Abstract:
Breast cancer (BC) has been studied in various models and cell types, yet patient-derived metastatic BC organoids remain to be further explored. Here, the impact of therapeutic
compounds on BC cell lines and patient-derived metastatic organoids was investigated, to
create a reliable application for in vitro drug screenings. In the first part of this study, different concentrations of various drugs were tested on two-dimensional BC cell lines to generate drug response curves and identify sensitive lines. Most studies on BC typically involve BC cell lines and primary tumor tissue for creating organoid models. Therefore, metastatic BC was addressed in the second part of this study. As metastasized BC has a poor outcome, there is a requirement for dependable precision oncology and models representing the heterogeneous nature of BC. A frequent symptom of metastasized BC includes the accumulation of fluid and metastatic cells in the peritoneum called malignant ascites (MA) or in the pleural cavity referred to as malignant pleural effusion (MPE). In this thesis, metastatic tumor cells derived from MA or MPE from advanced BC patients were cultured applying organoid technology. The resulting organoid cultures were referred to as metastatic BC patient-derived organoids (MBC-PDO). The characteristics of MBC-PDO lines were identified by applying immunohistochemistry and mutation analysis of hotspot regions. MBC-PDO lines could preserve their expression patterns and hotspot mutations when compared to their original metastatic counterpart and are therefore a suitable in vitro model for metastasized BC. Subsequently, MBC-PDO lines were treated with various inhibitors and chemotherapy drugs to implement a reliable application for drug screenings of metastasized BC cells. These three-dimensional drug assays demonstrated that drug responses are not always in accord with expression patterns and hotspot mutations. This discrepancy highlights the importance of combining immunohistochemistry stainings and mutational analysis of MBC-PDO lines with in vitro drug assays. Our MBC-PDO lines recapitulate the characteristics of their original sample derived from MA and MPE and serve as a valuable model that can be used in a preclinical setting for guiding therapy decisions. For instance, hematologic malignancies have been shown to be effectively treated with chimeric antigen receptor (CAR) T cell therapy. Yet, CAR-T efficacy in
battling solid tumors, like BC, still needs improvement. Lastly, the capability of utilizing MBC-PDO lines for Adapter CAR (AdCAR) T cell-mediated cytotoxicity assays was therefore investigated. MBC-PDO lines were transduced to express luciferase and GFP. Tumor-associated antigens of MBC-PDO lines were characterized by flow cytometry. An assay for the treatment of metastasized BC cells with AdCAR-T cells was implemented to assess responses in a personalized manner. MBC-PDO lines were used as a screening platform to evaluate AdCAR-T cell-based precision immunotherapy for flexible targeting of tumor-associated antigens. Based on their antigen expression, MBC-PDO lines were targeted with AdCAR-T cells and biotinylated monoclonal antibodies against CD276, HER2, EGFR, TROP2, and EpCAM. Flow cytometry data and co-culture assays demonstrated that cytolysis by AdCAR-T cells correlated with the antigen expression of MBC-PDO lines. The results suggest that MBC-PDO lines can serve as a reliable model for assessing the efficacy of AdCAR-T on metastatic BC. Overall, MBC-PDOs have the potential to serve as a tool to study metastasized BC and help prolong patients’ survival time and improve their quality of life.
Drug response