Potential Cancer Stem Cell Marker Research Articles

Abstract 1405: Novel biomarkers for gastric cancer stem cells utilizing comparative proteomics analysis

Abstract Purpose: Cancer stem cells (CSCs) are responsible for cancer progression, metastasis, and recurrence. The identification of CSC markers may open a new therapeutic perspective on the basis of selectively targeting this small population of cancer cells, however, the specific markers of CSCs remain undiscovered. The aim of this study was to identify novel biomarkers of gastric CSCs for clinical diagnosis using proteomics technology. Materials and Methods: Since the side population (SP) cells of gastric cancer possess CSC-like properties, we used CSC-like SP cells, OCUM-12/SP cells and OCUM-2MD3/SP cells in this study. Their parent OCUM-12 cells and OCUM-2MD3 cells were also used. Protein lysates from each cell line were analyzed using QSTAR Elite Liquid Chromatography with Tandem Mass Spectrometry, coupled with isobaric tags for relative and absolute quantitation technology. Proteins overexpressed in SP cells were determined by comparing the proteomes among parent cells and CSC-like SP cells. Candidate proteins detected by proteomics technology were validated by RT-PCR, western blot, invasion assay, and immunohistochemical analysis of 300 gastric cancers. Results: Based on the results of LC-MS/MS, eight proteins, including RBBP6, GLG1, VPS13A, DCTPP1, HSPA9, HSPA4, ALDOA, and KRT18, were up-regulated in both OCUM-12/SP cells and OCUM-2MD3/SP cells when compared to their corresponding parent cells. RT-PCR analysis indicated that the expression level of RBBP6, HSPA4, DCTPP1, HSPA9, VPS13A, ALDOA, GLG1, and CK18 was high in OCUM-12/SP and OCUM-2MD3/SP, in compared with the control of parent OCUM-12 and OCUM-2MD3. These proteins were significantly associated with advanced invasion depth, lymph node metastasis, distant metastasis, or advanced clinical stage. RBBP6, DCTPP1, HSPA4, and ALDOA expression in particular were significantly associated with a poor prognosis in the 300 gastric cancer patients. RBBP6 was determined to be an independent prognostic factor. The motility-stimulating ability of OCUM-12/SP cells and OCUM-2MD3/SP cells was inhibited by RBBP6 siRNA. Conclusion: The eight proteins, RBBP6, GLG1, VPS13A, DCTPP1, HSPA9, HSPA4, ALDOA, and KRT18, utilizing comparative proteomics analysis, were perceived to be potential CSC markers of gastric cancer. Of the eight candidate proteins, RBBP6 was suggested to be a promising prognostic biomarker and a therapeutic target for gastric cancer. Citation Format: Masakazu Yashiro, Tamami Morisaki, Tsuyoshi Hasegawa, Anna Kakehashi, Haruhito Kinoshita, Tatsunari Fukuoka, Hiroaki Kasashima, Go Masuda, Katsunobu Sakurai, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Hideki Wanibuchi, Kosei Hirakawa. Novel biomarkers for gastric cancer stem cells utilizing comparative proteomics analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1405. doi:10.1158/1538-7445.AM2015-1405

Abstract 1393: CD44v3-positive (CD44v3+) and CD24-negative (CD24−) cells possess cancer stem cell (CSC)-like properties in a human oral squamous cell carcinoma cell line

Abstract Background: CSCs are defined as minor cell population which have higher tumor-initiating ability, self-renewal ability and multilineage potential. Although CSCs in oral squamous cell carcinoma (OSCC) were identified by several surface antigen (e.g., CD44, CD133) or ALDEFLOUR assay, it is still elusive which are universal markers for CSC in OSCC. The aim of this study is to clarify potential CSC markers for a OSCC cell line using a combination of cell surface markers, CD44v3 and CD24. Materials and Methods: A OSCC cell line, SAS, was used in the experiment. CD44v3+/CD24− cell fraction was sorted from SAS cells with a FACSAria II and compared with the other three fractions (CD44v3+/CD24+, CD44v3− /CD24−, CD44v3−/CD24+ cell fraction) in cell proliferation, drug resistance and sphere forming ability. mRNA expression levels of various genes, such as stemness genes, anti-apoptosis genes and hypoxia-related genes, were compared among the fractions using quantitative real-time reverse transcriptase-polymerase chain reaction. Results: The proportion of CD44v3+/CD24−, CD44v3+/CD24+, CD44v3−/CD24−, CD44v3−/CD24+ cells was 34.9%, 39.8%, 14.2% and 11.0%, respectively. CD44v3+/CD24− cells showed a higher sphere forming ability, and drug resistance for CDDP, 5-FU and Cetuximab and expressed higher mRNA levels of CSC property-related genes (ABCG2, Oct-4, Nanog) and a hypoxia-related gene (HIF-1α) than the other cell fractions. There were no significant differences in cell proliferation among the fractions. Conclusion: The results suggest that CD44v3+/CD24− cell fractions in SAS possess CSC-like properties. The investigation of tumorigenic capacity in NOD/SCID mice is now under way. Citation Format: Keita Todoroki, Sachiko Ogasawara, Jun Akiba, Masamichi Nakayama, Yoshiki Naito, Jingo Kusukawa, Hirohisa Yano. CD44v3-positive (CD44v3+) and CD24-negative (CD24−) cells possess cancer stem cell (CSC)-like properties in a human oral squamous cell carcinoma cell line. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1393. doi:10.1158/1538-7445.AM2015-1393

Abstract A25: WWOX phosphorylation at Ser14 enhances melanoma docking and growth in the lung and liver in mice

Abstract Understanding of the fundamental cell behavior would help develop strategies for cancer stem cell eradication. Metastatic cancer cells tend to have epigenetically inactivated WWOX gene and are highly aggressive. Time-lapse microscopy revealed that they sense the surrounding WWOX-expressing normal or cancer cells, run away from them, and divide rapidly in order to survive. With time of growth and increased space pressure, these cancer cells grow as spheres with the expression of pluripotent stem cell markers such as Oct4, SSEA-4, TRA-1-60, and others. WWOX is re-expressed in the cancer stem cell spheres with S14 phosphorylation (pS14), suggesting that pS14WWOX is a potential cancer stem cell marker. Notably, organs, expressing pS14WWOX, are susceptible for melanoma cell docking and homing in vivo. Suppression of WWOX phosphorylation at Ser14 by micromolar levels of full-length Zfra1-31 or truncated Zfra4-10 prevents melanoma growth in the lung and liver in mice. Zfra stimulates non-T and non-B memory Hyal-2+ CD3- CD19- lymphocytes in the mouse spleens by binding to membrane hyaluronidase Hyal-2 and probably suppressing the TGF-beta/Hyal-2/WWOX/Smad4 signaling. In vitro education of Hyal-2+ CD3- CD19- cells suppresses melanoma growth in vivo. Citation Format: Wan-Pei Su, Wan-Jen Wang, Nan-Shan Chang. WWOX phosphorylation at Ser14 enhances melanoma docking and growth in the lung and liver in mice. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A25.

Tenascin-C: a novel candidate marker for cancer stem cells in glioblastoma identified by tissue microarrays.

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor, with dismal survival outcomes. Recently, cancer stem cells (CSCs) have been demonstrated to play a role in therapeutic resistance and are considered to be the most likely cause of cancer relapse. The identification of CSCs is an important step toward finding new and effective ways to treat GBM. Tenascin-C (TNC) protein has been identified as a potential marker for CSCs in gliomas based on previous work. Here, we have investigated the expression of TNC in tissue microarrays including 17 GBMs, 18 WHO grade III astrocytomas, 15 WHO grade II astrocytomas, 4 WHO grade I astrocytomas, and 7 normal brain tissue samples by immunohistochemical staining. TNC expression was found to be highly associated with the grade of astrocytoma. It has a high expression level in most of the grade III astrocytomas and GBMs analyzed and a very low expression in most grade II astrocytomas, whereas it is undetectable in grade I astrocytomas and normal brain tissues. Double-immunofluorescence staining for TNC and CD133 in GBM tissues revealed that there was a high overlap between theses two positive populations. The results were further confirmed by flow cytometry analysis of TNC and CD133 in GBM-derived stem-like neurospheres in vitro. A limiting dilution assay demonstrated that the sphere formation ability of CD133+/TNC+ and CD133–/TNC+ cell populations is much higher than that of the CD133+/TNC– and CD133–/TNC– populations. These results suggest that TNC is not only a potential prognostic marker for GBM but also a potential marker for glioma CSCs, where the TNC+ population is identified as a CSC population overlapping with part of the CD133– cell population.

Epigenetic regulation of CD271, a potential cancer stem cell marker associated with chemoresistance and metastatic capacity.

Cancer stem cells (CSCs) are considered to be the cause of tumor initiation, metastasis and recurrence. Additionally, CSCs are responsible for the failure of chemotherapy and radiotherapy. The isolation and identification of CSCs is crucial for facilitating the monitoring, therapy or prevention of cancer. We aimed to identify esophageal squamous cell carcinoma (ESCC) stem-like cells, the epigenetic mechanism and identify novel biomarkers for targeting ESCC CSCs. Sixty-three paired ESCC tissues and adjacent non-cancerous tissues were included in this study. CD271, which was identified as the CSC marker for melanoma, was assessed using quantitative PCR (qPCR). Using flow cytometry, we isolated CD271+ cells comprising 7.5% of cancer cells from the KYSE70 cell line. Sphere formation and anchorage-independent growth were analyzed in CD271+ and CD271− cancer cells, respectively. qPCR was used to detect stem-related genes and CCK-8 was performed to analyze the sensitivity to chemotherapy in the two groups. Bisulfite genomic sequencing was used to analyze the methylation status. CD271 expression was significantly higher in ESCC tissues than in adjacent non-cancerous tissues. Compared with CD271− cancer cells, CD271+ cancer cells showed a higher ability of sphere and colony formation, a high level expression of stem-related gene, and resistance to chemotherapy. The expression of CD271 was induced by a demethylation agent. In conclusion, CD271+ ESCC cells possess stem-like properties. CD271 can potentially act as a prognostic marker for ESCC, whose expression is regulated epigenetically.

Abstract 3476: Profiling CD antigens signature in human hepatocellular carcinoma (HCC) by cell-based high-throughput screening flow cytometry (HTS-FC)

Abstract Accumulating evidence supports the cancer stem cell (CSC) hypothesis, which posits that CSCs are the source of many solid tumor types including hepatocellular carcinoma (HCC). In HCC, CSCs have been identified by a variety of CD antigens, including CD133, CD326 (EpCAM), CD90, CD44, CD24, CD13, which could serve as potential therapeutic targets. However, due to the heterogeneity of HCC, these markers do not account for CSC activity in all tumors, and there is thus a need to identify novel HCC CSC markers. We have developed a cell-based high-throughput screening flow cytometry (HTS-FC) platform to robustly characterize expression profiles of CD antigens on primary human HCC cells. We titrated all commercial fluorescence conjugated CD antibodies (CD1 to CD363) into four 96-well microplates, isolated and stained HCC cells with CD45 antibody then divided and incubated these cells with different CD antibodies in microplates (1X10^5 cells/well) followed by a LIVE/DEAD® reactive dye treatment. Stained plates were fixed and subjected to BD™ LSR II flow cytometry using High Throughput Sampler Unit. Each CD antigen expression percentage was analyzed within the CD45 negative subpopulation by FlowJo software. After investigating 10 HCC tumors, we performed heat-map and cluster analysis using Multi Experiment Viewer software. Our analysis of primary human HCC cells demonstrated that previously identified index markers of HCC CSC are expressed on a highly variable fraction of tumor cells as follows: CD13 (76.10 ± 8.72%), CD24 (7.79 ± 5.33%), CD90 (5.29 ± 0.84%), EpCAM (0.50 ± 0.46%), CD44 (0.041 ± 0.019%) and CD133 (0.0051 ± 0.0034%). Based on the heat-map, we classified all CD markers into five groups: 20 CD13-like high expressions (30%∼100%), 12 median expressions (10%∼30%), 32 CD90-like low expressions (1.0%∼10%), 60 EpCAM-like rare expressions (0.03%∼1.0%) and 249 nonsense expressions. We focused on the CD90- and EpCAM-like groups (92 CD candidates) for novel potential CSC markers. Using quantitative real-time RT-PCR, we determined the mRNA level of 92 antigens in reference to the EpCAM+/CD133+ CSC subpopulations sorted from 4 human HCC cell lines (HepG2, Hep3B, PLC/PRF/5 and Huh7) and identified 20% of these 92 candidates with stem/progenitor mRNA phenotype. We then measured the mRNA level of these remaining candidates in 40 pairs of primary human HCC tumor/adjacent normal tissues and identified two novel CD antigens showing high tumor specificity. We are now purifying these cells from primary HCC tumors in order to determine whether these small populations identify cells with CSC activity in xenograft models and sphere-forming assays. These studies demonstrate that cell-based high-throughput screening flow cytometry screening can be used in combination with other cell/molecular biological techniques to identify novel populations of tumor cells with unique functional characteristics. Citation Format: Kui Chen, Laurie Ailles, John E. Dick, Anand Ghanekar. Profiling CD antigens signature in human hepatocellular carcinoma (HCC) by cell-based high-throughput screening flow cytometry (HTS-FC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3476. doi:10.1158/1538-7445.AM2014-3476

The prognostic value of CD133 expression in non-small cell lung cancer: a meta-analysis.

CD133 has been identified as a potential cancer stem cell (CSC) marker in non-small cell lung cancer (NSCLC). However, the clinical and prognostic significance of CD133 in NSCLC remains controversial. In this study, a meta-analysis with a total number of 13 studies was performed to clarify the association between CD133 expression and clinical outcomes in publications up to June 2013. Odds ratios (ORs) and their 95 % confidence intervals (CIs) were used to assess the association between CD133 expression and the clinicopathological characteristics of NSCLC. Hazard ratios (HRs) and their 95 % CI were used to quantify the predictive ability of CD133 on NSCLC prognosis. Analysis of these data showed that CD133 expression was not associated with any clinicopathological parameters except for histology (pooled OR = 1.35, 95%CI = 1.04-1.76, P = 0.024) and tumor differentiation (pooled OR = 3.19, 95%CI = 1.10-9.21, P = 0.032). Simultaneously, we also found that positive CD133 expression was not associated with disease-free survival (DFS) (pooled HR = 1.76, 95 % CI = 0.87-3.57, P = 0.114, random-effect) but was associated with overall survival (OS) (pooled HR = 2.06, 95 % CI = 1.08-3.91, P = 0.027, random-effect). Overall, it is appropriate to regard CD133 expression as a potential prognostic factor for the OS of NSCLC patients.

Cancer stem cells profile and clinical outcome in stage III colon cancer patients receiving adjuvant oxaliplatin-based chemotherapy.

474 Background: Although an oxaliplatin-based adjuvant treatment is a widely accepted option for radically resected stage III colorectal cancer patients, it is not uncommon to observe an heterogeneous clinical outcome independently from stage. Cancer stem cells (CSC) profile has been suggested to regulate key steps leading to progression, relapse and metastasis and therefore may be responsible for this otherwise unpredictable heterogeneity. ALCAM, CD133, CD24, LRG5, SOX2, ALDH1A1 have been reported as potential markers for CSC, particularly in digestive system cancer. Aim of our study was to assess the role of these markers in determining clinical outcome for stage III colon cancer patients receiving adjuvant chemotherapy. Methods: Patients undergoing radical surgical resection for stage III colon cancer and receiving adjuvant oxaliplatin in combination with either 5FU or capecitabine were eligible for our analysis. CSC profile (ALCAM, CD133, CD24, LGR5, SOX2, ALDH1A1) was analysed by RT-PCR in primary tumour samples. The multivariate analysis also included adjustments for other variables such as T and N stage, sex, age and tumour location. Results: Seventy-eight patients were eligible for analysis. Only high ALCAM expression was associated with clinical outcome. In particular in colon tumours overexpressing ALCAM we observed a significant interaction for a higher risk of relapse (p = 0.04) and a reduced time to relapse (not reached vs. 33.24 months, HR:0.46, 95%CI:0.23-0.89, p = 0.02). There were no significant differences according to main stratification factors between the high ALCAM group vs. low ALCAM group. In our series N stage (N1 vs. N2-3) resulted also relevant for clinical outcome. Conclusions: Our analysis supports the hypothesis that ALCAM expression may represent a crucial marker for a better risk stratification in stage III colon cancer patients receiving adjuvant chemotherapy. According to our data we also suggest that other potential markers of CSC are probably ineffective for patients stratification. Patients expressing high ALCAM levels may be optimal candidates for therapy intensification and possibly different treatment options.

Stem cell profiling in head and neck cancer reveals an Oct-4 expressing subpopulation with properties of chemoresistance

In the past decade cancer, including head and neck squamous cell cancer (HNSCC), is increasingly being regarded as a stem cell associated disease which arises from cells with the property of stemness. According to the cancer stem cell (CSC) theory, only a specific subpopulation of cancer cells has the ability to initiate and perpetuate cancer growth, especially under treatment. In this article we describe a subpopulation of cells within HNSCC that expresses the stemness factor Oct-4, which leads to apoptotic resistance after exposure to chemotherapeutic agents. Permanent cell lines and HNSCC tissue were analyzed for expression of stem cell markers using flow cytometric, immunohistochemical approaches and an antibody based protein array. Chemotherapeutic agent-induced growth inhibition, also known as "enrichment", was determined by the colorimetric cell proliferation assay (MTT-based) and putative stem cell markers were investigated by flow cytometry. Various potential CSC markers were identified in heterogenic expression profiles in permanent cell lines and solid tumors. Our data suggest the Oct-4A isoform as a marker of stemness in HNSCC and the enrichment of cancer stem-like cells by various chemotherapeutic agents was associated with a significantly higher expression of Oct-4. This data suggests that many potential CSC markers are expressed on different expression levels in HNSCC. Among these markers Oct-4(A) plays a pivotal role in the detection of cancer cells with enhanced chemoresistance and provide evidence for the existence of cancer stem-like cells in HNSCC.

Identification of CD90 as a marker for lung cancer stem cells in A549 and H446 cell lines

Accumulating evidence supports that cancer stem cells (CSCs) are responsible for tumor initiation, progression, distal metastasis and even drug resistance. Although CD90 has been identified as a marker for several types of stem cells, such as liver CSCs, the potential role of CD90 as a marker for lung CSCs has yet to be fully characterized. Our previous study demonstrated that the lung cancer stem-like cells isolated from A549 tumor spheres, which were cultured in serum-free conditioned medium, had stronger proliferation and self-renewal abilities, and expressed higher levels of the stem cell markers Sox2 and Oct4 as compared to A549 adherent cells. In the present study, we identified CD90 as a novel surface marker of CSCs in lung cancer cells. Furthermore, we isolated CD90+ CSCs from lung cancer cell lines A549 and H446. Our results revealed that the CD90+ cells, but not the CD90- cells, from lung cancer cells displayed higher tumorigenic capacity. These findings suggest that CD90 could be a potential marker of lung CSCs and thus provide new insight into further therapeutic strategies of lung cancer.

Immune-phenotype assessment of ovarian cancer stem cells.

e22200 Background: Current evidence suggests the presence of cancer stem cells (CSC) in epithelial ovarian cancer (EOC) and their role in chemoresistance and relapse. CD44+/CD24-, CD117+ and CD133+ expression have been reported as potential markers for CSC. Methods: Tumor cells from 5 patients with chemo-naive EOC and cells of ovarian and tubal epithelium from 5 healthy menopausal women undergoing surgery for benign pathology were cultured in standard medium (mediumA) and medium for embrionic cells (mediumB).Cells forming non-adherent spheres where considered CSC-like, and adherent cells as non-CSC-like. The percentages of CD24+, CD44+, CD117+, CD133+ and VEGF-R+ cells were determined by flow-cytometry. Results: After 1-7 week sphere formation was observed in all EOC-cells cultured in mediumB but in none of the healthy controls. Mean percentages of markers expression are shown below. Conclusions: Contrary to published data, expression of CD44+/CD24- was lower in CSClike; in Non-CSClike CD133+ was higher in EOC-cells compared to controls. Inmunophenotype of CSClike is still lacking and further investigation is warranted. [Table: see text] [Table: see text]

ABCG2 as a potential cancer stem cell marker in breast cancer.

e12007 Background: The identification of cancer stem cell (CSC) markers gives opportunity to develop new target therapies. ABCG2 protein is reported as a predictive marker for breast cancer resistance to chemotherapy and as a potential marker for CSC in solid tumors. Methods: We included prospectively 41 patients with locally advanced or metastatic (LAMBC) invasive ductal carcinomas treated with NACT. The expression of CD44/CD24 and ABCG2 was analyzed by flow cytometry after fresh tumor sample digestion. The mammosphere assay (Mammocult) was carried out in 23 samples. We analyzed the correlation between the percentage of ESA+/ABCG2+ and ESA+/CD44+/CD24- cells within the primary tumor with the number of spheres in culture and their relationship with clinical and pathological parameters. ABCG2+ and ABCG2- cells from four primary tumors and from MDA-MB231 cell line were sorted and cultured in triplicate in Mammocult medium for 7 days. The ability to form sphere was analyzed. Patients’ mean age was 52.8 ± 10.3 yo. The ER, PgR and HER2 positive expression rates were 65%, 58% and 45%, respectively. Patients were treated with docetaxel/anthracycline combination (n = 33) of FEC75 combination (n = 8). Results: Complete pathological response was observed in 10 patients (24%). ER negative patients and patients with lower percentage of ABCG2+ cells within the tumor were more likely to achieve near pCR (44% versus 11%, p = 0.04; and 50% versus 14%, p = 0.04, respectively). We did not observe a significant association between pCR and PgR and HER2 expression or the percentage of CD44+/CD24- cells within the tumor. There was a strong correlation between the percentage of ESA+/ABCG2+ cells and the number of mammospheres in culture (r = 0.63; p= 0.0007). This correlation was not significant comparing to CD44+/CD24- cells. Additionally, ABCG2+ cells from primary tumors and MB-231 cell line form a higher number of spheres in culture than ABCG2- (mean difference = 1.15±0.45 /1000 cells, p = 0.04; mean difference = 0.48±0.04 /1000cells, p = 0.008, respectively). Conclusions: ABCG2 is a potential marker for CSC in breast cancer, and the percentage of ABCG2+ cancer cells within the tumor is a predictive factor for pCR in LAMBC patients subjected to NACT.

Abstract 4892: Hypoxia enhances tumor vascularization by endothelial differentiation of endometrial cancer stem cells.

Abstract Emerging evidence has shown that a small subpopulation of cancer cells plays critical roles in tumorigenesis, establishing the cancer stem cells (CSCs) theory. CSCs have properties of tumorigenesis, self-renewal and multilineage differentiation. Previous our study demonstrated that CD133+ enodometrial cancer cells showed tumorigenic and self-renewal ability, suggesting that CD133 was a potential CSC marker in endometrial cancer. In this study, we focused on multilineage differentiation and investigated an ability of endothelial differentiation in endometrial CSCs. Immunohistochemistry of subcutaneous xenografts showed that human endometrial tumors contained human vessels labeled by human-specific anti-CD31 and vWF antibodies. Endothelial tube formation assay (in vitro angiogenesis) revealed that isolated CD133+ endometrial cancer cells could show tube formation while CD133- cells could not. CD133+ cells expressed CD31 and VEGFR-1 higher than CD133- cells. In some cell types of endometrial cancer cells, flow cytometric analysis showed expression of CD31 was high ratio under hypoxia compared to normoxia. Furthermore, in vitro angiogenesis assay demonstrated hypoxic condition could promote and keep tube formation in CD133+ cells, but not CD133- cells. These results suggest that CSCs have potential of endothelial differentiation and hypoxia enhances endothelial differentiation of CSCs. These findings shed light on the establishment of new target therapy for endometrial cancer. Citation Format: Mitsuhiro Nakamura, Xiuzhi Zhang, Masahiro Takakura, Yoshiko Maida, Yasunari Mizumoto, Yukiko Bono, Toshiyuki Sasagawa, Satoru Kyo. Hypoxia enhances tumor vascularization by endothelial differentiation of endometrial cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4892. doi:10.1158/1538-7445.AM2013-4892

Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma

Background. Despite improvement in treatment, the prognosis of hepatocellular carcinoma (HCC) remains disastrous. Cancer stem cells (CSCs) may be responsible for cancer malignant behaviors. ATP-binding cassette, subfamily G, member 2 (ABCG2) is widely expressed in both normal and cancer stem cells and may play an important role in cancer malignant behaviors. Methods. The expression of ABCG2 in HCC tissues and SMMC-7721 cells was examined, and the relevance of ABCG2 expression with clinical characteristics was analyzed. ABCG2+ and ABCG2− cells were sorted, and the potential of tumorigenicity was determined. Expression level of ABCG2 was manipulated by RNA interference and overexpression. Malignant behaviors including proliferation, drug resistance, migration, and invasion were studied in vitro. Results. Expression of ABCG2 was found in a minor group of cells in HCC tissues and cell lines. ABCG2 expression showed tendencies of association with unfavorable prognosis factors. ABCG2 positive cells showed a superior tumorigenicity. Upregulation of ABCG2 enhanced the capacity of proliferation, doxorubicin resistance, migration, and invasion potential, while downregulation of ABCG2 significantly decreased these malignant behaviors. Conclusion. Our results indicate that ABCG2 is a potential CSC marker for HCC. Its expression level has a close relationship with tumorigenicity, proliferation, drug resistance, and metastasis ability.

Expression of the potential cancer stem cell markers, CD133, CD44, ALDH1, and β‐catenin, in primary lung adenocarcinoma—their prognostic significance

The present study investigated expression profiles of the potential CSC markers including CD133, CD44, ALDH1, and β-catenin, and evaluated their prognostic value in lung adenocarcinomas. One-hundred-and-seventy-seven tumors (stage I) were immunohistochemically examined for the expression of these markers, and thresholds to subdivide expression levels were determined using receiver operating characteristics curves. Tumors with high levels of CD133 (adjusted hazard ratio (HR) 4.55 (95% confidence interval (CI) 1.26-16.40, P = 0.021), CD44 (HR 3.73, 95% CI 1.20-11.58, P = 0.023) or ALDH1 (HR 3.61, 95% CI 1.09-12.3, P = 0.036), but not β-catenin (HR 2.43, 95% CI 0.59-10.8, P = 0.220), showed a significantly higher risk of recurrence than the corresponding low expressers. In conclusion, levels of CD133, CD44, and ALDH1 had independent prognostic value to predict the recurrence of lung adenocarcinoma.

Blockade of interleukin-6 receptor suppresses the proliferation of H460 lung cancer stem cells

IL-6/6R signaling is closely associated with tumor growth and poor prognosis. Although there is evidence that interleukin-6 receptor (IL-6R)-mediated signaling promotes the growth and malignancy of cancer, the role of IL-6R in cancer stem cells (CSCs) is poorly defined. This study investigated the role of IL-6R in the proliferation of CSCs. Sphere-forming cells were isolated from the H460 non-small cell lung cancer (NSCLC) cell line and identified as CSCs using confocal microscopy, RT-PCR and WST-1 assay. The H460 spheres demonstrated the typical characteristics of CSCs, including CD133 expression, upregulation of Nanog, self-renewal, and drug resistance to methotrexate (MTX) and fluorouracil (5-FU). The release of IL-6R and its ligand, IL-6, were quantitatively determined and compared between CSCs and non-CSCs. The concentration of soluble IL-6R (sIL-6R) was remarkably high in CSCs compared to that in non-CSCs. Furthermore, significant upregulation of the IL-6R gene was also observed in the CSCs. The growth of CSCs was significantly inhibited by transfection with IL-6R small-interfering RNA (siRNA), as well as with the IL-6R monoclonal antibody (mAb). In addition, blocking both IL-6R and IL-6 using siRNA or mAbs intensified the inhibition of CSC proliferation. These findings indicate that IL-6R is present in CSCs and has an important role in the proliferation of CSCs in the H460 lung cancer cell line. Therefore, we suggest that IL-6R is both a viable target for the development of CSC-directed lung cancer therapeutics and a potential CSC marker in NSCLC.

The CD133 expression levels and its role as potential cancer stem cells marker in gastrointestinal stromal tumor

Dear Editor, We read with interest the article entitled ‘‘Expression profiling of GIST: CD133 is associated with KIT exon 11 mutations, gastric location and poor prognosis’’ by Arne et al., who used a tissue macro arrays (TMAs)-based approach to demonstrate that the expression of CD133 in gastrointestinal stromal tumor (GIST) segregates with c-kit exon 11 deletion and poor prognosis, and suggested that over-expressing CD133 cells might be GIST cancer stem cells (CSCs). The results of our own flow cytometry (FC) analysis of the expression of CD133 in naive and imatinib-treated GISTs were partially different and led us to different conclusions. Like Arne et al., we observed a trend toward higher CD133 expression in naive GISTs carrying c-kit exon 11 mutations in aggregate in a gastric location than in those with an intestinal location but, because of the small number of c-kit deleted cases (n 1⁄4 4), we can neither confirm nor deny the association between high CD133 levels and the c-kit exon 11 deletions. However, unlike Arne et al. (who found CD133 expression in only 28% of their cases), we observed CD133 expression in all our (untreated and treated) samples, even though with

H+-myo-Inositol Transporter SLC2A13 as a Potential Marker for Cancer Stem Cells in an Oral Squamous Cell Carcinoma

Cancer Stem Cells (CSCs) from tumors of different phenotypes possess a marked capacity for proliferation, self-renewal, and differentiation. They also play a critical role in cancer recurrence. Although CSC has been regarded as a new target for cancer therapy, the fundamental questions in the CSC study have not been resolved mainly due to the lack of proper CSC markers. To find new CSC markers for oral squamous cell carcinoma (OSCC), we cultured the primary tumor cells from OSCC patients the regular culture condition and the sphere-forming culture condition to enrich primary tumor cells and potential CSCs. We compared gene expression profiles between sphere-forming and non-forming cells, thus identifying that 23 membrane protein-coding genes were over-expressed in the sphere-forming cells. Among them, 8 belonged to the solute carrier (SLC) protein family. H⁺-myo-inositol transporter SLC2A13 and monocarbohydrate transporter SLC16A6 genes that were consistently increased in the sphere-forming cells in the primary cultures of OSCC samples. Confocal microscopy revealed that SLC2A13-expressing cells were embedded in the limited areas of tumor tissue as a cluster, while SLC16A6 was uniformly detected in hyperplastic epithelium. Moreover, SLC2A13 an expression was induced in human breast adenocarcinoma MCF7 cells after serum starvation. Taken together, our results suggest that SLC2A13 can be a potential markers for CSC in various tumors.

CD133(−) Cells, Derived From a Single Human Colon Cancer Cell Line, Are More Resistant to 5-Fluorouracil (FU) Than CD133(+) Cells, Dependent on the β1-Integrin Signaling

Recently, the cancer stem cells (CSCs) theory has been proposed, and CD133 has been suggested as a potential marker of CSCs in various cancer types. In the present study, we aimed evaluate CD133 as a potential marker of colorectal CSCs and, for this purpose, isolated CD133(+) and CD133(-) cells from a single colorectal cancer cell line, and compared their features, especially related to the tumor-forming and differentiation abilities, and the sensitivity to chemotherapy. CD133(+) cells had higher invivo tumor-forming ability than CD133(-) cells, and in culture, they progressively differentiated into CD133(-) cells, but not vice-versa. On the other hand, CD133(-) cells were more resistant to 5-fluorouracil (FU) treatment than CD133(+) cells, and it was found to be dependent on the higher expression of ß1-integrins, and consequently, higher ability to bind collagen. Disruption of the ß1-integrin function abrogated the chemoresistance. From the present results, we concluded that colorectal cancer CD133(+) cells, although showing some features of CSCs, are not more resistant to 5-FU than CD133(-) cells. Therefore, definite conclusions can not be drawn yet, but it is strongly suggestive that CD133 should not be used as a single CSC marker of colorectal cancer.

Abstract 4382: Matricellular protein Cyr61/CCN1 regulates the stemness of oral cancer stem-like cells

Abstract Background: Cysteine-rich 61 (Cyr61 [CCN1]), a matricellular and plasma membrane protein that has disparate function dependent on cell type-specific manner, is closely related to proliferation, invasion, angiogenesis and epithelial-mesenchymal transition (EMT). Besides, EMT promotes stemness properties in normal and neoplastic cells. Therefore, we want to examine whether CCN1 plays a role in our previous enriched oral cancer stem-like cells (OC-SLCs). Methods: Initially, the expression of CCN1 in OC-SLCs translationally was evaluated. Further, the cell surface CCN1 positive cells from oral cancer cells or OC-SLCs were sorted for further study. We will compare the difference of stemness properties and tumorigenicity between CCN1 positive and negative cells. Further, downregulation of CCN1 in oral cancer cells was achieved to evaluate the function of CCN1 on oral cancer tumorigenesis in vitro and in vivo. The molecular mechanisms mediated by CCN1 on regulating the oral cancer tumorigenicity will also be elucidated Results: We found that CCN1 protein was upregulated in OC-SLCs. Comparing to CCN1 negative cells, CCN1 positive cells exhibited higher sphere formation ability and highly expressed stem cell markers, Nanog and Oct-4. Flow cytometry analyses showed that cytoplasmic membrane CCN1 positive OC-SLCs also co-expression with cancer stem-like cell makers, ABCG2, Grp78 and CD133. In addition, CCN1 knockdown cells decreased sphere formation ability and displayed morphological alternation with loss of EMT. Analysis of CCN1 knockdown cells found that EMT related markers were significantly changed. Overall, we demonstrated that CCN1 is involved in maintenance of stemness and acquisition of epithelial-mesenchymal transition in OC-SLCs. Conclusion: Our data suggest that Cyr61 could be a potential oral cancer stem cell marker, and also the key molecule to regulate the stemness of oral cancer stem-like cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4382. doi:10.1158/1538-7445.AM2011-4382