Epithelial Ovarian Cancer Spheroids Research Articles

The Transcoelomic Ecosystem and Epithelial Ovarian Cancer Dissemination.

Epithelial ovarian cancer (EOC) is considered the deadliest gynecological disease and is normally diagnosed at late stages, at which point metastasis has already occurred. Throughout disease progression, EOC will encounter various ecosystems and the communication between cancer cells and these microenvironments will promote the survival and dissemination of EOC. The primary tumor is thought to develop within the ovaries or the fallopian tubes, both of which provide a microenvironment with high risk of causing DNA damage and enhanced proliferation. EOC disseminates by direct extension from the primary tumors, as single cells or multicellular aggregates. Under the influence of cellular and non-cellular factors, EOC spheroids use the natural flow of peritoneal fluid to reach distant organs within the peritoneal cavity. These cells can then implant and seed distant organs or tissues, which develop rapidly into secondary tumor nodules. The peritoneal tissue and the omentum are two common sites of EOC metastasis, providing a microenvironment that supports EOC invasion and survival. Current treatment for EOC involves debulking surgery followed by platinum-taxane combination chemotherapy; however, most patients will relapse with a chemoresistant disease with tumors developed within the peritoneum. Therefore, understanding the role of the unique microenvironments that promote EOC transcoelomic dissemination is important in improving patient outcomes from this disease. In this review article, we address the process of ovarian cancer cellular fate at the site of its origin in the secretory cells of the fallopian tube or in the ovarian surface epithelial cells, their detachment process, how the cells survive in the peritoneal fluid avoiding cell death triggers, and how cancer- associated cells help them in the process. Finally, we report the mechanisms used by the ovarian cancer cells to adhere and migrate through the mesothelial monolayer lining the peritoneum. We also discuss the involvement of the transcoelomic ecosystem on the development of chemoresistance of EOC.

Metastatic Voyage of Ovarian Cancer Cells in Ascites with the Assistance of Various Cellular Components.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy and has a unique metastatic route using ascites, known as the transcoelomic root. However, studies on ascites and contained cellular components have not yet been sufficiently clarified. In this review, we focus on the significance of accumulating ascites, contained EOC cells in the form of spheroids, and interaction with non-malignant host cells. To become resistant against anoikis, EOC cells form spheroids in ascites, where epithelial-to-mesenchymal transition stimulated by transforming growth factor-β can be a key pathway. As spheroids form, EOC cells are also gaining the ability to attach and invade the peritoneum to induce intraperitoneal metastasis, as well as resistance to conventional chemotherapy. Recently, accumulating evidence suggests that EOC spheroids in ascites are composed of not only cancer cells, but also non-malignant cells existing with higher abundance than EOC cells in ascites, including macrophages, mesothelial cells, and lymphocytes. Moreover, hetero-cellular spheroids are demonstrated to form more aggregated spheroids and have higher adhesion ability for the mesothelial layer. To improve the poor prognosis, we need to elucidate the mechanisms of spheroid formation and interactions with non-malignant cells in ascites that are a unique tumor microenvironment for EOC.

Suppression of the ABCA1 Cholesterol Transporter Impairs the Growth and Migration of Epithelial Ovarian Cancer.

Simple SummaryEpithelial ovarian cancer (EOC) is the most lethal gynaecological cancer. Over 80% of cases have already spread at diagnosis, and these patients face a five-year survival rate of 35%. EOC cells often spread to the greater omentum, an abdominal fat pad. Here, EOC cells take-up cholesterols. Excessive amounts of cholesterol are lethal; thus, we proposed that the ABCA1 cholesterol transporter exports cholesterol from serous EOC cells to maintain cholesterol balance. Indeed, we found that reducing the level of ABCA1 could suppress serous EOC growth in two-dimensional as well as three-dimensional cell culture and also hindered their migration, a key process required for cancer spread. We also identified drugs that impair EOC cell growth by inhibiting cholesterol export. Our data demonstrate that disrupting the cholesterol balance by targeting ABCA1 may be an effective treatment strategy for EOC patients.Background: Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy with over 80% of cases already disseminated at diagnosis and facing a dismal five-year survival rate of 35%. EOC cells often spread to the greater omentum where they take-up cholesterol. Excessive amounts of cholesterol can be cytocidal, suggesting that cholesterol efflux through transporters may be important to maintain homeostasis, and this may explain the observation that high expression of the ATP-binding cassette A1 (ABCA1) cholesterol transporter has been associated with poor outcome in EOC patients. Methods: ABCA1 expression was silenced in EOC cells to investigate the effect of inhibiting cholesterol efflux on EOC biology through growth and migration assays, three-dimensional spheroid culture and cholesterol quantification. Results: ABCA1 suppression significantly reduced the growth, motility and colony formation of EOC cell lines as well as the size of EOC spheroids, whilst stimulating expression of ABCA1 reversed these effects. In serous EOC cells, ABCA1 suppression induced accumulation of cholesterol. Lowering cholesterol levels using methyl-B-cyclodextrin rescued the effect of ABCA1 suppression, restoring EOC growth. Furthermore, we identified FDA-approved agents that induced cholesterol accumulation and elicited cytocidal effects in EOC cells. Conclusions: Our data demonstrate the importance of ABCA1 in maintaining cholesterol balance and malignant properties in EOC cells, highlighting its potential as a therapeutic target for this disease.

MiRNA-Dependent Regulation of AKT1 Phosphorylation.

The phosphoinositide-3-kinase (PI3K)/AKT pathway regulates cell survival and is over-activated in most human cancers, including ovarian cancer. Following growth factor stimulation, AKT1 is activated by phosphorylation at T308 and S473. Disruption of the AKT1 signaling pathway is sufficient to inhibit the epithelial-mesenchymal transition in epithelial ovarian cancer (EOC) cells. In metastatic disease, adherent EOC cells transition to a dormant spheroid state, characterized previously by low S473 phosphorylation in AKT1. We confirmed this finding and observed that T308 phosphorylation was yet further reduced in EOC spheroids and that the transition from adherent to spheroid growth is accompanied by significantly increased levels of let-7 miRNAs. We then used mechanistic studies to investigate the impact of let-7 miRNAs on AKT1 phosphorylation status and activity in cells. In growth factor-stimulated HEK 293T cells supplemented with let-7a, we found increased phosphorylation of AKT1 at T308, decreased phosphorylation at S473, and enhanced downstream AKT1 substrate GSK-3β phosphorylation. Let-7b and let-7g also deregulated AKT signaling by rendering AKT1 insensitive to growth factor simulation. We uncovered let-7a-dependent deregulation of PI3K pathway components, including PI3KC2A, PDK1, and RICTOR, that govern AKT1 phosphorylation and activity. Together, our data show a new role for miRNAs in regulating AKT signaling.

Chondroitin sulfate proteoglycan 4, a targetable oncoantigen that promotes ovarian cancer growth, invasion, cisplatin resistance and spheroid formation

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease encompassing several distinct molecular subtypes and clinical entities. Despite the initial success of surgical debulking and adjuvant chemotherapy, recurrence with chemotherapy resistant tumors is common in patients with EOC and leads to poor overall survival. The extensive genetic and phenotypic heterogeneity associated with ovarian cancers has hindered the identification of effective prognostic and predictive biomarkers in EOC patients. In the current studies, we identify a tumor cell surface oncoantigen, chondroitin sulfate proteoglycan 4 (CSPG4), as an independent risk factor for decreased survival of patients with EOC. Our results show that CSPG4 promotes EOC cell invasion, cisplatin resistance and spheroid formation in vitro and tumor expansion in vivo. Mechanistically, spheroid formation and tumor cell invasion are due to CSPG4-stimulated expression of the mesenchymal transcription factor ZEB1. Furthermore, we have developed a novel monoclonal anti-CSGP4 antibody against the juxtamembrane domain of the core protein that limits CSPG4-stimulated ZEB1 expression, tumor cell invasion and promotes EOC apoptosis within spheroid cultures. We therefore propose that CSPG4 expression drives phenotypic heterogeneity and malignant progression in EOC tumors. These studies further demonstrate that CSPG4 expression levels are a potential diagnostic biomarker in EOC and indicate that targeting cells which express this oncoantigen could limit recurrence and improve outcomes in patients with EOC.

Inhibition of the mTOR pathway and reprogramming of protein synthesis by MDM4 reduce ovarian cancer metastatic properties

Epithelial ovarian cancer (EOC) is a highly heterogeneous disease with a high death rate mainly due to the metastatic spread. The expression of MDM4, a well-known p53-inhibitor, is positively associated with chemotherapy response and overall survival (OS) in EOC. However, the basis of this association remains elusive. We show that in vivo MDM4 reduces intraperitoneal dissemination of EOC cells, independently of p53 and an immune-competent background. By 2D and 3D assays, MDM4 impairs the early steps of the metastatic process. A 3D-bioprinting system, ad hoc developed by co-culturing EOC spheroids and endothelial cells, showed reduced dissemination and intravasation into vessel-like structures of MDM4-expressing cells. Consistent with these data, high MDM4 levels protect mice from ovarian cancer-related death and, importantly, correlate with increased 15 y OS probability in large data set analysis of 1656 patients. Proteomic analysis of EOC 3D-spheroids revealed decreased protein synthesis and mTOR signaling, upon MDM4 expression. Accordingly, MDM4 does not further inhibit cell migration when its activity towards mTOR is blocked by genetic or pharmacological approaches. Importantly, high levels of MDM4 reduced the efficacy of mTOR inhibitors in constraining cell migration. Overall, these data demonstrate that MDM4 impairs EOC metastatic process by inhibiting mTOR activity and suggest the usefulness of MDM4 assessment for the tailored application of mTOR-targeted therapy.

A novel role for NUAK1 in promoting ovarian cancer metastasis through regulation of fibronectin production in spheroids.

Epithelial ovarian cancer (EOC) has a unique mode of metastasis, where cells shed from the primary tumour, form aggregates called spheroids to evade anoikis, spread through the peritoneal cavity, and adhere to secondary sites. We previously showed that the master kinase Liver kinase B1 (LKB1) is required for EOC spheroid viability and metastasis. We have identified novel (nua) kinase 1 (NUAK1) as a top candidate LKB1 substrate in EOC cells and spheroids using a multiplex inhibitor beads-mass spectrometry approach. We confirmed that LKB1 maintains NUAK1 phosphorylation and promotes its stabilization. We next investigated NUAK1 function in EOC cells. Ectopic NUAK1-overexpressing EOC cell lines had increased adhesion, whereas the reverse was seen in OVCAR8-NUAK1KO cells. In fact, cells with NUAK1 loss generate spheroids with reduced integrity, leading to increased cell death after long-term culture. Following transcriptome analysis, we identified reduced enrichment for cell interaction gene expression pathways in OVCAR8-NUAK1KO spheroids. In fact, the FN1 gene, encoding fibronectin, exhibited a 745-fold decreased expression in NUAK1KO spheroids. Fibronectin expression was induced during native spheroid formation, yet this was completely lost in NUAK1KO spheroids. Co-incubation with soluble fibronectin restored the compact spheroid phenotype to OVCAR8-NUAK1KO cells. In a xenograft model of intraperitoneal metastasis, NUAK1 loss extended survival and reduced fibronectin expression in tumours. Thus, we have identified a new mechanism controlling EOC metastasis, through which LKB1-NUAK1 activity promotes spheroid formation and secondary tumours via fibronectin production.

Activated CAMKK\u03b2-AMPK signaling promotes autophagy in a spheroid model of ovarian tumour metastasis

BackgroundA hallmark of epithelial ovarian cancer (EOC) metastasis is the process of spheroid formation, whereby tumour cells aggregate into 3D structures while in suspension in the peritoneal cavity. EOC spheroids are subjected to bioenergetic stress, thereby activating AMP-activated protein kinase (AMPK) signaling to enter a metabolically quiescent state, which can facilitate cell survival under nutrient-limiting conditions. Independently, we have also demonstrated that EOC spheroids induce autophagy, a process that degrades and recycles intracellular components to restore energy and metabolites. Herein, we sought to examine whether AMPK controls autophagy induction as a cell survival mechanism in EOC spheroids.ResultsWe observed a co-ordinate increase in phosphorylated AMPK and the autophagy marker LC3-II during EOC spheroid formation. Reduced AMPK expression by siRNA-mediated knockdown of PRKAA1 and PRKAA2 blocked autophagic flux in EOC spheroids as visualized by fluorescence microscopy using the mCherry-eGFP-LC3B reporter. A complementary approach using pharmacologic agents Compound C and CAMKKβ inhibitor STO-609 to inhibit AMPK activity both yielded a potent blockade of autophagic flux as well. However, direct activation of AMPK in EOC cells using oligomycin and metformin was insufficient to induce autophagy. STO-609 treatment of EOC spheroids resulted in reduced viability in 7 out of 9 cell lines, but with no observed effect in non-malignant FT190 cell spheroids.ConclusionsOur results support the premise that CAMKKβ-mediated AMPK activity is required, at least in part, to regulate autophagy induction in EOC spheroids and support cell viability in this in vitro model of EOC metastasis.

Ionizable lipid nanoparticles are effective at penetrating the core of epithelial ovarian cancer spheroids

Ionizable lipid nanoparticles are effective at penetrating the core of epithelial ovarian cancer spheroids

Epithelial ovarian cancer stem‑like cells are resistant to the cellular lysis of cytokine‑induced killer cells via HIF1A‑mediated downregulation of ICAM‑1.

Epithelial ovarian cancer(EOC) is the most lethal of all gynecologic tumors. Cancer spheroid culture is a widely used model to study cancer stem cells. Previous studies have demonstrated the effectiveness of cytokine‑induced killer(CIK) cell‑based therapies against cancer and cancer stem cells. However, it is not clear how EOC spheroid cells respond to CIK‑mediated cellular lysis, and the mechanisms involved have never been reported before. A flow cytometry‑based method was used to evaluate the anti‑cancer effects of CIK cells against adherent A2780 cells and A2780 spheroids. To demonstrate the association between hypoxia inducible factor‑1α(HIF1A) and intercellular adhesion molecule‑1(ICAM‑1), two HIF1A short hairpin RNA(shRNA) stable transfected cell lines were established. Furthermore, the protein expression levels of hypoxia/HIF1A‑associated signaling pathways were evaluated, including transforming growth factor‑β1(TGF‑β1)/mothers against decapentaplegic homologs(SMADs) and nuclear factor‑κB(NF‑κB) signaling pathways, comparing A2780 adherent cells and cancer spheroids. Flow cytometry revealed that A2780 spheroid cells were more resistant to CIK‑mediated cellular lysis, which was partially reversed by an anti‑ICAM‑1 antibody. HIF1A was significantly upregulated in A2780 spheroids compared with adherent cells. Using HIF1A shRNA stable transfected cell lines and cobalt chloride, it was revealed that hypoxia/HIF1A contributed to downregulation of ICAM‑1 in A2780 spheroid cells and adherent cells. Furthermore, hypoxia/HIF1A‑associated signaling pathways, TGF‑β1/SMADs and NF‑κB, were activated in A2780 spheroid cells by using western blotting. The findings indicate that EOC stem‑like cells resist the CIK‑mediated cellular lysis via HIF1A‑mediated downregulation of ICAM‑1, which may be instructive for optimizing and enhancing CIK‑based therapies.

Abstract A12: The metabolic stress mediator LKB1 is required for ovarian cancer metastasis

Abstract Most epithelial ovarian cancer (EOC) patients with metastatic disease initially respond to cytotoxic chemotherapy, yet almost all will relapse with resistant disease. Thus, improving patient outcomes will require novel approaches to limit metastasis and overcome chemo-resistance. Liver Kinase B1 (LKB1), encoded by the STK11 gene, is a key intracellular regulator of metabolic stress and is considered a putative tumor suppressor in some cancers. However, we have demonstrated that LKB1 is intact and required for EOC cell viability and growth in an in vitro spheroid model of ovarian cancer metastasis. We propose that LKB1 signaling enables malignant EOC cells to maintain viability and survive in metabolically challenging environments like that encountered during intraperitoneal metastasis. To further investigate the therapeutic potential of targeting LKB1 activity in metastatic EOC, we generated STK11-knockout cell lines—normal FT190 cells, and EOC cell lines OVCAR8, HeyA8, and iOvCa147—using CRISPR technology. STK11KO resulted in decreased malignant properties of EOC cells in vitro, including clonogenicity and anchorage-independent growth; however, loss of LKB1 in FT190 cells had no effect on cell proliferation, clonogenicity, or anchorage-independent growth, indicating LKB1 does not likely act as a tumor suppressor in EOC. Loss of LKB1 sensitized EOC cells to the growth-inhibiting effects of specific metabolic stresses. OVCAR8-STK11KO cells were more sensitive to nutrient deprivation in adherent culture, and to carboplatin and paclitaxel treatment in spheroid culture, as compared with OVCAR8 cells. Among the three EOC cell lines, STK11KO yielded variable sensitivity to inhibition of mitochondrial ATP production via oligomycin treatment. Interestingly, STK11KO did not affect induction of AMP-activated protein kinase (AMPK) phosphorylation in EOC spheroids, indicating that metabolic stress signaling to support EOC cell survival in spheroids during metastasis may occur via alternative pathways. In support of our previous knockdown results, EOC spheroids completely lacking LKB1 had markedly impaired growth in suspension culture compared to parental cell controls. In contrast, FT190 spheroids exhibited rapid cell attrition in spheroid culture regardless of LKB1 status. These results indicate that LKB1 may be specifically required in EOC cells to evade anoikis during metastatic spread. Finally, to test directly whether loss of LKB1 activity affects the metastatic potential of EOC cells, we performed intraperitoneal injections of OVCAR8-STK11KO and HeyA8-STK11KO cells with their respective parental cell controls. Loss of LKB1 in both aggressive EOC cell lines exhibited a dramatic reduction on tumor burden. STK11KO significantly decreased the establishment of large, solid tumor masses, reduced adhesion of OVCAR8 tumor nodules, and even changed the metastatic trajectory of HeyA8 cells with evidence of tumor cell growth only as a thin layer on peritoneal walls. Histologic analysis revealed evidence of extensive necrosis in STK11KO tumors, which was likely the major contributor to reduced tumor burden. These results strongly indicate that loss of LKB1 activity abrogates the metabolic stress response necessary during EOC metastasis both in spheroids and establishment of intraperitoneal tumors. Overall, LKB1 or its AMPK-independent signaling mediators represent unique yet very potent therapeutic vulnerabilities in metastatic EOC. Citation Format: Adrian Buensuceso, Yudith R. Valdes, Rene Figueredo, Gabriel E. DiMattia, Trevor G. Shepherd. The metabolic stress mediator LKB1 is required for ovarian cancer metastasis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A12.

Abstract B64: The novel ZIP4 regulation and its role in cancer stem cell-related activities in ovarian cancer

Abstract The role and regulation of ZIP4, a zinc (Zn) transporter, have been studied in the context of extracellular Zn transporting. Factors other than Zn with ZIP4 regulatory effects are essentially unknown. In addition, its role in cancer stem cell (CSC) has not been reported in any type of cancer. RNA-seq analyses were conducted to compare genes differentially expressed in less and more aggressive epithelial ovarian cancer (EOC) cells. ZIP4 expression and its regulation was assessed by immunoblotting, quantitative PCR, or immunofluorescence staining in human ovarian tissues. CSC-related activities were examined to evaluate the role of ZIP4 in human high-grade serous ovarian cancer (HGSOC) cells. RNAi and CRISPR techniques were used to knock down or knock out ZIP4 and related genes. EOC tumors overexpressed ZIP4 when compared with normal and benign tissues. ZIP4 knockout significantly reduced several CSC-related activities in EOC cells, including proliferation, anoikis resistance, colony formation, spheroid formation, drug resistance, and side-population (SP). Only ZIP4-expressing SP highly expressed known CSC markers ALDH1 and OCT4. ZIP4 knockout dramatically reduced tumorigenesis and ZIP4 overexpression increased tumorigenesis in vivo. In addition, the ZIP4-expressing SP population had the tumor initiating activity. Moreover, the oncolipid lysophosphatic acid (LPA) effectively upregulated ZIP4 expression via the nuclear receptor peroxisome proliferator-activated receptor gamma, and LPA’s promoting effects in CSC-related activities in HGSOC cells was at least partially mediated by ZIP4 in an extracellular Zn-independent manner. Our critical data imply that ZIP4 is a new and important cancer stem cell regulator in EOC. Our data also provide an innovative interpretation for the apparent disconnection between low levels of zinc and upregulation of ZIP4 in EOC tissues. Citation Format: Yan Xu, Qipeng Fan, Qingchun Cai, Kenneth P. Nephew. The novel ZIP4 regulation and its role in cancer stem cell-related activities in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B64.

The mechanical microenvironment regulates ovarian cancer cell morphology, migration, and spheroid disaggregation

There is growing appreciation of the importance of the mechanical properties of the tumor microenvironment on disease progression. However, the role of extracellular matrix (ECM) stiffness and cellular mechanotransduction in epithelial ovarian cancer (EOC) is largely unknown. Here, we investigated the effect of substrate rigidity on various aspects of SKOV3 human EOC cell morphology and migration. Young’s modulus values of normal mouse peritoneum, a principal target tissue for EOC metastasis, were determined by atomic force microscopy (AFM) and hydrogels were fabricated to mimic these values. We find that cell spreading, focal adhesion formation, myosin light chain phosphorylation, and cellular traction forces all increase on stiffer matrices. Substrate rigidity also positively regulates random cell migration and, importantly, directional increases in matrix tension promote SKOV3 cell durotaxis. Matrix rigidity also promotes nuclear translocation of YAP1, an oncogenic transcription factor associated with aggressive metastatic EOC. Furthermore, disaggregation of multicellular EOC spheroids, a behavior associated with dissemination and metastasis, is enhanced by matrix stiffness through a mechanotransduction pathway involving ROCK, actomyosin contractility, and FAK. Finally, this pattern of mechanosensitivity is maintained in highly metastatic SKOV3ip.1 cells. These results establish that the mechanical properties of the tumor microenvironment may play a role in EOC metastasis.

Stemness and chemoresistance are imparted to the OC cells through TGFβ1 driven EMT.

Ovarian cancer (OC) is the fourth most common gynecological malignancy due to its highly aggressive, recurrent, and drug-resistant nature. The last two features are rendered by the presence of cancer stem cells (CSCs). Factors like TGFβ1 and their downstream signaling pathways are upregulated in most cancers and are known to induce EMT and stemness, but the exact mechanisms underlying the process remain unelucidated. In our study, TGFβ1 induced enhanced stem-like properties like high expression of the pluripotent markers SOX2, OCT4a, and NANOG, along with CD44, and CD117 in the OC cells. In addition, increased activity of the aldehyde dehydrogenase enzyme, formation of compact spheroids, and a quiescent phenotype were observed. In deciphering the mechanism behind it, our data propose ZEB1 transcription factor to play a substantial role in inducing the EMT-mediated stemness and chemoresistance. Further, in our study, we elucidated the significant contribution of both Smad and non-Smad pathways like ERK, JNK, and P38 MAPK pathways in the induction of stem-like characteristics. The novelty of the study also resides with the fact in the expression of different lineage-specific markers, like CD31, CD45, and CD117 along with CD44 in the TGFβ1-induced epithelial ovarian cancer spheroids. This suggests a tendency of the spheroidal cells towards differentiating into heterogenic populations, which is a distinctive feature of a stem cell. Taken together, the present study provides an insight to the molecular cues involved in the acquisition of stemness and chemoresistance along with tumor heterogeneity in TGFβ1-induced OC cells.

Re-purposing of curcumin as an anti-metastatic agent for the treatment of epithelial ovarian cancer: in vitro model using cancer stem cell enriched ovarian cancer spheroids.

Malignant epithelial ovarian cancer (EOC) spheroids high frequently are detected in the malignant ascites of the patients with the extensive peritoneal metastasis of ovarian cancer, which represent a significant obstacle to efficacious treatment. Clinical data also suggested that EOC spheroids play a putative role in the development of chemoresistance. Since standard surgery and conventional chemotherapy is the only available treatment, there is an urgent need to identify a more effective therapeutic strategy. Recent studies demonstrated that curcumin exerts an anticancer effect in a variety of human cancers including ovarian cancer. This study evaluates anti-peritoneal metastasis and chemoresistance of curcumin related to the EOC spheroids. In this study, we confirm that the high invasive EOC cells forming the spheroids express a high level of a cancer stem cell (CSC) marker, aldehyde dehydrogenase 1 family member A1 (ALDH1A1), which was significantly down-regulated by curcumin treatment. Curcumin treatment markedly enhances the sensitivity of EOC spheroids to cisplatin in a dose-dependent manner. Our experiments provided evidence that curcumin could abolish the sphere-forming capacity of EOC cells in a dose-dependent manner. Moreover, curcumin substantially suppressed the growth of the pre-existed EOC spheroids, inhibited the adhesion of EOC spheroids to ECM as well as the invasion of EOC spheroids to the mesothelial monolayers. We propose to re-purpose curcumin as anti-metastatic and chemoresistant agent for EOC management in combination with conventional regimen. Further preclinical studies are necessary to validate the anti-cancer effect of curcumin in patients with EOC.

Mesothelial cells interact with tumor cells for the formation of ovarian cancer multicellular spheroids in peritoneal effusions.

Epithelial ovarian cancer (EOC) dissemination is primarily mediated by the shedding of tumor cells from the primary site into ascites where they form multicellular spheroids that rapidly lead to peritoneal carcinomatosis. While the clinical importance and fundamental role of multicellular spheroids in EOC is increasingly appreciated, the mechanisms that regulate their formation and dictate their cellular composition remain poorly characterized. To investigate these important questions, we characterized spheroids isolated from ascites of women with EOC. We found that in these spheroids, a core of mesothelial cells was encased in a shell of tumor cells. Analysis further revealed that EOC spheroids are dynamic structures of proliferating, non-proliferating and hypoxic regions. To recapitulate these in vivo findings, we developed a three-dimensional co-culture model of primary EOC and mesothelial cells. Our analysis indicated that, compared to the OVCAR3 cell line, primary EOC cells isolated from ascites as well as mesothelial cells formed compact spheroids. Analysis of heterotypic spheroid microarchitecture revealed a structure that grossly resembles the structure of spheroids isolated from ascites. Cells that formed compact spheroids had elevated expression of β1 integrin and low expression of E-cadherin. Addition of β1 integrin blocking antibody or siRNA-mediated downregulation of β1 integrin resulted in reduced tightness of the spheroids. Interestingly, the loss of MUC16 and E-cadherin expression resulted in the formation of more compact spheroids. Therefore, our findings support the heterotypic nature of spheroids from malignant EOC ascites. In addition, our data describe an unusual link between E-cadherin expression and less compact spheroids. Our data also emphasize the role of MUC16 and β1 integrin in EOC spheroid formation.

SMIFH2-mediated mDia formin functional inhibition potentiates chemotherapeutic targeting of human ovarian cancer spheroids.

Due to a lack of effective screening or prevention protocol for epithelial ovarian cancer (EOC), there is a critical unmet need to develop therapeutic interventions for EOC treatment. EOC metastasis is unique. Initial dissemination is not primarily hematogenous, yet is facilitated through shedding of primary tumor cells into the peritoneal fluid and accumulating ascites. Increasingly, isolated patient spheroids point to a clinical role for spheroids in EOC metastasis. EOC spheroids are highly invasive structures that disseminate upon peritoneal mesothelium, and visceral tissues including liver and omentum. Selection for this subset of chemoresistant EOC cells could influence disease progression and/or recurrence. Thus, targeting spheroid integrity/structure may improve the chemotherapeutic responsiveness of EOC. We discovered a critical role for mammalian Diaphanous (mDia)-related formin-2 in maintaining EOC spheroid structure. Both mDia2 and the related mDia1 regulate F-actin networks critical to maintain cell-cell contacts and the integrity of multi-cellular epithelial sheets. We investigated if mDia2 functional inhibition via a small molecule inhibitor SMIFH2 combined with chemotherapeutics, such as taxol and cisplatin, inhibits the viability of EOC monolayers and clinically relevant spheroids. SMIFH2-mediated mDia formin inhibition significantly reduced both ES2 and Skov3 EOC monolayer viability while spheroid viability was minimally impacted only at the highest concentrations. Combining either cisplatin or taxol with SMIFH2 did not significantly enhance the effects of either drug alone in ES2 monolayers, while Skov3 monolayers treated with taxol or cisplatin and SMIFH2 showed significant additive inhibition of viability. ES2 spheroids were highly responsive with clear additive anti-viability effects with dual taxol or cisplatin when combined with SMIFH2 treatments. While combined taxol with SMIFH2 in spheroids showed an additive effect relative to single treatments, Skov3 spheroids showed no additive effects from combined cisplatin and SMIFH2 treatments. Our data indicate that mDia formin inhibition combined with taxol to drive enhanced and/or additive anti-viability effects targeting 3D EOC structures, including ES2 and Skov3 spheroids. Combined mDia formin inhibition with cisplatin may be most effective in EOC spheroids where cisplatin sensitivity is retained at moderate levels, such as ES2 cells.

Abstract B51: Stress management: LKB1 controls growth and survival of dormant epithelial ovarian cancer spheroid cells.

Abstract Aberrant cell metabolism is rapidly establishing itself as a critical hallmark of human malignancies. Cancer cells are faced with huge metabolic demands to support rapid tumor growth, yet are commonly starved for nutrients. In response, cancer cells hijack alternative signaling pathways during these times of energy and metabolic stress to sustain viability. Metastatic epithelial ovarian cancer (EOC) cells are faced with additional stressors during transcoelomic spread, such as detachment from a matrix substratum and inhospitable conditions in peritoneal fluid. However, EOC cells have a natural ability to aggregate when in suspension to form multicellular aggregates, or spheroids, which supports a survival advantage for cells when transiting the peritoneal space during metastatic progression. Therefore, we utilize an in vitro spheroid culture model system to investigate signaling pathways altered in EOC cells that may be implicated in ovarian cancer pathobiology and promote metastasis. For example, we have shown that spheroid formation induces cellular quiescence and autophagy, two disparate processes which promote EOC cell survival and resistance to platinum-based chemotherapeutics. In addition, we discovered that EOC spheroids have significantly reduced mitochondrial activity and ATP levels compared with matched proliferating adherent cells. Liver kinase B1 (LKB1) acts as a chief responder to intracellular stress due to reduced energy and nutrients by eliciting general growth suppression during these starvation-like conditions. Thus, we hypothesized that LKB1 activity is increased in EOC spheroids to promote tumor cell dormancy and cell survival. Although the STK11 gene encoding LKB1 is heterozygously deleted in 84% of serous ovarian tumors, we demonstrate that almost all ovarian tumor cells and established EOC cell lines retain intact LKB1 expression. In fact, LKB1 protein expression increases when EOC cells form spheroids and this coincides with induced serine-428 phosphorylation, a modification that is required for LKB1 growth suppressive activity. To address the potential functional requirement of LKB1 in EOC spheroids, we first performed transient knockdown of STK11 to block LKB1 expression. Indeed, reduced LKB1 renders spheroids susceptible to cell death and increases sensitivity to carboplatin. Next, we utilized Cas9-mediated genome editing of the STK11 locus to completely ablate LKB1 expression in HEYA8 and OVCAR8 cells. STK11-knockout HEYA8 and OVCAR8 cells yielded significantly decreased spheroid number and viability compared with parental cell lines. In a reciprocal fashion, forced re-expression of LKB1 in CaOV3 and TOV21G cells—two EOC cell lines which harbor inactivating STK11 mutations—reduces cell growth and colony formation in proliferating adherent culture. Proliferation and dispersion of CaOV3-LKB1 and TOV21G-LKB1 cells from re-attached spheroids are also reduced. Taken together, LKB1 has growth suppressive effects in EOC cells, yet it serves the additional purpose to promote cell survival in spheroids. The canonical target of LKB1 is AMP-activated protein kinase (AMPK), which is thought to elicit the majority of LKB1 growth suppressive effects during stress metabolism signaling. As expected, AMPK phosphorylation at threonine-172 is also induced upon spheroid formation. Using STK11-knockout EOC cells, however, we demonstrate that LKB1 is not required to phosphorylate AMPK in spheroids; more importantly, knockdown of PRKAA1 encoding the catalytic alpha-subunit of AMPK has no effect on spheroid cell viability. Thus, our results suggest that LKB1 utilizes alternative mechanisms to regulate the dormancy phenotype in EOC spheroids. Future work will entail direct experiments testing whether LKB1-mediated stress metabolism signaling has the potential to promote EOC metastasis and recurrence of chemo-resistant disease. Citation Format: Trevor G. Shepherd, Yudith Ramos Valdes, Teresa Peart, Meera Shah, Gabriel E. DiMattia. Stress management: LKB1 controls growth and survival of dormant epithelial ovarian cancer spheroid cells. [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 B51.

TGFβ signaling regulates epithelial-mesenchymal plasticity in ovarian cancer ascites-derived spheroids.

Epithelial-mesenchymal transition (EMT) serves as a key mechanism driving tumor cell migration, invasion, and metastasis in many carcinomas. Transforming growth factor-beta (TGFβ) signaling is implicated in several steps during cancer pathogenesis and acts as a classical inducer of EMT. Since epithelial ovarian cancer (EOC) cells have the potential to switch between epithelial and mesenchymal states during metastasis, we predicted that modulation of TGFβ signaling would significantly impact EMT and the malignant potential of EOC spheroid cells. Ovarian cancer patient ascites-derived cells naturally underwent an EMT response when aggregating into spheroids, and this was reversed upon spheroid re-attachment to a substratum. CDH1/E-cadherin expression was markedly reduced in spheroids compared with adherent cells, in concert with an up-regulation of several transcriptional repressors, i.e., SNAI1/Snail, TWIST1/2, and ZEB2. Treatment of EOC spheroids with the TGFβ type I receptor inhibitor, SB-431542, potently blocked the endogenous activation of EMT in spheroids. Furthermore, treatment of spheroids with SB-431542 upon re-attachment enhanced the epithelial phenotype of dispersing cells and significantly decreased cell motility and Transwell migration. Spheroid formation was significantly compromised by exposure to SB-431542 that correlated with a reduction in cell viability particularly in combination with carboplatin treatment. Thus, our findings are the first to demonstrate that intact TGFβ signaling is required to control EMT in EOC ascites-derived cell spheroids, and it promotes the malignant characteristics of these structures. As such, we show the therapeutic potential for targeted inhibition of this pathway in ovarian cancer patients with late-stage disease.

Intact LKB1 activity is required for survival of dormant ovarian cancer spheroids.

Metastatic epithelial ovarian cancer (EOC) cells can form multicellular spheroids while in suspension and disperse directly throughout the peritoneum to seed secondary lesions. There is growing evidence that EOC spheroids are key mediators of metastasis, and they use specific intracellular signalling pathways to control cancer cell growth and metabolism for increased survival. Our laboratory discovered that AKT signalling is reduced during spheroid formation leading to cellular quiescence and autophagy, and these may be defining features of tumour cell dormancy. To further define the phenotype of EOC spheroids, we have initiated studies of the Liver kinase B1 (LKB1)-5'-AMP-activated protein kinase (AMPK) pathway as a master controller of the metabolic stress response. We demonstrate that activity of AMPK and its upstream kinase LKB1 are increased in quiescent EOC spheroids as compared with proliferating adherent EOC cells. We also show elevated AMPK activity in spheroids isolated directly from patient ascites. Functional studies reveal that treatment with the AMP mimetic AICAR or allosteric AMPK activator A-769662 led to a cytostatic response in proliferative adherent ovarian cancer cells, but they fail to elicit an effect in spheroids. Targeted knockdown of STK11 by RNAi to reduce LKB1 expression led to reduced viability and increased sensitivity to carboplatin treatment in spheroids only, a phenomenon which was AMPK-independent. Thus, our results demonstrate a direct impact of altered LKB1-AMPK signalling function in EOC. In addition, this is the first evidence in cancer cells demonstrating a pro-survival function for LKB1, a kinase traditionally thought to act as a tumour suppressor.