Background Peritoneal metastases (PM) in colorectal cancer (CRC) are associated with therapy resistance and poor survival. Oxaliplatin monotherapy is widely applied in the intraperitoneal treatment of PM, but fails to yield clinical benefit. We aimed to identify the mechanism(s) underlying PM resistance to oxaliplatin and to develop strategies overcoming such resistance. Experimental design We generated a biobank consisting of 35 primary tumour regions and 59 paired PM from 12 patients. All samples were analysed by RNA sequencing. We also generated a series of PM-derived organoid (PMDO) cultures and used these to design and test strategies to overcome resistance to oxaliplatin. Results PM displayed various hallmarks of aggressive CRC biology. The vast majority of PM and paired primary tumours belonged to the Consensus Molecular Subtype 4 (CMS4). PMDO cultures were resistant to oxaliplatin and expressed high levels of glutamate-cysteine ligase (GCLC) causing detoxification of oxaliplatin through glutathione synthesis. Genetic or pharmacological targeting of GCLC sensitised PMDOs to a 1-h exposure to oxaliplatin, through increased platinum-DNA adduct formation. Conclusions These results link oxaliplatin resistance of colorectal PM to their CMS4 status and high reducing capacity. Inhibiting the reducing capacity of PM may be an effective strategy to overcome PM resistance to oxaliplatin.

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CD95 is best known for its ability to induce apoptosis via a well-characterized pathway involving caspase-mediated proteolytic events. However, in apoptosis-resistant cell lines of diverse cancer types stimulation of CD95 primarily has pro-tumorigenic effects that affect many of the hallmarks of cancer. For instance, in colon cancer cells with a mutant KRAS gene CD95 primarily promotes invasion and metastasis. In the current study, we further investigated the context dependency of the consequences of CD95 activation in colon cancer. We used a series of patient-derived three-dimensional colon cancer cultures and studied their response to stimulation with CD95 ligand (CD95L). CD95L had a strong inhibitory effect on the clone-forming capacity of five out of nine cultures. In line with previous work, these cultures all had a wild-type KRAS gene and expressed high levels of CD95. Furthermore, the most sensitive cultures were characterized by microsatellite instability (MSI) and deficient mismatch repair. The reduced clonogenic growth of MSI-type colonospheres resulting from chronic CD95 stimulation was only partly due to apoptosis as many tumor cells survived treatment, yet were unable to regenerate clones. CD95 stimulation caused an irreversible cell cycle arrest, which was associated with cytokine secretion, similar to the senescence-associated secretory phenotype (SASP), and expression of senescence-associated β-galactosidase. In human colon cancer cohorts, CD95 expression was strongly correlated with the recently identified consensus molecular subtype 1 (CMS1), which mainly consists of MSI-high tumors, and with two independent SASP signatures. Mechanistically, CD95-induced senescence was caused by chronic DNA damage via caspase-activated DNAse resulting in p53 activation and p21 expression, with a minor contribution of the SASP. We conclude that induction of senescence is a hitherto unrecognized consequence of high CD95 expression, which appears to be most relevant for CMS1.

DNA mismatch repair deficiency (dMMR) in metastatic colorectal cancer (mCRC) is associated with poor survival and a poor response to systemic treatment. However, it is unclear whether dMMR results in a tumor cell-intrinsic state of treatment resistance, or whether alternative mechanisms play a role. To address this, we generated a cohort of MMR-proficient and -deficient Patient-Derived Organoids (PDOs) and tested their response to commonly used drugs in the treatment of mCRC, including 5-fluorouracil (5-FU), oxaliplatin, SN-38, binimetinib, encorafenib, and cetuximab. MMR status did not correlate with the response of PDOs to any of the drugs tested. In contrast, the presence of activating mutations in the KRAS and BRAF oncogenes was significantly associated with resistance to chemotherapy and sensitivity to drugs targeting oncogene-activated pathways. We conclude that mutant KRAS and BRAF impact the intrinsic sensitivity of tumor cells to chemotherapy and targeted therapy. By contrast, tumor cell-extrinsic mechanisms—for instance signals derived from the microenvironment—must underlie the association of MMR status with therapy response. Future drug screens on rationally chosen cohorts of PDOs have great potential in developing tailored therapies for specific CRC subtypes including, but not restricted to, those defined by BRAF/KRAS and MMR status.

Colorectal tumors are hierarchically organized: A small cancer stem cell (CSC) pool drives the formation of proliferating and differentiating tumor cells that make up the bulk of the tumor but lack tumor-initiating capacity. CSCs are thought to be derived from normal intestinal stem cells (ISCs) through the acquisition of multiple pro-tumorigenic genetic alterations. The maintenance of ISCs requires ‘niche’ signals that are provided, at least in the small intestine, by neighboring Paneth cells. CSC maintenance may also depend on niche signals although little is known about the nature of such signals and their potential source. Here we tested the hypothesis that differentiated tumor cells (DTC) could provide CSC-maintaining signals by making use of CSC-enriched colonosphere cultures that were freshly established from human colorectal tumors. We found that DTCs established from multiple colorectal tumors greatly increased the clone-forming capacity of CSCs from their own and from other tumors. This effect was reproduced by DTC-conditioned medium, suggesting the involvement of a secreted factor. By using a cytokine array and three CSC-supporting DTC cultures we identified 24 factors that were secreted by all three DTCs. Analysis of the gene expression profiles of two independent cohorts of primary colorectal tumors showed that only one of these secreted factors, stem cell factor (SCF), was significantly correlated with metastasis formation and poor overall survival. In human colonospheres SCF was mainly produced by DTCs, while the receptor for SCF, c-Kit, was expressed by CSCs. Indeed, recombinant SCF enhanced clone-forming capacity to a similar extent as DTC-conditioned medium. Furthermore, basal and DTC-stimulated clone-forming capacity was strongly reduced in the presence of an SCF-neutralizing antibody or in the presence of the cKit inhibitors Imatinib or ISCK03. qPCR analysis showed that inhibition of cKit caused a strong reduction in the expression of stem cell genes, including LGR5, CD133, OLMF4 and SOX2. In addition, both purified SCF and DTC-conditioned medium induced cKit-dependent expression of EMT and mesenchymal genes, including ZEB1, SNAIL1, fibronectin and vimentin. Our results identify a critical role for non-tumorigenic DTCs in the support of colorectal CSCs via paracrine SCF-cKit signaling. We conclude that cKit inhibitors that are widely used in the clinic to treat gastrointestinal stromal tumors (GIST) and acute myeloid leukemia (AML), may also have therapeutic value in the treatment of (a subgroup of) colorectal tumors. Citation Format: Szabolcs Fatrai, Susanne J. van Schelven, Inne H.M. Borel Rinkes, Onno Kranenburg. Stem Cell Factor (SCF) secreted by differentiated tumor cells helps maintaining the cancer stem cell phenotype in colorectal tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 269. doi:10.1158/1538-7445.AM2013-269