Glioblastoma extracellular vesicles modulate immune PD-L1 expression in accessory macrophages upon radiotherapy.

Glioblastoma (GBM) is the most aggressive brain tumor, presenting major challenges due to limited treatment options. Standard care includes radiation therapy (RT) to curb tumor growth and alleviate symptoms, but its impact on GBM is limited. In this study, we investigated the effect of RT on immune suppression and whether extracellular vesicles (EVs) originating from GBM and taken up by the tumor microenvironment (TME) contribute to the induced therapeutic resistance. We observed that (1) ionizing radiation increases immune-suppressive markers on GBM cells, (2) macrophages exacerbate immune suppression in the TME by increasing PD-L1 in response to EVs derived from GBM cells which is further modulated by RT, and (3) RT increases CD206-positive macrophages which have the most potential in inducing a pro-oncogenic environment due to their increased uptake of tumor-derived EVs. In conclusion, RT affects GBM resistance by immuno-modulating EVs taken up by myeloid cells in the TME.

Platelet RNA enables accurate detection of ovarian cancer: an intercontinental, biomarker identification study.

Platelets are reprogrammed by cancer via a process called education, which favors cancer development. The transcriptional profile of tumor-educated platelets (TEPs) is skewed and therefore practicable for cancer detection. This intercontinental, hospital-based, diagnostic study included 761 treatment-naïve inpatients with histologically confirmed adnexal masses and 167 healthy controls from nine medical centers (China, n = 3; Netherlands, n = 5; Poland, n = 1) between September 2016 and May 2019. The main outcomes were the performance of TEPs and their combination with CA125 in two Chinese (VC1 and VC2) and the European (VC3) validation cohorts collectively and independently. Exploratory outcome was the value of TEPs in public pan-cancer platelet transcriptome datasets. The AUCs for TEPs in the combined validation cohort, VC1, VC2, and VC3 were 0.918 (95% CI 0.889-0.948), 0.923 (0.855-0.990), 0.918 (0.872-0.963), and 0.887 (0.813-0.960), respectively. Combination of TEPs and CA125 demonstrated an AUC of 0.922 (0.889-0.955) in the combined validation cohort; 0.955 (0.912-0.997) in VC1; 0.939 (0.901-0.977) in VC2; 0.917 (0.824-1.000) in VC3. For subgroup analysis, TEPs exhibited an AUC of 0.858, 0.859, and 0.920 to detect early-stage, borderline, non-epithelial diseases and 0.899 to discriminate ovarian cancer from endometriosis. TEPs had robustness, compatibility, and universality for preoperative diagnosis of ovarian cancer since it withstood validations in populations of different ethnicities, heterogeneous histological subtypes, and early-stage ovarian cancer. However, these observations warrant prospective validations in a larger population before clinical utilities.

Nanotechnology-Inspired Extracellular Vesicles Theranostics for Diagnosis and Therapy of Central Nervous System Diseases.

Shuttling various bioactive substances across the blood-brain barrier (BBB) bidirectionally, extracellular vesicles (EVs) have been opening new frontiers for the diagnosis and therapy of central nervous system (CNS) diseases. However, clinical translation of EV-based theranostics remains challenging due to difficulties in effective EV engineering for superior imaging/therapeutic potential, ultrasensitive EV detection for small sample volume, as well as scale-up and standardized EV production. In the past decade, continuous advancement in nanotechnology provided extensive concepts and strategies for EV engineering and analysis, which inspired the application of EVs for CNS diseases. Here we will review the existing types of EV-nanomaterial hybrid systems with improved diagnostic and therapeutic efficacy for CNS diseases. A summary of recent progress in the incorporation of nanomaterials and nanostructures in EV production, separation, and analysis will also be provided. Moreover, the convergence between nanotechnology and microfluidics for integrated EV engineering and liquid biopsy of CNS diseases will be discussed.

Mitochondrial fission induces immunoescape in solid tumors through decreasing MHC-I surface expression.

Mitochondrial dynamics can regulate Major Histocompatibility Complex (MHC)-I antigen expression by cancer cells and their immunogenicity in mice and in patients with malignancies. A crucial role in the mitochondrial fragmentation connection with immunogenicity is played by the IRE1α-XBP-1s axis. XBP-1s is a transcription factor for aminopeptidase TPP2, which inhibits MHC-I complex cell surface expression likely by degrading tumor antigen peptides. Mitochondrial fission inhibition with Mdivi-1 upregulates MHC-I expression on cancer cells and enhances the efficacy of adoptive T cell therapy in patient-derived tumor models. Therefore mitochondrial fission inhibition might provide an approach to enhance the efficacy of T cell-based immunotherapy.

A direct high-throughput protein quantification strategy facilitates discovery and characterization of a celastrol-derived BRD4 degrader.

We describe a generalizable time-resolved Förster resonance energy transfer (TR-FRET)-based platform to profile the cellular action of heterobifunctional degraders (or proteolysis-targeting chimeras [PROTACs]) that is capable of both accurately quantifying protein levels in whole-cell lysates in less than 1 h and measuring small-molecule target engagement to endogenous proteins, here specifically for human bromodomain-containing protein 4 (BRD4). The detection mix consists of a single primary antibody targeting the protein of interest, a luminescent donor-labeled anti-species nanobody, and a fluorescent acceptor ligand. Importantly, our strategy can readily be applied to other targets of interest and will greatly facilitate the cell-based profiling of small-molecule inhibitors and PROTACs in a high-throughput format with unmodified cell lines. We furthermore validate our platform in the characterization of celastrol, a p-quinone methide-containing pentacyclic triterpenoid, as a broad cysteine-targeting E3 ubiquitin ligase warhead for potent and efficient targeted protein degradation.

Immune Checkpoint Inhibition in GBM Primed with Radiation by Engineered Extracellular Vesicles.

The lack of safe and effective delivery across the blood-brain barrier and the profound immune suppressive microenvironment are two main hurdles to glioblastoma (GBM) therapies. Extracellular vesicles (EVs) have been used as therapeutic delivery vehicles to GBM but with limited efficacy. We hypothesized that EV delivery to GBM can be enhanced by (i) modifying the EV surface with a brain-tumor-targeting cyclic RGDyK peptide (RGD-EV) and (ii) using bursts of radiation for enhanced accumulation. In addition, EVs were loaded with small interfering RNA (siRNA) against programmed cell death ligand-1 (PD-L1) for immune checkpoint blockade. We show that this EV-based strategy dramatically enhanced the targeting efficiency of RGD-EV to murine GBM, while the loaded siRNA reversed radiation-stimulated PD-L1 expression on tumor cells and recruited tumor-associated myeloid cells, offering a synergistic effect. The combined therapy significantly increased CD8+ cytotoxic T cells activity, halting tumor growth and prolonging animal survival. The selected cell source for EVs isolation and the presented functionalization strategy are suitable for large-scale production. These results provide an EV-based therapeutic strategy for GBM immune checkpoint therapy which can be translated to clinical applications.

Olfactory receptor 5B21 drives breast cancer metastasis.

Olfactory receptors (ORs), responsible for the sense of smell, play an essential role in various physiological processes outside the nasal epithelium, including cancer. In breast cancer, however, the expression and function of ORs remain understudied. We examined the significance of OR transcript abundance in primary and metastatic breast cancer to the brain, bone, and lung. Although 20 OR transcripts were differentially expressed in distant metastases, OR5B21 displayed an increased transcript abundance in all three metastatic sites compared with the primary tumor. Knockdown of OR5B21 significantly decreased the invasion and migration of breast cancer cells as well as metastasis to different organs especially the brain, whereas increasing of OR5B21 transcript abundance had the opposite effect. Mechanistically, OR5B21 expression was associated with epithelial to mesenchymal transition through the STAT3/NF-κB/CEBPß signaling axis. We propose OR5B21 (and potentially other ORs) as a novel oncogene contributing to breast cancer metastasis and a potential target for adjuvant therapy.

A Pan-Histone Deacetylase Inhibitor Enhances the Antitumor Activity of B7-H3-Specific CAR T Cells in Solid Tumors.

PURPOSE: The limited efficacy of chimeric antigen receptor (CAR) T-cell therapies with solid malignancies prompted us to test whether epigenetic therapy could enhance the antitumor activity of B7-H3.CAR T cells with several solid cancer types. EXPERIMENTAL DESIGN: We evaluated B7-H3 expression in many human solid cancer and normal tissue samples. The efficacy of the combinatorial therapy with B7-H3.CAR T cells and the deacetylase inhibitor SAHA with several solid cancer types and the potential underlying mechanisms were characterized with in vitro and ex vivo experiments. RESULTS: B7-H3 is expressed in most of the human solid tumor samples tested, but exhibits a restricted expression in normal tissues. B7-H3.CAR T cells selectively killed B7-H3 expressing human cancer cell lines in vitro. A low dose of SAHA upregulated B7-H3 expression in several types of solid cancer cells at the transcriptional level and B7-H3.CAR expression on human transgenic T-cell membrane. In contrast, the expression of immunosuppressive molecules, such as CTLA-4 and TET2, by T cells was downregulated upon SAHA treatment. A low dose of SAHA significantly enhanced the antitumor activity of B7-H3.CAR T cells with solid cancers in vitro and ex vivo, including orthotopic patient-derived xenograft and metastatic models treated with autologous CAR T-cell infusions. CONCLUSIONS: Our results show that our novel strategy which combines SAHA and B7-H3.CAR T cells enhances their therapeutic efficacy with solid cancers and justify its translation to a clinical setting.

Gene therapy for tuberous sclerosis complex type 2 in a mouse model by delivery of AAV9 encoding a condensed form of tuberin.

Tuberous sclerosis complex (TSC) results from loss of a tumor suppressor gene - TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins formed a complex to inhibit mTORC1-mediated cell growth and proliferation. Loss of either protein leads to overgrowth lesions in many vital organs. Gene therapy was evaluated in a mouse model of TSC2 using an adeno-associated virus (AAV) vector carrying the complementary for a "condensed" form of human tuberin (cTuberin). Functionality of cTuberin was verified in culture. A mouse model of TSC2 was generated by AAV-Cre recombinase disruption of Tsc2-floxed alleles at birth, leading to a shortened lifespan (mean 58 days) and brain pathology consistent with TSC. When these mice were injected intravenously on day 21 with AAV9-cTuberin, the mean survival was extended to 462 days with reduction in brain pathology. This demonstrates the potential of treating life-threatening TSC2 lesions with a single intravenous injection of AAV9-cTuberin.

Sustained subcutaneous delivery of secretome of human cardiac stem cells promotes cardiac repair following myocardial infarction.

AIMS: To establish pre-clinical proof of concept that sustained subcutaneous delivery of the secretome of human cardiac stem cells (CSCs) can be achieved in vivo to produce significant cardioreparative outcomes in the setting of myocardial infarction. METHODS AND RESULTS: Rats were subjected to permanent ligation of left anterior descending coronary artery and randomized to receive subcutaneous implantation of TheraCyte devices containing either culture media as control or 1 × 106 human W8B2+ CSCs, immediately following myocardial ischaemia. At 4 weeks following myocardial infarction, rats treated with W8B2+ CSCs encapsulated within the TheraCyte device showed preserved left ventricular ejection fraction. The preservation of cardiac function was accompanied by reduced fibrotic scar tissue, interstitial fibrosis, cardiomyocyte hypertrophy, as well as increased myocardial vascular density. Histological analysis of the TheraCyte devices harvested at 4 weeks post-implantation demonstrated survival of human W8B2+ CSCs within the devices, and the outer membrane was highly vascularized by host blood vessels. Using CSCs expressing plasma membrane reporters, extracellular vesicles of W8B2+ CSCs were found to be transferred to the heart and other organs at 4 weeks post-implantation. Furthermore, mass spectrometry-based proteomic profiling of extracellular vesicles of W8B2+ CSCs identified proteins implicated in inflammation, immunoregulation, cell survival, angiogenesis, as well as tissue remodelling and fibrosis that could mediate the cardioreparative effects of secretome of human W8B2+ CSCs. CONCLUSIONS: Subcutaneous implantation of TheraCyte devices encapsulating human W8B2+ CSCs attenuated adverse cardiac remodelling and preserved cardiac function following myocardial infarction. The TheraCyte device can be employed to deliver stem cells in a minimally invasive manner for effective secretome-based cardiac therapy.

The natural compound obtusaquinone targets pediatric high-grade gliomas through ROS-mediated ER stress.

BACKGROUND: Pediatric high-grade gliomas (pHGGs) are aggressive primary brain tumors with local invasive growth and poor clinical prognosis. Treatment of pHGGs is particularly challenging given the intrinsic resistance to chemotherapy, an absence of novel therapeutics, and the difficulty of drugs to reach the tumor beds. Accumulating evidence suggests that production of reactive oxygen species (ROS) and misfolded proteins, which typically leads to endoplasmic reticulum (ER) stress, is an essential mechanism in cancer cell survival. METHODS: Several cell viability assays were used in 6 patient-derived pHGG cultures to evaluate the effect of the natural compound obtusaquinone (OBT) on cytotoxicity. Orthotopic mouse models were used to determine OBT effects in vivo. Immunoblotting, immunostaining, flow cytometry, and biochemical assays were used to investigate the OBT mechanism of action. RESULTS: OBT significantly inhibited cell survival of patient-derived pHGG cells in culture. OBT inhibited tumor growth and extended survival in 2 different orthotopic xenograft models. Mechanistically, OBT induced ER stress through abnormal ROS accumulation. CONCLUSION: Our data demonstrate the utility and feasibility of OBT as a potential therapeutic option for improving the clinical treatment of pHGGs.

Mesenchymal Transformation: The Rosetta Stone of Glioblastoma Pathogenesis and Therapy Resistance.

Despite decades of research, glioblastoma (GBM) remains invariably fatal among all forms of cancers. The high level of inter- and intratumoral heterogeneity along with its biological location, the brain, are major barriers against effective treatment. Molecular and single cell analysis identifies different molecular subtypes with varying prognosis, while multiple subtypes can reside in the same tumor. Cellular plasticity among different subtypes in response to therapies or during recurrence adds another hurdle in the treatment of GBM. This phenotypic shift is induced and sustained by activation of several pathways within the tumor itself, or microenvironmental factors. In this review, the dynamic nature of cellular shifts in GBM and how the tumor (immune) microenvironment shapes this process leading to therapeutic resistance, while highlighting emerging tools and approaches to study this dynamic double-edged sword are discussed.

Regulatory T cells engineered with TCR signaling-responsive IL-2 nanogels suppress alloimmunity in sites of antigen encounter.

Adoptive cell transfer of ex vivo expanded regulatory T cells (Tregs) has shown immense potential in animal models of auto- and alloimmunity. However, the effective translation of such Treg therapies to the clinic has been slow. Because Treg homeostasis is known to require continuous T cell receptor (TCR) ligation and exogenous interleukin-2 (IL-2), some investigators have explored the use of low-dose IL-2 injections to increase endogenous Treg responses. Systemic IL-2 immunotherapy, however, can also lead to the activation of cytotoxic T lymphocytes and natural killer cells, causing adverse therapeutic outcomes. Here, we describe a drug delivery platform, which can be engineered to autostimulate Tregs with IL-2 in response to TCR-dependent activation, and thus activate these cells in sites of antigen encounter. To this end, protein nanogels (NGs) were synthesized with cleavable bis(N-hydroxysuccinimide) cross-linkers and IL-2/Fc fusion (IL-2) proteins to form particles that release IL-2 under reducing conditions, as found at the surface of T cells receiving stimulation through the TCR. Tregs surface-conjugated with IL-2 NGs were found to have preferential, allograft-protective effects relative to unmodified Tregs or Tregs stimulated with systemic IL-2. We demonstrate that murine and human NG-modified Tregs carrying an IL-2 cargo perform better than conventional Tregs in suppressing alloimmunity in murine and humanized mouse allotransplantation models. In all, the technology presented in this study has the potential to improve Treg transfer therapy by enabling the regulated spatiotemporal provision of IL-2 to antigen-primed Tregs.

Tumor-Educated Platelet RNA for the Detection and (Pseudo)progression Monitoring of Glioblastoma.

Tumor-educated platelets (TEPs) are potential biomarkers for cancer diagnostics. We employ TEP-derived RNA panels, determined by swarm intelligence, to detect and monitor glioblastoma. We assessed specificity by comparing the spliced RNA profile of TEPs from glioblastoma patients with multiple sclerosis and brain metastasis patients (validation series, n = 157; accuracy, 80%; AUC, 0.81 [95% CI, 0.74-0.89; p < 0.001]). Second, analysis of patients with glioblastoma versus asymptomatic healthy controls in an independent validation series (n = 347) provided a detection accuracy of 95% and AUC of 0.97 (95% CI, 0.95-0.99; p < 0.001). Finally, we developed the digitalSWARM algorithm to improve monitoring of glioblastoma progression and demonstrate that the TEP tumor scores of individual glioblastoma patients represent tumor behavior and could be used to distinguish false positive progression from true progression (validation series, n = 20; accuracy, 85%; AUC, 0.86 [95% CI, 0.70-1.00; p < 0.012]). In conclusion, TEPs have potential as a minimally invasive biosource for blood-based diagnostics and monitoring of glioblastoma patients.

Small but Fierce: Tracking the Role of Extracellular Vesicles in Glioblastoma Progression and Therapeutic Resistance.

Glioblastoma is the most common and aggressive brain tumor in adults. Most patients die within a year and long-term survival remains rare, owing to a combination of rapid progression/degeneration, lack of successful treatments, and high recurrence rates. Extracellular vesicles are cell-derived membranous structures involved in numerous physiological and pathological processes. In the context of cancer, these biological nanoparticles play an important role in intercellular communication, allowing cancer cells to exchange information with each other, the tumor microenvironment as well as distant cells. Here, light is shed on the role of extracellular vesicles in glioblastoma heterogeneity, tumor microenvironment interactions, and therapeutic resistance, and an overview on means to track their release, uptake, and cargo delivery is provided.

A cancer drug atlas enables synergistic targeting of independent drug vulnerabilities.

Personalized cancer treatments using combinations of drugs with a synergistic effect is attractive but proves to be highly challenging. Here we present an approach to uncover the efficacy of drug combinations based on the analysis of mono-drug effects. For this we used dose-response data from pharmacogenomic encyclopedias and represent these as a drug atlas. The drug atlas represents the relations between drug effects and allows to identify independent processes for which the tumor might be particularly vulnerable when attacked by two drugs. Our approach enables the prediction of combination-therapy which can be linked to tumor-driving mutations. By using this strategy, we can uncover potential effective drug combinations on a pan-cancer scale. Predicted synergies are provided and have been validated in glioblastoma, breast cancer, melanoma and leukemia mouse-models, resulting in therapeutic synergy in 75% of the tested models. This indicates that we can accurately predict effective drug combinations with translational value.

Extracellular Vesicles Induce Mesenchymal Transition and Therapeutic Resistance in Glioblastomas through NF-κB/STAT3 Signaling.

Glioblastoma (GBM) is the most common primary malignant brain tumor and despite optimal treatment, long-term survival remains uncommon. GBM can be roughly divided into three different molecular subtypes, each varying in aggressiveness and treatment resistance. Recent evidence shows plasticity between these subtypes in which the proneural (PN) glioma stem-like cells (GSCs) undergo transition into the more aggressive mesenchymal (MES) subtype, leading to therapeutic resistance. Extracellular vesicles (EVs) are membranous structures secreted by nearly every cell and are shown to play a key role in GBM progression by acting as multifunctional signaling complexes. Here, it is shown that EVs derived from MES cells educate PN cells to increase stemness, invasiveness, cell proliferation, migration potential, aggressiveness, and therapeutic resistance by inducing mesenchymal transition through nuclear factor-κB/signal transducer and activator of transcription 3 signaling. The findings could potentially help explore new treatment strategies for GBM and indicate that EVs may also play a role in mesenchymal transition of different tumor types.

CXCR4 antagonist AMD3100 (plerixafor): From an impurity to a therapeutic agent.

AMD3100 (plerixafor), a CXCR4 antagonist, has opened a variety of avenues for potential therapeutic approaches in different refractory diseases. The CXCL12/CXCR4 axis and its signaling pathways are involved in diverse disorders including HIV-1 infection, tumor development, non-Hodgkin lymphoma, multiple myeloma, WHIM Syndrome, and so on. The mechanisms of action of AMD3100 may relate to mobilizing hematopoietic stem cells, blocking infection of X4 HIV-1, increasing circulating neutrophils, lymphocytes and monocytes, reducing myeloid-derived suppressor cells, and enhancing cytotoxic T-cell infiltration in tumors. Here, we first revisit the pharmacological discovery of AMD3100. We then review monotherapy of AMD3100 and combination use of AMD3100 with other agents in various diseases. Among those, we highlight the perspective of AMD3100 as an immunomodulator to regulate immune responses particularly in the tumor microenvironment and synergize with other therapeutics. All the pre-clinical studies support the clinical testing of the monotherapy and combination therapies with AMD3100 and further development for use in humans.

Obtusaquinone: A Cysteine-Modifying Compound That Targets Keap1 for Degradation.

We have previously identified the natural product obtusaquinone (OBT) as a potent antineoplastic agent with promising in vivo activity in glioblastoma and breast cancer through the activation of oxidative stress; however, the molecular properties of this compound remained elusive. We used a multidisciplinary approach comprising medicinal chemistry, quantitative mass spectrometry-based proteomics, functional studies in cancer cells, and pharmacokinetic analysis, as well as mouse xenograft models to develop and validate novel OBT analogs and characterize the molecular mechanism of action of OBT. We show here that OBT binds to cysteine residues with a particular affinity to cysteine-rich Keap1, a member of the CUL3 ubiquitin ligase complex. This binding promotes an overall stress response and results in ubiquitination and proteasomal degradation of Keap1 and downstream activation of the Nrf2 pathway. Using positron emission tomography (PET) imaging with the PET-tracer 2-[18F]fluoro-2-deoxy-d-glucose (FDG), we confirm that OBT is able to penetrate the brain and functionally target brain tumors. Finally, we show that an OBT analog with improved pharmacological properties, including enhanced potency, stability, and solubility, retains the antineoplastic properties in a xenograft mouse model.