WO2015017626A1 - Three dimensional fibrous scaffolds for cell culture - Google Patents
Three dimensional fibrous scaffolds for cell culture Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0693—Tumour cells; Cancer cells
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
- C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
Definitions
- the invention described herein relates to the field of three-dimensional cell culture scaffolds.
- the disclosure encompasses embodiments of a 3D nanofibrous scaffold produced by electrospinning a mixture of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of polylactic acid (PLA) and mono-methoxypoiyethylene glycol (mPEG) designated as 3P.
- Cancer cells cultured on the 3P scaffold formed tight aggregates, termed tumoroids, similar to in vivo tumors that depend on the topography and net charge of the scaffold.
- 3P scaffolds induced tumor cells to undergo the epithelial-to-mesenchymal transition (EMT) as demonstrated by up-regulation of vimentin and loss of E-cadherin expression.
- EMT epithelial-to-mesenchymal transition
- the 3P- based tumoroids showed higher resistance to anticancer drugs than the same tumor cells grown as monolayers. Inhibition of ERK and PI3K signal pathways prevented EMT conversion and reduced tumoroid formation, diameter and number.
- Fine needle aspirates collected from tumors in mice, when cultured on the 3P scaffolds of the disclosure formed tumoroids, but showed an even greater decrease in sensitivity to anticancer drugs, compared to tumoroids formed by direct seeding.
- the 3P scaffolds provide an excellent platform for producing tumoroids from tumor cell lines and from biopsies and that the platform can be used to culture patient biopsies, test for anticancer compounds, and thereby allow the tailoring of a personalized cancer treatment.
- Three dimensional (3D) cultures provide innovative approaches to study processes that contribute to tumorigenesis because they recapitulate cancer cells in their native in vivo environment.
- tumorigenesis has been obtained using two dimensional (2D) cell culture systems.
- Cells in 2D are subjected to unnatural mechanical and geometric constraints that do not represent the three dimensional (3D) milieu of a tumor.
- the complex interplay between biochemical, and mechanical properties may be undermined or compromised in 2D cultures and may affect many important functions such as gene and protein expression.
- Considerations on the mechanical, biochemical and physical properties of any 3D system aim to mimic the native ECM.
- One of the major advantages is the potential for rapid experimental manipulation achieved by controlling these parameters that can permit development of sophisticated cancer models.
- Tailored 3D cell culture scaffolds combining relevant platforms with multiple bio-functionalities will allow for the specific induction of signal transduction pathways, the sorting of different cell types, or the control of cancer cell differentiation.
- the most commonly used 3D model are spheroids that are used for a variety of experimental studies in chemotherapy and radiotherapy and are being pursued in high throughput screening (HTS) programs for drug development, candidate efficacy and safety.
- Spheroids impart functional and mass transport properties similar to those observed in micrometastases or poorly vascularized regions in solid tumors (Hirschhaeuser et al., (2010) J. Biotechnol. 148: 3-15).
- the interaction of mammalian cells with sub cellular topography has proven to be an important signaling modality in controlling cell function via mechanotransductive cues.
- the tumor microenvironment consisting of tumor cells and corresponding stroma intimately associate with the physical structures of the ECM during all stages of tumorigenesis.
- Synthetic substrate topography has been shown to influence cell migration, differentiation, and gene expression.
- SAL/N cancer fibroblasts cultured on micropatterned PDMS and C6 glioma cells cultured on polystyrene periodic structures exhibit differences in morphology, proliferation and migration in response to various topographical cues
- Electrospinning is a versatile technique used to produce polymeric fibrous scaffolds for cell culture applications. It allows for the preparation of unique matrices of aligned or non-woven meshes containing nano to micrometer sized fibers using diverse materials and fabrication techniques (Zanatta et al., (2012) Brazilian J. Med. Biol. Res. 45: 125-130;
- Fig. 1 shows a scanning electron micrograph (SEM) of naked unmodified scaffold and 3P scaffold. Scaffolds consist of fibers arranged as randomly aligned mats.
- Fig. 2A shows an FTIR of the 3P scaffold.
- the peak at 1760cm "1 can be assigned to the carbonyl group of PLA 1087 and the peak at 1 184 cm "1 can be assigned to the -C-O-C bond of mPEG, PLA and PLGA.
- Fig. 2B shows a 1 H NMR of the 3P scaffold which confirms that PLA was
- the integral of the signal at 3.3 ppm is attributed to the three equivalent hydrogen atoms of the methyl group on mPEG-OH and used as the internal standard.
- the molecular weight of the PLA block is 23.1 kDa.
- Fig. 3 shows the swelling volume ratio (SWR) of FMN (3P), CFMN (3PC), and naked scaffolds as measured after the scaffolds were immersed in water for 24 hours and were considered to have reached the equilibrium of water uptake.
- SWR indicates the hydrophilic potential of the scaffolds.
- Fig. 4B is a graph showing the average number of spheroids per scaffold. Spheroids that were placed in 96 well plates grew on 3P scaffolds of average size 5mm 2 . Confocal images of a 20 day spheroid showed the inner necrotic core surrounded by a tight layer of proliferating cells.
- Figs. 5A and 5B show that the 3P scaffold induces spheroid formation and EMT.
- the figures shows flourescent images of LLC cells cultured on monolayer, PLGA, and 3P scaffolds immunostained with E-cadherin and vimentin antibodies and Dapi. Cells were cultured for three days then stained for markers. Spheroids stained positive for the mesynchymal marker vimentin and negative for the epithelial marker E-cadherin while cells on the monolayer and PLGA scaffold maintained epithelial marker expressions.
- Figs. 6A-6C show the dose dependent cytotoxic response of MCF-7 cells to inhibitors doxorubicin, LY 294002, and U0126.
- the IC50 of each inhibitor is shown after
- MCF-7 cells cultured on the 3P scaffold MCF cells (7x10 3 ) were cultured on monolayer and 3P scaffold for 7 days then treated with different concentration of LY294002 and U0126 for 48 hours. Cells were stained with calcein/am and ethydium bromide then counted. Mean percentage of live cells (+/- SEM) were plotted as a function of drug concentration on and represents 4 replicates in 3 separate experiments. Box data points indicate monolayer and diamond data points indicate 3P scaffold.
- Fig. 6A shows the dose dependent cytotoxic response of MCF-7 cells to inhibitor doxorubicin.
- Fig. 6B shows the dose dependent cytotoxic response of MCF-7 cells to inhibitor LY 294002.
- Fig. 6C shows the dose dependent cytotoxic response of MCF-7 cells to inhibitor
- Figs. 7A-7B show graphs showing the comparison of EMT gene expression associated with 3D vs. 2D culture. Gene expression differences in cells cultured on 3P scaffold vs. monolayer and 3P scaffold vs. PLGA at 48 hours culture. Shown are a selected group of genes that showed a greater than one-fold difference in differentiation and development (A) and cell growth and proliferation (B).
- Fig. 7A shows the comparison of EMT gene expression associated with 3D vs. 2D culture with a selected group of genes that showed a greater than one-fold difference in differentiation and development.
- Fig. 7B shows the comparison of EMT gene expression associated with 3D vs. 2D culture with a selected group of genes that showed a greater than one-fold difference in cell growth and proliferation.
- Figs. 8A-8B provides graphs showing the comparison of EMT gene expression associated with 3D vs. 2D culture. Gene expression differences in cells cultured on 3P scaffold vs. monolayer and 3P scaffold vs. PLGA at 48 hours culture.
- Fig. 8A shows the comparison of EMT gene expression associated with 3D vs. 2D culture with a selected group of genes that showed a greater than one-fold difference in extracellular matrix and cell adhesion.
- Fig. 8B shows the comparison of EMT gene expression associated with 3D vs. 2D culture with a selected group of genes that showed a greater than one-fold difference in signaling pathways.
- Fig. 9 provides a graph showing EMT gene expression associate with growth of tumor spheroids on 3P scaffold versus monolayer after 2 days.
- Fig. 10 provides a graph showing EMT gene expression associate with growth of tumor spheroids on 3P scaffold versus PLGA after 2 days.
- Fig. 1 1 provides a graph showing EMT gene expression associate with growth of tumor spheroids on 3P scaffold versus monolayer after 3 days.
- Fig. 12 provides a graph showing EMT gene expression associate with growth of tumor spheroids on 3P scaffold versus PLGA after 3 days.
- Figs. 13-17 illustrate the culturing tumor biopsies on 3P scaffold. FNA of implanted
- FIG. 13 illustrates the culturing tumor biopsies on 3P scaffold.
- FNA of implanted LLC1 mouse tumors were cultured on 3P scaffolds for the indicated time.
- Cells were stained with calcein AM/EthD-1.
- Fig. 14 illustrates the average size of biopsy tumoroids on 3P scaffolds from day 2 to day 5.
- Fig. 15 illustrates biopsy tumoroids fixed and immunostained for E-cadherin, vimentin and DAPI.
- Fig. 16 illustrates LLC-1 tumoroids and biopsy tumoroids dual-immunostained for CEA and either CD31 , F4/80 or SMA and DAPI.
- Fig. 17 illustrates biopsy tumoroids (day 2) treated with 1 ⁇ each of LY294002 or
- a three-dimensional scaffold composition comprising randomly oriented fibers, wherein the fibers each comprise a polyethylene glycol-polylactic acid block copolymer (PEG-PLA) and a poly(lactic-co-glycolic acid) (PLGA). Also provided are methods for using the three-dimensional scaffolds described herein. Term definitions used in the specification and claims are as follows:
- a cell includes a plurality of cells, including mixtures thereof.
- active derivative and the like means a modified PEG-PLA or PLGA composition that retains an ability to form a three-dimensional scaffold composition that can be used to grow cancer cell spheroids. Assays for testing the ability of an active derivative to perform in this fashion are provided herein.
- administering refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir.
- parenteral includes subcutaneous, intravenous, intramuscular, intraarticular, intra-peritoneal, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
- cancer As used herein, the terms "cancer,” “cancer cells,” “neoplastic cells,” “neoplasia,”
- tumor and “tumor cells” (used interchangeably) refer to cells which exhibit relatively autonomous growth so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation (i.e., de-regulated cell division).
- Neoplastic cells can be malignant or benign.
- a metastatic cell or tissue means that the cell can invade and destroy neighboring body structures.
- the cancer can be selected from astrocytoma, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, brain stem glioma, breast cancer, cervical cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, ductal cancer, endometrial cancer, ependymoma, Ewing sarcoma, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal cancer, germ cell tumor, glioma, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma,
- macroglobulinemia melanoma, mesothelioma, mouth cancer, multiple myeloma, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary cancer, prostate cancer, rectal cancer, renal cell cancer, retinoblastoma,
- the cancer is prostate cancer.
- cancer cell spheroid refers to an aggregate of cancer cells.
- the terms “cell,” “cell line,” and “cell culture” include progeny. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included.
- the "host cells” used in the present invention generally are prokaryotic or eukaryotic hosts.
- chitosan refers to a linear polysaccharide composed of randomly distributed p-(1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit).
- the molecular weight of commercially produced chitosan is between 3,800 and 20,000 Daltons.
- the chitosan has a molecular weight of approximately 3-12 kDa.
- the chitosan is water soluble and has a molecular weight of approximately 10 kDa.
- coating does not require a complete coverage of the coated object and that partial coverage is encompassed by the term.
- compositions and methods include the recited elements, but not excluding others.
- Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
- a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
- Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the
- compositions of this invention Embodiments defined by each of these transition terms are within the scope of this invention.
- an effective amount is an amount sufficient to effect beneficial or desired results.
- An effective amount can be administered in one or more administrations, applications, or dosages.
- fibrous scaffold refers herein to a three dimensional structure formed by randomly oriented fibers.
- electrospining methods are used to achieve the randomly oriented fiber construction.
- isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof are normally associated with in nature.
- an isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof are normally associated with in nature.
- polynucleotide is separated from the 3' and 5' contiguous nucleotides with which it is normally associated with in its native or natural environment, e.g., on the chromosome.
- a non-naturally occurring polynucleotide, peptide, polypeptide, protein, or antibody, or fragments thereof does not require "isolation” to distinguish it from its naturally occurring counterpart.
- a "concentrated" does not require "isolation" to distinguish it from its naturally occurring counterpart.
- polynucleotide, peptide, polypeptide, protein, or antibody, or fragments thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than "concentrated” or less than “separated” than that of its naturally occurring counterpart.
- a polynucleotide, peptide, polypeptide, protein, or antibody, or fragments thereof, which differs from the naturally occurring counterpart in its primary sequence or for example, by its glycosylation pattern, need not be present in its isolated form since it is distinguishable from its naturally occurring counterpart by its primary sequence, or alternatively, by another characteristic such as glycosylation pattern.
- a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide.
- a protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eukaryotic cell in which it is produced in nature.
- "Mammal” for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
- pill refers to powders, granular substances, and the like.
- a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
- pharmaceutically acceptable carrier or excipient means a carrier or excipient that is useful in preparing a pharmaceutical composition that is generally safe, non- toxic and neither biologically nor otherwise undesirable and includes a carrier or excipient that is acceptable for veterinary use as well as human pharmaceutical use.
- pharmaceutically acceptable carrier or excipient includes both one and more than one such carrier or excipient.
- pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
- the compositions also can include stabilizers and preservatives.
- pharmaceutically acceptable salts refers to any acid or base addition salt whose counter-ions are non-toxic to the subject to which they are administered in pharmaceutical doses of the salts.
- pharmaceutically effective amount refers to the amount of a compound that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
- PLGA refers to poly(lactic-co-glycolic acid) that is synthesized by means of random ring-opening co-polymerization of two different monomers, the cyclic dimers (1 ,4- dioxane-2,5-diones) of glycolic acid and lactic acid.
- cyclic dimers (1 ,4- dioxane-2,5-diones
- different forms of PLGA can be obtained: these are usually identified in regard to the monomers' ratio used (e.g. PLGA 75:25 identifies a copolymer whose composition is 75% lactic acid and 25% glycolic acid).
- controlled release sustained release
- extended release extended release
- timed release any drug-containing formulation in which release of the drug is not immediate, i.e., with a “controlled release” formulation, oral administration does not result in immediate release of the drug into an absorption pool.
- a “subject,” “individual,” or “patient,” used interchangeably herein, refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
- the term "therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent one or more of the symptoms of, the condition or disorder being treated. The therapeutically effective amount will vary depending on the compound, the disorder or conditions and their severity, the route of administration, time of administration, rate of excretion, drug combination, judgment of the treating physician, dosage form, and the age, weight, general health, sex and/or diet of the subject to be treated.
- treat include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating or impeding one or more causes of a disorder or condition.
- Treatments according to the invention may be applied preventively, prophylactically, pallatively or remedially.
- a three-dimensional scaffold composition comprising randomly oriented fibers, wherein the fibers comprise a polyethylene glycol-polylactic acid block copolymer (PEG-PLA) and a poly(lactic-co-glycolic acid) (PLGA).
- PEG-PLA polyethylene glycol-polylactic acid block copolymer
- PLGA poly(lactic-co-glycolic acid)
- PEG polyethylene oxide
- POE polyoxyethylene
- PEG usually refers to oligomers and polymers with a molecular mass below 20,000 g/mol.
- PEGs are prepared by polymerization of ethylene oxide and are commercially available over a wide range of molecular weights from 300 g/mol to 10,000,000 g/mol.
- Different forms of PEG are also available, depending on the initiator used for the polymerization process - the most common initiator is a monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), abbreviated mPEG.
- Lower-molecular-weight PEGs are also available as purer oligomers, referred to as monodisperse, uniform, or discrete.
- the PEG used to prepare the 3P and 3PC scaffolds described herein is a monomethoxy glycol (mPEG) having a molecular weight between approximately 0.5 and 20 kDa. Included herein are embodiments wherein the molecular weight of the PEG or mPEG is approximately 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 kDa. In one embodiment, the molecular weight of PEG is approximately 2 kDa.
- Polylactic acid or polylactide ((0 3 ⁇ 4 0 2 ) ⁇ ) is a thermoplastic aliphatic polyester derived from renewable resources, such as corn starch, tapioca roots, chips or starch, or sugarcane. Polymerization of a racemic mixture of L- and D-lactides usually leads to the synthesis of poly-DL-lactide (PDLLA), which is amorphous. Use of stereospecific catalysts can lead to heterotactic PLA which has been found to show crystallinity. The degree of crystallinity, and hence many important properties, is largely controlled by the ratio of D to L enantiomers used, and to a lesser extent on the type of catalyst used.
- poly-L-lactide is the product resulting from polymerization of L,L-lactide (also known as L-lactide).
- PLLA has a crystallinity of around 37%, a glass transition temperature between 60-65 °C, a melting temperature between 173-178 °C and a tensile modulus between 2.7-16 Gpa.
- the 3P and 3PC scaffolds provided herein can comprise a PLA composition having only D- enantiomers, only L-enantiomers or a mixture of D- and L-enantiomers.
- the PLA composition used to prepare the 3P or 3PC composition contains a racemic mixture of D- and L-enantiomers.
- PLGA is synthesized by means of random ring-opening co-polymerization of two different monomers, the cyclic dimers (1 ,4-dioxane-2,5-diones) of glycolic acid and lactic acid.
- Common catalysts used in the preparation of this polymer include tin(ll) 2- ethylhexanoate, tin(ll) alkoxides, or aluminum isopropoxide.
- successive monomeric units (of glycolic or lactic acid) are linked together in PLGA by ester linkages, thus yielding a linear, aliphatic polyester as a product.
- PLGA 75:25 identifies a copolymer whose composition is 75% lactic acid and 25% glycolic acid.
- the PLGA contains approximately 85% lactic acid and 15% glycolic acid. Also included herein are embodiments, where the lactic acid:glycolic ratio of PLGA is
- the 3P scaffold is composed predominantly of poly(lactide-co- glycolide) (PLGA) random copolymer and a poly-Iactide-poly(ethylene glycol) (PLA-PEG) block copolymer.
- the 3P scaffold also comprises chitosan.
- the chitosan can be coated onto the 3P scaffold. Chitosan coated scaffolds are referred to herein as 3PC scaffolds.
- the fiber polymer can be constructed by open ring polymerization of mPEG and PLA mixed with PLGA and electrospun.
- PEG poly(lactide-co-glycolide)-based electrospun nanofibrous scaffolds. Journal of Controlled Release. 2004;98:47-56.]
- PEG is used to modify and enhance the hydrophilicity of the fibers; in addition it is nontoxic and non- immunogenic.
- PEG'S protein-resistant properties arise from imparted nonionic charges, and a high excluded volume which facilitate steric repulsion thus minimizing the adsorption of proteins.
- Typical methods for spheroid formation employ similar non adherent surface modifications.
- the ratio of PEG-PLA to PLGA in each scaffold fiber is approximately 1 :4. In other embodiments, the ratio of PEG-PLA to PLGA in each scaffold fiber is approximately 1 :10. In still other embodiments, the ratio of PEG-PLA to PLGA in each scaffold fiber is approximately 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10 or 1 :20.
- the PEG-PLA and PLGA can be formed into fibers via any method known to those of skill in the art.
- solutions of PEG-PLA and PLGA are electrospun to form PEG-PLA-PLGA fibers.
- the scaffold fibers can be electrospun at any voltage, flow rate, and distance that provide for a fiber diameter between approximately 0.3 and 10 ⁇ , or more preferably a fiber diameter between approximately 0.69 to 4.18 pm. In one
- solutions of PEG-PLA and PLGA are electrospun at a positive voltage of 16 kV at a flow rate of 0.2 ml/hour and a distance of 13 cm using a high voltage power supply.
- the fibers were collected onto an aluminum covered copper plate at a fixed distance of approximately 70 mm.
- the present invention further includes a 3P or 3PC scaffold prepared by collecting the electrospun fibers at a fixed distance between approximately 60 mm and 80 mm.
- the resulting 3P or 3PC scaffold is a three-dimensional fibrous scaffold having pores.
- the scaffold comprises pores having a diameter of less than approximately 20 pm.
- the 3P or 3PC scaffold comprises pores having a diameter of less than approximately 15, 10 or 5 pm.
- the ability of the scaffold to monitor pharmacological efficacy was also analyzed by using known anti-tumor agents as control modulators of EMT to abrogate growth and differentiation of cancer cells. Accordingly, the examples demonstrate that the 3P scaffolds described herein can be utilized as a platform to study processes in tumorigenesis and can be used to evaluate anti-cancer therapies as an intermediate decision-making step in high throughput screening of drugs to treat cancer. Accordingly, provided herein are methods for screening pharmaceuticals for cancer treatment efficacy using the 3P scaffolds. In these methods, the 3P scaffolds are seeded with cancer cells, spheroids are allowed to form, and pharmaceuticals are administered to the cells in the scaffold for a given period of time. Dead and live cancer cells are then quantitated and the efficacy of the pharmaceutical for cancer treatment is determined. Also provided herein are methods for growing cancer cell spheroids, methods for tissue culture, methods for tissue regeneration, methods for treating arthritis, and methods for wound therapy.
- the 3P scaffold was constructed by electrospinning the co-block polymer mPEG/LA and PLGA dissolved in appropriate organic solvents.
- a PLGA unmodified scaffold was also constructed to compare the effects of the 3D environment. More specifically, methoxy PEG-PLA was prepared by ring-opening polymerization. Briefly, 3,6-Dimethyl-1 ,4-dioxane-2,5-dione (LA) (Fisher) was dried in a vacuum oven at 40 °C overnight.
- mPEG mono-methoxy poly(ethylene glycol)
- Solid products of the diblock copolymers were obtained by addition of the polymer solution to ice cold diethyl ether. The products were dissolved in dichloromethane and precipitated in cold diethyl ether twice, for purification. The final copolymer was dried in a vacuum oven at 50°C for 48 hours. Poly(lactic co glycolic acid) (MW 50-70 kDa, 85:15, Sigma) and the mPEG-PLA polymer described above were then dissolved in a solution of dichloromethane and chloroform (80/20 v/v). Briefly, 1.2 grams of PLGA and 0.3 grams of mPEG-PLA were used to construct the 3P scaffold.
- the solutions were electrospun at a positive voltage of 16 kV at a flow rate of 0.2 ml/hour and a distance of 13 cm using a high voltage power supply (Gamma High Voltage research, USA).
- the fibers were collected onto an aluminium covered copper plate at a fixed distance of 70 mm.
- both mPEG and PLA were placed under vacuum and dried (mPEG was dried at 800C and PLA at 500 ° C). Temperatures were maintained for at least 1 hour to remove all moisture.
- PEG PEG: PLA
- concentration 0.5 mPEG and 5 grams of PLA to 20 ml of Toluene and 0ul of TEH.
- 1 gram of mPEG and 4 grams PLA were used. The mixture was stirred under argon at 140 ° C for 5 hours at room temperature. The solution was allowed to cool to room temperature overnight.
- the mPEG/PLA copolymer was precipitated using 60 ml of diethyl ether that was cooled on ice.
- the mPEG/PLA solution was added to the cold diethyl ether drop wise. The solution turned from clear to cloudy. The solution was then stirred on ice for 2.5 hours. The sticky precipitate that formed at the bottom of the flask was then removed and dissolved in 10 ml chloroform, then precipitated in ether. This was repeated twice. The product was collected in a glass vial and left to dry for at least two days in a vacuum oven at 50 °C1 .375 grams of PLGA and 0.075 grams of the 1 :10 mPEG-PLA polymer was added to 5 ml of a solution of dichloromethane and chloroform (80:20 v/v).
- the 3P scaffold was deep coated with chitosan (90% deacylation). More specifically, the 3P scaffolds were cut into 8mm 2 squares and sterililized by UV. The 3P scaffolds were then deep coated by soaking overnight in 0.1 % Chitosan in 0.5% acetic acid solution. The scaffolds were washed in PBS three times prior being used for cell cultures.
- chitosan-coated scaffolds are referred to herein as 3PC, CMN or CFMN scaffolds. Each of these designations refers to the 3PC scaffold.
- the scaffolds provided good spatial interconnectivity between cells, a high surface to volume ratio and good porocity for fluid transport.
- the parameters that affect the pore size, diameter and thickness of the scaffold included voltage, needle collection distance and concentration of the polymer in the solvent.
- Scanning electron microscopy (SEM) of the 3P scaffold showed randomly aligned fibers that combine to form a highly porous mesh (Fig. 1 ).
- the diameter of the fibers ranged from 0.69 to 4.18 pm and naked scaffold range from 0.61 to 4.95um with pores of mainly subcellular sizes ( ⁇ 10um).
- the stretch of the C-O-C band is shown at 1087 and 1 184 cm “1 .
- the peaks at 2850 and 2950 represent -CH 2 stretching (Fig. 2A).
- the FTIR of mPEG/PLA and PLGA have similar characteristic peaks since they basically have the same functional groups.
- the molecular weight of the di-block copolymer was determined by 1 H nuclear magnetic resonance spectroscopy using the intensity of the terminal methoxy proton signal of LA at Q 3.39 ppm. The weight ratio of the repeated PEG- LA units was calculated to be 7.08 from the integral values of characteristic peaks, and the molecular weight was determined to be 23,100 Da.
- the molecular weight of the diblock copolymer was determined by 1 H nuclear magnetic resonance spectroscopy using the intensity of the terminal methoxy proton signal of mPEG at Q 3.39 ppm.
- the weight ratio of the repeated PEG-LA units was calculated to be from the integral values of characteristic peaks.
- the 1 H NMR confirmed that PLA was copolymerized with mPEG as shown in Fig. 2B.
- the swelling volume ratio (SWR) of MN (3P), CMN (3PC) and naked scaffolds was measured after the scaffolds were immersed in water for 24 hours and was considered to reach the equilibrium of water uptake.
- the SWR indicates the hydrophilic potential of the scaffolds. The results are shown in Fig. 3.
- spheroids on 3P scaffolds All of MCF-7, MDA-MB, MCF-10A breast cancer, PC3 prostate cancer, B16 melanoma, BG-1 ovarian and LLC Lewis lung cancer cells, grew spheroids on the 3P scaffolds.
- LLC cells (5x10 3 ) were cultured on monolayer, naked and 3P scaffolds from day 1 to day 5. Cells on monolayer and naked scaffolds did not form spheroids, but cells on 3P scaffolds formed spheroids beginning at day 3 and progressively increased in size at day 5. Cells were stained with calceinAM/EthD-1 for live (green) and dead (red) cells to demonstrate multiple spheroids of live cells on the 3P scaffolds (data not shown).
- the scaffolds were washed in cocadalate buffer then further dehydrated in an ascending series of ethanol at concentrations 10%, 35%, 50%, 70%, 95% and 100% for ten minutes. Final dehydration was done in hexamethyldisilazane (HMDS) for 10 minutes. Samples were air dried then sputter coated with gold at a density of 19.32/cm3 for 30 seconds under argon gas. SEM of the scaffold was viewed on a Jeol JSM 6490 scanning electron microscope. To calculate the diameters of the spheroids, the entire scaffold was scanned for full planar images. All spheroids were measured for diameter using Image J software.
- HMDS hexamethyldisilazane
- MCF-7 spheroids Analysis of MCF-7 spheroids in particular revealed a smooth surface, tight cell junction and indistinguishable cellular boundaries. The spheroids appeared flattened on the surface with intertwining fibers that form a spheroid/fiber composite. Notably, cells proliferated on unmodified PLGA scaffold and monolayer but did not form spheroids.
- Spheroids that grew on 3P scaffolds had an average size 5mm 2 that were placed in 96 well plates.
- a confocal image of a 20 day spheroid showed the inner necrotic core surrounded by a tight layer of proliferating cells (data not shown).
- the parameters essential for spheroid formation depended on cell type, concentration and time from initial seeding of the cells. It was observed that the higher the concentrations of cells, the faster the spheroids were formed with subsequent increase in diameter and numbers over time and this was observed for all cell types (Fig. 4).
- SEM of the spheroids indicated tight aggregates of cells with cellular boundaries that were indistinguishable at the periphery.
- tumor spheroids typically exhibit a spherical proliferative geometry defined by inward and outward diffusion gradients. Beyond the diffusion capacity of oxygen and fresh growth media, the innermost cells become quiescent and die from apoptosis or necrosis. Such results were observed in day 20 spheroids that had attained diameters above 500 pm. The viability of the cells within the spheroids was assessed using calcein AM/EthD-1 live/dead assay that stain dead cells red and live cells green, and this assay demonstrated that cells on the periphery appeared to encompass an inner core of dead cells (data not shown).
- LLC cells were cultured on PLGA, PLA and PLGA/PEG, PLA/PEG, and PLGA/PLA/PEG coated glass coverslips. It was observed that cells proliferated on all of the substrates but only self-assembled to spheroids on the PLGA/PLA/PEG coverslips. These spheroids however were fewer and easily dissociated when the substrate separated from the coverslip in day 3. Cells on the
- PLA/PEG construct grew large disorganized aggregates of cells that lacked the defined shape and structure observed in the 3P spheroids.
- the composite 3P/Chitosan scaffold was constructed as described above (referred to herein as the 3PC or CMN scaffold). It was observed that cells proliferated but did not form spheroids (data not shown). Chitosan was used because it is a naturally occurring polysaccharide that imparts a positive charge at physiological pH and increases hydrophilic properties of the scaffold.
- spheroids Formation and maintenance of spheroids on the 3P scaffolds support the observation that spheroid formation was directed by topographical and chemical cues transferred dynamically between the 3P ECM-like scaffold and the cells. It was observed that spheroids transferred to a regular tissue culture plate (TCP) coated for monolayer culture, adhered to the plate and grew out from the spheroid. LLC cells gradually migrated away from the spheroid from day 1 to day 4 at 0, 0.67 +/- 0.1 , 1.4 +/- 0.14, 2.1 +/- 0.29 mm, respectively, and ultimately formed a confluent monolayer (data not shown). Spheroids transferred to new scaffolds maintained their morphology and shape over the same time period.
- TCP tissue culture plate
- EMT epithelial-mesenchymal transition
- LLC spheroids were cultured for 4 days (seeding density 5x10 3 ). Spheroids were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.1 % Triton X- 00 for 20 minutes at 25 °C, and blocked using 3% BSA. Cells were incubated overnight with vimentin or E-cadherin Anatibody followed by Alexa Fluor 555 or 488 cojugated anti-mouse secondary Ab and Dapi. Cells were viewed using an Olympus BX51 microscope.
- spheroids grown on the scaffold at day 3 showed expression of vimentin while no vimentin expression was observed in the monolayer culture or on cells cultured on PLGA scaffold. Additionally, spheroids exhibited a loss of E-Cadherin expression on the 3P scaffold, while cells cultured on monolayer and PLGA scaffold retain this expression (Fig. 5B). This suggests that spheroid formation correlates with enhanced invasive potential and tumorgenicity, characteristics that define EMT and that appears to be manifested within the context of the 3P environment. To determine the timeline of EMT induction, LLC cells were cultured on the 3P scaffold and stained for vimentin and E- cadherin from dayl to day 4 culture. It was observed that the onset of EMT occurred at day 3 and continued into day 4 correlating with the self-assembly of the cells into spheroids (data not shown).
- PI3K phosphotidyl inositol -3 kinase
- Ly294002 and the mitogen activated protein kinase (MAPK) pathway inhibitor U0126 were treated after 24 hours of culture on the 3P scaffolds with Ly294002 and U0126 and observed for spheroid formation on day 4. All anti-tumor agents prevented spheroid formation and proliferation on 3P scaffolds (data not shown). In addition, these compounds were efficient at down regulating vimentin while maintaining E-cadherin expression in cells similar to that observed with cells cultured on PLGA scaffold alone and on the monolayer.
- Drug efficacy analysis using 3P scaffold To assess the ability of the 3P scaffold to monitor therapeutic efficacy to treat tumors, LLC spheroids were treated with different concentrations of the PI3K pathway inhibitor Ly294002 (1 .0 ⁇ , 0.1 ⁇ , and 0.01 ⁇ ) and spheroid viability was assessed over a time period of 24 hours to 96 hours. Measurements of spheroid size and numbers revealed a dose dependent cytotoxic response in treated spheroids compared to untreated spheroids.
- spheroids treated with Ly294002 at a concentration of 1 .0 ⁇ , 0.1 ⁇ or 0.01 ⁇ demonstrated an average decrease of 37.3 +/- 8.4%, 22.0+/-10.9% and 17+/-12.5% in size, respectively, compared to untreated spheroids.
- MCF-7 breast cancer cells were treated with Doxorubicin and Ly294002 and U0126 inhibitors to prevent spheroid formation and examined for a differential dose- dependent response which was compared with response in monolayer 2D culture.
- treatment of MCF-7 spheroids with inhibitors abrogated spheroid formation and on the 3P scaffold.
- the IC-50 of a drug is defined as the median lethal dose of a drug to inhibit biological or biochemical function. Successive viability measurements of treated cells revealed significantly different cytotoxic responses between cells on the 3P scaffold and on monolayer culture.
- MCF-7 cell viability was significantly lower and indicated a higher sensitivity in 2D monolayer culture than cells cultured on the 3P scaffold.
- the IC-50 of doxorubicin on monolayer was 0.6 ⁇ and on 3P scaffold was 12.0 ⁇ .
- the IC-50 of U0126 on the monolayer was 10.6 nm, whereas the IC- 50 U0126 on the 3P scaffold 105.9 nm.
- the IC-50 for LY294002 for the monolayer was 0.13 ⁇ , while it was 1 .15 ⁇ for the 3P scaffold (Fig. 6A-C).
- EMT is a key process that is implicated in tumor metastasis, and potentially involves a global change in gene expression. Since spheroid formation induced EMT on the 3P scaffold and not on PLGA or monolayer culture, gene expression analysis was performed to determine the underlying molecular mechanisms involved. In this analysis, a focused EMT microarray was used to test for expression of 84 genes encoding cell surface receptors, extracellular matrix proteins, cytoskeletal proteins that mediate cell migration, motility, and morphogenesis; proteins that control cell differentiation,
- EMT involves multiple intracellular signal transduction pathways that form highly interconnected networks.
- the network pathways highlight the expected relationships between differentially expressed genes.
- the transcriptional repression of key epithelial markers such as E-cadherin (Cdh1 ), and up regulation of mesenchymal markers such as vimentin (Vim) and fibronectin (Fn1 ) were observed.
- EMT is a continuum whereby induction of genes appears to occur 24 hours prior to the expression of related proteins as observed in 48 hours in the timeline experiment described above. Up regulation of the TGF- ⁇ , Notch and WNT genes was observed, which genes drive the three major pathways that control EMT expression and are responsible for activating key mediators such as Stat3, Gsk3b, and transcription factors Twist and Snail .
- transcription factors 1 and 4 transcription factors 1 and 4
- ESR1 estrogen receptorl
- TCFI1 and TCF4 transcription factors 1 and 4
- ESR1 estrogen receptorl
- Activation of these pathway genes further regulate the expression of genes that play a role in extracellular matrix and cell adhesion, integrin (Itgav) and metalloprotease (MMP9), and genes involved in cytoskeletal remodeling such as keratin (Krt7) and pleckstrin homology (Plek2).
- 3PC Scaffolds SEM showed that tumor cells maintained their basic morphology when grown on the electrospun 3PC scaffolds. Further, a Ki-67 assay and a viability assay showed that tumor cells proliferate and remain viable when grown on the 3PC scaffolds (for up to three days). Tumor cells grown on 3PC scaffolds were less sensitive to doxorubicin compared to cells grown on monolayer, suggesting that results from 2D studies cannot be relied upon. It was also determined that PLGA naked scaffolds are biocompatible and show increased tumorigenesis potential. From these observations, one can conclude that these 3D matrices can be used as a model to study cancer.
- FNAs fine needle aspirates
- tumor formation was monitored for 2 weeks after which tumor biopsies were collected by fine needle aspiration. Briefly, a 23-gauge needle was inserted into the tumor using a rotating motion and the tissue samples were flushed from the needle by attaching a syringe filled with tissue culture medium and expelling the contents into a sterile tube. This process was repeated twice. The three collected tumor samples were pooled and single cell suspensions were cultured on the scaffold.
- TME tumor microenvironment
- BITs were found positive for the tumor cell marker, carcinoma embryonic antigen (CEA), macrophage marker F4/80, endothelial progenitor cell marker CD31 and cancer associated fibroblast marker smooth muscle actin (SMA), indicating the presence of stromal components of the TME (Fig. 6D). LLC1 tumoroids, used as control expressed only CEA.
- FNA tumoroids were cultured in the presence or absence of the inhibitors, LY294002 and U0126. Results showed that inhibitors were effective in modulating growth of BITs as revealed by decreases in tumoroid size and a dose-dependent cytotoxicity. However, the IC 50 s of LY294002 and U0126 were greater than 1 mM (Fig. 6E) indicating a greater resistance of BITs to the drugs than were the LLC1 -based tumoroids. Increased drug resistance of biopsy tumoroids in the 3P system was not due to a lack of drug uptake.
- the present disclosure encompasses embodiments of a 3P fibrous scaffold that induces the formation of a micrometastatic compact aggregate of tumor cells that is termed a 'tumoroid'. Cancer cells seeded onto these scaffolds with growth medium containing the appropriate factors grow as tumoroids that show the EMT characteristic of in vivo tumorigenesis. In addition, this platform further allows the co-culture of tumor cells and stromal cells to identify changes in specific factors, gene expression and invasion potential.
- Tumoroids are able to respond to the same biochemical, nanotopographical and mechanical cues that drive tumor progression in the native ECM (Fischbach ef al., (2007) Nat. Methods 4: 855-860).
- the 3P platform of the disclosure can be used to evaluate therapeutic strategies for simultaneously targeting tumor cells and the stromal cells that are components of the stem cell niche (Ballangrud ef al. , (1999) Clin. Cancer Res. 5: 3171 s- 3176s).
- the 3P platform can further be adapted for use in a point-of-care device to culture a patient's own cells from a biopsy, test them ex vivo with different anticancer compounds and tailor a personalized treatment by selecting the most effective therapeutic agent and determining an effective dose for the particular tumor cells from that patient.
- the in vivo drug-resistant variant, EMT-6, of mouse mammary tumor cells lost resistance when cultured as a monolayer but regained it when regrown in vivo as a solid tumor or in vitro as tumoroids (Teicher ef al. (1990) Science 247: 1457-1461 ).
- the reasons for the differences between 3D and monolayer cultures remain unknown.
- Pathophysiological gradients to nutrients, oxygen and drugs and the concentric arrangement of heterogeneous cell populations within the 3P tumoroids may affect RNA and protein expression.
- 3P scaffold of the disclosure was an appropriate in vitro model for anticancer drug screening tumor biopsy specimens were cultured on the scaffold. 3P scaffolds supported the growth of both tumor and stromal cells from the biopsy and this in vitro platform therefore mimics in vivo tumor growth. Moreover, it was unexpectedly found that tumoroids derived from the biopsies (BITs) were more resistant to MEK and PI3K inhibitors than the tumoroids from cancer cell lines.
- BITs biopsies
- tumoroids derived from a cell line exhibit a distinct drug sensitivity profile, they do not accurately reflect the in vivo condition.
- results with the biopsy-derived tumoroids cultured on the 3P scaffold of the disclosure suggest that co-cultures of tumor cells with stromal cells, as obtained from the biopsied tumors will produce tumoroids that better reflect the in vivo context.
- These tumor/stroma effects on chemosensitivity underscore the advantage of using a 3D platform such as the 3P scaffold for growth of actual patient tumor biopsies to test the efficacy of anticancer drugs.
- the scaffold-based platform of the disclosure therefore, provides an approach useful for developing customized, or personalized, anticancer treatments.
- tumor biopsies can be cultured on a 3P scaffold, differentiated into BITs and then screened with an array of anticancer drugs to determine the most effective drug
- the 3P scaffold are useful for drug screening and customized cancer therapy by providing tumoroids that closely mimic the physiological and pharmaceutical responses seen in a tumor within an affected animal.
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