Radiation-induced immune changes in normal tissues also include acute and chronic immune effects that will be discussed below. While the effects of radiation-induced normal tissue inflammation are well described (100–106), the contribution of the complex immune mechanisms that support chronic, pathological changes e.g., fibrosis, are less investigated and still not yet completely understood.

Similar to the situation in tumors, radiation-induced acute damage and cell death in normal tissues also results in the release of DAMPs as well as pro-inflammatory cytokines and chemokines which have the capacity to modulate immune responses (105, 107). These "danger signals" trigger the activation and influx of innate and adaptive immune cells at the site of damage resulting in normal tissue inflammation. An excessive inflammation during the acute phase after radiation as a result of an overwhelming secretion of pro-inflammatory cytokines and the release of reactive oxygen species (ROS) supports normal tissue toxicity and severe side effects in treated patients (108).

In addition DAMPs can activate tissue regeneration in normal tissues as well as in tumor tissues. It is known that the extracellular DAMPs HMGB1 and ATP can activate and recruit cells, thus stimulating tissue repair (109). Of these, innate immune cells invade into the damaged tissue to clear dead cells and cellular debris (110). Moreover, stem cells and tissueassociated cells, e.g., fibroblasts, keratinocytes, endothelial cells, and vascular smooth muscle cells, are stimulated by these DAMPs to support angiogenesis and tissue regeneration (111–117). In addition, several DAMPs (e.g., HMGB1, S100A4, uric acid) can also enhance the expression of immunosuppressive mediators like interleukin (IL)-10 and indoleamine 2,3-dioxygenase (IDO) in stem cells, thereby inhibiting lymphocyte responses and contributing to tumor promotion (118). Excellent detailed reviews about the role of DAMPs in mediating regenerative pathways can be found elsewhere (109, 119).

Radiation-induced damage to normal tissues furthermore triggers chronic environmental changes e.g., hypoxia and senescence, that are reminiscent of the changes observed in the tumor microenvironment. These changes support chronic inflammation and repair processes, promote alternative polarization of recruited immune cells, pathologic immune cell interactions and excessive tissue remodeling, and thereby trigger not only the development of tissue scaring and fibrosis but also the development of secondary tumors (120, 121). For more detailed reviews about the acute and chronic events during radiation-induced normal tissue toxicity please refer to Wirsdorfer and Jendrossek (73), McKelvey et al. (79), Schaue et al. (105), Stone et al. (122), Barnett et al. (123), Kim et al. (124), and Ruhle and Huber (125).

The dual face of radiotherapy-induced immune changes in normal tissues can be nicely demonstrated in murine models of RILD. The acute phase of pneumonitis and the chronic event of fibrosis dramatically reveal how complex the radiation-induced alterations of the tissue micromilieu and the immune system impact disease pathogenesis (73).

Own studies in the murine C57BL/6 model of RILD revealed that whole thorax irradiation (WTI) with 15 Gy triggered an acute early immune suppression characterized by a pronounced reduction in the number of lymphocytes and myeloid cells that was followed by an influx of CD3+ T cell lymphocytes into the lung tissue during the pneumonitic phase Interestingly, WTI also enhanced numbers of regulatory T cells (Treg) in the lung tissue of irradiated animals both, during the early inflammatory state as well as at the time of fibrosis development. Of note, radiationinduced pulmonary fibrosis was more severe in recombinationactivating gene 2 (RAG2)-deficient mice that lack mature T- and B-lymphocytes suggesting that lymphocytes may have beneficial effects (126). Instead, depletion of CD4+ T cells during the pneumonitic phase decreased radiation-induced lung fibrosis in rats pointing to a contribution of CD4+ T cells to disease pathogenesis (68). These data suggest a causal link between the recruitment or local expansion of specific T-lymphocyte populations and the course of RILD that are also observed in other fibrosis models (127). But further work is required to define the beneficial and adverse effects of recruited and induced T cell subsets during the course of RILD (128).

Thoracic irradiation induces not only changes in the T cell compartment but also in the myeloid compartment and the lung environment. For example, others and we detected a significant reduction in the levels of total pulmonary macrophages and an almost complete eradication of alveolar macrophages early after irradiation as well as time-dependent changes in the macrophage phenotype with increased expression of markers for alternative macrophage activation [e.g., macrophage mannose receptor and de Leve et al. CD73 in the RT Response

Arginase-1] particularly during the fibrotic phase (102, 126, 129– 132). Further, macrophages accumulated in organized clusters and expressed pro-fibrotic mediators such as alpha smooth muscle actin (α-SMA) and transforming growth factor beta (TGF-β) at ≥25 weeks post-irradiation (131). Importantly, a recent report confirmed the formation of organized clusters of CD163+ macrophages also in lung tissue of irradiated patients. Intriguingly, pharmacologic inhibition of colony stimulating factor-1 (CSF-1) inhibited macrophage influx and attenuated RTinduced lung fibrosis in mice supporting a pathologic relevance of macrophages in RT-induced lung fibrosis (132). Similarly, pharmacological treatment with as connective tissue growth factor (CTGF) antibody before or after 20 Gy thoracic irradiation reduced acute and chronic radiation toxicity in mice and abrogated M2-like macrophage infiltration (133). The combined inhibition of TGF-β and Platelet-Derived Growth Factor (PDGF) blockade in a pre-clinical murine model attenuated radiationinduced pneumonitis and lung fibrosis and was accompanied by reduced osteopontin expression and leukocyte infiltration (134). Instead strategies using anti- Vascular Endothelial Growth Factor (VEGF) to target the tumor vasculature in combination with radiotherapy turned out to be highly toxic to normal lung tissue in pre-clinical murine models (21).

# THE IMMUNOMODULATORY CD73/ADENOSINE SYSTEM AS THERAPEUTIC TARGET FOR IMPROVING RADIOTHERAPY OUTCOME

Various observations from pre-clinical and clinical studies summarized in the former paragraphs suggest that targeting tumor-induced or radiation-induced immune deviation may offer novel and attractive opportunities for improving the outcome of radiotherapy by modulating the tumor radiation response, radiation-induced adverse late effects, or both. But the complexity of the tumor-induced and radiation-induced changes in the microenvironment as well as the time- and tissue-dependent "dual face" of radiotherapy-induced immune changes highlight the importance to identify strategies suited to balance adverse pro-inflammatory and immunosuppressive effects of radiotherapy and outweigh the beneficial effects of radioimmunotherapy with optimal tumor control and normal tissue protection. In this context, the purinergic CD73/adenosine system recently moved into the focus of research as it is an important endogenous regulator of the innate and adaptive immune systems with a documented role in tumor immune escape but also in adverse late effects of radiotherapy (36, 38, 131, 135–138).

We therefore hypothesized that the purinergic system might offer novel opportunities to interfere with normal tissue and tumor responses to radiotherapy and radiation-induced immune deviation. Extracellular ATP is a danger signal released by dying and damaged cells, and belongs to the earlier mentioned DAMPs, that function as immunostimulatory pro-inflammatory signals (139). In contrast, extracellular adenosine mostly exerts antiinflammatory, immunosuppressive or regulatory functions and is a critical mediator for the maintenance of tissue homeostasis in various tissues including the lung and to avoid overwhelming inflammation for example in response to infection (140–143). But balancing pro-inflammatory ATP and anti-inflammatory adenosine might also to be crucial for maintaining or reestablishing immune homeostasis and to orchestrate tissue inflammation and repair under conditions of damage-induced sterile inflammation (73, 144).

## CD73 and Adenosine Have Physiological Roles in Maintaining and Restoring Tissue Homeostasis

The purinergic signaling pathway is an evolutionary conserved mechanism that regulates immune homeostasis by the conversion of extracellular ATP to extracellular adenosine by using the sequential degradation via the ectoenzymes ectoapyrase (CD39, ectonucleoside triphosphate diphosphohydrolase 1) and CD73. Adenosine is either released from stressed or injured cells, or generated from extracellular adenine nucleotides by the concerted action of CD39 and CD73. While CD39 catalyzes the breakdown of ATP and ADP to AMP, CD73 converts AMP to adenosine. But the action of CD39 in degrading ATP can alternatively be executed by ectonucleotide pyrophosphatase (ENPP1, phosphodiesterase 1) (145).

CD39 and CD73 are expressed on the surface of specific lymphocyte subpopulations such as Treg and regulatory B cells (Breg) and endothelial cells and are important to their regulatory functions (143, 146–149). But CD73 is also expressed on stromal cells, mesenchymal stem cells (MSCs), and tumor-associated stem cells (150–153). Pre-clinical studies demonstrated that CD73 on stromal cells and tumor cells participates in the suppression of immune-mediated responses (152) as well as in homing and stemness of cancer stem cells (151, 154, 155). Furthermore, CD73 on MSCs promoted their immunosuppressive function and MSC were even able to upregulate CD73 expression on T cells (150). Inhibition of CD73 in a pre-clinical model of pancreatic neuroendocrine tumors led to reduced tumor growth and metastatic potential of cancer stem cells (151). Thus, stem cellmediated immunosuppressive or regenerative processes might help cancer cells to escape natural anti-tumor immune responses, anti-cancer immunotherapies, or both. **Table 1** shows detailed information on the expression of CD73 on multiple cell types in various tissues and their reported prognostic findings. Adenosine suppresses inflammatory functions of cells from innate and adaptive immune system and triggers expansion or differentiation of myeloid-derived suppressor cells (MDSC), M2-like macrophages as well as Treg and Breg and thereby participates in the creation of regulatory environments (144, 146, 149, 180–184). In addition, CD39/CD73-dependent generation of adenosine may also affect other processes in T-cell biology such as naive T-cell homeostasis, memory cell survival, and potentially T cell differentiation (168).

Extracellular adenosine can either be removed by enzymatic inactivation or cellular uptake or exert its actions through

#### TABLE 1 | CD73 expression on various cell types and tissues and its prognostic finding.

(Continued)

TABLE 1 | Continued

receptor binding. Adenosine deaminase (ADA) is responsible for the conversion of adenosine to inosine, a process that can happen either extracellularly or intracellularly (144). Adenosine may also be transported into its target cells via four different adenosine transporters, the so-called equilibrative nucleoside transporters (ENT) 1-4. Instead, adenosine exerts its actions by binding to one of four different G-protein-coupled adenosine receptors (ADORA1, ADORA2A, ADORA2B, and ADORA3) that are widely expressed on immune cells and resident tissue cells (185). ADORA1 and ADORA2A are high-affinity receptors responding to low concentrations of extracellular adenosine, while ADORA2B and ADORA3 are low affinity receptors and are mainly activated if the extracellular adenosine concentration rises above physiological levels (186). The adenosine receptors have various biological functions aimed at maintaining or restoring tissue homeostasis by triggering context-dependent pro- or anti-inflammatory effects (187–189).

#### Role of CD73 and Adenosine in Radiation-Induced Adverse Late Effects in the Lung

There is evidence from pre-clinical studies in models of injuryinduced sterile inflammation that an acute CD73-dependent increase in adenosine mostly exerts tissue protective functions (142, 181, 190, 191). Herein, the role of purinergic signaling to self-terminate TLR-responses in macrophages might contribute to the observed effects (187, 192).

In contrast, chronically increased adenosine-levels induced for example by genetic deficiency of the adenosinedegrading enzyme ADA or chronic treatment with the chemotherapeutic drug Bleomycin (BLM) can promote pathologic remodeling processes in various tissues leading to fibrosis development (136, 193–200). The pathologic effects of BLM-induced chronic adenosine-accumulation in the lung have been attributed to alternatively activated myeloid cells (201, 202).

So far the role of purinergic signaling for radiation-induced adverse late effects in the lung has only been addressed in own investigations (131, 138) while others investigated its role the skin (136). Our work demonstrated a pathologic role of chronically increased CD73/adenosine signaling in irradiated lungs of C57BL/6 mice, presumably by promoting or amplifying profibrotic signaling cascades. Pathologic signaling involved a time-dependent increase in the expression and activity of the CD73 in the lung tissue that could be confined to resident CD45− cells as well as CD45+ immune cells (CD4+ T cells including Treg, alveolar macrophages) and was associated with a progressive increase in adenosine levels in the bronchioalveolar lavage fluid C57BL/6 mice with a knockout of CD73 (CD73−/−) failed to accumulate high levels of adenosine in response to WTI resulting in decreased levels of fibrosis-associated proteins and mediators, reduced recruitment/formation of Treg, and attenuated pulmonary fibrosis with absence of clusters with alternatively activated macrophages. A similar protective effect was obtained by treatment of irradiated C57BL/6 mice either with pegylated ADA (PEG-ADA) to catabolize adenosine, or with the CD73 monoclonal antibody (mAb) TY/23 as of week 16 post-irradiation (131, 138).

Taken together, the progressive up-regulation of CD73/adenosine signaling in the irradiated lung environment promotes the accumulation of immunosuppressive cell types of the innate and adaptive immune system, e.g., Treg and M2-like macrophages and supports a pro-fibrotic cross-talk between damaged resident cells and infiltrating immune cells. Thereby, CD73/adenosine signaling helps to amplify radiation-induced lung fibrosis as a late normal tissue complication (**Figure 1**). In support of our findings, adenosine also promoted radiationinduced skin fibrosis; but here the pro-fibrotic effects had mainly been attributed to T-cell infiltrates and signaling via ADORA2A, without a role for alternatively activated macrophages (136).

Although radiation-induced intestinal injury models exist, the role of CD73/adenosine has only been studied so far in acute inflammatory disease models where CD73/adenosine executes tissue protective functions (203–208). Moreover, while CD73/adenosine had protective effects in acute renal disease models, chronic kidney injury in patients and murine studies was again linked to up-regulation of CD73 and ADORA2B (179, 198, 209, 210).

In summary, these studies point to disease-promoting effects of chronic CD73/adenosine signaling with tissue-specific and damage-specific mediators and immune changes.

FIGURE 1 | Purinergic signaling shapes the microenvironment in irradiated normal and tumor tissues. Exposure of normal tissues to ionizing radiation induces damage to tissue resident cells, e.g., endothelial cells and epithelial lung cells, as well as in resident immune cells. Equally exposure of tumor tissue results in radiation-induced damage to tumor cells and stromal cells. The resulting cell damage initiates stress responses and/or cell death with subsequent release of damage associated molecular patterns (DAMP). Release of ATP from dying cells is one component of radiation-induced tissue damage. Extracellular ATP acts as a potent inflammatory mediator that promotes inflammation and subsequent further damage to normal tissues. In tumor tissues extracellular ATP is an important mediator of anti-tumor CD8+ T cell responses as it participates in activation of dendritic antigen presenting cells (APC). To avoid excessive inflammation in normal tissues pro-inflammatory ATP is rapidly removed from the extracellular room by a two-step enzymatic conversion into adenosine, involving CD39 (or alternatively ectonucleotide pyrophosphatase) and CD73. Extracellular adenosine is an important endogenous regulator of inflammatory and repair processes as well as vascular functions. Adenosine exerts its pleiotropic actions in a tissue- and context-dependent manner through 4 different adenosine receptors that are expressed on various resident cells and immune cells (not shown). The immunosuppressive actions of adenosine involve the polarization of recruited immune cells toward regulatory or alternatively activated phenotypes, e.g., regulatory T cells (Treg), or M2-like macrophages. Moreover, adenosine mediates the inhibitory action of Treg and other regulatory cell types on proliferation and activation of cytotoxic T cells. By regulating endothelial cell activity CD73 and adenosine impact not only endothelial cell proliferation/angiogenesis but also vascular barrier function and the transmigration of leukocytes into damaged tissues. The expression of CD39 and CD73 thus balances the levels of pro-inflammatory ATP and immunosuppressive adenosine in normal tissues and tumors. The chronic activation of adenosine-driven processes observed in irradiated normal tissues promotes pathologic tissue remodeling and fibrosis development. Tumors coopt the CD73/adenosine system as a mechanism for promoting tumor growth and progression, angiogenesis, and immune escape. ADO, adenosine; CD39, ectonucleoside triphosphate diphosphohydrolase 1; CD73, 5 ′ ectonucleotidase; TAM, tumor associated macrophages.

#### Role of CD73 and Adenosine in the Control of Tumor Growth and Response to Therapy

Analyses of patient biopsies have shown that immune cell infiltrates in human tumors exhibit pronounced differences in cell types and numbers, not only intratumorally but also between patients and different tumor entities (211, 212). Interestingly, distribution and type of infiltrating immune cells turned out to have prognostic relevance; for example, the presence of infiltrating T cells was mostly linked to a favorable clinical outcome (213–218). Further pre-clinical and clinical studies showing that tumors can be strongly or poorly immunogenic supported these findings (31, 219). Moreover, the degree of immunogenicity positively correlated with reduced tumor growth and increased survival of tumor-bearing mice in response to immunotherapy indicating that the immune status can be seen as a predictive factor for therapy outcome (220).

High numbers of tumor-infiltrating cytotoxic lymphocytes were also predictive for the response of head and neck cancer patients to treatments involving radiotherapy whereas relapse after chemoradiotherapy and early recurrence correlated to infiltration with myeloid cells (217, 221–224). Local or systemic increases in Treg, high numbers of tumor associated macrophages, or recruitment of CD11b+ myeloid cells have also been associated with poor tumor response to radiotherapy and tumor relapse in pre-clinical models (88, 225). As a proof of concept for the synergistic interaction of radiotherapy and immunotherapy it has been shown that the combination with cancer vaccines, immune checkpoint blockade or inhibition of CD11b+ cell recruitment can improve the outcome of radiotherapy (52, 62, 89, 226–228).

Of note, tumors coopt the activities of the purinergic CD39/CD73/adenosine system to shape the immune landscape in the tumor microenvironment at multiple levels (**Figure 1**): For example, tumor cells and tumor-associated Treg use CD73-dependent adenosine generation to dampen intratumoral immune responses, particularly in hypoxic tumors (229, 230). The re-direction of the immune response involved suppression of T cell effector functions through CD73-dependent production of extracellular adenosine by CD39+/CD73+ Treg and signaling via stimulation of the ADORA2A on effector T cells (229). Adenosine and ADORA2A thus participate in shaping an immunosuppressive tumor microenvironment by negatively regulating CD8+ T cells (231–233). An adenosinedependent suppression of immunosurveillance via IFN-γ, NK cells, and CD8+ T cells had also been demonstrated in other pre-clinical models (35, 162). Finally, the creation of an immunosuppressive tumor microenvironment involved the expansion of immunosuppressive myeloid cells, e.g., myeloidderived suppressor cells, M2-like macrophages, and potentially N2-like neutrophils (234–236). More details about the various effects of CD73 and adenosine on cells from the innate and adaptive immune systems in the tumor microenvironment and the involved ADOR receptors can be found in the following reviews: (137, 143).

In addition, the CD73/adenosine system also supports tumor growth-promoting neovascularization, tumor metastasis, and chemotherapy resistance though part of these actions could also be attributed to the CD73/adenosine-induced modulation of immune cell types in the tumor microenvironment (36, 143, 229, 237–244).

For example, CD73−/− mice were strongly resistant to growth of subcutaneous MC38-ova colon and EG7 lymphoma tumors as well as carcinogen-induced or de novo growth of endogenous prostate tumors in transgenic TRAMP mice (162, 245, 246). These interesting observations pointed to a role of CD73+ host cells in tumor growth. However, CD73−/− mice were less resistant to growth of AT-3 mammary and B16F10 melanoma tumors revealing that the effect of host CD73 on the growth of experimental tumors also depends on the tumor type (245, 246). Of note, treatment with an anti-CD73 mAb reduced the growth of experimental 4T1.2 and E0771 breast tumors in wildtype mice, but not in severe combined immunodeficient (SCID) mice, suggesting a role of the adaptive immune system (245, 246). Anti-CD73 treatment also inhibited growth of carcinogeninduced fibrosarcoma tumors and of transgenic prostate tumors in transgenic TRAMP mice (162). The authors could further attribute the efficient tumor rejection to the action of CD8+ T cells whereas CD4+ T cells and NK cells were not involved (162, 246). These data highlight immunosuppressive CD73+ Treg as an important component of the tumor growth-promoting effects of CD73 and adenosine (162, 246).

Interestingly, CD73−/− mice also developed less lung metastases after intravenous injection of B16F10 or TRAMP-C1 cells (162, 246) suggesting that host CD73 also supports metastasis. In line with these observations treatment with an anti-CD73 mAb (TY/23) strongly reduced the lung metastases after injection of 4T1.2 or TRAMP-C1 tumor cells (162, 245). However, the suppression of metastasis formation was observed in both, immunocompetent and in SCID mice, and turned out to be independent of CD8+ T cells and NK cells (162, 245). Thereby the authors revealed a role of CD73+ non-hematopoietic host cells in metastasis formation, potentially endothelial cells, they could further link the pro-metastatic effect to signaling of tumorderived extracellular adenosine via ADORA2B activation, at least in the 4T1.2 model (245, 246).

In further studies, tumor-derived adenosine attracted myeloid cells and promoted their differentiation into adenosinegenerating tumor-associated macrophages (TAM) to amplify adenosine-dependent tumor-immune escape (247). In support of these findings, in vitro exposure to adenosine promoted alternative activation of macrophages and enhanced the immunosuppressive responses of macrophages to danger signals, particularly if stimulated in the presence of TLR ligands (141, 187). Interestingly, tumor-derived CD73-dependent adenosine promoted growth, neovascularization, and metastasis of subcutaneous B16F10 melanoma tumors and this was linked to infiltration and polarization of macrophages: genetic or pharmacologic inhibition of CD73 on the B16F10 melanoma cells significantly reduced the number of tumor-infiltrating macrophages recruited to subcutaneous B16F10 melanoma tumors on CD73−/− mice when compared to untreated B16F10 wildtype tumors on CD73−/− mice. Cytokine measurements in CD73+ B16F10 wildtype tumor lysates grown on CD73−/− mice revealed a down-regulation of pro-inflammatory cytokines [Granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFN-γ] and enhanced expression of anti-inflammatory/proangiogenic cytokines (IL-4, IL-10, IL-13, M-CSF) (248). Although the number of infiltrating macrophages did not change in CD73+ B16F10 WT tumors on CD73−/− mice, less MMR+ macrophages were found inside the tumor. Only a pharmacological CD73 inhibition or knockdown of CD73 in the tumor host reduced the amount of infiltrating macrophages (248, 249). The results indicate a role for CD73 in activation and polarization of macrophages that promote tumor progression. Furthermore, it was shown, that the recruitment and activation of tumor-infiltrating macrophages was dependent on ADORA1, ADORA2A, and ADORA3 (250).

Taken together, CD73-dependent adenosine from host cells and tumor cells participates in the support of tumor growth amongst others by promoting tumor immune escape whereas loss of CD73/adenosine signaling enhances tumor immunity. As nicely summarized in a recent review from Allard et al. CD73/adenosine has become an attractive therapeutic target in (immuno)-oncology (38). Several early-phase clinical trials currently evaluate the therapeutic potential of CD73/adenosine inhibitors to inhibit tumor growth and increase tumor immunity. Besides the direct inhibition of CD73 the identification of the respective ADOR involved in promoting tumor immune escape will offer additional opportunities for therapeutic intervention (38, 251, 252).

Intriguingly, co-inhibition of adenosine signaling via CD73 and ADORA2A achieved better anti-tumor immune responses compared to single treatments, at least in pre-clinical models of breast and colon cancer (253). These effects were associated with improved immune cell infiltration, DC-priming and CD8+ T cell expansion. In line with these findings, Young et al. also observed increased tumor growth delay in CD73/ADORA2A double knockout mice (254). Furthermore, investigations with the human monoclonal anti-CD73 antibody MEDI9447 that is currently in Phase I clinical trials, showed high efficacy in inhibiting CD73 in vitro and potent inhibition of pre-clinical syngeneic tumor models in vivo as well as additive activity in combination with immune checkpoint inhibitors. Interestingly, MEDI9447 efficiently modulated the tumor microenvironment with significant alterations in the number of both, CD8+ effector T cells and activated macrophages (255).

The immunosuppressive actions of CD73 and adenosine in the microenvironment of established tumors also attract major attention as an interesting target for combined treatment approaches, particularly with immunotherapy. In this context, inhibition of CD73 enhanced efficacy of immunotherapy with α-PD-1 or α-CTLA4 in pre-clinical models (251, 256). The synergistic effect of the combined treatment involved improved T cell effector function as well as reduced CD73 expression on tumor-infiltrating lymphocytes and was dependent on interferon gamma (IFN-γ) and perforin (253, 256). Therapeutic inhibition of ADORA2A was also able to modulate expression of T cell co-inhibitory receptors and to improve effector function for enhanced efficacy of immune checkpoint blockade and adoptive cell therapy in murine cancer models (251, 252).

Since the focus of this review is to highlight the therapeutic potential of CD73 and adenosine inhibition to improve the therapeutic gain in radiotherapy we will not discuss such approaches in more detail here. For further information please refer to reviews discussing the therapeutic potential of the purinergic pathway in immunotherapy in more detail (38, 251, 252).

Instead the therapeutic potential of combining radiotherapy or radioimmunotherapy with CD73/adenosine-inhibition in cancer has been highlighted as an attractive approach but sound data are missing so far (41, 137). Pre-clinical studies are now underway to test such approaches including investigations in our own laboratory (257).

Several pre-clinical studies addressing the role of CD39 in cancer revealed that genetic deficiency of CD39 in mice promotes resistance to metastasis of melanoma and colorectal cancer models (258). Similarly, inhibition of angiogenesis in a CD39-deficient background resulted in reduced growth and pulmonary metastasis of LLC and B16F10 tumors (259). Expression of CD39 was important for angiogenesis and the suppression of NK cell-mediated antitumor activity (260, 261). In line with these findings, overexpression of CD39 enhanced the metastatic potential in pre-clinical models whereas the pharmacological inhibition of CD39 reduced metastasis and enhanced antitumor immunity (261). Of note, clinical data also support a correlation of high CD39 expression with poor prognosis indicating that CD39 might be another promising target for cancer therapy (262–264). But CD39 is much less investigated as a cancer target compared to CD73 underlining the need for further pre-clinical studies.

Taken together various pre-clinical studies highlight the potential of CD39/CD73/adenosine-signaling as promising therapeutic target in immuno-oncology. So far, the observed effects have been associated in multiple studies with activation of T-cell dependent tumor immunity. However, it is important to consider further immunoregulatory actions of CD73 and adenosine in the tumor microenvironment, particularly their influence on the biology of myeloid cells and macrophages, respectively.

#### Targeting CD73 in Lung Cancer

Only limited data are available so far of the role of CD73 and adenosine in lung cancer. Herein, CD73 was found to be expressed in tumor tissue from NSCLC patients on tumor cells, tumor-promoting mesenchymal stromal cells and myeloid-derived suppressor cells, respectively (265–267). Tumor-derived TGF-β stimulated CD39 and CD73 expression in CD11b+CD33+ MDSC in tumor tissues and peripheral blood of NSCLC patients, thereby inhibiting activity of T cells and NK cells and protecting tumor cells from the cytotoxic effect of chemotherapy (267).

Moreover, the prognostic value of high CD73 expression for the survival in lung cancer patients remains controversial: Although one study reported a correlation of high CD73 gene expression and improved overall survival of NSCLC patients (268) another study identified high CD73 protein expression as an independent prognostic marker for poor overall survival and shorter recurrence free survival in NSCLC (269). Interestingly, in the same study, high ADORA2A gene expression was an independent predictor of favorable prognosis for overall survival (269). Own in silico analyses of publicly available datasets for gene expression of CD73 in lung cancer confirmed the positive correlation between high CD73 gene expression and better overall survival of NSCLC patients. Of note, if radiotherapy-treated patients were excluded from the analysis the correlation to an improved overall survival was abrogated. In addition, the in silico analyses revealed poorer overall survival in lung cancer patients with high gene expression of ADORA1, ADORA2A, and ADORA2B (**Figure 2**). Again, the results about the prognostic value of ADORA2A using immunohistochemical data revealed opposite results (269, 270). The discrepancy in the above findings may be due to the use of gene expression analyses vs. immunohistochemical data as CD73 expression in tumor samples turned out to be highly heterogenous (269). We speculate that the heterogeneity in CD73 protein expression in distinct tumor areas might be linked to heterogeneous tumor oxygenation and make the acquisition of representative gene expression data challenging. So far, CD39 inhibitors are not yet involved in clinical trials for cancer patients but such studies are underway (259).

Taken together, adenosine released in an inflammatory milieu or generated by the CD39/CD73 axis will impact the

immune landscape of lung tumors presumably by limiting T cell immunity and promoting immunosuppressive and tumorpromoting lymphoid and myeloid immune cell phenotypes (**Figure 1**). We thus speculate that modulating CD73/adenosine signaling in the lung tumor microenvironment is an attractive strategy to limit tumor progression, improve antitumor immune responses, and avoid escape from therapy in combination with radiotherapy and potentially radioimmunotherapy. On the other hand, the pathologic role of the radiation-induced increase in CD73/adenosine signaling in promoting chronic inflammation and fibrosis in the normal lung tissue strongly suggest that pharmacologic inhibition of CD73/adenosine offers the opportunity for widening the therapeutic window by reducing radiation-induced lung toxicity, particularly in CD73-rich thoracic tumors with a high risk for CD73-dependent normal tissue toxicity.

Targeting the CD73/adenosine pathway or the involved receptors may thus provide a clear therapeutic gain in the treatment of lung cancer and other CD73/adenosine-rich thorax-associated neoplasms: we expect that inhibition of CD73/adenosine signaling will limit lung toxicity during thoracic irradiation without protecting the tumor or even reinstall antitumor immunity when applied during therapeutic irradiation of adenosine-rich tumors with high radioresistance such as NSCLC (138). However, a tight regulation of pro- and antiinflammatory actions of resident and immune cells is necessary to protect the lung from inflammation-induced loss in its vital function (271–273). For example, immunosuppressive Treg are also to be part of a protective response limiting inflammationinduced collateral normal tissue damage after radiotherapy (44, 274). Therefore, pharmacologic strategies targeting the CD73/adenosine pathway in combination with radiotherapy or combined radioimmunotherapy will require careful validation of potential normal tissue complications. Such complications might include excessive inflammation or autoimmunity by abrogating protective signals mediated by various ADORAs, particularly during acute disease stages. Moreover, the dual effects of acute and chronic CD73 activation as well as spatiotemporal heterogeneity of CD73 and ADOR expression in normal and tumor tissues need to be considered when designing combination treatments for therapeutic intervention.

#### CURRENT RESEARCH AND FUTURE PERSPECTIVES

So far work from our group identified CD73/adenosine signaling as a novel mechanism promoting RILD through local and systemic actions. Consequences of pathologic CD73/adenosine signaling involved amongst others the accumulation and/or alternative activation of macrophages in organized clusters, their expression of pro-fibrotic mediators, or both. We speculate that the radiation-induced increase in CD73/adenosine is necessary to amplify pro-fibrotic signaling in the irradiated lung environment by fueling the multifaceted cross-talk between damaged resident cells, local and infiltrating immune cells, immunosuppressive Treg and other pro-fibrotic mediators such as hyaluronic acid and TGF-β.

Though immunomodulatory effects of adenosine had been linked to CD73/adenosine-induced adverse effects in other injury models (136, 202) the tissue specific effector and target cells of CD73/adenosine-signaling in response to genotoxic treatment (BLM, radiotherapy) are still controversial and need to be further investigated (73, 200). In this context radiation-induced normal tissue toxicity had also been linked to endothelial cell damage and dysfunction as well as endothelial cell loss as long-term complication (275, 276). As a direct consequence of impaired vascular function, WTI increased numbers of total CD45+ leukocytes, particularly profibrotic CD11b+ myeloid cells and Ly6C+ inflammatory monocytes, in lungs of irradiated mice. However, on the long term, persistence of an activated pro-coagulant endothelial cell type, thickening of the basement membrane, endothelial loss, and collapse of microvessels will contribute to the creation of a hypoxic, pro-inflammatory disease-promoting environment. We assume that the pathologic environment involves a hypoxiainduced up-regulation of CD73 and pathology-associated ADOR on resident cells and immune cells. It is tempting to speculate that therapeutic inhibition of CD73 might also impact adverse late effects in the lung by reducing radiation-induced vascular impairment, but this remains to be determined. Interestingly, further work demonstrates that locally irradiated MSC play a role in the pathogenesis of radiotherapy-induced pulmonary fibrosis by acquiring a pro-fibrotic myofibroblast-like phenotype that promotes extracellular matrix deposition, tissue remodeling, and the development of pulmonary fibrosis upon WTI (276). Since CD73 is expressed on endothelial cells and on MSC of healthy lungs (153) future studies should explore whether the expression of CD73 on the surface of endothelial cells or resident MSC impacts the development of RILD. The same holds true for the expression of CD39 and CD73 on cancer exosomes, which have also been shown to suppress T cells through adenosine production (239).

Adenosine released in an inflammatory milieu or generated by the CD39/CD73 axis impacts the tumor microenvironment and limits tumor immunity at multiple levels. Thus, modulating cancer-derived adenosine in the tumor microenvironment emerges as an attractive strategy to limit tumor progression and improve antitumor immune responses and our own studies suggest that this might be possible without excessively increasing late normal tissue complications (36, 187, 242, 243, 277). Fortunately, multiple approaches for pharmacologic modulation of adenosine levels exist or are being developed and multiple clinical studies have been initiated to evaluate the use of novel inhibitors of CD73 or ADORA2A signaling in cancer therapy alone and in combination with immune checkpoint blockade (38, 39, 143, 188). These studies will give insight into efficacy, compatibility, and potential side effects.

Herein, major attention in oncology has so far been attributed to adenosine signaling via ADORA2A as it is known to effectively dampen immune responses in tumors and normal tissues. However, it has to be taken into account that depending on the tissue of origin and the molecular and immune signature of the tumor, other ADOR may be more important. Moreover, the role of purinergic signaling in the radiation response of malignant tumors and the potential of CD73 or ADOR inhibitors to enhance the efficacy of RT alone and in combination with immunotherapy is still largely unknown. Finally, no reliable biomarkers for the prediction or diagnosis for the individual risk of RILD upon treatment are available to date. Thus, further studies are needed that correlate the gene and protein expression of CD73 and the ADORAs to the outcome after radio(chemo)therapy or immunotherapy. Moreover, as mentioned before, the receptors differ in their affinity for adenosine and extracellular adenosine levels will vary depending on the tissue, the treatment modality and intensity in a spatiotemporal manner. It would therefore be highly beneficial to perform an immunoscore of tissues from pre-clinical studies and test association of high or low expression of CD73 and the ADORAs with the presence of immunosuppressive lymphoid and myeloid cell subsets, and potentially tissue hypoxia. Such knowledge could later be translated into patient samples. Here, it was an intriguing observation that a high expression of CD73 in normal tissues was indicative for a poor infiltration of prostate tumors with CD8+ T cells whereas high CD73 expression in the tumor stroma was indicative for a longer recurrence-free survival (278). This highlights that CD73 expression in both, normal and tumor tissue should be evaluated.

#### FINAL REMARKS

Nowadays it is increasingly recognized that strategies for a biology-based optimization and individualization of radiotherapy should include not only the available knowledge about tumor promoting mutations, tumor heterogeneity, tumor cell plasticity, and unfavorable gene expression profiles indicative of the individual radiosensitivity of tumor and normal tissues, but also consider knowledge about the modulation of the radiation response by the immune system and vice-versa. Such a comprehensive view shall allow to harness the combined potential of high precision local radiotherapy, cytotoxic chemotherapy, molecularly targeted small molecule signal transduction inhibitors, and immunotherapy approaches for biologically optimized therapeutic strategies with acceptable safety profile and durable responses in the future (22, 30, 41, 279–284).

The observation that radiotherapy can help to reactivate anti-tumor immunity in immunogenic tumors or increase the potential of immunotherapy has attracted major attention to the use of radiotherapy in combination with various immunotherapies, particularly immune checkpoint blockade immunostimulatory antibodies, and cancer vaccines (24–26, 28– 30, 62, 67). However, tumors have evolved effective strategies to escape from immune surveillance and therapy-induced enhancement of tumor immunity is balanced by feed-back inhibition of immune activation in residual tumors, the mobilization of tissue regeneration mechanisms with tumor promoting actions, or both (41, 88, 89, 285–287).

We believe that the identification of mediators driving both, adverse immune changes in irradiated normal tissues and tumor immune escape, will allow us to uncover attractive new therapeutic targets for improving the outcome of radiotherapy. The CD73/adenosine pathway is such a signaling system that regulates adverse immune responses in tumors and normal tissues to microenvironmental stress (e.g., tumor hypoxia) and radiotherapy. So far, CD73/adenosine is mostly considered as a metabolic immune checkpoint that supports immunosuppressive signaling of Treg via ADORA2A. However, there is evidence that CD39, CD73 and adenosine are involved in further immunosuppressive and tumorpromoting signals in the tumor microenvironment beyond modulating Treg function. Intriguingly, radiochemotherapy was also shown to trigger up-regulation of CD73 and CD39 in circulating immune cells of cancer patients (288). This suggests that a radiotherapy-induced systemic upregulation of CD73/adenosine signaling may additionally dampen systemic anti-tumor immune responses during standard fractionated radiotherapy.

Thus, pharmacologic inhibition of CD73/adenosine signaling is an attractive approach to increase the therapeutic ratio in the RT of thoracic tumors with high risk of adverse late effects in the highly radiosensitive normal lung tissue by (i) dampening growth and metastasis of lung tumors, (ii) enhancing the radiation-induced activation of the antitumor immune response, (iii) by restricting the immunosuppressive action of CD39/CD73 on circulating immune cells, and (iv) attenuating adverse late effects in the lung. Moreover, pharmacologic modulation of CD73, adenosine or the four adenosine receptors might offer opportunities to enhance the potential of combined radioimmunotherapy to mount efficient and durable responses with acceptable safety profile.

But the complexity of the tumor-induced and radiationinduced changes in the microenvironment and the multifaceted interactions between damaged resident cells and recruited immune cells outlined above underline the necessity of further work suited to identify strategies that achieve the required balance between pro-immunogenic and immunosuppressive effects of radiotherapy and outweigh the beneficial effects of radioimmunotherapy with optimal tumor control and normal tissue protection. Moreover, further work is required to gain a better mechanistic understanding of the tissue-, injury-, and disease stage-dependent beneficial or adverse effects of CD73/adenosine as well as the identification of involved ADORAs and effector cells for a successful restriction of lung damage during therapeutic lung irradiation by targeting CD73, adenosine or specific ADORAs (73).

Finally, it remains to be determined which approach for targeting the CD73/adenosine axis might be best suited to be used in combination with RT. Above all, the immune effects of RT also depend on physical parameters such as total dose, fractionation schemes (43, 57, 225, 289) and potentially the quality of radiation (290). Thus, attention has also to be given to the best sequence of application, as well as appropriate radiation doses and fractionation-schemes as they may largely impact the effects of radiotherapy on microvessels, immunogenic cell death, immune cell infiltration, the production of immune modulatory mediators, and the activation of CD73/adenosine signaling in both, normal and tumor tissues. Here, the major challenge will be to therapeutically redirect the immune response toward anti-tumor action and avoid tumor recurrence without enhancing collateral normal tissue damage.

# AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

# FUNDING

The work was supported by grants of the DFG (GRK1739/2; JE275/1) and the BMBF (ZISStrans 02NUK047D). We acknowledge support by the Open Access Publication Fund of the University of Duisburg-Essen.

# REFERENCES

induction of IL-10+/IDO+ lymphocytes. J Immunol. (2014) 192:6102–10. doi: 10.4049/jimmunol.1303144

enhances radiation-induced lung fibrosis. Cancer Res. (2016) 76:3045–56. doi: 10.1158/0008-5472.CAN-15-2310

experimental rheumatoid arthritis. Sci Transl Med. (2012) 4:146ra108. doi: 10.1126/scitranslmed.3003717

3 ′ ,5′ -monophosphate and phosphatases. J Immunol. (2004) 173:932–44. doi: 10.4049/jimmunol.173.2.932

angiogenesis and macrophage infiltration. PLoS ONE. (2016) 11:e0151420. doi: 10.1371/journal.pone.0151420

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 de Leve, Wirsdörfer and Jendrossek. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Potential Role of CXCR4 Targeting in the Context of Radiotherapy and Immunotherapy of Cancer

Franziska Eckert 1 \*, Karin Schilbach2 , Lukas Klumpp1,3, Lilia Bardoscia1,4 , Efe Cumhur Sezgin1 , Matthias Schwab3,5, Daniel Zips 1 and Stephan M. Huber 1

*1 Department of Radiation Oncology, University Hospital Tuebingen, Tuebingen, Germany, 2 Department of General Pediatrics/Pediatric Oncology, University Hospital Tuebingen, Tuebingen, Germany, 3 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, 4 Department of Radiation Oncology, University of Brescia, Brescia, Italy, 5 Departments of Clinical Pharmacology, Pharmacy and Biochemistry, University Hospital and University Tuebingen, Tuebingen, Germany*

#### Edited by:

*Christian Ostheimer, Martin Luther University of Halle-Wittenberg, Germany*

#### Reviewed by:

*Luis De La Cruz-Merino, Hospital Universitario Virgen Macarena, Spain Magdalena Plebanski, RMIT University, Australia Viktor Umansky, German Cancer Research Center (DKFZ), Germany*

> \*Correspondence: *Franziska Eckert franziska.eckert@ med.uni-tuebingen.de*

#### Specialty section:

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology*

> Received: *29 August 2018* Accepted: *06 December 2018* Published: *21 December 2018*

#### Citation:

*Eckert F, Schilbach K, Klumpp L, Bardoscia L, Sezgin EC, Schwab M, Zips D and Huber SM (2018) Potential Role of CXCR4 Targeting in the Context of Radiotherapy and Immunotherapy of Cancer. Front. Immunol. 9:3018. doi: 10.3389/fimmu.2018.03018* Cancer immunotherapy has been established as standard of care in different tumor entities. After the first reports on synergistic effects with radiotherapy and the induction of abscopal effects—tumor shrinkage outside the irradiated volume attributed to immunological effects of radiotherapy—several treatment combinations have been evaluated. Different immunotherapy strategies (e.g., immune checkpoint inhibition, vaccination, cytokine based therapies) have been combined with local tumor irradiation in preclinical models. Clinical trials are ongoing in different cancer entities with a broad range of immunotherapeutics and radiation schedules. SDF-1 (CXCL12)/CXCR4 signaling has been described to play a major role in tumor biology, especially in hypoxia adaptation, metastasis and migration. Local tumor irradiation is a known inducer of SDF-1 expression and release. CXCR4 also plays a major role in immunological processes. CXCR4 antagonists have been approved for the use of hematopoietic stem cell mobilization from the bone marrow. In addition, several groups reported an influence of the SDF-1/CXCR4 axis on intratumoral immune cell subsets and anti-tumor immune response. The aim of this review is to merge the knowledge on the role of SDF-1/CXCR4 in tumor biology, radiotherapy and immunotherapy of cancer and in combinatorial approaches.

Keywords: immunotherapy, cancer radiotherapy, CXCR4, SDF-1 (CXCL12), T cells, dendritic cells, NK cells, regulatory T cells

# INTRODUCTION

In radiation oncology, chemokine receptor CXCR4 and its ligand SDF-1 (stromal cell derived factor-1, CXCL12) have been described as prognostic factor for head and neck squamous cell carcinoma [e.g., (1)]. Functional data in glioblastoma models point to a role in migration and invasion of cancer cells (2). These and other observations strongly suggest SDF-1/CXCR4 signaling as promising target in anti-cancer therapy, in particular, in combination with radiation therapy (3). However, clinical development of CXCR4 antagonists has mainly focussed on mobilization of hematopoietic stem cells from the bone marrow to peripheral blood (4).

Radiation therapy has proven to elicit both pro-inflammatory, immunostimulatory activities, and anti-inflammatory, immunosuppressive mechanisms. These effects are dependent on radiation dose, tumor biology and the host predisposition (5). As immunotherapy for cancer has been established as standard of care for several cancer entities, such as melanoma (6) and lung cancer (7), the immunologic effects of standard anti-cancer treatment, such as radiation therapy and targeted therapies are of major interest. Radiation-induced immune modulation has been described as direct effects on irradiated tumor cells ("on-target" immunogenic effects) as well as indirect effects in the tumor immune microenvironment ("off-target" effects) (8). Remarkably, recent data also link CXCR4 blockade with antitumor immunity in the tumor immune microenvironment suggesting SDF-1/CXCR4 targeting as a therapeutic tool to interfere with the immune system.

The present article, therefore, aims to give an overview about the plethora of functions of SDF-1/CXCR4 signaling in tumor biology and immune responses in the context of combined radiotherapy and immunotherapy. The knowledge about these functions is indispensable for developing new concepts of anticancer therapy that comprise radiotherapy, immunomodulation and SDF-1/CXCR4 targeting.

#### INTERFERENCE OF IONIZING RADIATION WITH IMMUNE RESPONSES

Radiotherapy effects on cancer had been mostly attributed to direct cytotoxic effects on cancer cells (especially DNA damage) (9). With the advance of cancer immunotherapies preclinical and clinical observations pointed toward synergistic effects. The so-called "abscopal effect" describes responses to radiotherapy (mostly in combination with immunotherapy) outside the irradiated volume and has been linked to immune mechanisms (10). The combination of immune checkpoint inhibitors with local tumor irradiation seems to induce synergistic effects and is currently tested in multiple clinical trials (11, 12). In addition, theoretical rationales and preclinical data provide the basis for also combining radiotherapy with other immunotherapy strategies, such as anticancer-vaccines and cytokine-based therapies (13).

#### Immune Effects of Tumor Irradiation

The mechanisms of radiation-induced immune effects have been summarized as immune-stimulating and immunosuppressive either directly in tumor cells or in the microenvironment (8). Radiation triggers anti-tumor immune responses directly in the cancer cells by upregulation of MHC-I molecules (14, 15) and possible induction of new tumor associated antigens (14). Cell death mechanisms induced by tumor irradiation lead to "immunogenic cell death" (ICD) (16, 17) characterized by the ability to stimulate the innate immune system and thus indirectly also adaptive immune responses (18, 19). ICD is characterized by the release of danger associated molecular patterns (DAMPs), such as calreticulin (20), high-mobility-group-box 1 (HMGB1) (21) and extracellular adenosine-tri-phosphate (ATP) (22). Additional mechanisms include cytokine release, such as CXCL10 (23), and type-1 interferon (24). Indirect immune stimulation has been attributed to increase and activation of tumor-infiltrating lymphocytes (25, 26), as well as maturation of dendritic cells (DCs) (27). The fact that clinically relevant anti-tumor responses (e.g., abscopal effects after palliative radiotherapy in metastatic cancer patients) are rare despite of these strong immunestimulating effects is most probably due to simultaneously induced immunosuppression by tumor irradiation. Irradiated tumor cells upregulate immune checkpoint molecules, such as PD-L1 (28, 29) and release immunosuppressive cytokines, such as TGFβ (30, 31). Immunosuppressive cells increased in the tumor immune microenvironment upon local irradiation include regulatory T cells (32, 33) and myeloid-derived suppressor cells (MDSC) (34–36).

### Combination Therapies of Irradiation and Immunotherapy for Cancer

These mechanisms have become the basis for combining radiotherapy and immunotherapy in order to exploit pro-immunogenic properties of irradiation and block immunosuppressive actions. Clinical trials ongoing with combinatorial approaches include immune checkpoint inhibition, cytokine based therapies and vaccination strategies (37, 38). Combination of radiotherapy with immune checkpoint inhibition has been recently summarized (39). Besides its use in metastatic cancer patients, durvalumab as adjuvant treatment after definitive radiochemotherapy for non-small cell lung cancer has shown significantly improved disease free survival (40). Vaccination strategies used in combination with radiotherapy include peptide and mRNA based approaches (41–43) which showed promising results in syngeneic mouse xenograft models. IL2 and IL12 as tumor targeted immunocytokines have been tested in combination with tumor irradiation in in vivo models showing promising results (44–47).

In conclusion, the strong rationale and promising results led to an increasing use of immunotherapeutics in combination with local tumor irradiation in standard of care treatment of palliative cancer patients as well as in numerous clinical trials with high expectations of the oncological field to improve survival and prognosis of cancer patients.

### SDF-1/CXCR4 FUNCTION IN TUMOR BIOLOGY

SDF-1/CXCR4 signaling has been shown to contribute to virtually all processes in tumor biology. As described in this section, SDF-1/CXCR4 signaling reportedly contributes to neoplastic transformation, malignant tumor progression, infiltration, metastasis, angiogenesis and vasculogenesis, and consequently therapy resistance of many different tumor entities.

# CXCR4, a Marker of Cancer Stem(-Like) Cells or Tumor-Initiating Cells

CXCR4 chemokine receptors are expressed by hematopoietic stem cells and are required for the trapping of these cells within the stem cell niches of the bone marrow. CXCR4 antagonists, such as AMD3100 (Plerixafor), therefore, can be used to mobilize stem cells into the peripheral blood for hematopoietic stem cell donation (see below). Beyond that, SDF-1/CXCR4 signaling has been shown to be functional in neural progenitor cells and to direct neural cell migration during embryogenesis (48). Notably, CXCR4 expression is further upregulated when neural progenitor cells differentiate into neuronal precursors whereas SDF-1 is upregulated during maturation of neural progenitor cells into astrocytes. While CXCR4 is localized in the cell body of neuronal precursors, expression is primarily restricted to axons and dendrites in mature neurons (49). In addition, SDF-1/CXCR4 signaling has been reported to contribute to chemotaxis and differentiation into oligodendrocytes of engrafted neural stem cells resulting in axonal remyelination in a mouse model of multiple sclerosis (50). Together this suggests that neurogenesis requires functional SDF-1/CXCR4 signaling and CXCR4 as marker of especially the neuronal lineage of neural stem cells.

Primary glioblastoma multiforme (GBM) develops directly by neoplastic transformation of neural stem cells and not by malignant progression from astrocytic gliomas or oligodendroglomas (the latter two are characterized by mutations in the IDH genes). Not unexpectedly, stem(-like) subpopulations of GBM functionally express SDF-1/CXCR4 signaling (51–56). Notably, auto-/paracrine SDF-1/CXCR4 signaling is required for maintenance of stemness and self-renewal capacity (57–59) since SDF-1/CXCR4 targeting leads to loss of stem cell markers and differentiation of stem(-like) cells into "differentiated" tumor bulk.

Besides glioblastoma, SDF-1/CXCR4 signaling has been shown to be functional in stem(-like) subpopulations of retinoblastoma (60), melanoma (61), pancreatic ductal adenocarcinoma (62), non-small cell lung cancer (63), cervical carcinoma (64), prostate cancer (65), head and neck squamous cell carcinoma (66), rhabdomyosarcoma (67, 68), synovial sarcoma (56), and leukemia (69). In summary, these data might hint to an ontogenetically early onset of SDF-1/CXCR4 signaling in mesenchymal and epithelial primordia of the different organs which might be the reason for SDF-1/CXCR4 expression in stem(-like) subpopulations of many different tumor entities.

Transition of stem(-like) cells and differentiated tumor bulk and vice versa seems to be highly dynamic and regulated by the reciprocal crosstalk with untransformed stroma cells of the tumor microenvironment (70–72). Beyond that, this crosstalk seems to induce phenotypical changes of cancer stem(-like) cells as deduced from the following observation. Sorted CD133+ stem(-like) cells and CD133− differentiated bulk cells of GBM did not differ in repair of radiationinduced DNA double strand breaks in vitro. Upon orthotopic transplantation in immunocompromized mice, however, CD133+ cells repaired more efficiently than CD133− cells indicating tumor-microenvironment-mediated upregulation of DNA repair selectively in CD133+ GBM cells (73). The next paragraph introduces the impact of SDF-1/CXCR4 signaling on the crosstalk of tumor cells with non-transformed stroma cells and its function for the cancer stem(-like) cell phenotype.

### SDF-1/CXCR4 Signaling in the Crosstalk of Cancer Stem(-Like) Cells With Non-transformed Stroma Cells

The functional significance of SDF-1/CXR4 signaling between tumor cells and the tumor stroma is suggested by a retrospective analysis of genetic SDF-1 variants in patients with colorectal cancer where a certain SDF-1 polymorphism in fibroblasts is associated with higher stromal SDF-1 expression and increased risk for lymph node metastases in stage T3 colorectal cancer (74). Moreover, diffuse-type gastric cancer probably develops from quiescent Mist1+ stem cells upon Kras and APC mutation and loss of E-cadherin. Most importantly, this seems to be dependent on inflammatory processes triggered by SDF-1 expressing endothelial cells and CXR4-expressing gastric innate lymphoid cells that form the perivascular gastric stem cell niche (75).

Likewise, GBM cells co-opt vessels and home to perivascular stem cell niches. Reciprocal signaling between endothelial and GBM cells within these niches has been shown to induce and maintain a stem(-like) cell phenotype of GBM cells on the one hand and to promote angiogenesis on the other [for review see (76)]. Moreover, trans-differentiation of GBM stem-like cells into endothelial cells (77, 78) and pericytes (79) contributes to the adaptation of the tumor microvasculature to the needs of the GBM cells. SDF-1/CXCR4 signaling reportedly exerts pivotal functions in these processes. In particular, CXCR4 on GBM cells and SDF-1 produced by endothelial cells direct perivascular invasion as demonstrated in vitro and in orthotopic glioma mouse models (79–81). Accordingly, SDF-1-degradation by the cysteine protease cathepsin K facilitates evasion of GBM cells out of the niches (82). In addition to chemotaxis, CXCR4 stimulation by SDF-1 induces the production of vascular endothelial growth factor (VEGF) in GBM (83) and especially in CD133+ GBM stem-like cells (84). VEGF, in turn, stimulates beyond angiogenesis upregulation of CXCR4 (85) and SDF-1 (86) in microvascular endothelial cells. Moreover, VEGF is required for trans-differentiation of GBM-derived progenitor cells into endothelial cells (77). The significance of targeting VEGF and SDF-1/CXCR4 signaling for stem cell niche formation can be deduced from the observation that targeting of both, VEGF and CXCR4, decreases the number of perivascular GBM cells expressing stem cell markers in an orthotopic glioma mouse model, which was associated with improved survival of the tumor-bearing mice (87).

A further example of up-regulation of a stem(-like) cell phenotype mediated by SDF-1 signaling was reported for breast cancer cells where SDF-1 release from tumor-associated fibroblasts is required for the maintenance of tumor initiation capability (88). Finally, leukemia cells have been demonstrated to be trapped in stem cell niches of the bone marrow (89– 91), and follicular lymphoma stem(-like) cells to follicular DCs in the germinal center of lymph nodes (92) by SDF-1/CXCR4 signaling. Combined, these data suggest that SDF-1 directed chemotaxis to certain microenvironmental stem cell niches is a general phenomenon of CXCR4-expressing hematopoietic and non-hematopoietic cancer cells. Of utmost importance, interactions with stromal cells within these niches contribute to a malignant and therapy-resistant phenotype of niche-residing cancer cells as outlined in the next paragraph.

## SDF-1/CXCR4 Signaling in Tumor Microenvironment-Induced Therapy Resistance of Cancer Stem(-Like) Cells

Subventricular zones (SVZs) of the brain accommodate neural stem cells and have been shown to attract human GBM stem(-like) cells through SDF-1/CXCR4 signaling in an orthotopic glioma mouse model (93). Importantly, SVZ residence induces radioresistance of GBM stem(-like) cells in direct dependence on SDF-1 release by the SVZ stromal cells (94). Evidence for radioresistance conferred by SDF-1/CXCR4 dependent residency in perivascular niches was further provided by the observation that CXCR4 knockdown in mouse GBM cells resulted in both, diminished perivascular invasion and increased radiosensitivity (81).

Likewise, in mouse models of acute myeloid leukemia CXCR4 antagonism mobilized leukemia cells out of the bone marrow niches and, at the same time, enhanced chemosensitivity (90, 91). Mechanistically, bone marrow mesenchymal cells have been demonstrated to upregulate a signaling complex in the leukemia cells comprising CXCR4 and activating pro-survival signals via extracellular signal-related kinase 1/2 (ERK1/2) and the phosphoinositide 3-kinase (PI3K)/Akt pathway (95). Moreover, bone marrow disseminated xenografted head and neck squamous cell carcinoma (HNSCC) exhibits a higher cisplatin resistance than lung metastases ex vivo. This difference critically depends on TGF-β-triggered SDF-1/CXCR4 signaling (96). In summary, these preclinical in vivo and ex vivo data strongly suggest that SDF-1/CXCR4-mediated residency of tumor cells in stem cell niches induces resistance to chemo- and/or radiation therapy probably by inducing expression of a therapy-resistant cancer stem(-like) cell phenotype. The maintenance of the latter as discussed above and impressively demonstrated by the ex vivo comparison of bone marrow and lung disseminated HNSCC—itself crucially depends on SDF-1/CXCR4. Beyond cancer stem(-like) cell induction, SDF-1/CXCR4 signaling has been demonstrated to trigger tumor invasion and metastasis as discussed in the next chapter.

### SDF-1/CXCR4 Signaling in Triggering Tumor Invasion and Distant Metastasis

Associations between SDF-1/CXCR4 polymorphisms or SDF-1/CXCR4 abundance in tumor specimens and clinical outcome in several but not all studies might suggest a role of SDF-1/CXCR4 signaling in metastatic progression in a variety of tumor entities, such as renal cell carcinoma (97), prostate cancer (98), HNSCC (99–102), esophagogastric cancer (103), colorectal cancer (74), hepatocellular carcinoma (104), or osteosarcoma (105). In preclinical studies, overexpression of CXCR4 has been demonstrated to dramatically increase lung and liver metastases of murine pancreatic cancer in tail vein metastasis assays in nude mice (106).  Intra-arterially injected circulating CXCR4-expressing melanoma cells require SDF-1 signaling by mesenchymal stem cells that act as pericytes for extravasation to bone and liver and perivascular niche formation as demonstrated by humanized heterotopic bone formation assay (110). Combined, these examples suggest that CXCR4 expression by cancer cells contribute to their tropism to metastatic niches.

Along those lines, CXCR4 downregulation by overexpression of miR-613 reportedly inhibits lung metastasis of osteosarcoma orthotopically xenografted in nude mice (105). Notably, epigenetic downregulation of SDF-1 has been demonstrated to boost metastases of CXCR4-expressing sarcoma in mouse models (111). Likewise, a SDF-1 polymorphism with low SDF-1 expression in breast cancer has been proposed to associate with susceptibility to metastases (112). It is, therefore, tempting to speculate that SDF-1−/CXCR4+ tumor cells are particularly prone to metastasize. As a matter of fact, high CXCR4 and low SDF-1 expression by the tumor has been associated with poor overall survival in osteosarcoma (111) and metastasis-free survival in head and neck squamous cell carcinoma (113). The latter association, however, was not confirmed by a recent study (114). Nevertheless, these reports strongly suggest a pro-metastatic function of SDF-1/CXCR4 signaling in several cancer entities.

In GBM which usually does not metastasize outside the central nervous system, SDF-1/CXCR4 signaling has been demonstrated in vitro to exert pivotal function in cell migration and chemotaxis (115–117). Most probably, SDF-1/CXCR4 dependent migration/chemotaxis does not only contribute to the above discussed homing of GBM cells to protective perivascular stem cell niches (see above) but also to deep infiltration of the brain parenchyma by highly migratory GBM stem(-like) cells. One driver of glioblastoma dissemination might be hypoxia through HIF-1α mediated up-regulation of SDF-1 and CXCR4 in GBM cells (85, 86). Unexpectedly, VEGF- or VEGF-Rtargeting has been demonstrated in vitro to directly up-regulate CXCR4 expression and chemotaxis toward SDF-1 in VEGF-Rexpressing GBM cells in a TGFβ-dependent manner (118). In accordance with these observations, anti-angiogenic therapy of orthotopic mouse glioma promotes GBM invasion by CXCR4 upregulation. Additional CXCR4-targeting blunts this effect (119). Consistently, combined VEGF- or VEGF-R- and CXCR4 antagonism prolongs survival of mice bearing orthotopically xenografted GBM as compared to only VEGF/VEGF-R-targeted mice (87, 118, 120). Also along those lines, anti-angiogenic therapy, such as Bevacizumab which has been demonstrated in large clinical trials not to improve overall survival of GBM patients is under suspicion to foster distant spread of the tumor at recurrence (121). Even if the tumor spread-promoting effect of Bevacizumab is under debate (122), nevertheless, these data bear witness to a close interaction between tumor hypoxia and SDF-1/CXCR4 signaling as introduced in more detail in the next chapter.

### SDF-1/CXCR4-Signaling and its Function for Vasculogenesis

Hypoxia-induced up-regulation of SDF-1 secretion in tumors reportedly stimulates homing and engraftment of bone marrowderived myeloid cells, as well as mesenchymal stem cell-derived endothelial and pericyte progenitor cells. This recruitment promotes neovascularization of the hypoxic tumor by transition of the myeloid and progenitor cells into endothelium and pericytes. Such SDF-1/CXCR4-dependent vasculogenesis has been demonstrated in mouse models of several tumor entities, such as GBM (123–127), HNSCC (128), lung adenocarcinoma (129), hepatocellular carcinoma (130) or breast cancer (131). Importantly, irradiation has been shown to induce SDF-1 expression and thus may boost vasculogenesis and tumor re-growth after end of therapy (125, 131–133) suggesting a radioresistance-conferring action of SDF-1/CXCR4 signaling as discussed in the next paragraph.

### SDF-1/CXCR4-Signaling and Radioresistance

In many tumor entities radiation therapy is part of standard of care. Ionizing radiation has been demonstrated in vitro as well as in vivo to stimulate SDF-1/CXCR4 signaling in different human and mouse tumor entities either directly by S-nitrosylation and stabilization of HIF-1α (134) or indirectly via radiationinduced endothelial cell killing and resulting hypoxia (135) or HIF-1α-independent mechanisms (136). Radiation-induced modifications in SDF-1/CXCR4 signaling, in turn, have been reported in gliomas (116, 125, 127, 137), mesotheliomas (138), prostate (139), cervical (140), lung (131, 141) and breast cancer (131). Aside from the direct effect on cancer cells, radiationinduced SDF-1 secretion is also observed in different normal tissues/cells (94, 136, 142–147) or cancer-associated fibroblasts (144).

Importantly, radiation-modulated SDF-1/CXCR4 signaling has been shown to stimulate tumor re-growth (142, 148), EMT (144), migration (116), invasiveness (81, 127, 138, 141, 144) and metastases (138, 145), as well as homing of hematopoietic progenitor cells and accelerated vasculogenesis (125, 127, 131– 133, 136, 137, 142). Thus, radiation-induced SDF-1/CXCR4 signaling may foster radioresistance, malignant progression and recurrence of tumors (94, 125, 139, 149–151). Preclinical evidence shows reduced metastases in orthotopic murine models of cervical cancer with Cisplatin-based radiochemotherapy and AMD3100 (152).

Thus, as CXCR4 is a prognostic marker for local control after curative radiotherapy and irradiation interferes with SDF-1/CXCR4 signaling, there is a strong rationale to develop translational and clinical interventional studies combining CXCR4 targeting with curative radio(chemo)therapy. The roles of SDF-1/CXCR4 signaling in tumor biology are summarized in **Table 1**.

# SDF-1/CXCR4 Signaling as Druggable Target in Anti-cancer Therapy

As already touched upon, retrospective clinical data might hint to associations between SDF-1 polymorphisms or SDF-1/CXCR4 expression levels with susceptibility to neoplastic transformation, malignant progression or therapy response in a variety of tumor entities, such as renal cell carcinoma (97), prostate cancer (98), HNSCC (1, 100, 102, 113, 114), esophagogastric cancer (103), hepatocellular carcinoma (104), colorectal cancer (74), breast cancer (153), osteosarcoma (111), low grade glioma (154, 155), or GBM (156, 157). Beyond cancer SDF-1 genetics has been associated with e.g., the pathogenesis of multiple sclerosis (158) or prognosis in patients with cardiovascular disease (159).

Apart from genetic variants, SDF-1 as well as CXCR4 were shown to be regulated epigenetically by DNA methylation. DNA methylation status of the genes was suggested as prognostic biomarkers for e.g., breast or pancreatic cancer and GBM (160–162). Such prognostic or predictive value of SDF-1/CXCR4 might be expected from the plethora of SDF-1/CXCR4 functions in tumor biology mentioned above. These functions contribute to malignancy, progression and therapy resistance of the tumors and render SDF-1/CXCR4 signal to an ideal target in anti-cancer therapy. In particular, a combination of SDF-1/CXCR4-targeting and radiotherapy seems to be promising given the above mentioned radioprotective functions of SDF-1/CXCR4 signaling (**Figure 1**). Moreover, combinatorial treatment of conventional chemotherapy with CXCR4 inhibitors might be an approach to overcome cancer therapy resistance (163).

In fact, several SDF-1- or CXCR4-targeting drugs have been applied in preclinical models [e.g., Ulocuplumab (164), ALT-1188 (165), POL5551 (166), PRX177561 (167)], were well-tolerated in clinical trials [e.g., AMD070 (168), Balixafortide (POL6326, Polyphor) (169)] or are FDA-approved [AMD3100, Plerixafor (170)] indicating that SDF-1/CXCR4 targeting is clinically feasible. Overall, Plerixafor used for stem cell mobilization does not induce severe side effects (171, 172). A randomized phase 3 trial comparing G-CSF with plerixafor vs. placebo reported mainly fatigue, gastrointestinal side effects like nausea and diarrhea and injection site reactions (173).

CXCR4, however, is expressed on immune cells suggesting that SDF-1/CXCR4-targeted anti-cancer therapy at the same time interferes with the immune response to cancers and cancer cells in e.g., circulation or micrometastases. In order to explore these functions and develop a rationale if trimodal therapy combining CXCR4 targeting with immunotherapy and radiotherapy might be of benefit, it is crucial to understand the function of SDF-1/CXCR4 signaling in immune cells and the effects of CXCR4 inhibition on the immune response to cancer.

# PHYSIOLOGIC ROLE OF CXCR4 IN THE IMMUNE SYSTEM

In addition to its function in tumor biology, SDF-1/CXCR4 signaling controls multiple physiological processes in

#### TABLE 1 | (Patho)physiological role of SDF1/CXCR4 signaling and targeting in cancer.

*BC, breast cancer; BM, bone marrow; CRC, colorectal cancer; CSC, cancer stem(like) cell; EMT, epithelial mesenchymal transition; GBM, glioblastoma multiforme; VEGF, vascular endothelial growth factor.*

FIGURE 1 | SDF-1/CXCR-4 signaling in tumors and its contribution to maintenance of tumor stemness, recruiting of distant stroma cells, angio- and vasculogenesis, and metastasis (for details see text).

hematopoiesis, T, B and NK cell development and the organization of the immune system. Ablation of either of the components of the SDF-1/CXCR4 axis generates a similar phenotype of deficient B lymphopoiesis and myelopoiesis, disturbed immune responses leading to cancers, autoimmunity and inflammatory diseases (174–176). Recently a 16 amino acid fragment of serum albumin (EPI-X4) was identified as an effective and highly specific endogenous CXCR4 antagonist (177). Peptide EPI-X4 is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. It antagonizes SDF-1-induced tumor cell migration and suppresses inflammatory responses in mice. In human the peptide is abundant in the urine of patients with inflammatory diseases. EPI-X4 mobilizes stem cells, which explains in part why stem cells can directly respond to inflammation with their migration into the periphery.

#### Hematopoietic Stem Cell Niche

HSCs (Hematopoietic stem cells) are a rare cell population that can give rise to all lineages of the immune system. HSCs reside in the undifferentiated state in the bone marrow, where the binding of their CXCR4 receptor to its ligand SDF-1—constitutively provided by the bone marrow (BM) niche—promotes their survival (178, 179) while negatively regulating their proliferation (180–182). In addition to direct effects on HSCs, SDF-1/CXCR4 signaling also promotes survival and growth of human bone marrow stromal stem cells (183). Inhibiting the interaction between CXCR4 receptor and SDF-1 leads to the migration of hematopoietic stem and progenitor cells (HSPC) into the periphery, a process termed mobilization, which is required for harvesting stem cells for transplantation [either autologous from the patient (184) or in healthy donors (185)]. A dramatic increase in mobilization efficiency and yields of progenitor cells compared to standard G-CSF is achieved when using CXCR4 antagonists, such as AMD3100, Mozobil R (184, 186, 187). CXCR4 antagonist BL-8040 in a recent phase I clinical study (NCT02073019) besides highly efficient mobilization of pluripotent hematopoietic progenitors showed also superior yields of CD4+ and CD8+ T cells, NKT, NK, and DCs, suggesting increased engraftment ability of CXCR4 mobilized populations, a higher anti-tumor effect and faster immune reconstitution potential. Moreover, mobilization as a 1-day procedure is less wearing for the donor and allows faster access to the stem cells (188). Since HSCs maintain hematopoiesis throughout life, qualitative and quantitative effects through prolonged pharmacologic blockade of the SDF-1/CXCR4 axis need to be investigated. Concerns that the HSC pool in the bone marrow would decrease were not confirmed, as CXCR4-blockade led to higher repopulating capacity and exceptional mobilization along with an expansion of the BM HSC pool, which unexpectedly suggests reversible inhibition of the SDF-1/CXCR4 axis also as a novel strategy to restore damaged BM (189). BM HSCs during reversible long-term CXCR4/SDF1 long term blockade increase their cycling activity 2- to 3-fold [only 10–20% of Lin-Sca1+Kit- (LSK) and 30–40% of LSK SLAM cells being quiescent (G0 phase)] compared to 50–60% of LSK and 70% of LSK SLAM under homeostatic conditions (190, 191). Thus, these findings together with mounting evidence for direct cytolytic and specific anti-leukemic effects of CXCR4 inhibition (192–194) suggests prolonged CXCR4 blockade as a novel safe therapeutic scheme for treatment of (hematologic) malignancies either alone or in conjunction with chemotherapy.

## Dendritic Cells

The priming of naïve T cells is dependent on efficient antigen presentation and a strong costimulatory signal both provided by dendritic cells during Th1 polarized immune responses. Th1 polarization is thought to be critical for immune rejection of tumors, while activated T cells polarized to Th2 cytokine and cell profiles might induce even tumor immune evasion (195). Plasmacytoid DCs (pDCs) as type I interferon (IFN) producing cells play a central role in antiviral and anti-tumor immunity. pDCs are produced from hematopoietic stem cells in the bone marrow where they nestle down with reticular cells in the intersinal space which abundantly provides SDF-1. Concordantly, the number of pDCs and their earliest progenitors is severely reduced in the absence of CXCR4 in vitro and in vivo, underlining the function of SDF-1/CXCR4 axis as a key regulator of pDC development and the importance of provision of SDF-1 by cellular niches (196, 197). Upon activation, CXCR4 expressing DCs migrate into SDF-1 expressing lymphatic vessels where they initiate immune responses, a process that is severely blocked by systemic CXCR4 antagonist application (198). Since the dysregulated expression of SDF-1/CXCR4 is associated with the pathology of various autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis, targeting SDF-1/CXCR4 axis with 4-F-Benzoyl-TN14003 may be beneficial for prevention of autoimmune disease (198–201). It is not clear, whether these effects on DCs might decrease the efficacy of anti-cancer immune responses upon CXCR4 inhibition.

# Myeloid Derived Suppressor Cells

As reviewed in (202), MDSCs are highly immunosuppressive cells in the tumor microenvironment and mainly suppress intratumoral T cells. SDF-1 secreted by tumor associated fibroblasts induces MDSCs and impairs anti-tumor immune responses as shown in a hepatic carcinoma model (203). Another liver cancer model (metastases of colorectal carcinoma) showed less MDSC infiltrates in the metastases after treatment with AMD3100, accompanied by reduced metastases growth (204). Patient samples of ascites also showed that CXCR4 signaling is involved in MDSC recruitment. SDF-1 release of cancer cells as well as CXCR4 signaling in MDSCs could be abrogated by COX2 inhibition.

# Regulatory T Cells (nTreg and iTreg)

Regulatory T cells (Tregs) constitute 5–10% of peripheral CD4+ T cells in humans (205, 206). Tregs maintain immune homeostasis, peripheral tolerance and prevent autoimmunity by suppressing and terminating immune responses. They constitute a major barrier for an effective antitumor immunity, and the number of peripheral and intratumoral Treg cells is an independent prognostic factor in malignancies (207). Cancer cell- and M2 macrophages derived SDF-1 attracts Treg cells into the tumor lesion where they robustly induce FOXP3 and other Treg signature molecules in human naïve CD4+ T cells which display enhanced FOXP3 stability and low expression of pro-inflammatory cytokines (208). Treg cells limit immune effector cell function via cytokines (209–212), direct lysis (213), inhibitory receptors on their cell surface (214– 217), via metabolic disruption (218), by starving effector cells via depletion of local IL-2 (219) or indirectly by turning secondary cell types into suppressive ones i.e., IDO (220) and tolerogenic cytokine producing DCs with low costimulatory potential (221). Treg depletion dramatically reduced tumor growth or induced full remission (222–224). In contrast to conventional chemotherapeutic agents which also deplete T effector cells and may induce autoimmunity due to the systemic elimination of T-regs (225), CXCR4 targeting allows the specific targeting of Tregs, since intratumoral Tregs consistently express higher CXCR4 levels than CD4+CD25− and CD8+ cells (226). In intraperitoneal papillary epithelial ovarian cancer, CXCR4 antagonism increased tumor cell apoptosis and necrosis, reduced intraperitoneal dissemination, and selectively reduced intratumoral FoxP3 Tregs (226). Superior immune responses as shown for CXCR4 antagonist BL-8040 is not solely owing to a selectively reduced recruitment of Treg cells into the tumor, and an increase in the number of immune and progenitor cells (227). CXCR4 antagonists have shown to also reverse Tregs' suppressive activity. Plerixafor and the antagonistic CXCR4 peptide R29 (228) inhibited Treg-suppressive activity significantly (by 10 fold) in Tregs from primary renal cancer specimens in which high numbers of activated Tregs (expressing CTLA-4/CXCR-4/PD-1/ICOS) were detected. A possible mechanistic explanation

involves the demethylation of Treg-FOXP3 promoter (229). Thus, inhibition of Tregs by blocking SDF-1/CXCR4 is one of the major rationales for a better anti-tumor immune response via CXCR4 inhibition.

# Effector Cells

T effector (Teff) cells also constitutively express the chemokine receptor CXCR4. Besides T cell migration along SDF-1 gradients, CXCR4 after T cell receptor crosslinking is recruited to and accumulates at the immunological synapse, resulting in stronger T cell-APC interaction, shutdown of T cell responsiveness to chemotactic gradients, and in higher levels of T cell proliferation and IFN-γ production (230). Vice versa, the presence of competing external chemokine signals has been shown to disrupt the stability of T-APC conjugates as a result of impaired recruitment of the receptor to the immunologic synapse (231). CXCR4 confers the homing of antiviral T cell responses to bone marrow. Ablation of CXCR4 thus impairs memory cell maintenance due to defective homeostatic proliferation in the bone marrow niche, yet allows fully functional asymmetric cell fates after antigenic rechallenge in CD8+ T cells (232). Antitumoral activity was shown for CXCR4 antagonist BL-8040 in tumor bearing mice, where it induced robust mobilization of CD4+ and CD8+ T lymphocytes and DC in numbers that were significantly higher compared to tumor free naïve counterparts. The authors did not discriminate the lymphocytic population with respect of Teff/Treg ratio or CD8+ content though (233), yet showed in pre-clinical in vivo pancreatic cancer models, immune cells mobilized from the bone marrow into the circulation accumulate within the tumor lesion where they inhibit tumor growth. Such a dramatic effect on the

*APC, antigen presenting cell; BM, bone marrow; MDSC, myeloid derived suppressor cells.*

intratumoral T cell compartment function is reflected in a study by Elda Righi (226) where CXCR4 antagonist AMD3100 by favorably modulating the intratumoral Teff/Treg ratio 6-fold, created a phenotype reminiscent of two studies that—although in different contexts—depleted intratumoral T-regs which highly significantly improved cytotoxic T-cell function in the tumor tissue and prolonged survival (234, 235). Along those lines, epigenetic down-regulation of SDF-1 expression in osteosarcoma has been demonstrated to impair cytotoxic T-cell homing to the tumor site (111). In contrast, SDF-1 overexpression by melanoma cells in the B16-ova melanoma model has been shown to chemo-repel antigen-specific cytotoxic T cells (236) suggesting a complex and fine-tuned control of Teff infiltration by SDF-1/CXCR4 signaling.

Chemokines control also the trafficking of developing and mature natural killer cells (NK) in the bone marrow (237).  Administration of the CXCR4 antagonist, AMD3100, induced strong reduction of mature NK and immature NK cells in the BM in a murine model and increased their number in blood and spleen, which suggests that this chemokine axis also regulates NK cell subsets localization in the bone marrow niche and their migration to the periphery for their maturation (238). Notably, genetic deletion of CXCR4 in myeloid cells in a melanoma mouse model fostered NK cell-mediated antitumor immunity suggesting indirect suppression of NK cell activity by CXCR4 signaling (239).

In summary, SDF-1/CXCR4 signaling affects most subsets of immune cells, the most prominent and clinically applied effect being the mobilization of HSCs by blocking CXCR4 as summarized in **Table 2**.

# Combined Immunotherapy and CXCR4 Targeting

The promotion of antitumor immunity by CXCR4-antagonists was reported for a mouse model of ovarian cancer (226) and in an orthotopic preclinical hepatocellular carcinoma (HCC) model where a CXCR4 antagonist was combined with a checkpoint inhibitor. In this HCC model multi-kinaseinhibitor sorafenib treatment-induced hypoxia fostered SDF-1 production, leading to the recruitment of immunosuppressive tumor-associated macrophages, myeloid-derived suppressive cells, and Tregs all with increased PD-L1 expression. CXCR4 antagonist plerixafor combined with anti-PD-1 therapy showed the most pronounced tumor growth delay, and was associated with increased intratumoral penetration and activation of CD8+ T lymphocytes (241). A novel strategy for the treatment

FIGURE 2 | Immunosuppressive and immunostimulatory action of SDF-1/CXCR4 signaling in tumors induced by radiation-therapy and hypoxia (for details see text; DAMPs, danger-associated molecular patterns; DC, dendritic cells; MDSC, myeloid derived suppressor cell; PD-1, programmed cell death protein-1; PD-L1, PD-1 ligand; TAA, tumor-associated antigens; TAM, tumor-associated macrophage; Treg, regulatory T-cell).

of drug-resistant ovarian cancer combines chemotherapy to increase immunogenic cell death and virally delivered CXCR4 to reverse the immunosuppressive tumor microenvironment (242). Ovarian cancer of murine and human ovarian tumor variants resistant to paclitaxel and carboplatin were infected with oncolytic vaccinia virus expressing a CXCR4 antagonist and were +/– treated in combination with doxorubicin. The chemo-resistant variants' augmented expression of CXCR4 was associated with an increased susceptibility to viral infection and doxorubicin-mediated killing compared to parental counterparts in vitro and in tumor-challenged mice. Antitumor immune responses in this model culminated in the control of metastatic tumor growth and tumor-free survival. Mechanistically, the authors showed combination treatment increased apoptosis and phagocytosis of tumor material by DCs which efficiently induced adaptive antitumor immunity, reflected by increased intratumoral infiltration of antitumor CD8+ T cells and reduced immunosuppressive Tregs (242). Based on these results (**Figure 2**), the MORPHEUS clinical trials were started including treatment arms combining immune-checkpoint inhibitors with CXCR4 inhibition (NCT03193190, NCT03281369 and NCT03337698 for pancreatic cancer, gastric cancer and non-small cell lung cancer, respectively).

#### CONCLUDING REMARKS

With combinatorial approaches of radiotherapy and immunotherapy on the rise, it is important to evaluate novel treatment strategies in radiation oncology with respect to tumor and radiation biology as well as immunologic effects. For SDF-1/CXCR4 targeting both perspectives provide a strong rationale for combination therapies. The SDF-1/CXCR4 axis plays pivotal roles in various aspects of tumor biology, and in particular in the stress response of tumors to ionizing radiation. In preclinical in vivo models CXCR4 targeting increases the efficacy of radiation therapy and blunts adverse effects, such as radiation-stimulated metastases and vasculogenesis.

#### REFERENCES

Mobilization of HSCs, a significant increase of immune and progenitor cells in the periphery that are able to migrate into the tumor and the selective targeting of Treg cells in the tumor lesion provide the rationale for an increased antitumor immune response upon CXCR4 inhibition. Preclinical mechanistic studies as well as translational and clinical evaluation of the role of the SDF-1/CXCR4 axis in the context of cancer radiotherapy and immunotherapy might lead to novel treatment strategies implementing SDF-1/CXCR4 targeting in this context using the small molecule inhibitors already approved for the use in patients and healthy donors for HSC mobilization.

# AUTHOR CONTRIBUTIONS

FE and SH designed the concept and wrote the manuscript. LK wrote chapter SDF-1/CXCR4-Signaling and Radioresistance. MS wrote chapter SDF-1/CXCR4 Signaling as Druggable Target in Anti-cancer Therapy. LB wrote chapter Interference of Ionizing Radiation With Immune Responses. ES contributed to chapter SDF-1/CXCR4 Function in Tumor Biology. KS wrote chapter Physiologic Role of CXCR4 in the Immune System. DZ and all authors read and approved the manuscript.

### FUNDING

FE was partly funded by the Else-Kroener-Fresenius Research Foundation under Grant 2015\_Kolleg.14. LK was supported by the ICEPHA program of the University of Tübingen and the Robert Bosch Stiftung, Bosch-Gesellschaft für Medizinische Forschung, Stuttgart, MS was partly supported by a grant from the German Cancer Aid (70112564) and by the Robert Bosch Stiftung, Stuttgart and ES by a scholarship from the DAAD. SH was partly funded by grants from the German Cancer Aid (70112872, 70113144). We acknowledge support by Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of University of Tübingen.

radioresistance of embryonal rhabdomyosarcoma stem-like cell population. Mol Cancer (2016) 15:16. doi: 10.1186/s12943-016-0501-y

explanation of Scherer's structures. Am J Pathol. (2008) 173:545–60. doi: 10.2353/ajpath.2008.071197

pathway in malignant mesothelioma cells. Oncotarget (2017) 8:68001–11. doi: 10.18632/oncotarget.19134

and multipotent progenitors. Cell Stem Cell. (2013) 13:102–16. doi: 10.1016/j.stem.2013.05.014

T cells are inhibited by CTLA-4 or IL-35 blockade. J Immunol. (2012) 189:5590–601. doi: 10.4049/jimmunol.1201744

**Conflict of Interest Statement:** FE has a research collaboration with Merck KgAa. SH has a research collaboration with Novocure. DZ, FE have research and educational grants from Elekta, Philips, Siemens, Sennewald.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest

Copyright © 2018 Eckert, Schilbach, Klumpp, Bardoscia, Sezgin, Schwab, Zips and Huber. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Antitumor Potential of Extracellular Vesicles Released by Genetically Modified Murine Colon Carcinoma Cells With Overexpression of Interleukin-12 and shRNA for TGF-β1

#### Edited by:

*Patrik Andersson, Massachusetts General Hospital, Harvard Medical School, United States*

#### Reviewed by:

*Robin Parihar, Baylor College of Medicine, United States Roberto Bei, University of Rome Tor Vergata, Italy Tomer Cooks, Ben-Gurion University of the Negev, Israel*

> \*Correspondence: *Joanna Rossowska<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology*

> Received: *26 October 2018* Accepted: *24 January 2019* Published: *13 February 2019*

#### Citation:

*Rossowska J, Anger N, Wegierek K, Szczygieł A, Mierzejewska J, Milczarek M, Szermer-Olearnik B and Pajtasz-Piasecka E (2019) Antitumor Potential of Extracellular Vesicles Released by Genetically Modified Murine Colon Carcinoma Cells With Overexpression of Interleukin-12 and shRNA for TGF-*β*1. Front. Immunol. 10:211. doi: 10.3389/fimmu.2019.00211* Joanna Rossowska\*, Natalia Anger, Katarzyna Wegierek, Agnieszka Szczygieł, Jagoda Mierzejewska, Magdalena Milczarek, Bozena Szermer-Olearnik and ˙ Elzbieta Pajtasz-Piasecka ˙

*Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland*

Recent developments demonstrate that tumor-derived extracellular vesicles (EVs) could become a highly effective tool for delivery of antitumor factors. The main objective of the study was to determine whether EVs secreted by MC38 colon carcinoma cells genetically engineered for overproduction of interleukin (IL-)12 and/or shRNA targeting TGF-β1 are effectively loaded with these molecules and whether the obtained EVs could be an efficient tool for antitumor therapy. Fractions of EVs released by genetically modified MC38 cells [both modified tumor-derived exosomes (mTEx) and modified microvesicles (mTMv)] and those released by unmodified, wild-type MC38 cells were characterized in terms of loading efficacy, using real-time PCR and ELISA, as well as their antitumor potential. In order to examine the therapeutic potential of mTEx, they were applied in the form of sole treatment as well as in combination with dendritic cell (DC)-based vaccines stimulated with mTMv in the therapy of mice with subcutaneously growing MC38 tumors. The results demonstrated that genetic modification of wild-type MC38 tumor cells is an effective method of loading the molecules of interest into extracellular vesicles secreted by the cells (both TEx and TMv). The results also showed that mTEx secreted by cells engineered for overproduction of IL-12 and/or shRNA for TGF-β1 are able to induce tumor growth inhibition as opposed to TEx from unmodified MC38 cells. Additionally, antitumor therapy composed of mTEx (especially those deprived of TGF-β1) and DC-based vaccines allowed for regeneration of antitumor immunity and induction of the systemic Th1 response responsible for the sustained effect of the therapy. In conclusion, tumor-derived exosomes loaded with IL-12 and/or deprived of TGF-β1 could become an efficient adjuvant supporting induction of a specific antitumor response in both immuno- and chemotherapeutic schemes of treatment.

Keywords: tumor-derived exosomes, tumor-derived microvesicles, dendritic cells, interleukin 12, TGF-β1 silencing, lentivectors, colon carcinoma, immunotherapy

# INTRODUCTION

Tumor development is dependent on reliable communication and interaction between tumor cells and other cellular components of the tumor microenvironment (TME) (1). Recent scientific reports indicate that both tumor and tumorinfiltrating cells secrete large amounts of extracellular vesicles (EVs), which were proved to be an important element of intercellular communication within and beyond the tumor (2, 3). Scientific literature distinguishes at least two major classes of EVs: exosomes and microvesicles. Exosomes, which have an approximate size of 30–200 nm, are formed from the membrane of late endosomes [multivesicular bodies (MVBs)] by its inward budding. They are released from the endosome to the extracellular space in the process of MVB fusion with the plasma membrane. By contrast, microvesicles (200–1,000 nm) are formed through direct budding of the plasma membrane (1, 4). Both of them are potent vectors capable of transporting biologically active molecules (proteins, lipids, RNA, DNA) to target cells. Depending on the final cargo they can induce a wide range of processes that support cancer development, including immune suppression, cell proliferation, angiogenesis, epithelial-to-mesenchymal transition (EMT) and metastasis (5–7). However, given that tumor-derived exosomes (TEx) as well as microvesicles (TMv) are an easily accessible source of tumor antigens, express tumor-specific integrins which direct their migration toward the tumor site or predicted metastatic sites (8), and act as efficient carriers of different biological structures, they seem to be ideal vehicles for delivery of a broad range of therapeutic agents including non-coding RNAs, mRNAs, proteins, and synthetic drugs. The passive loading through the physical mixing of EVs with drugs (i.e., paclitaxel, cisplatin, curcumin), as well as active loading of molecules such as small interfering RNA by electroporation or sonication, are the most common methods to obtain EVs with desired cargo (9–12). EVs with modified content can also be obtained from genetically engineered cells with overexpression of desired molecules (13–15).

Disorders of immune cells caused by highly immunosuppressive TME are the most common reasons for poor results of cancer immunotherapy (16–18). Hence, therapeutic strategies allowing for efficient reprogramming of the hostile TME abundant in suppressive cytokines such as TGF-β, IL-10, or VEGF are intensively studied (19–21). TME can be modulated both by delivery of inflammatory cytokines, i.e., IL-12 or IL-2 and by elimination of suppressing factors. In our work we focused attention on TGF-β1 and IL-12, which play opposite functions in a tumor development. TGF-β1 is an anti-inflammatory cytokine associated with tumor progression and metastasis, often correlated with poor prognosis in patients (22). By contrast, IL-12 is a proinflammatory cytokine with high antitumor potential but also with high toxicity when applied in the recombinant form (23). The main purpose of our study was to develop and analyze TGF-β1-deprived EVs with IL-12 cargo. The TEx and TMv with modified content, referred to here generally as mTEx and mTMv, were obtained from genetically engineered murine colon carcinoma MC38 cells with overexpression of IL-12 and/or shRNA for TGF-β1 (MC38/IL12, MC38/shTGFβ1, or MC38/IL12shTGFβ1). The particles released by unmodified, wild-type MC38 cells, referred to here as TEx MC38 or TMv MC38, were applied as a control. Since EVs are released by cells in response to stress conditions such as hypoxia, acidosis, or oxidation stress (24), we decided to induce EVs secretion by culture of MC38 cells in hypoxic conditions. TEx and TMv fractions were isolated from the wild-type or genetically modified MC38 culture supernatant and separated on the basis of size using sequential centrifugation. The gathered data indicate that genetic modification of tumor cells is an efficient method to change the tumor-derived EVs' cargo to a therapeutic one. Moreover, immunotherapy composed of mTEx, especially those obtained from MC38/shTGFβ1 or MC38/IL12shTGFβ1, and dendritic cells (DCs) stimulated with mTMv from MC38/shTGFβ1 or MC38/IL12shTGFβ1 cells, applied in the treatment of mice with a subcutaneously growing MC38 tumor, induced tumor growth inhibition accompanied by a reduced number of MDSCs in the tumor and enhanced local and systemic Th1-type antitumor activity.

### MATERIALS AND METHODS

#### Mice

Female C57BL/6 mice were obtained from the Center for Experimental Medicine of the Medical University of Bialystok (Bialystok, Poland). All experiments were performed in accordance with EU Directive 2010/63/EU for animal experiments and were approved by the 1st Local Ethics Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland (authorization number 33/2018). After the experiments, the mice were sacrificed by cervical dislocation.

#### Cell Culture

The in vivo growing cell line of MC38 murine colon carcinoma from the Tumor Bank of the TNO Radiobiology Institute, Rijswijk, Holland, was adapted to in vitro conditions as described by Pajtasz-Piasecka et al. (25). The cell culture was maintained in RPMI 1640 (Gibco) supplemented with 100 U/ml penicillin (Polfa), 100 mg/ml streptomycin (Polfa), 1 mM sodium pyruvate (Sigma-Aldrich), 2-mercaptoethanol (Sigma-Aldrich) here called complete medium (CM), and 5% fetal bovine serum (FBS, Sigma-Aldrich). The genetically modified, stable MC38 cell lines with overexpression of murine IL-12 (MC38/IL12) and/or shRNA targeting mRNA for TGF-β1 (MC38/IL12shTGFβ1, MC38/shTGFβ1) were obtained after transduction of the wildtype MC38 cell line with lentiviral vectors encoding murine interleukin 12 (mil12) genes (VectorBuilder) or shRNA for TGF-β1 (EzBiolab). Transduced MC38/IL12 or MC38/shTGFβ1 cells were maintained in standard culture medium for MC38 cells supplemented with Geneticin 418 (Gibco, 1 mg/ml) or puromycin (Gibco, 10 µg/ml), respectively. The double

**Abbreviations:** EVs, extracellular vesicles; mTEx, tumor-derived exosomes secreted by genetically modified cells; mTMv, tumor-derived microvesicles secreted by genetically modified cells; DCs, dendritic cells; MC38, murine colon carcinoma; MDSCs, myeloid-derived suppressor cells.

transduced MC38/IL12shTGFβ1 cells were selected using two antibiotics simultaneously. The efficacy of overexpression of IL-12 and silencing of TGF-β1 in MC38 cells cultured in normoxic or hypoxic conditions was estimated by real-time PCR. Total RNA was isolated using a NucleoSpin RNA kit (Macherey-Nagel) and reverse-transcribed with a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher). Realtime PCR was performed using TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay primers for IL-12 and TGF-β1 (Applied Biosystems) in reference to the HPRT gene expression. Production of IL-12 or TGF-β1 was measured by ELISA (eBioscience) in supernatant harvested from MC38 cells cultured for 24 or 48 h (0.5 × 106 cells/ml). DCs were differentiated from bone marrow of C57BL/6 mice according to the protocol described in our previous publication (26). The cells were cultured in CM supplemented with 10% FBS in the presence of rmGM-CSF (ImmunoTools, 40 ng/ml) and rmIL-4 (ImmunoTools, 10 ng/ml). After 6 days the loosely attached immature dendritic cells were stimulated with exosomes or microvesicles isolated from wild-type or transduced MC38 cell lines and used for further tests.

### Isolation and Characteristics of MC38 Tumor-Derived Particles

The production of TEx and TMv by MC38 cells was induced by culture of the cells in hypoxic conditions. Wild-type and genetically modified MC38 cells were seeded on multilayer flasks (Merck-Millipore) at the final density of 250 × 103 cells/ml and cultured in the CM supplemented with 5% exosome-deprived FBS (Sigma-Aldrich) in hypoxic conditions (1% O2) for 48 h. Then the culture supernatants were harvested. TEx and TMv were isolated from the supernatants according to the procedure described by Felicetti (27) using sequential centrifugation at 2,000, 10,000 and 100,000 × g (**Figure 2A**). The TMv fraction was collected after centrifugation at 10 000 × g, while TEx fraction was collected after ultracentrifugation. Both fractions were then washed in PBS (IIET) filtered through 0.2 µm filters (Merck Millipore). To determine the number of TEx and TMv in the final suspension we used the flow cytometry method under the control of Absolute Counting Beads (Thermo Fisher) and 1 µm beads (Polysciences INC). After isolation particles were re-suspended in PBS (IIET) filtered through 0.2 µm filters (Merck Millipore). During the analysis the TEx and TMv were separated from flow cytometer- and PBS-derived debris using CFSE staining (Thermo Scientific, 2.5 µM). The quality of the obtained fractions of TEx and TMv was evaluated using transmission electron microscopy (TEM), dynamic light scattering (DLS), flow cytometry (FC), and western blotting (WB).

#### The Dynamic Light Scattering Method

The dynamic light scattering method was used for measurement of the particle size distribution and the purity of obtained fractions. Isolated particles were resuspended in filtered PBS and then the suspension was evaluated using a DLS Zetasizer (Malvern).

#### TEM

TEx and TMv fractions were fixed in 2% paraformaldehyde (Serva) and allowed to adsorb onto formvar carbon-coated grids for 20 min. The grids were then washed in PBS (IIET), fixed in 1% glutaraldehyde (Sigma-Aldrich) for 5 min and washed with water (7 × 2 min). Then each grid was transferred to a drop of uranyloxalate (4% uranyl acetate and 0.15 M oxalic acid in 1:1 v:v ratio; Sigma-Aldrich) at pH 7 for 5 min. At this stage, samples were counterstained using two protocols: 1. with uranyl acetate or 2. with methylcellulose. 1: The grid was transferred to a drop of 2% uranyl acetate (Chemapol) for 5 min and washed with a drop of water 3 times. Then the grids were allowed to air dry for 10 min. 2: The grids were then embedded in 2% methylcellulose (Sigma-Aldrich) with uranyl acetate (9:1 v:v ratio) for 10 min on ice. The excess of methylcellulose was removed from grids by filter paper and grids were allowed to air dry for 20 min. Preparations were visualized using a JEOL JEM-1200 EX 80 kV TEM.

#### Western Blotting

MC38 cells, TEx and TMv were washed twice in PBS (IIET) and lysed in RIPA buffer supplemented with protease inhibitor cocktail (both Sigma-Aldrich). Lysates were purified by centrifugation for 10 min at 4◦C and 10,000 x g followed by supernatant transfer to new tubes. Total protein concentration in lysates was analyzed using the modified Lowry method (Bio-Rad) according to the manufacturer's protocol. Samples containing 20 µg (cell lysates), 100 µg (TEx lysates), or 10 µg (TMv lysates) of proteins were denatured in Laemmli Sample Buffer (Bio-Rad) supplemented with β-mercaptoethanol (Sigma-Aldrich) for 5 min at 100◦C and then separated in 4–20% miniprotean TGX gels (Bio-Rad, USA). After the electrophoresis, proteins were transferred from gels to polyvinylidene difluoride (PVDF) membranes (0.45 µm; Merck Millipore). After blocking in 5% non-fat dry milk in 0.1% TBS/Tween-20 (TBST) at room temperature for 1 h, membranes were washed three times for 5 min in 0.1% TBST and then incubated overnight at 4 ◦C with the following primary rabbit antibodies: monoclonal anti-CD81, monoclonal anti-TSG101, monoclonal anti-calnexin, monoclonal anti-CD9 (all from Abcam) or polyclonal anti-GM130 (Proteintech). After incubation with a primary antibody, the membrane was washed four times for 5 min in 0.1% TBST and then incubated at room temperature for 1 h with horseradish peroxidase conjugated secondary anti-rabbit antibody (DAKO). After incubation, the membranes were washed four times for 5 min in 0.1% TBST and protein bands were detected using luminol-based enhanced chemiluminescent substrate according to the manufacturer's protocol (Thermo Scientific). Images were acquired with G:BOX iChemi XR (Syngene).

#### Flow Cytometry

The flow cytometry method was applied to determine the expression of CD63, CD9, and CD81 on the surface of isolated particles. Isolated TEx or TMv were resuspended in PBS filtered through 0.2 µm filters (Merck Millipore) and then labeled with monoclonal antibodies conjugated with fluorochromes: anti-CD63 APC, anti-CD9 APC, anti-CD81 APC, rat IgG2aκ APC and Armenian hamster IgG APC isotype controls (all from BioLegend). The expression of cell surface markers was analyzed using FACS Fortessa with FACSDiva software (Becton Dickinson).

#### Determination of TGF-β1 and IL-12 Levels in Isolated Particles

Levels of mRNA for IL-12 and TGF-β1 in particles isolated from MC38 cells were measured by real-time PCR. Total RNA was isolated using the NucleoSpin RNA XS kit (Macherey-Nagel) and reverse-transcribed with the SuperScript III First-Strand Synthesis System (Thermo Fisher). Real-time PCR was performed using TaqMan Universal PCR Master Mix and TaqMan Gene Expression Assay primers for IL-12 and TGF-β1 (Applied Biosystems) in reference to the HPRT gene expression. The IL-12 and TGF-β1 proteins inside the TEx and TMv were evaluated by measurement of the cytokine concentration by ELISA (eBioscience) in lysate prepared from isolated particles using Lysis Buffer for ELISA kits (RayBiotech).

# Characteristics of Dendritic Cells Stimulated With MC38-Derived Particles

Immature DCs were stimulated with TEx and TMv obtained from wild-type or genetically modified MC38 tumor cells (5 × 106 particles/1 × 106 cells) in the presence of GM-CSF (40 ng/ml) for 24 h. After stimulation dendritic cells were harvested and labeled with monoclonal antibodies conjugated with fluorochromes: anti-CD40 PE (BD Biosciences), anti-CD80 PerCP-Cy5.5, anti-CD86 PE-Cy7, anti-MHC II FITC, anti-PD-L1 APC, and anti-CD11c BV650 (all from BioLegend). The expression of cell surface markers was analyzed using FACS Fortessa with FACSDiva software (Becton Dickinson). Moreover, the ability of TEx or TMv-stimulated DCs to activate naïve lymphocytes was evaluated. Stimulated DCs were co-cultured with splenocytes obtained from healthy mice in the final ratio of 1:10 for 5 days in CM supplemented with 10% FBS and 200 U/ml of recombinant human IL-2. After 5 days, the cells and supernatants were collected. Cytotoxic activity of primary stimulated splc toward DiO-labeled MC38 target cells, as well as the ability of the effector cells to secrete lytic granules, was measured as previously described by Rossowska et al. (28). The cytotoxic effector cells were identified by flow cytometry (FACS Fortessa) using the following monoclonal antibodies: anti-CD49b PE-CF594 (BD Biosciences), anti-CD8 PE-Cy7, and anti-CD107a APC (both from BioLegend). Production of IFN-γ by primed spleen cells was evaluated using commercially available ELISA kits (eBioscience) according to the manufacturer's instructions. A direct effect of EVs on splenocyte activity was evaluated as a concentration of IFN-γ in supernatants from 5-day culture of splenocytes in the presence of TEx or TMv.

#### Therapeutic Treatment Schedule

Eight- to ten-week old female C57BL/6 mice were subcutaneously inoculated in the right flank with MC38 cells (1.1 × 106 /0.2 ml/mouse). The mice were treated according to the scheme presented in **Figure 4A**. Tumorderived exosomes obtained from the wild-type MC38 cell line (TEx MC38) or genetically modified MC38/IL12 (TEx MC38/IL12), MC38/shTGFβ1 (TEx MC38/shTGFβ1), or MC38/IL12shTGFβ1 (TEx MC38/IL12shTGFβ1) cell lines were inoculated peritumorally (p.t.), twice per week for three consecutive weeks in a dose of 2 × 106 particles/100 µl NaCl/mouse. On the 16th, 23rd, and 30th days, dendritic cellbased vaccines stimulated with wild-type or genetically modified MC38-derived TMv (DC/TMv MC38, DC/TMv MC38/IL12, DC/TMv MC38/shTGFβ1, or DC/TMv MC38/IL12shTGFβ1) were applied p.t. in the final number of 0.7 × 106 /0.2 ml/mouse. On the 35th day, mice were sacrificed, and their spleens and tumor nodules were dissected, homogenized and stored in liquid nitrogen for further analyses. The procedure of tumor growth monitoring was presented by Rossowska et al. (29). The therapeutic effect of the treatment was evaluated using tumor growth inhibition (TGI). Statistically significant differences were calculated using Friedman and Dunn's multiple comparison tests.

# Analysis of MC38 Tumor-Infiltrating Immune Cells

Tumor cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described by Rossowska et al. (30). Briefly, tumor-derived cells were stained with LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher) and then stained with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 AlexaFluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD86 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE, anti-CD44 PE-Cy7, anti-CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using the FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).

#### Evaluation of Systemic Antitumor Response

In order to determine the polarization of the systemic immune response followed by applied treatment, Tbet and FoxP3 expression, and IFN-γ production by T cells was measured. Spleen cells, obtained from treated and control mice, were stimulated with ConA (0.5 µg/ml; Sigma-Aldrich) and IL-2 (200 U/ml) for 48 h. Then cells were harvested and after staining with the fluorochrome-conjugated antibodies anti-CD4 FITC and anti-CD8 APC/Fire 750 (BioLegend) were fixed and permeabilized for intracellular staining of Tbet, FoxP3 and IFNγ with the following antibodies: anti-Tbet PE-Cy7, anti-FoxP3 APC, anti-IFN-γ PE (eBioscience). Flow cytometry analyses

were performed using FACS Fortessa with FACSDiva software (Becton Dickinson).

#### Statistics

All the data were analyzed using GraphPad Prism 6 software. The cytometric data presentations were prepared using NovoExpress software. The statistical significance in kinetics of tumor growth was calculated using the Friedman test followed by Dunn's multiple comparison post-hoc test. In all remaining analyses the statistical differences were calculated using the nonparametric Kruskal-Wallis test for multiple independent groups followed by Dunn's multiple comparison post-hoc test. Differences with a p-value <0.05 were regarded as significant.

# RESULTS

#### Isolation and Characteristics of TEx and TMv Released by Genetically Modified MC38 Cells

Tumor-derived EVs with modified content (mTEx and mTMv) were obtained from the murine colon carcinoma MC38 cell line with silenced expression of murine TGF-β1 and/or with overexpression of murine IL-12. The MC38/IL12 and MC38/shTGFβ1 cell lines were established by transduction with lentiviral vectors encoding il12 genes or shRNA for TGF-β1 followed by geneticin or puromycin antibiotic selection, respectively. The MC38/IL12shTGFβ1 cell line was obtained by double transduction followed by selection in the presence of both antibiotics. The quality of the transduction process was monitored using real-time PCR and ELISA (**Figures 1A,B**). In order to stimulate TEx and TMv production by genetically modified or wild-type MC38 cell lines, the cells were cultured in hypoxic conditions for 48 h. To confirm that genetically engineered cells retain their properties in hypoxia we also monitored the IL-12 and TGF-β1 expression after culture of the cells in hypoxic conditions for 48 h (**Figures 1C,D**). Isolation of TEx and TMv from the culture supernatant was carried out according to the scheme presented in **Figure 2A**. After the final washing in PBS the sample from each isolated fraction was collected for further qualitative and quantitative analyses. The quantitative analysis was performed using the flow cytometric method. For this purpose, the samples from TEx and TMv fractions were stained with CFSE dye, fluorescence of which depends on the esterase activity inside the cells or, as in this case, inside particles. This method makes it possible to distinguish the TEx and TMv from flow cytometer and PBS-derived debris during cytometric analysis. The analysis was performed in the presence of counting beads to count the total number of CFSElabeled TEx or TMv in suspension and reference 1 µm beads to show the approximate size of isolated particles. Representative density plots showing the example of cytometric analysis of TEx and TMv isolated from wild-type MC38 cells are presented in

FIGURE 2 | presented for the example of particles isolated from unmodified MC38 cells. TEM analysis of TEx (C,E) and TMv (D,F) counterstained with uranyl acetate (C,D) or with methylcellulose (E,F). Magnification 100,000x. (G,H) Representative histograms showing the measurement of MC38-derived TEx and TMv particle size distribution using the DLS Zetasizer (Malvern). (I) WB analysis of CD81, CD9, TSG101, GM130, and calnexin in lysates from MC38 cell lines, TEx and TMv fractions. (J,K,N,O) Relative expression of mRNA for IL-12 or TGF-β1 in TEx and TMv isolated from wild-type or genetically modified MC38 cell lines. (L,M,P,Q) Concentration of IL-12 and TGF-β1 in lysates prepared from TEx and TMv isolated from wild-type or genetically modified MC38 cell lines measured using the ELISA. The results are given as the mean ± SD calculated for at least two repeats in two independent experiments. The differences between the groups were estimated using the nonparametric Kruskal-Wallis test followed by Dunn's multiple comparison test (\**p* < 0.05, \*\**p* < 0.01).

**Figure 2B**. All presented plots represent the result of acquiring 1,000 count beads per sample. Thus, the relation between control plots showing beads and debris and plots showing TEx or TMv is actual. The differences in the number of EVs secreted by the wild-type and genetically modified MC38 cells are presented in **Supplementary Figure 1**. However, it should be stressed that the final number of TEx in preparation may depend on differences in the TEx release as well as differences between proliferation rate of particular cell lines. The quality and size of particles obtained after isolation were measured using TEM and the DLS Zetasizer. The visualization of the EVs with TEM was performed using two protocols of counterstaining: the first with uranyl acetate (**Figures 2C,D**) and second with methylcellulose (**Figures 2E,F**). Both of them showed that the TMv fractions are contaminated by TEx, while the TEx fractions seems to be pure. The TEM analysis showed that the approximate size of TEx is 100 nm, whereas the size of TMv is >200 nm. Histograms presented in **Figures 2G,H** show differences in a size distribution, measured by DLS, between the TMv fraction obtained after centrifugation at 10,000 × g for 30 min and the TEx fraction achieved after ultracentrifugation at 100,000 × g for 60 min. Using this method we confirmed that the TMv fraction (mean size: 399 nm) was partially contaminated with small TEx particles (mean size: 123 nm), while the TEx fraction was more homogeneous and its mean size was 186 nm. We noted that the size of particles differs when measured by TEM and DLS. The TEM analysis revealed that there are a lot of aggregates in TEx or TMv suspensions. Thus, it seems probable that DLS measurements, which show the mean size of particles, are overstated due to the presence of aggregates in the suspension. There were no significant differences in size distribution between fractions from wild-type and genetically modified cells, thus only representative histograms for TEx and TMv isolated from the wild-type MC38 cell line were presented. In the next step we evaluated the expression of specific markers for exosomes by western blotting and flow cytometry. The western blotting method was used for analysis of both proteins specific for exosomes (CD9, CD81, and TSG101) and cell organelle proteins that exosomes should not contain (GM130 and calnexin). The comparative analysis of lysates from MC38 cell lines, TEx and TMv fractions revealed that GM130 and calnexin were not visible in the TEx-derived lysates, while TSG101, CD81, and CD9 were enriched in the TEx and TMv fractions (**Figure 2I**). The results indicate that TEx fractions were not contaminated with TMv or cell debris. Moreover, expression of CD81 and CD9 in the TMv fractions confirmed our previous observations showing contamination of TMv with TEx. Unfortunately, the comparative analysis of the TEx marker expression between particular cell lines were not possible, because we were not able to determine the precise concentration of total protein in the TEx fractions. We observed that TEx fractions were considerably contaminated with FBSderived proteins like albumin and the final protein level in a sample was very high, although the concentration of TEx-derived proteins was certainly much lower. For this reason, we decided to perform additional analysis of expression of TEx specific markers using flow cytometry. The analysis showed that the expression of CD63, CD9, and CD81 on the TEx is higher than in TMv. We also noted the difference in expression of these molecules between TEx isolated from genetically modified and wild-type MC38 cells (**Supplementary Figure 2**).

Further studies demonstrated the changes in the content of TEx and TMv following genetic modifications of the MC38 cell line. We observed increased expression of IL-12, measured as mRNA and protein level, in TEx and TMv isolated from cells transduced with lentivectors encoding murine il12 genes (**Figures 2J,L,N,P**), as well as diminished expression of mRNA and protein for TGF-β1 both in TEx and TMv isolated from MC38 cells with silenced expression of TGF-β1 (**Figures 2K,M,O,Q**). The obtained results indicate that the content of particles produced by tumor cells may be effectively changed following genetic modification of wild-type tumor cells.

#### The Influence of mTEx and mTMv on Bone Marrow-Derived Dendritic Cell Activity

After 24 h stimulation with mTEx or mTMv the changes in the phenotype of dendritic cells as well as their effectiveness in primary activation of naïve T cells isolated from spleen were evaluated. The results were related to the phenotype and activity of unstimulated DCs, DCs stimulated with lysate prepared by repeated freezing and thawing of wild-type MC38 cells (DC/TAg) or DCs stimulated with TEx or TMv isolated from wild-type MC38 cells (DC/TEx MC38 or DC/TMv MC38). The phenotype analysis showed that all types of TEx strongly affected the maturation of DCs (**Figures 3A,B**). Although after stimulation with TAg, TEx MC38 cells, and mTEx we observed a significant decrease in MHC II expression, the CD40, CD80, and CD86 co-stimulatory molecule expression on the DCs stimulated with TEx MC38 or mTEx considerably increased compared to DC or DC/TAg. The highest maturity stage was shown by DCs stimulated with TEx MC38/shTGFβ1 or with TEx MC38/IL12. Interestingly, TEx isolated from wild-type cells and TEx MC38/IL12shTGFβ1 induced similar phenotypical changes in DCs. Further analyses showed that DCs stimulated with TEx from unmodified MC38 cells or mTEx were more potent to activate cytotoxicity of T lymphocytes and NK cells