to redirect T cells to a selected tumor antigen. This non-physiologic T cell activation bypasses the need for tumor antigen presentation to major histocompatibility complex (MHC) Class I molecules, which are often downregulated in cancer, and allows antigen-expressing malignant cells to be recognized and destroyed by the CAR-redirected T cells. Different generations of CAR T cells exist. The first generations carry a single-chain fragment variable region (scFv) or activation domain against a TAA [85], while the second and third generations involve the addition of one or two co-stimulatory molecules, such as CD28, CD137 (4-1 BB), and/or CD134 (OX-40), and show improved T cell proliferation and survival and anti-tumor effects [86–88].

The majority of studies involving CAR T cells in the pediatric population were aimed at hematological malignancies, with fewer designed for malignant solid tumors [89]. Neuroblastoma was the first pediatric solid tumor on which CAR T cells have been tested in clinical trials [90,91]. In a phase I trial using first-generation CAR T cells targeting GD2 in refractory neuroblastoma, there was a 45% response rate in patients with active disease. Three of 11 (27%) patients achieved complete remission, with 2 achieving sustained remission for more than 5 years [90]. Currently, third-generation anti-GD2 CAR T cells which integrate the CD28 and OX-40 costimulatory domains are undergoing a phase I study (NCT01822652) for patients with refractory neuroblastoma [92]. The development of CAR T cells targeting ALK has also been suggested. Although no clinical trials have yet been initiated, human anti-ALK CAR T cells were shown to eradicate ALK-positive neuroblastoma tumors in a xenogeneic immunodeficient murine model [93]. However, the efficacy of these CAR T cells was dependent on both target tumor antigen and CAR receptor density [93].

HER2/Neu, which is highly expressed in medulloblastoma, osteosarcoma, and nephroblastoma, has also been incorporated into CAR T cells [94,95]. HER2-specific CAR T cells efficiently recognize and eliminate tumor cells even with modest levels of HER2 expression [96,97] and have been tested in a preclinical model of osteosarcoma [98]. In a phase I clinical trial utilizing anti-HER2 CAR T cells in 19 patients with advanced pediatric sarcoma (NCT00902044), 4 had stable disease for 3 to 14 months, and the median overall survival was 10.3 months [99].

Other CAR T cells being evaluated in clinical trials include those targeting interleukin 13 receptor alpha (IL-13Rα), which is shown to be overexpressed in gliomas and other pediatric brain tumors [100,101]. IL13Rα2-specific CAR T cells targeted and killed high-grade glioma cells and glioma stem-like initiating cells in vitro [102], as well as caused the regression of established human glioblastoma orthotopic xenografts [103]. Currently, no trials utilizing IL13Rα2-specific CAR T cells are actively enrolling pediatric patients.

While dramatic clinical responses were seen in clinical trials, significant potential toxicities were associated with the use of CAR T cell therapy. Cytokine release syndrome (CRS) has been the most commonly described severe toxicity and is characterized by fever, tachycardia, hypotension, and hypoxia. Reports of CRS ranged from mild flu-like symptoms to life-threatening multi-organ system failure [104]. This constellation of inflammatory symptoms results from the release of cytokines from the CAR T cells and other immune cells. A variety of neurotoxicities have also been reported with CAR T cell therapy, ranging from somnolence, tremors, and seizures to cerebral edema and death [104]. Models utilizing serum cytokine levels after CAR T cell infusion are being developed to predict those at risk for severe CRS or neurotoxicity and may guide future interventions with immunosuppression or cytokine-directed therapy [105]. Approaches to ameliorate CRS or neurotoxicity while maintaining treatment efficacy include directly targeting specific cytokines, such as IL6 blockade by tocilizumab [106], as well as the use of an inducible caspase suicide safety switch that may be activated, leading to programmed cell death to prevent unanticipated toxicities [107]. The latter has been tested in a clinical trial for pediatric patients and led to the elimination of 90% of CAR T cells within 30 minutes of the infusion [108]. Another type of toxicity associated with CAR T cell therapy is agammaglobulinemia, which may be corrected with gammaglobulin replacement.

#### *3.4. Natural Killer Cell-Based Immunotherapy*

NK cells have been investigated as potential immunotherapeutics due to their anti-tumor effects, through either direct cytotoxicity or antibody-dependent cellular toxicity. A major component of NK cell target recognition depends on the surveillance of human leukocyte antigen (HLA) class I molecules by killer immunoglobulin-like receptors (KIRs) [109]. KIRs are expressed on the surface of NK cells and transmit immune inhibitory signals to maintain tolerance to NK cells. Cancer cells without an inhibitory HLA ligand may trigger NK cell activation.

The potential for the therapeutic application of NK cells was primarily tested in hematologic malignancies, such as AML and ALL [110]. In these studies, reduced risk of relapse and improved survival were observed after allogeneic hematopoietic stem cell transplantation (HSCT) when HLA ligands against the inhibitory KIRs present in the donor were absent in the recipient. HSCT has also been proposed as a potential curative alternative in children with refractory solid tumors, such as Ewing sarcoma [111], neuroblastoma [112], melanoma [113], and hepatoblastoma [114]. Perez-Martinez et al. suggested that the clinically beneficial graft-versus-tumor (GVT) effect seen after HSCT may be mediated by donor–recipient inhibitory KIR–HLA mismatched NK cells [115]. That study examined three children with refractory solid tumors and observed a clinical response in the two patients with a KIR–HLA mismatched donor during the time when NK cells were the major lymphocyte population. In addition, the degree of tumor response appeared to correlate with the number of KIR-activating receptors [115].

A few ongoing early clinical trials are investigating the role of autologous and allogeneic NK cells in pediatric solid tumors. NCT01875601 is employing ex vivo activated and expanded autologous NK cells with recombinant human IL-15 in children with brain tumors, sarcoma, Wilms tumor, and rhabdomyosarcoma after lympho-depleting chemotherapy. Two trials (NCT01576692 and NCT01857934) are exploring the safety and feasibility of allogenic NK cell infusions from haploidentical donors in children with high-risk neuroblastoma in combination with the humanized anti-GD2 antibody (hu14.18K322A) and standard chemotherapy. NCT01287104 is assessing the feasibility and toxicity of infusing escalating doses of donor-derived activated NK cell donor lymphocyte infusions (NK-DLI) following HLA-matched T cell-depleted (TCD) peripheral blood stem cell transplant (PBSCT) in patients with metastatic or recurrent pediatric solid tumors. NCT00640796 was recently completed. This phase I study was designed to determine the safety of infusing expanded NK cells, obtained from a patient's family member with partial HLA mismatch, into pediatric patients with Ewing sarcoma family of tumors (ESFT) and rhabdomyosarcoma. The results of this trial are not yet available.

The efficacy of NK cell-based immunotherapy may be reduced by numerous factors such as limited in vivo proliferation and the immunosuppressive milieu of the TME. Furthermore, tumor cells develop various strategies to evade NK cell attack or to impair the activity and function of NK cell therapy. For example, tumor cells often upregulate the expression of KIR ligands, such as HLA-G [116]. HLA-G may inhibit the proliferation and cytotoxicity of NK cells [117]. Ectopic HLA-G expression on Ewing sarcoma suppressed the activity of GD2-specific CAR-expressing NK cells [118]. In addition, blocking of HLA-G on tumor cells in patients with chronic lymphocytic leukemia (CLL) increased their susceptibility to NK cell-mediated cytotoxicity [119]. Strategies to augment the anti-tumor efficacy of NK cells, prolong their survival and persistence in vivo, and restore their functions from exhaustion in the TME will maximize the effects of this novel therapy. Furthermore, NK cells have not been associated

with significant off-target effects, graft-versus-host disease, or CRS [120], making this therapy an attractive modality to explore.

#### *3.5. Cancer Vaccines*

Vaccines are some of the oldest means for modulating the immune response. The idea behind this therapy is that an exposure to a pathogen will allow for the generation of an adaptive immune response toward future re-exposure to that pathogen. For cancer, this same idea has also been explored in hopes of generating an anti-tumor response and cell-mediated immunity [5]. In comparison to other immunotherapies, this manipulation of the immune system shows promise specifically for pediatric solid tumors but has had limited study.

Anti-cancer vaccines typically exploit DCs. These antigen-presenting cells serve an important role as a bridge between the adaptive and the innate immune response, allowing for both an active and a passive attack on the tumor [121,122]. Different mechanisms used to stimulate T cell responses from DCs include mRNA strands, cell surface receptors, and lysed intracellular proteins. Researchers proved this point by pulsing DCs with sarcoma cell lysate and priming with cytokines. These DCs were administered to mice for immunization. They found these cells adept at producing a primary T cell response as well as significantly decreased pulmonary metastasis of the sarcoma tumor cells [123]. In pediatrics, a rhabdomyosarcoma cell line known as M3-9-M was grown in vivo and tested similarly with a vaccine. The authors were able to demonstrate a decrease in tumor growth in vivo, and upon depletion of CD4+ and CD8+ T cells, a lack of tumor cell response, indicating the necessity of a T cell response for anti-tumor effects [124].

In clinical trials, vaccines have been an area of focus for the treatment of gliomas, neuroblastoma, sarcoma, and Wilms tumor. In one clinical trial, dendritic vaccines were administered to pediatric patients with solid tumors, with one patient achieving a significant decrease in tumor size, and two patients showing undetectable disease. The tumor lysates were tested for immune response. Compared to pre-vaccine samples, the post-vaccine tumor lysates had a significantly higher level of IFN-γ, thus indicating the effectiveness of this vaccine at producing an immune response [125]. This study also demonstrated that dendritic cell-based vaccines could be administered in the outpatient setting and were not associated with significant toxicities in children [125]. Many of the future directions of the use of dendritic cell-based vaccines are toward improving efficacy through further immunomodulation [121]. Other clinical trials are studying the combination of vaccines with chemotherapeutics. A phase I/II trial for relapsed or refractory neuroblastoma and sarcoma used decitabine and DC/MAGE-A1, MAGE-A3, and NY-ESO-1 peptide vaccines (NCT01241162). Using CD137 as a T cell marker, 6 out of the 10 patients given the vaccine had a T cell response. Of those six patients, one had a complete tumor response, while one remains disease-free two years after the trial [126,127]. Such trials are the groundbreaking work that is needed to further implement these therapies and determine what is most effective in the pediatric population.

#### **4. Conclusions**

Immunotherapeutics with either direct utilization or modulation of the immune system provide novel treatment approaches for the treatment of children with solid tumors (Table 1). Although these therapies have shown promising clinical results, they are currently utilized in a limited number of pediatric cancer diagnoses. A more generalized pediatric use will require further studies to firmly establish the safety and treatment efficacy of these approaches and identify ways to integrate them with current conventional treatment regimens for a greater impact in pediatric solid tumors.

**Table 1.** Immunotherapy clinical trials for pediatric solid tumors discussed in this review.

#, number; HSV, Herpes simplex Virus; DIPG, diffuse intrinsic pontine glioma; NK, natural killer; hu3F8, humanized 3F8; mAB, monoclonal antibody; GM-CSF, granulocyte-macrophage colony stimulating factor; CTLA-4, cytotoxic T-lymphocyte-associated antigen-4; PD-1, programmed cell death receptor 1; IFN, interferon; RANKL, receptor activator of nuclear factor-κB ligand; TRAIL-R, tumor necrosis factor-related apoptosis-inducing ligand receptor; CAR, chimeric antigen receptor; RMS, rhabdomyosarcoma; rhIL-15, recombinant human interleukin 15; DC, dendritic cell.

**Author Contributions:** Writing the manuscript, R.M. and C.H.Q.; Review and editing, E.A.B.

**Funding:** All work was performed at the University of Alabama at Birmingham. This work was funded, in part, by National Institutes of Health T32 CA229102 (R.M.), and T35 HL007473 (C.H.Q.).

**Conflicts of Interest:** The authors declare that there are no conflicts of interest regarding the publication of this paper.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Immune Dysfunctions and Immunotherapy in Colorectal Cancer: The Role of Dendritic Cells**

**Sandra Gessani 1,\* and Filippo Belardelli <sup>2</sup>**

Received: 13 September 2019; Accepted: 27 September 2019; Published: 3 October 2019

**Abstract:** Colorectal cancer (CRC), a multi-step malignancy showing increasing incidence in today's societies, represents an important worldwide health issue. Exogenous factors, such as lifestyle, diet, nutrition, environment and microbiota, contribute to CRC pathogenesis, also influencing non neoplastic cells, including immune cells. Several immune dysfunctions were described in CRC patients at different disease stages. Many studies underline the role of microbiota, obesity-related inflammation, diet and host reactive cells, including dendritic cells (DC), in CRC pathogenesis. Here, we focused on DC, the main cells linking innate and adaptive anti-cancer immunity. Variations in the number and phenotype of circulating and tumor-infiltrating DC have been found in CRC patients and correlated with disease stages and progression. A critical review of DC-based clinical studies and of recent advances in cancer immunotherapy leads to consider new strategies for combining DC vaccination strategies with check-point inhibitors, thus opening perspectives for a more effective management of this neoplastic disease.

**Keywords:** colorectal cancer; dendritic cells; immunotherapy; pathogenesis; risk factors

#### **1. Introduction**

Colorectal cancer (CRC) is one of the major leading cause of cancer-associated mortality worldwide, thus representing an important public health issue, with a great impact in terms of human suffering and costs for the clinical management of patients [1]. The rate of CRC incidence is particularly high in populations living a Western lifestyle, but it is currently increasing in other geographic areas, including low income countries, thus representing a global health challenge [2].

The pathogenesis of CRC exhibits a great level of complexity, being characterized by several multi-step disease events, associated with the accumulation of both genetic and epigenetic alterations of the genome. In fact, the development of CRC is a long process taking several years to progress from barely detectable small neoplastic foci to adenomas and subsequently to malignant carcinomas endowed with metastatic behavior [3].

CRC is characterized by a high heterogeneity given the remarkable genomic instability [4]. Moreover, there is evidence that exogenous factors, such as lifestyle, diet, nutrition, environment and microbiota, contribute to the pathogenesis of CRC, also influencing non neoplastic cells, including immune cells, and leading to further heterogeneity [5,6].

Host immune dysfunctions are important factors contributing to CRC development. Indeed, a significant impairment of the host anti-tumor immunity has been reported during initiation of CRC mostly relying on escape mechanisms adopted by transformed cells to create a favorable growth environment [7–9]. During the initial stages of neoplastic transformation and progression, several changes occur within the tumor microenvironment to initially promote neoplastic cell proliferation, subsequently leading either to tumor progression and metastasis or to an immune-mediated cancer

inhibition. In particular, the tumor microenvironment can dictate the recruitment of inflammatory and immune cells playing complex roles in either controlling tumor growth or inducing a chronic inflammation status, thus promoting CRC progression by induction of immune suppressive mechanisms [10,11].

Today, an ensemble of data support the statement that inflammation plays an important role in CRC pathogenesis and progression [10]. The low-grade chronic inflammation characterizing obesity, a major risk factor for CRC development, and the anti-inflammatory drug benefits in lowering CRC risk and retarding intestinal tumors in ulcerative colitis patients provide compelling evidence for a link between inflammation and cancer [12]. In this regard, diet is nowadays recognized to play a key role in CRC initiation and progression due to its potential to contribute to a chronic inflammatory condition, either locally in the adipose tissue (AT) or systemically by regulating a variety of immune and inflammatory pathways. In addition, diet strongly controls the composition of the intestinal microbiota that not only maintains the immune homeostasis but can also be involved in colorectal carcinogenesis [13–15].

Information stemming from both mouse models and studies in patients points to a key role of immune cells and soluble factors with immunosuppressive activity in the CRC disease process [11]. Among the many cells of the immune system exerting important functions in the host response to neoplastic transformation, dendritic cells (DC) deserve a special attention, since these cells, which are an highly heterogeneous cell population present in the blood, in the lymphoid organs as well as in the tumor microenvironment, represent the major actors in linking innate and adaptive anti-cancer immunity [16].

The clinical management of CRC is firstly based on surgical resection, but the optimal treatments in patients with advanced metastatic disease is still matter of debate. Different protocols of chemotherapy and immunotherapy, including combination therapies, have been used in metastatic patients with variable success [17]. Of note, CRC is one of the first human cancer where a stringent correlation was found between tumor infiltrating CD8<sup>+</sup> T cells and clinical outcome [18], thus supporting the rationale for evaluating the efficacy of immunotherapy protocols in this neoplastic disease. In fact, the clinical research for implementing the management of CRC patients in an advanced disease stage often included the use of cytokines (i.e., IFN, IL-2), adoptive cell therapy and DC-based vaccines, but variable and inconclusive results were obtained so far. Today, we are facing a momentum of enthusiasm on cancer immunotherapy in the light of the emerging great clinical impact of check-point inhibitors (CPI) [19]. However, major research challenges are to fully understand the mechanisms of the response and to obtain clinical efficacy in non-responding and poorly responding patients by designing more sounded combination therapies. While the role of immunosurveillance in the control of CRC growth and progression is assumed to be of great importance [17,18], patients appear to be resistant to the blockade of immunological checkpoints with monoclonal antibodies (mAbs) specific for cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed cell death 1 (PDCD1, best known as PD-1) and the PD-1 ligand CD274 (best known as PD-L1), with the exception of a minority of subjects characterized by microsatellite instability (MSI) lesions [20,21]. This has been considered as an apparent paradox and an intriguing issue demanding further research efforts for fully understanding the mechanisms of the resistance to CPI and developing new and more effective therapeutic strategies [22]. In this review, we intend to specifically address the role of DC in the pathogenesis and progression of CRC as well as in the response to immunotherapy. A special attention will be given to the role of microbiota, obesity-related inflammation, diet and host reactive cells, including DC, in CRC pathogenesis, then discussing how we can translate the research progress in this field in strategies of prevention and management of CRC. Likewise, we will review the ensemble of studies reporting the variations of different DC subsets in CRC patients and their correlation with disease stages and clinical outcome. Lastly, we will provide a brief critical overview of the results of DC-based clinical trials in CRC patients, discussing new perspectives for their combination with CPI and some current research challenges for the management of this neoplastic disease.

#### **2. Diet, Inflammation and Microbiota in the Pathogenesis of CRC**

#### *2.1. Diet and Obesity as Important Factors in the Pathogenesis of CRC*

Excess adiposity is associated with increased incidence of several cancers and represents an important indicator of survival, prognosis, recurrence and response to therapy in CRC. Notably, patterns and trends in CRC incidence and mortality correlate with geographical location, societal and economic changes and their increase may reflect the obesity epidemic and the adoption of more Western lifestyles. Both genetic and a range of environmental, largely modifiable, lifestyle factors play an important role in CRC etiology. Among these, the links between body weight, dietary patterns and CRC risk are some of the strongest for any type of cancer with profound implications for prevention strategies. It is now well-recognized that CRC risk is highly modifiable through lifestyle, particularly diet and physical activity; recent reports suggest that up to 47% of CRC cases could be prevented by staying physically active, maintaining a healthy body weight and eating a healthy diet (available at https://www.wcrf.org continuous-update-project 2017). Obese subjects have a 1.5–3.5-fold increased risk of developing CRC compared with lean individuals, and epidemiologic evidence indicates that abdominal rather than overall obesity may be more predictive of CRC risk [23]. Multiple changes arising in condition of chronic positive energy balance are likely to contribute to the increased CRC risk and worse outcomes in obesity. In particular, during the progression to obesity, the AT undergoes profound structural and functional modifications [24] tightly coupled with dramatic changes in the immune cell repertoire and functions [25,26], that shift the balance of cell subsets and soluble mediators toward a pro-inflammatory profile. Growing evidence indicates that the chronic low-grade inflammatory state characterizing obesity contributes to the impairment of immune functions, thus representing a key determinant in the development of obesity-related morbidities including cancer [27]. Furthermore, lipids, especially fatty acids (FA), the main components of AT, are recognized to play an important role not only in obesity development but also in the interplay between excessive adiposity and development of associated diseases [28]. In this regard, qualitative changes, rather than quantitative, in the FA composition of AT have been reported to influence tissue dysfunctions and are associated with an enhanced STAT3 activation and concomitant down-regulation of anti-inflammatory pathways such as PPARγ and its downstream target adiponectin [29].

The metabolic disturbances characterizing obesity lead to chronic immune activation as unraveled by the presence of elevated levels of plasmatic inflammatory markers in obese subjects [30]. The bulk of immune alterations observed in obesity may provide an explanation for the higher rate of vaccine failure and infectious disease [31]. In this regard, the white AT, particularly visceral fat, is now well-recognized as a complex immunocompetent organ, homing adipocytes and resident immune cells exhibiting secretory as well as immunological, metabolic and endocrine regulatory activities. Furthermore, AT is a medium- to long-term indicator of FA dietary intake. Among the different factors potentially influencing visceral AT microenvironment and immune cell distribution, the relative composition of ω3/ω6 polyunsaturated fatty acid (PUFA) might play a pivotal role, since these molecules are capable to markedly modulate inflammation and to influence immune functions [32,33]. In this regard, visceral fat adipocytes from obese and CRC subjects exhibit distinct secretory and ω6 PUFA profiles characterized by a prevalence of pro-inflammatory factors and inflammation-promoting FA [34]. Of note, we recently reported that obese and CRC subjects share inadequate dietary habits and altered lipid metabolism, suggesting that the quality of the diet consumed, regardless the quantity of energy intake, is an important aspect to preserve human health [35]. CRC and obese subjects were found to be more prone to follow a saturated fatty acid (SFA)-rich diet and exhibit a reduced content of monounsaturated fatty acids (MUFA) (especially in oleic acid). The composition of AT, in particular in FA, may thus represent an important determinant in shaping the immune cell phenotype and in influencing processes/events occurring in distal tissues that may set the basis for CRC carcinogenesis.

#### *2.2. Relationships Between Diet, Microbiota and Immune Dysfunctions in CRC Pathogenesis*

The key role of diet in CRC initiation and progression as well as in prevention is not only linked to the capacity of some nutritional components to contribute to a chronic inflammatory condition by regulating a variety of immune and inflammatory pathways, but also to strongly control the composition of the intestinal microbiota. The human microbiota, a collection of commensal microorganisms colonizing gastro-intestinal, genitourinary, oral, respiratory and cutaneous tracts, interacts with the host in different ways and contribute to many important processes such as nutrient absorption, metabolism, tissue development, immunity and tumorigenesis [13–15]. It is now well-known that microbiota is influenced by several factors of genetic, dietary and environmental nature. Likewise, some of the metabolic effects of diet rely on gut microbiota. Examples of how diet can influence microbiota emerged from studies in populations consuming different diets, leading to the conclusion that dietary patterns defined as "healthy" (e.g. Mediterranean diet) are associated with higher microbial richness [36]. Among the best associations between diet and gut microbiota are dietary fibers, polyphenols and fats. In particular, different dietary fats may exert different effects on gut microbiota (diversity, alterations of specific microorganisms and their functions) with metabolic consequences such as regulation of systemic low-grade inflammation [37].

In obesity, the gut microbiota displays distinctive features and most studies have demonstrated a reduction in diversity and richness—termed dysbiosis—which has been associated with low-grade inflammation, increased body weight and fat mass, as well as type 2 diabetes (T2D). Nevertheless, the exact microbial signature of a healthy or an obese gut microbiota is still matter of debate. Dysbiosis is associated with a large array of diseases including cancer, where it is implicated in different ways [6]. In addition, microbiota can be directly oncogenic by favoring local mucosal inflammation or systemic metabolic/immune dysregulation or can act indirectly by virtue of its capacity to modulate anti-tumor immunity or the efficacy of anti-cancer therapy. In this regard it is of interest that the abundance of *Akkermansia muciniphila* has been positively associated with the antitumor effect of PD-1 blockade in epithelial tumors [38] and hepatocellular carcinoma [39]. Interestingly, decreased amounts of this bacterium have been linked to obesity, insulin resistance, T2D and other cardiometabolic disorders in rodents and in humans [40]. In addition, alterations of fecal and mucosal microbiota with reduction of bacterial diversity have been reported in CRC patients at different cancer stages [41,42].

In the following chapters, we provide a brief overview of the role of DC in the regulation of inflammatory and immune responses, of their functional changes in CRC patients and of their exploitation in immunotherapy protocols against CRC.

#### **3. The Role of DC in the Regulation of the Inflammatory and Immune Responses in CRC**

DC represent a heterogeneous group of innate immune cells endowed with the unique capacity to initiate and coordinate the immune response. They are professional antigen presenting cells (APC) and comprise a variety of subsets, of both myeloid and lymphoid origin, as either resident or migrating cells, in lymphoid and non-lymphoid organs. They are able to recognize, capture and process antigens and to present them to naïve T lymphocytes. DC are nowadays recognized as a family comprising several subtypes that differ in ontogeny, gene expression profile, anatomical location, phenotypic and functional features [43]. In this regard, consensus has been recently achieved on the recognition of five major DC types: plasmacytoid DC (pDC), type 1 conventional DC (cDC1), type 2 cDC (cDC2), Langerhans cells and monocyte-derived DC (MoDC). In the steady state, DC are largely present as immature cells exhibiting a high capacity to capture antigen, and a low expression of co-stimulatory molecules and secretion of effector cytokines. The exposure to different stimuli including microorganisms or damaged cells/tissues promotes DC activation, a functional state characterized by a decreased capacity to capture antigen, enhanced expression of MHC class I and II antigens as well as costimulatory molecules, active production of effector cytokines and migration to lymph nodes, where they interact with naïve CD4<sup>+</sup> and CD8<sup>+</sup> T lymphocytes.

It is currently thought that DC play an important role in presenting tumor antigens to T cells and in shaping an antitumor immune response, which may result in an effective control of tumor growth [43,44]. However, many studies have revealed how the phenotype and functions of these cells can markedly be affected by several molecular and cellular actors playing complex and even opposite roles within the tumor microenvironment. As an example, there is plenty of evidence indicating that tumors can not only suppress DC maturation, but can also induce the generation of DC endowed with immunosuppressive activities [45,46].

Dietary habits and excessive adiposity can not only influence cancer growth but also shape host immune response [47]. Myeloid DC, but not pDC, have been described to accumulate in the subcutaneous AT of obese subjects. While the number of CD11c+/CD141+DC is the same in lean with respect to obese subjects, the number of CD11c+/CD1c<sup>+</sup> cells positively correlates with the body mass index (BMI). This accumulation parallels an enhanced presence of Th17 lymphocytes in AT, suggesting a role of DC infiltrating AT in the regulation of tissue inflammation and Th17 cell expansion [31]. Of note, studies carried out in mouse models of obesity suggest that the presence of CD131<sup>+</sup> DC in the AT of lean mice can be important for the local expansion of T regulatory cells providing anti-inflammatory signals to maintain AT homeostasis [31]. Interestingly, the exposure of immune cells to visceral adipocyte conditioned media from obese and CRC affected subjects favors IL-10 production, reduces the immunostimulatory activity of DC and hampers their capacity to generate γδ T cell-mediated responses induced ex vivo, further highlighting the existence of a regulatory/suppressive AT microenvironment in both obesity and CRC [34]. Furthermore, distinct alterations of the immune cell repertoire in the periphery with respect to the AT uniquely characterize or are shared by obesity and CRC [48].

#### **4. Changes in the Phenotype and Function of DC in CRC Patients**

Several groups have described qualitative as well as quantitative changes of DC in the blood as well as in the tumor microenvironment of CRC patients at different stages of disease and their possible correlation with the clinical response of patients [46]. The interpretation of the overall results is not always easy, since contradictory data were reported in some cases, possibly due to differences in the clinical settings as well and in the methodologies used to identify specific DC subsets. Here, we will restrict our review to the discussion of only some studies, selected in view of their special potential clinical relevance. The possible correlations between the presence and maturation phenotype of tumor-infiltrating DC with the patient prognosis and clinical response have been investigated by Gulubova and colleagues [49]. These authors found that the presence of CD83<sup>+</sup> mature DC was lower in the tumor stroma of patients in an advanced disease stage. In general, we can state that negative correlations between the detection of these tumor infiltrating DC and the number of lymph node metastases as well as the survival time of CRC patients were frequently documented [49–51]. Notably, by comparing human primary CRC specimens with respect to normal colon mucosa, Schwaab and co-workers found that the number of infiltrating mature DC was higher in the CRC samples, while the DC density in metastases was markedly lower than in CRC primary tumors [52]. Of interest, Michielsen and colleagues reported that tumor conditioned-media from cultured human CRC tissue can impair DC maturation process, possibly by releasing chemokines and other soluble factors capable of inhibiting IL-12p70 secretion by DC [53,54]. Of note, Bauer and colleagues [55] reported that infiltration with mature DC was more elevated in MSI-high (MSI-H) tumors as compared to microsatellite-stable CRC. This observation is interesting since it can provide an explanation for the preferential clinical response of MSI-H CRC patients to novel immunotherapies, including CPI [20,21]. Some groups have also investigated the number and phenotype of DC in the peripheral blood of CRC patients with respect to healthy individuals as well to the disease stage and progression [56–59]. In particular, it has been found that the number and functions of different blood DC subsets were impaired in CRC patients, demonstrating that the magnitude of these effects positively correlated with the disease stage and prognosis [59]. Similar results were obtained by Orsini et al. [57], who described a significant reduction

of the DC number in total and advanced stage-CRC patients compared to healthy controls, and reported that this reduction was totally recovered after complete tumor resection, further supporting the concept of the importance of systemic immunosuppressive effects exerted by the tumor toward circulating blood immune cells. Of interest, some authors have also reported that the reduction in DC was mostly due to changes in pDC population [57].

A useful in vitro model to investigate the biology of DC and the mediators and mechanisms important in shaping their functions is represented by MoDC, generated from monocytes by in vitro treatments with GM-CSF and various cytokines, such as IL-4, IFN and other activation/maturation factors. Thus, some published studies where the phenotype and functions of MoDC from CRC patients were compared to those detectable in control subjects are available [56,60–62]. In particular, Orsini and colleagues showed an impaired in vitro differentiation of CRC patients' monocytes into immature DC, compared to healthy subjects [57]. Of note, CRC MoDC exhibited a reduced expression of costimulatory molecules and an impaired ability to present antigens to allogenic T lymphocytes and to stimulate proliferation, together with an immunosuppressive cytokine profile, mostly characterized by increased IL-10 and reduced IL-12 secretion [57]. Of interest, it was reported that the maturation status of the MoDC from CRC patients was phenotypically and functionally superior to the in vivo blood DC recovered from the same individuals. This observation somehow supported the potential value of using MoDC from CRC patients for clinical studies of cancer immunotherapy [63].

#### **5. DC and Immunotherapy of CRC**

Since the early study by William Coley in cancer patients treated with killed bacterial vaccine in 1891, for more than 120 years, the history of cancer immunotherapy has been characterized by alternate cycles of optimism and discouragement. The clinical use of certain cytokines (i.e., IFN-α and IL-2), the subsequent identification of the first set of human tumor antigens, the progress on cancer vaccines and in the development of protocols based on adoptive cell therapy have all represented important milestones in the field of cancer immunotherapy. However, it is only in recent years that we have registered a fundamental progress, which today leads to consider cancer immunotherapy as the latest revolution in cancer therapy. This is mainly due to the impressive results achieved in patients with different type of malignancies after treatment with CPI [19]. With regard to CRC, however, only modest clinical effects have been observed so far in patients treated with these new immunotherapy drugs (including anti-CTLA-4, anti-PD1 and anti-PD-L1 antibodies) [64], which instead proved to be highly effective in other human malignancies (including melanoma, Hodgkin lymphoma and non-small lung cell cancer).

In view of their crucial role in linking innate and adaptive antitumor immunity, DC have extensively been used in cancer immunotherapy clinical trials over the last two decades [44,65]. Notably, the large majority of DC-based studies involved the use of patient-derived DC generated from peripheral blood monocytes differentiated in vitro by the addition of cytokines (generally GM-CSF and IL-4), loaded with tumor-derived antigens by different experimental procedures and subjected to a further step of in vitro maturation, before their injection in therapeutic vaccination protocols [65]. In 2011, the registration of the DC-based Provenge vaccine for patients with prostate cancer led to a transient momentum of special optimism for the clinical development of DC-based cancer vaccines. However, in the following years, this cancer vaccine was not further developed and, in view of the limited response observed in hundreds of clinical trials, the clinical development of DC-based vaccines was regarded with a lower attention with respect to that devoted to new emerging tools in cancer immunotherapy, such as CPI and CAR-T adoptive cell transfer. There are recent and comprehensive reviews reporting the results of DC-based clinical trials, which also critically discuss the major challenges for their clinical development [44,65]. While the lack of any relevant toxicity represents a good starting point, there are still several critical issues to be addressed, including identification of the optimal DC to be used, reliable criteria to characterize the quality and potency of these cell products, the source/loading of tumor antigens, the modalities of injection and the possible combinations with other drugs/treatments

to increase their clinical efficacy. Today, we are facing a renovated interest in the development of new generation DC-based vaccines, as a result of a better understanding of the DC biology and of the discovery of new immunomodulatory molecules expected to enforce cancer immunotherapies [66–68].

In considering new and potentially more effective DC types to be used in cancer immunotherapy protocols, we may consider to use DC generated by monocytes by a short-term in vitro exposure to IFN-α and GM-CSF [69]. In fact, these DC (named as IFN-DC) exhibit a unique attitude to take-up tumor apoptotic bodies and induce a potent tumor specific T cell immunity in preclinical models [70] as well as in cancer patients, as suggested by results in pilot clinical trials where IFN-DC have been inoculated intratumorally in patients with metastatic melanoma [71] and indolent lymphomas [72].

Table 1 reports the main published data of clinical trials based on the use of DC in CRC patients. The general messages stemming from an overview of the main results published so far can be summarized as follows: (i) the large majority of studies reported results of pilot phase I-II trials in metastatic CRC patients with a relatively small number of patients; (ii) different methodologies were used for the in vitro generation of DC-based vaccines from monocytes, including the use of various cytokines and other activation/maturation factors, rendering difficult the comparison of the results; (iii) different methods of tumor antigen loading of DC were utilized and, in a few cases, unloaded DC were used; (iv) the regimen and route of DC administration as well as the number of DC injected markedly differed among the published studies; (v) in some cases, the patients were also treated with either conventional (for instance chemotherapy) or additional experimental cell therapies; (vi) there were marked differences in the protocol design as well as in the immunomonitoring methods to evaluate DC-induced immunogenicity. All this suggests that, even though some of these trials have represented important proof-of-principles for the lack of toxicity and potential efficacy of DC-based vaccines in inducing antitumor immune responses in CRC, the translation of the possible use of DC for the development of new-generation strategies of CRC immunotherapy needs further and coordinated research efforts.

Abbreviations: N: Patients' number; TCL: tumor cell lysate; rCEA: recombinant CEA; s.c.: subcutaneous; i.d.: intradermal; i.v.: intravenous; i.t.: intratumoral; CIK.: cytokine-induced killer cell. ˆ6 out of 12 patients injected with DC-loaded TCL, 6 with CEA.

One of the major reason for the limited response of CRC to the immunotherapy is thought to be represented by the immunosuppressive tumor microenvironment which generally occurs in patients with advanced disease. As a matter of fact, the major challenge for developing an effective protocol of cancer immunotherapy is indeed to counteract the several and complex immunosuppressive mechanisms activated in the tumor microenvironment of cancer patients. The role of several cancer-induced immunosuppressive mediators in CRC prognosis and treatment response has been reviewed elsewhere [9–11]. These mediators include cells endowed with immunosuppressive activity, such as regulatory T cells and certain macrophage populations, as well as soluble factors. Notably, modulations of the local production of certain cytokines as well as in their response can play a role in shaping the type of antitumor response [85]. Of interest, loss of type I IFN receptor has recently been identified as an important key factor linked to tumor microenvironment immunosuppression in CRC patients [86]. Thus, we may assume that a local production of and response to cytokines such as type I IFN can exhibit a beneficial role in shaping the response towards an effective immune control of CRC.

Today, in the new era of CPI, major research challenges are to fully understand the mechanisms of the response and how to increase the clinical efficacy in poorly responding patients by designing more sounded combination therapies, which may also include DC. Of interest, a recent study showed that an effective antitumor response to anti-PD1 mAbs strictly requires the occurrence of intratumoral DC [68]. Likewise, some recent studies have added further evidence underscoring a previously underestimated role of intratumoral DC in the tumor microenvironment in mediating the clinical response to immunotherapy regimens in cancer patients [87,88]. Of interest, the intratumoral DC involved in the generation of an antitumor response to anti-PD1 mAbs were characterized as mature DC producing high levels of IL-12 [68]. Notably, IFN-DC, which undergo a rapid and complete maturation after peripheral blood lymphocyte co-cultivation, are high producers of IL-12 [89] and therefore may represent good candidates for potentiating anti-PD1-based therapies. Of interest, we had previously shown that IFN-DC are highly efficient APC in inducing both CD8<sup>+</sup> and CD4<sup>+</sup> T-cell-mediated responses against the colon tumor antigen-1 in CRC patients at different stages of the disease [90]. Thus, on the basis of the overall preclinical and clinical data on IFN-DC obtained by our group [69–72,89,90], we consider these DC as valuable autologous cell products for the development of new-generation DC products to be used in clinical trials in CRC patients. For these DC-based therapies, we may envisage therapeutic scenarios where CRC patients are treated with autologous DC, either as unloaded APC injected intratumorally (endogenous tumor vaccination) in combination with agents either inducing or enhancing tumor cell death [71,72], or as in vitro antigen loaded DC, and subsequently injected with anti-PD1 antibodies or other CPI to increase the antitumor response in selected combination therapies.

#### **6. Conclusions**

CRC represents one of the human malignancies where promotion of prevention strategies can play a major role in reducing cancer development and tumor burden and progression. In fact, primary prevention, based on special attention to reduce exposure to environmental and lifestyle risk factors (including diet and physical exercise) is indeed of great importance for reducing CRC incidence, with enormous impact in terms of public health and reduction of costs for the national health services. In addition, in view of the long and multi-step processes involved in CRC development, strategies of secondary prevention, including the promotion of the use of early diagnostic platforms, can be very important for prevention and control of CRC. In spite of all this, the optimal therapeutic management of patients with metastatic CRC remains an important issue in clinical oncology. While surgery and chemo-radiotherapy interventions continue to represent essential therapeutic options depending on the stage of the disease and the clinical settings, immunotherapy has recently emerged as a powerful tool for tertiary immune prevention.

Recently, we have learned new rationales and modalities for combining different immunotherapy regimens with both conventional and target therapies. Likewise, we have recently started to understand

the importance of sex- and gender-specific differences in several pathologies including cancer. In fact, gender disparities have been reported in different aspects that can collectively influence CRC pathogenesis and therapy [91]. Thus, CRC incidence, outcome and survival as well as microbiota composition exhibit a different trend in men and women [92]. Likewise, some of the main risk factors for CRC, such as obesity and lifestyle-related aspects (i.e. diet and physical activity), are strongly linked to gender [93,94]. Worth of note, differences in the immune response have also been reported in women and men [95]. However, at the moment there are no studies describing gender differences in DC functions in CRC patients. Studies on gender related immune dysfunctions in CRC taking into consideration the DC biology are expected to contribute to our understanding of the pathogenesis and to the clinical management of this neoplastic disease.

Figure 1 summarizes the main DC dysfunctions observed in a specific non-neoplastic tissue relevant for CRC pathogenesis (i.e., AT), blood and tumor microenvironment of CRC patients, along with the risk factors playing a role in the disease process. It also depicts some main strategies and challenges for the development of DC-based immunotherapy strategies in CRC patients. Such strategies are aimed at considering DC either as in vivo targets for tumor antigen delivery and/or for recruiting and activating DC within the tumor microenvironment, or as autologous cell products generated from monocytes by different in vitro manipulations and then reinfused into the patients. In any case, new generation immunotherapy strategies should consider what is the impact and possible role of DC, which represent important cell actors in CRC pathogenesis and antitumor immune-based control.

**Figure 1.** A schematic representation of the main DC dysfunction in adipose tissue, peripheral blood and tumor tissue highlighting the main strategies to restore DC functions and to enhance anticancer immune response.

How to reverse DC dysfunctions occurring at different disease stages and in various tissues in CRC patients still remains a complex issue deserving further research efforts. In principle, intervention strategies for restoring DC functions in the very early stages of the disease could also be considered, but we need to reach a better knowledge of the role of this highly heterogeneous cell population in the pathogenesis and progression of CRC. The recent advances on the cross-talk between gut microbiota and human health and on the potential of lifestyle, food components and/or dietary patterns

to modulate this functional interplay has opened new perspectives for diet-based interventions in the modulation of the antitumor immune response [96].

With regard to the still critical issue of implementing the management of advanced metastatic CRC, a great importance is currently given to combination therapies, since we now have a much better knowledge on how different therapeutic tools and strategies should be associated. With the advent of next-generation sequencing methodologies, we have now the unprecedented ability to identify tumor, host, and microbial genomes. The growing application of these novel technologies to finely characterize patient's tumor and driver mutations as well as the immune repertoire for evaluating genetic responses to current immunotherapies has opened new ways to maximize patient benefits through cancer precision medicine strategies. We conclude by stating that, taking into account some recent findings [68,87,88], new generation DC-based strategies can represent a promising added value for enhancing the response to the new therapeutic regimens, including CPI, in CRC patients.

**Author Contributions:** S.G. and F.B. conceived, designed and wrote the review article.

**Funding:** This research was funded in part by Italian Association for Cancer Research (AIRC), grants 14185 (SG) and 16891 (FB).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **The Interplay of Autophagy and Tumor Microenvironment in Colorectal Cancer—Ways of Enhancing Immunotherapy Action**

#### **Evangelos Koustas 1, Panagiotis Sarantis 1, Georgia Kyriakopoulou 1, Athanasios G. Papavassiliou <sup>1</sup> and Michalis V. Karamouzis 1,2,\***

Received: 25 March 2019; Accepted: 11 April 2019; Published: 14 April 2019

**Abstract:** Autophagy as a primary homeostatic and catabolic process is responsible for the degradation and recycling of proteins and cellular components. The mechanism of autophagy has a crucial role in several cellular functions and its dysregulation is associated with tumorigenesis, tumor–stroma interactions, and resistance to cancer therapy. A growing body of evidence suggests that autophagy is also a key regulator of the tumor microenvironment and cellular immune response in different types of cancer, including colorectal cancer (CRC). Furthermore, autophagy is responsible for initiating the immune response especially when it precedes cell death. However, the role of autophagy in CRC and the tumor microenvironment remains controversial. In this review, we identify the role of autophagy in tumor microenvironment regulation and the specific mechanism by which autophagy is implicated in immune responses during CRC tumorigenesis and the context of anticancer therapy.

**Keywords:** Autophagy; colorectal cancer; immunotherapy; tumor stroma; tumor microenvironment

#### **1. Introduction**

Colorectal cancer (CRC) is the third most frequently diagnosed malignancy and the second leading cause of cancer-related deaths in the U.S.A. and worldwide [1]. By 2030, the estimated global burden of CRC is expected to reach more than 2.2 million new cases and 1.1 million deaths [2]. Despite significant advances in standard of care therapies, the 5-year survival rate for patients diagnosed with metastatic CRC remains very poor, at approximately 12% [1]. Among others, autophagy is a major mechanism which is strongly associated with tumorigenesis in different types of cancer, including CRC.

The mechanism of autophagy has been identified as a catabolic process with an essential role to digest proteins and dysfunctional cellular organelles [3]. Numerous steps related to autophagy include membrane trafficking vesicles, essential autophagy proteins, a double membrane organelle, which is called an autophagosome, and fusion with lysosomes to create the autophagolysosome. Autophagolysosome is a fundamental structure responsible for degrading the luminal content [4]. The role of autophagy is extended from cellular homeostasis to tumor development [5,6].

Many genes and proteins are crucial for the initiation and progression of autophagy. Genes, like *Beclin-1*, *LC3*, *ATG5*, and *ATG6*, have a crucial role for autophagy from normal function to CRC, where these genes have been reported with high expression. Furthermore, these autophagy gene-markers are associated with a more aggressive CRC phenotype [7].

Various morphological changes characterize the autophagy process. In the first step of autophagy, which is called initiation or nucleation, the phagophore, a double membrane structure, is formed through the activation of the class-III PI3K-Beclin-1 complex. Elongation is the next step in the autophagy process. This step is characterized by the arising of the phagophore from different double membrane organelles, such as the Endoplasmic Reticulum (ER), Golgi, and mitochondria. The phagophore starts to enclose the cytosolic cargos, leading to the formation of the autophagosome. The formation of the phagophore is highlighted by different Atgs, p62/ SQSTM1 (an adaptor protein responsible for the docking of specific cargoes), and the lipid-modification of LC3I to LC3II. The maturation step and the following fuse step include the autophagosome formation, which eventually fuses with lysosomes to form autolysosomes. Finally, during the degradation step, lysosomal/vacuolar hydrolases digest autolysosomal products and release them in the cytosol [4].

Over the last years, many studies have been conducted that support the dual role of autophagy in CRC. Autophagy appears to be responsible for maintaining the energy homeostasis in cells, which is required for several cellular functions, such as proliferation [8], angiogenesis, migration [9], and EMT (epithelial-mesenchymal transition) phenotype [10]. Autophagy is identified to be upregulated in a hypoxic region of already established tumors, where the energy demands are increased [11]. Moreover, cancer cells of high graded tumors appear to be addicted to autophagy to maintain their energy balance [12,13]. Numerous studies report the impact of autophagy in cancer patients' response to chemotherapy. Increasing levels of autophagy are linked with inadequate response to chemotherapeutic drugs and dismal survival rates [14].

In different cancer types, such as CRC, a single-nucleotide polymorphism, in autophagy-related genes, like *ATG16L1*, is associated with a reduction of autophagy and a significant negative predictive value for patients' survival with metastatic disease [15,16]. Besides, monoallelic deletion of other crucial autophagy genes, such as *Beclin-1*, which leads to autophagy reduction, has been identified in several diseases, such as cancer and Alzheimer's [17–19]. Other studies highlight the positive impact of monoallelic deletion or total loss of other genes, such as *ATG5*, *ATG7*, and *ATG4C*, in cancer development [20]. In addition, *KRAS*, an essential oncogene in CRC development, is strongly associated with autophagy [21]. Cancer cells of *KRAS*-dependent tumors use autophagy in order to support the growth of cancer cells under stressful conditions in hypoxic regions of tumors [8]. All these studies highlight the dual role of autophagy as a tumor promoter or tumor suppressor mechanism. The accumulation of dysfunctional proteins and cellular organelles through the reduction of autophagy increases the risk for malignant transformation. Furthermore, low basic levels of autophagy are required for cell survival as was identified through experiments with a knockout of different autophagy genes, such as *ATG* genes, *Beclin-1*, or *AMBRA1* [22,23]. Autophagy is responsible for recycling cellular components and producing energy and pro-oncogenic factors [24]. Different stage of tumors, anti-cancer treatment, mutations in *ATGs*, and oncogenes are closely associated with autophagy and its controversial role in tumorigenesis. Further study is required in order to address the link between autophagy and hallmarks of cancer.

Furthermore, the increasing levels of autophagy, in these regions, are strongly associated with the regulation of the immune response in the tumor microenvironment [11,25]. The microenvironment of different malignant tumors, including CRC, is characterized by numerous cell types (including immune, tumor, and other types of cells). All these stroma cell types utilize a different extent of autophagy. Therefore, focusing on autophagy and its role in the tumor microenvironment for the discovery of novel anti-cancer therapeutic targets should be further elucidated [11,26]. The role of autophagy in developing an immune response against tumor cells is far more complex. Therefore, autophagy may be a promising therapeutic target in combination with other anti-neoplastic drugs and immunotherapy in the context of this unique cellular composition of the tumor microenvironment.

#### **2. The Major Players in the Tumor Microenvironment**

For years, solid cancers were considered as a mass of homogenous cancer cells [27]. Cancer evolution and resistance to treatment is caused by tumor heterogeneity. Over the past decade, it has become increasingly clear that there is a wild diversity of cells with tangled and branching pedigrees in the same tumor. One section of a tumor might be dense with cells containing a particular oncogene mutation, whereas another section might have vastly different mutation backgrounds driving their growth [28]. Tumors should be perceived as separate tissues with a different and more complex cellular network with specialized or dedifferentiated malignant cell types, fibroblasts, tumor stem cells, immune, and endothelial cells. This complex network is characterized as a tumor stroma with unique potential for anticancer therapy [29].

#### *2.1. The Heterogeneity of the Tumor Microenvironment*

The vast majority of solid tumors are composed of not only malignant cells, but also of fibroblasts. It is widely accepted that tumorigenesis is a multistep process, the progression of which depends on a sequential accumulation of mutations within tissue cells. Moreover, tumor initiation is associated with the activation of different stromal, endothelial and mesenchymal cells, fibroblasts, and immunogenic cells [30,31]. It is well known that tumor heterogeneity is associated with the more aggressive phenotype and a lack of response against anti-cancer therapy in different types of cancer, including CRC [32].

#### *2.2. The Role of T-Lymphocytes*

The major effectors of the immune response against tumor cells are the cytotoxic CD8+ T-lymphocytes or T-cells (CTL). The abundance of T-cells is a decisive prognostic factor for the response of chemotherapy and immunotherapy in cancer patients especially at early stages of the disease, where patients have a strong effector T cell response and more frequently present a high Immunoscore [33,34]. CTLs are responsible for killing hostile cells, such as tumor cells [35]. Type 1 of T-helper cells (Th-1) regulates the activation of CTL and Th-2 initiates humoral immunity [36]. In many studies, the activation of the immune system and tumor-infiltrating lymphocytes (TILs) are used for the grading of the tumor and as a putative prognostic marker for CRC patients. The characterization is based on TILs, tumor invasion, spread to the lymph nodes, and the tumor staging system [33,35].