Cell Line MC38 Research Articles

Relationship of KIAA1199 on neutrophil recruitment and promotion of colorectal cancer liver metastasis via TGF-β signaling.

e16005 Background: Metastasis is the main cause of cancer-related death governed by both cancer cell-intrinsic properties and extrinsic immune contexture. Our previous reports have suggested critical roles of KIAA1199 in tumor invasion and metastasis in colorectal cancer (CRC). In the present study, we explored the potential effects of KIAA1199 on pre-metastatic niche formation. Methods: The CIBERSORT algorithm analysis for 241 patients with colorectal cancer from the Cancer Genome Atlas was performed to investigate the relationship between KIAA1199 expression and neutrophil accumulation. Neutrophils and T cells were isolated, stimulated and/or cultured for in vitro function assays. Proteins were knocked down in MC38 and HCT116 colorectal cancer cell lines, which were analyzed by immunoblotting and chemotaxis assays. Results: We demonstrate that patients with high KIAA1199 showed an increased level of liver-infıltrating neutrophils, which is further validated using flow cytometry analyses in mouse metastasis model. The increased influx of neutrophils is associated with KIAA1199-driven CRC liver metastasis. Neutrophils isolated from pre-metastatic niche decreased CD8+ T cells infiltration and IFN-r+ CD8+ T cells proportion in vivo. Moreover, they showed a N2-like phenotype and expressed higher level immunosuppressive molecules when exposed to the supernatant of normal CRC cell compared to KIAA1199-knock down cell. In CRC cells, we also found a positive correlation between expression of KIAA1199 and genes that ELR+ chemotaxis of neutrophil (CXCL1/CXCL3/CXCL5), which recruited neutrophils to CRC tumor via CXCR2. Importantly, LY2109761, a TGF-β receptor inhibitor, is sufficient to reduce the mRNA levels of these genes. Furthermore, there is a positive correlation between expression of KIAA1199 and expression of Transforming Growth Factor beta pathway genes (TGFBR1, TGFBR2, and pSMAD3), and further co-immunoprecipitation analysis suggests that KIAA1199 may activate the TGF-β signaling pathway via interacting with the TGFBR1 or TGFBR2. Conclusions: Our data indicate that the upregulation of KIAA1199 may increase the expression of cytokines mediated by TGF-β signaling, which can recruit metastasis-supporting neutrophils to promote colorectal cancer liver metastasis. Blockade of the TGF-β signaling could be a novel therapeutic approach against KIAA1199-high expression CRC.

Abstract 3206: Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors

Abstract Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that employs a targeted monoclonal antibody-photo-absorber conjugate (APC). Following antibody localization of the APC to a tumor cell surface antigen, NIR light is used to induce highly selective cytolysis. Extensive pre-clinical evidence demonstrates that NIR-PIT is effective at inducing tumor cell lysis using a number of different antibody-APC conjugates and NIR-PIT targeting EGFR with a cetuximab-APC conjugate has shown promising results in phase 2 clinical evaluation for the treatment of recurrent head and neck squamous cell carcinoma. NIR-PIT induces rapid, necrotic cell death that yields innate immune ligands that activate dendritic cells, consistent with immunogenic cell death. Yet, NIR-PIT treatment of syngeneic tumors in wild-type mice has mostly failed to induce durable regression of established tumors, suggesting the presence of one or more mechanisms of resistance to formation of meaningful anti-tumor immunity. In this study, we hypothesized that NIR-PIT could induce anti-tumor immunity that was being restricted by the PD-1/PD-L1 signaling axis, and that PD-1 could reverse innate immune resistance to induce durable, effective anti-tumor immune responses. Using a CD44-targeting APC, we demonstrated the ability of PD-1 immune checkpoint blockade to significantly enhance antigen-specific anti-tumor immunity induced by NIR-PIT in multiple syngeneic tumor models (MC38, LLC, and MOC1 cancer cell line). In two of three models, NIR-PIT monotherapy halted tumor growth, enhanced dendritic cell tumor infiltration, and induced de novo tumor antigen-specific T-cell responses absent at baseline. The addition of PD-1 blockade appeared to reverse adaptive immune resistance, resulting in both enhanced pre-existing tumor antigen-specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT. Enhanced immune responses appeared to correlate with shared tumor antigen expression, suggesting that antigenicity is a major determinant of response to combination NIR-PIT and PD-1 blockade. Combination treatment induced complete rejection of MC38 tumors treated with NIR-PIT as well as untreated, distant tumors. Accordingly, tumor antigen-specific T-cell responses were measured in both treated and untreated tumors, validating the development of systemic anti-tumor immunity. Cured mice resisted tumor challenge, indicating the presence of systemic immune memory. Cumulatively these results demonstrate reversal of adaptive immune resistance following induction of innate and adaptive immunity by NIR-PIT, resulting in high rates of tumor rejection and/or significant tumor growth control in highly antigenic syngeneic models of cancer. Citation Format: Tadanobu Nagaya, Jay Friedman, Yasuhiro Maruoka, Fusa Ogata, Shuhei Okuyama, Paul E. Clavijo, Peter L. Choyke, Clint Allen, Hisataka Kobayashi. Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors [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 3206.

Abstract 2268: Agonistic T cell non depleting ICOS antibody strongly enhances anti-tumor activity with CTLA4 blocking monoclonal antibody without exacerbating colitis

Abstract Background: ICOS is a member of the CD28 superfamily mainly expressed on activated T cells. An up-regulation of ICOS+ T cells is observed in patients treated with ipilimumab. These findings prompt further studies examining the synergy between CTLA-4 blockade and ICOS stimulation in generating optimal anti-tumor T cell immunity. Methods: Peripheral blood mononuclear cells (PBMC) were collected from French patients with metastatic melanoma and treated with ipilimumab at baseline (V1) and after 1-2 ipilimumab infusion and restimulated in vitro with anti-CD3 plus anti-human non-depleting agonist ICOS IgG4 isotype antibodies. T cell proliferation and cytokine secretion was determined after 24 and 48h incubation. For in vivo experiments, C57Bl/6 and BALB/c mice were inoculated subcutaneously with MC38 and CT26 tumor cell lines respectively. Mice received intraperitoneal injections of anti-CTLA4 or its isotype control along with anti-mouse ICOS or corresponding isotype at D7, D10 and D13 with tumor growth assessments three times a week. Results: PBMC collected from patients that respond to anti-CTLA-4 mAb exhibited stronger activation of T cells at baseline compared to patients with poor benefit. Treatment with a non-depleting agonist ICOS IgG4 mAb was able to rescue T cell activation in PBMC from patients with poor benefit. Furthermore, ICOS IgG4 with concurrent TCR engagement favored IL-10 secretion in patients that do not develop colitis during ipilimumab treatment suggesting that these patients may be protected from colitis due to a stronger capacity to secrete IL-10 after ipilimumab blockade particularly when ICOS is stimulated. In both mouse tumor models, combination with agonist non-depleting ICOS mAb increased the anti-CTLA-4 tumor activity without any colitis development. Conclusion: This study provides evidence in mice and in humans that non-depleting agonistic ICOS mAb may increase anti-CTLA-4 tumor activity particularly in patients that do not benefit from anti-CTLA-4 alone without exacerbation of colitis. Citation Format: Clélia Coutzac, Jean Jouniaux, Aurore Vozy, Marc Ballas, Sara Brett, Sapna Yadavilli, Eric Angevin, Axel Hoos, Nathalie Chaput. Agonistic T cell non depleting ICOS antibody strongly enhances anti-tumor activity with CTLA4 blocking monoclonal antibody without exacerbating colitis [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 2268.

Abstract LB-018: Orally active small molecule PD-L1 inhibitor demonstrating similar efficacy as Durvalumab in human knock-in MC38 model

Abstract Cancer immunotherapy with PD-1/PD-L1 antibody drugs has made tremendous progress in clinical application. It was also recognized by the scientific community with awarding of the 2018 Nobel Price. However, the discovery of orally active, small molecule drugs targeting PD-1/PD-L1 pathway remains highly challenging. We wish to report for the first time the similar efficacy as measured by TGI between small molecule inhibitor Max-10181 and Durvalumab in the head to head test of human PD-L1 knock-in MC38 cell line in human PD-1 knock-in mice. In addition, we would also like to report the superior efficacy of Max-10181 to mouse anti-PD-1 Ab in PD-L1 expression low syngeneic mouse 4T1 model. Our data indicated that small molecule drugs may have different dependency of PD-L1 expression level. As a result, they may have different therapeutic application in clinics. Furthermore, the data related to the mechanistic aspect of these observed results will also be discussed. Citation Format: Yuguang Wang, Nong Zhang, Feilan Wang, Qiang Zhao, Zenggang Li. Orally active small molecule PD-L1 inhibitor demonstrating similar efficacy as Durvalumab in human knock-in MC38 model [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 LB-018.

Abstract 3926: MSLN-TTC (BAY 2287411) induces immunogenic cell death and secretion of pro-inflammatory cytokines in vitro and triggers an immune memory effect against a mouse tumor model

Abstract Mesothelin (MSLN)-targeted thorium conjugate (MSLN-TTC; BAY 2287411) is the first targeted alpha therapy in clinical development for the treatment of patients suffering from MSLN-positive mesothelioma and ovarian cancer (NCT03507452). It consists of the MSLN-targeting antibody anetumab, covalently attached to a 3,2-HOPO chelator complexing the alpha-emitter thorium-227. The main mode of action of MSLN-TTC is the induction of clustered DNA double-strand breaks upon alpha decay of thorium-227, resulting in cell death. The preclinical efficacy of MSLN-TTC has been demonstrated previously. The tumor control achieved by external beam radiation is partly driven by immune-stimulatory effects (Vatner et al; Frontiers in Oncology 2014). The essential mechanisms can be attributed to different pathways, including (a) induction of immunogenic cell death and (b) activation of the STING-pathway resulting in the secretion of pro-inflammatory cytokines (Vanpouille-Box C et al; NatComm 2017). We therefore investigated whether the targeted alpha therapy MSLN-TTC is also able to induce both of these pathways in vitro. The human ovarian cancer MSLN-expressing cell line OVCAR-3 was exposed to MSLN-TTC resulting in a dose dependent upregulation of markers of immunogenic cell death, e.g. calreticulin and HMGB-1. Further, exposure of OVCAR-3 cells to MSLN-TTC resulted in the secretion of pro-inflammatory cytokines, including IFN-β, IL-6 and IP-10. The efficacy of MSLN-TTC was evaluated in a syngeneic subcutaneous tumor model. As MSLN-TTC is not cross-reactive to murine MSLN, an MC38 cell line stably transfected with human MSLN was established (MC38-hMSLN). In vitro, MSLN-TTC induced specific reduction of MC38-hMSLN cell viability. Following single dose administration to MC38-hMSLN tumor-bearing mice, MSLN-TTC induced dose dependent antitumor activity with complete tumor eradication in 10 out of 36 treated animals. Interestingly, when tumor-free animals were re-inoculated with MC38-hMSLN cells 121 days post treatment, no tumor growth was observed. In contrast, tumors grew in animals inoculated with B16F10 cells, suggesting the development of an immune memory response against MC38-hMSLN cells. In summary, the data presented demonstrate that MSLN-TTC is able to induce immunogenic cell death and secretion of pro-inflammatory cytokines in vitro. Further, MSLN-TTC monotherapy evokes an immune-stimulatory effect in vivo. Citation Format: Urs B. Hagemann, Pascale LeJeune, Jenny Karlsson, Christoph A. Schatz, Alan S. Cuthbertson, Hartwig Hennekes, Karl Ziegelbauer, Dominik Mumberg. MSLN-TTC (BAY 2287411) induces immunogenic cell death and secretion of pro-inflammatory cytokines in vitro and triggers an immune memory effect against a mouse tumor model [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 3926.

Interleukin-2 boosts local and abscopal antitumor effect of radiotherapy combined with anti-PD-1: A translational research from clinical radio-memory effect.

e14244 Background: Preclinical and clinical studies have shown that prior receipt of radiotherapy enhances antitumor immune responses, a phenomenon we call the “radio-memory effect.” However, all of the evidence regarding this effect to date comes from work with anti-PD-1 or anti-PD-L1 inhibitors. Here we explore whether this effect also occurs with other forms of immune therapy, specifically interleukin-2 (IL-2). If so, whether this triple combination therapy could produce synergetic anti-tumor effect. Methods: 1. Clinical work: we retrospectively assessed outcomes in patients with malignant peritoneal effusion (MPE) who had previously received radiotherapy for colon or rectum cancer within 18 months before the intrapleural infusion of IL-2 or cisplatin. All patients received intrapleural infusion of IL-2 or cisplatin, and most had had several cycles of standard chemotherapy for colon or rectum cancer. 2. Translational work: MC38 cell line (5×106) was subcutaneously inoculated into the right lower extremity of C57/BL mice to construct a primary tumor model. Six days later, MC38 cell line (1×106) was subcutaneously inoculated into the left lower limb to construct a second tumor model. The main research is as follows: To explore the optimal radiation dose and fraction for radiotherapy; Whether the addition of IL-2 will further enhance the efficacy of radiotherapy combined with anti-PD-1; To explore the optimal combination model of radiation, anti-PD-1 and IL-2; Whether the addition of IL-2 will increase the abscopal effect produced by radiotherapy and anti-PD-1. Results: 1. We identified 3,747 patients with MPE (median age 66 years [range 24–81)) treated at one of several institutions from August 2009 through May 2016; 412 patients had been treated with IL-2 and 592 with cisplatin and had survived for at least 3 months afterward. 2. Hypofractionated radiotherapy (12Gy × 2) combined with IL-2 is better; IL-2 will enhance the efficacy of radiotherapy combined with anti-PD-1; IL-2 and anti-PD-1 followed by radiotherapy might maximize the anti-tumor effect; IL-2 might increase the abscopal effect produced by radiotherapy combined with anti-PD-1. Conclusions: We speculate that previous radiotherapy could enhance the efficacy of IL-2 and the anti-tumor effect produced by radiotherapy combined with anti-PD-1 could be enhanced by IL-2. The “radio-memory” effect could be beneficial in future studies.

Disruption of sialic acid metabolism drives tumor growth by augmenting CD8+ T cell apoptosis.

Sialylated glycan structures are known for their immunomodulatory capacities and their contribution to tumor immune evasion. However, the role of aberrant sialylation in colorectal cancer and the consequences of complete tumor desialylation on anti‐tumor immunity remain unstudied. Here, we report that CRISPR/Cas9‐mediated knock out of the CMAS gene, encoding a key enzyme in the sialylation pathway, in the mouse colorectal cancer MC38 cell line completely abrogated cell surface expression of sialic acids (MC38‐Sianull) and, unexpectedly, significantly increased in vivo tumor growth compared to the control MC38‐MOCK cells. This enhanced tumor growth of MC38‐Sianull cells could be attributed to decreased CD8+ T cell frequencies in the tumor microenvironment only, as immune cell frequencies in tumor‐draining lymph nodes remained unaffected. In addition, MC38‐Sianull cells were able to induce CD8+ T cell apoptosis in an antigen‐independent manner. Moreover, low CMAS gene expression correlated with reduced recurrence‐free survival in a human colorectal cancer cohort, supporting the clinical relevance of our work. Together, these results demonstrate for the first time a detrimental effect of complete tumor desialylation on colorectal cancer tumor growth, which greatly impacts the design of novel cancer therapeutics aimed at altering the tumor glycosylation profile.

Alteration of Adaptive Immunity in a Colorectal Peritoneal Carcinomatosis Model.

Colorectal cancer (CRC) usually gives rise to transcoelomic spread and ultimately causes peritoneal carcinomatosis (PC). However, mechanism studies, especially the immunological basis of colorectal PC, are rarely revealed due to lack of a suitable PC model. Here we selected a mouse colorectal cancer cell line MC-38 for intraperitoneal inoculation in the C57BL/6 mice to mimic the development of colorectal PC. We demonstrated that the injected CRC cells preferentially and rapidly migrated and colonized in the visceral fat tissues, but not in other visceral organs. With flow cytometric analysis, we found the proportions of spleen T cells and B cells were not affected by PC progression, while the ratios of blood CD4+ and CD8+ T cells were largely influenced. Especially, the quantity or activity of CD4+ and CD8+ T cells in visceral fats were intimately regulated by PC development. Taken together, we successfully constructed a colorectal PC model in immune-competent mice and revealed the alteration of adaptive immunity in PC development. Our study might potentiate the research and therapy strategies of colorectal PC.

Intrinsic Expression of Immune Checkpoint Molecule TIGIT Could Help Tumor Growth in vivo by Suppressing the Function of NK and CD8+ T Cells.

TIGIT, an immune checkpoint molecule widely expressed on NK cells, activated T cells and Tregs, has been involved in delivering inhibitory signals through the interaction with PVR. The blockade of TIGIT/PVR interaction is a promising approach in cancer immunotherapy. Here, we unexpectedly discovered the expression of TIGIT in murine tumor cells. To elucidate the mechanism of such intrinsic expression, TIGIT knockout murine colorectal CT26 and MC38 cell lines were generated by using CRISPR/Cas9 system. Although TIGIT knockout showed no effects on proliferation and colony formation of tumor cells in vitro, the tumor growth in mice was considerably inhibited. TIGIT knockout led to the increase of IFN-γ secretion by NK and CD8+ T cells. Further, in BABL/c nude mice, CD8+ T cells depleting mice and NK cells depleting nude mice, the promotion of tumor growth was significantly diminished, suggesting that both NK cells and CD8+ T cells were involved in the tumor promoting process mediated by intrinsic TIGIT. In addition, blocking TIGIT/PVR interaction by the antibody or recombinant PVR protein could elicit anti-tumor effects by facilitating the tumor infiltration and restoring the function of CD8+ T cells, and the antibody-mediate TIGIT blockade could inhibit MC38 tumor growth through blocking TIGIT expressed on tumor cells. We therefore propose a novel TIGIT/PVR interaction mode that tumor intrinsic TIGIT delivers inhibitory signals to CD8+ T cells and NK cells by engaging with PVR.

Alteration of CD47 Expression in Human and Murine Tumor Models Changes M1/M2 Phagocytosis Effect in Vitro and In Vivo

IntroductionCD47 expression in human tumor cell represents a resistance mechanism escaping the macrophage surveillance system. To reinstate macrophage function in recognizing and engulfing tumor cells, unblocking CD47-SIRPalpha interaction and thereby re-activating macrophage activity has been an important strategy in immune-oncology therapy. To validate the strategy, we profiled a large panel of tumor models including tumor cell lines, PDX models in Crown tissue bank and clinical samples for CD47 expression. Despite of its expression in the majority of tumor cell models, the expression level of CD47 vary in flow cytometry examination. Interestingly a portion of tumor cells do not express CD47 at all.To facilitate in vitro screening for effective macrophage therapy, we established an in vitro assay platform using CD14+ monocytes as starting material. We also engineered human and murine cell models to overexpress human CD47 at one end and delete endogenous CD47 at the other end, and observed robust phagocytosis effect in these models. Finally, the findings in the in vitro assays were validated in MC38-hCD47 homograft in vivo.Material and MethodsTumor cell lines were CFSE-labeled and incubated with human monocyte-derived M1 or M2 macrophages (T:M 1:1) in the presence of 20μg/ml IgG1 isotype control, or anti-CD47 blocking antibodies B6H12 and BRIC126 and non-blocking antibody 2D3, for 2hrs. Cells were then harvested and stained with a macrophage marker (CD14+). Phagocytosed tumor cells were identified by flow cytometry as CD14+CFSE+. The phagocytosis index was calculated by dividing the number of macrophages engulfing tumor cells by the total number of macrophages. CD47 overexpression and deletion clones (KARPAS299, SK-OV3, and MC38 background) were developed in house. The CD14+ cell isolation kit was from Miltenyi. M1/M2 differentiation reagents were purchased from R&D and Stem Cell. Anti-CD47 antibodies B6H12 and 2D3 and mouse IgG1 were purchased from Thermo Fisher, and BRIC126 from GeneTex. B6H12 for animal study was sourced from Bioxcell.ResultsTo facilitate in vitro screening for effective macrophage therapy, we sought to establish a robust yet reproducible assay platform using CD14+ monocytes as starting material. Through macrophage differentiation we were able to achieve > 80% of M1 and M2 populations, respectively, though variation exists. Differentiated M1/M2 were then co-cultured with CFSE-labeled target tumor cells for subsequent flow cytometry analysis. Strong phagocytosis effect was observed with CD47 expression tumor cells when anti-CD47 antibody BRICK126, which blocks the CD47-SIRPalpha interaction, was introduced. In contrast, aCD274 (PD-L1) antibody didn't yield more than background phagocytosis effect in the same assay suggesting the specific activation effect of BRICK126 triggering M1/M2 functionality.To prove the CD47-specific effect, we engineered CARPAS299 and SK-OV3 tumor cell line models and MC38 murine cell model to overexpress human CD47 at one end and delete endogenous CD47 at the other end. The recombinant and isogenic models were then subject to the CD47 biology examination and more profound changes in phagocytosis responses were observed.To validate our findings in vivo, we introduced engineered MC38-hCD47 clone in to C57/BL6 mice to measure homograft tumor growth kinetics and pharmacological responses to B6H12, another CD47 blocking agent. Details of results will be presented.ConclusionDisrupting the CD47-SIRPa anti-phagocytic axis is a novel immunotherapy approach against cancer. This study reveals that CD47 expression level varies in different tumor cell models. Our in vitro screen demonstrated that M1 and M2 macrophages resulted in comparable anti-CD47 phagocytic activities, and suggests that Karpas299 with higher CD47 expression level may be a better model than SK-OV3 in cell engulfing study. We also developed Karpas299-hCD47, SK-OV-3-hCD47 and MC38-hCD47 stable clones to facilitate in vitro and in vivo screening towards macrophage therapy, and discovered strong positive correlation between CD47 expression level and phagocytic activity in in vitro and in vivo systems. DisclosuresNo relevant conflicts of interest to declare.

A role for paracrine interleukin-6 signaling in the tumor microenvironment in prostate tumor growth.

Interleukin-6 (IL-6) is a mediator of inflammation that can facilitate prostate cancer progression. We previously demonstrated that IL-6 is present in the prostate tumor microenvironment and is restricted almost exclusively to the stromal compartment. The present study examined the influence of paracrine IL-6 signaling on prostate tumor growth using allograft models of mouse prostate cancer (TRAMP-C2), colon cancer (MC38), and melanoma (B16) cell lines in wildtype (WT) and IL-6 knockout (IL-6-/- ) mice. Cells were implanted into WT or IL-6-/- mice and tumor sizes were measured at a 3 to 4 day interval. Serum, tumors, and other organs were collected for IL-6 analysis by ELISA and RNA in situ hybridization (RISH). There was a significant reduction in TRAMP-C2 and B16 tumor size grown in IL-6-/- mice versus WT mice (P = 0.0006 and P = 0.02, respectively). This trend was not observed for the MC38 cell line. RISH analysis of TRAMP-C2 tumors grown in WT mice showed that cells present in the tumor microenvironment were the primary source of IL-6 mRNA, not the TRAMP-C2 cells. Serum IL-6 ELISA analyses showed an increase in the circulating levels of IL-6 in WT mice bearing TRAMP-C2 tumors. Similar phospho-STAT3 expression and tumor vascularization were observed in TRAMP-C2 tumors grown in WT and IL-6-/- mice. Our results are consistent with previous studies in prostate cancer patients demonstrating that paracrine IL-6 production in the tumor microenvironment may influence tumor growth. Additionally, these data provide evidence that elevated systemic IL-6 levels may be involved in tumor growth regulation in prostate cancer, and are not simply caused by or indicative of tumor burden.

IL-33 facilitates proliferation of colorectal cancer dependent on\xa0COX2/PGE2

BackgroundInterleukin-33 (IL-33) participates in various types of diseases including cancers. Previous studies of this cytokine in cancers mainly focused on its regulation on immune responses by which IL-33 modulated cancer progression. The IL-33 triggered signals in cancer cells remain unclear.MethodsWe analyzed IL-33 gene expression in human colorectal cancer (CRC) tissues and carried out gene enrichment analysis with TCGA Data Portal. We studied CRC proliferation in vivo by inoculating MC38 tumors in IL-33 transgenic mice. We investigated the cell proliferation in vitro with primary CRC cells isolated from fresh human CRC tissues, human CRC cell line HT-29 and mouse CRC cell line MC38. To evaluate the proliferation modulating effects of recombinant IL-33 incubation and other administrated factors, we measured tumor growth, colony formation, cell viability, and the expression of Ki67 and proliferating cell nuclear antigen (PCNA). We used several inhibitors, prostaglandin E2 (PGE2) neutralizing antibody, ST2 blocking antibody and specific shRNA expressing plasmid to study the pathway mediating IL-33-induced CRC proliferation. The IL-33 receptor ST2 in human CRC tissues was detected by immunohistochemistry staining and western blotting. The ST2-positive or negative subsets of primary CRC cells were acquired by flow cytometry sorting.ResultsWe found that IL-33 expression was correlated with the gene signature of cell proliferation in 394 human CRC samples. The MC38 tumors grew more rapidly and the tumor Ki67 and PCNA were expressed at higher levels in IL-33 transgenic mice than in wild-type mice. IL-33 promoted cell growth, colony formation and expression of Ki67 and PCNA in primary CRC cells as well as CRC cell lines. IL-33 activated cycloxygenase-2 (COX2) expression and increased PGE2 production, whereas the COX2 selective inhibitor and PGE2 neutralizing antibody abolished the proliferation promoting effect of IL-33. ST2 blockade, ST2-negative sorting, NF-κB specific inhibitor and NF-κB specific shRNA (shP65) abrogated the COX2 induction caused by IL-33.ConclusionIL-33 facilitates proliferation of colorectal cancer dependent on COX2/PGE2. IL-33 functions via its receptor ST2 and upregulates COX2 expression through NF-κB signaling. Understanding the IL-33 signal transduction in CRC cells provides potential therapeutic targets for clinical treatment.

Abstract 4705: The novel oncolytic peptide, DTT-304, induces regression of colorectal cancer tumors in vivo

Abstract Colorectal cancer (CRC) is the third most common cancer in the world, and approximately 50% of the patients develop liver metastasis, which is a major cause of disease mortality. New drugs and treatment options are therefore necessary to improve the survival of these patients. Lately, different types of immunotherapy, such as check point inhibitors, have shown great promise in the treatment of metastatic cancer, although for most patients with CRC the efficacy has been restricted to cases with microsatellite instable disease. Another novel immunotherapeutic strategy is oncolytic immunotherapy, which has been shown to cause immunogenic cancer cell death. Following cancer cell lysis, damage associated molecular patterns (DAMPs) are released, which are recognized by immune cell receptors. Pro-inflammatory responses will then be triggered to induce release of tumor specific antigens that are presented to the immune system, leading to tumor-specific immune responses. In the present study, we have investigated the efficacy of a novel oncolytic peptide, DTT-304, using two mouse models. Subcutaneous tumors were established in Balb c mice using the cell line CT26 and in C57Bl6 mice using the cell line MC38, both murine CRC cell lines. In both models, intra-tumoral injection of the peptide induced tumor necrosis and complete regression a few days after initiation of treatment, thus showing the potential of this peptide for use in colorectal cancer. In ongoing experiments we are investigating if the peptide has the potential to induce long lasting protective tumor-specific immune responses by re-challenging previously treated mice with the same cell line in a liver metastasis model. In addition, the possibility of treating liver tumors by direct peptide injection will be investigated. Citation Format: Karianne G. Fleten, Johannes J. Eksteen, Brynjar Mauseth, Ketil Camilio, Øystein Rekdal, Meng Yu Wang, Gunhild M. Mælandsmo, Kjersti Flatmark. The novel oncolytic peptide, DTT-304, induces regression of colorectal cancer tumors in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4705.

Abstract LB-151: Prophylactic TNFα blockade unplugs toxicity and efficacy in immunotherapy anti-PD-1 + anti-CTLA-4 combinations

Abstract The combination of anti-CTLA-4 and anti-PD-1 checkpoint inhibitors ipilimumab and nivolumab (DIT) has improved survival of patients with metastatic melanoma and renal cell carcinoma in exchange for frequent immune-mediated serious side effects (mainly gastrointestinal), leading to treatment cessation in about one third of the patients and the need to reduce doses of these immunotherapy agents when used in combination. Tumor necrosis factor-α (TNF-α) is an important actionable mediator in inflammatory bowel disease (both ulcerative colitis and Crohn's disease) and its blockade with monoclonal antibodies has been recommended to treat steroid-resistant colitis following checkpoint inhibitors. We have studied whether prophylactic TNFα blockade is able to reduce immune toxicity associated with DIT without altering antitumor activity. Experiments were performed in C57BL/6 mice harboring subcutaneous tumors derived from the MC38 and B16OVA cell lines. Acute immune-mediated colitis was induced by exposure to 3% dextran sulfate sodium (DSS) in drinking water for 3 days. For TNFα blockade etanercept (hTNFR2-IgG chimera) or a mouse anti-TNF-α monoclonal antibody were used. Anti-tumor efficacy was tested following individual tumor sizes upon combined treatment with anti-mPD-1 + anti-mCTLA4 mAbs. Severity of DSS colitis was assessed and scored by daily weight, ultrasound evaluation of intestinal wall thickness and histological assessment on day 7. We observed that the antitumoral efficacy of PD-1+CTLA-4 blockade against MC38 of B16OVA established tumors was not reduced with the prophylactic association of TNF-α blockade. Intriguingly, an improvement of antitumor activity was observed in those mice in which DSS colitis was induced. Importantly, in mice suffering DSS-induced colitis that were co-treated with DIT and TNF-α blockade, digestive toxicity ameliorates as shown by body weight, ultrasound examination and histology. Therefore, TNF-α blockade can be prophylactically associated with anti-PD1 + anti-CTLA-4 mAb combination regimens to prevent toxicity without hindrance of anti-tumor efficacy. These results in mouse models warrant a clinical trial testing profilactic etanercept to improve the safety profile of the nivolumab + ipilimumab combination widening its therapeutic window. Citation Format: ELISABETH PEREZ-RUIZ, LUNA MINUTE, MARIA CARMEN OCHOA, ARANTXA AZPILICUETA, SARAY GARASA, M E RODRIGUEZ-RUIZ, VIRGINIA BELSUE, CARMEN MOLINA, PEDRO BERRAONDO, IGNACIO MELERO. Prophylactic TNFα blockade unplugs toxicity and efficacy in immunotherapy anti-PD-1 + anti-CTLA-4 combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-151.

1183 Surgical damage to the lymphatic system promotes tumor growth via impaired adaptive immune response

Adaptive immunity plays a crucial role in tumor clearance. Both lymph nodes (LNs) and lymphatic channels from primary sites to regional LNs are critical for initiation of adaptive immunity. However, as LNs are common metastatic sites in skin cancers, LN biopsies or dissections are frequently performed. In addition, reconstructive skin flaps after tumor resection may damage lymphatic flow from primary sites to regional LNs. In this study, we developed a murine model that simulates LNs dissection or skin flap that blocks lymphatic flow from primary sites to regional LNs and monitored tumor progression. As a poor immunogenic tumor line, the growth of inoculated B16F10 melanoma into syngeneic C57BL/6 mice was not affected by these surgeries. However, the growth of the same cell line in allogenic Balb/c mice was significantly accelerated by flap surgeries or LN dissection (p=0.0016 and p=0.0001, respectively). In addition, immune cell infiltration including CD4+ and CD8+ T cells into the tumor was reduced by these surgeries (CD4+ T cells: p=0.0185 and p=0.0235, respectively, CD8+ T cells: p=0.0122 and p=0.0104, respectively). Moreover, both cytotoxicity against B16F10 melanoma (p=0.05 and p=0.02, respectively) and numbers of apoptotic tumor cells (p=0.0237 and p=0.0055, respectively) were significantly diminished by these surgeries. Similarly, tumor growth of the immunogenic MC38 cell line in syngeneic C57BL/6 mice was significantly accelerated by these surgeries (p=0.0125 and p=0.0413, respectively). In this model, immune cell infiltration were also reduced by these surgeries (CD4+ T cells: p=0.0147 and p=0.0201, respectively, CD8+ T cells: p=0.0424 and p=0.0124, respectively). Moreover, apoptotic tumor cells were diminished by these surgeries (p=0.0431 and p=0.0434, respectively). These results strongly indicate that surgical damage of the lymphatic system may promote tumor progression via impaired adaptive immune response.

Surgical damage to the lymphatic system promotes tumor growth via impaired adaptive immune response

BackgroundBoth lymph nodes (LNs) and lymphatic channels from primary sites to regional LNs are critical for initiation of adaptive immunity. However, as LNs are common metastatic sites in skin cancers, LN biopsies or dissections are frequently performed. In addition, reconstructive skin flaps after tumor resection may damage lymphatic flow from primary sites to regional LNs. ObjectiveThis study was designed to investigate the effect on tumor progression by such surgeries. MethodsWe developed a mouse model that simulates LNs dissection or skin flap that blocks lymphatic flow from primary sites to regional LNs and monitored tumor progression. ResultsAs a poor immunogenic tumor line, the growth of inoculated B16F10 melanoma into syngeneic C57BL/6 mice was not affected by these surgeries. However, the growth of the same cell line in allogenic Balb/c mice was accelerated while immune cell infiltration (CD4+ and CD8+ T cells) into the tumor was reduced by these surgeries. In addition, both cytotoxicity against B16F10 melanoma and numbers of apoptotic tumor cells were diminished by these surgeries. Similarly, tumor growth of the immunogenic MC38 cell line in syngeneic C57BL/6 mice was accelerated and immune cell infiltration and apoptotic tumor cells were reduced by these surgeries. ConclusionThese results strongly indicate that surgical damage of the lymphatic system may promote tumor progression via impaired adaptive immune response.

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.

Molecular characterization of pro-metastatic functions of β4-integrin in colorectal cancer.

The β4-integrin subunit has been implicated in development and progression of several epithelial tumor types. However, its role in metastases of colorectal cancer (CRC) remains elusive. To study CRC metastasis, we generated a highly invasive, metastatic cell line MC38-LM10 (LM10) by passaging mouse CRC MC38 cells ten times, using a splenic injection model of liver metastasis. Affymetrix microarray analyses of LM10 and MC38 cell lines and their corresponding liver metastases generated a gene signature for CRC metastasis. This signature shows strong upregulation of β4-integrin in LM10 cells and corresponding metastases. Upregulation of β4-integrin in highly aggressive LM10 cells is associated with increased migration, invasion, and liver metastases. Furthermore, stable knockdown of β4-integrin in human CRC SW620 cells reduces Bcl-2 expression, increases apoptosis, and decreases invasion, tumorigenicity, and liver metastasis, thus resulting in significantly increased survival of mice (hazard ratio = 0.32, 95% confidence interval = 0.15-0.66, P<0.01). Patients with CRC tumors display higher β4-integrin levels in stages 1-4 and significantly lower survival rate. Collectively, β4-integrin plays a critical role in CRC progression, invasion, and metastasis, suggesting that it could be a potential therapeutic target for CRC patients.

Abstract LB-218: Validation of a novel microfluidic device for screening of immune checkpoint inhibitors using 3D organotypic tumor spheroids

Abstract Immune checkpoint blockade, including anti-PD-1 and PD-L1 therapies, is revolutionizing cancer care for melanoma, lung and other malignancies. However, pre-clinical models in which to rapidly and robustly evaluate the efficacy of these treatment approaches, including the development of combination therapies and/or relevant biomarkers, are lacking. We have developed a novel approach for evaluating ex vivo response to immune checkpoint blockade using murine- and patient derived organotypic tumor spheroids (mDOTS/pDOTS) cultured in a new 3D microfluidic system. We have demonstrated that spheroids isolated from fresh mouse and human tumor samples retain autologous lymphoid and myeloid cell populations. Methods: Using MC38 and B16F10 syngeneic mouse cancer models with reported responsiveness or resistance to PD-1 blockade respectively, we treated mDOTS in the 3D microfluidic device with either control IgG or increasing concentrations of anti-PD-1 for up to 6 days and quantified cell viability by dual labeling fluorescence microscopy. To confirm tumor cell-specific death and survival of mDOTS immune component we applied multi-color immunofluorescent staining of CD8 as well as CD11b+ and CD11c+ myeloid cells in parallel with viability dye calcein AM. Tumor cell-specific death was assessed by absence of calcein AM and pyknotic nuclear staining in CD45 negative mDOTS component. Results: We demonstrated dose-dependent killing following treatment with anti-PD-1 in MC38 spheroids. We showed presence of viable cytotoxic lymphocytes as well as other immune cells for up to 5 days of ex vivo growth, when we observed the beginning of tumor but not immune cell killing. In contrast, mDOTS derived from B16F10 melanoma, which is reported to be minimally sensitive to single agent PD-1 blockade, exhibited little cell death despite identical treatment and comparable immune profiles (at highest anti-PD-1 dose of 10ug/mL viability was 98.2%+0.04 versus 29.7%+1.68 in MC38) . No significant cell death (viability 88.2%+2.18) was also noted in spheroids generated from MC38 cell line alone, conceivably due to the absence of autologous cytotoxic T cells. Additional analysis of immune dynamics will be discussed. Conclusions: These data demonstrate the ability to recapitulate sensitivity and innate resistance to PD-1 blockade ex vivo. By being able to study the impact of immunotherapy and targeted therapy in a rapid ex vivo system the development of drug combinations and their associated biomarkers can be significantly enhanced. The preclinical studies can be used to develop and guide the next generation clinical trials for lung cancer patients, and hopefully improving their success rate. Citation Format: Elena Ivanova, Amir Aref, Russel Jenkins, Patrick Lizotte, Wei Huang, Sangeetha Palakurthi, Paul Kirschmeier, David Barbie, Cloud Paweletz, Pasi Janne, Kwok-Kin Wong. Validation of a novel microfluidic device for screening of immune checkpoint inhibitors using 3D organotypic tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-218. doi:10.1158/1538-7445.AM2017-LB-218

Combination therapy with dendritic cells and lenalidomide is an effective approach to enhance antitumor immunity in a mouse colon cancer model.

In this study, we investigated efficacy of lenalidomide in combination with tumor antigen-loaded dendritic cells (DCs) in murine colon cancer model. MC-38 cell lines were injected subcutaneously to establish colon cancer-bearing mice. After tumor growth, lenalidomide (50 mg/kg/day) was injected intraperitoneally on 3 consecutive days in combination with tumor antigen-loaded DC vaccination on days 8, 12, 16, and 20. The tumor antigen-loaded DCs plus lenalidomide combination treatment exhibited a significant inhibition of tumor growth compared with the other groups. These effects were associated with a reduction in immune suppressor cells, such as myeloid-derived suppressor cells and regulatory T cells, with the induction of immune effector cells, such as natural killer cells, CD4+ T cells and CD8+ T cells in spleen, and with the activation of cytotoxic T lymphocytes and NK cells. This study suggests that a combination of tumor antigen-loaded DC vaccination and lenalidomide synergistically enhanced antitumor immune response in the murine colon cancer model, by inhibiting the generation of immune suppressive cells and recovery of effector cells, and demonstrated superior polarization of Th1/Th2 balance in favor of Th1 immune response. This combination approach with DCs and lenalidomide may provide a new therapeutic option to improve the treatment of colon cancer.