Assessing human anti-tumor T-cell responses in real-time 3D co-culture

Details

Supervisors

Pierre van der Bruggen ; Annika Malin Bruger

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

In mice, tumor-associated macrophages (TAM) promote tumorigenesis and impair the efficacy of immunotherapy. However, gaps remain in understanding the impact of TAM on immunotherapy in humans. This project aims to establish a co-culture model to study the influence of tumor-associated macrophages on anti-tumor T-cell responses with real-time 3D microscopy. The in vitro co-culture model will be complementing a humanized mouse model established by our collaborators in Stanislas Goriely’s laboratory at ULB that uses the same tumor cells and T-cells. We selected two CD8 T-cell clones for the project: LB33-CTL.D4 recognizes the human tumor antigen MUM-3 and LB2369-CTL736/89 recognizes the human tumor antigen MAGE-A1. We expanded the T-cell clones and confirmed their functional activity via cytokine secretion assays and lytic capacity assays. Part of the expanded T-cell clones were provided to the Goriely laboratory for in vivo experiments. We selected four tumor cell lines, which express either the MUM-3 or the MAGE-A1 human tumor antigens, and would thus be targeted by the T-cell clones. We assessed their ability to form spheroids without the aid of Matrigel, which led to the confirmation of the melanoma cell line LB33.MEL.A.1 (expressing MUM-3) and the squamous cell carcinoma head-and-neck cell line LB1017-SCCHN (expressing MAGE-A1). We also identified the melanoma cell line SK29-MEL as a control tumor cell line. SK29-MEL expresses the HLA-A molecules that present MUM-3 or MAGE-A1 but neither antigen. SK29-MEL cells also form spheroids without Matrigel support. The cell lines were tagged with GFP and mCherry to better visualize them in real-time microscopy. Finally, we set up the real-time 3D microscopy for (mCherry-tagged) tumor spheroid and (green tracker-dye-tagged) T-cell co-cultures. This system allowed us to track how T-cell clones kill tumor spheroids over time.