Cancer Explant Models.

Overcoming the challenges of understanding and treating cancer requires reliable patient-derived models of cancer (PDMCs). For decades, cancer research and therapeutic development relied primarily on cancer cell lines because of their prevalence, reproducibility, and simplicity to maintain. However, findings from research conducted in cell lines are rarely recapitulated in vivo and seldom directly translatable to patients. The tumor microenvironment (TME), tumor-stromal interactions, and associations with host immune cells produce profound changes in tumor phenotype and complexity not captured in traditional monolayer cell culture. In this chapter, we present various cancer explant models and discuss their applicability based on specific research aims. We discuss the appropriateness of these models for basic science questions, drug screening/development, and for personalized, precision medicine. We also consider logistical factors such as resource cost, technical difficulty, and accessibility. We finish this chapter with a practical guide intended to help the reader select the cancer explant model system(s) that best address their research aims.

Lipid microsphere bound oxycodone for pain management in patients receiving radiotherapy for head and neck cancer.

BACKGROUND: Wax microsphere bound oxycodone was developed as an abuse-deterrent opioid and maintains a similar pharmacokinetic profile whether administered with or without an intact capsule. We hypothesized that microsphere oxycodone could be utilized for extended release analgesia in patients undergoing radiation (RT) for head-and-neck cancer (HNC) and would not need to be discontinued due to dysphagia or gastrostomy tube dependence. METHODS AND MATERIALS: We performed a prospective trial that enrolled participants > 18 years with histologically confirmed HNC who were scheduled to receive RT. Analgesia was prescribed in accordance with the WHO pain ladder. Microsphere oxycodone was initiated when total daily opioid dose exceeded 30 mg of morphine sulfate equivalent and was titrated weekly during RT. Pain level and effect on quality of life were assessed using the Brief Pain Inventory. The primary feasibility endpoint was frequency of microsphere oxycodone discontinuation within 3 months of RT for reasons other than pain resolution. RESULTS: Twenty-six eligible patients were enrolled. Microsphere oxycodone was initiated in 16 (61.5%) patients. Six (23.1%) patients utilized a gastrostomy tube to administer microsphere oxycodone during all or part of RT. Microsphere oxycodone was discontinued in 1 (7.6%) patient due to perceived inefficacy. No patients were discontinued due to toxicity or difficulty with administration. Ratings for average pain was 3.1 (± 3.4) at enrollment, 4.0 (± 2.4) at week 6 of RT, and 1.8 (± 2.2) at 3-month follow-up. CONCLUSIONS: These results support the feasibility and safety of microsphere oxycodone for extended release analgesia among patients with HNC undergoing RT.

BEERE: a web server for biomedical entity expansion, ranking and explorations.

BEERE (Biomedical Entity Expansion, Ranking and Explorations) is a new web-based data analysis tool to help biomedical researchers characterize any input list of genes/proteins, biomedical terms or their combinations, i.e. 'biomedical entities', in the context of existing literature. Specifically, BEERE first aims to help users examine the credibility of known entity-to-entity associative or semantic relationships supported by database or literature references from the user input of a gene/term list. Then, it will help users uncover the relative importance of each entity-a gene or a term-within the user input by computing the ranking scores of all entities. At last, it will help users hypothesize new gene functions or genotype-phenotype associations by an interactive visual interface of constructed global entity relationship network. The output from BEERE includes: a list of the original entities matched with known relationships in databases; any expanded entities that may be generated from the analysis; the ranks and ranking scores reported with statistical significance for each entity; and an interactive graphical display of the gene or term network within data provenance annotations that link to external data sources. The web server is free and open to all users with no login requirement and can be accessed at http://discovery.informatics.uab.edu/beere/.

Surface guided imaging during stereotactic radiosurgery with automated delivery.

PURPOSE: To report on the use of surface guided imaging during frameless intracranial stereotactic radiotherapy with automated delivery via HyperArcTM (Varian Medical Systems, Palo Alto, CA). METHODS: All patients received intracranial radiotherapy with HyperArcTM and were monitored for intrafraction motion by the AlignRT® (VisionRT, London, UK) surface imaging (SI) system. Immobilization was with the EncompassTM (Qfix, Avondale, PA) aquaplast mask device. AlignRT® log files were correlated with trajectory log files to correlate treatment parameters with SI reported offsets. SI reported offsets were correlated with gantry angle and analyzed for performance issues at non-zero couch angles and during camera-pod blockage during gantry motion. Demographics in the treatment management system were used to identify race and determine if differences in SI reported offsets are due to skin tone settings. RESULTS: A total of 981 fractions were monitored over 14 months and 819 were analyzed. The median AlignRT® reported motion from beginning to the end of treatment was 0.24 mm. The median offset before beam on at non-zero couch angles was 0.55 mm. During gantry motion when camera pods are blocked, the median magnitude was below 1 mm. Median magnitude of offsets at non-zero couch angles was not found to be significantly different for patients stratified by race. CONCLUSIONS: Surface image guidance is a viable alternative to scheduled mid-treatment imaging for monitoring intrafraction motion during stereotactic radiosurgery with automated delivery.

HPAanalyze: an R package that facilitates the retrieval and analysis of the Human Protein Atlas data.

BACKGROUND: The Human Protein Atlas (HPA) aims to map human proteins via multiple technologies including imaging, proteomics and transcriptomics. Access of the HPA data is mainly via web-based interface allowing views of individual proteins, which may not be optimal for data analysis of a gene set, or automatic retrieval of original images. RESULTS: HPAanalyze is an R package for retrieving and performing exploratory analysis of data from HPA. HPAanalyze provides functionality for importing data tables and xml files from HPA, exporting and visualizing data, as well as downloading all staining images of interest. The package is free, open source, and available via Bioconductor and GitHub. We provide examples of the use of HPAanalyze to investigate proteins altered in the deadly brain tumor glioblastoma. For example, we confirm Epidermal Growth Factor Receptor elevation and Phosphatase and Tensin Homolog loss and suggest the importance of the GTP Cyclohydrolase I/Tetrahydrobiopterin pathway. Additionally, we provide an interactive website for non-programmers to explore and visualize data without the use of R. CONCLUSIONS: HPAanalyze integrates into the R workflow with the tidyverse framework, and it can be used in combination with Bioconductor packages for easy analysis of HPA data.

Differential escape mechanisms in cetuximab-resistant head and neck cancer cells.

Acquired cetuximab resistance is a challenge for oncologists treating advanced head and neck carcinoma (HNC). While intrinsic cetuximab resistance mechanism in colorectal cancer is known, resistance in HNC is unclear. We established two different cetuximab resistant HNC cell lines by culturing epidermal growth factor (EGFR) expressing UM-SCC-1 and UM-SCC-6 cell lines in the presence of 5 µg/ml cetuximab. We then explored potential mechanisms of resistance. We found that the 2 cell lines developed resistance by different mechanisms. Specifically, we found that UM-SCC-1 resistant cells (UM-SCC-1R) showed enhanced EGF-induced downstream signals while UM-SCC-6 resistant cells (UM-SCC-6R) demonstrated EGF-independent signaling. Global kinase activity (kinomic) profiling revealed unique signaling differences in the two resistant cell lines. However, both of the resistant lines demonstrated increased phospho-serine 727 and total STAT3 expression compared to the parental lines. STAT3 knockdown promoted increased cytotoxicity both in the presence and absence of cetuximab in the resistant lines suggesting that STAT3 may be a common target in cetuximab resistance.

Optical surface guidance for submillimeter monitoring of patient position during frameless stereotactic radiotherapy.

PURPOSE: To evaluate the accuracy of monitoring intrafraction motion during stereotactic radiotherapy with the optical surface monitoring system. Prior studies showing a false increase in the magnitude of translational offsets at non-coplanar couch positions prompted the vendor to implement software changes. This study evaluated two software improvements intended to address false offsets. METHODS: The vendor implemented two software improvements: a volumetric (ACO) rather than planar calibration and, approximately 6 months later, an improved calibration workflow (CIB) designed to better compensate for thermal drift. Offsets relative to the reference position, obtained at table angle 0 following image-guided setup, were recorded before beam-on at each table position and at the end of treatment the table returned to 0° for patients receiving SRT. RESULTS: Prior to ACO, between ACO and CIB, and after CIB, 223, 155, and 436 fractions were observed respectively. The median magnitude of translational offsets at the end of treatment was similar for all three intervals: 0.29, 0.33, and 0.27 mm. Prior to ACO, the offset magnitude for non-zero table positions had a median of 0.79 mm and was found to increase with increasing distance from isocenter to the anterior patient surface. After ACO, the median magnitude was 0.74 mm, but the dependence on surface-to-isocenter distance was eliminated. After CIB, the median magnitude for non-zero table positions was reduced to 0.57 mm. CONCLUSION: Ongoing improvements in software and calibration procedures have decreased reporting of false offsets at non-zero table angles. However, the median magnitude for non-zero table angles is larger than that observed at the end of treatment, indicating that accuracy remains better when the table is not rotated.

Stable Phenotypic Changes of the Host T Cells Are Essential to the Long-Term Stability of Latent HIV-1 Infection.

UNLABELLED: The extreme stability of the latent HIV-1 reservoir in the CD4(+) memory T cell population prevents viral eradication with current antiretroviral therapy. It has been demonstrated that homeostatic T cell proliferation and clonal expansion of latently infected T cells due to viral integration into specific genes contribute to this extraordinary reservoir stability. Nevertheless, given the constant exposure of the memory T cell population to specific antigen or bystander activation, this reservoir stability seems remarkable, unless it is assumed that latent HIV-1 resides exclusively in memory T cells that recognize rare antigens. Another explanation for the stability of the reservoir could be that the latent HIV-1 reservoir is associated with an unresponsive T cell phenotype. We demonstrate here that host cells of latent HIV-1 infection events were functionally altered in ways that are consistent with the idea of an anergic, unresponsive T cell phenotype. Manipulations that induced or mimicked an anergic T cell state promoted latent HIV-1 infection. Kinome analysis data reflected this altered host cell phenotype at a system-wide level and revealed how the stable kinase activity changes networked to stabilize latent HIV-1 infection. Protein-protein interaction networks generated from kinome data could further be used to guide targeted genetic or pharmacological manipulations that alter the stability of latent HIV-1 infection. In summary, our data demonstrate that stable changes to the signal transduction and transcription factor network of latently HIV-1 infected host cells are essential to the ability of HIV-1 to establish and maintain latent HIV-1 infection status. IMPORTANCE: The extreme stability of the latent HIV-1 reservoir allows the infection to persist for the lifetime of a patient, despite completely suppressive antiretroviral therapy. This extreme reservoir stability is somewhat surprising, since the latently HIV-1 infected CD4(+) memory T cells that form the structural basis of the viral reservoir should be exposed to cognate antigen over time. Antigen exposure would trigger a recall response and should deplete the reservoir, likely over a relatively short period. Our data demonstrate that stable and system-wide phenotypic changes to host cells are a prerequisite for the establishment and maintenance of latent HIV-1 infection events. The changes observed are consistent with an unresponsive, anergy-like T cell phenotype of latently HIV-1 infected host cells. An anergy-like, unresponsive state of the host cells of latent HIV-1 infection events would explain the stability of the HIV-1 reservoir in the face of continuous antigen exposure.

A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium.

Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either ß-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium.

The protective effect of p16(INK4a) in oral cavity carcinomas: p16(Ink4A) dampens tumor invasion-integrated analysis of expression and kinomics pathways.

A large body of evidence shows that p16(INK4a) overexpression predicts improved survival and increased radiosensitivity in HPV-mediated oropharyngeal squamous cell carcinomas.(OPSCC). Here we demonstrate that the presence of transcriptionally active HPV16 in oral cavity squamous cell carcinomas does not correlate with p16(INK4a) overexpression, enhanced local tumor immunity, or improved outcome. It is interesting that HPV-mediated oropharyngeal squamous cell carcinomas can be categorized as having a 'nonaggressive' invasion phenotype, whereas aggressive invasion phenotypes are more common in HPV-negative squamous cell carcinomas. We have developed primary cancer cell lines from resections with known pattern of invasion as determined by our validated risk model. Given that cell lines derived from HPV-mediated oropharyngeal squamous cell carcinomas are less invasive than their HPV-negative counterparts, we tested the hypothesis that viral oncoproteins E6, E7, and p16(INK4a) can affect tumor invasion. Here we demonstrate that p16(INK4a) overexpression in two cancer cell lines (UAB-3 and UAB-4), derived from oral cavity squamous cell carcinomas with the most aggressive invasive phenotype (worst pattern of invasion type 5 (WPOI-5)), dramatically decreases tumor invasiveness by altering expression of extracellular matrix remodeling genes. Pathway analysis integrating changes in RNA expression and kinase activities reveals different potential p16(INK4a)-sensitive pathways. Overexpressing p16(INK4a) in UAB-3 increases EGFR activity and increases MMP1 and MMP3 expression, possibly through STAT3 activation. Overexpressing p16(INK4a) in UAB-4 decreases PDGFR gene expression and reduces MMP1 and MMP3, possibly through STAT3 inactivation. Alternatively, ZAP70/Syk might increase MUC1 phosphorylation, leading to the observed decreased MMP1 expression.

Enhancement of Cetuximab-Induced Radiosensitization by JAK-1 Inhibition.

BACKGROUND: It is known that cetuximab (an epidermal growth factor receptor [EGFr] inhibitor) is a radiosensitizer. Also, cetuximab is known to only partially inhibit the signal transducer and activator of transcription - 3 (STAT-3); a mediator of protection from apoptosis. Studies were performed to determine if the radiosensitizing effects of cetuximab could be enhanced with the addition of an inhibitor of STAT-3. METHODS/RESULTS: The interaction of JAK-STAT-3 inhibition ([JAK1i]; Calbiochem, LaJolla, CA) and EGFr inhibition (cetuximab) was assessed with and without radiation. Four human head and neck cell lines were studied: UM-SCC-1 and UM-SCC-5, and two modified UM-SCC-5 lines; a STAT-3 knockdown line (STAT-3-2.4) and control (NEG-4.17). Exposure to either 0.5 µg/ml of cetuximab or 1 µM JAK1i for 8 or 24 h resulted in reduced activated STAT-3 (immunoblot), and the combination treatment showed greater reduction in activated STAT-3 compared to the individual treatments. The use of either post-radiation JAK1i (1 µM for 72 h) or post-radiation cetuximab (0.5 µg/ml) enhanced radiation-induced anti-proliferative and apoptotic effects but the greatest enhancement was seen when cells were exposed to both JAK1i and cetuximab post-radiation. Similar results were seen for radiosensitization as assessed by colony formation. Finally, the combination treatment of JAK1i (1 µM) and cetuximab (0.5 µg/ml), following radiation, resulted in an increase of unrepaired radiation-induced DNA double strand breaks at 6 and 24 h after radiation compared to the use of post-radiation JAK1i or cetuximab alone as delineated by neutral comet assay. CONCLUSIONS: These findings suggest that dual inhibition of EGFr (cetuximab) and JAK-STAT-3 (JAK1i) leads to greater radiosensitization than with either cetuximab or JAK1i alone and suggests that this combination treatment may be clinically relevant even for tumors with a marked range of STAT-3 activity.

Explorative Discovery of Gene Signatures and Clinotypes in Glioblastoma Cancer Through GeneTerrain Knowledge Map Representation.

This study introduces the GeneTerrain Knowledge Map Representation (GTKM), a novel method for visualizing gene expression data in cancer research. GTKM leverages protein-protein interactions to graphically display differentially expressed genes (DEGs) on a 2-dimensional contour plot, offering a more nuanced understanding of gene interactions and expression patterns compared to traditional heatmap methods. The research demonstrates GTKM's utility through four case studies on glioblastoma (GBM) datasets, focusing on survival analysis, subtype identification, IDH1 mutation analysis, and drug sensitivities of different tumor cell lines. Additionally, a prototype website has been developed to showcase these findings, indicating the method's adaptability for various cancer types. The study reveals that GTKM effectively identifies gene patterns associated with different clinical outcomes in GBM, and its profiles enable the identification of sub-gene signature patterns crucial for predicting survival. The methodology promises significant advancements in precision medicine, providing a powerful tool for understanding complex gene interactions and identifying potential therapeutic targets in cancer treatment.

Exploring Gut Microbiota Alterations with Trimethoprim-Sulfamethoxazole and Dexamethasone in a Humanized Microbiome Mouse Model.

Along with the standard therapies for glioblastoma, patients are commonly prescribed trimethoprim-sulfamethoxazole (TMP-SMX) and dexamethasone for preventing infections and reducing cerebral edema, respectively. Because the gut microbiota impacts the efficacy of cancer therapies, it is important to understand how these medications impact the gut microbiota of patients. Using mice that have been colonized with human microbiota, this study sought to examine how TMP-SMX and dexamethasone affect the gut microbiome. Two lines of humanized microbiota (HuM) Rag1-/- mice, HuM1Rag and HuM2Rag, were treated with either TMP-SMX or dexamethasone via oral gavage once a day for a week. Fecal samples were collected pre-treatment (pre-txt), one week after treatment initiation (1 wk post txt), and three weeks post-treatment (3 wk post txt), and bacterial DNA was analyzed using 16S rRNA-sequencing. The HuM1Rag mice treated with TMP-SMX had significant shifts in alpha diversity, beta diversity, and functional pathways at all time points, whereas in the HuM2Rag mice, it resulted in minimal changes in the microbiome. Likewise, dexamethasone treatment resulted in significant changes in the microbiome of the HuM1Rag mice, whereas the microbiome of the HuM2Rag mice was mostly unaffected. The results of our study show that routine medications used during glioblastoma treatment can perturb gut microbiota, with some microbiome compositions being more sensitive than others, and these treatments could potentially affect the overall efficacy of standard-of-care therapy.

Patient-Derived Models of Cancer in the NCI PDMC Consortium: Selection, Pitfalls, and Practical Recommendations.

For over a century, early researchers sought to study biological organisms in a laboratory setting, leading to the generation of both in vitro and in vivo model systems. Patient-derived models of cancer (PDMCs) have more recently come to the forefront of preclinical cancer models and are even finding their way into clinical practice as part of functional precision medicine programs. The PDMC Consortium, supported by the Division of Cancer Biology in the National Cancer Institute of the National Institutes of Health, seeks to understand the biological principles that govern the various PDMC behaviors, particularly in response to perturbagens, such as cancer therapeutics. Based on collective experience from the consortium groups, we provide insight regarding PDMCs established both in vitro and in vivo, with a focus on practical matters related to developing and maintaining key cancer models through a series of vignettes. Although every model has the potential to offer valuable insights, the choice of the right model should be guided by the research question. However, recognizing the inherent constraints in each model is crucial. Our objective here is to delineate the strengths and limitations of each model as established by individual vignettes. Further advances in PDMCs and the development of novel model systems will enable us to better understand human biology and improve the study of human pathology in the lab.

Tumor Treating Fields Alter the Kinomic Landscape in Glioblastoma Revealing Therapeutic Vulnerabilities.

Treatment for the deadly brain tumor glioblastoma (GBM) has been improved through the non-invasive addition of alternating electric fields, called tumor treating fields (TTFields). Improving both progression-free and overall survival, TTFields are currently approved for treatment of recurrent GBMs as a monotherapy and in the adjuvant setting alongside TMZ for newly diagnosed GBMs. These TTFields are known to inhibit mitosis, but the full molecular impact of TTFields remains undetermined. Therefore, we sought to understand the ability of TTFields to disrupt the growth patterns of and induce kinomic landscape shifts in TMZ-sensitive and -resistant GBM cells. We determined that TTFields significantly decreased the growth of TMZ-sensitive and -resistant cells. Kinomic profiling predicted kinases that were induced or repressed by TTFields, suggesting possible therapy-specific vulnerabilities. Serving as a potential pro-survival mechanism for TTFields, kinomics predicted the increased activity of platelet-derived growth-factor receptor alpha (PDGFRα). We demonstrated that the addition of the PDGFR inhibitor, crenolanib, to TTFields further reduced cell growth in comparison to either treatment alone. Collectively, our data suggest the efficacy of TTFields in vitro and identify common signaling responses to TTFields in TMZ-sensitive and -resistant populations, which may support more personalized medicine approaches.

Correction: Chen et al. Targeted Extracellular Vesicles Delivered Verrucarin A to Treat Glioblastoma. Biomedicines 2022, 10, 130.

In the original publication [...].

Understanding the Effect of Prescription Isodose in Single-Fraction Stereotactic Radiosurgery on Plan Quality and Clinical Outcomes for Solid Brain Metastases.

BACKGROUND AND OBJECTIVES: There is wide variation in treatment planning strategy for central nervous system (CNS) stereotactic radiosurgery. We sought to understand what relationships exist between intratumor maximum dose and local control (LC) or CNS toxicity, and dosimetric effects of constraining hotspots on plan quality of multiple metastases volumetric modulated arc therapy radiosurgery plans. METHODS: We captured brain metastases from 2015 to 2017 treated with single-isocenter volumetric modulated arc therapy radiosurgery. Included tumors received single-fraction stereotactic radiosurgery, had no previous surgery or radiation, and available follow-up imaging. Our criterion for local failure was 25% increase in tumor diameter on follow-up MRI or pathologic confirmation of tumor recurrence. We defined significant CNS toxicity as Radiation Therapy Oncology Group irreversible Grade 3 or higher. We performed univariate and multivariate analyses evaluating factors affecting LC. We examined 10 stereotactic radiosurgery plans with prescriptions of 18 Gy to all targets originally planned without constraints on the maximum dose within the tumor. We replanned each with a constraint of Dmax 120%. We compared V50%, mean brain dose, and Dmax between plans. RESULTS: Five hundred and thirty tumors in 116 patients were available for analysis. Median prescription dose was 18 Gy, and median prescription isodose line (IDL) was 73%. Kaplan-Meier estimate of 12-month LC only tumor volume (HR 1.43 [1.22-1.68] P < .001) was predictive of local failure on univariate analysis; prescription IDL and histology were not. In multivariate analysis, tumor volume impacted local failure (HR 1.43 [1.22-1.69] P < .001) but prescription IDL did not (HR 0.95 [0.86-1.05] P = .288). Only a single grade 3 and 2 grade 4 toxicities were observed; tumor volume was predictive of CNS toxicity (HR 1.58 [1.25-2.00]; P < .001), whereas prescription IDL was not (HR 1.01 [0.87-1.17] P = .940). CONCLUSION: The prescription isodose line had no impact on local tumor control or CNS toxicity. Penalizing radiosurgery hotspots resulted in worse radiosurgery plans with poorer gradient. Limiting maximum dose in gross tumor causes increased collateral exposure to surrounding tissue and should be avoided.

Carmofur prevents cell cycle progression by reducing E2F8 transcription in temozolomide-resistant glioblastoma cells.

Sphingolipid metabolism is dysregulated in many cancers, allowing cells to evade apoptosis through increased sphingosine-1-phosphate (S1P) and decreased ceramides. Ceramidases hydrolyze ceramides to sphingosine, which is phosphorylated by sphingosine kinases to generate S1P. The S1P allows cells to evade apoptosis by shifting the equilibrium away from ceramides, which favor cell death. One tumor type that exhibits a shift in the sphingolipid balance towards S1P is glioblastoma (GBM), a highly aggressive brain tumor. GBMs almost always recur despite surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Understanding sphingolipid metabolism in GBM is still limited, and currently, there are no approved treatments to target dysregulation of sphingolipid metabolism in GBM. Carmofur, a derivative of 5-fluorouracil, inhibits acid ceramidase (ASAH1), a key enzyme in the production of S1P, and is in use outside the USA to treat colorectal cancer. We find that the mRNA for ASAH1, but not other ceramidases, is elevated in recurrent GBM. When TMZ-resistant GBM cells were treated with carmofur, decreased cell growth and increased apoptosis were observed along with cell cycle perturbations. RNA-sequencing identified decreases in cell cycle control pathways that were specific to TMZ-resistant cells. Furthermore, the transcription factor and G1 to S phase regulator, E2F8, was upregulated in TMZ-resistant versus parental GBM cells and inhibited by carmofur treatment in TMZ-resistant GBM cells, specifically. These data suggest a possible role for E2F8 as a mediator of carmofur effects in the context of TMZ resistance. These data suggest the potential utility of normalizing the sphingolipid balance in the context of recurrent GBM.

Using 3D-bioprinted models to study pediatric neural crest-derived tumors.

The use of three-dimensional (3D) bioprinting has remained at the forefront of tissue engineering and has recently been employed for generating bioprinted solid tumors to be used as cancer models to test therapeutics. In pediatrics, neural crest-derived tumors are the most common type of extracranial solid tumors. There are only a few tumor-specific therapies that directly target these tumors, and the lack of new therapies remains detrimental to improving the outcomes for these patients. The absence of more efficacious therapies for pediatric solid tumors, in general, may be due to the inability of the currently employed preclinical models to recapitulate the solid tumor phenotype. In this study, we utilized 3D bioprinting to generate neural crest-derived solid tumors. The bioprinted tumors consisted of cells from established cell lines and patient-derived xenograft tumors mixed with a 6% gelatin/1% sodium alginate bioink. The viability and morphology of the bioprints were analyzed via bioluminescence and immunohisto chemistry, respectively. We compared the bioprints to traditional twodimensional (2D) cell culture under conditions such as hypoxia and therapeutics. We successfully produced viable neural crest-derived tumors that retained the histology and immunostaining characteristics of the original parent tumors. The bioprinted tumors propagated in culture and grew in orthotopic murine models. Furthermore, compared to cells grown in traditional 2D culture, the bioprinted tumors were resistant to hypoxia and chemotherapeutics, suggesting that the bioprints exhibited a phenotype that is consistent with that seen clinically in solid tumors, thus potentially making this model superior to traditional 2D culture for preclinical investigations. Future applications of this technology entail the potential to rapidly print pediatric solid tumors for use in high-throughput drug studies, expediting the identification of novel, individualized therapies.

Targeted Extracellular Vesicles Delivered Verrucarin A to Treat Glioblastoma.

Glioblastomas, accounting for approximately 50% of gliomas, comprise the most aggressive, highly heterogeneous, and malignant brain tumors. The objective of this study was to develop and evaluate a new targeted therapy, i.e., highly potent natural compound verrucarin A (Ver-A), delivered with monoclonal antibody-directed extracellular vesicle (mAb-EV). First, the high surface expression of epidermal growth factor receptor (EGFR) in glioblastoma patient tissue and cell lines was confirmed using immunohistochemistry staining, flow cytometry, and Western blotting. mAb-EV-Ver-A was constructed by packing Ver-A and tagging anti-EGFR mAb to EV generated from HEK293F culture. Confocal microscopy and the In Vivo Imaging System demonstrated that mAb-EV could penetrate the blood-brain barrier, target intracranial glioblastoma xenografts, and deliver drug intracellularly. The in vitro cytotoxicity study showed IC50 values of 2-12 nM of Ver-A. The hematoxylin and eosin staining of major organs in the tolerated dose study indicated minimal systemic toxicity of mAb-EV-Ver-A. Finally, the in vivo anti-tumor efficacy study in intracranial xenograft models demonstrated that EGFR mAb-EV-Ver-A effectively inhibited glioblastoma growth, but the combination with VEGF mAb did not improve the therapeutic efficacy. This study suggested that mAb-EV is an effective drug delivery vehicle and natural Ver-A has great potential to treat glioblastoma.