Decoding of the surfaceome and endocytome in primary glioblastoma cells identifies potential target antigens in the hypoxic tumor niche.

Immunotherapies with antibody-drug-conjugates (ADC) and CAR-T cells, targeted at tumor surface antigens (surfaceome), currently revolutionize clinical oncology. However, target identification warrants a better understanding of the surfaceome and how it is modulated by the tumor microenvironment. Here, we decode the surfaceome and endocytome and its remodeling by hypoxic stress in glioblastoma (GBM), the most common and aggressive brain tumor in adults. We employed a comprehensive approach for global and dynamic profiling of the surfaceome and endocytosed (endocytome) proteins and their regulation by hypoxia in patient-derived GBM cultures. We found a heterogeneous surface-endocytome profile and a divergent response to hypoxia across GBM cultures. We provide a quantitative ranking of more than 600 surface resident and endocytosed proteins, and their regulation by hypoxia, serving as a resource to the cancer research community. As proof-of-concept, the established target antigen CD44 was identified as a commonly and abundantly expressed surface protein with high endocytic activity. Among hypoxia induced proteins, we reveal CXADR, CD47, CD81, BSG, and FXYD6 as potential targets of the stressed GBM niche. We could validate these findings by immunofluorescence analyses in patient tumors and by increased expression in the hypoxic core of GBM spheroids. Selected candidates were finally confronted by treatment studies, showing their high capacity for internalization and ADC delivery. Importantly, we highlight the limited correlation between transcriptomics and proteomics, emphasizing the critical role of membrane protein enrichment strategies and quantitative mass spectrometry. Our findings provide a comprehensive understanding of the surface-endocytome and its remodeling by hypoxia in GBM as a resource for exploration of targets for immunotherapeutic approaches in GBM.

Landscape of surfaceome and endocytome in human glioma is divergent and depends on cellular spatial organization.

Therapeutic strategies directed at the tumor surfaceome (TS), including checkpoint inhibitor blocking antibodies, antibody drug conjugates (ADCs), and chimeric antigen receptor T (CAR-T) cells, provide a new armament to fight cancer. However, a remaining bottleneck is the lack of strategies to comprehensively interrogate patient tumors for potential TS targets. Here, we have developed a platform (tumor surfaceome mapping [TS-MAP]) integrated with a newly curated TS classifier (SURFME) that allows profiling of primary 3D cultures and intact patient glioma tumors with preserved tissue architecture. Moreover, TS-MAP specifically identifies proteins capable of endocytosis as tractable targets for ADCs and other modalities requiring toxic payload internalization. In high-grade gliomas that remain among the most aggressive forms of cancer, we show that cellular spatial organization (2D vs. 3D) fundamentally transforms the surfaceome and endocytome (e.g., integrins, proteoglycans, semaphorins, and cancer stem cell markers) with general implications for target screening approaches, as exemplified by an ADC targeting EGFR. The TS-MAP platform was further applied to profile the surfaceome and endocytome landscape in a cohort of freshly resected gliomas. We found a highly diverse TS repertoire between patient tumors, not directly associated with grade and histology, which highlights the need for individualized approaches. Our data provide additional layers of understanding fundamental to the future development of immunotherapy strategies, as well as procedures for proteomics-based target identification and selection. The TS-MAP platform should be widely applicable in efforts aiming at a better understanding of how to harness the TS for personalized immunotherapy.

Hypoxia Attenuates Trastuzumab Uptake and Trastuzumab-Emtansine (T-DM1) Cytotoxicity through Redistribution of Phosphorylated Caveolin-1.

The antibody-drug conjugate trastuzumab-emtansine (T-DM1) offers an additional treatment option for patients with HER2-amplified 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+ 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.

Global extracellular vesicle proteomic signature defines U87-MG glioma cell hypoxic status with potential implications for non-invasive diagnostics.

PURPOSE: Glioblastoma 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. METHODS: We 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. RESULTS: In 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. CONCLUSIONS: We 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.

Ultrasensitive Immunoprofiling of Plasma Extracellular Vesicles Identifies Syndecan-1 as a Potential Tool for Minimally Invasive Diagnosis of Glioma.

PURPOSE: Liquid biopsy has great potential to improve the management of brain tumor patients at high risk of surgery-associated 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 assay-based ultrasensitive immunoprofiling of plEV proteins that was applied on a well-defined glioma study cohort (n = 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 plEVSDC1 originating from GBM tumors, as plEVSDC1 correlated with SDC1 protein expression in matched patient tumors, and plEVSDC1 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.

Tumor antigen glycosaminoglycan modification regulates antibody-drug conjugate delivery and cytotoxicity.

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.

Viral aetiology and clinical outcomes in hospitalised infants presenting with respiratory distress.

AIM: To determine the prevalence of various types of viruses in infants hospitalised due to respiratory distress, compare molecular diagnostic tests and evaluate symptom severity. METHODS: All 136 nasopharyngeal aspirates from infants hospitalised for respiratory distress over a 9-month period were analysed for virus type by in-house respiratory syncytial virus (RSV) polymerase chain reaction (PCR) microarray-based and/or Luminex-based multiplex molecular tests. Medical records were reviewed retrospectively for clinical data. RESULTS: Viral aetiology was confirmed in 126 subjects (92.6%) with 26 infected by more than one virus. RSVA/B was the most common (50.9%), followed by entero/rhinovirus (21.6%), human metapneumovirus (10.5%), parainfluenza virus (5.9%) and influenza (3.3%). RSV-infected infants had significantly lower saturation levels (89% versus 92%, p < 0.001), higher demand for oxygen (42.7% versus 21.6%, p = 0.021) and fluids (28% versus 9.8%; p = 0.014) and longer hospital stays (4 versus 3 days, <0.001) than other viruses. Luminex assays gave repeatable, slightly less sensitive results than in-house RSV PCR. Microarray-based assays were more sensitive, however, producing some unrepeatable results. CONCLUSION: Respiratory syncytial virus dominates as the viral cause in hospitalised infants with respiratory distress in Sweden during the winter season, resulting in a clinical course that is significantly more severe. The multiplex assays produced reasonably concordant results.

Monitoring hepatitis C infection in a major Swedish nephrology unit and molecular resolution of a new case of nosocomial transmission.

Hepatitis C virus (HCV) infection is a frequent problem in hemodialysis units. The prevalence and incidence of HCV infection over a decade were studied in a nephrology unit affected by previous nosocomial HCV transmission. The HCV non-structural 5B protein gene was sequenced to achieve phylogenetic analysis of a new (incident) case of infection. Proportions of patients who were and were not infected with HCV remained similar over the period, as did the inflow and outflow of patients infected previously. In 1997, 12/157 (8%) of patients at the unit (treatment: hemodialysis, peritoneal dialysis, and renal transplant recipients) were positive in HCV RNA, whereas in 2007 the overall number was 9/239 (4%). One patient acquired an HCV infection, and the NS5B sequence in that case clustered with genotype 2b sequences found in patients from an earlier outbreak. Comparing the HCV from the incident patient with several stored longitudinal samples and cloned PCR products from the most likely source patient revealed close phylogenetic relationship with an HCV quasispecies member from the possible source. The source patient and the incident newly infected patient were not scheduled on the same dialysis shift, although the records showed that simultaneous treatment occurred on two occasions during the months preceding transmission. In conclusion, over the 10-year period, the proportion of HCV-infected patients at the unit was unchanged. Only one new infection occurred, which originated from a fellow patient's quasispecies. This establishes phylogenetic analysis as a valuable tool for tracing patient sources of HCV transmission.

Evolution of hepatitis C virus variants following blood transfusion from one infected donor to several recipients: a long-term follow-up.

Variants of hepatitis C virus (HCV) from a single infected blood donor and 13 viraemic recipients who were traced were examined by sequencing and cloning to determine the extent of virus diversity in hypervariable region 1. Serum-derived viral isolates were studied from the donor when his HCV infection was discovered in 1993, in his recipients that year (0.3-5 years post-transfusion) and 5 years later in the donor and six viraemic recipients who were still alive. Viral variants of broad diversity were readily demonstrated in the baseline samples of the donor (nucleotide p-distance 0.130), but significantly less (P<0.00003) diversity was observed in the recipients' first samples (p-distances within recipients 0.003-0.062). In the first blood samples of the recipients, many of the viral variants identified were closely related to a strain variant from the donor. In follow-up samples drawn 5 years later from the donor and six recipients, the p-distance among donor clones had increased (0.172, P<0.0005) compared with the recipients, who displayed significantly narrower quasispecies (0.011-0.086). A common finding was that recipients of blood components processed from the same donation differed substantially in persisting HCV infectious sequence. Markedly few changes leading to changes of amino acids had occurred during follow-up in four of six recipients. These results question the significance of the development of viral variants as a necessary phenomenon in the evolution of HCV and pathogenesis of the disease.