WO2013155114A1 - Scaffold and method for proliferation and enrichment of cancer stem cells - Google Patents

Abstract

Methods for culturing cancer cells in vitro using a three-dimensional scaffold to provide cultured cancer cells having a population of cancer cells enriched with cancer stem cells, scaffolds that include the cultured cancer cells, and methods for using the cultured cancer cells and the scaffolds that include the cultured cancer cells in anticancer therapeutic drug development.

Description

SCAFFOLD AND METHOD FOR PROLIFERATION AND ENRICHMENT OF

CANCER STEM CELLS

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Patent Application No. 61/621,683, filed April 9, 2012, expressly incorporated herein by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is 40866_SEQ_FINAL_2013-03- 27.txt. The text file is 8 KB; was created on March 27, 2013; and is being submitted via EFS-Web with the filing of the specification.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with Government support under 1T32CA138312-01, RO1EB006043, and R01CA134213, each awarded by the National Institutes of Health (NIH). The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Cancer stem cells (CSCs) constitute a minority subpopulation of tumor cells characterized by the capacity of self-renewal, unlimited proliferation, and giving rise to tumor cells with a more differentiated phenotype. Compared to the majority of cells in a tumor, CSCs are more malignant as evidenced by greater invasiveness, metastatic potential, and resistance to standard therapeutic interventions. These findings indicate that CSCs are primary determinants of tumor clinical behavior, making them attractive targets for novel, more efficacious treatments. According to the current CSC, or hierarchical, model of cancer biology, drugs that can eradicate the CSC population in a tumor would likely be a highly effective therapy. Tumors with all CSCs removed would become a benign mass of cells. However, development of drugs specific for CSCs is hindered by the difficulty in isolating and propagating these cells in vitro since they represent such a small proportion of the total cells in tumor tissue and in monolayer cultures of tumor cell lines.

The most common method for isolating CSCs utilizes fluorescence-activated (FACS) or magnetic-activated (MACS) cell sorting of cells bound with antibodies specific for CSC surface markers (e.g., CD133, prominin-1). This approach requires costly antibody, dedicated equipment, and yields low numbers of viable cells. CSCs can also be isolated and propagated in vitro using serum-free, defined media as suspension cultures of tumorspheres. However, tumorsphere growth is slow, requires large volumes of expensive specialized media and is frequently not successful due to the small percentage of CSC cells in the tumor of origin. A major limitation of suspension cultures is the absence of a three dimensional (3D) environment required for cell-extracellular matrix interactions that facilitate proliferation and promote malignancy. Assays employing soft agar or agar microbeads have been used to grow isolated CSCs, but collection and subsequent analysis of cells is impaired by the high density and small pore sizes typical of polymerized agar.

Human glioblastoma (GBM) and hepatocellular carcinoma cell lines cultured on 3D CA scaffolds develop a more malignant phenotype, evidenced, in part, by increased tumorigenicity in nude mice, than those cultured as monolayers. The greater malignant potential of scaffold-grown cells has been attributed to the composition, microstructure, and mechanical properties of the CA matrix that mimic in vivo tumor niches. This conclusion is in accord with observations that the tumor microenvironment has a significant effect on the maintenance and self-renewal of CSCs and with the report that CA scaffolds support the proliferation of human embryonic stem cells.

A need exists for a simple, low cost method for readily producing CSCs. The present invention seeks to fulfill this need and provides further related advantages.

SUMMARY OF THE INVENTION

The present invention provides methods for culturing cancer cells in vitro using a three-dimensional scaffold, scaffolds that include cultured cancer cells, and methods for using the cultured cancer cells and the scaffolds that include cultured cancer cells in anticancer therapeutic drug development.

In one aspect, the invention provides a method for three-dimensional cell culture in vitro.

In one embodiment, the method includes seeding a porous chitos an- alginate scaffold with cancer cells to provide a scaffold comprising cancer cells; and then culturing the cancer cells in the scaffold for a time sufficient to provide a scaffold comprising cultured cancer cells. The cultured cancer cells produced by the method of the invention are a population of cancer cells enriched in cancer stem cells. In another embodiment, the invention provides a method for inducing the expression of epithelial-to-mesenchymal transition genes in cancer cells. In the method, a porous three-dimensional chitosan-alginate scaffold is seeded with cancer cells to provide a scaffold comprising cancer cells; and the cancer cells in the scaffold are cultured to provide cancer cells expressing epithelial-to-mesenchymal transition genes.

In a further embodiment, the invention provides a method for inducing the expression of cancer stem cell surface markers in cancer cells. In the method, a porous three-dimensional chitosan-alginate scaffold is seeded with cancer cells to provide a scaffold comprising cancer cells; and the cancer cells in the scaffold are cultured to provide cancer cells expressing a cancer stem cell surface marker.

In yet another embodiment, the invention provides a method for inducing the surface expression of CD133 on cancer cells. In the method, a porous three-dimensional chitosan-alginate scaffold is seeded with cancer cells to provide a scaffold comprising cancer cells; and the cancer cells in the scaffold are cultured to provide cancer cells expressing CD 133.

In the methods of the invention, the chitosan-alginate scaffold comprises a chitosan, an alginate, and a divalent metal cations, wherein the chitosan is ionically linked to the alginate.

In another aspect of the invention, a three-dimensional scaffold is provided. In one embodiment, the scaffold comprises a porous chitosan-alginate scaffold; and cultured cancer stem cells.

In a further aspect, the invention provides a method for producing a cancerous tumor in a subject. In the method, cultured cancer cells produced by a method of the invention are implanted in a subject. Suitable subjects include animals for cancer model studies. In one embodiment, implanting cultured cells comprises implanting the scaffold comprising cultured cancer cells.

In another aspect of the invention, a method for screening a candidate chemotherapeutic agent in vitro is provided. In the method, cultured cells obtained from the method of the invention are contacted with a candidate chemotherapeutic agent. In one embodiment, contacting cultured cells obtained from the method of the invention comprises contacting the candidate chemotherapeutic agent with the scaffold of the invention comprising cultured cancer cells. In certain embodiments, the method further comprises measuring cell proliferation inhibition, measuring the cell viability, and/or measuring protein expression levels.

In further aspect of the invention, a method for screening a candidate chemotherapeutic agent in vivo is provided. In the method, cultured cells obtained from the method of the invention are implanted in a subject and a candidate chemotherapeutic agent is administered to the subject. In one embodiment, implanting cultured cells obtained from the method of the invention comprises implanting the scaffold of the invention comprising cultured cancer cells. In the method, administering the candidate chemotherapeutic drug comprises administering the drug after a pre-determined period of time. In certain embodiments, the method further comprises comparing the tumor mass or volume measured prior to drug administration and after a pre-determined period of time after drug administration and/or harvesting the tumor mass after a pre-determined period of time after drug administration and analyzing the tumor.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.

FIGURE 1 compares SEM images of human U-118 MG GBM cells cultured on 3D CA scaffolds (CA) and 2D monolayers (2D). Scale bar corresponds to 10 μιη. Cells were seeded directly in 6-well plates containing 22 x 22 mm cover slips (2D) or 12-well plates containing CA scaffolds (CA) at 50,000 cells per sample in fully supplemented culture media. After 5, 10, and 15 days of culture, cells were fixed, dehydrated, and super-critically dried for SEM imaging.

FIGURE 2 compares the change in fraction of CD133⁺ cells in U-118 MG cell population grown for 15 days on CA scaffolds (CA) and monolayers (2D). Immunopositivity for CD 133 was determined by flow cytometry.

FIGURE 3 compares CD133 mRNA expression determined by real time PCR in U-87 MG GBM cells grown for 10 days on CA scaffolds (CA) and monolayer cultures (2D). CD 133 mRNA content was normalized to the monolayer condition.

FIGURE 4 shows immuno staining for CD133 and SEM imaging of CA-scaffold cultured U-118 MG GBM cells at day 5 (counter-staining of nuclei with DAPI): solitary U-118 MG cells generally showed no CD133 staining. The boxed regions in the top-row images correspond to the areas of the bottom images. Scale bars correspond to 50 μιη for the upper row and 25 μιη for the lower row.

FIGURE 5 shows immuno staining for CD 133 and SEM imaging of CA-scaffold cultured U-118 MG GBM cells at day 5 (counter-staining of nuclei with DAPI): small clusters of U-118 MG cells showed faint CD133 staining. The boxed regions in the top- row images correspond to the areas of the bottom images. Scale bars correspond to 50 μιη for the upper row and 25 μιη for the lower row.

FIGURE 6 shows immuno staining for CD 133 and SEM imaging of CA-scaffold cultured U-118 MG GBM cells at day 10 (counter- staining of nuclei with DAPI): intensity of CD133 staining increases as clusters of U-118 MG cells grow larger. The boxed regions in the top-row images correspond to the areas of the bottom images. Scale bars correspond to 50 μιη for the upper row and 25 μιη for the lower row.

FIGURE 7 shows immuno staining for CD133 and SEM imaging of CA-scaffold cultured U-118 MG GBM cells at day 15 (counter- staining of nuclei with DAPI): intensity of CD133 staining continues to increase as clusters of U-118 MG cells continue to grow. The boxed regions in the top-row images correspond to the areas of the bottom images. Scale bars correspond to 50 μιη for the upper row and 25 μιη for the lower row.

FIGURE 8 compares the fold-expression of the neural progenitor intermediate filament nestin and the level of mRNA for GFAP for CA scaffold- grown U-118 MG GBM cells. mRNA content was determined by real time PCR. Relative to 2D monolayer cultures (2D), cells grown on CA scaffolds (CA) show elevated expression of the neural progenitor intermediate filament nestin while the level of mRNA for GFAP, the intermediate filament of mature glia, is unchanged.

FIGURE 9 compares the fold-expression of mRNA of genes associated with normal neural cell development (Frizzled 4, GLI, HES, Snail, Notch) and the genesis of GBM in cells grown on CA scaffolds (CA) and cells grown as monolayers (2D). Immunofluorescence images in inset shows that enhanced protein expression accompanied elevation of mRNA level for Frizzled-4. Scale bars are 10 μιη.

FIGURE 10 compares flank tumor volume over time in nude mice injected subcutaneously with 50,000 cells harvested from CA scaffolds (CA) or monolayers (2D).

FIGURE 11 compares immunopositivity for CD 133 for tumors grown from monolayer cells (2D) and tumors grown from CA scaffold-grown cells (CA). Darkness reflects increased staining (immunopositivity). FIGURE 12A compares images of flank tumors in nude mice injected with 500 CD133⁺ or CD133^" U-118 MG glioblastoma cells sorted by FACS after culture in CA scaffolds. The tumors (circles) were evident only in mice injected with CD133⁺ cells.

FIGURE 12B compares tumorigenicity in nude mice injected subcutaneously with either 500 or 2,000 FACS-isolated CD133⁺ or CD 133^" cells grown on CA scaffolds for 10 days. Tumors grew only in all mice implanted with CD133⁺ cells.

FIGURE 13 compares SEM images of cells cultured in 2D plates (2D), commercially available polycaprolactone (PCL scaffold) and polystyrene (PS scaffold) scaffolds, CA scaffolds (CA Scaffold), and PCL coated CA scaffolds (PCL coated CA Scaffold). Scale bars: 25 μηι at 500x and 5 μηι at 2000x.

FIGURE 14 compares the fraction of CD133⁺ cells determined by flow cytometry after growth for 10 days on 2D plates (2D), PCL, PS, CA, and PCL coated CA (PCL/CA) scaffolds.

FIGURE 15 compares growth curves for CD133⁺ cells on CA scaffolds (CA) and in monolayer cultures (2D). CD133⁺ cell number was assessed by the Alamar blue and flow cytometry.

FIGURE 16 is a schematic illustration of a mechanism for CSC enrichment in CA scaffolds.

FIGURE 17 compares the fold-expression change in mRNA content for EMT related genes (CD44, SNAIl, SNAI2, Twist2) and CD133 during growth on CA scaffolds for 6 days.

FIGURE 18 compares the fold-expression of N-cadherin and E-cadherin mRNA in scaffold- grown cells at days 5 and 10 determined by real time PCR.

FIGURE 19 are SEM images comparing the morphology of SK-Hep-1 (liver), MDA-MB-231 (breast), and TRAMP-C2 (prostate) cancer cells grown as monolayers (2D) or on CA scaffolds (CA Scaffold).

FIGURE 20 compares flow cytometry histograms illustrating the increase in immunopositivity for CD 133 of SK-Hep-1 (liver), MDA-MB-231 (breast), and TRAMP- C2 (prostate) cancer cells grown on CA scaffolds (Day 0, Day 5, Day 10 and Day 15).

FIGURE 21 compares expression of mRNA for the stem cell marker CD133 in

SK-Hep-1 (liver), MDA-MB-231 (breast), and Tramp-C2 (prostate) cancer cells grown for 10 days on CA scaffolds (CA) and corresponding cells grown as monolayers (2D). FIGURE 22 compares expression of mRNA for the stem cell marker NANOG in SK-Hep-1 (liver), MDA-MB-231 (breast), and Tramp-C2 (prostate) cancer cells grown for 10 days on CA scaffolds (CA) and corresponding cells grown as monolayers (2D).

FIGURE 23A compares the growth of CD133⁺ SK-Hep-1 (liver) cancer cells as monolayers (2D) or on CA scaffolds (CA).

FIGURE 23B compares the growth of CD133⁺ MDA-MB-231 (breast) cancer cells as monolayers (2D) or on CA scaffolds (CA).

FIGURE 23C compares the growth of CD133⁺ TRAMP-C2 (prostate) cancer cells as monolayers (2D) or on CA scaffolds (CA).

FIGURE 24 compares fold-increase in CD133⁺ U-118 MG, U-87 MG (GBM),

SK-Hep-1 (liver cancer), MDA-MB-231 (breast cancer) and Tramp-C2 (prostate cancer) cells grown as monolayers (2D) or on CA scaffolds (CA) as a function of time (Day 5, Day 10, Day 15).

FIGURE 25 summarizes cell proliferation on CA scaffolds (U-118 MG, U-87 MG, SK-Hep-1, MDA-MB-231, Tramp-C2 cancer cells).

FIGURE 26 summarizes primers used for qPCR.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for culturing cancer cells in vitro using a three-dimensional scaffold, scaffolds that include cultured cancer cells, and methods for using the cultured cancer cells and the scaffolds that include cultured cancer cells in anticancer therapeutic drug development. The cultured cancer cells produced by the methods of the invention are a population of cancer cells enriched in cancer stem ells.

As noted above, tumor cells cultured on standard two dimension (2D) tissue culture flasks are exposed to a dramatically altered structural microenvironment as compared to in vivo tumors, and thus display altered cell function and response to drug treatment. The methods of the invention provide cultured cancer cells in which the population of cancer cells enriched in cancer stem cells.

As used herein, the term "a population of cancer cells enriched in cancer stem cells" refers to a population of cancer cells in which the number of cancer stem cells in the total population is greater than in a comparable population cultured by 2D culture methods.

Cancer stems cells are characterized by exhibiting phenotypic characteristics of cancer stem cells, by expressing characteristic cancer stem cell markers, by expressing genes that mediate epithelial-to-mesenchymal transition (EMT), and/or by production of proteins characteristic of cancer stem cells. As used herein, the term "cancer stem cells" refers to cancer stem cells and cancer cells having the characteristics of cancer stem cells.

The present invention provides an in vitro model that can more closely mimic the structure of the tumors comprising cancer stem cells and therefore can dramatically improve the translation of novel chemo therapeutics from in vitro to in vivo testing.

In one aspect, the invention provides a method for three-dimensional cell culture in vitro. In one embodiment, the method includes seeding a porous chitosan-alginate scaffold with cancer cells to provide a scaffold comprising cancer cells; and then culturing the cancer cells in the scaffold for a time sufficient to provide a scaffold comprising cultured cancer cells. The cultured cancer cells produced by the method of the invention are a population of cancer cells enriched in cancer stem cells. In this embodiment, the invention provides a method for proliferating and enriching a population of cancer cells with cancer stem cells. FIGURES 10-12B show CA scaffold-grown U- 118 MG GBM cells exhibiting phenotypic characteristics of CSCs.

In another embodiment, the invention provides a method for inducing the expression of epithelial-to-mesenchymal transition genes in cancer cells. In the method, a porous three-dimensional chitosan-alginate scaffold is seeded with cancer cells to provide a scaffold comprising cancer cells; and the cancer cells in the scaffold are cultured to provide cancer cells expressing epithelial-to-mesenchymal transition genes. Representative expressed EMT genes include N-cadherin, Twist, Notch, and Snail genes. FIGURES 15-18 show that proliferation of CD133⁺ U-87 MG CSC GBM cells on CA scaffolds reflects expression of genes that mediate EMT.

In a further embodiment, the invention provides a method for inducing the expression of cancer stem cell surface markers in cancer cells. In the method, a porous three-dimensional chitosan-alginate scaffold is seeded with cancer cells to provide a scaffold comprising cancer cells; and the cancer cells in the scaffold are cultured to provide cancer cells expressing a cancer stem cell surface marker. Representative surface markers include CD133, GFAP, CD44, SSEA1, 01ig2, and L1CAM. Expressed marker genes are selected from STAT3, β-catenin, and frizzled 4 for the Wnt pathway, GLI and Snail for the hedgehog pathway, Notch and HES for the Notch pathway, and BMPR2 for the BMP pathway. FIGURES 8 and 9 show that CA scaffold-grown U-118 MG GBM cells express characteristic neural stem cell markers. In yet another embodiment, the invention provides a method for inducing the surface expression of CD133 on cancer cells. In the method, a porous three-dimensional chitosan-alginate scaffold is seeded with cancer cells to provide a scaffold comprising cancer cells; and the cancer cells in the scaffold are cultured to provide cancer cells expressing CD 133. FIGURES 1-7 illustrates growth of CD133⁺ GBM cells on CA scaffolds. FIGURES 13 and 14 show that proliferation of CD133⁺ U-118 MG CSC GBM cells is promoted by the physicochemical environment of the CA scaffolds.

In the methods of the invention, culture of cancer cells in the scaffolds does not require any conditions beyond standard tissue culture conditions.

Chitosan-Alginate Scaffold. The scaffolds useful in the compositions and methods of the invention advantageously support cancer cell proliferation. These scaffolds are porous scaffolds that include a chitosan and an alginate. In these scaffolds, the chitosan is ionically linked to the alginate. In certain embodiments, the scaffolds are further crosslinked by divalent metal atoms. The porous scaffolds useful in the compositions and methods of the invention that include chitosan and alginate are referred to herein as "chitosan-alginate" scaffolds or "CA" scaffolds.

Chitosan and alginate are biocompatible, non-mammalian sourced natural polymers with properties ideal for cell culture scaffold formation. The chitosan and alginate can be used to create a 3D interconnected, CA complex porous structure.

Chitosans, natural polysaccharides derived from the partial deacetylation of chitin, shares structural similarities to glycosaminoglycans present in the native ECM. Chitosans are linear polysaccharides composed of randomly distributed P-(l-4)-linked D- glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitosans useful for making the scaffolds have an average molecular weight from about 5 kDa to about 1000 kDa. Generally, scaffolds made from higher molecular weight chitosans have greater mechanical strength than scaffolds made from lower molecular weight chitosans. An exemplary range of percentage deacetylation of chitosan useful for making the scaffolds is from about 80% to about 100% deacetylation. Alginates are a family of polyanionic copolymers derived from brown sea algae. Alginates are linear, 1,4-linked polysaccharides of β-D-mannuronic acid and cc-L-guluronic acid. In these scaffolds, chitosan is ionically linked to alginate. Alginate useful for making the scaffolds have an average molecular weight from about 5 kDa to about 1000 kDa. As used herein, the term "ionically linked" refers to a non-covalent chemical bond or associative interaction between two ions having opposite charges (e.g., electrostatic association between a chitosan amine group and an alginate carboxylic acid group present on alginate).

The scaffolds comprising chitosan and alginate may be crosslinked to increase their mechanical strength. In one embodiment, the porous chitosan/alginate scaffold is crosslinked with divalent metal ions. Thus, in one embodiment, in addition to the ionic linkages between chitosan and alginate, the scaffolds include ionic linkages formed between alginate carboxylic acid groups and divalent metal ions (e.g., Ca²⁺, Ba²⁺, Mg²⁺, Sr²⁺). While not wishing to be bound by theory, it is believed that the divalent metal cations form ionic linkages between adjacent alginate chains, thereby ionically crosslinking adjacent alginate molecules. In one embodiment, the divalent metal ions are Ca²⁺ ions.

In one embodiment, the scaffold further comprises one or more growth factors or inhibitory factors effective for cancer cell proliferation and cancerous tumor formations.

Suitable scaffolds have a porosity of from about 85 to about 96 percent. In one embodiment, the scaffold has a porosity of from about 91 to about 95 percent. In another embodiment, the scaffold has a porosity of from about 94 to about 96 percent.

Suitable scaffolds have an average pore size diameter of from about 50 to about 200 μιη. In one embodiment, the scaffold has an average pore size diameter of from about 40 to about 90 μιη. In another embodiment, the scaffold has an average pore size diameter of from about 60 to about 150 μιη. In one embodiment, the scaffold has a porosity of from about 85 to about 96 percent and an average pore size diameter of from about 50 to about 200 μιη.

The porous scaffold possesses mechanical strength. In one embodiment, the scaffold has a compressive yield strength of from about 0.15 MPa to about 0.5 MPa. The scaffold has a compressive modulus of from about 0.5 MPa to 10 MPa. In one embodiment, the scaffold has a compressive yield strength of from about 0.15 MPa to about 0.5 MPa and a compressive modulus of from about 0.5 MPa to 10 MPa.

In one embodiment, the scaffold has a porosity of from about 85 to about 96 percent, an average pore size diameter of from about 50 to about 200 μιη, a compressive yield strength of from about 0.35 MPa to about 0.5 MPa, and a compressive modulus of from about 0.5 MPa to 10 MPa. In one embodiment, the scaffold useful in the invention is a porous structure comprising a chitosan, an alginate, and divalent metal cations, wherein the chitosan is ionically linked to the alginate; and wherein the alginate is further crosslinked with divalent metal cations. In one embodiment, the ratio of the chitosan to the alginate is from 1: 1 to 4: 1.

The preparation of a representative chitosan/alginate scaffold useful in the methods of the invention is described in Example 2. Chitosan/alginate scaffolds useful in the methods of the invention are also described in U.S. Patent No. 7,736,669, expressly incorporated herein by reference in its entirety.

Methods for Producing Cancerous Tumors

In another aspect, the invention provides a method for producing a cancerous tumor in a subject. In the method, cultured cells (i.e., a population of cancer cells enriched in cancer stem cells) obtained from the methods of the invention are implanted in a subject. Representative subjects include animals such as mice, rats, and dogs. Cultured cancer cells (i.e., a population of cancer cells enriched in cancer stem cells) can be separated from the scaffold and implanted or the scaffolds comprising cancer cells can be implanted directed. In one embodiment, implanting cultured cells obtained from the method of the invention, comprises implanting a scaffold comprising cultured cancer cells. Implant of cultured cancer cells (i.e., a population of cancer cells enriched in cancer stem cells) can be done between 1-45 days (or even longer if cells are still growing) of culture on the scaffolds. Time depends on the cell line and how it responds to culture in the scaffold. Typically, cells are implanted after 10 days of culture.

Methods for Candidate Therapeutic Drug Screening

In further aspect of the invention, methods for screening candidate anticancer therapeutic drugs are provided.

In one embodiment, the invention provides a method for screening a candidate chemo therapeutic drug in vitro. In a representative method, a candidate chemotherapeutic drug is contacted with cultured cells obtained from the methods of the invention. In one embodiment of this method, contacting cultured cells with a candidate chemotherapeutic drug comprises contacting the candidate chemotherapeutic agent with the scaffold comprising cultured cancer cells. In vitro drug screening can be conducted between 3-45 days (or even longer if cells are still growing) of culture on the scaffolds. Typically, cells are cultured for 10 days before in vitro drug screening. To determine drug efficacy, in one embodiment, the method further comprises measuring cell proliferation inhibition; in another embodiment, the method further comprises measuring the cell viability; and in a further embodiment, the method further comprises measuring protein expression levels.

In another embodiment, the invention provides a method for screening a candidate chemo therapeutic agent in vivo. In a representative method, cultured cells obtained from the methods of the invention are implanted in a subject. After implantation, a candidate chemotherapeutic agent is administered to the subject. In one embodiment of this method, implanting cultured cells comprises implanting the scaffold comprising the cultured cancer cells. Drug candidates can be administered before tumor implant (tumor vaccine type studies), within 1-2 weeks of implant (growth inhibition studies), or once the tumor has reached a certain size, typically 100 mm after 2-8 weeks (cell kill and growth inhibition studies). In one embodiment, administering the candidate chemotherapeutic drug comprises administering the drug after a pre-determined period of time.

To determine drug efficacy, in one embodiment, the method further comprises comparing the tumor mass or volume measured prior to drug administration and after a pre-determined period of time after drug administration. In another embodiment, the method further comprising harvesting the tumor mass after a pre-determined period of time after drug administration and analyzing the tumor.

In Vitro Cancerous Tumor Model

In another aspect, the invention provides an in vitro cancerous tumor model. The model includes cultured cancer cells in a three-dimensional (3D) scaffold comprising chitosan and alginate. As noted above, the cultured cancer cells are a population of cancer cells enriched in cancer stem cells produced by the methods of the invention.

In a related aspect, scaffolds comprising cultured cells are provided. In one embodiment, the invention provides a three-dimensional scaffold comprising a porous chitosan-alginate scaffold and cultured cancer cells (i.e., a population of cancer cells enriched in cancer stem cells) produced by the method of the invention. The following is a description of a representative method of the invention for producing CSCs.

CA scaffold culture enriches CD133⁺ cells. Human U-118 MG GBM cells grown on CA scaffolds display pronounced differences in morphology and expression of CD133, a marker of GBM CSCs, compared to cells grown as monolayers. Scanning electron microscopy (SEM) revealed that growth on scaffolds produced aggregations of spherical- or ovoid-shaped cells (tumor spheroids) while growth in monolayer yielded sheets of flat, epithelioid cells with numerous, extended processes (FIGURE 1). Similar differences in cellular morphology were also observed for the human GBM line U-87 MG as well as for human liver and breast and mouse prostate cancer cells (FIGURE 19). As illustrated in FIGURES 2 and 25, growth on CA scaffolds for 15 days was accompanied by a 1226-fold increase in the population of CD133 immunopositive U-118 MG cells and an increase in fraction of CD133⁺ cells in GBM cell population from 1.5% to 62.7%. In contrast, the fraction of CD133⁺ cells in monolayer cultures remained unchanged (about 1%) in this interval. Notably, CD133 mRNA abundance in U-87 MG grown in CA scaffolds was 12-fold higher than grown in monolayers (FIGURE 3), suggesting that scaffolds stimulate de novo CD133 gene expression rather than the acquisition of a spherical morphology exposing occult CD 133 protein on the cell surface. Comparable differences in immunopositivity and mRNA expression of CD133, also a marker of CSCs in liver, breast, and prostate cancers, were also observed between scaffold and monolayer grown liver, breast, and prostate cancer cell lines (FIGURES 20 and 23A-23C), indicative of the preferential growth of CSCs being broadly applicable to various cancer cell types. Moreover, scaffold-grown liver, breast, and prostate cancer cell lines displayed greater abundance of mRNA for NANOG (FIGURE 22), a transcription factor essential for self-renewal expressed in undifferentiated embryonic stem cells and CSCs. Finally, examination of U-118 MG CD133 immunopositivity in fixed, paraffin- embedded sections of scaffolds revealed that solitary cells expressed no detectable CD133 protein (FIGURE 4), while cells that formed clusters showed apparent CD133 immuno staining, and the intensity of the immuno staining increased with the size of the tumor cell clusters (FIGURES 5-7). These results suggest that CD 133^" cells were unable to grow or grow very slowly whereas CD133⁺ cells preferentially grew into large clusters in the CA scaffolds, resulting in CSC enrichment.

CA scaffold cultured cells display the characteristics of CSCs. Considerable evidence indicates that transformation of normal neural stem cells underlies the genesis of GBM and is accompanied by the aberrant expression of genes that promote the normal development of neural cells. The expression of mRNA was compared for a panel of genes found in normal neural progenitor cells in scaffold and monolayer cultures of U- 118 MG. Ten days after cell seeding, cells harvested from scaffolds showed a 3.5-fold higher abundance of mRNA for nestin, a cytoskeletal protein specific to neural progenitor cells, compared to monolayer cells (FIGURE 8). In contrast, scaffold-grown cells showed no increase in mRNA expression for GFAP, a cytoskeletal protein that replaces nestin as neural progenitors differentiate into mature glial cells. As shown in FIGURE 9, growth on scaffolds was accompanied by elevated expression of mRNA for other neural development genes that have been implicated in the genesis of GBM, including Frizzled 4 of the WNT signaling pathway, GLI and Snail of the hedgehog pathway, and HES of the Notch pathway. These data indicate that growth of U-118 MG on scaffolds is accompanied by elevated expression of a host of genes characteristic of undifferentiated GBM CSCs. Similarly, in liver, breast, and prostate cancer cells the expression of the stem cell marker, CD 133 and NANOG, was significantly increased after culture in CA scaffolds for 10 days (FIGURE 22) indicating the broad applicability of the CA scaffolds for enriching CSCs in cancer cell populations.

CSCs are characterized by their ability to readily form tumors in nude mice. As shown in FIGURE 10, subcutaneous flank tumors were detectable earlier and grew to a larger size in animals inoculated with U-118 MG cells grown on scaffolds compared to those grown as monolayers. The tumors from scaffold- grown cells also expressed a higher level of CD 133 (FIGURE 11), indicating that the enhanced tumorigenicity of cells from scaffolds reflected expression and maintenance of the CSC phenotype. Notably, tumors were readily formed in all animals 9 weeks after receiving injection of 500 or 2,000 CD133⁺ cells harvested from scaffolds while no tumors formed in animals injected with CD133^" cells harvested from scaffolds (FIGURES 12A and 12B). This stringent test of tumorigenicity provides additional evidence that scaffold- grown CD133⁺ cells possess the hallmark properties of CSCs.

CSC enrichment is unique to CA scaffolds. To assess if the enrichment of GBM CSCs is a consequence of growth in any 3D structure, morphology and CD 133 immunopositivity was compared for the human glioma line U-87 MG cultured for 10 days on commercially available polycaprolactone (PCL) and polystyrene (PS) scaffolds with cells grown on CA scaffolds and as monolayers. As shown in FIGURE 13, the morphology of U-87 MG cells grown on the commercial scaffolds differed little from that of monolayer cells, in contrast to the clusters of spherical or ovoid cells on the CA scaffolds. Additionally, cells cultured on CA scaffolds coated with a thin layer of PCL did not develop the clusters of spherical cells inherent to CA scaffold culture. These data suggest that it was not simply the 3D structure of the scaffolds that promoted formation of the tumor spheroids; rather, it was likely a combination of the 3D and chemical structure of the scaffolds. Moreover, near 20% of cells on the CA scaffold were immunopositive for CD 133 after 10 days of culture compared to only 1-2% of CD133⁺ cells grown on the other substrates (FIGURE 14).

The greater proliferation of U-87 MG CSCs on CA scaffolds compared to monolayer culture is illustrated in FIGURE 15. After a delay of 5 days, U-87 MG CSCs grew rapidly on CA scaffolds through day 15. Comparable proliferation profiles were also observed for CSCs from liver, breast and prostate cancer cell lines (FIGURE 23 A- 23C). By 15 days after cell seeding the CSC fraction increased from 0.3% to 42% (FIGURE 25); the total number of U-87 MG CD133⁺ CSCs on CA scaffolds increased 2188-fold while those grown as monolayers showed little increase (FIGURE 24). Similar trends were observed for U-l 18 MG and for the liver, breast and prostate cancer cell lines (FIGURES 24 and 25). These results indicate that the proliferation and enrichment of CD133⁺ cells on the CA scaffold reflects the chemistry of the substrate as well as its geometry and that culture on CA scaffolds is a facile method of producing large numbers of CSC for subsequent study.

CA scaffold-induced EMT mediates CSC enrichment. The delay that precedes the rapid proliferation of CD133⁺ CSCs observed above suggests that changes in gene expression are necessary for cancer cells to proliferate on CA scaffolds. Emerging evidence indicates that the pathway that mediates the epithelial-to-mesenchymal transition (EMT) in cancer can also promote reversion of non-CSC tumor cells to CSCs. As illustrated in FIGURE 16, EMT is mediated by signaling cascades induced by the interaction of the transmembrane glycoprotein CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA). Activation of CD44 signaling is also associated with enhanced proliferation, invasion, and chemoresistance in cancer cells. Importantly, HA is composed of alternating monomers of uronic acid and acetylglucosamine, the components of alginate and chitosan, respectively, which suggests that cancer cells grown on CA scaffolds may acquire a CSC phenotype via the EMT pathway. To address this question, the expression of CD44 and other genes that participate in EMT in scaffold- grown U-87 MG cells was examined. As shown in FIGURE 18, CD44 mRNA content was elevated within a day of culture on scaffolds. Elevation of mRNA for Twist2, Snail and Snai2, genes that participate in EMT, was subsequently detected beginning at day 2. Overexpression of CD44 upon interaction with HA-mimicking CA induced the overexpression of Snail, Snai2, and Twist2 through downstream signaling which resulted in overexpression of CD133 between days 4-6. Another hallmark of EMT, enhanced expression of the cell adhesion molecule N-cadherin accompanied by suppression of E- cadherin, occurred between days 5 and 10 (FIGURE 17). In toto, these findings indicate that activation of at least some elements that participate in EMT accompany the increased proliferation of CD133⁺ CSCs on CA scaffolds.

Conclusions. The present invention demonstrates that culturing GBM and other cancer cells on CA scaffolds promotes the proliferation of cells expressing the hallmarks of CSCs. After an initial period, there was an exponential increase in the number of CD133⁺ cells growing on CA scaffolds, reaching about 70% after 15 days. The sternness of these CD133⁺ cells was verified using the gold-standard tumor formation assay where cells are implanted into nude mice at low inocula and without Matrigel. Only CD133⁺ cells from CA scaffolds grew tumors whereas CD 133^" cells showed no tumor formation. Additionally, cells cultured in CA scaffolds displayed increased expression of stem cell related genes such as Nestin, Frizzled 4, GLI, HES, Snail, and Notch.

The acquisition of the CSC phenotype appears to be specific to the chemistry of the CA scaffolds. Cells cultured in other 3D scaffolds showed no increase in CD133 immunopositivity. Importantly, cells cultured in PCL coated CA scaffolds, in which the 3D structure of the scaffold was conserved but the surface chemistry changed, did not show tumor spheroid formation not CD133⁺ cell enrichment. Therefore, the results demonstrate that both the structural and chemical environment of the CA scaffolds promote rapid enrichment of CSCs. The surface chemistry of CA scaffolds mimics that of HA, an extracellular matrix glycosaminoglycan that has been shown to promote expression of CSC-related genes. In fact, CD44, a transmembrane glycoprotein that interacts with HA, was found to be upregulated in CA scaffold cultured cells at early time points and likely promoted the downstream expression of CD133 through SNAI1, SNAI2, and Twist2. The enrichment of CSCs was found to be mediated by expression of genes involved in EMT, and most importantly generated a cadherin switch.

Cells cultured in CA scaffolds were able to revert to a more stem-like state and displayed the properties of CSCs. The fact that non-CSC can become CSCs under the right microenvironmental cues implies that CSC-targeted therapies may not be as effective as once thought. If non-CSCs in the tumor microenvironment can become CSCs, then cells targeted by anti-CSC therapies could simply be replaced by non-CSCs through genes involved in EMT. The methods of the invention provide a medium for the conversion of non-CSCs into CSCs to generate microenvironmental therapies and provide an in vitro model for the discovery of more efficacious anti-cancer therapies.

The following examples are provided for the purpose of illustrating, not limiting, the invention.

EXAMPLES

Example 1

Materials and Methods

Cell lines and tissue culture. The human cell lines U-87 MG and U-118 MG (glioblastoma), MDA-MB-231 (breast carcinoma), SK-Hep-1 (hepatocellular carcinoma), and the mouse prostate carcinoma line TRAMP-C2 were purchased from American Type Culture Collection (Manassas, VA) as was Minimum Essential Media (MEM). Cells were maintained according to manufacturer's instructions in MEM containing 10% FBS (Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA) at 37 °C and 5% C0₂ in a fully humidified incubator. Cells were seeded onto PBS damp CA scaffolds in 12-well plates at 50,000 cells per scaffold in 50 μΐ_^ of supplemented medium. Cells were allowed to attach to the scaffold for 1 h before adding 1 mL of medium to each well. For PCL and PS scaffolds, 50,000 cells were seeded per scaffold following the manufacturer's protocol. For 2D cultures, 12-well plates were inoculated with 1 mL medium containing 50,000 cells. Medium were replaced every 2 days or as required.

Cell proliferation analysis. Cell proliferation was determined using the Alamar Blue assay following the manufacturer's protocol. Briefly, cells were washed with PBS before adding 1 mL of Alamar Blue solution (110 μg Resazurin per 1 mL medium) to each well. After continuing incubation for 1.5 h, the solution was transferred to a black- bottom 96-well plate to measure fluorescence. The cell number was calculated based on standard curves created for each cell line grown as monolayers. For time course determinations, cells were washed with D-PBS to remove Alamar Blue and returned to fresh medium.

Scanning electron microscopy. Samples in medium were fixed with 2.5% glutaraldehyde for 30 min at 37°C, followed by incubation in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer overnight at 4°C. After dehydration by serial washing in increasing ethanol concentrations (0%, 30%, 50%, 70%, 85%, 95%, 100%) with each wash performed twice, samples were critical point dried, sectioned, mounted, and sputter coated with platinum before imaging with a JSM-7000 SEM (JEOL, Tokyo, Japan).

Flow cytometry analysis. Cells were detached from scaffolds with Versene and washed into FACS buffer (2% FBS in D-PBS) at 1 million cells per mL. Cells were incubated with anti-human/mouse/rat CD133 primary antibodies (1:50) for 1 hr on ice, washed thrice with FACS buffer, and incubated with FITC conjugated secondary antibodies to rabbit IgG (1: 1000) for 30 min on ice and washed thrice with FACS buffer. Secondary only stained cells were used as a background control. Cells were analyzed on a FACSCanto flow cytometer (Beckton Dickinson, Franklin Lakes, NJ), and data processed using the Flow Jo package (Tree Star, Ashland, OR).

Immuno stainin g . Cell cultured scaffolds were fixed overnight in 4% formaldehyde, embedded in paraffin, sectioned into 15 μιη sections, and affixed to slides. Slides were deparaffinized with xylenes and rehydrated followed by antigen retrieval using a double boiler and Tris-based antigen retrieval buffer. Slides were blocked with 10% BSA for 2 hr, incubated with primary antibody (1: 100) overnight at 4°C. For immunofluorescence imaging slides were washed thrice with 10% BSA before incubation with FITC-conjugated secondary antibody for 2 hr at 4°C (1: 1000 dilution in 10% BSA). Slides were mounted using Prolong Gold anti-fade reagent containing DAPI as a counter stain to visualize cell nuclei. Images were obtained on an inverted fluorescent microscope (Nikon Instruments, Melville, NY) with the appropriate filters using a Nikon Ril Color Cooled Camera System and 60x Oil Objective Lens (Nikon Instruments, Melville, NY). For immunohistochemistry, slides were sequentially incubated with biotinylated secondary antibody, peroxidase-labeled avidin, and diaminobenzidine/hydrogen peroxide chromogen substrate and counterstained with hematoxylin before mounting.

PCR. Cells were detached from samples with versene and cell pellets stored at -80°C before RNA extraction using the Qiagen RNeasy kit (Qiagen, Valencia, CA) following the manufacturer's protocol. Following reverse transcription (iScript cDNA synthesis kit, Bio-Rad, Hercules, CA), DNA transcripts were probed using BioRad iQ SYBR Green Supermix with the primers listed in FIGURE 26. A BioRad CFX96 Real- Time Detection System was used for PCR analysis and expression levels was normalized to GAPDH.

Tumorigenesis assay. Cells cultured on CA scaffolds for 15 days were detached using versene, and immunostained for CD 133 as described above. CD133⁺ and CD 133^" cells were sorted into PBS containing 2% FBS using an Aria flow cytometer (Beckton Dickinson, Franklin Lakes, NJ). Cells (500 or 2000) were injected subcutaneously into the flanks of athymic nude mice (Charles River Labs, Wilmington, MA) of 6-8 weeks of age. All animal experiments were conducted in accordance with the University of Washington Internal Animal Care and Use Committee approved protocols. For unsorted cell tumor growth studies, scaffolds containing cells cultured for 10 days were implanted into the flanks of athymic nude mice. 2D cultured cells were implanted with growth factor reduced matrigel at the same cell number as cells on CA scaffolds. Tumors were measured using calipers and the volume was calculated using established methods (Kievit, F.M. et al., Chitosan-alginate 3D scaffolds as a mimic of the glioma tumor microenvironment, Biomaterials 31, 5903-5910 (2010); Leung, M. et al., Chitosan- alginate scaffold culture system for hepatocellular carcinoma increases malignancy and drug resistance, Pharm Res 27, 1939-1948 (2010)).

Example 2

The Preparation and Seeding of a Representative Chitosan- Alginate Scaffold

Representative chitosan-alginate (CA) scaffolds were prepared and seeded as described below.

CA scaffold preparation. Chitosan (practical grade, > 75% deacetylated, MW = 190,000-375,000) and sodium alginate (alginic acid from brown seaweed) powders were purchased from Sigma- Aldrich (St. Louis, MO) and used without additional purification. CA scaffolds were prepared as follows. Briefly, a solution of 4 wt% chitosan and 2 wt% acetic acid was mixed under constant stirring for 7 min to obtain a homogeneous solution. A 4 wt% alginate solution in deionized water was then added and mixed for 10 min, followed by constant mixing in a blender for 5 min to obtain a homogeneous CA solution. Approximately 3-4 ml of the CA solution was cast in each 24-well cell culture plate and frozen at -20°C overnight. The samples were then lyophilized, sectioned into 2 mm thick, 13 mm diameter disks, then crosslinked with 0.2 M CaCl₂ for 10 min under vacuum, washed with deionized water several times to remove any excess salt, and sterilized in 70% (vol/vol) ethanol for 2 h under vacuum. The scaffolds were then washed three times with sterile PBS and placed on an orbital shaker for at least 12 h to remove any excess ethanol.

PCL coated CA scaffolds. To coat CA scaffolds with polycaprolactone (PCL), CA scaffolds were first dehydrated through two washes in excess tetrahydrofuran (THF). PCL was dissolved in THF at 10 mg/mL and added to dehydrated CA scaffolds for 2 hr to allow PCL to adsorb. CA scaffolds were then removed from PCL in THF and immediately dried with an air gun and washed in excess PBS overnight before culturing cells. A uniform PCL coating on CA scaffolds was confirmed using FTIR and SEM.

Cell seeding on scaffolds. Cells were seeded onto PBS damp CA scaffolds in 12- well plates at 50,000 cells per scaffold in 50 μΐ_^ of supplemented medium. Cells were allowed to attach to the scaffold for 1 h before adding 1 mL of medium to each well. For PCL and PS scaffolds, 50,000 cells were seeded per scaffold following the manufacturer's protocol. For 2D cultures, 12-well plates were inoculated with 1 mL medium containing 50,000 cells. Medium were replaced every 2 days or as required.

While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for proliferating and enriching a population of cancer cells with cancer stem cells, comprising,

(a) seeding a porous three-dimensional chitosan-alginate scaffold with cancer cells to provide a scaffold comprising cancer cells; and

(b) culturing the cancer cells in the scaffold to provide a population of cancer cells enriched with cancer stem cells.

2. An method for inducing the expression of epithelial-to-mesenchymal transition genes in cancer cells, comprising,

(a) seeding a porous three-dimensional chitosan-alginate scaffold with cancer cells to provide a scaffold comprising cancer cells; and

(b) culturing the cancer cells in the scaffold to provide cancer cells expressing epithelial-to-mesenchymal transition genes.

3. The method of Claim 2, wherein the genes are selected from the group consisting of N-cadherin, Twist, Notch, and Snail genes.

4. A method for inducing the expression of cancer stem cell surface markers in cancer cells, comprising:

(a) seeding a porous three-dimensional chitosan-alginate scaffold with cancer cells to provide a scaffold comprising cancer cells; and

(b) culturing the cancer cells in the scaffold to provide cancer cells expressing a cancer stem cell surface marker.

5. The method of Claim 4, wherein the surface marker is selected from the group consisting of CD133, GFAP, CD44, SSEA1, 01ig2, and L1CAM.

6. An method for inducing the surface expression of CD133 on cancer cells, comprising,

(a) seeding a porous three-dimensional chitosan-alginate scaffold with cancer cells to provide a scaffold comprising cancer cells; and (b) culturing the cancer cells in the scaffold to provide cancer cells expressing

CD133.

7. The method of any one of Claims 1-6, wherein the chitosan-alginate scaffold comprising a chitosan, an alginate, and a divalent metal cations, wherein the chitosan is ionically linked to the alginate.

8. The method of Claim 7, wherein the divalent metal cations are selected from the group consisting of Ca²⁺, Ba²⁺, Mg²⁺, and Sr²⁺.

9. A three-dimensional scaffold, comprising:

(a) a porous chitosan-alginate scaffold; and

(b) cultured cancer stem cells.

10. A method for producing a cancerous tumor in a subject, comprising implanting in a subject cultured cells obtained from the methods of any one of Claim 1-8.

11. The method of Claim 10, wherein implanting cultured cells comprises implanting the scaffold comprising cultured cancer cells.

12. A method for screening a candidate chemotherapeutic agent in vitro, comprising contacting cultured cells obtained from the methods of any one of Claims 1-8 with a candidate chemotherapeutic agent.

13. The method of Claim 12, wherein the cultured cells comprises contacting the candidate chemotherapeutic agent with the scaffold comprising cultured cancer cells.

14. The method of Claims 12 or 13 further comprising measuring cell proliferation inhibition, measuring the cell viability, or measuring protein expression levels.

15. A method for screening a candidate chemotherapeutic agent in vivo, comprising:

(a) implanting in a subject cultured cells obtained from the methods of any one of Claims 1-8; and

(b) administering a candidate chemotherapeutic agent to the subject.

16. The method of Claim 15, wherein implanting cultured cells comprises implanting the scaffold comprising cultured cancer cells.

17. The method of Claims 15 or 16 further comprising comparing the tumor mass or volume measured prior to drug administration and after a pre-determined period of time after drug administration, or harvesting the tumor mass after a pre-determined period of time after drug administration and analyzing the tumor.