By Jamshed Arslan, Pharm. D., PhD.
Colorectal cancer stem cells are a rare subpopulation of colorectal cancer cells that can self-renew and initiate and sustain tumor growth when transplanted into an animal host.1,2 Colorectal cancer stem cells are identified by CD133, CD166, CD44, nuclear-beta-catenin, ALDH1, EphB2 and Lgr5.2 Studying the molecular and phenotypic shifts in colorectal cancer stem cells in different tissue environments can help understand colorectal cancer development and metastasis. Cancer stem-like cells are part of the circulating tumor cells, but the currently used and established colorectal cancer circulating tumor cell lines do not adequately exhibit this reality.3 So, a team of researchers from Italy developed stable lines from human colorectal cancer that show all the necessary characteristics of colorectal cancer stem cells.2 Using a xenograft mouse model, they reported the presence of colorectal cancer stem cells in blood and in the metastatic lesions. After studying colorectal cancer stem cells in various stages of colorectal cancer, from tumor onset to metastasization, they concluded that stemness underlies all steps of human colorectal cancer.
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beta Catenin was detected in immersion fixed SW480 human colorectal adenocarcinoma cell line using Goat Anti-Human/Mouse/Rat beta Catenin Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1329) at 15 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm and nuclei.
Characterizing colorectal cancer stem cells obtained from patients
To characterize stem cells in colorectal cancer, the team obtained specimens from colorectal cancer patients with liver metastasis.2 Plating these colorectal cancer cells unraveled many CD133+ and Lgr5+ cells with long-term proliferation potential. The isolated cells showed strong and wide immunoreactivity against vimentin and CXCR4, indicating invasiveness and metastasis. In these stem cells, SNP arrays and targeted sequencing revealed mutations in several colorectal cancer-related genes including KRAS, EGFR, PDGFRA, PIK3CA, MGMT, TP53, ERBB2, NF1 and MET. When examined for chemotherapy-induced cell death, colorectal cancer stem cells were more resilient than their progeny. This means that colorectal cancer contains self-renewing cells that have bona fide colorectal cancer stem cells properties.
EpCAM/TROP 1 was detected in immersion fixed HT-29 human colon adenocarcinoma cell line using Mouse Anti-Human EpCAM/TROP 1 Monoclonal Antibody (Catalog # MAB960) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 493-conjugated Anti-Mouse IgG Secondary Antibody (green; Catalog # NL009) and counterstained with DAPI (blue).
The colorectal cancer stem cells were then injected into immunocompromised mice to see any tumorigenic potential. As expected, the mice developed tumors with histopathology similar to human disease: EpCAM immunoreactivity, several mitotic figures, abnormal appearance of cell nuclei, necrosis, and vascular proliferation. Injecting immunocompromised mice with Luc-tagged colorectal cancer stem cells resulted in tumors at various distant places, indicating the role of these cells in colorectal cancer metastasis.
This indicates that colorectal cancer stem cells from human colorectal cancer possess stemness. The next step was to identify circulating tumor cells in the blood of xenografted mice since cancer stem cells form part of circulating tumor cells.
Stemness and tumorigenicity of the circulating colorectal cancer stem cells
The xenografted colorectal cancer mice contained human circulating tumor cells positive for adenocarcinoma markers (CDX2 and CK20) and EpCAM. Tumors derived from circulating colorectal cancer stem cells were also immunoreactive to CDX2, EpCAM and CK20 and exhibited histological features of original human colorectal cancer specimens. When Luc-tagged circulating colorectal cancer stem cells were isolated and injected into the tail-vain of immunocompromised mice, metastatic lesions were evident in liver, spleen and lung, from which metastasizing circulating colorectal cancer stem cells could further be generated. The ex vivo functional characteristics of the circulating colorectal cancer stem cells revealed stemness, such as exponential growth, Aldeﬂuor-positivity in cell population, ability to be subcloned, and increased expression of genes related to the epithelial-to-mesenchymal transition pathway.
In short, stemness is responsible for initiating and sustaining primary lesions in human colorectal cancers. Colorectal cancer stem cells take part in colorectal cancer dissemination into the systemic circulation and subsequent metastasis to distant organs.
Significance for the development of colorectal cancer biomarkers
The model of human colorectal cancer presented in this paper provides antigenic, molecular and genetic characteristics of circulating colorectal cancer stem cells.2 This work also highlights the role of circulating and metastasizing tumor cells in colorectal cancer. This research can help in developing biomarkers and therapies specific to various stages of colorectal cancer.
Jamshed Arslan, Pharm D, PhD
Dr Arslan is an Assistant Professor at DowUniversity of Health Sciences, Pakistan,
where he teaches Pharmacology to future pharmacists.
- Yu, Zuoren, et al. "Cancer Stem Cells". International Journal of Biochemistry & Cell Biology, vol. 44, no. 12, 2012, pp. 2144–2151. https://doi.org/10.1016/j.biocel.2012.08.022
- Visioli, Alberto, et al. "Stemness Underpinning All Steps of Human Colorectal Cancer Deﬁnes the Core of Effective Therapeutic Strategies." EBioMedicine, 2019, n.pag. https://doi.org/10.1016/j.ebiom.2019.04.049
- Toloudi, Maria, et al. "Correlation between Cancer Stem Cells and Circulating Tumor Cells and Their Value." Case Reports in Oncology, vol. 4, no. 1, 2011, pp. 44-54. https://doi.org/10.1159/000324403