Cancer stem cells (CSCs) are the subpopulation of cells within a tumor that can self-renew, differentiate into multiple lineages, and drive tumor growth. Here we describe a two-pronged approach for the identification and characterization of CSCs from colorectal cancer cell lines, using a Matrigel-based differentiation assay, and cell surface markers CD44 and CD24. About 20 to 30% of cells from the SW1222 cell line form megacolonies in Matrigel that have complex 3D structures resembling colonic crypts. The megacolonies' capacity to self-renew in vitro is direct evidence that they contain the CSCs. Furthermore, just 200 cells from SW1222 megacolonies initiate tumors in NOD/SCID mice. We also showed that CD44 + CD24 + cells enriched for colorectal CSCs in the HT29 and SW1222 cell lines, which can self-renew and reform all four CD44/CD24 subpopulations, are the most clonogenic in vitro and can initiate tumors in vivo. A single SW1222 CD44 + CD24 + CSC, when grown in Matrigel, can form large megacolonies that differentiate into enterocyte, enteroendocrine, and goblet cell lineages. The HCT116 line does not differentiate or express CDX1, nor does it contain subpopulations of cells with greater tumor-forming capacity, suggesting that HCT116 contains mainly CSCs. However, forced expression of CDX1 in HCT116 leads to reduced clonogenicity and production of differentiating crypt-containing colonies, which can explain the selection for reduced CDX1 expression in many colorectal cancers. In summary, colorectal cancer cell lines contain subpopulations of CSCs, characterized by their cell surface markers and colony morphology, which can self-renew and differentiate into multiple lineages.clonogenicity | matrigel | CD44 | goblet cells

Brain-specific homing and direct interactions with the neural substance are prominent hypotheses for brain metastasis formation and a modern manifestation of Paget's “seed and soil” concept. However, there is little direct evidence for this “neurotropic” growth in vivo. In contrast, many experimental studies have anecdotally noted the propensity of metastatic cells to grow along the exterior of pre-existing vessels of the CNS, a process termed vascular cooption. These observations suggest the “soil” for malignant cells in the CNS may well be vascular, rather than neuronal. We used in vivo experimental models of brain metastasis and analysis of human clinical specimens to test this hypothesis. Indeed, over 95% of early micrometastases examined demonstrated vascular cooption with little evidence for isolated neurotropic growth. This vessel interaction was adhesive in nature implicating the vascular basement membrane (VBM) as the active substrate for tumor cell growth in the brain. Accordingly, VBM promoted adhesion and invasion of malignant cells and was sufficient for tumor growth prior to any evidence of angiogenesis. Blockade or loss of the β1 integrin subunit in tumor cells prevented adhesion to VBM and attenuated metastasis establishment and growth in vivo. Our data establishes a new understanding of CNS metastasis formation and identifies the neurovasculature as the critical partner for such growth. Further, we have elucidated the mechanism of vascular cooption for the first time. These findings may help inform the design of effective molecular therapies for patients with fatal CNS malignancies.

Tumour hypoxia renders cancer cells resistant to cancer therapy, resulting in markedly worse clinical outcomes. To find clinical candidate compounds that reduce hypoxia in tumours, we conduct a high-throughput screen for oxygen consumption rate (OCR) reduction and identify a number of drugs with this property. For this study we focus on the anti-malarial, atovaquone. Atovaquone rapidly decreases the OCR by more than 80% in a wide range of cancer cell lines at pharmacological concentrations. In addition, atovaquone eradicates hypoxia in FaDu, HCT116 and H1299 spheroids. Similarly, it reduces hypoxia in FaDu and HCT116 xenografts in nude mice, and causes a significant tumour growth delay when combined with radiation. Atovaquone is a ubiquinone analogue, and decreases the OCR by inhibiting mitochondrial complex III. We are now undertaking clinical studies to assess whether atovaquone reduces tumour hypoxia in patients, thereby increasing the efficacy of radiotherapy.

Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis.