Illuminating radionuclide therapy response: Monitoring cancer cell and CAF survival in a direct 2D co-culture model

Targeted radionuclide therapy (TRT) directed at fibroblast activation protein (FAP), highly expressed on cancer-associated fibroblasts (CAFs), is a promising approach for treating stroma-rich tumors such as pancreatic ductal adenocarcinoma (PDAC) and breast cancer (BC). To better assess the efficacy of FAP-TRT and to get more insight into the potential of crossfire effects from target expressing CAFs to neighboring cancer cells, more representative preclinical models are needed. Therefore, we established a direct 2D co-culture model consisting of both CAFs and cancer cells.

We developed a clinically representative PDAC and BC 2D co-culture model by co-seeding human CAFs and matching cancer cells. First, the CAFs and cancer cells were transduced with distinct fluorescent and bioluminescent reporter genes (RFP-CBG99 and GFP-CBR2, respectively). Fluorescent microscopy enabled the optimization of the seeding densities. Patient tumor samples were analyzed to guide the co-culture design, ensuring realistic representation of the patient situation. Co-cultures were optimized and subsequently treated with external beam radiation therapy and radionuclide therapy (i.e. lutetium-177). The luciferase expressed by the reporter gene enabled monitoring of CAF and cancer cell viability. Cell survival was assessed using dual-color bioluminescence imaging (BLI) at 540SP, 600LP, and open filter settings.

The established PDAC and BC co-culture models closely mimicked patient tumors with regard to CAF localization, stroma density, and FAP expression. The dual-color BLI allowed for reliable discrimination of CAF and cancer cell viability. We observed a drastic decrease in cell viability after EBRT in all four cell lines, whereas only three out of four cell lines responded to lutetium-177 therapy. In addition, all cell lines exhibited similar treatment responses in the co-culture and monoculture settings.

We successfully established 2D co-culture models of PDAC and BC that include CAFs neighboring the cancer cells, as is the case in the patient tumor microenvironment. The use of dual-color reporter genes enabled efficient, cell-type-specific analysis of radionuclide therapy efficacy via BLI. The developed models provide a user-friendly platform for evaluating therapeutic responses and can be further refined into 3D systems for even higher patient resemblance.