Immunotherapy offers numerous advantages in comparison to the conventional standard cancer treatment available nowadays [52,53]. Of those, when appropriately stimulated, tumor-specific immune cells can target a microscopic disease, disseminate metastasis, and long-term control might completely remove cancer due to the memory cells [54–56].

Although immunotherapy is efficient to treat different types of cancer, still there is a certain challenge in delivery of immunotherapeutic agents which is expected to be resolved using NPs [57]. A major goal of the utilization of NPs in cancer immunotherapy is to improve therapeutic index by enhancing deliver of immunotherapeutic agents directly to the site of interests only, enhancing accumulation and potency at a region of interest, while simultaneously minimize the dose-dependent systemic toxicity [58]. Unlike delivering chemotherapeutic agents to tumor cells, which necessitates a high dose of nanoformulated drugs to kill all the target cells to be effective, lower concentrations of immune-stimulating drugs can be used to initiate an immune cell or organs (such leukocytes or lymphoid organs) response [59]. For example, Schmid et al. developed antibody-targeted NPs that bind to CD8+ T cells in the blood, lymphoid tissues, and tumors of mice [60]. Synthesized NPs encapsulated with a SD-208, TGFβR1 inhibitor, or a TLR7/TLR8 agonist. Both in vitro and in vivo mice studies showed, successful targeting of PD-1+ T cells in the circulation and in the tumor. Compared to the free drugs, NPs-encapsulated SD-208 enhances survival of mouse bearing colorectal cancer. In addition, synthesized NPs enabled PD-1-targeted delivery of a TLR7/8 agonist to the TME and tumor-infiltrating CD8+ T cells were increased. Overall, this result shows that targeting tumor-infiltrating immune cells in the blood, rather than direct tumor cell targeting, is a better way to improve immunotherapeutic localization in tumors and to stimulate an antitumor response.

In summary, NPs based cancer treatment has a numerous advantage compared to conventional cancer therapy due to: (1) Nanoscale size with several surface characteristics to enhance drug accumulation at the site of interest via EPR effects, (2) Target tumor cells via active targeting which will minimize off-target normal cell toxicity, (3) Protect a therapeutic payload (such as protein, gene, small peptide) from biological degradation, (4) Enhance solubility of hydrophobic drugs and improves there bioavailability, (5) enhance in vivo stability and bioavailability, (6) prevents premature drug

release, (7) used as theranostic, combined imaging and therapeutic applications and (8) stimuli, internal or external, programmed to release its cargo at the site of interest.

Furthermore, due to their effectiveness at eliciting cellular and humoral immune responses, NPs can be designed to activate the immune system that could form a gorgeous basis for cancer vaccine development [61,62]. As the result, several NPs are synthesized to deliver different types of immunotherapeutic agents to enhance their therapeutic efficacy, and some of them already shown satisfactory results in clinical trials [63].

#### 2.1.1. Nanoparticles as the Carrier of Immunotherapeutic Agents

Over the last few decades, numerous studies and a large number of papers (Figure 1) have been published on nano-based therapies for cancer treatments. In the last two decades, the total number of papers related to 'nanoparticle + immunotherapy' on PubMed approximately doubled every two years which will be expected a rise similarly in the future.

**Figure 1.** A number of published papers for the last two decades (i.e., 2000–2020) by searching on PubMed using key words "nanoparticles + immunotherapy".

NPs should be precisely designed to target region of interest preferentially from site of administration (common vaccine administration routes are mucosal or parenteral) in order to enhance the efficacy of immunotherapeutic agents [64,65]. NPs targeting lymphoid tissues, where the majority of immune cells are concentrated, would enhance the efficacy of immunotherapeutic agents due to direct access to immune cells [66–69].

Depending on their physicochemical characteristics, including particle size, hydrophobicity, shape, and surface charge, NPs can directly drain to the nearest lymph node, or stay in the injection site and attract migratory DC or macrophages [70]. Several kinds of the literature show that NPs with particle sizes > 100 nm tend to form depot and taken up by APCs and then draining to lymph nodes [71,72]. However, NPs with moderate particle size < 100 nm drained to lymph nodes via lymphatics and retained relatively for a long time, while NPs with small particle size (<10 nm) drain to blood capillaries [73,74]. Regarding the surface charge, negatively charged NPs drain to the lymph node was reported due to charge repulsion with negatively charged ECM, while, cationic NPs tend to form a depot, taken up by peripheral and migratory APCs or gradually draining to lymph node [75]. NPs with nearly neutral charge exhibited potential vector for tumor antigen because they targeted the draining lymph nodes after subcutaneous injection. However, they were weakly immune-stimulatory. In addition, the presence of PEG (PEGylation) on Surface of NPs significantly enhanced large particle size (~200 nm) drain to lymph node and uptake by DCs [76].

Moreover, targeted and stimuli sensitive biocompatible NP can be synthesized to deliver immunotherapeutic agents' in their native conformations to increase antigen uptake, processing, and presentation. For example, NPs can be synthesized for facilitating the cytosolic delivery of antigens, increasing cross-presentation via the MHC-I pathway, and thus inducing cytotoxic T-cell responses. In addition, in the drug delivery system, it is possible to load or conjugate two or more than two drugs in the single nanocarriers as the co-delivery which will minimize dose related toxicity and enhance the activation of the immune response. For example, Song et al. developed the combined delivery of immunogenic chemotherapy and PD-L1 trap fusion protein using liposomal NPs [77]. They reported that PD-L1 trap is produced transiently and locally in the TME and oxaliplatin (OxP) boosts anti-PD-L1 therapy against murine colorectal cancer and exhibited reduced toxicity compared with non-nanoformulated ones (i.e., free PD-L1 antibodies and oxaliplatin).

In general, NP based drug delivery systems encompass a wide variety of nano-scale size materials including inorganic and Organic NPs in different forms [16].

#### 2.1.2. Antigens and Adjuvants Delivery to Antigen Presenting Cells (APCs)

APCs used as the link between innate and adaptive immune responses by interacting with T cells [78]. APCs are primarily used to recognize and present tumorigenic antigens on their surface via MHC complexes to T cells to initiate an effective adaptive response [79]. However, due to enzymes susceptible of antigens in the body, they are not easily transferred to APCs which decreases its immunogenicity. Hence by using NPs it is possible to overcome this limitation. NPs can encapsulate and deliver cancer antigens to APCs without tumor antigen degradation by the intracellular enzyme. In addition, nanoformulated antigens are more efficiently taken up and processed by APCs than soluble vaccines to amplify T-cell responses due to intradermally or subcutaneously injected NPs drain to lymph nodes, in which APCs are in closeness to T cells [80].

Shen et al. encapsulated ovalbumin (OVA) antigen in the PLGA and deliver successfully to primary mouse bone marrow-derived dendritic cells (BMDCs) [81]. Their result showed that the MHC class I presentation of PLGA-encapsulated OVA stimulated T cell IL-2 secretion at a 1000-fold lower concentration than soluble antigen and 10-fold lower than antigen-coated latex beads.

Kranz et al. precisely designed RNA-lipoplexes (RNA-LPX) NPs with the particle size of ~200–320 nm, by optimally adjusting lipid: RNA ratio to precisely target DC using intravenous injection (Figure 2) [82]. The lipoplexes protect antigen-encoding RNA degradation by ribonucleases. In addition, RNA-LPX enhances cellular uptake and expression of the encoded antigens. Moreover, two transient waves of IFN-α were observed after the NP vaccine injection that led to better T-cell responses and produced vigorous and long-term antitumor effects.

Several kinds of literature showed that the targeted delivery of NPs formulated antigen into DC would enhance antigen presentation to T cells [83–86]. For example, Cruz et al. designed Pegylated PLGA NPs functionalized with TLR3/7 ligand to encapsulate OVA to target surface receptors of DC (i.e., CD40, DEC-205, and CD11c) to accomplish an effective cytotoxic T cell response [87]. In vitro cellular uptake study showed that TLR3/7 ligand targeted NP was more taken up by DC compared to non-targeted NP. Furthermore, high expression of IL-12, IFN-γ, and co-stimulatory molecules were observed in the ligand targeted NP in comparison to non-targeted NPs. Moreover, in vivo vaccination studies showed that ligand targeted NP consistently showed higher efficacy than non-targeted NP in stimulating CD8+ T cell responses.

Some research finding shows that immune response will be enhanced by co-delivering of adjuvants along with tumor antigens due to efficient antigen cross-presentation and vigorous T-cell response for tumor immunotherapy [88–91]. For example, Kuai et al. synthesized high-density lipoprotein-mimicking nanodiscs for co-delivery of CpG adjuvant and neoantigens [92]. They reported that, synthesized nanodiscs elicited up to 47-fold greater frequencies of neoantigen-specific CTLs than soluble vaccines and 31-fold greater antigen-specific T-cell response compared to Montanide. Moreover, nanodiscs in combination with anti-PD-1 and anti-CTLA-4 therapy revealed better eradication of established cancer cells.

**Figure 2.** RNA-lipoplexes (RNA-LPX) delivery to DCs. (**A**) Mechanism action of RNA-LPX to induce anti-tumor immune responses, (**B**) Bioluminescence imaging of BALB/c mice, (**C**) Splenic localization of CD11c and Cy3 double-positive cells in BALB/c mice after 1 h of Cy3-labelled RNA-LPX i.v. injection, (**D**) in vivo studies in CT26 tumor bearing BALB/c mice immunized with gp70-LPX, and (**E**) Clinically administered RNA-LPX vaccines induce systemic INFα in dose-dependently manner. Reproduced with permission from [82]. Copyright 2016, Springer Nature.

Similarly, Liu et al. have been synthesized cell-penetrating peptide (CPP) decorated uniform-sized pristine NPs to deliver GM-CSF and IL-2 into tumor cells [93]. In vitro and in vivo (Figure 3) results revealed the programed promotions of multi-adjuvants on DC recruitment, antigen presentation, and T-cell activation. Furthermore, in vivo assessments revealed the satisfactory effects on tumor growth suppression, metastasis inhibition, and recurrence prevention.

**Figure 3.** (**A**) Scheme of multi-adjuvant WCTV to initiate anti-tumor immunity, (**B**) bioavailability and cellular up take of GM-CSF and IL-2 in LLC cells after incubating with nanoparticles (NPs) for 24 h, (**C**) relative expressions of CD80, CD86, MHC II, and MHC-I molecules after treatment with whole tumor cell lysate protein (WPro), p-NP, and CNP for 24 h and (**D**) Relative tumor volume of LLC tumor bearing mice after immunization with multi-adjuvant WCTVs compared with other vaccine groups. Reproduced with permission from [93]. Copyright 2013, Elsevier Ltd.

#### 2.1.3. Antigens and Adjuvants Delivery to Tumor Microenvironment (TME)

The TME comprised proliferating tumor cells, the tumor stroma, infiltrating inflammatory cells, apoptotic cancer cells, cancer-associated fibroblasts, myeloid-derived suppressor cells, tumor-associated macrophages, and a variety of associated tissue cells which are participating in the suppression of antitumor immunity [94]. These immunosuppressive cells secrete numerous soluble mediators including, Transforming growth factor-beta (TGF-β), Indoleamine 2,3-dioxygenase (IDO), arginase, prostaglandin E2 and nitric oxide synthase 2 (NOS2) [95–97]. By reducing the supply of indispensable amino acids (such as arginine (R) and Tryptophan (W)), IDO and arginase directly suppress T cell proliferation and differentiation [98]. The activity of arginase and IDO translates not only into amino acid deprivation but also in the production of metabolites (such as l-kynurenine and spermidine) capable of numerous physiologic effects [99,100]. For example, l-kynurenine derived from W, favors the differentiation of Treg cells and induces IDO expression in DCs. Similarly, TGF-β also alters activation, maturation, and differentiation of DCs, CD4+, and CD8+ T cells. In addition, PD-L1/PD-L2 expressed on tumor cells can engage PD-1 receptor on the surface of activated T cell and sends inhibitory signals via activating phosphatases, resulting in dephosphorylation of key elements in the T cell, leading to down-regulating proliferation, survival, and cytokine production [101]. Furthermore, the CTLA-4 receptor on tumor cells binds to co-stimulatory molecules on DCs and decreases antigen presentation. Moreover, there are an abundant accumulation of acellular components such as fibrosis, collagen, secreted protein acidic and rich in Cysteine (SPARC), and hyaluronan which alters the physicochemical properties of TME (including physical barriers, physical pressure (i.e., increase interstitial fluid pressure, change in metabolism, etc.) [102].

As mentioned above, although several immune effector cells are recruited to the TME, their anti-tumor activity is suppressed principally in response to tumor-derived signals [103]. Compared with normal tissue, TME has some unique characteristics, such as vascular abnormalities, hypoxia, increases in proteolytic activity, and an acidic microenvironment, which leads to treatment resistance [104]. Therefore, new approaches are demanded to overcome TME related immunosuppressive situations. Hence, targeting immunosuppressive cells (such as Treg) or Tumor-associated macrophage (TAM) in the TME using NPs could be the best tactics to prevent immunosuppression.

Sacchetti et al. designed ligand guided PEG-modified single-walled carbon nanotubes (PEG-SWCNTs) to target Treg-specific receptors in the TME [105]. They found that ligand targeted PEG-SWCNTs were preferentially up taken by Treg cell residing in the TME via glucocorticoid-induced TNFR-related receptor (GITR).

Similarly, Zhu et al. synthesized mannose targeted PEG-sheddable NPs to target TAM [106]. They reported that mannose-modified PEG-sheddable NPs was effectively targeted TAMs via the mannose-mannose receptor. As a result, more PEG-sheddable NPs accumulation was observed in TAM in comparison to non-sheddable PEG. This is maybe due to PEGylation which minimizes NPs opsonization and enhances its bioavailability.

In addition, NPs can be used to deliver anti-immunosuppressive factors, such as anti-TGF-β or TGF-β receptor inhibitor to the TME to increases the activation of the immune system. Park et al. synthesized liposomal polymeric gels (nLGs) to deliver IL-2 and TGF-β inhibitors (Figure 4) [107]. The author reported that IL-2 and TGF-β inhibitors were successfully delivered to the TME. In vivo results showed that nLGs treatment suppresses a tumor growth, improved survival rates, and enhanced the activity of NK cells and intratumoral-activated CD8+ CTLs.

In summary, NPs can enhance anticancer immunity by regulating the TME either by inhibiting immunosuppression or by endorsing immune activation which could synergize with clinically established immunotherapeutic agents such as Immune Checkpoint Inhibitors (ICIs). Hence, targeting immune cells in the TME using nanoformulated therapeutic agents is the best tactic to activate antitumor immunity.

**Figure 4.** (**A**) The synthesis approach of theliposomal polymeric gel (nLG) particle system. (**B**) Plot of tumor area versus time. Red arrows indicate treatments (via intratumoral injection). (*p* < 0.05, \*, *p* < 0.001, \*\*\*, By ANOVA with Turkey's multiple comparison test. *p* < 0.05, #, by two-tailed t-test. (**C**) Tumor masses vs nLG-treated groups, *p* < 0.001, \*\*\*, *p* < 0.01, \*\*, *p* < 0.05, \*, By ANOVA using Turkey's post-test. (**D**) Images of lung immediately before collection of lung-infiltrating lymphocytes from mice, (**E**) Uptake of lipid carrier (green) and rhodamine payload (red) around individual lung tumors at 2 h post injection. Reproduced with permission from [107]. Copyright 2012, Nature Publishing Group.

#### 2.1.4. Immune Checkpoint Inhibitors (ICIs) Delivery

Immune checkpoints are surface proteins on immune cells that act as negative regulators of immune activation by various antigens, including tumor antigens [108]. Immune checkpoint molecules include PD-1, PD-L1/2, CTLA-4, T-cell immunoglobulin and mucin domain-containing-3 (TIM-3), and lymphocyte-activation gene 3 (LAG-3) [109,110]. Immune checkpoint molecules are widely expressed on both tumor cells and immune cells, which might be negatively regulated by tumor-specific T cells via receptor–ligand interactions, causing T-cell anergy or exhaustion [111,112]. Tumor cells evade destruction from the immune system by triggering immune checkpoint receptors, such as CTLA-4, PD-1, or PD-L1, that are expressed on T-cells and whose engagement inhibits T-lymphocyte function [113].

ICIs are monoclonal antibody that inhibits the receptors-ligands interaction and enhance immune-mediated cancer eradication. The development of ICIs lays a key foundation in cancer immunotherapy [114]. In 2018, James P. Allison and Tasuku Honjo were awarded a Nobel prize in physiology or medicine for the discovery of cytotoxic T-lymphocyte-associated antigen (CTLA-4), and programmed cell death protein 1/programmed cell death protein ligand 1 (PD-1/PD-L1), respectively [115]. According to literature report, anti-CTLA-4 antibody overcomes a block in essential costimulatory signals (i.e., CTLA-4 and CD28 competes for the same ligands CD80 and CD86; CTLA-4 has a higher affinity than CD28) that are required for activation of both naive T cells and resting clones, whereas PD-1/PD-L1 blockade seems to remove a barrier and enable T cell effector function at the tumor site [116,117]. As the result, ICIs including anti-CTLA-4 and anti-PD-1/PD-L1 Abs were developed to block these inhibitory pathways [118–120]. Currently, some of the ICIs including the anti-CTLA-4 agent, ipilimumab, Tremelimumab; anti-PD-1 agents, nivolumab and pembrolizumab; and anti-PDL-1 agent, Atezolizumab, Avelumab, Cemiplimab, Durvalumab, ipilimumab, and atezolizumab [121–123] have been approved for the treatment of certain types of cancer [124]. Mechanism action of ICIs are briefly summarized in Figure 5, [124]. Furthermore, in their

current review paper, Vaddepally et al. have been briefly reviewed the majority of FDA-approved ICIs per national comprehensive cancer network guidelines [125].

**Figure 5.** Mechanism action of Immune Checkpoint Inhibitors (ICIs). Reproduced with permission from the Journal of Cell Biology [124].

Even though a promising clinical data was obtained using ICIs, still, it shows certain limitations including an occurrence of immune-related adverse events, low response rate, and acquired resistance which is expected to be improved using NPs.

Wang et al. designed pH-sensitive microneedle (MN) patch for the sustained delivery of anti-PD1 (aPD1) (Figure 6) [126]. Glucose oxidase was used to generate acidic environments by converting glucose to glucuronic acid, leading to NPs self-dissociation, which in turn facilitates sustained aPD1 releases. The authors found that, at the same dose, pH-sensitive MN patch induces more immune responses compared to non-sensitive MN or free aPD1 using B16F10 mouse melanoma model. Furthermore, the author demonstrated that the aCTLA-4 and aPD1 co-loaded in MN patch shows synergistic effects.

Similarly, Wang et al. have been designed inflammation-triggered CpG DNA-based "nano-cocoons" for co-delivery of anti-PD-1 Ab and CpG oligodeoxynucleotides (CpG ODNs) (Figure 7) [127].The author

reported that in comparison to free CpG nucleotides and aPD1, bioresponsive controlled release of CpG and aPD1 showed a considerable immune response and better therapeutic efficacy.

**Figure 6.** (**A**) Scheme of aPD1 delivery via microneedle (MN) patch, (**B**) Mechanism action of aPD1 to activate T-cell, (**C**) aPD1 release (%) from the MN patches in the presence of 100 mg/dL glucose solution at 37 ◦C, (**D**) Immunofluorescence staining of tumors treated with MN-GOx-aPD1 or free aPD1 at different time points (green: aPD1, blue: nucleus), (**E**) Bioluminescence signals vs. time after treatment with different groups, and (**F**) % Survival plot of mice after MN patch-assisted delivery of aPD1 therapy. P value: \*, *p* < 0.05. Reproduced with permission from [126]. Copyright 2016, American Chemical Society.

**Figure 7.** Schematic illustration of (**a**) aPD1 and caged restriction enzyme loaded DNA nanococoon (DNC), (**b**) In vivo tumor immunotherapy after primary tumor resection, local injection, and treatment of DNC-based delivery system and (**c**) Activation of DCs by CpG which in turn activates T cell response with aPD1 for PD 1 blockade. Reproduced with permission from [127]. Copyright 2016, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Several researchers reported that patients with advanced cancer poorly respond to PD1/PD-L1 inhibitory therapy due to low TAA expression [128,129]. Epigenetic alteration like DNA hypermethylation, which is commonly seen at TAA promoter regions, plays an essential role in immune evasion of cancer cells during tumorigenesis [130]. Hence, epigenetic modulators, such as hypomethylation agents (HMAs), play a key role in the induction of TAA expression, which in turn increase antitumor immune response [131]. Ruan et al. synthesized a pH and reactive oxygen species (ROS) sensitive bioresponsive gel depot for co-delivery of aPD1 and Zebularine (Zeb), HMA [132]. The author reported that combination therapy enhances the immunogenicity of cancer cells and plays a crucial role in converting immunosuppressive TME.

Preclinical animal studies using cancer nanovaccines, nanoformulated TAA, or tumor-specific neoantigens, revealed promising therapeutic efficacy [133]. However, the clinical use of these nanovaccines has been limited due to immune evasion and suppression in the TME [134,135]. Some literature showed that high expression of immune checkpoints such as PD-L1 is responsible for the occurrence of tumor resistance to vaccine-mediated immune responses. Hence, it possible to overwhelm this limitation by combining with ICIs such as anti-PD-1, anti-PD-L1 or anti-CTLA4 Ab.

Kim et al. developed a small lipid nanoparticle (SLNP)-based nanovaccines embedded with antigen/adjuvant (OVAPEP-SLNP@CpG), Figure 8 [136]. Synthesized nanovaccine showed high potent antitumor efficacy in both prophylactic and therapeutic E.G7 tumor models but induced T cell exhaustion by increasing PD-L1 expression, leading to tumor recurrence. However, by using mice that showed a good therapeutic response after the first cycle of immunization with the nanovaccine the author underwent a second cycle together with anti-PD-1 therapy. Their result revealed tumor relapse of suppressed, treatment sequence, and the timing of each modality is crucial in order to enhance antitumor efficacy using combinations of nanovaccines with ICIs.

**Figure 8.** (**A**) Scheme and mechanism action of OVAPEP-SLNP@CpG nanovaccine, (**B**) Therapeutic efficacy of OVAPEP-SLNP@CpG nanovaccine in an established tumor model, (**C**) representative image of tumors. Scale bar = 1 cm, (**D**) First cycle and second cycle of immunization, (**E**) Overall process of sequential and timely combination strategy between cancer nanovaccine. *p* < 0.001, \*\*\*, *p* < 0.05, \*, Reproduced with permission from [136]. Copyright 2020, Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Similarly, Fontana et al. have designed and assessed biohybrid nanovaccines in combination with anti-CTLA4 antibody [137]. The author observed, an increased activation of APCs and increased priming of CD8+ T cells after nanovaccine injection. Most interestingly, treatment efficacy was increased (87.5% of the animals responding, with 2 remissions) in the co-administration (nanovaccine with anti-CTLA4 antibody) compared to the checkpoint inhibitor alone in the B16.OVA model.

#### 2.1.5. Nanoparticles as the Direct Immunomodulators

Immunomodulatory compounds such as cytokines, monoclonal antibodies and adjuvants have been used to reshape the TME and to initiate anti-tumor immunity; although there are certain limitations such as therapeutic efficacy and unwanted side effects during systemic administration, to use in clinics [138].

Immunomodulatory NPs can readily improve the therapeutic effects by enhancing immune stimulation and minimizing off-target side effects. As the result, more research works are undergoing to understand the mechanisms of NPs-Immuno-interactions which is highly important to know the immunomodulating potential of NPs, as the immunostimulating or as immunosuppression [139]. The function of NP in the immunomodulation depends on several factors that are intrinsic to NPs, such as surface chemistry, charge, size, and shape, besides extrinsic factors such as route of administration [140].

Several researchers have widely explored the immunomodulating effects of both polymeric and inorganic NPs [28,141,142]. Different evidence suggests that the immune system cells interact with NPs through Toll-like receptors (TLRs) [143,144]. TLRs are transmembrane proteins, expressed on APCs such as DCs and macrophages, which recognize specific molecular patterns that act as danger signals to the immune system [145]. Depending on the type of receptor and the type of stimuli, TLR engagement plays a great role both in the innate and adaptive immune response by altering several gene expressions.

Inorganic NPs such as Gold nanoparticles (AuNP), Titanium nanoparticles (TiNPs), iron nanoparticles (FeNPs), Zinc nanoparticles (ZnNPs), and silver nanoparticles (AgNPs) are the most stable and promising particles to modulate immune systems [146–148].

Vasilichin et al. investigated the influence of metal oxide NPs on innate immunity by testing TLR-4/6 mRNAs in the human monocyte cell line [149]. They found that all studied NPs activated TLR-6 expression, while AlOOH enhanced both TLR-4 and -6 expression.

Moreover, in human peripheral blood mononuclear cells, the administration of AuNPs activates immune-related genes depends on its physicochemical properties [150]. Lee et al. reported that gold nanorods (GNRs) and SiO2-coated GNRs has a tendency to penetrate into macrophages to induce the release of inflammatory mediators (calcium (Ca), hydrogen peroxide, nitric oxide (NO), cytokines, prostaglandins, etc.) and the activation of immune response genes [151]. Both GNRs and SiO2-coated GNRs have an immunostimulatory property to reinforce immune reactions via calcium—transcription factors pathway.

Fallarini et al. synthesized mono- and disaccharides coated AuNPs with a particle size of ~2 and 5 nm [152]. Their in vitro results showed that synthesized NPs initiate the immune response by activating the macrophages. However, unlike monosaccharide coated AuNPs, disaccharide coated tends to induce T cell proliferation and an increase in IL-2 levels. According to this report, the immunoactivity is strongly dependent on size, 5 nm AuNPs perform far better than 2 nm ones.

Lin et al. also reported that CpG modified AuNP induced macrophage and DC tumor infiltration and suppresses tumor growth compared with free CpG [153]. Similarly, Ahn et al. also reported that AuNP facilitates tumor-associated self-antigen delivery to DC and then activates the cells to facilitate cross-presentation and induce antigen-specific cytotoxic T cell responses [154].

AgNPs also trigger inflammatory reactions cascade involving the activation of macrophages, neutrophils, and helper T cells [155]. Subsequently, AgNPs enhance the expression of numerous types of cytokines [156,157]. Furthermore, different researchers have been investigated the effect of AgNPs as the immunological adjuvant using both in vitro and in vivo studies [158,159].

Xu et al. have investigated an adjuvant effect of AgNPs [160]. The in vivo result showed that serum antigen-specific IgG and IgE levels were increased, showing that AgNPs elicited CD4+-mediated immune response. After 48h treatment with AgNPs, both the number of leukocytes and levels of cytokines TNF-α and IFN-γ was increased in abdominal lavage fluid of mice. Furthermore, the expression of the MHC complex class II molecule on the surface of peritoneal macrophages was significantly increased.

In addition, NPs can be designed as artificial APCs (aAPCs), that express surface features, that can activate immune cells or modulate the expression of pro- or anti-inflammatory genes [161,162]. This immunomodulatory behavior of NPs can enhance the therapeutic response of injected NPs by directly generating cytotoxic T cells. For example, Mandal et al. designed biocompatible and less-toxic anti-CD3 antibodies-modified artificial APCs based on poly (isocyano peptide) [163]. They found that synthesized aAPCs induce a more robust T cell response in comparison to free antibodies or PLGA particles. Similarly, Kosmides et al. designed and investigated the synergy between a PLGA-based aAPC and an aPD1 mAb [164]. Their in vitro results revealed that the combination of antigen-specific aAPC and aPD1 mAb induced IFN-γ secretion by CD8+ T cells. In addition, in vivo results showed that combination treatment synergistically inhibits tumor growth, while either treatment alone had no effect.

#### **3. Clinical Translation of Nano-Immunotherapy**

In the last few decades, several researchers have deeply explored a regulatory mechanism of antitumor immunity, particularly the immune checkpoint pathways, which lays a basic foundation for the invention of ICIs, that have revolutionized cancer treatment [165,166]. However, different literature showed that the activity of ICIs as monotherapy is not satisfactory for all cancer patients [167]. To address this clinical challenge, the different researchers tried to combine NPs with immunotherapeutic agents or conventional cancer treatment with ICIs [168,169]. Several kind of the literature showed that, conventional cancer treatments such as chemotherapy, photodynamic therapy, and radiotherapy can initiate the immune system to elicit a specific antitumor immunity, due to its ability to induce immunogenic cell death, in addition, to directly killing cancer cells, which can induce a release of certain damage-associated molecular patterns (DAMPs) that can activate APCs [170]. Activated APCs in turn phagocytose dying tumor cells and present tumor antigens to initiate T cell responses [171]. By taking this into consideration, NPs-based chemotherapeutic agents or photosensitizer delivery can be used to exploit the ICD inducing properties to achieve potent antitumor efficacy in combination with immunotherapeutic agents such as ICIs [172]. Most importantly, NPs based drug delivery can enhance selective target delivery and reduce off-target cytotoxicity of chemotherapeutic or immunotherapeutic agents which in turn extends the therapeutic index, especially for combination therapy.

As briefly discussed above, targeting APCs, cancer cells or TME clearly indicates that NPs significantly improved the therapeutic efficacy of immunotherapeutic agents. Based on the progress made so far, nano-immunotherapy has been achieving remarkable results, some of them were approved by the FDA, and the majority of them are in the preclinical stage, for the treatment of cancer. The first nano-immunotherapy approved for the treatment of advanced triple-negative breast cancer (TNBC) was Atezolizumab (Tecentriq®), an ICI against PD-L1, in combination with albumin-bound paclitaxel NP (nab-paclitaxel) [173,174]. The result showed that atezolizumab plus nab-paclitaxel significantly prolonged progression-free survival (PFS) compared to nab-paclitaxel in the intent-to-treat population and the PD-L1 positive subgroup.

Furthermore, Hensify®/NBTXR3, 50 nm crystalline hafnium oxide (HfO2) NP, received European market approval (CE Mark) in April 2019 for the treatment of locally advanced soft tissue sarcoma in combination with radiation therapy [175]. Hensify® is designed by Nanobiotix to physically destroy tumors and stimulate the immune system locally [176]. Nanobiotix is also running several clinical trials and has received US FDA approval to launch a combination trial with NBTXR3 and PD-1 antibodies to treat lung cancer (NCT03589339).

Similarly, the multicentre, randomized, open-label, phase 3 trial study was conducted as a first-line treatment for metastatic non-squamous non-small-cell lung cancer (IMpower130, NCT02367781) using Atezolizumab in combination with carboplatin plus nab-paclitaxel chemotherapy compared with chemotherapy alone [177]. The result revealed that there were significant improvements in median overall survival (OS), 18.6 months in the atezolizumab plus chemotherapy group, 13.9 months in the chemotherapy group, median PFS 7.0 months in the atezolizumab plus chemotherapy group, and 5.5 months in the chemotherapy group.

Furthermore, there is the first randomized phase 3 JAVELIN Ovarian 200 trial (NCT02580058) study which is designed to demonstrate that Avelumab (human immunoglobulin G1 anti-PD-L1 monoclonal antibody) alone or in combination with Pegylated liposomal doxorubicin (PLD) is superior to PLD alone in prolonging OS in patients with platinum-resistant/platinum refractory ovarian cancer [178]. The results revealed that PLD combined with avelumab slightly improved OS (15.7), PFS (3.7), and objective response rate (ORR) (13.3) compared to either PLD (13.1, 3.5, and 4.2 for OS, PFS, and ORR, respectively) or avelumab (11.8, 1.9, and 3.7 for OS, PFS, and ORR, respectively) alone (Reference: ClinicalTrials.gov; NCT0258005). In addition, RNA formulated NPs alone or in combination with immunotherapeutic agents, such as ICIs, were also explored and the majority of them are under clinical trials as listed in Table 1. Moreover, in his recent review, Yang Shi was briefly reviewed several studies that are FDA approved or under clinical trials using nano-immunotherapy, such as NPs albumin-bound paclitaxel, Pegylated liposomal doxorubicin, mRNA nanovaccines, and WDVAX [179].

**Table 1.** FDA approved nano-Immunotherapy and studies under clinical trials to treat cancer [180–182].

In summary, several clinical and preclinical study results demonstrate that NPs are highly important in immunotherapy as the delivery of immunotherapeutic agents or as the direct immunomodulators. However, due to the multifactorial nature of cancer-immune interactions, identifying unique biomarkers are crucial to designing multifunctional NPs (i.e., which have a diagnostic and theranostic application). Hence, in order to design a novel biomarker-guided multifunctional and

biocompatible NPs to enhance the efficacy and to promote clinical translation of nano-immunotherapy, a unique biomarker must be identified to distinguish which immune-activating or immunosuppressive cells or pathways are targeted.

#### **4. Conclusions**

Cancer immunotherapy is emerging as a beneficial tool for cancer treatment by activating the immune system to produce antitumor effects. However, there are some limitations to immunotherapy including poor therapeutic targeting and unwanted side effects. Currently, one of the emerging tracks in this field area is NPs-based immunotherapy which has a considerable potential to solve limitations related to immunotherapy. NPs plays a great role in cancer immunotherapy as the carrier of immunotherapeutic agents and as the direct immunomodulator. NPs based delivery of immunotherapeutic agents offers a great opportunity to minimize unwanted cytotoxicity through controlled release, dose-sparing, or enhanced tumor targeting capabilities. Hence, in the near future, as our knowledge enhanced to understand the detailed molecular mechanism of NPs-immune interaction, NP-based therapies will revolutionize and place NP-based immunotherapy at the forefront of immune-modulating therapeutics.

**Author Contributions:** T.A.D., conceived, designed, wrote and edited the review paper; C.-F.Y., funding and edited revised manuscript, W.-P.S., supervise, funding and edited the review paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Ministry of Science and Technology, Taiwan; grants number: MOST- 110-2811-B-006-501, 108-2811-B-006-501, 108-2811-B-006-525, 109-2314-B-006-078, 109-2314-B-006-084-MY3, CMNCKU10806 & Headquarters of University Advancement, National Cheng Kung University, grant number: HUA 109-25-18-133.

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

#### **References**

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

© 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/).

## *Review* **Intercellular Mitochondrial Transfer in the Tumor Microenvironment**

#### **Hana Sahinbegovic 1,2,3,4, Tomas Jelinek 1,2, Matous Hrdinka 1,2, Juli R. Bago 1,2, Marcello Turi 1,3, Tereza Sevcikova 1,2,3, Amina Kurtovic-Kozaric 4, Roman Hajek 1,2 and Michal Simicek 1,2,3,\***

Received: 12 June 2020; Accepted: 2 July 2020; Published: 4 July 2020

**Abstract:** Cell-to-cell communication is a fundamental process in every multicellular organism. In addition to membrane-bound and released factors, the sharing of cytosolic components represents a new, poorly explored signaling route. An extraordinary example of this communication channel is the direct transport of mitochondria between cells. In this review, we discuss how intercellular mitochondrial transfer can be used by cancer cells to sustain their high metabolic requirements and promote drug resistance and describe relevant molecular players in the context of current and future cancer therapy.

**Keywords:** cancer; mitochondria; mitochondrial transfer; tunneling nanotubes; tumor microenvironment

#### **1. Introduction**

The majority of human cells use mitochondria as the main source of energy and metabolites. A typical cancer cell, however, tends to upregulate glycolysis, as postulated by Otto Warburg more than 100 years ago [1,2]. At first glance, this might seem counterproductive, as glycolysis produces fewer ATP molecules and causes constant acidification of the extracellular space by increased production of lactate [3]. On the other hand, an enhanced glycolytic rate contributes to the development of several cancer hallmarks, such as the ability to evade apoptosis by inhibition of oxidative phosphorylation (OXPHOS) [4] and the promotion of metastatic dissemination by the degradation of the extracellular matrix and tissue outgrowth [5]. Moreover, tumor cells often reside in a hypoxic environment that favors the use of anoxygenic production of energy. Therefore, the idea of forcing tumor cells to use OXPHOS instead of glycolysis has emerged as a promising therapeutic strategy [6,7].

Even though most cancers have impaired mitochondrial respiration, recent discoveries indicate that certain solid tumors, such as pancreatic ductal adenocarcinoma and endometrial carcinoma, and many hematological neoplasms rely heavily on OXPHOS and upregulated mitochondrial metabolism [8,9]. In line with these observations, a number of studies highlighted the importance of mitochondria-dependent metabolic reprogramming in boosting proliferation and in the development of drug resistance in several types of cancers [6,10]. Consequently, the clinical relevance of biological processes involving active and healthy mitochondria, initially meant to have a rather tumor suppressive role, is now being revised.

Historically, cancer research has been mostly done by using 2D in vitro models of established cell lines [11,12]. Although this is a powerful and valuable approach, it completely neglects the presence of neighboring non-tumor cells supporting or suppressing the cancer tissue. The influence of the microenvironment on tumor cells is very complex and often includes the direct involvement of tumor mitochondria. Cancer cells can release (e.g., upon necrosis) entire mitochondria or their components, such as mitochondrial DNA (mtDNA), ATP, cytochrome C, or formylated peptides, to the extracellular space [13]. These then serve as Damage-Associated Molecular Patterns (DAMPs) that activate the immune cells [14,15]. Resulting pro-inflammatory and immunosuppressive responses then either inhibit or stimulate the growth and/or metastatic capacity of the tumor [16,17].

The modulation of tumor mitochondria is an important mechanism that aids cancer cells to escape from the immune system control and develop drug resistance [6,10]. In addition to neoplastic and immune cells, the tumor microenvironment contains many different cell types that can control the state of the mitochondria in a tumor both directly, by cell–cell contacts [18], and indirectly, by secretion of soluble factors and a variety of extracellular vesicles [19]. Recently, a novel mechanism of intercellular communication based on a horizontal transfer of mitochondria between non-tumor and malignant cells was described [20–24]. This paradigm-breaking discovery has led to the question of whether the phenomenon of direct mitochondria sharing could also contribute to the aversion of malignant cells to existing drug combinations and possibly further promote tumor growth. We still know very little about this new, exciting way of sharing intracellular molecules and organelles. A deeper understanding of the underlying molecular mechanisms and consequences on cell physiology will likely explain many therapeutic failures and ultimately lead to novel, more efficient drug combinations.

In this review, we provide an overview of the current knowledge of intercellular mitochondrial transfer, with a particular focus on its relevance in cancer initiation, progression, and drug resistance. We present a summary of the known molecular players involved in sharing mitochondria and show examples of mitochondrial exchange in both solid and hematological tumors. Finally, we place all findings in the context of the current therapeutic strategies.

#### **2. Means of Mitochondrial Transfer**

The first observation of mitochondrial transfer in 2006 demonstrated that mitochondria from bone marrow stromal cells (BMSCs), but not free mitochondria or mtDNA from the medium, were able to relocate to mitochondria-deficient A549 lung cancer cells and rescue their aerobic respiration [18]. A follow-up study further supported the regulated directionality of the exchange as donor, non-irradiated PC12 cells with faulty mitochondria could not nourish recipient PC12 cells, leading them back to life [25]. Importantly, the transfer of mitochondria was also observed in vivo in mouse melanoma cells injected into mice expressing a fluorescently labelled mitochondrial protein [24].

Fundamental research on the physiological relevance of mitochondrial transfer suggested its importance in the regeneration of damaged or infected tissue [26,27]. The "mito-healing" theory was subsequently supported by additional studies in a variety of tissues, including vascular [28], brain [19], lung [29], cornea [30], and several other tissues. Transfer of mitochondria was also described in the immune system to combat bacterial infections [31]. The presence of pathogens inside immune cells is usually accompanied by a switch from glycolytic metabolism to OXPHOS as a means of triggering a fast anti-microbial response [32]. A striking example of infection-induced metabolic switch is seen in macrophages during acute respiratory distress syndrome, when macrophages are fed additional mitochondria by surrounding mesenchymal stem cells (MSCs), boosting their anti-inflammatory and phagocytic capacity [31]. Thus, the importance of mitochondria exchange in maintaining tissue homeostasis is clearly not disputable. However, the shuttling of mitochondria between cells could also have severe pathological consequences, particularly in cancer, where malignant cells tend to take advantage of the surrounding environment (Figure 1) [23,33,34].

**Figure 1.** Putative effects of mitochondrial transfer on an injured cell and a cancer cell. ROS, reactive oxygen species.

The precise mechanism of mitochondrial transfer remains unknown, and so far, only a few crucial molecules involved in the process have been described. High OXPHOS demand and/or severe mitochondria damage are typical features of the recipient cells [23,35,36]. To initiate the transfer, the providing cells should not only possess non-damaged, healthy mitochondria [37] but also be specifically activated [22]. Metalloproteinase-1 (MMP-1), nestin, and proinflammatory cytokines have been identified as essential factors stimulating donor cells to dispatch mitochondria [22]. When cultured with leukemic cells, donor BMSCs exhibited increased levels of PGC1α, a master regulator of mitochondrial biogenesis necessary for efficient mitochondrial transfer [38]. The activation of donor functions often correlates with a rise in intracellular reactive oxygen species (ROS) [22,37] generated by the recipient cells [33]. The main trigger regulating the directional release of mitochondria is still unknown, but the signal is likely multifactorial and, at least partially, mediated by ROS.

#### **3. Tunneling Nanotubes Are the Main Delivery Route for Mitochondria**

Neighboring cells can share mitochondria through several mechanisms, including (i) the formation of extracellular vehicles (EVs), (ii) tunneling nanotubes (TNTs) formed at the sites of physical contact, (iii) mitochondrial ejection, or (iv) cytoplasmic fusion [39]. Multiple studies have shown that TNTs, ultrafine cytoplasmic bridges between cells, are the main delivery system for mitochondria in both healthy and tumor tissues (Figure 2) [23,33]. TNT-independent mitochondria sharing in certain tumors was also described [19,29,37]. However, there seems to be a rather scarce number of examples, and more investigation of the transfer mechanisms is needed.

**Figure 2.** Schematic representation of mitochondrial transfer via tunneling nanotubes. The donor cell, usually a non-cancerous cell, sends mitochondria to the recipient cell. In certain cases, the transfer is possible in both directions. Key regulatory molecules and triggers are listed.

TNTs, discovered by Rustom et al. in 2004, serve as direct communication channels between neighboring cells to exchange a wide variety of molecules and organelles [40]. Their diameter ranges from 50 to 200 nm, and their length may reach up to 150 μm. TNTs are formed de novo in a matter of minutes and can connect multiple cells at once. Specific structural features or biomarkers unique to TNTs have not been identified. TNTs lack any attachment to the substrate, but their structure is enforced by actin [40] and microtubule filaments [25,41]. In experimental settings, the actin-disrupting drug cytochalasin B is often used as an efficient inhibitor of TNTs formation [23,33].

For the efficient transport of mitochondria, the presence of functional microtubules and associated molecular motors seems to be required [25]. Specifically, Myosin X and Myosin Va have been co-localized with mitochondria inside TNTs [40,42,43]. Some donor cells exhibit high expression of the small GTPase Miro1 localized on the outer mitochondrial membrane [44]. When Miro1 was depleted in MSCs cultured with LA-4 epithelial cells, mitochondrial transfer was ineffective, whereas Miro1 overexpression increased the ability of MSCs to donate mitochondria [45]. Mechanistically, Miro1 seems to co-ordinate mitochondrial movement along microtubules by promoting the assembly of a complex molecular motor machinery [46].

The role of TNT-localized actin in mitochondria exchange is less clear. It is known that filamentous actin impedes the passive transfer of soluble cytoplasmic molecules through TNTs [40] and serves as a scaffold that stabilizes TNT structure [47]. The actin-binding protein M-Sec has been shown to be important for the formation of TNTs and the intercellular propagation of calcium (Ca2+) in macrophages [48,49]. Increased levels of Ca2+ activate mitochondria-localized Miro1 that further binds to microtubule-associated motor proteins [46].

Recently, CD38, an ectoenzyme involved in transmembrane signaling and cell adhesion, was identified as one of the key players in mitochondrial transfer [19,23]. In addition to its receptor function, CD38 modulates intracellular Ca2+ levels by generating cyclic ADP-ribose [50,51]. The Ca2+ regulatory role of CD38 seems important for mitochondrial delivery from BMSCs to myeloma cells [23] and from astrocytes to neurons in brain tissue damaged by stroke [19], even though the transfer occurs via a different mechanism (TNTs vs. EVs, respectively). Thus, an increase in intracellular Ca2+ might be a general mechanism priming mitochondria for transfer. However, it is unclear how CD38 enzymatic activity is initiated to raise cytosolic Ca2+ levels and further promote mitochondrial transfer. It is

possible that an increase in extracellular NAD+, the substrate of CD38, induced by changes in the cellular redox state could play a role [52].

Interestingly, the presence of gap junction (GJ) proteins, especially connexin 43 [53], in TNTs has been confirmed [54]. Connexins are important for Ca2+ propagation between neighboring cells via TNTs [48,55,56]. Similarities in the composition of GJ and TNTs have also been pointed out [40]. In spite of this, GJ have been functionally distinguished from TNTs [55]. While GJ perform short-range cell-to-cell interactions and allow the transfer of molecules up to 1.2 kDa only [57,58], TNTs mediate long-range cell-to-cell interactions and allow the transfer of significantly bigger cargos [59,60]. More research will be needed to fully understand the contribution of GJ proteins to TNT formation and mitochondrial transfer.

#### **4. Mitochondrial Transfer in Solid Cancers**

The current knowledge on mitochondrial transfer in solid cancers is limited, and the results of various studies are often difficult to compare due to the use of different experimental systems (Table 1). Cells of mesenchymal origin or fibroblasts are the most commonly used mitochondria donors [18,21,36]. However, the tumor tissue is a complex environment composed of many different cell types, and competition between cells could have a significant effect on mitochondrial transfer [61]. Indeed, in the co-culture of BMSCs, endothelial cells, and MCF7 cells, the formation of TNTs between the three cell types was observed, but mitochondria were sent only from endothelial cells to MCF7 cells [21]. Recently, TNT-mediated mitochondrial transfer was also observed between natural killer T cells and breast cancer cells [62]. These studies indicate that the ability to donate mitochondria might be affected by the presence of a particular cell type in the tumor microenvironment. Therefore, results of mitochondrial transfer experiments using cells usually not present in the solid tumor microenvironment (e.g., BMSCs or umbilical cord Wharton's jelly cells) should be carefully interpreted. Nevertheless, the current data still provide interesting views of the general mechanisms of mitochondria shuttling.

**Table 1.** Mitochondrial transfer studies.

**Table 1.** *Cont.*

ND: not defined; iPSC: induced pluripotent stem cell; BM-MSCs: bone marrow mesenchymal stem cells; NKT: natural killer T cell; AML: acute myeloid leukemia; T-ALL: T cell acute lymphoblastic leukemia; ALL: acute lymphoblastic leukemia; MM: multiple myeloma; TNTs: tunneling nanotubes; MSCs: mesenchymal stem cells; BMSCs: bone marrow stromal cells; OXPHOS: oxidative phosphorylation; ROS: reactive oxygen species.

Investigations of the mechanism of intercellular mitochondrial transfer in solid tumors point to TNTs as the main delivery route [21,45,63]. The very first mitochondrial transfer was seen between mesenchymal and lung cancer cells with severely damaged or completely missing mitochondria [18]. To promote mitochondrial transfer, many studies used inhibitors such as rotenone to block the electron transport in the respiratory chain [23,33,37,45,65]. Thus, the OXPHOS status in the recipient cells seems to be a crucial factor mediating mitochondrial transfer. However, no mitochondrial transfer occurred between BMSCs and osteosarcoma cells with pathogenic mutations in mtDNA, which encodes critical components of the OXPHOS system [35].  This suggests that not only the state of mitochondria but also specific metabolic requirements could play a role in promoting mitochondrial transfer.

#### **5. Mitochondrial Transfer in Hematological Malignancies**

The tumor microenvironment is critical for progression and drug resistance also in hematological cancers [11,59,60]. Acquiring new mitochondria in the bone marrow niche was recently suggested as a way by which leukemic cells can achieve drug resistance [22,23,37]. Up to date, mitochondrial transfer was observed in several types of hematological malignancies, where it seems to have a pro-tumor function [32,33,61].

#### **6. Mitochondrial Transfer in Acute Lymphoblastic Leukemia**

The cross-talk between acute lymphoblastic leukemia (ALL) cells and their niche proved that mitochondria are delivered via TNTs from MSCs to primary B-cell precursor ALL cells [22]. The presence of TNTs facilitated the signaling from ALL cells towards MSCs, affecting the release of cytokines and chemokines in the microenvironment, supporting the survival and chemoresistance of ALL cells [34]. In addition to B-cell ALL, transport of mitochondria from MSCs was later shown in T-cell ALL (T-ALL), where mitochondrial transfer was mediated by T-ALL cell/MSC adhesion and occurred through TNTs. However, in contrast to B-ALL cells, mitochondria were exported from malignant T-ALL cells to the surrounding MSCs, probably due to the preferential use of glycolysis in T-ALL cells [64]. This example provides a unique model that can help to uncover the signals and mechanisms driving the transfer directionality. Moreover, it would be interesting to see whether T-ALL cells could donate mitochondria to other cell types and whether the increase in mitochondria number in MSCs would directly support the tumoral properties of T-ALL cells.

#### **7. Mitochondrial Transfer in Acute Myeloid Leukemia**

Acute myeloid leukemia (AML) is a typical hematological malignancy highly dependent on OXPHOS [66]. Not surprisingly, AML cells are more prone to receive new mitochondria as compared to healthy CD34+ hematopoietic stem/progenitors or lymphoid CD3+ cells [37]. When co-cultured with human BMSCs, AML cells can gain additional mitochondria in a TNT-dependent process [33]. However, in other study, endocytic inhibitors blocked the mitochondria exchange between murine MS-5 BMSCs and human AML cells [37], suggesting a TNT-independent delivery.

Commonly used chemotherapeutics such as cytarabine [67], etoposide [68], and doxorubicin [69] are factors that promote mitochondrial uptake by AML cells [70]. Consequently, the treatment could have a pro-tumor effect by stimulating oxidative metabolism in resistant AML clones [12]. The surface molecule CD38 is another clinically relevant target in AML, critical for the transport of mitochondria from BMSCs to AML cells [71]. Daratumumab, a monoclonal anti-CD38 antibody approved for the treatment of multiple myeloma (MM) [72,73], was shown to block the delivery of mitochondria to AML cells under both in vitro and in vivo conditions, decrease the oxygen consumption rate (OCR), and inhibit the growth of leukemic cells [74,75]. These studies suggest a novel, previously unexpected anti-tumor mechanism of anti-CD38 therapy.

#### **8. Mitochondrial Transfer in Multiple Myeloma**

Aberrant myeloma cells reside in the hypoxic environment of the bone marrow [76]. Unexpectedly, primary multiple myeloma (MM) cells isolated from patients' biopsies were shown to have a higher basal OCR compared to long-term in vitro cultured MM cell lines [23]. Similarly, when established MM cell lines were injected into a mouse or co-cultured with BMSCs, their OCR and ATP production significantly increased [23]. Moreover, the presence of BMSCs enhanced mitochondrial metabolism and drug resistance in MM cells [23]. This suggests that the bone marrow microenvironment might stimulate aerobic respiration in MM cells. A deeper investigation of the responsible mechanism indicated the presence of TNT-mediated mitochondrial transfer from BMSCs to MM cells, and ROS-inducing compounds, including commonly used proteasome inhibitors, significantly potentiated this process [23,77].

CD38, a crucial player in mitochondrial transfer, is currently one of the most attractive molecules for targeted therapy in MM patients [74,78–80]. Treatment with an anti-CD38 antibody or genetic deletion of CD38 were shown to inhibit mitochondrial transfer from BMSCs to MM cells and induce tumor shrinkage in xenografts models [23]. Lower mitochondrial activity, particularly a drop in OXPHOS, was previously associated with increased sensitivity of MM cells to proteasome inhibitors [77]. In line

with these observations are clinical data indicating high efficacy of a combinatory therapy using an anti-CD38 antibody and proteasome inhibitors [81]. On the other hand, proteasome inhibitors were shown to downregulate the expression of vascular cell adhesion molecule 1 (VCAM-1) on BMSCs [82], a major ligand for VLA-4 on MM cells, and thus impede the binding of BMSCs and MMs [83], that is required for efficient mitochondrial transfer.

#### **9. Conclusions**

The direct transfer of mitochondria from one cell to another has emerged as a thrilling mechanism, whose potential targeting offers great opportunities for both cancer therapy and tissue regeneration. The fact that mitochondrial transfer seems to be executed in a similar way in both solid and hematological cancers further multiplies the importance of this process. It also underlines the significance of tumor microenvironment and cellular plasticity in cancer progression and drug resistance. Furthermore, the involvement of mitochondrial transfer may offer an explanation for the yet unclear mechanisms of action of certain anti-cancer drugs. Although the entire signaling machinery driving mitochondrial transfer is still unknown, the discovery of key molecular players such as Miro1, connexin 43, and CD38 has already opened the doors for possible therapeutic targeting. Future research of the molecular processes governing mitochondria shuttling in both normal and pathological settings will likely bring many exciting discoveries and provide new therapeutic possibilities to improve tissue regeneration and cancer therapy.

**Author Contributions:** H.S. and M.S. wrote the original draft and designed the figures. M.T. prepared the figures. T.J.; M.H.; T.S.; A.K.-K., J.R.B. and R.H. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by The Czech Science Foundation (GA CR 19-25354Y), Institutional Development Plan of University of Ostrava (project no. IRP03\_2018-2020), ERDF-Project ENOCH (No. CZ.02.1.01/ 0.0/0.0/16\_019/0000868), and New Directions of Biomedical Research in the Ostrava Region (No. CZ.02.1.01/ 0.0/0.0/18\_069/0010060), SGS03/LF/2019-2020, SGS03/PrF/2019-2020.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

© 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/).

## *Review* **Current Progresses and Challenges of Immunotherapy in Triple-Negative Breast Cancer**

#### **Karan Mediratta 1,2,3, Sara El-Sahli 1,2,3, Vanessa D'Costa 1,2,\* and Lisheng Wang 1,2,3,\***

Received: 5 November 2020; Accepted: 24 November 2020; Published: 26 November 2020

**Simple Summary:** The breakthrough of immunotherapy in melanoma has generated a glimmer of hope for lethal triple negative breast cancer (TNBC). This review summarizes the recent advances, challenges and potential new approaches of immunotherapy in TNBC.

**Abstract:** With improved understanding of the immunogenicity of triple-negative breast cancer (TNBC), immunotherapy has emerged as a promising candidate to treat this lethal disease owing to the lack of specific targets and effective treatments. While immune checkpoint inhibition (ICI) has been effectively used in immunotherapy for several types of solid tumor, monotherapies targeting programmed death 1 (PD-1), its ligand PD-L1, or cytotoxic T lymphocyte-associated protein 4 (CTLA-4) have shown little efficacy for TNBC patients. Over the past few years, various therapeutic candidates have been reviewed, attempting to improve ICI efficacy on TNBC through combinatorial treatment. In this review, we describe the clinical limitations of ICI and illustrate candidates from an immunological, pharmacological, and metabolic perspective that may potentiate therapy to improve the outcomes of TNBC patients.

 Introduction**

Breast cancer alone accounts for 30% of all female cancers and remains one of the leading causes of cancer-related deaths globally, with 626,679 deaths in 2018 alone [1,2]. Triple-negative breast cancer (TNBC) is the most refractory subtype, which accounts for 11.2% of new breast cancer cases, but disproportionately accounts for the majority of breast cancer-related deaths [3]. Chemotherapy remains the current mainstay treatment due to the lack of specific targets for TNBC. However, it is often associated with short-lived clinical responses [4], systemic toxicity [5] and enrichment of cancer stem cell (CSC) populations. CSCs are capable of self-renewal, differentiation, metastasis, and regeneration of a new tumor [6]. Moreover, CSCs possess an abundance of drug resistance mechanisms, including the expression of several ATP-binding cassette (ABC) transporters that contribute to the poor clinical outcomes associated with TNBC [7,8].

It is evident that effective treatment of TNBC would depend upon the elimination of CSC populations. In recent years, the emergence of immunotherapy (IT) has offered new perspectives in the treatment and management of TNBC. Despite the lack of targeted therapies for TNBC, recent studies suggest that TNBC is the most immunogenic breast cancer subtype [9–11]. In one study, TNBC was reported to have a higher expression of Programmed Death-Ligand 1 (PD-L1), an immune checkpoint molecule that contributes to immune evasion [12]. Through immunohistochemistry staining, TNBC tumors have been shown to exhibit higher numbers of intratumoral and stromal tumor-infiltrating lymphocytes (TILs) [13]. PD-L1 expression has been correlated with high levels of TILs, an association that has been considered as a favorable indicator for TNBC patients' prognosis [14,15]. Evaluating PD-L1 expression using immunohistochemistry assays such as VENTANA SP142 or Dako 22C3 has made it possible to identify patients who may benefit from immune checkpoint inhibition [16]. Other immune modulating receptors have also been identified as attractive targets for PD-L1− and/or TIL− tumors. Hallmarks of TNBC include dysregulated tumor vasculature, genomic instability, aberrant cell signaling, and deregulation of cellular energetics, each of which could be potential pharmacological targets for combination with immunotherapy. In this review, we discuss the progresses and challenges associated with the present modalities of immunotherapy with respect to TNBC and CSCs.

#### **2. Immune Checkpoint Inhibition: Therapeutic Strategies**

#### *2.1. PD-1*/*PD-L1 Axis*

Immune checkpoint inhibitors (ICIs) have been considered as viable candidates for the treatment of TNBC. Effector T-cells express the Programmed Death 1 (PD-1) cell surface receptor, which interacts with its ligand, PD-L1 (Figure 1). PD-L1 is normally expressed on the surfaces of dendritic cells and macrophages, and binding to PD-1 leads to the inhibition of cytotoxic T-cells [17]. By targeting tumors enriched with TILs that express PD-L1, T-cells within the tumor microenvironment (TME) can be activated [18]. Interestingly, higher rates of PD-L1 expression were found in TNBC patients than with other types of breast cancers [19]. Increased PD-L1 expression can occur via amplification of the PD-L1 gene at 9p24.1. While PD-L1 is normally amplified by 0.7% across most human cancers, this was elevated to 2.0% in TNBC and HER-2-positive breast cancers [20,21]. Sabatier et al. also demonstrated that higher PD-L1 expression resulted in a 50% pathological complete response (pCR), while normal PD-L1 expression resulted in a 21% pCR in response to neoadjuvant chemotherapy [22].

The anti-PD-L1 antibody Atezolizumab has been extensively studied and tested as first-line therapy in a phase I clinical trial (NCT01375842).  Contrarily, women who received Atezolizumab as second- or third-line therapy exhibited a median PFS of 1.8 months (95% CI, 1.4–2.3), a median OS of 7.3 months (95% CI, 6.1–10.8) and an incidence of trAEs of 43% [23]. The subsequent IMpassion130 phase III trial (NCT02425891) investigated whether Atezolizumab, combined with the chemotherapeutic agent nab-Paclitaxel, would generate improved clinical outcomes relative to chemotherapy alone. Blockade of PD-1/PD-L1 interactions in untreated metastatic TNBC patients significantly reduced tumor growth [24]. The median PFS prolongated to 7.2 months from 5.5 months (HR, 0.80; 95% CI, 0.69 to 0.92; *p* = 0.002) in the intention-to-treat population (with patients being randomized according to randomized treatment), and to 7.5 months from 5.0 months (HR, 0.62; 95% CI, 0.49 to 0.78; *p* < 0.001) among patients with PD-L1+ tumors [24]. Furthermore, the median OS prolongated to 21.3 months from 17.6 months (HR, 0.84; 95% CI, 0.69 to 1.02; *p* = 0.008) in the intention-to-treat population, and to 25.0 months from 15.5 months (HR, 0.62; 95% CI, 0.45 to 0.86) among patients with PD-L1+ tumors [24]. As such, Atezolizumab in combination with nab-Paclitaxel was FDA-approved for the treatment of locally advanced or metastatic PD-L1+ TNBC in March 2019.

**Figure 1.** Possible mechanism of immune checkpoint inhibitors in triple-negative breast cancer (TNBC). Tumors consist of heterogenous cell populations including different types of immune infiltrating cells that undergo proliferation and apoptosis. (**A**) Tumor-infiltrating antigen-presenting cells (APCs) display tumor-associated antigens (TAAs) on their major histocompatibility complexes (MHC), which activate antigen-specific naïve T-cells. (**B**) Cluster of differentiation 80 (CD80) on the APC and cluster of differentiation 28 (CD28) are both co-signaling molecules necessary for T-cell activation and expansion. (**C**) CD80 molecules on tumor-infiltrating APCs preferentially bind to cytotoxic T lymphocyte protein-4 (CTLA-4), constitutively expressed on regulatory T cells (Treg) that commonly recruit myeloid-derived suppressor cells (MDSCs) to the tumor microenvironment (TME) to inhibit T-cell activation. (**D**) Anti-CTLA-4 antibodies inhibit Treg activation and enhance anti-tumor activity. (**E**) Tumor cells express programmed death ligand-1 (PD-L1) and tumor-infiltrating lymphocytes express its receptor, PD-1. The interaction between PD-1 and PD-L1 inactivates T-cell activation/expansion within the TME. (**F**) Anti-PD-1 or anti-PD-L1 antibodies prevent PD-1/PD-L1 engagement, thereby inhibiting the suppressive signals and promoting anti-tumor immunity. Figure created with BioRender.com.

Similarly, the anti-PD-1 antibody Pembrolizumab was assessed in the KEYNOTE-086 phase II trial (NCT02447003), administered as a monotherapy to cohorts of previously treated metastatic TNBC and untreated PD-L1+ metastatic TNBC [25]. The median PFS was 2.0 months (95% CI, 1.9 to 2.0) in the previously treated population and 2.1 months (95% CI, 2.0 to 2.2) for the patients with PD-L1+ tumors [25,26]. The median OS was 9.0 months (95% CI, 7.6 to 11.2) in the previously treated population and 18.0 months (95% CI, 12.9 to 23.0) for the patients with PD-L1+ tumors [25,26]. The incidence of trAEs was 60.6% in the previously treated population and 63.1% in the PD-L1+ population [25,26], comparable to anti-PD-L1 monotherapy. This study concluded that Pembrolizumab showed durable anti-tumor activity for patients with PD-L1+ metastatic TNBC and was followed by the KEYNOTE-335 phase III trial (NCT02819518). This study investigated whether Pembrolizumab, in combination with

various chemotherapeutic agents, would provide improved clinical outcomes relative to chemotherapy with placebo. The results indicated that Pembrolizumab plus chemotherapy increased the median PFS from 5.6 months (95% CI, 5.3–7.5) to 9.7 months (95% CI, 7.6–11.3), relative to chemotherapy alone [27]. The objective response rate (ORR) increased from 40% (95% CI, 30%–50%, chemotherapy alone) to 53% (95% CI, 46–60) for the combinatorial treatment [27]. Fatal adverse events occurred in only 2.5% of patients receiving the combinatorial treatment, which was discontinued in 11% of patients due to trAEs [27]. As such, Pembrolizumab in combination with chemotherapy was granted accelerated FDA approval for patients with locally recurrent, unresectable, or metastatic TNBC in November 2020.

Conversely, several reports showed that anti-PD-1 or anti-PD-L1 antibodies did not exhibit better outcomes than chemotherapy in TNBC, while contributing to neurotoxicity [28,29]. Atezolizumab monotherapy demonstrated a low PFS of 1.9 months (95% CI, 1.4–2.5) and a high incidence of trAEs at 68% [23], leading to questions regarding its clinical efficacy. In the IMpassion130 trial, only a subset of patients with PD-L1+ tumors benefited from the treatment compared to the intention-to-treat population (Table 1). Despite the importance of PD-L1 and TILs as predictive biomarkers, a clinical method for TIL assessment needs to be standardized, which may impact patient outcomes of ICIs [30]. Furthermore, the IMpassion130 trial failed to incorporate an Atezolizumab monotherapy arm, whereas the IMpower110 phase III clinical trial (NCT02409342) compared single-agent Atezolizumab to chemotherapy (Table 1). Preliminary results showed a modest increase in median OS from 14.1 months with chemotherapy to 17.5 months with Atezolizumab [31]. Results from the recent IMpassion131 phase III trial (NCT03125902) showed ineffectiveness in the combination of Atezolizumab and chemotherapy for the patients with metastatic TNBC. The median PFS increased insignificantly from 5.7 months (95% CI, 5.4–6.5) to 6.0 months (95% CI, 5.6–7.4), and the median OS increased slightly from 22.1 months (95% CI, 19.2–30.5) to 22.8 months (95% CI, 17.1–28.3) in the placebo plus Paclitaxel and Atezolizumab plus Paclitaxel treatment arms, respectively [32]. Another concern with the IMpassion130 trial is that patients previously treated with adjuvant chemotherapy and relapsed within 12 months were excluded from the trial. It is expected that this will be clarified in the IMpassion132 phase III trial (NCT03371017), which is studying an anti-PD-1 and chemotherapy combinatorial treatment [33]. With respect to Pembrolizumab, the KEYNOTE-119 phase III clinical trial compared single-agent Pembrolizumab to chemotherapy in metastatic TNBC patients (Table 1). The median PFS decreased from 3.3 months (95% CI, 2.7–4.0) with chemotherapy to 2.1 months (95% CI, 2.0–2.1) with Pembrolizumab monotherapy [34]. The median OS also decreased from 10.8 months (95% CI, 9.1–12.6) with chemotherapy to 9.9 months (95% CI, 8.3–11.4) with Pembrolizumab monotherapy, concluding that Pembrolizumab monotherapy did not provide clinical improvements relative to chemotherapy [34]. With conflicting trial results regarding the clinical efficacy of Pembrolizumab, further research is warranted. In addition, researchers have reported associations between ICI monotherapy and immune-mediated neurotoxicity due to potential molecular mimicry or hidden autoimmunity with neuronal antigens in the peripheral nervous system [29,35,36]. As such, improvements to PD-1/PD-L1 axis-associated immunotherapy in TNBC are needed.

**Table 1.** List of completed or ongoing clinical trials using PD-1/PD-L1 immune checkpoint inhibitors.

PFS: progression-free survival; OS: overall survival; ORR: objective response rate; trAEs: treatment-related adverse events.

#### *2.2. CTLA-4 and Dual Checkpoint Inhibition*

The cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is an inhibitory receptor, constitutively expressed on regulatory T cells (Treg) and upregulated on the surface of activated CD4+ and CD8+ T-cells [37,38]. CD-80/B7-1 and CD-86/B7-2 ligands expressed on antigen-presenting cells (APCs) can bind to CD-28 on T-cells to induce T-cell activation and cytokine secretion [39]. CTLA-4 competes with CD-28 to bind CD-80/B7-1 and CD-86/B7-2 ligands, and negatively regulates T-cell function [37–40]. CTLA-4 is essential for neutralizing potential naïve autoreactive T-cells in the secondary lymphoid organs [40]. The inhibition of CTLA-4 can prevent T-cell inhibition and enhance T-cell anti-tumor activity, making it an attractive target for antibody-based therapy (Figure 1). The anti-CTLA-4 antibody Ipilimumab, in combination with the anti-PD-1 antibody Nivolumab, has been FDA-approved for the treatment of melanoma, lung cancer, and renal cell carcinoma, among other cancers [41,42]. The CheckMate032 phase 1/2 trial (NCT01928394) showed modest improvements in ORR for non-small cell lung cancer, from 10% of patients receiving Nivolumab alone to 33% of patients receiving Nivolumab plus Ipilimumab in combination [43]. More recently, the CheckMate-9LA phase III trial

(NCT03215706) demonstrated the superior clinical effectiveness of Nivolumab and Ipilimumab in combination with chemotherapy for metastatic or recurrent non-small-cell lung cancer. Combinatorial treatment increased the median PFS from 5.0 months to 6.7 months, increased the median OS from 10.9 months to 15.6 months, and slightly increased the incidence of trAEs from 38% to 47% relative to chemotherapy alone [44].

Since anti-PD-1/PD-L1 monotherapy was shown to benefit only a subset of the population, additional clinical trials combining anti-CTLA-4 monotherapy have been performed in an effort to improve clinical outcomes but failed to do so. A randomized phase II trial (NCT02519322) testing the clinical efficacy of the Nivolumab as monotherapy in combination with the anti-CTLA-4 antibody Ipilimumab in patients with high-risk resectable melanoma was terminated early because of the high incidence of trAEs. Grade 3 trAEs were reported in 8% of patients in the Nivolumab monotherapy treatment arm and 73% of patients in the combinatorial treatment arm [45]. In a separate phase II clinical trial (NCT02536794), the anti-PD-1 antibody Durvalumab and anti-CTLA-4 antibody Tremelimumab were combined in a single treatment arm without comparison to monotherapy. The drugs were administered to estrogen receptor-positive (ER+) breast cancer and TNBC patients. Preliminary data suggested that clinical benefit was derived in 71% of TNBC patients, but in none of the ER+ breast cancer patients [46]. This trial was also discontinued due to ORRs not meeting the required criteria. The termination of both trials suggested that the risks associated with dual immune checkpoint inhibition may not exceed the benefits.

The use of anti-CTLA-4 antibodies in monotherapy and in combination with anti-PD-1/PD-L1 antibodies have been discredited, due to the lack of significant clinical response and high incidence of autoimmunity. Its ORR in combination with Nivolumab was modest relative to the results seen in the trial by inhibition of the PD-1/PD-L1 axis. Alternatively, anti-CTLA-4 antibodies may be complemented by the stimulator of interferon genes (STING) agonists that promote intratumoral T-cell infiltration and sensitize tumor cells to NK cell killing [47,48]. Harding et al. reported minimal tumor regression following anti-CTLA-4 therapy in B16 murine melanoma models with STING knockout [49]. However, CTLA-4 antibodies may lead to autoimmunity, due to its role in maintaining self-tolerance [50,51]. In a study by Tivol et al., CTLA-4 −/− mice demonstrated excessive proliferation of the lymph nodes, severely destructive myocarditis and pancreatitis, suggesting the role of CTLA-4 in deleting autoreactive T-cells in the periphery [52]. Consistent with this, a study by Gough et al. showed that polymorphisms within the human CTLA-4 gene are associated with autoimmune diseases [53]. This was also consistent with the CheckMate238 clinical trial (NCT02388906), in which grade 3 or 4 trAEs were reported among 45.9% of participants in the Ipilimumab group, followed by discontinued treatment for 42.6% of the patients [54,55]. Interestingly, the efficacy of immune checkpoint inhibition was found to depend on the composition of commensal bacteria. Introducing strains such as *Bacteroides fragilis* to germ-free or antibiotic-treated mice helped to overcome the poor response of CTLA-4 blockades and further induced polarization of T helper cell 1 (TH1) [56]. As such, anti-CTLA-4 alone has not been considered as a viable front-line treatment option for TNBC to date.

#### *2.3. Next Generation Immune Modulatory Targets*

Many immune checkpoint molecules beyond PD-1/PD-L1 and CTLA-4 are currently under clinical investigation to identify additional drug targets for PD-L1- and TIL− patients or to enhance ICI monotherapy. The immunosuppressive protein sialic acid binding Ig-like lectin-15 (Siglec-15), normally expressed on myeloid cells, was shown to be upregulated in many human cancers [57]. With similar structural homology to PD-L1, Siglec-15 is targeted using the anti-Siglec-15 monoclonal antibody, NC318 [58]. In the phase I/II clinical trial (NCT03665285), NC318 was administered to patients with advanced or metastatic solid tumors, with results expected in 2021. T-cell Ig and ITIM domain (TIGIT) is another inhibitory receptor expressed on lymphocytes and upregulated upon activation [59]. Its ligands include CD112, CD113, and CD155, all of which are over-expressed in TNBC [60–62]. Interestingly, pre-clinical experiments on TIGIT−/− mice suggested higher safety and fewer trAEs than

anti-PD1/PD-L1 or anti-CTLA-4 monotherapies [63]. Lymphocyte-activation gene 3 (LAG-3) and T-cell Ig and mucin domain-containing protein 3 (TIM-3) are also attractive immunosuppressive targets being actively investigated in breast cancer. Saleh et al. reported that the co-inhibition of PD-1 and PD-L1 further upregulated LAG-3 and TIM-3 in T-cells and Tregs when co-cultured with TNBC cells, but not with other breast cancer cell lines [64].

In contrast to co-inhibitory immune molecules, co-stimulatory molecules are equally attractive targets in immunotherapy. OX40 is a positive immune checkpoint molecule involved in T-cell proliferation following activation, and Treg suppression [65]. Several clinical trials are investigating the efficacy of anti-OX40 antibodies in combination with ICI monotherapy for TNBC patients (NCT02528357, NCT03971409, NCT03241173). The inducible co-stimulatory receptor 4-1BB is expressed on activated T-cells and NK cells, and can be exploited to improve anti-tumor immunity [66,67]. However, the therapeutic value of 4-1BB in TNBC patients remains open to investigation. The glucocorticoid-induced TNFR-related (GITR) and inducible co-stimulator of T-cells (ICOS) are also attractive stimulatory targets. The ICOS monoclonal antibody, JTX-2011, was administered to TNBC patients in the phase I/II clinical trial (NCT02904226). Preliminary results reported a disease control rate of 25% with JTX-2011 monotherapy, and 29% in combination with Nivolumab (anti-PD-1) [68]. Notably, two grade 5 trAEs were observed among patients in the combinatorial treatment arm, potentially due to the simultaneous expression of ICOS on immunosuppressive Tregs [68]. In this regard, agonist antibodies in immunotherapy have been approached with caution and require additional research before implementation in the clinic.

#### **3. Factors A**ff**ecting the E**ffi**cacy of Immune Checkpoint Inhibitors**

Blockade of PD-1/PD-L1 and/or CTLA-4 has been demonstrated to be effective and durable in certain types of cancers. However, fewer than 10% of patients respond to single-agent treatments [69]. Co-administration of ICIs with chemotherapeutic agents, as described above, contributes to the enhanced immune priming [24,27,70]. Combinations with other therapies or factors may also increase efficacy, as described below.

#### *3.1. Dysregulated Tumor Vasculature*

Poor clinical outcomes associated with TNBC are partly attributed to the dysregulated angiogenesis that results in hypoxic conditions within the TME. As tumors expand over time, tumor cells within the tumor core become increasingly hypoxic, such that there is an upregulation of angiogenic growth factors associated with the expression of hypoxia-induced transcription factor (HIF-1) [71]. Angiogenic growth factors including vascular endothelial growth factor (VEGF), endothelial growth factor (EGF), and platelet-derived growth factor (PDGF) promote the migration of endothelial cells towards the tumor core, through which tumors acquire nutrients for growth and a route to metastasize into systemic circulation [71–73]. Under normal conditions, angiogenic growth factors are balanced by the metabolic demands of the surrounding tissue. However, the hypoxic conditions of the TME hijack this balance in favor of dysregulated angiogenesis [72,73].

Tumor-associated capillaries contribute to immunosuppression by reducing trafficking and activation of effector T-cells and restricting entry of cytotoxic drugs [74,75]. Tumor-associated endothelial cells may release interferon-γ (IFNγ) that upregulates PD-L1 expression to inhibit the anti-tumor activity of T-cells [76]. This is consistent with a study by Kammertoens et al., who showed that intratumoral injection of IFNγ caused rapid loss of tumor-associated vessels but also impeded anti-tumor activity of effector T-cells [77]. As IFNγ is mainly expressed by activated infiltrating T-cells, the IFNγ-mediated upregulation of PD-L1 can be exploited in a combinatorial therapy for non-responders to anti-PD-1 monotherapy [78]. Tian et al. reported an increase in pericyte coverage and a decrease in pulmonary metastasis (indicators of vasculature normalization) following immune checkpoint blockade in mice bearing TNBC 4T1 tumors [79,80].

To inhibit the growth and metastasis of TNBC and promote the anti-tumor activity of effector T-cells, a combination of immunotherapy with anti-angiogenic factors has been investigated. In the IMbrave150 phase III clinical trial (NCT03434379), the anti-PD-1 agent Atezolizumab was combined with anti-angiogenic agent Bevacizumab and compared with protein kinase inhibitor Sorafenib alone (Table 2). There was a clinically significant improvement in median PFS, which was 6.8 months (HR, 0.58; 95% CI, 5.7–8.3) in the Atezolizumab/Bevacizumab group and 4.3 months (HR, 0.59; 95% CI, 4.0–5.6) in the Sorafenib group [81]. Of note, grade 3 or 4 trAEs occurred in 56.5% of patients receiving Atezolizumab/Bevacizumab and 55.1% of patients receiving Sorafenib [81]. Furthermore, Huang et al. showed that lower doses of anti-VEGFR2 antibody improve tumor-associated vessel perfusion and reduce tumor hypoxia more effectively than the immunoglobulin-G (IgG) control and high-dose anti-VEGFR2 treatment groups [82]. This highlights the importance of further examining dosage and timing to optimize the combinatorial efficacy of anti-angiogenic and ICI treatments. The excessive use of anti-angiogenic agents may impede drug delivery and limit the infiltration of effector T-cells in the tumor [82,83]. Wu et al. proposed using angiopoietin-2 as a biomarker in addition to as a therapeutic target for predicting the clinical outcome of Bevacizumab monotherapy, due to its important role in treatment resistance [84]. Therefore, the combination of ICI with anti-angiogenesis therapy may represent a promising avenue for the future of TNBC treatment.

**Table 2.** List of completed or ongoing clinical trials using immune checkpoint inhibitors in combinatorial therapy.

PFS: progression-free survival; OS: overall survival; trAEs: treatment-related adverse events.

#### *3.2. Interleukin-8 and CXCR1*/*CXCR2*

Interleukin-8 (IL-8) is a chemokine responsible for the recruitment of neutrophils to areas of inflammation, infection, or injury [90]. IL-8 is secreted by macrophages, epithelial cells, airway smooth muscle cells, and endothelial cells [90,91]. IL-8 binds to CXCR1 and CXCR2 G-protein coupled receptors on granulocytes, monocytes, and endothelial cells [90–92].  Cheng et al. reported an overexpression of IL-8, CXCR1, and CXCR2 in breast, prostate, lung, and colon cancers [93]. The binding of IL-8 to CXCR1/CXCR2 was shown to induce the transition from an epithelial-like to mesenchymal-like status, thus promoting the migration, invasion, and reconstitution of a secondary tumor [94,95]. CXCR2 signaling also promotes the migration of human endothelial cells and angiogenesis, forming a positive feedback loop to further promote epithelial-to-mesenchymal transition (EMT) [96,97]. IL-8 inhibitors might play an important role as anti-angiogenic agents in combination with ICIs. In addition, IL-8 signaling directly promotes immunosuppression in the TME via the recruitment of myeloid-derived suppressor cells (MDSCs, Figure 2). MDSCs are capable of depleting nutrients such as L-arginine, L-tryptophan, and L-cysteine, all of which are essential for T-cell expansion [98,99]. MDSCs also inhibit anti-tumor activity by producing reactive oxygen species and peroxynitrite that can directly inactivate T-cell receptors [100], and by producing reactive nitrogen species that hinder the infiltration of cytotoxic T-cells into the tumor core [101]. Highfill et al. showed that early treatments with anti-PD-1 agents prevented tumor growth, but late treatments showed less benefit due to the presence of MDSCs in the TME [102]. They further showed that anti-CXCR2 monoclonal antibody therapy led to significant anti-tumor activity, even after delayed anti-PD1 treatment [102]. Sanmamed et al. also suggested using IL-8 as a prognostic biomarker to predict the clinical benefit of ICI therapy [103]. They observed that serum IL-8 levels decreased significantly among patients responding to anti-PD-1 checkpoint inhibition (*p* < 0.001) [103]. Serum IL-8 levels also increased significantly among non-responders to anti-PD-1 blockade (*p* = 0.013) [103]. Together, these results suggest that IL-8 inhibition may represent a potential candidate for combinatorial therapy with ICIs.

The anti-IL-8 antibody HuMax-IL8 (also known as BMS-986253) was developed for the successful depletion of tumor-secreted IL-8 and inhibition of CSC mesenchymal properties [85]. HuMax-IL8 also reduced the recruitment of polymorphonuclear MDSCs to the tumor core by preventing IL-8 from binding to CXCR2 receptors on the MDSCs [85]. The phase I trial for HuMax-IL8 (NCT02536469) concluded that serum IL-8 was significantly reduced after the third day of treatment relative to control (*p* = 0.0004) [86]. The incidence of trAEs was 33%, which was much lower than that of anti-angiogenic agents observed in clinical trials [86]. Additionally, the MAGIC-8 phase Ib/II clinical trial (NCT03689699) combined the anti-PD-1 agent Nivolumab with HuMax-IL8 for the treatment of hormone-sensitive prostate cancer [104]. The results of this study are expected in 2022. Thus, IL-8 inhibition may provide a benefit among non-responders of ICIs in TNBC patients, warranting further exploration.

**Figure 2.** Various tumor-promoting mechanisms that can be exploited in combination with immune checkpoint inhibition. Overexpression of IL-8 and CXCR1/2 in TNBC generates an autocrine positive feedback loop that promotes EMT, HIF-1-mediated angiogenesis and endothelial migration, and recruitment of immunosuppressive MDSCs to the tumor microenvironment (TME). Transcription of NF-κB, HIF-1, AP-1, and STAT3, in combination with Wnt and YAP signaling, upregulates CD73 on TNBC cells. Immunogenic cell death in the TME releases ATP in abundance, which is subsequently converted to adenosine by CD39 and CD73. Excess adenosine in the TME bind to adenosine type 1 purinergic G-protein coupled receptors that further facilitate MDSC expansion, CTLA-4 upregulation, inhibition of TIL activation, inhibition of DC maturation, and M2-type TAM polarization. Activation of adenosine receptors on tumor-associated endothelial cells promotes angiogenesis via upregulation of VEGF and HIF-1α, along with a simultaneous downregulation of adhesion molecules essential for diapedesis. Finally, activation of adenosine receptors on the TNBC cell further upregulate cytosolic cyclic AMP (cAMP), Erk1/2, PI3K, and MEK1, all of which further enhance the IL-8 signaling pathway. Figure created with BioRender.com.

#### *3.3. CD73 Expression*

Cluster of differentiation 73 (CD73), normally expressed on Treg cells, is an ectonucleotidase that dephosphorylates extracellular AMP to adenosine [105]. Its expression on bulk tumor cells and mesenchymal-like CSCs generates excess adenosine in the TME, which binds to the adenosine type 1 purinergic G-protein coupled receptor family (denoted A1, A2A, A2B, and A3), some of which are involved in inhibiting effector T-cell responses [106,107]. Adenosine acting on the A2A receptors has a suppressive effect on effector T-cells and an obligatory role in tumor immunomodulation [105,106,108]. In conjunction with IL-8 signaling, the excess adenosine levels facilitate MDSC expansion in the TME via the activation of A2B receptors to enhance immunosuppression [109]. Jin et al. showed that the knockdown of tumor CD73 and subsequent transfer of tumor-specific T-cells significantly enhanced tumor-free survival in tumor-bearing mice [108]. Hypoxic conditions within the tumor core, along with Wingless (Wnt) signaling, upregulate CD73 expression [110,111]. In addition to increasing immunosuppressive adenosine levels in the TME, CD73 modulates cell adhesion molecules within the endothelium, whereas upregulated CD73 promotes the attachment of lymphocytes and reduces their migration into the lymph nodes [112]. CD73 overexpression is also associated with poor prognosis in TNBC [113], highlighting the potential for targeting CD73 as part of a combinatorial treatment (Figure 2). In the phase I trial (NCT02754141), it was shown that the anti-CD73 agent BMS-986189 in combination with the anti-PD-1 antibody Nivolumab was well-tolerated in patients with advanced solid tumors [87]. TrAEs were observed in 58% of the patients receiving the combination, of which 15% were grade 3 in nature [87]. Alternatively, the SYNERGY phase I/II clinical trial (NCT03616886) compared the anti-CD73 agent Oleclumab with the anti-PD-1 agent Durvalumab, and chemotherapeutic agents, in single and combinatorial treatment arms (Table 2). The results of this study are expected in 2022.

#### *3.4. Long Non-Coding RNAs and Microsatellite Instability*

Long non-coding RNAs (lncRNAs) are ~200 nucleotides long and do not code for protein products [114]. Their role in disease regulation was only brought to light recently, when the lncRNA urothelial carcinoma-associated 1 (UCA1) was shown to contribute to resistance against tamoxifen therapy in ER+ breast cancers [115]. The lncRNA ROR was associated with a decrease in the expression of E-cadherin, an epithelial marker, and an increase in the expression of the mesenchymal markers vimentin, zeb1, and zeb2 [116]. ROR promoted metastasis via the EMT process and contributed to tamoxifen therapy resistance [116]. LncRNAs (e.g., the nuclear-enriched autosomal transcript1 (NEAT1)) were also associated with immunosuppression. Yan et al. reported that NEAT1 inhibition suppressed CD8+ T-cell apoptosis and enhanced anti-tumor activity [117]. Metastasis-associated lung adenocarcinoma transcript1 (MALAT1) is another lncRNA that was observed to upregulate the expression of PD-1 and CD-47 [118]. Wang et al. showed that knock-down of MALAT1 by shRNA decreased the expression of PD-1, and also suppressed the EMT process [119]. Despite the growing evidence regarding the potential role of lncRNAs in immunotherapy resistance, targeting them in the clinic remains a challenge. LncRNAs would be considered in combinatorial therapy, however, targeting them through RNAi-mediated gene silencing therapy, antisense oligonucleotide-based therapy, or small molecule inhibitors remains expensive and inconvenient, defies precision medicine, and may contribute to unforeseen systemic adverse events or trAEs [120,121]. Further research is therefore required before investigating their potential in the clinic.

Microsatellite instability (MSI) status has also been considered to impact efficacy of ICIs and is therefore used as a reliable biomarker. Microsatellites are short repetitive sequences scattered throughout the human genome as a result of aberrant DNA mismatch repair mechanisms [122]. Recent reports have suggested that a high MSI status, indicative of a hypermutation phenotype, may sensitize patients to ICIs [123,124]. Interestingly, 6.9% of TNBC cases showed a complete loss of relevant mismatch repair proteins, which were correlated with significantly greater PD-L1 expression [125]. Other contributors to MSI status may include tumor mutational burden, which is a measure of nonsynonymous mutations in tumor cells [126], and BRCA1 mutation status. BRCA1 expression modulates the silencing mechanisms in satellite DNA, such that BRCA1-mutated TNBC exhibit higher microsatellite instability than BRCA1-wildtype TNBC [127,128]. The phase II trial (NCT01876511) comparing Pembrolizumab efficacy in MSI-positive and MSI-negative colorectal cancer patients demonstrated an immune-related PFS of 78% among MSI-positive patients and 11% among MSI-negative patients [129]. Pembrolizumab was subsequently FDA-approved for unresectable or metastatic solid tumors with high MSI.

#### *3.5. Wnt and YAP Signaling*

Many components of the Wnt signaling pathway are involved in tumorigenesis via EMT and tumor regeneration [130–133]. Cytoplasmic stabilization and nuclear translocation of β-catenin into the nucleus allows for the expression of Wnt target genes [132]. The enrichment of β-catenin, nuclear translocation, and dysregulatedWnt signaling are all associated with poor clinical outcomes of TNBC [134]. The inhibition of Wnt signaling leads to the suppression of CSCs and bulk tumor cells in both TNBC cell lines and patient-derived xenograft (PDX) models [135]. Additionally, activation of β-catenin in tumor cells prevents spontaneous T-cell priming and infiltration of effector T-cells into the TME [136]. A mouse model for hepatocellular carcinoma also demonstrated the role of β-catenin in immune escape and resistance to anti-PD-1 monotherapy [137]. This was consistent with a study by Castagnoli et al., which reported a strong correlation between downstream Wnt signaling effector expression and PD-L1 expression in TNBC [138]. Furthermore, several studies have shown that Wnt inhibitors reduce PD-L1 expression and Wnt agonists enhance PD-L1 expression, suggesting Wnt inhibitors as possible adjuvants to anti-PD-1, anti-PD-L1, or anti-CTLA4 therapies [138,139]. In addition to modulating PD-L1 expression, Wnt/β-catenin signaling has been shown to inhibit T-cell maturation and activation and inhibit dendritic cells (DCs) from secreting chemokines essential for T-cell activation [140–142].

Yes-associated protein (YAP) signaling has also been shown to contribute to the EMT process. A study by Cordenonsi et al. demonstrated a strong correlation between YAP expression and mesenchymal-like CSC surface markers [143,144]. Moreover, YAP knockdown in TNBC cell lines led to a loss of cell proliferation and invasiveness [145]. Besides the role of YAP in modulating Wnt signaling via the β-catenin destruction complex [146], YAP was also shown to directly modulate PD-L1 expression in human TNBC [147]. Interestingly, recent studies have reported that the major transcription factor in YAP signaling, TEAD, has a binding site located close to the PD-L1 promoter. As such, YAP can bind directly to the enhancer region of PD-L1 [148,149]. YAP signaling has also been implicated in recruiting MDSCs to the TME, contributing to the suppression of anti-tumor activity in a similar manner to IL-8 [150]. Moreover, YAP signaling regulates the recruitment of tumor-associated macrophages (TAMs) to the TME. TAMs can differentiate into M1 and M2 phenotypes. M1-type TAMs secrete pro-inflammatory interleukin-1 (IL-1) and tumor necrosis factor-α (TNFα), which promote the expression of inducible nitric oxide synthase (iNOS) to enhance the antigen presentation process [151]. In contrast, M2-type TAMs secrete IL-10, IL-4, arginase-1, and other cytokines involved in resolving inflammation, wound-healing, and facilitating tumor growth [151]. High transcriptional levels of YAP have been reported to disproportionately promote the expression of M2-type TAMs in the TME to enhance tumor growth, drug resistance, and metastasis [152,153].

Wnt and YAP signaling might be important pharmacological targets for eliminating mesenchymal and epithelial CSC populations while simultaneously enhancing ICI immunotherapy outcomes. However, it remains unclear whether they can be implemented in combinatorial treatments, as molecular mechanisms remain unclear. Furthermore, the complexity of Wnt signaling and its broad involvement in normal stem cell self-renewal, differentiation, and organ homeostasis [154] suggest that inhibiting this pathway may be counterproductive and yield intolerable toxicity. However, a phase I clinical trial (NCT01302405) using the Wnt signaling inhibitor PRI-724 concluded an acceptable toxicity profile, with 11% of patients experiencing grade 3 trAEs [88]. Inhibition of YAP signaling poses its own challenges, as Ni et al. reported that YAP is essential for the differentiation of Treg cells, which prevent autoimmune disease [155]. Our recent report suggested that dual inhibition of Wnt and YAP signaling, but neither alone, is required for suppressing both mesenchymal and epithelial CSC populations and diminishing Paclitaxel-induced CSC enrichment in immune-deficient mice [156]. Although combination therapies typically result in better drug responsiveness and synergism than single-agent monotherapies [157], further studies are needed to clarify the efficacy and toxicity of combination therapy with Wnt and/or YAP inhibitors and ICIs.

#### *3.6. Nanoparticle Platforms as a Delivery System*

Traditional ICIs are delivered systemically as monoclonal antibodies, which have the potential of activating self-reactive T-cells [158,159]. Immune-related adverse effects may be amplified in combinatorial ICI therapies and may be reduced or resolved by the use of corticosteroids. However, this can increase the risk of other complications and diminish the therapeutic potential of ICIs [158]. Nanoparticle platform-based therapies have revolutionized drug delivery owing to their ability to accumulate in solid tumors, reduce toxicity to vital organs, and increase the therapeutic index [160– 162]. Studies have demonstrated the ability of nanoparticles (NPs) to deliver both hydrophobic and hydrophilic drugs, small molecule drugs, and antibodies to the TME, with minimal toxicity to surrounding tissues [160,163]. Studies have also demonstrated the ability of NPs to efficiently interact with and activate dendritic cells and macrophages within the TME [164]. For example, synthetic high-density lipoprotein (sHDL) nanodiscs loaded with Doxorubicin and combined with anti-PD-1 ICIs resulted in a seven-fold increase in IFNγ-positive CD8+ T-cells in the TME, compared with Doxorubicin treatment alone [165]. Additional benefits of the sHDL nanodiscs included the complete regression of colon carcinoma tumors in 80–88% of the treated animals and no apparent cardiotoxicity post-treatment [165]. In another report, poly(lactic-co-glycolic acid) (PLGA) NPs co-loaded with Paclitaxel, combined with detoxified bacterial lipopolysaccharide, resulted in a significant increase in TH1 cells in B16F10 mouse models, in comparison to Paclitaxel treatment alone [166]. PLGA NP-treated mice exhibited a 40% lower tumor volume and a higher degree of retention in biological activities of both co-encapsulated drugs [166]. In a third study, nanoscale coordination of polymer (NCP) core-shell nanoparticles co-encapsulated with Oxaliplatin and pyrolipids for photodynamic therapy demonstrated synergism with anti-PD-1 ICI therapy in CT26 and HT29 mouse models [167]. This treatment also reduced tumor volume to 2.9% of their original size when combined with anti-PD-1 therapy, and to 39.1% of their original size in the absence of anti-PD-1 therapy [167]. Such synergy with anti-PD-1 therapy was also observed with peptide-based structure-transformable NPs [168], dendrimers used for siRNA delivery [169], and inorganic NPs composed of gold, titanium dioxide, or iron oxide [170,171]. It is evident that various NP platforms can be used to enhance tumor immunogenicity, favor ICI therapies, and promote the pharmacokinetics and pharmacodynamics of combinatorial treatments.

#### **4. Cancer Cell Antigens: Potential Therapeutic Targets**

Since ICI immunotherapy for TNBC benefits only a subset of patients, developing treatment strategies for TNBC patients with lower immunogenic tumors remains an unmet medical need. An ideal therapy would be one with target antigens expressed on bulk tumor cells and also overexpressed in CSC populations [172]. To effectively boost anti-tumor immunity, antigen expression should be evaluated on both CSCs and bulk tumor cells. Interestingly, cancer-testis antigens (CTAs) have been shown to be overexpressed in CSC populations [173]. CTAs are normally expressed in germ-line tissues such as the testis, placenta, and ovaries, but are also highly expressed across several cancer types [174]. In the following section, we will describe some CTAs and two other tumor antigens that have shown potential as biomarkers or immunotherapeutic targets in TNBC.

#### *4.1. Cancer-Testis Antigens*

The progression from primary tumor to metastasis is somewhat resembled in the gonads, where trophoblasts invade and burrow into the endometrium [175]. Placenta-specific protein 1 (Plac1) normally plays an important role in trophoblast invasion and migration but is also found to be expressed in a large range of human cancers [176,177]. As such, trophoblast-specific pathways could be reactivated, contributing to the activation of lymphocyte-mediated tumor growth [177]. It has been hypothesized that placental mammals have a certain degree of placental invasiveness that is positively correlated with the incidence of metastatic tumors [178]. Females with a lower degree of placental invasiveness in some species have evolved mechanisms to counter trophoblast invasion, and thus

cancer metastasis [179]. Koslowski et al. showed that siRNA inhibition of Plac1 effectively suppressed tumor migration and invasion in breast cancer cell lines [180]. However, the correlation between Plac1 expression and clinical prognosis of TNBC remains unknown [180], and more research is required.

Another strong CTA candidate for TNBC immunotherapy is the New York esophageal squamous cell carcinoma 1 (NY-ESO-1). NY-ESO-1 is normally expressed in primary spermatocytes and rapidly declines in female oogonia [181]. NY-ESO-1 expression is believed to be involved in the proliferation of stem cells and epithelial CSC populations [182]. Ademuyiwa et al. reported NY-ESO-1 expression in 16% of TNBC patients, and antibody responses against NY-ESO-1 were observed in 73% of TNBC patients who were NY-ESO-1-positive [183]. It was also reported that NY-ESO-1-positive patients had higher CD8+ T-cell infiltration in TNBC tumors [184]. NY-ESO-1-specific CD8+ T-cells showed upregulated PD-1 expression, suppressing anti-tumor immunity [184]. As such, NY-ESO-1 may be an attractive candidate for a combinatorial therapy with anti-PD-1 ICI.

The MAGE-A family is also one of the CTAs that renders TNBC highly immunogenic. Raghavendra et al. reported MAGE-A expression in 47% of TNBC cases, and the majority of NY-ESO-1-positive TNBC tumors were also MAGE-A-positive [185]. MAGE-A is normally involved in chromosomal alignment and centrosome duplication [186]. In TNBC, the expression of MAGE-A, however, was positively correlated with the expression of mesenchymal-like CSC markers such as vimentin, but negatively correlated with the expression of epithelial-like CSC markers such as E-cadherin and β-catenin [187]. Targeting MAGE-A would likely enhance anti-tumor immunity by suppressing EMT. MAGE-A12 has been shown to enhance tumor cell proliferation and CSC maintenance [188]. Since targeting multiple CTAs would provide more benefit than targeting a single CTA in anti-tumor immunotherapy, clinical trials have looked at MAGE-A and NY-ESO-1-based vaccines. However, the MAGRIT phase III trial (NCT00480025, targeting MAGE-A3 in non-small cell lung cancer patients) was terminated due to the lack of clinical benefit [189]. Inter-tumoral heterogeneity could partially explain the extent to which certain CTAs are expressed in TNBC versus other tumors [190]. Further characterization of CTA expression in different types of tumor is essential for developing CTA-based therapies for a given tumor type. Although CTAs represent potential therapeutic targets, their expression remain elusive and appear limited to a small subset of patients [191,192].

#### *4.2. Tumor Antigens, Cancer Vaccine, and Oncolytic Virus*

The expression of tumor antigens susceptible to immunotherapies in CSC populations have been poorly characterized [186]. While conventional approaches to dendritic cell (DC) vaccines involve using bulk tumor cells as the antigen source, Ning et al. reported that DC vaccines loaded with the lysates of CSCs induced significantly better anti-tumor humoral and cellular immunity than those loaded with bulk tumor cells in mice [193]. Whole-tumor lysates, tumor-antigen-derived peptides, or antigen-encoding RNA/DNA have been used in cancer vaccines [194]. The resultant epitopes from vaccination were presented on major histocompatibility complexes (MHC) I or II by DCs for presentation to CD8+ or CD4+ T-cells, respectively [195].

Tumor-associated antigens (TAAs) have been considered as a possible solution for targeting CSCs, which are molecules expressed at high levels on cancer cells and low levels on healthy cells [196]. However, TAAs such as gp100 and tyrosinase have the potential for off-target toxicity, due to their systemic expression in normal tissues [196,197]. The challenge to select the appropriate antigen renders tumor vaccinations as a less favorable treatment option. One of the strategies used to overcome this challenge in the clinical setting is to use toll-like receptor (TLR) agonists, to potentiate the innate immune system [198]. In a phase II clinical trial (NCT00960752), the TLR-7/8 agonist Resiquimod was combined with gp100 and MAGE-3 peptide vaccines, with results expected later this year [199]. Other reports showed that TLR-7/8 agonists, among others, increase PD-L1 expression on DCs [200]. More studies will be needed to further consolidate the role of TAAs and CTAs in cancer vaccine and ICI immunotherapy for TNBC.

As an alternative approach to DC vaccinations, oncolytic viruses (OVs) have a well-characterized role in inducing anti-tumor immunity. OVs are naturally or genetically modified vectors that are able to selectively replicate in tumor cells, as tumor cells often have impaired antiviral defenses that make them susceptible to OV infections [201–208]. As OVs replicate in the tumor cells, they trigger an inflammatory response leading to immunogenic cell death (ICD) [204]. Following ICD, damage-associated molecular patterns (DAMPs) are released into the TME, which can be recognized by antigen-presenting cells that secrete cytokines including IFNα, IFNγ, TNFα, IL-6, and IL-12 to recruit innate immune cells [204,205]. Furthermore, ICD results in the release of TAAs and tumor-specific antigens (TSAs) into the TME, which activate antigen-specific CD4+ and CD8+ T-cells as part of adaptive immunity [206,207]. By stimulating both innate and adaptive immunity, OVs are able to maintain anti-tumor immunological memory to protect against tumor reconstitution. Similar to TLR agonists, a 2017 study revealed that the OV talimogene laherparepvec (T-VEC) increased PD-1 expression [208]. When T-VEC OV therapy was combined with the anti-PD-1 agent Pembrolizumab, the ORR increased by 62% [209]. In a phase I/II trial (NCT02779855), T-VEC OV therapy combined with neoadjuvant chemotherapy was compared with chemotherapy alone. Preliminary data showed an increase in pCR from 30% with chemotherapy alone to 55% in the combinatorial treatment for non-metastatic TNBC patients [210]. As OVs allow for the exploitation of DAMPs and tumor antigens, inflammation induced by the adenovirus primes the tumor for subsequent DC vaccination, which elicits an anti-tumor CD8+ T-cell response in mice with lung cancer [211]. Furthermore, the Maraba MG1 rhabdovirus, boosted with adenovirus, led to MAGE-3-specific CD4+ and CD8+ T-cell expansion that persisted for several months in mice with MAGE-3-positive solid malignancies [212]. Despite some benefits of OVs in immunotherapy, the main challenge is the systemic antiviral mechanism, which has the potential to block OV replication and infection of tumor cells [212].

#### **5. Chimeric Antigen Receptor T-Cell Therapy**

Chimeric antigen receptor T-cell (CAR-T) therapy involves cytotoxic T-cells that are engineered to express fusion proteins that are capable of recognizing and binding to TAAs expressed by tumor cells. These fusion proteins commonly consist of an extracellular single chain variable fragment (scFv) domain for TAA recognition, a transmembrane domain, and an intracellular T-cell coactivation domain [213]. Engineered CAR-T cells offer personalized immunotherapy but are not subject to the same regulatory signaling as endogenous T-cells [214]. This may contribute to trAEs such as cytokine release syndrome, in which the rapid activation and proliferation of CAR-T cells contributes to the excess production of pro-inflammatory cytokines [215]. Zhou et al. showed that the TAB004 monoclonal antibody, capable of recognizing the tumor variant of mucin1 glycoprotein (tMUC1), can be used to make the scFv domain of their MUC28z CAR-T cells [216]. As tMUC1 is expressed in 95% of malignant tissues (including TNBC); IFNγ levels increased from 2.6 to 18.7 pg/mL among HCC70 cells upon the introduction of MUC28z CAR-T cells [216]. Tumor endothelial marker 8 (TEM8)-specific CAR-T cells have also shown to eliminate TEM8+-TNBC tumor cells, and also target tumor-associated endothelial cells [217]. Selection of the right CAR scFv domain dictates the therapeutic potential of CAR-T cells against tumors.

Despite FDA approval, Singh et al. showed relatively poor results of CAR-T therapy against solid tumors, including TNBC, as they are unable to survive in the harsh TME [213]. This has not stopped research groups from exploring CAR-T therapy in TNBC. Based on reports of c-Met overexpression in 52% of TNBC tumors [218], a phase I trial (NCT01837602) demonstrated that c-Met-CAR T-cells did not induce cytokine release syndrome and exhibited on-target effects for c-Met-positive TNBC patients [219]. Previous reports also showed that various TNBC cells lines exhibit moderate to high levels of NKG2D ligand (NKG2DL) [220,221]. Accordingly, the use of the natural killer (NK) cell-activating receptor NKG2D CAR constructs in vivo led to significant MDA-MB-231 tumor regression in mice [220]. Furthermore, a phase I trial (NCT04107142) administered NKG2DL-targeting γ/δ CAR T-cells to

patients of varying tumor types, including TNBC. The results for this study are expected in 2021. Similar to the strategies above, further CAR-T research may lead to novel therapeutic options for TNBC.

#### **6. Immunotherapy and Metabolism**

#### *6.1. Metabolic Reprogramming in TNBC*

It has long been established that tumor cells exhibit an altered cellular metabolism where they shift their metabolic reliance to sustain their proliferative and competitive needs. Metabolic reprogramming is now recognized as a hallmark of malignancy in various different cancers [222,223]. In a phenomenon called the Warburg effect, cancer cells tend to undergo aerobic glycolysis where they rely on glycolysis instead of oxidative phosphorylation, even in the presence of oxygen [223]. This dysregulated increase in glucose influx and glycolytic rate is thought to provide energy on a large scale while depleting the TME of nutrients other cells needed by other cells. While this "Warburg" phenotype is tumor-dependent, TNBC has been shown to be more dependent on glycolysis compared to other breast cancer subtypes, as they overexpress glycolytic components such as lactate dehydrogenase (LDHA), glucose transporter 1 (GLUT1), and monocarboxylate transporters (MCT1/4) [224–226]. Some TNBC tumors overexpressed the GTPase-activating protein USP6NL which is involved in regulating signal transduction and upregulation of GLUT1 via the Wnt/β-catenin pathway [227]. The knockdown of USP6NL has been shown to inhibit TNBC cell growth, motility, and EMT [228]. TNBC cells are also known to be reliant on an increase in fatty acid oxidation and glutamine metabolism as an alternative energy source and to sustain the increased rate of cell growth [229,230].

#### *6.2. Aerobic Glycolysis and Immunosuppression*

Perhaps, one of the most intriguing advantages for tumor cell metabolic redirection is its influence on immune cell infiltration, where an immunosuppressive environment is created within the TME. The increased efflux of lactate that is typical in a glycolytic phenotype, for example, results in the acidification of the TME, which has been shown to inhibit CD8+ T-cell activity and TH1 cell IFNγ production [231,232], while the depletion of glucose due to the increased competitive uptake by cancer cells leads to cytotoxic immune cell dysfunction. In TNBC, LDHA expression was shown to increase the number of Tregs and reduce the infiltration of CD8+ T-cells [233]. In the same study, Haung et al. showed through a Kaplan–Meier survival analysis that co-expression of PD-L1 and LDHA in TNBC was linked to poor outcomes in patients with shorter OS and DFS [233]. Interestingly, they show that an over-expression of PD-L1 on TNBC cells results in an increase in LDHA and vice versa, identifying a therapeutic strategy to simultaneously inhibit metabolic and immunologic aspects of tumorigenesis by co-targeting LDHA and PD-L1 [233]. Feng et al. uncovered a role for TAZ, a YAP paralogous transcription cofactor and downstream effector of the Hippo pathway in the interplay between immunosuppression and aerobic glycolysis [234]. In their study, they show that a lactate-mediated increase in PD-L1 was dependent on TAZ in glycolytic cancer cells [234]. Furthermore, inhibiting the CSC-related Wnt pathway could aid in the decreased acidification of the TME and increased immune filtration [235]. The interplay of aerobic glycolysis and immunology in TNBC remains largely unexplored, with more players yet to be identified.

#### *6.3. Glutamine Metabolism in Immunosuppression*

Cancer cells also rely on glutamine metabolism for cell growth and anabolic processes. TNBC cells showed an increased reliance on glutamine uptake and metabolism where the glutamine transporters alanine, serine, cysteine-preferring transporter 2 (ASCT2), and L-type amino acid transporter 1 (LAT1) are over-expressed [236]. Once in the cell, glutamine is converted to glutamate and α-ketoglutarate, which could be converted to malate and then to pyruvate, effectively supplementing aerobic glycolysis and contributing to the Warburg phenotype. Lampa et al. reported that suppression of glutaminase synergized the inhibitory effect of mammalian target of rapamycin (mTOR) on the growth of TNBC

cell lines [237]. However, a recent study by Leone et al. also used a novel glutaminase antagonist, JHU083, that inhibits glutamine-requiring enzymes [238]. They found that treatment with JHU083 reverted the Warburg effect and inhibited glycolysis, thus increasing the glucose and glutamine content in the tumor as well as increasing the infiltration of CD8+ T-cells [238]. Metabolic analysis showed that the glutamine antagonist increased oxidative phosphorylation through an upregulation of mitochondrial proteins in T-cells but suppressed overall metabolism in cancer cells. While targeting glutamine uptake seems to be a plausible therapeutic strategy, it is limited by the fact that glutamine uptake is also essential for immune cell function [239]. However, combining JHU083 with immunotherapy led to a great response in vivo, where the mice treated with the glutamine antagonist and anti-PD-1 generated significant antitumor effects, with complete response rates close to 100% [238]. While the safety of the glutamine antagonist has yet to be determined, this work provides a glimmer of hope for simultaneously inhibiting metabolic reprogramming and activating the anti-tumor response as a means of therapy.

#### *6.4. Lipid Metabolism in Immunosuppression*

TNBC cells exhibit an increase in fatty acid oxidation (FAO) and a decrease in fatty acid synthesis (FAS) compared to other subtypes [239]. Specifically, FAO seems to be crucial in the maintenance of breast CSCs. Studies have reported a higher FAO rate in TNBC CSCs than non-CSCs [240,241]. Wang et al. found that FAO in breast CSCs is dependent on STAT3 signaling, identifying a possible avenue to target lipid metabolic rewiring through the inhibition of JAK/STAT3 [241]. Their work also establishes a link between chemoresistance and FAO levels, where blocking FAO re-sensitized cells to chemotherapy in vivo [241]. This was consolidated by Casciano et al., who reported a link between the highly amplified MYC transcription factor (which occurred in up to 50% of TNBC cases) and its role in promoting FAO [242]. While the role of MYC in TNBC metabolism remains largely unknown, it may be considered as a potential therapeutic avenue in the future.

Lipid metabolism also plays a role in immune cell development and activation. Treg cells adapt to the nutrient depleted hypoxic TME by metabolically depending on fatty acids. Tregs relies on FAO for energy to proliferate and exert an immunosuppressive function [243], while T-cell activation is dependent on FAS [244]. Furthermore, increased lipid uptake upregulated PD-1 in CD8+ T-cells, while PD-1 blockade activated these T-cells. Given that lipid metabolism is a crucial aspect of TNBC tumorigenicity and Treg function, targeting enzymes involved with FAO could potentially lead to a reduction in TNBC tumor burden, CSC enrichment, and enhance anti-tumor immunity. While pharmacologically targeting FAO in TNBC has garnered preclinical success, more work is still required to decipher the effect on immune cell infiltration [245].

Emerging evidence points to cholesterol as another culprit in the interplay between immune evasion and metabolic reprogramming, as well as CSC enrichment. Cholesterol is a key component in the cell membrane and acts as an important signaling molecule essential to cell growth and survival [246]. Breast CSCs seem to rely heavily on cholesterol synthesis, possibly for the maintenance of the desired level of membrane fluidity. Reduced membrane cholesterol levels are associated with metastasis, whereas high membrane cholesterol levels and further changes in membrane biophysical properties are associated with increased chemoresistance in breast cancer cells [247]. Increased cholesterol synthesis was associated with shorter relapse-free survival, and a recent study showed that inhibition of cholesterol synthesis pathway reduced breast CSC enrichment [248]. Some reports have shown that the inhibition of cholesterol synthesis pathways using statins, or inhibition of its master regulator RAR-related orphan nuclear receptor γ (RORγ), induced TNBC tumor regression [249,250]. The cholesterol synthesis pathway also overlaps with CSC-related pathways, such as YAP signaling. The cholesterol-lowering drug Simvastatin is currently in clinical trials to treat breast cancer, which indirectly inhibits YAP through the inhibition of HMG-CoA-Reductase [251]. Proprotein convertase subtilisin/kexin 9 (PCSK9) monoclonal antibodies or vaccinations work to reduce cholesterol and have also been proposed to improve clinical outcomes in breast cancer patients [252]. Cholesterol metabolism also plays a role in

immune cell activity, as they depend on their membrane to function [253]. A study by Ma et al. showed that cholesterol in the TME influences CD8+ T-cells, leading to the expression of immune checkpoint molecules, such as PD-1 [254]. High levels of cholesterol were associated with low anti-tumor immunity, which was restored upon reducing cholesterol [255]. The effect of drugs such as Simvastatin on immune cell infiltration remain open to investigation.

#### *6.5. Autophagy in TNBC*

One of the major players in the immunometabolic landscape of TNBC may be autophagy. Autophagy is a process in which intracellular constituents are degraded or recycled to regulate metabolic pathways under nutrient deprivation to maintain cell survival [256]. A marker of autophagy, microtubule-associated protein 1 light chanin 3B (LC3B) is highly expressed in TNBC and associated with poor clinical outcomes [257]. Glycolysis or GLUT1 inhibitors have been shown to induce autophagy deficiency and eventual cell death in TNBC cells [258,259]. Interestingly, the anti-CD73 antibody 1D7 was shown to mediate autophagy and inhibit the motility of TNBC cells [260]. Wen et al. further demonstrated that the inhibition of autophagy sensitized TNBC cells to chemotherapeutic agents [261]. Glutamine antagonsists have also been discussed as potential therapeutic targets; however, glutaminase inhibition accelerates autophagy and upreuglates FAO as a means for tumor cell survival [262]. Autophagy directly promotes FAO by providing the mitochondria with free fatty acids, leading to the accumulation of lipid droplets [263]. Furthermore, autophagy has been shown to hinder T-cell-mediated anti-tumor activity against TNBC both in vitro and in vivo [264]. As such, the interplay between metabolic pathways and autophagy in TNBC requires further investigation.

#### *6.6. Interplay of HIF-1*α *in Cancer Metabolism and Immunosurveillance*

In addition to the previously described importance of HIF-1α in TNBC angiogenesis, its role in metabolic reprogramming provides another therapeutic avenue in breast cancer cells and CSCs. HIF-1α activity in response to hypoxia leads to the expression of glycolytic enzymes and contributes to the Warburg effect, which increases the acidification of the TME and decreases immune cell infiltration [255,265]. Work by Bharti et al. used high-resolution 1H MRS (in vivo proton magnetic resonance spectroscopy) imaging in the aqueous and lipid phases of HIF-silenced tumors, after which the metabolic profiles were elucidated to determine the effect of HIF-1/2α inhibition [255]. They found that with HIF-1α silencing in TNBC, amino acids such as glutamine were decreased, along with lipid signals and droplets, suggesting that HIF-1α plays a role in TNBC metabolic adaptation [265]. Additionally, a study by Lee et al. discovered a strategy whereby silencing the oxidative stress master regulator NRF2 reduced HIF-1α accumulation and hindered HIF-1α induction of glycolysis-related genes [266]. In breast cancer cell lines, HIF-1α was found to increase the expression of adenosine receptor 2B (A2BR), which, as mentioned in earlier sections, plays a role in MDSC expansion and immunosuppression [267].

In breast cancer, HIF-1α also controls the expression of cluster of differentiation 47 (CD47), an integrin membrane protein expressed on many different cell types for the regulation of a wide range of cellular processes [268,269]. Specifically, cancer cells have been shown to overexpress CD47, where it forms a complex with signal-regulatory protein α (SRP- α) on phagocytes and inhibits macrophage-mediated phagocytosis of the TNBC cells [270]. CD47 expression is a well-known strategy by which tumor cells escape immunosurveillance. Researchers have explored blocking CD47 to induce a wide range of anti-tumor immune function [271–274]. High expression of CD47 in TNBC was associated with unfavorable prognosis, EMT signals, and metastasis [270]. Furthermore, a study by Kaur et al. showed that the blockade of CD47 was effective in TNBC CSC suppression and downregulation of stem-cell related pathways [274]. The preclinical success of targeting CD47 led to the therapy moving on to phase I clinical trials [275]. Interestingly, CD47 was recently shown to promote a Warburg phenotype by protecting the ubiquitin mediated degradation of ENO1, a glycolytic enzyme, providing another role of CD47 in cancer metabolic rewiring, in addition to its established