Murine Ovarian Cells Research Articles

An ovarian phenotype of alpha 7 nicotinic receptor knockout mice.

Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is expressed by various murine ovarian cells. Morphological and molecular investigations, including a proteomic study of adult Chrna7 knockout (KO) mouse ovaries, reveal the roles of these receptors in the local regulation of the ovary. Nicotinic acetylcholine receptor alpha 7 (nAChRa7), encoded by Chrna7, is involved in cellular functions ranging from synaptic transmission in neurons to regulation of inflammation, cell growth and metabolism to cell death in other cells. Our qPCR results and other studies indicated that nAChRa7 is expressed in the adult mouse ovary, while in situ hybridization and single-cell sequencing data suggested this expression may be shared by several ovarian cells, including fibroblast-like and steroidogenic stroma cells, macrophages and oocytes of small follicles. To explore a possible involvement of nAChRa7 in ovarian functions, we evaluated ovarian morphology of Chrna7-null mutant adult mice (KO) and wildtype mice (WT; 3 months, metestrus) by performing immunohistochemistry, qPCR studies, measurements of serum progesterone and proteomic analyses. The evaluation of serial sections indicated fewer primordial follicles but similar numbers of primary, secondary and tertiary follicles, as well as corpora lutea in KO and WT mice. Atresia was unchanged. Serum progesterone and mRNA levels of proliferation and most apoptosis markers were not changed, yet two typical macrophage markers were elevated. Furthermore, the proteomes of KO ovaries were significantly altered with 96 proteins increased and 32 decreased in abundance in KOs compared to WTs. Among the elevated proteins were markers for stroma cells. Hence, the lack of nAChRa7 causes changes in small follicle counts and alterations of the ovarian stroma cells. The ovarian phenotype of Chrna7 mutant mice links this channel protein to the local regulation of ovarian cells, including stroma cells.

A single-cell atlas of the cycling murine ovary.

The estrous cycle is regulated by rhythmic endocrine interactions of the nervous and reproductive systems, which coordinate the hormonal and ovulatory functions of the ovary. Folliculogenesis and follicle progression require the orchestrated response of a variety of cell types to allow the maturation of the follicle and its sequela, ovulation, corpus luteum formation, and ovulatory wound repair. Little is known about the cell state dynamics of the ovary during the estrous cycle and the paracrine factors that help coordinate this process. Herein, we used single-cell RNA sequencing to evaluate the transcriptome of >34,000 cells of the adult mouse ovary and describe the transcriptional changes that occur across the normal estrous cycle and other reproductive states to build a comprehensive dynamic atlas of murine ovarian cell types and states.

Abstract 3244: Exploring the mechanisms of olaparib resistance in resistant-BRCA1 deficient murine ovarian cells

Abstract Background: High-grade serous ovarian carcinoma (HGSOC) is the most frequent histotype of ovarian cancer. More than half of HGSOC is characterized by homologous recombination deficiency (HRD) due to mutations in genes involved in this pathway, including BRCA1/2. Olaparib is a poly(ADP-ribose) polymerase inhibitor (PARPi) recently approved in front line and maintenance therapy in platinum-sensitive BRCA mutated ovarian cancer patients. Despite olaparib clinical benefits, this treatment is associated with the development of resistance. A better understanding of the mechanisms at its basis could help in finding strategies to delay or overcome/counteract it, possibly translatable in the clinical setting. Methods: We generated an olaparib resistant cell line (BRCA1-/- OlaR), starting from the BRCA1 and TP53 deleted murine ID8 cells (BRCA1-/-) by step wise increasing drug concentrations. Sensitive and resistant cell lines were biologically and pharmacologically characterized and the molecular mechanisms at the basis of olaparib resistance investigated. Cellular viability was assessed by MTS assay, for molecular studies real-time PCR, western blot and RAD51-foci immunofluorescence were performed. Results: BRCA1-/- OlaR cells were 24 fold time more resistant than BRCA1-/- cells and displayed a similar in vitro cell growth compared to the parental cells. Olaparib treatment at IC50 dose caused a longer and stronger G2-M block of the cell cycle in sensitive as compared to resistant cells. BRCA1-/- OlaR cells were cross-resistant to the other PARPis tested, suggesting a common mechanism of resistance. Platinum compounds showed similar cytotoxic activity in sensitive and resistant cells, while a partial cross-resistance was observed with doxorubicin. Interestingly, resistant cells were less responsive to a panel of DNA damage response (DDR) inhibitors (i.e. ATR, WEE1 and CHK1 inhibitors) compared to parental ones; however their combinations with olaparib were synergic in both the sensitive and resistant lines. A partial restoration of the HR pathway (increase in RAD51 foci formation after olaparib and IR treatment), a downregulation of PARP1 protein levels without mutation in PARP1 gene and an upregulation of the MDR transcript could be found in BRCA1-/- resistant cells, strongly supporting multiple heterogeneous mechanisms of resistance. Conclusions: The HR deficient cell line resistant to olaparib we generated showed multiple mechanisms of resistance. The combinations of olaparib with different DDR inhibitors were equally active in sensitive and resistant cells. Citation Format: Michela Chiappa, Martina Anselmi, Luca Russo, Monica Lupi, Nicolò Panini, Federica Guffanti, Giovanna Damia. Exploring the mechanisms of olaparib resistance in resistant-BRCA1 deficient murine ovarian cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3244.

Combinations of ATR, Chk1 and Wee1 Inhibitors with Olaparib Are Active in Olaparib Resistant Brca1 Proficient and Deficient Murine Ovarian Cells.

Simple SummaryPoly(ADP-ribose) polymerases inhibitors (PARPis), including olaparib, have been recently approved for ovarian carcinoma treatment and PARPi resistance has already been observed in the clinics. With the aim of dissecting the molecular mechanisms of PARPi resistance, we generated olaparib resistant cells lines, both in a homologous recombination (HR)-deficient and -proficient background by continuous in vitro drug treatment. In the HR proficient background, olaparib resistance was caused by overexpression of multidrug resistance 1 gene (MDR1), while multiple heterogeneous co-existing mechanisms were found in olaparib resistant HR-deficient cells, including overexpression of MDR1, a decrease in PARP1 protein level and partial reactivation of HR repair. We found that combinations of ATR, Chk1 and Wee1 inhibitors with olaparib were synergistic in sensitive and resistant sublines, regardless of the HR status. These new olaparib resistant models will be instrumental to screen new therapeutic options for PARPi-resistant ovarian tumors.Background. Poly(ADP-ribose) polymerases inhibitor (PARPi) have shown clinical efficacy in ovarian carcinoma, especially in those harboring defects in homologous recombination (HR) repair, including BRCA1 and BRCA2 mutated tumors. There is increasing evidence however that PARPi resistance is common and develops through multiple mechanisms. Methods. ID8 F3 (HR proficient) and ID8 Brca1-/- (HR deficient) murine ovarian cells resistant to olaparib, a PARPi, were generated through stepwise drug concentrations in vitro. Both sensitive and resistant cells lines were pharmacologically characterized and the molecular mechanisms underlying olaparib resistance. Results. In ID8, cells with a HR proficient background, olaparib resistance was mainly caused by overexpression of multidrug resistance 1 gene (MDR1), while multiple heterogeneous co-existing mechanisms were found in ID8 Brca1-/- HR-deficient cells resistant to olaparib, including overexpression of MDR1, a decrease in PARP1 protein level and partial reactivation of HR repair. Importantly, combinations of ATR, Chk1 and Wee1 inhibitors with olaparib were synergistic in sensitive and resistant sublines, regardless of the HR cell status. Conclusion. Olaparib-resistant cell lines were generated and displayed multiple mechanisms of resistance, which will be instrumental in selecting new possible therapeutic options for PARPi-resistant ovarian tumors.

Abstract 2066: Modulation of homologous recombination repair pathway gene expression by a dual EZH2 and EHMT2 histone methyltransferase inhibitor and synergy with PARP inhibitors in ovarian cancer

Abstract Defective DNA damage response (DDR) is a hallmark of high grade serous ovarian cancer (HGSOC). Genetic and epigenomic aberrations in DDR pathways including homologous recombination (BRCA1/BRCA2) and non-homologous end joining (PARP-1) make some HGSOC vulnerable to cisplatin and Poly (ADP-ribose) polymerase inhibitors (PARPi). However, mechanisms behind primary and acquired resistance to these agents remain unknown. We hypothesise aberrant epigenomic changes at DDR-associated genes contribute to PARPi resistance and can be reversed using a novel histone methyltransferase inhibitor (HKMT-1-005) which targets two histone methyltransferases (EZH2 and EHMT2) frequently upregulated in HGSOC. MTS assays were used to calculate the IC50 values for HKMT-1-005 and the PARPi Rucaparib in a panel of human HGSOC cell lines (n=8). Isobologram analysis using Combenefit software was used to determine synergy. RNA-seq and ATAC-seq was performed on cell lines derived from the same patient before (PEO1) and after acquired resistance to chemotherapy (PEO4) pre/post treatment with HKMT-1-005. Pathway analysis was used to identify pathways enriched in genes with altered expression and/or chromatin conformation after treatment with HKMT-1-005 and validated with qRT-PCR. Synergy between HKMT-1-005 and Rucaparib and changes in chromatin conformation with HKMT-1-005 treatment were further examined in CRIPSR modified ID8 murine ovarian cell lines with the genotypes: WT, P53-/-, P53-/-BRCA1-/- and P53-/-BRCA2-/-. Isobologram analysis revealed HKMT-1-005 in combination with Rucaparib was synergistic in multiple cell lines. Synergy was greater in PEO1 compared to PEO4. RNA-seq and ATAC-seq identified a significant (FDR <0.05) enrichment of genes associated with DDR pathways which had altered expression and/or chromatin accessibility in PEO1 compared to PEO4, including BRCA1 and BRCA2. qRT-PCR analysis confirmed treatment with HKMT-1-005 reduced expression of BRCA1 and BRCA2 in PEO1 but not PEO4. Synergy between HKMT-1-005 and Rucaparib was greater in P53-/-BRCA1-/- and P53-/-BRCA2-/- compared to WT or P53-/- ID8 mouse cell lines. ATAC-seq identified multiple DDR-associated genes had significantly (FDR <0.05) altered chromatin accessibility in P53-/-BRCA2-/- compared with P53-/- alone. HKMT-1-005 treatment in ID8 cell lines revealed changes in chromatin at multiple DDR-associated genes including BRCA2. HKMT-1-005 is synergistic with the PARP inhibitor Rucaparib and can modulate the expression and chromatin conformation of DDR-associated genes in multiple human and mouse ovarian cell lines. Depletion of BRCA2 may additionally regulate chromatin conformation of genes including those associated with DDR pathways and explain why greater synergy between HKMT-1-005 and Rucaparib was observed in BRCA deficient lines. Citation Format: Ian M. Garner, Zhuyan Su, Sawyer Hu, Yue Wu, Iain A. McNeish, Matthew J. Fuchter, Robert Brown. Modulation of homologous recombination repair pathway gene expression by a dual EZH2 and EHMT2 histone methyltransferase inhibitor and synergy with PARP inhibitors in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2066.

Abstract 3650: Preclinical evaluation of the PARP inhibitor rucaparib in combination with PD-1 and PD-L1 inhibition in a syngeneic BRCA1 mutant ovarian cancer model

Abstract Background: Rucaparib (CO-338) is an oral small molecule inhibitor of poly(ADP-ribose) polymerase (PARP)-1, PARP-2 and PARP-3 that has shown clinical activity in patients with BRCA1 and BRCA2 mutated advanced ovarian cancer. Monoclonal antibodies against programmed death receptor-1 (PD-1) and programmed death-ligand (PD-L1) have also shown efficacy in advanced ovarian cancer patients. It has been reported that BRCA1 and BRCA2 mutated tumors have a higher mutational load and increased CD8+ T cell infiltration, suggesting that the combination of rucaparib and immune checkpoint inhibition may be complementary. However, PARP inhibition has also been reported to have an immunosuppressive effect in preclinical studies. Methods: Subcutaneous syngeneic models using the BRCA1 wild-type C2Km (P53-/-, myc, Kras-G12D, Akt-myr) and BRCA1 mutant BrKras (BRCA1-/-; P53-/-; myc; Kras-G12D; Akt-myr) murine ovarian cell lines were developed in the murine FVB/N background. Antibodies targeting PD-1 (RMP1-14) and PD-L1 (10F.9G2) were dosed by intraperitoneal injection at 5-10 mg/kg twice weekly, while rucaparib was administered by oral gavage at 150 mg/kg twice daily. Treatment was initiated at a tumor volume of ~150mm3 (n=15/group). Animals were dosed for 21 days, and tumors were allowed to regrow to day 76. Results: In vitro cytotoxicity assays demonstrated that rucaparib was 155-fold more potent in the BRCA1 deficient BrKras cell line (IC50 = 84 nM) than the isogenic BRCA1 wild-type C2Km cell line (IC50 = 13 μM). An in vivo study using the syngeneic BrKras model was performed in mice treated with: vehicle, rucaparib, PD-1, PD-L1, rucaparib+PD-1, and rucaparib+PD-L1. All monotherapy and combination groups resulted in significant tumor growth inhibition and were followed for survival analysis. The median survival time (MST) and % cures (defined as undetectable growth at Day 76 post-tumor implantation) for vehicle, PD-L1, PD-1 and rucaparib monotherapy treated animals was 34 days (0%), 41 days (13%), 76 days (40%) and >76 days (56%), respectively. The rucaparib+PD-1 and rucaparib+PD-L1 combination groups demonstrated greater efficacy than the monotherapies, with a MST of >76 days (100%) and >76 days (88%), respectively. Dose response and immune profiling studies are ongoing. In vivo studies were also performed in the BRCA1/2 wild-type models EMT-6, Pan02, and MC38. As expected, as a single agent, rucaparib showed limited activity in these homologous recombination competent models, whereas a range of tumor growth inhibition was observed with monotherapy PD-L1 treatment. No impact on anti-tumor activity was observed in animals treated with rucaparib+PD-L1 as compared to PD-L1 monotherapy in the BRCA1/2 wild-type syngeneic models examined. Conclusions: The combination of rucaparib with PD-1 and PD-L1 inhibition improved survival in a BRCA1 mutant syngeneic model. Citation Format: Liliane Robillard, Minh Nguyen, Andrea Loehr, Sandra Orsulic, Rebecca S. Kristeleit, Kevin Lin, Mitch Raponi, Thomas C. Harding, Andrew D. Simmons. Preclinical evaluation of the PARP inhibitor rucaparib in combination with PD-1 and PD-L1 inhibition in a syngeneic BRCA1 mutant ovarian cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3650. doi:10.1158/1538-7445.AM2017-3650

Tumor-Intrinsic PD-L1 Signals Regulate Cell Growth, Pathogenesis, and Autophagy in Ovarian Cancer and Melanoma.

PD-L1 antibodies produce efficacious clinical responses in diverse human cancers, but the basis for their effects remains unclear, leaving a gap in the understanding of how to rationally leverage therapeutic activity. PD-L1 is widely expressed in tumor cells, but its contributions to tumor pathogenicity are incompletely understood. In this study, we evaluated the hypothesis that PD-L1 exerts tumor cell-intrinsic signals that are critical for pathogenesis. Using RNAi methodology, we attenuated PD-L1 in the murine ovarian cell line ID8agg and the melanoma cell line B16 (termed PD-L1lo cells), which express basal PD-L1. We observed that PD-L1lo cells proliferated more weakly than control cells in vitro As expected, PD-L1lo cells formed tumors in immunocompetent mice relatively more slowly, but unexpectedly, they also formed tumors more slowly in immunodeficient NSG mice. RNA sequencing analysis identified a number of genes involved in autophagy and mTOR signaling that were affected by PD-L1 expression. In support of a functional role, PD-L1 attenuation augmented autophagy and blunted the ability of autophagy inhibitors to limit proliferation in vitro and in vivo in NSG mice. PD-L1 attenuation also reduced mTORC1 activity and augmented the antiproliferative effects of the mTORC1 inhibitor rapamycin. PD-L1lo cells were also relatively deficient in metastasis to the lung, and we found that anti-PD-L1 administration could block tumor cell growth and metastasis in NSG mice. This therapeutic effect was observed with B16 cells but not ID8agg cells, illustrating tumor- or compartmental-specific effects in the therapeutic setting. Overall, our findings extend understanding of PD-L1 functions, illustrate nonimmune effects of anti-PD-L1 immunotherapy, and suggest broader uses for PD-L1 as a biomarker for assessing cancer therapeutic responses. Cancer Res; 76(23); 6964-74. ©2016 AACR.

Interleukin-12 (IL-12) has potential roles in regulation of lutenization of murine ovarian cells

Interleukin-12 (IL-12) has potential roles in regulation of lutenization of murine ovarian cells

Gonadotrophin-induced paracrine regulation of human oocyte maturation by BDNF and GDNF secreted by granulosa cells

In mammalian ovaries, diverse paracrine factors have been identified to mediate or modulate LH-induced changes during ovulation. Due to the difficulty in obtaining non-stimulated granulosa cells during IVF, little is known about the LH-induced paracrine factors in the human ovary. Based on earlier studies using murine ovarian cells showing the paracrine roles of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in promoting oocyte maturation, we investigated the expression of these ligands in human granulosa cells and their regulation of human oocyte development. Non-stimulated granulosa cells were obtained from non-stimulated IVM (in vitro maturation) patients after oocyte retrieval. Women undergoing non-stimulated IVM treatment at a mean age of 30.8 ± 1.3 (n = 10) were recruited for this study. Immature oocytes and granulosa cells were collected from IVF patients undergoing gonadotrophin stimulation and ICSI. Immunocytochemical analyses of granulosa cells were carried out to investigate expression profiles of BDNF and GDNF, together with real-time RT-PCR to analyze the gonadotrophin regulation of BDNF and GDNF transcript levels. In addition, immature oocytes were cultured to analyze the regulation of oocyte maturation by BDNF and GDNF. BDNF and GDNF were found to be expressed in non-stimulated granulosa cells. After gonadotrophin (FSH and/or hCG) treatment, transcripts levels for BDNF and GDNF were significantly increased (P < 0.05). In cultured immature oocytes, treatment with BDNF or GDNF promoted total yields of metaphase II oocytes. These findings demonstrate that FSH and hCG treatments augment the expression of BDNF and GDNF by granulosa cells and that these granulosa-cell-derived factors are candidate paracrine factors capable of promoting oocyte maturation.

Efficient gene delivery into murine ovarian cells by intraovarian injection of plasmid DNA and subsequent in vivo electroporation.

We describe the use of direct injection of circular plasmid DNA and subsequent in vivo electroporation (EP) for efficient gene delivery to the ovarian cells, including follicular cells and oocytes of mice. When Trypan blue (TB) was injected into the central portion of an ovary by a glass micropipette, rapid dispersion of TB to each preantral and antral follicle was observed. Injections of lacZ-expressing plasmid DNA and subsequent in vivo EP resulted in transfection of follicles with efficiencies ranging from 8-60%, together with cells in the thecal portion of the ovary. Of the lacZ-positive follicles, some oocytes were also positive for lacZ activity. These findings suggest that a solution introduced inside the ovary is rapidly dispersed to each follicle. With this technique, we expect great progress in genetic engineering in murine ovary.

An aminopeptidase inhibitor, bestatin, enhances gonadotrophin-stimulated ovulation in mice.

The role of aminopeptidases in follicular growth and/or ovulation in vivo was examined. We injected an inhibitor of cell-surface aminopeptidases, bestatin (4 mg/ml, 100 microliters), i.p. four times during 2 days into 20 day old female ICR mice, in which follicular growth and ovulation were stimulated by pregnant mare's serum gonadotrophin (PMSG, 5 IU) and human chorionic gonadotrophin (HCG, 5 IU). The number of ovulated oocytes was estimated by counting the number of oocytes in the oviduct 19 h after HCG injection. The mean +/- SD number of ovulated oocytes in bestatin-treated mice was significantly higher than that in control mice [47.00 +/- 18.13 (n = 26) versus 35.90 +/- 10.14 (n = 28), P < 0.01]. To confirm the direct effect of bestatin on the ovary, bestatin (2 mg/ml, 3 microliters) or its stereoisomer (2 mg/ml, 3 microliters) with very weak inhibitory activity was unilaterally injected into the ovarian bursa 24 h before the administration of PMSG. As a control, buffer (3 microliters) was injected into the contralateral bursa. In some experiments, bestatin (2 mg/ml, 3 microliters) was injected just before HCG administration. The administration of bestatin via the ovarian bursa prior to PMSG administration significantly increased the number of ovulated oocytes per oviduct from the treated compared with the contralateral ovary [23.70 +/- 9.61 versus 17.10 +/- 5.83 (n = 25), P < 0.01], whereas its stereoisomer elicited no significant effects. The administration of bestatin just before HCG administration also had no effect. These findings indicate that membrane-bound peptidase(s) present on murine ovarian cells is an important regulating factor(s) of follicular growth and/or ovulation.

Anti‐asialo‐GM1 inhibits vaccinia virus infection of murine ovaries: Asialo‐GM1 as an additional virus receptor?

The neurovirulent strain of vaccinia virus, VV-WR, or recombinants derived from VV-WR, cause highly productive infection of murine ovaries, but infection could be partially inhibited in vivo using an antiserum to asialo-GM1 (as-GM1). In vitro analysis by flow cytometry revealed that murine ovarian cells expressed a cell surface antigen identical to or cross-reactive with as-GM1. The capacity of VV-WR to infect murine ovaries appears to depend in part upon as-GM1 expression on ovarian cells.