Non-canonical NF-κB Antagonizes STING Sensor-Mediated DNA Sensing in Radiotherapy.

The NF-κB pathway plays a crucial role in supporting tumor initiation, progression, and radioresistance of tumor cells. However, the role of the NF-κB pathway in radiation-induced anti-tumor host immunity remains unclear. Here we demonstrated that inhibiting the canonical NF-κB pathway dampened the therapeutic effect of ionizing radiation (IR), whereas non-canonical NF-κB deficiency promoted IR-induced anti-tumor immunity. Mechanistic studies revealed that non-canonical NF-κB signaling in dendritic cells (DCs) was activated by the STING sensor-dependent DNA-sensing pathway. By suppressing recruitment of the transcription factor RelA onto the Ifnb promoter, activation of the non-canonical NF-κB pathway resulted in decreased type I IFN expression. Administration of a specific inhibitor of the non-canonical NF-κB pathway enhanced the anti-tumor effect of IR in murine models. These findings reveal the potentially interactive roles for canonical and non-canonical NF-κB pathways in IR-induced STING-IFN production and provide an alternative strategy to improve cancer radiotherapy.

TSLP enhances progestin response in endometrial cancer via androgen receptor signal pathway.

BACKGROUND: The enriched proteins within in vitro fertilisation (IVF)-generated human embryonic microenvironment could reverse progestin resistance in endometrial cancer (EC). METHODS: The expression of thymic stromal lymphopoietin (TSLP) in EC was evaluated by immunoblot and IHC analysis. Transcriptome sequencing screened out the downstream pathway regulated by TSLP. The role of TSLP, androgen receptor (AR) and KANK1 in regulating the sensitivity of EC to progestin was verified through a series of in vitro and in vivo experiments. RESULTS: TSLP facilitates the formation of a BMP4/BMP7 heterodimer, resulting in activation of Smad5, augmenting AR signalling. AR in turn sensitises EC cells to progestin via KANK1. Downregulation of TSLP, loss of AR and KANK1 in EC patients are associated with tumour malignant progress. Moreover, exogenous TSLP could rescue the anti-tumour effect of progestin on mouse in vivo xenograft tumour. CONCLUSIONS: Our findings suggest that TSLP enhances the sensitivity of EC to progestin through the BMP4/Smad5/AR/KANK1 axis, and provide a link between embryo development and cancer progress, paving the way for the establishment of novel strategy overcoming progestin resistance using embryo original factors.

Expression and Prognostic Significance of LAG-3, TIGIT, VISTA, and IDO1 in Endometrial Serous Carcinoma.

Endometrial serous carcinoma (ESC) is an uncommon, aggressive type of endometrial cancer. While immune checkpoint blockade has emerged as a promising treatment option for endometrial carcinomas, research on the expression of immune checkpoints that could serve as prospective immunotherapy targets in ESC is limited. We examined the prevalence and prognostic value of lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and ITIM domain (TIGIT), V-domain immunoglobulin (Ig) suppressor of T-cell activation (VISTA), and indoleamine 2,3-dioxygenase 1 (IOD1) in 94 cases of ESC and correlated their expression with CD8+ and FOXP3+ tumor-infiltrating lymphocytes (TILs). We observed a positive correlation among LAG-3, TIGIT, and VISTA expressed on immune cells, and among these markers and CD8+ and FOXP3+ TIL densities. In Kaplan-Meier survival analysis, tumors with high levels of LAG-3 and TIGIT expression had better progression-free survival (PFS) and overall survival (OS) than those with lower levels of expression (LAG-3: PFS, P = .03, OS, P = .04; TIGIT: PFS, P = .01, OS, P = .009). In multivariate analysis, only high TIGIT expression was of independent prognostic value for better OS. VISTA expression in immune or tumor cells, and IDO1 expression in tumor cells, did not show a significant association with survival. Our data indicate that LAG-3, TIGIT, and VISTA immune checkpoints have roles in the microenvironment of ESC, and their expression patterns highlight the complex interactions among the different components of this system. High levels of these markers, together with high CD8+ TIL, suggest the potential immunogenicity of a subset of these tumors. Further studies are needed to elucidate the roles of various immune components in the ESC microenvironment and their association with intrinsic tumor properties.

Assisted reproductive technology treatment failure and the detection of intrauterine HPV through spent embryo transfer media sample.

Cervical human papillomavirus (HPV) infection is believed to increase the risks of pregnancy failure and abortion, however, whether the uterine cavity HPV infection reduces pregnancy rate or increases miscarriage rate remains unclarified in infertile women undergoing assisted reproductive technology (ART) treatment. Therefore, we aimed to assess ART outcomes in the presence of intrauterine HPV. This was a hospital-based multicenter (five reproductive medicine centers) matched cohort study. This study involved 4153 infertile women undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection treatment in five reproductive medicine centers between October 2018 and 2020. The spent embryo transfer media sample with endometrium tissue were collected and performed with flow-through hybridization and gene chips to detect HPV DNA. According to basic characteristics, HPV-positive and negative patients were matched in a ratio of 1:4 by age, body mass index transfer timing, transfer type, and number of embryos transferred. The primary outcome was pregnancy and clinical miscarriage rates in the transfer cycle underwent HPV detection. 92 HPV-positive and 368 HPV-negative patients were screened and analyzed statistically. Univariate analysis showed uterine cavity HPV infection resulted in lower rates of ongoing pregnancy (31.5% vs. 44.6%; p = 0.023), implantation (32.3% vs. 43.1%; p = 0.026), biochemical pregnancy (47.8% vs. 62.5%; p = 0.010), and clinical pregnancy (40.2% vs. 54.3%; p = 0.015) compared with HPV negative group. The infertile female with positive HPV also had a slightly higher frequency of biochemical miscarriage (15.9% vs. 13.0%; p = 0.610) and clinical miscarriage (24.3% vs. 15.5%; p = 0.188). These findings suggest that HPV infection in the uterine cavity is a high risk for ART failure. HPV screening is recommended before ART treatment, which may be benefit to improving pregnancy outcome.

KCNE4 is a crucial host factor for Orf virus infection by mediating viral entry.

The orf virus (ORFV) poses a serious threat to the health of domestic small ruminants (i.e., sheep and goats) and humans on a global scale, causing around $150 million in annual losses to livestock industry. However, the host factors involved in ORFV infection and replication are still elusive. In this study, we compared the RNA-seq profiles of ORFV-infected or non-infected sheep testicular interstitial cells (STICs) and identified a novel host gene, potassium voltage-gated channel subfamily E member 4 (KCNE4), as a key host factor involved in the ORFV infection. Both RNA-seq data and RT-qPCR assay revealed a significant increase in the expression of KCNE4 in the infected STICs from 9 to 48 h post infection (hpi). On the other hand, the RT-qPCR assay detected a decrease in ORFV copy number in both the STICs transfected by KCNE4 siRNA and the KCNE4 knockout (KO) HeLa cells after the ORFV infection, together with a reduced fluorescence ratio of ORFV-GFP in the KO HeLa cells at 24 hpi, indicating KCNE4 to be critical for the ORFV infection. Furthermore, the attachment and internalization assays showed decreased ORFV attachment, internalization, replication, and release by the KO HeLa cells, demonstrating a potential inhibition of ORFV entry into the cells by KCNE4. Pretreatment with the KCNE4 inhibitors such as quinidine and fluoxetine significantly repressed the ORFV infection. All our findings reveal KCNE4 as a novel host regulator of the ORFV entry and replication, shedding new insight into the interactive mechanism of ORFV infection. The study also highlights the K+ channels as possible druggable targets to impede viral infection and disease.

ß-catenin, PAX2, and PTEN Aberrancy Across the Spectrum of Endometrioid Ovarian Lesions.

Endometriosis is a common condition, with the ovary being the most common anatomic site. Endometriosis-particularly in the ovary-is associated with a risk of malignant progression, with a histologic spectrum of lesions from benign to malignant. Recently, a panel of 3 markers consisting of ß-catenin, PAX2, and PTEN has been described as a potentially useful diagnostic adjunct in the diagnosis of intrauterine endometrioid neoplasia, where aberrancy for one or more of the markers is strongly associated with neoplasia. Here, we applied the panel to ovarian endometrioid lesions, including endometriosis, endometriosis with flat cytologic atypia, endometrioid borderline tumors, and endometrioid adenocarcinoma (n=85 cases in total). The incidence of aberrancy for the 3 markers increased along this putative neoplastic spectrum, arguing for a role of each of the markers in the neoplastic transformation of ovarian endometriosis. Just 1/32 (3%) of cases of nonatypical endometriosis was marker-aberrant, and this case was aberrant only for PAX2. One of 5 cases (20%) of endometriosis with atypia was marker-aberrant (both PAX2 and PTEN), supporting prior findings that some cases of flat atypia may represent bona fide precursor lesions. Of 19 endometrioid borderline tumors, 10 (53%) were aberrant for one or more markers, with PAX2 being the most frequently aberrant. Of 29 endometrioid adenocarcinomas, 28 (96.6%) were aberrant for at least 1 marker, with PAX2 again the most frequently aberrant. Patterns of aberrancy were well-preserved in areas of nonatypical endometriosis adjacent to borderline tumor or adenocarcinoma, supporting a biological origin in a common marker-aberrant precursor. The findings show that the biomarker panel could be of some diagnostic utility in the characterization of ovarian endometrioid neoplasia, such as in the diagnosis of endometrioid borderline tumor, distinguishing endometrioid from nonendometrioid lesions, or in identifying other types of early precursors at a higher risk of malignant transformation.

Predicting Progestin Therapy Response With PTEN, PAX2, and ß-Catenin in Patients With Endometrioid Precancer.

This study investigates the predictive value of biomarkers PTEN, PAX2, and ß-catenin for therapeutic outcomes in patients with atypical endometrial hyperplasia or endometrioid intraepithelial neoplasia undergoing progestin therapy. In a retrospective study of 128 patients, we analyzed a total of 351 endometrial biopsy samples and categorized outcomes into responders (absence of residual disease) and nonresponders (presence of residual disease). We found aberrant biomarker expression in pretreatment cases: 48% for PTEN, 65% for PAX2, and 36% for ß-catenin. Approximately 77.3% of patients responded to progestin treatment, with nonresponders showing significantly higher initial PTEN loss (75.86% vs 39.79%, P < 0.001). Nonresponders also demonstrated significant PTEN loss (53.33% vs 20.55%, P < 0.001), PAX2 loss (57.33% vs 41.22%, P < 0.05), and ß-catenin nuclear staining (53.45% vs 27.91%, P < 0.01) in follow-up samples. In addition, nonresponders exhibited lower recovery of intact PTEN and PAX2, along with higher ß-catenin aberrancy in cases initially showing normal ß-catenin levels. We conclude that persistent aberrant PTEN and PAX2 expression, coupled with emerging aberrant ß-catenin in follow-ups, indicates a greater likelihood of treatment failure. Conversely, the absence of these aberrations suggests successful progestin therapy. Our findings highlight the utility of this 3-marker panel in assessing residual disease status and predicting progestin treatment outcomes, thus offering critical insights for patient management.

Systematic review and meta-analysis on the effect of adjuvant gonadotropin-releasing hormone agonist (GnRH-a) on pregnancy outcomes in women with endometriosis following conservative surgery.

BACKGROUND: Endometriosis frequently results in pain and infertility. While conservative surgery offers some relief, it often falls short of ensuring satisfactory pregnancy outcomes. Adjuvant GnRH-a is administered post-surgery to mitigate recurrence; however, its impact on pregnancy outcomes remains debated. This study endeavors to assess the efficacy of adjuvant GnRH-a in enhancing pregnancy outcomes post-conservative surgery in endometriosis patients. METHODS: Databases including PubMed, Embase, the Cochrane Library, Medline (Ovid), Web of Science, and Scopus were rigorously searched up to 02 August 2023, without linguistic constraints. Identified articles were screened using strict inclusion and exclusion criteria. Evaluated outcomes encompassed pregnancy rate, live birth rate, miscarriage rate, ectopic pregnancy rate, multiple pregnancy rate, mean postoperative pregnancy interval, recurrence rate, and adverse reaction rate. The Cochrane risk of bias tool and the Jadad score evaluated the included studies' quality. Subgroup and sensitivity analysis were implemented to analyze the pooled results. A meta-analysis model expressed results as standardized mean difference (SMD) and Risk ratio (RR). RESULTS: A total of 17 studies about 2485 patients were assimilated. Meta-analysis revealed that post-surgery, the GnRH-a cohort experienced a marginally elevated pregnancy rate (RR = 1.20, 95% CI = 1.02-1.41; P = 0.03) and a reduced mean time to conceive (RR = -1.17, 95% CI = -1.70- -0.64; P < 0.0001). Contrarily, other evaluated outcomes did not exhibit notable statistical differences. CONCLUSIONS: Incorporating adjuvant GnRH-a following conservative surgery may be deemed beneficial for women with endometriosis, especially before Assisted Reproductive Technology (ART). Nonetheless, owing to pronounced heterogeneity, subsequent research is warranted to substantiate these potential advantages conclusively. REGISTRATION NUMBER: CRD42023448280.

Identification of AGO2 and PLEC genes polymorphisms in Hu sheep and their relationship with body size traits.

The body size traits are major traits in livestock, which intuitively displays the development of the animal's bones and muscles. This study used PCR amplification, Sanger sequencing, KASPar genotyping, and quantitative real-time reverse transcription PCR (qRT-PCR) to analyze the Single-nucleotide polymorphism and expression characteristics of Argonaute RISC catalytic component 2 (AGO2) and Plectin (PLEC) genes in Hu sheep. Two intron mutations were found in Hu sheep, which were AGO2 g.51700 A > C and PLEC g.23157 C > T, respectively. Through association analysis of two mutation sites and body size traits, it was found that AGO2 g.51700 A > C mainly affects the chest and cannon circumference of Hu sheep of while PLEC g.23157 C mainly affects body height and body length. The combined genotypes of AGO2 and PLEC genes with body size traits showed SNPs at the AGO2 g.51700 A > C and PLEC g.23157 C > T loci significantly improved the body size traits of Hu sheep. In addition, the AGO2 gene has the highest expression levels in the heart, rumen, and tail fat, and the PLEC gene is highly expressed in the heart. These two loci can provide new research ideas for improving the body size traits of Hu sheep.

Regulation of cashmere fineness traits by noncoding RNA in Jiangnan cashmere goats.

BACKGROUND: Cashmere has long been used as the raw material for wool textiles. The diameter of the cashmere fibre determines its quality and economic value. However, the regulatory role of noncoding RNAs (ncRNAs) in cashmere fineness remains unclear, especially regarding the interaction between ncRNAs and coding RNAs. RESULTS: Transcriptome sequencing was used to identify the expression profiles of long noncoding RNAs (lncRNAs), circular RNAs (circRNAs) and microRNAs (miRNAs) in the skin tissues of Jiangnan cashmere goats with different cashmere fineness levels. Integration analysis of ncRNA and coding RNA was performed in combination with previous research results. The results showed that 16,437 lncRNAs, 2234 circRNAs, and 1322 miRNAs were identified in 8 skin samples of cashmere goats. A total of 403 differentially expressed (DE) lncRNAs, 62 DE circRNAs and 30 DE miRNAs were identified in the skin tissues of the fine groups (Fe) and coarse groups (Ce). We predicted the target gene of DE lncRNA, the target gene of DE miRNA and the host gene of DE circRNA. Based on functional annotation and enrichment analysis of target genes, we found that DE lncRNAs could be involved in regulating the fineness traits of cashmere. The most potential lncRNAs were MSTRG.42054.1, MSTRG.18602.3, and MSTRG.2199.13. CONCLUSIONS: The data from this study enriched the cashmere goat noncoding RNA database and helped to supplement the annotation of the goat genome. The results provided a new direction for the breeding of cashmere characters.

Shift in prevalence and systemic inflammation levels from NAFLD to MAFLD: a population-based cross-sectional study.

BACKGROUND: Variations in the prevalence and systemic inflammatory (SI) status between non-alcoholic fatty liver disease (NAFLD) and newly defined metabolic dysfunction-associated fatty liver disease (MAFLD) have only been reported by few studies. Hence, this study aimed to compile data on the prevalence and the systemic inflammation levels of MAFLD and NAFLD in a general population from Southeast China was summarized to explore the potential effect of the transformation of disease definition. METHODS: A total of 6718 general population participants aged 35-75 were enrolled. Logistic regression and restricted cubic spline (RCS) models were used to examine the relationship between 15 SI indicators and NAFLD and MAFLD. The predicted values of MAFLD and NAFLD were analyzed using the receiver operating characteristic (ROC) curve. RESULTS: The prevalence of MAFLD and NAFLD was 34.7% and 32.4%, respectively. Their overlapping rate was 89.7%, while only 8.3% and 1.9% of participants were MAFLD-only and NAFLD-only. Among three FLD groups, the MAFLD-only group had the highest levels of 8 SI indicators, including CRP, WBC, LYMPH, NEUT, MONO, ALB, NLR, and SIRI. The non-FLD group had the lower levels of all 15 SI indicators compared with all FLD subgroups. The odds ratios (ORs) of 10 SI indicators were significant in both multivariable-adjusted logistic regression and RCS analyses of MAFLD or NAFLD, including CRP, WBC, LYMPH, NEUT, MONO, ALB, PLR, LMR, ALI and CA. ROC analysis showed that the AUC values of all SI were lower than 0.7 in both MAFLD and NAFLD. CONCLUSIONS: MAFLD could cover more FLD than NAFLD, and the MAFLD-only group had a more severe inflammation status, whereas the NAFLD-only exhibited lower levels. Moreover, there was not a high AUC and a high sensitivity of SI indicators, suggesting that SI indicators are not good indicators to diagnose NAFLD/MAFLD.

Detection of single nucleotide polymorphism in HTR4 and its relationship with growth traits in sheep.

In this study, a single nucleotide polymorphism of HTR4 (hydroxytryptamine receptor 4) was detected using DNA sequencing and KASPar (Kompetitive Allele-Specific PCR) technique with the aim of analyzing its effect on growth traits in 1102 sheep. A synonymous mutation g.101220 C > T located on the fifth intron of the ovis HTR4 gene was detected, and association analysis showed that this mutation was significantly associated with growth traits in sheep (p ＜.05), with TT genotypes having significantly lower body weight, height, length and chest circumference than TC and CC genotypes. It showed that the polymorphism of this locus was significantly associated with growth traits in sheep. In addition, qRT-PCR results showed that HTR4 was expressed in different tissues of sheep. It is highly expressed in the liver, spleen and duodenum. As important metabolic, immune and digestive absorption organs in animals, the above tissues can regulate the excitability of intestinal smooth muscle by participating in the body metabolism and nutrient metabolism of sheep, so that sheep can show better growth characteristics. In conclusion, the polymorphic locus identified in HTR4 gene can be used as candidate molecular marker in sheep breeding.

Polymorphism and expression level of the FADS3 gene and associated with the growth traits in Hu sheep.

Growth traits are the economically important traits of sheep, and screening for genes related to growth and development is helpful for the genetic improvement of ovine growth traits. The fatty acid desaturase 3 (FADS3) is one of the important genes affecting the synthesis and accumulation of polyunsaturated fatty acids in animals. In this study, the expression levels of the FADS3 gene and polymorphism of the FADS3 gene associated with growth traits in Hu sheep were detected using quantitative real-time PCR (qRT-PCR), Sanger sequencing, and KAspar assay. The result showed that the expression levels of the FADS3 gene were widely expressed in all tissues, and the expression level of FADS3 in the lung was significantly higher than in other tissues (p < .05). Then, the polymorphism locus g. 2918 A > C was detected in intron 2 of the FADS3 gene, and associated analysis showed that the mutation in the FADS3 gene was associated significantly with growth traits (including body weight, body height, body length, and chest circumference, p < .05). Therefore, individuals with AA genotype showed significantly better growth traits than those with CC genotype, and FADS3 gene could be a candidate gene for improving growth traits in Hu sheep.

HPF Modulates the Differentiation of BMSCs into HLCs and Promotes the Recovery of Acute Liver Injury in Mice.

Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into hepatocyte-like cells (HLCs) to alleviate acute liver injury (ALI). Herpetfluorenone (HPF), as an active ingredient in the dried, mature seeds Herpetospermum caudigerum Wall, used in Tibetan medicine, has been proven to effectively alleviate ALI. Therefore, the purpose of this study was to determine whether HPF can promote the differentiation of BMSCs into HLCs and promote ALI recovery. Mouse BMSCs were isolated, and the BMSCs' differentiation into HLCs was induced by HPF and hepatocyte growth factor (HGF). Under the induction of HPF and HGF, the expression of hepatocellular specific markers and the accumulation of glycogen and lipids in the BMSCs increased, indicating that BMSCs successfully differentiated into HLCs. Then, the ALI mouse model was established, using carbon tetrachloride, followed by an intravenous injection of BMSCs. Then, only HPF was injected intraperitoneally, in order to verify the effect of HPF in vivo. In vivo imaging was used to detect the homing ability of HPF-BMSCs, and it was detected that HPF-BMSCs significantly increased the levels of serum AST, ALT and ALP in the liver of ALI mice, and alleviated liver cell necrosis, oxidative stress and liver pathology. In conclusion, HPF can promote the differentiation of BMSCs into HLCs and promote the recovery of ALI in mice.

Scalp hair loss is not random across follicular units: A new insight into human hair ageing.

OBJECTIVES: Scalp hair has the greatest number of hairs (typically 1-5) per follicular unit but is also the most susceptible body site to hair loss with age. Hence, we set-out to determine the degree to which scalp hair parameters change with age in women and men, any sex differences thereof and whether hair loss is random across follicular units. METHODS: A retrospective cross-sectional study of 200 Chinese men and 200 Chinese women (30-69 years). Image analysis and manual counting methods were used to measure occipital located hair parameters from 6 × 8 mm shaved scalp photographs and plucked hair microscopy images. RESULTS: Of the five hair parameters, the number of hairs per follicular unit had the greatest (negative) correlation with age in both men and women. Men had a greater number of hairs and follicular units than women on average but had a greater decrease in the number of hairs per follicular unit with age, particularly for the loss of multi-hair (3+) follicular units. The loss of hairs with age was significantly different to that expected by a random loss of hairs across follicular units and better described by a model of increased hair loss risk the greater number of hairs per follicular unit. CONCLUSIONS: We have found evidence of hair loss preferentially occurring in multi-hair follicular units, which was more pronounced in men. These data suggest that part of the reason scalp hair is more susceptible to hair loss than on other body sites is due to the greater presence of multi-hair follicular units on the scalp.

OBJECTIFS: Le cuir chevelu possède le plus grand nombre de cheveux (généralement de 1 à 5) par unité folliculaire, mais c'est aussi le site le plus sensible à la perte de cheveux avec l'âge. Nous avons donc entrepris de déterminer dans quelle mesure les paramètres des cheveux du cuir chevelu changent avec l'âge chez les femmes et les hommes, quelles sont les différences entre les sexes et si la perte de cheveux est aléatoire entre les unités folliculaires. MÉTHODES: Étude transversale rétrospective portant sur 200 hommes et 200 femmes chinois (30-69 ans). Des méthodes d'analyse d'image et de comptage manuel ont été utilisées pour mesurer les paramètres des cheveux situés dans la région occipitale à partir de photographies du cuir chevelu rasé de 6x8 mm et d'images microscopiques de cheveux arrachés. RÉSULTATS: Parmi les 5 paramètres capillaires, le nombre de cheveux par unité folliculaire présentait la corrélation la plus forte (négative) avec l'âge, tant chez les hommes que chez les femmes. Les hommes avaient en moyenne un plus grand nombre de cheveux et d'unités folliculaires que les femmes, mais le nombre de cheveux par unité folliculaire diminuait davantage avec l'âge, en particulier pour la perte d'unités folliculaires à plusieurs cheveux (3+). La perte de cheveux avec l'âge était significativement différente de celle attendue par une perte aléatoire de cheveux dans les unités folliculaires, et mieux décrite par un modèle d'augmentation du risque de perte de cheveux plus le nombre de cheveux par unité folliculaire est élevé. CONCLUSIONS: Nous avons trouvé des preuves que la perte de cheveux se produit préférentiellement dans les unités folliculaires à plusieurs cheveux, ce qui était plus prononcé chez les hommes. Ces données suggèrent qu'une partie de la raison pour laquelle les cheveux du cuir chevelu sont plus sensibles à la perte de cheveux que sur d'autres sites du corps est due à la plus grande présence d'unités folliculaires à cheveux multiples sur le cuir chevelu.

[Killing effect of anti-MSLN-iCAR-NK cells derived from induced pluripotent stem cells on ovarian epithelial cancer cells].

Objective: To investigate the cytotoxic effects of induced pluripotent stem (iPS) cells of anti-mesothelin (MSLN)-chimeric antigen receptor natural killer (CAR-NK) cells (anti-MSLN-iCAR-NK cells) on ovarian epithelial cancer cells. Methods: Twenty cases of ovarian cancer patients who underwent surgical treatment at Henan Provincial People's Hospital from September 2020 to September 2021 were collected, and 20 cases of normal ovarian tissues resected during the same period due to other benign diseases were also collected. (1) Immunohistochemistry and immunofluorescence were used to verify the expression of MSLN protein in ovarian cancer tissues. (2) Fresh ovarian cancer tissues were extracted and cultured to obtain primary ovarian cancer cells. Recombinant lentiviral vectors targeting anti-MSLN-CAR-CD244 were constructed and co-cultured with iPS cells to obtain anti-MSLN-iCAR cells. These cells were differentiated into anti-MSLN-iCAR-NK cells using cytokine-induced differentiation method. The cell experiments were divided into three groups: anti-MSLN-iCAR-NK cell group, natural killer (NK) cell group, and control group. (3) Flow cytometry and live cell staining experiment were used to detect the apoptosis of ovarian cancer cells in the three groups. (4) Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granzyme B (GZMB), perforin 1 (PRF1), interleukin (IL)-6, and IL-10 in the three groups of ovarian cancer cells. Results: (1) Immunohistochemistry analysis showed that a positive expression rate of MSLN protein in ovarian cancer tissues of 65% (13/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=4.912, P=0.027). Immunofluorescence analysis revealed that the positive expression rate of MSLN protein in ovarian cancer tissues was 70% (14/20), while normal ovarian tissues had a positive rate of 30% (6/20). The comparison between the two groups was statistically significant (χ2=6.400, P=0.011). (2) Flow cytometry analysis showed that the apoptotic rate of ovarian cancer cells in the anti-MSLN-iCAR-NK cell group was (29.27±0.85)%, while in the NK cell group and control group were (8.44±0.34)% and (6.83±0.26)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.01). Live cell staining experiment showed that the ratio of dead cells to live cells in the anti-MSLN-iCAR-NK cell group was (36.3±8.3)%, while in the NK cell group and control group were (5.4±1.4)% and (2.0±1.3)% respectively. There were statistically significant differences in the comparisons between the three groups (all P<0.001). (3) ELISA analysis revealed that the expression levels of IFN-γ, TNF-α, GZMB, PRF1, IL-6, and IL-10 in ovarian cancer cells of the anti-MSLN-iCAR-NK cell group were significantly higher than those in the NK cell group and the control group (all P<0.05). Conclusion: The anti-MSLN-iCAR-NK cells exhibit a strong killing ability against ovarian cancer cells, indicating their potential as a novel immunotherapy approach for ovarian cancer.

Brusatol sensitizes endometrial hyperplasia and cancer to progestin by suppressing NRF2-TET1-AKR1C1-mediated progestin metabolism.

Progestin resistance is the main obstacle for the conservative therapy to maintain fertility in women with endometrial cancer. Brusatol was identified as an inhibitor of the NRF2 pathway; however, its impact on progestin resistance and the underlying mechanism remains unclear. Here, we found that brusatol sensitized endometrial cancer to progestin by suppressing NRF2-TET1-AKR1C1-mediated progestin metabolism. Brusatol transcriptionally suppressed AKR1C1 via modifying the hydroxymethylation status in its promoter region through TET1 inhibition. Suppression of AKR1C1 by brusatol resulted in decreased progesterone catabolism and maintained potent progesterone to inhibit endometrial cancer growth. This inhibition pattern has also been found in the established xenograft mouse and organoid models. Aberrant overexpression of AKR1C1 was found in paired endometrial hyperplasia and cancer samples from the same individuals with progestin resistance, whereas attenuated or loss of AKR1C1 was observed in post-treatment samples with well progestin response as compared with paired pre-treatment tissues. Our findings suggest that AKR1C1 expression pattern may serve as an important biomarker of progestin resistance in endometrial cancer.

Dissection of transcriptome dysregulation and immune characterization in women with germline BRCA1 mutation at single-cell resolution.

BACKGROUND: High-grade serous carcinoma (HGSC) is the most frequent and lethal type of ovarian cancer. It has been proposed that tubal secretory cells are the origin of ovarian HGSC in women with familial BRCA1/2 mutations. However, the molecular changes underlying malignant transformation remain unknown. METHOD: We performed single-cell RNA and T cell receptor sequencing of tubal fimbriated ends from 3 BRCA1 germline mutation carriers (BRCA1 carriers) and 3 normal controls with no high-risk history (non-BRCA1 carriers). RESULTS: Exploring the transcriptomes of 19,008 cells, predominantly from BRCA1+ samples, we identified 5 major cell populations in the fallopian tubal mucosae. The secretory cells of BRCA1+ samples had differentially expressed genes involved in tumor growth and regulation, chemokine signaling, and antigen presentation compared to the wild-type BRCA1 controls. There are several novel findings in this study. First, a subset of the fallopian tubal secretory cells from one BRCA1 carrier exhibited an epithelial-to-mesenchymal transition (EMT) phenotype, which was also present in the mucosal fibroblasts. Second, we identified a previously unreported phenotypic split of the EMT secretory cells with distinct evolutionary endpoints. Third, we observed increased clonal expansion among the CD8+ T cell population from BRCA1+ carriers. Among those clonally expanded CD8+ T cells, PD-1 was significantly increased in tubal mucosae of BRCA1+ patients compared with that of normal controls, indicating that T cell exhaustion may occur before the development of any premalignant or malignant lesions. CONCLUSION: These results indicate that EMT and immune evasion in normal-looking tubal mucosae may represent early events leading to the development of HGSC in women with BRCA1 germline mutation. Our findings provide a probable molecular mechanism explaining why some, but not all, women with BRCA1 germline mutation present with early development and rapid dissemination of HGSC.

Prevalence and prognostic significance of PD-L1, TIM-3 and B7-H3 expression in endometrial serous carcinoma.

Endometrial serous carcinoma (ESC) is an aggressive type of endometrial carcinoma with a poor prognosis. Immune checkpoint blockade has evolved as a novel treatment option for endometrial cancers; however, data on expression of immune checkpoints that may be potential targets for immunotherapy in ESC are limited. We analyzed the prevalence and prognostic significance of PD-L1, TIM-3 and B7-H3 immune checkpoints in 99 ESC and evaluated their correlation with CD8 + tumor infiltrating lymphocytes. Applying the tumor proportion score (TPS) with a cutoff of 1%, PD-L1, TIM-3 and B7-H3 expression was present in 17%, 10% and 93% of cases, respectively. Applying the combined positive score (CPS) with a cutoff of 1, PD-L1, TIM-3 and B7-H3 expression was present in 63%, 67% and 94% of cases, respectively. Expression of these markers was largely independent of one another. PD-L1 correlated with higher CD8 + T-cell density when evaluated by either TPS (p = 0.02) or CPS (p < 0.0001). TIM-3 correlated with CD8 + T-cell density when evaluated by CPS (p < 0.0001). PD-L1 positivity was associated with improved overall survival (p = 0.038) when applying CPS. No association between PD-L1 expression and survival was found using TPS, and there was no association between TIM-3 or B7-H3 positivity and survival by either TPS or CPS. Using TPS, PD-L1 correlated with a higher tumor stage but not with survival, whereas the converse was true when PD-L1 was evaluated by CPS, suggesting that PD-L1 expression in immune cells correlates with prognosis and is independent of tumor stage. In conclusion, PD-L1, TIM-3 and B7-H3 may be potential therapeutic targets in selected patients with ESC. Further investigation of their roles as predictive biomarkers is needed.

Endometrial polyps are non-neoplastic but harbor epithelial mutations in endometrial cancer drivers at low allelic frequencies.

Endometrial polyps (EMPs) are common exophytic masses associated with abnormal uterine bleeding and infertility. Unlike normal endometrium, which is cyclically shed, EMPs persist over ovulatory cycles and after the menopause. Despite their usual classification as benign entities, EMPs are paradoxically associated with endometrial carcinomas of diverse histologic subtypes, which frequently arise within EMPs. The etiology and potential origins of EMPs as clonally-derived neoplasms are uncertain, but previous investigations suggested that EMPs are neoplasms of stromal origin driven by recurring chromosomal rearrangements. To better define benign EMPs at the molecular genetic level, we analyzed individual EMPs from 31 women who underwent hysterectomy for benign indications. The 31 EMPs were subjected to comprehensive genomic profiling by exome sequencing of a large panel of tumor-related genes including oncogenes, tumor suppressors, and chromosomal translocation partners. There were no recurring chromosomal rearrangements, and copy-number analyses did not reveal evidence of significant chromosome-level events. Surprisingly, there was a high incidence of single nucleotide variants corresponding to classic oncogenic drivers (i.e., definitive cancer drivers). The spectrum of known oncogenic driver events matched that of endometrial cancers more closely than any other common cancer. Further analyses including laser-capture microdissection showed that these mutations were present in the epithelial compartment at low allelic frequencies. These results establish a link between EMPs and the acquisition of endometrial cancer driver mutations. Based on these findings, we propose a model where the association between EMPs and endometrial cancer is explained by the age-related accumulation of endometrial cancer drivers in a protected environment that-unlike normal endometrium-is not subject to cyclical shedding.