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The cardiac conduction system (CCS) is a network of specialized cardiomyocytes that coordinates electrical impulse generation and propagation for synchronized heart contractions. Although the components of the CCS, including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers, were anatomically discovered more than 100 years ago, their molecular constituents and regulatory mechanisms remain incompletely understood. Here, we demonstrate the transcriptomic landscape of the postnatal mouse CCS at a single-cell resolution with spatial information. Integration of single-cell and spatial transcriptomics uncover region-specific markers and zonation patterns of expression. Network inference shows heterogeneous gene regulatory networks across the CCS. Notably, region-specific gene regulation is recapitulated in vitro using neonatal mouse atrial and ventricular myocytes overexpressing CCS-specific transcription factors, Tbx3 and/or Irx3. This finding is supported by ATAC-seq of different CCS regions, Tbx3 ChIP-seq, and Irx motifs. Overall, this study provides comprehensive molecular profiles of the postnatal CCS and elucidates gene regulatory mechanisms contributing to its heterogeneity.
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Sistema de Conducción Cardíaco , Proteínas de Homeodominio , Miocitos Cardíacos , Proteínas de Dominio T Box , Animales , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Sistema de Conducción Cardíaco/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Redes Reguladoras de Genes , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Regulación de la Expresión Génica , Animales Recién Nacidos , Análisis de la Célula Individual , Transcriptoma , Ramos Subendocárdicos/metabolismo , Ramos Subendocárdicos/fisiología , Nodo Atrioventricular/metabolismo , Nodo Sinoatrial/metabolismo , Fascículo Atrioventricular/metabolismoRESUMEN
High-grade serous ovarian carcinoma (HGSC) remains a disease of poor prognosis that is unresponsive to current immune checkpoint inhibitors. Although PI3K pathway alterations, such as PTEN loss, are common in HGSC, attempts to target this pathway have been unsuccessful. We hypothesized that aberrant PI3K pathway activation may alter the HGSC immune microenvironment and present a targeting opportunity. Single-cell RNA sequencing identified populations of resident macrophages specific to Pten-null omental tumors in murine models, which were confirmed by flow cytometry. These macrophages derived from peritoneal fluid macrophages and had a unique gene expression program, marked by high expression of the enzyme heme oxygenase-1 (HMOX1). Targeting resident peritoneal macrophages prevented the appearance of HMOX1hi macrophages and reduced tumor growth. Furthermore, direct inhibition of HMOX1 extended survival in vivo. RNA sequencing identified IL33 in Pten-null tumor cells as a likely candidate driver leading to the appearance of HMOX1hi macrophages. Human HGSC tumors also contained HMOX1hi macrophages with a corresponding gene expression program. Moreover, the presence of these macrophages correlated with activated tumoral PI3K/mTOR signaling and poor overall survival in HGSC patients. In contrast, tumors with low numbers of HMOX1hi macrophages were marked by increased adaptive immune response gene expression. These data suggest targeting HMOX1hi macrophages as a potential therapeutic strategy for treating poor prognosis HGSC.
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While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced efficacy of oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 promoted a metabolic shift characterized by enhanced glycolysis rate that resulted in increased lactate production. In turn, lactate inhibited type I IFN responses. In addition to adding mechanistic insight into PD-L1 intrinsic function, our results will also help guide the numerous ongoing efforts to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.
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Antígeno B7-H1 , Interferón Tipo I , Viroterapia Oncolítica , Virus Oncolíticos , Animales , Femenino , Humanos , Ratones , Antígeno B7-H1/metabolismo , Antígeno B7-H1/inmunología , Antígeno B7-H1/genética , Línea Celular Tumoral , Glucólisis , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Ácido Láctico/metabolismo , Neoplasias/inmunología , Neoplasias/terapia , Neoplasias/metabolismo , Viroterapia Oncolítica/métodos , Virus Oncolíticos/fisiología , Transducción de Señal , MasculinoRESUMEN
RATIONALE: The chronic lung disease bronchopulmonary dysplasia (BPD) is the most severe complication of extreme prematurity. BPD results in impaired lung alveolar and vascular development and long-term respiratory morbidity, for which only supportive therapies exist. Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) improve lung structure and function in experimental BPD. Results of clinical trials with MSCs for many disorders do not yet match the promising preclinical studies. A lack of specific criteria to define functionally distinct MSCs persists. OBJECTIVES: To determine and correlate single-cell UC-MSC transcriptomic profile with therapeutic potential. METHODS: UC-MSCs from five term donors and human neonatal dermal fibroblasts (HNDFs, control cells of mesenchymal origin) transcriptomes were investigated by single-cell RNA sequencing analysis (scRNA-seq). The lung-protective effect of UC-MSCs with a distinct transcriptome and control HNDFs was tested in vivo in hyperoxia-induced neonatal lung injury in rats. MEASUREMENTS AND MAIN RESULTS: UC-MSCs showed limited transcriptomic heterogeneity, but were different from HNDFs. Gene ontology enrichment analysis revealed distinct - progenitor-like and fibroblast-like - UC-MSC subpopulations. Only the treatment with progenitor-like UC-MSCs improved lung function and structure and attenuated pulmonary hypertension in hyperoxia-exposed rat pups. Moreover, scRNA-seq identified major histocompatibility complex class I as a molecular marker of non-therapeutic cells and associated with decreased lung retention. CONCLUSIONS: UC-MSCs with a progenitor-like transcriptome, but not with a fibroblast-like transcriptome, provide lung protection in experimental BPD. High expression of major histocompatibility complex class I is associated with reduced therapeutic benefit. scRNA-seq may be useful to identify subsets of MSCs with superior repair capacity for clinical application.
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The tumour microenvironment is infiltrated by immunosuppressive cells, such as regulatory T cells (Tregs), which contribute to tumour escape and impede immunotherapy outcomes. Soluble fibrinogen-like protein 2 (sFGL2), a Treg effector protein, inhibits immune cell populations, via receptors FcγRIIB and FcγRIII, leading to downregulation of CD86 in antigen presenting cells and limiting T cell activation. Increased FGL2 expression is associated with tumour progression and poor survival in several different cancers, such as glioblastoma multiforme, lung, renal, liver, colorectal, and prostate cancer. Querying scRNA-seq human cancer data shows FGL2 is produced by cells in the tumour microenvironment (TME), particularly monocytes and macrophages as well as T cells and dendritic cells (DCs), while cancer cells have minimal expression of FGL2. We studied the role of FGL2 exclusively produced by cells in the TME, by leveraging Fgl2 knockout mice. We tested two murine models of cancer in which the role of FGL2 has not been previously studied: epithelial ovarian cancer and melanoma. We show that absence of FGL2 leads to a more activated TME, including activated DCs (CD86+, CD40+) and T cells (CD25+, TIGIT+), as well as demonstrating for the first time that the absence of FGL2 leads to more activated natural killer cells (DNAM-1+, NKG2D+) in the TME. Furthermore, the absence of FGL2 leads to prolonged survival in the B16F10 melanoma model, while the absence of FGL2 synergizes with oncolytic virus to prolong survival in the ID8-p53-/-Brca2-/- ovarian cancer model. In conclusion, targeting FGL2 is a promising cancer treatment strategy alone and in combination immunotherapies.
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Fibrinógeno , Melanoma , Neoplasias Ováricas , Animales , Femenino , Humanos , Ratones , Células Presentadoras de Antígenos , Carcinoma Epitelial de Ovario , Melanoma/genética , Melanoma/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Microambiente TumoralRESUMEN
Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer. The disease is often diagnosed after wide-spread dissemination, and the standard treatment combines aggressive surgery with platinum-based chemotherapy; however, most patients experience relapse in the form of peritoneal carcinomatosis, resulting in a 5-year mortality below 45%. There is clearly a need for the development of novel treatments and cancer immunotherapies offering a different approach. Immunotherapies have demonstrated their efficacy in many types of cancers; however, only <15% of EOC patients show any evidence of response. One of the main barriers behind the poor therapeutic outcome is the reduced expression of Major Histocompatibility Complexes class I (MHC I) which occurs in approximately 60% of EOC cases. This review aims to gather and enhance our current understanding of EOC, focusing on its distinct cancer characteristics related to MHC I expression, immunogenicity, antigen presentation, epithelial-to-mesenchymal transition, and various ongoing immunotherapeutic strategies designed to stimulate antitumor immunity.
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Cancer cells often metastasize by undergoing an epithelial-mesenchymal transition (EMT). Although abundance of CD8+ T-cells in the tumor microenvironment correlates with improved survival, mesenchymal cancer cells acquire greater resistance to antitumor immunity in some cancers. We hypothesized the EMT modulates the immune response to ovarian cancer. Here we show that cancer cells from infiltrated/inflamed tumors possess more mesenchymal cells, than excluded and desert tumors. We also noted high expression of LGALS3 is associated with EMT in vivo, a finding validated with in vitro EMT models. Dissecting the cellular communications among populations in the tumor revealed that mesenchymal cancer cells in infiltrated tumors communicate through LGALS3 to LAG3 receptor expressed by CD8+ T cells. We found CD8+ T cells express high levels of LAG3, a marker of T cell exhaustion. The results indicate that EMT in ovarian cancer cells promotes interactions between cancer cells and T cells through the LGALS3 - LAG3 axis, which could increase T cell exhaustion in infiltrated tumors, dampening antitumor immunity.
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Linfocitos T CD8-positivos , Neoplasias Ováricas , Humanos , Femenino , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Galectina 3/genética , Galectina 3/metabolismo , Agotamiento de Células T , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Microambiente TumoralRESUMEN
Ovarian cancers exhibit high rates of recurrence and poor treatment response. Preclinical models that recapitulate human disease are critical to develop new therapeutic approaches. Syngeneic mouse models allow for the generation of tumours comprising the full repertoire of non-malignant cell types but have expanded in number, varying in the cell type of origin, method for transformation, and ultimately, the properties of the tumours they produce. Here we have performed a comparative analysis of high-grade serous ovarian cancer models based on transcriptomic profiling of 22 cell line models, and intrabursal and intraperitoneal tumours from 12. Among cell lines, we identify distinct signalling activity, such as elevated inflammatory signalling in STOSE and OVE16 models, and MAPK/ERK signalling in ID8 and OVE4 models; metabolic differences, such as reduced glycolysis-associated expression in several engineered ID8 subclones; and relevant functional properties, including differences in EMT activation, PD-L1 and MHC class I expression, and predicted chemosensitivity. Among tumour samples, we observe increased variability and stromal content among intrabursal tumours. Finally, we predict differences in the microenvironment of ID8 models engineered with clinically relevant mutations. We anticipate that this work will serve as a valuable resource, providing new insight to help select models for specific experimental objectives.
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Neoplasias Ováricas , Animales , Ratones , Humanos , Femenino , Neoplasias Ováricas/patología , Perfilación de la Expresión Génica , Transducción de Señal , Microambiente Tumoral/genéticaRESUMEN
Clinical trials are currently exploring combinations of PARP inhibitors and immunotherapies for the treatment of ovarian cancer, but their effects on the ovarian tumour microenvironment (TME) remain unclear. Here, we investigate how olaparib, PD-L1 monoclonal antibodies, and their combination can influence TME composition and survival of tumour-bearing mice. We further explored how BRCA deficiencies can influence the response to therapy. Olaparib and combination therapies similarly improved the median survival of Brca1- and Brca2-deficient tumour-bearing mice. Anti-PD-L1 monotherapy improved the survival of mice with Brca1-null tumours, but not Brca2-null tumours. A detailed analysis of the TME revealed that olaparib monotherapy resulted in a large number of immunosuppressive and immunomodulatory effects in the more inflamed Brca1-deficient TME but not Brca2-deficient tumours. Anti-PD-L1 treatment was mostly immunosuppressive, resulting in a systemic reduction of cytokines and a compensatory increase in PD-L1 expression. The results of the combination therapy generally resembled the effects of one or both of the monotherapies, along with unique changes observed in certain immune populations. In-silico analysis of RNA-seq data also revealed numerous differences between Brca-deficient tumour models, such as the expression of genes involved in inflammation, angiogenesis and PD-L1 expression. In summary, these findings shed light on the influence of novel therapeutics and BRCA mutations on the ovarian TME.
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Antígeno B7-H1 , Neoplasias Ováricas , Femenino , Humanos , Animales , Ratones , Antígeno B7-H1/genética , Microambiente Tumoral/genética , Genes BRCA2 , Proteína BRCA1/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Proteína BRCA2/genéticaRESUMEN
Many traditional cancer treatments such as radiation and chemotherapy are known to induce cellular DNA damage as part of their cytotoxic activity. The cGAS-STING signaling axis, a key member of the DNA damage response that acts as a sensor of foreign or aberrant cytosolic DNA, is helping to rationalize the DNA-damaging activity of these treatments and their emerging immunostimulatory capacity. Moreover, cGAS-STING, which is attracting considerable attention for its ability to promote antitumor immune responses, may fundamentally be able to address many of the barriers limiting the success of cancer immunotherapy strategies, including the immunosuppressive tumor microenvironment. Herein, we review the traditional cancer therapies that have been linked with cGAS-STING activation, highlighting their targets with respect to their role and function in the DNA damage response. As part of the review, an emerging "chemoimmunotherapy" concept whereby DNA-damaging agents are used for the indirect activation of STING is discussed as an alternative to the direct molecular agonism strategies that are in development, but have yet to achieve clinical approval. The potential of this approach to address some of the inherent and emerging limitations of cGAS-STING signaling in cancer immunotherapy is also discussed. Ultimately, it is becoming clear that in order to successfully employ the immunotherapeutic potential of the cGAS-STING axis, a balance between its contrasting antitumor and protumor/inflammatory activities will need to be achieved.
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Aberrant neurodevelopment is a core deficit of autism spectrum disorder (ASD). Here we ask whether a non-genetic factor, prenatal exposure to the environmental pollutant methylmercury (MeHg), is a contributing factor in ASD onset. We showed that adult mice prenatally exposed to non-apoptotic MeHg exhibited key ASD characteristics, including impaired communication, reduced sociability, and increased restrictive repetitive behaviors, whereas in the embryonic cortex, prenatal MeHg exposure caused premature neuronal differentiation. Further single-cell RNA sequencing (scRNA-seq) analysis disclosed that prenatal exposure to MeHg resulted in cortical radial glial precursors (RGPs) favoring asymmetric differentiation to directly generate cortical neurons, omitting the intermediate progenitor stage. In addition, MeHg exposure in cultured RGPs increased CREB phosphorylation and enhanced the interaction between CREB and CREB binding protein (CBP). Intriguingly, metformin, an FDA-approved drug, can reverse MeHg-induced premature neuronal differentiation via CREB/CBP repulsion. These findings provide insights into ASD etiology, its underlying mechanism, and a potential therapeutic strategy.
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Mammalian folliculogenesis is a complex process that involves the regulation of chromatin structure for gene expression and oocyte meiotic resumption. The SWI/SNF complex is a chromatin remodeler using either Brahma-regulated gene 1 (BRG1) or BRM (encoded by Smarca4 and Smarca2, respectively) as its catalytic subunit. SMARCA4 loss of expression is associated with a rare type of ovarian cancer; however, its function during folliculogenesis remains poorly understood. In this study, we describe the phenotype of BRG1 mutant mice to better understand its role in female fertility. Although no tumor emerged from BRG1 mutant mice, conditional depletion of Brg1 in the granulosa cells (GCs) of Brg1fl/fl;Amhr2-Cre mice caused sterility, whereas conditional depletion of Brg1 in the oocytes of Brg1fl/fl;Gdf9-Cre mice resulted in subfertility. Recovery of cumulus-oocyte complexes after natural mating or superovulation showed no significant difference in the Brg1fl/fl;Amhr2-Cre mutant mice and significantly fewer oocytes in the Brg1fl/fl;Gdf9-Cre mutant mice compared with controls, which may account for the subfertility. Interestingly, the evaluation of oocyte developmental competence by in vitro culture of retrieved two-cell embryos indicated that oocytes originating from the Brg1fl/fl;Amhr2-Cre mice did not reach the blastocyst stage and had higher rates of mitotic defects, including micronuclei. Together, these results indicate that BRG1 plays an important role in female fertility by regulating granulosa and oocyte functions during follicle growth and is needed for the acquisition of oocyte developmental competence.
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Cromatina , Neoplasias , Animales , Femenino , Ratones , Ensamble y Desensamble de Cromatina , Fertilidad/genética , MamíferosRESUMEN
Introduction: Epithelial ovarian cancer (OC) stands as one of the deadliest gynecologic malignancies, urgently necessitating novel therapeutic strategies. Approximately 60% of ovarian tumors exhibit reduced expression of major histocompatibility complex class I (MHC I), intensifying immune evasion mechanisms and rendering immunotherapies ineffective. NOD-like receptor CARD domain containing 5 (NLRC5) transcriptionally regulates MHC I genes and many antigen presentation machinery components. We therefore explored the therapeutic potential of NLRC5 in OC. Methods: We generated OC cells overexpressing NLRC5 to rescue MHC I expression and antigen presentation and then assessed their capability to respond to PD-L1 blockade and an infected cell vaccine. Results: Analysis of microarray datasets revealed a correlation between elevated NLRC5 expression and extended survival in OC patients; however, NLRC5 was scarcely detected in the OC tumor microenvironment. OC cells overexpressing NLRC5 exhibited slower tumor growth and resulted in higher recruitment of leukocytes in the TME with lower CD4/CD8 T-cell ratios and increased activation of T cells. Immune cells from peripheral blood, spleen, and ascites from these mice displayed heightened activation and interferon-gamma production when exposed to autologous tumor-associated antigens. Finally, as a proof of concept, NLRC5 overexpression within an infected cell vaccine platform enhanced responses and prolonged survival in comparison with control groups when challenged with parental tumors. Discussion: These findings provide a compelling rationale for utilizing NLRC5 overexpression in "cold" tumor models to enhance tumor susceptibility to T-cell recognition and elimination by boosting the presentation of endogenous tumor antigens. This approach holds promise for improving antitumoral immune responses in OC.
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Neoplasias Ováricas , Vacunas , Humanos , Femenino , Animales , Ratones , Proteínas NLR , Dominio de Reclutamiento y Activación de Caspasas , Microambiente Tumoral , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Antígenos de Histocompatibilidad Clase I , Neoplasias Ováricas/genética , Antígenos de NeoplasiasRESUMEN
Targeted therapy resistance frequently develops in melanoma due to intratumor heterogeneity and epigenetic reprogramming. This also typically induces cross-resistance to immunotherapies. Whether this includes additional modes of therapy has not been fully assessed. We show that co-treatments of MAPKi with VSV-based oncolytics do not function in a synergistic fashion; rather, the MAPKis block infection. Melanoma resistance to vemurafenib further perturbs the cells' ability to be infected by oncolytic viruses. Resistance to vemurafenib can be induced by the loss of SOX10, a common proliferative marker in melanoma. The loss of SOX10 promotes a cross-resistant state by further inhibiting viral infection and replication. Analysis of RNA-seq datasets revealed an upregulation of interferon-stimulated genes (ISGs) in SOX10 knockout populations and targeted therapy-resistant cells. Interestingly, the induction of ISGs appears to be independent of type I IFN production. Overall, our data suggest that the pathway mediating oncolytic resistance is due to the loss of SOX10 during acquired drug resistance in melanoma.
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Melanoma , Virus Oncolíticos , Virus ARN , Humanos , Virus Oncolíticos/genética , Melanoma/terapia , Vemurafenib , Epigenómica , Interferones , ARNRESUMEN
Natural Killer (NK) cell cytotoxicity and interferon-gamma (IFNγ) production are profoundly suppressed postoperatively. This dysfunction is associated with increased morbidity and cancer recurrence. NK activity depends on the integration of activating and inhibitory signals, which may be modulated by transforming growth factor-beta (TGF-ß). We hypothesized that impaired postoperative NK cell IFNγ production is due to altered signaling pathways caused by postoperative TGF-ß. NK cell receptor expression, downstream phosphorylated targets, and IFNγ production were assessed using peripheral blood mononuclear cells (PBMCs) from patients undergoing cancer surgery. Healthy NK cells were incubated in the presence of healthy/baseline/postoperative day (POD) 1 plasma and in the presence/absence of a TGF-ß-blocking monoclonal antibody (mAb) or the small molecule inhibitor (smi) SB525334. Single-cell RNA sequencing (scRNA-seq) was performed on PBMCs from six patients with colorectal cancer having surgery at baseline/on POD1. Intracellular IFNγ, activating receptors (CD132, CD212, NKG2D, DNAM-1), and downstream target (STAT5, STAT4, p38 MAPK, S6) phosphorylation were significantly reduced on POD1. Furthermore, this dysfunction was phenocopied in healthy NK cells through incubation with rTGF-ß1 or POD1 plasma and was prevented by the addition of anti-TGF-ß immunotherapeutics (anti-TGF-ß mAb or TGF-ßR smi). Targeted gene analysis revealed significant decreases in S6 and FKBP12, an increase in Shp-2, and a reduction in NK metabolism-associated transcripts on POD1. pSmad2/3 was increased and pS6 was reduced in response to rTGF-ß1 on POD1, changes that were prevented by anti-TGF-ß immunotherapeutics. Together, these results suggest that both canonical and mTOR pathways downstream of TGF-ß mediate phenotypic changes that result in postoperative NK cell dysfunction.
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Células Asesinas Naturales , Neoplasias , Factor de Crecimiento Transformador beta , Humanos , Leucocitos Mononucleares/metabolismo , Neoplasias/cirugía , Receptores de Células Asesinas Naturales/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Anticuerpos MonoclonalesRESUMEN
Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer with an imperative need for new treatments. Immunotherapy has had marked success in some cancer types; however, clinical trials studying the efficacy of immune checkpoint inhibitors for the treatment of EOC benefited less than 15% of patients. Given that EOC develops from multiple tissues in the reproductive system and metastasizes widely throughout the peritoneal cavity, responses to immunotherapy are likely hindered by heterogeneous tumor microenvironments (TME) containing a variety of immune profiles. To fully characterize and compare syngeneic model systems that may reflect this diversity, we determined the immunogenicity of six ovarian tumor models in vivo, the T and myeloid profile of orthotopic tumors and the immune composition and cytokine profile of ascites, by single-cell RNA sequencing, flow cytometry and IHC. The selected models reflect the different cellular origins of EOC (ovarian and fallopian tube epithelium) and harbor mutations relevant to human disease, including Tp53 mutation, PTEN suppression, and constitutive KRAS activation. ID8-p53-/- and ID8-C3 tumors were most highly infiltrated by T cells, whereas STOSE and MOE-PTEN/KRAS tumors were primarily infiltrated by tumor associated macrophages and were unique in MHC class I and II expression. MOE-PTEN/KRAS tumors were capable of forming T cell clusters. This panel of well-defined murine EOC models reflects some of the heterogeneity found in human disease and can serve as a valuable resource for studies that aim to test immunotherapies, explore the mechanisms of immune response to therapy, and guide selection of treatments for patient populations.
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Neoplasias Ováricas , Proteínas Proto-Oncogénicas p21(ras) , Ratones , Humanos , Femenino , Animales , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias Ováricas/terapia , Carcinoma Epitelial de Ovario/terapia , Inmunoterapia , Microambiente TumoralRESUMEN
Ovarian fibrosis is a pathological condition associated with aging and is responsible for a variety of ovarian dysfunctions. Given the known contributions of tissue fibrosis to tumorigenesis, it is anticipated that ovarian fibrosis may contribute to ovarian cancer risk. We recently reported that diabetic postmenopausal women using metformin had ovarian collagen abundance and organization that were similar to premenopausal ovaries from nondiabetic women. In this study, we investigated the effects of aging and metformin on mouse ovarian fibrosis at a single-cell level. We discovered that metformin treatment prevented age-associated ovarian fibrosis by modulating the proportion of fibroblasts, myofibroblasts, and immune cells. Senescence-associated secretory phenotype (SASP)-producing fibroblasts increased in aged ovaries, and a unique metformin-responsive subpopulation of macrophages emerged in aged mice treated with metformin. The results demonstrate that metformin can modulate specific populations of immune cells and fibroblasts to prevent age-associated ovarian fibrosis and offers a new strategy to prevent ovarian fibrosis.
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Metformina , Animales , Femenino , Fibroblastos , Fibrosis , Humanos , Metformina/farmacología , Metformina/uso terapéutico , Ratones , Miofibroblastos , OvarioRESUMEN
In adult tissue, the paired box 2 (PAX2) protein is expressed in healthy oviductal, but not normal ovarian surface epithelial cells. PAX2 is expressed in a subset of cases of serous ovarian carcinoma; however, the role of PAX2 in the initiation and progression of ovarian cancer remains unknown. The aim of the present study was to determine the biological effects of PAX2 expression in normal and cancerous epithelial cells. By culturing the normal and cancerous ovarian cells that express PAX2 in 3D culture and staining the cells with vasculogenic mimicry markers such as CD31 and PAS, it was shown that PAX2 overexpression in both normal and cancerous ovarian epithelial cells induced formation of vascular-like structures both in vitro and in vivo. These results indicated a potential role of PAX2 in ovarian cancer progression by increasing the presence of vascular-like structures to promote the supply of nutrients to tumor cells and facilitate cancer cell proliferation and invasion.
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Late lung development is a period of alveolar and microvascular formation, which is pivotal in ensuring sufficient and effective gas exchange. Defects in late lung development manifest in premature infants as a chronic lung disease named bronchopulmonary dysplasia (BPD). Numerous studies demonstrated the therapeutic properties of exogenous bone marrow and umbilical cord-derived mesenchymal stromal cells (MSCs) in experimental BPD. However, very little is known regarding the regenerative capacity of resident lung MSCs (L-MSCs) during normal development and in BPD. In this study we aimed to characterize the L-MSC population in homeostasis and upon injury. We used single-cell RNA sequencing (scRNA-seq) to profile in situ Ly6a+ L-MSCs in the lungs of normal and O2-exposed neonatal mice (a well-established model to mimic BPD) at 3 developmental timepoints (postnatal days 3, 7, and 14). Hyperoxia exposure increased the number and altered the expression profile of L-MSCs, particularly by increasing the expression of multiple pro-inflammatory, pro-fibrotic, and anti-angiogenic genes. In order to identify potential changes induced in the L-MSCs transcriptome by storage and culture, we profiled 15 000 Ly6a+ L-MSCs after in vitro culture. We observed great differences in expression profiles of in situ and cultured L-MSCs, particularly those derived from healthy lungs. Additionally, we have identified the location of Ly6a+/Col14a1+ L-MSCs in the developing lung and propose Serpinf1 as a novel, culture-stable marker of L-MSCs. Finally, cell communication analysis suggests inflammatory signals from immune and endothelial cells as main drivers of hyperoxia-induced changes in L-MSCs transcriptome.
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Displasia Broncopulmonar , Hiperoxia , Células Madre Mesenquimatosas , Animales , Animales Recién Nacidos , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/terapia , Células Endoteliales , Humanos , Hiperoxia/genética , Hiperoxia/metabolismo , Recién Nacido , Pulmón/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: Invasive lobular carcinoma (ILC) is the second most common type of breast cancer. As few tools exist to study ILC metastasis, we isolated ILC cells with increased invasive properties to establish a spontaneously metastasising xenograft model. METHODS: MDA-MB-134VI ILC cells were placed in transwells for 7 days. Migrated cells were isolated and expanded to create the VIVA1 cell line. VIVA1 cells were compared to parental MDA-MB-134VI cells in vitro for ILC marker expression and relative proliferative and invasive ability. An intraductally injected orthotopic xenograft model was used to assess primary and metastatic tumour growth in vivo. RESULTS: Similar to MDA-MB-134VI, VIVA1 cells retained expression of oestrogen receptor (ER) and lacked expression of E-cadherin, however showed increased invasion in vitro. Following intraductal injection, VIVA1 and MDA-MB-134VI cells had similar primary tumour growth and survival kinetics. However, macrometastases were apparent in 7/10 VIVA1-injected animals. Cells from a primary orthotopic tumour (VIVA-LIG43) were isolated and showed similar proliferative rates but were also more invasive than parental cells. Upon re-injection intraductally, VIVA-LIG43 cells had more rapid tumour growth with similar metastatic incidence and location. CONCLUSIONS: We generated a new orthotopic spontaneously metastasising xenograft model for ER+ ILC amenable for the study of ILC metastasis.