Abstract
While primary colorectal cancer (CRC) can usually be treated well through surgical removal of the affected tissue parts, the threat of this disease lies in the spread to other organs and the formation of secondary lesions, called metastases. Metastasis formation in a multi-step process that is highly complex and thus
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very inefficient. With this work, we have focused on the last step of this process, the colonization of an organ, which describes the outgrowth of cancer cells into clinically detectable lesions at a secondary site that eventually jeopardise the patient’s health.
To study this process from a novel angle, we have exploited two reporter systems that allow distinguishing between cancer cells of varying properties using fluorescent labelling. In particular, these reporters tell apart cells with stem versus non-stem cell behaviour as well as dividing versus non-dividing cells.
Using these tools in combination with patient-derived CRC organoids, we first addressed how cancer cells change during colonization of the liver (prime organ for CRC metastasis). We found that colonization starts with cells displaying behaviour reminiscent of tissue regeneration (YAP signalling), followed by active proliferation, and finally re-appearance of a cellular hierarchy similar to the homeostatic setting. Next, we identified that only those metastases that fail to grow out (micrometastases) are devoid of cancer stem cells (CSCs) and displaying persistent YAP activity, the latter actively supressing stemness.
To further profile the transcriptomic differences between micrometastasis and efficiently growing macrometastasis, we developed a liver metastasis isolation protocol with which we can stratify metastases by size. Macrometastases were characterized by TNFα signalling and epithelial-to-mesenchymal transition traits, while micrometastasis were enriched for fatty acid metabolism and oxidative phosphorylation. In continuous metastasis seeding experiments, we found that metastasis of microscopic size can be either entirely quiescent or proliferating. In contrast, macrometastasis always contain both quiescent and proliferating cells. Furthermore, the transcriptomic differences associated with metastasis size were reflected when comparing quiescent (or proliferating) cells in micro- versus macrometastasis. This suggests fundamental transcriptomic changes during metastatic outgrowth that might be (partially) instructed by the tumour microenvironment.
One of the pathways active in non-epithelial intestinal cells is Hedgehog signalling that contributes to intestinal homeostasis through paracrine signalling from the epithelium towards the mesenchyme. Also in CRC, most evidence hints at paracrine signalling, yet individual studies have suggested that Hedgehog signalling might also act in an autocrine manner. While CSCs are known to drive metastatic growth, we found that the class of Hedgehog ligands is enriched in non-CSCs, raising the question if Hedgehog ligands affect CRC through non-CSCs. To address this, Hedgehog signalling was altered in CRC organoids either through pharmacological perturbation of the pathway or by generating Hedgehog ligand knockout lines through CRISPR/Cas9 technology. Our data did not indicate autocrine signalling in our human CRC model. Furthermore, metastasis formation was not significantly affected by the lack of Hedgehog ligands in the cancer cells.
Taken together, we studied phenotypic and transcriptomic heterogeneity in CRC metastasis to unravel cellular characteristics that co-determine success and failure of CRC liver colonization.
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