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Introduction: Patients with ovarian cancer are usually diagnosed late, when tumors have already spread to the surrounding tissues. Hematogenous dissemination of ovarian cancer cells can also occur and lung metastasis can be found in 12% of ovarian cancer patients. Human mucin1 (MUC1) is a large transmembrane glycoprotein with roles in cell adhesion and migration. More than 80% of ovarian cancer overexpress MUC1, regardless of histology. MUC1 is a widely used vaccine target for numerous adenocarcinomas, including ovarian tumors. Approach and results: We have recently described the triple transgenic (Tg) mice with conditional (cre-loxP) mutations in Kras and Pten pathways that progress to human MUC1-expressing endometrioid ovarian tumors. Using orthotopic tumors from these mice we generated here several new murine cell lines. The MKP-L cell line was derived from an ovarian tumor implant attached to the liver surface. Intraperitoneal (IP) injection of MKP-L cells into syngeneic mice triggers IP tumors and multiple lung metastatic masses. Cells isolated from lung metastases (MKP-Lung cell line) triggered peritoneal tumors as well as lung metastases, suggesting that they retain the original homing capacity. Compared to the parental, endometrioid-looking and low-MUC1 expressing MKP-L cells, the metastatic MKP-Lung cells showed morphological changes consistent to epithelial-to-mesenchymal transition (EMT) and are homogeneously high in MUC1. Mouse WG6 microarray analysis of MKP-L and MKP-Lung cells revealed expression changes of several EMT-associated transcription factors as well as their downstream proteins. Western blot confirmed upregulation of ZEB1, vimentin and downregulation of cytokeratins. To dissect the mechanistic roles of Kras and Pten pathways in EMT and MUC1 regulation, we also generated a series of ovarian surface epithelial (OSE)-derived cell lines with conditional (Cre-loxP) oncogenic mutations in Kras (MKOSE), deletion in Pten (MPOSE) or both (MKPOSE). Using these cell lines we recapitulated in vitro the EMT phenotype and MUC1 upregulation acquired by MKP-Lung cells in vivo. Furthermore, small molecule inhibitors of oncogenic Kras (AZD6244) and Pi3K/Pten pathway (BEZ235) demonstrated that MUC1 expression is significantly upregulated in response to Pten deletion and that Pi3K inhibitor (BEZ235) downregulated MUC1. In contrast, Kras activation slightly lowered MUC1 expression while cell exposure to AZD6244 upregulated MUC1. Conclusions: We report here the first transgenic mouse metastatic ovarian cancer model that expresses human MUC1.Usig this model we identified a gene signature consistent with lung metastasis and EMT and identified mechanisms of MUC1 upregulation during these processes. These findings have important consequences for MUC1 immunobiology in metastatic ovarian tumors with mutations in Kras, Pten/Pi3K or both. Citation Format: Lixin Zhang, Tianzhou Ma, Joan Brozick, Kathlene Babalola, Raluca Budiu, George Tseng, Anda Vlad. Lung metastasis of ovarian cancer in a transplantable MUC1.Tg mouse model is accompanied by upregulation of MUC1 expression and epithelial to mesenchymal transition. [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 4081. doi:10.1158/1538-7445.AM2015-4081

Introduction: Ovarian epithelial tumors are highly heterogeneous. Patients are diagnosed late, when tumors have spread to the surrounding tissues, most often to the omentum. Despite optimal removal and response to therapy, the tumors often recur. Personalized medicine approaches often require molecular profiling of tumors, for patient selection and to predict response to therapy. Given intratumor heterogeneity, identification of tumor sites that are best to select for profiling is still up for debate. Approach: We recently developed triple transgenic (MUC1KrasPten.Tg) mice that develop orthotopic MUC1-expressing endometrioid ovarian tumors. The tumors develop in the ovaries and spread throughout the peritoneal cavity, closely mirroring the human disease. Using tumor cells isolated from either the MUC1KrasPten primary ovarian tumor (MKP-T) or the liver implant (MKP-L), we generated several new murine ovarian cancer cell lines which express human MUC1 as self, while simultaneously carrying constitutively active oncogenic Kras and defective Pten tumor suppressor. Results: MKP-T and MKP-L showed variable expression pattern of MUC1, EpCAM, folate receptor, ALDH, cytokeratin and vimentin, demonstrating the relationship between the anatomical location of each originating tumor and the cell line's in vitro phenotype. Unlike the MKP-L cells, the MKP-T cells display loss of K-ras heterozygosity, suggesting that migration to a metastatic site was an early event. Although both cell lines can grow IP tumors in syngeneic triple transgenic MUC1KrasPten.Tg, they carry distinct in vivo phenotypes. MKP-T cells multiply at higher rate and generate larger loco- regional tumors. In contrast, the MKP-L cells, derived from a metastatic site, have a slower growth rate but spread throughout the peritoneal cavity, undergo epithelial-mesenchymal transition (EMT) and trigger distant metastases to the lungs(MKP-Lng). In line with their Kras status, MEK 1/2 inhibitor AZD6244 (currently tested in several Phase II clinical trials) is more effective on MKP-T cells than on MKP-L and MKP-Lng cells in vitro, suggesting that metastatic tumor cells may be more resistant to MEK1/2 inhibitiors. Conclusions: Using novel murine tumor cell populations, we reproduce intratumoral heterogeneity and demonstrate that in vitro and in vivo biological properties vary with the location of originating tumor. Most importantly, we show that cells derived from metastatic sites, which are more likely to reproduce the characteristic of the recurrent tumors, have an increased metastatic potential and reduced response to therapeutic drugs. These results suggest that molecular profiling of tumors needs to focus on multiple tumor sites and that in ovarian cancer, distant tumor implants may be particularly important. Supported by NIH R01 CA163462 and DOD OC093429. Citation Format: Lixin Zhang, Raluca Budiu, Joan Brozick, Anda M. Vlad. Novel ovarian cancer transplantable models generated from MUC1KrasPten tumors show in vitro and in vivo heterogeneity. [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 4976. doi:10.1158/1538-7445.AM2014-4976

Introduction: Endometriosis is a largely benign, chronic inflammatory disease defined by the presence of endometrial-like glands surrounded by stroma. Epidemiologic studies suggest that endometriosis is an independent risk factor for endometrioid and clear cell epithelial ovarian tumors, collectively called endometriosis-associated ovarian cancers (EAOC). Histopathology findings demonstrate that EAOC occur in the presence of atypical endometriosis (AE), often found in direct continuity with the tumor, suggesting AE as the transitioning entity from benign lesions to malignant variants. Although it is widely accepted that chronic inflammation drives cancer, immune deregulation in chronic precursor lesions to ovarian cancer have not been studied in a systematic way. Experimental procedure: We extracted RNA from 135 paraffin tissue blocks comprising of normal endometrium (n=32), benign endometriosis consisting of ovarian and extra ovarian endometriosis cases (n=30,) atypical endometriosis (n=15) and EAOC, (n=43). Serous tumors (n=15) were included as non-endometriosis associated controls. Using Nanostring and the nCounter® GX Human Immunology Kit, we profiled a total of 511 immune genes. Cluster analyses and differential expressions (DE) were calculated using EdgeR. Protein expression of candidate proteins was validated with immunohistochemistry (IHC). To identify mechanisms of complement activation during early stages of genomic instability, we used murine cell lines derived from mice with conditional mutations in Kras and Pten pathways. Results: Nanostring immune gene expression profile eveal the predominant role of adaptive immunity and of the adaptive-innate immune cross-talk. The complement pathway was most prominently expressed, suggesting its roles as one of the major immune pathways involved in the transition from chronic endometriosis to atypical (premalignant) endometriosis and to EAOC. Complement pathway genes were upregulated in human endometriosis and atypical endometriosis. Tissue deposition of several complement components and changes in peripheral blood were confirmed by IHC and ELISA, respectively. Genomic instability in murine ovarian cancer cell lines with conditional mutations resulted in upregulation of complement genes expression in epithelial cells, but without an advantage on cell death. These findings further support a paradigm shift on complement roles in cancer, suggesting its pro-tumorigenic roles. Conclusions: We performed the first comprehensive gene profile of the tissue immune microenvironment in benign endometriosis, EAOC and precursor lesions and identified the previously unrecognized roles for the complement pathway. Complement activation may be an early trigger of inflammation and an early link between epithelium and immune environment. These findings have high translational potential for immune therapy and prevention in EAOC. Citation Format: Swati Maruti Suryawanshi, Xin Huang, Raluca Budiu, SungHwan Kim, George Tseng, Esther Elishaev, Marcia Klein-Patel, Ted Lee, Suketu Mansuria, Robert Edwards, Anda Vlad. Complement roles in endometriosis and endometriosis-associated ovarian cancer. [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 1653. doi:10.1158/1538-7445.AM2014-1653

Background: Despite improvements in surgical techniques and chemotherapy approaches, ovarian cancer continues to be associated with a low 5 year survival. Sustained improvement in the development of new therapeutic approaches depends in part on the availability of adequate preclinical models for in vivo testing of treatment efficacy. MUC1 oncoprotein is a tumor -associated antigen expressed by all epithelial ovarian tumors and a valuable target for immunotherapy. Objective: To test the therapeutic efficacy of a MUC1-based vaccine using dendritic cells matured in type 1 polarizing conditions (DC1). This vaccine was administered to novel triple transgenic conditional tumor-bearing mice, which we recently generated and characterized. Methods: Our new triple transgenic mice (MUC1+/−LSLKrasG12D/+PtenloxP/loxP) co-express a Kras oncogenic mutation and a Pten deficiency (previously described) and carry the MUC1 transgene encoding for the human MUC1 tumor-associated antigen, driven by the endogenous Muc1 promoter. We induced tumors in n=21 double and n=18 triple transgenic (Tg) mice by AdCre injection under the ovarian bursa, during survival surgery. Five of the 18 mice were vaccinated with 3x 103 DC1, grown in GM-CSF, matured with IL-4, IFNγ, LPS and Poly I:C (DC1 cocktail) and pulsed with 10ug/ml 100mer MUC1 peptides (DC1-MUC1). The vaccine was administered s.c 3 times, 2 weeks apart starting at week 5 after AdCre injection. We measured natural and vaccine- induced immune responses via ELISA, multicolor flow cytometry, and real-time PCR arrays. Results: Intrabursal administration of AdCre in the double (KrasPten) and triple (MUC1KrasPten) Tg mice leads to de novo ovarian epithelial tumors. The tumors occur in the injected ovary only, spread throughout the abdominal cavity and on the diaphragm and may be accompanied by hemorrhagic ascites. Characterization of the tumor environment revealed increase PGE2 accumulation in ascites and overexpression of COX2 at the tumor site and throughout the injected side of the female genital tract. Despite the histomorphologic similarities of MUC1KrasPten and KrasPten mice neoplasms, only the triple Tg tumors express MUC1, mimicking even closer the human disease. The overall survival was shorter in triple Tg compared to double Tg mice (average of 65 and 82 days, respectively, p = 0.105) pointing out to important MUC1 roles in ovarian tumor biology. DC1-TnMUC1 vaccine significantly increases overall survival (p = 0.003) and decreases CD4+FOXP3+ Treg cells in spleen, compared with non-vaccinated tumor-bearing mice (p < 0.001). Conclusions: MUC1KrasPten mice represent the first preclinical model for orthotopic ovarian carcinogenesis expressing the human MUC1 antigen. This preclinical data provides a platform for using MUC 1 vaccine as a form of immunotherapy in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 751. doi:10.1158/1538-7445.AM2011-751

Background: Ovarian cancer continues to be associated with a very high mortality rate. Improvements on the development of new therapeutic approaches, including antigen-specific immune therapies, depend in part on the availability of adequate preclinical models for in vivo testing of treatment efficacy. MUC1 mucin is a tumor associated antigen and a potential target for cancer immunotherapy. Human and murine mucin 1 share little homology in the extracellular region and transgenic mouse models with de novo lesions expressing the human antigen are needed. Objective: To identify MUC1 antigen-specific immune regulation in mice with de novo malignant ovarian lesions. Methods: In order to generate a human MUC1 antigen expressing ovarian cancer mouse model, we crossed the MUC1 transgenic mice which express human MUC1 under the endogenous promoter with the previously described KrasG12D/+PtenloxP/loxP mice that develop orthotopic ovarian tumors. Results: The newly generated MUC1+/−KrasG12D/+ PtenloxP/loxP triple transgenic mice constitute a valuable Cre-loxP conditional in vivo model for MUC1 expressing ovarian tumors. Upon injection of Cre-encoding adenovirus under the ovarian bursa, the triple transgenic mice develop aggressive ovarian tumors with clear cell histology. The lesions express high levels of MUC1 and are estrogen receptor and cytokeratin 7-positive, markers classically used to diagnose malignant epithelial proliferation. The tumors are accompanied by ascites and numerous peritoneal implants, mimicking late stage disease clinically seen in women. We identified increased accumulation of Treg cells in the para-aortic (draining) lymph nodes and presence of Th17 cells in ascites, mimicking closely the immune phenotype of the human disease. In addition, using quantitative PCR T cell gene profiler arrays, we identified 5 significantly up- and 16 down-regulated genes in the spleen T cells of tumor-bearing mice. One of the upregulated genes was osteopontin (Spp1), recently validated as a marker in detecting human recurrent ovarian cancer. Among the down-regulated genes we identified, Cd4, Cd40ligand (Cd40lg), Interferon-g (Ifnγ), IL-23 receptor (IL-23ra) all suggestive of a shift in immune effectors, some of which may promote the chronic inflammatory background. Conclusions: Our results reveal important immune regulatory mechanisms in mice with de novo MUC1-expressing ovarian tumors and provide unique opportunities for future in vivo testing of MUC1 vaccines as ovarian cancer therapy and potentially immune prevention. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4793.

Objective: Examine the expression and immunogenicity of a known ovarian tumor-associated antigen, MUC1, in ectopic endometriosis lesions and epithelial ovarian cancer (EOC). Determine whether an imbalance in CD4 T cell subsets contributes towards endometriosis pathogenesis and ovarian carcinogenesis. Methods: Biologic specimens were obtained from women with endometriosis, endometriosis-related (endometrioid and clear cell) or serous EOC, and healthy controls, according to University of Pittsburgh IRB protocols. MUC1 and immune cell expression were determined via immunohistochemistry (IHC). Anti-MUC1 IgM and IgG antibody levels were determined via ELISA. Peripheral blood mononuclear cells and cryopreserved cell suspensions from tissue lesions were immunophenotyped using multicolor flow cytometry. Stroma and epithelial cells from tissue biopsies were collected by laser capture microdissection (LCM) and examined with immune-based quantitative RT-PCR arrays. Results: MUC1 was overexpressed in 67% of endometriosis and 89% of EOC lesions. Women with early or late stages of endometriosis had significantly higher anti-MUC1 IgM responses when compared to those women with endometriosis-related EOC (25.3 % of positive control vs.14.2%, p=0.03; 26.3 vs. 14.2%, p=0.04) and to those with serous EOC (25.3 vs. 14.6%, p=0.04; 26.3 vs.14.6%, p=0.04). Women without disease also had significantly higher anti-MUC1 IgM responses compared to endometriosis-related (29.2 vs.14.2%, p=0.01) and serous EOC (29.2 vs.14.6%, p=0.02). Immune cells, including CD3, CD4, and CD8 T cells, are present in endometriotic lesions and appear more abundant than in eutopic endometrium. Flow cytometric analysis of endometriotic lesion-infiltrating T cells revealed a decrease in the frequency of IFN-γ CD4 and CD8 T cells, similar to that seen in patients with EOC. Conclusions: MUC1 is present and appears to be overexpressed in ectopic endometriotic and EOC lesions. Women with endometriosis-related EOC or with serous EOC had significant lower anti-MUC1 IgM levels, suggesting that a compression of antigen-specific humoral (i.e., Th2 driven) immunity may play a role in carcinogenesis. Endometriotic and EOC lesions also appear to have diminished Th1 immunity, suggesting a possible role for local immune suppression. Quantitative analyses of several immune genes in the lesion microenvironment are currently ongoing and will potentially contribute to immune biomarker discovery and improvements in the early detection and prevention of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1335.