CRP and soluble CTLA4 are determinants of anti-PD1 resistance in gastrointestinal cancer.

Targeting immune inhibitory checkpoint (IC) pathways have attracted great attention as a promising strategy for treating gastrointestinal (GI) cancer. However, the therapeutic efficacy is low in most cases, and little progress has been made in establishing biomarkers that predict the possible responses, and combination regimens that enhance the therapeutic efficacy. As a predictive biomarker, soluble forms of IC molecules have been recently highlighted. However, little is known about which IC molecules is most critically associated with the treatment resistance, and also about the biological and immunological roles of the IC molecules in GI cancer. In this study, we analyzed sera obtained from advanced gastric cancer patients before and one month after treatment with anti-PD1 nivolumab for soluble IC molecules by ELISA. We found that decrease of soluble CTLA4 (sCTLA4) at posttreatment were significantly associated with a better prognosis, and combination with low level of CRP at posttreatment more clearly defined anti-PD1 responders with long-term survival. Indeed, in the in vitro setting, CRP stimulation upregulated CTLA4 expression in tumor cells followed by generation of sCTLA4 that suppressed CTL induction, and simultaneously conferred high self-renewal and invasive abilities on the tumor cells accompanied by increase of EMT-related gene expressions. In the in vivo setting, CRP injection elevated sCTLA4 level in sera of mouse tumor metastasis models, leading to failure of anti-PD1 therapy. However, treatment with anti-CTLA4 mAb or a PPARγ agonist that can reduce in vivo CRP successfully elicited anti-tumor efficacy in the anti-PD1 resistant models. These suggest that targeting CRP and sCTLA4 may be a promising strategy for improving clinical outcomes in the treatments, including anti-PD1 therapy, of GI cancer.

IL25+ macrophages are a key determinant of treatment resistance of IL17RB+ breast cancer.

Recurrence and metastasis are resistant to multimodal treatments, and are the major causes of death in breast cancer. Accumulating evidence suggests that the IL17RB signaling pathway plays a key role in progression and metastasis of breast cancer. Clinical significance of the IL17RB positivity in tumor tissues has been also reported as a poor prognostic factor in breast cancer. However, the molecular mechanisms underlying the poor prognosis of patients with IL17RB+ breast cancer, particularly the immunological aspects, remain to be fully elucidated, and elimination of the IL17RB+ tumors has not been practically achieved in clinical settings. In this study, we identified a distinct molecular mechanism underlying the intractability of the IL17RB+ tumors through tumor biological and immunological investigation using mouse and human breast cancer cells transduced with il17rb gene. IL17RB overexpression in tumor cells confers cancer stemness, including high invasive and self-renewal abilities, and high resistance to CDK4/6 inhibitors that have been considered as a promising agent for treating breast cancer despite the limited efficacy. In the mice implanted with the IL17RB+ tumors, IL25+ macrophages (Møs) are expanded locally in tumor tissues and systemically in spleen, and promote the IL17RB+ tumor progression directly by intensifying the tumor functions, and indirectly via impairment of anti-tumor effector CTLs and NK cells utilizing the secreted IL25. Blocking IL25 with the specific mAb, however, interferes the adverse events, and successfully elicits significant anti-tumor efficacy in combination with CDK4/6 inhibitors providing better survival in murine mammary tumor models. These results suggest that the IL25+ Mø is a key determinant of building the solid treatment resistance of the IL17RB+ breast cancer. Targeting the IL17RB-IL25 axis may be a promising strategy to improve clinical outcomes in the treatment of breast cancer patients, particularly with IL17RB+ tumors.

Targeting myeloid villains in the treatment with immune checkpoint inhibitors in gastrointestinal cancer.

Despite the clinical outcomes being extremely limited, blocking immune inhibitory checkpoint pathways has been in the spotlight as a promising strategy for treating gastrointestinal cancer. However, a distinct strategy for the successful treatment is obviously needed in the clinical settings. Myeloid cells, such as neutrophils, macrophages, dendritic cells, and mast cells, are the majority of cellular components in the human immune system, but have received relatively less attention for the practical implementation than T cells and NK cells in cancer therapy because of concentration of the interest in development of the immune checkpoint blocking antibody inhibitors (ICIs). Abnormality of myeloid cells must impact on the entire host, including immune responses, stromagenesis, and cancer cells, leading to refractory cancer. This implies that elimination and reprogramming of the tumor-supportive myeloid villains may be a breakthrough to efficiently induce potent anti-tumor immunity in cancer patients. In this review, we provide an overview of current situation of the IC-blocking therapy of gastrointestinal cancer, including gastric, colorectal, and esophageal cancers. Also, we highlight the possible oncoimmunological components involved in the mechanisms underlying the resistance to the ICI therapy, particularly focusing on myeloid cells, including unique subsets expressing IC molecules. A deeper understanding of the molecular and cellular determinants may facilitate its practical implementation of targeting myeloid villains, and improve the clinical outcomes in the ICI therapy of gastrointestinal cancer.

Blocking FSTL1 boosts NK immunity in treatment of osteosarcoma.

Osteosarcoma (OS) is the most common primary bone malignancy. Many patients develop relapse and metastasis after treatments, and more effective treatments are needed for improving the clinical outcome. FSTL1 overexpression has been reported in murine and human OS, while the functional roles of FSTL1 remain unclear. Here, we elucidated tumor biological and immunological mechanisms underlying the refractory OS using mouse and human OS cell lines, mouse OS models, and clinical specimens. FSTL1 knockout in OS cells significantly suppressed cellular functions, including proliferation, invasion, sphere colony formation, and ALCAM expression. The FSTL1-ablated tumor cells were completely rejected due to generation of potent NK cells in the in vivo setting. Indeed, FSTL1 stimulation suppressed NK activity partly via apoptosis induction, but blocking FSTL1 or CD6, a receptor for ALCAM, significantly restored NK activity. Anti-FSTL1 therapy significantly suppressed tumor growth and metastasis in mouse OS models, and synergized with anti-CD6 therapy in providing significantly better prognosis. These suggest that blocking FSTL1 is a promising strategy for successfully treating OS. This study demonstrates a rationale of targeting the FSTL1-ALCAM axis in the treatment of OS in clinical settings.

Targeting AURKA in treatment of peritoneal tumor dissemination in gastrointestinal cancer.

Intraperitoneal (i.p.) tumor dissemination and the consequent malignant ascites remain unpredictable and incurable in patients with gastrointestinal (GI) cancer, and practical advances in diagnosis and treatment are urgently needed in the clinical settings. Here, we explored tumor biological and immunological mechanisms underlying the i.p. tumor progression for establishing more effective treatments. We established mouse tumor ascites models that murine and human colorectal cancer cells were both i.p. and subcutaneously (s.c.) implanted in mice, and analyzed peritoneal exudate cells (PECs) obtained from the mice. We then evaluated anti-tumor efficacy of agents targeting the identified molecular mechanisms using the ascites models. Furthermore, we validated the clinical relevancy of the findings using peritoneal lavage fluids obtained from gastric cancer patients. I.p. tumor cells were giant with large nuclei, and highly express AURKA, but less phosphorylated TP53, as compared to s.c. tumor cells, suggesting polyploidy-like cells. The i.p. tumors impaired phagocytic activity and the consequent T-cell stimulatory activity of CD11b+Gr1+PD1+ myeloid cells by GDF15 that is regulated by AURKA, leading to treatment resistance. Blocking AURKA with MLN8237 or siRNAs, however, abrogated the adverse events, and induced potent anti-tumor immunity in the ascites models. This treatment synergized with anti-PD1 therapy. The CD11b+PD1+ TAMs are also markedly expanded in the PECs of gastric cancer patients. These suggest AURKA is a determinant of treatment resistance of the i.p. tumors. Targeting the AURKA-GDF15 axis could be a promising strategy for improving clinical outcome in the treatment of GI cancer.

CD11b+DIP2A+LAG3+ cells facilitate immune dysfunction in colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide, and tumor metastasis is the leading cause of death. Targeting immune inhibitory checkpoint inhibitory pathways has attracted great attention, since the therapeutic efficacy induced by the specific blocking antibodies has been demonstrated even in metastatic CRC patients. However, the clinical outcome is low in many cases, and thus more effective treatments are needed in the clinical settings. A SPARC family member follistatin-like 1 (FSTL1) is known as a key driver of tumor metastasis in various types of cancer. However, the immunological roles of the FSTL1 in the CRC pathogenesis remain to be elucidated. In this study, we investigated the molecular mechanisms underlying the refractory FSTL1+ CRC using murine and human FSTL1-transduced CRC cells. Also, based on the results, we evaluated anti-tumor efficacy induced by agents targeting the identified molecules using murine CRC metastasis models, and validated the clinical relevancy of the basic findings using tumor tissues and peripheral blood obtained from CRC patients. FSTL1 transduction conferred EMT-like properties, such as low proliferative (dormant) and high invasive abilities, on tumor cells. When the transfectants were subcutaneously implanted in mice, CD11b+DIP2A+LAG3+ cells were abundantly expanded locally and systemically in the mice. Simultaneously, apoptotic T cells increased and were lastly excluded from the tumor tissues, allowing tumor aggravation leading to resistance to anti-PD1/PDL1 treatment. Blocking FSTL1 and LAG3, however, significantly suppressed the apoptosis induction, and successfully induced anti-tumor immune responses in the CRC metastasis models. Both treatments synergized in providing better prognosis of the mice. FSTL1 was significantly upregulated in tumor tissues and peripheral blood of CRC patients, and the CD11b+DIP2A+LAG3+ cells were significantly expanded in the PBMCs as compared to those of healthy donors. The expansion level was significantly correlated with decrease of potent Ki67+GZMB+ CTLs. These results suggest that the FSTL1-induced CD11b+DIP2A+LAG3+ cells are a key driver of immune dysfunction in CRC. Targeting the FSTL1-LAG3 axis may be a promising strategy for treating metastatic CRC, and anti-FSTL1/LAG3 combination regimen may be practically useful in the clinical settings.

CD11b+CTLA4+ myeloid cells are a key driver of tumor evasion in colorectal cancer.

BACKGROUND: Tumor metastasis is the major cause of death of colorectal cancer (CRC), and metastatic CRC remains incurable in many cases despite great advances in genetic and molecular profiling, and clinical development of numerous drugs, including immune checkpoint inhibitors. Thus, more effective treatments are urgently needed for the patients in clinical settings. METHODS: We used mouse CRC metastasis models that murine Colon26 cells were subcutaneously and intravenously implanted and attempted to elucidate the tumor biological and immunological mechanisms underlying cancer metastasis. Then, we evaluated in vivo antitumor efficacy induced by agents targeting the identified molecular mechanisms using the mouse models. We validated the clinical relevancy of the findings using peripheral blood mononuclear cells obtained from stage IV metastatic CRC patients. RESULTS: CD11b+CTLA4+ myeloid cells were systemically expanded in the metastatic settings and facilitated tumor progression and metastasis directly via generating lipid droplets in tumor cells and indirectly via inducing immune exhaustion. These events were mediated by IL1B produced via the CTLA4 signaling from the increased myeloid cells. Blocking CTLA4 and IL1B with the specific mAbs significantly suppressed tumor progression and metastasis in the mouse models resistant to anti-PD1 therapy, and the therapeutic efficacy was optimized by blocking cyclooxygenases with aspirin. CONCLUSIONS: The CD11b+CTLA4+ cells are a key driver of tumor evasion, and targeting the CTLA4-IL1B axis could be a promising strategy for treating metastatic CRC. The triple combination regimen with anti-CTLA4/IL1B mAbs and aspirin may be useful in clinical settings.

Targeting Oncoimmune Drivers of Cancer Metastasis.

Residual metastasis is a major cause of cancer-associated death. Recent advances in understanding the molecular basis of the epithelial-mesenchymal transition (EMT) and the related cancer stem cells (CSCs) have revealed the landscapes of cancer metastasis and are promising contributions to clinical treatments. However, this rarely leads to practical advances in the management of cancer in clinical settings, and thus cancer metastasis is still a threat to patients. The reason for this may be the heterogeneity and complexity caused by the evolutional transformation of tumor cells through interactions with the host environment, which is composed of numerous components, including stromal cells, vascular cells, and immune cells. The reciprocal evolution further raises the possibility of successful tumor escape, resulting in a fatal prognosis for patients. To disrupt the vicious spiral of tumor-immunity aggravation, it is important to understand the entire metastatic process and the practical implementations. Here, we provide an overview of the molecular and cellular links between tumors' biological properties and host immunity, mainly focusing on EMT and CSCs, and we also highlight therapeutic agents targeting the oncoimmune determinants driving cancer metastasis toward better practical use in the treatment of cancer patients.

IL33 Is a Key Driver of Treatment Resistance of Cancer.

Recurrence and treatment resistance are major causes of cancer-associated death. There has been a growing interest in better understanding epithelial-mesenchymal transition, stemness of cancer cells, and exhaustion and dysfunction of the immune system for which numerous genomic, proteomic, microenvironmental, and immunologic mechanisms have been demonstrated. However, practical treatments for such patients have not yet been established. Here we identified IL33 as a key driver of polyploidy, followed by rapid proliferation after treatment. IL33 induction transformed tumor cells into polyploid giant cells, showing abnormal cell cycle without cell division accompanied by Snail deregulation and p53 inactivation; small progeny cells were generated in response to treatment stress. Simultaneously, soluble IL33 was released from tumor cells, leading to expansion of receptor ST2-expressing cells including IL17RB+GATA3+ cells, which promoted tumor progression and metastasis directly and indirectly via induction of immune exhaustion and dysfunction. Blocking IL33 with a specific mAb in murine IL33+ metastatic tumor models abrogated negative consequences and successfully elicited antitumor efficacy induced by other combined treatments. Ex vivo assays using tumor tissues and peripheral blood mononuclear cells of patients with cancer validated the clinical relevancy of these findings. Together, these data suggest that targeting the IL33-ST2 axis is a promising strategy for diagnosis and treatment of patients likely to be resistant to treatments in the clinical settings. SIGNIFICANCE: These findings indicate that the functional role of IL33 in cancer polyploidy contributes to intrinsic and extrinsic mechanisms underlying treatment failure.

Blocking the FSTL1-DIP2A Axis Improves Anti-tumor Immunity.

Immune dysfunction is a strong factor in the resistance of cancer to treatment. Blocking immune checkpoint pathways is a promising approach to improve anti-tumor immunity, but the clinical efficacies are still limited. We previously identified follistatin-like 1 (FSTL1) as a determinant of immune dysfunction mediated by mesenchymal stromal/stem cells (MSCs) and immunoregulatory cells. Here, we demonstrate that blocking FSTL1 but not immune checkpoint pathways significantly suppresses cancer progression and metastasis in several mouse tumor models with increased MSCs. Expression of DIP2A (the receptor of FSTL1) in tumor cells is critical for FSTL1-induced immunoresistance. FSTL1/DIP2A co-positivity in tumor tissues correlates with poor prognosis in NSCLC patients. Thus, breaking the FSTL1-DIP2A axis may be a useful strategy for successfully inducing anti-tumor immunity.

Determination of poor prognostic immune features of tumour microenvironment in non-smoking patients with lung adenocarcinoma.

We have previously demonstrated that the prognostic significance of tumour-infiltrating CD8+ T cells significantly differs according to histological type and patient smoking habits in non-small cell lung cancer (NSCLC). This work suggested that infiltrating CD8+ T cells may not be activated sufficiently in the immunosuppressive microenvironment in non-smokers with adenocarcinoma. To understand the immunogenic microenvironment in NSCLC, we characterised immune cells comprehensively by performing an immunohistochemical evaluation using an alternative counting method and multicolour staining method (n = 234), and assessed immune-related gene expression by using genetic analytical approaches (n = 58). We found that high infiltration of activated CD8+ T cells expressing interferon gamma (IFN-γ) and granzyme was correlated with postoperative survival in patients with non-adenocarcinoma. On the contrary, CD8+ T-cell accumulation was identified as a worse prognostic factor in patients with adenocarcinoma, particularly in non-smokers. Infiltrating CD8+ T cells were significantly less activated in this microenvironment with high expression of various immunoregulation genes. Potentially immunoregulatory CD8+ FOXP3+ T cells and immunodysfunctional CD8+ GATA3+ T cells were increased in adenocarcinoma of non-smokers. CD4+ FOXP3+ regulatory T cells expressing chemokine receptor-4 (CCR4)- and chemokine ligand (CCL17)-expressing CD163+ M2-like macrophages also accumulated correlatively and significantly in adenocarcinoma of non-smokers. These characteristic immune cells may promote tumour progression possibly by creating an immunosuppressive microenvironment in non-smoking patients with lung adenocarcinoma. Our findings may be helpful for refining the current strategy of personalised immunotherapy including immune-checkpoint blockade therapy for NSCLC.

Novel Treatment of Chronic Graft-Versus-Host Disease in Mice Using the ER Stress Reducer 4-Phenylbutyric Acid.

Chronic graft-versus-host disease (cGVHD) is a notorious complication of allogeneic hematopoietic stem cell transplantation and causes disabling systemic inflammation and fibrosis. In this novel study, we focused on a relationship between endoplasmic reticulum (ER) stress and cGVHD, and aimed to create effective treatment of cGVHD. A series of experiments were conducted using a mouse model of cGVHD. Our data suggested (1) that ER stress was elevated in organs affected by cGVHD and (2) that 4-phenylbutyric acid (PBA) could reduce cGVHD-induced ER stress and thereby alleviate systemic inflammation and fibrosis. Because fibroblasts are thought to be implicated in cGVHD-elicited fibrosis and because macrophages are reported to play a role in the development of cGVHD, we investigated cGVHD-triggered ER stress in fibroblasts and macrophages. Our investigation demonstrated (1) that indicators for ER stress and activation markers for fibroblasts were elevated in cGVHD-affected lacrimal gland fibroblasts and (2) that they could be reduced by PBA. Our work also indicated that splenic macrophages from PBA-dosed mice exhibited the lower levels of ER stress and M2 macrophage markers than those from cGVHD-affected mice. Collectively, this study suggests that the reduction of ER stress utilizing PBA can be a clinically translatable method to treat systemic cGVHD.

Targeting ALCAM in the cryo-treated tumour microenvironment successfully induces systemic anti-tumour immunity.

Cryoablative treatment has been widely used for treating cancer. However, the therapeutic efficacies are still controversial. The molecular mechanisms of the cryo-induced immune responses, particularly underlying the ineffectiveness, remain to be fully elucidated. In this study, we identified a new molecular mechanism involved in the cryo failure. We used cryo-ineffective metastatic tumour models that murine melanoma B16-F10 cells were subcutaneously and intravenously implanted into C57BL/6 mice. When the subcutaneous tumours were treated cryoablation on day 7 after tumour implantation, cells expressing activated leucocyte cell adhesion molecule (ALCAM/CD166) were significantly expanded not only locally in the treated tumours but also systemically in spleen and bone marrow of the mice. The cryo-induced ALCAM(+) cells including CD45(-) mesenchymal stem/stromal cells, CD11b(+)Gr1(+) myeloid-derived suppressor cells, and CD4(+)Foxp3(+) regulatory T cells significantly suppressed interferon γ production and cytotoxicity of tumour-specific CD8(+) T cells via ALCAM expressed in these cells. This suggests that systemic expansion of the ALCAM(+) cells negatively switches host-immune directivity to the tumour-supportive mode. Intratumoural injection with anti-ALCAM blocking monoclonal antibody (mAb) following the cryo treatment systemically induced tumour-specific CD8(+) T cells with higher cytotoxic activities, resulting in suppression of tumour growth and metastasis in the cryo-resistant tumour models. These suggest that expansion of ALCAM(+) cells is a determinant of limiting the cryo efficacy. Further combination with an immune checkpoint inhibitor anti-CTLA4 mAb optimized the anti-tumour efficacy of the dual-combination therapy. Targeting ALCAM may be a promising strategy for overcoming the cryo ineffectiveness leading to the better practical use of cryoablation in clinical treatment of cancer.

Cancer-associated mesenchymal stem cells aggravate tumor progression.

Mesenchymal stem cells (MSCs) have both stemness and multi-modulatory activities on other cells, and the immunosuppressive and tumor-promotive mechanisms have been intensively investigated in cancer. The role of MSCs appears to be revealed in tumor aggravation, and targeting MSCs seems to be a promising strategy for treating cancer patients. However, it is still impractical in clinical therapy, since the precise MSCs are poorly understood in the in vivo setting. In previous studies, MSCs were obtained from different sources, and were prepared by ex vivo expansion for a long term. The inconsistent experimental conditions made the in vivo MSCs obscure. To define the MSCs in the host is a priority issue for targeting MSCs in cancer therapy. We recently identified a unique subpopulation of MSCs increasing in mice and human with cancer metastasis. These MSCs are specifically expanded by metastatic tumor cells, and promote tumor progression and dissemination accompanied by immune suppression and dysfunction in the host, more powerfully than normal MSCs growing without interference of cancer. In this review, we summarize current knowledge of the role of MSCs in tumor aggravation, along with our new findings of the bizarre MSCs.

Induction of immunoregulatory CD271+ cells by metastatic tumor cells that express human endogenous retrovirus H.

Human endogenous retroviruses (HERV) are associated with many diseases such as autoimmune diseases and cancer. Although the frequent expression of a variety of HERVs in tumor cells has been demonstrated, their functional contributions in cancer are as yet unclear. Intriguingly, HERVs and other retroviruses include an immunosuppressive domain in their transmembrane envelope proteins, but its mechanism of action and cancer relevance are obscure. In this study, we demonstrate that the human endogenous retrovirus HERV-H has a critical role in tumor metastasis and immune escape. We found that expression of herv-h mRNA was elevated in metastatic tumor cells undergoing epithelial-to-mesenchymal transition (EMT) and in primary tumor tissues from advanced colon cancer. The immunosuppressive peptide H17 derived from HERV-H was sufficient to induce EMT in tumor cells that expressed low levels of HERV-H, and it amplified this event within the tumor microenvironment. H17 also stimulated CCL19 expression in tumor cells, which in turn recruited and expanded a population of pluripotent immunoregulatory CD271(+) cells, which included mesenchymal stem cells and myeloid-derived suppressor cells. In tumor tissues from patients with advanced colon cancer, we confirmed that CD271(+) cells were increased in HERV-H(+)CCL19(+) tumor tissues. Notably, RNAi-mediated change of HERV-H or CCL19, or depletion of CD271(+) cells, improved immune responses in vitro and in vivo accompanied by tumor regression. Together, our results argued that HERV-H is a critical determinant of immune escape in cancer, suggesting its candidacy as a promising therapeutic target to treat patients with advanced cancer.

Targeting FSTL1 prevents tumor bone metastasis and consequent immune dysfunction.

Bone metastasis greatly deteriorates the quality of life in patients with cancer. Although mechanisms have been widely investigated, the relationship between cancer bone metastasis and antitumor immunity in the host has been much less studied. Here, we report a novel mechanism of bone metastasis mediated by FSTL1, a follistatin-like glycoprotein secreted by Snail(+) tumor cells, which metastasize frequently to bone. We found that FSTL1 plays a dual role in bone metastasis-in one way by mediating tumor cell invasion and bone tropism but also in a second way by expanding a population of pluripotent mesenchymal stem-like CD45(-)ALCAM(+) cells derived from bone marrow. CD45(-)ALCAM(+) cells induced bone metastasis de novo, but they also generated CD8(low) T cells with weak CTL activity in the periphery, which also promoted bone metastasis in an indirect manner. RNA interference-mediated attenuation of FSTL1 in tumor cells prevented bone metastasis along with the parallel increase in ALCAM(+) cells and CD8(low) T cells. These effects were accompanied by heightened antitumor immune responses in vitro and in vivo. In clinical specimens of advanced breast cancer, ALCAM(+) cells increased with FSTL1 positivity in tumor tissues, but not in adjacent normal tissues, consistent with a causal connection between these molecules. Our findings define FSTL1 as an attractive candidate therapeutic target to prevent or treat bone metastasis, which remains a major challenge in patients with cancer.

Improvement of cancer immunotherapy by combining molecular targeted therapy.

In human cancer cells, a constitutive activation of MAPK, STAT3, ß-catenin, and various other signaling pathways triggers multiple immunosuppressive cascades. These cascades result in the production of immunosuppressive molecules (e.g., TGF-ß, IL-10, IL-6, VEGF, and CCL2) and induction of immunosuppressive immune cells (e.g., regulatory T cells, tolerogenic dendritic cells, and myeloid-derived suppressor cells). Consequently, immunosuppressive conditions are formed in tumor-associated microenvironments, including the tumor and sentinel lymph nodes. Some of these cancer-derived cytokines and chemokines impair immune cells and render them immunosuppressive via the activation of signaling molecules, such as STAT3, in the immune cells. Thus, administration of signal inhibitors may inhibit the multiple immunosuppressive cascades by acting simultaneously on both cancer and immune cells at the key regulatory points in the cancer-immune network. Since common signaling pathways are involved in manifestation of several hallmarks of cancer, including cancer cell proliferation/survival, invasion/metastasis, and immunosuppression, targeting these shared signaling pathways in combination with immunotherapy may be a promising strategy for cancer treatment.

Cancer-induced immunosuppressive cascades and their reversal by molecular-targeted therapy.

Immunological status in tumor tissues varies among patients. Infiltration of memory-type CD8(+) T cells into tumors correlates with prognosis of patients with various cancers. However, the mechanism of the differential CD8(+) T cell infiltration has not been well investigated. In general, tumor-associated microenvironments, including tumor and sentinel lymph nodes, are under immunosuppressive conditions such that the immune system is not able to eliminate cancer cells without immune-activating interventions. Constitutive activation of various signaling pathways in human cancer cells triggers multiple immunosuppressive cascades that involve various cytokines, chemokines, and immunosuppressive cells. Signaling pathway inhibitors could inhibit these immunosuppressive cascades by acting on either cancer or immune cells, or both. In addition, common signaling mechanisms are often utilized for multiple hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression). Therefore, targeting these common signaling pathways may be an attractive strategy for cancer therapy including immunotherapy.

CCL2 is critical for immunosuppression to promote cancer metastasis.

We previously found that cancer metastasis is accelerated by immunosuppression during Snail-induced epithelial-to-mesenchymal transition (EMT). However, the molecular mechanism still remained unclear. Here, we demonstrate that CCL2 is a critical determinant for both tumor metastasis and immunosuppression induced by Snail(+) tumor cells. CCL2 is significantly upregulated in various human tumor cells accompanied by Snail expression induced by snail transduction or TGFß treatment. The Snail(+) tumor-derived CCL2 amplifies EMT events in other cells including Snail(-) tumor cells and epithelial cells within tumor microenvironment. CCL2 secondarily induces Lipocalin 2 (LCN2) in the Snail(+) tumor cells in an autocrine manner. CCL2 and LCN2 cooperatively generate immunoregulatory dendritic cells (DCreg) having suppressive activity accompanied by lowered expression of costimulatory molecules such as HLA-DR but increased expression of immunosuppressive molecules such as PD-L1 in human PBMCs. The CCL2/LCN2-induced DCreg cells subsequently induce immunosuppressive CD4(+)FOXP3(+) Treg cells, and finally impair tumor-specific CTL induction. In murine established tumor model, however, CCL2 blockade utilizing the specific siRNA or neutralizing mAb significantly inhibits Snail(+) tumor growth and metastasis following systemic induction of anti-tumor immune responses in host. These results suggest that CCL2 is more than a chemoattractant factor that is the significant effector molecule responsible for immune evasion of Snail(+) tumor cells. CCL2 would be an attractive target for treatment to eliminate cancer cells via amelioration of tumor metastasis and immunosuppression.

FSTL1 promotes bone metastasis by causing immune dysfunction.

In spite of significant advances in our understanding of the metastatic process, the relationship between the dissemination of primary neoplasms to the bones and antitumor immunity remains poorly understood. We have recently identified follistatin-like 1 (FSTL1), a soluble protein secreted by snail family zinc finger 1 (SNAI1)-expressing cancer cells, as a key determinant of bone metastasis that operates by inducing a systemic state of immune dysfunction.