Inactivation of interferon signaling promotes the establishment of an immunosuppressive microenvironment in colorectal cancers
ABSTRACT: Tumors arise and grow despite anti-cancer immune responses. These responses can be stimulated by immunotherapies such as immune checkpoint inhibitors (e.g. anti-PD1 antibodies) and chimeric antigen receptors (CAR). Efficacy of these agents in solid tumors including colorectal cancers (CRC) is limited by immunosuppressive tumor microenvironment (TME) that prevents killing of malignant cells by cytotoxic T lymphocytes (CTL). Understanding the nature of TME-generated immunosuppression is of paramount importance. Here we report that TME elicited immunosuppression via eliminating activated CTL; this elimination required TME stress-induced downregulation of the IFNAR1 chain of type I interferon (IFN) receptor and attenuation of its signaling. Downregulation of IFNAR1 was observed in human colorectal cancers (CRC) and in mouse CRC models where it was required for efficient tumor development and progression. Stabilization of IFNAR1 on CTL improved their survival and increased anti- tumor activities of CAR T cells and PD1 inhibitors thereby providing a rationale for targeting IFNAR1 degradation for immunotherapies optimization. Two genotypes of mice were examined either 9 or 21 days post injection of MC38 colon cancer cells or MC38mRFP cells, respectively. 2-3 replicate mice were analyzed on separate arrays for each condition. 6 conditions in total were analyzed.
Project description:The low immunogenicity of many cancer cells and the immunosuppression by various cancers and anti-cancer therapies have been an obstacle in the development of efficacious immunotherapies. Our goal was to test if TLR agonists and anti-cancer chemotherapeutic agents synergize in rendering tumor cells more immunogenic.
Project description:Mouse tumour organoids (MTOs) derived from a compound mutant (LAKTP) intestinal cancer model were orthoptopically transplanted into syngeneic C57BL/6J mice. Tumour-bearing mice were treated with TGF-beta inhibitor Galunisertib or vehicle control. Whole tumour mRNA was extracted from primary tumours and expression profiling was performed with the objective to characterize the tumour microenvironment (TME). As a TGF-beta-activated TME has been associated to a poor prognosis and the CRC consensum molecular subtype CMS4, and the mouse model was found to have such an activated TME, we used the array data to classify these tumours in this mouse model system as CMS4-like. Furthermore, treatment with TGF-beta inhibitor reduced the fibroblast- and T cell-specific TGF-beta response signatures, also associated to poor prognosis in human CRC. This treatment was associated to a strong reduction/prevention of liver metastasis, as well as a reduction of primary tumour (and local carcinomatosis) size. Overall design: For every MTO, two biological replicates were included: every strip was set up with 2 control samples vs 2 treated samples
Project description:Most immunotherapies benefit only subsets of cancer patients, except those with autoimmune diseases due to dysregulation of their immune system. Here, the ARE-del mouse model featuring IFNg-autoimmunity allowed us to examine the relationship between cancer, autoimmunity and immunotherapy. We demonstrate that systemic levels of IFNg below 20 pg/ml in ARE-del+/- (HET) mice exerts moderate anti-tumor effects and increase (p=0.02; ANOVA) overall survival (OS); while higher levels did not that replicate in vivo the anti-tumor “bell shape” response of IFNg. Having confirmed that PD1 and CD40 are expressed in tumor-bearing ARE-del mice, anti-PD1 and anti-CD40 alone or combined were used to improve the antitumor effects of IFNg in ARE-del+/- (HET) mice. In ARE-del+/+ (WT) mice, PD1/CD40 inhibited tumor growth and improve OS because it prevented increased infiltration and trapping of tumor associated macrophages (TAMs) into necrotic areas, an event also decreased by anti-CD40 but promoted by anti-PD1. Conversely, in ARE-del+/- (HET) mice, MHC class II expressing TAM infiltration associated with severe hematological and liver adverse effects hampered the efficiency of PD1/CD40, despite significant tumor growth inhibition and strong induction of IFNg signaling. Thus, excessive activation of IFN negatively impacted immune outcome. Given the lack of consensus on the right levels of IFNg, our data show the upper limit of IFNg that define the efficacy of checkpoint inhibitors in tumor-bearing hosts with autoimmune disease. Overall design: Subcutaneous B16F10 tumor mRNA profiles from wild type (WT) and ARE-del heterozigous female mice treated with anti-PD1, anti-CD40 or anti-PD1/CD40 mAB were generated by deep RNA sequencing.
Project description:Activation of oncogenes often leads to induction of the DNA damage responses and onset of the cell senescence. Given that DNA damage can also trigger production of type I interferons (IFN) that contribute to senescence development, we sought to determine the role of IFN in the oncogene-induced senescence. Our data in mouse model demonstrate that inactivation of IFN signaling is sufficient for inducing melanomas in melanocytes harboring mutant Braf. Restoration of IFN signaling in IFN-deficient melanoma cells induces cell senescence and suppresses melanoma progression. In addition, data in human patients that received high dose IFN therapy and in mouse transplanted tumor models strongly suggest the importance of the non-cell-autonomous IFN signaling. Suppression of IFN signaling mediated by the downregulation of IFN receptor IFNAR1 invariably occurs during development of mouse melanoma. Mice harboring the IFNAR1 mutant, which is relatively resistant to downregulation, delay melanoma development, suppress the metastatic disease, and better respond to treatment with BRAF or PD1 inhibitors. These results suggest that IFN signaling is an important tumor suppressive pathway that inhibits melanoma development and progression. Accordingly, the inhibition of IFN pathway via IFNAR1 downregulation plays a key role in melanoma pathogenesis. Conversely, these data also argue for targeting IFNAR1 downregulation to prevent the metastatic disease and improve the efficacy of molecularly targeted and immune-targeted therapies. Two genotypes of mice were examined at 2 to 3 times after tamoxifen adminstration, with 2 replicates for each condition, yielding 8 samples in total.
Project description:Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) dramatically limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists leading to a nearly tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME thus rendering tumors more responsive to immunotherapy. We treated KP orthotopic tumor-bearing mice with vehicle and FAK inhibitor (FAKi) for 14 days, then extracted total RNA from tumor tissues.
Project description:Recent success in cancer immunotherapy has come from the blockade of inhibitory receptors on T cells, such as programmed cell death-1, which can induce a state of T cell exhaustion upon constant antigen stimulation. Understanding miRNA regulation of PD1 can be useful to discover miRNAs for use in therapy or as prognostic markers in various diseases including cancer, autoimmunity and transplantation. We used microarrays to discover global miRNA expression changes upon PD1 upregulation and identified miRNAs that are both up- and down-regulated. B16F10 cells were injected subcutaneously into C57BL/6 mice and 16 days later CD4+PD1+ and CD4+PD1- were sorted from the lymph nodes and spleen for RNA extraction and hybridization on Affymetrix miRNA array.
Project description:Transcriptome analysis of CD4+ PD1+ T cells during LCMV CL13 infection Gene expression in WT and ERt2-cre;TGFbRII flox virus specific CD4 T cells Mixed chimeras of WT:ERt2cre+TGFbRII flox/flox were infected 9 days with LCMV and splenic CD4+ PD1+CD49d+ Cd8a- T cells sorted from each compartment by congenic marker
Project description:We have identified a CD57+PD1- CD4 T cell phenotype at the time of transplantation that strongly correlates with subsequesnt development of belatacept-resistant rejection. In this study, we used microarray to determine which genes were upregulated in CD57+ compared to CD57- CD4 T cells. Peripheral blood obtained from 5 healthy controls was processed and sorted into CD4+CD57+PD1- and CD4+CD57-PD1- populations. Total RNA was extracted from the sorted populations and quality assessed. cDNA synthesis and amplification was performed, and fragmented and biotinylated samples were hybridized to the Affymetrix Human U133 plus 2.0 probe array. The arrays were scanned and probe intensity measurements were normalized across the samples using the robust multichip average (RMA) algorithm.
Project description:The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. To determine whether Apc disruption is required for tumor maintenance, we developed a mouse model of CRC whereby Apc can be conditionally suppressed using a doxycycline-regulated shRNA. Apc suppression produces adenomas in both the small intestine and colon that, in the presence of Kras and p53 mutations, can progress to invasive carcinoma. In established tumors, Apc restoration drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. Tumor regression is accompanied by the re-establishment of normal crypt-villus homeostasis, such that once aberrantly proliferating cells reacquire self-renewal and multi-lineage differentiation capability. Our study reveals that CRC cells can revert to functioning normal cells given appropriate signals, and provide compelling in vivo validation of the Wnt pathway as a therapeutic target for treatment of CRC Analysis of RNA isolated from colon polyps that presented in shAPC or shAPC/Kras mice as compared to shRenilla (neutral) mouse colon mucosa
Project description:Through a diversity of functional lineages, cells of the innate and adaptive immune system either drive or constrain immune reactions within tumors. Thus, while the immune system has a powerful ability to recognize and kill cancer cells, this function is often suppressed preventing clearance of disease. The transcription factor (TF) BACH2 controls the differentiation and function of multiple innate and adaptive immune lineages, but its role in regulating tumor immunity is not known. Here, we demonstrate that BACH2 is required to establish immunosuppression within tumors. We found that growth of subcutaneously implanted tumors was markedly impaired in Bach2-deficient mice and coincided with intratumoral activation of both innate and adaptive immunity but was dependent upon adaptive immunity. Analysis of tumor-infiltrating lymphocytes in Bach2-deficient mice revealed high frequencies of CD4+ and CD8+ effector cells expressing the inflammatory cytokine IFN-γ. Lymphocyte activation coincided with reduction in the frequency of intratumoral CD4+ Foxp3+ regulatory T (Treg) cells. Mechanistically, Treg-dependent inhibition of CD8+ T cells was required for BACH2-mediated tumor immunosuppression. These findings demonstrate that BACH2 is a key component of the molecular programme of tumor immunosuppression and identify a new target for development of therapies aimed at reversing immunosuppression in cancer. Analysis of tumor-infiltrating lymphocytes in Bach2-deficient mice revealed high frequencies of CD4+ and CD8+ effector cells expressing the inflammatory cytokine IFN-γ. Lymphocyte activation coincided with reduction in the frequency of intratumoral CD4+ Foxp3+ regulatory T (Treg) cells. Mechanistically, Treg-dependent inhibition of CD8+ T cells was required for BACH2-mediated tumor immunosuppression.