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Abstract
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).
Initial culturing of four primary MFS tumor cases with complete media (CM) over 4 weeks. Ten total cases were selected, five cases for each UPS and MFS sarcoma subtypes. To date, four MFS cases #164, 207, 214, and 225 have been processed. TIL populations were identified and categorized based on their growth rates and labelled as ‘1’ or ‘2’ representing ‘fast’ or ‘slow’ growing TILs, respectively. ‘A’ and ‘B’ represent technical replicates. Population TIL 164 ‘2’ has no replicates. 15 populations were derived from the four MFS cases. TILs were cultured and expanded from tumor fragments in CM over 4 weeks in duration. CM consisted of Iscove’s Modified Dulbecco’s Medium, 6000 IU/mL IL-2, 10% human serum albumin, 25 mmol/L HEPES, 2mmol L-glutamine, 5.5x10-5 mol/L β-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin. At week 4, cells were collected and counted with a hemocytometer. Only 6 populations achieved ≥ 1x106 cells and are categorized as high initial cell count populations. 9 populations achieved <1x106 cells and are categorized as low initial cell count populations. The 9 low initial cell count populations were further numbered with specific cell counts in figure 1 for clarity. These cell yields, with the exception of TIL 207 1A and 1B, are insufficient for characterization experiments.
REP treatment of populations with high initial cell count over 3 weeks. This graph corresponds to table 1. 6 out of 15 populations achieved ≥ 1x106 cells after 4 weeks of initial culturing and were subsequently REP-treated with anti-CD3/anti-CD28 coated magnetic Dynabeads (Life Technologies) on 24-well plates. REP-treated populations have trendlines with positive slopes across the 3 weeks of REP expansion, indicating positive growth rates. Negative controls (-) without REP treatments have near-flat trendlines indicating lack of growth. REP was successful in expanding all 6 TIL populations. At week 3 post-REP, cells were collected and counted via hemocytometer.
REP treatment of populations with low initial cell count over 4 weeks. This graph corresponds to table 2. 9 out of 15 populations achieved <1x106 cells after 4 weeks of initial culturing and were subsequently REP-treated with anti-CD3/anti-CD28 coated beads on 96-well plates. REP-treated populations have trendlines with positive slopes across the 4 weeks of REP expansion, indicating positive growth rates. REP was successful in expanding 8 of 9 TIL populations. TIL 225 2B failed to expand (data not shown) due to cell fatigue and apoptosis during the initial tumor fragment culturing and not due to REP’s capabilities or robustness. Negative controls were not established due to constraints with initial cell availability. At week 4 post-REP, cells were collected and counted via hemocytometer.
REP treatment of populations with high initial cell count over 3 weeks. Table 1 corresponds to graphed analysis in figure 2. 6 populations that achieved ≥ 1x106 cells after 4 weeks of initial culturing were treated with anti-CD3/anti-CD28 coated beads on 24-well plates over 3 weeks. Total cells seeded before REP, total cells collected after REP, and fold-expansion calculated are shown. At week 0, all populations were seeded at a density of 1x106 cells per well, except for TIL 164 1A and TIL 164 1B which were seeded at 1.1x106 cells and 1.4x106 cells per well, respectively. Populations were expanded via addition of beads at a bead to cell ratio of 1:1. At week 3 post-REP, cells were collected, counted, and fold-expansion was determined. Cell counts performed via hemocytometer. Negative controls (-) were only allocated for certain populations with sufficient initial cell availability.
REP treatment of populations with low initial cell count over 4 weeks. Table 2 corresponds to graphed analysis in figure 3. 9 populations that achieved <1x106 cells after 4 weeks of initial culturing were treated with anti-CD3/anti-CD28 coated beads on 96-well plates over 4 weeks. Total cells seeded before REP, total cells collected after REP, and fold-expansion calculated are shown. At week 0, populations were seeded at a density of 8x104 cells per well, except for populations with <8x104 cells in total, which were seeded at all available cells per well. For example, TIL214 1B was seeded at 6x104 cells per well. Populations were expanded via addition of beads at a bead to cell ratio of 1:1; once at week 0 and once more as re-stimulation at week 2. At week 4 post-REP, cells were collected, counted, and fold-expansion was determined. TIL225 2B did not yield any growth under 10X light microscope observation. Lack of cells and cell debris was observed; hence this population was not collected. Cell counts performed via hemocytometer. Negative controls were not established due to constraints with initial cell availability.
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.
References
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