Abstract:
After years of extensive research, cancer is still a devastating disease. Even though the knowledge on cancer and its development is growing, a valid close-to-in vivo test system for new drugs is still missing. The aim of this thesis was the successful isolation of 3D patient derived microtumors (PDMs) from various cancer entities and the establishment of a PDM based test system using RCC and malignant melanoma PDMs improving the mentioned shortcomings in cancer drug discovery.
PDMs from 11 different tumor entities could be successfully isolated and cultured. The isolated PDMs of RCC and malignant melanoma models are characterized for their viability as well as compared to the patient tumor tissue using Immunohistochemistry. An interesting finding is the presence of cancer stem cells in the PDMs, making it feasible to test agents targeting this subset of cancer cells. By RPPA protein analysis, pathway mutations in the PDMs of different patients were determined and clustered. This information could be used to decide on appropriate PDM models for the testing of novel chemotherapeutic compounds. In future, possible treatment options for patients could be decided on using the RPPA information, ranging from approved SOCs for the specific cancer type to off-label use of approved drugs.
Treating single PDMs with different E:T ratios of antigen-specific T cells resulted in disintegration of the PDM and diminished live cell dye fluorescence, reflecting cell death. Based on these findings, a quantitative way to measure cell death was found by using the CellTox Green reagent, which starts to fluoresce green upon binding to free DNA. The established CytoTox assay can be used to test standard of care agents as well as novel chemotherapeutic compounds alone or in combination as well as in an immune cell co-culture. Furthermore, using autologous T cells the effects of checkpoint inhibitors on the ability to target the cancer cells could be investigated. Despite the complex assay condition due to the co-culture set-up, the assay results were reproducible and significant effects were detected with a low standard deviation.
During the isolation of the PDMs from patient tissue, TILs can be isolated and cultured for subsequent testing. Furthermore, the isolated TILs can be characterized using flow cytometry analysis to plan the treatment options to be verified using the CellTox™ Green cytotoxicity assay.
Moreover, not only cell death could be quantified using the PDMs, but also the PDM infiltration by T cells could be investigated and whether the infiltration can be enhanced by chemotherapeutic compounds or checkpoint inhibitors.
Taken together, the presented PDM-based assay is a valid test system for new anti-cancer or immunomodulatory drugs and, in future, if a large enough cohort could be tested, a possible predictive tool for treatment decisions in the clinic.
tumor biology