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 ¹³C₆-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 ¹³C₆-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.

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.

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

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.—Burtey, A., Wagner, M., Hodneland, E., Skaftnesmo, K. O., Schoelermann, J., Mondragon, I. R., Espedal, H., Golebiewska, A., Niclou, S. P., Bjerkvig, R., Kögel, T., Gerdes, H.‐H. Intercellular transfer of transferrin receptor by a contact‐, Rab8‐dependent mechanism involving tunneling nanotubes. FASEB J. 29, 4695‐4712 (2015). www.fasebj.org

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.

Summary: Polymer gel is frequently used for conformance control in fractured reservoirs, where it is injected to reside in fractures or high-permeability streaks to reduce conductivity. With successful polymer-gel conformance control in place, increased pressure gradients across matrix blocks may be achieved during chase floods, diverting water, gas, or enhanced oil recovery (EOR) chemicals into the matrix to displace oil. Knowledge of gel behavior during placement and chase floods is important because it largely controls the success of subsequent injections. Polymer-gel behavior is often studied in corefloods, where differential pressure and effluents from fracture and matrix outlets give information about gel deposition during placement and flow paths during chase floods. The work presented in this paper uses complementary positron emission tomography (PET) chromatographic tomography (CT) imaging to quantify the behavior and blocking capacity of Cr(III)-acetate hydrolyzed polyacrylamide (HPAM) gel during chase waterflooding. In-situ imaging provides information about changes that may not be extracted from pressure measurements and material balance only, such as changes in local fluid saturations and dynamic spatial flow within the fracture and within the structure of the gel network. Polymer gel was placed in core plugs with longitudinal fractures that connected the inlet and outlet, and chase water was subsequently injected to measure the gel blocking capacity. The water phase was labeled with a positron emitting radiopharmaceutical (F-18) to visualize and quantify local flows with PET during gel rupture and subsequent flooding. By use of PET, we study gel rupture and the development of wormholes during gel erosion after rupture as a function of flow rate. A particular strength with access to dynamic, local flow patterns is the direct comparison to global measurements, such as differential pressure and production rate, to verify existing gel-behavior models.