ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline.

H3K9 trimethylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that in male meiosis, ATF7IP2 amasses on autosomal and X-pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X-pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global up-regulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.

PRC1 directs PRC2-H3K27me3 deposition to shield adult spermatogonial stem cells from differentiation.

Spermatogonial stem cells functionality reside in the slow-cycling and heterogeneous undifferentiated spermatogonia cell population. This pool of cells supports lifelong fertility in adult males by balancing self-renewal and differentiation to produce haploid gametes. However, the molecular mechanisms underpinning long-term stemness of undifferentiated spermatogonia during adulthood remain unclear. Here, we discover that an epigenetic regulator, Polycomb repressive complex 1 (PRC1), shields adult undifferentiated spermatogonia from differentiation, maintains slow cycling, and directs commitment to differentiation during steady-state spermatogenesis in adults. We show that PRC2-mediated H3K27me3 is an epigenetic hallmark of adult undifferentiated spermatogonia. Indeed, spermatogonial differentiation is accompanied by a global loss of H3K27me3. Disruption of PRC1 impairs global H3K27me3 deposition, leading to precocious spermatogonial differentiation. Therefore, PRC1 directs PRC2-H3K27me3 deposition to maintain the self-renewing state of undifferentiated spermatogonia. Importantly, in contrast to its role in other tissue stem cells, PRC1 negatively regulates the cell cycle to maintain slow cycling of undifferentiated spermatogonia. Our findings have implications for how epigenetic regulators can be tuned to regulate the stem cell potential, cell cycle and differentiation to ensure lifelong fertility in adult males.

Epigenetic programming in the ovarian reserve.

The ovarian reserve defines female reproductive lifespan, which in humans spans decades. The ovarian reserve consists of oocytes residing in primordial follicles arrested in meiotic prophase I and is maintained independent of DNA replication and cell proliferation, thereby lacking stem cell-based maintenance. Largely unknown is how cellular states of the ovarian reserve are established and maintained for decades. Our recent study revealed that a distinct chromatin state is established during ovarian reserve formation in mice, uncovering a novel window of epigenetic programming in female germline development. We showed that an epigenetic regulator, Polycomb Repressive Complex 1 (PRC1), establishes a repressive chromatin state in perinatal mouse oocytes that is essential for prophase I-arrested oocytes to form the ovarian reserve. Here we discuss the biological roles and mechanisms underlying epigenetic programming in ovarian reserve formation, highlighting current knowledge gaps and emerging research areas in female reproductive biology.

Cytokine-armed vaccinia virus promotes cytotoxicity toward pancreatic carcinoma cells via activation of human intermediary CD56dimCD16dim natural killer cells.

Pancreatic ductal adenocarcinoma (PDAC) remains a particularly aggressive disease with few effective treatments. The PDAC tumor immune microenvironment (TIME) is known to be immune suppressive. Oncolytic viruses can increase tumor immunogenicity via immunogenic cell death (ICD). We focused on tumor-selective (vvDD) and cytokine-armed Western-reserve vaccinia viruses (vvDD-IL2 and vvDD-IL15) and infected carcinoma cell lines as well as patient-derived primary PDAC cells. In co-culture experiments, we investigated the cytotoxic response and the activation of human natural killer (NK). Infection and virus replication were assessed by measuring virus encoded YFP. We then analyzed intracellular signaling processes and oncolysis via in-depth proteomic analysis, immunoblotting and TUNEL assay. Following the co-culture of mock or virus infected carcinoma cell lines with allogenic PBMCs or NK cell lines, CD56+ NK cells were analyzed with respect to their activation, cytotoxicity and effector function. Both, dose- and time-dependent release of danger signals following infection were measured. Viruses effectively entered PDAC cells, emitted YFP signals and resulted in concomitant oncolysis. The proteome showed reprogramming of normally active core signaling pathways in PDAC (e.g., MAPK-ERK signaling). Danger-associated molecular patterns were released upon infection and stimulated co-cultured NK cells for enhanced effector cytotoxicity. NK cell subtyping revealed enhanced numbers and activation of a rare CD56dimCD16dim population. Tumor cell killing was primarily triggered via Fas ligands rather than granule release, resulting in marked apoptosis. Overall, the cytokine-armed vaccinia viruses induced NK cell activation and enhanced cytotoxicity toward human PDAC cells in vitro. We could show that cytokine-armed virus targets the carcinoma cells and thus has great potential to modulate the TIME in PDAC.

The chromatin remodeler CHD8 governs hematopoietic stem/progenitor survival by regulating ATM-mediated P53 protein stability.

The Chd8 gene encodes a member of the chromodomain helicase DNA-binding (CHD) family of SNF2H-like adenosine triphosphate (ATP)-dependent chromatin remodeler, the mutations of which define a subtype of autism spectrum disorders. Increasing evidence from recent studies indicates that ATP-dependent chromatin-remodeling genes are involved in the control of crucial gene-expression programs in hematopoietic stem/progenitor cell (HSPC) regulation. In this study, we identified CHD8 as a specific and essential regulator of normal hematopoiesis. Loss of Chd8 leads to severe anemia, pancytopenia, bone marrow failure, and engraftment failure related to a drastic depletion of HSPCs. CHD8 forms a complex with ATM and its deficiency increases chromatin accessibility and drives genomic instability in HSPCs causing an activation of ATM kinase that further stabilizes P53 protein by phosphorylation and leads to increased HSPC apoptosis. Deletion of P53 rescues the apoptotic defects of HSPCs and restores overall hematopoiesis in Chd8-/- mice. Our findings demonstrate that chromatin organization by CHD8 is uniquely necessary for the maintenance of hematopoiesis by integrating the ATM-P53-mediated survival of HSPCs.

Internal drainage for interdisciplinary management of anastomotic leakage after pancreaticogastrostomy.

BACKGROUND: Anastomotic leakage and postoperative pancreatic fistula (POPF) may occur after pancreatic head resection, also in the setting of pancreato-gastric reconstruction. For adequate complication management, a variety of non-standardized treatments are available. Still, data on clinical evaluation of endoscopic methods remain scarce. Based on our interdisciplinary experience on endoscopic treatment of retro-gastric fluid collections after left-sided pancreatectomies, we developed an innovative endoscopic concept with internal peri-anastomotic stent placement for patients with anastomotic leakage and/or peri-anastomotic fluid collection. METHODS: Over the period of 6 years (2015-2020) we retrospectively evaluated 531 patients after pancreatic head resections at the Department of Surgery, Charité-Unversitätsmedizin Berlin. Of these, 403 received reconstruction via pancreatogastrostomy. We identified 110 patients (27.3%) with anastomotic leakage and/or peri-anastomotic fluid collection and could define four treatment groups which received either conservative treatment (C), percutaneous drainage (PD), endoscopic drainage (ED), and/or re-operation (OP). Patients were grouped in a step-up approach for descriptive analyses and in a stratified, decision-based algorithm for comparative analyses. The study's primary endpoints were hospitalization (length of hospital stay) and clinical success (treatment success rate, primary/secondary resolution). RESULTS: We characterized an institutional, post-operative cohort with heterogenous complication management following pancreato-gastric reconstruction. The majority of patients needed interventional treatments (n = 92, 83.6%). Of these, close to one-third (n = 32, 29.1%) were treated with endoscopy-guided, peri-anastomotic pigtail stents for internal drainage as either primary, secondary and/or tertiary treatment modality. Following a decision-based algorithm, we could discriminate superior primary-(77,8% vs 53.7%) and secondary success rates (85.7% vs 68.4%) as well as earlier primary resolutions (11.4 days, 95%CI (5.75-17.13) vs 37.4 days, 95%CI (27.2-47.5)] in patients receiving an endoscopic compared to percutaneous management. CONCLUSION: This study underscores the importance of endoscopy-guided approaches for adequate treatment of anastomotic leakage and/or peri-anastomotic fluid collections after pancreatoduodenectomy. We herein report a novel, interdisciplinary concept for internal drainage in the setting of pancreato-gastric reconstruction.

Relationship of arterial tonometry and exercise in patients with chronic heart failure: a systematic review with meta-analysis and trial sequential analysis.

BACKGROUND: Arterial stiffness is a common characteristic in patients with chronic heart failure (CHF), and arterial tonometric technologies related to arterial stiffness are novel and effective methods and have an important value in the diagnosis and prognosis of CHF. In terms of ameliorating arterial stiffness in patients with CHF, exercise training is considered an adjuvant treatment and also an effective means in the diagnosis and judgment of prognosis. However, there are huge controversies and inconsistencies in these aspects. The objective of this meta-analysis was to systematically test the connection of arterial tonometry and exercise in patients with CHF. METHODS: Databases, including MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, were accessed from inception to 7 March 2022. The meta-analysis was then conducted, and trial sequential analysis (TSA) was performed jointly to further verify our tests and reach more convincing conclusions by using RevMan version 5.4 software, STATA version 16.0 software, and TSA version 0.9.5.10 Beta software. RESULTS: Eighteen articles were included, with a total of 876 participants satisfying the inclusion criteria. The pooling revealed that flow-mediated dilation (FMD) was lower in basal condition [standardized mean difference (SMD): - 2.28%, 95% confidence interval (CI) - 3.47 to - 1.08, P < 0.001] and improved significantly after exercise (SMD: 5.96%, 95% CI 2.81 to 9.05, P < 0.001) in patients with heart failure with reduced ejection fraction (HFrEF) compared with healthy participants. The high-intensity training exercise was more beneficial (SMD: 2.88%, 95% CI 1.78 to 3.97, P < 0.001) than the moderate-intensity training exercise to improve FMD in patients with CHF. For augmentation index (AIx), our study indicated no significant differences (SMD: 0.50%, 95% CI - 0.05 to 1.05, P = 0.074) in patients with heart failure with preserved ejection fraction (HFpEF) compared with healthy participants. However, other outcomes of our study were not identified after further verification using TSA, and more high-quality studies are needed to reach definitive conclusions in the future. CONCLUSIONS: This review shows that FMD is lower in basal condition and improves significantly after exercise in patients with HFrEF compared with healthy population; high-intensity training exercise is more beneficial than moderate-intensity training exercise to improve FMD in patients with CHF; besides, there are no significant differences in AIx in patients with HFpEF compared with the healthy population. More high-quality studies on this topic are warranted.

An Individualized EMT-Related Gene Signature to Predict Recurrence-Free Survival in Stage II/III Colorectal Cancer Patients.

BACKGROUND: Approximately 30% of stage II and 50-60% of stage III colorectal cancer (CRC) patients who have undergone surgery will develop recurrence within 5 years. Thus, more reliable prognostic biomarkers are urgently needed to identify the high-risk subset of patients who will benefit from postoperative adjuvant therapy. METHODS: We retrospectively analyzed 911 stage II/III CRC patients in multiple cohorts. Using a series of bioinformatic and statistical approaches, an individualized prognostic signature was established in the training cohort and validated in four other independent cohorts. An integrated decision tree was generated to improve risk stratification, and a nomogram was built to quantify risk assessment for individual patients. RESULTS: Epithelial-mesenchymal transition (EMT) was identified as a dominant risk factor for recurrence-free survival (RFS) in stage II/III CRC patients. The EMT-related gene signature could discriminate high-risk subsets in a training cohort and four independent validation cohorts (with 473, 89, 130, 74 and 145 patients, respectively). Survival analyses demonstrated that the EMT-related gene signature served as an independent risk factor for RFS in different subgroups. The decision tree could optimize the risk stratification, and the nomogram could predict the 5-year RFS probability accurately. CONCLUSION: The proposed EMT-related prognostic signature is a useful biomarker to predict RFS and identify the high-risk subset in stage II/III CRC patients.

HGF/c-MET pathway contributes to cisplatin-mediated PD-L1 expression in hepatocellular carcinoma.

Cisplatin has been reported to promote the expression of programmed cell death ligand-1 (PD-L1) in some cancer cells. However, the underlying mechanism through which PD-L1 is transcriptionally regulated by cisplatin in hepatocellular carcinoma (HCC) cells remains largely unknown. In the present study, we found that the expression of hepatocyte growth factor (HGF), p-Akt, p-ERK, and PD-L1 was increased in cisplatin-treated SNU-368 and SNU-739 cells. HGF stimulation also increased PD-L1 expression in these cells. Moreover, Inhibition of HGF/c-MET, PI3K/Akt, and MEK/ERK signaling pathways can dramatically block cisplatin or HGF-induced PD-L1 expression in SNU-368 and SNU-739 cells. In vivo, combination PHA665752 with cisplatin significantly reduced tumor weight with increased infiltration of CD8+ T cells in the tumor. Taken together, our study suggested that HGF/c-Met axis-induced the activation of PI3K/Akt and MEK/ERK pathways contributes to cisplatin-mediated PD-L1 expression. These findings may provide an alternative avenue for the treatment of HCC.

Protein Phosphatase 6 Protects Prophase I-Arrested Oocytes by Safeguarding Genomic Integrity.

Mammalian oocytes are arrested at prophase of the first meiotic division in the primordial follicle pool for months, even years, after birth depending on species, and only a limited number of oocytes resume meiosis, complete maturation, and ovulate with each reproductive cycle. We recently reported that protein phosphatase 6 (PP6), a member of the PP2A-like subfamily, which accounts for cellular serine/threonine phosphatase activity, functions in completing the second meiosis. Here, we generated mutant mice with a specific deletion of Ppp6c in oocytes from the primordial follicle stage by crossing Ppp6cF/F mice with Gdf9-Cre mice and found that Ppp6cF/F; GCre+ mice are infertile. Depletion of PP6c caused folliculogenesis defects and germ cell loss independent of the traditional AKT/mTOR pathway, but due to persistent phosphorylation of H2AX (a marker of double strand breaks), increased susceptibility to DNA damage and defective DNA repair, which led to massive oocyte elimination and eventually premature ovarian failure (POF). Our findings uncover an important role for PP6 as an indispensable guardian of genomic integrity of the lengthy prophase I oocyte arrest, maintenance of primordial follicle pool, and thus female fertility.

Loss of protein phosphatase 6 in oocytes causes failure of meiosis II exit and impaired female fertility.

Dynamic protein phosphorylation and dephosphorylation, mediated by a conserved cohort of protein kinases and phosphatases, regulate cell cycle progression. Among the well-known PP2A-like protein phosphatases, protein phosphatase 6 (PP6) has been analyzed in mammalian mitosis, and Aurora A has recently been identified as its key substrate. However, the functions of PP6 in meiosis are still entirely unknown. To identify the physiological role of PP6 in female gametogenesis, Ppp6c(F/F) mice were first generated and crossed with Zp3-Cre mice to selectively disrupt Ppp6c expression in oocytes. Here, we report for the first time that PP6c is dispensable for oocyte meiotic maturation but essential for exit from meiosis II (MII) after fertilization. Depletion of PP6c caused an abnormal MII spindle and disrupted MII cytokinesis, resulting in zygotes with high risk of aneuploidy and defective early embryonic development, and thus severe subfertility. We also reveal that PP6 inactivation interferes with MII spindle formation and MII exit owing to increased Aurora A activity, and that Aurora A inhibition with MLN8237 can rescue the PP6c depletion phenotype. In conclusion, our findings uncover a hitherto unknown role for PP6 as an indispensable regulator of oocyte meiosis and female fertility.

Oocyte-specific deletion of N-WASP does not affect oocyte polarity, but causes failure of meiosis II completion.

STUDY QUESTION: There is an unexplored physiological role of N-WASP (neural Wiskott-Aldrich syndrome protein) in oocyte maturation that prevents completion of second meiosis. SUMMARY ANSWER: In mice, N-WASP deletion did not affect oocyte polarity and asymmetric meiotic division in first meiosis, but did impair midbody formation and second meiosis completion. WHAT IS KNOWN ALREADY: N-WASP regulates actin dynamics and participates in various cell activities through the RHO-GTPase-Arp2/3 (actin-related protein 2/3 complex) pathway, and specifically the Cdc42 (cell division cycle 42)-N-WASP-Arp2/3 pathway. Differences in the functions of Cdc42 have been obtained from in vitro compared to in vivo studies. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: By conditional knockout of N-WASP in mouse oocytes, we analyzed its in vivo functions by employing a variety of different methods including oocyte culture, immunofluorescent staining and live oocyte imaging. Each experiment was repeated at least three times, and data were analyzed by paired-samples t-test. MAIN RESULTS AND THE ROLE OF CHANCE: Oocyte-specific deletion of N-WASP did not affect the process of oocyte maturation including spindle formation, spindle migration, polarity establishment and maintenance, and homologous chromosome or sister chromatid segregation, but caused failure of cytokinesis completion during second meiosis (P < 0.001 compared to control). Further analysis showed that a defective midbody may be responsible for the failure of cytokinesis completion. LIMITATIONS, REASONS FOR CAUTION: The present study did not include a detailed analysis of the mechanisms underlying the results, which will require more extensive further investigations. WIDER IMPLICATIONS OF THE FINDINGS: N-WASP may play an important role in mediating and co-ordinating the activity of the spindle (midbody) and actin (contractile ring constriction) when cell division occurs. The findings are important for understanding the regulation of oocyte meiosis completion and failures in this process that affect oocyte quality. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by the National Basic Research Program of China (No. 2012CB944404) and the National Natural Science Foundation of China (Nos 30930065, 31371451, 31272260 and 31530049). There are no potential conflicts of interests.

Overexpression of SETß, a protein localizing to centromeres, causes precocious separation of chromatids during the first meiosis of mouse oocytes.

Chromosome segregation in mammalian oocyte meiosis is an error-prone process, and any mistake in this process may result in aneuploidy, which is the main cause of infertility, abortion and many genetic diseases. It is now well known that shugoshin and protein phosphatase 2A (PP2A) play important roles in the protection of centromeric cohesion during the first meiosis. PP2A can antagonize the phosphorylation of rec8, a member of the cohesin complex, at the centromeres and thus prevent cleavage of rec8 and so maintain the cohesion of chromatids. SETß is a protein that physically interacts with shugoshin and inhibits PP2A activity. We thus hypothesized that SETß might regulate cohesion protection and chromosome segregation during oocyte meiotic maturation. Here we report for the first time the expression, subcellular localization and functions of SETß during mouse oocyte meiosis. Immunoblotting analysis showed that the expression level of SETß was stable from the germinal vesicle stage to the MII stage of oocyte meiosis. Immunofluorescence analysis showed SETß accumulation in the nucleus at the germinal vesicle stage, whereas it was targeted mainly to the inner centromere area and faintly localized to the interchromatid axes from germinal vesicle breakdown to MI stages. At the MII stage, SETß still localized to the inner centromere area, but could relocalize to kinetochores in a process perhaps dependent on the tension on the centromeres. SETß partly colocalized with PP2A at the inner centromere area. Overexpression of SETß in mouse oocytes caused precocious separation of sister chromatids, but depletion of SETß by RNAi showed little effects on the meiotic maturation process. Taken together, our results suggest that SETß, even though it localizes to centromeres, might not be essential for chromosome separation during mouse oocyte meiotic maturation, although its forced overexpression causes premature chromatid separation.

Deletion of Mylk1 in oocytes causes delayed morula-to-blastocyst transition and reduced fertility without affecting folliculogenesis and oocyte maturation in mice.

The mammalian oocyte undergoes two rounds of asymmetric cell divisions during meiotic maturation and fertilization. Acentric spindle positioning and cortical polarity are two major factors involved in asymmetric cell division, both of which are thought to depend on the dynamic interaction between myosin II and actin filaments. Myosin light chain kinase (MLCK), encoded by the Mylk1 gene, could directly phosphorylate and activate myosin II. To determine whether MLCK was required for oocyte asymmetric division, we specifically disrupted the Mylk1 gene in oocytes by Cre-loxP conditional knockout system. We found that Mylk1 mutant female mice showed severe subfertility. Unexpectedly, contrary to previously reported in vitro findings, our data showed that oocyte meiotic maturation including spindle organization, polarity establishment, homologous chromosomes separation, and polar body extrusion were not affected in Mylk1(fl/fl);GCre(+) females. Follicular development, ovulation, and early embryonic development up to compact morula occurred normally in Mylk1(fl/fl);GCre(+) females, but deletion of MLCK caused delayed morula-to-blastocyst transition. More than a third of embryos were at morula stage at 3.5 Days Postcoitum in vivo. The delayed embryos could develop further to early blastocyst stage in vitro on Day 4 when most control embryos reached expanded blastocysts. Our findings provide evidence that MLCK is linked to timely blastocyst formation, though it is dispensable for oocyte meiotic maturation.

Scaffold subunit Aalpha of PP2A is essential for female meiosis and fertility in mice.

Ppp2r1a encodes the scaffold subunit Aalpha of protein phosphatase 2A (PP2A), which is an important and ubiquitously expressed serine threonine phosphatase family and plays a critical role in many fundamental cellular processes. To identify the physiological role of PP2A in female germ cell meiosis, we selectively disrupted Ppp2r1a expression in oocytes by using the Cre-Loxp conditional knockout system. Here we report for the first time that oocyte-specific deletion of Ppp2r1a led to severe female subfertility without affecting follicle survival, growth, and ovulation. PP2A-Aalpha was essential for regulating oocyte meiotic maturation because depletion of PP2A-Aalpha facilitated germinal vesicle breakdown, causing elongation of the MII spindle and precocious separation of sister chromatids. The resulting eggs had high risk of aneuploidy, though they could be fertilized, leading to defective embryonic development and thus subfertility. Our findings provide strong evidence that PP2A-Aalpha within the oocyte plays an indispensable role in oocyte meiotic maturation, though it is dispensable for folliculogenesis in the mouse ovary.

Chromatin remodeler CHD4 establishes chromatin states required for ovarian reserve formation, maintenance, and germ cell survival.

The ovarian reserve defines female reproductive lifespan, which in humans spans decades due to the maintenance of meiotic arrest in non-growing oocytes (NGO) residing in primordial follicles. Unknown is how the chromatin state of NGOs is established to enable long-term maintenance of the ovarian reserve. Here, we show that a chromatin remodeler, CHD4, a member of the Nucleosome Remodeling and Deacetylase (NuRD) complex, establishes chromatin states required for formation and maintenance of the ovarian reserve. Conditional loss of CHD4 in perinatal mouse oocytes results in acute death of NGOs and depletion of the ovarian reserve. CHD4 establishes closed chromatin at regulatory elements of pro-apoptotic genes to prevent cell death and at specific genes required for meiotic prophase I to facilitate the transition from meiotic prophase I oocytes to meiotic arrested NGOs. In addition, CHD4 establishes closed chromatin at the regulatory elements of pro-apoptotic genes in male germ cells, allowing male germ cell survival. These results demonstrate a role for CHD4 in defining a chromatin state that ensures germ cell survival, thereby enabling the long-term maintenance of both female and male germ cells.

Clinical prognosticators and targets in the immune microenvironment of intrahepatic cholangiocarcinoma.

Background: Intrahepatic cholangiocarcinoma (ICC) is a disease with poor prognosis and limited therapeutic options. We investigated the tumor immune microenvironment (TIME) to identify predictors of disease outcome and to explore targets for therapeutic modulation. Methods: Liver tissue samples were collected during 2008-2019 from patients (n = 139) diagnosed with ICC who underwent curative intent surgery without neoadjuvant chemotherapy. Samples from the discovery cohort (n = 86) were immunohistochemically analyzed on tissue microarrays (TMAs) for the expression of CD68, CD3, CD4, CD8, Foxp3, PD-L1, STAT1, and p-STAT1 in tumor core and stroma areas. Results were digitally analyzed using QuPath software and correlated with clinicopathological characteristics. For validation of TIME-related biomarkers, we performed multiplex imaging mass cytometry (IMC) in a validation cohort (n = 53). Results: CD68+ cells were the predominant immune cell type in the TIME of ICC. CD4+high T cell density correlated with better overall survival (OS). Prediction modeling together with validation cohort confirmed relevance of CD4+ cells, PD-L1 expression by immune cells in the stroma and N-stage on overall disease outcome. In turn, IMC analyses revealed that silent CD3+CD4+ clusters inversely impacted survival. Among annotated immune cell clusters, PD-L1 was most relevantly expressed by CD4+FoxP3+ cells. A subset of tumors with high density of immune cells ("hot" cluster) correlated with PD-L1 expression and could identify a group of candidates for immune checkpoint inhibition (ICI). Ultimately, higher levels of STAT1 expression were associated with higher lymphocyte infiltration and PD-L1 expression. Conclusions: These results highlight the importance of CD4+ T cells in immune response against ICC. Secondly, a subset of tumors with "hot" TIME represents potential candidates for ICI, while stimulation of STAT1 pathway could be a potential target to turn "cold" into "hot" TIME in ICC.

The tumor immune microenvironment (TIME) plays a critical role in the immune response In many cancers, including intrahepatic cholangiocarcinoma (ICC). Molecular subtyping of the ICC microenvironment already revealed inter-tumoral heterogeneity with variant profiles of immune cell infiltrates. A recent study created an in-depth immune cell atlas of the TIME in biliary tract cancers and could demonstrate the relevance of specific immune cell subpopulations on patient outcome. We are able to provide a distinctive characterization of TIME, separating tumor epithelial- and stroma areas, in a large and representative ICC cohort using digitalized image analysis on tissue microarrays (TMA) as well as multiplex imaging mass cytometry (IMC). The study was designed for identification of immune cell prognosticators allocating institutional ICC patients into a discovery (2008­15) and a validation (2010­19) cohort. Immune cell subpopulations were correlated with clinicopathological characteristics and patient outcome. Our results highlight: i. The important role of CD4+ T cell infiltration in ICC patients; ii. ICC tumors with high density of immune cells associated with PD-L1 expression identifies a subset of patients with variant tumor biology; iii. Stimulation of STAT1 pathway may be a relevant target to turn "cold" into "hot" tumors.

PRC1 suppresses a female gene regulatory network to ensure testicular differentiation.

Gonadal sex determination and differentiation are controlled by somatic support cells of testes (Sertoli cells) and ovaries (granulosa cells). In testes, the epigenetic mechanism that maintains chromatin states responsible for suppressing female sexual differentiation remains unclear. Here, we show that Polycomb repressive complex 1 (PRC1) suppresses a female gene regulatory network in postnatal Sertoli cells. We genetically disrupted PRC1 function in embryonic Sertoli cells after sex determination, and we found that PRC1-depleted postnatal Sertoli cells exhibited defective proliferation and cell death, leading to the degeneration of adult testes. In adult Sertoli cells, PRC1 suppressed specific genes required for granulosa cells, thereby inactivating the female gene regulatory network. Chromatin regions associated with female-specific genes were marked by Polycomb-mediated repressive modifications: PRC1-mediated H2AK119ub and PRC2-mediated H3K27me3. Taken together, this study identifies a critical Polycomb-based mechanism that suppresses ovarian differentiation and maintains Sertoli cell fate in adult testes.

Correction: Felsenstein M. et al. Perineural Invasion in Pancreatic Ductal Adenocarcinoma (PDAC): A Saboteur of Curative Intended Therapies? J. Clin. Med. 2022, 11, 2367.

In the original publication [...].

ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline.

H3K9 tri-methylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that, in male meiosis, ATF7IP2 amasses on autosomal and X pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase I. Analyses of ATF7IP2-deficient meiosis reveal the protein's essential roles in the maintenance of MSCI, suppression of retrotransposons, and global upregulation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.