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Background The metzincin family of metalloproteinases and the tissue inhibitors of metalloproteinases (TIMPs) are essential proteins required for biological processes during cancer progression. This study aimed to determine the role of TIMP-2 in ovarian cancer progression and chemoresistance by reducing TIMP-2 expression in vitro in Fallopian tube secretory epithelial (FT282) and ovarian cancer (JHOS2 and OVCAR4) cell lines. Methods FT282, JHOS2 and OVCAR4 cells were transiently transfected with either single or pooled TIMP-2 siRNAs. The expression of different genes after TIMP-2 knock down (T2-KD) or in response to chemotherapy was determined at the mRNA level by quantitative real time PCR (qRT-PCR) and at the protein level by immunofluorescence. Sensitivity of the cell lines in response to chemotherapy after TIMP-2 knock down was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Ethynyl-2′-deoxyuridine (EdU) assays. Cell invasion in response to TIMP-2 knockdown was determined by xCELLigence. Results Sixty to 90 % knock down of TIMP-2 expression was confirmed in FT282, OVCAR4 and JHOS2 cell lines at the mRNA and protein levels. TIMP-2 knock down did not change the mRNA expression of TIMP-1 or TIMP-3. However, a significant downregulation of MMP-2 in T2-KD cells occurred at both the protein and activation levels, compared to Control (Cont; scrambled siRNA) and Parental cells (P, transfection reagent only). In contrast, membrane bound MT1-MMP protein levels were significantly upregulated in T2-KD compared to Cont and P cells. T2-KD cells exhibited enhanced proliferation and increased sensitivity to cisplatin and paclitaxel treatments. Enhanced invasion was observed in the T2-KD-JOSH2 and OVCAR4 cells but not in T2-KD-FT282 cells. Treatment with cisplatin or paclitaxel significantly elevated the expression of TIMP-2 in Cont cells but not in T2-KD cells, consistent with significantly elevated expression of chemoresistance and CSC markers and activation of STAT3. Furthermore, a potent inhibitor of STAT3 activation, Momelotinib, suppressed chemotherapy-induced activation of P-STAT3 in OVCAR4 cells with concomitant reductions in the expression of chemoresistance genes and CSC markers. Conclusions The above results suggest that TIMP-2 may have a novel role in ovarian cancer proliferation, invasion and chemoresistance.

Epithelial ovarian cancer (EOC) is most frequently diagnosed at an advanced stage, and is characterized by its high recurrence rate and almost-universal acquisition of chemoresistance. Metastasis of EOC is driven by heterogenous spheroids that passively disseminate throughout the peritoneal cavity, promoting tumor spread and recurrence. We previously identified a sub-population of highly motile and invasive “Leader cells” (LCs) that express the basal epithelial protein KRT14 as an absolute determinant of invasive potential. We demonstrated KRT14 knockout in EOC cells does not impact viability or proliferation, however completely ablates their ability to invade through Matrigel in 2D and 3D format in vitro. Although a role for LCs in mesothelial clearance and invasion has been established, these mechanisms remain poorly understood. Here we show a critical role for KRT14+ LCs in immunosuppression and tumor-immune privilege in vivo. In the ID8 syngeneic EOC mouse model, tumor-specific LC (KRT14) loss (LCKO) impaired ovarian intrabursal primary tumor engraftment and subsequent metastatic spread. Immune phenotyping by flow cytometry revealed that mice bearing LCKO tumors displayed decreased proportions of immunosuppressive tumor-adjacent Tregs and M2 macrophages, and improved CD8+ T-cell/Treg ratios. Conversely, forced LC-overexpression (LCOE) in EOC tumors accelerated metastasis, and promoted tumor-immune evasion via upregulated secretion of suppressive chemokines including CCL22 and CCL5. In addition to their critical role in migration and invasion, the current study provides in vivo evidence that leader cells are mediators of immune suppression in ovarian cancer. This work supports the basis that defining novel strategies to target LCs to promote anti-tumor immunity and limit metastatic progression may be a useful strategy for improved epithelial ovarian cancer therapy. Citation Format: Amy L. Wilson, Brittany R. Doran, Bashirah Basri, Laura R. Moffitt, Magdalena Plebanski, Andrew N. Stephens, Maree Bilandzic. Leader cells mediate tumor engraftment and promote immunosuppression to drive ovarian cancer progression in vivo [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B040.

Background Leader cells are a subset of cancer cells that coordinate the complex cell-cell and cell-matrix interactions required for ovarian cancer migration, invasion, tumour deposition and are negatively associated with progression-free survival and response to therapy. Emerging evidence suggests leader cells may be enriched in response to chemotherapy, underlying disease recurrence following treatment. Methods CRISPR was used to insert a bicistronic T2A-GFP cassette under the native KRT14 (leader cell) promoter. 2D and 3D drug screens were completed in the presence of chemotherapies used in ovarian cancer management. Leader cell; proliferative (Ki67); and apoptotic status (Cleaved Caspase 3) were defined by live cell imaging and flow cytometry. Quantitative real-time PCR defined “stemness” profiles. Proliferation was assessed on the xCELLigence real time cell analyser. Statistical Analysis was performed using unpaired non-parametric t-tests or one-way ANOVA and Tukey’s multiple comparison post hoc. Results Leader cells represent a transcriptionally plastic subpopulation of ovarian cancer cells that arise independently of cell division or DNA replication, and exhibit a “stemness” profile that does not correlate with epithelial-to-mesenchymal transition. Chemotherapeutics increased apoptosis-resistant leader cells in vitro, who retained motility and expressed known chemo-resistance markers including ALDH1, Twist and CD44v6. Functional impairment of leader cells restored chemosensitivity, with leader cell-deficient lines failing to recover following chemotherapeutic intervention. Conclusions Our data demonstrate that ovarian cancer leader cells are resistant to a diverse array of chemotherapeutic agents, and are likely to play a critical role in the recurrence of chemo-resistant disease as drivers of poor treatment outcomes.

OBJECTIVE: To establish a model of human implantation that responds to hormonal stimuli and can differentiate between endometrium from fertile women and those with idiopathic infertility. DESIGN: A trophoblast stem cell (trophectodermal) line (TSC; derived from human pre-implantation embryo) was used to form trophectodermal spheroids (TS). TS attachment to monolayers of endometrial epithelial cell lines or primary endometrial epithelial cells (pHEECs) was determined. SETTING: Independent Medical Research Institute with close clinical linkages INTERVENTIONS: Spheroid attachment and outgrowth was determined with added hormones (estradiol 17β (E), E + medroxyprogesterone acetate (MPA) or E + MPA + human chorionic gonadotropin (hCG)). Spheroid attachment to E/MPA treated pHEEC prepared from fertile women or those with idiopathic infertility tested. MAIN OUTCOME MEASURE: Firmly attached spheroids counted after co-culture for 6 h. Outgrowth was determined by quantitation of area covered by spheroid after firm adhesion. RESULTS: Functional adhesion of TS to two endometrial epithelial cell lines, Ishikawa and ECC-1 cells, was hormonally responsive, with adhesion/outgrowth increased by E/MPA (ECC-1; p < 0.01, Ishikawa; p < 0.01) and E/MPA/hCG (ECC-1; p < 0.001, Ishikawa p < 0.01) versus E alone. The same pattern of hormone responsiveness was observed in pHEEC obtained from fertile women (E vs, E/MPA; p < 0.01, E vs. E/MPA/hCG; p < 0.001). TS adhered to 85% of pHEEC obtained from fertile women (11/13) and 11% of pHEEC obtained from women with unexplained infertility (2/18, p < 0.001). CONCLUSION: This new model of “embryo” implantation largely discriminates between endometrial epithelial cells obtained from fertile vs. infertile women based on adhesion; this holds potential as an in vitro “diagnostic” tool of endometrial infertility.

Majority of ovarian cancers are diagnosed at advanced stages with intra-peritoneal spread as the most common mode of disease metastasis. The formation of cancer spheroids is essential for the collective migration process, where shed tumour cells from the primary tumour form aggregates rather than disseminating as individual cells and seed within the peritoneal cavity. These cancer spheroids consist of leader cells (LC) and follower cells (FC), with the LC subset as key drivers of cellular movement and invasion. LCs have stem cell-like properties and are highly chemo-resistant with a specific survival addiction to several cell signalling pathways, such as the PI3K/AKT/mTOR pathway. We explore in this book chapter, the evidence supporting the role of LC in OC metastasis and the suppression of LC as an attractive therapeutic option for the treatment of advanced OC.

Simple Summary The role of immunity in the development and progression of epithelial ovarian cancer (EOC) is well established. Poor T-cell infiltration is associated with mortality in EOC patients, and recent evidence has suggested that the enzyme DPP4 plays a role in this process. The aim of our study was to evaluate the potential of the clinically-approved DPP4-inhibitor sitagliptin to improve immune responses in mice with EOC. We showed that sitagliptin improved CD8+ T-cell responses in an EOC mouse model, consequently reducing metastatic burden and prolonging survival. These data provide a rationale for the use of DPP4-inhibitors as a second-line treatment for EOC. Abstract Immunity plays a key role in epithelial ovarian cancer (EOC) progression with a well-documented correlation between patient survival and high intratumoral CD8+ to T regulatory cell (Treg) ratios. We previously identified dysregulated DPP4 activity in EOCs as a potentially immune-disruptive influence contributing to a reduction in CXCR3-mediated T-cell infiltration in solid tumours. We therefore hypothesized that inhibition of DPP4 activity by sitagliptin, an FDA-approved inhibitor, would improve T-cell infiltration and function in a syngeneic ID8 mouse model of EOC. Daily oral sitagliptin at 50 mg/kg was provided to mice with established primary EOCs. Sitagliptin treatment decreased metastatic tumour burden and significantly increased overall survival and was associated with significant changes to the immune landscape. Sitagliptin increased overall CXCR3-mediated CD8+ T-cell trafficking to the tumour and enhanced the activation and proliferation of CD8+ T-cells in tumour tissue and the peritoneal cavity. Substantial reductions in suppressive cytokines, including CCL2, CCL17, CCL22 and IL-10, were also noted and were associated with reduced CD4+ CD25+ Foxp3+ Treg recruitment in the tumour. Combination therapy with paclitaxel, however, typical of standard-of-care for patients in palliative care, abolished CXCR3-specific T-cell recruitment stimulated by sitagliptin. Our data suggest that sitagliptin may be suitable as an adjunct therapy for patients between chemotherapy cycles as a novel approach to enhance immunity, optimise T-cell-mediated function and improve overall survival.

The treatment of ovarian cancer has not significantly changed in decades and it remains one of the most lethal malignancies in women. The serine protease dipeptidyl peptidase 4 (DPP4) plays key roles in metabolism and immunity, and its expression has been associated with either pro- or anti-tumour effects in multiple tumour types. In this study, we provide the first evidence that DPP4 expression and enzyme activity are uncoupled under hypoxic conditions in ovarian cancer cells. Whilst we identified strong up-regulation of DPP4 mRNA expression under hypoxic growth, the specific activity of secreted DPP4 was paradoxically decreased. Further investigation revealed matrix metalloproteinases (MMP)-dependent inactivation and proteolytic shedding of DPP4 from the cell surface, mediated by at least MMP10 and MMP13. This is the first report of uncoupled DPP4 expression and activity in ovarian cancer cells, and suggests a previously unrecognized, cell- and tissue-type-dependent mechanism for the regulation of DPP4 in solid tumours. Further studies are necessary to identify the functional consequences of DPP4 processing and its potential prognostic or therapeutic value.

Additional file 6: Table S1. Microscope filters used for the Immunofluorescence study

Epithelial ovarian cancer metastasis is driven by spheroids, which are heterogeneous cancer cell aggregates released from the primary tumour mass that passively disseminate throughout the peritoneal cavity to promote tumour spread, disease recurrence, and acquired chemoresistance. Despite their clinical importance, the molecular events that control spheroid attachment and invasion into underlying healthy tissues remain poorly understood. We examined a novel in vitro invasion model using imaging mass spectrometry to establish a &ldquo, snapshot&rdquo, of the spheroid/mesothelial interface. Amongst numerous adhesion-related proteins, we identified a sub-population of highly motile, invasive cells that expressed the basal epithelial marker KRT14 as an absolute determinant of invasive potential. The loss of KRT14 completely abrogated the invasive capacity, but had no impact on cell viability or proliferation, suggesting an invasion-specific role. Our data demonstrate KRT14 cells as an ovarian cancer &ldquo, leader cell&rdquo, phenotype underlying tumor invasion, and suggest their importance as a clinically relevant target in directed anti-tumour therapies.

This study tested the hypothesis that inhibins act in an autocrine manner on Leydig cells using a pre-pubertal Leydig cell line, TM3, as a model of immature Leydig cells. The expression of Inha, Inhba, and Inhbb in TM3 cells was determined by RT-PCR and the production of the inhibin-alpha subunit was confirmed by western blot. Knockdown of Inha expression resulted in significant decreases in the expression of Leydig cell markers Cyp17a1, Cyp11a1, Nr5a1, and Insl3. Western blot showed that activin A, TGFβ1 and TGFβ2 activated SMAD2, and that knockdown of Inha expression in TM3 cells enhanced both activin A- and TGFβ-induced SMAD2 activation. SB431542, a chemical inhibitor of the TGFβ/activin type I receptors, blocked ligand-induced SMAD2 activation and the downregulation of Cyp17a1 expression. Our findings demonstrate that TGFβs and activin A negatively regulate steroidogenic gene expression in TM3 cells via ALK4/5 and SMAD2 and endogenous inhibins can counter this regulation.

Despite its relatively low incidence ovarian cancer is one of the most lethal gynecological malignancies, characterized by high recurrence rate following primary therapy and almost universal acquisition of chemoresistance. Recently, we identified a subpopulation of “Leader Cells” (LCs) that control invasion, progression and acquired chemoresistance. Therapies targeting leader cells are likely to be pivotal to improving prognosis, particularly in patients who are refractory to standard-of-care. To investigate the role of LCs in chemoresistance, we used CRISPR to integrate an in-frame T2A-GFP expression cassette downstream of the native LC marker gene, Keratin 14 (KRT14). Genomic integration drives the LC specific expression of GFP, facilitating visualization and quantitative tracking of the LC subset. The status of GFP as a surrogate marker of LCs in vitro was confirmed by immunostaining and expression analyses. Chemotherapeutic intervention studies and co-staining with the apoptosis marker cleaved caspase 3, demonstrated a significant enrichment of LCs that represent a non-apoptotic subset of cells resistant to standard chemotherapy. High-throughput drug screening, combining large functional drug libraries with high-throughput viability and invasion assays, was used to identify drugs that could inhibit LC function in vitro. Ovarian cancer cell lines (SKOV3 and COV362.4 expressing KRT14-T2A-GFP) were screened against 1450 FDA-approved drugs, and 218 hits identified that inhibited LC viability, invasive capacity or both. Amongst the hits that we identified, the neural signaling pathway emerged as a potential novel mediator for LC function. Further validation experiments confirmed that inhibition of the neural signaling pathway did not affect viability but synergized with chemotherapy to reduce cisplatin IC50 by 10-20-fold. Our data demonstrate that the high-throughput screen developed is a viable approach to identify opportunities for drug repurposing. Moreover, the neural signaling pathway appears to be important in LC function and inhibiting this pathway results in increased sensitivity to chemotherapy in vitro. Ongoing work will evaluate the applicability of this new tool for clinical screening programs, and further, validate existing drugs for potential repurposing. Citation Format: Nazanin Karimnia, Maree Bilandzic, Magdalena Plebanski, Andrew N. Stephens. High-throughput screening program to target ovarian cancer leader cells [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 6338.

Ovarian cancers are the most lethal gynecologic malignancy, with high levels of relapse and acquired chemoresistance. Immunity clearly plays a role in ovarian tumor growth, but the inability to accurately correlate progression with stage-specific changes in immunity—both over time and in response to therapy—has hampered our understanding of antitumor immune responses. There is a clear need for improved methods to accurately model and monitor ovarian cancer progression and study its interplay with immunity in vivo. We have generated and characterized an ovarian cancer mouse model allowing noninvasive, real-time tumor imaging with stage-specific tracking of progression over time. Using CRISPR, we integrated the near infra-red phytochrome iRFP720 into the ROSA26 locus of ID8 mouse ovarian cancer cells, and intrabursally implanted these into syngeneic C57BL/6 mice. Noninvasive fluorescence imaging was correlated with post-mortem tumor deposition, and immune populations were assessed at each stage to link immune status with disease progression. Finally, we tested whether this model could be used to monitor therapeutic responses in vivo in real time. Fluorescence imaging accurately identified four distinct stages of cancer progression; (i) primary tumor establishment and growth over 3 weeks; (ii) initial dissemination; (iii) development and expansion of multiple metastases; and (iv) end-stage accumulation of peritoneal ascites. These observations were validated post-mortem. Weight and circumference increase, the standard parameters for tumor burden, did not occur until extensive disease dissemination (week 8), demonstrating their inadequacy for early tumor monitoring. There were no significant differences in immune populations between wild-type and iRFP+ tumor-bearing mice by flow cytometry, indicating iRFP expression by itself does not alter immune profiles. Tumor progression, by contrast, was clearly correlated with decreased frequencies of T-helper 1 (Th1) cells, increased Th2 cells, and a stage-specific decrease in Teff:Treg cell ratios in the peritoneal cavity. Treatment with paclitaxel reduced the tumor volume detected by fluorescence, with tumor recurrence over time. Moreover, paclitaxel depleted peritoneal CD8+ Teff cells, dendritic cells and Th1 cells. Our data demonstrate the effective use of noninvasive fluorescence imaging to discretely stage and quantify ovarian tumor progression, and to correlate therapeutic responses in real time. Moreover, the model mimics the progression of human disease, despite lacking genetic equivalence to human ovarian cancer, and hence has utility for the stage-specific analysis of immune-mediated function. Citation Format: Amy L. Wilson, Kirsty L. Wilson, Maree Bilandzic, Laura R. Moffitt, Magdalena Plebanski, Andrew N. Stephens. Noninvasive tumor tracking and characterization of stage-specific immunity in a syngeneic mouse model of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B69.

Ovarian cancers (OCs) are the most lethal gynaecological malignancy, with high levels of relapse and acquired chemo-resistance. Whilst the tumour&ndash, immune nexus controls both cancer progression and regression, the lack of an appropriate system to accurately model tumour stage and immune status has hampered the validation of clinically relevant immunotherapies and therapeutic vaccines to date. To address this need, we stably integrated the near-infrared phytochrome iRFP720 at the ROSA26 genomic locus of ID8 mouse OC cells. Intrabursal ovarian implantation into C57BL/6 mice, followed by regular, non-invasive fluorescence imaging, permitted the direct visualization of tumour mass and distribution over the course of progression. Four distinct phases of tumour growth and dissemination were detectable over time that closely mimicked clinical OC progression. Progression-related changes in immune cells also paralleled typical immune profiles observed in human OCs. Specifically, we observed changes in both the CD8+ T cell effector (Teff):regulatory (Treg) ratio, as well as the dendritic cell (DC)-to-myeloid derived suppressor cell (MDSC) ratio over time across multiple immune cell compartments and in peritoneal ascites. Importantly, iRFP720 expression had no detectible influence over immune profiles. This new model permits non-invasive, longitudinal tumour monitoring whilst preserving host&ndash, tumour immune interactions, and allows for the pre-clinical assessment of immune profiles throughout disease progression as well as the direct visualization of therapeutic responses. This simple fluorescence-based approach provides a useful new tool for the validation of novel immuno-therapeutics against OC.

Purpose For the vast majority of ovarian cancer patients, optimal surgical debulking remains a key prognostic factor associated with improved survival. A standardized, biomarker-based test, to preoperatively discriminate benign from malignant disease and inform appropriate patient triage, is highly desirable. However, no fit-for-purpose biomarkers have yet been identified. Experimental design We conducted a pilot study consisting of 40 patient urine samples (20 from each group), using label-free quantitative (LFQ) mass spectrometry, to identify potential biomarker candidates in urine from individual ovarian cancer patients. To validate these changes, we used parallel reaction monitoring (PRM) to investigate their abundance in an independent validation cohort (n = 20) of patient urine samples. Results LFQ analyses identified 4394 proteins (17 027 peptides) in a discovery set of 20 urine samples. Twenty-three proteins were significantly elevated in the malignant patient group compared to patients with benign disease. Several proteins, including LYPD1, LYVE1, PTMA, and SCGB1A1 were confirmed to be enriched in the urine of ovarian cancer patients using PRM. We also identified the established ovarian cancer biomarkers WFDC2 (HE4) and mesothelin (MSLN), validating our approach. Conclusions and clinical relevance This is the first application of a LFQ-PRM workflow to identify and validate ovarian cancer-specific biomarkers in patient urine samples.

Ovarian cancer is the seventh most commonly diagnosed cancer amongst women and has the highest mortality rate of all gynaecological malignancies. It is a heterogeneous disease attributed to one of three cell types found within the reproductive milieu: epithelial, stromal, and germ cell. Each histotype differs in etiology, pathogenesis, molecular biology, risk factors, and prognosis. Furthermore, the origin of ovarian cancer remains unclear, with ovarian involvement secondary to the contribution of other gynaecological tissues. Despite these complexities, the disease is often treated as a single entity, resulting in minimal improvement to survival rates since the introduction of platinum-based chemotherapy over 30 years ago. Despite concerted research efforts, ovarian cancer remains one of the most difficult cancers to detect and treat, which is in part due to the unique mode of its dissemination. Ovarian cancers tend to invade locally to neighbouring tissues by direct extension from the primary tumour, and passively to pelvic and distal organs within the peritoneal fluid or ascites as multicellular spheroids. Once at their target tissue, ovarian cancers, like most epithelial cancers including colorectal, melanoma, and breast, tend to invade as a cohesive unit in a process termed collective invasion, driven by specialized cells termed “leader cells”. Emerging evidence implicates leader cells as essential drivers of collective invasion and metastasis, identifying collective invasion and leader cells as a viable target for the management of metastatic disease. However, the development of targeted therapies specifically against this process and this subset of cells is lacking. Here, we review our understanding of metastasis, collective invasion, and the role of leader cells in ovarian cancer. We will discuss emerging research into the development of novel therapies targeting collective invasion and the leader cell population.

The therapeutic efficacy of two bis(thiosemicarbazonato) copper complexes, glyoxalbis[N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(gtsm)] and diacetylbis[N4-methylthiosemicarbazonato]Cu(II) [Cu(II)(atsm)], for the treatment of prostate cancer was assessed in cell culture and animal models. Distinctively, copper dissociates intracellularly from Cu(II)(gtsm) but is retained by Cu(II)(atsm). We further demonstrated that intracellular H2gtsm [reduced Cu(II)(gtsm)] continues to redistribute copper into a bioavailable (exchangeable) pool. Both Cu(II)(gtsm) and Cu(II)(atsm) selectively kill transformed (hyperplastic and carcinoma) prostate cell lines but, importantly, do not affect the viability of primary prostate epithelial cells. Increasing extracellular copper concentrations enhanced the therapeutic capacity of both Cu(II)(gtsm) and Cu(II)(atsm), and their ligands (H2gtsm and H2atsm) were toxic only toward cancerous prostate cells when combined with copper. Treatment of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model with Cu(II)(gtsm) (2.5 mg/kg) significantly reduced prostate cancer burden (∼70%) and severity (grade), while treatment with Cu(II)(atsm) (30 mg/kg) was ineffective at the given dose. However, Cu(II)(gtsm) caused mild kidney toxicity in the mice, associated primarily with interstitial nephritis and luminal distention. Mechanistically, we demonstrated that Cu(II)(gtsm) inhibits proteasomal chymotrypsin-like activity, a feature further established as being common to copper-ionophores that increase intracellular bioavailable copper. We have demonstrated that increasing intracellular bioavailable copper can selectively kill cancerous prostate cells in vitro and in vivo and have revealed the potential for bis(thiosemicarbazone) copper complexes to be developed as therapeutics for prostate cancer.

Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas.

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

STUDY QUESTION Do human blastocysts which subsequently implant release factors that regulate endometrial epithelial cell gene expression and adhesion to facilitate endometrial receptivity? SUMMARY ANSWER Blastocysts which subsequently implanted released factors that altered endometrial epithelial gene expression and facilitated endometrial adhesion while blastocysts that failed to implant did not. WHAT IS KNOWN ALREADY Human preimplantation blastocysts are thought to interact with the endometrium to facilitate implantation. Very little is known of the mechanisms by which this occurs and to our knowledge there is no information on whether human blastocysts facilitate blastocyst attachment to the endometrium. STUDY DESIGN, SIZE, DURATION We used blastocyst-conditioned medium (BCM) from blastocysts that implanted (n = 28) and blastocysts that did not implant (n = 28) following IVF. Primary human endometrial epithelial cells (HEECs) (n = 3 experiments) were treated with BCM and the effect on gene expression and adhesion to trophoblast cells determined. We compared the protein production of selected genes in the endometrium of women with normal fertility (n = 40) and infertility (n = 6) during the receptive phase. PARTICIPANTS/MATERIALS, SETTING, METHODS We used real-time RT-PCR arrays containing 84 genes associated with the epithelial to mesenchymal transition. We validated selected genes by real-time RT-PCR (n = 3) and immunohistochemistry in the human endometrium (n = 46). Adhesion assays were performed using HEECs and a trophoblast cell line (n = 3). MAIN RESULTS AND THE ROLE OF CHANCE Blastocysts that implanted released factors that differentially altered mRNA levels for six genes (>1.5 fold) compared with blastocysts that did not implant. A cohort of genes was validated at the protein level: SPARC and Jagged1 were down-regulated (P < 0.01), while SNAI2 and TGF-B1 were up-regulated (P < 0.05) by implanted compared with non-implanted BCM. Jagged-1 (P < 0.05) and Snai-2 protein (P < 0.01) showed cyclical changes in the endometrium across the cycle, and Jagged-1 staining differed in women with normal fertility versus infertility (only) (P < 0.01). HEEC adhesion to a trophoblast cell line was increased after treatment with implanted BCM compared with untreated control (P < 0.05). LIMITATIONS, REASONS FOR CAUTION This is an in vitro study and it would be beneficial to validate our findings using a physiological model, such as mouse. WIDER IMPLICATIONS OF THE FINDINGS This new strategy has identified novel pathways that may be important for human preimplantation blastocyst-endometrial interactions and opens the possibility of examining and manipulating specific pathways to improve implantation and pregnancy success. STUDY FUNDING/COMPETING INTEREST This study was supported by the National Health and Medical Research Council of Australia (Fellowship support #550905, #611827) and project grants by Monash IVF, Australia. There are no conflicts of interest to be declared.

The molecular pathways controlling granulosa cell tumor (GCT) survival are poorly understood. In many cell types, nuclear factor-κB (NFκB) and TGFβ coordinately regulate cell survival to maintain tissue homeostasis. Because GCT cell lines exhibit constitutively activated NFκB, we hypothesized that NFκB blocks TGFβ-mediated cell death in GCT cells. To test this hypothesis, we used the human GCT cell line KGN, which exhibits loss of betaglycan, a TGFβ co-receptor. After inhibition of NFκB in KGN cells, re-expression of betaglycan resulted in a decrease in cell viability, which was further decreased by TGFβ2. Intriguingly, TGFβ2 increased NFκB reporter activity in control cells, but betaglycan expression suppressed both basal and TGFβ2-stimulated NFκB activity. Chemical inhibition of Mothers against decapentaplegic homolog 2/3 (SMAD2/3) signaling or SMAD2/3 gene silencing revealed that both SMADs contributed to cell survival. Furthermore, inhibiting NFκB activity resulted in a specific reduction in SMAD3 expression. Conversely, overexpression of SMAD3 increased basal NFκB activity and countered betaglycan-mediated suppression of NFκB activity. Finally, ERK1/2 activation emerged as the point of convergence of NFκB, SMAD3, and TGFβ2/betaglycan governance of GCT cell viability. Key findings in KGN cells were reproduced in a second GCT cell line, COV434. Collectively, our data establish that both SMAD2/3 and NFκB signaling pathways support GCT cell viability and suggest the existence of a positive feedback loop between NFκB and SMAD3 signaling in late-stage GCT. Furthermore, our data suggest that loss of betaglycan during tumor progression in GCT alters the functional outcomes generated by NFκB and TGFβ pathway cross talk.

Tumor cells in ascites are a major source of disease recurrence in ovarian cancer patients. In an attempt to identify and profile the population of ascites cells obtained from ovarian cancer patients, a novel method was developed to separate adherent (AD) and non-adherent (NAD) cells in culture. Twenty-five patients were recruited to this study; 11 chemonaive (CN) and 14 chemoresistant (CR). AD cells from both CN and CR patients exhibited mesenchymal morphology with an antigen profile of mesenchymal stem cells and fibroblasts. Conversely, NAD cells had an epithelial morphology with enhanced expression of cancer antigen 125 (CA125), epithelial cell adhesion molecule (EpCAM) and cytokeratin 7. NAD cells developed infiltrating tumors and ascites within 12-14 weeks after intraperitoneal (i.p.) injections into nude mice, whereas AD cells remained non-tumorigenic for up to 20 weeks. Subsequent comparison of selective epithelial, mesenchymal and cancer stem cell (CSC) markers between AD and NAD populations of CN and CR patients demonstrated an enhanced trend in mRNA expression of E-cadherin, EpCAM, STAT3 and Oct4 in the NAD population of CR patients. A similar trend of enhanced mRNA expression of CD44, MMP9 and Oct4 was observed in the AD population of CR patients. Hence, using a novel purification method we demonstrate for the first time a distinct separation of ascites cells into epithelial tumorigenic and mesenchymal non-tumorigenic populations. We also demonstrate that cells from the ascites of CR patients are predominantly epithelial and show a trend towards increased mRNA expression of genes associated with CSCs, compared to cells isolated from the ascites of CN patients. As the tumor cells in the ascites of ovarian cancer patients play a dominant role in disease recurrence, a thorough understanding of the biology of the ascites microenvironment from CR and CN patients is essential for effective therapeutic interventions.

Betaglycan is a co-receptor for the TGFβ superfamily, particularly important in establishing the potency of its ligands on their target cells. In recent years, new insights have been gained into the structure and function of betaglycan, expanding its role from that of a simple co-receptor to include additional ligand-dependent and ligand-independent roles. This review focuses on recent advances in the betaglycan field, with a particular emphasis on its newly discovered actions in mediating the trafficking of TGFβ superfamily receptors and as a determinant of the functional output of TGFβ superfamily signalling. In addition, this review encompasses a discussion of the emerging roles of the betaglycan/inhibin pathway in reproductive cancers and disease.

Betaglycan is a co-receptor for the TGFβ superfamily, particularly important in establishing the potency of its ligands on their target cells. In recent years, new insights have been gained into the structure and function of betaglycan, expanding its role from that of a simple co-receptor to include additional ligand-dependent and ligand-independent roles. This review focuses on recent advances in the betaglycan field, with a particular emphasis on its newly discovered actions in mediating the trafficking of TGFβ superfamily receptors and as a determinant of the functional output of TGFβ superfamily signalling. In addition, this review encompasses a discussion of the emerging roles of the betaglycan/inhibin pathway in reproductive cancers and disease.

Betaglycan is a type III TGFβ receptor that modulates cellular sensitivity to inhibins and TGFβ. Previous studies have suggested that betaglycan acts as a tumor suppressor in certain human epithelial cancers. However, the roles of betaglycan in ovarian granulosa cell tumors (GCTs) are poorly understood. The objective of this study was to determine whether human GCTs exhibit betaglycan expression and, if so, what impact this receptor has on tumor biology. Real-time PCR was used to quantify betaglycan transcripts in human GCTs (n = 17) and normal premenopausal ovaries (n = 11). This analysis established that GCTs exhibited a significant 2-fold lower mean betaglycan mRNA level as compared with the normal ovary (P < 0.05). Similarly, two human GCT cell lines, KGN and COV434, exhibited low betaglycan expression and poor responsiveness to TGFβ and inhibin A in luciferase reporter assays, which was restored by stable transfection of wild-type betaglycan. Betaglycan significantly increased the adhesion of COV434 (P < 0.05) and KGN (P < 0.0001) cells, decreased cellular invasion through Matrigel, and inhibited wound healing. Expression of mutant forms of betaglycan that are defective in TGFβ and/or inhibin binding in each GCT cell line revealed that the inhibitory effects of betaglycan on wound healing were most strongly linked to the inhibin-binding region of betaglycan. Furthermore, knockdown of INHA mRNA expression abrogated the betaglycan-mediated inhibition of wound healing and invasion, whereas both INHA silencing and TGFβ neutralization abolished the betaglycan-mediated increase in adhesion to substrate. These data suggest that loss of betaglycan contributes to the pathogenesis of GCTs.

Background and Aims: Ovarian cancer is the most common cause of death from gynaecological cancer worldwide (1). Often called the “silent killer”, the high mortality rate is associated with the late stage in which the disease is diagnosed (2). Unlike other solid cancers, ovarian cancer cells rarely spread via the vasculature, instead shed from the primary tumor and disseminate within the peritoneal fluid or ascites (2). The formation of spheroids within the non-adherent peritoneal environment is a barrier to effective treatment, due to the enhanced ability of spheroids to survive chemotherapies and seed distal metastases (3-5). Invading ovarian cancer cells interact with mesothelial cells lining the peritoneal surface, attach to, and invade the underlying basement membrane to establish secondary lesions (6). The molecular events at the interface between invading ovarian cancer and peritoneal cells at the onset of invasion have been poorly studied, existing methods capture events after invasion is well-underway. Therefore, the regulatory signals which govern the initiation of a metastatic lesion are poorly understood. We aimed: i) to develop an innovative method to co-culture ascites-derived primary ovarian cancer cells to measure invasion through peritoneal mesothelial cell layers in real time (7); ii) identify the unique protein expression profile at the ovarian cancer spheroid-peritoneal interface during early invasion using proteomics. Methods: Real Time Cell Analysis (RTCA) technology (xCELLigence, ACEA) was adapted to establish a three-dimensional co-culture model of the tumor microenvironment of the peritoneum (7). RTCA wells were coated with Matrigel (basement membrane matrix) and a confluent monolayer of human mesothelial cells. Multicellular spheroids were generated from primary ovarian cancer lines (high-grade serous carcinoma, leiomysarcoma, adenocarcinoma, carcinosarcoma) or benign ascites derived cells cultured under non-adherent conditions. Spheroids were added to the RTCA plate and real time measurements were conducted to determine the precise period when invasion commenced. Parallel cultures were histologically-preserved at invasion initiation and processed for assessment by Matrix-Assisted Laser Desorption/Ionization (MALDI) imaging. Results: We assessed invasion in a panel of ovarian cancer samples, benign ascites cells and the mesothelial cell line. Continuous real time measurements revealed that individual ovarian cancer samples exhibited differences in the timing of the onset of invasion. High-grade serous carcinoma samples initiated invasion rapidly after addition, with some samples detected in under 30 min, a time period not captured by traditional Transwell assays. The benign and mesothelial cells themselves did not invade. MALDI imaging allowed for the identification of peptides expressed at the tumor-mesothelial interface during early invasion. These molecules represent novel molecules involved in the initiation of the metastatic process and are the subject of further characterisation. Conclusion: This method represents a high-throughput quantitative analysis of ovarian cancer spheroid invasion of mesothelial and ECM barriers. The study has provided an unprecedented insight into the molecular mechanisms and molecules involved in the initiation of ovarian cancer invasion of the mesothelium, identifying new therapeutic targets. References: 1. Marsha Rivkin Center for Ovarain Cancer Research - Facts and Figures Survival Rates. 2014. 2. Ahmed N et al., Front Oncol. 2013;3(256). 3. Aguilar-Gallardo C et al., Stem Cell Rev. 2012 Sep;8(3):994-1010. 4. Burleson KM et al., Gynecol Oncol. 2004 Apr;93(1):170-81. 5. Liao J et al., PLoS One. 2014;9(1):e84941 6. Kenny HA et al., Int J Cancer. 2007 Oct 1;121(7):1463-72. 7. Bilandzic M et al., Journal of Visual Experimentation. 2014 20/05/2014 (87). Citation Format: Maree Bilandzic, Adam Rainczuk, Andrew Stephens, Nicole Fairweather, Thomas Jobling, Kaye L Stenvers. Assessment of ovarian cancer spheroid attachment and invasion of mesothelial cells in real time to profile the molecular signature of the invasive interface [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-TECH-1105.