Feasibility and utility of MRI and dynamic 18F-FDG-PET in an orthotopic organoid-based patient-derived mouse model of endometrial cancer.

BACKGROUND: Pelvic magnetic resonance imaging (MRI) and whole-body positron emission tomography-computed tomography (PET-CT) play an important role at primary diagnostic work-up and in detecting recurrent disease in endometrial cancer (EC) patients, however the preclinical use of these imaging methods is currently limited. We demonstrate the feasibility and utility of MRI and dynamic 18F-fluorodeoxyglucose (FDG)-PET imaging for monitoring tumor progression and assessing chemotherapy response in an orthotopic organoid-based patient-derived xenograft (O-PDX) mouse model of EC. METHODS: 18 O-PDX mice (grade 3 endometrioid EC, stage IIIC1), selectively underwent weekly T2-weighted MRI (total scans = 32), diffusion-weighted MRI (DWI) (total scans = 9) and dynamic 18F-FDG-PET (total scans = 26) during tumor progression. MRI tumor volumes (vMRI), tumor apparent diffusion coefficient values (ADCmean) and metabolic tumor parameters from 18F-FDG-PET including maximum and mean standard uptake values (SUVmax/SUVmean), metabolic tumor volume (MTV), total lesion glycolysis (TLG) and metabolic rate of 18F-FDG (MRFDG) were calculated. Further, nine mice were included in a chemotherapy treatment study (treatment; n = 5, controls; n = 4) and tumor ADCmean-values were compared to changes in vMRI and cellular density from histology at endpoint. A Mann-Whitney test was used to evaluate differences between groups. RESULTS: Tumors with large tumor volumes (vMRI) had higher metabolic activity (MTV and TLG) in a clear linear relationship (r2 = 0.92 and 0.89, respectively). Non-invasive calculation of MRFDG from dynamic 18F-FDG-PET (mean MRFDG = 0.39 µmol/min) was feasible using an image-derived input function. Treated mice had higher tumor ADCmean (p = 0.03), lower vMRI (p = 0.03) and tumor cellular density (p = 0.02) than non-treated mice, all indicating treatment response. CONCLUSION: Preclinical imaging mirroring clinical imaging methods in EC is highly feasible for monitoring tumor progression and treatment response in the present orthotopic organoid mouse model.

Comparison of bone regenerative capacity of donor-matched human adipose-derived and bone marrow mesenchymal stem cells.

Adipose-derived stem cells (ASC) have been used as an alternative to bone marrow mesenchymal stem cells (BMSC) for bone tissue engineering. However, the efficacy of ASC in bone regeneration in comparison with BMSC remains debatable, since inconsistent results have been reported. Comparing ASC with BMSC obtained from different individuals might contribute to this inconsistency in results. Therefore, this study aimed to compare the bone regenerative capacity of donor-matched human ASC and BMSC seeded onto poly(L-lactide-co-ε-caprolactone) scaffolds using calvarial bone defects in nude rats. First, donor-matched ASC and BMSC were seeded onto the co-polymer scaffolds to evaluate their in vitro osteogenic differentiation. Seeded scaffolds and scaffolds without cells (control) were then implanted in calvarial defects in nude rats. The expression of osteogenesis-related genes was examined after 4 weeks. Cellular activity was investigated after 4 and 12 weeks. Bone formation was evaluated radiographically and histologically after 4, 12, and 24 weeks. In vitro, ASC and BMSC demonstrated mineralization. However, BMSC showed higher alkaline phosphatase activity than ASC. In vivo, human osteogenesis-related genes Runx2 and collagen type I were expressed in defects with scaffold/cells. Defects with scaffold/BMSC had higher cellular activity than defects with scaffold/ASC. Moreover, bone formation in defects with scaffold/BMSC was greater than in defects with scaffold/ASC, especially at the early time-point. These results suggest that although ASC have the potential to regenerate bone, the rate of bone regeneration with ASC may be slower than with BMSC. Accordingly, BMSC are more suitable for bone regenerative applications.

Intercellular transfer of transferrin receptor by a contact-, Rab8-dependent mechanism involving tunneling nanotubes.

Intercellular communication between cancer cells, especially between cancer and stromal cells, plays an important role in disease progression. We examined the intercellular transfer of organelles and proteins in vitro and in vivo and the role of tunneling nanotubes (TNTs) in this process. TNTs are membrane bridges that facilitate intercellular transfer of organelles of unclear origin. Using 3-dimensional quantitative and qualitative confocal microscopy, we showed that TNTs contain green fluorescent protein (GFP)-early endosome antigen (EEA) 1, GFP Rab5, GFP Rab11, GFP Rab8, transferrin (Tf), and Tf receptor (Tf-R) fused to mCherry (Tf-RmCherry). Tf-RmCherry was transferred between cancer cells by a contact-dependent but secretion-independent mechanism. Live cell imaging showed TNT formation preceding the transfer of Tf-RmCherry and involving the function of the small guanosine triphosphatase (GTPase) Rab8, which colocalized with Tf-RmCherry in the TNTs and was cotransferred to acceptor cells. Tf-RmCherry was transferred from cancer cells to fibroblasts, a noteworthy finding that suggests that this process occurs between tumor and stromal cells in vivo. We strengthened this hypothesis in a xenograft model of breast cancer using enhanced (e)GFP-expressing mice. Tf-RmCherry transferred from tumor to stromal cells and this process correlated with an increased opposite transfer of eGFP from stromal to tumor cells, together pointing toward complex intercellular communication at the tumor site.

Bevacizumab treatment induces metabolic adaptation toward anaerobic metabolism in glioblastomas.

Anti-angiogenic therapy in glioblastoma (GBM) has unfortunately not led to the anticipated improvement in patient prognosis. We here describe how human GBM adapts to bevacizumab treatment at the metabolic level. By performing (13)C6-glucose metabolic flux analysis, we show for the first time that the tumors undergo metabolic re-programming toward anaerobic metabolism, thereby uncoupling glycolysis from oxidative phosphorylation. Following treatment, an increased influx of (13)C6-glucose was observed into the tumors, concomitant to increased lactate levels and a reduction of metabolites associated with the tricarboxylic acid cycle. This was confirmed by increased expression of glycolytic enzymes including pyruvate dehydrogenase kinase in the treated tumors. Interestingly, L-glutamine levels were also reduced. These results were further confirmed by the assessment of in vivo metabolic data obtained by magnetic resonance spectroscopy and positron emission tomography. Moreover, bevacizumab led to a depletion in glutathione levels indicating that the treatment caused oxidative stress in the tumors. Confirming the metabolic flux results, immunohistochemical analysis showed an up-regulation of lactate dehydrogenase in the bevacizumab-treated tumor core as well as in single tumor cells infiltrating the brain, which may explain the increased invasion observed after bevacizumab treatment. These observations were further validated in a panel of eight human GBM patients in which paired biopsy samples were obtained before and after bevacizumab treatment. Importantly, we show that the GBM adaptation to bevacizumab therapy is not mediated by clonal selection mechanisms, but represents an adaptive response to therapy.

MRI radiomics captures early treatment response in patient-derived organoid endometrial cancer mouse models.

Background: Radiomics can capture microscale information in medical images beyond what is visible to the naked human eye. Using a clinically relevant mouse model for endometrial cancer, the objective of this study was to develop and validate a radiomic signature (RS) predicting response to standard chemotherapy. Methods: Mice orthotopically implanted with a patient-derived grade 3 endometrioid endometrial cancer organoid model (O-PDX) were allocated to chemotherapy (combined paclitaxel/carboplatin, n=11) or saline/control (n=13). During tumor progression, the mice underwent weekly T2-weighted (T2w) magnetic resonance imaging (MRI). Segmentation of primary tumor volume (vMRI) allowed extraction of radiomic features from whole-volume tumor masks. A radiomic model for predicting treatment response was derived employing least absolute shrinkage and selection operator (LASSO) statistics at endpoint images in the orthotopic O-PDX (RS_O), and subsequently applied on the earlier study timepoints (RS_O at baseline, and week 1-3). For external validation, the radiomic model was tested in a separate T2w-MRI dataset on segmented whole-volume subcutaneous tumors (RS_S) from the same O-PDX model, imaged at three timepoints (baseline, day 3 and day 10/endpoint) after start of chemotherapy (n=8 tumors) or saline/control (n=8 tumors). Results: The RS_O yielded rapidly increasing area under the receiver operating characteristic (ROC) curves (AUCs) for predicting treatment response from baseline until endpoint; AUC=0.38 (baseline); 0.80 (week 1), 0.85 (week 2), 0.96 (week 3) and 1.0 (endpoint). In comparison, vMRI yielded AUCs of 0.37 (baseline); 0.69 (w1); 0.83 (week 2); 0.92 (week 3) and 0.97 (endpoint). When tested in the external validation dataset, RS_S yielded high accuracy for predicting treatment response at day10/endpoint (AUC=0.85) and tended to yield higher AUC than vMRI (AUC=0.78, p=0.18). Neither RS_S nor vMRI predicted response at day 3 in the external validation set (AUC=0.56 for both). Conclusions: We have developed and validated a radiomic signature that was able to capture chemotherapeutic treatment response both in an O-PDX and in a subcutaneous endometrial cancer mouse model. This study supports the promising role of preclinical imaging including radiomic tumor profiling to assess early treatment response in endometrial cancer models.

Lactate dehydrogenases promote glioblastoma growth and invasion via a metabolic symbiosis.

Lactate is a central metabolite in brain physiology but also contributes to tumor development. Glioblastoma (GB) is the most common and malignant primary brain tumor in adults, recognized by angiogenic and invasive growth, in addition to its altered metabolism. We show herein that lactate fuels GB anaplerosis by replenishing the tricarboxylic acid (TCA) cycle in absence of glucose. Lactate dehydrogenases (LDHA and LDHB), which we found spatially expressed in GB tissues, catalyze the interconversion of pyruvate and lactate. However, ablation of both LDH isoforms, but not only one, led to a reduction in tumor growth and an increase in mouse survival. Comparative transcriptomics and metabolomics revealed metabolic rewiring involving high oxidative phosphorylation (OXPHOS) in the LDHA/B KO group which sensitized tumors to cranial irradiation, thus improving mouse survival. When mice were treated with the antiepileptic drug stiripentol, which targets LDH activity, tumor growth decreased. Our findings unveil the complex metabolic network in which both LDHA and LDHB are integrated and show that the combined inhibition of LDHA and LDHB strongly sensitizes GB to therapy.

Current landscape and future perspectives in preclinical MR and PET imaging of brain metastasis.

Brain metastasis (BM) is a major cause of cancer patient morbidity. Clinical magnetic resonance imaging (MRI) and positron emission tomography (PET) represent important resources to assess tumor progression and treatment responses. In preclinical research, anatomical MRI and to some extent functional MRI have frequently been used to assess tumor progression. In contrast, PET has only to a limited extent been used in animal BM research. A considerable culprit is that results from most preclinical studies have shown little impact on the implementation of new treatment strategies in the clinic. This emphasizes the need for the development of robust, high-quality preclinical imaging strategies with potential for clinical translation. This review focuses on advanced preclinical MRI and PET imaging methods for BM, describing their applications in the context of what has been done in the clinic. The strengths and shortcomings of each technology are presented, and recommendations for future directions in the development of the individual imaging modalities are suggested. Finally, we highlight recent developments in quantitative MRI and PET, the use of radiomics and multimodal imaging, and the need for a standardization of imaging technologies and protocols between preclinical centers.

Repurposing 18F-FMISO as a PET tracer for translational imaging of nitroreductase-based gene directed enzyme prodrug therapy.

Nitroreductases (NTR) are a family of bacterial enzymes used in gene directed enzyme prodrug therapy (GDEPT) that selectively activate prodrugs containing aromatic nitro groups to exert cytotoxic effects following gene transduction in tumours. The clinical development of NTR-based GDEPT has, in part, been hampered by the lack of translational imaging modalities to assess gene transduction and drug cytotoxicity, non-invasively. This study presents translational preclinical PET imaging to validate and report NTR activity using the clinically approved radiotracer, 18F-FMISO, as substrate for the NTR enzyme. Methods: The efficacy with which 18F-FMISO could be used to report NfsB NTR activity in vivo was investigated using the MDA-MB-231 mammary carcinoma xenograft model. For validation, subcutaneous xenografts of cells constitutively expressing NTR were imaged using 18F-FMISO PET/CT and fluorescence imaging with CytoCy5S, a validated fluorescent NTR substrate. Further, examination of the non-invasive functionality of 18F-FMISO PET/CT in reporting NfsB NTR activity in vivo was assessed in metastatic orthotopic NfsB NTR expressing xenografts and metastasis confirmed by bioluminescence imaging. 18F-FMISO biodistribution was acquired ex vivo by an automatic gamma counter measuring radiotracer retention to confirm in vivo results. To assess the functional imaging of NTR-based GDEPT with 18F-FMISO, PET/CT was performed to assess both gene transduction and cytotoxicity effects of prodrug therapy (CB1954) in subcutaneous models. Results:18F-FMISO retention was detected in NTR+ subcutaneous xenografts, displaying significantly higher PET contrast than NTR- xenografts (p < 0.0001). Substantial 18F-FMISO retention was evident in metastases of orthotopic xenografts (p < 0.05). Accordingly, higher 18F-FMISO biodistribution was prevalent ex vivo in NTR+ xenografts. 18F-FMISO NfsB NTR PET/CT imaging proved useful for monitoring in vivo NTR transduction and the cytotoxic effect of prodrug therapy. Conclusions:18F-FMISO NfsB NTR PET/CT imaging offered significant contrast between NTR+ and NTR- tumours and effective resolution of metastatic progression. Furthermore, 18F-FMISO NfsB NTR PET/CT imaging proved efficient in monitoring the two steps of GDEPT, in vivo NfsB NTR transduction and response to CB1954 prodrug therapy. These results support the repurposing of 18F-FMISO as a readily implementable PET imaging probe to be employed as companion diagnostic test for NTR-based GDEPT systems.

Patient-derived organoids reflect the genetic profile of endometrial tumors and predict patient prognosis.

Background: A major hurdle in translational endometrial cancer (EC) research is the lack of robust preclinical models that capture both inter- and intra-tumor heterogeneity. This has hampered the development of new treatment strategies for people with EC. Methods: EC organoids were derived from resected patient tumor tissue and expanded in a chemically defined medium. Established EC organoids were orthotopically implanted into female NSG mice. Patient tissue and corresponding models were characterized by morphological evaluation, biomarker and gene expression and by whole exome sequencing. A gene signature was defined and its prognostic value was assessed in multiple EC cohorts using Mantel-Cox (log-rank) test. Response to carboplatin and/or paclitaxel was measured in vitro and evaluated in vivo. Statistical difference between groups was calculated using paired t-test. Results: We report EC organoids established from EC patient tissue, and orthotopic organoid-based patient-derived xenograft models (O-PDXs). The EC organoids and O-PDX models mimic the tissue architecture, protein biomarker expression and genetic profile of the original tissue. Organoids show heterogenous sensitivity to conventional chemotherapy, and drug response is reproduced in vivo. The relevance of these models is further supported by the identification of an organoid-derived prognostic gene signature. This signature is validated as prognostic both in our local patient cohorts and in the TCGA endometrial cancer cohort. Conclusions: We establish robust model systems that capture both the diversity of endometrial tumors and intra-tumor heterogeneity. These models are highly relevant preclinical tools for the elucidation of the molecular pathogenesis of EC and identification of potential treatment strategies.

Erratum: Author Correction: Patient-derived organoids reflect the genetic profile of endometrial tumors and predict patient prognosis.

[This corrects the article DOI: 10.1038/s43856-021-00019-x.].

Novel clearance of muscle proteins by muscle cells.

Blood levels of cardiac troponins (cTn) and myoglobin are analysed when myocardial infarction (MI) is suspected. Here we describe a novel clearance mechanism for muscle proteins by muscle cells. The complete plasma clearance profile of cTn and myoglobin was followed in rats after intravenous or intermuscular injections and analysed by PET and fluorescence microscopy of muscle biopsies and muscle cells. Compared with intravenous injections, only 5 % of cTnT, 0.6 % of cTnI and 8 % of myoglobin were recovered in the circulation following intramuscular injection. In contrast, 47 % of the renal filtration marker FITC-sinistrin and 81 % of cTn fragments from MI-patients were recovered after intramuscular injection. In addition, PET and biopsy analysis revealed that cTn was taken up by the quadriceps muscle and both cTn and myoglobin were endocytosed by cultured muscle cells. This local clearance mechanism could possibly be the dominant clearance mechanism for cTn, myoglobin and other muscle damage biomarkers released by muscle cells.

Trifluoperazine prolongs the survival of experimental brain metastases by STAT3-dependent lysosomal membrane permeabilization.

Brain metastasis is a major cause of mortality in melanoma patients. The blood-brain barrier (BBB) prevents most anti-tumor compounds from entering the brain, which significantly limits their use in the treatment of brain metastasis. One strategy in the development of new treatments is to assess the anti-tumor potential of drugs currently used in the clinic. Here, we tested the anti-tumor effect of the BBB-penetrating antipsychotic trifluoperazine (TFP) on metastatic melanoma. H1 and Melmet1 human metastatic melanoma cell lines were used in vitro and in vivo. TFP effects on viability and toxicity were evaluated in proliferation and colony formation assays. Preclinical, therapeutic efficacy was evaluated in NOD/SCID mice, after intracardial injection of tumor cells. Molecular studies using immunohistochemistry, western blots, immunofluorescence and transmission electron microscopy were used to gain mechanistic insight into the biological activity of TFP. Our results showed that TFP decreased cell viability and proliferation, colony formation and spheroid growth in vitro. The drug also decreased tumor burden in mouse brains and prolonged animal survival after injection of tumor cells (53.0 days vs 44.5 days), TFP treated vs untreated animals, respectively (P < 0.01). At the molecular level, TFP treatment led to increased levels of LC3B and p62 in vitro and in vivo, suggesting an inhibition of autophagic flux. A decrease in LysoTracker Red uptake after treatment indicated impaired acidification of lysosomes. TFP caused accumulation of electron dense vesicles, an indication of damaged lysosomes, and reduced the expression of cathepsin B, a main lysosomal protease. Acridine orange and galectin-3 immunofluorescence staining were evidence of TFP induction of lysosomal membrane permeabilization. Finally, TFP was cytotoxic to melanoma brain metastases based on the increased release of lactate dehydrogenase into media. Through knockdown experiments, the processes of TFP-induced lysosomal membrane permeabilization and cell death appeared to be STAT3 dependent. In conclusion, our work provides a strong rationale for further clinical investigation of TFP as an adjuvant therapy for melanoma patients with metastases to the brain.

Near-Infrared Fluorescent Imaging for Monitoring of Treatment Response in Endometrial Carcinoma Patient-Derived Xenograft Models.

Imaging of clinically relevant preclinical animal models is critical to the development of personalized therapeutic strategies for endometrial carcinoma. Although orthotopic patient-derived xenografts (PDXs) reflecting heterogeneous molecular subtypes are considered the most relevant preclinical models, their use in therapeutic development is limited by the lack of appropriate imaging modalities. Here, we describe molecular imaging of a near-infrared fluorescently labeled monoclonal antibody targeting epithelial cell adhesion molecule (EpCAM) as an in vivo imaging modality for visualization of orthotopic endometrial carcinoma PDX. Application of this near-infrared probe (EpCAM-AF680) enabled both spatio-temporal visualization of development and longitudinal therapy monitoring of orthotopic PDX. Notably, EpCAM-AF680 facilitated imaging of multiple PDX models representing different subtypes of the disease. Thus, the combined implementation of EpCAM-AF680 and orthotopic PDX models creates a state-of-the-art preclinical platform for identification and validation of new targeted therapies and corresponding response predicting markers for endometrial carcinoma.

Imaging of Preclinical Endometrial Cancer Models for Monitoring Tumor Progression and Response to Targeted Therapy.

Endometrial cancer is the most common gynecologic malignancy in industrialized countries. Most patients are cured by surgery; however, about 15% of the patients develop recurrence with limited treatment options. Patient-derived tumor xenograft (PDX) mouse models represent useful tools for preclinical evaluation of new therapies and biomarker identification. Preclinical imaging by magnetic resonance imaging (MRI), positron emission tomography-computed tomography (PET-CT), single-photon emission computed tomography (SPECT) and optical imaging during disease progression enables visualization and quantification of functional tumor characteristics, which may serve as imaging biomarkers guiding targeted therapies. A critical question, however, is whether the in vivo model systems mimic the disease setting in patients to such an extent that the imaging biomarkers may be translatable to the clinic. The primary objective of this review is to give an overview of current and novel preclinical imaging methods relevant for endometrial cancer animal models. Furthermore, we highlight how these advanced imaging methods depict pathogenic mechanisms important for tumor progression that represent potential targets for treatment in endometrial cancer.

Pretreatment of Glioblastoma with Bortezomib Potentiates Natural Killer Cell Cytotoxicity through TRAIL/DR5 Mediated Apoptosis and Prolongs Animal Survival.

Background: Natural killer (NK) cells are potential effectors in anti-cancer immunotherapy; however only a subset potently kills cancer cells. Here, we examined whether pretreatment of glioblastoma (GBM) with the proteasome inhibitor, bortezomib (BTZ), might sensitize tumour cells to NK cell lysis by inducing stress antigens recognized by NK-activating receptors. Methods: Combination immunotherapy of NK cells with BTZ was studied in vitro against GBM cells and in a GBM-bearing mouse model. Tumour cells were derived from primary GBMs and NK cells from donors or patients. Flow cytometry was used for viability/cytotoxicity evaluation as well as in vitro and ex vivo phenotyping. We performed a Seahorse assay to assess oxygen consumption rates and mitochondrial function, Luminex ELISA to determine NK cell secretion, protein chemistry and LC-MS/MS to detect BTZ in brain tissue. MRI was used to monitor therapeutic efficacy in mice orthotopically implanted with GBM spheroids. Results: NK cells released IFNγ, perforin and granzyme A cytolytic granules upon recognition of stress-ligand expressing GBM cells, disrupted mitochondrial function and killed 24-46% of cells by apoptosis. Pretreatment with BTZ further increased stress-ligands, induced TRAIL-R2 expression and enhanced GBM lysis to 33-76% through augmented IFNγ release (p < 0.05). Blocking NKG2D, TRAIL and TRAIL-R2 rescued GBM cells treated with BTZ from NK cells, p = 0.01. Adoptively transferred autologous NK-cells persisted in vivo (p < 0.05), diminished tumour proliferation and prolonged survival alone (Log Rank10.19, p = 0.0014, 95%CI 0.252-0.523) or when combined with BTZ (Log Rank5.25, p = 0.0219, 95%CI 0.295-0.408), or either compared to vehicle controls (median 98 vs. 68 days and 80 vs. 68 days, respectively). BTZ crossed the blood-brain barrier, attenuated proteasomal activity in vivo (p < 0.0001; p < 0.01 compared to vehicle control or NK cells only, respectively) and diminished tumour angiogenesis to promote survival compared to vehicle-treated controls (Log Rank6.57, p = 0.0104, 95%CI 0.284-0.424, median 83 vs. 68 days). However, NK ablation with anti-asialo-GM1 abrogated the therapeutic efficacy. Conclusions: NK cells alone or in combination with BTZ inhibit tumour growth, but the scheduling of BTZ in vivo requires further investigation to maximize its contribution to the efficacy of the combination regimen.

Improved Drug Delivery to Brain Metastases by Peptide-Mediated Permeabilization of the Blood-Brain Barrier.

Patients with melanoma have a high risk of developing brain metastasis, which is associated with a dismal prognosis. During early stages of metastasis development, the blood-brain barrier (BBB) is likely intact, which inhibits sufficient drug delivery into the metastatic lesions. We investigated the ability of the peptide, K16ApoE, to permeabilize the BBB for improved treatment with targeted therapies preclinically. Dynamic contrast enhanced MRI (DCE-MRI) was carried out on NOD/SCID mice to study the therapeutic window of peptide-mediated BBB permeabilization. Further, both in vivo and in vitro assays were used to determine K16ApoE toxicity and to obtain mechanistic insight into its action on the BBB. The therapeutic impact of K16ApoE on metastases was evaluated combined with the mitogen-activated protein kinase pathway inhibitor dabrafenib, targeting BRAF mutated melanoma cells, which is otherwise known not to cross the intact BBB. Our results from the DCE-MRI experiments showed effective K16ApoE-mediated BBB permeabilization lasting for up to 1 hour. Mechanistic studies showed a dose-dependent effect of K16ApoE caused by induction of endocytosis. At concentrations above IC50, the peptide additionally showed nonspecific disturbances on plasma membranes. Combined treatment with K16ApoE and dabrafenib reduced the brain metastatic burden in mice and increased animal survival, and PET/CT showed that the peptide also facilitated the delivery of compounds with molecular weights as large as 150 kDa into the brain. To conclude, we demonstrate a transient permeabilization of the BBB, caused by K16ApoE, that facilitates enhanced drug delivery into the brain. This improves the efficacy of drugs that otherwise do not cross the intact BBB.

Lack of functional normalisation of tumour vessels following anti-angiogenic therapy in glioblastoma.

Neo-angiogenesis represents an important factor for the delivery of oxygen and nutrients to a growing tumour, and is considered to be one of the main pathodiagnostic features of glioblastomas (GBM). Anti-angiogenic therapy by vascular endothelial growth factor (VEGF) blocking agents has been shown to lead to morphological vascular normalisation resulting in a reduction of contrast enhancement as seen by magnetic resonance imaging (MRI). Yet the functional consequences of this normalisation and its potential for improved delivery of cytotoxic agents to the tumour are not known. The presented study aimed at determining the early physiologic changes following bevacizumab treatment. A time series of perfusion MRI and hypoxia positron emission tomography (PET) scans were acquired during the first week of treatment, in two human GBM xenograft models treated with either high or low doses of bevacizumab. We show that vascular morphology was normalised over the time period investigated, but vascular function was not improved, resulting in poor tumoural blood flow and increased hypoxia.

Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cells.

The cancer stem cell model suggests that glioblastomas contain a subpopulation of stem-like tumor cells that reproduce themselves to sustain tumor growth. Targeting these cells thus represents a novel treatment strategy and therefore more specific markers that characterize glioblastoma stem cells need to be identified. In the present study, we performed transcriptomic analysis of glioblastoma tissues compared to normal brain tissues revealing sensible up-regulation of CD9 gene. CD9 encodes the transmembrane protein tetraspanin which is involved in tumor cell invasion, apoptosis and resistance to chemotherapy. Using the public REMBRANDT database for brain tumors, we confirmed the prognostic value of CD9, whereby a more than two fold up-regulation correlates with shorter patient survival. We validated CD9 gene and protein expression showing selective up-regulation in glioblastoma stem cells isolated from primary biopsies and in primary organotypic glioblastoma spheroids as well as in U87-MG and U373 glioblastoma cell lines. In contrast, no or low CD9 gene expression was observed in normal human astrocytes, normal brain tissue and neural stem cells. CD9 silencing in three CD133+ glioblastoma cell lines (NCH644, NCH421k and NCH660h) led to decreased cell proliferation, survival, invasion, and self-renewal ability, and altered expression of the stem-cell markers CD133, nestin and SOX2. Moreover, CD9-silenced glioblastoma stem cells showed altered activation patterns of the Akt, MapK and Stat3 signaling transducers. Orthotopic xenotransplantation of CD9-silenced glioblastoma stem cells into nude rats promoted prolonged survival. Therefore, CD9 should be further evaluated as a target for glioblastoma treatment.

Engraftment of Human Glioblastoma Cells in Immunocompetent Rats through Acquired Immunosuppression.

Transplantation of glioblastoma patient biopsy spheroids to the brain of T cell-compromised Rowett (nude) rats has been established as a representative animal model for human GBMs, with a tumor take rate close to 100%. In immunocompetent littermates however, primary human GBM tissue is invariably rejected. Here we show that after repeated passaging cycles in nude rats, human GBM spheroids are enabled to grow in the brain of immunocompetent rats. In case of engraftment, xenografts in immunocompetent rats grow progressively and host leukocytes fail to enter the tumor bed, similar to what is seen in nude animals. In contrast, rejection is associated with massive infiltration of the tumor bed by leukocytes, predominantly ED1+ microglia/macrophages, CD4+ T helper cells and CD8+ effector cells, and correlates with elevated serum levels of pro-inflammatory cytokines IL-1α, IL-18 and TNF-α [corrected]. We observed that in nude rat brains, an adaptation to the host occurs after several in vivo passaging cycles, characterized by striking attenuation of microglial infiltration. Furthermore, tumor-derived chemokines that promote leukocyte migration and their entry into the CNS such as CXCL-10 and CXCL-12 are down-regulated, and the levels of TGF-ß2 increase. We propose that through serial in vivo passaging in nude rats, human GBM cells learn to avoid and or/ suppress host immunity. Such adapted GBM cells are in turn able to engraft in immunocompetent rats without signs of an inflammatory response.

Melanoma brain metastasis is independent of lactate dehydrogenase A expression.

BACKGROUND: The key metabolic enzyme lactate dehydrogenase A (LDHA) is overexpressed in many cancers, and several preclinical studies have shown encouraging results of targeted inhibition. However, the mechanistic importance of LDHA in melanoma is largely unknown and hitherto unexplored in brain metastasis. METHODS: We investigated the spatial, temporal, and functional features of LDHA expression in melanoma brain metastasis across multiple in vitro assays, in a robust and predictive animal model employing MRI and PET imaging, and in a unique cohort of 80 operated patients. We further assessed the genomic and proteomic landscapes of LDHA in different cancers, particularly melanomas. RESULTS: LDHA expression was especially strong in early and small brain metastases in vivo and related to intratumoral hypoxia in late and large brain metastases in vivo and in patients. However, LDHA expression in human brain metastases was not associated with the number of tumors, BRAF(V600E) status, or survival. Moreover, LDHA depletion by small hairpin RNA interference did not affect cell proliferation or 3D tumorsphere growth in vitro or brain metastasis formation or survival in vivo. Integrated analyses of the genomic and proteomic landscapes of LDHA indicated that LDHA is present but not imperative for tumor progression within the CNS, or predictive of survival in melanoma patients. CONCLUSIONS: In a large patient cohort and in a robust animal model, we show that although LDHA expression varies biphasically during melanoma brain metastasis formation, tumor progression and survival seem to be functionally independent of LDHA.