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Cell surface carbohydrate antigens sialyl Lewis X (sLeX) and Lewis Y (LeY) are paramount glycoconjugates and are abundantly expressed in the receptive endometrium. Furthermore, among the important biological functions of both antigens is their role in leukocytes adhesion and extravasation. Interleukin-1 beta (IL-1β) is involved in the process of human embryo implantation and placenta development. Here, we used an in vitro model to investigate whether sLeX and LeY are playing a role in the embryo implantation process mediated by IL-1β. Our results are showing that the expression of cell surface sLeX was enhanced in endometrial RL95-2 cells after exposure to IL-1β. RT-qPCR detection indicated that the transcript level of glycosyltransferase gene fucosyltransferase 3 (FUT3) was significantly elevated and that of FUT4/7 and ST3 beta-galactoside alpha-2,3-sialyltransferase 3/4 (ST3GAL3/4) were decreased by treatment with IL-1β. Modulatory role of glycosyltransferase FUT3 on sLeX biosynthesis was determined by FUT3 siRNA transfection in RL95-2 cells. Results showed that the expression level of sLeX was suppressed, but no change was observed in regard to LeY. Moreover, IL-1β promoted the HTR-8/SVneo trophoblast spheroids attachment to the RL95-2 endometrial monolayer, which was partially blocked by anti-sLeX antibody and FUT3 knockdown. Gene expression analysis of the RNA-seq transcriptome data from human secretory endometrium demonstrated a significantly higher level of FUT3 in the mid-secretory phase compared to the early secretory phase, which was correlated with the expression of IL1B. In summary, the inflammatory microenvironment at the fetomaternal interface can regulate the glycosylation pattern of endometrial cells at the time of implantation. SLeX can be significantly induced by IL-1β via increasing FUT3 expression, which facilitates the trophoblast adhesion during embryo implantation.

Background: Tumour heterogeneity in high-grade serous ovarian cancer (HGSOC) is a proposed cause of acquired resistance to treatment and high rates of relapse. Among the four distinct molecular subtypes of HGSOC, the mesenchymal subtype (MES) has been observed with high frequency in several study cohorts. Moreover, it exhibits aggressive characteristics with poor prognosis. The failure to adequately exploit such subtypes for treatment results in high mortality rates, highlighting the need for effective targeted therapeutic strategies that follow the idea of personalized medicine (PM). Methods: As a proof-of-concept, bulk and single-cell RNA data were used to characterize the distinct composition of the tumour microenvironment (TME), as well as the cell–cell communication and its effects on downstream transcription of MES. Moreover, transcription factor activity contextualized with causal inference analysis identified novel therapeutic targets with potential causal impact on transcription factor dysregulation promoting the malignant phenotype. Findings: Fibroblast and macrophage phenotypes are of utmost importance for the complex intercellular crosstalk of MES. Specifically, tumour-associated macrophages were identified as the source of interleukin 1 beta (IL1B), a signalling molecule with significant impact on downstream transcription in tumour cells. Likewise, signalling molecules tumour necrosis factor (TNF), transforming growth factor beta (TGFB1), and C-X-C motif chemokine 12 (CXCL12) were prominent drivers of downstream gene expression associated with multiple cancer hallmarks. Furthermore, several consistently hyperactivated transcription factors were identified as potential sources for treatment opportunities. Finally, causal inference analysis identified Yes-associated protein 1 (YAP1) and Nuclear Receptor Subfamily 2 Group F Member 6 (NR2F6) as novel therapeutic targets in MES, verified in an independent dataset. Interpretation: By utilizing a sophisticated bioinformatics approach, several candidates for treatment opportunities, including YAP1 and NR2F6 were identified. These candidates represent signalling regulators within the cellular network of the MES. Hence, further studies to confirm these candidates as potential targeted therapies in PM are warranted.

Background The therapy concepts that target several members of krüppel like factor (KLF) family have been achieved in breast cancer (BC). However, the role of KLF11 in BC remains unclear. This study explored the prognostic significance of KLF11 in BC patients and investigated its functional roles in this malignancy. Methods Immunohistochemistry (IHC) staining of KLF11 in 298 patients’ samples was performed to determine the prognostic role of the KLF11. Then the protein level was correlated to clinicopathological characteristics and survival outcomes. Afterward, the function of KLF11 was explored in vitro with siRNA-mediated loss-of-function of cell viability, proliferation, and apoptosis. Results From the cohort study, we found that the expression of KLF11 was positively associated with highly proliferative BC of BC. Furthermore, prognostic analysis demonstrated that KLF11 was an independent negative factor for disease-free survival (DFS) and distant-metastasis-free survival (DMFS) of BC. The KLF11-related prognostic model for DFS and DMFS showed high accuracy in predicting the 3-,5- and 10 -year survival probability of BC patients. Additionally, the knockdown of KLF11 inhibited cell viability and proliferation, as well as induced cell apoptosis in MCF7 and MDA-MB-231 cells, while only inhibited cell viability and induced cell apoptosis in SK-BR-3 cells. Conclusions Our study indicated that targeting KLF11 is an interesting therapeutic concept and further research could lead to a new therapeutic improvement in BC, especially in highly aggressive molecular subtypes.

Purpose Endometrial cancer (EC) is the most common gynecological cancer worldwide. Treatment has been improved in recent years, but, in advanced stages, therapeutical options are still limited. It has been reported that the expression of the blood group antigens Sialyl Lewis X (SLeX), Sialyl Lewis A (SLeA) and Lewis Y (LeY) is associated with prognosis in several tumors. Large studies on endometrial and cervical cancer are still pending. Methods Specimens of 234 patients with EC were immunohistochemically stained with antibodies for SLeX, SLeA and LeY. Expression was correlated to histopathological variables. Results High expression of SLeX was correlated to low pT-stage (p = 0.013), low grade (p p = 0.006) and better overall survival rates (OS; p = 0.023). High expression of SLeA was associated with low pT-stage (p = 0.013), low grade (p = 0.001) and better progression-free survival (PFS; p = 0.043). LeY staining was correlated to pN + (p = 0.038), low grade (p = 0.005) and poorer PFS (p = 0.022). Conclusion This is the first study examining the expression of SLeX, SLeA and LeY in EC, which can serve as additional future prognostic markers. Further studies are necessary to understand the underlying mechanisms. The study was approved by the local ethics committee of the Ludwig-Maximilians University Munich (reference number 19-249).

Purpose The significance of the non-classical G-protein-coupled estrogen receptor (GPER) as positive or negative prognostic factor for ovarian cancer patients remains still controversial. Recent results indicate that an imbalance of both co-factors and co-repressors of nuclear receptors regulates ovarian carcinogenesis by altering the transcriptional activity through chromatin remodeling. The present study aims to investigate whether the expression of the nuclear co-repressor NCOR2 plays a role in GPER signaling which thereby could positively impact overall survival rates of ovarian cancer patients. Methods NCOR2 expression was evaluated by immunohistochemistry in a cohort of 156 epithelial ovarian cancer (EOC) tumor samples and correlated with GPER expression. The correlation and differences in clinical and histopathological variables as well as their effect on prognosis were analyzed by Spearman’s correlation, Kruskal–Wallis test and Kaplan–Meier estimates. Results Histologic subtypes were associated with different NCOR2 expression patterns. More specifically, serous and mucinous EOC demonstrated a higher NCOR2 expression (P = 0.008). In addition, high nuclear NCOR2 expression correlated significantly with high GPER expression (cc = 0.245, P = 0.008). A combined evaluation of both high NCOR2 (IRS > 6) and high GPER (IRS > 8) expression revealed an association of a significantly improved overall survival (median OS 50.9 versus 105.1 months, P = 0.048). Conclusion Our results support the hypothesis that nuclear co-repressors such as NCOR2 may influence the transcription of target genes in EOC such as GPER. Understanding the role of nuclear co-repressors on signaling pathways will allow a better understanding of the factors involved in prognosis and clinical outcome of EOC patients.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilises the angiotensin-converting enzyme 2 (ACE2) transmembrane peptidase as cellular entry receptor. However, whether SARS-CoV-2 in the alveolar compartment is strictly ACE2-dependent and to what extent virus-induced tissue damage and/or direct immune activation determines early pathogenesis is still elusive.MethodsSpectral microscopy, single-cell/-nucleus RNA sequencing or ACE2 “gain-of-function” experiments were applied to infected human lung explants and adult stem cell derived human lung organoids to correlate ACE2 and related host factors with SARS-CoV-2 tropism, propagation, virulence and immune activation compared to SARS-CoV, influenza and Middle East respiratory syndrome coronavirus (MERS-CoV). Coronavirus disease 2019 (COVID-19) autopsy material was used to validateex vivoresults.ResultsWe provide evidence that alveolar ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation and virus-induced tissue damage in the human alveolus. Instead,ex vivoinfected human lungs and COVID-19 autopsy samples showed that alveolar macrophages were frequently positive for SARS-CoV-2. Single-cell/-nucleus transcriptomics further revealed nonproductive virus uptake and a related inflammatory and anti-viral activation, especially in “inflammatory alveolar macrophages”, comparable to those induced by SARS-CoV and MERS-CoV, but different from NL63 or influenza virus infection.ConclusionsCollectively, our findings indicate that severe lung injury in COVID-19 probably results from a macrophage-triggered immune activation rather than direct viral damage of the alveolar compartment.

SARS-CoV-2 is the causative agent of the immune response-driven disease COVID-19 for which new antiviral and anti-inflammatory treatments are urgently needed to reduce recovery time, risk of death and long COVID development. Here, we demonstrate that the immunoregulatory kinase p38 MAPK is activated during viral entry, mediated by the viral spike protein, and drives the harmful virus-induced inflammatory responses. Using primary human lung explants and lung epithelial organoids, we demonstrate that targeting p38 signal transduction with the selective and clinically pre-evaluated inhibitors PH-797804 and VX-702 markedly reduced the expression of the pro-inflammatory cytokines IL6, CXCL8, CXCL10 and TNF-α during infection, while viral replication and the interferon-mediated antiviral response of the lung epithelial barrier were largely maintained. Furthermore, our results reveal a high level of drug synergism of both p38 inhibitors in co-treatments with the nucleoside analogs Remdesivir and Molnupiravir to suppress viral replication of the SARS-CoV-2 variants of concern, revealing an exciting and novel mode of synergistic action of p38 inhibition. These results open new avenues for the improvement of the current treatment strategies for COVID-19.

Purpose Endometrial cancer is the most common gynecological malignancy. The helicase RIG-I, a part of the innate immune system, and EFTUD2, a splicing factor which can upregulate RIG-I expression, are shown to influence tumor growth and disease progression in several malignancies. For endometrial cancer, an immunogenic cancer, data about RIG-I and EFTUD2 are still missing. The aim of this study was to examine the expression of RIG-I and EFTUD2 in endometrial cancer. Methods 225 specimen of endometrial cancer were immunohistochemically stained for RIG-I and EFTUD2. The results were correlated to clinicopathological data, overall survival (OS) and progression-free survival (PFS). Results High RIG-I expression correlated with advanced tumor stages (FIGO: p = 0.027; pT: p = 0.010) and worse survival rates (OS: p = 0.009; PFS: p = 0.022). High EFTUD2 expression correlated to worse survival rates (OS: p = 0.026; PFS: p Conclusion Our data suggest that the expression of RIG-I and EFTUD2 correlates with survival data, which makes both a possible therapeutic target in the future.

BackgroundBreast cancer (BC) is a heterogeneous disease characterized by distinct molecular subtypes. Krüppel like Factor (KLF) family members in BC have been widely studied. However, the roles of KLFs as tumor-promoters or tumor-suppressors are context-dependent. KLF11, as a member of this family, the expression pattern and prognostic relevance of which in BC remains unclear.Methods To clarify whether KLF11 acts as a tumor-promoter or a tumor-suppressor in BC, we explored the prognostic role of the KLF11 in BC patients by performing immunohistochemistry (IHC) staining of KLF11 in 298 patients samples. Then we correlated the expression to clinicopathological characteristics and survival outcomes. To investigate the function of KLF11 in tumor cells, we performed different assays of cell viability, cell proliferation, and cell apoptosis assays in three distinct molecular subtypes of BC cell lines with siRNA-mediated loss-of-function of KLF11.ResultsIn the BC patient cohort, We demonstrated that KLF11 was higher in highly proliferative BC and lowest in luminal A-like BC. Furthermore, "KLF11-high" BC patients had shorter disease-free survival (DFS), distant-metastasis-free survival (DMFS), and local recurrence-free survival (LRFS). High KLF11 expression remained an independent indicator for DFS and DMFS of BC. The multivariate cox regression-dependent, KLF11-related nomograms of DFS and DMFS showed high accuracy in predicting BC patients' 3-,5- and 10 -year survival probability. Our results indicate that KLF11 might play a pro-tumor role in BC.From the KLF11-BC tumor cells perspective, the cellular functional assays confirmed that KLF11 acts as a pro-tumor transcription factor (TF) in BC cells via promoting proliferation and/or decreasing cell apoptosis. However, due to neoplastic intratumor heterogeneity, different cancer molecular subtypes could have different underlying mechanisms.ConclusionsPrior research has demonstrated that KLF11 expression is lower in BC compared to healthy breast tissue. However, we now could show that high KLF11 expression in BC is probably pro-tumorigenic and is associated with an impaired prognosis. Our study indicates that KLF11, as a transcriptional factor, acts as a new prognostic biomarker and might also be potentially a synergistic or an alternative therapeutic target for conventional targeting treatments of human BC.

Organ models have received widespread attention in the study of SARS-CoV-2, the pathogen causing the current COVID-19 pandemic. Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human lung organoids with SARS-CoV-2 in this protocol collection. We also prepared a detailed description on state-of-the-art methodologies to assess the infection impact and the analysis of relevant host factors in organoids. This protocol collection consists of five different sets of protocols. Set 1 describes the protein extraction from human lung organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), as an exemplary host factor of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human lung organoids and the subsequent RT-qPCR to quantify the expression level of e.g., ACE2or other host factors of SARS-CoV-2 on RNA level. Protocol set 3 contains an in-depth explanation on how to infect human lung organoids with SARS-CoV-2 and how to quantify the viral replication by plaque assay and viral E gene-based RT-qPCR. Set 4 provides a step-by-step protocol for the isolation of single cells from infected human lung organoids for further processing in single-cell RNA sequencing or flow cytometry. Set 5 presents a detailed protocol on how to perform the fixation of human lung organoids and guides through all steps of immunohistochemistry and in situ hybridization to visualize SARS-CoV-2 and its host factors. The infection and all subsequent analytical methods have been successfully validated by biological replications with human lung organoids based on material from different donors.

ObjectiveBecause mucinous carcinomas are rare tumors that affect several organ sites and are known to originate from different tissues, leading to frequent misdiagnoses, the objective was to characterize the differences between primary mucinous tumors of the ovary and metastatic mucinous cancer to the ovary by studying the expression pattern of several candidate biomarkers.MethodsTissue samples of mucinous histology were obtained between 1985 and 2015. Individual ovary and colon tissue samples were analyzed, including standard (PAX8, CK20, CK7, CDX2, SATB2, estrogen/progesterone) and new (MUC1, MUC5AC) biomarkers, which were then scored for immunoreactivity semi-quantitatively.ResultsThe study cohort included 98 mucinous tumor samples, including benign mucinous cystadenoma (n=24), mucinous borderline tumors (n=24), mucinous carcinomas (n=40), and metastatic mucinous ovarian carcinomas (n=10). A strong positive correlation was found between PAX8 scoring (p=0.003), CK7 scoring (p=0.0001), and MUC1 scoring (p=0.001) in primary mucinous ovarian cancer. Tumors of increasing invasiveness were analyzed and a significant decrease in the scoring of MUC5AC (p=0.001) was observed, with a stronger expression in adenomas (87%) and borderline tumors (75%), and a lower expression in mucinous cancers (42%). Patients survived significantly longer when their tumors expressed high PAX8 and showed an expansile invasion pattern (p=0.005 and p=0.015, respectively) compared with patients with PAX8-negative tumors and destructive invasion pattern.ConclusionThe study data support the diagnostic value of MUC1 as a new biomarker to differentiate between primary and metastatic mucinous ovarian cancer. In addition, the tumor growth pattern along with the PAX8 immunophenotype might represent potential prognostic biomarkers for primary mucinous ovarian carcinomas.

(1) Purpose: Endometrial cancer (EC) is the most common gynecological cancer worldwide. Treatment has been improved in recent years, but, in advanced stages, therapeutical options are still limited. It has been reported that the expression of the blood group antigens Sialyl Lewis X (SLeX), Sialyl Lewis A (SLeA) and Lewis Y (LeY) is associated with prognosis in several tumors. Large studies on endometrial and cervical cancer are still pending. (2) Methods: Specimens of 234 patients with EC were immunohistochemically stained with antibodies for SLeX, SLeA and LeY. Expression was correlated to histopathological variables. (3) Results: High expression of SLeX was correlated to low pT-stage (p=0.013), low grade (p

This protocol describes the working steps to infect human lung organoids with SARS-CoV-2 and quantify the viral replication at defined time points post infection via plaque assay and viral E gene RT-qPCR.

PurposeEndometrial cancer (EC) is one of the most common gynaecologic malignancies. Tumor infiltrating regulatory T-cells (Treg) have been reported to have a prognostic impact in many malignancies. Immunotherapeutic strategies are gaining interest for advanced and recurrent EC cases, where treatment options are rare. Our study was aimed at determining the value of Treg in EC progression.MethodsEC specimens from 275 patients and 28 controls were screened immunohistochemically for the presence of Treg represented by FoxP3. Correlations with clinicopathological and survival parameters were performed. Functional assays were performed using EC cell lines Ishikawa + and RL95-2 after co-culturing with isolated CD4 + CD25 + CD127dim Treg. To assess the influence of EC on the composition of peripheral blood mononuclear cells (PBMC), flow cytometric analyses were performed.ResultsWe found that an increased infiltration of Treg was associated with high grades and a reduced overall survival. Treg were almost absent in endometrium tissues from healthy control patients. Co-culture of tumor cells with CD4 + CD25 + CD127dim Treg led to functional changes: enhanced invasion, migration and viability indicated that increased levels of Treg in the tumor microenvironment may promote tumor growth. Furthermore, we found that the presence of EC cells led to phenotypic changes in PBMC, showing significantly increased levels of CD25 and FoxP3.ConclusionOur results indicate that the presence of Treg in the EC tumor environment is associated with a poorer outcome. A remarkable impact of Treg on tumor cell behaviour and vice versa of tumor cells on PBMC subpopulations support this notion mechanistically. Our findings provide a basis for focusing on Treg as potential future therapeutic targets in EC.

Forkhead Box Protein 3 (FoxP3) is known as a key mediator in the immunosuppressive function of regulatory T-cells (Tregs). The aim of our study was to investigate whether FoxP3-positive Tregs have the potential to act as an independent predictor in progression as well as in regression of cervical intraepithelial neoplasia, especially in patients with intermediate cervical intraepithelial neoplasia (CIN II). Nuclear FoxP3 expression was immunohistochemically analysed in 169 patient samples (CIN I, CIN II with regressive course, CIN II with progressive course, CIN III). The median numbers were calculated for each slide and correlated with the histological CIN grade. Statistical analysis was performed by SPSS 26 (Mann–Whitney U test, Spearman’s rank correlation). An increased FoxP3 expression in CIN II with progression could be detected in comparison to CIN II with regression (p = 0.003). Total FoxP3 expression (epithelium and dysplasia-connected stroma) was higher in more advanced CIN grades (p

In contrast to genetic abnormalities which are well known to be responsible for around 50 % of human miscarriages, there is very few data about epigenetic alterations in spontaneous and recurrent miscarriages (SM, RM). The aim of this study was to analyze the histone modification marks H3K9ac and H3K4me3 in SM and RM.The abundance of histone modifications H3K4me3/H3K9ac was analyzed by western blot in frozen abortion material of SM and RM compared to a control group of legal pregnancy terminations. Further, to characterize placental tissue cells expressing H3K4me3/H3K9ac immunohistochemistry (IHC) and immunofluorescence was performed in 20 SM, 19 RM and 26 controls.The western blot data showed a tendency to an overall reduction of H3K4me3/H3K9ac, in the placental tissue of particularly SM. Further we differentiated between syncytiotrophoblast, cytotrophoblast and decidual cells and found a significant decrease of H3K4me3 in SM in cytotrophoblast cells and syncytial stroma. In RM H3K4me3 was downregulated exclusively in the syncytiotrophoblast. H3K9ac was reduced in SM and RM in all evaluated compartments, except from the syncytiotrophoblast.Our study showed an overall reduced histone modification of H3K4me3 and H3K9ac in the placental tissue of SM. Concerning RM, particularly the reduction of H3K9ac was detected in the placental tissue, indicating that RM group has distinct profile in epigenetic regulation. Whether these histone modifications are part of a possible pathophysiologic cascade during SM and RM or are merely indicating a defective placentation, cannot be concluded from this study.

Forkhead Box Protein 3 (FoxP3) is known as a key mediator in the immunosuppressive function of regulatory T-cells (Tregs). The aim of our study was to investigate whether FoxP3-positive Tregs have the potential to act as an independent predictor in progression as well as in regression of cervical intraepithelial neoplasia, especially in patients with intermediate cervical intraepithelial neoplasia (CIN II).Nuclear FoxP3 expression was immunohistochemically analysed in 169 patient samples (CIN I, CIN II with regressive course, CIN II with progressive course, CIN III). The median numbers were calculated for each slide and correlated with the histological CIN grade. Statistical analysis was performed by SPSS 26 (Mann-Whitney U test, Spearman's rank correlation).An increased FoxP3 expression in CIN II with progression could be detected in comparison to CIN II with regression (p = 0.003). Total FoxP3 expression (epithelium and dysplasia-connected stroma) was higher in more advanced CIN grades (p 0.001 for CIN I vs. CIN II; p = 0.227 for CIN II vs. CIN III). A positive correlation could be detected between FoxP3-positive cells in epithelium and total FoxP3 expression (Spearman's Rho: 0,565; p 0.01).Expression of FoxP3 could be a helpful predictive factor to assess the risks of CIN II progression. As a prognosticator for regression and progression in cervical intraepithelial lesions it might thereby help in the decision process regarding surgical treatment vs. watchful waiting strategy to prevent conisation-associated risks for patients in child-bearing age. In addition, the findings support the potential of Tregs as a target for immune therapy in cervical cancer patients.

High-grade serous ovarian cancer (HGSOC) is the deadliest gynecological malignancy, with a 5-year survival rate of 30–40%, caused by late detection, high recurrence rate, and pervasive resistance to platinum chemotherapy. Moreover, the heterogeneous clinical presentation of this occult disease and failure to determine actionable molecular and genetic subtypes hampers the development of targeted novel therapies. A majority of HGSOC cases originate from the neighboring fallopian tube epithelium (FT), but the exact mechanisms of cellular transformation and early metastasis to the ovary remain elusive, and our knowledge about the interplay of risk factors remains rudimentary. Recent data from the novel in vitro models of chronic Chlamydia trachomatis infection in human fallopian tube organoids and patient-derived cultures from cancer tissue deposits suggest there is a molecular link between the regulation of stemness and differentiation in the epithelium and tumor drivers of HGSOC. The chapter focuses on this complex relationship between pathogens, tissue renewal, and inflammation in the upper genital tract and provides an overview of current knowledge about the cellular mechanism of cancer development. The inflammatory microenvironment influences both early carcinogenesis and the progression of advanced disease, thus these discoveries have important implications for the development of diagnostic tools and innovative lines of therapy.

Introduction Ovarian cancer remains the most lethal gynecologic cancer in women. Despite achievements in surgical and systemic therapy, most patients develop platinum resistance. Thus, new strategies to increase or reverse the platinum sensitivity in ovarian cancer patients are urgently needed. In this study, we aimed to investigate the anti-cancer effect of the leaves of Pistacia lentiscus L medicinal plants used in Algerian traditional medicine on ovarian cancer cells in vitro. Methods Four newly established primary cell lines derived from ascites of patients with high-grade serous and clear cell ovarian cancer were used to test the anti-cancer effect of the leaves of Pistacia lentiscus L. An experimental study was performed to study the therapeutic effects of the plant leaves substances in patient derived models. The anti- proliferative activity of ethanolic, acetonic and methanolic plant leaves extracts was measured by cell viability assays, and the apoptotic effect assessed using flow cytometry analysis. The impact on constitutive active oncogenic pathways and cytokine release in cell culture supernatant were monitored by Western blotting and ELISA, respectively. Results Sequencing analysis confirmed the presence of mutations in several genes, such as TP53, RB1, PIK3C, which are commonly mutated in high-grade serous ovarian cancer. Obtained results indicated a cytotoxic effect of the methanolic extract of P. lentiscus L (MEPL) on primary cell line cultures, inhibiting PI3K/AKT and MAPK/ERK signaling pathways, and decreasing the release of IL6 and VEGF by the malignant cells. Moreover, treatment with MEPL enhanced the sensitivity to platinum-based chemotherapy in our primary cell lines of patients. Conclusion Methanolic extract of P. lentiscus L might be a promising candidate for novel therapeutic approaches in combination with classic chemotherapy for patients with high-grade serous ovarian cancer.

High‐grade serous ovarian cancer (HGSOC) likely originates from the fallopian tube (FT) epithelium. Here, we established 15 organoid lines from HGSOC primary tumor deposits that closely match the mutational profile and phenotype of the parental tumor. We found that Wnt pathway activation leads to growth arrest of these cancer organoids. Moreover, active BMP signaling is almost always required for the generation of HGSOC organoids, while healthy fallopian tube organoids depend on BMP suppression by Noggin. Fallopian tube organoids modified by stable shRNA knockdown of p53, PTEN, and retinoblastoma protein (RB) also require a low‐Wnt environment for long‐term growth, while fallopian tube organoid medium triggers growth arrest. Thus, early changes in the stem cell niche environment are needed to support outgrowth of these genetically altered cells. Indeed, comparative analysis of gene expression pattern and phenotypes of normal vs. loss‐of‐function organoids confirmed that depletion of tumor suppressors triggers changes in the regulation of stemness and differentiation., Reversion of niche factor signaling in the fallopian tube epithelium allows for long‐term growth of ovarian cancer tissue.

SARS-CoV-2 utilizes the ACE2 transmembrane peptidase as essential cellular entry receptor. Several studies have suggested abundant ACE2 expression in the human lung, inferring strong permissiveness to SARS-CoV-2 infection with resultant alveolar damage and lung injury. Against this expectation, we provide evidence that ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation in the human alveolus. Instead, spectral imaging of ex vivo infected human lungs and COVID-19 autopsy samples depicted that alveolar macrophages were frequently positive for SARS-CoV-2, indicating viral phagocytosis. Single-cell transcriptomics of SARS-CoV-2 infected human lung tissue further revealed strong inflammatory and anti-viral activation responses in macrophages and monocytes, comparable to those induced by MERS-CoV, but with virus-specific gene expression profiles. Collectively, our findings indicate that severe lung injury in COVID-19 likely results from an overwhelming immune activation rather than direct viral damage of the alveolar compartment. Funding: ACH, LES, SH were supported by Berlin University Alliance GC2 Global Health (Corona Virus Pre-Exploration Project). ACH, SH, TW and CD were supported by BMBF (RAPID) and ACH, SH by BMBF (alvBarriereCOVID-19). KH, LB, SL, SH, CD, TW, ACH were funded by BMBF (NFN-COVID 19, Organo-Strat). KH, NS, LES, MW, SH, ADG, CD, TW and ACH were supported by DFG (SFB-TR 84). ACH was supported by BIH, Charite 3R, and Charite-Zeiss MultiDim. KH was supported by BMBF (Camo-COVID-19). MW, NS and SH was supported by BMBF (PROVID). MW and NS was supported by BIH and BMBF (SYMPATH, CAPSyS, NAPKON). BO and DB were funded through the BIH Clinical Single Cell Bioinformatics Pipeline. LB was supported by the BMBF (CoIMMUNE), the DFG (KFO 342) and the IZKF of the Medical Faculty of the WWU. Conflict of Interest: The authors declare no competing interests. Ethical Approval: The study was approved by the ethics committee at the Charite clinic (projects EA2/079/13) and Arztekammer Westfalen-Lippe and of the Westfalischen Wilhelms-Universitat (AZ: 2016-265-f-S). Written informed consent was obtained from all patients.

Background High-grade serous ovarian cancer (HGSOC) causes 80% of all ovarian cancer (OC) deaths. In this setting, the role of cancer stem-like cells (CSCs) is still unclear. In particular, the evolution of CSC biomarkers from primary (pOC) to recurrent (rOC) HGSOCs is unknown. Aim of this study was to investigate changes in CD133 and aldehyde dehydrogenase-1 (ALDH1) CSC biomarker expression in pOC and rOC HGSOCs. Methods Two-hundred and twenty-four pOC and rOC intrapatient paired tissue samples derived from 112 HGSOC patients were evaluated for CD133 and ALDH1 expression using immunohistochemistry (IHC); pOCs and rOCs were compared for CD133 and/or ALDH1 levels. Expression profiles were also correlated with patients' clinicopathological and survival data. Results Some 49.1% of the patient population (55/112) and 37.5% (42/112) pOCs were CD133+ and ALDH1+ respectively. CD133+ and ALDH1+ samples were detected in 33.9% (38/112) and 36.6% (41/112) rOCs. CD133/ALDH1 coexpression was observed in 23.2% (26/112) and 15.2% (17/112) of pOCs and rOCs respectively. Pairwise analysis showed a significant shift of CD133 staining from higher (pOCs) to lower expression levels (rOCs) (p < 0.0001). Furthermore, all CD133 + pOC patients were International Federation of Gynaecology and Obstetrics (FIGO)-stage III/IV (p < 0.0001) and had significantly worse progression-free interval (PFI) (p = 0.04) and overall survival (OS) (p = 0.02). On multivariate analysis, CD133/ALDH1 coexpression in pOCs was identified as independent prognostic factor for PFI (HR: 1.64; 95% CI: 1.03–2.60; p = 0.036) and OS (HR: 1.71; 95% CI: 1.01–2.88; p = 0.045). Analysis on 52 pts patients with known somatic BRCA status revealed that BRCA mutations did not influence CSC biomarker expression. Conclusions The study showed that CD133/ALDH1 expression impacts HGSOC patients' survival and first suggests that CSCs might undergo phenotypic change during the disease course similarly to non stem-like cancer cells, providing also a first evidence that there is no correlation between CSCs and BRCA status.

SummaryHigh-grade serous ovarian cancer (HGSOC) likely originates from the fallopian tube (FT) epithelium. Here, we established 15 organoid lines from HGSOC primary tumor deposits that closely match the parental tumor mutational profile and phenotype. We found that Wnt pathway activation leads to growth arrest of these cancer organoids. Moreover, active BMP signaling is almost always required for generation of HGSOC organoids, while healthy FT organoids depend on BMP suppression by Noggin. Interestingly, FT organoids modified by stable shRNA knockdown (KD) of p53, PTEN, and Retinoblastoma (RB), also require a low Wnt environment for long-term growth, while FT organoid medium triggers growth arrest. Thus, early changes in the stem cell niche environment are needed to support outgrowth of these genetically altered cells. Indeed, comparative analysis of gene expression pattern and phenotype of normal and KD organoids confirmed that depletion of tumor suppressors triggers changes in the regulation of stemness and differentiation.

Chronic infections of the fallopian tubes with Chlamydia trachomatis (Ctr) cause scarring and can lead to infertility. Here we use human fallopian tube organoids and genital Ctr serovars D, K and E for long-term in vitro analysis. The epithelial monolayer responds with active expulsion of the bacteria into the lumen and with compensatory cellular proliferation—demonstrating a role of epithelial homeostasis in the defense against this pathogen. In addition, Ctr infection activates LIF signaling, which we find to be an essential regulator of stemness in the organoids. Infected organoids exhibit a less differentiated phenotype with higher stemness potential, as confirmed by increased organoid forming efficiency. Moreover, Ctr increases hypermethylation of DNA, which is an indicator of accelerated molecular aging. Thus, the chronic organoid infection model suggests that Ctr has a long-term impact on the epithelium. These heritable changes might be a contributing factor in the development of tubal pathologies, including the initiation of high grade serous ovarian cancer., Chronic infections of the fallopian tubes with Chlamydia trachomatis can cause scarring and infertility. Here, the authors show that the pathogen alters stem cell differentiation and DNA methylation in human fallopian tube organoids, suggesting a potential link to cellular ageing and malignant transformation.

BACKGROUND: High-grade serous ovarian cancer (HGSOC) intratumoural vasculature evolution remains unknown. The study investigated changes in tumour microvessel density (MVD) in a large cohort of paired primary and recurrent HGSOC tissue samples and its impact on patients' clinico-pathological outcome. METHODS: A total of 222 primary (pOC) and recurrent (rOC) intra-patient paired HGSOC were assessed for immunohistochemical expression of angiogenesis-associated biomarkers (CD31, to evaluate MVD, and VEGF-A). Expression profiles were compared between pOCs and rOCs and correlated with patients' data. RESULTS: High intratumoural MVD and VEGF-A expression were observed in 75.7% (84/111) and 20.7% (23/111) pOCs, respectively. MVDhigh and VEGF(+) samples were detected in 51.4% (57/111) and 20.7% (23/111) rOCs, respectively. MVDhigh/VEGF(+) co-expression was found in 19.8% (22/111) and 8.1% (9/111) of pOCs and rOCs, respectively (p = 0.02). Pairwise analysis showed no significant change in MVD (p = 0.935) and VEGF-A (p = 0.121) levels from pOCs to rOCs. MVDhigh pOCs were associated with higher CD3(+) (p = 0.029) and CD8(+) (p = 0.013) intratumoural effector TILs, while VEGF(+) samples were most frequently encountered among BRCA-mutated tumours (p = 0.019). Multivariate analysis showed VEGF and MVD were not independent prognostic factors for OS. CONCLUSIONS: HGSOC intratumoural vasculature did not undergo significant changes during disease progression. High concentration of CD31(+) vessels seems to promote recruitment of effector TILs. The study also provides preliminary evidence of the correlation between VEGF-positivity and BRCA status. ispartof: BRITISH JOURNAL OF CANCER vol:119 issue:3 pages:330-338 ispartof: location:England status: published