Stem cell transdetermination is a targetable driver of therapeutic response to neoadjuvant chemoradiotherapy in rectal cancer

Neoadjuvant chemoradiotherapy (nCRT) is used in about one third of rectal cancer cases in the UK, but treatment response is heterogeneous, leading to difficulties in response prediction and sensitization. Phenotypic plasticity, driven by both cell-intrinsic mutations and cell-extrinsic signals from the tumour microenvironment (TME) has been shown to influence therapy resistance in colorectal cancer. This study explores the role of proliferative cancer stem cells (proCSCs) and revival cancer stem cells (revCSCs) in CRT response, and how phenotypic plasticity can be targeted to improve CRT sensitivity. Patient-derived organoids (PDOs) from a cohort of 10 patients with microsatellite-stable rectal cancer were subject to combinatorial perturbations of radiation, chemotherapy, exploratory radiosensitizers, and co-culture with cancer associated fibroblasts (CAFs). PDOs were analysed 3 hours and 48 hours after radiation in triplicate, totalling 2,400 experimental conditions. PDOs were analysed using single-cell thiol-organoid barcoding in situ mass cytometry (TOB is MC), enabling high-dimensional characterisation of CSC states, DNA damage responses, canonical signalling pathways, cell cycle, and apoptosis. PDOs displayed heterogeneous, patient-specific responses to CRT, and could be classified in the following manner: 1) high signalling response, high apoptosis; 2) high signalling response, low apoptosis; and 3) low signalling response, low apoptosis. Therapy-induced apoptosis did not correlate with on-target DNA damage, but instead strongly aligned with baseline stem cell index (SCI), a metric that describes the relative proCSC-revCSC ratio of each PDO. Higher rates of apoptosis were seen in PDOs that were proCSC-dominant at baseline (high-SCI), whereas revCSC-dominant PDOs (low-SCI) were more resistant apoptosis after treatment. In certain low-SCI PDOs, the proCSC population could be enriched using a PI3Ka activator before radiation, resulting in increased apoptosis after radiation with little toxicity. After CRT, a significant reduction in SCI was seen across the cohort. Blockade of revCSC transdetermination with a YAP/TEAD1 inhibitor also resulted in increased radiation-induced apoptosis. Highly plastic PDOs exhibiting large differences SCI following CRT were identified by high DACH1 expression. This study suggests that the initial level of on-target DNA-damage following CRT does not dictate cell death, rather, it is due to what state the cancer cell is in when the damage occurs. We find that rectal cancer cells in a proCSC state typically enter apoptosis in response to DNA damage, whereas cancer cells in the revCSC state survive therapy. We also find that pharmacologically enriching proCSCs and blocking access to the revCSC state can sensitize PDOs to CRT, and may have therapeutic importance in treating rectal cancer.