Flow cytometry assessment of residual melanoma cells in tumor‐infiltrating lymphocyte cultures

Abstract

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) is in development for the treatment of metastatic melanoma. In phase II clinical trials, patients with metastatic melanoma that received TIL after preconditioning had a 50-70% clinical response rate. The current approach to generate TIL is to culture melanoma enzyme digests in the presence of IL-2 for a 10-to 20-day period followed by 2 weeks of rapid expansion (REP). Prior to administration, cell therapies are characterized and tested for purity. TIL are characterized by CD3 surface marker expression, and purity is assessed by the amount of tumor remaining in culture. Evaluating TIL purity has traditionally been done by immunohistochemistry, which is often considered semiquantitative. To generate a quantitative assay, we used multiparameter flow cytometry to evaluate the presence of viable tumor cells by staining TIL populations with a viability dye and an antibody cocktail that detects intracellular tumor-antigens gp100, Mart-1, tyrosinase, S100, and surface tumor-antigen melanoma chondroitin sulfate proteoglycan (MCSP), and CD3 on T cells. Tumors were identified by gating on the viable CD3 2 population. Antigens in tumors were initially optimized with individual antibodies using both immunohistochemistry and flow cytometry. When eight different tumor cell lines were spiked into an activated T cell culture, flow cytometry was able to distinguish lymphocytes from tumors in all samples tested. Most importantly, the assay was able to detect melanoma cells in all enzyme digests (9/9) from patient samples. After IL-2-induced TIL expansion, there was a significant decrease in tumor cells; tumor cells were detected in only 2 of 12 samples. In eight IL-2-induced TIL samples that were further expanded in REP, no tumor cells were detected. We have demonstrated that flow cytometry is an alternative to immunohistochemistry for defining the purity of a TIL population. ' 2012 International Society for Advancement of Cytometry

Single Cell Analysis of Human Tissues and Solid Tumors with Mass Cytometry

“…Enzyme choice was based in part on prior use in several solid tumor types and preparation of single cell suspensions containing cancer cell and immune subsets for FACS (11)(12)(13)(14)(15)(16). DNase was also tested to determine its ability to enhance live cell yield from dissociation.…”

Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis

“…Additionally, flow cytometry has been used clinically to identify minimal residual disease and to detect disease progression in leukemia (Amir el et al, 2013; Borowitz et al, 2008; van Dongen et al, 2015). Fluorescence flow cytometry has also been applied to studies of solid tissues and tumors for research purposes (Al-Hajj et al, 2003; Chan et al, 2009; Donnenberg et al, 2013; Richards et al, 2012; Singh et al, 2004; Zimmerlin et al, 2011). …”

Single cell analysis of human tissues and solid tumors with mass cytometry

“…Six enzymes for cell separation were selected to compare in solid tumor preparation protocols for mass cytometry analysis: HyQTase, TrypLE, collagenase (Col) II, Col IV, Col V, and Col XI. Enzyme choice was based in part on prior use in several solid tumor types and preparation of single cell suspensions containing cancer cell and immune subsets for FACS . DNase was also tested to determine its ability to enhance live cell yield from dissociation.…”