Abstract

Background: Intra-tumor heterogeneity (ITH) poses a major obstacle in cancer therapy. In colorectal cancer (CRC), mutations in the transforming growth factor-β (TGF-β) pathway, especially in SMAD4 gene, have been correlated with decreased overall survival and are suspected to modulate chemoresistance. We have previously shown that SMAD4^R361H is associated with differential drug response towards EGFR, MEK and PI3K inhibitors. In the present study, we analyzed the mechanistic role of SMAD4^R361H using oncoproteomics in isogenic CRISPR-engineered SMAD4^R361H, CRC patient-derived organoids (PD3D®).

Methods: Here, we investigated multiple organoid cultures from a single CRC and its liver metastasis. Targeted amplicon sequencing identified the SMAD4^R361Hmutation in 2/5 subcultures.Using a CRISPR-Cas9 approach, we introduced the SMAD4^R361H into the wildtype organoids. Organoid cultureswere subjected to a comparative drug screeningand subsequent DigiWest® multiplex protein profiling analysis with a panel of >100 total and (phospho-) proteins on cancer-specific cellular pathways.

Results: SMAD4^R361H organoids demonstrated differential response to EGFR and MEK inhibition compared to their SMAD4 wildtype counterparts. DigiWest multiplex protein profiling revealed different levels of Wnt pathway activation under treatment, while showing only minor differences upon MEK inhibition in the downstream signaling cascade. Early results point towards an initial cell cycle arrest for growth inhibition.

Conclusions: PD3Ds recapitulate the genetic and phenotypic heterogeneity of the donor tumor tissue. We show that SMAD4^R361H contributes to the sensitivity towards trametinib in vitro. The approach taken in this study has a broad range of applications in understanding cancer biology, intra-tumor heterogeneity and drug response, and may also help to improve therapy response prediction in drug development studies.