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Abstract Background Metastasis is the leading cause of cancer-related death in non-small cell lung cancer (NSCLC) patients. We previously showed that low HERC5 expression predicts early tumor dissemination and a dismal prognosis in NSCLC patients. Here, we performed functional studies to unravel the mechanism underlying the “metastasis-suppressor” effect of HERC5, with a focus on mitochondrial metabolism pathways. Methods We assessed cell proliferation, colony formation potential, anchorage-independent growth, migration, and wound healing in NSCLC cell line models with HERC5 overexpression (OE) or knockout (KO). To study early tumor cell dissemination, we used these cell line models in zebrafish experiments and performed intracardial injections in nude mice. Mass spectrometry (MS) was used to analyze protein changes in whole-cell extracts. Furthermore, electron microscopy (EM) imaging, cellular respiration, glycolytic activity, and lactate production were used to investigate the relationships with mitochondrial energy metabolism pathways. Results Using different in vitro NSCLC cell line models, we showed that NSCLC cells with low HERC5 expression had increased malignant and invasive properties. Furthermore, two different in vivo models in zebrafish and a xenograft mouse model showed increased dissemination and metastasis formation (in particular in the brain). Functional enrichment clustering of MS data revealed an increase in mitochondrial proteins in vitro when HERC5 levels were high. Loss of HERC5 leads to an increased Warburg effect, leading to improved adaptation and survival under prolonged inhibition of oxidative phosphorylation. Conclusions Taken together, these results indicate that low HERC5 expression increases the metastatic potential of NSCLC in vitro and in vivo. Furthermore, HERC5-induced proteomic changes influence mitochondrial pathways, ultimately leading to alterations in energy metabolism and demonstrating its role as a new potential metastasis suppressor gene.

Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3+ T cells isolated from the peripheral blood (n = 20), malignant ascites (n = 16), and tumor tissue (n = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8+ T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1high CD8+ T cell population was detected, which differed from PD-1lowCD8+ T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8+ T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (n = 14) and malignant ascites (n = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the “don’t eat me” molecule CD24 on tumor cells. Additionally, ascites-derived CD24+EpCAM+ tumor cells showed a higher frequency of CD39+ or CD73+ cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8+ T cells.

IntroductionActin-binding proteins (ABPs) are essential for the regulation of morphological plasticity required for tumor cells to metastasize. The aim of this study was to perform an unbiased bioinformatic approach to identify the key ABPs significantly associated with the metastatic potential of breast cancer cells.MethodsMicroarray data from 181 primary breast cancer samples from our hospital were used, and all genes belonging to the Gene Ontology term actin cytoskeleton organization were obtained from QuickGO. Association with metastasis-free survival probability was tested using Cox proportional hazards regression, and pairwise co-expression was tested by Pearson correlations. Differential expression between different subgroups was analyzed using Wilcoxon tests for dichotomous traits and Kruskal–Wallis tests for categorical traits. Validation was performed using four publicly available breast cancer datasets.ResultsARHGAP25 was significantly associated with a low metastatic potential, and CFL1, TMSB15A, and ACTL8 were significantly associated with a high metastatic potential. A significantly higher expression of CFL1, TMSB15A, and ACTL8 mRNA was found in the more aggressive Her2-positive and triple-negative subtypes as well as in ER-negative samples. Also, these genes were co-expressed in the same tumors. However, only mRNA levels of CFL1 were increased in pN1 compared to pN0 patients. External validation revealed that CFL1 and TMSB15A had significant associations with consistent hazard ratios in two breast cancer cohorts, and among these, CFL1 exhibited the highest hazard ratios.ConclusionCFL1 showed the strongest correlation with the metastatic potential of breast tumors. Thus, targeted inhibition of CFL1 might be a promising approach to treat malignant breast cancer cells.

Abstract Most cancer patients ultimately die from the consequences of distant metastases. As metastasis formation consumes energy mitochondria play an important role during this process as they are the most important cellular organelle to synthesise the energy rich substrate ATP, which provides the necessary energy to enable distant metastasis formation. However, mitochondria are also important for the execution of apoptosis, a process which limits metastasis formation. We therefore wanted to investigate the mitochondrial content in ovarian cancer cells and link its presence to the patient’s prognosis in order to analyse which of the two opposing functions of mitochondria dominates during the malignant progression of ovarian cancer. Monoclonal antibodies directed against different mitochondrial specific proteins, namely heat shock proteins 60 (HSP60), fumarase and succinic dehydrogenase, were used in immunohistochemistry in preliminary experiments to identify the antibody most suited to detect mitochondria in ovarian cancer cells in clinical tissue samples. The clearest staining pattern, which even delineated individual mitochondria, was seen with the anti-HSP60 antibody, which was used for the subsequent clinical study staining primary ovarian cancers (n = 155), borderline tumours (n = 24) and recurrent ovarian cancers (n = 26). The staining results were semi-quantitatively scored into three groups according to their mitochondrial content: low (n = 26), intermediate (n = 50) and high (n = 84). Survival analysis showed that high mitochondrial content correlated with a statistically significant overall reduced survival rate In addition to the clinical tissue samples, mitochondrial content was analysed in ovarian cancer cells grown in vitro (cell lines: OVCAR8, SKOV3, OVCAR3 and COV644) and in vivo in severe combined immunodeficiency (SCID) mice. In in vivo grown SKOV3 and OVCAR8 cells, the number of mitochondria positive cells was markedly down-regulated compared to the in vitro grown cells indicating that mitochondrial number is subject to regulatory processes. As high mitochondrial content is associated with a poor prognosis, the provision of high energy substrates by the mitochondria seems to be more important for metastasis formation than the inhibition of apoptotic cell death, which is also mediated by mitochondria. In vivo and in vitro grown human ovarian cancer cells showed that the mitochondrial content is highly adaptable to the growth condition of the cancer cells.

Abstract The transcription factor ETV7 is an oncoprotein that is up-regulated in all breast cancer (BC) types. We have recently demonstrated that ETV7 promoted breast cancer progression by increasing cancer cell proliferation and stemness and was also involved in the development of chemo- and radio-resistance. However, the roles of ETV7 in breast cancer inflammation have yet to be studied. Gene ontology analysis previously performed on BC cells stably over-expressing ETV7 demonstrated that ETV7 was involved in the suppression of innate immune and inflammatory responses. To better decipher the involvement of ETV7 in these signaling pathways, in this study, we identified TNFRSF1A, encoding for the main receptor of TNF-α, TNFR1, as one of the genes down-regulated by ETV7. We demonstrated that ETV7 directly binds to the intron I of this gene, and we showed that the ETV7-mediated down-regulation of TNFRSF1A reduced the activation of NF-κB signaling. Furthermore, in this study, we unveiled a potential crosstalk between ETV7 and STAT3, another master regulator of inflammation. While it is known that STAT3 directly up-regulates the expression of TNFRSF1A, here we demonstrated that ETV7 reduces the ability of STAT3 to bind to the TNFRSF1A gene via a competitive mechanism, recruiting repressive chromatin remodelers, which results in the repression of its transcription. The inverse correlation between ETV7 and TNFRSF1A was confirmed also in different cohorts of BC patients. These results suggest that ETV7 can reduce the inflammatory responses in breast cancer through the down-regulation of TNFRSF1A.

Abstract Background The immunological composition of the tumor microenvironment has a decisive influence on the biological course of cancer and is therefore of profound clinical relevance. In this study, we analyzed the cooperative effects of integrin β4 (ITGB4) on tumor cells and E-/P-selectin on endothelial cells within the tumor stroma for regulating tumor growth by shaping the local and systemic immune environment. Methods We used several preclinical mouse models for different solid human cancer types (xenograft and syngeneic) to explore the role of ITGB4 (shRNA-mediated knockdown in tumor cells) and E-/P-selectins (knockout in mice) for tumor growth; effects on apoptosis, proliferation and intratumoral signaling pathways were determined by histological and biochemical methods and 3D in vitro experiments; changes in the intratumoral and systemic immune cell composition were determined by flow cytometry and immunohistochemistry; chemokine levels and their attracting potential were measured by ELISA and 3D invasion assays. Results We observed a very robust synergism between ITGB4 and E-/P-selectin for the regulation of tumor growth, accompanied by an increased recruitment of CD11b+ Gr-1Hi cells with low granularity (i.e., myeloid-derived suppressor cells, MDSCs) specifically into ITGB4-depleted tumors. ITGB4-depleted tumors undergo apoptosis and actively attract MDSCs, well-known to promote tumor growth in several cancers, via increased secretion of different chemokines. MDSC trafficking into tumors crucially depends on E-/P-selectin expression. Analyses of clinical samples confirmed an inverse relationship between ITGB4 expression in tumors and number of tumor-infiltrating leukocytes. Conclusions These findings suggest a distinct vulnerability of ITGB4Lo tumors for MDSC-directed immunotherapies. Graphical Abstract

Abstract Background The disruption of epithelial features represents a critical step during breast cancer spread. In this context, the dysregulation of desmosomal proteins has been associated with malignant progression and metastasis formation. Curiously, both tumour suppressive and pro-metastatic roles have been attributed to desmosomal structures in different cancer entities. In the present study, we describe the pro-metastatic role of the desmosomal protein desmocollin 2 (DSC2) in breast cancer. Methods We analysed the prognostic role of DSC2 at mRNA and protein level using microarray data, western blot analysis and immunohistochemistry. Functional consequences of DSC2 overexpression and DSC2 knock down were investigated in the triple negative breast cancer (TNBC) cell line MDA-MB-231 and its brain-seeking subline MDA-MB-231-BR, respectively in vitro and in vivo. Results We found a significantly higher DSC2 expression in the more aggressive molecular subtypes HER2-positive and TNBC than in luminal breast cancers, as well as a significant correlation between increased DSC2 expression and a shorter disease-free—also in multivariate analysis—and overall survival. Additionally, a significant association between DSC2 expression in the primary tumour and an increased frequency of cerebral and lung metastasis could be observed. In vitro, ectopic DSC2 expression or DSC2 down-regulation in MDA-MB-231 and MDA-MB-231-BR led to a significant tumour cell aggregation increase and decrease, respectively. Furthermore, tumour cells displaying higher DSC2 levels showed increased chemoresistance in 3D structures, but not 2D monolayer structures, suggesting the importance of cell aggregation as a means for reduced drug diffusion. In an in vivo brain dissemination xenograft mouse model, reduced expression of DSC2 in the brain-seeking TNBC cells led to a decreased amount of circulating tumour cells/clusters and, in turn, to fewer and smaller brain metastatic lesions. Conclusion We conclude that high DSC2 expression in primary TNBC is associated with a poorer prognosis, firstly by increasing tumour cell aggregation, secondly by reducing the diffusion and effectiveness of chemotherapeutic agents, and, lastly, by promoting the circulation and survival of tumour cell clusters, each of which facilitates distant organ colonisation.

Examinations of ovarian cancer cells require the ability to identify tumor cells. Array-based comparative genome hybridization (aCGH) on 30 ovarian carcinomas (OC) identified three genomic loci (8q24.23; 17p12; 18q22.3) over- or under-represented in OC. A fluorescence in situ hybridization (FISH) probe of these three loci is intended to identify tumor cells by their signal pattern deviating from a diploid pattern. Human DNA from these three loci is isolated from bacterial artificial chromosomes (BAC), amplified and labeled with fluorescent dyes. After a standard FISH procedure, 71 OC suspensions from primary tumors, three OC cell lines, three lymphocyte suspensions, and one mesenchymal cell line LP-3 are analyzed with a fluorescence microscope. On average, 15% of the lymphocytes deviate from the expected diploid signal pattern, giving a cut-off of 36%. If this value is exceeded, tumor cells are detected. The mesenchymal cell line LP-3 shows only 21% as a negative control. The OC cell lines as positive controls exceed this value at 38%, 67%, and 54%. Of the 71 OC primary cultures, four cases fell below this cut-off as false negatives. In the two-sample t-test, the percentages of conspicuous signal patterns differ significantly.

IntroductionTumor-associated macrophages (TAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype and function of these cells. The present study aims to characterize macrophages in high-grade serous ovarian cancer (HGSOC).MethodsPhenotype and expression of co-regulatory markers were assessed on TAMs derived from malignant ascites (MA) or peripheral blood (PB) by multiparametric flow cytometry. Samples were obtained from HGSOC patients (n=29) and healthy donors (HDs, n=16). Additional expression analysis was performed by RNAseq (n=192). Correlation with clinically relevant parameters was conducted and validated by a second patient cohort (n=517). Finally, the role of TIGIT in repolarization and phagocytosis was investigated in vitro.ResultsExpression of the M2-associated receptors CD163, CD204, and CD206, as well as of the co-regulatory receptors TIGIT, CD226, TIM-3, and LAG-3 was significantly more frequent on macrophages in HGSOC than in HDs. CD39 and CD73 were broadly expressed on (mainly M2) macrophages, but without a clear clustering in HGSOC. CD163 mRNA levels were higher in TAMs from patients with residual tumor mass after surgery and associated with a shorter overall survival. In addition, TIGIT expression was associated with a higher tumor grading, indicating a prognostic relevance of M2 infiltration in HGSOC. TIGIT blockade significantly reduced the frequency of M2 macrophages. Moreover, combined blockade of TIGIT and CD47 significantly increased phagocytosis of ovarian cancer cells by TAMs in comparison to a single blockade of CD47.ConclusionCombined blockade of TIGIT and CD47 represents a promising approach to enhance anti-CD47-facilitated phagocytosis.

IntroductionThe PI3K/AKT pathway is activated in 43-70% of breast cancer (BC)-patients and promotes the metastatic potential of BC cells by increasing cell proliferation, invasion and radioresistance. Therefore, AKT1-inhibition in combination with radiotherapy might be an effective treatment option for triple-negative breast cancer (TNBC)-patients with brain metastases.MethodsThe impact of AKT1-knockout (AKT1_KO) and AKT-inhibition using Ipatasertib on MDA-MB-231 BR cells was assessed using in vitro cell proliferation and migration assays. AKT1-knockout in MDA-MB-231BR cells was performed using CRISPR/Cas9. The effect of AKT1-knockout on radiosensitivity of MDA-MB-231BR cell lines was determined via colony formation assays after cell irradiation. To detect genomic variants in AKT1_KO MDA-MB-231BR cells, whole-genome sequencing (WGS) was performed.ResultsPharmacological inhibition of AKT with the pan-AKT inhibitor Ipatasertib led to a significant reduction of cell viability but did not impact cell migration. Moreover, only MDA-MB-231BR cells were sensitized following Ipatasertib-treatment. Furthermore, specific AKT1-knockout in MDA-MB-231BR showed reduced cell viability in comparison to control cells, with significant effect in one of two analyzed clones. Unexpectedly, AKT1 knockout led to increased cell migration and clonogenic potential in both AKT1_KO clones. RNAseq-analysis revealed the deregulation of CTSO, CYBB, GPR68, CEBPA, ID1, ID4, METTL15, PBX1 and PTGFRN leading to the increased cell migration, higher clonogenic survival and decreased radiosensitivity as a consequence of the AKT1 knockout in MDA-MB-231BR.DiscussionCollectively, our results demonstrate that Ipatasertib leads to radiosensitization and reduced cell proliferation of MDA-MB-231BR. AKT1-inhibition showed altered gene expression profile leading to modified cell migration, clonogenic survival and radioresistance in MDA-MB-231BR. We conclude, that AKT1-inhibition in combination with radiotherapy contribute to novel treatment strategies for breast cancer brain metastases.

Ovarian cancer disseminates primarily intraperitoneally. Detached tumor cell aggregates (spheroids) from the primary tumor are regarded as ‘metastatic units’ that exhibit a low sensitivity to classical chemotherapy, probably due to their unique molecular characteristics. We have analyzed the cellular composition of ascites from OvCa patients, using flow cytometry, and studied their behavior in vitro and in vivo. We conclude that ascites‐derived cultured cells from OvCa patients give rise to two subpopulations: adherent cells and non‐adherent cells. Here, we found that the AD population includes mainly CD90+ cells with highly proliferative rates in vitro but no tumorigenic potential in vivo, whereas the NAD population contains principally tumor cell spheroids (EpCAM+/CD24+) with low proliferative potential in vitro. Enriched tumor cell spheroids from the ascites of high‐grade serous OvCA patients, obtained using cell strainers, were highly tumorigenic in vivo and their metastatic spread pattern precisely resembled the tumor dissemination pattern found in the corresponding patients. Comparative transcriptome analyses from ascites‐derived tumor cell spheroids (n = 10) versus tumor samples from different metastatic sites (n = 30) revealed upregulation of genes involved in chemoresistance (TGM1, HSPAs, MT1s), cell adhesion and cell‐barrier integrity (PKP3, CLDNs, PPL), and the oxidative phosphorylation process. Mitochondrial markers (mass and membrane potential) showed a reduced mitochondrial function in tumoroids from tumor tissue compared with ascites‐derived tumor spheroids in flow cytometry analysis. Interestingly, response to OXPHOS inhibition by metformin and IACS010759 in tumor spheroids correlated with the extent of mitochondrial membrane potential measured by fluorescence‐activated cell sorting. Our data contribute to a better understanding of the biology of ovarian cancer spheroids and identify the OXPHOS pathway as new potential treatment option in advanced ovarian cancer.

Methylation of the BRCA1 promoter is an epigenetic gene expression regulator and is frequently observed in ovarian cancer; however, conversion of methylation status is thought to drive disease recurrence. Therefore, longitudinal monitoring of methylation status by liquid biopsy in cell‐free DNA may be a predictive marker. In total, 135 plasma samples were collected from 69 ovarian cancer patients before and during systemic treatment. Our liquid biopsy assay could detect down to a single molecule of methylated DNA in a high background of normal DNA (0.03%) with perfect specificity in control samples. We found that 60% of the cancer patients exhibited BRCA1 promoter hypermethylation at one point, although 24% lost hypermethylation during treatment. Multivariate survival analyses indicate that relapses are independent events and that hypermethylation and methylation conversion are independently correlated to longer relapse‐free survival. We present a highly sensitive and specific methylation‐specific quantitative PCR‐based liquid biopsy assay. BRCA1 promoter hypermethylation is frequently found in ovarian cancer and is often reversed upon recurrence, indicating the selection of therapy‐resistant clones and unfavorable clinical outcome.

Abstract Background The survival rate is poor in breast cancer patients with brain metastases. Thus, new concepts for therapeutic approaches are required. During metastasis, the cytoskeleton of cancer cells is highly dynamic and therefore cytoskeleton-associated proteins are interesting targets for tumour therapy. Methods Screening for genes showing a significant correlation with brain metastasis formation was performed based on microarray data from breast cancer patients with long-term follow up information. Validation of the most interesting target was performed by MTT-, Scratch- and Transwell-assay. In addition, intracellular trafficking was analyzed by live-cell imaging for secretory vesicles, early endosomes and multiple vesicular bodies (MVB) generating extracellular vesicles (EVs). EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Western blotting, mass spectrometry, and ingenuity pathway analysis (IPA). Effect of EVs on the blood-brain-barrier (BBB) was examined by incubating endothelial cells of the BBB (hCMEC/D3) with EVs, and permeability as well as adhesion of breast cancer cells were analyzed. Clinical data of a breast cancer cohort was evaluated by χ2-tests, Kaplan-Meier-Analysis, and log-rank tests while for experimental data Student’s T-test was performed. Results Among those genes exhibiting a significant association with cerebral metastasis development, the only gene coding for a cytoskeleton-associated protein was Tubulin Tyrosine Ligase Like 4 (TTLL4). Overexpression of TTLL4 (TTLL4plus) in MDA-MB231 and MDA-MB468 breast cancer cells (TTLL4plus cells) significantly increased polyglutamylation of β-tubulin. Moreover, trafficking of secretory vesicles and MVBs was increased in TTLL4plus cells. EVs derived from TTLL4plus cells promote adhesion of MDA-MB231 and MDA-MB468 cells to hCMEC/D3 cells and increase permeability of hCMEC/D3 cell layer. Conclusions These data suggest that TTLL4-mediated microtubule polyglutamylation alters exosome homeostasis by regulating trafficking of MVBs. The TTLL4plus-derived EVs may provide a pre-metastatic niche for breast cancer cells by manipulating endothelial cells of the BBB.

ObjectiveAt present, maintenance therapy with the antiangiogenic agent bevacizumab or with PARP-inhibitors represent two options for BRCA-wildtype ovarian cancer patients, after platinum-based first line chemotherapy. The identification of molecular markers to predict patient response to different maintenance therapies remains a major challenge. In the present study we analyzed the predictive potential of vascular endothelial growth factor C (VEGF-C) to identify ovarian cancer patients that might benefit from an antiangiogenic therapy.Methods101 patients with primary epithelial ovarian cancer were analyzed for serum levels of VEGF-A,-C and CA-125 by ELISA. Serum levels were compared between patients with low pT-stage (pT1a-pT2c n = 11), healthy individuals (n = 27) and patients with higher pT-stage (> = pT3 n = 90). Adjusted ROC curves and an adjusted logistic regression model were carried out to evaluate the potential impact of VEGF-A and -C, as well as CA-125 serum level concentration on bevacizumab-therapy response, under consideration of covariates such as FIGO, pM, pN and residual tumor after surgery.ResultsA patient which has in comparison twice the VEGF-C concentration in serum, has a significant increased chance of response to bevacizumab by a factor of 2.79. Further, only VEGF-C serum levels were significantly higher in the group of patients with lower pT-stage compared to healthy individuals, whereas VEGF-A or CA-125 serum levels could not discriminate between healthy individuals and patients with ovarian cancer at low pT-stages.ConclusionVEGF-C serum level might serve as as a biomarker to evaluate treatment response under bevacizumab.

Background: Hyaluronan (HA), a component of the extracellular matrix, is frequently increased under pathological conditions including cancer. Not only stroma cells but also cancer cells themselves synthesize HA, and the interaction of HA with its cognate receptors promotes malignant progression and metastasis. Methods: In the present study, HA deposition in tissue sections was analyzed by hyaluronan-binding protein (HABP) ligand histochemistry in 17 borderline tumors and 102 primary and 20 recurrent ovarian cancer samples. The intensity and, particularly, localization of the HA deposition were recorded: for the localization, the pericellular deposition around the ovarian cancer cells was distinguished from the deposition within the stromal compartment. These histochemical data were correlated with clinical and pathological parameters. Additionally, within a reduced subgroup of ovarian cancer samples (n = 70), the RNA levels of several HA-associated genes were correlated with the HA localization and intensity. Results: Both stroma-localized and pericellular tumor-cell-associated HA deposition were observed. Cancer-cell pericellular HA deposition, irrespective of its staining intensity, was significantly associated with malignancy, and in the primary ovarian cancer cohort, it represents an independent unfavorable prognostic marker for overall survival. Furthermore, a significant association between high CD44, HAS2 and HAS3 mRNA levels and a cancer-cell pericellular HA-deposition pattern was noted. In contrast, stromal hyaluronan deposition had no impact on ovarian cancer prognosis. Conclusions: In conclusion, the site of HA deposition is of prognostic value, but the amount deposited is not. The significant association of only peritumoral cancer-cell HA deposition with high CD44 mRNA expression levels suggests a pivotal role of the CD44–HA signaling axis for malignant progression in ovarian cancer.

Breast cancer (BC) is the second-most common cause of brain metastases (BM) and BCBM patients have a reduced quality of life and a poor prognosis. Hyaluronan (HA), and in particular the hyaluronidase Hyal-1, has been already linked to the development of BCBM, and therefore presents an interesting opportunity to develop new effective therapeutic options. HA metabolism was further discovered by the CRISPR/Cas9-mediated knockout of HYAL1 and the shRNA-mediated down-regulation of HA-receptor CD44 in the brain-seeking triple-negative breast cancer (TNBC) cell line MDA-MB-231-BR. Therefore, the impact of Hyal-1 on adhesion, disruption, and invasion through the brain endothelium, both in vitro and in vivo, was studied. Our analysis points out a key role of Hyal-1 and low-molecular-weight HA (LMW-HA) in the formation of a pericellular HA-coat in BC cells, which in turn promotes tumor cell adhesion, disruption, and migration through the brain endothelium in vitro as well as the extent of BM in vivo. CD44 knockdown in MDA-MB-231-BR significantly reduced the pericellular HA-coat on these cells, and, consequently, tumor cell adhesion and invasion through the brain endothelium. Thus, the interaction between Hyal-1-generated LMW-HA fragments and the HA-receptor CD44 might represent a potential target for future therapeutic options in BC patients with a high risk of cerebral metastases formation.

Abstract Background Ligands of the C-type lectin CLEC10A such as Tn and sialyl-Tn representing early intermediates of O-glycosylation are hallmarks of many human malignancies. A variety of regulatory mechanisms underlying their expression are being discussed. Methods CLEC10A ligands were detected in various tissues and cells using the recombinant glycan-binding domain of CLEC10A. In normal breast and endometrium, presence of ligands was correlated to the female cycle. Estrogen- and stress dependent induction of CLEC10A ligands was analyzed in MCF7 and T47D cells exposed to 4-hydroxy-tamoxifen (Tam), zeocin and hydrogen peroxide. The expression and localization of CLEC10A ligands was analyzed by Western blot and immunofluorescence. In breast cancer patients CLEC10A ligand expression and survival was correlated by Kaplan-Meyer analysis. Result We observed binding of CLEC10A in normal endometrial and breast tissues during the late phase of the female hormonal cycle suggesting a suppressive effect of female sex hormones on CLEC10A ligand expression. Accordingly, CLEC10A ligands were induced in MCF7- and T47D breast cancer cells after Tam treatment and accumulated on the cell surface and in the endosomal/lysosomal compartment. Phagocytosis experiments indicate that macrophages preferentially internalize CLEC10A ligands coated beads and Tam treated MCF7 cells. CLEC10A ligands were also expressed after the addition of zeocin and hydrogen-peroxide. Each substance induced the production of ROS indicating reactive oxygen species as a unifying mechanism of CLEC10A ligand induction. Mechanistically, increased expression of GalNAc-transferase 6 (GalNT6) and translocation of GalNT2 and GalNT6 from cis- towards trans-Golgi compartment was observed, while protein levels of COSMC and T-synthase remained unaffected. In breast cancer patients, positivity for CLEC10A staining in tumor tissues was associated with improved outcome and survival. Conclusion CLEC10A ligands are inducible by hormone depletion, 4-hydroxy-tamoxifen and agents inducing DNA damage and oxidative stress. Our results indicate that CLEC10A acts as a receptor for damaged and dead cells and may play an important role in the uptake of cell debris by macrophages and dendritic cells. Graphical Abstract

Long noncoding RNAs (lncRNAs) are frequently precursor RNAs of microRNAs (miRNAs) or act as competing endogenous RNAs (ceRNAs) to interact with miRNAs. To better understand the shared impact of lncRNAs and miRNAs in the regulatory post‐transcriptional network, we focused here on the relationships between (a) lncRNA H19 and miR‐675, (b) NEAT1 and miR‐204, and (c) HOTAIR and miR‐331 in plasma of early breast cancer (BC) patients. We quantified each RNA in plasma samples of 63 BC patients and 10 healthy women by quantitative real‐time PCR. In cell culture experiments, the influence of these noncoding RNAs (ncRNAs) on proliferation and apoptosis of BC cell line MCF‐7 was examined. Plasma levels of H19 (P = 0.030), NEAT1 (P = 0.030), and miR‐331 (P = 0.012) were deregulated in BC patients compared with healthy women. In both cohorts, the concentrations of H19 correlated with those of miR‐675 (P = 0.0001). Higher H19 (P = 0.001) along with lower miR‐675 (P = 0.007) levels and higher miR‐204 (P = 0.017) along with lower NEAT1 (P = 0.030) levels were detected in plasma of HER2‐positive patients compared with the other BC subgroups. Whereas the expression of HOTAIR was below the detection level, miR‐331 levels correlated with nodal status (P = 0.002) and recurrence (P = 0.012). In cell culture experiments, a competitive impact on cell proliferation and apoptosis by these ncRNAs was also documented. Our findings describe a relationship of the plasma levels of H19/miR‐675 and NEAT1/miR‐204 in the different BC subtypes; in addition, they reveal an interplay between these lncRNAs and miRNAs in the regulatory network in MCF‐7 cells, which should also be considered in the search for new diagnostic and therapeutic markers.

Phenotypic characterization of γδ T cells in the MALs (malignant ascites lymphocytes), TILs (tumor infiltrating lymphocytes), and PBLs (peripheral blood lymphocytes) of ovarian cancer (OvCA) patients is lacking. Therefore, we quantified γδ T cell prevalence in MAL, TIL, and PBL specimens from n = 18 OvCA patients and PBL from age-matched healthy donors (HD, n = 14). Multicolor flow cytometry was performed to evaluate the expression of inhibitory receptors (TIGIT, PD-1 and TIM-3), stimulatory receptors (Ox40), and purinergic ectoenzymes (CD39 and CD73) on γδ T cell subsets. We identified an abundant infiltration of Vδ1 T cells in the MALs and TILs. These cells varied in their differentiation: The majority of Vδ1 TILs displayed an effector memory (EM) phenotype, whereas Vδ1 MALs had a more mature phenotype of terminally differentiated effector memory cells (TEMRA) with high CD45RA expression. TIGIT and TIM-3 were abundantly expressed in both MALs and PBLs, whereas Vδ1 TILs exhibited the highest levels of PD-1, CD39, and Ox40. We also observed specific clusters on mature differentiation stages for the analyzed molecules. Regarding co-expression, Vδ1 TILs showed the highest levels of cells co-expressing TIGIT with PD-1 or CD39 compared to MALs and PBLs. In conclusion, the Vδ1 T cell population showed a high prevalence in the MALs and primary tumors of OvCA patients. Due to their (co-)expression of targetable immune receptors, in particular TIGIT with PD-1 and CD39 in TILs, Vδ1 T cell-based approaches combined with the inhibition of these targets might represent a promising strategy for OvCA.

Epithelial ovarian cancer (EOC) was previously shown to be associated with glycosylation changes of total serum and total IgG proteins. However, as a majority of previous studies analyzed released glycan profiles, still little is known about IgG subclass-specific alterations in ovarian cancer. Hence, in this study, we investigated EOC-related glycosylation changes of the three most abundant IgG subclasses, namely, IgG1, IgG2 and IgG3 isolated from sera of 87 EOC patients and 74 age-matched healthy controls. In order to separate IgG2 and IgG3, we performed a two-step affinity purification employing Protein A and Protein G Sepharose. After tryptic digestion, IgG glycopeptides were enriched and measured by MALDI-TOF-MS. Finally, EOC-related glycosylation changes were monitored at the level of total agalactosylation, monogalactosylation, digalactosylation, sialylation, bisection and fucosylation, which were calculated separately for each IgG subclass. Interestingly, aside from an EOC-related increase in agalactosylation/decrease in monogalactosylation and digalactosylation observed in all IgG subclasses, some subclass-specific trends were detected. Glycosylation of IgG1 was found to be most strongly affected in EOC, as it exhibited the highest number of significant differences between healthy controls and EOC patients. Specifically, IgG1 was the only subclass that showed a significant decrease in sialylation and a significant increase in fucosylation in EOC patients. Interestingly, IgG2 and IgG3 that were often investigated collectively in previous studies, were found to have distinct glycosylation patterns. IgG3 displayed stronger EOC-related increase in agalactosylation/decrease in digalactosylation and was characterized by notably higher sialylation, which consequently decreased in EOC patients. In conclusion, our study indicates that IgG subclasses exhibit subtly distinct glycosylation patterns of EOC-related alterations and that IgG1 and IgG3 agalactosylation show the strongest association with CA125, the routine diagnostic marker. Additionally, our results show that simultaneous analyses of IgG2 and IgG3 might lead to wrong conclusions as these two subclasses exhibit noticeably different glycosylation phenotypes.

Brain metastases (BM) represent a growing problem for breast cancer (BC) patients. Recent studies have demonstrated a strong impact of the BC molecular subtype on the incidence of BM development. This study explores the interaction between BC cells of different molecular subtypes and the blood–brain barrier (BBB). We compared the ability of BC cells of different molecular subtypes to overcome several steps (adhesion to the brain endothelium, disruption of the BBB, and invasion through the endothelial layer) during cerebral metastases formation, in vitro as well as in vivo. Further, the impact of these cells on the BBB was deciphered at the molecular level by transcriptome analysis of the triple-negative (TNBC) cells themselves as well as of hBMECs after cocultivation with BC cell secretomes. Compared to luminal BC cells, TNBC cells have a greater ability to influence the BBB in vitro and consequently develop BM in vivo. The brain-seeking subline and parental TNBC cells behaved similarly in terms of adhesion, whereas the first showed a stronger impact on the brain endothelium integrity and increased invasive ability. The comparative transcriptome revealed potential brain-metastatic-specific key regulators involved in the aforementioned processes, e.g., the angiogenesis-related factors TNXIP and CXCL1. In addition, the transcriptomes of the two TNBC cell lines strongly differed in certain angiogenesis-associated factors and in several genes related to cell migration and invasion. Based on the present study, we hypothesize that the tumor cell’s ability to disrupt the BBB via angiogenesis activation, together with increased cellular motility, is required for BC cells to overcome the BBB and develop brain metastases.

Specific microRNAs (miRNAs) are packaged in exosomes that regulate processes in tumor development and progression. The current study focuses on the influence of exosomal miRNAs in the pathogenesis of epithelial ovarian cancer (EOC). MiRNA profiles were determined in exosomes from plasma of 106 EOC patients, eight ovarian cystadenoma patients, and 29 healthy women by TaqMan real‐time PCR‐based miRNA array cards containing 48 different miRNAs. In cell culture experiments, the impact of miR‐200b and miR‐320 was determined on proliferation and apoptosis of ovarian cancer cell lines. We report that miR‐21 (P = 0.0001), miR‐100 (P = 0.034), miR‐200b (P = 0.008), and miR‐320 (P = 0.034) are significantly enriched, whereas miR‐16 (P = 0.009), miR‐93 (P = 0.014), miR‐126 (P = 0.012), and miR‐223 (P = 0.029) are underrepresented in exosomes from plasma of EOC patients as compared to those of healthy women. The levels of exosomal miR‐23a (P = 0.009, 0.008) and miR‐92a (P = 009, 0.034) were lower in ovarian cystadenoma patients than in EOC patients and healthy women, respectively. The exosomal levels of miR‐200b correlated with the tumor marker CA125 (P = 0.002) and patient overall survival (P = 0.019). MiR‐200b influenced cell proliferation (P = 0.0001) and apoptosis (P

Immune checkpoints are intensively investigated as targets in cancer therapy. T-cell immunoreceptor with immunoglobulin (Ig) and ITIM domains (TIGIT) and its ligand poliovirus receptor (PVR) are recently emerging as novel promising targets in immunotherapy. Here, we show that high expression of PVR represents an independent prognostic marker being associated with poor outcome for breast cancer patients. Furthermore, PVR mRNA, as well as protein expression, is associated with more aggressive breast cancer subtypes such as HER2 positive and triple-negative breast cancer. In vitro, blocking TIGIT or PVR resulted in enhanced immune cell-mediated lysis of breast cancer cell lines SKBR-3, MDA-MB-231, MDA-MB-468, and BT549 and additionally increased the cytotoxic effects of a bispecific T cell engager BiTE® antibody construct targeting EGFR. Taken together, our data identify the immune checkpoint factor PVR as a novel prognostic marker in breast cancer and indicate that blocking the TIGIT-PVR axis might represent a novel therapeutic option for the treatment of breast cancer patients.

During the last few years, diverse studies have shown that tumors can actively interact with the lymphatic system and promote metastases development. In order to examine the molecular mechanisms involved in this interaction, we co-cultured tumor and lymphatic endothelial cells (LEC) and subsequently analyzed the molecular alterations of LECs. Therefore, LECs were co-cultivated with either a highly or weakly metastatic breast cancer cell line using contact (mixture) and non-contact (transwell) co-cultures. mRNA profiles from LECs were subsequently analyzed for genes specifically induced by highly metastatic tumor cells (“metastatic specific”). Among the up-regulated “metastatic specific” genes, we found candidates involved in cell cycle, cell adhesion and motility (BST2, E-selectin, and HMMR), cytokines (CCL7, CXCL6, CXCL1, and CSF2) and factors of the complement system (C1R, C3, and CFB). Among the down-regulated genes, we detected the hyaluronan receptor STAB2, angiogenic factor apelin receptor (APLNR), and the glycosylation enzyme MAN1A1. In an additional prostate cancer co-culture model, we could confirm a “metastatic specific” upregulation of E-selectin and CCL7 in LECs after interaction with the prostate cancer cell lines LNCAP (highly metastatic) and DU145 (weakly metastatic). These data allowed us to identify a set of genes regulated in LECs during in vitro communication with cancer cells, which might subsequently facilitate lymphatic metastasis.

The Loeys-Dietz syndrome (LDS) is an inherited connective tissue disorder caused by mutations in the transforming growth factor β (TGF-β) receptors TGFBR1 or TGFBR2. Most patients with LDS develop severe aortic aneurysms resulting in early need of surgical intervention. In order to gain further insight into the pathophysiology of the disorder, we investigated circulating outgrowth endothelial cells (OEC) from the peripheral blood of LDS patients from a cohort of 23 patients including 6 patients with novel TGF-β receptor mutations.We performed gene expression profiling of OECs using microarray analysis followed by quantitative PCR for verification of gene expression. Compared to OECs of age- and sex-matched healthy controls, OECs isolated from three LDS patients displayed altered expression of several genes belonging to the TGF-β pathway, especially those affecting bone morphogenic protein (BMP) signalling including BMP2, BMP4 and BMPR1A. Gene expression of BMP antagonist Gremlin-1 (GREM1) showed the most prominent up-regulation. This increase was confirmed at the protein level by immunoblotting of LDS-OECs. In immunohistochemistry, abundant Gremlin-1 protein expression could be verified in endothelial cells as well as smooth muscle cells within the arterial media. Furthermore, Gremlin-1 plasma levels of LDS patients were significantly elevated compared to healthy control subjects.These findings open new avenues in the understanding of the pathogenesis of Loeys-Dietz syndrome and the development of new diagnostic serological methods for early disease detection.