Periodic Reporting for period 1 - DSMT16 (Deciphering causes and consequences of inflammation in subtypes of sporadic intestinal cancer)
Período documentado: 2018-02-15 hasta 2020-02-14
Colorectal cancer (CRC) is a heterogeneous disease with diverse morphological and molecular subtypes. This heterogeneity is considered as one of the principal obstacles to efficiently treat the disease and to predict patient outcome. Whereas next-generation sequencing efforts revealed the mutational landscape of CRC, little is known on how different oncogenes and mutation patterns in the primary tumour educate their specific microenvironment (composed of immune- and stroma cells as well as microbiota) and dictate the pro- and anti-tumourigenic immune response of the stroma. Hence, the overall objective of this study was to decipher the cellular composition of the tumour microenvironment (TME) and associated inflammatory signals, which may be induced by activation of distinct oncogenes (Kras(G12D), Braf(V637E), Pik3ca(H1047R)) or by inactivation of the tumour suppressor gene Apc.
Systematic immunophenotyping of mouse models, which express oncogenic Kras(G12D), Braf(V637E) and Pik3ca(H1047R) or harbour a loss-of function deletion of Apc, using multi-colour microscopy (histocytometry), flow cytometry and transcriptomic profiling, was performed to assess the cellular composition of the TME in molecular subtypes of intestinal cancer and different tumour stages (early lesions and advanced tumour stages). In order to understand how the activation of an oncogene or deletion of a tumour suppressor gene in intestinal epithelial cells (IEC) may influence the TME, it was important to delineate the molecular basis of the tumours. For this purpose, tumour cells from the different mouse models were isolated, 3D organoid (epithelial) cultures were generated and a living organoid biobank of murine intestinal tumours was set up.
Since the above-mentioned endogenous models of intestinal subtypes predominantly develop tumours in the small intestine, which is a rare location of human cancers, it was of great importance to study tumour formation also in its native anatomical location (colon). For this purpose, an orthotopic transplantation model that is based on colonoscopy-guided injections of intestinal organoids into the submucosa of the rectum/distal colon was established. Importantly, this model also enabled us to efficiently model CRC subtypes in different syngeneic immuno-competent and immuno-deficient models, to functionally analyse tumour development from initiation to metastasis and to assess the cross-talk of cancer cells with their microenvironment.
To conclude this action, our data suggest that the mutational make-up of the tumour dictates its specific immune microenvironment, and also drives microbial alterations in the gut, which in turn may determine tumour development and survival. The results of this study could provide a basis for the development of new personalised cancer immune therapies for CRC.
Systematic immunophenotyping of mouse models, which express oncogenic Kras(G12D), Braf(V637E) and Pik3ca(H1047R) or harbour a loss-of function deletion of Apc, using multi-colour microscopy (histocytometry), flow cytometry and transcriptomic profiling, was performed to assess the cellular composition of the TME in molecular subtypes of intestinal cancer and different tumour stages (early lesions and advanced tumour stages). In order to understand how the activation of an oncogene or deletion of a tumour suppressor gene in intestinal epithelial cells (IEC) may influence the TME, it was important to delineate the molecular basis of the tumours. For this purpose, tumour cells from the different mouse models were isolated, 3D organoid (epithelial) cultures were generated and a living organoid biobank of murine intestinal tumours was set up.
Since the above-mentioned endogenous models of intestinal subtypes predominantly develop tumours in the small intestine, which is a rare location of human cancers, it was of great importance to study tumour formation also in its native anatomical location (colon). For this purpose, an orthotopic transplantation model that is based on colonoscopy-guided injections of intestinal organoids into the submucosa of the rectum/distal colon was established. Importantly, this model also enabled us to efficiently model CRC subtypes in different syngeneic immuno-competent and immuno-deficient models, to functionally analyse tumour development from initiation to metastasis and to assess the cross-talk of cancer cells with their microenvironment.
To conclude this action, our data suggest that the mutational make-up of the tumour dictates its specific immune microenvironment, and also drives microbial alterations in the gut, which in turn may determine tumour development and survival. The results of this study could provide a basis for the development of new personalised cancer immune therapies for CRC.
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.
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.
The fellow learnt state-of-the-art techniques, i.e. multi-colour microscopy (histo-cytometry), flow-cytometry and organoid culture and acquired new expertise in genetic engineering, especially in CRISPR/Cas9-based editing of organoids. In addition, the fellow implemented an orthotopic organoid transplantation model, by which organoids can be implanted into the submucosa of the colon using colonoscopy.
The fellow acquired the competence to independently carry out a project. He was trained to think critically and independently as well as to evaluate his own findings and those of others critically. In this context, the fellow is also appointed as peer reviewer in several journals. Moreover, the fellow was successfully applying for a project within the Collaborative Research Centre 1371 (SFB1371; funded by the German Research Foundation), in which he was integrated as junior group leader. This funding provides the fellow personnel costs for 1 PhD student (65% position), consumables and costs for mouse housing.
The results of the project were presented at different institutional and departmental meetings as well as national and international conferences. The results were also presented to a non-science audience at the departmental open day and at a charity gala, which helped the fellow to promote the public understanding of his area of research and to bring awareness to the public to the topic of gastrointestinal cancer and intestinal inflammation.
Moreover, the experiments will be finalised and the results will be prepared for manuscript submission.
The fellow acquired the competence to independently carry out a project. He was trained to think critically and independently as well as to evaluate his own findings and those of others critically. In this context, the fellow is also appointed as peer reviewer in several journals. Moreover, the fellow was successfully applying for a project within the Collaborative Research Centre 1371 (SFB1371; funded by the German Research Foundation), in which he was integrated as junior group leader. This funding provides the fellow personnel costs for 1 PhD student (65% position), consumables and costs for mouse housing.
The results of the project were presented at different institutional and departmental meetings as well as national and international conferences. The results were also presented to a non-science audience at the departmental open day and at a charity gala, which helped the fellow to promote the public understanding of his area of research and to bring awareness to the public to the topic of gastrointestinal cancer and intestinal inflammation.
Moreover, the experiments will be finalised and the results will be prepared for manuscript submission.