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.

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.

BackgroundTumor 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.MethodsWe 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.ResultsCD11b+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.ConclusionsThe 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.

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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.

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Purpose: Distant recurrence following metastasis is a major cause of cancer-associated death. Interfering spread and re-growth of the disseminated tumor cells, which could be cancer stem-like cells (CSCs) via EMT, must be greatly helpful for improving prognosis of cancer patients. However, little is known about the molecular mechanisms underlying the refractoriness. In this study, we attempted to elucidate the mechanism how the dormant CSCs would wake up from the sleeping at the metastatic lesions. Results: C57BL/6 mice were subcutaneously and intravenously implanted with murine melanoma B16-F10 cells or B16-F10 cells transduced with snail gene as an EMT control, and 3 weeks later, the metastasized tumor cells (F10-BM or F10-BM-snail+) were harvested from the bone marrow, a niche for CSCs, or from the subcutaneous tumors as a control (F10-primary). We comprehensively analyzed these clones for EMT-related molecular expressions and biological functions in vitro and in vivo. The F10-primary cells were round and adhesive having high expression of epithelial markers such as E-cadherin, but not mesenchymal markers such as Snail, and the F10-BM-snail+ cells were small and spindle having high expression of mesenchymal markers, but not epithelial markers. In contrast, the F10-BM cells were giant and very adhesive with high expression of epithelial markers, but still keeping high Snail expression in the cytoplasm. Interestingly, these cells were polyploidy having a number of nuclei in a cell, and proliferated significantly slower than the F10-primary cells. However, once receiving stimulation with a low concentration of chemotherapeutics, the F10-BM cells aggressively proliferated through generation of many progeny cells, looking resistant to the treatment. In the in vivo settings, the F10-BM tumor growth was significantly slower after implantation in mice, and the mice survived for a longer term than the F10-primary-implanted mice. As seen in the in vitro culture, the F10-BM tumors quite rapidly grew in response to treatments such as chemotherapeutics and immune checkpoint inhibitors, and the treated mice died significantly earlier than the nontreated F10-BM-implanted or the F10-primary-implanted mice, suggesting hyperprogression that tumor cell propagation might exceed elimination in the mice. Conclusions: These data suggest that dormant and polyploidy giant cells could be a risk factor of hyperprogression of the metastatic tumors after treatments. We are now identifying a specific molecule regulating this mechanism utilizing cDNA microarray analysis. Citation Format: Chie Kudo-Saito, Yukinori Ozaki, Keiichi Kinowaki, Hidetaka Kawabata, Yamato Ogiwara. A mode of treatment resistance of metastatic tumor cells: Propagation exceeds elimination in number [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3751.

Purpose: Tumor heterogeneity is composed of genetic and phenotypic changes in tumor itself, and also of the consequence of reciprocal evolution together with the tumor microenvironment containing a variety of cells such as stromal cells, vascular cells and immune cells in the host. Imbalanced immunity is one of the major problems supporting tumor progression and then amplification of the heterogeneity. Many inhibitors specific for immune checkpoint molecules interfering induction of anti-tumor immunity have been recently developed in clinical settings, and the clinical responses have convinced us of its efficaciousness. However, good results are limited to a part of the treated patients, and additional but another strategy is needed for treating patients more efficiently and effectively. For the purpose, we have been investigating the complex network in the tumor milieu and the systemic responses in the whole body comprehensively and collectively, and previously identified a BMP family FSTL1, which is significantly secreted from Snail+ cancer stem-like cells, as a key effector molecule in the cancer-associated immune suppression and dysfunction. FSTL1 expands pluripotent mesenchymal stem/stromal cells (MSCs), which generate immunoregulatory cells such as Tregs and tolerogenic DCs, and functionally impaired CD8-low T cells. FSTL1 and its induced MSCs also confer higher invasive and metastatic properties on tumor cells leading to further promotion of tumor growth and metastasis. To prevent such FSTL1-caused vicious circulation, we established anti-FSTL1 blocking mAbs, and evaluated the anti-tumor efficacy in combination with immune checkpoint inhibitors (ICIs) using mouse FSTL1+ tumor models. Experimental Procedures: We generated monoclonal antibodies (mAbs) specific for both mouse and human FSTL1 utilizing the chicken immunization system followed by constructing cDNA phage library. After in vitro screening, the established anti-FSTL1 mAbs were tested for the in vivo anti-tumor activity using two syngeneic tumor models: a bone metastatic tumor model that C57BL/6 was implanted with snail-transduced melanoma B16-F10 cells, and a lung metastatic tumor model that BALB/c was implanted with colon cancer intrinsic FSTL1+ CT26 cells. The mAbs (10mg/kg) were intraperitoneally injected into these mice twice on day 4 and day 7 after tumor implantation. On day 14, immunological analysis was conducted using tumor-infiltrating cells, spleen cells and bone marrow cells obtained from the mice. Results: We established chicken/mouse chimeric anti-FSTL1 mAb capable of inhibiting the FSTL1-elicited events including MSC expansion, immunoregulatory cell induction, and tumor invasion in vitro. In both bone and lung metastatic tumor models, treatment with the anti-FSTL1 mAb induced tumor-specific CD8+ T cells with higher CTL activities by suppressing increase of MSCs, but not Tregs and MDSCs, and significantly suppressed subcutaneous tumor growth and metastasis in the treated mice as compared to those of the control mice. The ICI mono-therapy was insufficient to suppress increase of MSCs, although significantly reduced immunoregulatory cells, and the anti-tumor efficacy was almost similar. Combination with the anti-FSTL1 mAb synergistically and significantly enhanced the ICI mono-therapeutic efficacy, particularly anti-PDL1 mAb, by inducing potent tumor-specific CTLs, resulting in tumor-free in some of the treated mice. Conclusions: Blocking FSTL1 reprograms and properly induces anti-tumor immunity by switching the tumor-supportive immune directivity to the active mode against cancer. Anti-FSTL1 mAb having totally different molecular mechanisms from the conventional ICIs may be a promising partner for radically correcting cancer-manipulated immunity in clinical settings. Citation Format: Chie Kudo-Saito, Yamato Ogiwara, Marina Hennmi, Kazunori Aoki. Targeting FSTL1 augments therapeutic activities of immune checkpoint inhibitors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR12.