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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.

Mechanisms of epithelial renewal in the alveolar compartment remain incompletely understood. To this end, we aimed to characterize alveolar progenitors. Single-cell RNA-sequencing (scRNA-seq) analysis of the HTII-280+/EpCAM+ population from adult human lung revealed subclusters enriched for adult stem cell signature (ASCS) genes. We found that alveolar progenitors in organoid culture in vitro show phenotypic lineage plasticity as they can yield alveolar or bronchial cell-type progeny. The direction of the differentiation is dependent on the presence of the GSK-3β inhibitor, CHIR99021. By RNA-seq profiling of GSK-3β knockdown organoids we identified additional candidate target genes of the inhibitor, among others FOXM1 and EGF. This gives evidence of Wnt pathway independent regulatory mechanisms of alveolar specification. Following influenza A virus (IAV) infection organoids showed a similar response as lung tissue explants which confirms their suitability for studies of sequelae of pathogen-host interaction.

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.

Human-based organ models can provide strong predictive value to investigate the tropism, virulence, and replication kinetics of viral pathogens. Currently, such models have received widespread attention in the study of SARS-CoV-2 causing the COVID-19 pandemic. Applicable to a large set of organoid models and viruses, we provide a step-by-step work instruction for the infection of human alveolar-like 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 alveolar-like organoids and the determination of protein expression of angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and FURIN as exemplary host factors of SARS-CoV-2. Set 2 provides detailed guidance on the extraction of RNA from human alveolar-like organoids and the subsequent qPCR to quantify the expression level of ACE2, TMPRSS2, and FURIN as host factors of SARS-CoV-2 on the mRNA level. Protocol set 3 contains an in-depth explanation on how to infect human alveolar-like 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 alveolar-like 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 alveolar-like 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 alveolar-like organoids based on material from different donors.

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

International audience; The prognostic impact of tumor-infiltrating lymphocytes (TILs) is intensively investigated in breast cancer (BC). It is already known that triple-negative breast cancer (TNBC), the most aggressive type of BC, has the highest percentage of TILs. In addition, there is an influence of steroid hormone receptor expression (type I nuclear receptors) on TIL subpopulations in breast cancer tissue. The link between type II nuclear receptors and the level of TILs is unclear. Therefore, the aim of this study was to quantify TILs in a panel of 264 sporadic breast cancers and investigate the correlation of TIL levels with type I and II nuclear receptors expression. TIL levels were significantly increased in the subgroup of TNBC. By contrast, they decreased in estrogen (ER)- or progesterone receptor (PR)-positive cases. Moreover, TIL levels were correlated with type II nuclear receptors, including PPARγ, with a significant inverse correlation of the nuclear form (r = −0.727, p < 0.001) and a weak positive correlation of the cytoplasmic form (r = 0.202, p < 0.002). Surprisingly, BC cases with a TIL Salgado score of >15% showed a significantly decreased overall survival. In addition, peritumoral inflammation was also quantified in BC tissue samples. In our cohort, although the level of peritumoral inflammation was not correlated with OS, it determined the prognostic value of ER, PR, and PPARγ in BC. Altogether, the present study provides a differentiated overview of the relations between nuclear receptor expression, TIL levels, peritumoral inflammation, and prognosis in BC. View Full-Text

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.

Simple Summary Recent advances in our understanding of the stem cell potential in adult tissues have far-reaching implications for cancer research, and this creates new opportunities for the development of new therapeutic strategies. Here we outline changes in stem cell biology that characterize main gynaecological malignancies, ovarian, endometrial, and cervical cancer, and focus on specific differences between them. We highlight the importance of the local niche environment as a driver of malignant transformation in addition to mutations in key cancer-driving genes. Patient-derived organoids capture in vitro main aspects of cancer tissue architecture and stemness regulatory mechanisms, thus providing a valuable new platform for a personalized approach in the treatment of gynecological malignancies. This review summarizes the main achievement and formulates remaining open questions in this fast-evolving research field. Abstract Gynaecological malignancies represent a heterogeneous group of neoplasms with vastly different aetiology, risk factors, molecular drivers, and disease outcomes. From HPV-driven cervical cancer where early screening and molecular diagnostics efficiently reduced the number of advanced-stage diagnosis, prevalent and relatively well-treated endometrial cancers, to highly aggressive and mostly lethal high-grade serous ovarian cancer, malignancies of the female genital tract have unique presentations and distinct cell biology features. Recent discoveries of stem cell regulatory mechanisms, development of organoid cultures, and NGS analysis have provided valuable insights into the basic biology of these cancers that could help advance new-targeted therapeutic approaches. This review revisits new findings on stemness and differentiation, considering main challenges and open questions. We focus on the role of stem cell niche and tumour microenvironment in early and metastatic stages of the disease progression and highlight the potential of patient-derived organoid models to study key events in tumour evolution, the appearance of resistance mechanisms, and as screening tools to enable personalisation of drug treatments.

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

The intracellular human bacterial pathogen Chlamydia trachomatis pursues effective strategies to protect infected cells against death-inducing stimuli. Here, we show that Chlamydia trachomatis infection evokes 3-phosphoinositide-dependent protein kinase-1 (PDPK1) signaling to ensure the completion of its developmental cycle, further leading to the phosphorylation and stabilization of MYC. Using biochemical approaches and imaging we demonstrate that Chlamydia-induced PDPK1-MYC signaling induces host hexokinase II (HKII), which becomes enriched and translocated to the mitochondria. Strikingly, preventing the HKII interaction with mitochondria using exogenous peptides triggers apoptosis of infected cells as does inhibiting either PDPK1 or MYC, which also disrupts intracellular development of Chlamydia trachomatis. These findings identify a previously unknown pathway activated by Chlamydia infection, which exhibits pro-carcinogenic features. Targeting the PDPK1-MYC-HKII-axis may provide a strategy to overcome therapeutic resistance of infection., Highlights • MYC oncogene expression is induced and stabilized in Chlamydia-infected cells via activation of PDPK1. • Activation of the PDPK1-MYC axis enhances hexokinase II expression. • HKII-mitochondria association prevents apoptosis to support chlamydial growth. Activation of oncogenes promotes cell survival and metabolic reprogramming. Intracellular pathogens target oncogenes to exploit cellular machineries and inhibit apoptosis. Al-Zeer et al. now highlight the role of the oncogene MYC for inducing hexokinase II activation during Chlamydia infection, linking cell survival to energy metabolism.

SummaryAntimicrobial peptides are constituents of the first-line innate mucosal defense system that acts as a barrier to establishment of infection. The highly successful human gastric pathogen, Helicobacter pylori, is able to persistently colonize its host despite inducing expression of several antimicrobial peptides, including human β-defensin 3 (hBD3). We find that hBD3 is highly active against H. pylori in vitro and is rapidly induced during early infection via EGFR-dependent activation of MAP kinase and JAK/STAT signaling. However, during prolonged infection, hBD3 was subsequently downregulated by the H. pylori virulence determinant CagA. Upon translocation into host cells, CagA activated the cellular tyrosine phosphatase, SHP-2, terminating EGFR activation and downstream signaling and increasing bacterial viability. Chemical inhibition and knockdown of SHP-2 expression rescued hBD3 synthesis and bactericidal activity. Thus, we reveal how cagPAI-positive H. pylori strains use CagA to evade a key innate mucosal defense pathway to support the establishment of persistent infection.