Leveraging a Patient-Derived Tumoroid Platform for Precision Radiotherapy: Uncovering DNA Damage Repair Inhibitor-Mediated Radiosensitization and Therapeutic Resistance in Rectal Cancer

BackgroundPrecision radiation strategies that expand the therapeutic window by selectively sensitizing tumors and sparing normal tissues are needed. We developed a matched tumoroid-organoid preclinical platform to identify and characterize personalized radiosensitization strategies. MethodsWe established 15 rectal cancer-derived tumoroids and 3 matched normal rectal epithelial organoids. Whole exome sequencing characterized mutation profiles and phylogenetic relationships. Tumoroids were treated with one of four DNA damage repair inhibitors (DDRi; ATMi, DNA-PKi, PARPi, or ATRi), 5-fluorouracil, or a DMSO control, followed by escalating doses of radiation. Cell viability was measured, and intrinsic radiosensitivity as well as radiosensitizer efficacy were characterized using linear regression models. Four tumoroids were derived from one patient at distinct stages and disease sites, including pre-progression tumoroids (primary tumor, splenic metastasis) and post-progression tumoroids (rectal recurrence and vaginal recurrence). ResultsTumoroid radiosensitivity demonstrated variability, paralleling the spectrum of clinical responses seen in rectal cancer. Genomic analyses revealed two distinct mutational signatures (SBS14 and SBS17b) associated with radioresistance. Radiation sensitization by DDRis was highly heterogeneous, depending on both specific tumoroid and inhibitor choice. In the subset of four tumoroids derived pre- and post-progression, post-progression tumoroids demonstrated greater radioresistance and diminished DDRi-induced radiosensitization. Phylogenetic analysis revealed increased clonal and subclonal complexity in these radiation and DDRi-resistant tumoroids. Lastly, comparing matched patient-derived tumoroids and normal organoids established that tumoroids were generally more radiosensitive and exhibited enhanced DDRi-mediated radiosensitization compared to normal organoids. However, the optimal DDRi for maximizing therapeutic index varied among tumoroids. ConclusionTo our knowledge, this is the first ex vivo study to systematically quantify intrinsic radiation sensitivity and DDRi sensitization in a comprehensive, patient-specific tumoroid platform. Our findings underscore the utility of a patient-matched tumoroid-organoid model as a platform to quantify and predict personalized responses to radiation and DDR inhibitors. Moreover, by comparing tumoroids from a single patient across different disease stages, our model reveals how tumors adapt to therapeutic pressure and develop increased radioresistance, offering a valuable potential framework for guiding precision radiotherapy. Moreover, the data show that DDRi efficacy is largely tumor-selective and not solely predicted by mutational profiles, highlighting opportunities to refine treatment for radioresistant tumors, minimize injury to normal tissues, and adapt therapy over disease progression.