LAUSR

The importance of the immune system in cancer treatment is now recognized with a range of therapies utilizing its power to more effectively target autologous cancerous cells. Multiple immune cells actively infiltrate the tumor in response to chemokine signaling in an attempt to halt disease progression. Tumor infiltrating lymphocytes (TILs) elicit altered responses (anti- or pro-tumorigenic) dependent on the microenvironment and stage of disease. Individuals with high numbers of TILs are expected to be more responsive to neoadjuvant chemotherapy and have improved pathological complete response rates. Therefore the ability to study TIL response to immune and chemotherapies in vitro is ever more important. Here, we present an advanced 3D model of TILs, enabling the simultaneous quantification of phenotypes, cell type ratios and activation status of infiltrated and non-infiltrated cells. Single spheroids were formed in ultra-low attachment 96 well plates for 3 days from a variety of cell types, namely ovarian (SKOV3), breast (BT474) and lung (A549) cancer. PBMCs were added for a period of 24 h (pre-activated) or 40 h (in well activation) before non-infiltrated PBMCs were removed and the spheroids were dissociated. Analysis of subsets for both infiltrating and non-infiltrating T cells was performed using the T Cell Activation and Cytokine Profiling Kit (Sartorius) and the iQue3 Flow Cytometer (Sartorius). Tumor type-specific infiltration was observed, with A549 spheroids exhibiting 54% reduced infiltration of CD3+ TILs compared with spheroids formed of BT474. Despite this reduced infiltration, A549 exhibited a higher CD8:CD4 ratio of 1.4 ± 0.13 compared with 0.6 ± 0.04 for BT474. An altered ratio of CD8:CD4 T cells has been reported in multiple tumor types, with increasing CD4+ cells often recognized with poor outcome. Interestingly, infiltrating T cells were observed to have significantly increased activation markers (CD69, CD25 and HLA-DR) compared to their non-infiltrated counterparts. For example in BT474 spheroids, maximal CD69 expression of infiltrated CD3+ cells was reached with just 247 CD3/CD28 Dynabeads/well, compared with 20K/well for non-infiltrated CD3+ cells. T cell infiltration can be affected by stromal cells, which produce extracellular matrix components contributing to a stiffer ECM. This remodeling of the ECM affects the quantities and types of T cells that can infiltrate into a tumor. When including normal human dermal fibroblasts in BT474 spheroids at a 1:1 ratio, a 60% reduction of maximal infiltration of CD3+ cells was observed. Although infiltration declined, T cell activation levels were comparable to those observed in spheroids containing only BT474 cells. These data highlight the complexity of modelling TILs in vitro, with marked dependence on tumor cell type and the presence of stromal cells. Utilizing flow cytometry alongside advanced assays has the potential to offer translational models for the development of therapeutics modulating T cell infiltration. Citation Format: Donald Weldon, Lauren Kelsey, Kirsty McBain, Clare Szybut, Timothy Dale. Phenotyping tumor infiltrating lymphocytes (TILs) in vitro using 3D cell models and advanced flow cytometry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 311.