Background Sarcoma is a group of rare bone and soft tissue tumors with over 50 distinct subtypes. Survival rate ranges widely due to the lack of efficacious treatments. Immunotherapy, such as adoptive cell therapy (ACT), has drawn significant interest due to its minimal toxicity. In ACT, tumor infiltrating lymphocytes (TILs) are isolated from patients, expanded, and autologously infused back. We recently observed TILs’ presence in Undifferentiated Pleomorphic Sarcoma (UPS) and Myxofibrosarcoma (MFS) tumors and found that tumor’s PD-L1 overexpression is correlated with better clinical outcome in UPS but not MFS.1 The Th1 anti-tumoral inflammatory pathway was highly activated in the former cohort, which may explain the favorable outcome. We hypothesize that there are phenotypic and functional differences between TILs of UPS with differential PD-L1 expression, which may be related to clinical outcomes. However, sarcoma TILs are rare and challenging to culture, which significantly impedes their studies. We first aim to robustly expand sarcoma TILs to sufficient numbers.
Methods Tumors’ PD-L1 expression was determined by RT-qPCR (table 1). To initiate the tumor-fragment (TF) method of TIL culturing, primary tumors were fragmented and cultured in IMDM, IL-2, and 10% HSA. We further optimized the TF protocol to expand rare sarcoma TILs. Rapid expansion protocol (REP) with anti-CD3/anti-CD28 co-stimulating beads was employed for additional expansion. During REP, TILs were co-treated with gamma-chain cytokines (IL-2, 7, 15, 21).
Results Of the 15 MFS TIL populations expanded, only 40% achieved sufficient growth (1x106) for analysis (figure 1A). Our optimized TF protocol expanded TILs from 8 UPS cases with a 62.5% success rate (figure 1B). UPS TILs were further stimulated with REP and various gamma-chain cytokine treatments. In ACT, prolonged culturing with IL-2 is known to cause activation-induced cell death, problematic in clinical treatments. We demonstrated that treatments with a Trio-cocktail (IL-7, 15, and 21) or IL-15 alone can achieve TIL proliferation comparable to that of IL-2 (figure 2).
Conclusions Sarcoma infiltrates are difficult to culture, and their roles remain largely unstudied. By optimizing the TF protocol in conjunction with anti-CD3/CD28 treatments, we developed a robust in vitro pipeline to expand rare sarcoma TILs, enabling downstream characterization. We also demonstrated the potential for alternate gamma-chain cytokines to favorably replace IL-2 during TIL expansion. Future phenotypic and functional evaluation of UPS TILs would elucidate the impact of tumors’ differential PD-L1 expression on UPS patients‘ prognoses. These findings would inform the implementation of ACT in sarcoma treatments.
Wunder J, Lee M, Nam J, Lau B, Dickson B, Pinnaduwage D, Bull S, Ferguson P, Seto A, Gokgoz N, Andrulis I. Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation. Onco Immunology 2020;9: e1737385-1- e1737385-13.
Ethics Approval Patients provided signed consent before study entry, as approved by Mount Sinai Hospital’s Ethics Board (REB#01-0138-U).
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