Advanced bladder cancer patients have limited therapeutic options resulting in a median overall survival (OS) between 12 and 15 months. Adoptive cell therapy (ACT) using tumor infiltrating lymphocytes (TIL) has been used successfully in treating patients with metastatic melanoma, resulting in a median OS of 52 months. In this study, we investigated the feasibility of expanding TIL from the tumors of bladder cancer patients. Primary bladder tumors and lymph node (LN) metastases were collected. Tumor specimens were minced into fragments, placed in individual wells of a 24-well plate, and propagated in high dose IL-2 for four weeks. Expanded TIL were phenotyped by flow cytometry and anti-tumor reactivity was assessed after co-culture with autologous tumor digest and IFN-gamma ELISA. Of the 28 transitional cell bladder or LN tumors collected, 14/20 (70%) primary tumors and all of the LN metastases demonstrated TIL expansion. Expanded TIL were predominantly CD3+ (median 63%, range 10–87%) with a median of 30% CD8 + T cells (range 5–70%). TIL secreted IFN-gamma in response to autologous tumor. Addition of agonisitic 4-1BB antibody improved TIL expansion from primary bladder tumors regardless of pre-treatment with chemotherapy. This study establishes the practical first step towards an autologous TIL therapy process for therapeutic testing in patients with bladder cancer.
Immunotherapy, including adoptive cell transfer (ACT) with tumor-infiltrating lymphocytes (TIL) predominantly targets improvement in MHC Class I-mediated anti-tumor immune responses. The primary objective of this study is to better understand the role of CD4+ TIL in ACT as a complementary avenue for therapeutic efficacy. Briefly, CD4+ TIL were isolated by negative selection from metastatic melanoma patients who received TIL therapy at Moffitt Cancer Center on IRB approved protocols. Individual T cell clones were tracked by TCRbeta sequencing to quantify clonal persistence in patients. CD4+ TIL clones were found to be decidedly persistent in a candidate patient who achieved a complete response (CR) after infusion of 88% CD4+ T cells. CD4+ TIL from additional patients were stimulated with anti-CD3/CD28 in vitro and those who were clinical responders demonstrated a pleiotropic cytokine profile marked by an elevated ratio of Th1:Th2 cytokines (p=0.07, n=13). When cultured with APCs loaded with autologous tumor (AT), CD4+ TIL produced high levels of IFN-gamma in an MHC Class II-dependent manner. Induction of MHC Class II on melanoma cell lines and AT determined that CD4+ TIL secreted IFNg and TNFa directly in response to AT. ACT of tumor-reactive CD4+ TIL in immunodeficient (NSG) mice provided significant control of AT growth when compared to non-reactive CD4+ TIL. Preliminary data in syngeneic mouse models also suggests that antigen-specific CD4+ T cells aid in initial tumor rejection, memory formation and epitope spreading, resulting in an overall increased therapeutic efficacy during ACT. This data supports the conclusion that CD4+ TIL are tumor-reactive and instrumental to an effective anti-tumor immune response in cancer patients.
BackgroundThe tumor immune microenvironment comprises a heterogeneous collection of adaptive and innate immune cells that play a critical role in immune evasion and response to immunotherapeutic agents. cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway results in activation of various immune cells promoting innate immunity in addition to senescence of cancer cells. However, the mechanisms involved in response and resistance to cGAS-STING pathway activation is not well understood. Using Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq), we explored immunological heterogeneity of tumor microenvironment in colorectal cancer and analyzed transcriptional and compositional changes of the immune landscape in response to cGAS-STING pathway activation alone and in combination with a PD-1 inhibitor nivolumab.MethodsAll human tumor samples were obtained with proper patient consent and IRB approval. Fresh patient tumor tissue was processed to generate uniform sized live 3D tumoroids measuring 150 µm in size. Treatment groups included a STING agonist, ADU-S100, alone or in combination with nivolumab. Here, we applied multi-modal CITE-seq profiling using the 10X Genomics platform to interrogate cellular responses to ex vivo treatment. Culture supernatants were collected for multiplex analysis of cytokine release in media. Additionally, flow cytometry was used to assess the activation profile of resident immune cells.ResultsMultimodal analysis of transcriptomes or proteomics at the single-cell level provided an unprecedented view of cellular diversity and enabled better understanding of how activation of STING pathway alone and in combination with nivolumab affects the TME in colorectal cancer. Flow cytometric analysis of immune cell populations isolated from 3D tumoroids demonstrated treatment mediated activation of tumor resident T-cells and changes in the innate immune cells, which coincided with marked changes in pro-and anti-inflammatory cytokine profiles determined by multiplex analysis.ConclusionsThese results demonstrate that the 3D-EXplore ex vivo tumoroid model provides a unique platform to assess the efficacy of immunotherapeutic agents and to develop novel therapeutic combinations. Furthermore, implementation of this platform in the clinical studies may also allow identifying clinically relevant biomarkers to enable the most effective treatment strategies for individual patients.
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