The Prostate

BackgroundRisk stratification for prostate cancer (PCa) progression or aggressiveness is often based on clinicopathologic features, some of which may be influenced by genetic factors. We developed a novel, germline polygenic risk score (PRSagg) to predict likelihood of developing aggressive PCa. MethodsPRSagg was developed using data from 38,688 patients with PCa (case-only analysis) from the Million Veteran Program (MVP) through a genome-wide search for variants associated with PCa grade group at diagnosis. We tested associations of PRSagg with grade group using the entire MVP dataset using the .632 bootstrap method. In an MVP cohort with localized PCa that was initially monitored without treatment, we tested PRSagg for association with unfavorable outcomes (subsequent development of grade group 4-5, metastasis, and/or biochemical recurrence after definitive treatment). We performed external validation in data from patients in the PRACTICAL Consortium (n=45,214) and from participants in the ProtecT randomized trial who underwent active monitoring (n=316). Odds ratios (ORs) were calculated per standard deviation (SD) increase with 95% confidence intervals, while adjusting for age, genetic ancestry, a previously developed polygenic score for risk of PCa (PHS601), and a polygenic score for benign elevated prostate-specific antigen (PRSPSA). For the outcome of metastasis, we additionally adjusted for PSA at diagnosis. ResultsIn the MVP training dataset, PRSagg (172 variants) was associated with higher grade group at diagnosis (OR = 1.53 [1.51-1.56]) and with increased risk of unfavorable outcomes during monitoring (OR = 1.13 [1.09-1.18]). These findings were confirmed in the external datasets. PRSagg was associated with greater odds of higher grade group at diagnosis (OR = 1.09 [1.06-1.11]). Among ProtecT participants undergoing active monitoring, PRSagg was associated with higher risk of metastasis (OR = 2.15 [1.02-3.88]). Among MVP participants with high polygenic risk of developing any PCa, the risk of aggressive disease was highest in men with high PRSagg and low genetic risk of PSA elevation. ConclusionsAmong men who develop PCa, a weighted sum of common germline variants (PRSagg) is independently associated with PCa aggressiveness. These findings may inform future study of germline influence on tumor evolution and risk-stratified intensity of active surveillance.

The PRESERVE trial (NCT04972097) is a prospective, single-arm pivotal IDE study evaluating focal irreversible electroporation (IRE) using the NanoKnife System for intermediate-risk prostate cancer. Men with Gleason Grade Group 2-3 disease underwent focal IRE and were followed for durability of oncologic control and safety. At 24 months, 68 patients completed follow-up with no new treatment failures identified. PSA levels were below baseline in 97% of patients, and one clinically triggered biopsy was negative for cancer. No new device- or procedure-related adverse events occurred beyond 12 months. These findings demonstrate durable efficacy and sustained safety of focal IRE.

Aims Active surveillance (AS) allows selected men with localized prostate cancer to defer curative therapy and reduce treatment morbidity. Conversion from AS to treatment is commonly triggered by Gleason grade group (GGG) upgrading on confirmatory biopsy. We developed and validated a digital pathology artificial intelligence (DPAI) biomarker to predict GGG upgrading in AS-eligible patients. Materials & Methods The DPAI model was trained using histopathology image features from diagnostic biopsies of 998 patients and validated in an independent cohort of 296 patients meeting criteria for AS. Logistic regression estimated the probability of confirmatory-biopsy GGG increase, and feature selection identified the most predictive variables. Results AI-GUR (Artificial Intelligence-Gleason Upgrade Risk) predicted GGG reclassification at confirmatory biopsy (OR 1.60; p=0.0003), and provided information beyond conventional stratification (risk group, CAPRA) and cribriform morphology (all p<0.01). Predicted risks were similar across time from diagnosis (~10-15% to ~85% at 1, 1.5, or 2 years; p for time=0.50), consistent with initial biopsy mischaracterization rather than time-dependent progression. Conclusions AI-GUR provides individualized estimates of confirmatory-biopsy GGG upgrading for AS candidates. Using DPAI may improve shared decision-making by complementing standard clinicopathologic tools and molecular testing using the same biopsy specimen, while informing the likelihood of grade upgrade at confirmation.

Introduction Aquablation for surgical treatment of benign prostatic enlargement (BPE) causing bladder outflow obstruction (BOO) has demonstrated good functional outcomes, even for large glands, with high rates of ejaculatory preservation reported. This is a protocol for a study that aims to review real-world outcomes of ejaculatory preservation or restoration post-Aquablation in an unselected cohort and compare to published clinical trial outcomes. Methods Retrospective data will be collected from a prospectively maintained consecutive case series of patients who underwent Aquablation, in a single UK centre. The primary outcome is ejaculatory function subjectively reported by men post-operatively, and classified as: antegrade ejaculation, retrograde/low volume ejaculation, anejaculation or not sexually active. Secondary outcomes are International Prostate Symptom Severity (IPSS), Quality of Life (QoL) Score, post-void residual (PVR), and incontinence. Descriptive and comparative statistical tests will be performed. Conclusions This study will review real-world ejaculatory function and clinical outcomes following robotic Aquablation for prostatic bladder outflow obstruction and compare this to published clinical trial outcomes.

Suppressor of cytokine signaling 1 (SOCS1) negative regulates inflammatory cytokine production and attenuates oncogenic growth factor signaling pathways. Reduced SOCS1 protein expression in human prostate cancer correlates with greater disease severity. To define the physiological functions of SOCS1 functions in the prostate, we conditionally ablated Socs1 in prostate epithelial cells of C57BL/6 mice. These Socs1{Delta}PE mice exhibited normal prostate development, maturation and lobular architecture. However, adult Socs1{Delta}PEmice developed progressive epithelial hyperplasia and inflammatory cell infiltration that were temporally and spatially distinct. SOCS1-deficient prostate showed increased epithelial cell proliferation and elevated oxidative stress markers, and prostate organoids recapitulated this hyperplasia phenotype. Diet-induced obesity exacerbated both hyperplasia and inflammation in SOCS1-deficient prostate. Upon transurethral infection with uropathogenic Escherichia coli UPEC1677 expressing the genotoxin colibactin, Socs1{Delta}PE mice developed invasive prostate cancer with complete loss of lobular architecture, whereas control mice developed hyperplasia and pre-neoplastic lesions. In vitro, SOCS1-deficient prostate organoid-derived epithelial cells exhibited increased DNA damage following exposure to UPEC1677. Deletion of the colibactin biosynthetic gene clbP in UPEC1677 abolished its ability to induce DNA damage in SOCS1-deficient cells and to drive prostate cancer in vivo. Proteomic analysis of prostate organoids revealed dysregulation of basal and luminal epithelial lineage markers and signaling pathway proteins that could promote neoplasia in SOCS1-deficient cells. Collectively, these findings establish an essential, epithelial cell-intrinsic role for SOCS1 in maintaining prostate tissue homeostasis by restraining proliferation, regulating lineage plasticity, limiting inflammation and oxidative stress, and conferring protection against genotoxic injury and neoplastic transformation.

Background: Prostate cancer is the second most common cancer in men worldwide. The Prostate Specific Antigen (PSA) blood test is widely used for prostate cancer detection but suffers from high false-positive rates (up to 80%). Genetic risk scores (GRS/PRS) have a similar performance to PSA testing in predicting prostate cancer risk. Method: GRS269 for prostate cancer was derived using 269 known risk variants and applied to UK Biobank participants. We assessed whether GRS269 improved power to predict prostate cancer diagnosis on top of age and pre-prostatectomy PSA level among 17,380 cases. Longitudinal PSA measurements were processed as median, first, last (most recent), and random PSA. All models were adjusted for age. Results: Across all PSA measures, the integrated model combining GRS269, PSA, and age consistently outperformed models using GRS269 or PSA alone. The highest predictive performance was observed using the last PSA value combined with GRS269 (AUC = 0.82, 95% CI: 0.81-0.82), compared to GRS269 alone (AUC = 0.70, 95% CI: 0.68-0.72) or PSA alone (AUC = 0.73, 95% CI: 0.70-0.75). Conclusion: Combining genetic risk with PSA and age improves prostate cancer risk prediction in a population setting. These findings highlight the potential clinical implications of integrating GRS will enhance early prostate cancer prediction pathways in primary care.

Bladder outlet obstruction leads to pathological remodeling and emergence of lower urinary tract symptoms. Although relief of obstruction is associated with symptomatic improvement, it is not universally successful, reflecting persistent alterations in the bladder. Reliable surrogate biomarkers of obstruction are lacking, particularly early in the disease course before irreversible damage to the bladder may have occurred. In this study, re-analysis of publicly available transcriptomic datasets from diverse rodent models of obstruction identified tissue transcripts including Cthrc1, Grem1, Ltbp2 and Msn that were induced in response to injury. Candidate markers were validated experimentally in an independent model of neurogenic obstruction demonstrating time-dependent changes. Candidate markers were also attenuated with either surgical removal of obstruction or treatment with anticholinergic medication or inosine. Integrated analysis of tissue transcriptomics data and tissue and urine proteomics data from a model of neurogenic obstruction revealed significant concordance between markers observed in tissue and urine. Urinary proteomics analysis identified a statistically significant increase in MSN in patients with neurogenic bladder compared to unaffected controls. These findings identify tissue and urine biomarkers of both non-neurogenic and neurogenic obstruction that may reflect early changes in obstructive uropathy that could be monitored in a non-invasive manner.

Single-cell RNA sequencing (scRNA-seq) effectively captures the differences in transcriptomic landscape of cell types and cell states between benign and cancer tissues. Pooling publicly available datasets distributed across independent studies enables increased sample representation and cross-study comparisons. Here we present a harmonized scRNA-seq atlas of the human prostate constructed by integrating 17 available studies, comprising 163 samples from 106 donors. The dataset contains benign tissue, primary tumors, and metastatic disease profiles. Raw sequencing FASTQ data files were uniformly reprocessed to minimize technical variability. Study metadata were curated and standardized using a unified schema capturing donor identity, tissue site, disease context, and histologic grade. Post quality control, the integrated dataset contains 754,000 high-quality cells. Harmonized cell type annotations were generated using a pseudobulk correlation framework informed by multiple reference resources. The workflow identified 17 distinct cell types representing epithelial, mesenchymal, and immune compartments of the prostate. The processed expression matrices, standardized metadata, and analysis workflows are publicly available to support reproducible analysis and enable exploration of heterogeneity across prostate disease states.

While prostate cancer (PC) is defined as immunologically cold, limiting the efficacy of immune checkpoint inhibitors, therapeutic vaccination targeting tumor-associated antigens represents an attractive strategy to promote disease control in low volume metastatic patients. The UV1 cancer vaccine is based on immunization with tripeptide fragments from human telomerase reverse transcriptase (hTERT) and a phase II clinical trial demonstrated induction of robust T cell response in men with de novo metastatic castration-sensitive prostate cancer (mCSPC). Comparison with long-term survival data of non-metastatic CSPC patients as reference showed that despite metastatic disease at diagnosis, UV1-treated patients who mounted an early vaccine-induced immune response achieved progression-free and overall survival comparable to non-metastatic patients. We examined biological determinants of clinical benefit following UV1 vaccination including tumor transcriptome and T cell receptor (TCR) profiling from circulating and tissue resident T-cells of the 22 men enrolled. Analysis of diagnostic and post-UV1 treatment biopsies revealed that low baseline exhaustion of T cells and higher CD8+ T cell abundance are associated with early immune response to the vaccine and longer survival. Moreover, we identified specific TCR motifs relative to early responders, that can indicate potential benefit from UV1 vaccination. These findings indicate that baseline intratumoral T cell exhaustion state and repertoire shape responsiveness to hTERT vaccination and long-term outcome. Overall, our study underlines how baseline immune profiling may be used as a companion biomarker to predict mCSPC patients most likely to benefit from therapeutic vaccination.

Conventional semen cryopreservation involves equilibration at 4{degrees}C and optimum freezing rates. We hypothesized that a cholesterol-based semen extender obviates the need for equilibration, minimizing total processing time for semen cryopreservation. Experiments were conducted to determine the effects of semen extender (egg yolk- or cholesterol-based) and freezing method (routine or fast) on post-thaw sperm characteristics and fertility of beef and bison semen. In Experiment 1, beef semen diluted in tris-egg yolk-glycerol (TEYG) or cholesterol-cyclodextrin tris-glycerol (CCTG) extender underwent routine or fast freezing method. Cholesterol from animal and plant origins were compared. The routine method included 90-min equilibration at 4{degrees}C and routine freezing (RE-RF, total time 97 min) whereas the fast method included no equilibration and fast freezing (NE-FF, total time 14 min). Post-thaw sperm quality was assessed by CASA, and in vitro fertilization. Post-thaw sperm motility was not affected by the origin of cholesterol (animal or plant), but was lowest in the TEYG NE-FF group (24% vs 43-51%, P < 0.05). In vitro cleavage and blastocyst development rates did not differ between RE-RF and NE-FF groups. In Experiment 2, bison semen was diluted in TEYG or plant-CCTG extender and frozen as in Experiment 1. Post-thaw sperm motility was lowest in the TEYG NE-FF group (10% vs 39-51%, P < 0.05). In Experiment 3, beef semen diluted in TEYG or plant-CCTG extender underwent either a routine (RE-RF) or modified freezing (NE-RF, total time 25 min) method. Post-thaw sperm characteristics did not differ between extenders but were greater using routine freezing (RE-RF) compared to the modified method of freezing (NE-RF). Pregnancy rates were similar between extenders (TEYG vs plant-CCTG) using the modified freezing method without equilibration and insemination at 72 h after progesterone device removal. In conclusion, beef and bison semen diluted in cholesterol-based extender may be cryopreserved without equilibration.

Mitochondrial homeostasis, governed by the balance between biogenesis and mitophagy, is essential for steroidogenesis in adrenocortical cells. While the requirement of active mitochondria for steroid synthesis is well-established, the hormonal regulation of genes governing mitochondrial function remains poorly understood. This study investigated whether angiotensin II (Ang II) and the cAMP/PKA pathway modulate the expression of key regulatory factors involved in mitochondrial biogenesis and redox status in the human adrenocortical H295R cell line. Using real-time qPCR and Western blot, we show that Ang II and 8Br-cAMP --a permeant analogue of cAMP-- modulate NRF-1, Nrf2, UCP2, and ANT1 impacting on mitochondrial biogenesis, antioxidant defense, and respiratory activity. These molecular changes correlated with increased mitochondrial membrane polarization, as confirmed by MitoTracker red staining. Interestingly, Ang II stimulation promoted a time-dependent increase in TFAM levels, a key transcription factor in mitochondria, which correlates with the increase in mitochondrial DNA (mtDNA) content. The rate of oxygen consumption (OCR) and mitochondrial parameters were determined, with results showing that Ang II led to a significant increase in basal and maximum respiration, ATP production, and proton leak. These findings suggest that hormone stimulation favors mitochondrial activity, thereby enhancing the bioenergetic capacity of adrenocortical cells. Furthermore, treatment with the uncoupler CCCP triggered a retrograde signaling response, upregulating nuclear-encoded mitochondrial genes to counteract mitochondrial membrane depolarization. Our findings demonstrate for the first time that hormonal signals directly modulate the mitochondrial genetic program in H295R human adrenocortical cells, optimizing the bioenergetic platform required for efficient steroidogenic function.

Accurate prognostic assessment of prostate cancer (PCa) requires an integrated understanding of tissue morphology-encompassing cell structure, glandular architecture, and tissue organization-and the immune environment. We present Prostate-TriMod, a novel tri-modal histology dataset designed to integrate high-resolution visual morphology with spatial tissue maps, immune infiltration patterns, and clinical outcomes. This dataset, generated from the Cell DIVE multiplexed imaging platform, consists of three synchronized modalities: (1) multiscale virtual H&E tiles (224px, 256px, 512px, and 2040px) providing visual morphological context, (2) spatial tissue maps identifying cancerous/non-cancerous epithelial cells, stroma and immune cell populations (via TOPAZ and CAT models), and (3) text captions generated from single-cell data and patterns. The dataset includes comprehensive clinical annotations, including Grade Groups and biochemical recurrence (BCR) status. By providing high-fidelity alignment between visual features, spatial tissue maps, and textual descriptions, Prostate-TriMod empowers the development of advanced multimodal AI frameworks. We expect this resource to support reuse in multimodal representation learning, spatial analysis, and benchmarking studies that link histology morphology and immune context to clinical outcomes in prostate cancer.

The standard-of-care for patients with higher-risk non-muscle invasive bladder cancer (NMIBC) after tumour resection is intravesical administration of Bacillus Calmette-Guerin (BCG). While this form of adjuvant immunotherapy has improved recurrence-free and progression-free survival, a large proportion of patients experience recurrences within a year of diagnosis. The reasons for this high rate of early recurrence following BCG therapy remain unclear; however, inadequate activation of systemic immunity may be a contributing factor. To address this, we analysed the transcriptomic and chromatin accessibility profiles of peripheral blood mononuclear cells obtained from patients with NMIBC at single-cell resolution before BCG immunotherapy and after five induction doses of BCG. Monocytes from patients who experienced disease recurrence within a year of initiation of BCG therapy (BCG non-responders) exhibited a pro-inflammatory phenotype consistent with age-related immunosenescence prior to BCG immunotherapy. Moreover, inflammation-associated pathways that were active before initiation of BCG therapy in the BCG non-responders were down-regulated after five instillations of BCG. In contrast, these pathways were quiescent before BCG therapy in patients who remained disease-free for at least a year but were markedly up-regulated after five doses of BCG. Genomic regions with accessible chromatin were enriched in activator protein 1 (AP-1) binding sequences in monocytes from BCG-non-responders prior to BCG therapy. AP-1 is a central regulator of the inflammatory phenotype associated with immunosenescence. Our findings indicate that a pre-existing state of innate immunosenescence underlies early disease recurrence following BCG. Patients unlikely to benefit from BCG may be offered alternative therapies early in their disease journey. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/723215v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1f7c844org.highwire.dtl.DTLVardef@7cea65org.highwire.dtl.DTLVardef@1008d23org.highwire.dtl.DTLVardef@131f973_HPS_FORMAT_FIGEXP M_FIG C_FIG

PurposeBladder cancer (BLCA) is a heterogeneous tumor type. Only one third of muscle-invasive (MIBC) patients respond to immune checkpoint inhibitors (ICIs). Reliable resistance markers are needed to guide clinical decisions. We investigated the nerve growth factor receptor (NGFR) in BLCA and analyzed its correlation with disease progression and response to immunotherapy. Experimental DesignWe analyzed NGFR expression in BLCA cell lines, organoids, mouse models and patient samples. The cohorts used were The Cancer Genome Atlas (TCGA), enriched in muscle-invasive bladder cancer (MIBC) (n=407); IMvigor210, representing MIBC patients treated with ICIs (n=348); and UROMOL2, as a non-muscle-invasive bladder cancer (NMIBC)-specific cohort (n=535). IMvigor010 was also included (n=728). Patients were stratified by NGFR expression quartiles. We analyzed survival and tumor subtypes and performed stromal deconvolution and functional profiling. We assessed stemness- and invasion-related features in SCaBER cells. ResultsNGFR marks a basal tumor cell subcluster and is independently associated with poor prognosis in TCGA and IMvigor210. NGFR-high tumors show stromal content enriched in cancer-associated fibroblasts, lower neoantigen burden, higher CD8+ T effector signature together with an immune-excluded phenotype, and a CAF-specific TGF{beta} signature. In the immunotherapy-treated cohort, high NGFR expression was also associated with poorer outcome. Functionally, NGFR appears to promote a stem-like/pro-invasive program in BLCA cells. ConclusionsNGFR identifies a basal-like BLCA subpopulation linked to poor survival, while its association with immunotherapy response requires further validation. In addition, our in vitro analyses support a role of NGFR in stem-like and invasive traits, highlighting its relevance as a biomarker in BLCA.

ObjectiveTo 1) determine the expression and distribution of all PDE4 isozymes (A-D) along the length of the anterior urethra, 2) culture fibroblasts and epithelial cells from healthy and strictured urethras, 3) investigate an in vitro model of anterior urethral stricture disease (aUSD), and 4) assess the therapeutic potential of phosphodiesterase-4 (PDE4) inhibitors and testosterone compared to paclitaxel. MethodsThe presence and relative abundance of PDE4 isozymes (A-D) was confirmed using immunohistochemistry on 5 male cadaveric urethras. Human urethral fibroblasts (FBs) were cultured from healthy control urethras of patients undergoing vaginoplasty (n=3) and from idiopathic bulbar urethral strictures (L2S1E2) of patients undergoing urethroplasty (n=3). Epithelial cells (ECs) were cultured from a healthy control urethra and two urethral strictures. To investigate a model of aUSD, Control FBs were stimulated with TGF{beta}1 and compared to Stricture FBs on assays of cell proliferation and expression of genes relevant to aUSD pathophysiology. To test therapeutics, Stricture FBs were treated with the PDE4 inhibitor, roflumilast, testosterone (T), or paclitaxel and compared to Control FBs on the previously mentioned assays and cell viability. ResultsPDE4- A, B, and D were detected along the length of the urethra. Expression levels did not differ between urethral regions. TGF{beta}1 altered proliferation and gene expression in a dose-dependent manner. Roflumilast and T preserved cell viability and proliferation and decreased expression of genes positively associated with auSD. ConclusionUrethral FBs and ECs can be cultured from healthy and strictured surgical specimens, enabling in vitro research. PDE4 inhibitors and T may be non-cytotoxic alternatives or additions to paclitaxel for aUSD. HighlightsO_LIPDE4 isozymes A, B, and D are expressed in adult anterior urethras C_LIO_LIPDE4 is expressed equally from proximal bulbar to meatal urethra C_LIO_LIEpithelial cells and fibroblasts can be cultured from healthy and stricture urethra C_LIO_LITGF{beta}1 may not be an optimal method to model aUSD in vitro C_LIO_LIUnlike paclitaxel, roflumilast and testosterone are not toxic to urethral cells C_LI

Phthalates are pervasive endocrine-disrupting chemicals widely used in consumer products. The wide use of many phthalates results in chronic human exposure to complex mixtures rather than single compounds. Despite extensive studies on individual compounds, the combined effects of phthalate metabolites on oogenesis remain poorly understood. Here, we developed a precise microinjection-based single-oocyte toxicological assay to examine the impact of a defined phthalate metabolite mixture on meiotic progression. Phthalate mixture exposure markedly impaired oocyte maturation, as most oocytes failed to extrude the first polar body. Mechanistic analyses revealed severe meiotic defects, including disrupted spindle morphology, chromosome misalignment, disorganized actin cytoskeleton, and impaired mitochondrial function, accompanied by excessive reactive oxygen species (ROS) accumulation and DNA damage. Single-cell transcriptomic profiling further identified differentially expressed genes enriched in biological processes related to exocytosis, secretory pathway regulation, and cytoskeletal organization, as well as in MAPK, JAK-STAT, cGMP-PKG, and GnRH signaling pathways that are essential for follicular development and oocyte maturation. Together, these findings demonstrate that combined phthalate exposure directly compromises female gamete quality and underscore the importance of evaluating mixture effects when assessing risks to womens reproductive health.

The anticancer efficacy of radiotherapy (RT) is limited by acquired radioresistance (RR). Here, we aim to characterize prolonged responses of breast carcinoma cells to hypofractionated irradiation (hFI). To this end, murine mammary 4T1 tumor cells (4T1WT) were subjected to a clinically oriented hFI protocol (56 Gy cumulative dose) to select radioresistant cells in vitro (4T1RS). Furthermore, hFI of subcutaneously growing 4T1CTR tumors (hFI; 24 Gy cumulative dose) was performed to radioselected 4T1IR cells in vivo. Following single irradiation in vitro, radioselected 4T1RS cells revealed increased proliferation, attenuated G2/M arrest and reduced apoptosis as compared to parental 4T1WT cells. Moreover, 4T1RS cells showed increased expression of DNA-damage response (DDR)-related proteins (pKAP1, pCHK2, {gamma}H2AX) and improved DSB repair efficiency as demonstrated by nuclear {gamma}H2AX foci analyses. The mRNA expression of factors regulating cell cycle progression, DDR, apoptosis and oxidative stress was substantially different between both cell variants in vitro. Ten days after hFI of in vivo growing tumors, residual DNA damage and apoptosis were increased in the radioselected 4T1IR tumors, whereas proliferation was reduced as compared to non-irradiated 4T1CTR control tumors. Both irradiated and non-irradiated tumors revealed complex differences in the mRNA expression profile of susceptibility- and metastasis related genes, including GADD45a, DUSP1, CDKN1a and NQO1 as well as CD44 and Rho-related factors, respectively. Moreover, hFI stimulated the infiltration of MPO-positive immune cells into tumor tissue while the presence of CD3-positive cells was reduced in the tumor area. In addition, hFI in vivo resulted in a dysregulated mRNA expression of various immune cell markers, Rho-regulatory factors, tissue remodeling molecules and cell adhesion factors. Summarizing, we identified long-lasting adaptive changes following hFI in vitro and in vivo that are associated with DNA replication, DNA repair, senescence and apoptosis as well as immune cell infiltration and tissue remodeling.

Transcriptional bursts regulate gene expression by altering burst size or burst frequency. Here, we present a protocol that integrates fixed-cell smFISH and live-cell single-molecule imaging to analyze estrogen-responsive transcriptional bursting of the TFF1 gene in human breast cancer cell lines. This workflow enables measurement of burst size, burst initiation, and active allele frequency to determine how endocrine disruptor chemicals modulate transcriptional bursting dynamics. For complete details on the use and execution of this protocol, please refer to Day, Yasar et al.1

In the terminal segment of the seminiferous tubules, SOX17 expression in the rete testis (RT) epithelium plays a crucial role in the formation of the Sertoli valve (SV), as revealed by phenotypic analyses of RT-specific Sox17 conditional knockout (cKO) mouse testes. In these RT-specific Sox17 cKO testes, SV disruption leads to the backflow of RT fluid into the seminiferous tubules, resulting in defective spermiogenesis and male infertility. Although valve deformation in the Sox17 cKO testes is likely caused indirectly by impaired downstream actions of Sox17 in the RT, the mechanisms by which SOX17 in RT influences SV formation in the seminiferous tubules remain unclear. To address this, we generated a novel AMH-Sox17 transgenic (Tg) mouse line carrying a human AMH promoter-driven Sox17 cDNA cassette. We analyzed the phenotypes of the Sertoli valve and spermatogenesis in AMH-Sox17 Tg mice, as well as in RT-specific Sox17 cKO; AMH-Sox17 Tg double mutant mice. Ectopic SOX17 (SOX17+) expression in Sertoli cells resulted in excessive Sertoli valve structures with acetylated tubulin bundles in the terminal segment of the AMH-Sox17 Tg testes, along with enhanced WNT4/RSPO1 signaling, suggesting the enhanced valve formation of ectopic SOX17+ Sertoli cells by themselves. Moreover, the AMH-Sox17 Tg could partially rescue the SV deformation and infertility in RT-specific Sox17 cKO mice, leading to proper SV formation, normal spermiogenesis and a partial recovery of male fertility in AMH-Sox17 Tg; RT-specific Sox17 cKO double mutant mice. These findings genetically demonstrate that ectopic SOX17+ Sertoli cells can compensate for SOX17 paracrine signaling in the RT, underscoring a key shared downstream pathway between RT and SV. Summary statementThe paracrine actions downstream of ectopic SOX17 expression in the Sertoli cells not only promote the valve formation, but also partially rescue the defective spermiogenesis of the rete testis-specific Sox17-null mice.

Severe salivary gland injury can cause chronic xerostomia and persistent secretory dysfunction, yet the epithelial populations that support repair remain poorly defined. Here, we identify NGFR/Ngfr as a conserved surface marker for isolating organoid-forming epithelial stem/progenitor cells from human and mouse salivary glands and show that mouse Ngfr-lineage cells contribute to ductal-acinar regeneration after injury. Single-cell transcriptomic analysis of human salivary gland tissue identified a restricted NGFR-expressing basal duct epithelial subpopulation with progenitor-like features and early positions along inferred epithelial differentiation trajectories. Functionally, NGFR-expressing cells showed enhanced primary and secondary organoid-forming capacity, and NGFR-enriched human organoids engrafted after transplantation into injured salivary glands of immunodeficient mice. In mouse salivary glands, isolated Ngfr-expressing cells showed enriched organoid-forming activity, and Ngfr expression localized to injury-associated ductal regions after duct ligation and local inflammatory injury. Ngfr-CreERT2 lineage tracing further showed that Ngfr-lineage cells contribute to ductal and acinar compartments during post-injury regeneration. Together, these findings establish NGFR/Ngfr as a conserved surface marker for prospectively isolating basal duct epithelial stem/progenitor populations with organoid-forming activity and injury-responsive ductal-acinar regenerative potential.