Cryopreservation of Whole Tumor Biopsies from Rectal Cancer Patients Enable Phenotypic and In Vitro Functional Evaluation of Tumor-Infiltrating T Cells

SIMPLE SUMMARY: Colorectal cancer (CRC) remains the third most common malignancy. Tumor-infiltrating lymphocytes (TILs) have emerged as correlates to CRC patient outcome after treatment. The pro- or anti-tumor responses of TILs are usually assessed in cell suspensions of fresh tumors that were surgically removed a few hours earlier. We propose a platform for concurrent enumeration and in vitro functional evaluation of TILs in cryopreserved tumor biopsies, offering the benefit of postponing tumor processing and analyses of TILs in cell suspensions until clinical post-treatment responses are established. Our platform is practical considering the inconsistent time when patient samples become available for research purposes and can be readily utilized by other laboratories. With a fresh portion of tumor biopsies as benchmark, we validated the recovery of viable TILs capable of interferon (IFN)-γ responses in the cryopreserved portion of same biopsies. Ultimately, this platform could provide sufficient information on TILs, to also predict patient outcome after CRC treatments. ABSTRACT: TILs comprise functionally distinct conventional and unconventional T cell subsets and their role in responses to CRC treatments is poorly understood. We explored recovery of viable TILs from cryopreserved tumor biopsies of (chemo)-radiated patients with rectal cancer to establish a platform for retrospective TIL analyses of frozen tumors from pre-selected study cohorts. Frequencies of TIL subsets and their capacity to mount IFN-γ responses in cell suspensions of fresh vs. cryopreserved portions of the same tumor biopsies were determined for platform validation. The percentages and proportions of CD4+ TILs and CD8+ cytotoxic T lymphocytes (CTLs) among total TILs were not affected by cryopreservation. While recovery of unconventional γδ T cells and mucosal-associated invariant T cells (MAIT cells) was stable after cryopreservation, the regulatory T cells (Tregs) were reduced, but in sufficient yields for quantification. IFN-γ production by in vitro-stimulated CD4+ TILs, CTLs, γδ T cells, and MAIT cells were proportionally similar in fresh and cryopreserved tumor portions, albeit the latter displayed lower levels. Thus, the proposed platform intended for TIL analyses on cryopreserved tumor biobank biopsies holds promises for studies linking the quantity and quality of TIL subsets with specific clinical outcome after CRC treatment.