The ST6Gal-I glycosyltransferase, which adds α2-6-linked sialic acids to glycoproteins, is overexpressed in colon adenocarcinoma, and enzyme activity is correlated with tumor cell invasiveness. Previously we reported that forced expression of oncogenic ras in HD3 colonocytes causes upregulation of ST6Gal-I, leading to increased α2-6 sialylation of β1 integrins. To determine whether ras-induced sialylation is involved in promoting the tumor cell phenotype, we used shRNA to downregulate ST6Gal-I in ras-expressors, and then monitored integrin-dependent responses. Here we show that forced ST6Gal-I downregulation, leading to diminished α2-6 sialylation of integrins, inhibits cell adhesion to collagen-I, a β1 ligand. Correspondingly, collagen binding is reduced by enzymatic removal of cell surface sialic acids from ras-expressors with high ST6Gal-I levels (i.e., no shRNA). Cells with forced ST6Gal-I downregulation also exhibit decreased migration on collagen-I and diminished invasion through Matrigel. Importantly, GD25 cells, which lack β1 integrins (and ST6Gal-I), do not demonstrate differential invasiveness when forced to express ST6Gal-I, suggesting that the effects of variant sialylation are mediated specifically by β1 integrins. The observation that cell migration and invasion can be blocked in oncogenic ras-expressing cells by forcing ST6Gal-I downregulation implicates differential sialylation as an important ras effector, and also suggests that ST6Gal-I is a promising therapeutic target.
Ras; integrin; sialic acid; metastasis; collagen; colonocytes
Ovarian epithelial cancer is the most lethal gynecologic malignancy. The high mortality is attributed to the fact that most cases typically present in late stage when ovarian cancer (OC) has already spread beyond the ovary. Ovarian epithelial cancer cells are shed into intraperitoneal ascites and easily disseminate throughout the peritoneal cavity with preferential metastasis to the omentum, peritoneum, and local organs. Understanding how ovarian epithelial cells interact with and modulate their microenvironment can provide insight into the molecular mechanism(s) involved with malignant transformation and progression which may eventually identify novel diagnostic, prognostic, and therapeutic targets. The objective of this paper is to provide a brief consideration of ovarian surface epithelial-stromal interactions in regard to normal physiological function and tumor progression as influenced by two potentially key interleukins, interleukins-1 (IL-1) and -6 (IL-6), present in the microenvironment. Lastly, we will consider the clinical implications of IL-1 and IL-6 for OC patients.
Ovarian cancer is the most lethal gynecologic malignancy and the five-year survival rate is only 35% after diagnosis. Epithelial ovarian cancer is a highly metastatic disease characterized by widespread peritoneal dissemination and ascites. The death incidences from ovarian cancer could be significantly lowered by developing new methods for the early diagnosis and treatment of this fatal disease. Several potential markers have been identified recently. However, mucins are the most promising markers for ovarian cancer diagnosis. Mucins are large extracellular, heavily glycosylated proteins and their aberrant expression has been implicated in the pathogenesis of a variety of cancers, including ovarian cancer. This review will summarize known facts about the pathological and molecular characteristics of ovarian cancer, the current status of ovarian cancer markers, as well as general information about mucins, the putative role of mucins in the progression of ovarian cancer and their potential use for the early diagnosis and treatment of this disease.
Ovarian cancer is the deadliest of the gynecological diseases and the fifth cause of cancer death among American women. This is mainly due to the lack of prognostic tools capable of detecting early stages of ovarian cancer and to the high rate of resistance to the current chemotherapeutic regimens. In this scenario the overall 5-year survival rate for ovarian cancer patients diagnosed at late stage is less than 25%. Abnormalities associated with the malignant phenotype and the mechanisms of tumor progression are not clearly understood. In vitro studies are necessary, yet have been hampered due to the limitations accompanied with the use of ovarian cancer cell lines and the heterogeneity of the ovarian cancer cell population derived from ascites fluids. In this study we present a simple, rapid and reproducible method for the isolation and characterization of ovarian cancer cells from solid tumor tissue and show that enzymatic digestion for 30 minutes with dispase II results in the most effective recovery of viable epithelial ovarian cancer (EOC) cells. The resulting cancer (EOC) cell preparations demonstrate a significant yield, high levels of viability and are fibroblast-free. They grow for up to six passages and retain the capacity of forming spheroids-like structures in agarose. In addition, they can be genetically manipulated and used for drug screening, thus rendering them highly suitable for downstream applications. Notably, isolation of ovarian cancer cells from solid specimens using this method has the advantage of allowing for isolation of cancer cells from early stages of ovarian cancer as well as obtaining cells from defined either primary and/or metastatic ovarian cancer sites. Thus, these cells are highly suitable for investigations aimed at understanding ovarian cancer.
Human ovarian cancer, the leading cause of death from gynecologic malignancy, tends to remain localized to the peritoneal cavity until late in the disease. In established disease, ascitic fluid accumulates in the peritoneal cavity. We have previously demonstrated that this ascitic fluid is a potent source of in vitro mitogenic activity including at least one unique growth factor. We now report that the human ovarian adenocarcinoma line, HEY, can be induced to grow intraperitoneally in immunodeficient nude mice in the presence (23/28 mice), but not absence (0/21 mice) of ascitic fluid from ovarian cancer patients. Ascitic fluid from patients with benign disease did not have similar effects on intraperitoneal growth of HEY cells (1/15 mice). Once tumors were established by injections of exogenous ascitic fluid, they could progress in the absence of additional injections of ascitic fluid. The mice eventually developed ascitic fluid which contained potent growth factor activity, suggesting that the tumors eventually produced autologous growth factors. This nude mouse model provides a system to study the action of ovarian cancer growth factors on tumor growth in vivo and to evaluate preclinically, therapeutic approaches designed to counteract the activity of these growth factors.
Ovarian cancer is the most lethal gynecologic cancer mainly because of widespread peritoneal dissemination and malignant ascites. Key to this is the capacity of tumor cells to escape suspension-induced apoptosis (anoikis), which also underlies their resistance to chemotherapy. Here, we used a nonadherent cell culture model to investigate the molecular mechanisms of apoptotic resistance of ovarian cancer cells that may mimic the chemoresistance found in solid tumors. We found that ovarian cancer cells acquired a remarkable resistance to anoikis and apoptosis induced by exposure to clinically relevant doses of two front-line chemotherapeutic drugs cisplatin and paclitaxel when grown in three-dimensional than monolayer cultures. Inhibition of the hepatocyte growth factor (HGF) receptor c-Met, which is frequently overexpressed in ovarian cancer, by a specific inhibitor or small interfering RNA blocked the acquired anoikis resistance and restored chemosensitivity in three-dimensional not in two-dimensional cultures. These effects were found to be dependent on both phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) 1/2 signaling pathways. Inhibitors of PI3K/Akt abrogated ERK1/2 activation and its associated anoikis resistance in response to HGF, suggesting a signaling relay between these two pathways. Furthermore, we identified a central role of Ras as a mechanism of this cross talk. Interestingly, Ras did not lie upstream of PI3K/Akt, whereas PI3K/Akt signaling to ERK1/2 involved Ras. These findings shed new light on the apoptotic resistance mechanism of nonadherent ovarian cancer ascites cells and may have important clinical implications.
The interplay between peritoneal mesothelial cells and ovarian cancer cells is critical for the initiation and peritoneal dissemination of, and ascites formation in, ovarian cancer. The production of lysophosphatidic acid (LPA) by both peritoneal mesothelial cells and ovarian cancer cells has been shown to promote metastatic phenotype in ovarian cancer. Herein, we report that exogenous addition or ectopic overexpression of the matricellular protein SPARC (secreted protein acidic and rich in cysteine) significantly attenuated LPA-induced proliferation, chemotaxis, and invasion in both highly metastatic SKOV3 and less metastatic OVCAR3 ovarian cancer cell lines. SPARC appears to modulate these functions, at least in part, through the regulation of LPA receptor levels and the attenuation of extracellular signal-regulated kinase (ERK) 1/2 and protein kinase B/AKT signaling. Moreover, our results show that SPARC not only significantly inhibited both basal and LPA-induced interleukin (IL) 6 production in both cell lines but also attenuated IL-6-induced mitogenic, chemotactic, and proinvasive effects, in part, through significant suppression of ERK1/2 and, to a lesser extent, of signal transducers and activators of transcription 3 signaling pathways. Our results strongly suggest that SPARC exerts a dual inhibitory effect on LPA-induced mesothelial-ovarian cancer cell crosstalk through the regulation of both LPA-induced IL-6 production and function. Taken together, our findings underscore the use of SPARC as a potential therapeutic candidate in peritoneal ovarian carcinomatosis.
SPARC; LPA; IL-6; ovarian cancer; mesothelial cells
Ovarian cancer is one of the leading causes of death from gynecological cancers in the United States. Conventional therapies are unlikely to control advanced stage ovarian cancers, thus requiring innovative alternative therapies. In the current study, we characterized the therapeutic effect of tumor cell-based vaccines combined with the adjuvant, α-Galactosylceramide (α-GalCer) using two different mouse models. Our data suggests that treatment with α-GalCer led to an increase in the IFN-γ serum levels in the presence or absence of irradiated mouse ovarian surface epithelial tumor cells (MOSEC). Furthermore, administration of irradiated MOSEC tumor cells with adjuvant α-GalCer generated significant protective and therapeutic antitumor effects against MOSEC tumors in vaccinated C57BL/6 mice. In addition, immune cells expressing CD4, CD8 or NK1.1 markers were found to be important for the protective anti-tumor effects generated by irradiated tumor cell-based vaccines combined with adjuvant α-GalCer. We also found that treatment of a spontaneous ovarian cancer murine model, the Müllerian inhibiting substance type II receptor T antigen (TgMISIIR-TAg) transgenic mice with ovarian tumor cell-based vaccines combined with adjuvant α-GalCer led to prolonged survival as well as increased numbers of tumor-specific CD8+ T cells. Therefore, irradiated tumor cell-based vaccines in combination with α-GalCer are capable of breaking immune tolerance and generating significant antitumor effects in two different mouse tumor models. Our study serves as a foundation for future clinical translation.
α-Galactosyl Ceramide; tumor cell-based vaccines; adjuvant; MOSEC; ovarian
Epithelial ovarian cancer (EOC) is the leading cause of gynecologic cancer death in the USA. Recurrence rates are high after front-line therapy and most patients eventually die from platinum (Pt) - resistant disease. Cisplatin resistance is associated with increased nucleotide excision repair (NER), decreased mismatch repair (MMR) and decreased platinum uptake. The objective of this study is to investigate how a novel combination of sodium arsenite (NaAsO2) and hyperthermia (43°C) affect mechanisms of cisplatin resistance in ovarian cancer.
We established a murine model of metastatic EOC by intraperitoneal injection of A2780/CP70 human ovarian cancer cells into nude mice. We developed a murine hyperthermic intraperitoneal chemotherapy model to treat the mice. Mice with peritoneal metastasis were perfused for 1 h with 3 mg/kg cisplatin ± 26 mg/kg NaAsO2 at 37 or 43°C. Tumors and tissues were collected at 0 and 24 h after treatment.
Western blot analysis of p53 and key NER proteins (ERCC1, XPC and XPA) and MMR protein (MSH2) suggested that cisplatin induced p53, XPC and XPA and suppressed MSH2 consistent with resistant phenotype. Hyperthermia suppressed cisplatin-induced XPC and prevented the induction of XPA by cisplatin, but it had no effect on Pt uptake or retention in tumors. NaAsO2 prevented XPC induction by cisplatin; it maintained higher levels of MSH2 in tumors and enhanced initial accumulation of Pt in tumors. Combined NaAsO2 and hyperthermia decreased cisplatin-induced XPC 24 h after perfusion, maintained higher levels of MSH2 in tumors and significantly increased initial accumulation of Pt in tumors. ERCC1 levels were generally low except for NaAsO2 co-treatment with cisplatin. Systemic Pt and arsenic accumulation for all treatment conditions were in the order: kidney > liver = spleen > heart > brain and liver > kidney = spleen > heart > brain respectively. Metal levels generally decreased in systemic tissues within 24 h after treatment.
NaAsO2 and/or hyperthermia have the potential to sensitize tumors to cisplatin by inhibiting NER, maintaining functional MMR and enhancing tumor platinum uptake.
cisplatin; sodium arsenite; hyperthermia; HIPEC; metastatic human ovarian cancer; p53; XPA; XPC; MSH2; platinum accumulation
Epithelial ovarian cancer is the fourth leading cause of death from gynecologic malignancies in the United States. Most cases are diagnosed at late stages, with the solid tumor masses growing as peritoneal implants, or floating within the ascitic fluid (peritoneal ovarian carcinomatosis). Despite aggressive surgical “debulking,” recurrence of recalcitrant disease is frequent with poor patient survival. Efforts to improve survival rates are hindered by lack of biomarkers that can detect and effectively treat ovarian cancer in its early stages. Urokinase plasminogen activator receptor (uPAR) is a multifunctional receptor involved in a myriad of tumor cell processes. However, the role of host uPAR in ovarian cancer is still elusive. To define the potential proinflammatory role of uPAR in ovarian cancer, first, using a syngeneic murine model in uPAR-/- mice, we found that ablation of uPAR restrained tumor take and peritoneal implants and prolonged the survival of uPAR-/- mice compared with their uPAR+/+ counterparts. Ascitic fluid accumulation was significantly decreased in uPAR-/- mice with decreased macrophage infiltration. Second, in vitro mechanistic studies revealed that host uPAR is involved in the multiple steps of peritoneal metastatic cascade. Third, we evaluated the prognostic utility of tumor and stromal uPAR in human ovarian cancer tissue microarray. In summary, our studies indicated that uPAR plays a significant role in ovarian cancer cell-stromal crosstalk and contributes to increased vascular permeability and inflammatory ovarian cancer microenvironment. This provides a rationale for targeting the uPAR with either specific neutralizing antibodies or targeting its downstream inflammatory effectors in patients with ovarian cancer.
Ovarian cancer is the most lethal gynecologic malignancy. Despite advances in chemotherapy, the five-year survival rate of advanced ovarian cancer patients with peritoneal metastasis remains around 30%. The most significant prognostic factor is stage, and most patients present at an advanced stage with peritoneal dissemination. There is often no clearly identifiable precursor lesion; therefore, the events leading to metastatic disease are poorly understood. This article reviews metastatic suppressor genes, the epithelial-mesenchymal transition (EMT), and the tumor microenvironment as they relate to ovarian cancer metastasis. Additionally, novel chemotherapeutic agents targeting the metastasis-related biochemical pathways are discussed.
cancer; metastasis suppressor gene; EMT; tumor microenvironment
Dissemination of ovarian tumors involves the implantation of cancer spheroids into the mesothelial monolayer on the walls of peritoneal and pleural cavity organs. Biopsies of tumors attached to peritoneal organs show that mesothelial cells are not present under tumor masses. We have developed a live, image-based in vitro model in which interactions between tumor spheroids and mesothelial cells can be monitored in real time to provide spatial and temporal understanding of mesothelial clearance. Here we provide evidence that ovarian cancer spheroids utilize integrin – and talin - dependent activation of myosin and traction force to promote mesothelial cells displacement from underneath a tumor cell spheroid. These results suggest that ovarian tumor cell clusters gain access to the sub-mesothelial environment by exerting force on the mesothelial cells lining target organs, driving migration and clearance of the mesothelial cells.
ovarian cancer; mesothelium; myosin; integrins; force
A crucial step in the metastatic spread of ovarian cancer (OC) is the adhesion and implantation of tumor cells to the peritoneal mesothelium. In order to study this step in the cascade, we derived a pro-metastatic human ovarian carcinoma cell line (MFOC3) from the non-metastatic FOC3 line.
Molecular profiling of the isogeneic lines identified differentially expressed genes, and investigation for a role in dissemination for specific factors was achieved by development of a co-culture adhesion assay utilizing monolayers of human mesothelial cells.
After murine intraperitoneal inoculation, the FOC3 cell line formed no metastases, but the MFOC3 subline formed metastases in > 80% of SCID mice. MFOC3 cells also adhered 2-3 times more avidly to mesothelial monolayers. This adhesion was inhibited by neutralizing antibodies to IL-1β and enhanced by recombinant IL-1β (p < 0.01). IL-1β induced mesothelial cell β1-integrin, and an antibody to this subunit also inhibited the adhesion of MFOC3 to mesothelial cells in vitro and significantly reduced metastases in vivo. Immunohistochemical analysis of a cohort of 96 ovarian cancer cases showed that negative IL-1β expression was significantly associated with an improved overall survival rate.
These results suggest that a IL-1β/β1-integrin axis plays a role in ovarian tumor cell adhesion to mesothelia, a crucial step in ovarian cancer dissemination.
ovarian cancer; peritoneal dissemination; IL-1β; β1 integrin; mesothelial cell
An optical probe, RG-(gal)28GSA, was synthesized to improve the detection of peritoneal implants by targeting the β-d-galactose receptors highly expressed on the cell surface of a wide variety of cancers arising from the ovary, pancreas, colon, and stomach. Evaluation of RG-(gal)28GSA, RG-(gal)20GSA, glucose-analog RG-(glu)28GSA, and control RG-HSA, demonstrates specificity for the galactose, binding to several human adenocarcinoma cell lines, and cellular internalization. Studies using peritoneally disseminated SHIN3 xenografts in mice also confirmed a preference for galactose with the ability to detect submillimeter size lesions. Preliminary toxicity study for RG-(gal)28GSA using Balb/c mice reveal no toxic effects up to 100x of the standard imaging dose of 1mg/kg administered either intraperitoneally or intravenously. These data indicate that RG-(gal)28GSA can selectively target a variety of human adenocarcinoma, can improve intraoperative or endoscopic tumor detection and resection, and may have little or no toxic in vivo effects; hence, it may be clinically translatable.
lectin; targeted optical agent; galactosylated human serum albumin; fluorescence imaging; toxicity; ovarian cancer
Patients with metastatic ovarian cancer continue to have a dismal prognosis, emphasizing the need for new strategies to identify and develop new molecular targets for therapy. Chemokine CXCL12 and its receptor CXCR4 are upregulated in metastatic ovarian cancer cells and the intraperitoneal tumor microenvironment. CXCL12-CXCR4 signaling promotes multiple steps in proliferation and dissemination of ovarian cancer cells, suggesting that targeted inhibition of this pathway will limit tumor progression. To investigate CXCL12-CXCR4 signaling in ovarian cancer and establish effects of inhibiting this pathway on tumor progression and survival, we designed a Gaussia luciferase complementation imaging reporter system to detect CXCL12 binding to CXCR4 in ovarian cancer cells. In cell-based assays, we established that the complementation imaging reporter could detect CXCL12 binding to CXCR4 and quantify specific inhibition of ligand-receptor interaction. We monitored CXCL12-CXCR4 binding and inhibition in a mouse xenograft model of metastatic human ovarian cancer by imaging Gaussia luciferase complementation and assessed tumor progression with firefly luciferase. Bioluminescence imaging studies in living mice showed that treatment with AMD3100, a clinically approved inhibitor of CXCL12-CXCR4, blocked ligand-receptor binding and reduced growth of ovarian cancer cells. Treatment with AMD3100 also modestly improved overall survival of mice with metastatic ovarian cancer. The Gaussia luciferase complementation imaging reporter system will facilitate further preclinical development and optimization of CXCL12-CXCR4 targeted compounds for treatment of ovarian cancer. Our research supports clinical translation of existing CXCR4 inhibitors for molecular therapy for ovarian cancer.
Ovarian carcinoma is the leading cause of death among gynecologic cancers. Although transformation of the outer ovarian epithelium was linked with ovulation, the disease is significantly more prevalent and severe in postmenopausal women. We postulated that menopause could augment ovarian cancer progression through the effects of gonadotropins on multifocal seeding to the mesothelial layer lining the peritoneum. This seeding is mediated by integrins as well as by CD44 interaction with hyaluronan (HA). Here, we report the effect of gonadotropins on HA synthesis and degradation and on peritoneal adhesion. A significant concentration- and time-dependent induction in expression levels of HA synthases (HASs) and hyaluronidases (Hyals) was observed in vitro on stimulation of human epithelial ovarian carcinoma cells by gonadotropins. Hormonal regulation of HA-mediated adhesion was manifested in vivo as well, by fluorescence microscopy of stained MLS multicellular tumor spheroids. The number of spheroids adhered to the mesothelium of ovariectomized CD-1 nude mice 9.5 hours after intraperitoneal insertion was significantly higher than in nonovariectomized mice. Inhibition of HA synthesis by 6-diazo-5-oxo-1-norleucine (DON) both in spheroids and ovariectomized mice significantly reduced the number of adhered spheroids. Thus, the change in the hormonal environment during menopause assists in HA-dependent adherence of ovarian cancer spheroids onto the peritoneum. However, HA is antiangiogenic and it can significantly suppress tumor progression. Accordingly, angiogenesis of the adhered spheroids was significantly elevated in DON-treated tumors. These results can explain the selective pressure that can lead to simultaneously increased tumor expression of both HASs and Hyals.
Intraperitoneal metastases commonly recur after surgery because small tumor foci escape detection within the complex anatomy of the peritoneal cavity and mesentery. Accurate localization of peritoneal implants during surgery could improve the resection of ovarian cancer and other malignancies, but few practical techniques to enhance detectability are currently available. Here, we describe a targeted molecular imaging method that employs fluorescently labeled avidin to detect submillimeter peritoneal implants of ovarian cancer in mice. After binding to surface lectins on the tumor, fluorescein-conjugated avidin enabled thespectral fluorescence imaging of disseminated peritoneal implants. High spatial resolution and high tumor-to-background ratio allowed the visualization of implants as small as 0.3 mm (with 100% sensitivity and specificity; n = 150) and the identification of even smaller lesions ex vivo. These results suggest that targeted molecular imaging with a fluorescence-labeled lectin-ligand system is a promising technique for the detection of disseminated submillimeter foci of cancer.
Metastasis; optical imaging; ovarian cancer; target imaging; microfoci
Ovarian cancer is the most deadly gynecological cancer. The high rate of mortality is due to the large tumor burden with extensive metastatic lesion of the abdominal cavity. Despite initial chemosensitivity and improved surgical procedures, abdominal recurrence remains an issue and results in patients' poor prognosis. Transcriptomic and genetic studies have revealed significant genome pathologies in the primary tumors and yielded important information regarding carcinogenesis. There are, however, few studies on genetic alterations and their consequences in peritoneal metastatic tumors when compared to their matched ovarian primary tumors. We used high-density SNP arrays to investigate copy number variations in matched primary and metastatic ovarian cancer from 9 patients. Here we show that copy number variations acquired by ovarian tumors are significantly different between matched primary and metastatic tumors and these are likely due to different functional requirements. We show that these copy number variations clearly differentially affect specific pathways including the JAK/STAT and cytokine signaling pathways. While many have shown complex involvement of cytokines in the ovarian cancer environment we provide evidence that ovarian tumors have specific copy number variation differences in many of these genes.
α-Galactosylceramide (α-GalCer) can be presented by CD1d molecules of antigen-presenting cells, and is known to induce a potent NKT cell-dependent cytotoxic response against tumor cells. However, the main effector cells in α-GalCer-induced antitumor immunity are still controversial.
In order to elucidate the cell phenotype that plays the most important role in α-GalCer-induced antitumor immunity, we purified and analyzed tumor-infiltrating leukocytes (TILs) from liver metastatic nodules of a colon cancer cell line (Colon26), comparing α-GalCer- and control vehicle-treated mice. Flow cytometry was performed to analyze cell phenotype in TILs and IFN-γ ELISA was performed to detect antigen-specific immune response.
Flow cytometry analysis showed a significantly higher infiltration of NK cells (DX5+, T cell receptor αβ (TCR)-) into tumors in α-GalCer-treated mice compared to vehicle-treated mice. The DX5+TCR+ cell population was not significantly different between these two groups, indicating that these cells were not the main effector cells. Interestingly, the CD8+ T cell population was increased in TILs of α-GalCer-treated mice, and the activation level of these cells based on CD69 expression was higher than that in vehicle-treated mice. Moreover, the number of tumor-infiltrating dendritic cells (DCs) was increased in α-GalCer-treated mice. IFN-γ ELISA showed stronger antigen-specific response in TILs from α-GalCer-treated mice compared to those from vehicle-treated mice, although the difference between these two groups was not significant.
In α-GalCer-induced antitumor immunity, NK cells seem to be some of the main effector cells and both CD8+ T cells and DCs, which are related to acquired immunity, might also play important roles in this antitumor immune response. These results suggest that α-GalCer has a multifunctional role in modulation of the immune response.
NK cell; cytotoxic T lymphocyte; dendritic cell; tumor-infiltrating leukocyte
Most cases of ovarian cancer are epithelial in origin and diagnosed at advanced stage when the cancer is widely disseminated in the peritoneal cavity. The objective of this study was to establish an immunocompetent syngeneic mouse model of disseminated epithelial ovarian cancer (EOC) to facilitate laboratory-based studies of ovarian tumor biology and preclinical therapeutic strategies.
Individual lines of TgMISIIR-TAg transgenic mice were phenotypically characterized and backcrossed to inbred C57BL/6 mice. In addition to a previously described line of EOC-prone mice, two lines (TgMISIIR-TAg-Low) were isolated that express the oncogenic transgene, but have little or no susceptibility to tumor development. Independent murine ovarian carcinoma (MOVCAR) cell lines were established from the ascites of tumor-bearing C57BL/6 TgMISIIR-TAg transgenic mice, characterized and tested for engraftment in the following recipient mice: 1) severe immunocompromised immunodeficient (SCID), 2) wild type C57BL/6, 3) oophorectomized tumor-prone C57BL/6 TgMISIIR-TAg transgenic and 4) non-tumor prone C57BL/6 TgMISIIR-TAg-Low transgenic. Lastly, MOVCAR cells transduced with a luciferase reporter were implanted in TgMISIIR-TAg-Low mice and in vivo tumor growth monitored by non-invasive optical imaging.
Engraftment of MOVCAR cells by i.p. injection resulted in the development of disseminated peritoneal carcinomatosis in SCID, but not wild type C57BL/6 mice. Oophorectomized tumor-prone TgMISIIR-TAg mice developed peritoneal carcinomas with high frequency, rendering them unsuitable as allograft recipients. Orthotopic or pseudo-orthotopic implantation of MOVCAR cells in TgMISIIR-TAg-Low mice resulted in the development of disseminated peritoneal tumors, frequently accompanied by the production of malignant ascites. Tumors arising in the engrafted mice bore histopathological resemblance to human high-grade serous EOC and exhibited a similar pattern of peritoneal disease spread.
A syngeneic mouse model of human EOC was created by pseudo-orthotopic and orthotopic implantation of MOVCAR cells in a susceptible inbred transgenic host. This immunocompetent syngeneic mouse model presents a flexible system that can be used to study the consequences of altered gene expression (e.g., by ectopic expression or RNA interference strategies) in an established MOVCAR tumor cell line within the ovarian tumor microenvironment and for the development and analysis of preclinical therapeutic agents including EOC vaccines and immunotherapeutic agents.
Ovarian cancer remains difficult to treat mainly due to presentation of the disease at an advanced stage. Conditionally-replicating adenoviruses (CRAds) are promising anti-cancer agents that selectively kill the tumor cells. The present study evaluated the efficacy of a novel CRAd (Ad5/3-CXCR4-TIMP2) containing the CXCR4 promoter for selective viral replication in cancer cells together with TIMP2 as a therapeutic transgene, targeting the matrix metalloproteases (MMPs) in a murine orthotopic model of disseminated ovarian cancer. An orthotopic model of ovarian cancer was established in athymic nude mice by intraperitonal injection of the human ovarian cancer cell line, SKOV3-Luc, expressing luciferase. Upon confirmation of peritoneal dissemination of the cells by non-invasive imaging, mice were randomly divided into four treatment groups: PBS, Ad-ΔE1-TIMP2, Ad5/3-CXCR4, and Ad5/3-CXCR4-TIMP2. All mice were imaged weekly to monitor tumor growth and were sacrificed upon reaching any of the predefined endpoints, including high tumor burden and significant weight loss along with clinical evidence of pain and distress. Survival analysis was performed using the Log-rank test. The median survival for the PBS cohort was 33 days; for Ad-ΔE1-TIMP2, 39 days; for Ad5/3-CXCR4, 52.5 days; and for Ad5/3-CXCR4-TIMP2, 63 days. The TIMP2-armed CRAd delayed tumor growth and significantly increased survival when compared to the unarmed CRAd. This therapeutic effect was confirmed to be mediated through inhibition of MMP9. Results of the in vivo study support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of human patients with advanced ovarian cancer.
Commonly used in flow cytometry, multiplexed optical probes can diagnose multiple types of cell surface marker, potentially leading to improved diagnosis accuracy in vivo. Herein, we demonstrate the targeting of two different tumor markers in models of disseminated ovarian cancer. Two ovarian cancer cell lines (SKOV3 and SHIN3) were employed; both over-express D-galactose receptor (D-GalR), but only SKOV3 over-expresses HER2/neu. Additionally, fusion tumors composed of SKOV3 and SHIN3/RFP were evaluated. Both galactosyl serum albumin-rhodamine green (GSA-RhodG) which binds D-galR and trastuzumab-Alexa680 which binds HER2/neu were administered to tumor bearing mice for in vivo fluorescence imaging and in situ fluorescence microscopy. In vivo fluorescence imaging depicted 64 of 69 SKOV3 tumors (94.2%) based on their dual spectra corresponding to both RhodG and Alexa680, while all 71 SHIN3 tumors (100%) were detected based on their single spectrum corresponding only to RhodG. All 59 SHIN3 and 36 SKOV3 tumors were correctly diagnosed with in situ microscopy. Additionally, in the mixed tumor model, all tumors could be depicted using the RhodG spectrum, but only SKOV3 components also showed the Alexa680 spectrum. In conclusion, multi-targeted multi-color optical imaging enabled specific in vivo diagnosis of tumors expressing distinct patterns of receptors, leading to improved diagnostic accuracy.
Molecular imaging; Optical probe; Cancer; Multi-color; Multi-targeting
Epithelial ovarian cancer (EOC) is the second most common genital malignancy in women and is the most lethal gynecological malignancy, with an estimated five-year survival rate of 39%. Despite efforts to develop an effective ovarian cancer screening method, 60% of patients still present with advanced disease. Comprehensive management using surgical cytoreduction to decrease the tumor load to a minimum, and intraperitoneal chemotherapy to eliminate microscopic disease on peritoneal surface, has the potential to greatly improve quality of life and to have an impact on survival in ovarian cancer patients. Despite achieving clinical remission after completion of initial treatment, most patients (60%) with advanced EOC will ultimately develop recurrent disease or show drug resistance; the eventual rate of curability is less than 30%. Given the poor outcome of women with advanced EOC, it is imperative to continue to explore novel therapies.
Peritoneal carcinosis; Ovarian cancer; Intraperitoneal hyperthermic chemotherapy; Cytoreduction
Most deaths from ovarian cancer are due to metastases that are resistant to conventional therapies. But the factors that regulate the metastatic process and chemoresistance of ovarian cancer are poorly understood. In the current study, we investigated the aberrant expression of human sperm protein 17 (HSp17) in human epithelial ovarian cancer cells and tried to analyze its influences on the cell behaviors like migration and chemoresistance.
Immunohistochemistry and immunocytochemistry were used to identify HSp17 in paraffin embedded ovarian malignant tumor specimens and peritoneal metastatic malignant cells. Then we examined the effect of HSp17 overexpression on the proliferation, migration, and chemoresistance of ovarian cancer cells to carboplatin and cisplatin in a human ovarian carcinoma cell line, HO8910.
We found that HSp17 was aberrantly expressed in 43% (30/70) of the patients with primary epithelial ovarian carcinomas, and in all of the metastatic cancer cells of ascites from 8 patients. The Sp17 expression was also detected in the metastatic lesions the same as in ovarian lesions. None of the 7 non-epithelial tumors primarily developed in the ovaries was immunopositive for HSp17. Overexpression of HSp17 increased the migration but decreased the chemosensitivity of ovarian carcinoma cells to carboplatin and cisplatin.
HSp17 is aberrantly expressed in a significant proportion of epithelial ovarian carcinomas. Our results strongly suggest that HSp17 plays a role in metastatic disease and resistance of epithelial ovarian carcinoma to chemotherapy.
Achieving maximal cytoreduction during surgery is a critical prognostic factor for women with advanced stage ovarian cancer. Targeting optical imaging agents directly to ovarian cancer cells by attaching them to galactosyl (galactosamine-conjugated) serum albumin, whose sugar residues bind surface lectins that are expressed in certain ovarian adenocarcinomas, may improve metastatic tumor identification and resection. Thus, we sought to demonstrate that galactosyl serum albumin-conjugated fluorophores would be a robust mechanism through which to target ovarian cancer by evaluating its tumor-targeting capability in nine human ovarian adenocarcinoma cell lines. The optical fluorophore Rhodamine Green was conjugated to galactosyl serum albumin, a non-immunogenic targeting molecule. Galactosyl serum albumin-Rhodamine Green’s ability to target nine human ovarian adenocarcinoma cell lines was evaluated by flow cytometry, fluorescence microscopy, and in vivo optical fluorescence imaging using female athymic nu/nu mice. All nine cell lines tested bound galactosyl serum albumin-Rhodamine Green more effectively than non-glycosylated controls (p<0.0001). Fluorescence microscopy demonstrated that galactosyl serum albumin-Rhodamine Green was internalized into each cell line in a galactosamine-dependent manner. In vivo optical fluorescence images of intraperitoneal tumor-bearing mice acquired three hours after intraperitoneal injection of galactosyl serum albumin-Rhodamine Green successfully differentiated between tumor and normal tissue. This technique also allowed the visualization of submillimeter-sized ovarian tumor implants. These results indicate that galactosyl serum albumin-Rhodamine Green can selectively target a variety of human ovarian adenocarcinomas for optical fluorescence imaging and thus may improve intraoperative tumor detection and resection.