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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.

High grade serous carcinoma (HGSC) metastasises primarily intraperitoneally via cancer spheroids. Podocalyxin (PODXL), an anti-adhesive transmembrane protein, has been reported to promote cancer survival against chemotherapy, however its role in HGSC chemoresistance is unclear. This study investigated whether PODXL plays a role in promoting chemoresistance of HGSC spheroids. We first showed that PODXL was expressed variably in HGSC patient tissues (n = 17) as well as in ovarian cancer cell lines (n = 28) that are more likely categorised as HGSC. We next demonstrated that PODXL-knockout (KO) cells proliferated more slowly, formed less compact spheroids and were more fragile than control cells. Furthermore, when treated with carboplatin and examined for post-treatment recovery, PODXL-KO spheroids showed significantly poorer cell viability, lower number of live cells, and less Ki-67 staining than controls. A similar trend was also observed in ascites-derived primary HGSC cells (n = 6)—spheroids expressing lower PODXL formed looser spheroids, were more vulnerable to fragmentation and more sensitive to carboplatin than spheroids with higher PODXL. Our studies thus suggests that PODXL plays an important role in promoting the formation of compact/hardy HGSC spheroids which are more resilient to chemotherapy drugs; these characteristics may contribute to the chemoresistant nature of HGSC.

ObjectiveTo establish a model of human implantation that responds to hormonal stimuli and can differentiate between endometrium from fertile women and those with idiopathic infertility.DesignA 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.SettingIndependent Medical Research Institute with close clinical linkagesInterventionsSpheroid 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 measureFirmly attached spheroids counted after co-culture for 6 h. Outgrowth was determined by quantitation of area covered by spheroid after firm adhesion.ResultsFunctional 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).ConclusionThis 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.

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.

Background High grade serous carcinoma (HGSC) is the most common and lethal subtype of ovarian cancer, yet its prognosis has remained unchanged in the past 3 decades. HGSC is known to have evolved immune evasion strategies to promote survival, but these mechanisms are not well understood. Podocalyxin (PODXL), a CD34-related sialomucin, is often expressed in HGSC patients with poor prognosis. We have recently reported that PODXL promotes the formation of compact and chemoresistant HGSC spheroids to boost their survival. Methods In this current study, we investigated whether PODXL may also influence HGSC spheroid susceptibility to NK cell infiltration and cytotoxicity. We co-cultured HGSC spheroids with primary human NK cells isolated from peripheral blood mononuclear cells (PBMCs) and examined the impact on these spheroids following 24, 48 and 72 h of co-culture. We first used a cell line model of HGSC spheroids employing Kuramochi cells, which express the highest level of PODXL among known HGSC cell lines. To study the impact of PODXL levels, we compared spheroids of control and PODXL knockout (PODXL-KO) cells that we have previously engineered. We then validated the data in primary cancer spheroids derived from ascites of HGSC patients that express high and low levels of PODXL. Results In both the cell line and primary HGSC spheroid models, co-culture of spheroids expressing lower levels of PODXL resulted in more NK cell infiltration and cytotoxicity, while spheroids expressing higher levels of PODXL were resistant to destruction and showed more proliferation. Conclusions Collectively, these data suggest that PODXL may play an important role in aiding immune evasion in HGSC, at least partly by conferring resistance to NK cell infiltration and the related cytotoxicity.

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.

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 “snapshot” 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 “leader cell” phenotype underlying tumor invasion, and suggest their importance as a clinically relevant target in directed anti-tumour therapies.

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.

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.

Metastasis is the leading cause of cancer-related mortality; however, a complete understanding of the molecular programs driving the metastatic cascade is lacking. Metastasis is dependent on collective invasion—a developmental process exploited by many epithelial cancers to establish secondary tumours and promote widespread disease. The key drivers of collective invasion are “Leader Cells”, a functionally distinct subpopulation of cells that direct migration, cellular contractility, and lead trailing or follower cells. While a significant body of research has focused on leader cell biology in the traditional context of collective invasion, the influence of metastasis-promoting leader cells is an emerging area of study. This review provides insights into the expanded role of leader cells, detailing emerging evidence on the hybrid epithelial–mesenchymal transition (EMT) state and the phenotypical plasticity exhibited by leader cells. Additionally, we explore the role of leader cells in chemotherapeutic resistance and immune evasion, highlighting their potential as effective and diverse targets for novel cancer therapies.