Journal Articles

The molecular and functional landscape of resistance to immune checkpoint blockade in melanoma

Resistance to immune checkpoint inhibitor therapies in melanoma is common and remains an intractable clinical challenge. In this study, we comprehensively profile immune checkpoint inhibitor resistance mechanisms in short-term tumor cell lines and matched tumor samples from melanoma patients progressing on immune checkpoint inhibitors. Combining genome, transcriptome, and high dimensional flow cytometric profiling with functional analysis, we identify three distinct programs of immunotherapy resistance. Here we show that resistance programs include (1) the loss of wild-type antigen expression, resulting from tumor-intrinsic IFN_ signaling and melanoma de-differentiation, (2) the disruption of antigen presentation via multiple independent mechanisms affecting MHC expression, and (3) immune cell exclusion associated with PTEN loss. The dominant role of compromised antigen production and presentation in melanoma resistance to immune checkpoint inhibition highlights the importance of treatment salvage strategies aimed at the restoration of MHC expression, stimulation of innate immunity, and re-expression of wild-type differentiation antigens.

Author Info: (1) Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia. Melanoma Institute Australia, The University of Sydney,

Author Info: (1) Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia. Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. (2) Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia. Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. (3) Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia. Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. Department of Medical Oncology, Chris O'Brien Lifehouse, Sydney, NSW, Australia. (4) Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia. Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. (5) Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia. Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. (6) Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia. Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. (7) Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia. Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. (8) Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. Department of Neurosurgery, Chris O'Brien Lifehouse, Sydney, NSW, Australia. Department of Neurosurgery, Royal Prince Alfred Hospital, Sydney, NSW, Australia. (9) Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. Department of Melanoma and Surgical Oncology, Royal Prince Alfred Hospital, Sydney, NSW, Australia. Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia. (10) Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia. Department of Medical Oncology, Northern Sydney Cancer Centre, Royal North Shore Hospital, Sydney, NSW, Australia. Department of Medical Oncology, Mater Hospital, Sydney, NSW, Australia. (11) Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia. Department of Medical Oncology, Blacktown Cancer and Haematology Centre, Blacktown Hospital, Sydney, NSW, Australia. Department of Medical Oncology, Crown Princess Mary Cancer Centre, Westmead Hospital, Sydney, NSW, Australia. (12) Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia. Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital and NSW Health Pathology, Sydney, NSW, Australia. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia. (13) Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia. Department of Medical Oncology, Northern Sydney Cancer Centre, Royal North Shore Hospital, Sydney, NSW, Australia. Department of Medical Oncology, Mater Hospital, Sydney, NSW, Australia. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia. (14) Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, NSW, Australia. helen.rizos@mq.edu.au. Melanoma Institute Australia, The University of Sydney, Sydney, NSW, Australia. helen.rizos@mq.edu.au.

Guadecitabine increases response to combined anti-CTLA-4 and anti-PD-1 treatment in mouse melanoma in vivo by controlling T-cells, myeloid derived suppressor and NK cells

BACKGROUND: The combination of Programmed Cell Death 1 (PD-1) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) blockade has dramatically improved the overall survival rate for malignant melanoma. Immune checkpoint blockers (ICBs) limit the tumor's immune escape yet only for approximately a third of all tumors and, in most cases, for a limited amount of time. Several approaches to overcome resistance to ICBs are being investigated among which the addition of epigenetic drugs that are expected to act on both immune and tumor cells. Guadecitabine, a dinucleotide prodrug of a decitabine linked via phosphodiester bond to a guanosine, showed promising results in the phase-1 clinical trial, NIBIT-M4 (NCT02608437). METHODS: We used the syngeneic B16F10 murine melanoma model to study the effects of immune checkpoint blocking antibodies against CTLA-4 and PD-1 in combination, with and without the addition of Guadecitabine. We comprehensively characterized the tumor's and the host's responses under different treatments by flow cytometry, multiplex immunofluorescence and methylation analysis. RESULTS: In combination with ICBs, Guadecitabine significantly reduced subcutaneous tumor growth as well as metastases formation compared to ICBs and Guadecitabine treatment. In particular, Guadecitabine greatly enhanced the efficacy of combined ICBs by increasing effector memory CD8+ T cells, inducing effector NK cells in the spleen and reducing tumor infiltrating regulatory T cells and myeloid derived suppressor cells (MDSC), in the tumor microenvironment (TME). Guadecitabine in association with ICBs increased serum levels of IFN-_ and IFN-_-induced chemokines with anti-angiogenic activity. Guadecitabine led to a general DNA-demethylation, in particular of sites of intermediate methylation levels. CONCLUSIONS: These results indicate Guadecitabine as a promising epigenetic drug to be added to ICBs therapy.

Author Info: (1) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (2) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy

Author Info: (1) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (2) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (3) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. Department of Internal Medicine, University of Genova, Genova, Italy. (4) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (5) Immunology and Molecular Oncology Diagnostics, Istituto Oncologico Veneto IRCCS, Padova, Italy. (6) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (7) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (8) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (9) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (10) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (11) Department of Dermatology, University of Zurich, University Hospital of Zurich, Zurich, Switzerland. (12) Department of Dermatology, University of Zurich, University Hospital of Zurich, Zurich, Switzerland. (13) Functional Genomics Center Zurich, University of Zurich and ETH Zurich, Zurich, Switzerland. (14) Department of Interdisciplinary Medicine, University of Bari "Aldo Moro", Bari, Italy. (15) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (16) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (17) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. (18) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. Department of Internal Medicine, University of Genova, Genova, Italy. (19) Immunology and Molecular Oncology Diagnostics, Istituto Oncologico Veneto IRCCS, Padova, Italy. Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy. (20) Department of Dermatology, University of Zurich, University Hospital of Zurich, Zurich, Switzerland. (21) University of Siena, Siena, Italy. (22) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy. ulrich.pfeffer@hsanmartino.it. (23) IRCCS Ospedale Policlinico San Martino, Largo Rosanna Benzi, 10, 16132, Genova, Italy.

Tumor PD-L1 engages myeloid PD-1 to suppress type I interferon to impair cytotoxic T lymphocyte recruitment Featured  

Investigating the role of PD-L1 on tumor cells, Klement and Redd et al. found that it did not affect primary tumor growth or directly limit CTL activity, but did engage with PD-1 on myeloid cells, activating SHP2 and inhibiting IFN-I signaling at sites of metastasis. When this mechanism was interrupted, IFN-I signaling increased, leading to increased STAT1, increased MHC-I and MHC-II expression, and increased production of CTL-recruiting chemokines, CXCL9 and CXCL10. This increased CTL recruitment, which helped to control and limit metastatic tumor outgrowth. In patient data, responses to PD-1 blockade correlated with IFN-I responses in myeloid cells.

Investigating the role of PD-L1 on tumor cells, Klement and Redd et al. found that it did not affect primary tumor growth or directly limit CTL activity, but did engage with PD-1 on myeloid cells, activating SHP2 and inhibiting IFN-I signaling at sites of metastasis. When this mechanism was interrupted, IFN-I signaling increased, leading to increased STAT1, increased MHC-I and MHC-II expression, and increased production of CTL-recruiting chemokines, CXCL9 and CXCL10. This increased CTL recruitment, which helped to control and limit metastatic tumor outgrowth. In patient data, responses to PD-1 blockade correlated with IFN-I responses in myeloid cells.

ABSTRACT: The cellular and molecular mechanisms underlying tumor cell PD-L1 (tPD-L1) function in tumor immune evasion are incompletely understood. We report here that tPD-L1 does not suppress cytotoxic T lymphocyte (CTL) activity in co-cultures of tumor cells and tumor-specific CTLs and exhibits no effect on primary tumor growth. However, deleting tPD-L1 decreases lung metastasis in a CTL-dependent manner in tumor-bearing mice. Depletion of myeloid cells or knocking out PD-1 in myeloid cells (mPD-1) impairs tPD-L1 promotion of tumor lung metastasis in mice. Single-cell RNA sequencing (scRNA-seq) reveals that tPD-L1 engages mPD-1 to activate SHP2 to antagonize the type I interferon (IFN-I) and STAT1 pathway to repress Cxcl9 and impair CTL recruitment to lung metastases. Human cancer patient response to PD-1 blockade immunotherapy correlates with IFN-I response in myeloid cells. Our findings determine that tPD-L1 engages mPD-1 to activate SHP2 to suppress the IFN-I-STAT1-CXCL9 pathway to impair CTL tumor recruitment in lung metastasis.

Author Info: (1) Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Augusta, GA 30912, USA; Charlie Norwood VA Medical

Author Info: (1) Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Augusta, GA 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. (2) Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Augusta, GA 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. (3) Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Augusta, GA 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. (4) Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Augusta, GA 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. (5) Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Augusta, GA 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. (6) Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Augusta, GA 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. (7) Department of Pathology, Medical College of Georgia, Augusta, GA 30912, USA. (8) Georgia Cancer Center, Augusta, GA 30912, USA. (9) Georgia Cancer Center, Augusta, GA 30912, USA. (10) Trinity Biomedical Sciences Institute, School of Medicine, Trinity College Dublin, Dublin, Ireland. (11) Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA; Georgia Cancer Center, Augusta, GA 30912, USA; Charlie Norwood VA Medical Center, Augusta, GA 30904, USA. Electronic address: kliu@augusta.edu.

Eliciting effective tumor immunity against ovarian cancer by cancer stem cell vaccination

Advanced ovarian cancer (OC) patients have limited benefit from current relevant cytotoxic and targeted therapies following debulking surgery. Therefore, new therapeutic strategies are in urgent need. Immunotherapy has shown great potential in tumor treatment, especially in tumor vaccine development. The study objective was to evaluate the immune effects of cancer stem cells (CSCs) vaccines on OC. The CD44(+)CD117(+)CSCs were isolated from human OC HO8910 and SKOV3 cells using the magnetic cell sorting system; the cancer stem-like cells were selected from murine OC ID8 cell by no-serum formed sphere culture. The CSC vaccines were prepared by freezing and thawing these CSCs, which were then injected into mice followed by challenging the different OC cells. The in vivo antitumor efficacy of CSC immunization revealed the vaccines were capable of significantly provoking immune responses to autologous tumor antigens in vaccinated mice as the mice were found to have markedly inhibited tumor growth, prolonged survival, and decreased CSC counts in OC tissues when compared to mice without the CSC vaccination. The in vitro cytotoxicities of immunocytes toward SKOV3, HO8910 and ID8 cells indicated a significant killing efficacy compared with the controls. However, the antitumor efficacy was remarkably reduced whilst the mucin-1 expression in CSC vaccines was down-regulated by small interfering RNA. Overall, findings from this study provided the evidence that has deepened our understanding of CSC vaccine immunogenicity and anti-OC efficacy, particularly for the role of dominant antigen mucin-1. It is possible to turn the CSC vaccine into an immunotherapeutic approach against ovarian cancer.

Author Info: (1) Department of Pathogenic Biology and Immunology, School of Medicine, Southeast University, Nanjing 210009, China; Department of Gynecology & Obstetrics, Zhongda Hospital, Schoo

Author Info: (1) Department of Pathogenic Biology and Immunology, School of Medicine, Southeast University, Nanjing 210009, China; Department of Gynecology & Obstetrics, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China. (2) Department of Pathogenic Biology and Immunology, School of Medicine, Southeast University, Nanjing 210009, China. (3) Department of Gynecology & Obstetrics, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China. (4) Department of Gynecology & Obstetrics, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China. (5) Department of Pathogenic Biology and Immunology, School of Medicine, Southeast University, Nanjing 210009, China. (6) Department of Oncology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China. (7) Department of Gynecology & Obstetrics, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, China. (8) Department of Pathogenic Biology and Immunology, School of Medicine, Southeast University, Nanjing 210009, China. Electronic address: njdoujun@seu.edu.cn.

Antitumor effect of neoantigen-reactive T cells combined with PD1 inhibitor therapy in mouse lung cancer

PURPOSE: Neoantigens produced from mutations in tumors are important targets of T-cell-based immunotherapy and immune checkpoint blockade has been approved for treating multiple solid tumors. We investigated the potential benefit of adoptive neoantigen-reactive T (NRT) cells in combination with programmed cell death protein 1 inhibitor (anti-PD1) for treating lung cancer in a mouse model. METHODS: NRT cells were prepared by co-culturing T cells and neoantigen-RNA vaccine-induced dendritic cells. Then, adoptive NRT cells in combination with anti-PD1 were administered to tumor-bearing mice. Pre- and post-therapy cytokine secretion, antitumor efficacy, and tumor microenvironment (TME) changes were determined both in vitro and in vivo. RESULTS: We successfully generated NRT cells based on the 5 neoantigen epitopes identified in this study. NRT cells exhibited an enhanced cytotoxic phenotype in vitro and the combination therapy led to attenuated tumor growth. In addition, this combination strategy downregulated the expression of the inhibitory marker PD-1 on tumor-infiltrating T cells and promoted the trafficking of tumor-specific T cells to the tumor sites. CONCLUSION: The adoptive transfer of NRT cells in association with anti-PD1 therapy can exert an antitumor effect on lung cancer, and is a feasible, effective, and novel immunotherapy regimen for treating solid tumors.

Author Info: (1) Department of Pulmonary and Critical Care Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China. (2) Breast Disease Center, The Affiliated Hos

Author Info: (1) Department of Pulmonary and Critical Care Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China. (2) Breast Disease Center, The Affiliated Hospital of Qingdao University, Qingdao, 266071, China. (3) Department of Pulmonary and Critical Care Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China. (4) Department of Pulmonary and Critical Care Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China. (5) Department of Pulmonary and Critical Care Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China. (6) School of Basic Medicine, Wenzhou Medical University, Wenzhou Tea Mountain Higher Education Park, Wenzhou, 325000, China. (7) Department of Respiratory and Critical Care Medicine, The Affiliated Hospital of Qingdao University, Qingdao, China. jiaxing20022001@163.com. (8) Department of Pulmonary and Critical Care Medicine, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China. liqressh1962@163.com.

Blocking Dectin-1 prevents colorectal tumorigenesis by suppressing prostaglandin E2 production in myeloid-derived suppressor cells and enhancing IL-22 binding protein expression

Dectin-1 (gene Clec7a), a receptor for _-glucans, plays important roles in the host defense against fungi and immune homeostasis of the intestine. Although this molecule is also suggested to be involved in the regulation of tumorigenesis, the role in intestinal tumor development remains to be elucidated. In this study, we find that azoxymethane-dextran-sodium-sulfate-induced and Apc(Min)-induced intestinal tumorigenesis are suppressed in Clec7a(-/-) mice independently from commensal microbiota. Dectin-1 is preferentially expressed on myeloid-derived suppressor cells (MDSCs). In the Clec7a(-/-) mouse colon, the proportion of MDSCs and MDSC-derived prostaglandin E(2) (PGE(2)) levels are reduced, while the expression of IL-22 binding protein (IL-22BP; gene Il22ra2) is upregulated. Dectin-1 signaling induces PGE(2)-synthesizing enzymes and PGE(2) suppresses Il22ra2 expression in vitro and in vivo. Administration of short chain _-glucan laminarin, an antagonist of Dectin-1, suppresses the development of mouse colorectal tumors. Furthermore, in patients with colorectal cancer (CRC), the expression of CLEC7A is also observed in MDSCs and correlated with the death rate and tumor severity. Dectin-1 signaling upregulates PGE(2)-synthesizing enzyme expression and PGE(2) suppresses IL22RA2 expression in human CRC-infiltrating cells. These observations indicate a role of the Dectin-1-PGE(2)-IL-22BP axis in regulating intestinal tumorigenesis, suggesting Dectin-1 as a potential target for CRC therapy.

Author Info: (1) Department of Gastroenterology and Hepatology, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. tan

Author Info: (1) Department of Gastroenterology and Hepatology, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. tangc7@mail.sysu.edu.cn. Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. tangc7@mail.sysu.edu.cn. Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Yamazaki 2669, Noda-shi, Chiba, 278-0022, Japan. tangc7@mail.sysu.edu.cn. (2) Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Yamazaki 2669, Noda-shi, Chiba, 278-0022, Japan. (3) Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Yamazaki 2669, Noda-shi, Chiba, 278-0022, Japan. (4) Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Yamazaki 2669, Noda-shi, Chiba, 278-0022, Japan. (5) Department of Gastroenterology and Hepatology, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Yamazaki 2669, Noda-shi, Chiba, 278-0022, Japan. (6) Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Yamazaki 2669, Noda-shi, Chiba, 278-0022, Japan. (7) Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. (8) Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. (9) Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. (10) Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. (11) Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. (12) Department of Gastroenterology and Hepatology, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. (13) Department of Anesthesiology, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. (14) Department of Gastroenterology and Hepatology, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. Institute of Precision Medicine, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. (15) Department of Gastroenterology and Hepatology, The First Affiliated Hospital, Sun Yat-sen University, No.58, Zhong Shan Er Lu, 510080, Guangzhou, Guangdong Province, China. (16) Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, 192-0392, Japan. (17) Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, 192-0392, Japan. (18) Boehringer Ingelheim USA, 900 Ridgebury Rd, Ridgefield, CT, 06877, USA. (19) Center for Animal Disease Models, Research Institute for Biomedical Sciences, Tokyo University of Science, Yamazaki 2669, Noda-shi, Chiba, 278-0022, Japan. iwakura@rs.tus.ac.jp.

A Phase II Trial of Guadecitabine plus Atezolizumab in Metastatic Urothelial Carcinoma Progressing after Initial Immune Checkpoint Inhibitor Therapy

PURPOSE: Based on preclinical evidence of epigenetic contribution to sensitivity and resistance to immune checkpoint inhibitors (ICI), we hypothesized that guadecitabine (hypomethylating agent) and atezolizumab (anti-PD-L1) together would potentiate a clinical response in patients with metastatic urothelial carcinoma (UC) unresponsive to initial immune checkpoint blockade therapy. PATIENTS AND METHODS: We designed a single arm Phase II study (NCT03179943) with a safety run-in to identify the recommended phase II dose of the combination therapy of guadecitabine and atezolizumab. Patients with recurrent/advanced urothelial carcinoma who had previously progressed on ICI therapy with PD-1 or PD-L1 targeting agents were eligible. Pre-planned correlative analysis was performed to characterize peripheral immune dynamics and global DNA methylation, transcriptome, and immune infiltration dynamics of patient tumors. RESULTS: Safety run-in enrolled 6 patients and Phase II enrolled 15 patients before the trial was closed for futility. No dose-limiting toxicity was observed. Four patients, with best response of stable disease, exhibited extended tumor control (8-11 months) and survival (>14 months). Correlative analysis revealed lack of DNA demethylation in tumors after 2 cycles of treatment. Increased peripheral immune activation and immune infiltration in tumors after treatment correlated with progression-free survival and stable disease. Furthermore, high IL-6 and IL-8 levels in the patients' plasma associates with short survival. CONCLUSIONS: No RECIST responses were observed after combination therapy in this trial. Although we could not detect the anticipated tumor-intrinsic effects of guadecitabine, the addition of hypomethylating agent to ICI therapy induced immune activation in a few patients, which associated with longer patient survival.

Author Info: (1) Van Andel Institute, Grand Rapids, MI, United States. (2) Van Andel Institute, Grand Rapids, MI, United States. (3) Fox Chase Cancer Center, Philadelphia, PA, United States. (4

Author Info: (1) Van Andel Institute, Grand Rapids, MI, United States. (2) Van Andel Institute, Grand Rapids, MI, United States. (3) Fox Chase Cancer Center, Philadelphia, PA, United States. (4) Van Andel Institute, Grand Rapids, MI, United States. (5) Fox Chase Cancer Center, Philadelphia, PA, United States. (6) Van Andel Institute, Grand Rapids, United States. (7) Van Andel Institute, Grand Rapids, MI, United States. (8) Van Andel Institute, Grand Rapids, MI, United States. (9) Fox Chase Cancer Center, Philadelphia, PA, United States. (10) Fox Chase Cancer Center, Philadelphia, PA, United States. (11) Fox Chase Cancer Center, Philadelphia, PA, United States. (12) Fox Chase Cancer Center, Philadelphia, United States. (13) Fox Chase Cancer Center, Philadelphia, PA, United States. (14) Fox Chase Cancer Center, Philadelphia, PA, United States. (15) Fox Chase Cancer Center, Birmingham, AL, United States. (16) University of Southern California, Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA, United States. (17) Coriell Institute For Medical Research, Camden, NJ, United States. (18) Coriell Institute For Medical Research, Camden, NJ, United States. (19) Johns Hopkins School of Medicine, Baltimore, Maryland, United States. (20) University of Southern California, Los Angeles, CA, United States. (21) Coriell Institute For Medical Research, Camden, NJ, United States. (22) Johns Hopkins University, Baltimore, MD, United States. (23) Johns Hopkins University School of Medicine, Baltimore, MD, United States. (24) Van Andel Institute, Los Angeles, United States. (25) Fox Chase Cancer Center, Philadelphia, PA, United States. (26) Van Andel Institute, Grand Rapids, MI, United States. (27) Fox Chase Cancer Center, Philadelphia, PA, United States.

Tumour microenvironment as a predictive factor for immunotherapy in non-muscle-invasive bladder cancer

Bladder cancer (BC) can be divided into two subgroups depending on invasion of the muscular layer: non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Its aggressiveness is associated, inter alia, with genetic aberrations like losses of 1p, 6q, 9p, 9q and 13q; gain of 5p; or alterations in the p53 and p16 pathways. Moreover, there are reported metabolic disturbances connected with poor diagnosis-for example, enhanced aerobic glycolysis, gluconeogenesis or haem catabolism.Currently, the primary way of treatment method is transurethral resection of the bladder tumour (TURBT) with adjuvant Bacillus Calmette-GuŽrin (BCG) therapy for NMIBC or radical cystectomy for MIBC combined with chemotherapy or immunotherapy. However, intravesical BCG immunotherapy and immune checkpoint inhibitors are not efficient in every case, so appropriate biomarkers are needed in order to select the proper treatment options. It seems that the success of immunotherapy depends mainly on the tumour microenvironment (TME), which reflects the molecular disturbances in the tumour. TME consists of specific conditions like hypoxia or local acidosis and different populations of immune cells including tumour-infiltrating lymphocytes, natural killer cells, neutrophils and B lymphocytes, which are responsible for shaping the response against tumour neoantigens and crucial pathways like the PD-L1/PD-1 axis.In this review, we summarise holistically the impact of the immune system, genetic alterations and metabolic changes that are key factors in immunotherapy success. These findings should enable better understanding of the TME complexity in case of NMIBC and causes of failures of current therapies.

Author Info: (1) Oncology Department, Medical University of Warsaw, Warsaw, Poland. asemeniukwojtas@gmail.com. (2) Oncology Department, 4 Military Clinical Hospital with a Polyclinic, Wroclaw,

Author Info: (1) Oncology Department, Medical University of Warsaw, Warsaw, Poland. asemeniukwojtas@gmail.com. (2) Oncology Department, 4 Military Clinical Hospital with a Polyclinic, Wroclaw, Poland. (3) Pathomorphology Department, Medical University of Warsaw, Warsaw, Poland. (4) Oncology Department, Medical University of Warsaw, Warsaw, Poland. (5) Oncology Department, Medical University of Warsaw, Warsaw, Poland. (6) Department of General, Active and Oncological Urology, Military Institute of Medicine, Warsaw, Poland. (7) Pathomorphology Department, Medical University of Warsaw, Warsaw, Poland. (8) Oncology Department, 4 Military Clinical Hospital with a Polyclinic, Wroclaw, Poland. (9) Oncology Department, Medical University of Warsaw, Warsaw, Poland.

Regular Voluntary Running is Associated with Increased Tumor Vascularization and Immune Cell Infiltration and Decreased Tumor Growth in Mice

Tumors present dysfunctional vasculature that limits blood perfusion and hinders immune cells delivery. We aimed to investigate if regular voluntary running promotes tumor vascular remodelling, improves intratumoral immune cells infiltration and inhibits tumor growth. Tumors were induced in C57BL/6 male mice (n=28) by subcutaneous inoculation in the dorsal region with a suspension of RM1 cells (1.5_10(5) cells/500_µL PBS) and randomly allocated into two groups: sedentary (n=14) and voluntarily exercised on a wheel (n=14). Seven mice from each group were sacrificed 14 and 28 days after cells' inoculation to evaluate tumor weight, microvessel density, vessels' lumen regularity and the intratumoral quantity of NKG2D receptors, CD4(+)and CD8(+)T cells, by immunohistochemistry. The statistical inference was done through a two-way ANOVA. Exercised mice developed smaller tumors at 14 (0.17±0.1_g vs. 0.48±0.2_g, p<0.05) and 28 (0.92±0.7_g vs. 2.09±1.3_g, p<0.05) days, with higher microvessel density (21.20±3.2 vs. 15.86±4.0 vessels/field, p<0.05), more regular vessels' lumen (1.06±0.2 vs. 1.43±0.2, p<0.05), and higher CD8(+)T cells (464.95±48.0 vs. 364.70±49.4 cells/mm(2), p<0.01), after 28 days. NKG2D expression was higher in exercised mice at 14 (263.27±25.8 cells/mm(2), p<0.05) and 28 (295.06±56.2 cells/mm(2), p<0.001) days. Regular voluntary running modulates tumor vasculature, increases immune cells infiltration and attenuates tumor growth, in mice.

Author Info: (1) Instituto de Investiga‹o, Inova‹o e Desenvolvimento Fernando Pessoa (FP-I3ID), Escola Superior de Saude Fernando Pessoa, Porto, Portugal. Laboratory of Biochemistry and Exper

Author Info: (1) Instituto de Investiga‹o, Inova‹o e Desenvolvimento Fernando Pessoa (FP-I3ID), Escola Superior de Saude Fernando Pessoa, Porto, Portugal. Laboratory of Biochemistry and Experimental Morphology, CIAFEL, Porto, Portugal. (2) Laboratory of Biochemistry and Experimental Morphology, CIAFEL, Porto, Portugal. (3) Laboratory of Biochemistry and Experimental Morphology, CIAFEL, Porto, Portugal. (4) Clinical and Experimental Endocrinology, Unit for Multidisciplinary Research in Biomedicine, University of Porto Institute of Biomedical Sciences Abel Salazar, Porto, Portugal. (5) Clinical and Experimental Endocrinology, Unit for Multidisciplinary Research in Biomedicine, University of Porto Institute of Biomedical Sciences Abel Salazar, Porto, Portugal. (6) Communication Unit, Universidade do Porto Instituto de Investiga‹o e Inova‹o em Saœde, Porto, Portugal. (7) Clinical and Experimental Endocrinology, Unit for Multidisciplinary Research in Biomedicine, University of Porto Institute of Biomedical Sciences Abel Salazar, Porto, Portugal. (8) Clinical and Experimental Endocrinology, Unit for Multidisciplinary Research in Biomedicine, University of Porto Institute of Biomedical Sciences Abel Salazar, Porto, Portugal. (9) Laboratory of Biochemistry and Experimental Morphology, CIAFEL, Porto, Portugal. TOXRUN, University Institute of Health Sciences, CESPU, Gandra, Portuga.

Complexity and diversity of FOXP3 isoforms: Novel insights into the regulation of the immune response in metastatic breast cancer

FOXP3 is a key transcription factor in the regulation of immune responses, and recent studies have uncovered the complexity and diversity of FOXP3 isoforms in various cancers, including metastatic breast cancers (mBCs). It has dual role in the tumor microenvironment of mBCs. This review aims to provide novel insights into the complexity and diversity of FOXP3 isoforms in the regulation of the immune response in breast cancer. We discuss the molecular mechanisms underlying the function of FOXP3 isoforms, including their interaction with other proteins, regulation of gene expression, and impact on the immune system. We also highlight the importance of understanding the role of FOXP3 isoforms in breast cancer and the potential for using them as therapeutic targets. This review highlights the crucial role of FOXP3 isoforms in the regulation of the immune response in breast cancer and underscores the need for further research to fully comprehend their complex and diverse functions.

Author Info: (1) Cancer Biology Laboratory, Department of Biochemistry and Bioinformatics, GITAM School of Science, GITAM (Deemed to be University), Visakhapatnam 530045, Andhra Pradesh, India.

Author Info: (1) Cancer Biology Laboratory, Department of Biochemistry and Bioinformatics, GITAM School of Science, GITAM (Deemed to be University), Visakhapatnam 530045, Andhra Pradesh, India. Electronic address: rmalla@gitam.edu. (2) Department of Biotechnology, Sri Padmavathi Mahila Visvavidhyalayam, Tirupati 517502, Andhra Pradesh, India. (3) Radiation Biology Laboratory, UGC-DAE-CSR, Kolkata Centere, Kolkata 700098, West Bengal, India.

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