HERC5 downregulation in non-small cell lung cancer is associated with altered energy metabolism and metastasis.

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.

Detection of Multiple HPV Types in Liquid Biopsies of Cervical Neoplasia.

BACKGROUND: More than 95% of cervical cancers and their precancerous lesions are caused by human papillomavirus (HPV). Cell-free (cf) HPV DNA detection in blood samples may serve as a monitoring tool for cervical cancer. METHODS: In our methodological study, an HPV panel for simultaneous detection of 24 types using mass spectrometry-based analysis was developed for liquid biopsy approaches and tested on HPV positive cell lines, plasmid controls, and cervical high-grade squamous intraepithelial lesions (HSIL) in positive smear samples (n = 52). It was validated in cfDNA blood samples (n = 40) of cervical cancer patients. RESULTS: The HPV panel showed proficient results in cell lines and viral plasmids with a limit of detection of 1 IU (international units)/µL for HPV16/18 and 10GE/µL for HPV11/31/33/39/45/51/52/58/59 and a specificity of 100% for the tested HPV types. In cervical smear samples, HPV DNA was detected with a sensitivity of 98.14%. The overall agreement between the new HPV panel and clinical records was 97.2% (κ = 0.84). In cervical cancer cfDNA, 26/40 (65.0%) tested positive for any HPV type, with most infections due to hrHPV (24/26). HPV positive samples were found in all FIGO stages, with the highest positivity ratio in FIGO III and IV. Even the lowest stage, FIGO I, had 12/23 (52.2%) patients with a positive HPV plasma status. CONCLUSIONS: This proof-of-concept paper shows that the described assay produces reliable results for detecting HPV types in a multiplex mass spectrometry-based assay in cervical smear and cfDNA with high specificity and sensitivity in both cohorts. The assay shows potential for liquid biopsy-based applications in monitoring cervical cancer progression.

Ovarian cancer cells regulate their mitochondrial content and high mitochondrial content is associated with a poor prognosis.

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.

Immunosuppressive M2 TAMs represent a promising target population to enhance phagocytosis of ovarian cancer cells in vitro.

Introduction: Tumor-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). Methods: Phenotype 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. Results: Expression 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. Conclusion: Combined blockade of TIGIT and CD47 represents a promising approach to enhance anti-CD47-facilitated phagocytosis.

Ang-2 is a potential molecular marker for lymphatic metastasis and better response to bevacizumab therapy in ovarian cancer.

PURPOSE: In ovarian cancer, there are two main routes of metastasis, namely intraperitoneal and retroperitoneal. Their biologic background is poorly understood. Identifying molecular markers involved might enable the development of tailored therapy regimens. Moreover, no reliable markers for response to anti-angiogenic treatment with bevacizumab are yet established. Angiopoietin-2 (Ang-2) is an angiogenic growth factor, involved in lymphatic activation and is associated with tumor progression. Here, we assessed the potential of Ang-2 as a molecular marker in metastasis and treatment of ovarian cancer. METHODS: In our study, quantitative and qualitative protein Ang-2 expression in tumor tissue of ovarian cancer patients was analyzed by Western blot (n = 138) and immunohistochemistry (n = 58). Further, Ang-2 levels in blood samples were quantified in enzyme-linked immunosorbent assay (n = 38). Expression levels of different tumor spread patterns were evaluated, and survival analyses were made. RESULTS: We observed that Ang-2 expression is significantly higher in tumors with retroperitoneal dissemination (pT1a-pT3b, pN1) compared to those showing intraperitoneal tumor growth (pT3c, pN0). In addition, patients with high Ang-2 expression have significantly longer overall survival compared to patients with low Ang-2 expression. Patients with high Ang-2 expression benefit significantly from therapy with bevacizumab. CONCLUSION: All in all, Ang-2 may serve as a molecular marker for patients with tumors prone to spread to lymph nodes and for patients who might benefit from bevacizumab therapy.

EUSICA/COST IMMUNO-model workshop fostering collaboration to advance sinonasal cancer research: A meeting report.

Sinonasal cancer is a clinically and histologically heterogeneous group of rare tumors with generally poor clinical outcomes. Their low incidence hampers the advancement of clinical management as well as translational research, and calls for multicenter and multinational collaboration between physicians and researchers. This report describes the proceedings of a two-day conference organized by the European Network for Sinonasal Cancer Research (EUSICA) and COST Action 'IMMUNO-model', fostering such collaboration and focusing on preclinical tumor and immuno models, surgical and radio-oncological treatments, core facilities for genetic characterization and molecular tumor classification, and cancer registry.

Impact of AKT1 on cell invasion and radiosensitivity in a triple negative breast cancer cell line developing brain metastasis.

Introduction: The 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. Methods: The 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. Results: Pharmacological 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. Discussion: Collectively, 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.

ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis.

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.

Tumor cell integrin ß4 and tumor stroma E-/P-selectin cooperatively regulate tumor growth in vivo.

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 ß4 (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.

The role of the desmosomal protein desmocollin 2 in tumour progression in triple negative breast cancer patients.

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.

Ovarian Cancer-Cell Pericellular Hyaluronan Deposition Negatively Impacts Prognosis of Ovarian Cancer Patients.

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.

Key Role of Hyaluronan Metabolism for the Development of Brain Metastases in Triple-Negative Breast 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.

Combined Liquid Biopsy Methylation Analysis of CADM1 and MAL in Cervical Cancer Patients.

Cervical cancer is the fourth most common cancer in women, which is associated in >95% with a high-risk human papillomavirus (HPV) infection. Methylation of specific genes has been closely associated with the progress of cervical high-grade dysplastic lesions to invasive carcinomas. Therefore, DNA methylation has been proposed as a triage for women infected with high-risk HPV. Methylation analyses of cervical cancer tissue have shown that cell adhesion molecule 1 (CADM1) and myelin and lymphocyte protein (MAL) methylation are present in over 90% of all cervical high-grade neoplasias and invasive cervical cancers. Here, we established a liquid biopsy-based assay to detect MAL and CADM1 methylation in cell free (cf)DNA of cervical cancer. Methylation of the target gene was validated on bisulfite converted smear-DNA from cervical dysplasia patients and afterward applied to cfDNA using quantitative real-time PCR. In 52 smears, a combined analysis of CADM1 and/or MAL (CADM1/MAL) showed methylation in 86.5% of the cases. In cfDNA samples of 24 cervical cancer patients, CADM1/MAL methylation was detected in 83.3% of the cases. CADM1/MAL methylation was detected already in 81.8% of stage I-II patients showing the high sensitivity of this liquid biopsy assay. In combination with a specificity of 95.5% towards healthy donors (HD) and an area under the curve (AUC) of 0.872 in the receiver operating characteristic (ROC) analysis, CADM1/MAL cfDNA methylation detection might represent a novel and promising liquid biopsy marker in cervical cancer.

VEGF-C serum level is associated with response to bevacizumab maintenance therapy in primary ovarian cancer patients.

OBJECTIVE: At 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. METHODS: 101 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. RESULTS: A 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. CONCLUSION: VEGF-C serum level might serve as as a biomarker to evaluate treatment response under bevacizumab.

Tissue-Specific Expression of TIGIT, PD-1, TIM-3, and CD39 by γδ T Cells in Ovarian Cancer.

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.

Insights into the Steps of Breast Cancer-Brain Metastases Development: Tumor Cell Interactions with the Blood-Brain Barrier.

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.

Circulating Cellular Communication Network Factor 1 Protein as a Sensitive Liquid Biopsy Marker for Early Detection of Breast Cancer.

BACKGROUND: Despite recent progress in liquid biopsy technologies, early blood-based detection of breast cancer is still a challenge. METHODS: We analyzed secretion of the protein cellular communication network factor 1 (CCN1, formerly cysteine-rich angiogenic inducer 61) in breast cancer cell lines by an enzyme-linked immunosorbent assay (ELISA). Soluble CCN1 in the plasma (2.5 µL) of 544 patients with breast cancer and 427 healthy controls was analyzed by ELISA. The breast cancer samples were acquired at the time of primary diagnosis prior to neoadjuvant therapy or surgery. A classifier was established on a training cohort of patients with breast cancer and age-adapted healthy controls and further validated on an independent cohort comprising breast cancer patients and healthy controls. Samples from patients with benign breast diseases were investigated as additional controls. Samples from patients with acute heart diseases (n = 127) were investigated as noncancer controls. The diagnostic accuracy was determined by receiver operating characteristic using the parameters area under the curve, sensitivity, and specificity. RESULTS: CCN1 was frequently secreted by breast cancer cell lines into the extracellular space. Subsequent analysis of clinical blood samples from patients with breast cancer and age-adjusted healthy controls revealed an overall specificity of 99.0% and sensitivity of 80.0% for cancer detection. Remarkably, 81.5% of small T1 cancers were already CCN1-positive, while CCN1 concentrations in patients with benign breast lesions were below the threshold for breast cancer detection. CONCLUSIONS: Circulating CCN1 is a potentially novel blood biomarker for the detection of breast cancer at the earliest invasive stage.

Transcriptome Analysis in Vulvar Squamous Cell Cancer.

To date, therapeutic strategies in vulvar squamous cell carcinoma (VSCC) are lacking molecular pathological information and targeted therapy hasn't been approved in the treatment of VSCC, yet. Two etiological pathways are widely accepted: HPV induced vs. HPV independent, associated with chronic skin disease, often harboring TP53 mutations (mut). The aim of this analysis was to analyze the RNA expression patterns for subtype stratification on VSCC samples that can be integrated into the previously performed whole exome sequencing data for the detection of prognostic markers and potential therapeutic targets. We performed multiplex gene expression analysis (NanoString) with 770 genes in 24 prior next generation sequenced samples. An integrative data analysis was performed. Here, 98 genes were differentially expressed in TP53mut vs. HPV+ VSCC, in the TP53mut cohort, where 56 genes were upregulated and 42 were downregulated in comparison to the HPV+ tumors. Aberrant expression was primarily observed in cell cycle regulation, especially in HPV+ disease. Within the TP53mut group, a distinct cluster was identified that was correlated to a significantly worse overall survival (p = 0.017). The RNA expression profiles showed distinct patterns with regard to the known VSCC subtypes and could potentially enable further subclassification in the TP53mut groups.

Cargo-specific recruitment in clathrin- and dynamin-independent endocytosis.

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.