Characterisation of the TME showed that immune cell infiltration, but also the microbiota composition significantly differs between the different CRC subtypes. In order to assess tumour cell-intrinsic signalling pathways, we isolated tumour cells of each mouse model, generated epithelial organoid cultures and established a murine tumour organoid biobank covering different histological grades of the 4 different genotypes. We performed genomic characterisation, transcriptional and proteomic profiling of selected organoids. Notably, gene ontology and pathway enrichment analysis showed that the differentially expressed genes (compared to wildtype organoids) were significantly enriched in the inflammatory response, which suggests that the epithelial fraction of the tumour may directly influence its environment by generating an inflammatory milieu.
To implement a model that allows spatio-temporal monitoring of the TME formation during tumour progression, organoids were orthotopically transplanted in the colon of recipient syngeneic mice. Notably, organoids derived from normal (no morphological and histological alterations) and tumour tissues, which harboured CRC-relevant mutations, engrafted in the colon. Moreover, tumours generated by orthotopic injections of tumour-derived organoids did not only recapitulate the histology of the primary tumour, but also acquired new features (e.g. towards undifferentiated sarcomatoid phenotypes) and showed progression to more advanced tumour stages.
For the validation and dissecting the role of immune cells in intestinal tumours of CRC subtypes, different tumour-resident immune cells were isolated by fluorescence-activated cell sorting (FACS) and cultured in vitro. Also, organoid – immune cell co-culture systems were established to investigate the interaction and communication of specific immune cells and tumour cells in a simplistic approach with reduced complexity.
Moreover, immunodeficient Rag2;Il2rg–/– mice, which lack B-, T- and NK cells were used to elucidate the role of these immune cells in sporadic carcinogenesis of Kras(G12D), Braf(V637E), Pik3ca(H1047R) and Apcfl/wt mice. Furthermore, for a systematic investigation of tumour subtype specific immune mechanisms, we generated novel mouse models (dual recombinase systems) by combining classical Cre-loxP with Flp-frt recombination technologies, which allow us spatio-temporal targeting of different microenvironmental cell populations – in parallel and independently.
Dissemination of results:
The results achieved within the period covered by the report were presented at different institutional and departmental meetings/retreats as well as national and international conferences. Moreover, the data will be presented at the AACR Annual Meeting (American Association for Cancer Research), which is the biggest international conference for cancer-related topics, in San Diego/USA later this year (postponed due to SARS-CoV-2).
Some further experiments are needed to finalise the manuscript for submission to a peer-reviewed journal.
