Glioblastoma (GBM) is an aggressive brain tumor, with a highly immunosuppressive tumor immune microenvironment (TIME). In this work, we investigated the use of the STimulator of INterferon Genes (STING) pathway as an effective means to remodel the GBM TIME through the recruitment of both innate and adaptive immune cell populations. Using hyaluronic acid (HA), we developed a novel polymer-drug conjugate of a non-nucleotide STING agonist (MSA2), called HA-MSA2 for the in situ treatment of GBM. In JAWSII cells, HA-MSA2 exerted a greater increase of STING signaling and upregulation of STING-related downstream cyto-/chemokines in immune cells than the free drug. HA-MSA2 also elicited cancer cell-intrinsic immunostimulatory gene expression and promoted immunogenic cell death of GBM cells. In the SB28 GBM model, local delivery of HA-MSA2 induced a delay in tumor growth and a significant extension of survival. The analysis of the TIME showed a profound shift in the GBM immune landscape after HA-MSA2 treatment, with higher infiltration by innate and adaptive immune cells including dendritic, natural killer (NK) and CD8 T cell populations. The therapeutic potential of this novel polymer conjugate warrants further investigation, particularly with other chemo-immunotherapeutics or cancer vaccines as a promising combinatorial therapeutic approach.

Immunotherapy of advanced melanoma has encountered significant hurdles in terms of clinical efficacy. Here, we designed a clinically translatable hyaluronic acid (HA)-based vaccine delivering a combination of major histocompatibility complex (MHC) class I- and class II-restricted melanoma antigens (TRP2 and Gp100, respectively) conjugated to HA. HA-nanovaccine (HA-TRP2-Gp100 conjugate) exhibited tropism in the lymph nodes and promoted stimulation of the immune response (2.3-fold higher than the HA+TRP2+Gp100). HA-nanovaccine significantly delayed the growth of B16F10 melanoma and extended survival in both the prophylactic and therapeutic settings (median survival of 22 and 27, respectively, vs 17 days of the untreated group). Moreover, mice prophylactically treated with the HA-nanovaccine displayed significantly higher CD8+ and CD4+ T-cell/Treg ratios in both the spleen and tumor at day 16, suggesting that the HA-nanovaccine overcame the immunosuppressive tumor microenvironment. Superior infiltration of active CD4+ and CD8+ T cells was observed at the endpoint. This study supports the conclusion that HA potentiates the effect of a combination of MHC I and MHC II antigens via a potent immune response against melanoma.

Immunotherapy efficacy as monotherapy is negligible for glioblastoma (GBM). We hypothesized that combining therapeutic vaccination using a plasmid encoding an epitope derived from GBM-associated antigen (pTOP) with local delivery of immunogenic chemotherapy using mitoxantrone-loaded PEGylated PLGA-based nanoparticles (NP-MTX) would improve the survival of GBM-bearing mice by stimulating an antitumor immune response. We first proved that MTX retained its ability to induce cytotoxicity and immunogenic cell death of GBM cells after encapsulation. Intratumoral delivery of MTX or NP-MTX increased the frequency of IFN-γ-secreting CD8 T cells. NP-MTX mixed with free MTX in combination with pTOP DNA vaccine increased the median survival of GL261-bearing mice and increased M1-like macrophages in the brain. The addition of CpG to this combination abolished the survival benefit but led to increased M1 to M2 macrophage ratio and IFN-γ-secreting CD4 T cell frequency. These results highlight the benefits of combination strategies to potentiate immunotherapy and improve GBM outcome.

Orthotopic glioblastoma xenografts are paramount for evaluating the effect of innovative anti-cancer treatments. In longitudinal studies, tumor growth (or regression) of glioblastoma can only be monitored by noninvasive imaging. For this purpose, bioluminescence imaging (BLI) has gained popularity because of its low cost and easy access. In the context of the development of new nanomedicines for treating glioblastoma, we were using luciferase-expressing GL261 cell lines. Incidentally, using BLI in a specific GL261 glioblastoma model with cells expressing both luciferase and the green fluorescent protein (GL261-luc-GFP), we observed an apparent spontaneous regression. By contrast, the magnetic resonance imaging (MRI) analysis revealed that the tumors were actually growing over time. For other models (GL261 expressing only luciferase and U87 expressing both luciferase and GFP), data from BLI and MRI correlated well. We found that the divergence in results coming from different imaging modalities was not due to the tumor localization nor the penetration depth of light but was rather linked to the instability in luciferase expression in the viral construct used for the GL261-luc-GFP model. In conclusion, the use of multi-modality imaging prevents possible errors in tumor growth evaluation, and checking the stability of luciferase expression is mandatory when using BLI as the sole imaging modality.

Glioblastoma (GBM) treatment has remained almost unchanged for more than 20 years. The current standard of care involves surgical resection (if possible) followed by concomitant radiotherapy and chemotherapy. In recent years, immunotherapy strategies have revolutionized the treatment of many cancers, increasing the hope for GBM therapy. However, mostly due to the high, multifactorial immunosuppression occurring in the microenvironment, the poor knowledge of the neuroimmune system and the presence of the blood−brain barrier, the efficacy of immunotherapy in GBM is still low. Recently, new strategies for GBM treatments have employed immunotherapy combinations and have provided encouraging results in both preclinical and clinical studies. The lessons learned from clinical trials highlight the importance of tackling different arms of immunity. In this review, we aim to summarize the preclinical evidence regarding combination immunotherapy in terms of immune and survival benefits for GBM management. The outcomes of recent studies assessing the combination of different classes of immunotherapeutic agents (e.g., immune checkpoint blockade and vaccines) will be discussed. Finally, future strategies to ameliorate the efficacy of immunotherapy and facilitate clinical translation will be provided to address the unmet medical needs of GBM.

Glioblastoma is an unmet clinical need. Local treatment strategies offer advantages, such as the possibility to bypass the blood–brain barrier, achieving high drug concentrations at the glioblastoma site, and consequently reducing systemic toxicity. In this study, we evaluated the feasibility of using hyaluronic acid (HA) for the local treatment of glioblastoma. HA was conjugated to doxorubicin (DOX) with distinct bio-responsive linkers (direct amide conjugation HA-NH-DOX), direct hydrazone conjugation (HA-Hz-DOX), and adipic hydrazone (HA-AdpHz-DOX). All HA-DOX conjugates displayed a small size (less than 30 nm), suitable for brain diffusion. HA-Hz-DOX showed the best performance in killing GBM cells in both 2D and 3D in vitro models and displayed superior activity in a subcutaneous GL261 tumor model in vivo compared to free DOX and other HA-DOX conjugates. Altogether, these results demonstrate the feasibility of HA as a polymeric platform for the local treatment of glioblastoma and the importance of rationally designing conjugates.

Combination immunotherapy has emerged as a promising strategy to increase the immune response in glioblastoma (GBM) and overcome the complex immunosuppression occurring in its microenvironment. In this study, we hypothesized that combining DNA vaccines-to stimulate a specific immune response-and dual immune checkpoint blockade (ICB)-to decrease the immunosuppression exerted on T cells-will improve the immune response and the survival in an orthotopic unresectable GL261 model. We first highlighted the influence of the insertion position of a GBM epitope sequence in a plasmid DNA vaccine encoding a vesicular stomatitis virus glycoprotein (VSV-G) (here referred to as pTOP) in the generation of a specific and significant IFN-γ response against the GBM antigen TRP2 by inserting a CD8 epitope sequence in specific permissive sites. Then, we combined the pTOP vaccine with anti-PD-1 and anti-CTLA-4 ICBs. Immune cell analysis revealed an increase in effector T cell to Treg ratios in the spleens and an increase in infiltrated IFN-γ-secreting CD8 T cell frequency in the brains following combination therapy. Even if the survival was not significantly different between dual ICB and combination therapy, we offer a new immunotherapeutic perspective by improving the immune landscape in an orthotopic unresectable GBM model.

BackgroundStrategies to increase nucleic acid vaccine immunogenicity are needed to move towards clinical applications in oncology. In this study, we designed a new generation of DNA vaccines, encoding an engineered vesicular stomatitis virus glycoprotein as a carrier of foreign T cell tumor epitopes (plasmid to deliver T cell epitopes, pTOP). We hypothesized that pTOP could activate a more potent response compared with the traditional DNA-based immunotherapies, due to both the innate immune properties of the viral protein and the specific induction of CD4 and CD8 T cells targeting tumor antigens. This could improve the outcome in different tumor models, especially when the DNA-based immunotherapy is combined with a rational therapeutic strategy.MethodsThe ability of pTOP DNA vaccine to activate a specific CD4 and CD8 response and the antitumor efficacy were tested in a B16F10-OVA melanoma (subcutaneous model) and GL261 glioblastoma (subcutaneous and orthotopic models).ResultsIn B16F10-OVA melanoma, pTOP promoted immune recognition by adequate processing of both MHC-I and MHC-II epitopes and had a higher antigen-specific cytotoxic T cell (CTL) killing activity. In a GL261 orthotopic glioblastoma, pTOP immunization prior to tumor debulking resulted in 78% durable remission and long-term survival and induced a decrease of the number of immunosuppressive cells and an increase of immunologically active CTLs in the brain. The combination of pTOP with immune checkpoint blockade or with tumor resection improved the survival of mice bearing, a subcutaneous melanoma or an orthotopic glioblastoma, respectively.ConclusionsIn this work, we showed that pTOP plasmids encoding an engineered vesicular stomatitis virus glycoprotein, and containing various foreign T cell tumor epitopes, successfully triggered innate immunity and effectively promoted immune recognition by adequate processing of both MHC-I and MHC-II epitopes. These results highlight the potential of DNA-based immunotherapies coding for viral proteins to induce potent and specific antitumor responses.

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.