5-FU and the resistance of patient-derived rectal cancer organoids to irinotecan via activating the Hedgehog pathway.
Authors: LV Tao , Xu Xiaoya , Yao Ye , Mu Peiyuan , Shen Lijun , Zhang Hui , Wu Ruiyan , Wan Juefeng , Wang Yan , Zhang Long , Tang Peiyuan , Mo Shaobo , Wang Yaqi , Xia Fan , Li Xiaomeng , Guan Ruoyu , Hua Guoqiang , Zhang Zhen 
Published: June 2022

e15598 Background: 5-FU-based regimens are the mainstay treatment for colorectal cancer. However, the limited response rate to 5-FU-based regimens hampered the increase in the treatment efficacy of locally advanced rectal cancer. During the drug screening in the rectal cancer organoid biobank (n = 106), we found out that more than half of the organoids that were resistant to 5-FU monotherapy and sensitive to irinotecan maintained sensitive status to the treatment of 5-FU combined with irinotecan (combination-sensitive), while a quite proportion of them turn to become remarkably resistant to 5-FU plus irinotecan (combination-resistant). Some studies suggested the mechanism of 5-FU-based resistance was associated with the activation of some oncogenic pathways. However, the mechanism of the addition of 5-FU promoting the resistance to irinotecan is still unclear. Methods: To investigate the relationship between the combination-resistant organoids and corresponding patient responses, we collected patient clinical responses and survival outcomes. RNA sequencing and ATAC sequencing were performed to identify differential genes and their enrichment pathways. Moreover, small molecule inhibitors were used to try to reverse the resistance induced by 5-FU. Results: Twenty-one organoids showed sensitivity to irinotecan and resistance to 5-FU. Among them, 8 organoids (38.1%) were combination-resistant and 13 organoids (61.9%) showed sensitivity to combination treatment. Combination-resistant organoids corresponding patients had worse pathologic tumor regression grades (pTRGs) distribution (8/8, 100% had TRGs of 2-3 versus 100% had TRGs of 0-1 in combination-sensitive patients; p < 0.05) and disease-free survival (DFS) rates (HR:11.57, 95% IC:1.025-130.5; p = 0.0047) than patients whose organoids were combination-sensitive. Combination-resistant organoids had higher expression of Ki-67 and CD44 and reduced cleaved-caspase 3 in the combination treatment group and 5-FU group compared with the irinotecan group and control. Furthermore, we identified hedgehog pathway activation after adding 5-FU to irinotecan in combination-resistant organoids through RNA sequencing and ATAC sequencing. GANT-61, an inhibitor of the hedgehog pathway, increased sensitivity to the combination of 5-FU and irinotecan in combination-resistant organoids. Conclusions: We found that organoids which are sensitive to irinotecan turn to become resistant to 5-FU combined with irinotecan. Moreover, corresponding patients had worse responses and a lower DFS rate. The hedgehog pathway is activated in these organoids after the addition of 5-FU to irinotecan, and GANT-61 could reverse the resistance caused by 5-FU. However, more organoid-associated trials with large-scale patients are warranted to reverse the 5-FU-based resistance in the future.