Hypoxia promotes tumour aggressiveness and resistance of cancers to oncological treatment. The identification of cancer cell internalizing antigens for drug targeting to the hypoxic tumour niche remains a challenge of high clinical relevance. Here we show that hypoxia down-regulates the surface proteome at the global level and, more specifically, membrane proteome internalization. We find that hypoxic down-regulation of constitutive endocytosis is HIF-independent, and involves caveolin-1-mediated inhibition of dynamin-dependent, membrane raft endocytosis. Caveolin-1 overexpression inhibits protein internalization, suggesting a general negative regulatory role of caveolin-1 in endocytosis. In contrast to this global inhibitory effect, we identify several proteins that can override caveolin-1 negative regulation, exhibiting increased internalization at hypoxia. We demonstrate antibody-mediated cytotoxin delivery and killing specifically of hypoxic cells through one of these proteins, carbonic anhydrase IX. Our data reveal that caveolin-1 modulates cell-surface proteome turnover at hypoxia with potential implications for specific targeting of the hypoxic tumour microenvironment.

Hypoxia and acidosis are inherent stress factors of the tumor microenvironment and have been linked to increased tumor aggressiveness and treatment resistance. Molecules involved in the adaptive mechanisms that drive stress-induced disease progression constitute interesting candidates of therapeutic intervention. Here, we provide evidence of a novel role of heparan sulfate proteoglycans (HSPG) in the adaptive response of tumor cells to hypoxia and acidosis through increased internalization of lipoproteins, resulting in a lipid-storing phenotype and enhanced tumor-forming capacity. Patient glioblastoma tumors and cells under hypoxic and acidic stress acquired a lipid droplet (LD)-loaded phenotype, and showed an increased recruitment of all major lipoproteins, HDL, LDL, and VLDL. Stress-induced LD accumulation was associated with increased spheroid-forming capacity during reoxygenation in vitro and lung metastatic potential in vivo. On a mechanistic level, we found no apparent effect of hypoxia on HSPGs, whereas lipoprotein receptors (VLDLR and SR-B1) were transiently upregulated by hypoxia. Importantly, however, using pharmacologic and genetic approaches, we show that stress-mediated lipoprotein uptake is highly dependent on intact HSPG expression. The functional relevance of HSPG in the context of tumor cell stress was evidenced by HSPG-dependent lipoprotein cell signaling activation through the ERK/MAPK pathway and by reversal of the LD-loaded phenotype by targeting of HSPGs. We conclude that HSPGs may have an important role in the adaptive response to major stress factors of the tumor microenvironment, with functional consequences on tumor cell signaling and metastatic potential. Cancer Res; 76(16); 4828-40. Ó2016 AACR.

Purpose: Liquid biopsy has great potential to improve the management of brain tumor patients at high risk of surgeryassociated complications. Here, the aim was to explore plasma extracellular vesicle (plEV) immunoprofiling as a tool for noninvasive diagnosis of glioma. Experimental Design: PlEV isolation and analysis were optimized using advanced mass spectrometry, nanoparticle tracking analysis, and electron microscopy. We then established a new procedure that combines size exclusion chromatography isolation and proximity extension assaybased ultrasensitive immunoprofiling of plEV proteins that was applied on a well-defined glioma study cohort (n ¼ 82). Results: Among potential candidates, we for the first time identify syndecan-1 (SDC1) as a plEV constituent that can discriminate between high-grade glioblastoma multiforme (GBM, WHO grade IV) and low-grade glioma [LGG, WHO grade II; area under the ROC curve (AUC): 0.81; sensitivity: 71%; specificity: 91%]. These findings were independently validated by ELISA. Tumor SDC1 mRNA expression similarly discriminated between GBM and LGG in an independent glioma patient population from The Cancer Genome Atlas cohort (AUC: 0.91; sensitivity: 79%; specificity: 91%). In experimental studies with GBM cells, we show that SDC1 is efficiently sorted to secreted EVs. Importantly, we found strong support of plEV SDC1 originating from GBM tumors, as plEV SDC1 correlated with SDC1 protein expression in matched patient tumors, and plEV SDC1 was decreased postoperatively depending on the extent of surgery. Conclusions: Our studies support the concept of circulating plEVs as a tool for noninvasive diagnosis and monitoring of gliomas and should move this field closer to the goal of improving the management of cancer patients.

Aggressive cancers are characterized by hypoxia, which is a key driver of tumor development and treatment resistance. Proteins specifically expressed in the hypoxic tumor microenvironment thus represent interesting candidates for targeted drug delivery strategies. Carbonic anhydrase (CAIX) has been identified as an attractive treatment target as it is highly hypoxia specific and expressed at the cell-surface to promote cancer cell aggressiveness. Here, we find that cancer cell internalization of CAIX is negatively regulated by post-translational modification with chondroitin or heparan sulfate glycosaminoglycan chains. We show that perturbed glycosaminoglycan modification results in increased CAIX endocytosis. We hypothesized that perturbation of CAIX glycosaminoglycan conjugation may provide opportunities for enhanced drug delivery to hypoxic tumor cells. In support of this concept, pharmacological inhibition of glycosaminoglycan biosynthesis with xylosides significantly potentiated the internalization and cytotoxic activity of an antibody-drug conjugate (ADC) targeted at CAIX. Moreover, cells expressing glycosaminoglycan-deficient CAIX were significantly more sensitive to ADC treatment as compared with cells expressing wild-type CAIX. We find that inhibition of CAIX endocytosis is associated with an increased localization of glycosaminoglycan-conjugated CAIX in membrane lipid raft domains stabilized by caveolin-1 clusters. The association of CAIX with caveolin-1 was partially attenuated by acidosis, i.e. another important feature of malignant tumors. Accordingly, we found increased internalization of CAIX at acidic conditions. These findings provide first evidence that intracellular drug delivery at pathophysiological conditions of malignant tumors can be attenuated by tumor antigen glycosaminoglycan modification, which is of conceptual importance in the future development of targeted cancer treatments.

Tumor-specific delivery of ligand-directed prodrugs can increase the therapeutic window of chemotherapeutics by maintaining efficacy whilst decreasing toxic side effects. We have previously described a series of synthetic N-alkylated isatin cytotoxins that destabilize microtubules and induce apoptosis with 10-fold greater potency than conventional anti-mitotics in vitro. Here, we report the characterization, in vitro cytotoxicity and in vivo efficacy of a lead compound, 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (N-AI) conjugated via an esterase-labile linker (N-AIE) to two proven targeting ligands, transferrin (Tf) and plasminogen activator inhibitor type 2 (PAI-2/serpinB2). N-AI was released from N-AIE and the targeting ligands Tf/PAI-2 in an esterase-dependent manner at 37 C and both Tf- and PAI-2-N-AIE conjugates were stable at physiological pH. Human cancer cell lines which vary in their expression levels of Tf receptor (TfR/CD71) and PAI-2 target, receptor bound urokinase (uPA) selectively internalized the conjugates. Tf-N-AIE was up to 24 times more active than the free drug and showed clear selectivity patterns based on TfR levels. PAI-2-N-AIE showed equivalent activity compared to the parent drug and strong selectivity patterns for uPA levels. In preliminary in vivo experiments, the PAI-2- and Tf-N-AIE conjugates were efficacious at 1/20(th) and 1/10(th) of the dose of the free N-AI, respectively, in a metastatic, orthotopic human breast tumor xenograft mouse model. Thus, this strategy specifically delivers and concentrates a novel class of isatin-based, tubulin destabilizing agents to tumors in vivo and warrants further detailed preclinical investigation.

The antibody-drug conjugate trastuzumab-emtansine (T-DM1) offers an additional treatment option for patients with HER2amplified tumors. However, primary and acquired resistance is a limiting factor in a significant subset of patients. Hypoxia, a hallmark of cancer, regulates the trafficking of several receptor proteins with potential implications for tumor targeting. Here, we have investigated how hypoxic conditions may regulate T-DM1 treatment efficacy in breast cancer. The therapeutic effect of T-DM1 and its metabolites was evaluated in conjunction with biochemical, flow cytometry, and high-resolution imaging studies to elucidate the functional and mechanistic aspects of hypoxic regulation. HER2 and caveolin-1 expression was investigated in a well-annotated breast cancer cohort. We find that hypoxia fosters relative resistance to T-DM1 in HER2 þ cells (SKBR3 and BT474). This effect was not a result of deregulated HER2 expression or resistance to emtansine and its metabolites. Instead, we show that hypoxia-induced translocation of caveolin-1 from cytoplasmic vesicles to the plasma membrane contributes to deficient trastuzumab internalization and T-DM1 chemosensitivity. Caveolin-1 depletion mimicked the hypoxic situation, indicating that vesicular caveolin-1 is indispensable for trastuzumab uptake and T-DM1 cytotoxicity. In vitro studies suggested that HER2 and caveolin-1 are not coregulated, which was supported by IHC analysis in patient tumors. We find that phosphorylation-deficient caveolin-1 inhibits trastuzumab internalization and T-DM1 cytotoxicity, suggesting a specific role for caveolin-1 phosphorylation in HER2 trafficking. Implications: Together, our data for the first time identify hypoxic regulation of caveolin-1 as a resistance mechanism to T-DM1 with potential implications for individualized treatment of breast cancer.

Background Primary brain tumors, in particular glioblastoma (GBM), remain among the most challenging cancers. Like most malignant tumors, GBM is characterized by hypoxic stress that triggers paracrine, adaptive responses, such as angiogenesis and macrophage recruitment, rescuing cancer cells from metabolic catastrophe and conventional oncological treatments. The unmet need of strategies to efficiently target tumor “stressness” represents a strong clinical motivation to better understand the underlying mechanisms of stress adaptation. Here, we have investigated how lipid loading may be involved in the paracrine crosstalk between cancer cells and the stromal compartment of the hypoxic tumor microenvironment. Methods Regions from patient GBM tumors with or without the lipid loaded phenotype were isolated by laser capture microdissection and subjected to comparative gene expression analysis in parallel with cultured GBM cells with or without lipid loading. The potential involvement of extracellular lipids in the paracrine crosstalk with stromal cells was studied by immunoprofiling of the secretome and functional studies in vitro as well as in various orthotopic GBM mouse models, including hyperlipidemic ApoE−/− mice. Statistical analyses of quantitative experimental methodologies were performed using unpaired Student’s T test. For survival analyses of mouse experiments, log-rank test was used, whereas Kaplan-Meier was performed to analyze patient survival. Results We show that the lipid loaded niche of GBM patient tumors exhibits an amplified hypoxic response and that the acquisition of extracellular lipids by GBM cells can reinforce paracrine activation of stromal cells and immune cells. At the functional level, we show that lipid loading augments the secretion of e.g. VEGF and HGF, and may potentiate the cross-activation of endothelial cells and macrophages. In line with these data, in vivo studies suggest that combined local tumor lipid loading and systemic hyperlipidemia of ApoE−/− mice receiving a high fat diet induces tumor vascularization and macrophage recruitment, and was shown to significantly decrease animal survival. Conclusions Together, these data identify extracellular lipid loading as a potentially targetable modulator of the paracrine adaptive response in the hypoxic tumor niche and suggest the contribution of the distinct lipid loaded phenotype in shaping the glioma microenvironment. Electronic supplementary material The online version of this article (10.1186/s13046-019-1228-6) contains supplementary material, which is available to authorized users.

PurposeGlioblastoma multiforme (GBM) is the most common and lethal of primary malignant brain tumors. Hypoxia constitutes a major determining factor for the poor prognosis of high-grade glioma patients, and is known to contribute to the development of treatment resistance. Therefore, new strategies to comprehensively profile and monitor the hypoxic status of gliomas are of high clinical relevance. Here, we have explored how the proteome of secreted extracellular vesicles (EVs) at the global level may reflect hypoxic glioma cells.MethodsWe have employed shotgun proteomics and label free quantification to profile EVs isolated from human high-grade glioma U87-MG cells cultured at normoxia or hypoxia. Parallel reaction monitoring was used to quantify the identified, hypoxia-associated EV proteins. To determine the potential biological significance of hypoxia-associated proteins, the cumulative Z score of identified EV proteins was compared with GBM subtypes from HGCC and TCGA databases.ResultsIn total, 2928 proteins were identified in EVs, out of which 1654 proteins overlapped with the ExoCarta EV-specific database. We found 1034 proteins in EVs that were unique to the hypoxic status of U87-MG cells. We subsequently identified an EV protein signature, “HYPSIGNATURE”, encompassing nine proteins that strongly represented the hypoxic situation and exhibited close proximity to the mesenchymal GBM subtype.ConclusionsWe propose, for the first time, an EV protein signature that could comprehensively reflect the hypoxic status of high-grade glioma cells. The presented data provide proof-of-concept for targeted proteomic profiling of glioma derived EVs, which should motivate future studies exploring its utility in non-invasive diagnosis and monitoring of brain tumor patients.Electronic supplementary materialThe online version of this article (10.1007/s11060-019-03262-4) contains supplementary material, which is available to authorized users.