Gabriele Cantini

The natural killer cell response and tumor debulking are associated with prolonged survival in recurrent glioblastoma patients receiving dendritic cells loaded with autologous tumor lysates.

Recurrent glioblastomas (GBs) are highly aggressive tumors associated with a 6–8 mo survival rate. In this study, we evaluated the possible benefits of an immunotherapeutic strategy based on mature dendritic cells (DCs) loaded with autologous tumor-cell lysates in 15 patients affected by recurrent GB. The median progression-free survival (PFS) of this patient cohort was 4.4 mo, and the median overall survival (OS) was 8.0 mo. Patients with small tumors at the time of the first vaccination (< 20 cm3; n = 8) had significantly longer PFS and OS than the other patients (6.0 vs. 3.0 mo, p = 0.01; and 16.5 vs. 7.0 mo, p = 0.003, respectively). CD8+ T cells, CD56+ natural killer (NK) cells and other immune parameters, such as the levels of transforming growth factor β, vascular endothelial growth factor, interleukin-12 and interferon γ (IFNγ), were measured in the peripheral blood and serum of patients before and after immunization, which enabled us to obtain a vaccination/baseline ratio (V/B ratio). An increased V/B ratio for NK cells, but not CD8+ T cells, was significantly associated with prolonged PFS and OS. Patients exhibiting NK-cell responses were characterized by high levels of circulating IFNγ and E4BP4, an NK-cell transcription factor. Furthermore, the NK cell V/B ratio was inversely correlated with the TGFβ2 and VEGF V/B ratios. These results suggest that tumor-loaded DCs may increase the survival rate of patients with recurrent GB after effective tumor debulking, and emphasize the role of the NK-cell response in this therapeutic setting.

Immunotherapy against the radial glia marker GLAST effectively triggers specific antitumor effectors without autoimmunity

The glutamate-aspartate transporter GLAST is a radial glia marker that is highly expressed in GL261 stem-like cells (GSCs). To target GLAST, we treated glioma-bearing mice with three subcutaneous injections of four GLAST peptides emulsified with Montanide ISA-51 in association with granulocyte macrophage colony-stimulating factor (GM-CSF) injections. Vaccination with GLAST peptides significantly prolonged survival, effectively enhanced systemic T-cell and NK-cell responses and promoted robust antitumor cytotoxicity. GLAST expression significantly decreased in gliomas from immunized mice, as evaluated by histological analysis and real-time PCR (RT-PCR). Moreover, the immunization protocol led to the upregulation of interferonγ (IFNγ) and tumor necrosis factorα (TNFα) as well as to the downregulation of transforming growth factor (TGF) β1 and β2 in the tumor. Beyond these changes, gliomas from immunized mice exhibited an increased recruitment of NK cells and antigen-specific CD8+ T cells expressing the tumor homing molecule VLA-4, as well as a local chemotactic gradient featuring expression of CXCL10 (which may be responsible for the recruitment of CTLs), CCL3, CCL4 and CCL5 (which are involved in NK-cell migration), and NKG2D ligand on glioma cells. Importantly, although GLAST is expressed in the central nervous system, autoimmune reactions were not observed in immunized mice. Altogether, these results support the contention that GLAST may constitute a glioma antigen against which immune responses can be efficiently induced without major safety concerns.

Frequency of NFKBIA deletions is low in glioblastomas and skewed in glioblastoma neurospheres.

The NF-kB family of transcription factors is up-regulated in inflammation and different cancers. Recent data described heterozygous deletions of the NF-kB Inhibitor alpha gene (NFKBIA) in about 20% of glioblastomas (GBM): deletions were mutually exclusive with epidermal growth factor receptor (EGFR) amplification, a frequent event in GBM. We assessed the status of NFKBIA and EGFR in 69 primary GBMs and in corresponding neurospheres (NS). NFKBIA deletion was investigated by the copy number variation assay (CNV); EGFR amplification by CNV ratio with HGF; expression of EGFR and EGFRvIII by quantitative PCR or ReverseTranscriptase PCR. Heterozygous deletions of NFKBIA were present in 3 of 69 primary GBMs and, surprisingly, in 30 of 69 NS. EGFR amplification was detected in 36 GBMs: in corresponding NS, amplification was lost in 13 cases and reduced in 23 (10 vs 47 folds in NS vs primary tumors; p < 0.001). The CNV assay was validated investigating HPRT1 on chromosome X in females and males. Results of array-CGH performed on 3 primary GBMs and 1 NS line were compatible with the CNV assay. NS cells with NFKBIA deletion had increased nuclear activity of p65 (RelA) and increased expression of the NF-kB target IL-6. In absence of EGF in the medium, EGFR amplification was more conserved and NFKBIA deletion less frequent point to a low frequency of NFKBIA deletions in GBM and suggest that EGF in the culture medium of NS may affect frequency not only of EGFR amplifications but also of NFKBIA deletions.

The multidrug-resistance transporter Abcc3 protects NK cells from chemotherapy in a murine model of malignant glioma

ABSTRACT Abcc3, a member of the ATP-binding cassette transporter superfamily, plays a role in multidrug resistance. Here, we found that Abcc3 is highly expressed in blood-derived NK cells but not in CD8+ T cells. In GL261 glioma-bearing mice treated with the alkylating agent temozolomide (TMZ) for 5 d, an early increased frequency of NK cells was observed. We also found that Abcc3 is strongly upregulated and functionally active in NK cells from mice treated with TMZ compared to controls. We demonstrate that Abcc3 is critical for NK cell survival during TMZ administration; more importantly, Akt, involved in lymphocyte survival, is phosphorylated only in NK cells expressing Abcc3. The resistance of NK cells to chemotherapy was accompanied by increased migration and homing in the brain at early time points. Cytotoxicity, evaluated by IFNγ production and specific lytic activity against GL261 cells, increased peripherally in the later phases, after conclusion of TMZ treatment. Intra-tumor increase of the NK effector subset as well as in IFNγ, granzymes and perforin-1 expression, were found early and persisted over time, correlating with a profound modulation on glioma microenvironment induced by TMZ. Our findings reveal an important involvement of Abcc3 in NK cell resistance to chemotherapy and have important clinical implications for patients treated with chemo-immunotherapy.

Abstract A031: CD8+T cells fail to form an effector memory in glioblastoma patients treated with dendritic cell immunotherapy in combination with chemotherapy

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.

Altered function of the glutamate aspartate transporter GLAST in glioblastoma

In glioma patients, high levels of glutamate can cause brain edema and seizures. GLAST, a glutamate-aspartate transporter expressed by astrocytes with a role in glutamate uptake, is highly expressed on the plasma membrane of glioblastoma (GBM) cells, and its expression significantly correlates with shortened patient survival. Here, it was demonstrated that inhibition of GLAST expression limited the progression and invasion of GBM xenografts. Magnetic resonance spectroscopy was used to measure glutamate in GLAST-expressing gliomas showing that these tumors exhibit increased glutamate concentration. Despite their GLAST expression, GBM stem-like cells (GSCs) released rather than took up glutamate due to their lack of Na+/K+-ATPase. Overexpression of Na+/K+-ATPase in these cells restored glutamate uptake and induced apoptosis. The therapeutic relevance of targeting GLAST in gliomas was assessed using the inhibitor UCPH-101. In glioma-bearing mice, a single intratumoral injection of UCPH-101 significantly increased survival by decreasing GLAST expression and inducing apoptosis. Thus, GLAST has a novel role in GBM that appears to have crucial relevance in glutamate trafficking and may thus be a new therapeutic target.

Abstract 1343: Abcc3 up-regulation confers protection from chemotherapy to NK cells in a murine model of malignant glioma

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

P03.08IMPROVED YIELD OF STEM-LIKE CELLS USING GLIOBLASTOMA SUSPENSIONS FROM CAVITRON ULTRASONIC SURGERY ASPIRATOR (CUSA) BAGS AS STARTING MATERIAL

Absence of serum and presence of growth factors (mostly EGF and bFGF) has been used to culture cancer stem cells (CSC). In case of GB the term neurospheres (NS) has been considered as synonymous of glioma stem-like cells (GSCs). In our laboratories we isolate and culture GSC from fresh GB specimens growing in the absence of serum and in the presence of EGF and bFGF as neurospheres after mechanical and enzymatic dissociation. Previous data and our experience suggest that NS may mirror more closely than previous, serum-based glioma cell lines, the actual biology of GB. Tumor fragments were used to start the culture. The Cavitation Ultrasonics Surgical Aspirator (CUSA) is increasingly used for GB surgery, decreasing the availability of tumor fragments. CUSA delivers an irrigating solution that converts the fragmented tissue into an emulsion and then aspirates the particles directly into a sterile bag. The bag contains some larger pieces of tissue, debris and high amounts of erythrocytes, and occasionally necrotic or reactive tissue. Here we describe our experience with CUSA bags as the staring material for GSC preparation. Tissue fragments in CUSA bags after several rounds of spinning ad washing are dissociated using the GentleMacs Dissociator (Miltenyi Biotec) that provides a closed system and reproducible results. We have optimized a gentle and effective protocol starting from appropriate gentleMACS programs, allowing to obtain a high yield of viable tumor cells. After processing, cell suspensions are cultured as neurospheres in DMEM/F12, B27 supplement, human recombinant b-FGF and EGF. During the last year we obtained from the Department of Neurosurgery of Istituto Besta a total of 54 CUSA. Primary GB were the most represented brain tumors (87%). Using tumor fragments obtained from surgery and combining mechanical dissociation with enzymatic disaggregation on a series of primary GB we previously obtained GSC in 52% of the cases. This percentage is now increased to 71% with the use of surgical material from CUSA and the optimized protocol. Proliferation kinetics was studied by plating three primary cell lines obtained in parallel from GB specimen and from CUSA material at density of 15,000-30,000 cells/cm2. Cultures were collected every 5 days and the total number of viable cells assessed at each passage by Trypan Blue exclusion. A long term proliferation of cell lines at low sub-culturing stages (3 to 10 passages), showed that proliferation increased exponentially, but the proliferation index of NS from CUSA materials was higher that cells from specimens (1.32 vs 1.18 respectively, p = 0.03). These improvements and the use of a closed system providing a wider margin of safety support the incorporation of this process in a clinical trial protocol which plans to use GSC as a source of antigens for dendritic cell loading, replacing the whole lysate from GB specimens used in current immunotherapy protocols.

Abstract 2839: NK cell response and tumor debulking are associated to prolonged survival in recurrent glioblastoma treated by dendritic cells loaded with autologous tumor lysate.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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.

Abstract A60: NK cell activation and cytotoxicity can be enhanced with chemotherapy in a murine model of glioblastoma.

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 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.