Cancer cell plasticity and niche independency drive metastasis in colorectal cancer

Colorectal cancer (CRC) is one of the most common type of cancer worldwide. Surgical resection of the primary tumors is the main treatment for early-stage/localized colon cancers and often patients have a positive follow up. Unfortunately the relative survival of patients undergoing recurrence and development of distant metastasis is modest, and adjuvant therapies are most likely non curative in these cases. Over the past decades, a number of culture systems and in vivo murine approaches have been established with the aim to mimic CRC in the lab and gain better understanding of disease development, response to therapy and acquisition of resistance. While initial stages of CRC can be well studied with the current models, few approaches allow the visualization of metastatic colonization of distant organs. This thesis describes the development of a microsurgical approach, based on orthotopic transplantation of CRC organoids in mice. This technique allowed modelling of in situ CRC and its metastatic spread (Chapter 2). In Chapter 3 this technique was used to test the in vivo contribution of four of the most common CRC driver mutations to disease progression. We discovered that only combination of all these genetic alterations drive the onset of metastatic lesions. Stem cells maintenance in healthy intestine is guaranteed by factors secreted by the surrounding stem cell niche. Quadruple mutant organoids have the capacity to grow without a supporting niche, because they survive in vitro in absence of any stem cell-inducing factor. Therefore we concluded that niche independence is a feature sufficient to drive metastatic colonization of CRC cells. Chapter 4 describes the development of a novel fluorescent intestinal stem cell reporter (the STem cell Ascl2 Reporter - STAR) that can be easily integrated in CRC organoids to map cell fate decisions in vitro. Orthotopic transplantation of CRC organoids expressing fluorescent stem cell reporters (such as STAR or Lgr5) allowed visualization of cancer stem cells (CSCs) in both human and murine colorectal tumors in vivo (Chapter 4 and Chapter 5). In Chapter 5 we focused on dissecting the role of CSCs in the different steps of CRC metastasis. We found that CSCs are not the major contributors of metastatic seeding whilst Lgr5- more committed cancer cells are the main population of cancer cells we detected in the blood as circulating tumor cells. Once Lgr5- cancer cells reach the secondary site they undergo phenotypic transition and become CSCs (i.e. they start expressing the Lgr5 CSCs marker). Our experiments revealed that this plasticity can occur without the trigger of signals from the microenvironment and it is a required step for metastatic outgrowth. Ultimately, the transplantation approach presented in this thesis opens up opportunities for further in vivo characterization of the late stages of CRC, potentially providing a set up for preclinical models of metastatic CRC. The discovery that CSCs are not the main cancer cells taking part in CRC metastatic seeding calls upon a rethink on how to strategically try to battle CRC disease. Are the CSCs the only enemies we need to defeat