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Figure S3 shows co-localization of different fractions of bevacizumab or human IgG (hIgG) with Rab4 or LAMP1 in CD133+ GBM cells in vitro, and of rat anti-mouse VEGF-A IgG in an established tumor in the immune competent mouse model of GBM in vivo, show similar trafficking patterns.

Figure S1 shows that bevacizumab gains access to the perivascular tumor space and is internalized by tumor cells when administered to an orthotopic xenograft model of GBM and confirmation of cancer stem-like cell markers in vivo.

Purpose: Bevacizumab, a humanized monoclonal antibody to VEGF, is used routinely in the treatment of patients with recurrent glioblastoma (GBM). However, very little is known regarding the effects of bevacizumab on the cells in the perivascular space in tumors.Experimental Design: Established orthotopic xenograft and syngeneic models of GBM were used to determine entry of monoclonal anti-VEGF-A into, and uptake by cells in, the perivascular space. Based on the results, we examined CD133+ cells derived from GBM tumors in vitro. Bevacizumab internalization, trafficking, and effects on cell survival were analyzed using multilabel confocal microscopy, immunoblotting, and cytotoxicity assays in the presence/absence of inhibitors.Results: In the GBM mouse models, administered anti-mouse-VEGF-A entered the perivascular tumor niche and was internalized by Sox2+/CD44+ tumor cells. In the perivascular tumor cells, bevacizumab was detected in the recycling compartment or the lysosomes, and increased autophagy was found. Bevacizumab was internalized rapidly by CD133+/Sox2+-GBM cells in vitro through macropinocytosis with a fraction being trafficked to a recycling compartment, independent of FcRn, and a fraction to lysosomes. Bevacizumab treatment of CD133+ GBM cells depleted VEGF-A and induced autophagy thereby improving cell survival. An inhibitor of lysosomal acidification decreased bevacizumab-induced autophagy and increased cell death. Inhibition of macropinocytosis increased cell death, suggesting macropinocytosis of bevacizumab promotes CD133+ cell survival.Conclusions: We demonstrate that bevacizumab is internalized by Sox2+/CD44+-GBM tumor cells residing in the perivascular tumor niche. Macropinocytosis of bevacizumab and trafficking to the lysosomes promotes CD133+ cell survival, as does the autophagy induced by bevacizumab depletion of VEGF-A. Clin Cancer Res; 23(22); 7059–71. ©2017 AACR.

Figure S7 shows bevacizumab is transcytosed across normal brain ECs and TECs.

Figure S5 shows co-localization of a fraction of the bevacizumab-VEGF complex with Rab4 and a fraction with LAMP1 in CD133+ GBM tumor cells.

Figure S6 shows bevacizumab treatment causes a reduction in phospho-VEGFR2 and downregulation of VEGF-A induces autophagy in CD133+ GBM cells.

Figure S4 shows FcRn staining largely colocalizes with the endothelial cell marker vWf in xenograft GBM tumors.

Additional details regarding the methods for cells, reagents, animal studies, immunofluorescence, transcytosis, and ELISA assays.

Figure S2 shows the internalization of bevacizumab by CD133-negative non-stem GBM tumor cells was not inhibited by EIPA.

Macroautophagy/autophagy is considered to play key roles in tumor cell evasion of therapy and establishment of metastases in breast cancer. High expression of LC3, a residual autophagy marker, in primary breast tumors has been associated with metastatic disease and poor outcome. FIP200/Atg17, a multi-functional pro-survival molecule required for autophagy, has been implicated in brain metastases in experimental models. However, expression of these proteins has not been examined in brain metastases from patients with breast cancer. In this retrospective study, specimens from 44 patients with brain metastases of infiltrating ductal carcinoma of the breast (IDC), unpaired samples from 52 patients with primary IDC (primary-BC) and 16 matched-paired samples were analyzed for LC3 puncta, expression of FIP200/Atg17, and p62 staining. LC3-puncta+ tumor cells and FIP200/Atg17 expression were detected in greater than 90% of brain metastases but there were considerable intra- and inter-tumor differences in expression levels. High numbers of LC3-puncta+ tumor cells in brain metastases correlated with a significantly shorter survival time in triple-negative breast cancer. FIP200/Atg17 protein levels were significantly higher in metastases that subsequently recurred following therapy. The percentages of LC3 puncta+ tumor cells and FIP200/Atg17 protein expression levels, but not mRNA levels, were significantly higher in metastases than primary-BC. Meta-analysis of gene expression datasets revealed a significant correlation between higher FIP200(RB1CC1)/Atg17 mRNA levels in primary-BC tumors and shorter disease-free survival. These results support assessments of precision medicine-guided targeting of autophagy in treatment of brain metastases in breast cancer patients.

The normalized $^{238}$U(n,f)/$^{235}$U(n,f) cross section ratio has been measured using the NIFFTE fission Time Projection Chamber from the reaction threshold to $30$~MeV. The fissionTPC is a two-volume MICROMEGAS time projection chamber that allows for full three-dimensional reconstruction of fission-fragment ionization profiles from neutron-induced fission. The measurement was performed at the Los Alamos Neutron Science Center, where the neutron energy is determined from neutron time-of-flight. The $^{238}$U(n,f)/$^{235}$U(n,f) ratio reported here is the first cross section measurement made with the fissionTPC, and will provide new experimental data for evaluation of the $^{238}$U(n,f) cross section, an important standard used in neutron-flux measurements. Use of a development target in this work prevented the determination of an absolute normalization, to be addressed in future measurements. Instead, the measured cross section ratio has been normalized to ENDF/B-VIII.$\beta$5 at 14.5 MeV.

Purpose: Bevacizumab, a humanized monoclonal antibody to VEGF, is used routinely in the treatment of patients with recurrent glioblastoma (GBM). However, very little is known regarding the effects of bevacizumab on the cells in the perivascular space in tumors. Experimental Design: Established orthotopic xenograft and syngeneic models of GBM were used to determine entry of monoclonal anti-VEGF-A into, and uptake by cells in, the perivascular space. Based on the results, we examined CD133+ cells derived from GBM tumors in vitro. Bevacizumab internalization, trafficking, and effects on cell survival were analyzed using multilabel confocal microscopy, immunoblotting, and cytotoxicity assays in the presence/absence of inhibitors. Results: In the GBM mouse models, administered anti-mouse-VEGF-A entered the perivascular tumor niche and was internalized by Sox2+/CD44+ tumor cells. In the perivascular tumor cells, bevacizumab was detected in the recycling compartment or the lysosomes, and increased autophagy was found. Bevacizumab was internalized rapidly by CD133+/Sox2+-GBM cells in vitro through macropinocytosis with a fraction being trafficked to a recycling compartment, independent of FcRn, and a fraction to lysosomes. Bevacizumab treatment of CD133+ GBM cells depleted VEGF-A and induced autophagy thereby improving cell survival. An inhibitor of lysosomal acidification decreased bevacizumab-induced autophagy and increased cell death. Inhibition of macropinocytosis increased cell death, suggesting macropinocytosis of bevacizumab promotes CD133+ cell survival. Conclusions: We demonstrate that bevacizumab is internalized by Sox2+/CD44+-GBM tumor cells residing in the perivascular tumor niche. Macropinocytosis of bevacizumab and trafficking to the lysosomes promotes CD133+ cell survival, as does the autophagy induced by bevacizumab depletion of VEGF-A. Clin Cancer Res; 23(22); 7059–71. ©2017 AACR.

The circulating levels of soluble tumor necrosis factor receptor-1 (sTNF-R1) and sTNF-R2 are altered in numerous diseases, including several types of cancer. Correlations with the risk of progression in some cancers, as well as systemic manifestations of the disease and therapeutic side-effects, have been described. However, there is very little information on the levels of these soluble receptors in glioblastoma (GBM). Here, we report on an exploratory retrospective study of the levels of sTNF-Rs in the vascular circulation of patients with GBM. Banked samples were obtained from 112 GBM patients (66 untreated, newly-diagnosed patients and 46 with recurrent disease) from two institutions. The levels of sTNF-R1 in the plasma were significantly lower in patients with newly-diagnosed or recurrent GBM than apparently healthy individuals and correlated with the intensity of expression of TNF-R1 on the tumor-associated endothelial cells (ECs) in the corresponding biopsies. Elevated levels of sTNF-R1 in patients with recurrent, but not newly-diagnosed GBM, were significantly associated with a shorter survival, independent of age (p = 0.02) or steroid medication. In contrast, the levels of circulating sTNF-R2 were significantly higher in recurrent GBM than healthy individuals and there was no significant correlation with expression of TNF-R2 on the tumor-associated ECs or survival time. The results indicate that larger, prospective studies are warranted to determine the predictive value of the levels of sTNF-R1 in patients with recurrent GBM and the factors that regulate the levels of sTNF-Rs in the circulation in GBM patients.

Parvovirus B19 is a small single-stranded DNA virus of the Parvoviridae family. Depending on host factors, it may produce a wide array of clinical disease states. Disease severity can range from self-limited to severe, requiring significant supportive care. Immunocompromised patients are generally affected more severely but rarely develop prolonged and persistent infections. Here, we describe a patient who was diagnosed with parvovirus during maintenance therapy for acute lymphoblastic leukemia and required therapy with intravenous immunoglobulin; the patient remained parvovirus positive according to a polymerase chain reaction testing but had no clinical symptoms for 27 months off chemotherapy.

The secretion of soluble pro-angiogenic factors by tumor cells and stromal cells in the perivascular niche promotes the aggressive angiogenesis that is typical of glioblastoma (GBM). Here, we show that angiogenesis also can be promoted by a direct interaction between brain tumor cells, including tumor cells with cancer stem-like properties (CSCs), and endothelial cells (ECs). As shown in vitro, this direct interaction is mediated by binding of integrin αvβ3 expressed on ECs to the RGD-peptide in L1CAM expressed on CSCs. It promotes both EC network formation and enhances directed migration toward basic fibroblast growth factor. Activation of αvβ3 and bone marrow tyrosine kinase on chromosome X (BMX) is required for migration stimulated by direct binding but not for migration stimulated by soluble factors. RGD-peptide treatment of mice with established intracerebral GBM xenografts significantly reduced the percentage of Sox2-positive tumor cells and CSCs in close proximity to ECs, decreased integrin αvβ3 and BMX activation and p130CAS phosphorylation in the ECs, and reduced the vessel surface area. These results reveal a previously unrecognized aspect of the regulation of angiogenesis in GBM that can impact therapeutic anti-angiogenic targeting.

Histiocytic sarcoma (HS) is a malignant tumor composed of proliferating cells of histiocytic origin. True HS is exceedingly rare, particularly in pediatric patients. These tumors are frequently aggressive, and outcome for patients with HS has traditionally been poor. There is currently no consensus on the optimal management of these tumors, with the literature consisting largely of case reports and small case series utilizing a wide variety of therapies. We describe a case of HS in an 8-year-old female who was successfully treated with an abbreviated leukemia chemotherapy regimen.

Pneumatic irradiation facilities are used for neutron activation analysis, materials testing, electronics hardening, and radioisotope production. The application of these facilities can benefit from a greater understanding of the neutron energy profile inside the irradiation cavities as often only the cadmium ratio is known. A program to measure the energy-dependent neutron fields inside the pneumatic tube at the Ohio State University Research Reactor was undertaken using wires consisting of thermal activation materials and threshold reaction materials with and without thermal and resonance absorbers surrounding them. The measured activities were unfolded using the MAXED unfolding code to reconstruct the neutron energy spectra and compared to the MCNP-calculated flux.

Parvovirus B19 is a small single-stranded DNA virus of the Parvoviridae family. Depending on host factors, it may produce a wide array of clinical disease states. Disease severity can range from self-limited to severe, requiring significant supportive care. Immunocompromised patients are generally affected more severely but rarely develop prolonged and persistent infections. Here, we describe a patient who was diagnosed with parvovirus during maintenance therapy for acute lymphoblastic leukemia and required therapy with intravenous immunoglobulin; the patient remained parvovirus positive according to a polymerase chain reaction testing but had no clinical symptoms for 27 months off chemotherapy.

BACKGROUND: A histologic characteristic of glioblastoma tumors (GBM) is angiogenesis, which requires endothelial cell (EC) sprouting and migration. This is thought to be facilitated by signals in a specialized microenvironment, the perivascular niche, where glioma stem cells (GSC) reside in proximity to ECs. The goal of this study was to determine whether ECs and GSCs directly interact, the mechanism by which this occurs, and the effect on EC signaling and function. METHODS: We utilized multi-label immunofluorescence of GBM tissue and of co-cultured ECs and GSCs to evaluate the proximity of ECs to GSCs and effect of this interaction on signaling, respectively. Cell-cell binding assays, two-photon laser scanning microscopy of cells injected into brain slices in organotypic culture, live-video microscopy of 2D-migration analyzed by Cell Tracker and 3D-chemotactic migration assessed the mechanism of EC-GSC interaction and EC motility. In vivo effects of inhibiting EC interaction with GSCs were evaluated in an orthotopic mouse model of GBM. RESULTS: We found that GSCs were largely localized in very close proximity to ECs in GBM biopsies and bound to normal brain or GBM-derived ECs on Matrigel promoting EC network-formation. In multiple assays, we found that ECs and GSCs directly interact through a mechanism mediated by integrin αvβ3 on ECs binding the RGD-peptide in extracellular L1CAM on GSCs. Importantly, the direct interaction of ECs with GSCs increased the velocity and directional 2D motility of ECs, as well as the chemotactic migration of ECs. A cyclic RGD-peptide, neutralizing antibodies to integrin αvβ3 or L1CAM, and the downregulation of β3 on ECs or of L1CAM on GSCs all inhibited the EC-GSC direct interaction and the increased EC motility. Signaling studies showed that the EC-GSC interaction increased activation of integrin αvβ3, ERK and JNK in ECs and increased nuclear Akt in GSCs, suggesting bidirectional signaling. Soluble L1CAM also activated integrin αvβ3 on ECs. Cyclic RGD-peptide (Cilengitide) treatment of established mouse GBM tumors reduced the percent of Sox2-positive cells within 25-microns of vessels from 65%-to-29% and the percent Sox2-positive cells within 10-microns of vessels from 30%-to-17%. The ratio of Sox2-postiive cells/vessel was unaltered. CONCLUSIONS: These data suggest that (i) GSCs can promote EC motility and thereby angiogenesis through direct contact with ECs, and indicate a role for GSC L1CAM and EC αvβ3 in this interaction; and (ii) Soluble L1CAM activates integrin αvβ3 on ECs, thus circulating soluble L1CAM could interfere with the therapeutic effects of Cilengitide in patients. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Histiocytic sarcoma (HS) is a malignant tumor composed of proliferating cells of histiocytic origin. True HS is exceedingly rare, particularly in pediatric patients. These tumors are frequently aggressive, and outcome for patients with HS has traditionally been poor. There is currently no consensus on the optimal management of these tumors, with the literature consisting largely of case reports and small case series utilizing a wide variety of therapies. We describe a case of HS in an 8-year-old female who was successfully treated with an abbreviated leukemia chemotherapy regimen.

Members of the Src family kinases (SFK) can modulate diverse cellular processes, including division, death and survival, but their role in autophagy has been minimally explored. Here, we investigated the roles of Lyn, a SFK, in promoting the survival of human glioblastoma tumor (GBM) cells in vitro and in vivo using lentiviral vector-mediated expression of constitutively-active Lyn (CA-Lyn) or dominant-negative Lyn (DN-Lyn). Expression of either CA-Lyn or DN-Lyn had no effect on the survival of U87 GBM cells grown under nutrient-rich conditions. In contrast, under nutrient-deprived conditions (absence of supplementation with L-glutamine, which is essential for growth of GBM cells, and FBS) CA-Lyn expression enhanced survival and promoted autophagy as well as inhibiting cell death and promoting proliferation. Expression of DN-Lyn promoted cell death. In the nutrient-deprived GBM cells, CA-Lyn expression enhanced AMPK activity and reduced the levels of pS6 kinase whereas DN-Lyn enhanced the levels of pS6 kinase. Similar results were obtained in vitro using another cultured GBM cell line and primary glioma stem cells. On propagation of the transduced GBM cells in the brains of nude mice, the CA-Lyn xenografts formed larger tumors than control cells and autophagosomes were detectable in the tumor cells. The DN-Lyn xenografts formed smaller tumors and contained more apoptotic cells. Our findings suggest that on nutrient deprivation in vitro Lyn acts to enhance the survival of GBM cells by promoting autophagy and proliferation as well as inhibiting cell death, and Lyn promotes the same effects in vivo in xenograft tumors. As the levels of Lyn protein or its activity are elevated in several cancers these findings may be of broad relevance to cancer biology.

PURPOSE Guidelines encourage primary care clinicians to document smoking status when obtaining patients’ blood pressure, temperature, and pulse rate (vital signs), but whether this practice promotes cessation counseling is unclear. We examined whether the vital sign intervention influences patient-reported frequency and intensity of tobacco cessation counseling. METHODS This study was a cluster-randomized, controlled trial conducted in the Virginia Ambulatory Care Outcomes Research Network (ACORN). At intervention practices, nurses and medical assistants were instructed to assess the tobacco use status of every adult patient and record it with the traditional vital signs. Control practices did not use any systematic tobacco screening or identification system. Outcomes were the proportion of smokers reporting clinician counseling of any kind and the frequency of 2 counseling subcomponents: simple quit advice and more intensive discussion. RESULTS A total of 6,729 adult patients (1,149 smokers) at 18 primary care practices completed exit questionnaires during a 6-month comparison period. Among 561 smokers at intervention practices, 61.9% reported receiving any counseling, compared with 53.4% of the 588 smokers at control practices, for a difference of 8.6% (P = .04). The effect was largely restricted to simple advice, which was reported by 59.9% of intervention patients and 51.5% of control patients (P=.04). There was no significant increase in more extensive discussion, with 32.5% and 29.3% of patients at intervention and control practices, respectively, reporting this type of counseling (P=.18). CONCLUSIONS The vital sign intervention promotes tobacco counseling at primary care practices through a modest increase in simple advice to quit. When implemented as a stand-alone intervention, it does not appear to increase intensive counseling.

Sprouting angiogenesis requires endothelial cell (EC) migration. In glioblastoma (GBM), cancer stem cells (CSC) in the perivascular niche secrete factors that promote EC migration. Here, we identify a direct interaction between the CSCs and ECs that is mediated by RGD-peptide-dependent binding of L1CAM on CSCs with integrin αvβ3 on ECs. Using in vitro assays, we found that this interaction increased migration-associated signaling events in ECs, including activation of integrin αvβ3, ERK, JNK and Akt, and VCAM-1 and E-selectin mRNA expression. Comparison of the effects of co-culturing CSCs with ECs versus the effects of conditioned media from CSCs co-cultured with ECs indicated that the effects of the direct interaction and soluble factors differed quantitatively and qualitatively. The direct interactions stimulated the migration of ECs and primed them to the migratory effects of soluble factors produced by the CSCs. The direct interaction also enhanced expression of pro-survival genes in CSCs and ECs. In GBM biopsies, Sox2-positive tumor cells were localized in close proximity to ECs and injection of β3-negative ECs or L1CAM-negative CSCs into brain slices in organotypic culture resulted in reduced interactions between ECs and CSCs. RGD-peptide treatment of mice with established intracerebral GBM xenografts increased the mean distance of Sox2-positive tumor cells from ECs, decreased integrin αvβ3 activation and decreased vessel surface area. These data show that direct interactions between CSCs and ECs have potent effects on EC migration and angiogenesis in GBM and have implications for the design of angiogenesis-targeted therapies. Citation Format: Monica E. Burgett, Justin D. Lathia, Patrick Roth, Amy S. Nowacki, Ping Huang, Amit Vasanji, Meizhang Li, Tatiana Byzova, Tom Mikkelsen, Shideng Bao, Jeremy Rich, Michael Weller, Candece L. Gladson. L1CAM and integrin αvβ3 mediate direct cell contact between cancer stem cells and endothelial cells: Promotion of endothelial cell migration and survival. [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 4178. doi:10.1158/1538-7445.AM2015-4178

Angiogenesis, the development of a neovasculature, is a key histologic feature of malignant glioma. It occurs through multiple mechanisms, including sprouting and migration of existing endothelial cells (ECs), and is driven by signals in the microenvironment. Glioma stem cells (GSC) inhabit a region near ECs (perivascular niche). Here we examined the hypothesis that GSCs interact directly with ECs and promote EC motility. In 4 of 5 glioblastoma biopsies we found that ∼40% of GSCs were localized within 25 μm of ECs, based on double-labeling for Sox2 and vWf. We then examined the effect of GSCs on EC motility when plated on a 2D laminin substrate, and found a significant increase in EC motility in the presence of GSCs as well as a direct interaction. In a 3D assay, EC migration was enhanced by GSC-conditioned media, but the physical presence and interaction between ECs and GSCs further increased EC migration through a laminin-coated, 3-μm pore filter, as compared to GSC-conditioned media. Integrin αvβ3 and L1CAM have been reported to be upregulated on ECs and GSCs, respectively, in GBM tumors, and integrin αvβ3 can bind the RGD-peptide in L1CAM. We examined the effect of blocking or downregulating integrin αvβ3 or L1CAM on EC interaction with GSCs. In a cell-cell binding assay, blocking or downregulating the integrin β3 subunit significantly inhibited EC-GSC direct contact, as did an RGD peptide and an antibody to L1CAM. Furthermore, downregulation of integrin β3 on ECs significantly reduced EC migration in the presence of GSCs in the transwell migration assay and reduced EC association with GSCs in mouse brain slices in organotypic culture, analyzed by 2-Photon-LSM. These data suggest GSCs promote angiogenesis in part by promoting EC motility, and that the EC-GSC direct interaction mediated by integrin αvβ3 on ECs and L1CAM on GSCs also stimulates EC motility. Citation Format: Monica E. Burgett, Ping Huang, Russell S. Tipps, Amit Vasanji, Justin D. Lathia, Meizhang Li, Shideng Bao, Jeremy N. Rich, Candece L. Gladson. Glioma stem cells promote brain endothelial cell motility: a new mechanism for the direct interaction of glioma stem cells with endothelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3898. doi:10.1158/1538-7445.AM2013-3898

2014 Background: Patients with CNS met have a poor outcome with significant variation in overall survival. No marker to predict survival exists. Rb1 is a negative regulator of the cell cycle, and FiP200 is an upstream signaling node that is distributed between the nucleus and cytoplasm. Nuclear FIP200 inhibits cell proliferation by promoting expression of Rb1, whereas cytoplasmic FIP200 promotes cell survival through autophagy. Previously FIP200 deletion/mutation has been reported in 20% of primary invasive breast cancer [BR ca] patients. FIP200 cellular localization and genetic alterations have not been examined in CNS met. Methods: A retrospective analysis of BR ca and CNS met was performed based on tissue availability from 2 institutions. FIP200 and Rb1 expression and localization was evaluated by immunohistochemistry. Genetic alterations were evaluated by DNA array analysis. Results: 80 patients (42 BR ca and 38 CNS met) were identified. Overall CNS met samples had significantly higher levels of cytoplasmic FIP200 as compared to BR ca (52% vs 24% respectively, p=0.0002) and increased expression of FIP200 around areas of hypoxia/necrosis, consistent with increased autophagy. There was also a significant increase in expression of nuclear FIP200 and Rb1 in the CNS met compared to BR ca (p=0.0007 and p=0.0055 respectively). Median survival from development of CNS met was longer in the group with high levels of nuclear Rb1; 14 (3-27) vs 20 (12-33) months (low expression < 20% vs high ≥ 20%, p=0.1). The same finding was observed with FIP200, longer survival was observed with high nuclear expression; 16 (7-33) vs 19 (0.4-27) months (low expression

Objective. To determine volunteer preceptors’ perceived value and desired frequency of quality assurance visits by experiential education faculty members. Methods. An electronic survey instrument was sent to 235 volunteer preceptors. Results. A 71.5% response rate was achieved. Nearly 90% of respondents indicated that onsite visits met their needs. Approximately 50% of respondents preferred monthly onsite visits, 17% preferred every other month, and 32% preferred once per year. Conclusions. A quality assurance program for preceptors and experiential sites that includes onsite visits from experiential education faculty members meets multiple needs of the college and the preceptors. More research is needed to determine the impact of this method of quality assurance on experiential education.

Angiogenesis is a prominent characteristic of malignant glioma. Glioma stem cells (GSCs) reside adjacent to blood vessels in the perivascular niche, and recent evidence suggests a role for GSCs in promoting angiogenesis. Here we examined the interplay between GSCs and endothelial cells (ECs) on a laminin substrate, and the potential for GSC promotion of EC motility. In an in vitro 2D motility assay, we found that GSCs dramatically stimulated EC migration, and ECs also stimulated GSC migration. An antibody to integrin α6 blocked in part the motility of both GSCs and ECs. We also found that GSCs directly interact with ECs; therefore, we investigated which cell-adhesion molecules mediated the interaction. In a cell-cell binding assay, blocking antibodies to both L1-cell adhesion molecule (L1CAM) and integrin αvβ3 inhibited in part the direct contact between GSCs and ECs. In the motility assay, the combination of antibodies to L1CAM and αvβ3 significantly inhibited the motility and interaction of both cell types. These data suggest GSCs can promote angiogenesis by promoting EC migration, and indicate a role for the cell-adhesion receptors L1CAM and αvβ3 in the direct interaction between GSCs and ECs that may promote EC motility. 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 5288. doi:1538-7445.AM2012-5288

222 Background: BreastCA is the 2nd leading cause of cancer deaths in women in the US. Although the outcome for BreastCA with brain met remains poor, there is significant variation in overall survival (OS). Yet, there are no markers or models to predict OS. FIP200 regulates several pathways. In the nucleus, it inhibits cell proliferation by promoting Rb-1 and p21, and in the cytoplasm it promotes cell survival by inhibiting Pyk2 and regulating autophagy. It has been reported that FIP200 gene is deleted or mutated in 20% of primary BreastCAs, and its Exp is correlated with Rb1 levels. FIP200 Exp, its cellular localization, and gene alterations have not been examined in brain met of any cancer. Methods: Brain met tissues of 21 BreastCA patients (pts) biopsied in our institution between 8/2000 and 3/2010 were obtained and Exp of FIP200 and Rb1 were analyzed. Biopsies were selected based on tissue adequacy and prior diagnosis (Dx) of invasive intra-ductal BreastCA. Immunohistochemistry (IHC) was used to evaluate the localization and Exp levels of FIP200 and Rb1 which in turn were tested as predictors of OS. Genetic alterations in FIP200 and Rb1 were examined after DNA extraction. Results: Median time from Dx to met was 23 (range 0-245) months (mos), and median OS from Dx was 43 (range 6-264) mos. Nuclear Exp of Rb1 in < 30% vs. ≥ 30% of cells were seen in 11 vs. 10 pts. Median OS was 39 (range 6-264) mos for Rb1 Exp < 30% and 47 (range 20-190) mo for Rb1 Exp ≥ 30%. Nuclear Exp of FIP200 in Conclusions: The nuclear Exp of Rb1 in brain met from invasive intra-ductal BreastCA may be linked OS. An expanded study is underway to define whether nuclear FIP200 in >20% of cells correlates with nuclear Rb and OS in BreastCA.

e21152 Background: BreastCA is the most common non-cutaneous cancer and the 2nd leading cause of cancer deaths. Although the outcome for BreastCA patients (pts) with brain met remains poor, there i...