Rapid Biofabrication of an Advanced Microphysiological System Mimicking Phenotypical Heterogeneity and Drug Resistance in Glioblastoma.

Microphysiological systems (MPSs) reconstitute tissue interfaces and organ functions, presenting a promising alternative to animal models in drug development. However, traditional materials like polydimethylsiloxane (PDMS) often interfere by absorbing hydrophobic molecules, affecting drug testing accuracy. Additive manufacturing, including 3D bioprinting, offers viable solutions. GlioFlow3D, a novel microfluidic platform combining extrusion bioprinting and stereolithography (SLA) is introduced. GlioFlow3D integrates primary human cells and glioblastoma (GBM) lines in hydrogel-based microchannels mimicking vasculature, within an SLA resin framework using cost-effective materials. The study introduces a robust protocol to mitigate SLA resin cytotoxicity. Compared to PDMS, GlioFlow3D demonstrated lower small molecule absorption, which is relevant for accurate testing of small molecules like Temozolomide (TMZ). Computational modeling is used to optimize a pumpless setup simulating interstitial fluid flow dynamics in tissues. Co-culturing GBM with brain endothelial cells in GlioFlow3D showed enhanced CD133 expression and TMZ resistance near vascular interfaces, highlighting spatial drug resistance mechanisms. This PDMS-free platform promises advanced drug testing, improving preclinical research and personalized therapy by elucidating complex GBM drug resistance mechanisms influenced by the tissue microenvironment.

Coordinated Targeting of S6K1/2 and AXL Disrupts Pyrimidine Biosynthesis in PTEN-Deficient Glioblastoma.

Intrinsic resistance to targeted therapeutics in PTEN-deficient glioblastoma (GBM) is mediated by redundant signaling networks that sustain critical metabolic functions. Here, we demonstrate that coordinated inhibition of the ribosomal protein S6 kinase 1 (S6K1) and the receptor tyrosine kinase AXL using LY-2584702 and BMS-777607 can overcome network redundancy to reduce GBM tumor growth. This combination of S6K1 and AXL inhibition suppressed glucose flux to pyrimidine biosynthesis. Genetic inactivation studies to map the signaling network indicated that both S6K1 and S6K2 transmit growth signals in PTEN-deficient GBM. Kinome-wide ATP binding analysis in inhibitor-treated cells revealed that LY-2584702 directly inhibited S6K1, and substrate phosphorylation studies showed that BMS-777607 inactivation of upstream AXL collaborated to reduce S6K2-mediated signal transduction. Thus, combination targeting of S6K1 and AXL provides a kinase-directed therapeutic approach that circumvents signal transduction redundancy to interrupt metabolic function and reduce growth of PTEN-deficient GBM. SIGNIFICANCE: Therapy for glioblastoma would be advanced by incorporating molecularly targeted kinase-directed agents, similar to standard of care strategies in other tumor types. Here, we identify a kinase targeting approach to inhibit the metabolism and growth of glioblastoma.

VISTA Emerges as a Promising Target against Immune Evasion Mechanisms in Medulloblastoma.

BACKGROUND: Relapsed medulloblastoma (MB) poses a significant therapeutic challenge due to its highly immunosuppressive tumor microenvironment. Immune checkpoint inhibitors (ICIs) have struggled to mitigate this challenge, largely due to low T-cell infiltration and minimal PD-L1 expression. Identifying the mechanisms driving low T-cell infiltration is crucial for developing more effective immunotherapies. METHODS: We utilize a syngeneic mouse model to investigate the tumor immune microenvironment of MB and compare our findings to transcriptomic and proteomic data from human MB. RESULTS: Flow cytometry reveals a notable presence of CD45hi/CD11bhi macrophage-like and CD45int/CD11bint microglia-like tumor-associated macrophages (TAMs), alongside regulatory T-cells (Tregs), expressing high levels of the inhibitory checkpoint molecule VISTA. Compared to sham control mice, the CD45hi/CD11bhi compartment significantly expands in tumor-bearing mice and exhibits a myeloid-specific signature composed of VISTA, CD80, PD-L1, CTLA-4, MHCII, CD40, and CD68. These findings are corroborated by proteomic and transcriptomic analyses of human MB samples. Immunohistochemistry highlights an abundance of VISTA-expressing myeloid cells clustering at the tumor-cerebellar border, while T-cells are scarce and express FOXP3. Additionally, tumor cells exhibit immunosuppressive properties, inhibiting CD4 T-cell proliferation in vitro. Identification of VISTA's binding partner, VSIG8, on tumor cells, and its correlation with increased VISTA expression in human transcriptomic analyses suggests a potential therapeutic target. CONCLUSIONS: This study underscores the multifaceted mechanisms of immune evasion in MB and highlights the therapeutic potential of targeting the VISTA-VSIG axis to enhance anti-tumor responses.

Genome-wide loss of heterozygosity predicts aggressive, treatment-refractory behavior in pituitary neuroendocrine tumors.

Pituitary neuroendocrine tumors (PitNETs) exhibiting aggressive, treatment-refractory behavior are the rare subset that progress after surgery, conventional medical therapies, and an initial course of radiation and are characterized by unrelenting growth and/or metastatic dissemination. Two groups of patients with PitNETs were sequenced: a prospective group of patients (n = 66) who consented to sequencing prior to surgery and a retrospective group (n = 26) comprised of aggressive/higher risk PitNETs. A higher mutational burden and fraction of loss of heterozygosity (LOH) was found in the aggressive, treatment-refractory PitNETs compared to the benign tumors (p = 1.3 × 10-10 and p = 8.5 × 10-9, respectively). Within the corticotroph lineage, a characteristic pattern of recurrent chromosomal LOH in 12 specific chromosomes was associated with treatment-refractoriness (occurring in 11 of 14 treatment-refractory versus 1 of 14 benign corticotroph PitNETs, p = 1.7 × 10-4). Across the cohort, a higher fraction of LOH was identified in tumors with TP53 mutations (p = 3.3 × 10-8). A machine learning approach identified loss of heterozygosity as the most predictive variable for aggressive, treatment-refractory behavior, outperforming the most common gene-level alteration, TP53, with an accuracy of 0.88 (95% CI: 0.70-0.96). Aggressive, treatment-refractory PitNETs are characterized by significant aneuploidy due to widespread chromosomal LOH, most prominently in the corticotroph tumors. This LOH predicts treatment-refractoriness with high accuracy and represents a novel biomarker for this poorly defined PitNET category.

Phytochemical Modulation of Ion Channels in Oncologic Symptomatology and Treatment.

Modern chemotherapies offer a broad approach to cancer treatment but eliminate both cancer and non-cancer cells indiscriminately and, thus, are associated with a host of side effects. Advances in precision oncology have brought about new targeted therapeutics, albeit mostly limited to a subset of patients with an actionable mutation. They too come with side effects and, ultimately, 'self-resistance' to the treatment. There is recent interest in the modulation of ion channels, transmembrane proteins that regulate the flow of electrically charged molecules in and out of cells, as an approach to aid treatment of cancer. Phytochemicals have been shown to act on ion channels with high specificity regardless of the tumor's genetic profile. This paper explores the use of phytochemicals in cancer symptom management and treatment.

A Review of Approaches to Potentiate the Activity of Temozolomide against Glioblastoma to Overcome Resistance.

A glioblastoma (GBM) is one of the most aggressive, infiltrative, and treatment-resistant malignancies of the central nervous system (CNS). The current standard of care for GBMs include maximally safe tumor resection, followed by concurrent adjuvant radiation treatment and chemotherapy with the DNA alkylating agent temozolomide (TMZ), which was approved by the FDA in 2005 based on a marginal increase (~2 months) in overall survival (OS) levels. This treatment approach, while initially successful in containing and treating GBM, almost invariably fails to prevent tumor recurrence. In addition to the limited therapeutic benefit, TMZ also causes debilitating adverse events (AEs) that significantly impact the quality of life of GBM patients. Some of the most common AEs include hematologic (e.g., thrombocytopenia, neutropenia, anemia) and non-hematologic (e.g., nausea, vomiting, constipation, dizziness) toxicities. Recurrent GBMs are often resistant to TMZ and other DNA-damaging agents. Thus, there is an urgent need to devise strategies to potentiate TMZ activity, to overcome drug resistance, and to reduce dose-dependent AEs. Here, we analyze major mechanisms of the TMZ resistance-mediated intracellular signaling activation of DNA repair pathways and the overexpression of drug transporters. We review some of the approaches investigated to counteract these mechanisms of resistance to TMZ, including the use of chemosensitizers and drug delivery strategies to enhance tumoral drug exposure.

A Phase 0/I Pharmacokinetic and Pharmacodynamics and Safety and Tolerability Study of Letrozole in Combination with Standard Therapy in Recurrent High-Grade Gliomas.

PURPOSE: High-grade gliomas (HGG) carry a poor prognosis, with glioblastoma accounting for almost 50% of primary brain malignancies in the elderly. Unfortunately, despite the use of multiple treatment modalities, the prognosis remains poor in this population. Our preclinical studies suggest that the presence of aromatase expression, encoded by CYP19A1, is significantly upregulated in HGGs. Remarkably, we find that letrozole (LTZ), an FDA-approved aromatase inhibitor, has marked activity against HGGs. PATIENTS AND METHODS: We conducted a phase 0/I single-center clinical trial (NCT03122197) to assess the tumoral availability, pharmacokinetics (PK), safety, and tolerability of LTZ in recurrent patients with HGG. Planned dose cohorts included 2.5, 5, 10, 12.5, 15, 17.5, and 20 mg of LTZ administered daily pre- and postsurgery or biopsy. Tumor samples were assayed for LTZ content and relevant biomarkers. The recommended phase 2 dose (R2PD) was determined as the dose that resulted in predicted steady-state tumoral extracellular fluid (ECF; Css,ecf) >2 µmol/L and did not result in ≥33% dose-limiting adverse events (AE) assessed using CTCAE v5.0. RESULTS: Twenty-one patients were enrolled. Common LTZ-related AEs included fatigue, nausea, musculoskeletal, anxiety, and dysphoric mood. No DLTs were observed. The 15 mg dose achieved a Css,ecf of 3.6 ± 0.59 µmol/L. LTZ caused dose-dependent inhibition of estradiol synthesis and modulated DNA damage pathways in tumor tissues as evident using RNA-sequencing analysis. CONCLUSIONS: On the basis of safety, brain tumoral PK, and mechanistic data, 15 mg daily is identified as the RP2D for future trials.

Chaetocin-mediated SUV39H1 inhibition targets stemness and oncogenic networks of diffuse midline gliomas and synergizes with ONC201.

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPG/DMG) are devastating pediatric brain tumors with extraordinarily limited treatment options and uniformly fatal prognosis. Histone H3K27M mutation is a common recurrent alteration in DIPG and disrupts epigenetic regulation. We hypothesize that genome-wide H3K27M-induced epigenetic dysregulation makes tumors vulnerable to epigenetic targeting. METHODS: We performed a screen of compounds targeting epigenetic enzymes to identify potential inhibitors for the growth of patient-derived DIPG cells. We further carried out transcriptomic and genomic landscape profiling including RNA-seq and CUT&RUN-seq as well as shRNA-mediated knockdown to assess the effects of chaetocin and SUV39H1, a target of chaetocin, on DIPG growth. RESULTS: High-throughput small-molecule screening identified an epigenetic compound chaetocin as a potent blocker of DIPG cell growth. Chaetocin treatment selectively decreased proliferation and increased apoptosis of DIPG cells and significantly extended survival in DIPG xenograft models, while restoring H3K27me3 levels. Moreover, the loss of H3K9 methyltransferase SUV39H1 inhibited DIPG cell growth. Transcriptomic and epigenomic profiling indicated that SUV39H1 loss or inhibition led to the downregulation of stemness and oncogenic networks including growth factor receptor signaling and stemness-related programs; however, D2 dopamine receptor (DRD2) signaling adaptively underwent compensatory upregulation conferring resistance. Consistently, a combination of chaetocin treatment with a DRD2 antagonist ONC201 synergistically increased the antitumor efficacy. CONCLUSIONS: Our studies reveal a therapeutic vulnerability of DIPG cells through targeting the SUV39H1-H3K9me3 pathway and compensatory signaling loops for treating this devastating disease. Combining SUV39H1-targeting chaetocin with other agents such as ONC201 may offer a new strategy for effective DIPG treatment.

GABA(A) receptor activation drives GABARAP-Nix mediated autophagy to radiation-sensitize primary and brain-metastatic lung adenocarcinoma tumors.

In non-small cell lung cancer (NSCLC) treatment, targeted therapies benefit only a subset of NSCLC, while radiotherapy responses are not durable and toxicity limits therapy. We find that a GABA(A) receptor activator, AM-101, impairs viability and clonogenicity of NSCLC primary and brain metastatic cells. Employing an ex vivo 'chip', AM-101 is as efficacious as the chemotherapeutic docetaxel, which is used with radiotherapy for advanced-stage NSCLC. In vivo , AM-101 potentiates radiation, including conferring a survival benefit to mice bearing NSCLC intracranial tumors. GABA(A) receptor activation stimulates a selective-autophagic response via multimerization of GABA(A) Receptor-Associated Protein (GABARAP), stabilization of mitochondrial receptor Nix, and utilization of ubiquitin-binding protein p62. A targeted-peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis triggering autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity. Highlights: Activating GABA(A) receptors intrinsic to lung primary and metastatic brain cancer cells triggers a cytotoxic response. GABA(A) receptor activation works as well as chemotherapeutic docetaxel in impairing lung cancer viability ex vivo . GABA(A) receptor activation increases survival of mice bearing lung metastatic brain tumors.A selective-autophagic response is stimulated by GABA(A) receptor activation that includes multimerization of GABARAP and Nix.Employing a new nanomolar affinity peptide that abrogates autophagosome formation inhibits cytotoxicity elicited by GABA(A) receptor activation.

Influence of Dose, Particle Size and Concentration on Dermal Penetration Efficacy of Curcumin.

The influence of size, particle concentration and applied dose (finite vs. infinite dose) on the dermal penetration efficacy of curcumin was investigated in this study. For this, curcumin suspensions with different particle sizes (approx. 20 µm and approx. 250 nm) were produced in different concentrations (0.625-5% (w/w)). The dermal penetration efficacy was determined semi-quantitatively on the ex vivo porcine ear model. The results demonstrated that the presence of particles increases the dermal penetration efficacy of the active compounds being dissolved in the water phase of the formulation. The reason for this is the formation of an aqueous meniscus that develops between particles and skin due to the partial evaporation of water from the vehicle after topical application. The aqueous meniscus contains dissolved active ingredients, and therefore creates a small local spot with a locally high concentration gradient that leads to improved dermal penetration. The increase in penetration efficacy depends on the number of particles in the vehicle, i.e., higher numbers of particles and longer contact times lead to higher penetration efficacy. Therefore, nanocrystals with a high particle concentration were found to be the most suitable formulation principle for efficient and deep dermal penetration of poorly water-soluble active ingredients.

Factors associated with radiation necrosis and intracranial control in patients treated with immune checkpoint inhibitors and stereotactic radiotherapy.

BACKGROUND AND PURPOSE: Emerging data suggest immune checkpoint inhibitors (ICI) and stereotactic radiosurgery (SRS) or radiotherapy (SRT) may work synergistically, potentially increasing both efficacy and toxicity. This manuscript characterizes factors associated with intracranial control and radiation necrosis in this group. MATERIALS AND METHODS: All patients had non-small cell lung cancer, renal cell carcinoma, or melanoma and were treated from 2013 to 2021 at two institutions with ICI and SRS/SRT. Univariate and multivariate analysis were used to analyze factors associated with local failure (LF) and grade 2+ (G2 + ) radiation necrosis. RESULTS: There were 179 patients with 549 metastases. The median follow up from SRS/SRT was 14.7 months and the median tumor size was 7 mm (46 tumors ≥ 20 mm). Rates of LF and G2 + radiation necrosis per metastasis were 5.8% (32/549) and 6.9% (38/549), respectively. LF rates for ICI +/- 1 month from time of radiation versus not were 3% (8/264) and 8% (24/285) (p = 0.01), respectively. G2 + radiation necrosis rates for PD-L1 ≥ 50% versus < 50% were 17% (11/65) and 3% (5/203) (p=<0.001), respectively. PD-L1 ≥ 50% remained significantly associated with G2 + radiation necrosis on multivariate analysis (p = 0.03). Rates of intracranial failure were 54% (80/147) and 17% (4/23) (p = 0.001) for those without and with G2 + radiation necrosis, respectively. CONCLUSIONS: PD-L1 expression (≥50%) may be associated with higher rates of G2 + radiation necrosis, and there may be improved intracranial control following the development of radiation necrosis. Administration of ICIs with SRS/SRT is overall safe, and there may be some local control benefit to delivering these concurrently.

Advanced Age in Humans and Mouse Models of Glioblastoma Show Decreased Survival from Extratumoral Influence.

PURPOSE: Glioblastoma (GBM) is the most common aggressive primary malignant brain tumor in adults with a median age of onset of 68 to 70 years old. Although advanced age is often associated with poorer GBM patient survival, the predominant source(s) of maladaptive aging effects remains to be established. Here, we studied intratumoral and extratumoral relationships between adult patients with GBM and mice with brain tumors across the lifespan. EXPERIMENTAL DESIGN: Electronic health records at Northwestern Medicine and the NCI SEER databases were evaluated for GBM patient age and overall survival. The commercial Tempus and Caris databases, as well as The Cancer Genome Atlas were profiled for gene expression, DNA methylation, and mutational changes with varying GBM patient age. In addition, gene expression analysis was performed on the extratumoral brain of younger and older adult mice with or without a brain tumor. The survival of young and old wild-type or transgenic (INK-ATTAC) mice with a brain tumor was evaluated after treatment with or without senolytics and/or immunotherapy. RESULTS: Human patients with GBM ≥65 years of age had a significantly decreased survival compared with their younger counterparts. While the intra-GBM molecular profiles were similar between younger and older patients with GBM, non-tumor brain tissue had a significantly different gene expression profile between young and old mice with a brain tumor and the eradication of senescent cells improved immunotherapy-dependent survival of old but not young mice. CONCLUSIONS: This work suggests a potential benefit for combining senolytics with immunotherapy in older patients with GBM.

Screening Ion Channels in Cancer Cells.

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development of a broad range of disorders or channelopathies. Cancer cells utilize ion channels to drive their own development, as well as to improve as a tumor and to assimilate in a microenvironment that includes various non-cancerous cells. Furthermore, increases in levels of growth factors and hormones within the tumor microenvironment can result in enhanced ion channel expression, which contributes to cancer cell proliferation and survival. Thus, the pharmacological targeting of ion channels is potentially a promising approach to treating solid malignancies, including primary and metastatic brain cancers. Herein, protocols to characterize the function of ion channels in cancerous cells and approaches to analyze modulators of ion channels to determine their impact on cancer viability are described. These include staining a cell(s) for an ion channel(s), testing the polarized state of mitochondria, establishing ion channel function using electrophysiology, and performing viability assays to assess drug potency.

Advances in Focused Ultrasound for the Treatment of Brain Tumors.

Employing the full arsenal of therapeutics to treat brain tumors is limited by the relative impermeability of the blood-brain and blood-tumor barriers. In physiologic states, the blood-brain barrier serves a protective role by passively and actively excluding neurotoxic compounds; however, this functionality limits the penetrance of therapeutics into the tumor microenvironment. Focused ultrasound technology provides a method for overcoming the blood-brain and blood-tumor barriers through ultrasound frequency to transiently permeabilize or disrupt these barriers. Concomitant delivery of therapeutics has allowed for previously impermeable agents to reach the tumor microenvironment. This review details the advances in focused ultrasound in both preclinical models and clinical studies, with a focus on its safety profile. We then turn towards future directions in focused ultrasound-mediated therapies for brain tumors.

Capicua (CIC) mutations in gliomas in association with MAPK activation for exposing a potential therapeutic target.

Gliomas are the most prevalent neurological cancer in the USA and care modalities are not able to effectively combat these aggressive malignancies. Identifying new, more effective treatments require a deep understanding of the complex genetic variations and relevant pathway associations behind these cancers. Drawing connections between gene mutations with a responsive genetic target can help drive therapy selections to enhance patient survival. We have performed extensive molecular profiling of the Capicua gene (CIC), a tumor and transcriptional suppressor gene, and its mutation prevalence in reference to MAPK activation within clinical glioma tissue. CIC mutations occur far more frequently in oligodendroglioma (52.1%) than in low-grade astrocytoma or glioblastoma. CIC-associated mutations were observed across all glioma subtypes, and MAPK-associated mutations were most prevalent in CIC wild-type tissue regardless of the glioma subtype. MAPK activation, however, was enhanced in CIC-mutated oligodendroglioma. The totality of our observations reported supports the use of CIC as a relevant genetic marker for MAPK activation. Identification of CIC mutations, or lack thereof, can assist in selecting, implementing, and developing MEK/MAPK-inhibitory trials to improve patient outcomes potentially.

Ligand-Gated Ion Channels: Prognostic and Therapeutic Implications for Gliomas.

Gliomas are common primary brain malignancies that remain difficult to treat due to their overall aggressiveness and heterogeneity. Although a variety of therapeutic strategies have been employed for the treatment of gliomas, there is increasing evidence that suggests ligand-gated ion channels (LGICs) can serve as a valuable biomarker and diagnostic tool in the pathogenesis of gliomas. Various LGICs, including P2X, SYT16, and PANX2, have the potential to become altered in the pathogenesis of glioma, which can disrupt the homeostatic activity of neurons, microglia, and astrocytes, further exacerbating the symptoms and progression of glioma. Consequently, LGICs, including purinoceptors, glutamate-gated receptors, and Cys-loop receptors, have been targeted in clinical trials for their potential therapeutic benefit in the diagnosis and treatment of gliomas. In this review, we discuss the role of LGICs in the pathogenesis of glioma, including genetic factors and the effect of altered LGIC activity on the biological functioning of neuronal cells. Additionally, we discuss current and emerging investigations regarding the use of LGICs as a clinical target and potential therapeutic for gliomas.

Cancer stem cell assay-guided chemotherapy improves survival of patients with recurrent glioblastoma in a randomized trial.

Therapy-resistant cancer stem cells (CSCs) contribute to the poor clinical outcomes of patients with recurrent glioblastoma (rGBM) who fail standard of care (SOC) therapy. ChemoID is a clinically validated assay for identifying CSC-targeted cytotoxic therapies in solid tumors. In a randomized clinical trial (NCT03632135), the ChemoID assay, a personalized approach for selecting the most effective treatment from FDA-approved chemotherapies, improves the survival of patients with rGBM (2016 WHO classification) over physician-chosen chemotherapy. In the ChemoID assay-guided group, median survival is 12.5 months (95% confidence interval [CI], 10.2-14.7) compared with 9 months (95% CI, 4.2-13.8) in the physician-choice group (p = 0.010) as per interim efficacy analysis. The ChemoID assay-guided group has a significantly lower risk of death (hazard ratio [HR] = 0.44; 95% CI, 0.24-0.81; p = 0.008). Results of this study offer a promising way to provide more affordable treatment for patients with rGBM in lower socioeconomic groups in the US and around the world.

Lessons learned from evolving frameworks in adult glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant adult brain tumor. Significant effort has been directed to achieve a molecular subtyping of GBM to impact treatment. The discovery of new unique molecular alterations has resulted in a more effective classification of tumors and has opened the door to subtype-specific therapeutic targets. Morphologically identical GBM may have different genetic, epigenetic, and transcriptomic alterations and therefore different progression trajectories and response to treatments. With a transition to molecularly guided diagnosis, there is now a potential to personalize and successfully manage this tumor type to improve outcomes. The steps to achieve subtype-specific molecular signatures can be extrapolated to other neuroproliferative as well as neurodegenerative disorders.