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Title: A genome editing based approach to study tumor cell heterogeneity
Author: Turon, Gemma
Director/Tutor: Batlle, Eduard
Cortina, Carme
Keywords: Càncer colorectal
Cèl·lules mare
Colorectal cancer
Stem cells
Issue Date: 25-Apr-2019
Publisher: Universitat de Barcelona
Abstract: [eng] Colorectal tumors are not homogeneous entities but rather composed of a mixture of cells with different phenotypes, reminiscent of healthy intestinal epithelium cell types. Intestinal epithelium is one of the organs with highest self-renewal rate, maintained by a pool of highly proliferating stem cells located at the base of the crypts. Daughters of the stem cells abandon the crypts and differentiate as they move up the villi, in a process that takes no longer than six days. Recent findings suggest that colorectal cancers (CRCs), like normal intestine, rely on a stem cell hierarchy for its growth. Cancer stem cells, identified by LGR5 gene expression, are at the apex of this hierarchy, and are thought to be the drivers of CRC expansion and metastasis. This thesis is focused on characterizing the growth dynamics of the different tumor compartments and identifying the cells that fuel tumor growth. Moreover, we have also tried to elucidate which is the cell of origin of metastasis. To complete this project we have first developed new models to study human disease, as most of the previous work relied on genetically modified mouse models to reproduce the disease. Here, we have combined patient-derived organoid 3D cultures and CRISPR/Cas9 genome editing techniques to insert fluorescent reporter proteins under the control of our genes of interest. This has allowed us to visualize different tumor cell types in vivo using LGR5, KRT20 and EMP1 as markers for stemness, differentiation and invasion respectively. In addition, we have also set up a system to follow the progeny of the abovementioned populations in vivo in intact tumors. We have identified the different tumor compartments by subcutaneously injecting modified human organoids into immunosuppressed mice. Flow cytometry purification and reinjection into secondary hosts of stem-like and differentiated-like cells has enabled us to discover that cancer cells retaining stem cell characteristics are more proficient in tumor initiation than the rest of the tumor. Nevertheless, lineage tracing of the abovementioned genes in an intact tumor cell environment shows how both stem and differentiated progenies are able to give rise to long lasting clones and thus equally fuel tumor growth. Furthermore, we have observed plasticity arising from both cell types, indicative that the cellular hierarchy is disrupted during tumor progression. In addition, we have defined EMP1 as a putative gene marker for invasive cells. EMP1-High cells are a differentiated subset of tumor cells that harbor migratory properties and secrete myeloid recruiting chemokines to the tumor site. Myeloid cells have been shown to contribute to all steps of metastasis in several cancer types. We hypothesize that this EMP1+ subpopulation is the one that disseminates from the primary tumor and initiates metastasis. For metastatic studies, we have resorted to mouse derived tumor organoids that allow the growth of primary and metastatic disease in fully immunocompetent ice, and we have set up new models to study disease relapse in metastatic sites upon primary tumor removal Taken together, our data provides new insights on the mode of tumor growth in advanced human colorectal carcinomas and suggests that stem cell traits are not required for tumor growth neither metastatic spread, contrary to what was initially though based on mouse adenoma studies.
[cat] Els tumors colorectals no són una entitat homogènia sinó que estan formats per una barreja de cèl·lules de fenotips variats, reminiscents dels tipus cel·lulars de l’epiteli intestinal sa. Estudis recents suggereixen que el creixement del càncer colorectal (CCR), igual que el de l’intestí normal, està mediat per una jerarquia amb origen en cèl·lules mare. Les cèl·lules mare del càncer, identificades per l’expressió del gen LGR5, es troben a l’àpex de la jerarquia i impulsen l’expansió del CCR i la metàstasis. Aquesta tesi se centra en caracteritzar la dinàmica d’expansió dels diferents compartiments tumorals i en identificar les cèl·lules que en mantenen el creixement. També hem intentat elucidar quina és la cèl·lula d’origen de la metàstasi. Per a realitzar aquest projecte primer hem desenvolupat nous models per estudiar la malaltia humana, combinant el cultiu d’organoids derivats de pacients i l’edició genòmica mitjançant CRISPR/Cas9. Això ens ha permès visualitzar diferents tipus cel·lulars tumorals in vivo usant LGR5, KRT20 i EMP1 com a marcadors de cèl·lula mare, cèl·lula diferenciada i cèl·lula invasiva, respectivament. Addicionalment, també hem establert un sistema per seguir la progènie de les poblacions mencionades. Hem descobert que tant el compartiment de cèl·lules mare com el diferenciat són capaços de donar lloc a una progènie que persisteix en el temps, suggerint que ambdós tipus cel·lulars contribueixen al creixement tumoral. A més a més, hem observat plasticitat entre els dos compartiments, cosa que indica que la jerarquia cel·lular es perd durant el desenvolupament del tumor. Finalment, mitjançant l’ús d’EMP1 com a marcador de cèl·lules invasives hem identificat un subgrup de cèl·lules diferenciades amb propietats migratòries i amb potencial per reclutar cèl·lules mieloides. La nostra hipòtesi és que la població EMP1+ és la que dissemina del tumor primari i inicia la metàstasi. En resum , les nostres dades suposen una nova visió en l’estudi del mode de creixement del càncer de colon d’estadis avançats en humà, i suggereixen que els trets de cèl·lula mare no són necessaris per creixement tumoral ni la disseminació metastàtica, contràriament al que es pensava inicialment, degut als estudis realitzats en adenoma de ratolí.
Appears in Collections:Tesis Doctorals - Facultat - Biologia

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