Introduction: Sarcoma is a group rare soft tissue and bone tumors with over 50 distinct subtypes. Survival rate ranges widely due to the lack of efficacious treatments. Due to its low toxicity and autologous specificity, adoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) has drawn significant interest and may be of value in sarcoma. We recently observed the presence of TILs in Undifferentiated Pleomorphic Sarcoma (UPS) and Myxofibrosarcoma (MFS), and tumor’s PD-L1 overexpression is correlated with better clinical outcomes in UPS but not MFS patients. The T_h1 inflammatory pathway, responsible for anti-tumor T cell activity, was highly activated in the former subtype and may explain the better outcome. From these findings, we hypothesize that there are phenotypic and functional differences between TILs of UPS and MFS that may be related to clinical outcomes. We aim to improve ACT for sarcoma treatments with these explorations.

Methods: Sarcoma TILs are rare and difficult to culture. We first aim to robustly expand TILs to achieve sufficient yield. TILs are being isolated and initially expanded from primary UPS and MFS tumors with varying PD-L1 levels, determined by RT-qPCR. To initiate TIL culturing, primary bulk tumors were fragmented into 1mm, seeded at 1 fragment/well, and cultured with high dose interluekin-2 growth factor (6000IU/mL). To augment TIL expansion and yield, a modified rapid expansion protocol (REP) with anti-CD3/CD28 magnetic Dynabeads was employed.

Results: Of the 4 MFS cases processed to date, 15 TIL populations were derived from biological replicates and slow-and fast-growing TILs as defined by the Ohashi group. Over 4 weeks of initial expansion, 9 populations obtained <1x10^6 cells while only 6 populations obtained ≥1x10^6 cells. Employing the modified REP protocol with Dynabeads further expanded 14 out of 15 TIL populations, obtaining up to 268.0x10^6 cells. We also developed new cytokine treatment methods to proliferate TILs using a cocktail of gamma-chain cytokines containing IL-7, IL-15, and IL-21. Our results yielded growth comparable to the gold standard proliferation from IL-2.

Conclusion: Sarcoma infiltrates are challenging to culture, and their roles remain largely unstudied. Our results demonstrate anti-CD3/CD28-mediated co-stimulation’s capability to expand sarcoma TILs, and we established a robust method of expansion for downstream experimental analysis. We further showed that gamma chain cytokine cocktail (IL-7, IL-15, & IL21) is a potential alternative for TIL expansion, avoiding IL-2 mediated side effects such as activation-induced cell death and exhaustion. Future investigation of TIL’s lineage markers, cytokine profiles, and cytotoxicity will assess clinically relevant differences between UPS and MFS immune infiltrates.