Prognostic roles for IL‐2‐producing and CD69+ T cell subsets in colorectal cancer patients

Tumor infiltrating T cells are a predictor of patient outcome in patients with colorectal cancer (CRC). However, many T cell populations have been associated with both poor and positive patient prognoses, indicating a need to further understand the role of different T cell subsets in CRC. In this study, the T cell infiltrate from the tumor and nontumor bowel (NTB) was examined in 95 CRC patients using flow cytometry and associations with cancer stage and disease recurrence made. Our findings showed that IFN‐γ‐producing T cells were associated with positive patient outcomes, and CD69⁺ T cells were associated with disease recurrence. Inflammatory (IL‐17) and regulatory T cells were not associated with disease recurrence. Surprisingly, in a second cohort of 32 patients with long‐term clinical follow up data, tumor infiltrating IL‐2‐producing T cells correlated negatively with disease free survival (DFS) and a higher frequency of IL‐2‐producing T cells was found in the NTB of patients with poorly differentiated tumors. These results point toward the possibility of a negative impact of IL‐2 in tumor immune responses, which may influence future immunotherapy treatments in CRC patients.     

Tumor‐reactive CD4+CD8αβ+ CD103+ αβT cells: A prevalent tumor‐reactive T‐cell subset in metastatic colorectal cancers

High level of T‐cell infiltration in colorectal carcinomas (CRCs) is a good prognostic indicator, but the tumor reactivity of this infiltrate (tumor infiltrating lymphocytes [TIL]) is poorly documented. This study examined the presence, phenotype and functional features of tumor‐reactive lymphocytes in human CRC. Freshly dissociated TIL and T cell lines were isolated from CRC samples and from some paired normal colonic mucosa. Four tumor cell lines were obtained. Autologous tumor reactivity of CRC TIL and tumor‐reactive cell features were analyzed. We demonstrate the presence among CRC TIL of variable fractions (up to 18%) of double positive CD4⁺CD8αβ⁺ (DP) αβ T cells. Interestingly, a high proportion (16–20%) of this TIL subset displayed tumor reactivity, whilst this was the case for no or few single positive TIL. Low levels of DP TIL were found in most CRC samples and in normal colonic mucosa, but these cells were higher in metastatic CRC. Furthermore, we showed that DP TIL were polyclonal, restricted by HLA class‐I, proliferated poorly and secreted higher amounts of IL‐4 and IL‐13 than single positive T cells, on cognate or CD3 stimulation. DP CRC TIL also expressed CD103, confirming their mucosal origin. Increased frequencies of tumor‐reactive DP TIL in metastatic CRC suggest that these cells play a role in the metastatic process of this cancer. Based on their high secretion of IL‐4 and IL‐13 and on previously described roles of these cytokines in cancers, we postulate that DP TIL could favor CRC growth or metastasis and/or downmodulate immune responses to these tumors.     

Cancer-associated fibroblasts promote PD-L1 expression in mice cancer cells via secreting CXCL5

Cancer-associated fibroblasts (CAFs) play a key role in orchestrating the tumor malignant biological properties within tumor microenvironment and evidences demonstrate that CAFs are a critical regulator of tumoral immunosuppression of the T cell response. However, the functions and regulation of CAFs in the expression of programmed death-ligand 1 (PD-L1) in melanoma and colorectal carcinoma (CRC) are not completely understood. Herein, by scrutinizing the expression of α-SMA and PD-L1 in melanoma and CRC tissues, we found that CAFs was positive correlated with PD-L1 expression. Further analyses showed that CAFs promoted PD-L1 expression in mice tumor cells. By detecting a majority of cytokines expression in normal mice fibroblasts and CAFs, we determined that CXCL5 was abnormal high expression in CAFs and the immunohistochemistry and in situ hybridization confirmed that were CAFs which were expressing CXCL5. In addition, CXCL5 promoted PD-L1 expression in B16, CT26, A375 and HCT116. The silencing of CXCR2, the receptor of CXCL5, inhibited the PD-L1 expression induced by CAFs in turn. Functionally, CXCL5 derived by CAFs promoted PD-L1 expression in mice tumor cells through activating PI3K/AKT signaling. LY294002, the inhibitor of PI3K, confirmed that CXCL5 forested an immunosuppression microenvironment by promoting PD-L1 expression via PI3K/AKT signaling. Meanwhile, the B16/CT26 xenograft tumor models were used and both CXCR2 and p-AKT were found to be positively correlated with PD-L1 in the xenograft tumor tissues. The immunosuppressive action of CAFs on tumor cells is probably reflective of them being a potential therapeutic biomarker for melanoma and CRC.     

IL‐33 promotes growth and liver metastasis of colorectal cancer in mice by remodeling the tumor microenvironment and inducing angiogenesis

Liver metastasis is the major cause of death from colorectal cancer (CRC). Understanding its mechanisms is necessary for timely diagnosis and development of effective therapies. Interleukin‐33 (IL‐33) is an IL‐1 cytokine family member that uniquely functions as a cytokine and nuclear factor. It is released by necrotic epithelial cells and activated innate immune cells, functioning as an alarmin or an early danger signal. Its role in invoking type 2 immune response has been established; however, it has contrasting roles in tumor development and metastasis. We identified IL‐33 as a potently upregulated cytokine in a highly metastatic murine CRC cell line and examined its role in tumor growth and metastasis to the liver. IL‐33 was transgenically expressed in murine and human adenocarcinoma and carcinoma cell lines and their growth and spontaneous metastasis to the liver were assessed in orthotopic models of CRC in wild‐type C57Bl/6 and Il33 knockout mice. The results showed that increased expression of IL‐33 in CRC cells enhanced their tumor take, growth, and liver metastasis. Tumor‐ rather than host‐derived IL‐33 induced the enhanced recruitment of CD11b⁺ GR1⁺ and CD11b⁺F4/80⁺ myeloid cells to remodel the tumor microenvironment by increased expression of mobilizing cytokines, and tumor angiogenesis by activating endothelial cells. IL‐33 expression was elevated in patient tumor tissues, induced early in adenoma development, and activated by pro‐inflammatory cytokines derived from the tumor microenvironment. The data suggest that tumor‐derived IL‐33 modulates the tumor microenvironment to potently promote colon carcinogenesis and liver metastasis, underscoring its potential as a therapeutic target. © 2016 Wiley Periodicals, Inc.     

IL‐17A‐producing CD30+ Vδ1 T cells drive inflammation‐induced cancer progression

Although it has been suspected that inflammation is associated with increased tumor metastasis, the exact type of immune response required to initiate cancer progression and metastasis remains unknown. In this study, by using an in vivo tumor progression model in which low tumorigenic cancer cells acquire malignant metastatic phenotype after exposure to inflammation, we found that IL‐17A is a critical cue for escalating cancer cell malignancy. We further demonstrated that the length of exposure to an inflammatory microenvironment could be associated with acquiring greater tumorigenicity and that IL‐17A was critical for amplifying such local inflammation, as observed in the production of IL‐1β and neutrophil infiltration following the cross‐talk between cancer and host stromal cells. We further determined that γδT cells expressing Vδ1 semi‐invariant TCR initiate cancer‐promoting inflammation by producing IL‐17A in an MyD88/IL‐23‐dependent manner. Finally, we identified CD30 as a key molecule in the inflammatory function of Vδ1T cells and the blockade of this pathway targeted this cancer immune‐escalation process. Collectively, these results reveal the importance of IL‐17A‐producing CD30⁺ Vδ1T cells in triggering inflammation and orchestrating a microenvironment leading to cancer progression.     

Long‐term prognostic significance of interleukin‐17‐producing T cells in patients with non‐small cell lung cancer

The presence of interleukin (IL)‐17‐producing T cells has recently been reported in non‐small cell lung cancer (NSCLC) patients. However, the long‐term prognostic significance of these populations in NSCLC patients remains unknown. In the present study, we collected peripheral blood from 82 NSCLC patients and 22 normal healthy donors (NC). Percentages of IL‐17‐producing CD4⁺T (Th17), CD8⁺T (Tc17) and γδT cells (γδT17) were measured to determine their association with clinical outcomes and overall survival (OS) in NSCLC. All NSCLC patients were followed up until July 2018. Median follow‐up time was 13.5 months (range 1‐87 months). The 3‐ and 5‐year survival rate was 27% and 19.6%, respectively. We found that Th17 cells and γδT17 cells were significantly increased, whereas Tc17 cells were markedly decreased in patients with NSCLC compared with those in NC. In addition, Th17 cells were significantly positively associated with T helper type 1 cells (Th1), whereas γδT17 cells were significantly negatively associated with γδT ⁺ interferon (IFN)‐γ⁺ cells. High percentages of peripheral Tc17 cells were significantly associated with favorable 5‐year OS (P = .025), especially in patients with early TNM stage (P = .016). Furthermore, high percentages of peripheral Th17 cells were positively associated with favorable 5‐year OS in patients with late TNM stage (P = .002). However, no significant association was observed between γδT17 cells and OS, regardless of the TNM stage. In conclusion, our findings suggest that enhanced Th17 and reduced Tc17 cells in the peripheral blood could be a significant predictor of a favorable prognosis for NSCLC patients.     

Lack of interleukin‐6 in the tumor microenvironment augments type‐1 immunity and increases the efficacy of cancer immunotherapy

Conquering immunosuppression in tumor microenvironments is crucial for effective cancer immunotherapy. It is well known that interleukin (IL)‐6, a pleiotropic cytokine, is produced in the tumor‐bearing state. In the present study, we investigated the precise effects of IL‐6 on antitumor immunity and the subsequent tumorigenesis in tumor‐bearing hosts. CT26 cells, a murine colon cancer cell line, were intradermally injected into wild‐type and IL‐6‐deficient mice. As a result, we found that tumor growth was decreased significantly in IL‐6‐deficient mice compared with wild‐type mice and the reduction was abrogated by depletion of CD8⁺ T cells. We further evaluated the immune status of tumor microenvironments and confirmed that mature dendritic cells, helper T cells and cytotoxic T cells were highly accumulated in tumor sites under the IL‐6‐deficient condition. In addition, higher numbers of interferon (IFN)‐γ‐producing T cells were present in the tumor tissues of IL‐6‐deficient mice compared with wild‐type mice. Surface expression levels of programmed death‐ligand 1 (PD‐L1) and MHC class I on CT26 cells were enhanced under the IL‐6‐deficient condition in vivo and by IFN‐γ stimulation in vitro. Finally, we confirmed that in vivo injection of an anti‐PD‐L1 antibody or a Toll‐like receptor 3 ligand, polyinosinic‐polycytidylic acid, effectively inhibited tumorigenesis under the IL‐6‐deficient condition. Based on these findings, we speculate that a lack of IL‐6 produced in tumor‐bearing host augments induction of antitumor effector T cells and inhibits tumorigenesis in vivo, suggesting that IL‐6 signaling may be a promising target for the development of effective cancer immunotherapies.     

Multipotent mesenchymal stromal cells promote tumor growth in distinct colorectal cancer cells by a β1‐integrin‐dependent mechanism

Tumor–stroma interactions play an essential role in the biology of colorectal carcinoma (CRC). Multipotent mesenchymal stromal cells (MSC) may represent a pivotal part of the stroma in CRC, but little is known about the specific interaction of MSC with CRC cells derived from tumors with different mutational background. In previous studies we observed that MSC promote the xenograft growth of the CRC cell‐line DLD1. In the present study, we aimed to analyze the mechanisms of MSC‐promoted tumor growth using various in vitro and in vivo experimental models and CRC cells of different mutational status. MSC specifically interacted with distinct CRC cells and supported tumor seeding in xenografts. The MSC–CRC interaction facilitated three‐dimensional spheroid formation in CRC cells with dysfunctional E‐cadherin system. Stable knock‐downs revealed that the MSC‐facilitated spheroid formation depended on β1‐integrin in CRC cells. Specifically in α‐catenin‐deficient CRC cells this β1‐integrin‐dependent interaction resulted in a MSC‐mediated promotion of early tumor growth in vivo. Collagen I and other extracellular matrix compounds were pivotal for the functional MSC–CRC interaction. In conclusion, our data demonstrate a differential interaction of MSC with CRC cells of different mutational background. Our study is the first to show that MSC specifically compared to normal fibroblasts impact early xenograft growth of distinct α‐catenin deficient CRC cells possibly through secretion of extracellular matrix. This mechanism could serve as a future target for therapy and metastasis prevention.     

Inhibition of MK2 suppresses IL‐1β, IL‐6, and TNF‐α‐dependent colorectal cancer growth

Colorectal cancer (CRC) development and progression is associated with chronic inflammation. We have identified the MAPK‐activated protein kinase 2 (MK2) pathway as a primary mediator of inflammation in CRC. MK2 signaling promotes production of proinflammatory cytokines IL‐1β, IL‐6 and TNF‐α. These cytokines have been implicated in tumor growth, invasion and metastasis. For the first time, we investigate whether MK2 inhibition can improve outcome in two mouse models of CRC. In our azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis‐associated CRC, MK2 inhibitor treatment eliminated murine tumor development. Using the implanted, syngeneic murine CRC cell line CT26, we observe significant tumor volume reduction following MK2 inhibition. Tumor cells treated with MK2 inhibitors produced 80% less IL‐1β, IL‐6 and TNF‐α and demonstrated decreased invasion. Replenishment of downstream proinflammatory MK2‐mediated cytokines (IL‐1β, IL‐6 and TNF‐α) to tumors led to restoration of tumor proliferation and rapid tumor regrowth. These results demonstrate the importance of MK2 in driving proinflammatory cytokine production, its relevance to in vivo tumor proliferation and invasion. Inhibition of MK2 may represent an attractive therapeutic target to suppress tumor growth and progression in patients.     

Cancer‐associated fibroblast‐targeted strategy enhances antitumor immune responses in dendritic cell‐based vaccine

Given the close interaction between tumor cells and stromal cells in the tumor microenvironment (TME), TME‐targeted strategies would be promising for developing integrated cancer immunotherapy. Cancer‐associated fibroblasts (CAFs) are the dominant stromal component, playing critical roles in generation of the pro‐tumorigenic TME. We focused on the immunosuppressive trait of CAFs, and systematically explored the alteration of tumor‐associated immune responses by CAF‐targeted therapy. C57BL/6 mice s.c. bearing syngeneic E.G7 lymphoma, LLC1 Lewis lung cancer, or B16F1 melanoma were treated with an anti‐fibrotic agent, tranilast, to inhibit CAF function. The infiltration of immune suppressor cell types, including regulatory T cells and myeloid‐derived suppressor cells, in the TME was effectively decreased through reduction of stromal cell‐derived factor‐1, prostaglandin E₂, and transforming growth factor‐β. In tumor‐draining lymph nodes, these immune suppressor cell types were significantly decreased, leading to activation of tumor‐associated antigen‐specific CD8⁺ T cells. In addition, CAF‐targeted therapy synergistically enhanced multiple types of systemic antitumor immune responses such as the cytotoxic CD8⁺ T cell response, natural killer activity, and antitumor humoral immunity in combination with dendritic cell‐based vaccines; however, the suppressive effect on tumor growth was not observed in tumor‐bearing SCID mice. These data indicate that systemic antitumor immune responses by various immunologic cell types are required to bring out the efficacy of CAF‐targeted therapy, and these effects are enhanced when combined with effector‐stimulatory immunotherapy such as dendritic cell‐based vaccines. Our mouse model provides a novel rationale with TME‐targeted strategy for the development of cell‐based cancer immunotherapy.     

Distribution of Th17 cells and FoxP3(+) regulatory T cells in tumor‐infiltrating lymphocytes,tumor‐draining lymph nodes and peripheral blood lymphocytes in patients with gastric cancer

Although Th17 cells reportedly play critical roles in the development of autoimmunity and allergic reactions, information on Th17 cells in cancer‐bearing hosts is still limited. In the present study, we investigated the distribution of Th17 cells in relation to regulatory T cells (Treg) in the tumor‐infiltrating lymphocytes (TILs), regional lymph node lymphocytes, and peripheral blood lymphocytes of gastric cancer patients. Interleukin (IL)‐17‐producing CD4(+) cells as Th17 cells and CD4(+)CD25(+)FoxP3(+) cells as Treg were evaluated by flow cytometry and expressed as a percentage of the total CD4⁺ cells, in addition to performing a Th1/Th2 balance assay. Moreover, immunohistochemical staining for IL‐17 and FoxP3 were performed. In TILs from patients with early disease (n = 27), the frequency of Th17 cells was significantly higher than that in the normal gastric mucosa (23.7 ± 8.9 vs 4.5 ± 3.1%). In TILs from patients with advanced disease (n = 28), the frequency of Th17 cells was also significantly higher, but lower compared to early disease, than that in the normal gastric mucosa (15.1 ± 6.2 vs 4.0 ± 2.0%). This observation for Th17 cell‐distribution was also confirmed by immunohistochemistry. When the ratio of Th17/Treg in TILs was evaluated in individual cases, it was more markedly increased in early than in advanced disease. In conclusion, the accumulation of Th17 cells as well as Treg in the tumor microenvironment of gastric cancer occurred in early disease and then the infiltration of Th17 cells gradually decreased according to the disease progression, in contrast to increased Treg. (Cancer Sci 2010; 00: 00–00)     

NPPB is a novel candidate biomarker expressed by cancer‐associated fibroblasts in epithelial ovarian cancer

Most solid tumors contain cancer‐associated fibroblasts (CAFs) that support tumorigenesis and malignant progression. However, the cellular origins of CAFs in epithelial ovarian cancers (EOCs) remain poorly understood, and their utility as a source of clinical biomarkers for cancer diagnosis has not been explored in great depth. Here, we report establishing in vitro and in vivo models of CAFs in ovarian cancer development. Normal ovarian fibroblasts and mesenchymal stem cells cultured in the presence of EOC cells acquired a CAF‐like phenotype, and promoted EOC cell migration in vitro. CAFs also promoted ovarian cancer growth in vivo in both subcutaneous and intraperitoneal murine xenograft assays. Molecular profiling of CAFs identified gene expression signatures that were highly enriched for extracellular and secreted proteins. We identified novel candidate CAF‐specific biomarkers for ovarian cancer including NPPB, which was expressed in the stroma of 60% primary ovarian cancer tissues (n = 145) but not in the stroma of normal ovaries (n = 4). NPPB is a secreted protein that was also elevated in the blood of 50% of women with ovarian cancer (n = 8). Taken together, these data suggest that the tumor stroma is a novel source of biomarkers, including NPPB, that may be of clinical utility for detection of EOC.     

Blockade of TGF‐β enhances tumor vaccine efficacy mediated by CD8+ T cells

Though TGF‐β inhibition enhances antitumor immunity mediated by CD8⁺ T cells in several tumor models, it is not always sufficient for rejection of tumors. In this study, to maximize the antitumor effect of TGF‐β blockade, we tested the effect of anti‐TGF‐β combined with an irradiated tumor vaccine in a subcutaneous CT26 colon carcinoma tumor model. The irradiated tumor cell vaccine alone in prophylactic setting significantly delayed tumor growth, whereas anti‐TGF‐β antibodies alone did not show any antitumor effect. However, tumor growth was inhibited significantly more in vaccinated mice treated with anti‐TGF‐β antibodies compared to vaccinated mice without anti‐TGF‐β, suggesting that anti‐TGF‐β synergistically enhanced irradiated tumor vaccine efficacy. CD8⁺ T‐cell depletion completely abrogated the vaccine efficacy, and so protection required CD8⁺ T cells. Depletion of CD25⁺ T regulatory cells led to the almost complete rejection of tumors without the vaccine, whereas anti‐TGF‐β did not change the number of CD25⁺ T regulatory cells in unvaccinated and vaccinated mice. Though the abrogation of CD1d‐restricted NKT cells, which have been reported to induce TGF‐β production by MDSC through an IL‐13‐IL‐4R‐STAT6 pathway, partially enhanced antitumor immunity regardless of vaccination, abrogation of the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway did not enhance vaccine efficacy. Taken together, these data indicated that anti‐TGF‐β enhances efficacy of a prophylactic vaccine in normal individuals despite their not having the elevated TGF‐β levels found in patients with cancer and that the effect is not dependent on TGF‐β solely from CD4⁺CD25⁺ T regulatory cells or the NKT cell‐IL‐13‐IL‐4R‐STAT‐6 immunoregulatory pathway.     

IL17 producing γδT cells induce angiogenesis and are associated with poor survival in gallbladder cancer patients

Despite conventional treatment modalities, gallbladder cancer (GBC) remains a highly lethal malignancy. Prognostic biomarkers and effective adjuvant immunotherapy for GBC are not available. In the recent past, immunotherapeutic approaches targeting tumor associated inflammation have gained importance but the mediators of inflammatory circuit remain unexplored in GBC patients. In the current prospective study, we investigated the role of IL17 producing TCRγδ⁺ (Tγδ17), CD4⁺ (Th17), CD8⁺ (Tc17) and regulatory T cells (Tregs) in pathogenesis of GBC. Analysis by multi‐color flow cytometry revealed that compared to healthy individuals (HI), Tγδ17, Th17 and Tc17 cells were increased in peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TIL) of GBC patients. Tregs were decreased in PBMCs but increased in TILs of GBC patients. The suppressive potential of Tregs from GBC patients and HI were comparable. Serum cytokines profile of GBC patients showed elevated levels of cytokines (IL6, IL23 and IL1β) required for polarization and/or stabilization of IL17 producing cells. We demonstrated that Tγδ17 cells migrate toward tumor bed using CXCL9‐CXCR3 axis. IL17 secreted by Tγδ17 induced productions of vascular endothelial growth factor and other angiogenesis related factors in GBC cells. Tγδ17 cells promote vasculogenesis as studied by chick chorioallantoic membrane assay. Survival analysis showed that Tγδ17, Th17 and Treg cells in peripheral blood were associated with poor survival of GBC patients. Our findings suggest that Tγδ17 is a protumorigenic subtype of γδT cells which induces angiogenesis. Tγδ17 may be considered as a predictive biomarker in GBC thus opening avenues for targeted therapies.     

Up‐regulation of interleukin‐17 expression by human papillomavirus type 16 E6 in nonsmall cell lung cancer

BACKGROUND:

Human papillomavirus (HPV) 16/18 infection is associated with nonsmoking lung cancer. In this study, the authors investigated a putative correlation between interleukin (IL)‐17 expression and HPV infection in clinical nonsmall cell lung cancer (NSCLC) tissues and examined the effects of HPV infection on a human NSCLC cell line.

METHODS:

IL‐17 expression was investigated in 79 NSCLC tumor tissues by immunohistochemistry. Growth rate, IL‐17 mRNA, and secreting protein levels were also examined in HPV 16/18 E6‐transfected H1299 human NSCLC cells.

RESULTS:

Immunohistochemical data showed that 48.1% of lung tumors had IL‐17 staining, which was significantly associated with patients' sex (P = .03), HPV infection (P = .002), and tumor stage (P = .03). Significant correlations of IL‐17 with IL‐6 (P < .001) and IL‐17 with Mcl‐1 (P < .001) expression were also observed. Cell growth rate was increased, and IL‐17/Mcl‐1 expression levels were elevated in HPV 16 E6‐transfected H1299 cells. The transfected E6 oncoproteins can significantly up‐regulate expression levels of IL‐17 and antiapoptotic protein Mcl‐1.

CONCLUSIONS:

The study suggests that HPV infection‐induced IL‐17 levels can stimulate Mcl‐1 expression through the PI3K pathway and promote lung tumor cell progression through a p53‐ and IL‐6‐independent pathway. Cancer 2010. © 2010 American Cancer Society.     

Targeting tumor‐infiltrating Ly6G+ myeloid cells improves sorafenib efficacy in mouse orthotopic hepatocellular carcinoma

Sorafenib, a multikinase inhibitor with antiangiogenic activity, is an approved therapy for hepatocellular carcinoma (HCC). It is unclear whether the proinflammatory and immunosuppressive mechanisms may limit the therapeutic efficacy of sorafenib in HCC. We used a syngeneic mouse liver cancer cell line to establish orthotopic liver or subcutaneous tumors to study how proinflammatory and immunosuppressive mechanisms impact on the efficacy of sorafenib. We found sorafenib exhibited a potent therapeutic effect in subcutaneous tumors, but a less potent effect in orthotopic liver tumors. The protein levels of interleukin‐6 (IL‐6) and vascular endothelial growth factor A (VEGF‐A) were persistently elevated in orthotopic liver tumors, but not in subcutaneous tumors, treated with sorafenib. Likewise, the tumor‐infiltrating Ly6G⁺ myeloid‐derived suppressor cells (MDSCs) and immune suppressors were increased in orthotopic liver tumors, not in subcutaneous tumors, treated with sorafenib. The tumor‐infiltrating Ly6G⁺ MDSCs of sorafenib‐treated orthotopic liver tumors significantly induced IL‐10 and TGF‐β expressing CD4⁺ T cells, and downregulated the cytotoxic activity of CD8⁺ T cells. IL‐6, but not VEGF‐A, protected Ly6G⁺ MDSCs from sorafenib‐induced cell death in vitro. The combination of anti‐Ly6G antibody or anti‐IL‐6 antibody with sorafenib significantly reduced the cell proportion of Ly6G⁺ MDSCs in orthotopic liver tumors, enhanced the T cells proliferation and improved the therapeutic effect of sorafenib synergistically. Modulating tumor microenvironment through targeting tumor‐infiltrating Ly6G⁺MDSCs represents a potential strategy to improve the anti‐HCC efficacy of sorafenib.     

Involvement of local renin‐angiotensin system in immunosuppression of tumor microenvironment

To improve current cancer immunotherapies, strategies to modulate various immunosuppressive cells including myeloid derived suppressor cells (MDSC) which were shown to be negative factors in immune‐checkpoint blockade therapy, need to be developed. In the present study, we evaluated the role of the local renin‐angiotensin system (RAS) in the tumor immune‐microenvironment using murine models bearing tumor cell lines in which RAS was not involved in their proliferation and angiogenetic ability. Giving angiotensin II receptor blockers (ARB) to C57BL/6 mice bearing murine colon cancer cell line MC38 resulted in significant enhancement of tumor antigen gp70 specific T cells. ARB administration did not change the numbers of CD11b⁺ myeloid cells in tumors, but significantly reduced their T‐cell inhibitory ability along with decreased production of various immunosuppressive factors including interleukin (IL)‐6, IL‐10, vascular endothelial growth factor (VEGF), and arginase by CD11b⁺ cells in tumors. ARB also decreased expression of immunosuppressive factors such as chemokine ligand 12 and nitric oxide synthase 2 in cancer‐associated fibroblasts (CAF). Last, combination of ARB and anti‐programmed death‐ligand 1 (PD‐L1) antibodies resulted in significant augmentation of anti‐tumor effects in a CD8⁺ T cell‐dependent way. These results showed that RAS is involved in the generation of an immunosuppressive tumor microenvironment caused by myeloid cells and fibroblasts, other than the previously shown proliferative and angiogenetic properties of cancer cells and macrophages, and that ARB can transform the immunosuppressive properties of MDSC and CAF and could be used in combination with PD‐1/PD‐L1 immune‐checkpoint blockade therapy.