ABSTRACT
Considering the limited efficacy of current therapies in lung, colorectal, and pancreatic cancers, innovative combination treatments with diverse mechanisms of action are needed to improve patients' outcomes. Chitinase-3 like-1 protein (CHI3L1) emerges as a versatile factor with significant implications in various diseases, particularly cancers, fostering an immunosuppressive tumor microenvironment for cancer progression. Therefore, pre-clinical validation is imperative to fully realize its potential in cancer treatment. We developed phage display-derived fully human monoclonal CHI3L1 neutralizing antibodies (nAbs) and verified the nAbs-antigen binding affinity and specificity in lung, pancreatic and colorectal cancer cell lines. Tumor growth signals, proliferation and migration ability were all reduced by CHI3L1 nAbs in vitro. Orthotopic or subcutaneous tumor mice model and humanized mouse model were established for characterizing the anti-tumor properties of two CHI3L1 nAb leads. Importantly, CHI3L1 nAbs not only inhibited tumor growth but also mitigated fibrosis, angiogenesis, and restored immunostimulatory functions of immune cells in pancreatic, lung, and colorectal tumor mice models. Mechanistically, CHI3L1 nAbs directly suppressed the activation of pancreatic stellate cells and the transformation of macrophages into myofibroblasts, thereby attenuating fibrosis. These findings strongly support the therapeutic potential of CHI3L1 nAbs in overcoming clinical challenges, including the failure of gemcitabine in pancreatic cancer.
Subject(s)
Antibodies, Monoclonal , Cell Proliferation , Chitinase-3-Like Protein 1 , Colorectal Neoplasms , Fibrosis , Lung Neoplasms , Neovascularization, Pathologic , Pancreatic Neoplasms , Animals , Chitinase-3-Like Protein 1/metabolism , Chitinase-3-Like Protein 1/antagonists & inhibitors , Humans , Colorectal Neoplasms/pathology , Colorectal Neoplasms/immunology , Colorectal Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/drug therapy , Neovascularization, Pathologic/drug therapy , Mice , Cell Line, Tumor , Lung Neoplasms/pathology , Lung Neoplasms/immunology , Lung Neoplasms/drug therapy , Cell Proliferation/drug effects , Antibodies, Monoclonal/pharmacology , Tumor Microenvironment/drug effects , Xenograft Model Antitumor Assays , Antibodies, Neutralizing/pharmacology , Antineoplastic Agents, Immunological/pharmacology , AngiogenesisABSTRACT
Background: Chitinase 3-like-1 (CHI3L1) is a secretion glycoprotein associated with the immunosuppressive tumor microenvironment (TME). The secretory mode of CHI3L1 makes it a promising target for cancer treatment. We have previously reported that Rab37 small GTPase mediates secretion of IL-6 in macrophages to promote cancer progression, whereas the roles of Rab37 in the intracellular trafficking and exocytosis of CHI3L1 are unclear. Methods: We examined the concentration of CHI3L1 in the culture medium of splenocytes and bone marrow derived macrophages (BMDMs) from wild-type or Rab37 knockout mice, and macrophage or T cell lines expressing wild type, active GTP-bound or inactive GDP-bound Rab37. Vesicle isolation, total internal reflection fluorescence microscopy, and real-time confocal microscopy were conducted. We developed polyclonal neutralizing-CHI3L1 antibodies (nCHI3L1 Abs) to validate the therapeutic efficacy in orthotopic lung, pancreas and colon cancer allograft models. Multiplex fluorescence immunohistochemistry was performed to detect the protein level of Rab37 and CHI3L1, and localization of the tumor-infiltrating immune cells in allografts from mice or tumor specimens from cancer patients. Results: We demonstrate a novel secretion mode of CHI3L1 mediated by the small GTPase Rab37 in T cells and macrophages. Rab37 mediated CHI3L1 intracellular vesicle trafficking and exocytosis in a GTP-dependent manner, which is abolished in the splenocytes and BMDMs from Rab37 knockout mice and attenuated in macrophage or T cell lines expressing the inactive Rab37. The secreted CHI3L1 activated AKT, ß-catenin and NF-κB signal pathways in cancer cells and macrophages to foster a protumor TME characterized by activating M2 macrophages and increasing the population of regulatory T cells. Our developed nCHI3L1 Abs showed the dual properties of reducing tumor growth/metastases and eliciting an immunostimulatory TME in syngeneic orthotopic lung, pancreas and colon tumor models. Clinically, high plasma level or intratumoral expression of CHI3L1 correlated with poor survival in 161 lung cancer, 155 pancreatic cancer and 180 colon cancer patients. Conclusions: These results provide the first evidence that Rab37 mediates CHI3L1 secretion in immune cells and highlight nCHI3L1 Abs that can simultaneously target both cancer cells and tumor microenvironment.
Subject(s)
Chitinase-3-Like Protein 1/immunology , Immunotherapy/methods , Neoplasms , rab GTP-Binding Proteins/immunology , Animals , Cell Line, Tumor , Cohort Studies , Gene Expression Regulation, Neoplastic , Mice , Mice, Knockout , Neoplasms/immunology , Neoplasms/therapy , Tumor MicroenvironmentABSTRACT
Influenza A virus infections can result in severe respiratory diseases. The H7N9 subtype of avian influenza A virus has been transmitted to humans and caused severe disease and death. Nonstructural protein 1 (NS1) of influenza A virus is a virulence determinant during viral infection. To elucidate the functions of the NS1 encoded by influenza A H7N9 virus (H7N9 NS1), interaction partners of H7N9 NS1 in human cells were identified with immunoprecipitation followed by SDS-PAGE coupled with liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). We identified 36 cellular proteins as the interacting partners of the H7N9 NS1, and they are involved in RNA processing, mRNA splicing via spliceosome, and the mRNA surveillance pathway. Two of the interacting partners, cleavage and polyadenylation specificity factor subunit 2 (CPSF2) and CPSF7, were confirmed to interact with H7N9 NS1 using coimmunoprecipitation and immunoblotting based on the previous finding that the two proteins are involved in pre-mRNA polyadenylation machinery. Furthermore, we illustrate that overexpression of H7N9 NS1, as well as infection by the influenza A H7N9 virus, interfered with pre-mRNA polyadenylation in host cells. This study comprehensively profiled the interactome of H7N9 NS1 in host cells, and the results demonstrate a novel endotype for H7N9 NS1 in inhibiting host mRNA maturation.