Mouse Ovarian Surface Epithelial Cells Research Articles

Abstract 3077: Integrin expression during ovarian cancer progression

Abstract During peritoneal dissemination, ovarian cancer cells exfoliate from the primary tumor and aggregate in the peritoneal fluid. This elicits survival signals via changes in mitochondrial morphology and function. As spheroids adhere to secondary sites, mitochondrial function is restored in the form of recovery of mitochondrial morphology, increased mitochondrial membrane potential, glucose uptake and increased proliferation. These findings suggested a mitochondrial phenotypic switch from mitophagy in spheroids to upregulation of mitobiogenesis upon spheroids adhesion to secondary sites. Hence, our objective was to identify critical receptors that facilitate the adhesion of spheroids to secondary sites, focusing on integrins that facilitate cell-extracellular matrix (ECM) attachment and activate downstream signaling cascades. To investigate changes in integrin expression, we mimicked three major stages of ovarian cancer progression by developing adherent stage, spheroid stage and adherent spheroid stage using our mouse ovarian surface epithelial (MOSE) cell lines representing benign (MOSE-E) stage as well as slow-developing (MOSE-L) and fast-developing disease (MOSE-LTICv) in biologically relevant conditions. Using RT qPCR and Western blotting, we determined gene and protein expression levels of integrins and adhesion related genes. Confocal microscopy was used to determine protein localization during ovarian cancer progression. RT qPCR result indicated differential up-regulated expression of integrins in both spheroid and adherent spheroid stage, suggesting the role of different integrins during the stages of ovarian cancer progression. Specifically, we observed a decrease (integrin ɑ5, ɑ6) or increase (ɑ2𝛃;1) of integrins after aggregation and an upregulation of 𝛃;1, α2, αV, α3, α5 and immunomodulatory integrins such as αE and αM in adherent spheroids, indicating their role in mediating specific steps during progression of ovarian cancer from tumorigenesis to metastatic implantation. We also observed increased expression levels of extracellular matrix proteins and their modulators, and cell-to-cell adhesion genes during ovarian cancer progression that was more pronounced in the MOSE-LTICv cells which suggests their association with an aggressive cancer phenotype. Overall, this project will aid in the identification of adhesion targets to develop new anti-metastatic treatments that will reduce mortality in women with ovarian cancer. Citation Format: Nazia Bano, Eva M. Schmelz. Integrin expression during ovarian cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3077.

Potent and Targeted Sindbis Virus Platform for Immunotherapy of Ovarian Cancer

Our laboratory has been developing a Sindbis viral (SV) vector platform for treatments of ovarian and other types of cancers. In this study we show that SV.IL-12 combined with an agonistic OX40 antibody can eliminate ovarian cancer in a Mouse Ovarian Surface Epithelial Cell Line (MOSEC) model and further prevent tumors in mice rechallenged with tumor cells after approximately 5 months. Treatment efficacy is shown to be dependent upon T-cells that are transcriptionally and metabolically reprogramed. An influx of immune cells to the tumor microenvironment occurs. Combination of sequences encoding both IL-12 and anti-OX40 into a single SV vector, SV.IgGOX40.IL-12, facilitates the local delivery of immunoregulatory agents to tumors enhancing the anti-tumor response. We promote SV.IgGOX40.IL-12 as a safe and effective therapy for multiple types of cancer.

Epithelial tumor compartment- and adjacent stromal compartment-specific expression of PARP1 in different anatomical sites in patients with HGSOC

High-grade serous ovarian carcinoma (HGSOC) is responsible for the majority of ovarian cancer-associated deaths. PARP (poly-(ADP-ribose) polymerase) inhibitors (PARPi) (olaparib, rucaparib and niraparib) have been approved in recent years to be used in both the frontline setting as maintenance therapy and in the recurrent setting for patients with ovarian cancer (OC). Previous studies have reported an increased expression of PARP1, a sensor for DNA damage, in OC; however, its compartment (epithelial tumor vs stroma)-, anatomical site (fallopian tube, omentum, ovary and serous tubal in situ carcinoma)- and histotype-specific expression have not been studied. Here, we showed that PARP1 protein levels are higher in epithelial tumors compared to adjacent non-malignant stroma at four different anatomical locations in HGSOC, with the most significant difference in PARP1 levels in omental metastases. Furthermore, we found that PARP1 protein levels are elevated in tumors relative to normal tissue in the fallopian tube, omentum and ovary in patients with HGSOC, again with the most significant difference in the omentum. We also showed that PARP1 expression is increased in a malignant and invasive stage in mouse ovarian surface epithelium cells, and that PARP1 mRNA levels are lower in mucinous histotype compared to serous histotype of epithelial OC. These findings indicate that a better understanding of compartment-, anatomical location- and histotype-specific changes in PARP1 levels in OC is needed since PARP inhibitors are currently being used in the treatment of patients with OC.

Abstract 3641: Age-related changes in microenvironmental collagen affect ovarian cancer metastasis

Abstract Aging is the biggest risk factor for the development of ovarian cancer (OvCa), the deadliest cancer of the female reproductive system. Despite this, age is understudied in the OvCa field. Using a C57Bl/6 mouse model of aging, young (Y) mice ranging from 3-6 months of age and aged (A) mice ranging from 20-23 months of age were used to study the role aging has on metastasis. Fluorescently tagged C57Bl/6 syngeneic ID8trp53-/- mouse ovarian surface epithelial cancer cells were injected intraperitoneally in Y and A mice and disease progression was evaluated following 5.5 weeks. Organ-specific tumor burden was quantified with ImageJ, revealing increased tumor burden in A mice compared to their Y counterparts. These results were reproduced in the FVB mouse model using syngeneic PTENshRNA/KRASG12V oviductal epithelial cells. While in vitro assays showed no significant difference in adhesion or invasion of OvCa cells on Y vs A collagen, human cell lines showed increased invasion through Boyden invasion chambers lined with A collagen compared to Y. The relationship between tumors and aged collagen was further investigated by analysis of collagen hybridizing peptide (CHP) staining of paraffin-embedded omental tumor sections, which shows an increase of intratumoral collagen remodeling in A tumors despite no significant difference in overall collagen amount as shown by trichrome analysis of serial sections. However, second harmonic generation microscopy (SHG) and CHP fluorescence analysis of early tumor events shows increased peritumoral collagen remodeling in Y mice, showing a difference between intratumoral collagen and microenvironmental collagen. Immunohistochemical analysis of paraffin-embedded tumor sections showed similar amounts of advanced glycation end products (AGEs) and cancer-associated fibroblasts (CAFs) in Y and A tumors, suggesting intratumoral collagen is newly synthesized by CAFs whereas the collagen in the microenvironment is truly aged. To further investigate peritumoral collagen in the metastatic microenvironment, SHG was used to visualize collagen of common metastatic sites from Y and A tumor naïve C57Bl/6 mice. Distinct structural differences were shown in omental collagen in the Y vs A cohorts and validated with scanning electron microscopy (SEM). In conclusion, aging induces changes in the structure and properties of both intra- and peritumoral collagen, increasing OvCa metastatic success. Citation Format: Elizabeth Harper, Elizabeth Agadi, Emma Sheedy, Preston Carey, Paul Wilkinson, Michael Siroky, Tyvette Hilliard, Ethan Low, Annemarie Leonard, Yueying Liu, Jing Yang, M. Sharon Stack. Age-related changes in microenvironmental collagen affect ovarian cancer metastasis [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 3641.

Differential effects of nanosecond pulsed electric fields on cells representing progressive ovarian cancer

Nanosecond pulsed electric fields (nsPEFs) may induce differential effects on tumor cells from different disease stages and could be suitable for treating tumors by preferentially targeting the late-stage/highly aggressive tumor cells. In this study, we investigated the nsPEF responses of mouse ovarian surface epithelial (MOSE) cells representing progressive ovarian cancer from benign to malignant stages and highly aggressive tumor-initiating-like cells. We established the cell-seeded 3D collagen scaffolds cultured with or without Nocodazole (eliminating the influence of cell proliferation on ablation outcome) to observe the ablation effects at 3 h and 24 h after treatment and compared the corresponding thresholds obtained by numerically calculated electric field distribution. The results showed that nsPEFs induced larger ablation areas with lower thresholds as the cell progress from benign, malignant to a highly aggressive phenotype. This differential effect was not affected by the different doubling times of the cells, as apparent by similar ablation induction after a synergistic treatment of nsPEFs and Nocodazole. The result suggests that nsPEFs could induce preferential ablation effects on highly aggressive and malignant ovarian cancer cells than their benign counterparts. This study provides an experimental basis for the research on killing malignant tumor cells via electrical treatments and may have clinical implications for treating tumors and preventing tumor recurrence after treatment.

Abstract 384: T cell infiltration into tumors induced by DPX-Survivac combination immunotherapy demonstrated by PET/MRI imaging in an orthotopic ovarian cancer model

Abstract Introduction: Epithelial ovarian cancers are highly aggressive and often diagnosed at late stages. To date, a limited number of these patients benefit from immunotherapies, and recurrent ovarian cancer remains an area of unmet medical need. Novel immunotherapies that generate and drive activated T cells into tumors, such as DPX-Survivac, represent a promising approach. To better understand the underlying mechanisms of action of this novel immunotherapy and how it may correlate with clinical outcomes, we used an orthotopic ovarian cancer model and a dual approach using molecular imaging to track immune cells in vivo followed by biological assays. Methods: Humanized transgenic mice (HLA-A2.1-/HLA-DR1; H-2 class-I/II knockout) were implanted with 104 syngeneic mouse ovarian surface epithelial cells in the left ovarian bursa and were either untreated or treated with DPX-Survivac, intermittent low dose cyclophosphamide and anti-PD-1. The CD8+ cytotoxic T cells (CTLs) and myeloid cells (MCs) were isolated from disease matched mice, cultured in vitro, loaded with superparamagnetic iron oxide (SPIO) nanoparticles, and injected i.v. in recipient mice. The next day, recipient mice were injected with 500µCi of 18F-fluorodeoxyglucose and imaged by magnetic resonance (MRI) and positron emission tomography (PET). Anatomical images and tumor volumes were collected with a balance steady-state free precession sequence, cells were semi-quantified using R2* maps from a multi-echo single point imaging sequence (TurboSPI) and tumor metabolism was assessed by simultaneous acquisition of PET. Mice were imaged days 41, 49, and 56 post-implant. CTLs and MCs were characterized by flow cytometry (FC) before injection into recipient mice. Following imaging completion, tumors and lymph nodes (LN) were assayed for tumor infiltrating lymphocytes. Ascites were analyzed for tumor cell to lymphocyte content by FC. Results: PET/MRI images showed that the combination immunotherapy significantly decreased primary tumor burden and improved survival rates. The recruitment of isolated CTLs and MCs was detected in both tumors and LNs. CTL recruitment to tumors was increased in mice receiving combination immunotherapy while MCs recruitment was decreased. CTLs were recruited at a higher rate to the DPX-Survivac draining LN than tumor draining LN and MCs were equally recruited to both. The expression of checkpoint inhibitor molecules on CTL did not appear to vary with treatment. CTLs from treated mice expressed higher rates of Ki67 (a proliferative marker) than CTLs from untreated mice. The treatment increased the lymphocyte:tumor cell ratio within the tumors of treated animals and included a population of CD8/CD4 double positive (DP) T cells. Differences in tumor infiltrating lymphocyte phenotype correlated with tumor burden. Conclusions: DPX-Survivac combination immunotherapy induces the recruitment of CD8+ T cells into tumors, resulting in tumor control in an orthotopic ovarian cancel model. Citation Format: Marie-Laurence Tremblay, Caitrin Sobey-Skelton, Hailey Wyatt, Victoria Gonzalez, Andrea Nuschke, Christa Davis, Alecia Mackay, Kim Bobbit, Andrea West, Barbara Vanderhyden, Genevieve Weir, Alexandra Merkx-Jacques, Kimberly Brewer, Marianne Stanford. T cell infiltration into tumors induced by DPX-Survivac combination immunotherapy demonstrated by PET/MRI imaging in an orthotopic ovarian cancer model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 384.

Non-canonical WNT5a regulates Epithelial-to-Mesenchymal Transition in the mouse ovarian surface epithelium

The ovarian surface epithelium (OSE) is a monolayer that covers the ovarian surface and is involved in ovulation by rupturing and enabling release of a mature oocyte and by repairing the wound after ovulation. Epithelial-to-mesenchymal transition (EMT) is a mechanism that may promote wound healing after ovulation. While this process is poorly understood in the OSE, in other tissues wound repair is known to be under the control of the local microenvironment and different growth factors such as the WNT signaling pathway. Among WNT family members, WNT4 and WNT5a are expressed in the OSE and are critical for the ovulatory process. The objective of this study was to determine the potential roles of WNT4 and WNT5a in regulating the OSE layer. Using primary cultures of mouse OSE cells, we found WNT5a, but not WNT4, promotes EMT through a non-canonical Ca2+-dependent pathway, up-regulating the expression of Vimentin and CD44, enhancing cell migration, and inhibiting the CTNNB1 pathway and proliferation. We conclude that WNT5a is a stimulator of the EMT in OSE cells, and acts by suppressing canonical WNT signaling activity and inducing the non-canonical Ca2+ pathway.

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 (TGFβ1) is a cytokine commonly known to induce an EMT and is present throughout the ovarian microenvironment. We, therefore, hypothesized that TGFβ1 induces an EMT in OSE cells and activates signaling pathways important for wound repair. Treating primary cultures of mouse OSE cells with TGFβ1 induced an EMT mediated by TGFβRI signaling. The transcription factor Snail was the only EMT-associated transcription factor increased by TGFβ1 and, when overexpressed, was shown to increase OSE cell migration. A polymerase chain reaction array of TGFβ signaling targets determined Cyclooxygenase-2 (Cox2) to be most highly induced by TGFβ1. 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 TGFβ1 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 TGFβ1 increases Cox2 to promote wound repair by enhancing cell survival. These data support that TGFβ1 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.

Targeted Detection of Cyclooxygenase-1 in Ovarian Cancer.

Overexpression of cyclooxygenase-1 (COX-1) is associated with the initiation and progression of ovarian cancer, and targeted imaging of COX-1 is a promising strategy for early detection of this disease. We report the discovery of N-[(5-carboxy-X-rhodaminyl)but-4-yl]-3-(1-(4-methoxyphenyl)-5-(p-tolyl)-1H-pyrazol-3-yl)propenamide (CMP) as the first COX-1-targeted optical agent for imaging of ovarian cancer. CMP exhibits light emission at 604 nm (λmax), thereby minimizing tissue autofluorescence interference. In both purified enzyme and COX-1-expressing human ovarian adenocarcinoma (OVCAR-3) cells, CMP inhibits COX-1 at low nanomolar potencies (IC50 = 94 and 44 nM, respectively). CMP's selective binding to COX-1 in OVCAR-3 cells was visualized microscopically as intense intracellular fluorescence. In vivo optical imaging of xenografts in athymic nude mice revealed COX-1-dependent accumulation of CMP in COX-1-expressing mouse ovarian surface epithelial carcinoma (ID8-NGL) and OVCAR-3 cells. These results establish proof-of-principle for the feasibility of targeting COX-1 in the development of new imaging and therapeutic strategies for ovarian cancer.

Abstract 2805: DFMO and 5-azacytidine increase M1 macrophages in the tumor microenvironment of an ovarian cancer mouse model

Abstract Although ovarian cancer has a low incidence rate encompassing roughly 1% of all new cancers, it remains the most deadly gynecologic malignancy. Our lab has designed a novel combination therapy which combines a DNA methyl transferase inhibitor (DNMTi) and ornithine decarboxylase inhibitor that together, alter the tumor microenvironment to inhibit ovarian tumor growth. Previous work has demonstrated that the DNMTi 5-Azacytidine (AZA) activates type I interferon signaling (Li, 2014, Chiappinelli, 2015) to increase IFNγ+ T cells and NK cells and reduce the percentage of macrophages and myeloid derived suppressor cells (MDSCs) in the tumor microenvironment (Stone, 2017). To improve the efficacy of epigenetic therapy, we hypothesized that the addition of α-difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor may decrease activity of MDSCs through inhibition of arginase. Immunocompetent C57Bl/6 mice were injected with syngeneic mouse ovarian surface epithelial (MOSE) tumor cells (ID8-VEGF-Defensin) and subsequently treated with AZA, DFMO, or combination AZA+DFMO. After several weeks, mice developed hemorrhagic ascites that was drained, processed and stained for cell-surface markers and analyzed by flow cytometry to identify T cells, NK cells, and macrophages. We found that in vivo AZA, DFMO, and AZA+DFMO significantly increased survival in mice, decreased tumor burden, and caused recruitment of activated (IFNγ+) CD4+ T cells, CD8+ T cells, and NK Cells compared to vehicle. The combination therapy had a striking increase in survival when compared to single agent treatment; however, the difference in recruited lymphocytes was less striking. Instead, combination therapy led to the most dramatic decrease in immunosuppressive cells such as M2 polarized macrophages and increase in tumor-killing M1 macrophages. Using a CSF1R blocking antibody, we found that depleting macrophages in this model reduced the efficacy of AZA+DFMO treatment, and resulted in fewer M1 macrophages in the tumor microenvironment. We thus conclude that our novel combination therapy modifies macrophage polarization in the tumor microenvironment, recruiting M1 macrophages and prolonging survival. We hypothesize that this treatment regimen could have a widespread impact on macrophage-rich tumors in general, and plan to test this hypothesis in additional tumor models. Citation Format: Meghan Travers, Stephen Brown, Matthew Dunworth, Cassandra Holbert, Robert Casero, Cynthia Zahnow. DFMO and 5-azacytidine increase M1 macrophages in the tumor microenvironment of an ovarian cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2805.

Prognostic values of GMPS, PR, CD40, and p21 in ovarian cancer.

Early detection and prediction of prognosis and treatment responses are all the keys in improving survival of ovarian cancer patients. This study profiled an ovarian cancer progression model to identify prognostic biomarkers for ovarian cancer patients. Mouse ovarian surface epithelial cells (MOSECs) can undergo spontaneous malignant transformation in vitro cell culture. These were used as a model of ovarian cancer progression for alterations in gene expression and signaling detected using the Illumina HiSeq2000 Next-Generation Sequencing platform and bioinformatical analyses. The differential expression of four selected genes was identified using the gene expression profiling interaction analysis (http://gepia.cancer-pku.cn/) and then associated with survival in ovarian cancer patients using the Cancer Genome Atlas dataset and the online Kaplan–Meier Plotter (http://www.kmplot.com) data. The data showed 263 aberrantly expressed genes, including 182 up-regulated and 81 down-regulated genes between the early and late stages of tumor progression in MOSECs. The bioinformatic data revealed four genes (i.e., guanosine 5′-monophosphate synthase (GMPS), progesterone receptor (PR), CD40, and p21 (cyclin-dependent kinase inhibitor 1A)) to play an important role in ovarian cancer progression. Furthermore, the Cancer Genome Atlas dataset validated the differential expression of these four genes, which were associated with prognosis in ovarian cancer patients. In conclusion, this study profiled differentially expressed genes using the ovarian cancer progression model and identified four (i.e., GMPS, PR, CD40, and p21) as prognostic markers for ovarian cancer patients. Future studies of prospective patients could further verify the clinical usefulness of this four-gene signature.

Abstract 1405: PAX2 induces tubular structure in ovarian cancer cells

Abstract In adult tissues, PAX2 protein is expressed in normal oviductal epithelial cells (OVE) but not in normal ovarian surface epithelial cells (OSE). Recent studies reported that PAX2 is expressed in serous ovarian carcinoma cases but the role of PAX2 in enhancing ovarian cancer is unrevealed yet. The aim of this study is to understand the biological consequences after Pax2 overexpression in mouse ovarian surface epithelial (MOSE) cells. We found that Pax2 overexpression in MOSE cells induced the formation of vascular channels both in vitro and in vivo, which indicate a possible contribution of PAX2 to ovarian cancer progression by increasing the vascular channels to supply nutrients to the tumor cells. Citation Format: Kholoud Alwosaibai. PAX2 induces tubular structure in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1405.

Abstract TMEM-029: AGING INCREASES SUSCEPTIBILITY TO OVARIAN CANCER METASTASIS IN A MURINE ALLOGRAFT MODEL

Abstract Ovarian cancer (OvCa) is the leading gynecological malignancy in women in the United States. OvCa metastasizes uniquely, spreading through the peritoneal cavity and generating widespread metastatic sites. The vast majority of OvCa cases occur in women over 40 and the median age at diagnosis is 63 (SEER). Despite age being a significant risk factor for the development of OvCa, there is a paucity of studies addressing the role of aging in OvCa metastasis. To our knowledge, there are no reports utilizing old mice to investigate the effects of age on metastasis in vivo. We designed a study using a C57BL/6 model of aging where young (Y) mice are 3-6 months of age and aged (A) mice are 20-23 months of age, corresponding to young (20-30 years) and aged (60-67 years) humans. Using the C57BL/6 syngeneic ID8 mouse ovarian surface epithelial cell line, we tested the effect of aging on metastatic success in vivo. An allograft study was carried out with Y and A mice that were intraperitoneally injected with 3.7x106 ID8 RFP-tagged cells. The mice were imaged once a week starting at 4.5 weeks post injection and were sacrificed for dissection at 8 weeks post injection. Live imaging suggested OvCa metastasis was more efficient in the aged animals than in the young animals. After dissection, the abdominal organs were imaged ex vivo and tumor burden was quantified. The aged mice displayed heavier tumor burden in the gonadal fat compared to the young. Interestingly, no difference in metastasis to the omentum was detected. To investigate why gonadal fat is more receptive to metastasis in the aged animals, periovarian adipose from 4 young and 4 aged healthy non-tumor bearing mice was isolated for RNAseq analysis. Several immune pathways involving B cells were found to be significantly upregulated in the RNA from aged animals. Studies will be conducted to elucidate the status of B cells in aging periovarian adipose, including immunohistochemistry for CD45 and other B cell markers upregulated in the RNAseq dataset. Citation Format: Elizabeth Loughran, Annemarie Leonard, Ryan Phan, Laura Tarwater, Tyvette Hilliard, Marwa Asem, Yueying Liu, Jing Yang, Yuliya Klymenko, Jeff Johnson, Zonggao Shi, Matthew Leevy, Matthew Ravosa and M. Sharon Stack. AGING INCREASES SUSCEPTIBILITY TO OVARIAN CANCER METASTASIS IN A MURINE ALLOGRAFT MODEL [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-029.

Dysregulation of mitotic machinery genes precedes genome instability during spontaneous pre-malignant transformation of mouse ovarian surface epithelial cells.

BackgroundBased in epidemiological evidence, repetitive ovulation has been proposed to play a role in the origin of ovarian cancer by inducing an aberrant wound rupture-repair process of the ovarian surface epithelium (OSE). Accordingly, long term cultures of isolated OSE cells undergo in vitro spontaneous transformation thus developing tumorigenic capacity upon extensive subcultivation. In this work, C57BL/6 mouse OSE (MOSE) cells were cultured up to passage 28 and their RNA and DNA copy number profiles obtained at passages 2, 5, 7, 10, 14, 18, 23, 25 and 28 by means of DNA microarrays. Gene ontology, pathway and network analyses were focused in passages earlier than 20, which is a hallmark of malignancy in this model.ResultsAt passage 14, 101 genes were up-regulated in absence of significant DNA copy number changes. Among these, the top-3 enriched functions (>30 fold, adj p < 0.05) comprised 7 genes coding for centralspindlin, chromosome passenger and minichromosome maintenance protein complexes. The genes Ccnb1 (Cyclin B1), Birc5 (Survivin), Nusap1 and Kif23 were the most recurrent in over a dozen GO terms related to the mitotic process. On the other hand, Pten plus the large non-coding RNAs Malat1 and Neat1 were among the 80 down-regulated genes with mRNA processing, nuclear bodies, ER-stress response and tumor suppression as relevant terms. Interestingly, the earliest discrete segmental aneuploidies arose by passage 18 in chromosomes 7, 10, 11, 13, 15, 17 and 19. By passage 23, when MOSE cells express the malignant phenotype, the dysregulated gene expression repertoire expanded, DNA imbalances enlarged in size and covered additional loci.ConclusionPrior to early aneuploidies, overexpression of genes coding for the mitotic apparatus in passage-14 pre-malignant MOSE cells indicate an increased proliferation rate suggestive of replicative stress. Concomitant down-regulation of nuclear bodies and RNA processing related genes suggests altered control of nuclear RNA maturation, features recently linked to impaired DNA damage response leading to genome instability. These results, combined with cytogenetic analysis by other authors in this model, suggest that transcriptional profile at passage 14 might induce cytokinesis failure by which tetraploid cells approach a near-tetraploid stage containing primary chromosome aberrations that initiate the tumorigenic drive.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3068-5) contains supplementary material, which is available to authorized users.

Vitamin D postpones the progression of epithelial ovarian cancer induced by 7, 12-dimethylbenz [a] anthracene both in vitro and in vivo.

PurposeOvarian cancer is the most lethal malignancy of the female reproductive system, and the prevention and treatment of ovarian carcinoma are still far from optimal. Epidemiological studies reported that ovarian cancer risk was inversely associated with low level of 25-hydroxy vitamin D [25(OH)]. Therefore, this study focuses on exploring the chemoprevention of vitamin D on epithelial ovarian cancer induced by 7, 12-dimethylbenz [a] anthracene (DMBA).MethodsThe mouse ovarian surface epithelial cells were isolated from estrus mice by mild trypsinization and maintained in completed culture medium by repeated passaging. The malignant transformation of mouse ovarian surface epithelial cells was induced by DMBA in vitro. DMBA was directly injected into the bursa of mouse ovary to produce optimized in vivo ovarian cancer model.ResultsThe results indicate that 1α,25 dihydroxyvitamin D3 may delay malignant transformation of mouse ovarian surface epithelial cells induced by DMBA and significantly decreased the colony formation rate from 18.4% to 3.2% (P<0.05). There was a negative correlation between incidence of DMBA-induced tumor and 25-hydroxy vitamin D level (R2=0.978, P<0.05). Vitamin D3 can delay the progression of ovarian cancer induced by DMBA, and the administration of vitamin D3 during the whole process worked more effectively than the administration only during tumor initiation or promotion. Moreover, we found the vitamin D3 increased the expression of E-cadherin and vitamin D receptor while it decreased the expression of β-catenin.ConclusionWe succeeded in establishment of epithelial ovarian cancer models both in vitro and in vivo. The DMBA-implanted model in mice yields high incidence and specificity of epithelial derived tumors. We also found that vitamin D delays the progression of ovarian cancer. However, spontaneous epithelial ovarian carcinoma models are still to be explored for testing the preventive effects of vitamin D on epithelial ovarian cancer.

Abstract B67: The impact of parity on the metastatic success of ovarian cancer.

Abstract Ovarian cancer is the most fatal gynecological cancer. Epithelial ovarian cancer (OvCa), the most common subtype, usually goes undetected until metastatic and often fatal stages of the disease. OvCa follows a unique form of metastasis, spreading through the peritoneal cavity and forming metastatic sites on the peritoneum. Epidemiologic data suggest that child-bearing, or parity, reduces a woman's risk of developing ovarian cancer, with more births providing greater protection. Despite the association of parity with a decreased incidence of ovarian cancer, very few studies have explored the relationship between parity and metastatic success. A recent study compared metastatic success to the omentum in 12-month-old C57Bl/6 retired breeders and 5-month-old virgin mice, reporting that parous mice are less susceptible to metastasis due to the parity-associated differences in the immune compositional profile in the omental fat band (Cohen et al. 2013). This tumor study compared mice of different ages and did not report specific numbers of pregnancies. To further investigate the role of parity number in OvCa metastasis, we designed a study that controls for age and compares mice with specific parity number. Three age-matched C57Bl/6 groups were evaluated: nulliparous (V), parous 1 (P1), and parous 3 (P3) mice. We tested the effect of parity on metastatic success in vivo with an allograft study using the C57Bl/6 syngeneic ID8 mouse ovarian surface epithelial cell line. ID8 ovarian cancer cells (106) were injected into the peritoneal cavity of V, P1 and P3 mice. In contrast to the results of Cohen at al. that utilized a different syngeneic ovarian cancer cell line, we found no significant difference in metastasis to the omentum in the parous animals, but significantly reduced metastasis to the fat-enveloped ovaries and visceral fat pads in the P3 mice. This suggests that the visceral fat adjacent to the uterus and ovaries in multi-parous animals is a unique environment, resilient to metastasis. Factors in fat tissue responsible for this phenomenon are being investigated. Citation Format: Elizabeth A. Loughran, Ryan Phan, Annemarie K. Leonard, Laura Tarwater, Marwa Asem, Yuliya Klymenko, Yueying Liu, Jing Yang, Jeff Johnson, Matthew Ravosa, M. Sharon Stack. The impact of parity on the metastatic success of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B67.

Enhanced contactless dielectrophoresis enrichment and isolation platform via cell-scale microstructures.

We designed a new microfluidic device that uses pillars on the same order as the diameter of a cell (20 μm) to isolate and enrich rare cell samples from background. These cell-scale microstructures improve viability, trapping efficiency, and throughput while reducing pearl chaining. The area where cells trap on each pillar is small, such that only one or two cells trap while fluid flow carries away excess cells. We employed contactless dielectrophoresis in which a thin PDMS membrane separates the cell suspension from the electrodes, improving cell viability for off-chip collection and analysis. We compared viability and trapping efficiency of a highly aggressive Mouse Ovarian Surface Epithelial (MOSE) cell line in this 20 μm pillar device to measurements in an earlier device with the same layout but pillars of 100 μm diameter. We found that MOSE cells in the new device with 20 μm pillars had higher viability at 350 VRMS, 30 kHz, and 1.2 ml/h (control 77%, untrapped 71%, trapped 81%) than in the previous generation device (untrapped 47%, trapped 42%). The new device can trap up to 6 times more cells under the same conditions. Our new device can sort cells with a high flow rate of 2.2 ml/h and throughput of a few million cells per hour while maintaining a viable population of cells for off-chip analysis. By using the device to separate subpopulations of tumor cells while maintaining their viability at large sample sizes, this technology can be used in developing personalized treatments that target the most aggressive cancerous cells.

Abstract POSTER-THER-1415: Translational study of DPX-survivac peptide vaccine and metronomic cyclophosphamide using a novel HLA-A2/ survivin expressing ovarian tumor model to mimic phase I clinical data

Abstract Survivin, a member of the inhibitors of the apoptosis protein family, is involved in critical pathways of cancer cell growth and survival. Survivin overexpression is detected in 90% of epithelial ovarian cancers. DPX-Survivac is a peptide vaccine containing five survivin HLA class I peptides (A1, A2, A3, A24 and B7) and a universal HLA class II helper peptide derived from tetanus toxin (A16L) formulated in the novel adjuvanting vaccine platform DepoVaxTM. In a phase I clinical study DPX-Survivac was combined with a continuous (metronomic) low dose of cyclophosphamide (mCPA) to treat patients with advanced or recurrent ovarian cancer. In this study, we found mCPA significantly enhanced the immune responses induced by DPX-Survivac. In order to investigate this effect and to understand the mechanisms involved, we developed an ovarian tumor model in HLA-A2 transgenic mice. Vaccination of HLA-A2 transgenic mice generates an immune response towards the A2-restricted survivin peptide in DPX-Survivac. Mouse Ovarian Surface Epithelial (MOSE) cell lines are “natural” ovarian cancer models established by spontaneous in vitro transformation of primary ovarian surface epithelial cells. MOSE cell lines were confirmed to express HLA-A2 and overexpress survivin, and formed subcutaneous and intraperitoneal ovarian tumors when implanted into syngeneic recipients. HLA-A2 transgenic mice bearing MOSE tumors were treated with either DPX-Survivac, mCPA (administrated at 20 mg/kg/day in drinking water) or a combination of mCPA and DPX-Survivac. We found that combining DPX-Survivac with mCPA provided most effective control of tumor growth. Ex vivo IFN-γ ELISPOT revealed that mCPA increases immunogenicity of the DPX-Survivac in mice bearing MOSE tumors. Analysis of the blood and tumor microenvironment by RT-qPCR showed that mice treated with the combination therapy had an altered immune gene signature with increased cytotoxic T lymphocyte markers compared to mice treated with DPX-Survivac or metronomic cyclophosphamide alone. Our data indicates that the combination treatment enhances the infiltration of CD8+ T cells into tumors, enhancing tumor specific immune responses. In summary, we have established a new model of epithelial ovarian cancer in HLA-A2 transgenic mice. This model was used to investigate the immunogenicity and efficacy of DPX-Survivac, an HLA-restricted peptide vaccine with proven immunogenicity in human clinical trials, in combination with the immune modulator mCPA. This model can be used to explore mechanisms of establishment and progression of ovarian cancer as well as for evaluation of new treatment strategies including new immune modulator/ vaccine combinations. Citation Format: Olga Hrytsenko, Genevieve M. Weir, Mohan Karkada, Neil L. Berinstein, Marianne M. Stanford, Marc Mansour. Translational study of DPX-survivac peptide vaccine and metronomic cyclophosphamide using a novel HLA-A2/ survivin expressing ovarian tumor model to mimic phase I clinical data [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1415.

Genetic determinants of FOXM1 overexpression in epithelial ovarian cancer and functional contribution to cell cycle progression.

The FOXM1 transcription factor network is frequently activated in high-grade serous ovarian cancer (HGSOC), the most common and lethal subtype of epithelial ovarian cancer (EOC). We used primary human EOC tissues, HGSOC cell lines, mouse and human ovarian surface epithelial (OSE) cells, and a murine transgenic ovarian cancer model to investigate genetic determinants of FOXM1 overexpression in EOC, and to begin to define its functional contribution to disease pathology. The Cancer Genome Atlas (TCGA) data indicated that the FOXM1 locus is amplified in ~12% of HGSOC, greater than any other tumor type examined, and that FOXM1 amplification correlates with increased expression and poor survival. In an independent set of primary EOC tissues, FOXM1 expression correlated with advanced stage and grade. Of the three known FOXM1 isoforms, FOXM1c showed highest expression in EOC. In murine OSE cells, combined knockout of Rb1 and Trp53 synergistically induced FOXM1. Consistently, human OSE cells immortalized with SV40 Large T antigen (IOSE-SV) had significantly higher FOXM1 expression than OSE immortalized with hTERT (IOSE-T). FOXM1 was overexpressed in murine ovarian tumors driven by combined Rb1/Trp53 disruption. FOXM1 induction in IOSE-SV cells was partially dependent on E2F1, and FOXM1 expression correlated with E2F1 expression in human EOC tissues. Finally, FOXM1 functionally contributed to cell cycle progression and relevant target gene expression in human OSE and HGSOC cell models. In summary, gene amplification, p53 and Rb disruption, and E2F1 activation drive FOXM1 expression in EOC, and FOXM1 promotes cell cycle progression in EOC cell models.

Impact of MDSC emergence during ovarian cancer in cytokine-deficient mice. (TUM6P.1011)

Abstract We have been studying the emergence of myeloid-derived suppressor cells (MDSCs) following i.p. transfer of ID8 cells (mouse ovarian surface epithelial cell carcinoma) into intact, syngeneic, C57BL/6J (wild type, WT) mice. Our current work studies the impact of the cytokine (CK) environment on ID8 expansion and MDSC emergence in the peritoneal cavity (PerC). We wanted to determine if particular pro- (IFNg) or anti- (IL4, IL10) inflammatory CKs were drivers of disease in this model. We found that, relative to WT mice, the development of hemorrhagic ascites was accelerated in IL10-/-, IL4-/-, and IFNgR-/- mice. The greatest percentage and number of MDSCs was observed in IL10-/- mice, which also had the greatest expansion of ID8 cells. ID8 triggered a marked increase in PerC Mfs, particularly in the IL10-/- mice, and lymphocytes, particularly in the IFNgR-/- and IL4-/- mice. However, for all mice, the percent representation of B cells in the total PerC cell pool declined markedly, particularly in the IL10-/- mice (&amp;gt; 70%). Interestingly, beyond the peritoneal locus of disease, both B and T lymphocyte percentages dropped in the spleens of all mice. This was seen without a concomitant increase in the number of MDSCs or appearance of ID8 in the spleen. These observations reinforce that inflammation drives MDSC emergence and suggest that changes in lymphocyte populations could serve as biomarkers for assessing ovarian cancer.