Single-Cell RNA Sequencing Reveals Repair Features of Human Umbilical Cord Mesenchymal Stromal Cells.

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 profiles with therapeutic potential. Methods: UC-MSCs from five term donors and human neonatal dermal fibroblasts (HNDFs; control cells of mesenchymal origin) transcriptomes were investigated using single-cell RNA sequencing (scRNA-seq) analysis. 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 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 nontherapeutic 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.

Pevonedistat, a first-in-class NEDD8-activating enzyme inhibitor, sensitizes cancer cells to VSVΔ51 oncolytic virotherapy.

The clinical efficacy of VSVΔ51 oncolytic virotherapy has been limited by tumor resistance to viral infection, so strategies to transiently repress antiviral defenses are warranted. Pevonedistat is a first-in-class NEDD8-activating enzyme (NAE) inhibitor currently being tested in clinical trials for its antitumor potential. In this study, we demonstrate that pevonedistat sensitizes human and murine cancer cells to increase oncolytic VSVΔ51 infection, increase tumor cell death, and improve therapeutic outcomes in resistant syngeneic murine cancer models. Increased VSVΔ51 infectivity was also observed in clinical human tumor samples. We further identify the mechanism of this effect to operate via blockade of the type 1 interferon (IFN-1) response through neddylation-dependent interferon-stimulated growth factor 3 (ISGF3) repression and neddylation-independent inhibition of NF-κB nuclear translocation. Together, our results identify a role for neddylation in regulating the innate immune response and demonstrate that pevonedistat can improve the therapeutic outcomes of strategies using oncolytic virotherapy.

SWI/SNF chromatin remodeling subunit Smarca4/BRG1 is essential for female fertility†.

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.

Single-Cell RNA Sequencing-Based Characterization of Resident Lung Mesenchymal Stromal Cells in Bronchopulmonary Dysplasia.

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.

In vivo modeling of metastatic human high-grade serous ovarian cancer in mice.

Metastasis is responsible for 90% of human cancer mortality, yet it remains a challenge to model human cancer metastasis in vivo. Here we describe mouse models of high-grade serous ovarian cancer, also known as high-grade serous carcinoma (HGSC), the most common and deadliest human ovarian cancer type. Mice genetically engineered to harbor Dicer1 and Pten inactivation and mutant p53 robustly replicate the peritoneal metastases of human HGSC with complete penetrance. Arising from the fallopian tube, tumors spread to the ovary and metastasize throughout the pelvic and peritoneal cavities, invariably inducing hemorrhagic ascites. Widespread and abundant peritoneal metastases ultimately cause mouse deaths (100%). Besides the phenotypic and histopathological similarities, mouse HGSCs also display marked chromosomal instability, impaired DNA repair, and chemosensitivity. Faithfully recapitulating the clinical metastases as well as molecular and genomic features of human HGSC, this murine model will be valuable for elucidating the mechanisms underlying the development and progression of metastatic ovarian cancer and also for evaluating potential therapies.

VIVA1: a more invasive subclone of MDA-MB-134VI invasive lobular carcinoma cells with increased metastatic potential in xenograft models.

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.

Lats1 and Lats2 are required for the maintenance of multipotency in the Müllerian duct mesenchyme.

WNT signaling plays essential roles in the development and function of the female reproductive tract. Although crosstalk with the Hippo pathway is a key regulator of WNT signaling, whether Hippo itself plays a role in female reproductive biology remains largely unknown. Here, we show that conditional deletion of the key Hippo kinases Lats1 and Lats2 in mouse Müllerian duct mesenchyme cells caused them to adopt the myofibroblast cell fate, resulting in profound reproductive tract developmental defects and sterility. Myofibroblast differentiation was attributed to increased YAP and TAZ expression (but not to altered WNT signaling), leading to the direct transcriptional upregulation of Ctgf and the activation of the myofibroblast genetic program. Müllerian duct mesenchyme cells also became myofibroblasts in male mutant embryos, which impeded the development of the male reproductive tract and resulted in cryptorchidism. The inactivation of Lats1/2 in differentiated uterine stromal cells in vitro did not compromise their ability to decidualize, suggesting that Hippo is dispensable during implantation. We conclude that Hippo signaling is required to suppress the myofibroblast genetic program and maintain multipotency in Müllerian mesenchyme cells.

Preventing Surgery-Induced NK Cell Dysfunction Using Anti-TGF-ß Immunotherapeutics.

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.

Single-cell RNA-sequencing reveals transcriptional dynamics of estrogen-induced dysplasia in the ovarian surface epithelium.

Estrogen therapy increases the risk of ovarian cancer and exogenous estradiol accelerates the onset of ovarian cancer in mouse models. Both in vivo and in vitro, ovarian surface epithelial (OSE) cells exposed to estradiol develop a subpopulation that loses cell polarity, contact inhibition, and forms multi-layered foci of dysplastic cells with increased susceptibility to transformation. Here, we use single-cell RNA-sequencing to characterize this dysplastic subpopulation and identify the transcriptional dynamics involved in its emergence. Estradiol-treated cells were characterized by up-regulation of genes associated with proliferation, metabolism, and survival pathways. Pseudotemporal ordering revealed that OSE cells occupy a largely linear phenotypic spectrum that, in estradiol-treated cells, diverges towards cell state consistent with the dysplastic population. This divergence is characterized by the activation of various cancer-associated pathways including an increase in Greb1 which was validated in fallopian tube epithelium and human ovarian cancers. Taken together, this work reveals possible mechanisms by which estradiol increases epithelial cell susceptibility to tumour initiation.

LY75 Suppression in Mesenchymal Epithelial Ovarian Cancer Cells Generates a Stable Hybrid EOC Cellular Phenotype, Associated with Enhanced Tumor Initiation, Spreading and Resistance to Treatment in Orthotopic Xenograft Mouse Model.

The implications of the epithelial-mesenchymal transition (EMT) mechanisms in the initiation and progression of epithelial ovarian cancer (EOC) remain poorly understood. We have previously shown that suppression of the antigen receptor LY75 directs mesenchymal-epithelial transition (MET) in EOC cell lines with the mesenchymal phenotype, associated with the loss of Wnt/ß-catenin signaling activity. In the present study, we used the LY75-mediated modulation of EMT in EOC cells as a model in order to investigate in vivo the specific role of EOC cells, with an epithelial (E), mesenchymal (M) or mixed epithelial plus mesenchymal (E+M) phenotype, in EOC initiation, dissemination and treatment response, following intra-bursal (IB) injections of SKOV3-M (control), SKOV3-E (Ly75KD) and a mixed population of SKOV3-E+M cells, into severe combined immunodeficiency (SCID) mice. We found that the IB-injected SKOV3-E cells displayed considerably higher metastatic potential and resistance to treatment as compared to the SKOV3-M cells, due to the acquisition of a Ly75KD-mediated hybrid phenotype and stemness characteristics. We also confirmed in vivo that the LY75 depletion directs suppression of the Wnt/ß-catenin pathway in EOC cells, suggestive of a protective role of this pathway in EOC etiology. Moreover, our data raise concerns regarding the use of LY75-targeted vaccines for dendritic-cell EOC immunotherapy, due to the possible occurrence of undesirable side effects.

Ovarian cancer and the evolution of subtype classifications using transcriptional profiling†.

Ovarian cancer is a complex disease with multiple subtypes, each having distinct histopathologies and variable responses to treatment. This review highlights the technological milestones and the studies that have applied them to change our definitions of ovarian cancer. Over the past 50 years, technologies such as microarrays and next-generation sequencing have led to the discovery of molecular alterations that define each of the ovarian cancer subtypes and has enabled further subclassification of the most common subtype, high-grade serous ovarian cancer (HGSOC). Improvements in mutational profiling have provided valuable insight, such as the ubiquity of TP53 mutations in HGSOC tumors. However, the information derived from these technological advances has also revealed the immense heterogeneity of this disease, from variation between patients to compositional differences within single masses. In looking forward, the emerging technologies for single-cell and spatially resolved transcriptomics will allow us to better understand the cellular composition and structure of tumors and how these contribute to the molecular subtypes. Attempts to incorporate the complexities ovarian cancer has resulted in increasing sophistication of model systems, and the increased precision in molecular profiling of ovarian cancers has already led to the introduction of inhibitors of poly (ADP-ribose) polymerases as a new class of treatments for ovarian cancer with DNA repair deficiencies. Future endeavors to define increasingly accurate classification strategies for ovarian cancer subtypes will allow for confident prediction of disease progression and provide important insight into potentially targetable molecular mechanisms specific to each subtype.

COX2 is induced in the ovarian epithelium during ovulatory wound repair and promotes cell survival†.

The ovarian surface epithelium (OSE) is a monolayer of cells surrounding the ovary that is ruptured during ovulation. After ovulation, the wound is repaired, however, this process is poorly understood. In epithelial tissues, wound repair is mediated by an epithelial-to-mesenchymal transition (EMT). Transforming Growth Factor Beta-1 (TGFß1) is a cytokine commonly known to induce an EMT and is present throughout the ovarian microenvironment. We, therefore, hypothesized that TGFß1 induces an EMT in OSE cells and activates signaling pathways important for wound repair. Treating primary cultures of mouse OSE cells with TGFß1 induced an EMT mediated by TGFßRI signaling. The transcription factor Snail was the only EMT-associated transcription factor increased by TGFß1 and, when overexpressed, was shown to increase OSE cell migration. A polymerase chain reaction array of TGFß signaling targets determined Cyclooxygenase-2 (Cox2) to be most highly induced by TGFß1. Constitutive Cox2 expression modestly increased migration and robustly enhanced cell survival, under stress conditions similar to those observed during wound repair. The increase in Snail and Cox2 expression with TGFß1 was reproduced in human OSE cultures, suggesting these responses are conserved between mouse and human. Finally, the induction of Cox2 expression in OSE cells during ovulatory wound repair was shown in vivo, suggesting TGFß1 increases Cox2 to promote wound repair by enhancing cell survival. These data support that TGFß1 promotes ovulatory wound repair by induction of an EMT and activation of a COX2-mediated pro-survival pathway. Understanding ovulatory wound repair may give insight into why ovulation is the primary non-hereditary risk factor for ovarian cancer.

The polypeptide GALNT6 Displays Redundant Functions upon Suppression of its Closest Homolog GALNT3 in Mediating Aberrant O-Glycosylation, Associated with Ovarian Cancer Progression.

Epithelial ovarian cancer (EOC) represents the most lethal gynecologic malignancy; a better understanding of the molecular mechanisms associated with EOC etiology could substantially improve EOC management. Aberrant O-glycosylation in cancer is attributed to alteration of N-acetylgalactosaminyltransferases (GalNAc-Ts). Reports suggest a genetic and functional redundancy between GalNAc-Ts, and our previous data are indicative of an induction of GALNT6 expression upon GALNT3 suppression in EOC cells. We performed single GALNT3 and double GALNT3/T6 suppression in EOC cells, using a combination of the CRISPR-Cas9 system and shRNA-mediated gene silencing. The effect of single GALNT3 and double GALNT3/T6 inhibition was monitored both in vitro (on EOC cells roliferation, migration, and invasion) and in vivo (on tumor formation and survival of experimental animals). We confirmed that GALNT3 gene ablation leads to strong and rather compensatory GALNT6 upregulation in EOC cells. Moreover, double GALNT3/T6 suppression was significantly associated with stronger inhibitory effects on EOC cell proliferation, migration, and invasion, and accordingly displayed a significant increase in animal survival rates compared with GALNT3-ablated and control (Ctrl) EOC cells. Our data suggest a possible functional redundancy of GalNAc-Ts (GALNT3 and T6) in EOC, with the perspective of using both these enzymes as novel EOC biomarkers and/or therapeutic targets.

Characteristics and outcome of the COEUR Canadian validation cohort for ovarian cancer biomarkers.

BACKGROUND: Ovarian carcinoma is the most lethal gynecological malignancy due to early dissemination and acquired resistance to platinum-based chemotherapy. Reliable markers that are independent and complementary to clinical parameters are needed to improve the management of patients with this disease. The Canadian Ovarian Experimental Unified Resource (COEUR) provides researchers with biological material and associated clinical data to conduct biomarker validation studies. Using standards defined by the Canadian Tissue Repository Network (CTRNet), we have previously demonstrated the quality of the biological material from this resource. Here we describe the clinical characteristics of the COEUR cohort. METHODS: With support from 12 Canadian ovarian cancer biobanks in Canada, we created a central retrospective cohort comprised of more than 2000 patient tissue samples with associated clinical data, including 1246 high-grade serous, 102 low-grade serous, 295 endometrioid, 259 clear cell and 89 mucinous carcinoma histotypes. A two-step reclassification process was applied to assure contemporary histological classification (histotyping). For each histotypes individually, we evaluated the association between the known clinico-pathological parameters (stage, cytoreduction, chemotherapy treatment, BRCA1 and BRCA2 mutation) and patient outcome by using Kaplan-Meier and Cox proportional hazard regression analyses. RESULTS: The median follow-up time of the cohort was 45 months and the 5-year survival rate for patients with high-grade serous carcinomas was 34%, in contrast to endometrioid carcinomas with 80% at 5 years. Survival profiles differed by histotype when stratified by stage or cytoreduction. Women with mucinous or clear cell carcinomas at advanced stage or with non-optimally debulked disease had the worst outcomes. In high-grade serous carcinoma, we observed significant association with longer survival in women harboring BRCA1 or BRCA2 mutation as compared to patients without detectable mutation. CONCLUSIONS: Our results show the expected survival rates, as compared with current literature, in each histotype suggesting that the cohort is an unbiased representation of the five major histotypes. COEUR, a one stop comprehensive biorepository, has collected mature outcome data and relevant clinical data in a comprehensive manner allowing stratified analysis.

An Immunohistochemical Algorithm for Ovarian Carcinoma Typing.

There are 5 major histotypes of ovarian carcinomas. Diagnostic typing criteria have evolved over time, and past cohorts may be misclassified by current standards. Our objective was to reclassify the recently assembled Canadian Ovarian Experimental Unified Resource and the Alberta Ovarian Tumor Type cohorts using immunohistochemical (IHC) biomarkers and to develop an IHC algorithm for ovarian carcinoma histotyping. A total of 1626 ovarian carcinoma samples from the Canadian Ovarian Experimental Unified Resource and the Alberta Ovarian Tumor Type were subjected to a reclassification by comparing the original with the predicted histotype. Histotype prediction was derived from a nominal logistic regression modeling using a previously reclassified cohort (N=784) with the binary input of 8 IHC markers. Cases with discordant original or predicted histotypes were subjected to arbitration. After reclassification, 1762 cases from all cohorts were subjected to prediction models (χ Automatic Interaction Detection, recursive partitioning, and nominal logistic regression) with a variable IHC marker input. The histologic type was confirmed in 1521/1626 (93.5%) cases of the Canadian Ovarian Experimental Unified Resource and the Alberta Ovarian Tumor Type cohorts. The highest misclassification occurred in the endometrioid type, where most of the changes involved reclassification from endometrioid to high-grade serous carcinoma, which was additionally supported by mutational data and outcome. Using the reclassified histotype as the endpoint, a 4-marker prediction model correctly classified 88%, a 6-marker 91%, and an 8-marker 93% of the 1762 cases. This study provides statistically validated, inexpensive IHC algorithms, which have versatile applications in research, clinical practice, and clinical trials.

Akt confers cisplatin chemoresistance in human gynecological carcinoma cells by modulating PPM1D stability.

Ovarian cancer (OVCA) and cervical cancer (CECA) are lethal gynecological malignancies. Cisplatin (CDDP) and platinum derivatives are first line chemotherapeutics and their resistance impedes successful treatment. Understanding the molecular dysregulation underlying chemoresistance is important in developing rational therapeutic strategies. We have established that Protein Phosphatase Magnesium-dependent 1 D (PPM1D) confers CDDP resistance in gynecological cancer cells by deactivating p53. However, whether CDDP regulates intra-cellular PPM1D localization and whether this regulation is different between chemosensitive and chemoresistant cancer cells is unknown. Moreover, whether Akt regulates PPM1D in the context of CDDP resistance has not been studied. To illustrate the role of PPM1D in gynecological cancer cell chemoresistance and its regulation by Akt we have demonstrated that: (a) CDDP induced PPM1D down-regulation through proteasomal degradation in sensitive CECA cells; (b) CDDP induced PPM1D nuclear localization in resistant CECA cells, and nuclear exclusion in sensitive CECA cells and OVCA xenografts; (c) Over-expression of active Akt in sensitive CECA cells stabilized PPM1D content through inhibition of CDDP-induced PPM1D down-regulation; (d) Inhibition of Akt activity in resistant OVCA cells leads to decreased PPM1D stability and CDDP-induced down-regulation in resistant CECA cells; and (e) PPM1D is highly expressed in human ovarian tumor subtypes and in a tissue microarray panel of human ovarian tumors. In conclusion, we have established that PPM1D plays an important role in promoting CDDP resistance and as a novel downstream target of Akt, PPM1D mediates its action in conferring CDDP resistance in gynecological cancer cells.

Epithelial ovarian cancer stem cells: underlying complexity of a simple paradigm.

The lack of significant progress in the treatment of epithelial ovarian cancer (EOC) underscores the need to gain a better understanding of the processes that lead to chemoresistance and recurrence. The cancer stem cell (CSC) hypothesis offers an attractive explanation of how a subpopulation of cells within a patient's tumour might remain refractory to treatment and subsequently form the basis of recurrent chemoresistant disease. This review examines the literature defining somatic stem cells of the ovary and fallopian tube, two tissues that give rise to EOC. In addition, considerable research has been reviewed, that has identified subpopulations of EOC cells, based on marker expression (CD133, CD44, CD117, CD24, epithelial cell adhesion molecule, LY6A, ALDH1 and side population (SP)), which are enriched for tumour initiating cells (TICs). While many studies identified either CD133 or CD44 as markers useful for enriching for TICs, there is little consensus. This suggests that EOC cells may have a phenotypic plasticity that may preclude the identification of universal markers defining a CSC. The assay that forms the basis of quantifying TICs is the xenograft assay. Considerable controversy surrounds the xenograft assay and it is essential that some of the potential limitations be examined in this review. Highlighting such limitations or weaknesses is required to properly evaluate data and broaden our interpretation of potential mechanisms that might be contributing to the pathogenesis of ovarian cancer.

17ß-estradiol upregulates GREB1 and accelerates ovarian tumor progression in vivo.

Exogenous 17ß-estradiol (E2) accelerates the progression of ovarian cancer in the transgenic tgCAG-LS-TAg mouse model of the disease. We hypothesized that E2 has direct effects on ovarian cancer cells and this study was designed to determine the molecular mechanisms by which E2 accelerates ovarian tumor progression. Mouse ovarian cancer ascites (MAS) cell lines were derived from tgCAG-LS-TAg mice. Following intraperitoneal engraftment of two MAS cell lines, MASC1 and MASE2, into SCID mice, exogenous E2 significantly decreased the survival time and increased the tumor burden. Microarray analysis performed on MASE2-derived tumors treated with E2 or placebo showed that E2 treatment caused the upregulation of 197 genes and the downregulation of 55 genes. The expression of gene regulated by estrogen in breast cancer 1 (Greb1) was upregulated in mouse tumors treated with E2 and was overexpressed in human ovarian cancers relative to human ovarian surface epithelium, suggesting a role for GREB1 in human ovarian tumor progression. RNA interference-mediated knockdown of GREB1 in MASE2 cells decreased their proliferation rate in vitro and increased survival time in mice engrafted with the cells. These results emphasize the importance of E2 in ovarian tumor progression and identify Greb1 as a novel gene target for therapeutic intervention.

FGL2 promotes tumour growth and attenuates infiltration of activated immune cells in melanoma and ovarian cancer models.

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.

Transcriptional regulation of the postnatal cardiac conduction system heterogeneity.

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.