Intraperitoneal delivery of human natural killer cells for treatment of ovarian cancer in a mouse xenograft model

Background aimsThere is an urgent need for novel therapeutic strategies for relapsed ovarian cancer. Dramatic clinical anti-tumor effects have been observed with interleukin (IL)-2 activated natural killer (NK) cells; however, intravenous delivery of NK cells in patients with ovarian cancer has not been successful in ameliorating disease. We investigated in vivo engraftment of intraperitoneally (IP) delivered NK cells in an ovarian cancer xenograft model to determine if delivery mode can affect tumor cell killing and circumvent lack of NK cell expansion.MethodsAn ovarian cancer xenograft mouse model was established to evaluate efficacy of IP-delivered NK cells. Tumor burden was monitored by bioluminescent imaging of luciferase-expressing ovarian cancer cells. NK cell persistence, tumor burden and NK cell trafficking were evaluated. Transplanted NK cells were evaluated by flow cytometry and cytotoxicity assays.ResultsIP delivery of human NK cells plus cytokines led to high levels of circulating NK and was effective in clearing intraperitoneal ovarian cancer burden in xenografted mice. NK cells remained within the peritoneal cavity 54 days after injection and had markers of maturation. Additionally, surviving NK cells were able to kill ovarian cancer cells at a rate similar to pre-infusion levels, supporting that in vivo functionality of human NK cells can be maintained after IP infusion.ConclusionsIP delivery of NK cells leads to stable engraftment and antitumor response in an ovarian cancer xenograft model. These data support further pre-clinical and clinical evaluation of IP delivery of allogeneic NK cells in ovarian cancer.     

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women ^1,2,3. Serous tumors are the most widespread forms of ovarian cancer and ^4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter ⁶. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice. Objective: Although tumor imaging is possible ⁷, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses. Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm ^8,9,10. We orthotopically implanted MOV1^Kat in the ovary ^11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry. Results: Orthotopically implanted MOV1^Kat cells developed serous ovarian tumors. MOV1^Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers ¹⁵ (fig. 2). Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.Download video file.^{(63M, mov)}     

Copper content in ascitic fluid is associated with angiogenesis and progression in ovarian cancer

BackgroundAscites is associated with the poor prognosis of malignant tumors. The biological importance of the changes in the content of trace elements in the ascitic fluid is unknown. Herein, we analyzed trace elements in the ascitic fluid of patients with ovarian tumors and used cultured cells to determine the copper (Cu)-induced changes in gene expression in ovarian cancer.MethodsInductively coupled plasma mass spectrometry (ICP-MS) was used to compare ascitic fluid trace element levels in patients with benign ovarian tumors (n = 22) and borderline/malignant tumors (n = 5) for primary screening. Cu levels were validated using atomic absorption spectrometry (AAS) in 88 benign, 11 borderline, and 25 malignant ovarian tumor patients. To confirm Cu-induced gene expression changes, microarray analysis was performed for Cu-treated OVCAR3, A2780, and Met5A cells. The vascular endothelial growth factor (VEGF) concentration in the cell supernatant or ascitic fluid (ovarian cancer samples) was measured using ELISA.ResultsICP-MS showed that Co, Ni, Cu, Zn, As, Se, and Mo levels significantly increased in patients with malignant/borderline ovarian tumors compared to those in patients with benign ovarian tumors. AAS showed that malignant ovarian tumors were independently associated with elevated levels of Cu in ascites adjusted for age, body mass index, alcohol, smoking, and supplement use (p < 0.001). Microarray analysis of both Cu-treated ovarian cancer cell lines OVCAR3 and A2780 and the mesothelial cell line Met-5A revealed the upregulation of the angiogenesis biological process. Real-time polymerase chain reaction and ELISA demonstrated that an increased Cu content significantly enhanced VEGF mRNA expression and protein secretion in OVCAR3, A2780, and Met-5A cells. VEGF levels and clinical stages of the tumors correlated with the ascitic fluid Cu content in patients with malignant ovarian tumors (correlation coefficient 0.445, 95 % confidence interval [CI]: 0.069–0.710, p = 0.023 and correlation coefficient 0.406, 95 % CI: 0.022–0.686, p = 0.040, respectively).ConclusionCu levels significantly increased in patients with malignant ovarian cancer. Cu induced angiogenic effects in ovarian cancer and mesothelial cells, which affected ascites fluid production. This study clarifies the link between elevated Cu in ascites and malignant ovarian tumor progression. Strategies to decrease Cu levels in the ascitic fluid may help downregulate VEGF expression, thereby improving the prognosis of ovarian malignancies.     

Anti-Transforming Growth Factor ? Antibody Treatment Rescues Bone Loss and Prevents Breast Cancer Metastasis to Bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.     

Targeting CD24 for treatment of ovarian cancer by short hairpin RNA

Background aimsCD24 is markedly overexpressed in ovarian cancer and plays a critical role in ovarian cancer survival and metastasis, rendering it an interesting target for anti-tumor therapy. Using short hairpin RNA (shRNA) targeting CD24, we aimed to investigate the anti-tumor efficacy of CD24 knockdown in ovarian cancer cells in vitro and in vivo.MethodsCD24 shRNA vector (CD24–shRNA) and empty plasmid vector (EP) were transfected into ovarian cancer SKOV3 cells and the knockdown efficacy assessed by Western blot analysis. The effects of CD24 knockdown in SKOV3 cells in vitro, including cell viability and apoptosis, were determined using methyl thiazolyl blue tetrazolium bromide (MTT), flow cytometry and propidium iodide (PI) staining assays. The effects in vivo of CD24 knockdown on angiogenesis, cell proliferation and apoptosis were assessed using immunohistochemistry against CD31, proliferating cell nuclear antigen (PCNA) and terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assays.ResultsTransfection of CD24–shRNA effectively down-regulated CD24 expression in vitro and in vivo. Administration of CD24–shRNA into nude mice bearing ovarian cancer significantly suppressed tumor volume growth.ConclusionsKnockdown of CD24 expression by CD24–shRNA significantly inhibited cell viability and induced apoptosis of SKOV3 cells in vitro. Administration with CD24–shRNA in vivo suppressed tumor volume increase by microvessel density (MVD) decrease, cell proliferation inhibition and apoptosis induction. All the data suggested that knockdown of CD24 by shRNA might be a potential therapeutic approach against human ovarian cancer.     

卵巢高/低级别浆液性癌的定量蛋白质组学比较研究

目的:探讨卵巢高级别浆液性癌和低级别浆液性癌的差异表达蛋白,为阐明卵巢癌发生机制及寻找诊断和预后标志物的提供线索。方法:收集卵巢癌新鲜组织标本冻存于液氮中,经病理学确诊为高级别浆液性癌和低级别浆液性癌,两种类型各收集15例。应用i TRAQ定量蛋白质组学技术筛选及鉴定高/低级别浆液性癌的差异表达蛋白,并进行生物信息学分析。结果:卵巢高级别和低级别浆液性癌组织的定量蛋白质组学比较研究鉴定出差异表达蛋白314个,其中与低级别浆液性癌组比较,高级别浆液性癌组上调蛋白有97种,下调蛋白有217种。GO分析显示这些差异蛋白在分子功能、生物学功能、细胞成分方面均具有一定分布特点。KEGG分析显示这些差异蛋白涉及复杂的信号通路。结论:高/低级别浆液性癌之间存在差异表达蛋白,这些蛋白涉及复杂的功能和信号通路可能在两型卵巢癌发生机制及肿瘤生物学行为差异中具有重要意义。     

Decreased Peritoneal Ovarian Cancer Growth in Mice Lacking Expression of Lipid Phosphate Phosphohydrolase 1

Lysophosphatidic acid (LPA) is a bioactive lipid that enhances ovarian cancer cell proliferation, migration and invasion in vitro and stimulates peritoneal metastasis in vivo. LPA is generated through the action of autotaxin or phospholipases, and degradation begins with lipid phosphate phosphohydrolase (LPP)-dependent removal of the phosphate. While the effects of LPA on ovarian cancer progression are clear, the effects of LPA metabolism within the tumor microenvironment on peritoneal metastasis have not been reported. We examined the contribution of lipid phosphatase activity to ovarian cancer peritoneal metastasis using mice deficient in LPP1 expression. Homozygous deletion of LPP1 (LPP1 KO) results in elevated levels and decreased turnover of LPA in vivo. Within 2 weeks of intraperitoneal injection of syngeneic mouse ovarian cancer cells, we observed enhanced tumor seeding in the LPP1 KO mice compared to wild type. However, tumor growth plateaued in the LPP1 KO mice by 3 weeks while tumors continued to grow in wild type mice. The decreased tumor burden was accompanied by increased apoptosis and no change in proliferation or angiogenesis. Tumor growth was restored and apoptosis reversed with exogenous administration of LPA. Together, these observations demonstrate that the elevated levels of LPA per se in LPP1 KO mice do not inhibit tumor growth. Rather, the data support the notion that either elevated LPA concentration or altered LPA metabolism affects other growth-promoting contributions of the tumor microenvironment.     

Implications of Proprotein Convertases in Ovarian Cancer Cell Proliferation and Tumor Progression: Insights for PACE4 as a Therapeutic Target

Proprotein convertases are a family of kexin-like serine proteases that process proteins at single and multiple basic residues. Among the predicted and identified PC substrates, an increasing number of proteins having functions in cancer progression indicate that PCs may be potential targets for antineoplastic drugs. In support of this notion, we identified PACE4 as a vital PC involved in prostate cancer proliferation and progression, contrasting with the other co-expressed PCs. The aim of the present study was to test the importance of PCs in ovarian cancer cell proliferation and tumor progression. Based on tissue-expression profiles, furin, PACE4, PC5/6 and PC7 all displayed increased expression in primary tumor, ascites cells and metastases. These PCs were also expressed in variable levels in three model ovarian cell lines tested, namely SKOV3, CAOV3 and OVCAR3 cells. Since SKOV3 cells closely represented the PC expression profile of ovarian cancer cells, we chose them to test the effects of PC silencing using stable gene-silencing shRNA strategy to generate knockdown SKOV3 cells for each expressed PC. In vitro and in vivo assays confirmed the role of PACE4 in the sustainment of SKOV3 cell proliferation, which was not observed with the other three PCs. We also tested PACE4 peptide inhibitors on all three cell lines and observed consequent reduced cell proliferation which was correlated with PACE4 expression. Overall, these data support a role of PACE4 in promoting cell proliferation in ovarian cancer and provides further evidence for PACE4 as a potential therapeutic target.     

Tumor Mutation Burden Forecasts Outcome in Ovarian Cancer with BRCA1 or BRCA2 Mutations

Background

Increased number of single nucleotide substitutions is seen in breast and ovarian cancer genomes carrying disease-associated mutations in BRCA1 or BRCA2. The significance of these genome-wide mutations is unknown. We hypothesize genome-wide mutation burden mirrors deficiencies in DNA repair and is associated with treatment outcome in ovarian cancer.

Methods and Results

The total number of synonymous and non-synonymous exome mutations (Nmut), and the presence of germline or somatic mutation in BRCA1 or BRCA2 (mBRCA) were extracted from whole-exome sequences of high-grade serous ovarian cancers from The Cancer Genome Atlas (TCGA). Cox regression and Kaplan-Meier methods were used to correlate Nmut with chemotherapy response and outcome. Higher Nmut correlated with a better response to chemotherapy after surgery. In patients with mBRCA-associated cancer, low Nmut was associated with shorter progression-free survival (PFS) and overall survival (OS), independent of other prognostic factors in multivariate analysis. Patients with mBRCA-associated cancers and a high Nmut had remarkably favorable PFS and OS. The association with survival was similar in cancers with either BRCA1 or BRCA2 mutations. In cancers with wild-type BRCA, tumor Nmut was associated with treatment response in patients with no residual disease after surgery.

Conclusions

Tumor Nmut was associated with treatment response and with both PFS and OS in patients with high-grade serous ovarian cancer carrying BRCA1 or BRCA2 mutations. In the TCGA cohort, low Nmut predicted resistance to chemotherapy, and for shorter PFS and OS, while high Nmut forecasts a remarkably favorable outcome in mBRCA-associated ovarian cancer. Our observations suggest that the total mutation burden coupled with BRCA1 or BRCA2 mutations in ovarian cancer is a genomic marker of prognosis and predictor of treatment response. This marker may reflect the degree of deficiency in BRCA-mediated pathways, or the extent of compensation for the deficiency by alternative mechanisms.     

Metabolomic characterization of experimental ovarian cancer ascitic fluid

Introduction

Malignant ascites (MA) is a major cause of morbidity that occurs in 37% of ovarian cancer patients. The accumulation of MA in the peritoneal cavity due to cancer results in debilitating symptoms and extremely poor quality of life. There is an urgent unmet need to expand the understanding of MA to design effective treatment strategies, and to improve MA diagnosis.

Objective

Our purpose here is to contribute to a better characterization of MA metabolic composition in ovarian cancer.

Method

We determined the metabolic composition of ascitic fluids resulting from orthotopic growth of two ovarian cancer cell lines, the mouse ID8- vascular endothelial growth factor (VEGF)-Defb29 cell line and the human OVCAR3 cell line using high-resolution ¹H MRS. ID8-VEGF-Defb29 tumors induce large volumes of ascites, while OVCAR3 tumors induce ascites less frequently and at smaller volumes. To better understand the factors driving the metabolic composition of the fluid, we characterized the metabolism of these ovarian cancer cells in culture by analyzing cell lysates and conditioned culture media with ¹H NMR.

Results

Distinct metabolite patterns were detected in ascitic fluid collected from OVCAR3 and ID8-VEGF-Defb29 tumor bearing mice that were not reflected in the corresponding cell culture or conditioned medium.

Conclusion

High-resolution ¹H NMR metabolic markers of MA can be used to improve characterization and diagnosis of MA. Metabolic characterization of MA can provide new insights into how MA fluid supports cancer cell growth and resistance to treatment, and has the potential to identify metabolic targeting strategies to reduce or eliminate the formation of MA.

     

Serum S100A6 Concentration Predicts Peritoneal Tumor Burden in Mice with Epithelial Ovarian Cancer and Is Associated with Advanced Stage in Patients

Background

Ovarian cancer is the 5th leading cause of cancer related deaths in women. Five-year survival rates for early stage disease are greater than 94%, however most women are diagnosed in advanced stage with 5 year survival less than 28%. Improved means for early detection and reliable patient monitoring are needed to increase survival.

Methodology and Principal Findings

Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of S100A6 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. S100A6 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100A6 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as S100A6 was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease.

Conclusions

S100A6 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. S100A6 is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100A6 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers.     

Cystathionine Beta-Synthase (CBS) Contributes to Advanced Ovarian Cancer Progression and Drug Resistance

Background

Epithelial ovarian cancer is the leading cause of gynecologic cancer deaths. Most patients respond initially to platinum-based chemotherapy after surgical debulking, however relapse is very common and ultimately platinum resistance emerges. Understanding the mechanism of tumor growth, metastasis and drug resistant relapse will profoundly impact the therapeutic management of ovarian cancer.

Methods/Principal Findings

Using patient tissue microarray (TMA), in vitro and in vivo studies we report a role of of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme in ovarian carcinoma. We report here that the expression of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme, is common in primary serous ovarian carcinoma. The in vitro effects of CBS silencing can be reversed by exogenous supplementation with the GSH and H₂S producing chemical Na₂S. Silencing CBS in a cisplatin resistant orthotopic model in vivo by nanoliposomal delivery of CBS siRNA inhibits tumor growth, reduces nodule formation and sensitizes ovarian cancer cells to cisplatin. The effects were further corroborated by immunohistochemistry that demonstrates a reduction of H&E, Ki-67 and CD31 positive cells in si-RNA treated as compared to scrambled-RNA treated animals. Furthermore, CBS also regulates bioenergetics of ovarian cancer cells by regulating mitochondrial ROS production, oxygen consumption and ATP generation. This study reports an important role of CBS in promoting ovarian tumor growth and maintaining drug resistant phenotype by controlling cellular redox behavior and regulating mitochondrial bioenergetics.

Conclusion

The present investigation highlights CBS as a potential therapeutic target in relapsed and platinum resistant ovarian cancer.     

Anti-tumour effects of a specific anti-ADAM17 antibody in an ovarian cancer model in vivo

ADAM 17 (TNF-α converting enzyme, TACE) is a potential target for cancer therapy, but the small molecule inhibitors reported to date are not specific to this ADAM family member. This membrane-bound metalloproteinase is responsible for ectodomain shedding of pathologically significant substrates including TNF-α and EGFR ligands. The aim of this study was to evaluate the pharmacokinetics, pharmacodynamics and anti-tumour efficacy of the first specific inhibitor, an anti-human ADAM17 IgG antibody, clone D1(A12). We used intraperitoneal xenografts of the human ovarian cancer cell line IGROV1-Luc in Balb/c nude mice, chosen because it was previously reported that growth of these xenografts is inhibited by knock-down of TNF-α. In vitro, 200 nM D1(A12) inhibited shedding of ADAM17 substrates TNF-α, TNFR1-α, TGF-α, amphiregulin (AREG), HB-EGF and IL-6Rα, from IGROV1-Luc cells, (4.7 nM IC₅₀ for TNF-α shedding). In IGROV1-Luc xenografts in vivo, D1(A12) IgG showed pharmacokinetic properties suitable for efficacy studies, with a single i.p. dose of 10 mg/kg D1(A12) sufficient to maintain IgG plasma and ascites fluid concentrations above 100 nM for more than 7 days. The plasma half life was 8.6 days. Next, an efficacy study was performed, dosing D1(A12) or anti-human TNF-α antibody infliximab at 10 mg/kg q7d, quantifying IGROV1-Luc tumour burden by bioluminescence. D1(A12) IgG showed a significant reduction in tumour growth (p = 0.005), 56% of vehicle control. Surprisingly, D1(A12) did not reduce the concentration of circulating human TNF-α, suggesting that another enzyme may compensate for inhibition of ADAM17 in vivo (but not in vitro). However, D1(A12) did show clear pharmacodynamic effects in the mice, with significant inhibition of shedding from tumour of ADAM17 substrates TNFR1-α, AREG, and TGF-α (4–15-fold reductions, p<0.0001 for all three). Thus, D1(A12) has anti-ADAM17 activity in vivo, inhibits shedding of EGFR ligands and has potential for use in EGF ligand-dependent tumours.     

The O-Linked Glycome and Blood Group Antigens ABO on Mucin-Type Glycoproteins in Mucinous and Serous Epithelial Ovarian Tumors

Background

Mucins are heavily O-glycosylated proteins where the glycosylation has been shown to play an important role in cancer. Normal epithelial ovarian cells do not express secreted mucins, but their abnormal expression has previously been described in epithelial ovarian cancer and may relate to tumor formation and progression. The cyst fluids were shown to be a rich source for acidic glycoproteins. The study of these proteins can potentially lead to the identification of more effective biomarkers for ovarian cancer.

Methods

In this study, we analyzed the expression of the MUC5AC and the O-glycosylation of acidic glycoproteins secreted into ovarian cyst fluids. The samples were obtained from patients with serous and mucinous ovarian tumors of different stages (benign, borderline, malignant) and grades. The O-linked oligosaccharides were released and analyzed by negative-ion graphitized carbon Liquid Chromatography (LC) coupled to Electrospray Ionization tandem Mass Spectrometry (ESI-MSⁿ). The LC-ESI-MSⁿ of the oligosaccharides from ovarian cyst fluids displayed differences in expression of fucose containing structures such as blood group ABO antigens and Lewis-type epitopes.

Results

The obtained data showed that serous and mucinous benign adenomas, mucinous low malignant potential carcinomas (LMPs, borderline) and mucinous low-grade carcinomas have a high level of blood groups and Lewis type epitopes. In contrast, this type of fucosylated structures were low abundant in the high-grade mucinous carcinomas or in serous carcinomas. In addition, the ovarian tumors that showed a high level of expression of blood group antigens also revealed a strong reactivity towards the MUC5AC antibody. To visualize the differences between serous and mucinous ovarian tumors based on the O-glycosylation, a hierarchical cluster analysis was performed using mass spectrometry average compositions (MSAC).

Conclusion

Mucinous benign and LMPs along with mucinous low-grade carcinomas appear to be different from serous and high-grade mucinous carcinomas based on their O-glycan profiles.     

Potent Therapeutic Activity Against Peritoneal Dissemination and Malignant Ascites by the Novel Anti-Folate Receptor Alpha Antibody KHK2805

Many ovarian cancer patients often show peritoneal metastasis with malignant ascites. However, unmet medical needs remain regarding controlling these symptoms after tumors become resistant to chemotherapies. We developed KHK2805, a novel anti-folate receptor α (FOLR1) humanized antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The primary aim of the present study was to evaluate whether the anti-tumor activity of KHK2805 was sufficient for therapeutic application against peritoneal dissemination and malignant ascites of platinum-resistant ovarian cancer in preclinical models. Here, both the ADCC and CDC of KHK2805 were evaluated in ovarian cancer cell lines and patient-derived samples. The anti-tumor activity of KHK2805 was evaluated in a SCID mouse model of platinum-resistant peritoneal dissemination. As results, KHK2805 showed specific binding to FOLR1 with high affinity at a novel epitope. KHK2805 exerted potent ADCC and CDC against ovarian cancer cell lines. Furthermore, primary platinum-resistant malignant ascites cells were susceptible to autologous ADCC with KHK2805. Patient-derived sera and malignant ascites induced CDC of KHK2805. KHK2805 significantly reduced the total tumor burden and amount of ascites in SCID mice with peritoneal dissemination and significantly prolonged their survival. In addition, the parental rat antibody strongly stained serous and clear cell-type ovarian tumors by immunohistochemistry. Overall, KHK2805 showed cytotoxicity against both ovarian cancer cell lines and patient-derived cells. These translational study findings suggest that KHK2805 may be promising as a novel therapeutic agent for platinum-resistant ovarian cancer with peritoneal dissemination and malignant ascites.     

Antiangiogenic Therapy with Human Apolipoprotein(a) Kringle V and Paclitaxel in a Human Ovarian Cancer Mouse Model

INTRODUCTION: The present study compared the effect of combination therapy using human apolipoprotein(a) kringle V (rhLK8) to conventional chemotherapy with paclitaxel for human ovarian carcinoma producing high or low levels of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: Human ovarian carcinoma cells producing high (SKOV3ip1) or low (HeyA8) levels of VEGF were implanted into the peritoneal cavity of female nude mice. Seven days later, mice were randomized into four groups: control (vehicle), paclitaxel [5 mg/kg, weekly intraperitoneal (i.p.) injection], rhLK8 (50 mg/kg, daily i.p. injection), or the combination of paclitaxel and rhLK8. Mice were treated for 4 weeks and examined by necropsy. RESULTS: In mice implanted with SKOV3ip1 cells, rhLK8 treatment had no significant effect on tumor incidence or the volume of ascites but induced a significant decrease in tumor weight compared with control mice. Paclitaxel significantly reduced tumor weight and ascites volume, and combination treatment with paclitaxel and rhLK8 had an additive therapeutic effect. Similarly, in HeyA8 mice, the effect of combination treatment on tumor weight and tumor incidence was statistically significantly greater than that of paclitaxel or rhLK8 alone. Immunohistochemical analysis showed a significant decrease in microvessel density and a marked increase of apoptosis in tumor and tumor-associated endothelial cells in response to combination treatment with paclitaxel and rhLK8. CONCLUSION: Collectively, these results suggest that antiangiogenic therapy with rhLK8 in combination with taxane-based conventional chemotherapy could be effective for the treatment of ovarian carcinomas, regardless of VEGF status.     

Endonucleases induced TRAIL-insensitive apoptosis in ovarian carcinoma cells

TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18^®:DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.