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

Objective Inducing tumor cell apoptosis is a primary objective of chemotherapy but, to date, there are no validated biomarkers of apoptosis sensitivity or resistance. Our objective was to image multiple apoptosis pathway proteins at single cell level and determine multi- protein associations with recurrence risk and chemotherapy response in patients with stage II colorectal cancer (CRC). Methods and analysis Multiplexed imaging of 16 proteins in the intrinsic and extrinsic apoptosis pathways at single cell resolution on resected tissue from 194 patients with stage II CRC who either received adjuvant chemotherapy (n=108) or were treated with surgery only (n=86). K- means clustering of >600 000 cancer cells and cell level intensities of APAF1, procaspase- 9, procaspase- 3, XIAP, SMAC, BAX, BAK, BCL2, BCL- XL, MCL- 1, procaspase- 8, BID, FADD, FLIP, RIP3 and CIAP1 identified distinct cell cluster profiles. Results Chemotherapy- treated patients with a higher percentage of cell clusters with low procaspase- 3 and high XIAP had a higher risk of recurrence. This was validated in an independent cohort of adjuvant chemotherapy- treated high- risk patients with stage II CRC. We also applied two established system models of apoptosis initiation and execution to estimate cellular apoptosis sensitivity and show that these cell clusters do not appear to have impaired mitochondrial outer membrane permeabilisation sensitivity, but downstream procaspase- 3 cleavage is compromised. This represents a key characteristic of drug- tolerant ‘persister’ cells. Conclusion This study represents the most comprehensive analysis to date of apoptosis protein distribution at single cell level in CRC tumours. Our study identifies a subgroup of patients with stage II CRC with an apoptosis- resistant ‘persister’ cell profile who do not benefit from adjuvant chemotherapy.