Three-dimensional (3D) culture of cancer cell lines has long been advocated as a better model of the malignant phenotype that is most closely related to tumorigenicity in vivo. Moreover, new drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumors and to decrease the use of experimental animals. The induction of chemotherapy or concomitant chemoradiotherapy has been used to increasingly to improve survival, and organ preservation. This approach encounters significant morbidity and mortality. Therefore reliable chemosensitivity assays are needed to accurately predict the response to chemotherapy and guide the selection and treatment of cancer patients. The purpose of this study is to examine and evaluate optimum drug candidates in vitro chemosensitivity on patient tumor tissues directly in culture and on their Cancer stem cell cultures. The tumor samples obtained after surgery or biopsy, were placed immediately in Celprogen Tumor Transportation Media and shipped at 4-8 OC for processing. Tissues were washed with 1X PBS solution and aseptically cut into 0.5mm sections and cultured in 6 well tissue culture plates with an insert pre-coated with ECM. All cancer cell types remain viable and maintain their native architecture for at least 14 days and incorporated DNA measured by adding EdU(5-ethynyl-2’-deoxyuridine) to the culture. The efficacy of various therapeutic agents targeting major pathways (wnt,Notch,PI3K,MAPK,STAT) and chemotherapy agents were tested using DNA uptake and TUNNEL assay anti-cancer agents was calculated according to the inhibition index. The same compounds were tested for utilizing the patient's Pancreatic Cancer Stem Cell Cultures established with Celprogen's Media and ECM. Expression of PDX-1, SHH, CD24, CD44, CD133, EpCAM, CBX7, OCT4, SNAIL, SLUG, TWIST, Ki-67, E-cadherin, β-catenin and vimentin were quantified by qPCR or immunocytochemistry per cell culture. The epithelial-mesenchymal transition (EMT) is linked to induction of a stem-cell like phenotype. We cultured the cells in low oxygen since Tumor hypoxia induces EMT, which induces invasion and metastasis, and is linked to cancer stem cells (CSCs). Among the 600 compounds tested Gemcitabine, Taxol, Fluorouracil, Leucovorin, Irinotecan, and Oxaliptin were not effective against Pancreatic Cancer Stem cell (CSC) but were effective on tumor cells (differentiated CSCs). We were able to show 6 compounds that were effective against Pancreatic CSC targeting selected pathways. Citation Format: John P. Clery, Esteban Gomez, Michael Sharma, Aabha khemani, Cristian Sharma, Rubio Punzalan, miriam navel, Natalee Amezcua, Jitesh Jani, Jay Sharma. Optimal drug concentration screening and evaluation in cancer stem cells & 3D tumor stem cell cultures drug response assays in association with clinical efficacy for pancreatic cancer stem cell. [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 5545. doi:10.1158/1538-7445.AM2013-5545

Breast cancer is the most common malignancy among women in developed countries, affecting more than a million women per year worldwide. Of these, triple negative breast carcinoma represents 10-17 %. Triple negative breast carcinomas, characterized by estrogen, progesterone and HER2 receptor negativity are very aggressive tumors with poor prognosis. Breast Cancer Stem derived from triple negative parental Breast Cancer tumors, are a subpopulation of cells within the parental breast cancer population within the individual which are positive for the following markers: CD133+CD44+CD24+ESA+SSEA-1+TRA-1-61+& TRA1-81+ and Oct ¾ these Breast Cancer Stem cells are highly tumorigenic and possess the stem cell-like properties of self-renewal and the ability to produce differentiated progeny. Breast Cancer Stem cells also demonstrate up regulation of SSEA3+, SSEA4+ upon differentiation into parental cancer phenotype. Individualized treatment (tailored therapy) based on molecular biology markers of tumor and patient is the trend in clinical practice these days. However, molecular targets and predictors for the treatment of triple negative breast carcinoma do not currently exist. With the identification and characterization of Breast Cancer Stem Cells from parental triple negative tumors, enables one to screen novel drug candidates for potential development of therapeutics for triple negative Breast Cancer Patients. In this study we have utilized Breast Cancer Stem Cells from triple negative Tumors to screen potential drug candidates. The Breast Cancer Stem Cell based assay system may provide novel therapeutic approaches into treatment of triple negative breast cancer patients, which are resistant to standard chemotherapy and radiation. 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 3319.

Pancreatic cancer is the fourth leading cause of cancer mortality in the US, despite significant improvements in diagnostic imaging and operative modalities. The 5-year survival rate remains less than 6% because of microscopic or gross metastatic disease at time of diagnosis. Although the treatment of pancreatic cancer remains a huge challenge, it is entering a new era with the development of new strategies and trial designs. Because there is an increasing number of novel therapeutic agents and potential combinations available to test in patients with pancreatic cancer, the identification of robust prognostic and predictive markers and of new targets and relevant pathways is a top priority as well as the design of adequate trials incorporating molecular-driven hypothesis. Over the past decade, increasing evidence suggested that stem cells play a crucial role not only in the generation of complex multicellular organisms, but also in the development and progression of malignant diseases. Most tumors have been shown to contain a subset of distinct cancer cells that is responsible for tumor initiation and propagation. These cells are termed cancer stem cells or tumor-initiating cells and they are highly resistant to chemotherapeutic agents. Here, we examined the efficacy of combined treatments of CEP1101 and gemcitabine in human pancreatic cancer cells, and pancreatic CSCs (CD133, CD44, CXCR4, SSEA3/4, Oct4, ALDH, Telomerase & Nestin) from the same donors. CEP1101 inhibited the growth of CSCs, while gemcitabine suppressed the viability of non-CSCs. In vivo studies showed that CEP1101 combined with gemcitabine eliminate the engraftment of human pancreatic cancer and CSCs, more effectively than the individual agents. These data demonstrate that administration of CEP1101, which targets CSCs, may constitute a potential therapeutic strategy for improving the efficacy of gemcitabine to eradicate pancreatic cancer. This study shows potential molecular therapeutic targets to eradicate the tumor - and metastasis-initiating cells and their progenies for the evaluation of new effective combination therapies against locally advanced and metastatic pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4408. doi:1538-7445.AM2012-4408

Cancer stem cells (CSCs) are thought to be critical for engraftment and long-term growth of many tumors, including glioblastoma multiforme (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. Human glioblastoma multiforme is a heterogenous tumor composed from tumor cells and small portion of cancer stem cells, which have a high tumorigenic potential and a low proliferation rate. Glioblastoma cancer stem cells are phenotypically similar to the normal stem cells, they express CD133 gene and other genes characteristic of neural stem cells and posses self-renewal potential. The CD133+ GBM CSCs have been isolated with Celprogen Media and ECM and characterized as chemo-/radio-resistant tumor-initiating cells which are responsible as one of the many factors involved in post-treatment recurrence. In order to explore the molecular properties of tumorigenic CD133+ GBM CSCs that resist treatment, we isolated CD133+ GBM CSCs from tumors that are recurrent and have previously received chemo-/radio-therapy. We found that the purified CD133+ GBM CSCs sorted from the CD133+ GBM CSC spheres express SOX2,CD44 and are capable of clonal self-renewal. We generated in-vivo model systems in mice and rats to screen potential drug agents for the treatment of GBM patients. We were capable of generating brain tumors in mice and rats within 20 days by subcutaneously injecting 1000 GBM CSCs at the hind limbs of the rodents. Within day 5 the GBM CSCs were already present in the brain's of the rodents. At days 10, 15 & 20 the GBM CSCs labeled with GFP were located in the brain. We tested various compound candidates in this model system and we were able to show decrease in the migration of the GBM CSCs in the brain when we started the treatment at day 10 compared to non-treatment groups. We were able to demonstrate all the gene signatures associated with GBM CSCs in this model system. We do see a down regulation of certain cancer promotes genes in the treatment group when compared to non-treatment groups. The drug candidates were tested for TGFβ and STAT pathways. The chemotherapeutic agents tested were Among the 300 compounds tested Adria, Taxol, DTIC, Thalidomide, Paclitaxel, Abraxane, Ertibux and Velcade. We were able to show 4 compounds that were effective against GBM CSC targeting selected pathways. 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 3306. doi:10.1158/1538-7445.AM2011-3306

Melanoma is one of the most aggressive cancers, and its incidence and mortality rate are on rise since no treatment options are available for metastatic disease. Recent evidences from in vitro and in vivo studies have demonstrated that aberrant reactivation of the Sonic Hedgehog (SHH) signaling pathway regulates genes that promote cellular proliferation in various human cancer stem cells (CSC) 36118-45. In melanoma cells, RAS-MEK and AKT signaling has also been shown to regulate the nuclear localization and transcriptional activity of Gli-1. Therefore, the agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for pancreatic cancer. Through extensive structure-activity studies in our laboratory, based on naturally occurring isothiocyanates (ITCs) and their isosteric selenium analogs, we have recently identified phenylbutyl isoselecynate (ISC-4) as a promising agent that significantly retarded melanoma tumor growth without any systemic toxicity. We demonstrate here the effect of ISC-4 on the proliferation of CSC and 36118-45CTC (circulating tumor cell) cells isolated from a melanoma patient. ISC-4 treatments resulted in a dose-dependent inhibition of SHH signaling (SMO, PTCH-1, Gli1, Gli2, and Gli3) and induced significant growth inhibition of CSC and CTC cells. The data demonstrate that Hh signaling regulates proliferation of CSC and CTC of human melanoma and treatment with agents like ISC-4 might be a novel approach to prevent growth and metastasis of human melanoma. Citation Format: Arun K. Sharma, Jitesh Jani, Cristian Sharma, Patrick Cleary, Michael Sharma, Shruthi Satish, Esteban Gomez, Michael Prez, Natalee Amezcua, Mariam Navel, Deepkamal N. Karelia, Dhimant Desai, Shantu Amin, Jay Sharma. ISC-4, a novel inhibitor of hedgehog-Gli signaling, inhibits growth of CSC and CTC of melanoma patients. [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 2712. doi:10.1158/1538-7445.AM2014-2712