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Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.

XLS file - 12K, Mutations of PTEN, TP53, and NF1 genes in glioblastoma neurospheres

PDF file - 41K, Table S2: EGFR gene copy number in neurospheres and the corresponding original glioblastomas. Table S3: Immunophenotype of neurospheres. Table S4: EGFR gene amplification, Met expression, and MET gene alterations in glioblastomas and corresponding neurospheres. Table S5: Multiple transcriptional signatures in Met-neg and Met-pos neurospheres. Table S6: Immunohistochemistry on tumors derived from neurospheres. Table S8: Antibodies used in flow-cytometric analysis

Cancer stem–like cells (CSC) could be a novel target for cancer therapy, including dendritic cell (DC) immunotherapy. To address this, we developed experiments aimed at DC targeting of neurospheres (NS) from GL261 glioma cells because neurospheres can be enriched in CSC. We obtained murine neurospheres by growing GL261 cells in epidermal growth factor/basic fibroblast growth factor without serum. GL261-NS recapitulated important features of glioblastoma CSC and expressed higher levels of radial glia stem cell markers than GL261 cells growing under standard conditions (GL261 adherent cells, GL261-AC), as assessed by DNA microarray and real-time PCR. GL261-NS brain gliomas were highly infiltrating and more rapidly lethal than GL261-AC, as evidenced by survival analysis (P < 0.0001), magnetic resonance imaging and histology. DC from the bone marrow of syngeneic mice were then used for immunotherapy of GL261-NS and GL261-AC tumors. Strikingly, DC loaded with GL261-NS (DC-NS) cured 80% and 60% of GL261-AC and GL261-NS tumors, respectively (P < 0.0001), whereas DC-AC cured only 50% of GL261-AC tumors (P = 0.0022) and none of the GL261-NS tumors. GL261-NS expressed higher levels of MHC and costimulatory molecules (CD80 and CD86) than GL261-AC; the JAM assay indicated that DC-NS splenocytes had higher lytic activity than DC-AC splenocytes on both GL261-NS and GL261-AC, and immunohistochemistry showed that DC-NS vaccination was associated with robust tumor infiltration by CD8+ and CD4+ T lymphocytes. These findings suggest that DC targeting of CSC provides a higher level of protection against GL261 gliomas, a finding with potential implications for the design of clinical trials based on DC vaccination. (Cancer Res 2006; 66(21): 10247-52)

PDF file - 454KB, Supplementary Figure 1: Sox2 expression in tumors analyzed by qRT-PCR. Supplementary Figure 2: SOX2 peptides are able to induce a specific anti-tumor immune response in the peripheral and local immune effector cells. Supplementary Figure 3: Sox2-deleted cells expressing differentiation markers in transplantated brain.

PDF file - 2.3MB, Figure S1: Histopathology of primary glioblastomas and genetic lesions of neurospheres. Figure S2: Genome-wide expression profiling and unsupervised hierarchical clustering of neurospheres (classical, mesenchymal and proneural centroids). Figure S3: Expression of MET, EGFR, and a panel of self-renewal markers in neurospheres. Figure S4: Genome-wide expression profiling and unsupervised hierarchical clustering of neurospheres (multiple signatures). Figure S5: Proliferative response, activation of tyrosine kinase receptors, and differentiation pattern in Met-neg-NS and Met-pos-NS. Figure S6: Methigh and Metneg sorting parameters. Figure S7: The Methigh subpopulation of Met-pos-NS retains clonogenic and tumorigenic cells. Figure S8: H&E and Met immunohistochemical staining of histological sections from tumors formed by BT302 neurosphere and its sorted subpopulations. Figure S9: HGF sustains the stem-like and invasive phenotype of Met-pos-NS.

Supplementary Methods and Figures 1-8 from Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas

XLS file - 44K, List of primer pairs used for gene sequencing

Supplementary Table S2 from Neurospheres Enriched in Cancer Stem–Like Cells Are Highly Effective in Eliciting a Dendritic Cell–Mediated Immune Response against Malignant Gliomas

PDF file - 125K

XLS file - 231KB, Excel file with the DEG genes from microarrays analysis.

Despite the first classification of glioblastoma (GBM) as an immunologically “cold” tumor, evidence suggests that this cancer type is susceptible to T-cell infiltration, providing a glimmer of hope for developing effective immunotherapy approaches. However, whether the “exhausted” phenotype of tumor-infiltrating lymphocytes (TILs) and the immunosuppressive signaling in the GBM microenvironment (ME) can be therapeutically reprogrammed to unlock the anticancer potential of the immune system remains to be elucidated. We performed a histological analysis of GBM specimens collected pre-therapy from 24 newly-diagnosed GBM patients treated with dendritic cell (DC)-immunotherapy as part of the DENDR1 clinical trial (NCT04801147). Our preliminary results suggest an association between the distribution and density of CD8+ TIL subsets and patients’ responses to immunotherapy. TIL spatial patterns revealed either an immune-excluded or an immune-infiltrated scenario with either scattered or widespread TILs. A widespread CD8+ TIL pattern was significantly correlated with a better response to immunotherapy. It was also observed that the frequency of CD8+ TILs was significantly increased at recurrence compared to their primary tumors, suggesting that these patients are more likely to benefit from CD8+ T cell antitumor immunity. In light of these observations, we have hypothesized that tumor-reactive (TR) TILs isolated from GBM can be used as direct personalized treatment mediators exerting an effective antitumor activity with low side effects per se. TR-TILs were isolated from GBM surgical material using CD137 as a key marker, and successfully expanded from 50% of patients’ GBM showing specific reactivity against autologous tumor cells. For the remaining cases, TR-TILs failed to expand, suggesting terminal dysfunction. Single-cell sequencing of the transcriptome was performed on the immune contexture after CD137+ TILs enrichment revealing the presence of a specific signature related to exhausted, cytotoxic, and memory state. A gene signature highly upregulated in regulatory T cells (Treg) was related to a more immune-suppressive ME. Flow cytometry analysis on immune contexture confirmed the presence of FoxP3+ CD4+ T cells, expressing CD137 can be enriched and persist, dysregulating the expansion of TR-TILs during the early stage in culture. Our preliminary findings indicate that a high frequency of Treg can be investigated as a negative predictive indicator of ex-vivo expansion of TR-TILs, which should be taken into consideration when planning a personalized TIL therapy. Citation Format: Irene Sambruni, Silvia Musio, Natalia Di Ianni, Martina Maffezzini, Monica Patanè, Marica Eoli, Antonio Silvani, Bianca Pollo, Francesco DiMeco, Serena Pellegatta. Single-cell analysis of glioblastoma immune contexture identifies a subset of activated and memory tumor-reactive CD8+ TILs and a Treg signature contributing to TIL irreversible dysfunction. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4694.

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.

Although some progress has been made in understanding GBM biology, treatment remains a challenge. There is increasing evidence that radiotherapy (RT) not only provides immunomodulatory effects on tumor microenvironment but also influences systemic immune response, supporting the advantage of combinatorial strategies with immunotherapy (IT). Using immune-competent mice in which syngeneic glioma cells are grown intracranially and treated with local fractionated radiation, we assessed the effects of RT on both local innate and adaptive immune cells. GL261-glioma bearing mice were locally irradiated with a total dose of 15 Gy in three consecutive fractions of 5 Gy on day 7, 8, and 9 after the tumor implantation. Irradiation was delivered both as exclusive (RT) and concomitant treatment in combination with dendritic cell (DC) immunotherapy (RT-IT). DCs were injected subcutaneously on day 16, 23, and 30 after tumor implantation. The potential role of RT in reprogramming the glioma-associated microglia is still poorly characterized. Microglia were isolated and enriched using CD11b microbeads from both the brain tumor hemisphere and contralateral hemisphere of RT, RT-IT, and control mice. A gene expression signature was analyzed on isolated microglia. Expression of mmp14 and trem2, involved in enhancing glioma proliferation, decreased in microglia isolated from RT mice (2.3 and 2.0-fold lower than in controls, day 16 P=0.01). The frequency of CD45dim/CD11b+/CD172A+ microglia showed an early increase at day 16 in the tumor mass and contralateral hemisphere in RT mice, but not in controls (p Citation Format: Serena Pellegatta, Natalia Di Ianni, Martina Maffezzini, Maria Luisa Fumagalli, Valentina Pinzi, Silvia Musio, Laura Fariselli, Gaetano Finocchiaro. Radiotherapy treatment in combination with Dendritic Cell Immunotherapy promotes a microglia activation and a disruption of the SIRPα-CD47 signaling axis in the GL261 glioma model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO087.

The existence of treatment-resistant cancer stem cells contributes to the aggressive phenotype of glioblastoma. However, the molecular alterations that drive stem cell proliferation in these tumors remain unknown. In this study, we found that expression of the MET oncogene was associated with neurospheres expressing the gene signature of mesenchymal and proneural subtypes of glioblastoma. Met expression was almost absent from neurospheres expressing the signature of the classical subtype and was mutually exclusive with amplification and expression of the EGF receptor (EGFR) gene. Met-positive and Met-negative neurospheres displayed distinct growth factor requirements, differentiated along divergent pathways, and generated tumors with distinctive features. The Methigh subpopulation within Met-pos neurospheres displayed clonogenic potential and long-term self-renewal ability in vitro and enhanced growth kinetics in vivo. In Methigh cells, the Met ligand HGF further sustained proliferation, clonogenicity, expression of self-renewal markers, migration, and invasion in vitro. Together, our findings suggest that Met is a functional marker of glioblastoma stem cells and a candidate target for identification and therapy of a subset of glioblastomas. Cancer Res; 72(17); 4537–50. ©2012 AACR.

Chimeric Antigen Receptor-redirected T cells (CAR-Ts) remain challenging for the treatment of glioblastoma (GBM) due to the heterogeneous expression of targetable antigens, which leads to antigen-loss variants. In addition, the emerging role of the GBM-derived neurospheres (GBM-NS) as a critical cell subset in causing GBM recurrence highlights the needs for targeting these cells to achieve sustained responses. By exploiting a well-established culture system, we generated and expanded GBM-NS from 23 surgical samples, and tested using flow cytometry the expression of CSPG4, an antigen found to be overexpressed in GBM by mRNA profiling. We observed that 70% of GBM-NS displayed high expression of CSPG4 (71 to 99%), 17% moderate-high expression (51-70%), and 13% moderate-low expression (less than 50%). Based on these results, we hypothesized that CAR-Ts specific for the CSPG4 antigen would represent a broadly applicable strategy for the treatment of GBM. We generated CSPG4.CAR-Ts, encoding the 4-1BB endodomain, from six healthy donors. CSPG4.CAR-Ts efficiently eliminated 19 different GBM-NS, showing high to moderate-low expression of CSPG4, in co-culture experiments at the E:T ratios ranging from 2:5 to 1:5 (0.2±0.5% and 0.6±0.9% residual GBM-NS, respectively). By contrast, GBM-NS continued to grow in the presence of control T cells (60.7±17.6% residual GBM-NS). CSPG4.CAR-Ts, but not control T cells, also rapidly proliferated in response to GBM-NS as evaluated by the CFSE assay. CSPG4.CAR-Ts showed a Th1 cytokine profile in response to GBM-NS, releasing significantly more IFN-γ (3593.8±1718.1 pg/ml/2×10⁁5 cells) and IL-2 (258.8±153.3 pg/ml/2×10⁁5 cells) than control T cells (1.8±2.5 and 0.9±1.2 pg/ml/2×10⁁5 cells, respectively). For the in vivo experiments we compared CSPG4.CAR-Ts encoding three different co-stimulatory domains, specifically CD28, 4-1BB, and CD28-4-1BB and used CAR.CD19-Ts as negative control. Two different GBM-NS with moderate-low and high expression of CSPG4 were selected and transduced to express the FFluciferase gene to monitor the tumor growth by in vivo bioluminescence imaging. Both GBM-NS and CAR-Ts were intracranially injected in 5 wks old female nude mice. CSPG4.CAR-Ts were efficient in controlling tumor growth of both moderate and high CSPG4-expressing GBM-NS. In all CAR treated mice, we observed an early eradication of the tumor mass from high-CSPG4 expressing GBM-NS, and a significant improved survival in mice bearing moderate CSPG4-expressing GBM-NS. CAR-Ts encoding the 4-1BB were significantly more efficient than those encoding CD28 or CD28-4-1BB in prolonging survival (p = 0.04). Our data suggest that CSPG4 is a promising target for CAR-Ts in GBM and opens the path for pursuing this approach in the clinical setting. Citation Format: Serena Pellegatta, Barbara Savoldo, Chuang Su, Ferrone Soldano, Gaetano Finocchiaro, Dotti Gianpietro. Condroitin sulfate proteoglycan 4 (CSPG4)- redirected T cells eliminate glioblastoma-derived neurospheres. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-249.

A critical requirement of an efficient cancer immunotherapy is the generation of long-lasting, specific memory. In a clinical trial active at Istituto Besta, first diagnosis glioblastoma (GBM) patients, with post-surgery volume ≤10 cc, underwent leukapheresis before radiotherapy and chemotherapy with the alkylating agent temozolomide (TMZ). Three intradermal injections of mature dendritic cells (DCs) loaded by autologous tumor lysates were done before adjuvant chemotherapy. Subsequent 4 injections were performed in association with six cycles of adjuvant TMZ. Peripheral blood lymphocytes (PBLs) were analyzed by flow cytometry to characterize immune response before and after DC vaccines. The ratio of vaccination/baseline frequencies and counts (V/B ratio) of all of the immunological parameters for each patient was calculated, and the median of all of the observations used as the cut off value to separate patients. V/B ratio was correlated with the progression free survival (PFS) of each patient. Preliminary results from the interim analysis on 24 patients showed that an increased NK, but not CD8+ T cell response was significantly associated with prolonged survival (p In a group of responder patients (PFS>12), during the first three vaccines, CD8+ T cells underwent a rapid expansion and produced higher levels of IFN-γ compared to the baseline (p After the third vaccine and TMZ administration, primed CD8+ T cells failed to form an effector memory phenotype (CCR7negCD45RAnegCD62Llow/neg). Our results support a possible interference of adjuvant chemotherapy on effector memory formation. Further investigations are required to understand how memory T cells behave in response to repeated exposure to TMZ. Citation Format: Serena Pellegatta, Marica Eoli, Elena Anghileri, Sara Pessina, Carlo Antozzi, Simona Frigerio, Gabriele Cantini, Maria Grazia Bruzzone, Bianca Pollo, Eugenio A. Parati, Gaetano Finocchiaro. CD8+T cells fail to form an effector memory in glioblastoma patients treated with dendritic cell immunotherapy in combination with chemotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A031.

Growing evidence suggests that chemotherapy can influence the immune response by inducing lymphopenia or enhancing immunogenicity of dying tumor cells. In a clinical trial currently active in our Institution, patients with first diagnosis of glioblastoma are treated with dendritic cells (DC) loaded with autologous tumor lysate together with standard radio and chemotherapy with temozolomide. Peripheral blood lymphocytes from 22 patients were analyzed by flow cytometry to identify immune cell activation before and after DC vaccines. The ratio of vaccine/baseline frequencies (V/B ratio) was correlated with the survival of each patient. Increased V/B ratio for NK cells was significantly associated with prolonged PFS and OS (median 15.0 vs 8.0 mo, p = 0.003; 22.0 vs 12.0 mo, p = 0.02, respectively). Using the murine malignant glioma GL261, we investigated the molecular mechanisms induced by TMZ on NK and CD8 T cells. We treated mice 9 days after intracranial implantation of GL261 cells with intraperitoneal injections of TMZ (5 mg/kg) or vehicle (control mice) for 5 days. Mice were sacrificed at different time points and tumor and peripheral blood harvested and analyzed by flow cytometry. Gene expression profiling on peripheral blood lymphocytes revealed an up-regulation of multidrug resistance genes in NK cells, but not in CD8 T lymphocytes, in TMZ-treated mice compared to controls. The increased expression of the ATP transporter gene Abcc3 was confirmed by real time PCR (4.2-fold higher than controls, P = 0.006). Using eFluxx-ID multidrug resistance (MDR) assay based on specific inhibitors, we also demonstrated that Abcc3 was functionally active during TMZ treatment. NK cell resistance to chemotherapy was accompanied by an increase in migration and homing ability into the brain at early time point and in cytotoxicity at later phases (beyond the end of TMZ treatment). Our data show that murine NK cells are resistant to and activated by TMZ chemotherapy. Further investigations in patients treated by radio-chemotherapy with or without DC immunotherapy are warranted. Note: This abstract was not presented at the meeting. Citation Format: Serena Pellegatta, Sara Pessina, Gabriele Cantini, Emanuela Cazzato, Dimos Kapetis, Gaetano Finocchiaro. Abcc3 up-regulation confers protection from chemotherapy to NK cells in a murine model of malignant glioma. [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 1343. doi:10.1158/1538-7445.AM2015-1343

Recurrent glioblastoma (GB) are highly aggressive tumors allowing 6-8 month survival. Here we evaluated the possible benefits of immunotherapy with mature dendritic cells (DC) loaded with autologous tumor lysate in 15 patients with recurrent GB. After a median follow-up of 8 months median progression free survival (PFS) was 4.4 months and median overall survival (OS) 8.0 months. Patients with small tumors at first vaccination (< 20 cc; n = 8) had significantly longer PFS and OS than others: 6.0 vs 3.0 months (p = 0.01) and 16.5 vs 7.0 months (p = 0.003), respectively. Patients were analysed for CD8+ T cells, CD56+ NK cells and other relevant immunological parameters in peripheral blood before and after immunization, defining a vaccination/baseline ratio (V/B ratio). Increased V/B ratio of NK but not CD8+ T cells was significantly associated to longer PFS and OS. Patients showing NK cell responses had higher levels of IFN-γ and E4BP4, the NK cell transcription factor. Furthermore, NK V/B ratio was inversely correlated with TGF-β2 and VEGF V/B ratio. The results suggest that tumor-loaded DC may increase survival of recurrent GB after effective tumor debulking and emphasize the role of NK cell responses in this therapeutic setting. Citation Format: Serena Pellegatta, Marica Eoli, Simona Frigerio, Carlo Antozzi, Maria Grazia Bruzzone, Gabriele Cantini, Sara Nava, Elena Anghileri, Lucia Cuppini, Valeria Cuccarini, Emilio Ciusani, Marta Dossena, Bianca Pollo, Renato Mantegazza, Eugenio A. Parati, Gaetano Finocchiaro. NK cell response and tumor debulking are associated to prolonged survival in recurrent glioblastoma treated by dendritic cells loaded with autologous tumor lysate. [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 2839. doi:10.1158/1538-7445.AM2013-2839 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Chemotherapy is currently regarded as a potential ally for cancer immunotherapy. Several anticancer agents, including classical chemotherapeutic compounds, are able to enforce tumor specific immune responses either by inducing the immunogenic death of tumor cells or by modulating key cells for immune suppression or activation. To determine whether Temozolomide (TMZ), the standard chemotherapeutic agent for glioblastoma and malignant gliomas, could induce tumor immunogenicity, we treated murine GL261 cells with 50 and 150 microM TMZ in vitro for 2, 8 and 20 hours. Flow cytometry showed that the NKG2D ligand, involved in NKG2D-mediated NK cell recognition of tumor cells, was highly expressed by TMZ-treated GL261, whereas GL261 treated with vehicle showed only a weak expression (P < 0.01). B7-H3, an inhibitory molecule for NK cells, significantly decreased in TMZ-treated GL261 cells (P < 0.01). TGF-beta1 and TGF-beta2 concentrations in the supernatant from GL261 treated with TMZ significantly decreased, as shown by ELISA (from 23.2 ± 2.5 and 478.4 ± 19.3 in vehicle to 13.2 ± 2.2 and 283.8 ± 25.9 in TMZ-treated cells, respectively, P < 0.0001). We therefore studied the effects of TMZ on anti-tumor NK cell response by treating mice 7 days after intracranial implantation of GL261 gliomas with intraperitoneal injections of TMZ (5mg/kg) or vehicle (control mice) for 5 days. Five mice were sacrificed 20 hours after treatment on days 1-5: brain, spleen and blood were harvested and analyzed by flow cytometry. Trafficking of NKp46+ NK1.1+ CD3- NK cells in blood but not in spleen and their homing ability into the brain significantly increased in TMZ-treated compared to control mice after the second administration of TMZ (7.5 ± 1.4 vs 2.3 ± 1.2, P = 0.001; 10.9 ± 0.4 vs. 3.9 ± 0.6, P = 0.005, respectively). Notably TMZ led to an enrichment of CD11bhigh CD27high NK cells, the most potent effector cells. To verify NK anti-glioma activity, NK1.1 positive cells were isolated from blood and brain of treated and control mice using magnetic separation and incubated with GL261 cells. NK cell cytotoxicity from TMZ-treated mice was significantly higher than that NK cells from control mice. These results support the contention that chemotherapy may induce enhancement of NK cell response and open new opportunities to design novel combined therapies reversing the immune suppressive role of glioblastoma and unmasking the therapeutic potential of NK responses. Citation Format: Serena Pellegatta, Gabriele Cantini, Sara Pessina, Gaetano Finocchiaro. NK cell activation and cytotoxicity can be enhanced with chemotherapy in a murine model of glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A60.

The identification of antigens preferentially expressed in glioblastoma (GBM) and involved in its malignant phenotype is critical for for developing therapeutic strategies. We observed by DNA microarray analysis and confirmed by western blot and immunocytochemistry that the neural stem cells marker GLAST (Glutamate Aspartate Transporter), highly expressed in cells of the radial glia, is also expressed in the plasma membrane of murine and human GBM stem-like cells (GSC). GLAST is a membrane protein expressed by astrocytes with a relevant role in glutamate uptake. When we evaluated the uptake in different GSC lines we found that their glutamate uptake is up to 100 fold lower than in normal astrocytes. GSC are able to release glutamate in culture medium just as hypoxic astrocytes, showing that in pathological conditions such as ischemia, transporters can reverse uptake and release glutamate. We have used GLAST as a marker to isolate a GSC subpopulation by immunomagnetic sorting and test the capacity of GLAST-enriched cells for tumor formation in mice. Using the murine glioma model GL261, GLAST+ cells injected intracranially were significantly more tumorigenic than GLAST- or unsorted cells (p=0.00057 and p=0.00028, respectively). We confirmed these observations in nude mice using GSC from one human recurrent GBM. Histological analysis provided evidence of the invasive nature of GLAST+ GSC asin the contralateral hemisphere many tumor cellsexpress GLAST and the migration marker Double-Cortin. To evaluate the functional role of GLAST in GSC we decided to target GLAST expression using lentiviral particles expressing GLAST shRNA. GLAST inhibition impacts on cell proliferation and especially on Matrigel invasion in vitro and interferes with tumor progression, improving survival in injected nude mice (p = 0.0034 shGLAST vs Scrambled). As a consequence of GLAST interference we also found in vitro a significant reduction of glutamate release in silenced cells compared to control cells(non specific shRNA). We investigated the expression of GLAST in 60 human primary GBM and 15 low grade gliomas (LGG) by immunohistochemistry. We found marked expression of GLAST in the large majority of GBM, in contrast LGG were negative or showed nuclear expression of GLAST. When we correlated GLAST expression to overall survival of 51 GBM patients we found that high percentage of GLAST pos cells and moderate or strong reactivity correlate with decreased overall survival (p=0.02). Our data suggest that GLAST is expressed in GBM and may be involved in proliferation and invasion. In addition GLAST could be investigated as clinical marker in association with prognosis of GBM patients and for development of novel therapeutic strategies. 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 3403. doi:10.1158/1538-7445.AM2011-3403

Malignant brain tumours are among the most deadly cancers. Current treatment strategies, such as surgery, chemotherapy and radiotherapy only modestly improve patient survival. The limited success of these strategies is largely due to a high incidence of tumor recurrence after the treatment. Recent evidences suggested that a rare population of stem-like cells (or tumour initiating cells) present in brain tumours might be responsible for the aggressiveness and recurrence of these tumours. These cells, named brain tumor stem-like cells (BTSC), share many properties of normal neural stem cells, including self renewal, extended proliferation, formation of neurospheres (when cultured in vitro in the presence of growth factors) and potential to differentiate into neurons and glial cells. Further investigations have suggested that studying this BTSC population in glioblastoma (GBM) is a more relevant system for exploring glioma biology than the adherently growing glioma cell lines. In the current study we identified target genes and proteins that are differently expressed between adherent glioma cell lines and sphere growing BTSC, using microarray and differential proteomics, respectively. Fatty acid-binding protein 7 (FABP7) was identified as overexpressed in BTSC compared to glioma adherent cell lines both at the gene as well as at the protein level. FABP proteins are a family of small, highly conserved, cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. FABPs are thought to play important roles in fatty acid uptake, transport, and metabolism. Functionally, FABPS were described to play a role in gene regulation, cell signaling, cell growth and differentiation. The functional role of FABP7 in glioblastoma was investigated by assessing the effect of silencing FABP7 on migration and cell proliferation of glioblastoma cells. We observed that down-regulating FABP7 expression significantly reduced in vitro cell proliferation and even more the migration of BTSC. Since increased tumour infiltration was reported as a consequence of radiotherapy, the potential involvement of FABP7 in this process was evaluated. Irradiated BTSCs displayed significantly higher FABP7 protein levels and therefore we suggest that FABP7 may contribute to the radiotherapy-induced aggressive phenotype of BTSCs. Moreover, further mechanistic investigations indicated that directly or indirectly reducing the expression of FABP7 significantly impacts on the behaviour of glioblastoma derived BTSC. 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 5148.

IL-17, produced by Th17 cells, may play a dual role in antitumor immunity. Using the GL261 murine glioma model, we have investigated how Th17 cells can affect tumor growth and microenvironment. We first obtained TIL from fresh GL261-glioma 10 and 20 days after tumor implantation, and evaluated by flow cytometry infiltrating-Th17 as a percentage of total CD4+ cells. Th17 increased from 7.9±1.9 on day 10 to 13.1±5.2 on day 20 (p=0.03). To characterize the direct effects of Th17 cells on GL261-glioma and on tumor microenvironment, we isolated IL-17 producing cells enriched using Miltenyi technology from splenocytes derived from naïve or glioma-bearing mice (Gb) and pre-stimulated in vitro with or without TGF-ß. Th17 derived from naïve (nTh17) or Gb splenocytes (GbTh17) were co-injected subcutaneously with GL261 cells in immune-competent mice. Gliomas in mice co-injected with nTh17 were smaller than gliomas obtained from GL261 cells only (34±39.5mm3 vs 235.3±22.0mm3 respectively, p=0.003). In these gliomas, using RT-PCR we also found enhanced expression in IFN- and decreased expression of IL-10, TGF- and VEGF. Opposite results were obtained in gliomas of mice co-injected with Gb-Th17 (866±627 mm3, p=0.01 vs GL261, p By MTT assay and kinetic proliferation we found that IL-17 per se did not exert effects on GL261 proliferation. Because Th17 may differentiate under the influence of TGF-ß as well as regulatory T cells (Treg), we characterized both Treg and Th17. Treg were significantly higher in splenocytes from Gb than naïve mice (19.8±2.8% vs 3.5±0.05% respectively, p=0.01). nTh17 produced higher levels of IFN- than IL-10 (9.7±0.7% vs 2.2± 1.5, respectively, p=0.01) suggesting a suppressive function on tumor microenvironment. In contrast, GbTh17 produced more IL-10 than IFN- (9.3±1.4% vs 1.6±0.7%, respectively, p=0.02), suggesting a promoting function on tumor growth. To determine the potential influence of Treg in driving Th17 cytokine profile, using PC61 we depleted the Treg fraction in Gb splenocytes from 19.5±2.0% to 1.3±0.6% (p Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 LB-315.