New therapy development is critically needed for ovarian cancer. We used the chicken egg CAM assay to evaluate efficacy of anticancer drug delivery using recently developed biodegradable PMO (periodic mesoporous organosilica) nanoparticles. Human ovarian cancer cells were transplanted onto the CAM membrane of fertilized eggs, resulting in rapid tumor formation. The tumor closely resembles cancer patient tumor and contains extracellular matrix as well as stromal cells and extensive vasculature. PMO nanoparticles loaded with doxorubicin were injected intravenously into the chicken egg resulting in elimination of the tumor. No significant damage to various organs in the chicken embryo occurred. In contrast, injection of free doxorubicin caused widespread organ damage, even when less amount was administered. The lack of toxic effect of nanoparticle loaded doxorubicin was associated with specific delivery of doxorubicin to the tumor. Furthermore, we observed excellent tumor accumulation of the nanoparticles. Lastly, a tumor could be established in the egg using tumor samples from ovarian cancer patients and that our nanoparticles were effective in eliminating the tumor. These results point to the remarkable efficacy of our nanoparticle based drug delivery system and suggests the value of the chicken egg tumor model for testing novel therapies for ovarian cancer.

TWIST protein is critical to development and is activated in many cancers. TWIST regulates epithelial-mesenchymal transition, and is linked to angiogenesis, metastasis, cancer stem cell phenotype, and drug resistance. The majority of epithelial ovarian cancer (EOC) patients with metastatic disease respond well to first-line chemotherapy but most relapse with disease that is both metastatic and drug resistant, leading to a five-year survival rate under 20%. We are investigating the role of TWIST in mediating these relapses. We demonstrate TWIST-siRNA (siTWIST) and a novel nanoparticle delivery platform to reverse chemoresistance in an EOC model. Hyaluronic-acid conjugated mesoporous silica nanoparticles (MSN-HAs) carried siTWIST into target cells and led to sustained TWIST knockdown in vitro. Mice treated with siTWIST-MSN-HA and cisplatin exhibited specific tumor targeting and reduction of tumor burden. This platform has potential application for overcoming clinical challenges of tumor cell targeting, metastasis and chemoresistance in ovarian and other TWIST overexpressing cancers.

Objective: Diabetes compromises blood circulation and immune responses, as well as major metabolic pathways, which can lead to increased susceptibility to infection. In the present study, we examined this possibility using diabetic versus non-diabetic mice with systemic candidiasis caused by a Candida albicans azole sensitive(C.alb-S) or azole resistant(C.alb-R) strain. Infected mice were treated with fluconazole (Flu) or liposomal amphotericin B (AmBisome®, AmBi). Methods: ICR male mice (3-4 weeks old) were maintained on a high (60%) fat diet for 4 weeks. Diabetes was then chemically induced by one intraperitoneal nicotinamide (60 mg/kg) and streptozotocin (100 mg/kg) treatment; by day 7-14 mean blood glucose levels were ≥200 mg/dL Diabetic mice (n=9-14/gp) and nondiabetic mice (n=14-16/gp) were challenged intravenously (IV) with C.alb-R (ATCC 62342) (diabetic: 3.5x10ex6 cells/mouse; non-diabetic: 1.4x10ex6 cells/mouse) or C.alb-S (CP620) (diabetic: 1.5x10ex6 cells/mouse; non-diabetic: 6.9 x10ex6 cells/mouse) and 24h later, given 5 mg/kg AmBi or 5% dextrose in water (D5W) intravenously for 6 days or Flu 40 mg/kg PO 2X/day for 6 days. Tissues (kidneys, liver, spleen) were collected (n=3-6/group) on d4 (diabetic) and d7 (nondiabetic) post-challenge, homogenized and plated on Sabouraud's agar to determine fungal burden (CFU/g). Other mice (n=6-10/gp) were monitored for morbidity to d28. Results: With a C.alb-R infection, AmBi treated, non-diabetic and diabetic mice, had significantly better survival than Flu or D5W treated mice (AmBi 83-100% survival, Flu 0-30% survival, D5W (0-20% survival) (p≤0.01). Significant decreases in CFU/g kidneys were observed with AmBi treatment versus D5W or Flu in diabetic and non-diabetic mice (p≤ 0.04); no fungi were recovered in livers or spleens of AmBi treated mice (p≤ 0.04). In comparison, with C.alb-S infection, AmBi or Flu treated, non-diabetic mice had 100% and 80% survival, respectively, but in diabetic mice, survival was 100% for AmBi and only 16% for Flu even though this was a Flu sensitive strain (p=0.0021). D5W yielded 0% (non-diabetic) and 14% (diabetic) survival. The fungal burden in the livers and spleens was significantly lower with AmBi treatment versus Flu or D5W, with AmBi treatment reducing the liver and spleen fungal burdens to undetectable levels. In the kidneys, the fungal burden in non-diabetic mice was significantly lower for AmBi versus Flu or D5W, but there was no significant difference in kidney fungal burden in diabetic mice given AmBi or Flu. Conclusions: In non-diabetic mice, AmBi and Flu were effective in systemic candidiasis caused a C. albicans azole sensitive strain, while AmBi was effective in treating non-diabetic diabetic mice infected with a C. albicans azole sensitive or resistant strain. Notably, in diabetic mice challenged with a C. albicans azole sensitive strain, Flu was associated with poor survival and elevated tissue fungal burden, indicating that the diabetic condition enhanced susceptibility to systemic candidiasis whether it was cause...

Purpose: To develop rapidly operational CAM-PDX (chorioallantoic membrane-patient derived xenograft) tumor models that morphologically resemble human tumors in order to perform therapeutic/efficacy studies targeting TWIST, a transcription factor expressed in cancer stem cells critical to embryonic development, and aberrantly activated in many cancers. This CAM-PDX model was used to optimize mesoporous silica nanoparticle with hyaluronic acid (MSN-HA) and siTWIST in combination with cisplatin, as well as screen other drug therapies (e.g., doxorubicin) that will benefit ovarian cancer patients. Experimental Procedures: Fertilized eggs were obtained from AA Lab Eggs, Inc. The eggs were incubated for 10 days at 100°F and 60% humidity. At day 10, a small window was opened on the surface of the egg and the CAM was dropped. A teflon ring was inserted, 50µg/µL of cancer cells (1 million cells) were deposited, and the window was covered with transparent film dressing and replaced in the incubator. 120µL of siTWIST (with MSN-HA) and/or 100µL of cisplatin were administered intravenously (day 13 and 14, respectively). On day 17, the eggs were imaged using Leica microscope. The CAM-PDX models were developed first with standard EOC cell lines and second using primary cells from patient tissues transfected with eGFP/ffluc for imaging growth, histology, and tumor biodistribution studies. Toxicity studies were performed by administering doxorubicin at varying doses and with different nanoparticle delivery methods. Results: The study showed that we were able to grow patient-derived tumors in the egg CAM model. Moreove,r a histologic comparison of CAM tumors and ovarian patient tumors reveals highly similar morphology. Furthermore, when treated with chemotherapy, the tumor regressed, as it does in patients. Our toxicity study demonstrates that surrounding organs, as well as the embryo, were spared from detrimental off-target effects of doxorubicin when the drug was delivered inside nanoparticles, as opposed to free-drug, as demonstrated by final egg survivability count. Images show free-drug administration of doxorubicin damaged organs in the peritoneal cavity while nanoparticle administration of doxorubicin resulted in a clean cavity and sound organs. Our study also shows that our siTWIST MSN-HA sensitized drug-resistant cancer stem cells to cisplatin. Finally, our results also show that the optimal treatment for tumor dissemination in the PDX-CAM system is the combination of siTWIST MSN-HA and cisplatin compared to cisplatin alone. Conclusions: This study provides a much-needed, cost-effective means to grow PDX lines from patient tissues. The CAM-PDX system provides a faster means of collecting information on the efficacy of novel MSNs for the delivery of siRNA and drugs than expensive mouse models. Thus, the CAM-PDX model is beneficial toward moving us closer to translation and application in other cancers. Citation Format: Altagracia Contreras, Sophia Allaf Shahin, Laura E. Ratliff, Shirleen I. Simargi, Ruining Wang, Binh Vu, Thanh H. Dellinger, Jeffrey I. Zink, Fuyuhiko Tamanoi, Julia Unternaehrer-Hamm, Carlotta A. Glackin. Optimizing therapies for drug-resistant ovarian cancer stem cells using aCAM-PDX model [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 2152.

Purpose: TWIST is a transcription factor critical to embryonic development, and aberrantly activated in many cancers. It regulates epithelial-to-mesenchymal transition (EMT), the process underlying metastatic spread and drug resistance. The majority of epithelial ovarian cancer (EOC) patients respond well to chemotherapy, but relapse with metastatic and drug-resistant disease. We are investigating the role of TWIST-mediated relapse, with the goal of developing treatments that sensitize chemoresistant tumors and improve survival. Experimental Procedures: We employ siRNA to target TWIST mRNA and have created a mesoporous silica nanoparticle with hyaluronic acid (siTWIST-MSN-HA) as a delivery vehicle. Microscopy was conducted to ensure cancer stem cell (CSC) targeting via CD44, and efficient uptake of MSN-HAs. We tested TWIST knockdown combined with cisplatin in vitro and in vivo. At necropsy, total tumor, metastasis, and ascites were evaluated to determine effects of siTWIST-MSN-HA compared to cisplatin alone. qPCR was conducted on tumors to examine effect of siRNA against TWIST, and its target genes. Mice were imaged via bioluminescence to observe CSC localization of siTWIST-MSN-HA compared to siTWIST-MSN (No HA). Tissue sections were stained via IHC to determine MSN-HA tumor targeting. CD44 staining was conducted to reveal HA co-localization in tumor. Results: siTWIST-MSN-HAs significantly knocked down TWIST, and sensitized cells to cisplatin compared to control in vitro. Following necropsy, tumor quantification revealed mice treated with siTWIST-MSN-HAs plus cisplatin exhibited a startling 75% loss of overall tumor burden (p=0.0012), 88% loss of total metastasis (p=0.001), and further 86% loss of total ascites (p=0.002) compared to cisplatin-alone treatment groups. EMT target expression by qPCR analysis revealed loss of TWIST1, vimentin, N-cadherin, and gain of E-cadherin in tumors treated with siTWIST-MSN-HA compared to controls. Bioluminescent images of mice at necropsy revealed highly specific tumor localization of MSN-HA Dylight (680nm) compared to MSN without HA. MSN-HA exhibited CSC targeting at metastatic sites, where most of the signal was emitted from the primary tumor; and negligible quantities of MSN-HAs were detected elsewhere. MSN-HA RITC (576nm) reveals highly specific tumor targeting via IHC; MSN-HA nanoparticles localized primarily in tumor cells and not in control organs. CD44 staining of these tissues reveals MSN-HAs co-localized with CD44 in CSCs. Conclusions: These studies demonstrate TWIST as a promising target for metastasis and acquired drug resistance in EOC. MSN-HAs provide CSC specific CD44 targeting with high efficacy and reduce tumor burden via siTWIST therapy. Thus, siTWIST-MSN-HAs provide a promising platform to improve survival from metastatic, drug-resistant ovarian cancer. Citation Format: Carlotta A. Glackin, Sophia A. Shahin, Shirleen Simargi, Ruining Wang, Altagracia Contreras, Liliana Parra, Loiuse Qu, Wei Wen, Thanh Dellinger, Julia Unternaehrer, Fuyujiko Tamanoi, Jeffrey Zink. Hyaluronic acid conjugated nanoparticle delivery of siTWIST reduces tumor burden and enhances chemosensitivity in 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 2024.