Murine Ovarian Cancer Cell Line Research Articles

Preclinical studies of a PARP targeted, Meitner-Auger emitting, theranostic radiopharmaceutical for metastatic ovarian cancer

Advanced ovarian cancer currently has few therapeutic options. Poly(ADP-ribose) polymerase (PARP) inhibitors bind to nuclear PARP and trap the protein-inhibitor complex to DNA. This work investigates a theranostic PARP inhibitor for targeted radiopharmaceutical therapy of ovarian cancer in vitro and PET imaging of healthy mice in vivo. Methods[77Br]RD1 was synthesized and assessed for pharmacokinetics and cytotoxicity in human and murine ovarian cancer cell lines. [76Br]RD1 biodistribution and organ uptake in healthy mice were quantified through longitudinal PET/CT imaging and ex vivo radioactivity measurements. Organ-level dosimetry following [76/77Br]RD1 administration was calculated using RAPID, an in-house platform for absorbed dose in mice, and OLINDA for equivalent and effective dose in human. ResultsThe maximum specific binding (Bmax), equilibrium dissociation constant (Kd), and nonspecific binding slope (NS) were calculated for each cell line. These values were used to calculate the cell specific activity uptake for cell viability studies. The half maximal effective concentration (EC50) was measured as 0.17 (95 % CI: 0.13–0.24) nM and 0.46 (0.13–0.24) nM for PARP(+) and PARP(−) expressing cell lines, respectively. The EC50 was 0.27 (0.21–0.36) nM and 0.30 (0.22–0.41) nM for BRCA1(−) and BRCA1(+) expressing cell lines, respectively. When measuring the EC50 as a function of cellular activity uptake and nuclear dose, the EC50 ranges from 0.020 to 0.039 Bq/cell and 3.3–9.2 Gy, respectively. Excretion through the hepatobiliary and renal pathways were observed in mice, with liver uptake of 2.3 ± 0.4 %ID/g after 48 h, contributing to estimated absorbed dose values in mice of 19.3 ± 0.3 mGy/MBq and 290 ± 10 mGy/MBq for [77Br]RD1 and [76Br]RD1, respectively. Conclusion[77Br]RD1 cytotoxicity was dependent on PARP expression and independent of BRCA1 status. The in vitro results suggest that [77Br]RD1 cytotoxicity is driven by the targeted Meitner-Auger electron (MAe) radiotherapeutic effect of the agent. Further studies investigating the theranostic potential, organ dose, and tumor uptake of [76/77Br]RD1 are warranted.

Abstract 571: Inclusion of a Dap10 costimulatory domain decreases cytokine release syndrome in chimeric antigen-expressing gamma delta T cells

Abstract Adoptive transfer of T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be expressed in T cells. While CAR T cells can successfully treat hematopoietic malignancies, CAR T cells have not shown success in treating solid tumors. There are many factors that may enhance CAR T cell efficacy and safety for solid tumor treatment, including addition of costimulatory domains to increase T cell functions and switching the T cell type used from alpha beta to gamma delta T cells. We developed a novel CAR (chimeric PD-1, chPD1) that targets the programmed death 1 receptor (PD-1) ligands expressed on many types of solid tumors. The aim of this study was to compare anti-tumor efficacy and safety of chPD1-expressing gamma delta and alpha beta T cells in murine breast and ovarian cancer models. Inclusion of different costimulatory domains, CD28, 4-1BB, and Dap-10, in the chPD1 receptor was also compared. There were no differences in T cell viability and proliferation when comparing costimulatory domains or T cell type. Additionally, there were no significant differences in killing of murine breast and ovarian cancer cell lines. ChPD1-CD28 T cells had increased secretion of proinflammatory and cytokine release syndrome (CRS)-inducing cytokines, including IFN-gamma, IL-2, IL-6, GM-CSF, and nitric oxide, compared to chPD1-41BB and chPD1-Dap10 receptors. Additionally, alpha beta chPD1 T cells had increased cytokine secretion of inflammatory cytokines compared to chPD1 gamma delta T cells. Furthermore, while gamma delta and alpha beta T cells expressing chPD1 receptors with a Dap10 domain had similar anti-tumor efficacy in vivo, chPD1-Dap10 gamma delta T cells had the lowest serum levels of inflammatory cytokines IFN-gamma, IL-6, and GM-CSF. Therefore, inclusion of Dap10 domains in gamma delta T cells may be a safer and more efficacious option for CAR T cell therapy of solid tumors. Citation Format: Amorette E. Barber. Inclusion of a Dap10 costimulatory domain decreases cytokine release syndrome in chimeric antigen-expressing gamma delta T cells [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 571.

Abstract 2746: CD276 in tumor cells contributes to the M2 macrophage-rich immunosuppressive tumor microenvironment via upregulating the CCL2-CCR2 axis in ovarian cancer

Abstract [Objective] Increased infiltration of CD8+ T cells and decreased infiltration of M2 macrophages are favorable prognostic factors in high-grade serous ovarian cancer (HGSOC). CD276 (B7H3) is a transmembrane co-inhibitory protein belonging to the B7 family. CD276 expression is suppressed in CD8+ T cell-rich, PD-L1-high immune reactive tumors. This suggests that CD276 plays immunosuppressive roles in addition to acting as a molecular brake for countering the T cell activation. In this study, we aimed to elucidate the role of CD276 in HGSOC microenvironment toward M2 macrophages. [Methods] CD276 expression was evaluated in HGSOC clinical cases (n=62) by immunohistochemistry tests. The correlation between survival and the presence of tumor-infiltrating immune cells, including M2 macrophages, was assessed. CD276 knockout murine ovarian cancer cell lines (HM1 and ID8) were established and used for experiments. [Results] In HGSOC patients, higher CD276 expression associated with poor prognosis (p<0.001). CD276 expression positively correlated with the number of CD206+ M2 macrophages (Pearson's r=0.29, p=0.02), but not with that of CD8+ T cells. Although CD276 knockout did not affect in vitro proliferation of HM1 and ID8 cell lines, CD276 knockout tumors were less progressive than control tumors in immunocompetent mouse models (p<0.001). Lower infiltration of CCR2+/CD206+ M2 macrophages and higher infiltration of IFN-γ+ CD8+ T cells was observed in the knockout tumors (p<0.001 and P=0.02, respectively). The difference in progression between CD276 knockout tumors and control tumors was negated when tumor cells were inoculated into immunodeficient nude mice. RNA sequencing and protein analyses revealed decreased levels of CCL2, the ligand of CCR2, in CD276 knockout cells and tumors. In vitro, monocyte chemotaxis was reduced by CD276 knockout cells' supernatant (p<0.001), and the difference was partially negated with CCR2 antagonist pretreatment. Furthermore, CCR2 blocking in vivo reduced tumor-infiltrating M2 macrophages and tumor volume in the CD276 control (p=0.048 and p=0.02, respectively), but not in the CD276 knockout group, suggesting that CCL2-CCR2 axis-induced M2 macrophages partially contribute to CD276-mediated tumor progression. [Conclusion] CD276 contributes to an unfavorable immunosuppressive tumor microenvironment driven by M2 macrophages via the CCL2-CCR2 axis in HGSOC. Citation Format: Taito Miyamoto, Ryusuke Murakami, Masayo Ukita, Mana Taki, Koji Yamanoi, Kaoru Abiko, Ken Yamaguchi, Junzo Hamanishi, Takashi Kobayashi, Masaki Mandai. CD276 in tumor cells contributes to the M2 macrophage-rich immunosuppressive tumor microenvironment via upregulating the CCL2-CCR2 axis in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2746.

Abstract 1516: Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors

Abstract Adoptive transfer of T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be expressed in T cells. One receptor that is a prospective target for a new chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 receptor extracellular domain and the activation domain of CD3 zeta. In addition, current chimeric antigen receptor therapies utilize various costimulatory domains to enhance anti-tumor efficacy. Therefore, we also compared the inclusion of CD28, Dap10, 4-1BB, GITR, ICOS, or OX40 costimulatory domains in the chPD1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. To determine if this novel CAR could potentially target a wide variety of tumors, the anti-tumor efficacy of chPD1 T cells against murine lymphoma, melanoma, kidney, pancreatic, liver, colon, breast, ovarian, prostate, and bladder cancer cell lines was measured. Of the eighteen cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T cells. Regardless of the costimulatory domain in the CAR, all of the chPD1 T cells induced similar levels of T cell proliferation and tumor cell lysis. However, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the CAR. While most of the chPD1 T cell receptor combinations secreted both pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10), chPD1 T cells containing a Dap10 costimulatory domain secreted high levels of proinflammatory cytokines but did not secrete a significant amount of anti-inflammatory cytokines. Furthermore, T cells expressing chPD1 receptors with a Dap10 domain also had the strongest anti-tumor efficacy in vivo. ChPD1 T cells did not survive for longer than 14 days in vivo, however treatment with chPD1 T cells induced long-lived protective host-anti-tumor immune responses in tumor-bearing mice. Therefore, adoptive transfer of chPD1 T cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the Dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies. Citation Format: Amorette E. Barber. Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1516.

Hyperthermic intraperitoneal chemotherapy enhances antitumor effects on ovarian cancer through immune-mediated cancer stem cell targeting

Purpose We aimed to determine the effects and possible mechanisms of hyperthermic intraperitoneal chemotherapy (HIPEC) in targeting ovarian cancer stem-like cells (CSCs). Methods Murine ovarian cancer cell lines presenting CSC surface markers were grown intraperitoneally in both immunocompetent and immunodeficient mice, which were then treated by intraperitoneal hyperthermia with the chemotherapeutic agents: paclitaxel and cisplatin. Tumor growth was measured by non-invasive luminescent imaging. Intraperitoneal immune cells, such as CD4+, CD8+ T cells, macrophages, and dendritic cells, were evaluated through flow cytometry analysis. Results Combined hyperthermia and chemotherapy exhibited an efficient therapeutic effect in the immunocompetent mice. However, a similar effect was not observed in the immunodeficient mice. Intraperitoneal hyperthermia increased the number of Intraperitoneal macrophages and dendritic cells that were lost due to chemotherapy. Compared with ovarian cancer bulk cells, CSCs were more susceptible to phagocytosis by macrophages. Conclusion We demonstrated that the superior therapeutic efficacy and reduced proportion of CSCs associated with intraperitoneal hyperthermic chemotherapy were immune-related. Hyperthermia recruits the phagocytes that target surviving CSCs after chemotherapy. These results provide a novel mechanism for the efficacy of HIPEC in treating ovarian cancer.

An Extract of Taro (Colocasia esculenta) Mediates Potent Inhibitory Actions on Metastatic and Cancer Stem Cells by Tumor Cell-Autonomous and Immune-Dependent Mechanisms.

The taro plant, Colocasia esculenta, contains bioactive proteins with potential as cancer therapeutics. Several groups have reported anti-cancer activity in vitro and in vivo of taro-derived extracts (TEs). We reported that TE inhibits metastasis in a syngeneic murine model of Triple-Negative Breast Cancer (TNBC).Purpose:We sought to confirm our earlier studies in additional models and to identify novel mechanisms by which efficacy is achieved.Methods:We employed a panel of murine and human breast and ovarian cancer cell lines to determine the effect of TE on tumor cell viability, migration, and the ability to support cancer stem cells. Two syngeneic models of TNBC were employed to confirm our earlier report that TE potently inhibits metastasis. Cancer stem cell assays were employed to determine the ability of TE to inhibit tumorsphere-forming ability and to inhibit aldehyde dehydrogenase activity. To determine if host immunity contributes to the mechanism of metastasis inhibition, efficacy was assessed in immune-compromised mice.Results:We demonstrate that viability of some, but not all cell lines is inhibited by TE. Likewise, tumor cell migration is inhibited by TE. Using 2 immune competent, syngeneic models of TNBC, we confirm our earlier findings that tumor metastasis is potently inhibited by TE. We also demonstrate, for the first time, that TE directly inhibits breast cancer stem cells. Administration of TE to mice elicits expansion of several spleen cell populations but it was not known if host immune cells contribute to the mechanism by which TE inhibits tumor cell dissemination. In novel findings, we now show that the ability of TE to inhibit metastasis relies on immune T-cell-dependent, but not B cell or Natural Killer (NK)-cell-dependent mechanisms. Thus, both tumor cell-autonomous and host immune factors contribute to the mechanisms underlying TE efficacy. Our long-term goal is to evaluate TE efficacy in clinical trials. Most of our past studies as well as many of the results reported in this report were carried out using an isolation protocol described earlier (TE). In preparation for a near future clinical trial, we have now developed a strategy to isolate an enriched taro fraction, TE-method 2, (TE-M2) as well as a more purified subfraction (TE-M2F1) which can be scaled up under Good Manufacturing Practice (GMP) conditions for evaluation in human subjects. We demonstrate that TE-M2 and TE-M2F1 retain the anti-metastatic properties of TE.Conclusions:These studies provide further support for the continued examination of biologically active components of Colocasia esculenta as potential new therapeutic entities and identify a method to isolate sufficient quantities under GMP conditions to conduct early phase clinical studies.

Identification of key genes and pathways associated with topotecan treatment using multiple bioinformatics tools

The goal of this study is to determine critical genes and pathways associated with topotecan using publicly accessible bioinformatics tools. Topotecan signatures were downloaded from the Library of Integrated Network-Based Cellular Signatures (LINCS) database (http://www.ilincs.org/ilincs/). Differentially expressed genes (DEGs) were defined as genes that appeared at least three times with p values <0.05 and a fold change of ≥50% (|log2FC| ≥ 0.58). Hub genes were identified by evaluating the following parameters using a protein-protein interaction network: node degrees, betweenness, and eigenfactor scores. Hub genes and the top-40 DEGs by |log2FC| were used to generate a Venn diagram, and key genes were identified. Functional and pathway enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Information on ovarian cancer patients derived from The Cancer Genome Atlas (TCGA) database was analyzed, and the effect of topotecan on the protein expression was examined by Western blotting. Eleven topotecan signatures were downloaded, and 65 upregulated and 87 downregulated DEGs were identified. Twenty-one hub genes were identified. We identified eight key genes as upregulated genes, including NFKBIA, IKBKB, GADD45A, CDKN1A, and HIST2H2BE, while EZH2, CDC20, and CDK7 were identified as downregulated genes, which play critical roles in the cell cycle and carcinogenesis in KEGG analysis. In the TCGA analysis, the CDKN1A+/EZH2- group had the longest median survival, while the CDKN1A-/EZH2+ group had the shortest median survival. Topotecan-treated murine ovarian (MOSEC), colorectal (CT26), and lung (LLC) cancer cell lines displayed upregulated CDKN1A encoding p21 and downregulated Ezh2. Using publicly accessible bioinformatics tools, we evaluated key genes and pathways related to topotecan and examined the key genes using the TCGA database and in vitro studies.

Inhibiting WNT Ligand Production for Improved Immune Recognition in the Ovarian Tumor Microenvironment.

In ovarian cancer, upregulation of the Wnt/β–catenin pathway leads to chemoresistance and correlates with T cell exclusion from the tumor microenvironment (TME). Our objectives were to validate these findings in an independent cohort of ovarian cancer subjects and determine whether inhibiting the Wnt pathway in a syngeneic ovarian cancer murine model could create a more T-cell-inflamed TME, which would lead to decreased tumor growth and improved survival. We preformed RNA sequencing in a cohort of human high grade serous ovarian carcinoma subjects. We used CGX1321, an inhibitor to the porcupine (PORCN) enzyme that is necessary for secretion of WNT ligand, in mice with established ID8 tumors, a murine ovarian cancer cell line. In order to investigate the effect of decreased Wnt/β–catenin pathway activity in the dendritic cells (DCs), we injected ID8 cells in mice that lacked β–catenin specifically in DCs. Furthermore, to understand how much the effects of blocking the Wnt/β–catenin pathway are dependent on CD8+ T cells, we injected ID8 cells into mice with CD8+ T cell depletion. We confirmed a negative correlation between Wnt activity and T cell signature in our cohort. Decreasing WNT ligand production resulted in increases in T cell, macrophage and dendritic cell functions, decreased tumor burden and improved survival. Reduced tumor growth was found in mice that lacked β–catenin specifically in DCs. When CD8+ T cells were depleted, CGX1321 treatment did not have the same magnitude of effect on tumor growth. Our investigation confirmed an increase in Wnt activity correlated with a decreased T-cell-inflamed environment; a relationship that was further supported in our pre-clinical model that suggests inhibiting the Wnt/β–catenin pathway was associated with decreased tumor growth and improved survival via a partial dependence on CD8+ T cells.

1946P - Inhibiting Ehmt2 and Ezh2 histone methyltransferases alters the immune microenvironment in a Trp53-/- murine ovarian cancer model

BackgroundOvarian cancer prognosis is strongly dependent on the development of an anti-tumour immune response. However, tumours can epigenetically silence immunostimulatory genes in order to evade this response. We investigated whether a novel dual inhibitor of Ehmt2/Ezh2 methyltransferases (HKMT) was able to derepress expression of critical chemokines and augment immune responses in a murine ovarian cancer model. MethodsID8 Trp53-/- murine ovarian cancer cell line was previously generated, using CRISPR-Cas9 technique. Mice bearing intraperitoneal Trp53-/-tumours were treated with the novel Ehmt2/Ezh2 inhibitor, HKMTI-1-005, for 14 days (21-35d). Tumours were harvested for immune cell phenotyping by flow cytometry. HKMT1-1-005 was screened in vitrofor its ability to enhance expression of 84-chemokine genes in ID8 Trp53-/- ovarian cancer cells. ResultsIn vitro, HKMTI-1-005 treatment significantly (p<0.05) upregulated the expression of cxcl10 (3-fold), cxcl9 (22-fold) and ccl5 (14-fold), after stimulation with IFNγ. Mice treated with HKMTI-1-005 had longer survival (52 vs45d, p<.0001), less ascites (3.7 vs5.6ml, p=.0037) and trended towards tumour size reduction (weight 138 vs178mg, p=.10) compared to vehicle treatment. Tumours harvested 24hr post last HKMTI-1-005 dose had significantly more effector CD8+T cells (p=.03), natural killer (NK) cells (p<.0001) and dendritic cells (DCs, p=.02), and less naïve CD8+T cells (p=.02) and immunosuppressive CD4+Tregs (p=.02). Expression of the Cxcl9/Cxcl10 receptor Cxcr3 was increased in HKMTI-1-005-treated cohort tumours on CD8+[mean fluorescence intensity (MFI) 3959 vs2097, p<.0001], CD4+(MFI 2341 vs1099, p<.0001)and NK (MFI 1507 vs440, p<.0001) cells. ConclusionsInhibition of Ehmt2/Ezh2 HKMTs stimulates expression of chemokines involved in T cell, NK and DC recruitment. In vivo, HKMTI-1-005 alters the immune microenvironment and confers a small survival benefit. This suggests that HKMTI-1-005 could augment the anti-tumour immune response of current immunotherapies. Legal entity responsible for the studyImperial College London. FundingHas not received any funding. DisclosureP. Spiliopoulou: Honoraria (self): BMS; Travel / Accommodation / Expenses: Nucana. I.A. McNeish: Advisory / Consultancy: Clovis Oncology; Advisory / Consultancy: Takeda; Advisory / Consultancy: Tesaro; Advisory / Consultancy: AstraZeneca; Research grant / Funding (institution): AstraZeneca. All other authors have declared no conflicts of interest.

The effects of Wnt inhibition on tumor progression and the tumor microenvironment in a syngeneic mouse model of ovarian cancer.

e17078 Background: Alterations in the Wnt/β-catenin pathway have been associated with tumor progression in multiple cancers. Gene expression profiling in ovarian cancer has correlated Wnt upregulation to patterns of immune evasion in the tumor microenvironment (TME). We investigated effects of in vitro Wnt inhibition in a murine ovarian cancer cell line with p53 knocked out as a model to mimic human ovarian carcinoma. Methods: We used a small molecule inhibitor, CGX-1321, in C57Bl6 mice, an immunocompetent mouse model, injected intraperitoneally with either ID8 cells, a murine ovarian cancer cell line, or ID8-p53-/- cells. Mouse omentums were collected and weighed for evaluation of tumor growth. Representative histology slides were stained for H&amp;E. Western blots for β-catenin baseline levels were performed on the cell lines. TopFlash Assays with WNT3A stimulation were used for analysis of cell line nuclear β-catenin levels. Flow cytometry was performed on mouse omentums for TME evaluation. Results: Treatment with CGX-1321 decreased tumor size via omentum weight with both cell lines, p = 0.0098 with ID8 cells and p = 0.0855 with ID8-p53-/- cells. H&amp;E slides revealed a decreased tumor progression. Western blots confirmed a difference in baseline β-catenin levels between these two cell lines. However, TopFlash assays showed a response to stimulation. Changes in the TME between treated samples with ID8-p53-/- cells did now show significant differences via flow cytometry. Conclusions: In the syngeneic mouse model with a murine ovarian cancer cell line, with p53 knocked out, tumor size and proliferation were decreased with treatment with Wnt inhibition. Surprisingly, the TME changes were inconclusive via flow cytometry. Perhaps these cells do not rely upon the Wnt/ β-catenin pathway as heavily, as seen in changes to baseline β-catenin levels, or perhaps tumors progress quicker and the critical time point of TME change is being overlooked. More investigation needs to be performed to further elucidate these differences.

T cell trafficking within the peritoneal tumor environment and ovarian cancer progression.

5573 Background: Ovarian cancer is associated with high mortality due to detection at late stages with widespread peritoneal metastases at diagnosis in a majority of patients. Ovarian cancer recurrences are primarily found in the peritoneal cavity, and peritoneal disease is the primary cause of morbidity and mortality in this disease. This pattern of tumor engraftment and recurrence indicates that the peritoneal tumor environment is distinct from other niches. Our data indicate that selective peritoneal dissemination is immunologically mediated, based on functional differences in peritoneal and systemic T cells during ovarian cancer progression. This is consistent with data in other solid tumors demonstrating that tumor immunity is driven by regional lymphocyte populations. However, less is known about the mechanisms that establish a permissive immune environment in the peritoneal cavity. We hypothesize that pathways regulating T cell recruitment and retention in the peritoneal cavity (PC) are co-opted by ovarian cancer cells to enable intraperitoneal cancer dissemination and recurrence. Methods: We have developed a novel model that uses direct in vivo labeling of peritoneal cells in an established immune-competent high grade serous murine cancer model. This functional approach enables us to identify T cell subsets retained in the PC with tumor engraftment and progression. Results: We identified high expression of CD49d (a4 integrin) as the most prevalent cell surface marker on T cells retained in the peritoneal cavity, consistent with prior published data in healthy mice and people. We demonstrated a functional role for CD49dhi in T cell retention by showing preferential binding to VCAM. A role for tumor cells mediating this interaction was observed based on enhanced binding affinity in vitro with tumor monolayers. The importance of this mechanism is supported by high VCAM expression in multiple murine and human ovarian cancer cell lines, and T cell localization to VCAM rich areas within ovarian cancer tumors in vivo. Conclusions: CD49d not only defines a lymphocyte subset with a significant role in tumor immunity but presents itself as an important potential therapeutic target to modulate T cell trafficking.

Programmed cell death ligand 1 disruption by clustered regularly interspaced short palindromic repeats/Cas9-genome editing promotes antitumor immunity and suppresses ovarian cancer progression.

Programmed cell death ligand 1 (PD‐L1) on tumor cells suppresses anti‐tumor immunity and has an unfavorable prognostic impact in ovarian cancer patients. We herein report the pathophysiological and therapeutic impacts of PD‐L1 disruption in ovarian cancer. PD‐L1 was genetically disrupted in the murine ovarian cancer cell line ID8 using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated genome editing. PD‐L1 knockout (KO) and control ovarian cancer cells were intraperitoneally inoculated into syngeneic mice, and survival and tumor dissemination were evaluated. Survival times were significantly longer in the PD‐L1‐KO ID8‐inoculated groups than in their control groups, and its therapeutic benefit was enhanced in combination with the cisplatin treatment. Tumor weights and ascites volumes were significantly lower in the PD‐L1‐KO ID8 groups than in their control groups. Immunohistochemical and immunofluorescence analyses showed that intratumoral CD4+ T cells, CD8+ T cells, NK cells and CD11c+ M1 macrophages were significantly increased, whereas regulatory T cells were significantly decreased in the PD‐L1‐KO ID8 groups compared with those in their control groups. The intratumoral mRNA expression of interferon‐γ, tumor‐necrosis factor‐α, interleukin (IL)‐2, IL‐12a, CXCL9 and CXCL10 was significantly stronger, while that of IL‐10, vascular endothelial growth factor, CXCL1 and CXCL2 was significantly weaker in the PD‐L1‐KO ID8 groups. These results indicate that CRISPR/Cas9‐mediated PD‐L1 disruption on tumor cells promotes anti‐tumor immunity by increasing tumor‐infiltrating lymphocytes and modulating cytokine/chemokine profiles within the tumor microenvironment, thereby suppressing ovarian cancer progression. These results suggest that PD‐L1‐targeted therapy by genome editing may be a novel therapeutic strategy for ovarian cancer.

The oncogene KRAS promotes cancer cell dissemination by stabilizing spheroid formation via the MEK pathway

BackgroundPeritoneal dissemination is a critical prognostic factor in ovarian cancer. Although stabilized spheroid formation promotes cancer cell peritoneal dissemination in ovarian cancer, the associated oncogenes are unknown. In this study, we assessed the role of the KRAS oncogene in ovarian cancer cell dissemination, focusing on the stability of cells in spheroid condition, as well as the modulation of intracellular signaling following spheroid transformation.MethodsWe used ID8, a murine ovarian cancer cell line, and ID8-KRAS, an oncogenic KRAS (G12 V)-transduced ID8 cell line in this study. Spheroid-forming (3D) culture and cell proliferation assays were performed to evaluate the growth characteristics of these cells. cDNA microarray analysis was performed to identify genes involved in KRAS-associated signal transduction in floating condition. A MEK inhibitor was used to evaluate the effect on cancer peritoneal dissemination.ResultsCell viability and proliferation in monolayer (2D) cultures did not differ between ID8 and ID8-KRAS cells. However, the proportions of viable and proliferating ID8-KRAS cells in 3D culture were approximately 2-fold and 5-fold higher than that of ID8, respectively. Spheroid-formation was increased in ID8-KRAS cells. Analysis of peritoneal floating cells obtained from mice intra-peritoneally injected with cancer cells revealed that the proportion of proliferating cancer cells was approximately 2-fold higher with ID8-KRAS than with ID8 cells. Comprehensive cDNA microarray analysis revealed that pathways related to cell proliferation, and cell cycle checkpoint and regulation were upregulated specifically in ID8-KRAS cells in 3D culture, and that some genes partially regulated by the MEK-ERK pathway were upregulated only in ID8-KRAS cells in 3D culture. Furthermore, a MEK inhibitor, trametinib, suppressed spheroid formation in 3D culture of ID8-KRAS cells, although trametinib did not affect 2D-culture cell proliferation. Finally, we demonstrated that trametinib dramatically improved the prognosis for mice with ID8-KRAS tumors in an in vivo mouse model.ConclusionsOur data indicated that KRAS promoted ovarian cancer dissemination by stabilizing spheroid formation and that the MEK pathway is important for stabilized spheroid formation. Disruption of spheroid formation by a MEK inhibitor could be a therapeutic target for cancer peritoneal dissemination.

Abstract B29: Cisplatin is pro-immunogenic and promotes intrinsic and reactive immune suppression in inflamed and noninflamed ovarian cancer mouse models

Abstract Objective: Front-line therapy in ovarian cancer combines platinum/taxane chemotherapy and surgery. We studied here the role of cisplatin on PDL1 biology in vitro and in vivo in new ovarian tumor models. We also explored the in vivo efficacy of PDL1 blockade in combination with cisplatin. Methods: We analyzed in vitro the time and dose dependent effect of cisplatin on PD-L1 expression on human and murine cell lines with varying susceptibility to the drug. Cisplatin-induced molecular changes were detected via unbiased transcriptomics (RNAseq, Illumina). Mechanisms of tumor cell-intrinsic immune resistance were explored in a series of murine ovarian cancer cell lines with defined genetic traits, including conditional mutations in either Kras (activation), Pten (deletion), or both. The effect of cisplatin in combination with PDL1 blockade was tested in vivo in two syngeneic OC models (2F8 and 2F8cis) that mirror platinum sensitive and moderately resistant tumors, respectively. These models have different inflammation at baseline, with 2F8cis tumors being more inflamed (increased tumor-infiltrating lymphocytes [TILs]-increased necrosis) than 2F8 tumors. Results: Exposure of murine and human ovarian cancer cell lines to IC50 cisplatin triggers increased PD-L1 expression in a time- and dose-dependent manner. The effect is partly mediated by Erk/MAPK pathway, as MEK inhibitor AZD6244 inhibits both the baseline and the cisplatin-induced PD-L1 expression. Gene expression analyses (RNASeq) demonstrate that cisplatin upregulates a number of genes that contribute to tumor immunogenicity and increased tumor-reactive CD8 T cells. The cisplatin-induced proinflammatory effect was also observed in vivo, predominantly in platinum-sensitive 2F8 tumors that show increased T-cell infiltration of both CD8 and FoxP3 Tregs. Tumor PDL1 gene expression is also increased upon cisplatin treatment, as detected by qPCR. Mice with platinum-sensitive (2F8) tumors showed increased survival in response to cisplatin alone and anti-PDL1 alone, compared to the combination, which was not significantly different than control treated hosts. In contrast, cisplatin/anti-PDL1 treatment combination triggered significantly longer survival in mice with moderately resistant (2F8cis) tumors. Conclusion: Overall, these results suggest that exposure to cisplatin has the potential to increase tumor immunogenicity and recognition by CD8 TILs while also triggering immune evasion through PD-L1 upregulation either directly (through tumor cell-intrinsic mechanisms) or indirectly via adaptive immune resistance due to IFNγ secreting TILs. Inflamed and noninflamed respond to anti-PD-L1 alone. although the effect is generally modest. Inflamed tumors with moderate platinum resistance respond to cisplatin/anti-PDL1 combination, and the effect is better than for each agent alone. These results provide the rationale for the design of combination therapies that take into consideration tumor inflammation at baseline and drug-induced immunogenic effects. Citation Format: Shannon Grabosch, Mirna Bulatovic, Feitianzhi Zeng, Tianzhou Ma, Lixin Zhang, Malcolm Ross, Joan Brozick, George Tseng, Esther Elishaev, Robert P. Edwards, Anda Vlad. Cisplatin is pro-immunogenic and promotes intrinsic and reactive immune suppression in inflamed and noninflamed ovarian cancer mouse models. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B29.

Abstract 3582: Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors

Abstract Adoptive transfer of tumor-reactive T cells is a promising anti-tumor therapy for many cancers. To enhance tumor recognition by T cells, chimeric antigen receptors (CAR) consisting of signaling domains fused to receptors that recognize tumor antigens can be created and expressed in T cells. One receptor that is a prospective target for a new chimeric antigen receptor is PD1 because the ligands for the PD1 receptor are expressed on many cancer types. Therefore, we developed a murine chimeric PD1 receptor (chPD1) consisting of the PD1 receptor extracellular domain and the activation domain of CD3 zeta. In addition, current chimeric antigen receptor therapies utilize various costimulatory domains to enhance anti-tumor efficacy. Therefore, we also compared the inclusion of CD28, Dap10, 41BB, GITR, ICOS, or OX40 costimulatory domains in our chPD1 receptor to determine which costimulatory domain induced optimal anti-tumor immunity. To determine if this novel CAR could potentially target a wide variety of tumors, the anti-tumor efficacy of chPD1 T cells against murine lymphoma, melanoma, kidney, pancreatic, liver, colon, breast, ovarian, prostate, and bladder cancer cell lines was measured. Of the eighteen cell lines tested, all expressed PD1 ligands on their cell surface, making them potential targets for chPD1 T cells. Regardless of the costimulatory domain in the CAR, all of the chPD1 T cells induced similar levels of T cell proliferation and tumor cell lysis. However, differences were observed in the cytokine secretion profiles depending on which costimulatory receptor was included in the CAR. While most of the chPD1 T cell receptor combinations secreted both pro-inflammatory (IFNγ, TNFα, IL-2, GM-CSF, IL-17, and IL-21) and anti-inflammatory cytokines (IL-10 and IL-5) as determined by ELISA and LegendPlex analysis, chPD1 T cells containing a Dap10 costimulatory domain secreted high levels of proinflammatory cytokines, but did not secrete a significant amount of anti-inflammatory cytokines. Furthermore, T cells expressing chPD1 receptors with a Dap10 domain also had the strongest anti-tumor efficacy in vivo. ChPD1 T cells did not survive for longer than 14 days in vivo, however treatment with chPD1 T cells induced long-lived protective host-anti-tumor immune responses in tumor-bearing mice. Therefore, adoptive transfer of chPD1 T cells could be a novel therapeutic strategy to treat multiple types of cancer and inclusion of the Dap10 costimulatory domain in chimeric antigen receptors may induce a preferential cytokine profile for anti-tumor therapies. Citation Format: Amorette E. Barber, Geoffrey Parriott, Shane Crean, Hailey Kintz. Inclusion of a Dap10 costimulatory domain enhances anti-tumor efficacy of chimeric PD1-expressing T cells in multiple types of solid tumors [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 3582.

IL-12 Expressing oncolytic herpes simplex virus promotes anti-tumor activity and immunologic control of metastatic ovarian cancer in mice.

BackgroundDespite advances in surgical aggressiveness and conventional chemotherapy, ovarian cancer remains the most lethal cause of gynecologic cancer mortality; consequently there is a need for new therapeutic agents and innovative treatment paradigms for the treatment of ovarian cancer. Several studies have demonstrated that ovarian cancer is an immunogenic disease and immunotherapy represents a promising and novel approach that has not been completely evaluated in ovarian cancer. Our objective was to evaluate the anti-tumor activity of an oncolytic herpes simplex virus “armed” with murine interleukin-12 and its ability to elicit tumor-specific immune responses. We evaluated the ability of interleukin−12-expressing and control oncolytic herpes simplex virus to kill murine and human ovarian cancer cell lines in vitro. We also administered interleukin−12-expressing oncolytic herpes simplex virus to the peritoneal cavity of mice that had developed spontaneous, metastatic ovarian cancer and determined overall survival and tumor burden at 95 days. We used flow cytometry to quantify the tumor antigen-specific CD8+ T cell response in the omentum and peritoneal cavity.ResultsAll ovarian cancer cell lines demonstrated susceptibility to oncolytic herpes simplex virus in vitro. Compared to controls, mice treated with interleukin−12-expressing oncolytic herpes simplex virus demonstrated a more robust tumor antigen-specific CD8+ T-cell immune response in the omentum (471.6 cells vs 33.1 cells; p = 0.02) and peritoneal cavity (962.3 cells vs 179.5 cells; p = 0.05). Compared to controls, mice treated with interleukin−12-expressing oncolytic herpes simplex virus were more likely to control ovarian cancer metastases (81.2 % vs 18.2 %; p = 0.008) and had a significantly longer overall survival (p = 0.02). Finally, five of 6 mice treated with interleukin−12-expressing oHSV had no evidence of metastatic tumor when euthanized at 6 months, compared to two of 4 mice treated with sterile phosphate buffer solution.ConclusionOur pilot study demonstrates that an interleukin−12-expressing oncolytic herpes simplex virus effectively kills both murine and human ovarian cancer cell lines and promotes tumor antigen-specific CD8+ T-cell responses in the peritoneal cavity and omentum, leading to reduced peritoneal metastasis and improved survival in a mouse model.

Abstract 4030: Epigenetic induction of MHC-II pathway expression in murine ovarian cancer cell line

Abstract INTRODUCTION This study evaluated the potential of epigenetic treatments to induce the expression of the MHC-II antigen presentation pathway in ovarian cancer. Ovarian cancer escapes immune response allowing it to spread in the peritoneal cavity; however, patients with greater immune response to their tumors at baseline have improved survival. Typically, exogenous antigens from tumors are processed and presented via MHC-II to CD4 T cells by antigen-presenting cells. However, if ovarian cancer cells could be induced to express the MHC-II pathway, they could be converted into antigen presenting cells and stimulate an anti-tumor response. METHODS Murine epithelial ovarian cancer cells (ID8) were treated for 72 h with the histone deacetylase inhibitors (HDACi): entinostat (MS-275) (1.25, 2.5, 5, 10 μM) and quisinostat (20, 40, 80, 160 nM). Cells were treated with 5-azacytidine (5-aza), a DNA methytransferase inhibitor, alone and in combination with MS-275 (5 μM) at 17.25, 37.5, 75, and 150 nM. After treatment, the expression of CD74 (an MCH-II pathway protein) and MHC-II was evaluated by flow cytometry. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to measure CD74 and CIITA, a transcriptional coactivator that controls MHC-II gene expression. RNA was extracted using RNEasy Mini Kit (Qiagen), cDNA was created with SuperScript VILO Master Mix (ThermoFischer), and a 109bp amplicon was created using JumpStart REDTaq ReadyMix (Sigma-Aldrich). For statistical analysis, a Welch ANOVA was performed using JMP Pro 12. Tukey-Kramer analysis evaluated individual differences. The mean fluorescence intensities (MFI) for the combination treatments were compared using an unequal variance F-test. RESULTS Increased protein expression of CD74 and MHC-II was seen after treatment with MS-275, quisinostat, and 5-aza compared to untreated control. Increased expression of CD74 and MHC-II was seen with the combination treatment of 5-aza (17, 37.5, 75, 150 nM) and MS-275 (5 μM) compared to individual treatments (p &amp;lt; .0001). A dose dependent increased expression of CIITA was demonstrated by RT-PCR with 5-aza treatment alone and in combination with MS-275. Increased mRNA expression of CD74 was also seen for all treatments compared to a negative control. CONCLUSION Treatment with HDAC inhibitors and a DNA methyltransferase inhibitor induces expression of MHC-II in murine epithelial ovarian cancer cells. The conversion of ovarian cancer cells into antigen presenting cells provides a potential therapeutic model to augment the immune response against epithelial ovarian cancer, a disease known to suppress anti-tumor immunity. Citation Format: Taylor B. Turner, Rebecca C. Arend, Mei Li, Troy D. Randall, Andres Forero-Torres, Albert F. LoBuglio, J Michael Straughn, Donald J. Buchsbaum. Epigenetic induction of MHC-II pathway expression in murine ovarian cancer cell line. [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 4030.

Abstract 3208: Chemo-induced biology of PD-L1 and in vivo combination immune therapy for ovarian cancer

Abstract Rationale: Ovarian cancer patients receive platinum/taxane-based chemotherapy as standard of care. Five year survival has remained unchanged for several decades, at less than 45%, pointing to the need for new and improved therapies. We recently reported in vivo anti-tumor efficacy of PD-L1 immune checkpoint blockade in a new transplantable ovarian cancer mouse model. However, chemo-induced effects on ovarian tumor PD-L1 expression have not been addressed. We studied here the effect of platinum/taxane on tumor PD-L1 expression in vitro and in vivo and tested the efficacy of PD-L1 blockade in combination with cisplatin, using several treatment regimens. Methods and Results: Our results demonstrate that in vitro exposure of several human ovarian cancer cell lines, with various baseline susceptibilities to cisplatin and taxol, triggers PD-L1 upregulation. Similar effects were observed when newly tumor-derived, platinum sensitive (2F8) and platinum resistant (2F8cis) murine ovarian cancer cell lines were exposed to chemo drugs in vitro. Based on these findings we postulated that PD-L1 immune checkpoint blockade in combination with cisplatin may provide therapeutic benefit in ovarian cancer. In vivo, we have challenged n = 37 mice IP with 0.8 million 2F8 cells. Tumor-bearing mice were treated with cisplatin, anti-PD-L1 antibody, both drugs or isotype control every two weeks for three doses starting at day 14 post-inoculation. Study endpoint was overall survival. Secondary endpoints were tumor CD8 infiltration, changes in Th1/cytotoxic immunity. Tumors and ascites-derived cancer cells were analyzed with flow cytometry. RNA was extracted from splenocytes and analyzed with Nanostring using probes for n = 511 immune genes. In line with in vitro results, tumor cells isolated ex vivo from cisplatin-treated mice expressed increased PD-L1. Compared to control treated mice, both Cisplatin alone and anti-PD-L1 alone increase overall survival (p = 0.002 and p = 0.02, respectively). No increase in survival was observed in anti-PD-L1/Cisplatin-treated mice, due to an over-responsive immune cascade overwhelming the animal. Addition of celecoxib, a cyclooxygenase-2 inhibitor, to the anti-PD-L1/cisplatin combination was well tolerated and led to improved overall survival. Conclusion: Ovarian cancer cells upregulate PD-L1 in response to chemotherapy exposure in vivo and in vitro. Though effective independently, combination cisplatin and anti-PD-L1 blockade did not improve survival, likely due to cytokine release syndrome. Celecoxib added to cisplatin and anti-PD-L1 improves overall survival. Anti-tumor immunity in responder mice revealed a cytotoxic T cell mediated gene signature. These findings reveal benefits and pitfalls of chemotherapy in combination with immune checkpoint blockade and have high translational potential for ovarian cancer treatment. Citation Format: Shannon Grabosch, Feitianzhi Zeng, Lixin Zhang, Tianzhou Ma, George Tseng, Robert P. Edwards, Anda M. Vlad. Chemo-induced biology of PD-L1 and in vivo combination immune therapy for ovarian cancer. [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 3208.

Evaluation of microparticulate ovarian cancer vaccine via transdermal route of delivery

Ovarian cancer is the fifth most commonly occurring malignancy in women, with the highest mortality rate among all the gynecological tumors. Microparticulate vaccine can serve as an immunotherapeutic approach with a promising antigenic delivery system without a need for conventional adjuvants. In this study, a microparticulate vaccine using whole cell lysate of a murine ovarian cancer cell line, ID8 was prepared by spray drying. Further, the effect of interleukins (ILs) such as IL-2 and IL-12 was evaluated in a separate study group by administering them with vaccine particles to enhance the immune response. The vaccine microparticles were administered to C57BL/6 female mice via transdermal alone and in combination with the oral route. The transdermal vaccine was delivered using a metallic microneedle device, AdminPen™. Orally administered microparticles also included an M-cell targeting ligand, Aleuria aurantia lectin, to enhance the targeted uptake from microfold cells (M-cells) in Peyer's patches of small intestine. In case of combination of routes, mice were given 5 transdermal doses and 5 oral doses administered alternatively, beginning with transdermal dose. At the end of vaccination, mice were challenged with live tumor cells. Vaccine alone resulted in around 1.5 times tumor suppression in case of transdermal and combination of routes at the end of 15th week when compared to controls. Inclusion of interleukins resulted in 3 times tumor suppression when administered with transdermal vaccine and around 9 times tumor suppression for the combination route of delivery in comparison to controls. These results were further potentiated by serum IgG, IgG1 and IgG2a titers. Moreover, CD8+ T-cell, CD4+ T-cell and NK (natural killer) cell populations in splenocytes were elevated in case of vaccinated mice. Thus, vaccine microparticles could trigger humoral as well as cellular immune response when administered transdermally and via combination of route of delivery. However overall, vaccine administered with interleukins, via combination of route, was found to be the most efficacious to suppress the tumor growth and lead to a protective immune response.

Effects of Kras activation and Pten deletion alone or in combination on MUC1 biology and epithelial-to-mesenchymal transition in ovarian cancer.

Mucin1 (MUC1) is an epithelial glycoprotein overexpressed in ovarian cancer and actively involved in tumor cell migration and metastasis. Using novel in vitro and in vivo MUC1-expressing conditional (Cre-loxP) ovarian tumor models, we focus here on MUC1 biology and the roles of Kras activation and Pten deletion during cell transformation and epithelial-to-mesenchymal transition (EMT). We generated several novel murine ovarian cancer cell lines derived from the ovarian surface epithelia (OSE) of mice with conditional mutations in Kras, Pten or both. In addition, we also generated several tumor-derived new cell lines that reproduce the original tumor phenotype in vivo and mirror late stage metastatic disease.Our results demonstrate that de novo activation of oncogenic Kras does not trigger increased proliferation, cellular transformation or EMT and prevents MUC1 upregulation. In contrast, Pten deletion accelerates cell proliferation, triggers cellular transformation in vitro and in vivo and stimulates MUC1 expression. Ovarian tumor-derived cell lines MKP-Liver and MKP-Lung cells reproduce in vivo EMT and represent the first immune competent mouse model for distant hematogenous spread. Whole genome microarray expression analysis using tumor and OSE-derived cell lines reveals a 121 gene signature associated with EMT and metastasis. When applied to n=542 cases from the ovarian cancer TCGA dataset, the gene signature identifies a patient subset with decreased survival (p=0.04). Using an extensive collection of novel murine cell lines we have identified distinct roles for Kras and Pten on MUC1 and EMT in vivo and in vitro. The data has implications for future design of combination therapies targeting Kras mutations, Pten deletions and MUC1 vaccines.