Lindsey D. Goodman

Percentage of mesenchymal stem cells in high-grade glioma tumor samples correlates with patient survival.

Background Human mesenchymal stem cells (hMSCs) have been shown to reside as stromal cells in human gliomas as glioma-associated hMSCs (GA-hMSCs), but their biological role remains unclear. Because recent evidence indicates that GA-hMSCs drive tumor cell proliferation and stemness, we hypothesized that a higher percentage of GA-hMSCs in tumors predicts poor patient prognosis. Method We determined the percentage of cells coexpressing GA-hMSC markers CD105+/CD73+/CD90+ from patients with newly diagnosed high-grade glioma and analyzed the association between this percentage and overall survival (OS) in 3 independent cohorts: fresh surgical glioblastoma specimens (cohort 1, N = 9), cultured tumor specimens at passage 3 (cohort 2, N = 28), and The Cancer Genome Atlas (TCGA) database. Results In all cohorts, patient OS correlated with the percentages of GA-hMSCs in tumors. For cohort 1, the median OS of patients with tumors with a low percentage of triple-positive cells was 46 months, and for tumors with a high percentage of triple-positive cells, it was 12 months (hazard ratio [HR] = 0.24; 95% CI: 0.02-0.5, P = .02). For cohort 2, the median OS of patients with tumors with a low percentage of GA-hMSCs was 66 months, and for tumors with a high percentage, it was 11 months (HR = 0.38; 95% CI: 0.13-0.9, P = .04). In the database of TCGA, the median OS times in patients with high and low coexpression levels of CD105/CD73/CD90 were 8.4 months and 13.1 months (HR = 0.4; 95% CI: 0.1-0.88; P = .04), respectively. Conclusions The percentage of GA-MSCs inversely correlates with OS, suggesting a role for GA-MSCs in promoting aggressive behavior of gliomas.

ABT-888 restores sensitivity in temozolomide resistant glioma cells and xenografts

Background Temozolomide (TMZ) is active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced. However, even in responsive cases, its beneficial effect is undermined by the emergence of drug resistance. Here, we tested whether inhibition of poly (ADP-ribose) polymerase-1 and -2 (PARP) enhanced the effectiveness of TMZ. Methods Using patient derived brain tumor initiating cells (BTICs) and orthotopic xenografts as models of newly diagnosed and recurrent high-grade glioma, we assessed the effects of TMZ, ABT-888, and the combination of TMZ and ABT-888 on the viability of BTICs and survival of tumor-bearing mice. We also studied DNA damage repair, checkpoint protein phosphorylation, and DNA replication in mismatch repair (MMR) deficient cells treated with TMZ and TMZ plus ABT-888. Results Cells and xenografts derived from newly diagnosed MGMT methylated high-grade gliomas were sensitive to TMZ while those derived from unmethylated and recurrent gliomas were typically resistant. ABT-888 had no effect on the viability of BTICs or tumor bearing mice, but co-treatment with TMZ restored sensitivity in resistant cells and xenografts from newly diagnosed unmethylated gliomas and recurrent gliomas with MSH6 mutations. In contrast, the addition of ABT-888 to TMZ had little sensitizing effect on cells and xenografts derived from newly diagnosed methylated gliomas. In a model of acquired TMZ resistance mediated by loss of MMR gene MSH6, re-sensitization to TMZ by ABT-888 was accompanied by persistent DNA strand breaks, re-engagement of checkpoint kinase signaling, and interruption of DNA synthesis. Conclusion In laboratory models, the addition of ABT-888 to TMZ overcame resistance to TMZ.

Abstract 3314: Phenotypic plasticity in glioma stem cells mediated by NFκB signaling

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Here we report the identification of two major subtypes of glioma stem cells (GSCs) that closely mirror previously described glioblastoma (GBM) groups, namely proneural (PN) and mesenchymal (MES), based on gene expression patterns. Using flow cytometry, we found that PN GSCs express both CD15/SSEA1, a marker for neural stem cells, as well as CD44, a MES marker. In contrast MES GSCs were CD15low/CD44high. MES GSCs and CD44high subpopulations in PN GSCs showed increased expression of transcription factors previously reported to induce the MES phenotype (STAT3, C/EBP-α, and TAZ), and were -resistant to α-irradiation. By distilling cytokines that can induce MES differentiation, we found that TNF-α found in patient-derived microglial conditioned media or conditioned media from MES GSCs caused a PN-to-MES transition through activation of NFκB resulting in radio-resistance. We found that NFκB mediates MES transition indirectly by inducing the transcriptional circuitry of STAT3, C/EBP-α, and TAZ. Blockade of NFκB activation using minocycline abrogated the MES phenotype (both induced and constitutive), reduced tumor growth, and rendered GSCs radio-sensitive. Taken together, our findings reveal that phenotypic and surface marker plasticity is common in GSCs, and that the NFκB driven PN-to-MES transition provides an escape mechanism for GSCs to evade cytotoxic treatment. Based on our studies, we propose that minocycline, a commonly used antibiotic, could target GSCs and should be further evaluated in preclinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3314. doi:1538-7445.AM2012-3314

Abstract 2467: Podoplanin expressing cancer stem cells show increased resistance to DNA damage induced by ionizing radiation

The majority of patients with malignant gliomas, the most common and most lethal brain tumor, develop disease recurrence despite aggressive, multi-modality therapies. This treatment resistance has been attributed to a small population of tumor-initiating glioma cancer stem cells (GSCs). We recently described a new marker of GSCs, the transmembrane glycoprotein podoplanin (pdpn), based on analysis of gene expression profiling data and have found it to be more accurately identify the stem cell population than other described markers. Pdpn expression correlates with poor patient survival and decreased response to radiotherapy. Pdpn expressing cells form neurospheres at high rates in vitro, are more resistant to ionizing radiation, and initiate tumors in vivo. Silencing of pdpn leads to increased radiosensitivity. To identify the mechanism of radiation resistance in pdpn-expressing GSCs, we screened for putative binding partners and identified target of myb-like-1 (tom1l1), a known regulator of Src-pathway activation. We hypothesized that modulation of pdpn and/or tom1l1 expression would lead to alternations in the response of GSCs to radiation via src signaling. Mechanisms of radiation resistance in GSCs are poorly understood to date and the role of src family kinases in modulating radiosensitivity has not previously been reported in gliomas. Pdpn binding of tom1l1 was confirmed by co-immunoprecipation and src activation monitored by immunoblot. Pdpn and tom1l1 expression was downregulated by RNA interference and dsDNA damage and repair assayed by neutral comet assay and by scoring of γH2AX foci. Silencing of pdpn in primary culture, neurophere forming GSC-like cells resulted in increased DNA damage following 2 and 6 Gy ionizing radiation. We similarly examined effects of decreased tom1l1 to determine if the effects on DNA repair mediating by pdpn were acting via the src pathway. In summary, our data suggest that pdpn expression correlates with increased dsDNA repair following ionizing radiation and presents a possible mechanism by which GSCs resist treatment and regrow the tumor. 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 2467. doi:10.1158/1538-7445.AM2011-2467

Abstract 3313: A proliferating population of glioma stem-like cells as characterized by expression of the cell surface glycoprotein CD58

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Glioblastomas (GBMs) are among the most common and most lethal primary brain tumors, characterized by a high cellular proliferation and invasion into brain parenchyma. Glioma stem-like cells (GSCs) have been proposed as being responsible for tumor initiation and recurrence following conventional therapy with chemoradiation. Attempts to identify a single cell surface marker which specifically identifies GSCs within human tumors have had limited success. In an effort to refine GSC identification through the use of a panel of cell surface markers, we identified the glycoprotein CD58 as a marker which more selectively identifies GSCs when added to established markers, such as CD133, podoplanin (pdpn), or CD15. To characterize the frequency of CD58 expression in neurosphere forming primary culture cell lines and freshly obtained surgical specimens, cells were subjected to flow cytometry using an anti-CD58 monoclonal antibody. In vitro analysis of self-renewal, a principle feature of GSCs, was performed with the neurosphere assay. Treatment resistance was compared by radiating single cell suspensions with 2 Gy ionizing radiation prior to neurosphere formation. Cell cycle was measured by flow cytometry and S-phase by incorporation of EdU. Survival analysis was performed on archival tumor specimens following immunohistochemistry with an anti-CD58 monoclonal antibody. Using a panel of 14 GSC primary cultures, CD58 expression was found in all lines, with a median expression of 85.4% of cells, ranging from 8.7% to 100%. CD58 expression was found in 67% of fresh GBM specimens, with expression ranging from 5% to 89% of tumor cells (median 9.4). Culturing of cells beyond 5 passages led to further enrichment for CD58 expressing cells. Neurosphere assays demonstrated an increase in self-renewal in cells expressing CD58 compared to pdpn/CD133/CD15 alone. To investigate whether this difference was a result of increased proliferation of CD58 cells, we performed cell cycle analysis and found a significantly higher percentage of CD58 expressing cells in S-phase (mean of 23.2% vs. 7.9% for CD58 expressing vs. non-expressing, respectively). Immunofluorescence of archival specimens with MIB1/Ki-67 revealed preferential staining in CD58 expression cells. In cell mixing experiments, we observed that CD58 expressing cells rapidly outgrew CD58 non-expressing, highlighting the differences in proliferative capacity. Analysis of patients based on CD58 expression in their tumors revealed that patients with CD58 expression tumors had a poorer survival compared to patients with low or non-expressing tumors (median survival 64 vs 43 weeks, 2yr survival 3% vs 25%, p=0.0192, logrank). In summary, CD58 expression identifies a highly proliferative subpopulation of GSCs, improves upon established markers of these cells, and is an independent predictor of survival based on expression in patient tumors. 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 3313. doi:10.1158/1538-7445.AM2011-3313

Abstract 5157: Functional and regulatory role of src kinase in podoplanin expressing glioma cancer stem cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The majority of patients with malignant gliomas, the most common and most lethal brain tumor, develop disease recurrence despite aggressive, multi-modality therapies. This treatment resistance has been attributed to a small population of tumor-initiating glioma cancer stem cells (GSCs). We recently described a new marker of GSCs, podoplanin (pdpn), based on analyses of gene expression profiling data and have found it to more accurately identify the stem cell population than other described markers, such as CD133, and to play a functional role stem cell phenotype. Pdpn expressing cells formed neurospheres at high rates in vitro, were more resistant to ionizing radiation, and initiated tumors in vivo. Pdpn belongs to a mesenchymal gene signature that is characteritic of poor patients survival from malignant gliomas. To identify the mechasism of pdpn within GSCs, we evaluated putuative binding partners to this transmembrane glycoprotein. One such partner, target of myb-like-1 (tom1l1) has been shown to regulate the Src oncogene pathway. The cytoplasmic tyrosine kinases of the Src family (SFK) play important roles in signal transduction induced by growth factors leading to DNA synthesis, cytoskeletal rearrangement, and receptor endocytosis. We therefore hypothesized that pdpns role GSCs is due to its interaction with the Src pathway. Our aim for this study was to analyze downstream signaling events of Src in association with pdpn and its functional significance in glioma stem cell maintanence, invasion, and chemoradiation resistance. Using primary GSC and established glioma cell lines engineered to either overexpress or silence PDPN, we screened for SFK protein activation (phosphorylation) by immunblot. Changes in Src activation were correlated to in vitro invasion assays, in vivo tumor initiation, and radiation resistance. Preliminary data confirms binding of tom1l1 to pdpn and regulation of SFK activation. Engineered clones with shRNA targeting pdpn and/or tom1l1 were examined by expression profiling for downstream transcriptional changes. In summary, our data suggest a role of SFKs in the regulation of stemness by pdpn. These data integrate well with ongoing trials using Src-kinase inhibitors which may directly target the pdpn-expressing, GSC population in tumors and perhaps overcome the treatment resistance of these critical 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 5157.

Abstract 5147: Trancriptional activation of the glioma stem cell marker podoplanin by the homeobox transcription factor prox1

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The homeobox transcription factor Prox1 is related to the Drosophila prospero gene, which mediates cell fate decisions of neuroblasts. Prox1 is a regulator of lymphangiogenesis but it is also expressed in the developing central nervous system. Homeobox proteins are known to play essential role in the determination of cell fate and the development of the body plan. It has been shown in Drosophila that prospero-mediated transcriptional repression of stem cell genes and activation of differentiation genes prevents tumorigenic growth, suggesting that prospero is in fact a tumor suppressor. Due to its homology to prospero, we speculated that prox1 may play a role in glioma tumorigensis. We previously identified the transmembrane glycoprotein podoplanin (pdpn) as a prognostic marker in infiltrating gliomas, the most common and lethal of adult brain tumors, by analyses of gene expression profiling data. Subsequent functional studies revealed that pdpn identified a subpopulation of cells from primary neurophere cultures and fresh tumors that have characteristics of glioma cancer stem cells (GSCs), such as radiation resistance and tumor initiation, and that the protein played a functional role in maintaining the stem cell phenotype. Like prox1, pdpn is expressed both during lymphangiogenesis and neural development and mice null for either protein have a similar phenotype, including lymphedema and the lack of terminal alveoli formation in the lung. While other transcription factors have been implicated in cancer stem cells, such as Bmi-1, prox1 has note previously been implicated to have a role in this tumor-initiating, treatment resistant cell population. We therefore hypothesized that prox1 transcriptionally regulates PDPN expression in GSCs. To test this, we examined gene expression profiling data from a large glioma dataset for correlations between PDPN and PROX1 expression. No correlation was seen between expression of the two genes; however, PROX1 expression was predictive of patient survival independent of tumor grade (p=.0333). Next, we screened primary neurosphere cell lines for levels of prox1 and pdpn by immunoblot. Co-expression with pdpn was observed in all cell lines expressing prox1. To examine the role of prox1 in PDPN expression, we compared the levels of PDPN in cells for whom prox1 expression had been silenced. In preliminary results, real-time PCR for PDPN showed significant decreases in expression in the presence of PROX1-targeting siRNA. Ongoing promotor and functional analyses will validate the role of prox1 in the transcriptional activation of PDPN. Comparison of the expression profiles of isogenic clones differing only in their expression of PROX1 identifies additional transcriptional targets. In summary, we have identified prox1 as a candidate transcription factor in the regulation of the glioma stem cell phenotype. 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 5147.

Abstract 4305: Refinement of the glioma cancer stem cell marker profile

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Infiltrating gliomas are the most frequently occurring adult brain tumor and, among high-grade forms, typically result in poor patient survival. These tumors tend to be refractory to most treatments, including aggressive surgical resection and chemoradiation. This treatment resistance is hypothesized to be the result of a small population of cells within the tumor which are responsible for tumor initiation, called glioma cancer stem cells (GCSCs). As is true in other stem cell systems, such as hematopoiesis, multiple cell-surface protein markers are needed to accurately define the true stem cell population. We and others have previously identified the glycoprotein podoplanin (pdpn) as a marker of GCSC, which improves upon the established marker, CD133. To refine the GCSC marker profile beyond these two proteins, we examined a number of candidate markers in combination with pdpn that have been proposed as stem cell markers in gliomas or other tumors or tissues. Candidate markers were analyzed by flow cytometry using primary glioma cell lines maintained as neurosphere cultures and fresh tumor specimens. Markers identifying a unique cell population either alone or in combination with pdpn were further analyzed by in vitro neurosphere formation assay and clonogenic survival assay following 2 Gy ionizing radiation. Candidates showing enrichment for stem cells based on these assays were examine for in vivo tumor initiation using flow sorted subpopulations of cells with and without the expression of the candidate protein. As expression of putative stem cell markers will likely predict for poor patient survival, candidate markers were also examined in human tumors by immunohistochemsitry. Among the most promosing candidates were CD97, CXCR4, CD58 and CD44. Flow cytometry revealed CD97 expression in 75%, CD58 in 80%, CXCR4 in 100%, and CD44 in 100% of tumors examined to date. Examination of primary neurosphere cultures, found CD97 in 80%, CXCR4 in 83%, CD58 in 100%, and CD44 in 100% of lines examined. Both CD44+ and CD58+ cells exhibited increased neurophere formation rates and in vivo tumor initiation. These stem-like characteristics were enhanced in those subpopulations also expressing pdpn. RNAs collected from these subpopulations were used for expression profiling to determine if these subpopulation express a unique gene expression. Results thus far indicate that pdpn expression is needed for the stem-like phenotype and that this is enhanced in populations co-expressing CD44, CD15, and/or CD58. Further refinement of this profile will aide in the isolation of the “true” GCSC and allow for improved therapeutic targeting of these resistant 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 4305.