Serrated polyposis syndrome (SPS) presents with multiple sessile serrated lesions (SSL) in the large intestine and confers increased colorectal cancer (CRC) risk. However, the etiology of SPS is not known. SSL-derived organoids have not been previously studied but may help provide insights into SPS pathogenesis and identify novel biomarkers and chemopreventive strategies. This study examined effects of EGFR and COX pathway inhibition in organoid cultures derived from uninvolved colon and polyps of SPS patients. We also compared with organoids representing the hereditary gastrointestinal syndromes, Familial Adenomatous Polyposis (FAP) and Lynch syndrome (LS). Eighteen total organoid colon cultures were generated from uninvolved colon and polyps in SPS, FAP, LS, and non-syndromic screening colonoscopy patients. BRAF and KRAS mutation status was determined for each culture. Erlotinib (EGFR inhibitor) and sulindac (COX inhibitor) were applied individually and in combination. A 44-target gene custom mRNA panel (including WNT and COX pathway genes) and a 798-gene microRNA gene panel were used to quantitate organoid RNA expression by NanoString analysis. Erlotinib treatment significantly decreased levels of mRNAs associated with WNT and MAPK kinase signaling in organoids from uninvolved colon from all four patient categories and from all SSL and adenomatous polyps. Sulindac did not change the mRNA profile in any culture. Our findings suggest that EGFR inhibitors may contribute to the chemopreventive treatment of SSLs. These findings may also facilitate clinical trial design using these agents in SPS patients. Differentially expressed genes identified in our study (MYC, FOSL1, EGR1, IL33, LGR5 and FOXQ1) may be used to identify other new molecular targets for chemoprevention of SSLs.

Numerous demographic factors have been associated with colorectal cancer (CRC) risk. To better define biological mechanisms underlying these associations, we performed RNA sequencing of stem-cell-enriched organoids derived from the healthy colons of seven European Americans and eight African Americans. A weighted gene co-expression network analysis was performed following RNA sequencing. Module-trait relationships were determined through the association testing of each module and five CRC risk factors (age, body mass index, sex, smoking history, and race). Only modules that displayed a significantly positive correlation for gene significance and module membership were considered for further investigation. In total, 16 modules were associated with known CRC risk factors (p < 0.05). To contextualize the role of risk modules in CRC, publicly available RNA-sequencing data from TCGA-COAD were downloaded and re-analyzed. Differentially expressed genes identified between tumors and matched normal-adjacent tissue were overlaid across each module. Loci derived from CRC genome-wide association studies were additionally overlaid across modules to identify robust putative targets of risk. Among them, MYBL2 and RXRA represented strong plausible drivers through which cigarette smoking and BMI potentially modulated CRC risk, respectively. In summary, our findings highlight the potential of the colon organoid system in identifying novel CRC risk mechanisms in an ancestrally diverse and cellularly relevant population.

Genome engineering has become more accessible thanks to the CRISPR-Cas9 gene-editing system. However, using this technology in synthetic organs called \"organoids\" is still very inefficient. This is due to the delivery methods for the CRISPR-Cas9 machinery, which include electroporation of CRISPR-Cas9 DNA, mRNA, or ribonucleoproteins containing the Cas9-gRNA complex. However, these procedures are quite toxic for the organoids. Here, we describe the use of the \"nanoblade (NB)\" technology, which outperformed by far gene-editing levels achieved to date for murine- and human tissue-derived organoids. We reached up to 75% of reporter gene knockout in organoids after treatment with NBs. Indeed, high-level NB-mediated knockout for the androgen receptor encoding gene and the cystic fibrosis transmembrane conductance regulator gene was achieved with single gRNA or dual gRNA containing NBs in murine prostate and colon organoids. Likewise, NBs achieved 20%-50% gene editing in human organoids. Most importantly, in contrast to other gene-editing methods, this was obtained without toxicity for the organoids. Only 4 weeks are required to obtain stable gene knockout in organoids and NBs simplify and allow rapid genome editing in organoids with little to no side effects including unwanted insertion/deletions in off-target sites thanks to transient Cas9/RNP expression.

Early onset colorectal cancer (EOCRC) rates have increased in recent decades. While lowering the recommended age for routine colonoscopies to 45 may reduce this burden, such measures do not address those who develop CRC before that age. Additional measures are needed to identify individuals at-risk for CRC. To better define transcriptomic events that precede the development of CRC, we performed RNA-sequencing analysis in colon organoids derived from seven healthy and six familial adenomatous polyposis (FAP) patients. This led to the identification of 2635 significant differentially expressed genes (FDR < 0.05). Through secondary analysis of publicly available datasets, we found that these genes were enriched for significant genes also present in FAP CRC and non-hereditary CRC datasets, including a subset that were unique to EOCRC. By exposing FAP colon organoids to a three-day ethanol treatment, we found that two EOCRC-relevant genes were also targets of CRC related lifestyle factors. Our data provides unique insight into the potential, early mechanisms of CRC development in colon epithelial cells, which may provide biomarkers for patient monitoring. We also show how modifiable lifestyle factors may further alter genes relevant to EOCRC, adding weight to the hypothesis that such factors represent an important contributor to increased EOCRC incidence.

Organoids have revolutionized cancer research as highly adaptable models that enable an array of experimental techniques to interrogate tissue morphology and function. Because they preserve the genetic, phenotypic, and behavioral traits of their source tissue, organoids have gained traction as the most relevant models for drug discovery, tracking therapeutic response and for personalized medicine. As organoids are indisputably becoming a mainstay of cancer research, this review specifically addresses how colon-derived organoids can be perfected as multidimensional, scalable, reproducible models of healthy, pre-neoplastic and neoplastic conditions of the colon and for use in high-throughput \"Phase-0\" human clinical trials-in-a-dish.