Clinical Implications Of Cancer Cell Biology

Cancer Stem Cells and Therapy Resistance

Discuss About The Clinical Implications Of Cancer Cell Biology.

Pancreatic ductal adenocarcinoma (PDAC) and hepatocellular carcinoma (HCC) are the most deadly forms of cancer with the shortest expectations of life after diagnosis [1]. PDAC is the fourth leading cause of death in the western world and incidence of this cancer is increasing, probably due to the epidemic of obesity and diabetes which are predisposing factors [1]. HCC has the fifth highest incidence and third highest mortality rate of all malignancies worldwide [2]. Most cases of both cancers are inoperable and the only treatment that can be offered is chemotherapy or palliative procedures [1-3]. Targeted therapy has been of limited success. The epidermal growth factor (EGF) receptor kinase inhibitor, erlotininib in combination with gemcitabine modestly improves life expectancy in a sub-group of patients [4]. At the present time, only one drug, sorafenib, is FDA approved drug for treating advanced HCC. It blocks different receptors, particularly VEGFR and c-KIT [5].

While late diagnosis undoubtedly contributes to the lack of response of these cancers to therapy, it has become apparent that the presence of cancer stem cells (CSC), which is not easily eradicated by standard chemotherapeutics are ultimately responsible for the failure of existing strategies [6-8]. Recent understanding of the heterogeneous makeup of the cancer cells in a tumor has revealed the presence of CSCs [9-10]. CSCs can be characterized their self-renewing ability via asymmetric division, ability to differentiate into diverse phenotypes, ability to initiate tumors from minute numbers, and chemoresistance [9-10]. As expected, the discovery of CSCs in cancer development has reshaped our understanding of cancer biology and is revolutionizing the current efforts of developing new therapeutics. Recent studies have demonstrated the unique properties of CSCs in many tumor types, including breast, colon, melanoma, brain, bone, ovary, prostate as well as in PDAC [6,7], and HCC [8] By FACS analysis, Li et al isolated CD44+/CD24+/EpCAM+ (CD326) pancreatic CSCs which counted for 0.2–0.8% of total cancer cells, showed stem-cell like properties and had the capability of form tumors in animal models when as few as 100 cells [6]. Hermann et al characterized another subpopulation of pancreatic CSCs expressing the surface marker CD133 and described it to be exclusively tumorigenic and highly resistant to standard chemotherapy [Hermann et al., 2007]. Similarly, hepatic CSCs have been shown to be enriched via different cell surface markers, eg, CD13, CD24, CD44, CD90, CD133, and EpCAM (CD326). Functional assays such as screening cells with a high activity of aldehyde dehydrogenase could also be used to identify CSCs [8]. Being a viable cause of metastasis along with their high resistance to conventional chemotherapy has led to the growing belief that CSCs can be the ultimate foe in the combat against cancer. It is, therefore, becoming the common wisdom that therapies that would specifically eradicate those CSCs are being actively investigated. CSCs are known to contribute to tumor initiation, self-renewal, chemoresistance, and metastasis [10]. Indeed, they are the only cells from a tumor that are able to recapitulate the disease, indicating that their eradication is essential [10].

Targeting CSCs

Epithelial cell adhesion molecule (EpCAM) proves to be a very useful marker and target for CSCs, EpCAM is overexpressed in epithelial cell cancer stem cells, particularly in PDAC and HCC. Targeting EpCAM⁺ HCC cells has been shown to be possible using the antibodies, which inhibit the growth of pancreatic carcinomas.

Metformin is an AMP kinase inhibitor which has become the mainstay of therapy for type 2 diabetes, belongs to a class of compounds called biguanine that was first isolated from a medicinal plant known as Galega officinalis. The anticancer effects of metformin were first documented by one of the investigators on the present project, when this drug completely prevented the formation of pancreatic tumors in a hamster model of PDAC [24]. A recent epidemiological study revealed that diabetics taking metformin had a 46% and 78% decreased the risk of PDAC and HCC, respectively [Zhang et al., 2013]. Two theories have been proposed to explain metastasis: epithelial–mesenchymal transition (EMT) and CSC. Regardless which mechanism prevails, reports showing metformin-mediated transcriptional repression of EMT, a cellular phenotype associated with CSC, support the potential use of metformin in preventing metastasis [15]. Metformin has also been shown to inhibit colony and sphere formation in cancer stem cells from breast, ovary, prostate lung as well as pancreas and liver carcinomas [14-19, 26-27]. These findings have already been translated into clinical trials and there are currently at least 26 ongoing clinical trials with metformin in various cancers [15].

Other approaches to the eradication of cancer stem cells include targeting the hedgehog (Hh), Wnt-β-catenin, and notch pathways [12, 28-30].Reactivation of these developmental pathways is common in both PDAC and HCC [12, 28-30]. Recent studies have shown that both temporal and spatial control of Hh and Wnt activity is involved in specifying the lineage that can progress to cancer [31]. Activation of the Hh in PDAC and HCC has also been shown to regulate HCC invasiveness and the response to chemotherapy [28, 30-31]. Suppression of the Hh pathway by its inhibitor, vismodegib (GDC-0499), a smoothened (SMO) antagonist, not only decreased the number of progenitors but also caused regression of these tumors [32-33]. This agent recently received FDA approval having shown remarkable activity in clinical trials [30]. Vismodegib is currently undergoing clinical trials for the treatment of a variety of cancers including PDAC and HCC [34].

Another major hurdle in the treatment of solid malignancies; in particular PDAC and to a lesser extent in HCC is the stromal response to the tumor [35]. One of the hallmarks of PDAC is the presence of extensive desmoplasia consisting of stellate cells, fibroblasts, immune cells vasculature and extensive extracellular matrix [35]. The cancer cells exploit this microenvironment for support of growth, invasion and metastatic spread [35]. More importantly from the therapeutic point of view are that the stoma provides an effective barrier to prevent therapeutic agents penetrating the cancer cells [35-37]. Studies have shown that the stroma impairs vasculature and perfusion of the tumors in a mouse model of PDAC and that inhibition of hedgehog signaling that depleted the tumor stromal response markedly improved the tumor response to gemcitabine [37]. This was accompanied by a marked increase in intratumoral gemcitabine concentrations, indicating that the stroma was contributing to chemoresistance [37].

Strategies for Eradicating CSCs

Several approaches have been used to disrupt the peri-tumoral stroma in order to enhance the effect of conventional therapeutics. These include inhibitors of the hedgehog pathway, the TGFB pathway, targeting of specific matrix metalloproteinases, blocking angiogenesis, targeting the notch pathway, or enzymatically destroying the hyaluronan component of the extracellular matrix [38-41]. Of these approaches, the use of hyaluronidase appears to be the most effective method of destroying the extracellular matrix and enhancing the effects of conventional chemotherapy [39]. Since hyaluronidase has a short half-life in the circulation, a pegylated human recombinant form of the enzyme, PEGPH20 has been developed [40-41]. Administration of PEGPH20 rapidly normalizes the elevated interstitial fluid pressure surrounding tumors and markedly enhances the therapeutic effects of the standard chemotherapeutic agent, gemcitabine in a pancreatic cancer model [40]. In a separate study, PEGPH20 was shown to rapidly and sustainably deplete HA in the extratumoral matirix, induce the expansion of blood vessels and Increase the intratumoral concentrations of gemcitabine [41]. The combination of PEGPH20 with gemcitabine inhibited cancer growth and markedly prolonged survival in the animals [41]. Phase 2 clinical trials with PEGPH20 and gemcitabine are currently underway in pancreatic cancer [42].

It is clear from the failure of conventional chemotherapy to control PDAC and HCC, that novel approaches in the therapy and involvement of agents targeting several different aspects of the disease, particularly, the cancer stem cells and the peritumoral stroma are needed if we are going to make any impact on these devastating diseases. Here we plan to address this problem by targeting the CSCs using a novel nano-technological approach as well as targeting the peritumoral stroma.

The overall objective is to use a novel therapeutic approach to eradication of cancer stem cells (CSCs) by the use of targeted nanoparticles combined with another approach to eradicate the peritumoral stroma to enchanced exposure to the nanoparticle. The surface of the nanoparticles will include monoclonal antibodies to EpCAM to address them to the cancer stem cells and tomato red fluorescent protein to evaluate the effectiveness of targeting suing the IVIS animal imaging system. The payload of the particles will include metformin and the smoothened inhibitor, vismodegib, both of which will target the CSCs. Pegylated hyaluronidase, PEGPH20 will be used to destroy and prevent regrowth of the stroma. However, since the stromal response to the cancer also depends on the Hh pathway, release of vismodegib may also help reduce the stroma. This will be address by the following specific aims:

Disrupting the Peri-tumoral Stroma

Chitosan will be used as a carrier for the reagents that wil be investigated in the current study. Chitosan is selected for its known biocompatibility, biodegradability, anti-bacterial and anti-tumor activity. Chemically-functionalized chitosan (CF-chitosan) will be fabricated in the form of nanoparticles (NPs) to make use of its high surface area to include all targeting molecules. Structural stability and size monodispersity of chitosan NPs will be thoroughly studied. CF-Chitosan NPs will incorporate metformin and vismodegib to induce apoptosis in the CSCs, while its surfaces will be decorat with EpCAM monoclonal antibodies to target the CSCs and Rhodamine 123 as a fluorofore for identification.

We will optimize the nanoparticles in vitro using two pancreatic (S2013 and AsPC-1) and two hepatocellular cancer cell lines (MHCC-97H, and SK-Hep1), chosen because of their ability to form metastases in orthotopic transplant models. Using these cell lines we will investigate the effectiveness of the nanoparticles to reduce growth of the cancer cells alone and in combination with standard chemotherapeutic agents (gemcitabine for PDAC and sorafenib for HCC). Optimization will include determining the most effective drug concentrations within the nanoparticles on their ability to reduce the nubmer of cells carrying stem cell markers. We will evaluate the effectiveness of the individual components of the therapeutic approach, including the chitosan backbone, chitosan with EpCAM, metformin, vismodegib and the various combinations of these agents.

We will utilize two orthotopic transplant models of human pancreatic cancer (S2013 and AsPC-1 cells), with stable expression of firefly luciferase. This model mimics the human disease with invasion, metastases, biliary obstruction and cachexia [Hennig et al., 2005]. For hepatoceluular carcinoma MHCC-97H and SK-Hep1 cells wil be used to develop orthotopic nude mouse models where firefly luciferase will be similarly expressed. The proposed cells are known to highly metastatic HCC cell lines [44, 45]. Tumor growth and the development of metastases will be monitored in real time using the IVIS Spectrum animal visualization system. We will evaluate growth of the primary tumor, development of hepatic, lymph node and lung metastases. At the end of the experiment, we will evaluate the proportions of CSCs by immunocytochemistry and any toxicity by measurement of complete blood count and serum hepatic enzyme activities.

The proposed research plan is divided to three integrated phases, starting with the development of drugs-containing nanoparticles followed by their in vitro and in vivo evaluations of treating hepatic and pancreatic tumors. Aims of the project phases are explained in details in the following sections.

Recent advances have given rise to what is known collectively as cancer nanotechnology [46]. Multifunctional biocompatible nanoparticles (NPs) are loaded with bioactive reagents, markers, and targeting molecules and are injected intravenously into tumor-bearing animals. Compared with other types of cancer treatment modalities, targeted delivery of such molecules to the cancer site via loading them onto nanoparticles is currently believed to be the most direct approach to tackle cancer.

Various types of biocompatible NPs have been extensively studied. Their chemical structure ranges from inorganic, such as iron oxide [47] or gold [48] to organic, mostly biodegradable, such as poly(lactide-co-glycolide) [49], polycaprolactone [50], or chitosan [51]. In addition, a class of lipid-based NPs such as liposomes [52] and solid lipid NPs (SLN) [53] have been investigated. Inorganic NPs are usually coated with biocompatible organic-based layers to facilitate the immobilization of the bioactive and targeting molecules. On the other hand, biodegradable NPs are characterized by their built-in functionalities that facilitate the attachment of various types of molecules onto its surfaces. Moreover, their biodegradability can be pre-tailored to facilitate the delivery of the drugs, while assuring non-cyto toxicity by virtue of the biocompatibility of its degradation products.

Chitosan, a naturally occurring biodegradable polymer has been used as stand-alone biomaterial in the form of NPs, nanofibers and scaffolds in various tissue engineering and drug delivery applications [54-57]. Chitosan has been proven to have antimicrobial and antifungal activity [58]. Chitosan micro- and nanoparticles were evaluated as carriers of various types of drugs [59-60]. Chitosan NPs was also found to have antitumor activity against Sarcoma-180, and mouse hepatoma H22 even in the absence of a therapeutic drug payload [61]. In a recent study, chemically functionalized chitosan NPs were used as a drug carrier for Metformin for the treatment of pancreatic cancer [62]. Sustained release kinetics of Metformin was found to be pH-dependent, and the Metformin-loaded chitosan NPs were found successful in inducing a preferential toxicity on the pancreatic cancer cells [Wen et al., 2012].

Various methods are known for the preparation of chitosan NPs [63-65]. In the current study, chitosan NPs will be fabricated using an ionic gelation technique, which was previously established and fine-tuned by Fan et al in 2012. [61] This technique is characterized by the ability of fine tune the NPs size and distribution and to maintain stable suspended solutions of the NPs for a reproducible drug delivery application. In addition, the technique is also known for its convenience and the use of non-toxic reagents. It is based on the stabilization of chitosan NPS through immobilizing functional groups on its surface. Normally, chitosan is soluble in acidic media, and will therefore have positively charged amino groups on its surfaces. Polyanions such as sodium tripolyphosphate have negatively charged groups will be used for stabilization of the chitosan NPs through the electrostatic interaction between the oppositely charged groups.

Experimentally, acidic solutions of chitosan in acetic acid with a pH range of 4.7-4.8 will be pre-heated at 60^oC, then subjected to the sudden addition of the polyanion capping agent (such as sodium tripolyphopshate) under low temperature (2-4^oC) conditions. A stable chitosan NPs

suspension will be produced and will be uced for characterization and as a base for the inclusion of the bioactive ingredients to be used for treatment of hepatic and pancreas cancer.

In a parallel experiment, stable suspensions of Chitosan NPs stabilized by oxycarboxy methyl groups will be prepared by the same method mentioned above. This functionality was previously shown to support the inclusion of Metformin due to its interaction with the NH3⁺ group of Metformin [Snimaa et al., 2013]. In the current study, the inclusion of Metformin and vismodegib molecules will be attempted into chitosan NPs stabilized by sodium tripolyphosphate and oxycarboxy methyl functionalities. Inclusion of these molecules will take place in situ during the formation of the functionalized chitosan NPs. Moreover, a fluorescent dye; Rhodamine 123, will be used to label the decorated NPs and to be able to trace the drug-carrying NPs after injection and throughout the delivery process. For a directed delivery of the drug-containing NPs, a monoclonal antibody molecule (EpCAMmab) will be conjugated to the surface of the CF-chitosan NPs containing Metformin, Vismodegib and Rhodamine 123. Conjugation of EpCAMmab will take place by using a common linker; ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS). The antibody is a kind gift from Prof. Judith P. Johnson, Institute for Immunology, Munich, Germany. Concentrations of each of the bioactive molecules will be determined based on the knowledge of the optimum dose of each molecule as a result of preliminary in vitro experimentes.

Characterization of the bare, chemically functionalized and loaded chitosan NPs will take place by various techniques. Light scattering technique will be used to measure the size and size distribution of the NPs before and after chemical functionalization and after inclusion of the bioactive molecules. Scanning and transmission electron microscopies will be used to investigate the morphology of the nanoparticles and confirm the size monodispersity of the NPs. Surface area, porosity and pore size distribution of the NPs will be assessed using a N2-adsorption technique. Infrared spectroscopy, ultraviolet-spectroscopy will be used to investigate the attachment of each of the bioactive molecules into and onto the surfaces of the CF-chitosan NPs.

• Bare CF-chitosan NPs

• CF-chitosan NPs loaded with Metformin and decorated with EpCAMmab and Rhodamine 123

• CF-chitosan NPs loaded with Vismodegib and decorated with EpCAMmab and Rhodamine 123

• CF-chitosan incorporating Metformin and Vismodegib, and decorated with Rhodamine 123 and EpCAMmab

It should be mentioned that proper concentrations of each of the above mentioned reagents will be determined based on the FDA approved dosages of Metformin and Vismodegib (850 and 150 mg/day respectively). Corresponding concentrations of these reagents will be calculated based on the ultimate size and distribution of the NPs.

• Using proper capping agents, such as sodium tripolyphosphate and O-carboxymethyl molecules is expected to achieve this objective. If unstable suspensions are produced, we will switch to other known capping agents that will have the criteria of stabilizing the NPs in solution and help in the conjugation of the various molecules intended to be delivered to the cancer site.
• Formation  of  NPs  containing  the bioactive  molecules  mentioned above  is  the second

objective. Various techniques are mentioned above to be used for the confirmation of the immobilization of these molekules into and onto the surfaces of the NPs. If any of these molecules are proven not to attach as presumed, we will switch to alternative linker molecules.

• Concentrations of each of the bioactive molecules will be determined based on preliminary in vitro testing of each of the molecules on the corresponding cancer cells. Variation of the concentration of the effectively attached molecules than the calculated ones will direct us to more optimization of the immobilization reactions to achieve the target concentrations onto the NPs.

To determine the percentage of CSCs following treatment with NPs, Sorafenib or solvent control, single cells from the xenografts will be seeded in 6-well plates at a density of 30,000 cells/well and treated with sub-toxic concentrations for 72 hours. Cells will then be allowed to recover for another 72 hours in a medium without the drugs. The percentage of CD44+/CD90+ cells arising from each treatment will then be determined by flow cytometry. In addition, the percentage of CD133+ cells will be evaluated.

The functional presence of CSCs present after the above in vitro treatments will also be determined by tumor-seeding assay. Following the treatment and recovery from the exposure to the different drugs, the cells will be injected into nude mice in serial dilutions. The difference with regards to new tumor formation between NPs-treated cells, control, cells treated with Sorafenib alone or cells treated with both NPs and Sorafenib in mice will be determined.

The pancreatic cancer cell lines used in this study will be the same as in Specific Aim 1.

The hepatocellular carcinoma cell lines to be used are MHCC-97L, HCC-LM3, or SMMC7721.

Cells will be seeded at a density of 5,000 cells / well into 96-well plates. After 24 h, cells will be treated for another 24 or 48h with different concentrations of nanoparticles or their individual components in triplicate. Control cultures will be treated with vehicle alone. The effect of treatment on cell viability will be determined using a CellTiter-Glo Luminescent Cell Viability assay (Promega Corporation, Madison; US), based on quantification of ATP, which signals the presence of metabolically active cells. The luminescent signal will be measured using the GLOMAX Luminometer system. Data were presented as proportional viability (%) by comparing the treated group with the untreated cells, the viability of which is assumed to be 100%.

The cytotoxicity detection kitplus (Roche) will be used to colorimetricly and quantitatively determine cytotoxicity by measuring release of lactate dehydrogenase (LDH) activity from damaged cells. Cells will be cultured to 80% confluence. After trypsin digestion, the cells will be counted and pipette into 96-well plates at a density of 2000 cells/well. Background control (medium only), low controls (spontaneous LDH release), high controls (maximum LDH release), and experiments will be prepared on the same plate according to the manufacturer’s instructions. The 96-well plates will be incubated in a humidified incubator at 37?C in 5% CO2 for 4, 8, 12, 24, 48, and 72 h. Results will be expressed as the absorbance of each well at 492nm (OD492). Cytotoxicity (%) will be calculated using the equation: (experimental value − low control)/(high control − low control) x 100%.

Cells grown on the chambers (Lab-Tek® Chambered Cover glass System) at a density of 2x10⁴cells/well will be treated with NPs at different concentrations. For the competition/inhibition studies, a large amount of NPs will be added to the incubation medium. After incubation for 30 min, cells will be treated with NPs and continuous incubated. Cells will be washed with PBS, fixed with 4% paraformaldehyde, and stained with the hoechst for visualization of the nucleus. The samples will then be mounted by the DAKO® Fluorescent mounting medium before examination. The stained coverslips will be photographed using confocal laser scanning microscopy.

The expression level of CSC-related markers will be determined by flow cytometry. Briefly, cells will be grown to 80% confluence. After trypsin digestion, the cells will be re-suspended in

medium at a concentration of 1x10⁶ cells/mL and incubated with primary antibodies against CD90, CD133, CD24, EPCAM, and CD44 (diluted 1 : 11) at 4?C for 15 min. After multiple PBS washes, cells will be analyzed using a FACSC Flow Cytometer (Guava EasyCyte, Millipore).

Possible pitfalls and alternative approaches

None anticipated.

We hypothesize that the strategy of combining the CSC-targeted nanoparticles with PEGLH20 will substantially reduce the metastatic potential of the different cancer cell lines and also markedly reduce of eradicate the CSCs. The PEGPH20 is a kind gift from Dr. Gregory I Frost, Halozyme Therapeutics Inc., San Diego, CA, USA. The orthotopic models we have developed are good mimics of the human disease with metastatic spread, stromal reaction to the tumor, biliary obstruction and cachexia [43]. The pancreatic and hepatocellular cancer cell lines will be transfected with a lentivirus to induce stable expression of firefly luciferase. Thus, we are able to monitor tumor growth and metastasis dynamically, by the use of the IVIS Spectrum animal visualization system. By use of a separate Rhodamine 123 on the nanoparticles we will be able to monitor the time-course and effectiveness of the cancer targeting.

Two human PDAC (S2013, AsPC-1) and two HCC (MHCC-97L, HCC-LM3, or SMMC7721) cell lines will be used in this study. This cell lines provide time-courses with different therapeutic windows. For example, with S2103 cells hepatic metastases develop within one week, tumor burden is marked within one month and the animals are severely cachectic. With AsPC-1 cells the time-course is about two months.

Retroviral particles containing the luciferase gene will be obtained from Perkin Elmer. These are not currently available, but I assured that they will be available before August 2013. Transfection will be carried out following the manufacturer’s instructions. We previously established three cell lines (S2013, MiaPaCa2 and AsPC-1) with stable expression of GFP and so do not anticipate problems with this step.

For cancer xenografts 6-8 week old athymic NMRI nude mice (nu/nu, Charles River, Suizfeld, Germany) will be bred in the animal facility. The mice will be housed in micro-isolator cages in a filtered-air laminar flow cabinet (EuroBioConcept, Paris, France), handled under aseptic conditions and fed with autoclaved laboratory rodent food pellets. Each group will be comprised of 10 animals as determined by power analysis. For the pancreatic cancer cell lines, using a sterile 20 gauge needle, a suspension of 0.25x10⁶ cells in 10μl serum free media will be injected under the capsule in the pancreas adjacent to duodenum. For the hepatocellular cell lines, similar injections of 0.25x10⁶ cells in 10μl will be made into the left lobe of the liver. Imaging following transplantation will confirm that the injections form a discrete tumor without leakage from the injection site. The length of the experiment will depend on the cell line. For example, xenografts of S2013 cells grow and metastasize rapidly and end stage disease is reached within 4-5 weeks. For AsPC-1 cells the course is slower and end-stage disease is reached in 8-9 weeks. Suspensions of chitosan NPs loaded with 4 mg Metformin and 2 mg Vismodegib will be administered on daily basis. PEGPH20 will be administered at a dose of 4.5 mg/kg i.p. every 3^rd day. Gemcitabine will be administered to the pancreatic cancer animals at a dose of 20 mg/kg i.p. on days 3, 6, 9, 12 and 15, following tumor implantation. Sorafenib will be administered daily at a dose of 30 mg/kg i.p.

The Xenogen IVIS Lumina II animal visualization system will be used to monitor cancer growth and development of metastases. Tumor bearing mice will be monitored every 4^th day. Mice will be isoflurane anesthetized and luciferin (XenoLight Rediject D-Luciferin Ultra, Perkin Elmer)

adminisered i.p. Animals will be imaged three at a time. At the end of the experiment, the animals will be euthanized and pancreas, liver, mesentery and lungs harvested and fixed for histology. Sections will also be observed directly to detect micro metastases. Immunocytochemistry will be used to identify the proportion of cancer cells expressing CSC markers. Degree of expression will be confirmed by real-time RT-PCR in tumor tissue. Apoptosis in the tumor tissue will be assessed by the in situ TUNEL assay. The percentage of TUNEL-positive apoptotic cells will be counted at 400X magnification. In each section, 2000 cells will be evaluated. Blood samples will also be collected at autopsy for complete blood count and measurement of liver enzyme activities (ALT, AST, and LDH) to assess toxicity.

The proportion of surviving CSCs will be characterized in the cell cultures by immunophenotypic characterization. The cells will be trypsinized and the cellular suspension centrifuged (300g, 4 min). CSCs will be stained with antibodies conjugated with fluorescein isothiocyanate (FITC), allophycocyanin (APC) or phycoerythrin (PE): CD24-APC, CD44-PE, CD133-FITC (Abcam). A total of 5x10⁵ cells will be re-suspended in 0.2 ml PBS and incubated with FITC-, APC- or PE-conjugated antibodies for 20 minutes at room temperature and protected from light. The samples will then be analyzed by flow cytometry (Guava EasyCyte, Millipore) for identification of specific fluorescence channels of each antibody.

We anticipate that all of the cell lines proposed will reliably form pancreatic tumors in the nude mice based on the previous experience with this model. If we have any problems with development of metastases with any of the cell lines then we will switch to alternative lines.

We will investigate the effects of the various drugs on the growth of the pancreatic and hepatocellular cancer cells in culture in vitro and in vivo, with particular emphasis on the proportion of remaining cells with cancer stem cell markers.

Triplicate samples will be obtained for all cell growth studies in Aims 1 and 3. Experiments will be repeated at least four times. Results from the combined experiments will be expressed in graphic form with mean ± std. ANOVA will be used to calculate statistical difference between different groups, using the Dunnett or Bonferroni post-hoc tests, as appropriate. For the animal experiments, analysis will focus on outcomes such as size of the primary tumor and number of metastases in lymph nodes and liver, comparing results observed in two groups of animal, treated

• and controls (C). Each group will consist of 10 animals. It is expected that size of the primary tumor will be reduced by 50% in treated animals relative to the control group, and that the difference will increase gradually with time. Thus if index i denotes time points, i=1,2,3,..k; we expect Average (Ci-Ti) to increase in i. In addition, at the end of the study we expect Average [(Ck-Tk)/Ck] (average relative difference in tumor size) to be about 50% or greater. Comparisons will be made using nonparametric equivalent of two sample t-test, namely Wilcoxon Mann Whitney test. Similarly, the comparison in the number of metastases between T and C groups (separately for lymph nodes and separately for liver) will be made using exact multinomial (binomial) methods such as Fisher exact test.

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