The microenvironment (ME) plays a critical role in causing glioblastoma (GBM) to be a moving and incurable target. The main features governing the interaction between cancer cells and the ME include dependency, promotion, and in rare cases, even competition. In the original Stupp protocol, the alkylating agent temozolomide (TMZ) is the first-line chemotherapy drug to treat GBM, and it is broadly used together or after radiotherapy. Some studies have described TMZ as an adjuvant to other therapeutic approaches including immunotherapy because of its ability to induce an immunogenic death of cancer cells. TMZ also exerts immunomodulatory effects on the tumor and immune ME. These findings support the coexistence of two circuits, i.e., one that subverts local immunosuppressive mechanisms and another that exerts a harmful influence on the peripheral immune response. A bias toward the latter can drive the failure of treatments based on the combination of chemotherapy and immunotherapy approaches. In this review, we will reanalyze how intrinsic and acquired resistance to TMZ impacts the immunomodulatory effects previously described by way of inducing a functional alteration of local immune cells and promoting immunosuppression and how different components of the immune ME, with particular attention to tumor-associated macrophages and microglia, can cause TMZ resistance to circumvent potential local immunogenic mechanisms.
The interpretation of the presence and function of immune infiltration in glioblastoma (GBM) is still debated. Over the years, GBM has been considered a cold tumor that is less infiltrated by effector cells and characterized by a high proportion of immunosuppressive innate immune cells, including GBM-associated microglia/macrophages (GAMs). In this context, the failure of checkpoint inhibitors, particularly in recurrent GBM (rGBM), caused us to look beyond the clinical results and consider the point of view of immune cells. The tumor microenvironment in rGBM can be particularly hostile, even when exposed to standard immunomodulatory therapies, and tumor-infiltrating lymphocytes (TILs), when present, are either dysfunctional or terminally exhausted. However, after checkpoint blockade therapy, it was possible to observe specific recruitment of adaptive immune cells and an efficient systemic immune response. In this review article, we attempt to address current knowledge regarding the tumor and immune microenvironment in rGBM. Furthermore, immunosuppression induced by GAMs and TIL dysfunction was revisited to account for genetic defects that can determine resistance to therapies and manipulate the immune microenvironment upon recurrence. Accordingly, we reevaluated the microenvironment of some of our rGBM patients treated with dendritic cell immunotherapy, with the goal of identifying predictive immune indicators of better treatment response.
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<0.01). At later stages, the percentage of activated microglia was similar in all groups, including RT-IT mice. SIRPa can interact with CD47 on tumor cells inducing a “don’t me” signal and preventing phagocytosis. The percentage of CD47+ cells, investigated by flow cytometry, was high in gliomas from control mice (62.0±3.6%), and significantly decreased in gliomas from RT and RT-IT mice (14.9±2.2% and 9.3±0.8%, respectively, P<0.005 vs controls). In RT-IT gliomas the CD47 decreased expression correlated with a robust infiltration of CD8+ T cells (65.7±3.2% vs. 22.3±0.8 in RT, 16.9±1.3% in controls, P<0.005). Glioma-bearing mice treated with the combination of RT and IT survived longer than RT mice or controls (median of survival: 60 vs 19.5 vs 17 days respectively, P<0.004). These preliminary results suggest a key role of radiotherapy in activating microglia and favoring an anti-tumor response induced by dendritic cells. The depletion of the CD47 expression encourages us to investigate the potential role of radiotherapy in re-educating microglia to phagocytize glioma cells. 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.
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.
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