Cancer Stem Cells Cultures Research Articles

Abstract B44: Therapeutic response of primary glioblastoma cancer stem cells relative to patient-matched non-stem tumor cells

Abstract Background: Populations of cells within glioblastoma multiforme (GBM) tumors that have properties similar to neural stem cells are thought to be the source of tumor cell self-renewal. These cancer stem cells (CSCs) may also be particularly resistant to therapy, although support for this important concept is limited. We asked whether early passage primary CSC cultures respond differently than patient-matched non-CSC cultures to both standard-of-care (Temozolomide (TMZ) and Radiation (XRT)) and experimental epigenetic therapy targeting histone deactylases (HDACs). Experimental Design: We established primary cultures of CSCs as adherent glioma neural stem cell and patient-matched non-CSC monolayer cultures in serum from 10 primary glioblastomas. To test response to standard glioma therapy, we treated early passage cultures with single or multiple physiological doses of TMZ (5–50uM), with or without XRT (2Gy). To examine the relative efficacy of the HDAC inhibitor vorinostat, the paired cultures were treated with physiological doses (1–2uM). Response to therapy was assayed by cell cycle analysis on a BD FACSCalibur and for cell viability using CellTiter-Glo. MGMT methylation was assessed by methylation-specific PCR and genome-wide methylation analysis was performed on IIIumina Infinium arrays. Results: The majority of patient-derived primary cultures, including CSC and non-CSC cell lines, are resistant to clinically relevant doses of TMZ. This result is in line with the general lack of response in most but not all GBM patients. Furthermore, the in vitro response to TMZ correlated with the methylation status of MGMT, a robust prognostic marker of survival in GBM patients treated with TMZ. Surprisingly, we observed for two pairs of patient-matched cultures that the CSC cultures were more responsive to single and multi-fraction XRT treatment than the non-CSCs. To further understand this difference in response in patient-matched cultures, we performed genome-wide DNA methylation analysis and found over 1500 genes with differential methylation between CSC and non-CSC cultures, including PTEN and CDKN2A. We examined the status of CD133, a marker of stemness, for our CSC and non-CSC cultures and found that cultures that were enriched for CD133+ cells were not more resistant to either TMZ or XRT. Finally, we examined whether vorinostat, an HDAC inhibitor currently in clinical trials with TMZ/XRT for GBM patients, could sensitize cultures to standard of care therapy. Clinically relevant doses of vorinostat led to decreased cell viability but did not appear to have a synergistic effect when combined with either TMZ or XRT. Conclusions: Our study demonstrates that early passage primary gliomas cultures can be used to assess drug response in less time than the interval between surgery and onset of adjuvant therapy. Furthermore, we find that for our patient-matched cultures, the CSC population is not uniformly more resistant to therapy in vitro than the non-CSC population. If the in vitro response mirrors patient response in vivo, the in vitro cultures might be beneficial for making clinical decisions on a patient-specific basis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B44.

Comparative Proteomics of Colon Cancer Stem Cells and Differentiated Tumor Cells Identifies BIRC6 as a Potential Therapeutic Target

Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that "cell death " regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells.

Abstract 4371: Optimal drug concentration screening and evaluation in cancer stem cells & 3D tumor stem cell cultures drug response assays in association with clinical efficacy for melanoma cancer stem cell

Abstract Optimal drug concentration screening and evaluation in Cancer stem cells & 3D Tumor Stem Cell cultures Drug Response assays in association with Clinical efficacy for Melanoma Cancer Stem Cell. Three-dimensional (3D) culture of cancer cell lines has long been advocated as a better model of the malignant phenotype that is most closely related to tumorigenicity in vivo. Moreover, new drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumors and to decrease the use of experimental animals. The induction of chemotherapy or concomitant chemoradiotherapy has been used to increasingly to improve survival, and organ preservation. This approach encounters significant morbidity and mortality. Therefore reliable chemosensitivity assays are needed to accurately predict the response to chemotherapy and guide the selection and treatment of cancer patients. The purpose of this study is to examine and evaluate optimum drug candidates in vitro chemosensitivity on patient tumor tissues directly in culture and on their Cancer stem cell cultures. The tumor samples obtained after surgery or biopsy, were placed immediately in Celprogen Tumor Transportation Media and shipped at 4-8 OC for processing. Tissues were washed with 1X PBS solution and aseptically cut into 0.5mm sections and cultured in 6 well tissue culture plates with an insert pre-coated with ECM. All cancer cell types remain viable and maintain their native architecture for at least 14 days and incorporated DNA measured by adding EdU(5-ethynyl-2’-deoxyuridine) to the culture. The efficacy of various therapeutic agents targeting major pathways (wnt, Notch, PI3K, MAPK, STAT) and chemotherapy agents were tested using DNA uptake and TUNNEL assay anti-cancer agents was calculated according to the inhibition index. The same compounds were tested for utilizing the patient's Melanoma Cancer stem Cell Cultures established with Celprogen's Media and ECM. Among the 300 compounds tested Adria, Taxol, DTIC, Thalidomide, Paclitaxel, Abraxane, Ertibux and Velcade were not effective against Melanoma Cancer Stem cell (CSC). We were able to show 3 compounds that were effective against Melanoma CSC targeting selected pathways. 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 4371. doi:10.1158/1538-7445.AM2011-4371

Disulfiram modulated ROS–MAPK and NFκB pathways and targeted breast cancer cells with cancer stem cell-like properties

Background:Previous studies indicate that disulfiram (DS), an anti-alcoholism drug, is cytotoxic to cancer cell lines and reverses anticancer drug resistance. Cancer stem cells (CSCs) are the major cause of chemoresistance leading to the failure of cancer chemotherapy. This study intended to examine the effect of DS on breast cancer stem cells (BCSCs).Methods:The effect of DS on BC cell lines and BCSCs was determined by MTT, western blot, CSCs culture and CSCs marker analysis.Results:Disulfiram was highly toxic to BC cell lines in vitro in a copper (Cu)-dependent manner. In Cu-containing medium (1 μM), the IC50 concentrations of DS in BC cell lines were 200–500 nM. Disulfiram/copper significantly enhanced (3.7–15.5-fold) cytotoxicity of paclitaxel (PAC). Combination index isobologram analysis demonstrated a synergistic effect between DS/Cu and PAC. The increased Bax and Bcl2 protein expression ratio indicated that intrinsic apoptotic pathway may be involved in DS/Cu-induced apoptosis. Clonogenic assay showed DS/Cu-inhibited clonogenicity of BC cells. Mammosphere formation and the ALDH1+VE and CD24Low/CD44High CSCs population in mammospheres were significantly inhibited by exposure to DS/Cu for 24 h. Disulfiram/copper induced reactive oxygen species (ROS) generation and activated its downstream apoptosis-related cJun N-terminal kinase and p38 MAPK pathways. Meanwhile, the constitutive NFκB activity in BC cell lines was inhibited by DS/Cu.Conclusion:Disulfiram/copper inhibited BCSCs and enhanced cytotoxicity of PAC in BC cell lines. This may be caused by simultaneous induction of ROS and inhibition of NFκB.

Establishing a lung cancer stem cell culture using autologous intratumoral fibroblasts as feeder cells

Human LCSCs (lung cancer stem cells) were first isolated from lung cancer patients and cultured using serum-free culture methods. To recreate the intratumoural microenvironment to sustain LCSC growth, autologous intratumoral fibroblasts were used as feeder cells. In this study, we investigated the growth and maintenance of pluripotency in prolonged LCSCs culture on autologous intratumoural fibroblasts. LCSCs isolated from three clinical samples all showed vigorous growth on feeder cells for 16 weeks of continuous cultures with a doubling time of 41-47 h. The cells continued expressing stem cell marker CD133 and remained undifferentiated. Pluripotency was demonstrated by tumour formation in immunodeficient mice. In a feeder-free culture system, growth of LCSCs spheres was retarded and would cease when the diameter reached 100 μm if immediate passage was not performed. Moreover, spontaneous differentiation was more frequently seen in a serum-free culture system. In conclusion, we have successfully established a culture system using autologous intratumoural fibroblast cells as feeder cells for prolonged culture of undifferentiated LCSCs in vitro.

Abstract B38: Therapeutic response in primary glioblastoma neural stem cells relative to patient-matched non-stem tumor cells

Abstract Background: Glioblastoma multiforme (GBM) is the most common and most malignant form of primary brain cancer. The standard of care for GBM patients is surgical resection followed by radiation (XRT) and temozolomide (TMZ). Populations of cells within GBMs that have properties similar to neural stem cells are thought to be the source of tumor cell self-renewal. These cancer stem cells (CSCs) may also be particularly resistant to therapy, although support for this important concept is limited. Additionally, widespread epigenetic changes and their ensuing dysregulation of gene networks are known to contribute to GBM formation and to therapeutic response. In particular, the widespread deacetylation of histones occurs in cancers with poor prognosis. Histone deactylase inhibitors (HDACi) are in clinical trials for GBM patients and one particular agent, vorinostat, has modest effects as a single agent in recurrent GBM. We asked whether primary-derived CSC cultures might respond differently than patient-matched non-CSC cultures to both standard-of-care and experimental epigenetic therapy. Experimental Design: We established primary cultures of CSC as adherent glioma neural stem cell (GNS) and patient-matched non-CSC monolayer cultures in serum (ML) from each 10 primary glioblastomas. To test response to standard glioma therapy, we treated early passage cultures with physiological doses of TMZ (5-50 uM), with or without XRT (2Gy). To examine the relative efficacy of HDACi, the paired cultures were treated with physiological doses of vorinostat (1 −2 uM). Response to therapy was assayed by cell cycle analysis on a BD FACSCalibur. Results: The majority (90%) of patient-derived primary cultures, including GNS and ML cell lines, are resistant to physiological doses of TMZ, similar to the general lack of response in most but not all GBM patients. When treated with TMZ and XRT alone or in combination, one particular GNS exhibited a response to XRT and the combination with TMZ, but not TMZ alone, while the patient-matched ML culture did not exhibit sensitivity to XRT, contrary to expectations. Lastly, treatment with vorinostat resulted in G2 arrest and increased cell death for 90% of cultures. However, the response observed between patient-matched GNS and ML cultures was not uniform across the panel of GBMs, with some GNS lines being more sensitive and others more resistant compared to ML cultures. Conclusions: Most primary tumor-derived cultures are resistant to TMZ, irrespective of their enrichment for CSC. Furthermore, one GNS line was more sensitive to XRT than patient-matched non-CSC line suggesting that not all glioma CSCs are resistant to therapy as previously thought. The difference in response to vorinostat between GNS and non-CSC cultures reinforces this finding. Our study demonstrates that early passage primary gliomas cultures can be used to assess drug response in a timely manner. If the in vitro response mirrors in vivo response, this patient-specific model system might be beneficial for making clinical decisions on a patient-specific basis. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B38.

Abstract 3344: Two different stem cell populations exist in breast cancer to control tumor initiation

Abstract The cancer stem cell theory states that a determinate number of cells within a tumor have unique, stem cell-like features to regenerate the entire tumor mass. However, a cancer stem cell may need the right microenvironment to have its greatest tumor initiation potential. We have documented the presence of two stem cell populations within a tumor to yield consistent and most aggressive recapitulation of tumor initiation in vivo. To isolate cancer stem cells, we used the MMTV-PyMT transgenic model of breast cancer in mice to generate stable cell lines from spontaneous breast tumors. Stable in vitro cultures of FACS analyzed cancer stem cells and tumor-residing mesenchymal stem cells were established and tested for tumor initiating ability using two methods of implantation: subcutaneous (s.c.) injection and cleared mammary fat pad (CMFP) injection. To determine the limit of tumor initiation, a highly malignant cancer stem cell culture (419) was injected s.c. This cell culture produced tumors with 1×104, 1×103, and 1×102 cells with 100%, 93%, and 33% tumorigenicity, respectively. In comparison, 100% tumorigenicity was obtained with 1×102 419 cells implanted in the CMFP, a significant increase compared to s.c. injection (Chi-squared, p<0.001). S.c. injection of a less malignant cancer stem cell culture (145) produced tumors less efficiently, with 100% tumorigenicity with 1×104 cells but 13% tumorigenicity with 1×103 cells. To increase tumorigenicity at low cell numbers, cancer stem cell cultures were combined with tumor-residing mesenchymal stem cells just prior to injection. Cancer stem cell cultures were s.c. injected with 1×105 tumor-residing mesenchymal stem cells (383) at the tumor limiting dose, 1×101 for 419 and 1×103 for 145; tumorigenicity increased 160% in the highly malignant cells (419) and 133% in the less malignant cells (145). The tumor-residing mesenchymal stem cells increased tumorigenicity significantly compared to cancer stem cell injection alone (Chi-squared, p<0.002). Control experiments using non-tumorigenic mesenchymal cells did not produce any increase in tumorigenicity. Genome-wide expression profiling experiments were carried out to demonstrate the clinical relevance of the varying malignancy settings in the cancer stem cell and tumor-residing mesenchymal stem cell cultures. Our results suggest that the cellular composition of the local microenvironment within the breast tumors is the major determinant of efficiency of tumor initiation. The addition of tumor-residing mesenchymal stem cells markedly increases the tumor initiation potential of breast cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3344.

Abstract 4262: Characterization and regulation of a CXCR4-expressing subpopulation in head and neck cancer stem cells

Abstract The cancer stem cell (CSC) hypothesis proposes that only the CSCs within a tumor possess the ability to self-renew and to generate new tumors. Under this model, CSCs are presumed responsible for cellular invasion and metastasis, as well as the recurrence of cancers following chemotherapy. In head and neck squamous cell carcinoma (HNSCC), cells which are CD44 positive and lineage negative (CD44+/Lin-) have been shown to possess properties of CSCs. These cells form tumors in NOD/SCID mice at a much greater efficiency compared to CD44-/Lin- cells, and express the proto-oncogene BMI-1. More recently, it has been shown in pancreatic cancer that a distinct subpopulation of CSCs expressing the chemokine receptor CXCR4 is essential for tumor metastasis. Here we demonstrate the existence of a highly invasive CXCR4+ subpopulation in HNSCC cancer stem cells and elucidate the pathways through which this subpopulation could be regulated. For this study putative CSC cultures derived from primary HNSCC tissue were used from which CXCR4-expressing subpopulations were isolated. A matrigel invasion assay revealed that the CXCR4+ fraction of CSCs was significantly more invasive compared to the CXCR4- fraction. We also surveyed the mRNA expression of these two fractions by qRT-PCR, and found that expression of the proteinases MMP2 and MMP9 was markedly higher in CXCR4+ cells. Interestingly, treatment with nicotine increased the expression of CXCR4 and its ligand SDF-1 in the parental CSC culture, suggesting a possible mechanism by which CXCR4 could be regulated within the CSC population. Nicotine, a major component of tobacco, is known to have proliferative, anti-apoptotic, and angiogenic effects, and has been shown to promote epithelial-to-mesenchymal transition in a variety of human cancers. Mortality from HNSCC remains high due primarily to cancer invasion of vital structures and development of distant metastases. Our findings shed light on the mechanisms by which CXCR4 confers an invasive and metastatic CSC phenotype. In addition, the observed effects of nicotine could provide novel insights towards the regulation of the CXCR4/SDF-1 axis in HNSCC, for which tobacco use has been identified as the leading risk factor. Continued efforts in mapping the molecular mechanisms surrounding CXCR4-mediated invasion and metastasis of CSCs could lead to new and effective treatment strategies for HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4262.

Abstract 3342: Specific inhibition of IKKβ induces caspase-independent cell death in the ovarian cancer stem cells

Abstract Introduction: Current studies show that the cancer stem cells represent the chemo-resistant phenotype and therefore may survive chemotherapy to eventually rebuild the tumor. Our group previously identified and characterized the ovarian cancer stem cells using CD44 and MyD88 as a marker and showed that these cells have a constitutively active IKKβ/NFκB axis. We hypothesize that specific targeting of this pathway may affect survival of the ovarian cancer stem cells. Methods: A panel of CD44+/MyD88+ ovarian cancer stem cells were treated with increasing concentrations of the specific IKKβ inhibitor, BAY 11-7082 (0.1, 0.5 1, 5, and 10 μg/ml). Effect on cell growth was monitored using the Incucyte real time imaging system and cell viability was determined using the Celltiter 96 assay. Specific inhibition of IKKβ was determined by: (a) the effect on the mTOR pathway by analyzing levels of phospho-S6 kinase and (b) by determining the effect on TNFα-induced NFκB activity by measuring levels of IκB. Activity of caspases- 3/7, −8, and −9 was measured using Caspase-Glo assay and levels of XIAP monitored using western blot analysis. Effect on mitochondrial membrane potential was measured using flow cytometry after staining with JC1 dye. In vivo activity was monitored using a s.c. ovarian cancer xenograft model in nude mice. The effect of BAY 11-7082 (10 mg/kg every other day given i.p.) was compared to carboplatin (40mg/kg q7d given i.p.) and paclitaxel (10mg/kg q3d given i.p.). Results: Treatment with BAY 11-7082 inhibited cell growth and decreased cell viability in all ovarian cancer stem cell cultures tested in a dose and time dependent manner with GI50 at ∼ 5μg/ml. Specific inhibition of IKKβ was observed at this dose by a decrease in phospho-p70S6 kinase and inhibition of TNFα-induced IκB degradation. Cell death was caspase-independent with no significant increase in caspases 3/7, 8, nor 9 and no effect on the levels of XIAP. Cell death was associated with loss of mitochondrial membrane potential with more than 90% of cells showing a shift in JC1 fluorescence from red to green in 4h. In vivo studies showed that BAY 11-7082 is able to inhibit tumor growth with T/C of 64%, significantly better than carboplatin and paclitaxel (T/C 87% and 74%, p=0.009 and 0.0025, respectively). Conclusion: The capacity of the cancer stem cells to survive chemotherapy and rebuild the tumor represent a major target to inhibit recurrence in ovarian cancer patients. The demonstration that specific targeting of IKKβ can induce cell death in the ovarian cancer stem cells opens new therapeutic options for ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3342.

Cancer stem cells: Cell culture, markers, and targets for new therapies

A cancer stem cell (CSC) is defined as an undifferentiated cell with the ability to self-renew, differentiate to multiple lineages and initiate tumors that mimic the parent tumor. In this review, we focus on glioblastomas, describing recent progress and problems in characterizing these cells. There have been advances in CSC culture, but tumor cell heterogeneity has made purification of CSCs difficult. Indeed, it may be that CSCs significantly vary from tumor to tumor. We also discuss the proposal that CSCs are resistant to radiotherapy and chemotherapy and play a major role in repopulating tumors following treatment. To overcome their resistance to conventional therapies, we may be able to use our extensive knowledge of the signaling pathways essential for stem cells during development. These pathways have potential as targets for new glioblastoma therapies. Hence, although there is an ongoing debate on the nature of CSCs, the theory continues to suggest new ideas for both the lab and the clinic.

Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity

Colon carcinoma is one of the leading causes of death from cancer and is characterized by a heterogenic pool of cells with distinct differentiation patterns. Recently, it was reported that a population of undifferentiated cells from a primary tumor, so-called cancer stem cells (CSC), can reconstitute the original tumor on xenotransplantation. Here, we show that spheroid cultures of these colon CSCs contain expression of CD133, CD166, CD44, CD29, CD24, Lgr5, and nuclear beta-catenin, which have all been suggested to mark the (cancer) stem cell population. More importantly, by using these spheroid cultures or freshly isolated tumor cells from multiple colon carcinomas, we now provide compelling evidence to indicate that the capacity to propagate a tumor with all differentiated progeny resides in a single CSC. Single-cell-cloned CSCs can form an adenocarcinoma on xenotransplantation but do not generate the stroma within these tumors. Moreover, they can self-renew and are capable of multilineage differentiation. Further analysis indicated that the lineage decision is dictated by phosphoinositide 3-kinase (PI3K) signaling in CSCs. These data support the hypothesis that tumor hierarchy can be traced back to a single CSC that contains multilineage differentiation capacity, and provides clues to the regulation of differentiation in colon cancers in vivo.

Prognostic value of cancer stem cell expression and growth in glioblastoma

2004 Background: Cancer stem cells (CSCs) expressing the CD133 marker are a small fraction of tumorigenic cells responsible for glioblastoma development and spreading, and are relatively resistant to conventional radiotherapy and chemotherapy. To evaluate the prognostic value of CSC expression and growth. Methods: We investigated the relationship between patient death or disease progression and the in vitro growth potential of glioblastoma CSCs, CD133 and CD133/Ki67 expression by immunohistochemistry and immunofluorescence, in tumors of 44 glioblastoma patients treated with complete or partial tumorectomy, followed by radiotherapy combined with temozolomide treatment. Results: CSC cultures were obtained from 14 of the 44 tumors (32 percent). CSC generation was significantly associated with overall survival (OS) (p<0.001) and progression-free survival (PFS) (p<0.001). The time required for CSC expansion strongly correlated with OS (r=0.74; p=0.002) and PFS (r=0.85; p<0.001). The presence of ≥ 2% CD133+ cel...

High prognostic potential of glioblastoma stem cell analysis

10580 Background: Cancer stem cells (CSCs) are a rare cell population responsible for tumor development and maintenance. Recent studies have shown that glioblastoma stem cells express the CD133 marker and are resistant to radiotherapy and chemotherapy. However, clinical data based on the study of CSCs in patients with glioblastoma are not available yet. Methods: Glioblastoma samples from 44 patients treated with complete or partial tumorectomy, followed by radiotherapy and temozolomide were prospectively analyzed. Immunohistochemistry was performed to evaluate the prognostic value of the number of CD133+ cells present in tumors. Moreover, the relationship between the ability to generate long-term culture of tumorigenic cells in vitro and the clinical outcome of glioblastoma patients was tested. Results: CD133 expression did not show an overall prognostic value. CSC cultures were obtained from 14 of the 44 tumors (32%). The generation of CSCs emerged as significant independent prognostic factor by the Cox multivariate analyses, with an adjusted hazard ratio of 2.50 (95% CI, 1.04 to 6.06; P=0.004). The median overall survival among patients with tumors generating CSCs was 8.0 months (95% CI, 4.0 to 11.5), as compared with 15 months (95% CI, 11.0 to 19.0) among those without generation of CSCs (P=0.0002). The median progression-free survival was 3.5 months (95% CI, 2.0 to 6.0) for glioblastoma generating CSCs and 9.0 months (95% CI, 7.0 to 12.0) for glioblastoma not generating CSCs (P=0.0001). A higher CD133 expression significantly associated with tumors generating CSCs (P=0.006), and correlated with a higher risk of death in patients with tumors generating CSCs in vitro (hazard ratio of 1.65, 95% CI, 1.05 to 2.60; P=0.0285). Conclusions: Generation of long-term culture of tumorigenic CSCs in vitro from glioblastoma predicts a poor clinical outcome for patients, in terms of both overall and progression-free survival. In tumors generating CSCs, CD133 expression may have a prognostic value. No significant financial relationships to disclose.

Culture of human head and neck cancer stem cells using soft agar.

The soft-agar stem-cell assay was applied to head and neck cancer. We have successfully grown 23 (64%) of 36 head and neck tumors from both primary lesions and metastases. More poorly differentiated tumors had positive cultures more frequently than well-differentiated tumors. The plating efficiency (colonies per cells in the inoculum) averaged 0.006% (range, 0.001% to 0.08%). The system allows testing and sensitivity of individual tumors to cancericidal drugs, and our initial trials using methotrexate, bleomycin sulfate, and cisplatin (cisplatinum) show a high degree of variability between individual tumors.