Abstract 1116: Spatio-temporal analysis of the tumor microenvironment of colorectal cancer subtypes using an orthotopic organoid transplantation model
Authors: Tschurtschenthaler Markus , Silva Miguel G. , Jesinghaus Moritz , Sinha Ankit , Roper Jatin , Rad Roland , Saur Dieter 
Published: September 2020

Abstract
Colorectal cancer (CRC) can be classified in several molecular, morphological and histological subtypes. Modelling of these subtypes in genetically engineered mice contributed to a mechanistic understanding of CRC development. However, these models exhibit a late tumor onset and predominantly develop tumors in the small intestine, which is a rare location of human cancers. Furthermore, murine tumors rarely progress to advanced stages and poorly recapitulate the adenoma-carcinoma sequence observed in human CRC. Preliminary data suggest that the mutational make-up of the tumor may dictate its specific immune microenvironment, but may also drive microbial alterations in the gut, which in turn determine tumor development and survival. In order to study CRC in its native anatomical location (colon) and to elucidate the cross-talk between different molecular subtypes and the microenvironment in a spatio-temporal manner, we used an orthotopic transplantation model that is based on colonoscopy-guided injections of intestinal organoids into the submucosa of the rectum/distal colon. To investigate the role of genes altered in human CRC and their interactions, we performed CRISPR/Cas9-based gene editing in organoids followed by orthotopic allogenic transplantation into the colon of recipient syngeneic mice. Notably, organoids derived from normal and tumor tissues, which harbor CRC-relevant mutations, engraft in the colon. Tissues with activated oncogenes/inactivated tumor suppressors display the ability to form invasive tumors locally and seed to distant sites. Moreover, tumors generated by orthotopic injections of tumor-derived organoids not only recapitulate the histology of the primary tumor, but also show signs of tumor progression (e.g. towards undifferentiated sarcomatoid phenotypes). Whole exome sequencing data showed that organoids derived from tumors after implantation share common mutations with the parental organoids isolated from the endogenous tumor. Interestingly, after transplantation of the parental line specific mutations are selected for, as seen by their increased variant allelic frequency (VAF). Additionally, exclusive mutations were detected in both the parental- and the xenograft-derived organoid lines suggesting that in vivo tumor progression might be fuelled by a clonal selection event taking place after engraftment of the parental organoid line. In summary, these orthotopic transplantations allow us to efficiently model CRC subtypes in different syngeneic immuno-competent and immuno-deficient models, to functionally analyze tumour development from initiation to metastasis and to assess the combinatorial effect of mutations on tumor evolution, histomorphology and the cross talk of cancer cells with their microenvironment.
Citation Format: Markus Tschurtschenthaler, Miguel G. Silva, Moritz Jesinghaus, Ankit Sinha, Jatin Roper, Roland Rad, Dieter Saur. Spatio-temporal analysis of the tumor microenvironment of colorectal cancer subtypes using an orthotopic organoid transplantation model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1116.