Journal Articles

Tumor micro-environment

Composition, function and interactions of the tumor immune environment and strategies to modulate the tumor immune environment; Immune biomarkers

The Role of Immune Escape and Immune Cell Infiltration in Breast Cancer

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While detailed analysis of aberrant cancer cell signaling pathways and changes in cancer cell DNA has dominated the field of breast cancer biology for years, there now exists increasing evidence that the tumor microenvironment (TME) including tumor-infiltrating immune cells support the growth and development of breast cancer and further facilitate invasion and metastasis formation as well as sensitivity to drug therapy. Furthermore, breast cancer cells have developed different strategies to escape surveillance from the adaptive and innate immune system. These include loss of expression of immunostimulatory molecules, gain of expression of immunoinhibitory molecules such as PD-L1 and HLA-G, and altered expression of components involved in apoptosis. Furthermore, the composition of the TME plays a key role in breast cancer development and treatment response. In this review we will focus on i) the different immune evasion mechanisms used by breast cancer cells, ii) the role of immune cell infiltration in this disease, and (iii) implication for breast cancer-based immunotherapies.

Author Info: (1) Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany. (2) Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.

Author Info: (1) Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany. (2) Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.

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Targeting the Immune Microenvironment in Acute Myeloid Leukemia: A Focus on T Cell Immunity

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Immunotherapies, such as chimeric antigen receptor T cells, bispecific antibodies, and immune checkpoint inhibitors, have emerged as promising modalities in multiple hematologic malignancies. Despite the excitement surrounding immunotherapy, it is currently not possible to predict which patients will respond. Within solid tumors, the status of the immune microenvironment provides valuable insight regarding potential responses to immune therapies. Much less is known about the immune microenvironment within hematologic malignancies but the characteristics of this environment are likely to serve a similar predictive role. Acute myeloid leukemia (AML) is the most common hematologic malignancy in adults, and only 25% of patients are alive 5 years following their diagnosis. There is evidence that manipulation of the immune microenvironment by leukemia cells may play a role in promoting therapy resistance and disease relapse. In addition, it has long been documented that through modulation of the immune system following allogeneic bone marrow transplant, AML can be cured, even in patients with the highest risk disease. These concepts, along with the poor prognosis associated with this disease, have encouraged many groups to start exploring the utility of novel immune therapies in AML. While the implementation of these therapies into clinical trials for AML has been supported by preclinical rationale, many questions still exist surrounding their efficacy, tolerability, and the overall optimal approach. In this review, we discuss what is known about the immune microenvironment within AML with a specific focus on T cells and checkpoints, along with their implications for immune therapies.

Author Info: (1) Pediatric Hematology/Oncology, Seattle Children's Hospital, Seattle, WA, United States. (2) Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, United States.

Author Info: (1) Pediatric Hematology/Oncology, Seattle Children's Hospital, Seattle, WA, United States. (2) Molecular Microbiology and Immunology, Oregon Health & Science University, Portland, OR, United States.

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Tumor microenvironment: recent advances in various cancer treatments

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This is a review regarding different types of cancer treatments. We aimed at analyzing the tumor microenvironment and the recent trends for the therapeutic applications and effectiveness for several kinds of cancers. Traditionally the cancer treatment was based on the neoplastic cells. Methods such as surgery, radiation, chemotherapy, and immunotherapy, which were targeted on the highly proliferating mutated tumor cells, have been investigated. The tumor microenvironment describes the non-cancerous cells in the tumor and has enabled to investigate the behavior and response of the cancer cells to a treatment process; it consists in a tissue that may have a predictive significance for tumor behavior and response to therapy. These include fibroblasts, immune cells and cells that comprise the blood vessels. It also includes the proteins produced by all of the cells present in the tumor that support the growth of the cancer cells. By monitoring changes in the tumor microenvironment using its molecular and cellular profiles as the tumor progresses will be vital for identifying cell or protein targets for the cancer prevention and its therapeutic purposes.

Author Info: (1) Department of General Surgery, Chun'an First People's Hospital, (Zhejiang Provincial People's Hospital Chun'an Branch), Hangzhou, Zhejiang Province, China. 25852832@qq.com. (2) (3)

Author Info: (1) Department of General Surgery, Chun'an First People's Hospital, (Zhejiang Provincial People's Hospital Chun'an Branch), Hangzhou, Zhejiang Province, China. 25852832@qq.com. (2) (3)

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Nanoparticles Targeting and Remodeling Tumor Microenvironment for Cancer Theranostics

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The tumor microenvironment (TME) is featured with aberrant vasculatures, specific physiological parameters, viscoelastic extracellular matrix and stromal cells, which are important factors in tumor initiation, development and metastasis. The components in TME form physical/biological barriers for drug delivery and therapy, and also contribute to resistance to treatment and immunosuppression. The advances in nanobiotechnology have offered a myriad of nanoparticles for targeting and treating tumors through the passive or active targeting strategies. However, the barriers in TME always limit the drug delivery and therapeutic efficacy of nanoparticles. To cope with this, recent strategies have employed nanoparticles to target and remodel tumor microenvironment, while a variety of nanoparticles have been developed with different functions for this score. In this review, we have described the typical features of tumor microenvironment along with their roles in tumor progression, and then focused on recent progresses in development and application of nanoparticles to target and remodel TME for enhanced cancer therapy.

Author Info: (1) (2) (3)

Author Info: (1) (2) (3)

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A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments

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Intratumoral stimulatory dendritic cells (SDCs) play an important role in stimulating cytotoxic T cells and driving immune responses against cancer. Understanding the mechanisms that regulate their abundance in the tumor microenvironment (TME) could unveil new therapeutic opportunities. We find that in human melanoma, SDC abundance is associated with intratumoral expression of the gene encoding the cytokine FLT3LG. FLT3LG is predominantly produced by lymphocytes, notably natural killer (NK) cells in mouse and human tumors. NK cells stably form conjugates with SDCs in the mouse TME, and genetic and cellular ablation of NK cells in mice demonstrates their importance in positively regulating SDC abundance in tumor through production of FLT3L. Although anti-PD-1 'checkpoint' immunotherapy for cancer largely targets T cells, we find that NK cell frequency correlates with protective SDCs in human cancers, with patient responsiveness to anti-PD-1 immunotherapy, and with increased overall survival. Our studies reveal that innate immune SDCs and NK cells cluster together as an excellent prognostic tool for T cell-directed immunotherapy and that these innate cells are necessary for enhanced T cell tumor responses, suggesting this axis as a target for new therapies.

Author Info: (1) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA

Author Info: (1) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA. (2) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA. (3) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA. (4) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Madrid, Spain. Department of Biochemistry, Faculty of Medicine, Universidad Autonoma de Madrid, Madrid, Spain. (5) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. (6) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA. (7) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA. (8) UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA. Melanoma Clinical Research Unit, University of California San Francisco, San Francisco, CA, USA. Department of Dermatology, University of California San Francisco, San Francisco, CA, USA. (9) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA. (10) Department of Dermatology, University of California San Francisco, San Francisco, CA, USA. (11) Department of Dermatology, University of California San Francisco, San Francisco, CA, USA. (12) Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA. (13) Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (14) Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. (15) Department of Dermatology, University of California San Francisco, San Francisco, CA, USA. (16) Department of Otolaryngology, University of California San Francisco, San Francisco, CA, USA. (17) Department of Otolaryngology, University of California San Francisco, San Francisco, CA, USA. (18) Pionyr Immunotherapeutics, San Francisco, CA, USA. (19) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA. (20) UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA. (21) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA. (22) Department of Pathology, University of California San Francisco, San Francisco, CA, USA. matthew.krummel@ucsf.edu. UCSF Immunoprofiler Initiative, University of California San Francisco, San Francisco, CA, USA. matthew.krummel@ucsf.edu.

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Single-cell profiling of breast cancer T cells reveals a tissue-resident memory subset associated with improved prognosis

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The quantity of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is a robust prognostic factor for improved patient survival, particularly in triple-negative and HER2-overexpressing BC subtypes (1) . Although T cells are the predominant TIL population (2) , the relationship between quantitative and qualitative differences in T cell subpopulations and patient prognosis remains unknown. We performed single-cell RNA sequencing (scRNA-seq) of 6,311 T cells isolated from human BCs and show that significant heterogeneity exists in the infiltrating T cell population. We demonstrate that BCs with a high number of TILs contained CD8(+) T cells with features of tissue-resident memory T (TRM) cell differentiation and that these CD8(+) TRM cells expressed high levels of immune checkpoint molecules and effector proteins. A CD8(+) TRM gene signature developed from the scRNA-seq data was significantly associated with improved patient survival in early-stage triple-negative breast cancer (TNBC) and provided better prognostication than CD8 expression alone. Our data suggest that CD8(+) TRM cells contribute to BC immunosurveillance and are the key targets of modulation by immune checkpoint inhibition. Further understanding of the development, maintenance and regulation of TRM cells will be crucial for successful immunotherapeutic development in BC.

Author Info: (1) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria

Author Info: (1) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia. (2) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. (3) Bioinformatics Division, Walter & Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia. School of Medicine, Tsinghua University, Beijing, China. (4) Bioinformatics Division, Walter & Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia. Department of Mathematics and Statistics, La Trobe University, Melbourne, Victoria, Australia. (5) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. (6) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. (7) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. Department of Pathology, GZA Ziekenhuizen, Antwerp, Belgium. (8) Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. (9) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia. (10) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. (11) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. (12) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. (13) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. (14) Division of Cancer Surgery, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. (15) Division of Cancer Surgery, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. (16) Division of Cancer Surgery, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. Department of Surgery, Royal Melbourne Hospital and Royal Womens' Hospital, Melbourne, Victoria, Australia. (17) Division of Cancer Surgery, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. (18) Division of Cancer Surgery, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. (19) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia. (20) Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia. Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. (21) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia. (22) Bioinformatics Division, Walter & Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia. Department of Mathematics and Statistics, University of Melbourne, Melbourne, Victoria, Australia. (23) Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia. (24) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. paul.neeson@petermac.org. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia. paul.neeson@petermac.org. (25) Division of Research, Peter MacCallum Cancer Centre, University of Melbourne, Melbourne, Victoria, Australia. sherene.loi@petermac.org. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia. sherene.loi@petermac.org.

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A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

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Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Author Info: (1) Moores Cancer Center, University California San Diego. (2) Moores Cancer Center, University California San Diego. (3) Duke University School of Medicine. (4) Moores Cancer

Author Info: (1) Moores Cancer Center, University California San Diego. (2) Moores Cancer Center, University California San Diego. (3) Duke University School of Medicine. (4) Moores Cancer Center, University California San Diego; rewhite@ucsd.edu.

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Fever-Inspired Immunotherapy Based on Photothermal CpG Nanotherapeutics: The Critical Role of Mild Heat in Regulating Tumor Microenvironment

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Although there have been more than 100 clinical trials, CpG-based immunotherapy has been seriously hindered by complications in the immunosuppressive microenvironment of established tumors. Inspired by the decisive role of fever upon systemic immunity, a photothermal CpG nanotherapeutics (PCN) method with the capability to induce an immunofavorable tumor microenvironment by casting a fever-relevant heat (43 degrees C) in the tumor region is developed. High-throughput gene profile analysis identifies nine differentially expressed genes that are closely immune-related upon mild heat, accompanied by IL-6 upregulation, a pyrogenic cytokine usually found during fever. When treated with intratumor PCN injection enabling mild heating in the tumor region, the 4T1 tumor-bearing mice exhibit significantly improved antitumor immune effects compared with the control group. Superb efficacy is evident from pronounced apoptotic cell death, activated innate immune cells, enhanced tumor perfusion, and intensified innate and adaptive immune responses. This work highlights the crucial role of mild heat in modulating the microenvironment in optimum for improved immunotherapy, by converting the tumor into an in situ vaccine.

Author Info: (1) Shanghai East Hospital The Institute for Biomedical Engineering and Nano Science Tongji University School of Medicine Shanghai 200092 P. R. China. (2) Shanghai East

Author Info: (1) Shanghai East Hospital The Institute for Biomedical Engineering and Nano Science Tongji University School of Medicine Shanghai 200092 P. R. China. (2) Shanghai East Hospital The Institute for Biomedical Engineering and Nano Science Tongji University School of Medicine Shanghai 200092 P. R. China. (3) Shanghai East Hospital The Institute for Biomedical Engineering and Nano Science Tongji University School of Medicine Shanghai 200092 P. R. China. (4) Shanghai East Hospital The Institute for Biomedical Engineering and Nano Science Tongji University School of Medicine Shanghai 200092 P. R. China. (5) School of Materials Science and Engineering Tongji University 4800 Caoan Road Shanghai 201804 P. R. China. (6) School of Life Science and Technology Tongji University 1239 Siping Road Shanghai 200092 P. R. China. (7) School of Materials Science and Engineering Tongji University 4800 Caoan Road Shanghai 201804 P. R. China. (8) Shanghai East Hospital The Institute for Biomedical Engineering and Nano Science Tongji University School of Medicine Shanghai 200092 P. R. China. The Materials Science and Engineering Program Department of Mechanical and Materials Engineering College of Engineering and Applied Science University of Cincinnati Cincinnati OH 45221 USA. (9) Shanghai East Hospital The Institute for Biomedical Engineering and Nano Science Tongji University School of Medicine Shanghai 200092 P. R. China.

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Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

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The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Author Info: (1) Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine; Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine. (2)

Author Info: (1) Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine; Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine. (2) Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine. (3) Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine; Andrew.Sikora@bcm.edu.

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Cancer-Associated Fibroblasts Affect Intratumoral CD8(+) and FoxP3(+) T Cells via Interleukin 6 in the Tumor Microenvironment

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PURPOSE: Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) play a central role in tumor progression. We investigated whether CAFs can regulate tumor-infiltrating lymphocytes (TILs) and their role in tumor immunosuppression. EXPERIMENTAL DESIGN: 140 cases of esophageal cancer were analyzed for CAFs and CD8(+)or forkhead box protein 3 (FoxP3(+)) TILs by immunohistochemistry. We analyzed cytokines using murine or human fibroblasts and cancer cells. Murine-derived fibroblasts and cancer cells were also inoculated into BALB/c or BALB/c-nu/numice, and the tumors treated with recombinant interleukin 6 (IL-6) or anti-IL-6 antibody. RESULTS: CD8(+)TILs and CAFs were negatively correlated in intra-tumoral tissues (P< 0.001), while FoxP3(+)TILs were positively correlated (P< 0.001) in esophageal cancers. Co-cultured Colon26 cancer cells and fibroblasts resulted in accelerated tumor growth in BALB/c mice, along with decreased CD8(+)and increased FoxP3(+)TILs, compared with cancer cells alone. In vitro, IL-6 was highly secreted in both murine and human cancer cell/fibroblast co-cultures. IL-6 significantly increased Colon26 tumor growth in immune-competent BALB/c (P< 0.001) with fewer CD8(+)TILs than untreated tumors (P< 0.001), whereas no difference in BALB/c-nu/numice. In contrast, FoxP3(+)TILs increased in IL-6-treated tumors (P< 0.001). IL-6 antibody blockade of tumors co-cultured with fibroblasts resulted not only in regression of tumor growth but also in the accumulation of CD8(+)TILs in intra-tumoral tissues. CONCLUSIONS: CAFs regulate immunosuppressive TIL populations in the TME via IL-6. IL-6 blockade, or targeting CAFs, may improve pre-existing tumor immunity and enhance the efficacy of conventional immunotherapies.

Author Info: (1) Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences. (2) Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical

Author Info: (1) Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences. (2) Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences knoma@md.okayama-u.ac.jp. (3) Pathology & Experimental Medicine, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences. (4) Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences. (5) Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences. (6) Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences. (7) Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences. (8) Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences. (9) Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences. (10) Center for Innovative Clinical Medicine, Okayama University Hospital. (11) Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine. (12) Department of Gastroenterological Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences.

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