Clinical Implications of Colorectal Cancer Stem Cells in the Age of Single-Cell Omics and Targeted Therapies

The cancer stem cell (CSC) concept emerged from the recognition of inherent tumor heterogeneity and suggests that within a given tumor, in analogy to normal tissues, there exists a cellular hierarchy composed of a minority of more primitive cells with enhanced longevity (ie, CSCs) that give rise to shorter-lived, more differentiated cells (ie, cancer bulk populations), which on their own are not capable of tumor perpetuation. CSCs can be responsible for cancer therapeutic resistance to conventional, targeted, and immunotherapeutic treatment modalities, and for cancer progression through CSC-intrinsic molecular mechanisms. The existence of CSCs in colorectal cancer (CRC) was first established through demonstration of enhanced clonogenicity and tumor-forming capacity of this cell subset in human-to-mouse tumor xenotransplantation experiments and subsequently confirmed through lineage-tracing studies in mice. Surface markers for CRC CSC identification and their prospective isolation are now established. Therefore, the application of single-cell omics technologies to CSC characterization, including whole-genome sequencing, RNA sequencing, and epigenetic analyses, opens unprecedented opportunities to discover novel targetable molecular pathways and hence to develop novel strategies for CRC eradication. We review recent advances in this field and discuss the potential implications of next-generation CSC analyses for currently approved and experimental targeted CRC therapies. The cancer stem cell (CSC) concept emerged from the recognition of inherent tumor heterogeneity and suggests that within a given tumor, in analogy to normal tissues, there exists a cellular hierarchy composed of a minority of more primitive cells with enhanced longevity (ie, CSCs) that give rise to shorter-lived, more differentiated cells (ie, cancer bulk populations), which on their own are not capable of tumor perpetuation. CSCs can be responsible for cancer therapeutic resistance to conventional, targeted, and immunotherapeutic treatment modalities, and for cancer progression through CSC-intrinsic molecular mechanisms. The existence of CSCs in colorectal cancer (CRC) was first established through demonstration of enhanced clonogenicity and tumor-forming capacity of this cell subset in human-to-mouse tumor xenotransplantation experiments and subsequently confirmed through lineage-tracing studies in mice. Surface markers for CRC CSC identification and their prospective isolation are now established. Therefore, the application of single-cell omics technologies to CSC characterization, including whole-genome sequencing, RNA sequencing, and epigenetic analyses, opens unprecedented opportunities to discover novel targetable molecular pathways and hence to develop novel strategies for CRC eradication. We review recent advances in this field and discuss the potential implications of next-generation CSC analyses for currently approved and experimental targeted CRC therapies. Tumor development and progression have been associated, at the DNA level, with cumulative alterations in oncogenes, tumor suppressor genes, and repair/stability genes.1Futreal P.A. Coin L. Marshall M. et al.A census of human cancer genes.Nat Rev Cancer. 2004; 4: 177-183Crossref PubMed Google Scholar,2Vogelstein B. Kinzler K.W. 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Some of these technologies have already been used successfully to generate large data sets that enabled comprehensive molecular characterization of CRC and provided a foundation for several novel molecular-based cancer classifications, with the goal of further refining individualized therapeutic strategies.15The Cancer Genome Atlas NetworkComprehensive molecular characterization of human colon and rectal cancer.Nature. 2012; 487: 330-337Crossref PubMed Scopus (4691) Google Scholar, 16Perez-Villamil B. Romera-Lopez A. Hernandez-Prieto S. et al.Colon cancer molecular subtypes identified by expression profiling and associated to stroma, mucinous type and different clinical behavior.BMC Cancer. 2012; 12: 260Crossref PubMed Scopus (74) Google Scholar, 17Schlicker A. Beran G. Chresta C.M. et al.Subtypes of primary colorectal tumors correlate with response to targeted treatment in colorectal cell lines.BMC Med Genomics. 2012; 5: 66Crossref PubMed Scopus (146) Google Scholar, 18Budinska E. 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This classification provides the most comprehensive molecular CRC characterization to date and has recently been shown to serve as an independent prognostic marker in patients with metastatic CRC who undergo first-line chemotherapy in combination with bevacizumab or cetuximab.24Lenz H.-J. Ou F.-S. Venook A.P. et al.Impact of consensus molecular subtype on survival in patients with metastatic colorectal cancer: results from CALGB/SWOG 80405 (Alliance).J Clin Oncol. 2019; 37: 1876-1885Crossref PubMed Scopus (52) Google Scholar In addition, the emergence of novel single-cell analysis technologies provided an unprecedented opportunity to examine the cellular diversity within an individual tumor, which might contain clones of cells with more than 1 molecular subtype defined by Guinney et al. These analyses have already enabled a more detailed characterization of molecular and cellular tumor composition and revealed intra-tumor heterogeneity likely responsible for differential therapeutic responses and resistance patterns. In 1990, Fearon and Vogelstein25Fearon E.R. Vogelstein B. A genetic model for colorectal tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (9317) Google Scholar proposed that CRCs develop from colonic adenomas as a result of the successive accumulation of genetic mutations. This linear paradigm of CRC progression has been challenged by recent high-resolution genomic studies that uncovered early nonlinear development of intra-tumor mutational heterogeneity, resulting in simultaneous accumulation of genetically diverse cancer clones subsequent to an initial oncogenic transformation event.26Sottoriva A. Kang H. Ma Z. et al.A Big Bang model of human colorectal tumor growth.Nat Genet. 2015; 47: 209-216Crossref PubMed Scopus (493) Google Scholar Using genomic profiling of 349 individual tumor glands, each originating from a single stem cell, Sottoriva et al26Sottoriva A. Kang H. Ma Z. et al.A Big Bang model of human colorectal tumor growth.Nat Genet. 2015; 47: 209-216Crossref PubMed Scopus (493) Google Scholar discovered that after the initial transformation, CRC tumors grew predominantly as a single expansion populated by numerous intermixed sub-clones. Based on these observations, they proposed a novel “Big Bang” theory of CRC progression, which posits that after an initial common oncogenic event, the majority of mutations driving tumor growth occur during early tumor expansion and are responsible for the clonal diversity and intra-tumor heterogeneity. Hence, the Big Bang theory points to the existence of a common ancestor during cancer clonal evolution. The question arises, therefore, whether the Big Bang theory is consistent with or even supportive of the CSC theory. According to the latter, tumors are maintained through unlimited CSC self-renewal and differentiation capacity. Moreover, according to the CSC theory, CSCs are capable of fully regenerating original tumor heterogeneity upon transplantation. This functional definition therefore implies that CSCs contain cells representative of all genetic sub-clones within a given tumor (Figure 2A). Moreover, it implies that clones that do not contain CSCs would not self-renew and would ultimately be eliminated from heterogeneous tumors. Thus, observations of long-term co-existence of diverse mutational tumor clones along with heterogeneity for established CSC markers suggests the presence of cells with non-mutationally determined CSC phenotype responsible for the maintenance and persistence of each individual cancer clone in heterogeneous tumors. The Big Bang model is therefore consistent with, and in fact supports, the existence of CSCs as a mutationally heterogeneous, yet with regard to CSC marker expression homogeneous, subpopulation of self-renewing cells, which quite possibly arose from a common ancestor that sustained a first oncogenic mutation and gave rise to diverse clones within a single tumor27Clevers H. The cancer stem cell: premises, promises and challenges.Nat Med. 2011; 17: 313-319Crossref PubMed Scopus (1265) Google Scholar (Figure 2A). It is useful to recall here the original consensus definition of a CSC that was arrived at by Clarke et al28Clarke M.F. Dick J.E. Dirks P.B. et al.Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells.Cancer Res. 2006; 66: 9339-9344Crossref PubMed Scopus (2304) Google Scholar in 2006, that is, “a cell within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor.” This consensus view further stipulates that “cancer stem cells can thus only be defined experimentally by their ability to recapitulate the generation of a continuously growing tumor.”28Clarke M.F. Dick J.E. Dirks P.B. et al.Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells.Cancer Res. 2006; 66: 9339-9344Crossref PubMed Scopus (2304) Google Scholar It is therefore clear that this functional CSC definition makes no assumptions about a particular cell of origin of cancer and its evolutionary trajectory, but indeed allows for, as a specific point-in-time snapshot across an established tumor entity, for cellular conversion of mutationally heterogeneous cells of distinct clones toward CSC phenotype, through intrinsic or acquired plasticity induced by, for example, among other factors, metabolic restraints, microenvironmental signaling factors, and/or therapeutic selection pressures. Thus, the CSC theory accommodates both monoclonal and polyclonal tumor evolution. Single-cell genomics also enabled high-resolution analyses of the tumor evolutional hierarchy.29Gawad C. Koh W. Quake S.R. Single-cell genome sequencing: current state of the science.Nat Rev Genet. 2016; 17: 175-188Crossref PubMed Scopus (598) Google Scholar Wu et al30Wu H. Zhang X.Y. Hu Z. et al.Evolution and heterogeneity of non-hereditary colorectal cancer revealed by single-cell exome sequencing.Oncogene. 2017; 36: 2857-2867Crossref PubMed Scopus (25) Google Scholar employed DNA-based single-cell sequencing to examine the cancer tissues and noncancerous polyps isolated from 2 unrelated patients with sporadic CRC. They demonstrated the existence of common mutations in GPCR, PI3K-Akt, or FGFR signaling pathway genes among different cancer cell clones in individual patients and provided evidence for monoclonal CRC origin with subsequent sub-clonal evolution. This concept was challenged by Yu et al,31Yu C. Yu J. Yao X. et al.Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing.Cell Res. 2014; 24: 701-712Crossref PubMed Scopus (77) Google Scholar who detected 2 distinct clones in a single colonic adenocarcinoma: the predominant clone driven by antigen presenting cells and TP53 mutations and the minor independent clone characterized by early mutations in CDC27 and PABPC1. However, Wu et al disputed this conclusion upon reanalysis of the study and convincingly demonstrated that the second independent clone likely represented a noncancerous contaminant within the tumor, and colon cancer in this patient originated from a clone defined by antigen presenting cells and TP53 mutations.30Wu H. Zhang X.Y. Hu Z. et al.Evolution and heterogeneity of non-hereditary colorectal cancer revealed by single-cell exome sequencing.Oncogene. 2017; 36: 2857-2867Crossref PubMed Scopus (25) Google Scholar The existence of monoclonal CSC-driven CRC origin is also supported by the single-cell–level examination of somatic copy number alterations (SCNAs). A study of 2 rectal tumors revealed that the majority of SCNAs were shared by all cells within a given tumor and thus represented early events in malignant transformation.32Liu M. Liu Y. Di J. et al.Multi-region and single-cell sequencing reveal variable genomic heterogeneity in rectal cancer.BMC Cancer. 2017; 17: 787Crossref PubMed Scopus (10) Google Scholar In addition, the tumors also contained sub-clones with novel focal SCNAs, which defined the intra-tumor clonal heterogeneity. Notably, a follow-up study by the same authors found that both CD45– EpCAMhigh CD44+ CSCs and CD45– EpCAMhigh CD44– differentiated tumor cells had similar SCNA profiles,33Liu M. Di J. Liu Y. et al.Comparison of EpCAM(high)CD44(+) cancer stem cells with EpCAM(high)CD44(–) tumor cells in colon cancer by single-cell sequencing.Cancer Biol Ther. 2018; 19: 939-947Crossref PubMed Scopus (2) Google Scholar thus supporting the possibility of a monoclonal CSC phenotype. Single-cell genomic analyses have also been employed to study the clonal evolution of CRC metastases.34Heitzer E. Auer M. Gasch C. et al.Complex tumor genomes inferred from single circulating tumor cells by array-CGH and next-generation sequencing.Cancer Res. 2013; 73: 2965-2975Crossref PubMed Scopus (373) Google Scholar,35Leung M.L. Davis A. 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