Due to their self-renewal and tumorigenic properties, tumor-initiating cells (TICs) have been hypothesized to be important targets for colorectal cancer (CRC). However the study of TICs is hampered by the fact that the identification and culturing of TICs is still a subject of extensive debate. Floating three-dimensional spheroid cultures (SC) that grow in serum-free medium supplemented with growth factors are supposed to be enriched in TICs. We generated SC from fresh clinical tumor specimens and compared them to SC isolated from CRC cell-lines as well as to adherent differentiated counterparts. Patient-derived SC display self-renewal capacity and can induce serial transplantable tumors in immuno-deficient mice, which phenotypically resemble the tumor of origin. In addition, the original tumor tissue and established SC retain several similar CRC-relevant mutations. Primary SC express key stemness proteins such as SOX2, OCT4, NANOG and LGR5 and importantly show increased chemoresistance ability compared to their adherent differentiated counterparts and to cell line-derived SC. Strikingly, cells derived from spheroid or adherent differentiating culture conditions displayed similar self-renewal capacity and equally formed tumors in immune-deficient mice, suggesting that self-renewal and tumor-initiation capacity of TICs is not restricted to phenotypically immature spheroid cells, which we describe to be highly plastic and able to reacquire stem-cell traits even after long differentiation processes. Finally, we identified two genes among a sphere gene expression signature that predict disease relapse in CRC patients. Here we propose that SC derived from fresh patient tumor tissue present interesting phenotypic features that may have clinical relevance for chemoresistance and disease relapse and therefore represent a valuable tool to test for new CRC-therapies that overcome drug resistance.

In solid tumors, cancer stem cells (CSCs) or tumor-initiating cells (TICs) are often found in hypoxic niches. Nevertheless, the influence of hypoxia on TICs is poorly understood. Using previously established, TIC-enrichedpatient-derived colorectal cancer (CRC) cultures, we show that hypoxia increases the self-renewal capacity of TICs while inducing proliferation arrest in their more differentiated counterpart cultures. Gene expression data revealed macroautophagy/autophagy as one of the major pathways induced by hypoxia in TICs. Interestingly, hypoxia-induced autophagy was found to induce phosphorylation of EZR (ezrin) at Thr567 residue, which could be reversed by knocking down ATG5, BNIP3, BNIP3L , or BECN1 . Furthermore, we identified PRKCA/PKCα as a potential kinase involved in hypoxia-induced autophagy-mediated TIC self-renewal. Genetic targeting of autophagy or pharmacological inhibition of PRKC/PKC and EZR resulted in decreased tumor-initiating potential of TICs. In addition, we observed significantly reduced in vivo tumor initiation and growth after a stable knockdown of ATG5 . Analysis of human CRC samples showed that p-EZR is often present in TICs located in the hypoxic and autophagic regions of the tumor. Altogether, our results establish the hypoxia-autophagy-PKC-EZR signaling axis as a novel regulatory mechanism of TIC self-renewal and CRC progression. Autophagy inhibition might thus represent a promising therapeutic strategy for cancer patients. Abbreviations ATG: autophagy related; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CQ: chloroquine; CSC: cancer stem cells; CRC: colorectal cancer; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PRKC/PKC: protein kinase C; SQSTM1/p62: sequestosome 1; TICs: tumor-initiating cells.

Most cancers contain a subpopulation of highly tumorigenic cells, known as cancer stem cells (CSCs) or tumor-initiating cells (TICs). Targeting TICs may be essential to achieve cure, because of their self-renewal and tumorigenic properties as well as their resistance to conventional therapies. Despite significant advances in TIC biology, their isolation and identification remain largely disputed and incompletely established. In this review, we discuss the latest developments in isolation and culturing approaches of TICs, with focus on colorectal cancer (CRC). We feature recent findings on TIC-relevant signaling pathways and the metabolic identity of TICs, as well as their current clinical implications. Lastly, we highlight the influence of inter- and intra-tumoral heterogeneity on TIC function and targeting approaches.

Low oxygen concentrations (hypoxia) are known to affect the cellular metabolism and have been suggested to regulate a subpopulation of cancer cells with tumorigenic properties, the so-called tumor-initiating cells (TICs). To better understand the mechanism of hypoxia-induced TIC activation, we set out to study the role of hypoxia-responsive miRNAs in recently established colon cancer patient-derived TICs. We were able to show that low oxygen concentrations consistently lead to the upregulation of miR-210 in different primary TIC-enriched cultures. Both stable overexpression of miR-210 and knockdown of its target gene ISCU resulted in enhanced TIC self-renewal. We could validate the tumorigenic properties of miR- 210 in in vivo experiments by showing that ectopic expression of miR-210 results in increased tumor incidence. Furthermore, enhanced miR-210 expression correlated with reduced TCA cycle activity and increased lactate levels. Importantly, by blocking lactate production via inhibition of LDHA, we could reverse the promoting effect of miR-210 on self-renewal capacity, thereby emphasizing the regulatory impact of the glycolytic phenotype on colon TIC properties. Finally, by assessing expression levels in patient tissue, we could demonstrate the clinical relevance of the miR-210/ISCU signaling axis for colorectal carcinoma. Taken together, our study highlights the importance of hypoxia-induced miR-210 in the regulation of colon cancer initiation.

The vast majority of colorectal cancer-related deaths can be attributed to metastatic spreading of the disease. Therefore, deciphering molecular mechanisms of metastatic dissemination is a key prerequisite to improve future treatment options. With this aim, we took advantage of different colorectal cancer cell lines and recently established primary cultures enriched in colon cancer stem cells, also known as tumor-initiating cells (TIC), to identify genes and miRNAs with regulatory functions in colorectal cancer progression. We show here that metastasis-derived TICs display increased capacity for self-renewal, TGFβ signaling activity, and reduced expression of the miR-371∼373 cluster compared with nonmetastatic cultures. TGFβ receptor 2 () and aldehyde dehydrogenase A1 () were identified as important target genes of the miR-371∼373 cluster. In addition, TGFBR2 repression, either by direct knockdown or indirectly via overexpression of the entire miR-371∼373 cluster, decreased tumor-initiating potential of TICs. We observed significantly reduced self-renewal activity as well as lowered tumor initiation and metastatic outgrowth capacity following stable overexpression of the miR-371∼373 cluster in different colon TIC cultures. Inhibitor of DNA binding 1 (ID1) was affected by both TGFBR2 and miR-371∼373 cluster alterations. Functional sphere and tumor formation as well as metastatic dissemination assays validated the link between miR-371∼373 and ID1. Altogether, our results establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel regulatory mechanism of TIC self-renewal and metastatic colonization. These findings establish the miR-371∼373/TGFBR2/ID1 signaling axis as a novel mechanism regulating self-renewal of tumor-initiating cell and metastatic colonization, potentially opening new concepts for therapeutic targeting of cancer metastasis. http://cancerres.aacrjournals.org/content/canres/78/14/3793/F1.large.jpg .