Collagen deposition within brain metastases is associated with leptomeningeal failure after cavity-directed radiosurgery.

Background: Leptomeningeal failure (LMF) represents a devastating progression of disease following resection of brain metastases (BrM). We sought to identify a biomarker at time of BrM resection that predicts for LMF using mass spectrometry-based proteomic analysis of resected BrM and to translate this finding with histochemical assays. Methods: We retrospectively reviewed 39 patients with proteomic data available from resected BrM. We performed an unsupervised analysis with false discovery rate adjustment (FDR) to compare proteomic signature of BrM from patients that developed LMF versus those that did not. Based on proteomic analysis, we applied trichrome stain to a total of 55 patients who specifically underwent resection and adjuvant radiosurgery. We used competing risks regression to assess predictors of LMF. Results: Of 39 patients with proteomic data, FDR revealed type I collagen-alpha-1 (COL1A1, P = .045) was associated with LMF. The degree of trichrome stain in each block correlated with COL1A1 expression (ß = 1.849, P = .001). In a cohort of 55 patients, a higher degree of trichrome staining was associated with an increased hazard of LMF in resected BrM (Hazard Ratio 1.58, 95% CI 1.11-2.26, P = .01). Conclusion: The degree of trichrome staining correlated with COL1A1 and portended a higher risk of LMF in patients with resected brain metastases treated with adjuvant radiosurgery. Collagen deposition and degree of fibrosis may be able to serve as a biomarker for LMF.

A transformer-based deep-learning approach for classifying brain metastases into primary organ sites using clinical whole-brain MRI images.

Treatment decisions for brain metastatic disease rely on knowledge of the primary organ site and are currently made with biopsy and histology. Here, we develop a deep-learning approach for accurate non-invasive digital histology with whole-brain magnetic resonance imaging (MRI) data. Contrast-enhanced T1-weighted and fast spoiled gradient echo brain MRI exams (n = 1,582) were preprocessed and input to the proposed deep-learning workflow for tumor segmentation, modality transfer, and primary site classification into one of five classes. Tenfold cross-validation generated an overall area under the receiver operating characteristic curve (AUC) of 0.878 (95% confidence interval [CI]: 0.873,0.883). These data establish that whole-brain imaging features are discriminative enough to allow accurate diagnosis of the primary organ site of malignancy. Our end-to-end deep radiomic approach has great potential for classifying metastatic tumor types from whole-brain MRI images. Further refinement may offer an invaluable clinical tool to expedite primary cancer site identification for precision treatment and improved outcomes.

Advancements in drug delivery methods for the treatment of brain disease.

The blood-brain barrier (BBB) presents a formidable obstacle to the effective delivery of systemically administered pharmacological agents to the brain, with ~5% of candidate drugs capable of effectively penetrating the BBB. A variety of biomaterials and therapeutic delivery devices have recently been developed that facilitate drug delivery to the brain. These technologies have addressed many of the limitations imposed by the BBB by: (1) designing or modifying the physiochemical properties of therapeutic compounds to allow for transport across the BBB; (2) bypassing the BBB by administration of drugs via alternative routes; and (3) transiently disrupting the BBB (BBBD) using biophysical therapies. Here we specifically review colloidal drug carrier delivery systems, intranasal, intrathecal, and direct interstitial drug delivery methods, focused ultrasound BBBD, and pulsed electrical field induced BBBD, as well as the key features of BBB structure and function that are the mechanistic targets of these approaches. Each of these drug delivery technologies are illustrated in the context of their potential clinical applications and limitations in companion animals with naturally occurring intracranial diseases.

Solid Fiber Inside of Capillary and Modified Fusion-Spliced Fiber Optic Microneedle Devices for Improved Light Transmission Efficiency.

Convection-enhanced delivery (CED) is a drug delivery technique used to deliver therapeutics directly to the brain and is a continually evolving technique to treat glioblastoma. Early versions of CED have proven to result in inadequate drug volume dispersed (Vd), increasing the likelihood of tumor recurrence. Fiber optic microneedle devices (FMDs) with the ability to deliver fluid and thermal energy simultaneously have shown an ability to increase Vd, but FMDs have historically had low light transmission efficiency. In this study, we present a new fabrication method, solid fiber inside capillary (SFIC) FMD, and a modified fusion splicing (FS) method with the goal of increasing light delivery efficiency. The modified FS FMD resulted in an increase in light transmission efficiency between 49% and 173% compared to previous prototypes. However, the FS FMD resulted in significantly lower transmission efficiencies compared to the SFIC FMD (p ≤ 0.04) and FS FMDs perform much worse when light-absorptive materials, like black dye, are placed in the bore. The light absorption of a candidate cytotoxic agent, QUAD-CTX, appear to be similar to water, and light delivery through FS FMDs filled with QUAD-CTX achieves a transmission efficiency of 85.6 ± 5.4%. The fabrication process of the SFIC FMDs results in extremely fragile FMDs. Therefore, the use of a modified FS FMD fabrication process appears to be better suited for balancing the desire to increase light transmission efficiency while retaining a sturdy FMD construction.

Clinical outcomes of dose-escalated re-irradiation in patients with recurrent high-grade glioma.

Background: Re-irradiation for recurrent gliomas is a controversial treatment option with no clear standard dose or concurrent systemic therapy. Methods: This series represents a single-institution retrospective review of patients treated with re-irradiation for recurrent high-grade glioma. After 2012, patients were commonly offered concurrent bevacizumab as a cytoprotective agent against radiation necrosis. Kaplan-Meier method was used to estimate overall survival and progression-free survival. Cox proportional hazards regression was used to identify factors associated with overall survival and progression-free survival. Results: Between 2001 and 2021, 52 patients underwent re-irradiation for a diagnosis of recurrent high-grade glioma. 36 patients (69.2%) had a histologic diagnosis of glioblastoma at the time of re-irradiation. The median BED10 (biological equivalent dose 10 Gy) of re-irradiation was 53.1 Gy. Twenty-one patients (40.4%) received concurrent bevacizumab with re-irradiation. Median survival for the entire cohort and for glioblastoma at the time of recurrence patients was 6.7 months and 6.0 months, respectively. For patients with glioblastoma at the time of recurrence, completing re-irradiation (HR 0.03, P < .001), use of concurrent bevacizumab (HR 0.3, P = .009), and the BED10 (HR 0.9, P = .005) were predictive of overall survival. Nine patients developed grade 3-5 toxicity; of these, 2 received concurrent bevacizumab and 7 did not (P = .15). Conclusion: High dose re-irradiation with concurrent bevacizumab is feasible in patients with recurrent gliomas. Concurrent bevacizumab and increasing radiation dose may improve survival in patients with recurrent glioblastoma.

Metal-Based Nanostructured Therapeutic Strategies for Glioblastoma Treatment-An Update.

Glioblastoma (GBM) is the most commonly diagnosed and most lethal primary malignant brain tumor in adults. Standard treatments are ineffective, and despite promising results obtained in early phases of experimental clinical trials, the prognosis of GBM remains unfavorable. Therefore, there is need for exploration and development of innovative methods that aim to establish new therapies or increase the effectiveness of existing therapies. One of the most exciting new strategies enabling combinatory treatment is the usage of nanocarriers loaded with chemotherapeutics and/or other anticancer compounds. Nanocarriers exhibit unique properties in antitumor therapy, as they allow highly efficient drug transport into cells and sustained intracellular accumulation of the delivered cargo. They can be infused into and are retained by GBM tumors, and potentially can bypass the blood-brain barrier. One of the most promising and extensively studied groups of nanostructured therapeutics are metal-based nanoparticles. These theranostic nanocarriers demonstrate relatively low toxicity, thus they might be applied for both diagnosis and therapy. In this article, we provide an update on metal-based nanostructured constructs in the treatment of GBM. We focus on the interaction of metal nanoparticles with various forms of electromagnetic radiation for use in photothermal, photodynamic, magnetic hyperthermia and ionizing radiation sensitization applications.

Comparison of linear and volumetric criteria for the determination of therapeutic response in dogs with intracranial gliomas.

BACKGROUND: Brain tumor therapeutic responses can be quantified from magnetic resonance images (MRI) using 1- (1D) and 2-dimensional (2D) linear and volumetric methods, but few studies in dogs compare these techniques. HYPOTHESES: Linear methods will be obtained faster, but have less agreement than volumetric measurements. Therapeutic response agreement will be highest with the total T2W tumor volumetric (TTV) method. Therapeutic response at 6-weeks will correlate with overall survival (OS). ANIMALS: Forty-six dogs with intracranial gliomas. METHODS: Prospective study. Three raters measured tumors using 1D and 2D linear, TTV, and contrast-enhancing volumetric (CEV) techniques on 143 brain MRI to determine agreement between methods, define therapeutic responses, and assess relations with OS. RESULTS: Raters performed 1D the fastest (2.9 ± 0.57 minutes) and CEV slowest (17.8 ± 6.2 minutes). Inter- and intraobserver agreements were excellent (intraclass correlations ≥.91) across methods. Correlations between linear (1D vs 2D; ρ > .91) and volumetric (TTV vs CEV; ρ > .73) methods were stronger than linear to volumetric comparisons (ρ range, .26-.59). Incorporating clinical and imaging data resulted in fewer discordant therapeutic responses across methods. Dogs having partial tumor responses at 6 weeks had a lower death hazard than dogs with stable or progressive disease when assessed using 2D, CEV, and TTV (hazard ration 2.1; 95% confidence interval, 1.22-3.63; P = .008). CONCLUSIONS AND CLINICAL IMPORTANCE: One-dimensional, 2D, CEV, and TTV are comparable for determining therapeutic response. Given the simplicity, universal applicability, and superior performance of the TTV, we recommend its use to standardize glioma therapeutic response criteria.

NEDD4 degrades TUSC2 to promote glioblastoma progression.

Whether tumor suppressor candidate 2 (TUSC2) plays an important role in glioblastoma (GBM) progression is largely unknown. Whether TUSC2 undergoes polyubiquitination is unknown. Herein, we report that TUSC2 protein expression is reduced/lost in GBM compared to normal brain due to protein destabilization; TUSC2 mRNA is equally expressed in both tissues. NEDD4 E3 ubiquitin ligase polyubiquitinates TUSC2 at residue K71, and the TUSC2-K71R mutant is resistant to NEDD4-mediated proteasomal degradation. Analysis of GBM specimens showed NEDD4 protein is highly expressed in GBM and the level is inversely correlated with TUSC2 protein levels. Furthermore, TUSC2 restoration induces apoptosis and inhibits patient-derived glioma stem cells (PD-GSCs) in vitro and in vivo. Conversely, TUSC2-knockout promotes PD-GSCs in vitro and in vivo. RNA-Seq analysis and subsequent validations showed GBM cells with TUSC2-knockout expressed increased Bcl-xL and were more resistant to apoptosis induced by a Bcl-xL-specific BH3 mimetic. A TUSC2-knockout gene signature created from the RNA-seq data predicts poor patient survival. Together, these findings establish that NEDD4-mediated polyubiquitination is a novel mechanism for TUSC2 degradation in GBM and that TUSC2 loss promotes GBM progression in part through Bcl-xL upregulation.

HSP90-CDC37 functions as a chaperone for the oncogenic FGFR3-TACC3 fusion.

The FGFR3-TACC3 (F3-T3) fusion gene was discovered as an oncogenic molecule in glioblastoma and bladder cancers, and has subsequently been found in many cancer types. Notably, F3-T3 was found to be highly expressed in both untreated and matched recurrence glioblastoma under the concurrent radiotherapy and temozolomide (TMZ) treatment, suggesting that targeting F3-T3 is a valid strategy for treatment. Here, we show that the F3-T3 protein is a client of heat shock protein 90 (HSP90), forming a ternary complex with the cell division cycle 37 (CDC37). Deprivation of HSP90 or CDC37 disrupts the formation of the ternary complex, which destabilizes glycosylated F3-T3, and thereby suppresses F3-T3 oncogenic activity. Gliomas harboring F3-T3 are resistant to TMZ chemotherapy. HSP90 inhibitors sensitized F3-T3 glioma cells to TMZ via the inhibition of F3-T3 activation and potentiated TMZ-induced DNA damage. These results demonstrate that F3-T3 oncogenic function is dependent on the HSP90 chaperone system and suggests a new clinical option for targeting this genetic aberration in cancer.

Diversity in responses to oncolytic Lassa-vesicular stomatitis virus in patient-derived glioblastoma cells.

The difficulty of glioblastoma treatment makes it a good candidate for novel therapies, such as oncolytic viruses. Vesicular stomatitis virus expressing Lassa virus glycoprotein (Lassa-VSV) showed significant promise in animal models using established glioblastoma cell lines. These experiments were to determine the susceptibility of low-passage, patient-derived cell lines to Lassa-VSV oncolysis. Four patient-derived glioblastoma cell lines were infected with Lassa-VSV that expresses green fluorescent protein (GFP) and analyzed by fluorescence microscopy, flow cytometry, and cell viability assays. Cells were also analyzed as tumorspheres containing primarily glioma stem-like cells. Three low-passage, patient-derived cells were further analyzed with RNA sequencing (RNA-seq). Individual cell lines varied somewhat in their levels of viral gene expression and time course of Lassa-VSV-induced cell death, but each was susceptible to Lassa-VSV. Brain Tumor Center of Excellence (BTCOE) 4765 cells had the highest level of expression of interferon-stimulated genes but were most susceptible to Lassa-VSV-induced cell death, indicating that more susceptible cells do not necessarily have lower interferon pathway activation. Cells cultured as tumorspheres and infected with Lassa-VSV also showed variable susceptibility to Lassa-VSV, but BTCOE 4765 cells were least susceptible. Thus, patient-derived brain tumor cells show variable responses to Lassa-VSV infection, but each of the lines was susceptible to VSV oncolysis.

The Presence and Potential Role of ALDH1A2 in the Glioblastoma Microenvironment.

Glioblastoma (GBM) is the most aggressive malignant glioma. Therapeutic targeting of GBM is made more difficult due to its heterogeneity, resistance to treatment, and diffuse infiltration into the brain parenchyma. Better understanding of the tumor microenvironment should aid in finding more effective management of GBM. GBM-associated macrophages (GAM) comprise up to 30% of the GBM microenvironment. Therefore, exploration of GAM activity/function and their specific markers are important for developing new therapeutic agents. In this study, we identified and evaluated the expression of ALDH1A2 in the GBM microenvironment, and especially in M2 GAM, though it is also expressed in reactive astrocytes and multinucleated tumor cells. We demonstrated that M2 GAM highly express ALDH1A2 when compared to other ALDH1 family proteins. Additionally, GBM samples showed higher expression of ALDH1A2 when compared to low-grade gliomas (LGG), and this expression was increased upon tumor recurrence both at the gene and protein levels. We demonstrated that the enzymatic product of ALDH1A2, retinoic acid (RA), modulated the expression and activity of MMP-2 and MMP-9 in macrophages, but not in GBM tumor cells. Thus, the expression of ALDH1A2 may promote the progressive phenotype of GBM.

Phase I trial of convection-enhanced delivery of IL13RA2 and EPHA2 receptor targeted cytotoxins in dogs with spontaneous intracranial gliomas.

BACKGROUND: The interleukin-13 receptor alpha 2 (IL13RA2) and ephrin type A receptor 2 (EPHA2) are attractive therapeutic targets, being expressed in ~90% of canine and human gliomas, and absent in normal brain. Clinical trials using an earlier generation IL-13 based cytotoxin showed encouraging clinical effects in human glioma, but met with technical barriers associated with the convection-enhanced delivery (CED) method. In this study, IL-13 mutant and ephrin A1 (EFNA1)-based bacterial cytotoxins targeted to IL13RA2 and EPHA2 receptors, respectively, were administered locoregionally by CED to dogs with intracranial gliomas to evaluate their safety and preliminary efficacy. METHODS: In this phase I, 3 + 3 dose escalation trial, cytotoxins were infused by CED in 17 dogs with gliomas expressing IL13RA2 or EPHA2 receptors. CED was performed using a shape-fitting therapeutic planning algorithm, reflux-preventing catheters, and real-time intraoperative MRI monitoring. The primary endpoint was to determine the maximum tolerated dose of the cytotoxic cocktail in dogs with gliomas. RESULTS: Consistent intratumoral delivery of the cytotoxic cocktail was achieved, with a median target coverage of 70% (range, 40-94%). Cytotoxins were well tolerated over a dose range of 0.012-1.278 µg/mL delivered to the target volume (median, 0.099 µg/mL), with no dose limiting toxicities observed. Objective tumor responses, up to 94% tumor volume reduction, were observed in 50% (8/16) of dogs, including at least one dog in each dosing cohort >0.05 µg/mL. CONCLUSIONS: This study provides preclinical data fundamental to the translation of this multireceptor targeted therapeutic approach to the human clinic.

Multireceptor targeting of glioblastoma.

BACKGROUND: Treatment for glioblastoma (GBM) remains an unmet need in medicine. Novel therapies that address GBM complexity and heterogeneity in particular are warranted. To this end, we target 4 tumor-associated receptors at a time that span virtually all of the GBM microenvironment including bulk tumor cells, infiltrating tumor cells, neovasculature, and tumor-infiltrating cells with one pharmaceutical agent delivering a cytotoxic load. METHODS: We engineered multivalent ligand-based vector proteins termed QUAD with an ability to bind to 4 of the following GBM-associated receptors: IL-13RA2, EphA2, EphA3, and EphB2. We conjugated QUAD with a modified bacterial toxin PE38QQR and tested it in vitro and in vivo. RESULTS: The QUAD variants preserved functional characteristics of the respective ligands for the 4 receptors. The QUAD 3.0 variant conjugate was highly cytotoxic to GBM cells, but it was nontoxic in mice, and the conjugate exhibited strong antitumor effect in a dog with spontaneous GBM. CONCLUSION: The QUAD addresses, to a large extent, the issues of intra- and intertumoral heterogeneity and, at the same time, it targets several pathophysiologically important tumor compartments in GBM through multiple receptors overexpressed in tumors allowing for what we call "molecular resection." QUAD-based targeted agents warrant further pre- and clinical development.

Attenuating hypoxia driven malignant behavior in glioblastoma with a novel hypoxia-inducible factor 2 alpha inhibitor.

Hypoxia inducible factor (HIFs) signaling contributes to malignant cell behavior in glioblastoma (GBM). We investigated a novel HIF2α inhibitor, PT2385, both in vitro, with low-passage patient-derived cell lines, and in vivo, using orthotopic models of glioblastoma. We focused on analysis of HIF2α expression in situ, cell survival/proliferation, and survival in brain tumor-bearing mice treated with PT2385 alone and in combination with standard of care chemoradiotherapy. HIF2α expression increased with glioma grade, with over half of GBM specimens HIF2α positive. Staining clustered in perivascular and perinecrotic tumor regions. Cellular phenotype including proliferation, viability, migration/invasion, and also gene expression were not altered after PT2385 treatment. In the animal model, PT2385 single-agent treatment did improve median overall survival compared to placebo (p = 0.04, n = 21) without a bioluminescence correlate (t = 0.67, p = 0.52). No difference in animal survival was seen in combination treatment with radiation (RT)/temozolomide (TMZ)/PT2385 (p = 0.44, n = 10) or mean tumor bioluminescence (t 1.13, p = 0.32). We conclude that HIF2α is a reasonable novel therapeutic target as expressed in the majority of glioblastomas in our cohort. PT2385 as a single-agent was efficacious in vivo, however, an increase in animal survival was not seen with PT2385 in combination with RT/TMZ. Further study for targeting HIF2α as a therapeutic approach in GBM is warranted.

ß2-adrenoreceptor Signaling Increases Therapy Resistance in Prostate Cancer by Upregulating MCL1.

There is accumulating evidence that continuous activation of the sympathetic nervous system due to psychosocial stress increases resistance to therapy and accelerates tumor growth via ß2-adrenoreceptor signaling (ADRB2). However, the effector mechanisms appear to be specific to tumor type. Here we show that activation of ADRB2 by epinephrine, increased in response to immobilization stress, delays the loss of MCL1 apoptosis regulator (MCL1) protein expression induced by cytotoxic drugs in prostate cancer cells; and thus, increases resistance of prostate cancer xenografts to cytotoxic therapies. The effect of epinephrine on MCL1 protein depended on protein kinase A (PKA) activity, but was independent from androgen receptor expression. Furthermore, elevated blood epinephrine levels correlated positively with an increased MCL1 protein expression in human prostate biopsies. In summary, we demonstrate that stress triggers an androgen-independent antiapoptotic signaling via the ADRB2/PKA/MCL1 pathway in prostate cancer cells. IMPLICATIONS: Presented results justify clinical studies of ADRB2 blockers as therapeutics and of MCL1 protein expression as potential biomarker predicting efficacy of apoptosis-targeting drugs in prostate cancer.

Expression of the Androgen Receptor Governs Radiation Resistance in a Subset of Glioblastomas Vulnerable to Antiandrogen Therapy.

New approaches are needed to overcome intrinsic therapy resistance in glioblastoma (GBM). Because GBMs exhibit sexual dimorphism and are reported to express steroid hormone receptors, we reasoned that signaling through the androgen receptor (AR) could mediate therapy resistance in GBM, much as it does in AR-positive prostate and breast cancers. We found that nearly half of GBM cell lines, patient-derived xenografts (PDX), and human tumors expressed AR at the transcript and protein level-with expression levels overlapping those of primary prostate cancer. Analysis of gene expression datasets also revealed that AR expression is higher in GBM patient samples than normal brain tissue. Multiple clinical-grade antiandrogens slowed the growth of and radiosensitized AR-positive GBM cell lines and PDXs in vitro and in vivo Antiandrogens blocked the ability of AR-positive GBM PDXs to engage adaptive transcriptional programs following radiation and slowed the repair of radiation-induced DNA damage. These results suggest that combining blood-brain barrier permeable antiandrogens with radiation may have promise for patients with AR-positive GBMs.

Challenges to Successful Implementation of the Immune Checkpoint Inhibitors for Treatment of Glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant glioma, treatment of which has not improved significantly in many years. This is due to the unique challenges that GBM tumors present when designing and implementing therapies. Recently, immunotherapy in the form of immune checkpoint inhibition (ICI) has revolutionized the treatment of various malignancies. The application of immune checkpoint inhibition in GBM treatment has shown promising preclinical results. Unfortunately, this has met with little to no success in the clinic thus far. In this review, we will discuss the challenges presented by GBM tumors that likely limit the effect of ICI and discuss the approaches being tested to overcome these challenges.

Drug Conjugates for Targeting Eph Receptors in Glioblastoma.

Glioblastoma (GBM) is a complex and heterogeneous tumor that warrants a comprehensive therapeutic approach for treatment. Tumor-associated antigens offer an opportunity to selectively target various components of the GBM microenvironment while sparing the normal cells within the central nervous system. In this study, we conjugated a multivalent vector protein, QUAD 3.0, that can target four receptors: EphA3, EphA2, EphB2, and also IL-13RA2, spanning virtually 100% of the GBM microenvironment, to doxorubicin derivatives. The conjugates effectively bound to all four receptors, although to varying degrees, and delivered cytotoxic loads to both established and patient-derived GBM cell lines, with IC50 values in the low nM range. The conjugates were also non-toxic to animals. We anticipate that the QUAD 3.0 Dox conjugates will be further used in preclinical models and possibly clinics in the foreseeable future.