Discrimination Between Pre- and Postcapillary Pulmonary Hypertension Using Platelet RNA.

Background Appropriate treatment of pulmonary hypertension (PH) is critically dependent on accurate discrimination between pre- and postcapillary PH. However, clinical discrimination is challenging and frequently requires a right heart catheterization. Existing risk scores to detect postcapillary PH have suboptimal discriminatory strength. We have previously shown that platelet-derived RNA profiles may have diagnostic value for PH detection. Here, we hypothesize that platelet-derived RNAs can be employed to select unique biomarker panels for the discrimination between pre- and postcapillary PH. Methods and Results Blood platelet RNA from whole blood was isolated and sequenced from 50 patients with precapillary PH (with different PH subtypes) as well as 50 patients with postcapillary PH. RNA panels were calculated by ANOVA statistics, and classifications were performed using a support vector machine algorithm, supported by particle swarm optimization. We identified in total 4279 different RNAs in blood platelets from patients with pre- and postcapillary PH. A particle swarm optimization-selected RNA panel of 1618 distinctive RNAs with differential levels together with a trained support vector machine algorithm accurately discriminated patients with precapillary PH from patients with postcapillary PH with 100% sensitivity, 60% specificity, 80% accuracy, and 0.95 (95% CI, 0.86-1.00) area under the curve in the independent validation series (n=20). Conclusions This proof-of-concept study demonstrates that particle swarm optimization/support vector machine-enhanced classification of platelet RNA panels may be able to discriminate precapillary PH from postcapillary PH. This research provides a foundation for the development of a blood test with a high negative predictive value that would improve early diagnosis of precapillary PH and prevents unnecessary invasive testing in patients with postcapillary PH.

Organotypic-liver slide culture systems to explore the role of extracellular vesicles in pancreatic cancer metastatic behavior and guide new therapeutic approaches.

Introduction: Recent studies suggested that extracellular vesicles (EVs) play a role both in the metastatic niche formation and in the progression of several tumors, including pancreatic cancer. In particular, the effects of EVs on metastasis should be studied in model systems that take into account both the tumor cells and the metastatic site/tumor microenvironment. Studies with labeled EVs or EV-secreting cells in ex vivo models will reflect the physiological and pathological functions of EVs. The organotypic-tissue slide culture systems can fulfill such a role.Areas covered: This review provides an overview of available organotypic-culture slide systems. We specifically focus on the assay system of liver culture-slides in combination with pancreatic tumors, which can be modulated to test the efficacy of new therapeutic approaches.Expert opinion: The intercellular exchange of EVs has emerged as a biologically relevant phenomenon to drive cancer metastasis. However, further models need to be developed to better elucidate the functional roles of EVs. The use of novel organotypic slide culture systems provides the opportunity to explore the role of EVs in the metastatic behavior of pancreatic cancer, decreasing the use of costly and cumbersome organoid or animal models.

Inhalation anesthesia and shielding devices to allow accurate preclinical irradiation of mice with clinical linac-based systems: Design and dosimetric characteristics.

This technical note describes two devices to enable accurate irradiation of mice on clinical linac-based systems. To study the effects of radiation in murine, preclinical animal models, controlled and accurate dosing is important. This is not only important when specific volumes need to be irradiated, but also when the whole animal body is irradiated. To enable both purposes, we designed two devices. One device to administer Total Body Irradiation (TBI) simultaneously to six, free walking mice, and a second device, denoted as target box, in which we irradiate specific parts of the mice whilst organs-at-risk (OAR) are protected. In this latter device, we can position the mice in multiple ways. One configuration allows to sedate twelve mice simultaneously by isoflurane inhalation anesthesia and protect the body by lead shielding to allow radiation of the head only. Alternatively, the target box can be used to sedate maximal 4 mice simultaneously to irradiate the flank or paws only. All these setups allow high experimental throughput and thus a minimal occupation of the clinical equipment. As measured, the delivered radiation dosages in the regions of interest were accurate for both devices. In this technical note, we describe the design and build of these devices.

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.

Impact of hypoxia on chemoresistance of mesothelioma mediated by the proton-coupled folate transporter, and preclinical activity of new anti-LDH-A compounds.

BACKGROUND: Expression of proton-coupled folate transporter (PCFT) is associated with survival of mesothelioma patients treated with pemetrexed, and is reduced by hypoxia, prompting studies to elucidate their correlation. METHODS: Modulation of glycolytic gene expression was evaluated by PCR arrays in tumour cells and primary cultures growing under hypoxia, in spheroids and after PCFT silencing. Inhibitors of lactate dehydrogenase (LDH-A) were tested in vitro and in vivo. LDH-A expression was determined in tissue microarrays of radically resected malignant pleural mesothelioma (MPM, N = 33) and diffuse peritoneal mesothelioma (DMPM, N = 56) patients. RESULTS: Overexpression of hypoxia marker CAIX was associated with low PCFT expression and decreased MPM cell growth inhibition by pemetrexed. Through integration of PCR arrays in hypoxic cells and spheroids and following PCFT silencing, we identified the upregulation of LDH-A, which correlated with shorter survival of MPM and DMPM patients. Novel LDH-A inhibitors enhanced spheroid disintegration and displayed synergistic effects with pemetrexed in MPM and gemcitabine in DMPM cells. Studies with bioluminescent hypoxic orthotopic and subcutaneous DMPM athymic-mice models revealed the marked antitumour activity of the LDH-A inhibitor NHI-Glc-2, alone or combined with gemcitabine. CONCLUSIONS: This study provides novel insights into hypoxia/PCFT-dependent chemoresistance, unravelling the potential prognostic value of LDH-A, and demonstrating the preclinical activity of LDH-A inhibitors.

Combined Therapy of AXL and HDAC Inhibition Reverses Mesenchymal Transition in Diffuse Intrinsic Pontine Glioma.

PURPOSE: Diffuse intrinsic pontine glioma (DIPG) is an incurable type of pediatric brain cancer, which in the majority of cases is driven by mutations in genes encoding histone 3 (H3K27M). We here determined the preclinical therapeutic potential of combined AXL and HDAC inhibition in these tumors to reverse their mesenchymal, therapy-resistant, phenotype. EXPERIMENTAL DESIGN: We used public databases and patient-derived DIPG cells to identify putative drivers of the mesenchymal transition in these tumors. Patient-derived neurospheres, xenografts, and allografts were used to determine the therapeutic potential of combined AXL/HDAC inhibition for the treatment of DIPG. RESULTS: We identified AXL as a therapeutic target and regulator of the mesenchymal transition in DIPG. Combined AXL and HDAC inhibition had a synergistic and selective antitumor effect on H3K27M DIPG cells. Treatment of DIPG cells with the AXL inhibitor BGB324 and the HDAC inhibitor panobinostat resulted in a decreased expression of mesenchymal and stem cell genes. Moreover, this combination treatment decreased expression of DNA damage repair genes in DIPG cells, strongly sensitizing them to radiation. Pharmacokinetic studies showed that BGB324, like panobinostat, crosses the blood-brain barrier. Consequently, treatment of patient-derived DIPG xenograft and murine DIPG allograft-bearing mice with BGB324 and panobinostat resulted in a synergistic antitumor effect and prolonged survival. CONCLUSIONS: Combined inhibition of AXL and HDACs in DIPG cells results in a synergistic antitumor effect by reversing their mesenchymal, stem cell-like, therapy-resistant phenotype. As such, this treatment combination may serve as part of a future multimodal therapeutic strategy for DIPG.

Data-driven prioritization and preclinical evaluation of therapeutic targets in glioblastoma.

BACKGROUND: Patients with glioblastoma (GBM) have a dismal prognosis, and there is an unmet need for new therapeutic options. This study aims to identify new therapeutic targets in GBM. METHODS: mRNA expression data of patient-derived GBM (n = 1279) and normal brain tissue (n = 46) samples were collected from Gene Expression Omnibus and The Cancer Genome Atlas. Functional genomic mRNA profiling was applied to capture the downstream effects of genomic alterations on gene expression levels. Next, a class comparison between GBM and normal brain tissue was performed. Significantly upregulated genes in GBM were further prioritized based on (1) known interactions with antineoplastic drugs, (2) current drug development status in humans, and (3) association with biologic pathways known to be involved in GBM. Antineoplastic agents against prioritized targets were validated in vitro and in vivo. RESULTS: We identified 712 significantly upregulated genes in GBM compared to normal brain tissue, of which 27 have a known interaction with antineoplastic agents. Seventeen of the 27 genes, including EGFR and VEGFA, have been clinically evaluated in GBM with limited efficacy. For the remaining 10 genes, RRM2, MAPK9 (JNK2, SAPK1a), and XIAP play a role in GBM development. We demonstrated for the MAPK9 inhibitor RGB-286638 a viability loss in multiple GBM cell culture models. Although no overall survival benefit was observed in vivo, there were indications that RGB-286638 may delay tumor growth. CONCLUSIONS: The MAPK9 inhibitor RGB-286638 showed promising in vitro results. Furthermore, in vivo target engagement studies and combination therapies with this compound warrant further exploration.

MEK/MELK inhibition and blood-brain barrier deficiencies in atypical teratoid/rhabdoid tumors.

BACKGROUND: Atypical teratoid/rhabdoid tumors (AT/RT) are rare, but highly aggressive. These entities are of embryonal origin occurring in the central nervous system (CNS) of young children. Molecularly these tumors are driven by a single hallmark mutation, resulting in inactivation of SMARCB1 or SMARCA4. Additionally, activation of the MAPK signaling axis and preclinical antitumor efficacy of its inhibition have been described in AT/RT. METHODS: We established and validated a patient-derived neurosphere culture and xenograft model of sonic hedgehog (SHH) subtype AT/RT, at diagnosis and relapse from the same patient. We set out to study the vascular phenotype of these tumors to evaluate the integrity of the blood-brain barrier (BBB) in AT/RT. We also used the model to study combined mitogen-activated protein kinase kinase (MEK) and maternal embryonic leucine zipper kinase (MELK) inhibition as a therapeutic strategy for AT/RT. RESULTS: We found MELK to be highly overexpressed in both patient samples of AT/RT and our primary cultures and xenografts. We identified a potent antitumor efficacy of the MELK inhibitor OTSSP167, as well as strong synergy with the MEK inhibitor trametinib, against primary AT/RT neurospheres. Additionally, vascular phenotyping of AT/RT patient material and xenografts revealed significant BBB aberrancies in these tumors. Finally, we show in vivo efficacy of the non-BBB penetrable drugs OTSSP167 and trametinib in AT/RT xenografts, demonstrating the therapeutic implications of the observed BBB deficiencies and validating MEK/MELK inhibition as a potential treatment. CONCLUSION: Altogether, we developed a combination treatment strategy for AT/RT based on MEK/MELK inhibition and identify therapeutically exploitable BBB deficiencies in these tumors.

The human cytomegalovirus-encoded G protein-coupled receptor UL33 exhibits oncomodulatory properties.

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein-coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies.

Splicing modulation as novel therapeutic strategy against diffuse malignant peritoneal mesothelioma.

INTRODUCTION: Therapeutic options for diffuse malignant peritoneal mesothelioma (DMPM) are limited to surgery and locoregional chemotherapy. Despite improvements in survival rates, patients eventually succumb to disease progression. We investigated splicing deregulation both as molecular prognostic factor and potential novel target in DMPM, while we tested modulators of SF3b complex for antitumor activity. METHODS: Tissue-microarrays of 64 DMPM specimens were subjected to immunohistochemical assessment of SF3B1 expression and correlation to clinical outcome. Two primary cell cultures were used for gene expression profiling and in vitro screening of SF3b modulators. Drug-induced splicing alterations affecting downstream cellular pathways were detected through RNA sequencing. Ultimately, we established bioluminescent orthotopic mouse models to test the efficacy of splicing modulation in vivo. RESULTS: Spliceosomal genes are differentially upregulated in DMPM cells compared to normal tissues and high expression of SF3B1 correlated with poor clinical outcome in univariate and multivariate analysis. SF3b modulators (Pladienolide-B, E7107, Meayamycin-B) showed potent cytotoxic activity in vitro with IC50 values in the low nanomolar range. Differential splicing analysis of Pladienolide-B-treated cells revealed abundant alterations of transcripts involved in cell cycle, apoptosis and other oncogenic pathways. This was validated by RT-PCR and functional assays. E7107 demonstrated remarkable in vivo antitumor efficacy, with significant improvement of survival rates compared to vehicle-treated controls. CONCLUSIONS: SF3B1 emerged as a novel potential prognostic factor in DMPM. Splicing modulators markedly impair cancer cell viability, resulting also in potent antitumor activity in vivo. Our data designate splicing as a promising therapeutic target in DMPM.

The constitutive activity of the virally encoded chemokine receptor US28 accelerates glioblastoma growth.

Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251- and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8.

Cancer cells release extracellular vesicles (EVs) that contain functional biomolecules such as RNA and proteins. EVs are transferred to recipient cancer cells and can promote tumour progression and therapy resistance. Through RNAi screening, we identified a novel EV uptake mechanism involving a triple interaction between the chemokine receptor CCR8 on the cells, glycans exposed on EVs and the soluble ligand CCL18. This ligand acts as bridging molecule, connecting EVs to cancer cells. We show that glioblastoma EVs promote cell proliferation and resistance to the alkylating agent temozolomide (TMZ). Using in vitro and in vivo stem-like glioblastoma models, we demonstrate that EV-induced phenotypes are neutralised by a small molecule CCR8 inhibitor, R243. Interference with chemokine receptors may offer therapeutic opportunities against EV-mediated cross-talk in glioblastoma.

Three-dimensional histochemistry and imaging of human gingiva.

In the present study, 3D histochemistry and imaging methodology is described for human gingiva to analyze its vascular network. Fifteen human gingiva samples without signs of inflammation were cleared using a mixture of 2-parts benzyl benzoate and 1-part benzyl alcohol (BABB), after being immunofluorescently stained for CD31, marker of endothelial cells to visualize blood vessels in combination with fluorescent DNA dyes. Samples were imaged in 3D with the use of confocal microscopy and light-sheet microscopy and image processing. BABB clearing caused limited tissue shrinkage 13 ± 7% as surface area and 24 ± 1% as volume. Fluorescence remained intact in BABB-cleared gingiva samples and light-sheet microscopy was an excellent tool to image gingivae whereas confocal microscopy was not. Histochemistry on cryostat sections of gingiva samples after 3D imaging validated structures visualized in 3D. Three-dimensional images showed the vascular network in the stroma of gingiva with one capillary loop in each stromal papilla invading into the epithelium. The capillary loops were tortuous with structural irregularities that were not apparent in 2D images. It is concluded that 3D histochemistry and imaging methodology described here is a promising novel approach to study structural aspects of human gingiva in health and disease.

Identification of MEK162 as a Radiosensitizer for the Treatment of Glioblastoma.

Glioblastoma (GBM) is a highly aggressive and lethal brain cancer type. PI3K and MAPK inhibitors have been studied preclinically in GBM as monotherapy, but not in combination with radiotherapy, which is a key component of the current standard treatment of GBM. In our study, GBM cell lines and patient representative primary cultures were grown as multicellular spheroids. Spheroids were treated with a panel of small-molecule drugs including MK2206, RAD001, BEZ235, MLN0128, and MEK162, alone and in combination with irradiation. Following treatment, spheroid growth parameters (growth rate, volume reduction, and time to regrow), cell-cycle distribution and expression of key target proteins were evaluated. In vivo, the effect of irradiation (3 × 2 Gy) without or with MEK162 (50 mg/kg) was studied in orthotopic GBM8 brain tumor xenografts with endpoints tumor growth and animal survival. The MAPK-targeting agent MEK162 was found to enhance the effect of irradiation as demonstrated by growth inhibition of spheroids. MEK162 downregulated and dephosphorylated the cell-cycle checkpoint proteins CDK1/CDK2/WEE1 and DNA damage response proteins p-ATM/p-CHK2. When combined with radiation, this led to a prolonged DNA damage signal. In vivo data on tumor-bearing animals demonstrated a significantly reduced growth rate, increased growth delay, and prolonged survival time. In addition, RNA expression of responsive cell cultures correlated to mesenchymal stratification of patient expression data. In conclusion, the MAPK inhibitor MEK162 was identified as a radiosensitizer in GBM spheroids in vitro and in orthotopic GBM xenografts in vivo The data are supportive for implementation of this targeted agent in an early-phase clinical study in GBM patients. Mol Cancer Ther; 17(2); 347-54. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

Ape1 guides DNA repair pathway choice that is associated with drug tolerance in glioblastoma.

Ape1 is the major apurinic/apyrimidinic (AP) endonuclease activity in mammalian cells, and a key factor in base-excision repair of DNA. High expression or aberrant subcellular distribution of Ape1 has been detected in many cancer types, correlated with drug response, tumor prognosis, or patient survival. Here we present evidence that Ape1 facilitates BRCA1-mediated homologous recombination repair (HR), while counteracting error-prone non-homologous end joining of DNA double-strand breaks. Furthermore, Ape1, coordinated with checkpoint kinase Chk2, regulates drug response of glioblastoma cells. Suppression of Ape1/Chk2 signaling in glioblastoma cells facilitates alternative means of damage site recruitment of HR proteins as part of a genomic defense system. Through targeting "HR-addicted" temozolomide-resistant glioblastoma cells via a chemical inhibitor of Rad51, we demonstrated that targeting HR is a promising strategy for glioblastoma therapy. Our study uncovers a critical role for Ape1 in DNA repair pathway choice, and provides a mechanistic understanding of DNA repair-supported chemoresistance in glioblastoma cells.

Optical clearing and fluorescence deep-tissue imaging for 3D quantitative analysis of the brain tumor microenvironment.

BACKGROUND: Three-dimensional visualization of the brain vasculature and its interactions with surrounding cells may shed light on diseases where aberrant microvascular organization is involved, including glioblastoma (GBM). Intravital confocal imaging allows 3D visualization of microvascular structures and migration of cells in the brain of mice, however, with limited imaging depth. To enable comprehensive analysis of GBM and the brain microenvironment, in-depth 3D imaging methods are needed. Here, we employed methods for optical tissue clearing prior to 3D microscopy to visualize the brain microvasculature and routes of invasion of GBM cells. METHODS: We present a workflow for ex vivo imaging of optically cleared brain tumor tissues and subsequent computational modeling. This workflow was used for quantification of the microvasculature in relation to nuclear or cellular density in healthy mouse brain tissues and in human orthotopic, infiltrative GBM8 and E98 glioblastoma models. RESULTS: Ex vivo cleared mouse brain tissues had a >10-fold imaging depth as compared to intravital imaging of mouse brain in vivo. Imaging of optically cleared brain tissue allowed quantification of the 3D microvascular characteristics in healthy mouse brains and in tissues with diffuse, infiltrative growing GBM8 brain tumors. Detailed 3D visualization revealed the organization of tumor cells relative to the vasculature, in both gray matter and white matter regions, and patterns of multicellular GBM networks collectively invading the brain parenchyma. CONCLUSIONS: Optical tissue clearing opens new avenues for combined quantitative and 3D microscopic analysis of the topographical relationship between GBM cells and their microenvironment.

Preclinical evaluation of convection-enhanced delivery of liposomal doxorubicin to treat pediatric diffuse intrinsic pontine glioma and thalamic high-grade glioma.

OBJECTIVE Pediatric high-grade gliomas (pHGGs) including diffuse intrinsic pontine gliomas (DIPGs) are primary brain tumors with high mortality and morbidity. Because of their poor brain penetrance, systemic chemotherapy regimens have failed to deliver satisfactory results; however, convection-enhanced delivery (CED) may be an alternative mode of drug delivery. Anthracyclines are potent chemotherapeutics that have been successfully delivered via CED in preclinical supratentorial glioma models. This study aims to assess the potency of anthracyclines against DIPG and pHGG cell lines in vitro and to evaluate the efficacy of CED with anthracyclines in orthotopic pontine and thalamic tumor models. METHODS The sensitivity of primary pHGG cell lines to a range of anthracyclines was tested in vitro. Preclinical CED of free doxorubicin and pegylated liposomal doxorubicin (PLD) to the brainstem and thalamus of naïve nude mice was performed. The maximum tolerated dose (MTD) was determined based on the observation of clinical symptoms, and brains were analyzed after H & E staining. Efficacy of the MTD was tested in adult glioma E98-FM-DIPG and E98-FM-thalamus models and in the HSJD-DIPG-007-Fluc primary DIPG model. RESULTS Both pHGG and DIPG cells were sensitive to anthracyclines in vitro. Doxorubicin was selected for further preclinical evaluation. Convection-enhanced delivery of the MTD of free doxorubicin and PLD in the pons was 0.02 mg/ml, and the dose tolerated in the thalamus was 10 times higher (0.2 mg/ml). Free doxorubicin or PLD via CED was ineffective against E98-FM-DIPG or HSJD-DIPG-007-Fluc in the brainstem; however, when applied in the thalamus, 0.2 mg/ml of PLD slowed down tumor growth and increased survival in a subset of animals with small tumors. CONCLUSIONS Local delivery of doxorubicin to the brainstem causes severe toxicity, even at doxorubicin concentrations that are safe in the thalamus. As a consequence, the authors could not establish a therapeutic window for treating orthotopic brainstem tumors in mice. For tumors in the thalamus, therapeutic concentrations to slow down tumor growth could be reached. These data suggest that anatomical location determines the severity of toxicity after local delivery of therapeutic agents and that caution should be used when translating data from supratentorial CED studies to treat infratentorial tumors.

Blocking Tumor-Educated MSC Paracrine Activity Halts Osteosarcoma Progression.

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)-educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFß, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFß-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFß are increased in osteosarcoma patients.Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFß-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721-33. ©2017 AACR.

Bevacizumab Targeting Diffuse Intrinsic Pontine Glioma: Results of 89Zr-Bevacizumab PET Imaging in Brain Tumor Models.

The role of the VEGF inhibitor bevacizumab in the treatment of diffuse intrinsic pontine glioma (DIPG) is unclear. We aim to study the biodistribution and uptake of zirconium-89 ((89)Zr)-labeled bevacizumab in DIPG mouse models. Human E98-FM, U251-FM glioma cells, and HSJD-DIPG-007-FLUC primary DIPG cells were injected into the subcutis, pons, or striatum of nude mice. Tumor growth was monitored by bioluminescence imaging (BLI) and visualized by MRI. Seventy-two to 96 hours after (89)Zr-bevacizumab injections, mice were imaged by positron emission tomography (PET), and biodistribution was analyzed ex vivo High VEGF expression in human DIPG was confirmed in a publically available mRNA database, but no significant (89)Zr-bevacizumab uptake could be detected in xenografts located in the pons and striatum at an early or late stage of the disease. E98-FM, and to a lesser extent the U251-FM and HSJD-DIPG-007 subcutaneous tumors, showed high accumulation of (89)Zr-bevacizumab. VEGF expression could not be demonstrated in the intracranial tumors by in situ hybridization (ISH) but was clearly present in the perinecrotic regions of subcutaneous E98-FM tumors. The poor uptake of (89)Zr-bevacizumab in xenografts located in the brain suggests that VEGF targeting with bevacizumab has limited efficacy for diffuse infiltrative parts of glial brain tumors in mice. Translating these results to the clinic would imply that treatment with bevacizumab in patients with DIPG is only justified after targeting of VEGF has been demonstrated by (89)Zr-bevacizumab immuno-PET. We aim to confirm this observation in a clinical PET study with patients with DIPG. Mol Cancer Ther; 15(9); 2166-74. ©2016 AACR.