Integrated multiplex analysis of cell death regulators in stage II colorectal cancer suggests patients with ‘persister’ cell profiles fail to benefit from adjuvant chemotherapy

Abstract Apoptosis is regulated by intrinsic and extrinsic signaling orchestrated through multiple proteins that initiate or inhibit apoptosis. Inducing tumor cell apoptosis is a primary objective of chemotherapy, but to date, biomarkers of apoptosis sensitivity or resistance have shown mixed results. To understand the interplay between these proteins and whether the balance of apoptosis regulators influences chemotherapy responses and patient outcomes, we conducted multiplexed imaging of 16 proteins in the intrinsic and extrinsic apoptosis pathways at single cell resolution on resected tissue from 194 stage II colorectal cancer (CRC) patients who either received adjuvant chemotherapy ( n= 108) or were treated with surgery only (n=86). K-means clustering of >400,000 individual cancer cells and cell-level intensities of Apaf-1, procaspase-9, procaspase-3, XIAP, SMAC, BAX, BAK, Bcl-2, Bcl-xL, MCL-1, procaspase-8, BID, FADD, FLIP, RIP3 and cIAP1 identified distinct cell cluster profiles and demonstrated profound patient-to-patient heterogeneity. In chemotherapy-treated stage II CRC patients, those with a higher percentage of cell clusters with low procaspase-3 and high XIAP had a higher risk of recurrence. The high risk cell cluster (low-procaspase 3, high XIAP) was validated in an independent cohort of adjuvant chemotherapy-treated high-risk stage II CRC patients. To further interrogate the apoptosis sensitivity of the cell clusters, we also applied two established systems models of apoptosis initiation and execution, the BCL-2 pathway (DR_MOMP) and the caspase activation pathway (APOPTO-CELL). Here we showed that cell clusters associated with increased recurrence risk do not appear have impaired MOMP sensitivity, but downstream procaspase-3 cleavage is compromised. This represents a key characteristic of drug-tolerant ‘persister’ cells. Our study represents the most comprehensive, integrated analysis to date of apoptosis protein distribution at single-cell level in CRC tumors and identifies a subgroup of stage II patients with an apoptosis resistant, ‘persister’ cell profile who do not benefit from adjuvant chemotherapy.