Abstract 5001: Barriers to adoptive immunotherapy of pediatric cancer
Authors: Kumar Rajesh , Shivaprasad Nityashree , Hoglund Virginia , LeCeil Cosette , Wang Lingyan , Zhang Yue , Dimitrov Dimiter S. , Khan Javed , Orentas Rimas J. 
Published: September 2020

Abstract
As the adoptive immunotherapy with chimeric antigen modified T cells (CAR-T) has not proven effective in pediatric solid tumors, we established a model to define which tumor- and stromal-derived factors must be surmounted to obtain effective treatment. The human alveolar rhabdomyosarcoma cell line RH30 (fusion gene positive) was administered both i.v. and i.m. to NSG mice, and subsequently treated with a FGFR4-specifc CAR-T cells (expressing the M410 binder). Although some response was noted in the i.v. model, the i.m. tumor was entirely resistant. We observed that in both primary tumors and liver metastases RH30 was surrounded with a rich stromal element when CAR-T or control activated T cells were administered. Trichrome and H&E histochemical analysis of i.m. tumors confirmed the presence of a rich collagen-containing stromal element in T cell treated mice. This was not present in untreated tumors. We therefore analyzed cell surface and soluble factors produced by RH30 by RNA array analysis, flow cytometry and ELISA in cultured RH30 alone, or treated with interferon-gamma (IFNG) to model the presence of activated T cells. RH30 expressed transcripts for both canonical and non-canonical chemokines: CCL24, GPI, MIF, CCL26, CCL20, CCL2; as well as the cytokines: IL-15, IL-32, LIF, TGFB1, and CSF1. ELISA confirmed high expression of MIF, TGFB1, CCL24 and LIF. Transcripts for the chemokines CCL8, 9, 10, and 11 were expressed at low or undetectable levels, but were strongly induced by IFNG. And although class I MHC was present, and upregulated in the presence of IFNG, so was PDL1. Surface marker analysis by flow cytometry demonstrated that CD80, CD86 and CXCR2 are absent while the CAR-T targets CD276, EGFR, FGFR4 as well as CXCR4 are present. Flow cytometry also demonstrated consistent expression of PDL2, GAL-9, and CTLA4, and strong induction of HLA class I and PDL1 by IFNG treatment of RH30. We are currently evaluating CRISPR knock-out lines of each soluble factor for effects on gene expression, IFNG-induced marker expression, and tumor stroma in vivo. Our results indicate that in this model of rhabdomyosarcoma, tumor surface expression of glycoproteins that give an immune inhibitory signal, PDL1, PDL2, CTLA4, GAL-9, as well as soluble modulators known to be associated with tumor escape from immune control, MIF, TGFB, and LIF, all must be considered in armoring CAR-T for effective immune destruction of tumor. Moreover, the numerous chemokines produced by tumor alone, as well as induced by IFNG, indicate a complex network for recruitment of local and bone marrow-derived stromal cells that create a physical as well as cellular barrier to adoptive immunotherapy.
Citation Format: Rajesh Kumar, Nityashree Shivaprasad, Virginia Hoglund, Cosette LeCeil, Lingyan Wang, Yue Zhang, Dimiter S. Dimitrov, Javed Khan, Rimas J. Orentas. Barriers to adoptive immunotherapy of pediatric cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5001.