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.

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) 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.

Dendritic cells (DCs) are phagocytic innate immune cells specialized in antigen acquisition, processing, and presentation. The high expression of both major histocompatibility I and II complexes (M...

Tumor infiltration by dendritic cells (DCs) has been identified as a biomarker associated with favorable outcomes and improved responses to immunotherapies by T cell immune checkpoint blockade. However, tumor induced immunosuppressive pathways contrive efficient DC infiltration. This calls for the development of DC-targeted interventions purposed to stimulate DC infiltration and local expansion. Here we deliver a proof-of-concept for a cell-based immunotherapeutic approach promoting circulating DC progenitors infiltration in the tumor together with their local expansion. We report that engraftment of autologous mesenchymal stromal cells engineered to express the membrane bound form of FLT3L (eMSC-FLT3L) uniquely induce DC infiltration, T cells activation and anti-tumor immunity, when combined with an adjuvant like the poly(I:C) double stranded RNA mimic. Mechanistic experiments demonstrate that poly(I:C)-induced CXCR3 and CCR5 ligands (CXCL9, CCL5) are required for intra-tumoral DC and T cell recruitment and for the therapeutic effect of eMSC-FLT3L + poly(I:C) immunotherapy. Stromal cell-based delivery of CXCL9 and CCL5, together with FLT3L, by-passes the requirement for poly(I:C) agonist and is sufficient to induce T cell and DC recruitment within tumors. These findings are transferrable to human settings as we show that eMSCs-FLT3L-CXCL9-CCL5 stimulate tissue infiltration by human XCR1⁺ DC1s in mice harboring a ‘Human Immune System’ (HIS mice). Lastly, we report that engraftment of eMSC-FLT3L-CXCL9-CCL5 in the tumor induces anti-tumor immunity and partially overcomes resistance to immune checkpoint blockade. Altogether, these data support the therapeutic potential of expanding intra-tumoral DC1s using a continuous and local source of FLT3L, CXCL9 and CCL5. One sentence summary Intra-tumoral delivery of FLT3L synergizes with CXCL9 and CCL5 to activate the expansion of intra-tumoral DCs.