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Objectives: Dendritic cell (DC) based immunotherapies have been shown to be a potential treatment option for various cancers; however, the exact strategies in ovarian cancer remain unknown. This study aimed to evaluate the effectiveness of mouse CD8α+ dendritic cells (DCs), corresponding to human CD141+ DCs, derived from bone marrow (BM) hematopoietic stem cells (HSCs) in a syngeneic and orthotopic mouse ovarian cancer model. Methods: Stem-DCs from HSCs and Mono-DCs from monocytes were generated from the bone marrow mononuclear cells (BM-MNCs) of C57BL/6 mice in condition of pulsing with ID8 tumor cell lysates. C57BL/6 mice were intraperitoneally injected with 5 × 106 ID8 cells. They were treated with vehicle, low/medium/high dose pulsed Stem-DCs, Mono-DCs, and unpulsed Stem-DCs. At 8 to 9 weeks, the treated mice were sacrificed, and tumor responses and immune responses, such as lymphocyte proliferation and cytokine secretion, were analyzed. Results: Mono-DCs and Stem-DCs were characterized by CD11c+CD80+CD86+ and CD8α+Clec9a+ expression, respectively. They were confirmed by the secretion of immunostimulatory cytokines, such as interleukin (IL)-12 and interferon (IFN)-γ, and T cell proliferation was observed after maturation. Despite a lower dose compared with Mono-DCs, mice treated with pulsed Stem-DCs showed a reduced amount of ascitic fluid and lower body weights compared with those of vehicle treated mice (P = 0.0021 and P = 0.0092, respectively). These mice treated with pulsed Stem-DCs appeared to have fewer tumor implants which were usually confined in the epithelium of ovaries, diaphragm and peritoneum. All mice injected with DCs demonstrated longer survival than the vehicle group (P = 0.0187), especially the medium/high dose pulsed Stem-DC treatment groups. Moreover, these favorable tumor responses were associated with a low proportion of myeloid-derived suppressor cells (MDSCs) and regulatory T cells, high IL-12 and IFN-γ levels, and accumulation of several tumor-infiltrating lymphocytes. Conclusions: We demonstrated that mouse CD8α+ DCs derived from BM HSCs could decrease tumor progression and enhance antitumor immune responses against murine ovarian cancer. Further studies are necessary to develop potent DC vaccines using human CD141+ DCs. Citation Format: Shin-Wha Lee, Dasol Oh, Hyunah Lee, Kyung-Won Lee, Min-Je Kim, Sung Wan Kang, Young-Jae Lee, HyunSoo Kim, Yong-Man Kim. CD8α+ dendritic cells potentiate the antitumor and immune activities against murine ovarian cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6798.

Background: High-risk human papillomavirus (HPV) type 16 is the major etiological agent found in more than 60% of patients with cervical cancer and associated with cancer development and progression. Persistent expression HPV E6 and E7 oncoproteins are essential for the initiation and maintenance of cervical cancer. The therapeutic approaches targeting HPV E6 and E7 have been proved to be highly efficient in killing malignant cells. The suppression of HPV 16 E6/E7 expression by the short interfering RNA (siRNA) was more effective in combination therapy with cisplatin (CDDP) for cervical cancer. ENB101-LNP is an ionizable lipid nanoparticles (LNPs) encapsulating siRNA against E6/E7 of HPV 16 for delivery to the tumor cells. Methods: To investigate the anticancer efficacy and mechanism of ENB101-LNP combined with cisplatin in cervical cancer; the xenograft model was generated by the injection of CaSki cells subcutaneously into BALB/c nude mice. Mice were randomly divided into six groups: 1) blank control; 2) 1mg/kg ENB101-LNP; 3) 3mg/kg ENB101-LNP; 4) CDDP; 5) 1mg/kg ENB101-LNP-CDDP; 6) 3mg/kg ENB101-LNP-CDDP. ENB101-LNP was treated with intravenous injection three times weekly for 3 weeks and CDDP was treated with IP injection once a week for 3 weeks. Tumor growth curves were plotted to calculate the tumor inhibition rate. Mice were sacrificed one week after the last treatment and the tumor tissues were investigated using histopathology, immunohistochemical staining and western blotting. Results: Compared with the control and the other treatment groups, the tumor growth was significantly inhibited (P < 0.05). The tumor inhibition rate was 29.7% in 1mg/kg ENB101-LNP group, 29.6% in 3mg/kg ENB101-LNP group, 34.0% in CDDP group, 47.0% in 1mg/kg ENB101-LNP-CDDP group. At the dose of 3mg ENB101-LNP/kg combined with CDDP exhibited superior antitumor efficacy, the tumor inhibition rate was 68.8%. The successful ENB101-LNP mediated knockdown (with up to 80%) of HPV16 E6/E7 in tumors of both 1mg/kg and 3mg/kg groups was confirmed by RT-PCR. The ENB101-LNP increased p53 and p21 expression in tumors compared to control and CDDP alone groups and combination treatment with CDDP showed decreased expression of PD-L1 expression compared to CDDP alone group. The treatment of ENB101-LNP and combination with CDDP showed significant increase in apoptotic cells compared to control and single-agent groups respectively. Conclusions: The ENB101-LNP inhibiting E6 and E7 of HPV 16 in combination with CDDP showed promising anticancer activity in the cervical cancer cells xenograft mouse model. Citation Format: Sung Wan Kang, Shin-Wha Lee, Ji-young Lee, Ok Ju Kang, Hyejeong Kim, Yisak Kim, Yong-Man Kim. Evaluation of anti-cancer efficacy of lipid nanoparticles containing siRNA against HPV16 E6 and E7 combined with cisplatin in xenograft model of cervical cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3779.

Many studies have reported the efficacy of poly (ADP-ribose) polymerase (PARP) inhibitors in patients with epithelial ovarian cancer (EOC) with or without both germline/somatic BRCA mutations. However, resistance to PARP inhibitor occurs frequently in patients with advanced stage and recurrent. The receptor tyrosine kinase cMET, which is frequently overexpressed in EOC associates with PARP1 activity and cell survival pathway. Therefore, we investigated whether the combination of olaparib and savolitinib, a novel cMET inhibitor, could overcome PARP inhibitor resistance on resistant patient-derived xenografts (PDXs). Tumor tissues were obtained from patients with EOC operated at department of obstetrics and gynecology, Asan medical center, Seoul, South Korea. BRCA1 or BRCA2 mutated and cMET overexpressed tumors were selected and used for animal experiments. The tumor tissues were subcutaneous transplanted into right flank of NOD-scid IL2Rgammanull (NSG) mice. 60~90 days after transplantation, olaparib was administrated (80~100 mg/kg, daily, P.O.). Tumors were inhibited until volume decreased to 0~50 mm3. When the tumor volume was reached under 50 mm3, administration was stopped and tumors were allowed to regrowth. After tumor regrowth, to confirm resistance, administrated concentration of the olaparib was gradually increased 40 to 100 mg/kg (daily, P.O.). The resistant tumors were harvested and re-transplanted into 40 NSG mice. When tumor volumes reached at 100 mm3, mice were divided into 4 groups (Vehicle, Olapairb, Savolitinib, Combination) and olaparib (100 mg/kg, daily, P.O.) and savolitinib (25 mg/kg, daily, P.O.) were administrated. We established acquired resistant and innate resistant PDXs, which have BRCA mutation and cMET overexpression. These resistant models were used for evaluation of combined treatment of PARP inhibitor and cMET inhibitor. In case of the olaparib sensitive PDXs, tumor growth was suppressed to a similar level in combination group and olaparib single treatment group. Single treatment of cMET inhibitor also showed little inhibition in tumor growth. In case of the acquired resistant PDXs, tumor growth rate were highly increased in vehicle group than the sensitive PDXs. Contrary to sensitive group, combination treatment was more efficient to inhibit tumor growth rather than olaparib single treatment. The innate resistant PDXs were more tolerated to olapairb than the acquired resistant PDXs. Similar to acquired resistant PDXs, innate PDXs also showed rapid tumor growth in vehicle. As the acquired resistant PDXs, combination treatment was most efficient to inhibited tumor growth. In this study, cMET inhibition sensitized the resistant tumor to PARP inhibitor. These results indicated that savolitinib, novel cMET inhibitor, could have synergy with the PARP inhibitor for patients with PARP inhibitor resistant ovarian cancer. Citation Format: Min-Je Kim, Shin-Wha Lee, Young-Jae Lee, Su-Bin Park, Dong-Woo Kang, Yong-Man Kim. Combining PARP inhibitor with cMET Inhibition overcomes PARP inhibitor resistance in epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6185.

Immune checkpoint inhibitors (ICIs) have been applied in patients with various solid tumors since they were approved by U.S. FDA in 2011. However, only less than 20% of patients benefit from ICIs including anti-programmed cell death protein 1 (aPD-1). Recently, many studies to improve the response of ICIs are in progress, and especially, vascular endothelial growth factor receptors (VEGFR) pathway was emerged as major target. VEGFR inhibitors regulate the differentiation of tumor-associated macrophage, antigen presenting dendritic cell, and T cell infiltration, so they make a synergic antitumor effect with ICIs. In this study, we investigated the effect of lenvatinib combined with anti-mouse PD-1 in syngeneic murine cervical cancer models to show whether VEGFRi enhance the antitumor effect of ICIs. We established syngeneic mouse models of cervical cancer to confirm synergic effect by lenvatinib combined with aPD-1. 1x107 cells of U14 cells were injected subcutaneously into the flanks of BALB/c wild- type (immunocompetent) mice and BALB/c nude (immunocompromised) mice. They were treated with lenvatinib when the tumor volume reached at 200 mm3, subsequently, aPD-1 was administered in immunocompetent mice. We administrated lenvatinib (10 mg/kg, orally, daily) and aPD-1(200 µg per mouse, intraperitoneally (I.P), twice a week) for 3 weeks. Tumor volume was measured twice a week. After the end of the experiment, we harvested tumors and spleens, and performed histological analysis. All experiments were approved by the Institutional Animal Use and Care Committee (IACUC) in Asan Institute for Life Science.Although the tumor was increased despite lenvatinib treatment in immunocompromised model with BALB/c nude mice, on the 17th day after injection, the tumor growth was inhibited in lenvatinib group compared with vehicle group (2914 mm3 vs. 3663 mm3, respectively) (P=0.0014). On the other hand, the tumor volume was significantly reduced by lenvatinib in the immunocompetent model (278 mm3 in Len vs. 490 mm3 in Veh) (P=0.0347), particularly the tumor size was decreased since 2 weeks of injection. Subsequently, we investigated the synergistic effects of combination with lenvatinib and aPD-1 in immunocompetent model. Each single treatment group showed the reduction of tumor volume compared to vehicle group (278 mm3 in Len and 258 mm3 in aPD-1 vs. 490 mm3 in Veh). Furthermore, the lenvatinib plus aPD-1 combination group reduced tumor volume to 110mm3 on the 24th day after injection and it was significantly different compared to each single treatment group (P=0.13868 and P=0.27385, respectively).In this study, anti-tumor effect of aPD-1 was enhanced by the regulation of tumor microenvironment with lenvatinib in immunocompetent murine cervical cancer models. In conclusion, addition of lenvatinib is expected to increase the efficacy of ICIs in patients with cervical cancer who have the resistance or insensitivity to ICIs. Citation Format: Su-Bin Park, Ji-Sik Kang, Min-Je Kim, Shin-Wha Lee, Dong-Woo Kang, Yong-Man Kim. Anti-tumor effects of lenvatinib plus anti-PD-1 in syngeneic murine cervical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6190.

The RB1/E2F1 signaling pathway is frequently deregulated in colorectal cancer and has been suggested to intersect with Wnt/β-catenin and PI3K/Akt pathways, but molecular evidence for this link is lacking. In this study, we demonstrate that phospholipase D1 (PLD1), a transcriptional target of β-catenin/TCF4, orchestrates functional interactions between these pathways during intestinal tumor development. Overexpression of PLD1 in intestinal epithelial cells protected cells from apoptosis induced by PLD1 ablation in the Apcmin/+ mouse model of intestinal tumorigenesis. Mechanistic investigations revealed that genetic and pharmacologic targeting of PLD1 promote the E2F1-dependent apoptotic program via both miR-192/4465–mediated downregulation of RB1 and inhibition of Akt–TopBP1 pathways. Moreover, the miRNA–RB1 axis and Akt pathway also contributed to the PLD1-mediated self-renewal capacity of colon cancer–initiating cells. Finally, PLD1-driven E2F1 target gene expression positively correlated with tumor stage in patients with colorectal cancer. Overall, our findings suggest that PLD1 mediates cross-talk between multiple major signaling pathways to promote the survival and malignancy of colon cancer cells and may therefore represent an ideal signaling node for therapeutic targeting. Cancer Res; 77(1); 142–52. ©2016 AACR.

Background and Purpose: Ovarian cancer is the most frequent cause of death from gynecologic malignancy. Recently, immunotherapies have been suggested as ways to treat this malignancy. However, the ideal of immunotherapy has not been realized yet in ovarian cancer patients. Therefore, we investigated effects of monocyte-derived dendritic cells (mono DCs) and new-generation DCs subset (new DCs) on an ID8 orthotropic mouse model of ovarian cancer. Methods: Mono DCs were prepared from bone marrow of C57BL/6 mice. We isolated monocyte from bone marrow by monocyte isolation kit. Isolated monocyte was incubated for 7 days with IL-4 and GM-CSF. In addition, monocytes were pulsed with LPS and ID8 tumor lysate while incubating. Likewise new DCs were isolated from bone marrow of C57BL/6 mice. We isolated lineage negative cells from bone marrow using lineage cell depletion kit. Isolated cells were incubated with IL-4 and GM-CSF; also the cells were pulsed by ID8 tumor lysate and Rg3, which is a ginsenoside. Orthotropic model of ID8 was made on C57BL/6 mice. We injected 5x106 cells into peritoneal cavity. 4 weeks after cell injection, mice were grouped into three groups, which were vehicle, mono DCs and new DCs. Mice were injected with mono DCs and new DCs. 5x106 cells of DCs were injected 1 time/week for three weeks. One week later, mice were sacrificed and organs were harvested such as peritoneum, phren, ovary and tumor mass for histologic analysis. Results: DCs treated groups showed lower ascites formation than vehicle group. Ascites were fewest in new DCs group. Survival rate was lowest in vehicle group. Each survival rate is 0% in vehicle, 63% in new DCs and 80% in mono DCs. Also tumor growth at ovary was more decreased in mono DCs and new DCs group. In addition, mono DCs-treated mice showed low level of metastasis to other organs. Conclusion: Immunotherapy with dendritic cells showed inhibition of tumor growth and metastasis on mouse ovarian cancer. These results indicated that DCs treatments are able to be a potential therapy for ovarian cancer. It also could have synergy with the combined treatment of existing anticancer drugs. However, there are several limitations such as a relatively high dose of DCs and the procedure of isolating DCs needed large amount of bone marrow. We will continue to study and improve these issues. Citation Format: Min-Je Kim, Joo-won Kim, Ju-hyun Kim, Young-Jae Lee, Shin-Wha Lee, Yong-Man Kim. Immunotherapy with dendritic cells inhibited tumor growth on an ID8 orthotropic mouse model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 119.

Background: The analysis of single parameters alone may not provide sufficient insights about complex immune system-tumor interactions. This study is to validate the immune contexture as prognostic biomarkers in high-grade serous ovarian cancer (HGS-OC) and to find new era of immunoscore in HGS-OC. Methods: We collected FFPE samples from 187 patients with HSOC and produced TMA samples. We accomplished the OPAL multiplex IHC assay for the quantitative analysis of immune markers, including CD4, CD8, CD20, FoxP3, PD-L1, and CK. Multiplex Biomarker Imaging and inForm® Image Analysis Software was used. Results: FIGO stage III and IV patients were 84.5% (158/187). The optimal debulking surgery was done in 66.8% (125/187). The 3-year disease-free survival and 5-year overall survival were 35.1% and 50.0%, respectively. Any single marker was not related to the survival, including CD8, FoxP3, and PD-L1. However, high CD8:FoxP3 and CD8:PD-L1 ratios were correlated with good survival. In Cox regression model, the risk factors for HGS-OC survival were FIGO stage (HR 1.784, 95% CI: 1.295-2.457, p Conclusion: These findings indicate that although any single immune marker is not related to the survival, CD8:FoxP3 and CD8:PD-L1 ratios provide the positive correlation with the prognosis in HGS-OC; especially, CD8:PD-L1 ratio is prognostic biomarker that is comparable to clinical biomarkers. The next study for immunoscore is necessary to define immunoscore in ovarian cancer. Citation Format: Shin-Wha Lee, Min-Je Kim, Ju-hyun Kim, Young-Jae Lee, Yong-Man Kim, Yong-Man Kim. Immunoscore as prognostic biomarkers in patients with epithelial ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5703.

Objective: Most BRCA1/2 carriers do not undergo risk-reducing salpingo-oophorectomy (RRSO) by the recommended age. This study aimed to find the incidence of precursor lesions and cancer after RRSO. Methods: We retrospectively reviewed breast cancer patients identified as BRCA mutation carriers who underwent RRSO at Asan Medical Center, Seoul, Korea, from 2010 to 2014. From 2013, all cases were examined according to the SEE/FIM protocol and underwent immunohistochemically (IHC) staining. RRSO was performed in 63 patients, 27 in 2010-2012 and 36 in 2013-2014. Results: The median age at RRSO was 46.5 years (32-73 years). Occult invasive cancer was detected in 8 patients, of ovarian origin in 5 and of tubal origin in 3. All occult invasive cancer cases with metastasis were detected in patients older than 40 years. Of the 36 patients from the 2013-2014 cohort, 7 showed p53 overexpression, 1 showed Ki-67 overexpression, 2 showed serous tubal intraepithelial carcinoma (STIC), and 3 showed occult cancer. The detection rate of precursor lesions or cancer was 36.1% (13/36). In the analysis according to age, precursor lesions were more common in BRCA1 mutation carriers younger than 40 years old (66.7% vs 20.0%). In BRCA2 mutation carriers, precursor lesions were only detected in those older than 40 years of age, indicating the possible faster occurrence of precursor lesions in BRCA1 mutation carriers. Conclusions: Many patients still tend to delay RRSO until after they are 40 years old. Our findings support the significance of RRSO before the age of 40 in germline BRCA mutation carriers. Citation Format: YoungJae Lee, YongMan Kim, ShinWha Lee. Pathologic findings at RRSO in germline BRCA mutation carriers with breast cancer: significance of bilateral RRSO at the optimal age in germline BRCA mutation carriers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 493. doi:10.1158/1538-7445.AM2017-493

Background: Endometrial cancer is the most common malignancy of the female reproductive tract and the third most common cause of death from women's cancer. Risk factors of endometrial cancer include high levels of estrogen, obesity, diabetes mellitus, breast cancer, long term use of tamoxifen, late menopause, high blood pressure, and increasing age. Although the exact mechanisms in endometrial carcinogenesis due to chronic exposure are unclear, it is though that the pro-proliferative and DNA-damaging effects of estrogen and its metabolites, related with and insufficient counterbalance by progesterone, promote the hyper-proliferation and transformation of cells. Selective progesterone receptor modulator (SPRM) is an agent that acts on the progesterone receptor. Ulipristal acetate (UA) is one of SPRM and other SPRM, such as Mifepristone, showed inhibitory effect on various endometrial cancer cell lines. UA has been available as an emergency contraception and a treatment for uterine leiomyoma. The effect of UA on leiomyoma is decreasing neovascularization, proliferation, and viability. Objective: This study was undertaken to investigate antitumor effect of UA and to explore the effect of combination treatment with chemotherapy drug in endometrial cancer. Method: Inhibitory concentration (IC), proliferation assay, and migration assay were performed using Ishikawa, HEC-1-A, HEC-1-B, and/or patient-derived primary cancer cells (PCC). Different concentration of UA were treated in endometrial cancer cell lines and PCCs for the each assay. The CellTiter-Glo assay were performed to investigate IC50 in cell lines and PCCs. Crystal violet staining and wound healing assay were performed to examine cell proliferation and migration. Results: UA inhibited cell viability of endometrial cancer cell lines and PCCs at high concentration, most abviously 10uM in dose-dependent manner. In combination treatment assay with paclitaxel, UA showed synergistic anti-tumor effect with low-dose of paclitaxel on cell lines and PCCs. Conclusions: We showed that combination treatment of UA and paclitaxel effectively than single treatment by low dosage. In summary, we are actively exploring new effective applicable drugs for endometrial cancer, and our data suggest that UA may have therapeutic value with chemo-combination treatment of patients with endometrial cancer. Citation Format: Ha-Young Lee, A Ra Ko, Dong-Woo Kang, Yu-Seon Kim, Su-Min Kim, Tae-Wook Kang, Shin-Wha Lee, Yong-Man Kim. Anti-tumor effect of selective progesterone receptor modulator (ulipristal acetate) on endometrial cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1191.

Effect of hyaluronic acid and hydroethyl starch mixture in ovarian cancer cell Objective: Post-operative tissue adhesion is a very serious problem in patients with ovarian cancer. This study is to investigate the role of hyaluronic acid and hydroethyl starch mixture against the tissue adhesion through in vitro experimentations with ovarian cancer cell lines. Methods: We used different concentrations of hyaluronic acid and hydroethyl starch (HA/HES) mixture in SKOV3 cell lines. We performed the adhesion and proliferation inhibition assay in different method, mixing and spreading cells. For the migration inhibition assay, we evaluated the effect according to the concentration of HA/HES mixture. The wound-healing assay was performed after artificial injury and physical barrier of 100% HA/HES mixture was evaluated. Results: In adhesion and proliferation inhibition assay, the viable adherent cells were decreased according to the concentration of HA/HES mixture. The confluence increased over time, except for 100% HA/HES mixture. This result was more obvious in spreading method. The wound recovery was slow in high concentration of HA/HES mixture in the migration inhibition assay and wound-healing assay. The physical barrier of 100% HA/HES mixture inhibited the cell growth until 48 hours. Conclusion: The proliferation and recovery inhibition of SKOV3 cell lines using HA/HES mixture was confirmed and the inhibitory effect appeared to be in proportion of the concentration of HA/HES mixture. This growth inhibition effect of HA/HES mixture will be useful clinically in patients with ovarian cancer Citation Format: Ha-Young Lee, Hyun-Jung Cho, Shin-Wha Lee, Sang-Eun Lee, Dae-Yeon Kim, Jong-Hyeok Kim, Yong-Man Kim, Young-Tak Kim, Joo-Hyun Nam. Evaluation of the mixed solution of sodium hyaluronate and hydroxyethylstarch on gynecologic cancer cell line. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 333. doi:10.1158/1538-7445.AM2015-333

Objective: The objective of this study is to refine a simple, rapid and reproducible method for the isolation and characterization of epithelial ovarian cancer (EOC) cells from ovarian tumor tissues and to evaluate the intracellular expression of CK-7, CA-125, HE4 and FOLR-1 in primary ovarian cancer cells. Methods: Fourteen patients diagnosed with papillary serous adenocarcinoma of ovary were selected in ASAN Medical Center. Isolation of tumor EOC cells involves the mechanical disruption of the tumor tissue and placed directly into culture with very little manipulation. Primary ovarian cancer cells were monitored after initial plating and every day for following 15 days. Confocal laser scanning microscopy was used for the imaging of intracellular immunofluorescence of CK-7, CA-125, HE4 and FOLR-1. Results: Two days after plating primary ovarian cancer cells began to adhere to the plate by the presence of small adherent clusters. The primary ovarian cancer cells became a confluent monolayer and displayed the typical epithelial cobblestone morphology. The mean duration of passage was 8.5 days, it was revealed variously in the primary ovarian cancer cells. Immunofluorescent detection of CK-7 expression in EOC cells demonstrates the epithelial origin, however their expression was not constant. CA-125, HE-4 and FOLR-1 were expressed by all tissue-derived EOC cells. The expression pattern of CA-125, HE-4 and FOLR-1 was different in each patient and that was not correlated with the serum level of CA-125. Conclusions: The isolation and characterization of tissue-derived cancer cells from patients with EOC using the expression of CK-7, CA-125, HE4 and FOLR-1 were reasonable method. This result could provide clinically relevant models suitable for investigation of the ex vivo biological characteristics of ovarian cancers. Citation Format: Shin-Wha Lee, Sang-Eun Lee, Ha-Young Lee, Dae-Yeon Kim, Jong-Hyeok Kim, Yong-Man Kim, Young-Tak Kim, Joo-Hyun Nam. Evaluation of multi-marker in tissue-derived cancer cells from patients with epithelial ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5132. doi:10.1158/1538-7445.AM2015-5132

Objective: The quantitative and qualitative analysis of circulating tumor cells (CTCs) aids in diagnosis of disease, prognosis, disease recurrence, and therapeutic response in several cancers. However, detection and isolation of CTCs from the blood circulation is a difficult task because of their scarcity and the lack of reliable markers to identify these cells. Especially, in epithelial ovarian cancer, there is a little reported data for CTCs and their CTCs has been defined to the EpCAM+ cells, even though EpCAM+ cells are just a few in ovarian cancer. The aim of this study is to investigate the effective principles and methods to detect and isolate CTCs through novel markers in ovarian cancer. Methods: CTC analysis was performed in the spiking test to find cancer cells in the human serum mixed with WBCs. Four cell lines including OVCAR3, SKOV3, SNU8 and SNU251 were used and primary cancer cells from 4 patients were analyzed. Cancer cells were isolated on the basis of cell size by filtration through CytoGen capture device, followed by identification according to validated immunocytochemistry based on the expression of DAPI, CD45, EpCAM, CK, CA-125 and HE4. Results: We obtained cancer cells more than 90% based on the cell size by filtration. Immunocytochemistry confirmed the epithelial origin through the expression of CK and EpCAM in cancer cell lines. The rate of expression was 35-99 % for CK and 1-99 % for EpCAM. A small number of cancer cell lines expressed CD45 that is the marker of WBC. The expression of CA-125 and HE4 showed their ovarian origin. The rate of expression was 7-98 % for CA-125 and 40-99 % for HE4 in cancer cell lines. Primary cancer cells were detected most effectively as the method of combination with 4 markers, including DAPI, CD45, EpCAM and HE4. Conclusion: Our findings provide the first evidence for effective methods to detect and isolate CTCs in ovarian cancer. Combination of DAPI, CD45, EpCAM and HE4 could be a useful marker to support CTCs research in epithelial ovarian cancer. Citation Format: Shin-Wha Lee, Ha-Young Lee, Jin-Young Mo, Byung Chul Kim, Eun Hye Kim, Yong-Man Kim. Detection and isolation of circulating tumor cells in ovarian cancer: Principles and methods. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3073. doi:10.1158/1538-7445.AM2014-3073

Objectives : Ovarian cancer is the 7th most common malignancy and the 5th most common cause of death from cancers in women. While many patients initially respond to surgery and chemotherapy, the long-term prognosis is generally unfavorable, with recurrence and development of drug-resistant disease. Therefore, new strategies of treatment, for examples, immunotherapy or targeted therapy, are necessary. This study evaluated the efficacy and safety of activated human natural killer (NK) cells against ovarian cancer. Methods:Four Korean patients were selected from the Asan Medical Center. These patients were diagnosed with endometrioid adenocarcinoma (n = 2), papillary serous adenocarcinoma (n = 2). And two established human ovarian epithelial carcinoma cell lines (SKOV3 and OVCAR3) were used for all experiments. First of all, for the morphological analysis to detect ovarian cancer, cytokeratin (CK-7) was used on primary cells by fluorescence microscopy and nuclei were stained with DAPI. MTT assay to evaluate cell viability, the modified lactate dehydrogenase (LDH) release assay to determine the cytotoxic activity, and ELISA to detect the secretion levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and IL-12p70 were measured for the capacity of activated NK cells on the ovarian cancer cells. Results: Primary cells were confirmed using CK-7 as a tool of distinction of ovarian cancers by fluorescence microscopy. Secondly, an MTT assay was performed to determine the effect of NK cells on the proliferation of cancer cells after 4, 12, 24, and 48 h of induction in cell ratios of 0.3:1, 1:1, and 3:1. NK cells caused the loss of cancer cell viability and proliferation, although the time courses and cell ratios differed by cancer cell type. Thirdly, to investigate whether NK cells could have a cytotoxic effect on cancer cells, an LDH assay was performed in NK cells and target cells. SKOV3 showed higher cytotoxicity than OVCAR3 in cancer cell lines and cytotoxicity of papillary serous adenocarcinoma cells was much more pronounced that all other cell types. Finally, IFN-γ and TNF-α were significantly released at all ratios in SKOV3 and OVCAR3 cell lines and patient-derived cells, but IL-12 secretion was not detected in all ovarian cancer cells. Although NK cells were activated independently from IL-12, these results suggest that various cytokines play a pivotal role in NK-cell-induced immune responses and could be evidence for activated NK cells. Conclusion: Taken together, NK cells affect ovarian cancer cells upon treatment of direct killing malignant cells and producing cytokines that enhances both the innate and the antigen-specific anti-tumor response. Therefore, the results suggest that NK cell-based treatment could be an alternative immunotherapy for ovarian cancer patients. Citation Format: Mo Jin Young, Shin-Wha Lee, Yong-Man Kim. The effective activation of highly purified NK cells expanded ex vivo on identified epithelial ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2845. doi:10.1158/1538-7445.AM2013-2845