In situ administration of STING-activating hyaluronic acid conjugate primes anti-glioblastoma immune response.

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.

Local delivery of doxorubicin prodrug via lipid nanocapsule-based hydrogel for the treatment of glioblastoma.

Glioblastoma (GBM) recurrences appear in most cases around the resection cavity borders and arise from residual GBM cells that cannot be removed by surgery. Here, we propose a novel treatment that combines the advantages of nanomedicine and local drug delivery to target these infiltrating GBM cells. We developed an injectable lipid nanocapsule (LNC)-based formulation loaded with lauroyl-doxorubicin prodrug (DOXC12). Firstly, we demonstrated the efficacy of intratumoral administration of DOXC12 in GL261 GBM-bearing mice, which extended mouse survival. Then, we formulated an injectable hydrogel by mixing the appropriate amount of prodrug with the lipophilic components of LNC. We optimized the hydrogel by incorporating cytidine-C16 (CytC16) to achieve a mechanical stiffness adapted for an application in the brain post-surgery (DOXC12-LNCCL). DOXC12-LNCCL exhibited high DOXC12 encapsulation efficiency (95%) and a size of approximately 60 nm with sustained drug release for over 1 month in vitro. DOXC12-LNCCL exhibited enhanced cytotoxicity compared to free DOXC12 (IC50 of 349 and 86 nM, respectively) on GL261 GBM cells and prevented the growth of GL261 spheroids cultured on organotypic brain slices. In vivo, post-surgical treatment with DOXC12-LNCCL significantly improved the survival of GL261-bearing mice. The combination of this local treatment with the systemic administration of anti-inflammatory drug ibuprofen further delayed the onset of recurrences. In conclusion, our study presents a promising therapeutic approach for the treatment of GBM. By targeting residual GBM cells and reducing the inflammation post-surgery, we present a new strategy to delay the onset of recurrences in the gap period between surgery and standard of care therapy.

Improvement of Lipid Detection in Mouse Brain and Human Uterine Tissue Sections Using In Situ Matrix Enhanced Secondary Ion Mass Spectrometry.

The potential of mass spectrometry imaging, and especially ToF-SIMS 2D and 3D imaging, for submicrometer-scale, label-free molecular localization in biological tissues is undisputable. Nevertheless, sensitivity issues remain, especially when one wants to achieve the best lateral and vertical (nanometer-scale) resolution. In this study, the interest of in situ matrix transfer for tissue analysis with cluster ion beams (Bin+, Arn+) is explored in detail, using a series of six low molecular weight acidic (MALDI) matrices. After estimating the sensitivity enhancements for phosphatidylcholine (PC), an abundant lipid type present in almost any kind of cell membrane, the most promising matrices were softly transferred in situ on mouse brain and human uterine tissue samples using a 10 keV Ar3000+ cluster beam. Signal enhancements up to 1 order of magnitude for intact lipid signals were observed in both tissues under Bi5+ and Ar3000+ bombardment. The main findings of this study lie in the in-depth characterization of uterine tissue samples, the demonstration that the transferred matrices also improve signal efficiency in the negative ion polarity and that they perform as well when using Bin+ and Arn+ primary ions for analysis and imaging.

Assessment of Hyperosmolar Blood-Brain Barrier Opening in Glioblastoma via Histology with Evans Blue and DCE-MRI.

BACKGROUND: While the blood-brain barrier (BBB) is often compromised in glioblastoma (GB), the perfusion and consequent delivery of drugs are highly heterogeneous. Moreover, the accessibility of drugs is largely impaired in the margins of the tumor and for infiltrating cells at the origin of tumor recurrence. In this work, we evaluate the value of methods to assess hemodynamic changes induced by a hyperosmolar shock in the core and the margins of a tumor in a GB model. METHODS: Osmotic shock was induced with an intracarotid infusion of a hypertonic solution of mannitol in mice grafted with U87-MG cells. The distribution of fluorescent dye (Evans blue) within the brain was assessed via histology. Dynamic contrast-enhanced (DCE)-MRI with an injection of Gadolinium-DOTA as the contrast agent was also used to evaluate the effect on hemodynamic parameters and the diffusion of the contrast agent outside of the tumor area. RESULTS: The histological study revealed that the fluorescent dye diffused much more largely outside of the tumor area after osmotic shock than in control tumors. However, the study of tumor hemodynamic parameters via DCE-MRI did not reveal any change in the permeability of the BBB, whatever the studied MRI parameter. CONCLUSIONS: The use of hypertonic mannitol infusion seems to be a promising method to increase the delivery of compounds in the margins of GB. Nevertheless, the DCE-MRI analysis method using gadolinium-DOTA as a contrast agent seems of limited value for determining the efficacy of opening the BBB in GB after osmotic shock.

Glioblastoma-targeted, local and sustained drug delivery system based on an unconventional lipid nanocapsule hydrogel.

The objective of this work was to develop an implantable therapeutic hydrogel that will ensure continuity in treatment between surgery and radiochemotherapy for patients with glioblastoma (GBM). A hydrogel of self-associated gemcitabine-loaded lipid nanocapsules (LNC) has shown therapeutic efficacy in vivo in murine GBM resection models. To improve the targeting of GBM cells, the NFL-TBS.40-63 peptide (NFL), was associated with LNC. The LNC-based hydrogels were formulated with the NFL. The peptide was totally and instantaneously adsorbed at the LNC surface, without modifying the hydrogel mechanical properties, and remained adsorbed to the LNC surface after the hydrogel dissolution. In vitro studies on GBM cell lines showed a faster internalization of the LNC and enhanced cytotoxicity, in the presence of NFL. Finally, in vivo studies in the murine GBM resection model proved that the gemcitabine-loaded LNC with adsorbed NFL could target the non-resected GBM cells and significantly delay or even inhibit the apparition of recurrences.

Combination of local immunogenic cell death-inducing chemotherapy and DNA vaccine increases the survival of glioblastoma-bearing mice.

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.

Hyaluronic acid-antigens conjugates trigger potent immune response in both prophylactic and therapeutic immunization in a melanoma model.

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.

Multifunctional Self-Assembled Peptide Hydrogels for Biomedical Applications.

Self-assembly is a growth mechanism in nature to apply local interactions forming a minimum energy structure. Currently, self-assembled materials are considered for biomedical applications due to their pleasant features, including scalability, versatility, simplicity, and inexpensiveness. Self-assembled peptides can be applied to design and fabricate different structures, such as micelles, hydrogels, and vesicles, by diverse physical interactions between specific building blocks. Among them, bioactivity, biocompatibility, and biodegradability of peptide hydrogels have introduced them as versatile platforms in biomedical applications, such as drug delivery, tissue engineering, biosensing, and treating different diseases. Moreover, peptides are capable of mimicking the microenvironment of natural tissues and responding to internal and external stimuli for triggered drug release. In the current review, the unique characteristics of peptide hydrogels and recent advances in their design, fabrication, as well as chemical, physical, and biological properties are presented. Additionally, recent developments of these biomaterials are discussed with a particular focus on their biomedical applications in targeted drug delivery and gene delivery, stem cell therapy, cancer therapy and immune regulation, bioimaging, and regenerative medicine.

Mismatch between Bioluminescence Imaging (BLI) and MRI When Evaluating Glioblastoma Growth: Lessons from a Study Where BLI Suggested "Regression" while MRI Showed "Progression".

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.

Stimuli-Responsive Multifunctional Nanomedicine for Enhanced Glioblastoma Chemotherapy Augments Multistage Blood-to-Brain Trafficking and Tumor Targeting.

Minimal therapeutic advances have been achieved over the past two decades for glioblastoma (GBM), which remains an unmet clinical need. Here, hypothesis-driven stimuli-responsive nanoparticles (NPs) for docetaxel (DTX) delivery to GBM are reported, with multifunctional features that circumvent insufficient blood-brain barrier (BBB) trafficking and lack of GBM targeting-two major hurdles for anti-GBM therapies. NPs are dual-surface tailored with a i) brain-targeted acid-responsive Angiopep-2 moiety that triggers NP structural rearrangement within BBB endosomal vesicles, and ii) L-Histidine moiety that provides NP preferential accumulation into GBM cells post-BBB crossing. In tumor invasive margin patient cells, the stimuli-responsive multifunctional NPs target GBM cells, enhance cell uptake by 12-fold, and induce three times higher cytotoxicity in 2D and 3D cell models. Moreover, the in vitro BBB permeability is increased by threefold. A biodistribution in vivo trial confirms a threefold enhancement of NP accumulation into the brain. Last, the in vivo antitumor efficacy is validated in GBM orthotopic models following intratumoral and intravenous administration. Median survival and number of long-term survivors are increased by 50%. Altogether, a preclinical proof of concept supports these stimuli-responsive multifunctional NPs as an effective anti-GBM multistage chemotherapeutic strategy, with ability to respond to multiple fronts of the GBM microenvironment.

Synergistic effect of doxorubicin lauroyl hydrazone derivative delivered by α-tocopherol succinate micelles for the treatment of glioblastoma.

We hypothesized that tocopherol succinate (TOS) and D-α-tocopherol polyethylene2000 succinate (TPGS2000) micelles could work as a drug delivery system while enhancing the anti-cancer efficacy of doxorubicin lauryl hydrazone derivative (DOXC12) for the treatment of glioblastoma. The DOXC12-TOS-TPGS2000 micelles were formulated with synthesized DOXC12 and TPGS2000. They showed a high drug loading of hydrophobic DOXC12 (29%), a size of <100 nm and a pH sensitive drug release behaviour. In vitro, fast uptake of DOXC12-TOS-TPGS2000 micelles by GL261 cells was observed. For cytotoxicity, DOXC12-TOS-TPGS2000 micelles were evaluated on two glioblastoma cell lines and showed synergism between DOXC12 and TOS-TPGS2000. The higher cytotoxicity of DOXC12-TOS-TPGS2000 micelles was mainly caused by necrosis. The DOXC12-TOS-TPGS2000 micelles seem to be a promising delivery system for enhancing the anticancer efficacy of doxorubicin in glioblastoma (GBM).

Combination of hyaluronic acid conjugates with immunogenic cell death inducer and CpG for glioblastoma local chemo-immunotherapy elicits an immune response and induces long-term survival.

The efficacy of standard glioblastoma (GBM) treatments has been limited due to the highly immunosuppressive tumor immune microenvironment, interpatient tumor heterogenicity and anatomical barriers, such as the blood brain barrier. In the present work, we hypothesized that a new local therapy based on the combination of doxorubicin (DOX) as an immunogenic cell death (ICD) inducer and CpG, a Toll-like receptor (TLR)-9 agonist, would act synergistically to eradicate GBM. DOX and CpG were first tested in an orthotopic GL261 GBM model showing enhanced survival. To improve the outcome with a reduced dose, we designed bioresponsive hyaluronic acid (HA)-drug conjugates for effective in situ chemoimmunotherapy. HA was derivatized with CpG. The new HA-CpG conjugate showed high efficacy in re-educating protumoral M2-like microglia into an antitumoral M1-like phenotype, inducing the expression of immune-stimulatory cytokines. DOX was also conjugated to HA. DOX conjugation increased ICD induction in GL261 cells. Finally, a combination of the conjugates was explored in an orthotopic GL261 GBM model. The local delivery of combined HA-DOX + HA-CpG into the tumor mass elicited antitumor CD8+ T cell responses in the brain tumor microenvironment and reduced the infiltration of M2-like tumor-associated macrophages and myeloid-derived suppressor cells. Importantly, the combination of HA-DOX and HA-CpG induced long-term survival in >66% of GBM-bearing animals than other treatments (no long-term survivor observed), demonstrating the benefits of conjugating synergistic drugs to HA nanocarrier. These results emphasize that HA-drug conjugates constitute an effective drug delivery platform for local chemoimmunotherapy against GBM and open new perspectives for the treatment of other brain cancers and brain metastasis.

Dual-drug loaded nanomedicine hydrogel as a therapeutic platform to target both residual glioblastoma and glioma stem cells.

Glioblastoma (GBM) recurrences are inevitable, and mainly originate from residual tumor cells and the presence of glioma stem cells (GSC) around the resection cavity borders. We previously showed that the local treatment of GBM with nanomedicine-based Lauroyl-gemcitabine lipid nanocapsules (GemC12-LNC) hydrogel delayed tumor onset in various preclinical models and can be used as a scaffold to deliver multiple drugs. However, it does not inhibit tumor relapse in the long-term. In this work, we aim at encapsulating an anti-GSC molecule in the GemC12-LNC hydrogel to eliminate both GBM cells and GSC. We performed a screening on GBM cell lines (GL261 and U-87 MG) and patient-derived GSC (GBM9) to select the anti-GSC molecule that could act synergically with GemC12. Based on our results, salinomycin (Sal) and curcumin (Cur) were selected for further development. Both GemC12-Sal-LNC and GemC12-Cur LNC showed similar size (55 nm), zeta potential (- 2 mV) and viscoelastic properties compared to the GemC12-LNC hydrogel. Encapsulation efficiency was above 95 %. Moreover, the GemC12-Sal-LNC hydrogel was stable for at least 6 months and released both drugs over 30 days in vitro. Both hydrogels inhibited the growth of GL261 and U-87 MG spheroids. Flow cytometry analysis showed that Sal reduced the GSC population in GL261 and U-87 MG cells. Our results show that the co-encapsulation of Sal in the GemC12-LNC hydrogel can reduce both GBM cells and GSC, and therefore might be promising to avoid the onset of GBM recurrences.

Barriers to the Intestinal Absorption of Four Insulin-Loaded Arginine-Rich Nanoparticles in Human and Rat.

Peptide drugs and biologics provide opportunities for treatments of many diseases. However, due to their poor stability and permeability in the gastrointestinal tract, the oral bioavailability of peptide drugs is negligible. Nanoparticle formulations have been proposed to circumvent these hurdles, but systemic exposure of orally administered peptide drugs has remained elusive. In this study, we investigated the absorption mechanisms of four insulin-loaded arginine-rich nanoparticles displaying differing composition and surface characteristics, developed within the pan-European consortium TRANS-INT. The transport mechanisms and major barriers to nanoparticle permeability were investigated in freshly isolated human jejunal tissue. Cytokine release profiles and standard toxicity markers indicated that the nanoparticles were nontoxic. Three out of four nanoparticles displayed pronounced binding to the mucus layer and did not reach the epithelium. One nanoparticle composed of a mucus inert shell and cell-penetrating octarginine (ENCP), showed significant uptake by the intestinal epithelium corresponding to 28 ± 9% of the administered nanoparticle dose, as determined by super-resolution microscopy. Only a small fraction of nanoparticles taken up by epithelia went on to be transcytosed via a dynamin-dependent process. In situ studies in intact rat jejunal loops confirmed the results from human tissue regarding mucus binding, epithelial uptake, and negligible insulin bioavailability. In conclusion, while none of the four arginine-rich nanoparticles supported systemic insulin delivery, ENCP displayed a consistently high uptake along the intestinal villi. It is proposed that ENCP should be further investigated for local delivery of therapeutics to the intestinal mucosa.

Combination of DNA Vaccine and Immune Checkpoint Blockades Improves the Immune Response in an Orthotopic Unresectable Glioblastoma Model.

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.

Surface Modification of Lipid-Based Nanoparticles.

There is a growing interest in the development of lipid-based nanocarriers for multiple purposes, including the recent increase of these nanocarriers as vaccine components during the COVID-19 pandemic. The number of studies that involve the surface modification of nanocarriers to improve their performance (increase the delivery of a therapeutic to its target site with less off-site accumulation) is enormous. The present review aims to provide an overview of various methods associated with lipid nanoparticle grafting, including techniques used to separate grafted nanoparticles from unbound ligands or to characterize grafted nanoparticles. We also provide a critical perspective on the usefulness and true impact of these modifications on overcoming different biological barriers, with our prediction on what to expect in the near future in this field.

Layer-by-Layer Nanoarchitectonics Using Protein-Polyelectrolyte Complexes toward a Generalizable Tool for Protein Surface Immobilization.

Layer-by-layer (LbL) self-assembly is an attractive method for the immobilization of macromolecules at interfaces. Integrating proteins in LbL thin films is however challenging due to their polyampholyte nature. Recently, we developed a method to integrate lysozyme into multilayers using protein-polyelectrolytes complexes (PPCs). In this work, we extended this method to a wide range of protein-polyelectrolyte combinations. We demonstrated the robustness and versatility of PPCs as building blocks. LL-37, insulin, lysozyme, and glucose oxidase were complexed with alginate, poly(styrenesulfonate), heparin, and poly(allylamine hydrochloride). The resulting PPCs were then LbL self-assembled with chitosan, PAH, and heparin. We demonstrated that multilayers built with PPCs are thicker compared to the LbL self-assembly of bare protein molecules. This is attributed to the higher mass of protein in the multilayers and/or the more hydrated state of the assemblies. PPCs enabled the self-assembly of proteins that could otherwise not be LbL assembled with a PE or with another protein. Furthermore, the results also show that LbL with PPCs enabled the construction of multilayers combining different proteins, highlighting the formation of multifunctional films. Importantly, we show that the adsorption behavior and thus the multilayer growth strongly depend on the nature of the protein and polyelectrolyte used. In this work, we elaborated a rationale to help and guide the use of PPCs for protein LbL assembly. It will therefore be beneficial to the many scientific communities willing to modify interfaces with hard-to-immobilize proteins and peptides.

Design of Bio-Responsive Hyaluronic Acid-Doxorubicin Conjugates for the Local Treatment of Glioblastoma.

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.

Immunotherapy for glioblastoma: the promise of combination strategies.

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.

Colorectal cancer inhibition by BET inhibitor JQ1 is MYC-independent and not improved by nanoencapsulation.

Bromodomain and extraterminal domain protein inhibitors (BETi) for cancer treatment did not convince during their first clinical trials. Their epigenetic mechanism of action is still not well understood, even if MYC is generally considered as its main downstream target. In this context, we intended to assess two new nanoformulations of the BETi JQ1 for the treatment of colorectal cancer (CRC). JQ1 was encapsulated at 10 mg/mL in lipid nanocapsules (LNC) or polymeric micelles (PM), both compatible for an intravenous administration. Their effect was compared with free JQ1 on several CRC cell lines in vitro and with daily intraperitoneal cyclodextrin (CD)-loaded JQ1 on the CT26 CRC tumor model in vivo. We showed that LNC preferentially accumulated in tumor, liver, and lymph nodes. LNC-JQ1 and CD-JQ1 similarly delayed tumor growth and increased median survival from 15 to 23 or 20.5 days. JQ1 altered MYC in only two among four CRC cell lines. This MYC-independence found in CT26 was confirmed in vivo by PCR and immunohistochemistry. The main explanation of the JQ1 anticancer effect was an increase in apoptosis. The investigation of its impact on the tumor microenvironment did not show significant effects. Finally, JQ1 association with irinotecan did not synergize in vivo with JQ1 nanoformulations. In conclusion, we demonstrated that the JQ1 anticancer effect was not improved by nanoencapsulation even if their tumor delivery was probably higher. MYC inhibition was not associated to JQ1 efficacy in the case of the CT26 CRC murine model.