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 great interest due to its minimal toxicity. In ACT, tumor infiltrating lymphocytes (TILs) are isolated from patients, expanded, and autologously reinfused back. We recently observed TIL’s 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 Thelper1 inflammatory pathway was highly activated in the former subtype, which may explain the better outcome. These results illustrate the immunological differences where TILs may play a critical role. We hypothesize that there are phenotypic and functional differences between TILs of UPS and MFS that may be related to clinical outcomes. Sarcoma TILs are rare and challenging to culture which impedes their studies. We first aim to robustly expand TILs to sufficient numbers.
Methods TILs are being expanded and cultured from UPS and MFS primary tumors with various PD-L1 levels. To initiate TIL culturing, bulk tumors were fragmented into 1mm, seeded at 1 fragment/well, and cultured in interluekin-2 supplemented complete media. Due to insufficient cell yields for characterization, rapid expansion protocol (REP) with anti-CD3/anti-CD28 co-stimulating beads was subsequently employed for further expansion.
Results Of 4 MFS cases processed to date, 15 TIL populations were derived and cultured (figure 1). Only 6 in 15 TIL cultures obtained ≥1x106 cells and are considered high initial cell count populations. 9 in 15 cultures obtained <1x106 cells and are considered low initial cell count populations. REP successfully expanded 14 out of 15 TIL populations, each obtaining between 7.8 to 268.0 x106 cells (tables 1 and 2, figures 2 and 3).
Conclusions Sarcoma infiltrates are difficult to culture and their roles remain largely unstudied. Our results demonstrate anti-CD3/anti-CD28 co-stimulation’s capability in expanding 93.3% of TILs and established a robust method of expansion. Future investigation of lineage markers, cytokine profiles, and cytotoxicity aims to identify immunological differences between UPS and MFS. TILs will be primed with memory-inducing cytokines (IL-7, IL-12, IL-15, IL-21) to modulate differentiation state and enrich cellular stemness.2 This would enhance TIL’s in vivo anti-tumor activity and prolong their survival. Elucidating TILs and their relations with tumor‘s PD-L1 expression would allow clinicians to appropriately recognize sarcoma’s tumor immune environments and select the most desirable infiltrates for superior ACT.
Ethics Approval The study was approved by Mount Sinai Hospital’s Ethics Board, approval number 01-0138-U.
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. OncoImmunology 2020;9:e1737385-1- e1737385-13.
Yang S, Ji Y, Gattinoni L, Zhang L, Yu Z, Restifo N, Rosenberg S, Morgan R. Modulating the differentiation status of ex vivo-cultured anti-tumor T cells using cytokine cocktails. Cancer Immunol Immunother 2012;62:727–736.
This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/licenses/by/4.0/.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.