Final Report Summary - CSC PROGRAM (Intestinal stem cells and their role in colorectal cancer progression)
Overview of the results - part 1
During the last two years, we achieved to set up a mouse model system which allows investigation of intestinal stem cell (ISC)-like and differentiated-like phenotypes of large intestinal (colonic) tumour disease and which closely reproduces the complexity and aggressiveness of human colorectal cancer. The strategy that we have successfully followed is based on generating mice bearing conditional compound mutations which can be induced specifically in ISCs via a LGR5-driven CreERT2 (1). Importantly, by setting up an optimised protocol, induction of compound mutation-driven tumours is highly restricted to the colon and thus, the colon adeno-carcinoma development after treatment of animals with tamoxifen. In detail, we have been able to promote formation of malignant, invasive tumours starting from large ISCs via expression of the Ras oncogene (Ras-V12D) while inactivating at the same time the adenomatous polyposis coli (APC) tumour suppressor. To achieve this, we are using commercially available 129S4-Krastm4Tyj/J mice (Jackson laboratories). This strain carries a point mutation (G12D) whose expression is blocked by the presence of a loxP-flanked stop codon. Cre-mediated excision of the floxed stop element leads to an induction of GTP-bound and therefore permanently activated Kras allele. In addition, we also implicated B6.129P2-Trp53tm1Brn/J mice (Jackson laboratories) in which exons 2-10 of the p53 tumour-suppressor are flanked by lox-P sites. Cre-mediated excision of exons 2-10 leads to conditional ablation of the p53 tumour suppressor allele. By a sophisticated crossing strategy, we have generated transgenic mice with the genotype: Apcfl/fl; Lgr5+/Cre-ERt2; p53fl/fl; KrasLSL-G12/+. In these animals, cre-mediated recombination leads to ISC specific loss of both APC alleles, inactivation of p53, and concomitant expression of Ras-V12D.
Use of the project results / ongoing work - part 1
The mouse model described above is currently used to study the effects of compound genetic alterations on the cell heterogeneity of tumours (i.e. proportions of ISC-like and differentiated-like cells) as well as to understand how the compound mutations promote aggressiveness (invasion, metastasis) in each tumour cell population. Importantly, Lgr5-high and Lgr5-low cells (representing mouse colonic stem and transient amplifying cells, respectively) can be traced during tumour progression by measuring green fluorescent protein (GFP) expression pattern and intensity (1). Furthermore, we are currently testing the tumour-propagating capacity of ISC-like or differentiated-like cells after transplantation into the ceacum of syngeneic mice (2). In addition, by using a three-dimensional (3D) tissue culture system, we have succeeded to expand mouse colonic organoids with this genotype ex vivo (4) and cells have been equipped with ectopic expression of cherry-luciferase via lentiviral infection. This allows us now to trace injected or transplanted tumour cells in living animals over time using an IVIS imaging system (Caliper Life Sciences).
Overview of the results - part 2
As a complementary strategy to tackle the proposed objectives, we achieved to develop in vitro culture systems for human normal colonic stem cells (CoSCs) and human colorectal cancer-initiating cells. As outlined in the original application, ISCs have been shown previously to represent the origin of intestinal cancer disease in mice (4). Due to the much higher complexity of human colorectal cancer disease compared to classical mouse tumour models, we sought to establish in vitro culture systems for human normal colonic epithelial stem cells derived from normal colonic mucosa and cancer re-initiating cells derived from primary colorectal tumours. These model systems allow a direct comparison of normal CoSCs with their malignant counterparts coming from the same individual. Our work on characterisation and in vitro expansion of human CoSCs has been published in Nature Medicine (6). This project has been performed in cooperation with the laboratory of Hans Clevers (Hubrecht Institute, Utrecht, the Netherlands).
With the intention to study the tumour-initiating and metastatic potential of human colorectal cancer cells, we have adapted the culture conditions established for in vitro growth of normal CoSCs. Furthermore, we reported that high levels of EPHB2 characterise mouse small intestine ISCs as well as CRC stem cells (5). Importantly, as addressed the initial objective of the proposal, we could reveal a genetic program which is shared between normal CoSCs and the most aggressive tumours (5). Individuals with colorectal tumours characterised by this CoSCs transcriptional programme show a high risk of disease relapse after intended curative therapy whereas disease recurrence is rare in patients suffering from non-stem cell-like tumours (5).
Furthermore, we succeeded to isolate viable single EPHB2-high epithelial tumour cells from primary tumours by multi-colour fluorescence assisted cell sorting (FACS) sorting. These cells grow into compact tumour spheres when embedded in a 3D matrix. EPHB2-high tumour-spheres have been equipped with ectopic expression of luciferase enzyme via an adapted lentiviral infection procedure. These tumour-initiating cells have been already injected into immune-deficient mice, give rise to primary tumour growth and this process was followed in animals over time using an IVIS imaging system (Caliper Life Sciences). In addition, orthotopic micro-injection of EPHB2-high tumour spheres into the caecum-wall of immune-suppressed animals gave already detailed insight into the metastatic potential of EPHB2-high cells derived from different individuals. These experiments are ongoing and are performed in cooperation with the group of Ramon Mangues (Sant Pau Hospital. Barcelona) who has established this valuable tool in order to achieve induction of metastatic foci in clinically relevant sites (2).
Conclusions:
Taken together, the established model systems and currently performed studies are closely matching the principle objectives outlined in the original grant proposal. As discussed in the mid-term report, we had to adapt the experimental strategy due to technical difficulties encountered. Of note, the currently performed experimental analysis is at the final stage and has already yielded a significant amount of valuable results within the 2-year time period of the fellowship. Moreover, a part of our ongoing research has already been accepted for publication high-impact scientific journals (5, 6).
Socio-economic impact of the research performed
At the end of June 2011, we sent an abstract of my work on CoSCs, which has been published in Nature Medicine (6), to apply for the Cold Spring Harbor Conference on Stem Cell Biology which took place from September 20th-24th 2011. Importantly, my work was selected for a platform presentation at this conference and received a lot of positive feed-back from the audience. I had the great opportunity to discuss my research with scientist of leading international laboratories in the field of stem cell research, editors of scientific journals, and representatives of world-wide leading biotech companies.
Just recently, I attended the European Association of Cancer Research (EACR) meeting in Barcelona (7 - 10 July 2012) and our abstract on normal and cancer stem cells of the human colon had been chosen for poster presentation. Out of more than 1 080 presented posters, it received the poster award together with two other scientists, showing that the work I have performed during my time as a Marie Curie fellow is of broad interest for the scientific community and has a strong impact for the society.
Human CoSCs and cancer-initiating cells of colorectal cancer can be cultured in three dimensions for the first time with our protocols. Since this work has been published, many laboratories across Europe and beyond have contacted us in order to acquire the technical skills for robust long-term in vitro culture of normal human mucosal cells and cancer-initiating cells. Especially in the case of the human colon, we could proof that crypt EphB2-high cells are bona-fide colon stem cells (CoSCs). We characterised for the first time ever this cell type and worked out a protocol to expand these cells in a culture dish ex vivo (6). The tools we have established during the last two years are of broad interest and can now be used by medically orientated research laboratories across the world in order to explore a wide range of gastro-intestinal disease, such as inflammatory bowel disease and cancer. Our data and results provide the basis to ex vivo-culture normal human CoSCs and tumour-initiating colorectal cancer cells from the same individual thereby opening new avenues for drug-screening and customised treatment of CRC disease. Furthermore, our work on ex vivo long-term culture and expansion of normal colonic epithelium provides a valuable tool for researchers working in the field of regenerative medicine.
Reference List
(1) Barker, N., van Es, J. H., Kuipers, J., Kujala, P., van den, B. M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P. J. & Clevers, H. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003-1007 (2007).
(2) Cespedes, M. V., Espina, C., Garcia-Cabezas, M. A., Trias, M., Boluda, A., Gomez del Pulgar, M. T., Sancho, F. J., Nistal, M., Lacal, J. C. & Mangues, R. Orthotopic microinjection of human colon cancer cells in nude mice induces tumour foci in all clinically relevant metastatic sites. Am. J. Pathol. 170, 1077-1085 (2007).
(3) Sato, T., Stange, D. E., Ferrante, M., Vries, R. G., van Es, J. H., van den, B. S., Van Houdt, W. J., Pronk, A., Van, G. J., Siersema, P. D. & Clevers, H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762-1772 (2011).
(4) Barker, N., Ridgway, R. A., van Es, J. H., van de, W. M., Begthel, H., van den, B. M., Danenberg, E., Clarke, A. R., Sansom, O. J. & Clevers, H. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457, 608-611 (2009).
(5) Merlos-Suarez, A., Barriga, F. M., Jung, P., Iglesias, M., Cespedes, M. V., Rossell, D., Sevillano, M., Hernando-Momblona, X., da Silva-Diz, V., Munoz, P., Clevers, H., Sancho, E., Mangues, R. & Batlle, E. The ISC signature identifies colorectal cancer stem cells and predicts disease relapse. Cell Stem Cell 8, 511-524 (2011).
(6) Jung, P., Sato, T., Merlos-Suarez, A., Barriga, F. M., Iglesias, M., Rossell, D., Auer, H., Gallardo, M., Blasco, M. A., Sancho, E., Clevers, H. & Batlle, E. Isolation and in vitro expansion of human colonic stem cells. Nat. Med. 17, 1225-1227 (2011).
During the last two years, we achieved to set up a mouse model system which allows investigation of intestinal stem cell (ISC)-like and differentiated-like phenotypes of large intestinal (colonic) tumour disease and which closely reproduces the complexity and aggressiveness of human colorectal cancer. The strategy that we have successfully followed is based on generating mice bearing conditional compound mutations which can be induced specifically in ISCs via a LGR5-driven CreERT2 (1). Importantly, by setting up an optimised protocol, induction of compound mutation-driven tumours is highly restricted to the colon and thus, the colon adeno-carcinoma development after treatment of animals with tamoxifen. In detail, we have been able to promote formation of malignant, invasive tumours starting from large ISCs via expression of the Ras oncogene (Ras-V12D) while inactivating at the same time the adenomatous polyposis coli (APC) tumour suppressor. To achieve this, we are using commercially available 129S4-Krastm4Tyj/J mice (Jackson laboratories). This strain carries a point mutation (G12D) whose expression is blocked by the presence of a loxP-flanked stop codon. Cre-mediated excision of the floxed stop element leads to an induction of GTP-bound and therefore permanently activated Kras allele. In addition, we also implicated B6.129P2-Trp53tm1Brn/J mice (Jackson laboratories) in which exons 2-10 of the p53 tumour-suppressor are flanked by lox-P sites. Cre-mediated excision of exons 2-10 leads to conditional ablation of the p53 tumour suppressor allele. By a sophisticated crossing strategy, we have generated transgenic mice with the genotype: Apcfl/fl; Lgr5+/Cre-ERt2; p53fl/fl; KrasLSL-G12/+. In these animals, cre-mediated recombination leads to ISC specific loss of both APC alleles, inactivation of p53, and concomitant expression of Ras-V12D.
Use of the project results / ongoing work - part 1
The mouse model described above is currently used to study the effects of compound genetic alterations on the cell heterogeneity of tumours (i.e. proportions of ISC-like and differentiated-like cells) as well as to understand how the compound mutations promote aggressiveness (invasion, metastasis) in each tumour cell population. Importantly, Lgr5-high and Lgr5-low cells (representing mouse colonic stem and transient amplifying cells, respectively) can be traced during tumour progression by measuring green fluorescent protein (GFP) expression pattern and intensity (1). Furthermore, we are currently testing the tumour-propagating capacity of ISC-like or differentiated-like cells after transplantation into the ceacum of syngeneic mice (2). In addition, by using a three-dimensional (3D) tissue culture system, we have succeeded to expand mouse colonic organoids with this genotype ex vivo (4) and cells have been equipped with ectopic expression of cherry-luciferase via lentiviral infection. This allows us now to trace injected or transplanted tumour cells in living animals over time using an IVIS imaging system (Caliper Life Sciences).
Overview of the results - part 2
As a complementary strategy to tackle the proposed objectives, we achieved to develop in vitro culture systems for human normal colonic stem cells (CoSCs) and human colorectal cancer-initiating cells. As outlined in the original application, ISCs have been shown previously to represent the origin of intestinal cancer disease in mice (4). Due to the much higher complexity of human colorectal cancer disease compared to classical mouse tumour models, we sought to establish in vitro culture systems for human normal colonic epithelial stem cells derived from normal colonic mucosa and cancer re-initiating cells derived from primary colorectal tumours. These model systems allow a direct comparison of normal CoSCs with their malignant counterparts coming from the same individual. Our work on characterisation and in vitro expansion of human CoSCs has been published in Nature Medicine (6). This project has been performed in cooperation with the laboratory of Hans Clevers (Hubrecht Institute, Utrecht, the Netherlands).
With the intention to study the tumour-initiating and metastatic potential of human colorectal cancer cells, we have adapted the culture conditions established for in vitro growth of normal CoSCs. Furthermore, we reported that high levels of EPHB2 characterise mouse small intestine ISCs as well as CRC stem cells (5). Importantly, as addressed the initial objective of the proposal, we could reveal a genetic program which is shared between normal CoSCs and the most aggressive tumours (5). Individuals with colorectal tumours characterised by this CoSCs transcriptional programme show a high risk of disease relapse after intended curative therapy whereas disease recurrence is rare in patients suffering from non-stem cell-like tumours (5).
Furthermore, we succeeded to isolate viable single EPHB2-high epithelial tumour cells from primary tumours by multi-colour fluorescence assisted cell sorting (FACS) sorting. These cells grow into compact tumour spheres when embedded in a 3D matrix. EPHB2-high tumour-spheres have been equipped with ectopic expression of luciferase enzyme via an adapted lentiviral infection procedure. These tumour-initiating cells have been already injected into immune-deficient mice, give rise to primary tumour growth and this process was followed in animals over time using an IVIS imaging system (Caliper Life Sciences). In addition, orthotopic micro-injection of EPHB2-high tumour spheres into the caecum-wall of immune-suppressed animals gave already detailed insight into the metastatic potential of EPHB2-high cells derived from different individuals. These experiments are ongoing and are performed in cooperation with the group of Ramon Mangues (Sant Pau Hospital. Barcelona) who has established this valuable tool in order to achieve induction of metastatic foci in clinically relevant sites (2).
Conclusions:
Taken together, the established model systems and currently performed studies are closely matching the principle objectives outlined in the original grant proposal. As discussed in the mid-term report, we had to adapt the experimental strategy due to technical difficulties encountered. Of note, the currently performed experimental analysis is at the final stage and has already yielded a significant amount of valuable results within the 2-year time period of the fellowship. Moreover, a part of our ongoing research has already been accepted for publication high-impact scientific journals (5, 6).
Socio-economic impact of the research performed
At the end of June 2011, we sent an abstract of my work on CoSCs, which has been published in Nature Medicine (6), to apply for the Cold Spring Harbor Conference on Stem Cell Biology which took place from September 20th-24th 2011. Importantly, my work was selected for a platform presentation at this conference and received a lot of positive feed-back from the audience. I had the great opportunity to discuss my research with scientist of leading international laboratories in the field of stem cell research, editors of scientific journals, and representatives of world-wide leading biotech companies.
Just recently, I attended the European Association of Cancer Research (EACR) meeting in Barcelona (7 - 10 July 2012) and our abstract on normal and cancer stem cells of the human colon had been chosen for poster presentation. Out of more than 1 080 presented posters, it received the poster award together with two other scientists, showing that the work I have performed during my time as a Marie Curie fellow is of broad interest for the scientific community and has a strong impact for the society.
Human CoSCs and cancer-initiating cells of colorectal cancer can be cultured in three dimensions for the first time with our protocols. Since this work has been published, many laboratories across Europe and beyond have contacted us in order to acquire the technical skills for robust long-term in vitro culture of normal human mucosal cells and cancer-initiating cells. Especially in the case of the human colon, we could proof that crypt EphB2-high cells are bona-fide colon stem cells (CoSCs). We characterised for the first time ever this cell type and worked out a protocol to expand these cells in a culture dish ex vivo (6). The tools we have established during the last two years are of broad interest and can now be used by medically orientated research laboratories across the world in order to explore a wide range of gastro-intestinal disease, such as inflammatory bowel disease and cancer. Our data and results provide the basis to ex vivo-culture normal human CoSCs and tumour-initiating colorectal cancer cells from the same individual thereby opening new avenues for drug-screening and customised treatment of CRC disease. Furthermore, our work on ex vivo long-term culture and expansion of normal colonic epithelium provides a valuable tool for researchers working in the field of regenerative medicine.
Reference List
(1) Barker, N., van Es, J. H., Kuipers, J., Kujala, P., van den, B. M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P. J. & Clevers, H. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003-1007 (2007).
(2) Cespedes, M. V., Espina, C., Garcia-Cabezas, M. A., Trias, M., Boluda, A., Gomez del Pulgar, M. T., Sancho, F. J., Nistal, M., Lacal, J. C. & Mangues, R. Orthotopic microinjection of human colon cancer cells in nude mice induces tumour foci in all clinically relevant metastatic sites. Am. J. Pathol. 170, 1077-1085 (2007).
(3) Sato, T., Stange, D. E., Ferrante, M., Vries, R. G., van Es, J. H., van den, B. S., Van Houdt, W. J., Pronk, A., Van, G. J., Siersema, P. D. & Clevers, H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762-1772 (2011).
(4) Barker, N., Ridgway, R. A., van Es, J. H., van de, W. M., Begthel, H., van den, B. M., Danenberg, E., Clarke, A. R., Sansom, O. J. & Clevers, H. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457, 608-611 (2009).
(5) Merlos-Suarez, A., Barriga, F. M., Jung, P., Iglesias, M., Cespedes, M. V., Rossell, D., Sevillano, M., Hernando-Momblona, X., da Silva-Diz, V., Munoz, P., Clevers, H., Sancho, E., Mangues, R. & Batlle, E. The ISC signature identifies colorectal cancer stem cells and predicts disease relapse. Cell Stem Cell 8, 511-524 (2011).
(6) Jung, P., Sato, T., Merlos-Suarez, A., Barriga, F. M., Iglesias, M., Rossell, D., Auer, H., Gallardo, M., Blasco, M. A., Sancho, E., Clevers, H. & Batlle, E. Isolation and in vitro expansion of human colonic stem cells. Nat. Med. 17, 1225-1227 (2011).