EpCAM and its potential role in tumor-initiating cells

Epithelial cell adhesion molecule EpCAM is expressed on a subset of normal epithelia and overexpressed on malignant cells from a variety of different tumor entities. This overexpression is even more pronounced on so-called tumor-initiating cells (TICs) of many carcinomas. Taking this rather ubiquitous expression of EpCAM in carcinomas and TICs into account, the question arises how EpCAM can serve as a reliable marker for tumor-initiating cells and what might be the advantage for TICs to express this molecule. Furthermore, several approaches for therapeutic strategies targeting exclusively EpCAM on cancer cells were undertaken over the past decades and have recently been transferred to pre-clinical attempts to eradicate TICs. In the present review, we will depict potential functions of EpCAM in tumor cells with a special focus on TICs and therapeutic implications.     

Highly efficient elimination of colorectal tumor-initiating cells by an EpCAM/CD3-bispecific antibody engaging human T cells

With their resistance to genotoxic and anti-proliferative drugs and potential to grow tumors and metastases from very few cells, cancer stem or tumor-initiating cells (TICs) are a severe limitation for the treatment of cancer by conventional therapies. Here, we explored whether human T cells that are redirected via an EpCAM/CD3-bispecific antibody called MT110 can lyse colorectal TICs and prevent tumor growth from TICs. MT110 recognizes EpCAM, a cell adhesion molecule expressed on TICs from diverse human carcinoma, which was recently shown to promote tumor growth through engagement of elements of the wnt pathway. MT110 was highly potent in mediating complete redirected lysis of KRAS-, PI3 kinase- and BRAF-mutated colorectal TICs, as demonstrated in a soft agar assay. In immunodeficient mice, MT110 prevented growth of tumors from a 5,000-fold excess of a minimally tumorigenic TIC dose. T cells engaged by MT110 may provide a potent therapeutic means to eradicate TICs and bulk tumor cells derived thereof.     

Epithelial Cell Adhesion Molecule Regulates Tumor Initiation and Tumorigenesis via Activating Reprogramming Factors and Epithelial-Mesenchymal Transition Gene Expression in Colon Cancer

Epithelial cell adhesion molecule (EpCAM) is highly expressed in epithelial-transformed neoplasia and tumor-initiated cells (TICs), but the role that EpCAM plays in the stemness properties of TICs is still unclear. Here we show that EpCAM and reprogramming factors (c-Myc, Oct4, Nanog, and Sox2) were concomitantly elevated in TICs, which were shown to have superior self-renewal, invasiveness, and tumor-initiating abilities. Elevation of EpCAM enhanced tumorsphere formation and tumor initiation. Knockdown of EpCAM inhibited the expressions of reprogramming factors and epithelial-mesenchymal transition genes, thereby suppressing tumor initiation, self-renewal, and invasiveness. In addition, EpCAM, especially intracellular domain of EpCAM (EpICD), bound to and activated the promoter of reprogramming factors. Treatment with the inhibitor of γ-secretase (DAPT) led to the blockage of the expressions of reprogramming factors and epithelial-mesenchymal transition genes, which was accompanied by the reduction of tumor self-renewal and invasion. Furthermore, the increased release of EpEX enhanced production of EpICD and regulated the expression of reprogramming factors. Together, these findings suggest that EpCAM plays an important role in regulating cancer-initiating abilities in TICs of colon cancer. This discovery can be used in the development of new strategies for cancer therapy.     

Disulfiram Eradicates Tumor-Initiating Hepatocellular Carcinoma Cells in ROS-p38 MAPK Pathway-Dependent and -Independent Manners

Tumor-initiating cells (TICs) play a central role in tumor development, metastasis, and recurrence. In the present study, we investigated the effect of disulfiram (DSF), an inhibitor of aldehyde dehydrogenase, toward tumor-initiating hepatocellular carcinoma (HCC) cells. DSF treatment suppressed the anchorage-independent sphere formation of both HCC cells. Flow cytometric analyses showed that DSF but not 5-fluorouracil (5-FU) drastically reduces the number of tumor-initiating HCC cells. The sphere formation assays of epithelial cell adhesion molecule (EpCAM)⁺ HCC cells co-treated with p38-specific inhibitor revealed that DSF suppresses self-renewal capability mainly through the activation of reactive oxygen species (ROS)-p38 MAPK pathway. Microarray experiments also revealed the enrichment of the gene set involved in p38 MAPK signaling in EpCAM⁺ cells treated with DSF but not 5-FU. In addition, DSF appeared to downregulate Glypican 3 (GPC3) in a manner independent of ROS-p38 MAPK pathway. GPC3 was co-expressed with EpCAM in HCC cell lines and primary HCC cells and GPC3-knockdown reduced the number of EpCAM⁺ cells by compromising their self-renewal capability and inducing the apoptosis. These results indicate that DSF impaired the tumorigenicity of tumor-initiating HCC cells through activation of ROS-p38 pathway and in part through the downregulation of GPC3. DSF might be a promising therapeutic agent for the eradication of tumor-initiating HCC cells.     

Prowling wolves in sheep's clothing: the search for tumor stem cells

The importance of a subset of cells which have 'stem like' characteristics and are capable of tumor initiation has been reported for a range of tumors. Isolation of these tumor-initiating cells (TICs) has largely been based on differential cell surface protein expression. However, there is still much debate on the functional significance of these markers in initiating tumors, as many properties of tumor initiation are modified by cell-cell interactions. In particular, the relationship between TICs and their microenvironment is poorly understood but has therapeutic implications, as the microenvironment can maintain tumor cells in a prolonged period of quiescence. However, a major limitation in advancing our understanding of the crosstalk between TICs and their microenvironment is the lack of sensitive techniques which allow the in vivo tracking and monitoring of TICs. Application of new in vivo cellular and molecular imaging technologies holds much promise in uncovering the mysteries of TIC behavior at the three-dimensional level. This review will describe recent advances in our understanding of the TIC concept and how the application of in vivo imaging techniques can advance our understanding of the biological fate of TICs. A supplementary resource guide describing TICs from different malignancies is also presented.     

Hypoxia-induced autophagy drives colorectal cancer initiation and progression by activating the PRKC/PKC-EZR (ezrin) pathway

ABSTRACT

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.     

Cancer stem cell markers as potential targets for epithelial cancers

In recent years, the role of tumor-initiating cells (popularly known as cancer stem cells) in tumor development and availability of novel cancer stem cell/tumor initiating cell markers promises a new arena in understanding their role in developing novel targeted molecules. It is important to identify and understand the relevance of cancer stem cells (CSC)/tumor initiating cells (TIC) in tumor development and to design appropriate strategies for CSCs and TICs elimination, which is crucial to future cancer prevention and treatment. In this review, we attempt to define various potential markers of cancer stem cells and potential exploration as therapeutic targets for epithelial cancer prevention and treatment.     

Distinct types of tumor-initiating cells form human colon cancer tumors and metastases

Human colon cancer harbors a small subfraction of tumor-initiating cells (TICs) that is assumed to be a functionally homogeneous stem-cell-like population driving tumor maintenance and metastasis formation. We found unexpected cellular heterogeneity within the TIC compartment, which contains three types of TICs. Extensively self-renewing long-term TICs (LT-TICs) maintained tumor formation in serial xenotransplants. Tumor transient amplifying cells (T-TACs) with limited or no self-renewal capacity contributed to tumor formation only in primary mice. Rare delayed contributing TICs (DC-TICs) were exclusively active in secondary or tertiary mice. Bone marrow was identified as an important reservoir of LT-TICs. Metastasis formation was almost exclusively driven by self-renewing LT-TICs. Our results demonstrate that tumor initiation, self-renewal, and metastasis formation are limited to particular subpopulations of TICs in primary human colon cancer. We identify LT-TICs as a quantifiable target for therapies aimed toward eradication of self-renewing tumorigenic and metastatic colon cancer cells.     

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent ‘floater’ cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.     

CD49fhigh Cells Retain Sphere-Forming and Tumor-Initiating Activities in Human Gastric Tumors

Identification of gastric tumor-initiating cells (TICs) is essential to explore new therapies for gastric cancer patients. There are reports that gastric TICs can be identified using the cell surface marker CD44 and that they form floating spheres in culture, but we could not obtain consistent results with our patient-derived tumor xenograft (PDTX) cells. We thus searched for another marker for gastric TICs, and found that CD49f^high cells from newly-dissected gastric cancers formed tumors with histological features of parental ones while CD49f^low cells did not when subcutaneously injected into immunodeficient mice. These results indicate that CD49f, a subunit of laminin receptors, is a promising marker for human gastric TICs. We established a primary culture system for PDTX cells where only CD49f^high cells could grow on extracellular matrix (ECM) to form ECM-attaching spheres. When injected into immunodeficient mice, these CD49f^high sphere cells formed tumors with histological features of parental ones, indicating that only TICs could grow in the culture system. Using this system, we found that some sphere-forming TICs were more resistant than gastric tumor cell lines to chemotherapeutic agents, including doxorubicin, 5-fluorouracil and doxifluridine. There was a patient-dependent difference in the tumorigenicity of sphere-forming TICs and their response to anti-tumor drugs. These results suggest that ECM plays an essential role for the growth of TICs, and that this culture system will be useful to find new drugs targeting gastric TICs.     

Tumor-initiating cells are not enriched in cisplatin-surviving BRCA1;p53-deficient mammary tumor cells in vivo

Although many breast cancers respond to chemotherapy or hormonal therapy, lack of tumor eradication is a central clinical problem preceding the development of drug-resistant tumors. Using the K14cre;Brca1^{F5–13/F5–13};p53^{F2–10/F2–10} mouse model for hereditary breast cancer, we have previously studied responses of mammary tumors to clinically relevant anti-cancer drugs, including cisplatin. The BRCA1- and p53-deficient tumors generated in this model are hypersensitive to cisplatin and never become resistant to this agent due to the large, irreversible deletion in Brca1. We show here that even dose-dense treatment with a maximum tolerated dose of cisplatin does not result in complete tumor eradication. To explain this result we have addressed the hypothesis that the lack of eradication of drug-sensitive tumors is due to increased in vivo chemotherapy resistance of tumor-initiating cells (TICs). Using the CD24 and CD49f cell surface markers that detect normal mouse mammary stem cells, we have identified tumor-initiating cells in BRCA1- and p53-deficient tumors. In addition to the Lin⁻/CD24⁺/CD49f⁺ subpopulation, we show that a larger population of Lin⁻/CD24⁺/CD49f cells also has tumor-initiating capability in at least two serial orthotopic transplantations, suggesting that these are not more differentiated transit-amplifying cells. However, we did not find an enrichment of TICs in cisplatin-treated tumor remnants. We conclude that in this model the tolerance of the cisplatin-surviving cells cannot be attributed to special biochemical defense mechanisms of TICs.Key words: tumor-initiating cells, cisplatin, genetically-engineered mouse model, BRCA1, breast cancer     

Catumaxomab

Catumaxomab, a monoclonal bispecific trifunctional antibody, was approved in the European Union in April 2009 for the intraperitoneal treatment of patients with malignant ascites. The marketing authorization holder Fresenius Biotech GmbH developed catumaxomab (Removab®) together with its partner TRION Pharma GmbH, Germany. It is the first substance worldwide with a regulatory label for the treatment of malignant ascites due to epithelial carcinomas. Since the peritoneum is of mesothelial origin and therefore lacks EpCAM expression, the intraperitoneal administration of catumaxomab is an attractive targeted immunotherapeutic approach. Catumaxomab is able to destroy EpCAM positive tumor cells in the peritoneal cavity known as the main cause of malignant ascites. In addition, catumaxomab is a potential therapeutic option for several primary tumors since the EpCAM molecule is expressed on the majority of epithelial carcinomas. This review focuses on the clinical development of catumaxomab and indicates future directions.     

Tumor-initiating cells are not enriched in cisplatin-surviving BRCA1;p53-deficient mammary tumor cells in vivo

Although many breast cancers respond to chemotherapy or hormonal therapy, lack of tumor eradication is a central clinical problem preceding the development of drug resistant tumors. Using the K14cre;Brca1^F5-13/F5-13;p53^F2-10/F2-10mouse model for hereditary breast cancer, we have previously studied responses of mammary tumors generated in to clinically relevant anti-cancer drugs, including cisplatin. The BRCA1- and p53-deficient tumors generated in this model are hypersensitive to cisplatin and never become resistant to this agent due to the large, irreversible deletion in Brca1. We show here that even dose-dense treatment with a maximum tolerated dose of cisplatin does not result in complete tumor eradication. To explain this result we have addressed the hypothesis that the lack of eradication of drug-sensitive tumors is due to increased in vivo chemotherapy resistance of tumor-initiating cells (TICs). Using the CD24 and CD49f cell surface markers which detect normal mouse mammary stem cells, we have identified tumor-initiating cells in BRCA1- and p53-deficient tumors. In addition to the "OLE_LINK14">Lin^-/CD24⁺/CD49f⁺ subpopulation, we show that a larger population of Lin^-/CD24⁺/CD49f^- cells also has tumor-initiating capability in at least two serial orthotopic transplantations, suggesting that these are not more differentiated transit-amplifying cells. However, we did not find an enrichment of TICs in cisplatin-treated tumor remnants. We conclude that in this model the tolerance of the cisplatin-surviving cells cannot be attributed to special biochemical defense mechanisms of TICs.

     

Cancer stem cells: A review from origin to therapeutic implications

Cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), are elucidated as cells that can perpetuate themselves via autorestoration. These cells are highly resistant to current therapeutic approaches and are the main reason for cancer recurrence. Radiotherapy has made a lot of contributions to cancer treatment. However, despite continuous achievements, therapy resistance and tumor recurrence are still prevalent in most patients. This resistance might be partly related to the existence of CSCs. In the present study, recent advances in the investigation of different biological properties of CSCs, such as their origin, markers, characteristics, and targeting have been reviewed. We have also focused our discussion on radioresistance and adaptive responses of CSCs and their related extrinsic and intrinsic influential factors. In summary, we suggest CSCs as the prime therapeutic target for cancer treatment.     

Metformin,a Diabetes Drug,Eliminates Tumor-Initiating Hepatocellular Carcinoma Cells

Metformin has been widely used as an oral drug for diabetes mellitus for approximately 60 years. Interestingly, recent reports showed that metformin exhibited an anti-tumor action in a wide range of malignancies including hepatocellular carcinoma (HCC). In the present study, we investigated its impact on tumor-initiating HCC cells. Metformin suppressed cell growth and induced apoptosis in a dose-dependent manner. Flow cytometric analysis showed that metformin treatment markedly reduced the number of tumor-initiating epithelial cell adhesion molecule (EpCAM)⁺ HCC cells. Non-adherent sphere formation assays of EpCAM⁺ cells showed that metformin impaired not only their sphere-forming ability, but also their self-renewal capability. Consistent with this, immunostaining of spheres revealed that metformin significantly decreased the number of component cells positive for hepatic stem cell markers such as EpCAM and α-fetoprotein. In a xenograft transplantation model using non-obese diabetic/severe combined immunodeficient mice, metformin and/or sorafenib treatment suppressed the growth of tumors derived from transplanted HCC cells. Notably, the administration of metformin but not sorafenib decreased the number of EpCAM⁺ cells and impaired their self-renewal capability. As reported, metformin activated AMP-activated protein kinase (AMPK) through phosphorylation; however its inhibitory effect on the mammalian target of rapamycin (mTOR) pathway did not necessarily correlate with its anti-tumor activity toward EpCAM⁺ tumor-initiating HCC cells. These results indicate that metformin is a promising therapeutic agent for the elimination of tumor-initiating HCC cells and suggest as-yet-unknown functions other than its inhibitory effect on the AMPK/mTOR pathway.     

What Do We Learn from Spheroid Culture Systems? Insights from Tumorspheres Derived from Primary Colon Cancer Tissue

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.     

Ovarian tumor-initiating cells display a flexible metabolism

An altered metabolism during ovarian cancer progression allows for increased macromolecular synthesis and unrestrained growth. However, the metabolic phenotype of cancer stem or tumor-initiating cells, small tumor cell populations that are able to recapitulate the original tumor, has not been well characterized. In the present study, we compared the metabolic phenotype of the stem cell enriched cell variant, MOSE-L_FFLv (TIC), derived from mouse ovarian surface epithelial (MOSE) cells, to their parental (MOSE-L) and benign precursor (MOSE-E) cells. TICs exhibit a decrease in glucose and fatty acid oxidation with a concomitant increase in lactate secretion. In contrast to MOSE-L cells, TICs can increase their rate of glycolysis to overcome the inhibition of ATP synthase by oligomycin and can increase their oxygen consumption rate to maintain proton motive force when uncoupled, similar to the benign MOSE-E cells. TICs have an increased survival rate under limiting conditions as well as an increased survival rate when treated with AICAR, but exhibit a higher sensitivity to metformin than MOSE-E and MOSE-L cells. Together, our data show that TICs have a distinct metabolic profile that may render them flexible to adapt to the specific conditions of their microenvironment. By better understanding their metabolic phenotype and external environmental conditions that support their survival, treatment interventions can be designed to extend current therapy regimens to eradicate TICs.     

miR-130b Promotes CD133(+) liver tumor-initiating cell growth and self-renewal via tumor protein 53-induced nuclear protein 1

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor, called tumor-initiating cells (TICs) or cancer stem cells (CSCs). Here we describe the identification and characterization of such cells from hepatocellular carcinoma (HCC) using the marker CD133. CD133 accounts for approximately 1.3%-13.6% of the cells in the bulk tumor of human primary HCC samples. When compared with their CD133? counterparts, CD133(+) cells not only possess the preferential ability to form undifferentiated tumor spheroids in vitro but also express an enhanced level of stem cell-associated genes, have a greater ability to form tumors when implanted orthotopically in immunodeficient mice, and can be serially passaged into secondary animal recipients. Xenografts resemble the original human tumor and maintain a similar percentage of tumorigenic CD133(+) cells. Quantitative PCR analysis of 41 separate HCC tissue specimens with follow-up data found that CD133(+) tumor cells were frequently detected at low quantities in HCC, and their presence was also associated with worse overall survival and higher recurrence rates. Subsequent differential microRNA expression profiling of CD133(+) and CD133? cells from human HCC clinical specimens and cell lines identified an overexpression of miR-130b in CD133(+) TICs. Functional studies on miR-130b lentiviral-transduced CD133? cells demonstrated superior resistance to chemotherapeutic agents, enhanced tumorigenicity in vivo, and a greater potential for self renewal. Conversely, antagonizing miR-130b in CD133(+) TICs yielded an opposing effect. The increased miR-130b paralleled the reduced TP53INP1, a known miR-130b target. Silencing TP53INP1 in CD133? cells enhanced both self renewal and tumorigenicity in vivo. Collectively, miR-130b regulates CD133(+) liver TICs, in part, via silencing TP53INP1.     

CD133(+)EpCAM(+) phenotype possesses more characteristics of tumor initiating cells in hepatocellular carcinoma Huh7 cells

Background: EpCAM or CD133 has been used as the tumor initiating cells (TICs) marker in hepatocellular carcinoma (HCC). We investigated whether cells expressing with both EpCAM and CD133 surface marker were more representative for TICs in hepatocellular carcinoma Huh7 cells.Methods: Four different phenotypes of CD133⁺EpCAM⁺, CD133⁺EpCAM^-, CD133^-EpCAM⁺ and CD133^-EpCAM^- in Huh7 cells were sorted by flow cytometry. Then cell differentiation, self-renewal, drug-resistance, spheroid formation and the levels of stem cell-related genes were detected to compare the characteristics of TICs. The ability of tumorigenicity was measured in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to verify TICs.Results: CD133⁺EpCAM⁺ cells have many characteristics of TICs in Huh7 cells compared with CD133⁺EpCAM^-, CD133^-EpCAM⁺, CD133^-EpCAM^- cells, including enrichment in side population cells, higher differentiation capacity, increased colony-formation ability, preferential expression of stem cell-related genes, appearance of drug-resistant to some chemotherapeutics, more spheroid formation of culture cells and stronger tumorigenicity in NOD/SCID mice.Conclusion: CD133⁺EpCAM⁺ phenotype is precisely represented TICs in Huh7 cells. It might be useful for studying biology mechanism of TICs in hepatocellular carcinoma and screening new targets for cancer therapy.     

Calcium channel α2δ1 subunit is a functional marker and therapeutic target for tumor-initiating cells in non-small cell lung cancer

It is hypothesized that tumor-initiating cells (TICs) with stem cell-like properties constitute a sustaining force to drive tumor growth and renew fully established malignancy. However, the identification of such a population in non-small cell lung carcinoma (NSCLC) has been hindered by the lacking of reliable surface markers, and very few of the currently available surface markers are of functional significance. Here, we demonstrate that a subpopulation of TICs could be specifically defined by the voltage-gated calcium channel α2δ1 subunit from non-small cell lung carcinoma (NSCLC) cell lines and clinical specimens. The α2δ1⁺ NSCLC TICs are refractory to conventional chemotherapy, and own stem cell-like properties such as self-renewal, and the ability to generate heterogeneous tumors in NOD/SCID mice. Moreover, α2δ1⁺ NSCLC cells are more enriched for TICs than CD133⁺, or CD166⁺ cells. Interestingly, α2δ1 is functionally sufficient and indispensable to promote TIC properties by mediating Ca²⁺ influx into cells, which subsequently activate Calcineurin/NFATc2 signaling that directly activates the expression of NOTCH3, ABCG2. Importantly, a specific antibody against α2δ1 has remarkably therapeutic effects on NSCLC xenografts by eradicating TICs. Hence, targeting α2δ1 to prevent calcium influx provides a novel strategy for targeted therapy against TICs of NSCLC.Subject terms: Cancer stem cells, Predictive markers