Potential Cancer Stem Cells Research Articles

Do Increased Doses to Stem-Cell Niches during Radiation Therapy Improve Glioblastoma Survival?

Background and Purpose. The reasons for the inevitable glioblastoma recurrence are yet understood. However, recent data suggest that tumor cancer stem cells (CSCs) in the stem-cell niches, with self-renewing capacities, might be responsible for tumor initiation, propagation, and recurrence. We aimed to analyze the effect of higher radiation doses to the stem-cell niches on progression-free survival (PFS) and overall survival (OS) in glioblastoma patients. Materials and Methods. Sixty-five patients with primary glioblastoma treated with radiation therapy were included in this retrospective analysis. The SVZ and DG were segmented on treatment planning magnetic resonance imaging, and the dose distributions to the structures were calculated. The relationship of dosimetry data and survival was evaluated using the Cox regression analysis. Results. Conventionally fractionated patients (n = 54) who received higher doses (D mean ≥ 40 Gy) to the IL SVZ showed improved PFS (8.5 versus 5.2 months; p = 0.013). Furthermore, higher doses (D mean ≥ 30 Gy) to the CL SVZ were associated with increased PFS (10.1 versus 6.9 months; p = 0.025). Conclusion. Moderate higher IL SVZ doses (≥40 Gy) and CL SVZ doses (≥30 Gy) are associated with improved PFS. Higher doses to the DG, the second stem-cell niche, did not influence the survival. Targeting the potential cancer stem cells in the SVZ might be a promising treatment approach for glioblastoma and should be addressed in a prospective randomized trial.

Putative CD133 + melanoma cancer stem cells induce initial angiogenesis in vivo

Tumor angiogenesis is essential for tumor growth and metastasis, and is regulated by a complex network of various types of cells, chemokines, and stimulating factors. In contrast to sprouting angiogenesis, tumor angiogenesis is also influenced by hypoxia, inflammation, and the attraction of bone-marrow-derived cells. Recently, cancer stem cells have been reported to mimic vascularization by differentiating into endothelial cells and inducing vessel formation. In this study, the influence of cancer stem cells on initial angiogenesis was evaluated for the metastatic melanoma cell line D10. Following flow cytometry, CD133+ and CD133− cells were isolated using magnetic cell separation and different cell fractions were transferred to porcine gelatin sponges, which were implanted into the dorsal skinfold chamber of immunocompromised mice. Angiogenesis was analyzed based on microvessel density over a 10-day period using in vivo fluorescence microscopy, and the results were verified using immunohistology. CD133+ D10 cells showed a significant induction of early angiogenesis in vivo, contrary to CD133− D10 cells, unsorted D10 cells, and negative control. Neovascularization was confirmed by visualizing endothelial cells by immunohistology using an anti-CD31 antibody. Because CD133+ cells are rare in clinical specimens and hardly amenable to functional assays, the D10 cell line provides a suitable model to study the angiogenic potential of putative cancer stem cells and the leukocyte–endothelial cell interaction in the dorsal skinfold chamber in vivo. This cancer stem cell model might be useful in the development and evaluation of therapeutic agents targeting tumors.

ICAM1 Is a Potential Cancer Stem Cell Marker of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) accounts for about 90% of esophageal cancer diagnosed in Asian countries, with its incidence on the rise. Cancer stem cell (CSC; also known as tumor-initiating cells, TIC) is inherently resistant to cytotoxic chemotherapy and radiation and associates with poor prognosis and therapy failure. Targeting therapy against cancer stem cell has emerged as a potential therapeutic approach to develop effective regimens. However, the suitable CSC marker of ESCC for identification and targeting is still limited. In this study, we screened the novel CSC membrane protein markers using two distinct stemness characteristics of cancer cell lines by a comparative approach. After the validation of RT-PCR, qPCR and western blot analyses, intercellular adhesion molecule 1 (ICAM1) was identified as a potential CSC marker of ESCC. ICAM1 promotes cancer cell migration, invasion as well as increasing mesenchymal marker expression and attenuating epithelial marker expression. In addition, ICAM1 contributes to CSC properties, including sphere formation, drug resistance, and tumorigenesis in mouse xenotransplantation model. Based on the analysis of ICAM1-regulated proteins, we speculated that ICAM1 regulates CSC properties partly through an ICAM1-PTTG1IP-p53-DNMT1 pathway. Moreover, we observed that ICAM1 and CD44 could have a compensation effect on maintaining the stemness characteristics of ESCC, suggesting that the combination of multi-targeting therapies should be under serious consideration to acquire a more potent therapeutic effect on CSC of ESCC.

Bub1 is required for maintaining cancer stem cells in breast cancer cell lines.

Breast cancer is a leading cause of death among women worldwide due to therapeutic resistance and cancer recurrence. Cancer stem cells are believed to be responsible for resistance and recurrence. Many efforts to overcome resistance and recurrence by regulating cancer stem cells are ongoing. Bub1 (Budding uninhibited by benzimidazoles 1) is a mitotic checkpoint serine/threonine kinase that plays an important role in chromosome segregation. Bub1 expression is correlated with a poor clinical prognosis in patients with breast cancer. We identified that depleting Bub1 using shRNAs reduces cancer stem cell potential of the MDA-MB-231 breast cancer cell line, resulting in inhibited formation of xenografts in immunocompromised mice. These results suggest that Bub1 may be associated with cancer stem cell potential and could be a target for developing anti-breast cancer stem cell therapies.

Cancer stemness and progression: mitochondria on the stage

Cancer stemness and progression: mitochondria on the stage

MiRNA-regulated delivery of lincRNA-p21 suppresses β-catenin signaling and tumorigenicity of colorectal cancer stem cells.

Cancer stem cells (CSCs) are key cellular targets for effective cancer therapy, due to their critical roles in cancer progression and chemo/radio-resistance. Emerging evidence demonstrates that long non-coding RNAs (lncRNAs) are important players in the biology of cancers. However, it remains unknown whether lncRNAs could be exploited to target CSCs. We report that large intergenic non-coding RNA p21 (lincRNA-p21) is a potent suppressor of stem-like traits of CSCs purified from both primary colorectal cancer (CRC) tissues and cell lines. A novel lincRNA-p21-expressing adenoviral vector, which was armed with miRNA responsive element (MRE) of miR-451 (Ad-lnc-p21-MRE), was generated to eliminate CRC CSCs. Integration of miR-451 MREs into the adenovirus efficiently delivered lincRNA-p21 into CSCs that contained low levels of miR-451. Moreover, lincRNA-p21 inhibited the activity of β-catenin signaling, thereby attenuating the viability, self-renewal, and glycolysis of CSCs in vitro. By limiting dilution and serial tumor formation assay, we demonstrated that Ad-lnc-p21-MRE significantly suppressed the self-renewal potential and tumorigenicity of CSCs in nude mice. Importantly, application of miR-451 MREs appeared to protect normal liver cells from off-target expression of lincRNA-p21 in both tumor-bearing and naïve mice. Taken together, these findings suggest that lncRNAs may be promising therapeutic molecules to eradicate CSCs and MREs of tumor-suppressor miRNAs, such as miR-451, may be exploited to ensure the specificity of CSC-targeting strategies.

Profilin 2 promotes migration, invasion, and stemness of HT29 human colorectal cancer stem cells.

We investigated the role of profilin 2 in the stemness, migration, and invasion of HT29 cancer stem cells (CSCs). Increased and decreased levels of profilin 2 significantly enhanced and suppressed the self-renewal, migration, and invasion ability of HT29 CSCs, respectively. Moreover, profilin 2 directly regulated the expression of stemness markers (CD133, SOX2, and β-catenin) and epithelial mesenchymal transition (EMT) markers (E-cadherin and snail). CD133 and β-catenin were up-regulated by overexpression of profilin 2 and down-regulated by depletion of profilin 2. SOX2 was decreased by profilin 2 depletion. E-cadherin was not influenced by profilin 2- overexpression but increased by profilin 2- knockdown. The expression of snail was suppressed by profilin 2- knockdown. We speculated that stemness and the EMT are closely linked through profilin 2-related pathways. Therefore, this study indicates that profilin 2 affects the metastatic potential and stemness of colorectal CSCs by regulating EMT- and stemness-related proteins.

Antigenic assessment of a recombinant human CD90 protein expressed in prokaryotic expression system

Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas.Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform.In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15–Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody–antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90+ cells in a flow cytometry experiment.

Abstract 2807: Oral silibinin inhibits tumorigenic potential of colon cancer stem cells

Abstract Recent studies by our group have demonstrated that silibinin, a non-toxic, chemopreventive agent, has an inhibitory effect on the self-renewal capacity and differentiation potential of the cancer stem cell (CSC) population of CRC cell lines. However, these observations were under in vitro conditions. To overcome the limitations of these findings, in the present study, we assessed oral silibinin effect on tumorigenic potential of colon CSC. For this, we employed phenotypic cell isolation strategy to separate CD44+EpCAMhigh (stem cells enriched) HT-29 CRC cells by flow cytometry/magnetic activated sorting. These sorted and unsorted cells were used to generate ectopic xenograft tumors in NOD/SCID mice and silibinin effect on initiation, growth, and metabolism of these tumors was assessed. Briefly, unsorted and sorted population (CD44+EpCAMhigh) from CRC HT29 cells purified using FACS as per defined protocols were injected sub-cutaneously into NOD/SCID mice (6-8 weeks old), and animals were orally fed with vehicle or silibinin (200 mg/ kg body weight), starting 24 h post-injection (n = 14 per group). Unsorted cells were similarly injected as 2 x106 cells/ mouse. Once xenograft started growing, tumor sizes and volumes were calculated. Tumor latency (time from injection to detection of palpable tumor) were also noted. In general, experiment was terminated by 40 days post cell injections or terminated earlier if tumors reach a maximum diameter of 15 mm. To support tumor latency and tumor size results obtained visibly, anatomical proton-density magnetic resonance imaging (MRI) scans (day 2, 7 and 14) were acquired. Functional imaging (performed for 60-80 mm3 tumor and by end of study) was performed to report on tumor cellularity by diffusion-weighted (DW-), vascularity by dynamic contrast-enhanced (DCE-) MRI and glycolytic activity by 18Fluorine-2-Deoxy-Glucose (FDG) positron emission tomography (PET). Tumors harvested at the end of above study were subjected to live cell sorting analysis to determine the percentage of CSC population (CD44+EpCAMhigh cells) in xenografts. To determine oral silibinin effect on CSC self-renewal in serial transplantation studies, these CSC enriched fractions and the unsorted population of tumor cells were transplanted back into NOD/SCID mice and followed for initiation, growth, and tumor phenotype with or without silibinin treatment. Sorted CSC and unsorted tumor cells from both control and silibinin-treated mice were also subjected to in vitro experiments to determine their self-renewal and differentiation capacity. Results show that indeed silibinin feeding has an inhibitory effect on tumorigenic potential of colon CSC; a decrease in latency as well as size/ volume of tumor was observed. This effect was associated with a reduced number of CSC's in silibinin-fed tumors, a decrease in tumor metabolic activity and an inhibition of angiogenic feed to the tumors. Citation Format: Sushil Kumar, Dileep Kumar, Komal Raina, Natalie J. Serkova, Chapla Agarwal, Rajesh Agarwal. Oral silibinin inhibits tumorigenic potential of colon cancer stem cells. [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 2807. doi:10.1158/1538-7445.AM2015-2807

Abstract 4232: CAPE (caffeic acid phenethyl ester) induces a mammary stem cell lineage restriction to a luminal phenotype via chromatin remodeling

Abstract Propolis, and its active component, CAPE, is a widely available, safe, honeybee natural product with anti-inflammatory, antioxidant, and antitumor properties. We have previously shown that CAPE, inhibits 1) growth of ER+/PR+, ER-/PR- and Her2 + breast cancer cells, 2) the tumorigenic potential of breast cancer stem cells, and 3) mediates its effects through epigenetic modifications via HDAC inhibition, resulting among others, in increased ER in ER- cells. We postulated that CAPE may be useful in chemoprevention for women at high risk for triple-negative breast cancers (TNBC). The cell-of-origin hypothesis states that these cancers arise from transformation of mammary stem cells (SC). We tested the ability of CAPE to inhibit SC self-renewal in murine mammary epithelial cells (MEC) and a human breast epithelial cell line (MCF10A). MEC isolated from BALB/c mice were cultured as mammospheres (MMS) for 7 days in ULA 96 well plates using media containing 5% FBS and 5% Matrigel. MMS were dissociated into single cells, recultured in the presence of CAPE for 7 days and passaged in secondary and tertiary passages in media alone. Alternatively, MCF10A transfected with a Let7c-miRNA reporter were sorted for Let7c-negative cells, and similarly cultured with CAPE. Murine MMS were analyzed for luminal marker, K18, basal marker K14, or progesterone receptor (PR) using immunofluorescence. Open chromatin states were identified using ATAC-seq and peaks were independently called using PeakDeck. Open chromatin areas unique to CAPE treated murine MMS were used for pathway analysis performed by Ingenuity Pathway Analysis and confirmed by Integrative Genome Viewer. CAPE treatment resulted in a dose dependent decrease in mammosphere forming efficiency that persisted in secondary and tertiary passages, indicating reduced self-renewal. CAPE shifted the cells within murine MMS from predominantly K14 to K18-positive and increased PR expression. Interestingly, ATAC-seq showed enrichment of open chromatin of the SMARCA4 gene in the exonic region. SMARCA4 is a chromatin remodeling gene that is involved in SWI/SNF complex and alters the positioning of the nucleosome, it is thought to regulate transcription of certain genes by altering the chromatin structure around those genes. In addition, SMARCA4 can bind BRCA1, and regulate the expression of the tumorigenic breast cancer SC marker, CD44. These data indicate that CAPE both inhibited mammary SC self-renewal and shifted the lineage commitment to a luminal lineage. ATAC-seq demonstrated effects at the chromatin level that may regulate the SMARCA4 gene, which controls the transcription of many genes involved in stem cell renewal, pluripotency, and embryonic development and is differentially expressed in the luminal phenotype of human MEC. Thus, CAPE may have an effect on lineage commitment in agreement with our chemoprevention strategy to reduce TNBC breast cancer development. Citation Format: Coral O. Omene, Manan Patel, Kasthuri Kannan, Adriana Heguy, Mary Helen Barcellos-Hoff. CAPE (caffeic acid phenethyl ester) induces a mammary stem cell lineage restriction to a luminal phenotype via chromatin remodeling. [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 4232. doi:10.1158/1538-7445.AM2015-4232

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

Metformin and prostate cancer stem cells: a novel therapeutic target.

Prostate cancer is the second most frequently diagnosed cancer in the world. Localized disease can be effectively treated with radiation therapy or radical prostatectomy. However, advanced prostate cancer is more difficult to treat and if metastatic, is incurable. There is a need for more effective therapy for advanced prostate cancer. One potential target is the cancer stem cell (CSC). CSCs have been described in several solid tumors, including prostate cancer, and contribute to therapeutic resistance and tumor recurrence. Metformin, a common oral biguanide used to treat type 2 diabetes, has been demonstrated to have anti-neoplastic effects. Specifically, metformin targets CSCs in breast cancer, pancreatic cancer, glioblastoma and colon cancer. Metformin acts directly on the mitochondria to inhibit oxidative phosphorylation and reduce mitochondrial ATP production. This forces tumor cells to compensate by increasing the rate of glycolysis. CSCs rely heavily on mitochondrial oxidative phosphorylation for energy production. The glycolytic switch results in an energy crisis in these cells. Metformin could be used to exploit this metabolic weakness in CSCs. This would increase CSC sensitivity to conventional cancer therapies, circumventing treatment resistance and enhancing treatment efficacy. This review will explore the characteristics of prostate CSCs, their role in tumor propagation and therapeutic resistance and the role of metformin as a potential prostate CSC sensitizer to current anticancer therapies.

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.

P0111 Sub-ventricular zone irradiation in glioblastoma: Can it increase survival?

Background Glioblastoma multiforme are aggressive tumours with high relapse rates, which could be attributed to the neural progenitor stem cells in the sub-ventricular zone (SVZ). Irradiation of stem cell niche in the SVZ could translate into increased overall survival. We aimed to correlate the radiation dose received by the SVZ and its effect on overall survival. Methods We did a retrospective analysis of data from patients with glioblastoma multiforme (treated at our institute in 2008–10) whose treatment plans could be retrieved and for whom current clinical status was known. All adult patients who underwent maximum safe resection and concurrent chemoradiation followed by adjuvant chemotherapy, were included in the study. The planning CT were de-archived, SVZ was contoured in axial segments by taking a 3 mm and 5 mm auto-margin lateral to the lateral ventricle. Laterality of the SVZ was determined according to the tumour epicentre and was denoted as ipsilateral SVZ (IL SVZ), contralateral SVZ (CL SVZ) and combined volume of SVZ (Comb SVZ). Irradiation doses received by each structure were analysed. Findings 50 patients were included. The ratio of men to women was 66:34. Recursive partitioning analysis classified 28% of tumours as RPA class grade 3, 62% as grade 4, and 10% as grade 5. Gross total excision was done in 46%, sub-total resection was done in 54%. Median overall survival was 15.63 months (95% CI 8.83–22.43). Doses >50 Gy to IL SVZ (both 3 mm and 5 mm) were associated with a significant increase in overall survival (median OS 19.83 months versus 6.07 months; p = 0.031). Doses > 37 Gy to the CL SVZ (3 mm) were associated with a non-significant increase in overall survival (median OS 16.73 months versus 8.73 months; p = 0.305). Doses > 37 Gy to the CL SVZ (5 mm) were associated with a non-significant increase in overall survival (median 19.83 months versus 8.73 months; p = 0.118). Interpretation Irradiation of potential cancer stem cell niches in the SVZ influences overall survival in glioblastoma multiforme. Increased doses to the IL SVZ are associated with increased survival in glioblastoma multiforme. Further studies are needed to know what dose should be delivered to what portion of the SVZ to attain this survival advantage. Age, KPS, RPA, extent of surgical resection, and various other biological factors also play an important part.

Co-expression of CXCR4 and CD133 in gastric neoplastic tissue and their correlation with clinicopathological factors and prognosis in gastric cancer

Purpose: Worldwide Gastric carcinoma considered as the second most common cause of cancer related death. Cancer stem cell plays significant role in prognosis and invasion of gastric cancer. CXCR4 is a chemokine receptor and plays an important role in self renewal, differentiation potential, and cell adhesion of cancer stem cell (CSC). On the other hand CD133 is a cell surface glycoprotein and could serve as a prognostic indicator for tumor re-growth, malignant progression, and patient survival. The aim of this study was to establish the expression pattern of CXCR4 and CD133 in gastric cancer tissues; and their correlation with clinicopathological factors like age and gender of patients, position, size and depth of tumor, lymphatic invasion and node metastasis. Methods: Expression of CXCR4 and CD133 proteins were assessed by immunohistochemical and immunofluorescence staining of paraffin–embedded tissues, and followed by RT-PCR in 90 tumors (observed group) and 30 normal gastric samples. The clinical pathological data was statistically analyzed by chi-square methods. Results: The positive rate of CXCR4 and CD133 expression in the observed group was 94.44 (85/90) and 95.55 (86/90) respectively. The expression of CXCR4 and CD133 were correlated with age and gender of patients, and position, size, & depth of the tumor, lymphatic invasion and node metastasis ( p < 0.05). While CXCR4 was positive, CD133 had a positive rate of 92.22% but the positive rate was 2.22% when CXCR4 expression was negative (χ 2 = 58.657; p < 0.001). Conclusion: Overall this data suggests that increased expression of CXCR4 and CD133 might be attributed with disease progression and malignant transformation of gastric epithelium cells. A significant correlation was found in between CXCR4 and CD133 expression and their co-expression may play significant role in invasiveness of gastric cancer.

Selection of a breast cancer subpopulation-specific antibody using phage display on tissue sections.

Breast cancer tumors are composed of heterogeneous cell populations. These populations display a high variance in morphology, growth and metastatic propensity. They respond differently to therapeutic interventions, and some may be more prone to cause recurrence. Studying individual subpopulations of breast cancer may provide crucial knowledge for the development of individualized therapy. However, this process is challenged by the availability of biomarkers able to identify subpopulations specifically. Here, we demonstrate an approach for phage display selection of recombinant antibody fragments on cryostat sections of human breast cancer tissue. This method allows for selection of recombinant antibodies binding to antigens specifically expressed in a small part of the tissue section. In this case, a CD271+ subpopulation of breast cancer cells was targeted, and these may be potential breast cancer stem cells. We isolated an antibody fragment LH 7, which in immunohistochemistry experiments demonstrates specific binding to breast cancer subpopulations. The selection of antibody fragments directly on small defined areas within a larger section of malignant tissue is a novel approach by which it is possible to better target cellular heterogeneity in proteomic studies. The identification of novel biomarkers is relevant for our understanding and intervention in human diseases. The selection of the breast cancer-specific antibody fragment LH 7 may reveal novel subpopulation-specific biomarkers, which has the potential to provide new insight and treatment strategies for breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1007/s12026-015-8657-x) contains supplementary material, which is available to authorized users.

Therapeutic potential of cancer stem cells.

Cancer stem cells (CSCs) play an important role in cancer growth, self-renewal, metastasis, recurrence and radio/chemotherapy. However, the underlying mechanisms remain elusive. In this review, we explore the roles of CSCs in cancer's relapse and progression and discuss the biomarkers of CSCs to predict clinical outcome and their diagnostic potential. The different approaches of CSC therapies are also reviewed, including cytotoxic, radiation, differentiation and targeting signaling pathways. We also discuss the challenge of targeting CSCs in cancer therapy. In addition, non-coding RNAs in CSC therapies are also discussed.

MiR-135a Inhibits Cancer Stem Cell-Driven Medulloblastoma Development by Directly Repressing Arhgef6 Expression.

microRNAs (miRNAs) are short noncoding RNAs, which regulate gene expression post-transcriptionally and play crucial roles in relevant biological and pathological processes. Here, we investigated the putative role of miRNAs in modulating the tumor-initiating potential of mouse medulloblastoma (MB)-derived cancer stem cells (CSCs). We first subjected bona fide highly tumorigenic (HT) CSCs as well as lowly tumorigenic MB CSCs and normal neural stem cells to miRNA profiling, which identified a HT CSC-specific miRNA signature. Next, by cross-checking CSC mRNA/miRNA profiles, we pinpointed miR-135a as a potential tumor suppressor gene, which was strongly downregulated in HT CSCs as well as in the highly malignant experimental tumors derived from them. Remarkably, enforced expression of miR-135a in HT CSCs strongly inhibited tumorigenesis by repressing the miR-135a direct target gene Arhgef6. Considering the upregulation of Arhgef6 in human MBs and its involvement in mediating experimental medulloblastomagenesis, its efficient suppression by miR-135a might make available an effective therapeutic strategy to selectively impair the tumorigenic potential of MB CSCs. Stem Cells 2015;33:1377-1389.

P0239 : Tumorigenic potential of Cancer Stem Cells (CSCs) isolated from human cholangiocarcinoma (CCA) subtypes in cirrhotic microenvironment

P0239 : Tumorigenic potential of Cancer Stem Cells (CSCs) isolated from human cholangiocarcinoma (CCA) subtypes in cirrhotic microenvironment