Immune Response In Colorectal Cancer Research Articles

DDX10 Promotes the Proliferation and Metastasis of CRC Cells via RPL35

Colorectal cancer (CRC) has become the second deadliest cancer worldwide and severely threatens human health. More and more studies focus on the role of The RNA helicase DEAD-box (DDX) family in CRC. However, mechanism of DDX10 in CRC has not been elucidated. In the present study, we identified DDX10 as being highly expressed in CRC by TCGA and GEO databases. DDX10 expression was measured by immunochemical staining in 86 CRC patients. Biological roles of DDX10 were explored by cell proliferation, migration and invasion in vitro and in vivo, cell cycle assays, and using DDX56‐knockdown or overexpressed CRC cells. We found that DDX10 decreasing markedly reduced CRC cell proliferation, migration and invasion. We constructed the protein-protein interaction network (PPI) by STRING. Using LC-MS/MS and Co-IP experiments, we discovered that RPL35 is the downstream protein of DDX10. In addition, we conjecture RPL35 is related to E2F1 and immune response in CRC. In conclusion, our study helps us understand the molecular mechanisms of DDX10 in CRC, and provide candidate targets for diagnosis and treatment of CRC. Funding Information: This study was supported by the project of Suzhou Technology Bureau (No. SYS2018055), Suzhou health talent project (No. GSWS2020037), medical talent project of Jiangsu Commission of Health (No. QNRC2016873), the project of research and practice innovation plan for Postgraduates in Jiangsu Province (No. KYCX20-2721). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: The study was approved by the ethics committee of the Second Affiliated Hospital of Soochow University. All patients signed the informed consent form.

Molecular correlates of immune cytolytic subgroups in colorectal cancer by integrated genomics analysis.

Although immune checkpoint inhibition (ICI) has shown promising results in metastatic dMMR/MSI-H colorectal cancer (CRC), the majority of pMMR/MSS patients do not respond to such therapies. To systematically evaluate the determinants of immune response in CRC, we explored whether patients with diverse levels of immune cytolytic activity (CYT) have different patterns of chromothripsis and kataegis. Analysis of CRC genomic data from the TCGA, indicated an excess of chromothriptic clusters among CYT-low colon adenocarcinomas, affecting known cancer drivers (APC, KRAS, BRAF, TP53 and FBXW7), immune checkpoints (CD274, PDCD1LG2, IDO1/2 and LAG3) and immune-related genes (ENTPD1, PRF1, NKG7, FAS, GZMA/B/H/K and CD73). CYT-high tumors were characterized by hypermutation, enrichment in APOBEC-associated mutations and kataegis events, as well as APOBEC activation. We also assessed differences in the most prevalent mutational signatures (SBS15, SBS20, SBS54 and DBS2) across cytolytic subgroups. Regarding the composition of immune cells in the tumor milieu, we found enrichment of M1 macrophages, CD8+ T cells and Tregs, as well as higher CD8+ T-cells/Tregs ratio among CYT-high tumors. CYT-high patients had higher immunophenoscores, which is predictive of their responsiveness if they were to be treated with anti-PD-1 alone or in combination with anti-CTLA-4 drugs. These results could have implications for patient responsiveness to immune checkpoint inhibitors.

Tumor RNA-loaded nanoliposomes increases the anti-tumor immune response in colorectal cancer

Purpose Tumor RNA vaccines can activate dendritic cells to generate systemic anti-tumor immune response. However, due to easily degraded of RNA, direct RNA vaccine is less effective. In this study, we optimized the method for preparing PEGylated liposom-polycationic DNA complex (LPD) nanoliposomes, increased encapsulate amount of total RNA derived from CT-26 colorectal cancer cells. Tumor RNA LPD nanoliposomes vaccines improved anti-tumor immune response ability of tumor RNA and can effectively promote anti-tumor therapeutic effect of oxaliplatin. Methods Total tumor-derived RNA was extracted from colorectal cancer cells (CT-26 cells), and loaded to our optimized the LPD complex, resulting in the LPD nanoliposomes. We evaluated the characteristics (size, zeta potential, and stability), cytotoxicity, transfection ability, and tumor-growth inhibitory efficacy of LPD nanoliposomes. Results The improved LPD nanoliposomes exhibited a spherical shape, RNA loading efficiency of 9.07%, the average size of 120.37 ± 2.949 nm and zeta potential was 3.34 ± 0.056 mV. Also, the improved LPD nanoliposomes showed high stability at 4 °C, with a low toxicity and high cell transfection efficacy toward CT-26 colorectal cancer cells. Notably, the improved LPD nanoliposomes showed tumor growth inhibition by activating anti-tumor immune response in CT-26 colorectal cancer bearing mice, with mini side effects toward the normal organs of mice. Furthermore, the effect of the improved LPD nanoliposomes in combination with oxaliplatin can be better than that of oxaliplatin alone. Conclusion The improved LPD nanoliposomes may serve as an effective vaccine to induce antitumor immunity, presenting a new treatment option for colorectal cancer.

ZG16 regulates PD-L1 expression and promotes local immunity in colon cancer

CRC is a heterogeneous disease due to global molecular alterations, including mismatch-repair-deficient (dMMR) and microsatellite instability-high (MSI-H). Immunotherapy has achieved durable responses in a subset of patients with dMMR-MSI-H metastatic CRC. It has been showed that Loss of ZG16 is highly associated with colorectal cancer. However, whether ZG16 modulates tumor immunity in colorectal cancer is unclear. In this study, we demonstrated that the expression of ZG16 is associated with distant metastasis and lymphatic invasive in colorectal cancer. Besides, ZG16 is negatively correlated to PD-L1 expression in patient with CRC and overexpression of ZG16 blocks PD-L1 expression in colorectal cancer cells. In addition, overexpression of ZG16 promotes NK cells survival and proliferation, which is dependent on NKG2D expression. Our data demonstrate that ZG16 plays a role in modulation of immune response in colorectal cancer. The strong correlation between ZG16 and PD-L1 suggests that ZG16 may serve a biomarker to stratify patient who will benefit from immunotherapy.

The role of sex in the innate and adaptive immune environment of metastatic colorectal cancer

BackgroundWomen with colorectal cancer (CRC) have a significant survival advantage over men. Sex influences on the tumour microenvironment (TME) are not well characterised, despite the importance of immune response in CRC. We hypothesised that sex-divergent immune responses could contribute to survival.MethodsUsing a murine model of metastatic CRC, we examined T cells, macrophages, and cytokines locally and systemically. TME and serum cytokines were measured by multiplex bead-based arrays, while FCA was used to identify cells and phenotypes. IHC provided spatial confirmation of T cell infiltration.ResultsFemales had increased survival and T cell infiltration. CD8, CD4 and Th2 populations correlated with longer survival. Males had increased serum levels of chemokines and inflammation-associated cytokines. Within the TME, males had lower cytokine levels than females, and a shallower cytokine gradient to the periphery. Female tumours had elevated IL-10+ macrophages, which correlated with survival.ConclusionsThese data demonstrate survival-associated differences in the immune response of males and females to metastatic CRC. Females showed changes in cytokine production accompanied by increased immune cell populations, biased toward Th2-axis phenotypes. Key differences in the immune response to CRC correlated with survival in this model. These differences support a multi-faceted shift across the TME.

PD-1/PD-L1-dependent immune response in colorectal cancer.

Colorectal cancer (CRC) is still considered as the third most frequent cancer in the world. Microsatellite instability (MSI), inflammation, and microRNAs have been demonstrated as the main contributing factors in CRC. Subtype 1 CRC is defined by NK cells infiltration, induction of Th1 lymphocyte and cytotoxic T cell responses as well as upregulation of immune checkpoint proteins including programmed cell death-1 (PD-1). Based on the diverse features of CRC, such as the stage and localization of the tumor, several treatment approaches are available. However, the efficiency of these treatments may be decreased due to the development of diverse resistance mechanisms. It has been proven that monoclonal antibodies (mAbs) can increase the effectiveness of CRC treatments. Nowadays, several mAbs including nivolumab and pembrolizumab have been approved for the treatment of CRC. Immune checkpoint receptors including PD-1 can be inhibited by these antibodies. Combination therapy gives an opportunity for advanced treatment for CRC patients. In this review, an update has been provided on the molecular mechanisms involved in MSI colorectal cancer immune microenvironment by focusing on PD-ligand 1 (PD-L1) and treatment of patients with advanced immunotherapy, which were examined in the different clinical trial phases. Considering induced expression of PD-L1 by conventional chemotherapeutics, we have summarized the role of PD-L1 in CRC, the chemotherapy effects on the PD-1/PD-L1 axis and novel combined approaches to enhance immunotherapy of CRC by focusing on PD-L1.

66P - Dual inhibition of TGF-β and AXL as a novel treatment for colorectal cancer

BackgroundAXL and transforming growth factor β (TGF-β) signalling pathways play a key role in modulating tumour microenvironment by promoting epithelial to mesenchymal transition (EMT), stromal activation and regulating immune response in colorectal cancer (CRC). Here we have evaluated the effect of double blockade of TGF-β and AXL receptors, as an innovative therapeutic strategy for CRC. MethodsWe assessed the correlation of TGF-β and AXL by an in silico analysis of a human CRC gene expression profile database. Afterwards, an evaluation of AXL expression of in a panel of human CRC cell lines by Western Blot (WB) and Real time PCR was performed. The sensitivity of HCT116 and LOVO cells to treatment with galunisertib (LY21209761), a TGF-β R1 inhibitor, and R428, an AXL inhibitor, was assessed by MTT, cell migration, and colony forming assays. Furthermore we generated patient derived 3D spheroid cultures from primary CRC, and tested their susceptibility to galunisertib and R428 by MTT assay. Finally, in vivo experiments were done with HCT116 and LOVO tumour xenografts in immunodeficient mice. ResultsIn silico analysis showed an association between high expression levels of AXL and TGF-β R1 in CMS4 CRC. AXL was differently expressed on cell lines, and interestingly it resulted increased in HCT116 and LOVO after TGF-β inhibition, probably representing a mechanism of cancer cell escape. Dual blockade with galunisertib and R428 determined a reduction in cell migration of 50% in HCT116 and 37% in LOVO cells, respectively, and colony formation of approximately 90 % in both cell lines. Moreover, combined treatment displayed a strong synergistic activity in 3D cultures derived from five human CRC samples, resulting in the inhibition of proliferation ranging from 80 to 90%. The in vivo experiments of HCT116 and LOVO subcutaneous xenograft models are currently on going and results will be presented. ConclusionsCombined TGF-β and AXL inhibition showed a promising activity in preclinical models of CRC and could represent a novel potential therapeutic strategy for patients with CRC. Legal entity responsible for the studyUniversità degli studi della Campania "Luigi Vanvitelli". FundingICURE project, Università degli studi della Campania "Luigi Vanvitelli". DisclosureD. Melisi: Research grant/Funding (self): Lilly.

Harnessing the Differential Immune Signature of High Versus Low Linear Energy Transfer Protons to Elicit Potent Systemic Immune Responses in Colorectal Cancer

Harnessing the Differential Immune Signature of High Versus Low Linear Energy Transfer Protons to Elicit Potent Systemic Immune Responses in Colorectal Cancer

Prognostic significance of inflammatory cell response in patients with colorectal cancer.

Cancer cells are characterized by a low antigenic immunogenicity, a rapid growth and an immunosuppressive effect on the extracellular matrix. These properties induce a weak immune response in colorectal cancer (CRC) carcinogenesis. It is therefore crucial to determine the composition of the inflammatory mass, including neutrophils, macrophages and eosinophils in the tumor tissue of patients with CRC, and to analyze other clinicopathological parameters. The present study included 144 patients diagnosed with CRC. Tissue samples obtained from routine histopathological diagnosis were stained with hematoxylin and eosin. Inflammatory cells were assessed in the invasive front and in the center of the tumor by light microscopy under a high-power magnification. The percentage of neutrophils in the invasive front was significantly higher compared with that in the center of the tumor mass (P<0.01). Macrophages and eosinophils were present in the invasive front and in the center of tumor mass in most cases. The presence of neutrophils, macrophages and eosinophils was correlated with various clinicopathological features. Patients with macrophages present in the center of tumor mass had longer disease-free survival time (P=0.041). In conclusion, the present study demonstrated that the inflammatory cell infiltrate served a significant role in the immune response of patients with CRC. It should be noted that the presence of macrophages localized in the stroma of the central part of the primary tumor mass was associated with the survival time of patients with CRC.

The density of Tbet+ tumor-infiltrating T lymphocytes reflects an effective and druggable preexisting adaptive antitumor immune response in colorectal cancer, irrespective of the microsatellite status

ABSTRACTPurpose: The recent success of anti-PD1 antibody in metastatic colorectal cancer (CRC) patients with microsatellite instability (MSI), known to be associated with an upregulated Th1/Tc1 gene signature, provides new promising therapeutic strategies. However, the partial objective response highlights a crucial need for relevant, easily evaluable, predictive biomarkers. Here we explore whether in situ assessment of Tbet+ tumor infiltrating lymphocytes (TILs) reflects a pre-existing functional antitumor Th1/Tc1/IFNγ response, in relation with clinicopathological features, microsatellite status and expression of immunoregulatory molecules (PD1, PDL1, IDO-1).Methodology: In two independent cohorts of CRC (retrospective n = 80; prospective n = 27) we assessed TILs density (CD3, Tbet, PD1) and expression profile of PDL1 and IDO-1 by immunohistochemistry/image analysis. Furthermore, the prospective cohort allowed to perform ex vivo CRC explant cultures and measure by Elisa the IFNγ response, at baseline and upon anti-PD1 treatment.Results: The density of Tbet+ TILs was significantly higher in MSI CRC, especially in the medullary subtype but also in a subgroup of MSS (microsatellite stable), and positively correlated with PD1 and PDL1 expression, but not with IDO-1. Finally, a high number of Tbet+ TILs was associated with a favorable overall survival. These Tbet+ TILs were functional as their density positively correlated with basal IFNγ levels. In addition, the combined score of Tbet+ PD1+ TILs coupled with IDO-1 expression predicted the magnitude of the IFNγ response upon anti-PD1.Conclusion: Altogether, immunohistochemical quantification of Tbet+ TILs is a reliable and accurate tool to recapitulate a preexisting Th1/Tc1/IFNγ antitumor response that can be reinvigorated by anti-PD1 treatment.

STING signaling is a potential immunotherapeutic target in colorectal cancer.

Background: Stimulator of Interferon Genes (STING) is an innate immune sensor for cytosolic DNA. STING signaling activation is indispensable for type I interferon response and the anti-cancer immune response by CD8+ T cells. The aim of this study was to characterize intratumoral STING expression pattern and its clinical implication in colorectal cancer (CRC).Methods: We analyzed STING and CD8 expression in 225 CRC patients who underwent surgical resection. Clinicopathological variables and survival outcomes were analyzed according to STING expression levels. Mice with syngeneic MC38 tumors were also treated with a STING agonist, and tumor microenvironments were analyzed using immunofluorescent staining and flow cytometry.Results: Distinct STING expression was observed in the CRC tumor specimens. Patients with higher STING expression had early stage cancer with increased intratumoral CD8+ T cell infiltration and less frequent lymphovascular invasion. Compared to CRC patients with lower STING expression, those with higher STING expression had longer overall and recurrence-free survival. Multivariate Cox regression model also revealed higher STING expression to be an independent prognostic factor for better overall survival. When MC38 colon tumors were treated with intratumoral injection of STING agonist, tumor growth was remarkably suppressed with increased intratumoral CD8+ T cell infiltration. Moreover, T-cell activation markers, ICOS and IFN-γ, were also upregulated in CD8+ T cells, indicating enhanced effector T cell function after STING treatment.Conclusion: We confirmed the distinct STING expression in CRC and demonstrated its independent prognostic value in survival outcomes. STING could be a potential therapeutic target that enhances anti-cancer immune response in CRC.

Abstract 4061: The inflammasome of tumor cells modulates the biology of tumor-infiltrating T lymphocytes in colorectal cancer

Abstract In colorectal cancer (CRC), little is known about mechanisms by which tumor cells can influence the biology of Tumor Infiltrating T lymphocytes (TILs) present in the tumor microenvironment. One of these mechanisms could be modulation of the inflammasome of tumor cells. The inflammasome is a molecular platform present in normal intestinal epithelial cells, whose effector protein, caspase-1, can rapidly mature IL-18 and generate a mucosal Th1 (IFNγ) response. However, the inflammasome status of tumor cells in CRC and its potential role in the biology of TILs are unknown yet. This prospective study aimed to determine in CRC patients (n = 50) : i) the status of the inflammasome (caspase-1 / IL-18 axis) in tumor cells according to their microsatellite status [unstable (MSI) or stable (MSS)], in relation with the density of Th1/Tc1 TILs (T-bet+) and with the levels of the prototype Th1 cytokine IFNγ, secreted in an ex vivo explant culture model of CRC we developed and ii) the impact of the inflammasome-dependent cytokines potentially secreted by tumor cells on the biology of isolated TILs. Our study delineates two major groups of patients. The first group (33% of cases, mostly MSS with a low immunoscore) featured no active caspase-1 in tumor cells, no or low levels of mature IL-18 and IFNγ and only few T-bet+ TILs in the tumor microenvironment. This profile could correspond to an immune escape mechanism facilitating tumor progression. The second group (66% of cases, both MSI and MSS with high immunoscore) featured active caspase-1 in tumor cells associated with mature IL-18 secretion, high density of T-bet+ TILs expressing IL-18Rα and IFNγ release in most cases. In addition, isolated IL-18Rα+ TILs cultured with recombinant IL-18 were able to secrete IFNγ, either left unstimulated or stimulated with anti-CD3. In these CRC, the inflammasome of tumor cells, maintained and active, can contribute to a Th1/Tc1 antitumor response elicited by TILs present in the microenvironment that can modulate tumor growth. Interestingly, in a few cases of this second group (MSI CRC), the numerous T-bet+ TILs were unable to generate a Th1 response. Noticeably, these TILs express numerous immune checkpoints including PD1 and TIGIT, potentially responsible for their exhaustion. This study is the first to delineate functional interactions between tumor cells and TILs in CRC, using ex vivo explant cultures. Altogether, our findings support the inflammasome of tumor cells as a potential new therapeutic target to strengthen the Th1/Tc1 immune response in CRC, in association with immune checkpoint blockers. This work is supported by the “Ligue contre le Cancer Grand Ouest” Citation Format: Linda Bilonda, Delphine Dansette, Cecile Deleine, Romain Oger, Nicolas Jouand, Juliette Podevin, Pierre Fourquier, Emilie Thibaudeau, Jerome Chetritt, Jean-François Mosnier, Celine Bossard, Nadine Gervois, Anne Jarry. The inflammasome of tumor cells modulates the biology of tumor-infiltrating T lymphocytes in colorectal cancer [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 4061.

Evolving notions on immune response in colorectal cancer and their implications for biomarker development.

Colorectal cancer (CRC) still represents the third most commonly diagnosed type of cancer in men and women worldwide. CRC is acknowledged as a heterogeneous disease that develops through a multi-step sequence of events driven by clonal selections; this observation is sustained by the fact that histologically similar tumors may have completely different outcomes, including a varied response to therapy. In "early" and "intermediate" stage of CRC (stages II and III, respectively) there is a compelling need for new biomarkers fit to assess the metastatic potential of their disease, selecting patients with aggressive disease that might benefit from adjuvant and targeted therapies. Therefore, we review the actual notions on immune response in colorectal cancer and their implications for biomarker development. The recognition of the key role of immune cells in human cancer progression has recently drawn attention on the tumor immune microenvironment, as a source of new indicators of tumor outcome and response to therapy. Thus, beside consolidated histopathological biomarkers, immune endpoints are now emerging as potential biomarkers. The introduction of immune signatures and cellular and molecular components of the immune system as biomarkers is particularly important considering the increasing use of immune-based cancer therapies as therapeutic strategies for cancer patients.

The Clinical and Biomarker Association of Programmed Death Ligand 1 and its Spatial Heterogeneous Expression in Colorectal Cancer.

Background: Programmed death ligand 1 (PD-L1) expression has been shown to predict benefit from anti-PD-1 treatment in several cancers. However, its predictive value in colorectal cancer seems limited. This study was aimed to explore the clinical and biomarker association of programmed death ligand 1 and its spatial heterogeneous expression in colorectal cancer.Methods: Tissue microarrays of 422 primary colorectal cancers from our hospital were used for the interpretation of PD-L1 and programmed death 1 (PD-1) expression, cluster of differentiation 4 (CD4) and CD8 density and microsatellite instability (MSI) status by immunohistochemistry. To assess the spatial heterogeneity of PD-L1 expression, Tissue microarrays of 383 paired intra-primary-tumor tissues, and 105 paired lymph node metastatic tumors and 64 paired distant metastatic tumors were also used.Results: PD-L1 was positive in 188 (44.5%) primary colorectal cancers. PD-L1 expression was associated with less advanced N category (P<0.001), less advanced TNM stage (P<0.001) and less nervous invasion (P=0.04). Higher PD-L1 expression was associated with higher PD-1 expression (P<0.001), higher CD4 (P<0.001) and CD8 (P<0.001) density and DNA mismatch repair deficiency (P=0.01). PD-L1 expression was associated with better disease-free survival and overall survival, but it was only an independent prognostic factor for disease-free survival (hazard ratio and 95% confidence interval: 0.42 [0.25-0.72], P<0.001). The probability of inconsistent PD-L1 expression was respectively 17.8%, 31.4% and 39.1% within primary tumors, between primary tumors and lymph node metastatic tumors, and between primary tumors and distant metastatic tumors. All the three differences were statistically significant (P<0.001, P<0.001 and P=0.05, respectively).Conclusions: PD-L1 expression was a marker of pre-existing immune responses in colorectal cancer, however, it was heterogeneously expressed in colorectal cancer, especially between primary and metastatic tumors. This might partially explain the low-efficiency of its predictive value for benefit from anti-PD-1 treatment.

Why Don't Immune Checkpoint Inhibitors Work in Colorectal Cancer?

In recent years, immune checkpoint inhibitors have been shown to be effective in treating manifold types of cancer but less robust in colorectal cancer (CRC). While, the subgroup of CRC with microsatellite instability (MSI; also termed as mismatch repair deficient) showed a moderate response to Pembrolizumab in a single arm phase II clinical trial, microsatellite stable (MSS) cancers were unresponsive. Possible mechanisms that affect immune response in colorectal cancer will be reviewed in this article. We will also propose that histone deacetylase (HDAC) inhibition may reverse the immune editing commonly seen in advanced CRC and render them sensitive to immune checkpoint blockade.

Abstract 5018: Dissecting antigen-specific T-cell response in a novel mouse model of colon cancer

Abstract Immune checkpoint blockade therapies, which unleash the immune system’s ability to recognize and eliminate cancer cells, are currently among the most promising treatments for cancer. Antibodies that block the immune inhibitory receptors CTLA4 and PD1 have shown clinical success, with objective response rates over 20% and some durable responses exceeding 10 years. However, some cancers, such as colorectal cancer (CRC), have proven particularly refractory to immunotherapy. Although it is appreciated that the colon is an immunosuppressive environment, the mechanism and extent of immune suppression remains unclear. Studies of human CRC tumors have shown that T cell infiltration is an important predictor of clinical outcome, yet current mouse models of CRC are not particularly well suited to the mechanistic study of T cell responses against tumor-specific antigens. Syngeneic cell line transplants, by far the most widely used models to study adaptive immune response in CRC, are characterized by a highly complex tumor antigen landscape, and have not employed orthotopic transplant into the colon. In order to track highly specific T cell responses in CRC, we have employed a novel colon organoid-based system, in which transformed organoids expressing model antigens are orthotopically transplanted into the wall of the colon via colonscopy-guided injection, a method pioneered in the lab of Omer Yilmaz. Organoids were harvested from VillinCreER; Apcfl/fl mice and grown in the presence of Wnt ligand; addition of tamoxifen and removal of Wnt from the media facilitated expression of the colon-specifc Cre, deletion of Apc, and selection of transformed organoids capable of growing in the absence of Wnt. To induce expression of model antigens, organoids were infected with a lentiviral vector that, in addition to the fluorescent reporter FusionRed, expressed the T cell antigens SIYRYYGL and two antigens from ovalbumin - SIINFEKL and OVA323-339 - fused to the C-terminus of luciferase (FRLucOS), enabling us to monitor antigen expression in vivo. Organoids were then sorted and injected into the wall of the colon. Previous work by Michael DuPage, in which he expressed the LucOS construct in an autochthonous lung cancer and sarcoma mouse model, provided key insights into the dynamic nature of T cell response against cancer cells. The novel model system described here provides a powerful new tool to dissect antitumor T cell responses in the poorly understood context of CRC. Citation Format: Mary C. Beytagh, Peter M. Westcott, Olivia C. Smith, Jatin Roper, Omer Yilmaz, Tyler E. Jacks. Dissecting antigen-specific T-cell response in a novel mouse model of colon cancer [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 5018. doi:10.1158/1538-7445.AM2017-5018

Intratumoral FoxP3+Helios+ Regulatory T Cells Upregulating Immunosuppressive Molecules Are Expanded in Human Colorectal Cancer.

Regulatory T cells (Tregs) can be antitumorigenic or pro-tumorigenic in colorectal cancer (CRC) depending on the presence of different Treg subsets with various immunosuppressive molecules. Some studies reported the phenotypic characteristics of tumor-infiltrating immune cells in CRC, but limited studies have focused on the co-expression of suppressive molecules on immune cells. The aim of this study was to characterize immune cells in the tumor microenvironment (TME), compared to paired adjacent non-tumor colon tissue of CRC patients. Additionally, we investigated co-expression of immunosuppressive molecules on different Treg subsets in the TME, normal colon tissue, and peripheral blood of CRC patients and healthy donors. In this preliminary study, we report that the majority of CD3+ T cells in the TME are CD4+ T cells with high co-expression of programmed death 1 (PD-1)/cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and PD-1/CD39 molecules. Levels of CD4+FoxP3+Helios+ Tregs were significantly increased in the TME. Furthermore, we observed increased levels of PD-1/CTLA-4 and PD-1/CD39 co-expressing cells within FoxP3+Helios+ and FoxP3+Helios− Treg subsets, indicative of their potent immunosuppressive potential. These results suggest synergistic associations between PD-1/CTLA-4 and PD-1/CD39 in dampening T-cell activation and function along with suppressing tumor-specific immune responses, suggesting that dual blockade of these molecules could be a more effective strategy for inducing antitumor immune responses in CRC.

Role of the inflammasome of tumor cells in modulating the biology of Tumor Infiltrating T lymphocytes (TILs) in colorectal cancer.

e23087 Background: In colorectal cancer (CRC), little is known about mechanisms by which tumor cells can influence the phenotype and biology of Tumor Infiltrating T lymphocytes (TILs) of the tumor microenvironment. One of these mechanisms could be the inflammasome, a molecular platform present in normal intestinal epithelial cells, whose effector protein, caspase-1, can rapidly mature IL18 and generate a mucosal Th1/Tc1 (IFNγ) response. However, the inflammasome status of tumor cells in CRC and its potential role on TILs are unknown yet. Methods: Prospective and retrospective cohort studies aimed to determine in CRC patients: the status of the inflammasome in tumor cells (IL18 immunostaining on tissue microarrays (TMA) and in situ detection of active caspase-1 on frozen sections) and the density of TILs (CD8+, T-bet+) in relation with i) the microsatellite stable (MSS) or unstable (MSI) status of CRC, and ii) the levels of cytokines (IL18, IFNγ) secreted in an ex vivo explant culture model of CRC. Finally, we assessed the effect of recombinant human IL18 (rhIL18) on the IFNγ response of isolated TILs. Results: TMA analysis of the retrospective cohort showed that IL18 was significantly expressed (in more than 50% of tumor cells) in 80% of CRC, especially in MSI CRC, and correlated with a high density of T-bet+ and CD8+ intraepithelial TILs (IEL-TILs). Active caspase-1 was detected in tumor cells in 60% of CRC. In the prospective cohort, the presence of active caspase-1 in tumor cells was associated with high levels of mature IL18 secreted in explant cultures, with high density of T-bet+ TILs and with IFNγ release in most cases. In addition, isolated TILs expressing IL18 receptors (IL18Rα), cultured with rhIL18, were able to secrete IFNγ either unstimulated or stimulated with OKT3. Conclusions: The inflammasome of tumor cells, when maintained and active, can contribute to a Th1/Tc1 antitumor immune response elicited by TILs, that can modulate tumor growth. The inflammasome of tumor cells can thus be considered as a potential new therapeutic target to strengthen the antitumor immune response in CRC, in association with other immunotherapies.

Role of the inflammasome of tumor cells in modulating the biology of Tumor Infiltrating T lymphocytes (TILs) in colorectal cancer.

640 Background: In colorectal cancer (CRC), little is known about mechanisms by which tumor cells can influence the phenotype and biology of Tumor Infiltrating T lymphocytes (TILs) of the tumor microenvironment. One of these mechanisms could be the inflammasome, a molecular platform present in normal intestinal epithelial cells, whose effector protein, caspase-1, can rapidly mature IL18 and generate a mucosal Th1/Tc1 (IFNg) response. However, the inflammasome status of tumor cells in CRC and its potential role on TILs are unknown yet. Methods: Prospective and retrospective cohort studies aimed to determine in CRC patients: the status of the inflammasome in tumor cells (IL18 immunostaining on tissue microarrays (TMA) and in situ detection of active caspase-1 on frozen sections) and the density of TILs (CD8+, T-bet+) in relation with the microsatellite stable (MSS) or unstable (MSI) status of CRC, and the levels of cytokines (IL18, IFNg) secreted in an ex vivo explant culture model of CRC. Finally, we assessed the effect of recombinant human IL18 (rhIL18) on the IFNg response of isolated TILs. Results: TMA analysis of the retrospective cohort showed that IL18 was significantly expressed (in more than 50% of tumor cells) in 80% of CRC, especially in MSI CRC, and correlated with a high density of T-bet+ and CD8+ intraepithelial TILs (IEL-TILs). Active caspase-1 was detected in tumor cells in 60% of CRC. In the prospective cohort, the presence of active caspase-1 in tumor cells was associated with high levels of mature IL18 secreted in explant cultures, with high density of T-bet+ TILs and with IFNg release in most cases. In addition, isolated TILs expressing IL18 receptors (IL18Ra), cultured with rhIL18, were able to secrete IFNg either unstimulated or stimulated with OKT3. Conclusions: The inflammasome of tumor cells, when maintained and active, can contribute to a Th1/Tc1 antitumor immune response elicited by TILs, that can modulate tumor growth. The inflammasome of tumor cells can thus be considered as a potential new therapeutic target to strengthen the antitumor immune response in CRC, in association with other immunotherapies.

Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients.

A humoral immune response against aberrant tumor proteins can be elicited in cancer patients, resulting in the production of auto-antibodies (Abs). By serological proteome analysis we identified the surface membrane protein ADAM10, a metalloproteinase that has a role in epithelial-tumor progression and invasion, as a target of the immune response in colorectal cancer (Crc). A screening carried out on the purified protein using testing cohorts of sera (Crc patients n = 57; control subjects n = 39) and validation cohorts of sera (Crc patients n = 49; control subjects n = 52) indicated that anti-ADAM10 auto-Abs were significantly induced in a large group (74%) of colon cancer patients, in particular in patients at stage II and III of the disease. Interestingly, in Crc patients classified as stage III disease, the presence of anti-ADAM10 auto-Abs in the sera was associated with a favourable follow-up with a significant shifting of the recurrence-free survival median time from 23 to 55 months. Even though the ADAM10 protein was expressed in Crc regardless the presence of auto-Abs, the immature/non-functional isoform of ADAM10 was highly expressed in the tumor of anti-ADAM10-positive patients and was the isoform targeted by the auto-Abs. In conclusion, the presence of anti-ADAM10 auto-Abs seems to reflect the increased tumor expression of the immunogenic immature-ADAM10 in a group of Crc patients, and is associated with a favourable prognosis in patients at stage III of the disease.