Lindsay Holmes

Glioblastoma resistance to anti-VEGF therapy is associated with myeloid cell infiltration, stem cell accumulation, and a mesenchymal phenotype

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis. Inhibiting the VEGF-VEGF receptor (R) signal transduction pathway in glioblastoma has recently been shown to delay progression, but the relative benefit and mechanisms of response and failure of anti-VEGF therapy and VEGFR inhibitors are not well understood. The purpose of our study was to evaluate the relative effectiveness of VEGF sequestration and/or VEGFR inhibition on orthotopic tumor growth and the mechanism(s) of treatment resistance. We evaluated, not only, the effects of anti-VEGF therapy (bevacizumab), anti-VEGFR therapy (sunitinib), and the combination on the survival of mice bearing orthotopic gliomas, but also the differential effects of the treatments on tumor vascularity, cellular proliferation, mesenchymal and stem cell markers, and myeloid cell infiltration using flow cytometry and immunohistochemistry. Bevacizumab significantly prolonged survival compared with the control or sunitinib alone. Both antiangiogenic agents initially reduced infiltration of macrophages and tumor vascularity. However, multitargeted VEGFR inhibition, but not VEGF sequestration, rapidly created a vascular gradient and more rapidly induced tumor hypoxia. Re-infiltration of macrophages was associated with the induction of hypoxia. Combination treatment with bevacizumab and sunitinib improved animal survival compared with bevacizumab therapy alone. However, at the time of tumor progression, a significant increase in CD11b(+)/Gr1(+) granulocyte infiltration was observed, and tumors developed aggressive mesenchymal features and increased stem cell marker expression. Collectively, our results demonstrate a more prolonged decrease in tumor vascularity with bevacizumab than with sunitinib, associated with a delay in the development of hypoxia and sustained reduction of infiltrated myeloid cells.

Acquired Resistance to Anti-VEGF Therapy in Glioblastoma Is Associated with a Mesenchymal Transition

Purpose: Antiangiogenic therapy reduces vascular permeability and delays progression but may ultimately promote an aggressive treatment-resistant phenotype. The aim of the present study was to identify mechanisms responsible for glioblastoma resistance to antiangiogenic therapy. Experimental Design: Glioma stem cell (GSC) NSC11 and U87 cell lines with acquired resistance to bevacizumab were developed from orthotopic xenografts in nude mice treated with bevacizumab. Genome-wide analyses were used to identify changes in tumor subtype and specific factors associated with resistance. Results: Mice with established parental NSC11 and U87 cells responded to bevacizumab, whereas glioma cell lines derived at the time of acquired resistance to anti-VEGF therapy were resistant to bevacizumab and did not have prolongation of survival compared with untreated controls. Gene expression profiling comparing anti-VEGF therapy-resistant cell lines to untreated controls showed an increase in genes associated with a mesenchymal origin, cellular migration/invasion, and inflammation. Gene-set enrichment analysis showed that bevacizumab-treated tumors showed a highly significant correlation to published mesenchymal gene signatures. Mice bearing resistant tumors showed significantly greater infiltration of myeloid cells in NSC11- and U87-resistant tumors. Invasion-related genes were also upregulated in both NSC11 and U87 resistant cells which had higher invasion rates in vitro compared with their respective parental cell lines. Conclusions: Our studies identify multiple proinflammatory factors associated with resistance and identify a proneural to mesenchymal transition in tumors resistant to antiangiogenic therapy. Clin Cancer Res; 19(16); 4392–403. ©2013 AACR.

Neutrophils promote the malignant glioma phenotype through S100A4.

Purpose: Antiangiogenic therapy is effective in blocking vascular permeability, inhibiting vascular proliferation, and slowing tumor growth, but studies in multiple cancer types have shown that tumors eventually acquire resistance to blockade of blood vessel growth. Currently, the mechanisms by which this resistance occurs are not well understood. Experimental Design: In this study, we evaluated the effects of neutrophils on glioma biology both in vitro and in vivo and determined target genes by which neutrophils promote the malignant glioma phenotype during anti-VEGF therapy. Results: We found that an increase in neutrophil infiltration into tumors is significantly correlated with glioma grade and in glioblastoma with acquired resistance to anti-VEGF therapy. Our data demonstrate that neutrophils and their condition media increased the proliferation rate of glioblastoma-initiating cells (GIC). In addition, neutrophils significantly increased GICs Transwell migration compared with controls. Consistent with this behavior, coculture with neutrophils promoted GICs to adopt morphologic and gene expression changes consistent with a mesenchymal signature. Neutrophil-promoting tumor progression could be blocked by S100A4 downregulation in vitro and in vivo. Furthermore, S100A4 depletion increased the effectiveness of anti-VEGF therapy in glioma. Conclusions: Collectively, these data suggest that increased recruitment of neutrophils during anti-VEGF therapy promotes glioma progression and may promote treatment resistance. Tumor progression with mesenchymal characteristics is partly mediated by S100A4, the expression of which is increased by neutrophil infiltration. Targeting granulocytes and S100A4 may be effective approaches to inhibit the glioma malignant phenotype and diminish antiangiogenic therapy resistance. Clin Cancer Res; 20(1); 187–98. ©2013 AACR.

Suppression of RAF/MEK or PI3K synergizes cytotoxicity of receptor tyrosine kinase inhibitors in glioma tumor-initiating cells

BackgroundThe majority of glioblastomas have aberrant receptor tyrosine kinase (RTK)/RAS/phosphoinositide 3 kinase (PI3K) signaling pathways and malignant glioma cells are thought to be addicted to these signaling pathways for their survival and proliferation. However, recent studies suggest that monotherapies or inappropriate combination therapies using the molecular targeted drugs have limited efficacy possibly because of tumor heterogeneities, signaling redundancy and crosstalk in intracellular signaling network, indicating necessity of rationale and methods for efficient personalized combination treatments. Here, we evaluated the growth of colonies obtained from glioma tumor-initiating cells (GICs) derived from glioma sphere culture (GSC) in agarose and examined the effects of combination treatments on GICs using targeted drugs that affect the signaling pathways to which most glioma cells are addicted.MethodsHuman GICs were cultured in agarose and treated with inhibitors of RTKs, non-receptor kinases or transcription factors. The colony number and volume were analyzed using a colony counter, and Chou-Talalay combination indices were evaluated. Autophagy and apoptosis were also analyzed. Phosphorylation of proteins was evaluated by reverse phase protein array and immunoblotting.ResultsIncreases of colony number and volume in agarose correlated with the Gompertz function. GICs showed diverse drug sensitivity, but inhibitions of RTK and RAF/MEK or PI3K by combinations such as EGFR inhibitor and MEK inhibitor, sorafenib and U0126, erlotinib and BKM120, and EGFR inhibitor and sorafenib showed synergy in different subtypes of GICs. Combination of erlotinib and sorafenib, synergistic in GSC11, induced apoptosis and autophagic cell death associated with suppressed Akt and ERK signaling pathways and decreased nuclear PKM2 and β-catenin in vitro, and tended to improve survival of nude mice bearing GSC11 brain tumor. Reverse phase protein array analysis of the synergistic treatment indicated involvement of not only MEK and PI3K signaling pathways but also others associated with glucose metabolism, fatty acid metabolism, gene transcription, histone methylation, iron transport, stress response, cell cycle, and apoptosis.ConclusionInhibiting RTK and RAF/MEK or PI3K could induce synergistic cytotoxicity but personalization is necessary. Examining colonies in agarose initiated by GICs from each patient may be useful for drug sensitivity testing in personalized cancer therapy.

Abstract 3483: Synergistic combination therapy with molecular targeted drugs in glioma stem-like cells

[Introduction] The prognosis of patients with malignant gliomas is poor despite multimodality therapies underscoring the need for novel therapeutic strategies. The majority of glioblastomas have aberrant receptor tyrosine kinase (RTK)/RAS/phosphoinositide 3 kinase (PI3K) signaling pathways and malignant glioma cells are thought to be addicted to these aberrant signaling pathways for their survival and proliferation. However, a large number of clinical trials have demonstrated that monotherapies have limited efficacy. Tumor heterogeneities and signaling redundancy and crosstalk in intracellular signaling network may imply necessity of combination treatments. Recent studies also suggested that effective methods to personalize antitumor therapy are required. However, drug sensitivity testing using tumor cells from each patient, which is one of the potent methods for personalized tumor therapy, has been unsuccessful. One possible reason of this is a technical issue regarding evaluation of clonogenicity of glioma stem-like cells (GSCs) that are thought to be key players in gliomagenesis and the disease progression and recurrence and thus targets of glioma therapy. We previously presented an effective method to evaluate clonogenicity of GSCs by using agarose-based culture system. In this study, we tested the therapeutic effects of combination treatments on GSCs using targeted drugs that affect the signaling pathways to which most glioma cells are thought to be addicted. [Materials and Methods] Human GSCs were cultured in agarose and treated with inhibitors of RTKs, non-receptor kinase or transcription factor. The colony number and volume were analyzed using GelCountTM colony counter system (Oxford Optronix Inc., UK) and Chou-Talalay combination index was analyzed. Phosphorylation of proteins was evaluated by reverse phase protein array and immunoblotting. [Results] While GSCs showed diverse sensitivity to targeted therapies even in the cells of the same glioma subtype, combinations of EGFR inhibitors with sorafenib, EGFR inhibitors with MEK inhibitors, Sorafenib with U0126, and erlotinib with BKM120 showed synergy in different GSC lines, indicating effectiveness of suppressing RTK and its downstream molecule. Combination of erlotinib with sorafenib, synergistic in the GSC11 cells, induced apoptosis and autophagic cell death associated with synergistic suppression of Akt and ERK signaling pathways and with decreased nuclear PKM2 and beta-catenin in vitro, and significantly improved survival of nude mice bearing GSC11 brain tumors compared with control and monotherapy groups. [Conclusions] Inhibition of RTK and its downstream molecule induced synergistic antitumor effects but sensitivity of GSC lines to therapies was diverse. Examining colonies initiated by GSCs obtained from individual patients may be useful for drug sensitivity testing in personalized cancer therapy. Citation Format: Takashi Shingu, Lindsay Holmes, Verlene Henry, Khatri Latha, Anupama E. Gururaj, Laura A. Gibson, Tiffany Doucette, Frederick F. Lang, Ganesh Rao, Liang Yuan, Erik P. Sulman, Nicholas P. Farrell, Waldemar Priebe, Kenneth R. Hess, Yaoqi A. Wang, Jian Hu, Oliver Bogler. Synergistic combination therapy with molecular targeted drugs in glioma stem-like 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 3483. doi:10.1158/1538-7445.AM2015-3483

Abstract 1612: Targeting intercellular adhesion molecule-1 (ICAM-1) prolongs glioblastoma survival in combination with bevacizumab .

Glioblastoma is the most common malignant brain tumor and is characterized by cellular heterogeneity, vascular proliferation and extensive tissue infiltration. Analysis of gene expression data from orthotopic glioma stem cells with acquired resistance to bevacizumab therapy identified over expression of intercellular adhesion molecule 1 (ICAM1, CD54) in resistant tumors. ICAM1 is a cell adhesion glycoprotein of the immunoglobulin supergene family which interacts with β2 integrins, mediates leukocyte transendothelial migration and T cell activation during inflammatory processes. ICAM1 over expression in tumors resistant to antiangiogenic therapy was validated by real time PCR and Western blot. In a panel of glioma stem cell lines, ICAM1 expression was higher in mesenchymal compared to proneural cell lines. GFP-tagged ICAM1 shRNA lentivirus was used to knockdown ICAM1 in multiple cell lines. We injected shICAM1 NSC11 and scramble glioma stem cells into the brain of nude mice. Mice bearing tumors formed from shICAM1 NSC11 cells survived significantly longer than mice injected with scramble control cells. Tumor size was significantly decreased in mice bearing tumors formed from shICAM1 cells than that in mice bearing tumors from GFP-tagged NSC11 control cells. Knocking-down ICAM1 suppresses tumor invasion both in vitro and in vivo. We next examined the mechanism of ICAM1 over expression after treated with anti-VEGF therapy. We observed that ICAM1 over expression correlated with hypoxia-inducible factor by immnofluorescence analysis in tumor tissue. Furthermore, we found that ICAM1 protein expression was markedly increased after NSC11 and NSC17 cells under hypoxic condition in a time-dependent manner. Furthermore, hypoxia-induced p-STAT3 increased transcription of ICAM1, which was inhibited by an inhibitor of the JAK/STAT3 pathway. Our studies identify ICAM1 as a potentially important mediator of tumor migration/invasion in glioblastoma. Targeting ICAM1 may provide a new strategy to prolong the efficacy of antiangiogenic therapy and prevent the invasive phenotype. Citation Format: Yuji Piao, Ji Liang, Verlene Henry, Lindsay Holmes, John F. de Groot. Targeting intercellular adhesion molecule-1 (ICAM-1) prolongs glioblastoma survival in combination with bevacizumab . [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 1612. doi:10.1158/1538-7445.AM2013-1612

Abstract LB-314: CSF-1R inhibitor JNJ-28312141 reduces antiangiogenic therapy-induced myeloid cell infiltration and mesenchymal shift

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Glioblastoma is the most common and malignant primary brain tumor in adults. Despite maximal tumor resection, radiation and chemotherapy, the tumors high degree of infiltration leads to 100% recurrence and an average life expectancy after diagnosis of about 12 to 14 months. Vascular endothelial growth factor (VEGF) and other pro-angiogenic factors have been identified as critical mediators of angiogenesis in glioblastoma. Inhibition of angiogenesis results in a high radiographic response rate but ultimately all patients’ progress on this therapy. An increase in monocyte/macrophage recruitment has been associated with resistance to antiangiogenic therapy in preclinical mouse xenograft models and in patients. We hypothesized that combining antiangiogenic therapy with therapies inhibiting macrophage infiltration may delay resistance and prolong animal survival. In our glioma stem cell model, we evaluated the impact of bevacizumab alone and in combination with JNJ-28312141 (a novel colony-stimulating factor-1 receptor/FMS-related receptor tyrosine kinase-3 inhibitor) on the recruitment of myeloid cells, microvascular density, and animal survival. Four days after orthotopic implantation of 1× 105 NSC11 glioma stem cells, mice were randomized to treatment with bevacizumab, JNJ-28312141, or the combination of bevacizumab plus JNJ-28312141 and followed for survival (n=8-10 per group). JNJ-28312141 did not prolong survival compared to control. However, JNJ-28312141 combined with bevacizumab significantly reduced tumor vascularity (microvascular density measured using Factor VIII) and prolonged survival compared with bevacizumab alone (p=0.04). We compared the F4/80+ myeloid cell population in xenograft tumors using immunohistochemistry and immunofluorescence. The number of F4/80+ myeloid cells was significantly less in mice treated with JNJ-28312141 and bevacizumab compared with mice treated with bevacizumab alone (p<0.01). To evaluate the impact of treatment on the expression of mesenchymal markers, we quantified YKL-40 protein expression using immunofluorescence. Bevacizumab increased the expression of YKL-40 compared to controls and this increase in expression was significant blocked in the mice treated with the combination of bevacizumab and JNJ -28312141. These results suggest that resistance to antiangiogenic therapy can be partly overcome in glioblastoma by combining anti-VEGF therapy with an inhibitor of macrophages. In our orthotopic glioma model, CSF-1R inhibition is associated with a decrease in the recruitment and infiltration of F4/80+ cells into tumor and a decrease in mesenchymal marker expression. Our studies provide a new strategy to prolong the efficacy of antiangiogenic therapy and accelerate the integration of combination therapies into clinical trials. 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 LB-314. doi:1538-7445.AM2012-LB-314

Abstract 1388: Acquired resistance to anti-VEGF therapy in glioblastoma is associated with an inflammatory and mesenchymal phenotype

Glioblastoma is the most common malignant brain tumor and is characterized by rapid angiogenesis-dependent (re)growth, cell heterogeneity, and extensive local tissue infiltration. Antiangiogenic therapy, among the most promising treatment developments, rapidly reduces vascular permeability and delays glioblastoma progression but the therapy itself ultimately promotes an aggressive treatment-resistant phenotype. The aim of the present study was to identify mechanisms responsible for glioblastoma resistance to antiangiogenic therapy. When mice bearing U87 and NSC11 xenografts treated with bevacizumab became moribund, tumor tissue was extracted to obtain tumor cells with acquired resistance to bevacizumab. U87 and NSC11 cells were dissociated and cultured in serum-containing and stem cell media, respectively. To verify resistance, the resistant cells were expanded in vitro and then re-implanted orthotopically in nude mice. Mice were randomized to receive bevacizumab or vehicle and followed for survival. As anticipated, tumors formed by parental U87 and NSC11 cells were sensitive, whereas tumors obtained at the time of acquired resistance to anti-VEGF therapy were resistant to bevacizumab and did not have prolongation of survival compared to untreated controls. To elucidate the molecular mechanisms underlying resistance to antiangiogenic treatment, we performed gene expression profiling using Affymetrix U133Plus_2.0 arrays comparing untreated and bevacizumab-treated tumors. Comparison of anti-VEGF therapy-resistant cell lines to untreated controls demonstrated an increase in genes associated with a mesenchymal phenotype. Antiangiogenic therapy increased the expression of genes of mesenchymal origin, cellular migration/invasion, and inflammation including chemokine secretion, myeloid cell chemotaxis and multiple markers reflecting a pro-inflammatory environment. Consistent with these data, mice bearing U87 resistant tumors showed significantly greater infiltration of F4/80 + myeloid cells compared to wild-type tumors. Comparing the mesenchymal gene signature from Phillips et al with the gene changes in our experiments using Gene Set Enrichment Analysis (GSEA v2.07, Broad Institute, MIT), we show that NSC11 tumors treated with bevacizumab showed a highly significant correlation to the published mesenchymal gene signature. In addition to the pro-inflammatory changes, invasion-related genes were also upregulated. We found the U87 and NSC11 resistant cells have significantly higher invasion rates in vitro compared with U87 and NSC11 parental cell lines. Our studies identify prominent mechanisms of glioblastoma resistance to antiangiogenic therapy. A better understanding of resistance may provide new strategies to prolong the efficacy of this therapy and accelerate the integration of combination therapies into clinical trials. 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 1388. doi:1538-7445.AM2012-1388