Abstract 5609: Investigating the undifferentiated pleomorphic sarcoma microenvironment through cytokine profiles of primary tumor and immune infiltrates
Authors: Coward Victoria , Chen Jacky , Gokgoz Nalan , Tsoi Kim , Wunder Jay S. , Andrulis Irene L. 
Published: June 2022

Sarcoma is a heterogeneous group of connective tissue cancers with over 60 subtypes. Curative treatment relies on surgical resection combined with radiation. In the event of metastasis, chemotherapy is used but generally only with palliative intent. Given the heterogeneity of sarcomas, targeted therapies are limited, which has contributed to the relatively stagnant survival rates seen in the past decades.

Objective: Personalized treatments, such as adoptive cell therapy (ACT), may be promising for select sarcoma patients, specifically those with Undifferentiated Pleomorphic Sarcoma (UPS). To better understand ACT’s feasibility for UPS, we aim to investigate the intersection of tumor-infiltrating lymphocytes (TILs) and tumor cells by examining their cytokine output, whose role remains largely unstudied.

Methods: Tissue samples from untreated sarcoma patients have been collected and viably preserved by our group. The samples were processed for in vitro expansion of tumor or TIL cell lines. Tumor cultures were validated with Whole Exome Sequencing (WES) to ensure tumor-specific variations were retained. TILs were isolated with tumor fragment (TF) processing and magnetic bead coated anti-CD3/CD28 Rapid Expansion Protocol (REP). After validating tumor cultures, both tumor and TIL-secreted cytokines were evaluated using Luminex assays.

Results: Of the 11 primary UPS samples cultured, 10 generated viable cell cultures as defined by the presentation of stable growth beyond passage 5. A total of 3 cell lines were selected for further validation, and through WES, retained tumor specific variations beyond passage 5. On the other hand, the sarcoma-optimized TF and REP protocols expanded TILs from 7 of 8 UPS cases and yielded up to 6 x 10^7 cells. Although IL-2 is commonly used to facilitate TIL growth, it can also cause activation-induced cell death which is problematic for therapy. We demonstrated that an alternative γ-chain interleukin, IL-15, can support UPS TIL growth comparable to IL-2. Unbiased cytokine screening detected anti-tumoral CXCL9/IFN-γ and pro-tumoral IL-10/TGF-Beta as highly secreted by TIL cultures while some tumor cultures secreted relatively high concentrations of IL-6 and CCL2.

Conclusion: We developed a robust in vitro pipeline to expand sarcoma tumor cultures and TILs for downstream characterization. Functional assays aimed at dissecting the consequences of tumor-mediated IL-6 secretion are underway. These include siRNA-induced knock downs of genes hypothesized to augment the cytokine profile of tumor cells. Future evaluation of TILs will identify their clinical efficacy via flow cytometry and intracellular cytokine staining. Understanding cytokine secretion from both the tumor and immune components of sarcoma will aid in elucidating interactions within the microenvironment that may be therapeutically relevant.

Citation Format: Victoria Coward, Jacky Chen, Nalan Gokgoz, Kim Tsoi, Jay S. Wunder, Irene L. Andrulis. Investigating the undifferentiated pleomorphic sarcoma microenvironment through cytokine profiles of primary tumor and immune infiltrates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5609.