[go: up one dir, main page]

WO2012040587A2 - Compositions et procédés de modification de la rigidité d'une matrice destinés à réguler la croissance de cellules cancéreuses et phénotype - Google Patents

Compositions et procédés de modification de la rigidité d'une matrice destinés à réguler la croissance de cellules cancéreuses et phénotype Download PDF

Info

Publication number
WO2012040587A2
WO2012040587A2 PCT/US2011/053003 US2011053003W WO2012040587A2 WO 2012040587 A2 WO2012040587 A2 WO 2012040587A2 US 2011053003 W US2011053003 W US 2011053003W WO 2012040587 A2 WO2012040587 A2 WO 2012040587A2
Authority
WO
WIPO (PCT)
Prior art keywords
cell
substrate
composition
cell line
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2011/053003
Other languages
English (en)
Other versions
WO2012040587A3 (fr
Inventor
Robert Tilghman
J. Thomas Parsons
Daniel Tschumperlin
Brett R. Blackman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UVA Licensing and Ventures Group
Harvard University
Original Assignee
University of Virginia Patent Foundation
Harvard University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Virginia Patent Foundation, Harvard University filed Critical University of Virginia Patent Foundation
Publication of WO2012040587A2 publication Critical patent/WO2012040587A2/fr
Publication of WO2012040587A3 publication Critical patent/WO2012040587A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • At least a 2-fold difference between tumor cell number on the first substrate after culture for 5 days and tumor cell number on the second substrate after culture for 5 days indicates a rigidity dependent phenotype. In another embodiment, less than a 2- fold difference indicates a rigidity independent phenotype. In yet another example, at least a 10-fold difference between tumor cell number on the first substrate after culture for 5 days and tumor cell number on the second substrate after culture for 5 days indicates a rigidity dependent phenotype.
  • the method optionally includes culturing the tumor cell on a plurality of different substrates, e.g., a third, fourth, or fifth substrate (or more), wherein each substrate has a different elastic modulus.
  • the method further comprises culturing the tumor cell on five substrates, wherein the five substrates have an elastic modulus of 150 Pa, 1200 Pa, 2400 Pa, 4800 Pa and 9600 Pa, respectively.
  • each of the substrates is present on a single multi-well culture plate.
  • the substrates may differ in other way such as the composition of the substrate itself.
  • at least one substrate comprises collagen covalently coupled to a polyacrylamide gel.
  • at least one substrate comprises one or more extracellular matrix (ECM) proteins covalently coupled to a matrix, e.g., collagen, elastin, fibronectin and/or laminin.
  • ECM extracellular matrix
  • a method of identifying an anti-tumor composition is carried out by contacting a rigidity dependent cell, e.g., identified using the methods described above, with a candidate compound on a rigidity-matched substrate.
  • an amount or level of proliferation is detected and the difference between the level associated with culture on the first substrate compared to the level associated with culture on the second substrate (or a plurality of substrates, each of which has a different elastic modulus) is calculated, e.g., the gathered data is transformed using a machine or computer.
  • a decrease in cell proliferation in the presence of the candidate compound as compared to cell proliferation in the absence of the candidate compound indicates that the candidate compound comprises anti-tumor activity.
  • a method for identifying an anti-tumor composition includes the following steps: (a) preparing or obtaining a cell line derived from a cancer cell present in an in vivo environment in a tumor in a mammal; (b) identifying the elastic modulus (stiffness) of the in vivo environment of the cancer cell in the mammal; (c) identifying a rigidity-matched substrate for the cell line; (d) culturing the cell line on the rigidity-matched substrate, and measuring 5-day cell growth (or growth after about 2 or more days) of the cell line on the substrate in the presence of a candidate composition; (e) culturing the cell line on the rigidity-matched substrate, and measuring 5-day cell growth (or growth for the same amount of time as in (d)) of the cell line on the substrate in the absence of the candidate composition; (f) comparing the growth rates measured in (d) and (e), wherein a decrease by at least two-fold in 5 -day (or other time length of) growth on
  • a method for identifying a cancer cell line that exhibits a morphology or phenotype similar to that of a mammalian dormant cancer cell comprises: (a) preparing or obtaining a cancer cell line derived from a cancer cell present in a mammal; (b) culturing cancer cells from the cell line on a first soft substrate; (c) culturing cancer cells from the cell line on a second more rigid substrate; and (d) measuring in the cancer cells of (b) and (c) cultured for the same amount of time at least one selected from the group consisting of (i) cell cycle length, length of at least one cell cycle phase, or both; (ii) cellular ATP levels; and (iii) protein synthesis; and (e) identifying whether the cancer cell line exhibits a morphology or phenotype similar to that of a mammalian dormant cancer cell based on results obtained upon measuring in (d), wherein at least one of an increase in (i), a decrease in (ii)
  • Advantages of the methods described herein include reduced time and cost for screening. In addition to increased efficiency of the screening process, testing in this manner ultimately results in increased safety. Screening with this method increases the biofidelity of the culture environment, and permits identifying candidate compounds whose function is specific to certain substrate stiffness conditions that are relevant to in vivo environments. Hence, the method detects functional compounds that exert negligible effects in traditional rigid tissue culture environments but are functional in certain in vivo environments.
  • Culture refers to a process by which cells are grown under controlled conditions, such as in a tissue culture dish or plate in an incubator.
  • a “candidate compound” is a compound that is being tested for, or has or potentially has, certain effects on one or more cells, such as tumor/cancer cells in vivo or in vitro, when contacted with the cell, cells or tissue comprising cells, or when administered to a subject.
  • An “elastic modulus” is the measure of stiffness of a substance. It is the degree of strain that the object undergoes in response to a defined stress applied to it. It is measured in Pascal's (Pa), i.e., force per unit of area.
  • an “anti-tumor composition” is a composition that kills or inhibits growth of a tumor cell when contacted with the cell, cells or tissue comprising cells, or when administered to a subject.
  • Figure 1A is a diagram showing design of a typical 5-day growth assay using the SoftPlate96 yields a "growth profile," which reflects the effect of rigidity on the proliferation of the cell line.
  • Collagen- coated polyacrylamide gels of varying rigidity are attached to the bottom of the wells of a 96-well plate. Cells are cultured on the gels for 5 days, and growth is measured by quantitation of cellular DNA. The resulting "growth profile" is generated for each cell line and summarizes the response of that cell line to extracellular rigidity.
  • Figure IB is a bar graph showing a 5-day growth assay of four cancer cell lines on plastic.
  • Figure 1C is a bar graph showing 5-day growth assays of the four cancer cell lines on the SoftPlate96. Data are expressed as fold change over the number of cells initially plated. Results are representative of at least three experiments. These figures show growth of cancer cell lines on flexible substrates.
  • Figure 4A is a series of micrographs of A549, MDA-MB-231, PC-3, and mPanc96 cells that were plated on 150 or 4800 Pa gel substrates for 20 hours.
  • Figure 4B is a bar graph showing areas of cells that were plated for 20 hours on 150 or 4800 Pa gel substrates. Results show mean fold increase over an unspread cell + SEM of at least 20 cells counted for each condition.
  • Figure 4C is a bar graph showing cell velocity. A549, MDA-MB-231, PC-3, and mPanc96 cells were plated for 2 hours, then filmed for an additional 18 hours. Mean cell velocity + SEM in jim/hr was determined by tracing and measuring the paths of 15 cells per rigidity per cell line. * p ⁇ 0.05. These figures demonstrate rigidity-dependent changes in morphology and migration correlate with rigidity-dependent cell proliferation.
  • FIG. 5D is a bar graph showing the relative levels of Slug and E-cadherin mRNA in A549 cells cultured on PA gels for 3 days as measured by real-time RT-PCR. Results show mean + SEM of three independent experiments. These figures demonstrate that substrate rigidity regulates E-cadherin expression in A549 cells.
  • Figs. 6A-D are bar graphs showing that culturing rigidity-dependent cells on soft substrates does not select for a subpopulation of rigidity-independent cells.
  • A549 cells (A, B) or MDA-MB-231 cells (C, D) were cultured on plastic (A, C) or a 150 Pa substrate (B, D) for 15 days. The cells were then subjected to a 5-day growth assay on a Soft-Plate 96. Each cell line exhibited its typical Soft-Plate profile.
  • Figure 8 is a series of photographs of a western blots and a series of bar charts showing cyclin Dl expression and cell cycle analysis in rigidity-dependent cancer cells growing on soft and stiff gels.
  • Figure 8A is a photograph of a western blot, wherein A549 cells and MDA-MB-231 cells were cultured on 150 Pa, 4800 Pa, or 19200 Pa polyacrylamide gels for 2 or 5 days. Cells were lysed and analyzed by western blot for the expression of cyclin Dl (top panel). The expression of GAPDH was analyzed as a loading control (bottom panel). Despite a lack of FAK activity and a decrease in growth on soft gells, A549 and MDA-MB- 231 cells continue to express cyclin D.
  • Figure 8B is a photograph of a western blot showing cyclin Dl expression as compared to actin expression.
  • Figure 8C is a series of bar charts showing cell cycle profiles of A549 and MDA-MB-231 cells cultured on soft (150 Pa) or still (19200 Pa) gels for 2 or 5 days as measured by DNA staining and flow cytometry. Both cell lines show a slight accumulation of cells in the Gl stage of the cell cycle after 5 days. The A549 cells do not show an accumulation of cells in any one stage of the cell cycle after 2 days of culture on soft gels.
  • Figure 9 is a series of scatter plots and bar charts demonstrating BrdU pulse-chase of cell cycle progression.
  • Figure 9A shows that the cells are "pulsed" for 30 minutes with the nucleotide analog BrdU, resulting in the majority of cells in S phase incorporating the BrdU label (first row). The BrdU+ population is then tracked over time to calculate the rate at which the cells are progressing through the cell cycle (second row). A549 cells were pulsed with BrdU for 30 minutes following growth on soft or stiff gels for 2 days.
  • Figure 9A shows scatter plot histograms of BrdU-labeled cells on soft (third row) or stiff (fourth row) gels, stained for DNA content (X-axis) and BrdU (Y-axis). The times indicated are the times, in hours, after the BrdU pulse.
  • Figure 9B shows cell cycle progression analysis was performed on the scatter plot histograms from the cells grown on gels for 2 days (left) or 5 days (right).
  • Figure 10 is a series of bar charts showing ATP levels in rigidity-dependent cells cultured on soft or stiff gels. ATP levels were measured in A549 cells (left) or MDA-MB- 231 cells (right) following culture on a Softplate96 for 2 days. Data represents the average of two experiments performed in triplicate + S.E., of cells on soft (300 Pa) or stiff (19200 Pa) gels. Total ATP levels were normalized to cell numbers.
  • Figure 11 is a schematic and a scatter plot showing differential analysis of protein expression by "stable isotope labeling of amino acids in cell culture” (SILAC). Protein synthesis is decreased in rigidity-dependent cells cultured on soft gels. A549 cells were subjected to SILAC analysis to determine rates of protein synthesis on soft or stiff gels. Figure 11 A shows an overview of the SILAC procedure. A549 cells were cultured for 3-4 passages in media containing stable heavy isotopes of lysine and arginine, resulting in -100% of cellular proteins labeled with the heavy amino acids.
  • SILAC stable isotope labeling of amino acids in cell culture
  • FIG. 11B shows a scatter plot of heavy to light (H/L) ratios of proteins identified by SILAC/mass spectroscopy from A549 cells (left) or mPanc96 cells (right). Each dot represents an individual protein, and the dotted lines represent the means of the H/L ratios of proteins from cells on soft (blue) or stiff (orange) gels.
  • Figure 14 shows the cell cycle analysis of A549 cells cultured on soft (150 Pa) or stiff (19200 Pa) gels.
  • A549 cells were cultured on gels for 2 days and cell cycle analysis was performed by BrdU incorporation.
  • the cells on the soft gels exhibited longer Gl and S phases than the cells on the stiff gels.
  • the 1:4 ratio of growth of A549 cells on soft versus stiff gels is similar to what is seen experimentally.
  • Figure 15 shows the validation of the SILAC results presented above. Cells were grown for 5 days on gels, and plated on plastic for the indicated time.
  • the mechanical properties of the extracellular matrix have an important role in cell differentiation.
  • cancer cells responsiveness to changes in microenvironmental rigidity was unclear.
  • a 96-well assay system that arrays extracellular matrix-conjugated polyacrylamide gels that vary in stiffness by at least 2 fold across the plate was established. This assay was used to determine how changes in the rigidity of the ECM modulate the biological properties of tumor cells.
  • the cell lines tested fell into one of two categories based on their proliferation on substrates of differing stiffness: "rigidity dependent" (those which show an increase in cell growth as extracellular rigidity is increased), and “rigidity independent” (those which grow equally on both soft and stiff substrates).
  • ECM extra cellular matrix
  • EC proliferation is regulated by complex interactions with the surrounding microenvironment, including exposure to growth factors, contact with adjacent cells, and adhesion to components of the extracellular matrix (ECM). Alteration of the signaling pathways that regulate the response to these microenvironmental cues is a critical event in tumor initiation, progression and metastasis.
  • the mechanical properties of the ECM have been identified as an important factor regulating the differentiation and proliferation of a multitude of cell types both in vitro and in vivo.
  • the rigidity (“stiffness") of the ECM defined by its elastic modulus (E) in units of force per area (Pa) affects the growth, differentiation, and functionality of many cell types, including stem cells, fibroblasts, glial cells, and cardiomyocytes.
  • disease states are often accompanied by a local increase in ECM rigidity. Cancer progression in soft tissues is typically associated with an increase in rigidity due to local accumulation of a dense, crosslinked collagen matrix allowing detection of the tumor by physical palpation.
  • These attributes are considered hallmarks of tumor cells and are characterized as being an integral component of a transition from a relatively quiescent to a "malignant" phenotype, driven by a local increase in ECM rigidity.
  • Cancer cells are responsive to variations in microenvironmental rigidity. Fibroblasts transformed with oncogenic H-Ras no longer show inhibition of growth on soft substrates.
  • the growth properties of clonal populations of the breast cancer cell line MDAMB- 231 differ in response to rigidity, and they correlate with the ability to grow in the soft lung or stiff bone in vivo. This indicates that the growth properties of a particular cancer cell line in response to substrate rigidity may be determined by its genetic or epigenetic composition.
  • Analysis of human cancer cell lines is generally performed using cells cultured on rigid plastic, or in matrigel or soft agar, the mechanical properties of which are poorly defined and difficult to modulate.
  • a method for culturing cells on biologically relevant "soft" substrates using ECM-conjugated polyacrylamide (PA) gels that can span the stiffness range of 100 Pa - 150,000 Pa was developed.
  • PA polyacrylamide
  • This system was used to determine how changes in the rigidity of the ECM modulate the biological properties of tumor cells, including growth, morphology, and migratory properties.
  • the cell lines tested diverged into two categories based on their proliferation profiles: "rigidity dependent” lines generally exhibited increasing cell growth as extracellular rigidity increased, while “rigidity independent” lines grew equally well across the entire tested spectrum of matrix stiffness. Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions.
  • the growth of four representative cell lines selected from these two categories was assessed in vivo by introducing the cells into the soft tissue environment of the lung.
  • the two rigidity-independent cell lines (PC-3 and mPanc96) grew well in soft (lung) tissue, while the rigidity dependent cell lines (A549 and MDA-MB- 231) did not grow well in the lung.
  • the lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug.
  • Example 1 Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype
  • MDA-MB-231(SA) cells were a gift from Amy Bouton and Theresa Guise (UVa), VMM 18 and VMM39 were a gift from Victor Engelhard (UVa), and HPSC cells were a gift from Rosa Hwang (M.D. Anderson).
  • Cells were routinely cultured in RPMI supplemented with 10% fetal bovine serum (FBS), except for the BT549, 22Rvl, and mPanc96 cell lines, which were maintained in DMEM with 10% FBS.
  • FBS fetal bovine serum
  • the MCF-IOA human mammary cells were maintained (Debnath et al. , Methods, 30: 256-268, 2003).
  • Monoclonal antibodies to E-cadherin and FAK were purchased from Cell Signaling Technologies.
  • Monoclonal anti-actin and vimentin, and polyclonal anti-FAK pY397 were purchased from Sigma.
  • Cell growth and adhesion assays were performed using the Soft-plate96 (USSN: 12/675,882 and 12/675,839). Analysis of cell spreading, migration, cell cycle, and apoptosis were performed on gels on glass coverslips.
  • Cancer cell lines were fluorescently labeled by infecting with a lentivirus encoding GFP.
  • 1 x 106 cells in 200 ⁇ 1 PBS were injected into the tail vein of 6-8 week-old nude mice (Taconic). Lungs were removed at 2-24 hours or 14 days following tumor cell injection and digested in collagenase (0.5 mg/ml in growth media) overnight at 37° C. Lung homogenates were fixed for 20 minutes at room temperature with 2% paraformldehyde. Samples were analyzed by flow cytometry on a FACSCalibur Benchtop Analyzer and data acquired with Cell Quest software (Beckton Dickinson). 5x105 events were collected, and GFP positive cancer cells counted with FlowJo v.8.8.6.
  • Immunofluorescence staining and immunoblotting were performed using standard methods. Quantitative PCR was performed on cDNA generated from cells cultured for three days on PA gel-coated coverslips.
  • Soft-plate96 96- well assay system
  • the Soft-plates were comprised of five sections, each containing two columns of collagen-coated PA gels of a specific elastic modulus (Figure 1A), 150 Pa and 1200 Pa (comparable to lung and breast), 2400 Pa and 4800 Pa (comparable to a mammary tumor), and 9600 Pa (approximating striated muscle).
  • the growth profile of fourteen cancer cell lines was determined by plating the cells on the Soft-plate96 and measuring the fold change in cell number after five days using a fluorescent DNA-binding dye (Table 1).
  • the growth profiles of nontumorigenic mammary epithelial cells (MCF-IOA) and two fibroblast lines were determined. Cell growth on defined matrices generated a qualitative "growth profile" for each cell line ( Figure 1 A).
  • the growth profiles of the cell lines fell into one of two categories: "rigidity-dependent” cells, at least a 2-fold change in cell number across the range of extracellular rigidity tested (e.g., MDA-MB-231 breast cancer cells and A549 lung cancer cells), and "rigidity-independent” cells which grew equally well across the range of tested matrix stiffness (e.g., PC-3 prostate cancer cells and mPanc96 pancreatic cancer cells) (Table 1). There was no correlation between the shape of the stiffness-dependent growth profile and the tissue of origin, or whether the cells were originally cultured from the primary tumor or from a metastatic lesion.
  • Table 1 is a compilation of 5-day growth assays for 17 cell lines. Included in the table are original source of the cells (indicated by literature citations), the ability to grow on 150 Pa and 9600 Pa substrates (from SoftPlate96 assays), and the Soft-plate96 growth profile for each cell line. Grey profiles indicate rigidity-dependent lines and black profiles indicate rigidity- independent lines. Growth is measured as follows: - ⁇ 1 fold; + 1-5 fold; ++ 5-10 fold; +++ 10-15 fold; ++++ 15-20 fold; +++++ > 20 fold increase in cell number over 5 days.
  • A549 or MDA-MB-231 cells were cultured on plastic or 150 Pa substrates for 15 days. These cells were then harvested and subjected to a 5-day growth assay on a Soft-Plate 96. No change in the Soft-Plate growth profile was observed after prolonged culturing on soft substrates (Figure 6). These data establish cell line specific differences in the ability to grow on soft versus rigid substrates and indicate that the "rigidity profile" is an intrinsic property of each cell line.
  • a lack of adhesion signaling in anchorage-dependent cells results in a block at the Gl/S checkpoint of the cell cycle.
  • A549 cells cultured on a 150 Pa gel for five days showed a modest but significant accumulation in the Gl phase of the cell cycle with a corresponding decrease in the percentage of cells in the S phase ( Figure 2B), consistent with a block at the Gl/S checkpoint.
  • MDA-MB-231 cells exhibited no significant change in their cell cycle profile, similar to the rigidity-independent cell lines PC-3 and mPanc96 ( Figure 2B).
  • mice injected with the two rigidity-dependent cell lines contained fewer GFP-positive cells, compared to the lungs of mice injected with rigidity-independent cell lines (PC-3 and mPanc96) (Figure 3A).
  • Hematoxylin and eosin (H&E) staining of paraffin-embedded sections of the lungs two weeks post-injection of mPanc96 cells showed microcolonies within the alveoli, while the lungs of the mice that were injected with the A549 cells and MDA-MB-231 containied no detectable microcolonies (Figure 3B).
  • PC-3 cells were able to spread on 150 Pa gels to a similar extent as on the 4800 Pa gels, whereas mPanc96 cells (rigidity-independent) did not spread appreciably on either soft or rigid substrates (Figure 4A- B).
  • the ability to spread on more rigid substrates also correlated with the ability of A549 and MDA-MB-231 cells to migrate.
  • both PC-3 and mPanc96 cells failed to show significant differences in migration when plated on soft versus more rigid substrates (Figure 4C).
  • Focal adhesion kinase is a critical signaling component of integrin signaling and has been implicated in sensing the rigidity of the ECM (Provenzano et at, Oncogene, 28: 4326-4343, 2009; Tilghman et al. Semin.Cancer Biol., 18: 45-52, 2008).
  • FAK activity as measured by its autophosphorylation on tyrosine397, was only modestly activated as a function of matrix stiffness in A549 cells, and was not significantly altered in the other cell lines tested (Figure 7). These data emphasize that the behaviors of the different cancer cell lines on soft or rigid substrates cannot be simply attributed to alterations in general adhesion signaling through FAK activation.
  • EMT epithelial-to-mesenchymal transition
  • the transcription repressor Slug is a member of the Snail family of DNA-binding elements that regulates E-cadherin expression (Hayashida et at, J.Biol. Chem., 281: 32469- 32484, 2006) and has been shown to be critical for conferring a metastatic phenotype in an experimental model of melanoma ( Shibue et at, Proc.Natl.Acad.Sci.U.S.A, 106: 10290- 10295, 2009).
  • MDA-MB-231 cells while exhibiting rigidity-dependent proliferation, did not express detectable levels of E- cadherin at either 150 Pa or 19200 Pa, suggesting that these cells, while morphologically similar to A549 cells on soft and rigid substrates, do not alter the expression of this epithelial marker when exposed to a soft microenvironment.
  • the rigidity-dependent lines showed a marked decrease in cell spreading and migration when plated on soft versus rigid substrates, and at least one of the cell lines (A549) exhibited a rigidity-dependent regulation of E-cadherin expression and reversible modulation of epithelial and mesenchymal phenotype.
  • rigidity-dependent cell lines did not grow as well as rigidity-independent lines, indicating a correlation between the ability to grow on soft matrices in vitro and proliferative capacity in vivo in the lung.
  • the Soft-plate96 multiwell assay represents a relatively high-throughput approach to assess the role of substrate rigidity on the properties of cancer cells in culture.
  • the method of Pelham and Wang has been adapted to generate a multiwell plate in which the substrate is comprised of
  • polyacrylamide gels of varying stiffness that have been functionalized to provide a binding surface for extracellular matrix proteins, e.g., collagen.
  • extracellular matrix proteins e.g., collagen.
  • the plates were designed to encompass five levels of elastic moduli, ranging from 150 to 9600 Pa, however other formats are easily created.
  • the endpoint of the assay was cell proliferation, but other endpoints, e.g., cell survival are readily measured. As illustrated, the assay system provides a rapid and reproducible method to assess the role of matrix rigidity on cell growth and survival.
  • a panel of cancer cell lines was surveyed to determine how changes in the mechanical properties of the matrix influence cell proliferation.
  • Nine of the cancer cell lines exhibited a dependence on matrix rigidity for growth, growing significantly better on stiff/rigid matrices than on the less rigid/soft matrices.
  • Rigidity-independent cell lines exhibited virtually no changes in growth rate over the range of matrix stiffness used on the plates.
  • all of the cancer cell lines examined in this study were capable of proliferating on soft substrates, whereas normal fibroblasts, smooth muscle and epithelial cells exhibit a strict dependence on matrix rigidity for growth. This observation reflects the "oncogenic" transformation of the cancer cell lines relative to normal cells, events that reflect the multiple mutations that characterize cancer cells.
  • Extracellular rigidity affects the growth of certain cancer cell lines, and the ability of a cell line to grow on a soft substrate in vitro predicts its ability to grow in a soft environment in vivo.
  • a cell line's response to extracellular rigidity in vitro predicts its reaction to the desmoplastic response in vivo, i.e., whether an increase in rigidity of the microenvironment in vivo will favor growth of the tumor cells.
  • a cell line's Soft-plate growth profile also predicts its sensitivity to therapeutic drugs in soft tissue. For example, the DNA-crosslinker mitomycin C has been shown to inhibit proliferation of mesenchymal stem cells more efficiently on rigid versus soft substrates.
  • pancreatic cancer cell hnes that express epithelial markers such as E-cadherin and lower levels of the mesenchymal marker vimentin are more responsive to erlotinib treatment. Therefore, if a cell line (such as A549) were to become more epithelial-like when cultured in a soft environment, it would be predicted to be more sensitive to erlotinib.
  • Cellular plasticity or the ability to transition back and forth between a sessile epithelial cell and a migratory mesenchymal cell, is a phenomenon that is critical to several physiological processes, including embryonic development, wound healing, and cancer progression.
  • a common feature to EMT is a downregulation of cell-cell adhesion, primarily through inhibition of E-cadherin expression, and the acquirement of a motile phenotype along with increased expression of certain infrastructural components such as vimentin.
  • this transition may not always be complete, as there are many examples of cells which undergo a "partial" EMT (p-EMT) in which cells become motile by transiently acquiring some but not all of the mesenchymal cell characteristics.
  • p-EMT partial EMT
  • E-cadherin expression alone has been linked to inhibition of migration and Gl/S cell cycle arrest.
  • the observations described herein indicate that certain cancer cell lines, while they may have mesenchymal characteristics when they are cultured on rigid substrates, when placed in a soft microenvironment they may respond accordingly by activating an epithelial-type program.
  • the data described herein demonstrate the response of cancer cell lines to changes in the rigidity of their microenvironment.
  • the substrates and methods are useful to identify for tumors based on the mechanical milieu in which they thrive.
  • rigidity-dependent cancer cells identified by the methods described herein by virtue of the fact that when grown on soft substrates (e.g., 100- 300 Pa), rigidity-dependent cancer cells exhibit longer cell cycles, less active metabolism, and reduced protein synthesis, as compared to the same cells when grown on a rigid/stiff substrate for the same amount of time (e.g., 1-5 days).
  • soft substrates e.g. 100- 300 Pa
  • rigidity-dependent cancer cells When grown on soft substrates, rigidity-dependent cancer cells continue to synthesize, at normal rates, proteins that are necessary to sustain cell growth— i.e., protein machinery needed to keep dormant cells alive even if growth and metabolism is slowed. See Table 5.
  • rigidity-dependent cancer cells grown on soft substrates such those identified using the SoftPlate96 assay, provide an excellent tool for the study of dormant cancer cells, such as cancer stem cells.
  • Described herein is the utilization of "softplate96" technology to identify the properties of rigidity-dependent cancer cell lines that regulate their differential growth on soft and rigid substrates.
  • softplate96 Compared to cells growing on more rigid/stiff substrates, cells on soft substrates (100-300 Pa) exhibited a longer cell cycle, due predominantly to an extension of the Gl phase of the cell cycle, and were metabolically less active, showing decreased levels of intracellular ATP and a marked reduction in protein synthesis.
  • the rates of protein synthesis of over 1200 cellular proteins under conditions of growth on soft and rigid/stiff substrates were measured using stable isotope labeling of amino acids in culture (SILAC) and mass spectrometry.
  • SILAC stable isotope labeling of amino acids in culture
  • the former category included proteins that regulate cytoskeletal structures (e.g. , tubulins) whereas the latter category included proteins that regulate key metabolic pathways required to sustain cell growth, e.g.. nicotinamide phosphoribosyltransferase, a regulator of the NAD salvage pathway.
  • the cellular properties of rigidity-dependent cancer cells growing on soft matrices are reminiscent of the properties of cancer stem cells or dormant cancer cells, e.g. , slow growth rate and reduced metabolism.
  • the soft plate technology provides a unique platform for the study of stem cells and dormant cancer cells, allowing the growth and molecular analysis of cells in different mechanical environments that reflect the changing microenvironment of cancers in experimental animal models and in patients.
  • ECM extracellular matrix
  • Tumor dormancy is defined as a stage in cancer progression in which residual disease is present but is asymptomatic (Aguirre-Ghiso, J.A. 2007. Models, mechanisms and clinical evidence for cancer dormancy. Nat Rev Cancer. 7:834-46). Twenty to forty-five percent of breast and prostate cancers will relapse years or decades later. Most cancer types are associated with disseminated disease that, after treatment, might persist as minimal residual disease. For example, many dormant cells survive chemotherapy, and disseminated breast cancer cells are growth- arrested and resistant to doxorubicin. It is hypothesized that dormant cells survive because they are not dividing; however, prior to the invention described herein, it was unknown whether the cells are not dividing because they have
  • PLoS One. 5:el2905) In the assay described herein, a 96-well assay system that arrays PA gels of varying stiffness in user defined increments across the plate was utilized (Mih, J.D., A.S. Sharif, F. Liu, A. Marinkovic, M.M. Symer, and D.J. Tschumperlin. A multiwell platform for studying stiffness-dependent cell biology. PLoS One. 6:el9929). This experiment has been used to assess how changes in the rigidity of the ECM modulate the biological properties of tumor cells, including growth, morphology, and migratory properties.
  • bisacrylamide (all from Bio-rad) were delivered into the well plate with a multichannel pipettor.
  • A549 cells were grown for two passages in SILAC media (Lysine and Arginine replaced with Lys 13C6 and Arg 13C6, respectively) to incorporate the heavy isotopes into the cellular proteins.
  • the labeled cells were then cultured on soft (150 Pa) or stiff (19200 Pa gels for 4 days In the presence of heavy amino acids. On the fourth day, the cells were washed twice in PBS, and incubated for 24 hours in unlabeled ("light”) media. The cells were then collected by trypsinization, counted, and lysed in sample buffer. Lysates were separated by SDS-PAGE. Protein bands were cut from the gel and digested with trypsin.
  • the resulting peptides were analyzed and identified by mass spectrometry and for each individual protein the number of heavy (H) or light (L) peptides was determined.
  • the mean H/L ratios of peptides derived from the A549 cell lysates were normalized by dividing each mean by itself, and proteins were identified as being at least one standard deviation away from the normalized mean.
  • Rigidity-dependent A549 (lung carcinoma)or MDA-MB-231 (breast caracinoma) cells were cultured on 150 Pa, 4800 Pa, or 19200 Pa gels for 2 or 5 days, and cyclin Dl expression was measured by western blot. As shown in Figure 8, both cell lines still expressed cyclin Dl even when cultured on the soft (150 Pa) gels. These data indicate that rigidity-dependent cells do not exit the cell cycle, even on soft gels where the cells exhibit slower growth.
  • Figure 14 shows the cell cycle analysis of A549 cells cultured on soft (150 Pa) or stiff (19200 Pa) gels.
  • A549 cells were cultured on gels for 2 days and cell cycle analysis was performed by BrdU incorporation as described above.
  • the cells on the soft gels exhibited longer Gl and S phases than the cells on the stiff gels.
  • the 1 :4 ratio of growth of A549 cells on soft versus stiff gels is similar to what is seen experimentally.
  • Soft Ratio « Stiff Ratio have the largest decreases (green) or the least changes (red) in their rates of synthesis when the cells are cultured on soft compared to stiff substrates, respectively.
  • proteins whose synthesis was most sensitive to the shift from stiff to soft matrix (largest change in heavy/light ratio ( Figure 12, green circles) are associated with cytoskeletal structures or glucose/sugar metabolism, whereas the proteins that exhibit the least change in rate of protein synthesis (smallest change in heavy to light ratio, red circles, Figure 12) are proteins involved in the metabolism of cellular macromolecules required to sustain growth on soft matrices.
  • Figure 15 shows the validation of the SILAC results presented above. Cells were grown for 5 days on gels, and plated on plastic for the indicated time. These results indicate that suitable targets for dormant cancer cells include tubulin, nicotinamide
  • phosphoribosyltransferase phosphofructokinase
  • epoxide hydrolase phosphoribosyltransferase
  • rigidity-dependent cells A549 and MDA321
  • SILAC proteomic analysis
  • Proteins whose synthesis is most affected by rigidity are significantly decreased on soft substrates:
  • IPI00455383 Isoform 2 of Clathrin heavy chain 1
  • IPI00294578 Isoform 1 of Protein-glutamine gamma- glutamy transferase 2
  • IPI00939174 Isoform 1 of Ubiquitin thioesterase OTUB 1
  • IPI00019502 Isoform 1 of Myosin-9
  • IPI00793930 TUBA1B protein (tubulin, alpha)
  • IPI00376215 Isoform 2 of DNA-dependent protein kinase catalytic subunit
  • Proteins whose synthesis is least affected by rigidity are relatively sustained on soft substrates:
  • Figure 13 shows a schematic illustrating potential signaling processes regulated by extracellular rigidity.
  • rigidity-dependent cell cycle progression is regulated by the mechanosensor FAK through Rac activity and cyclin D expression.
  • adhesion-dependent regulation of gene expression occurs through the MAPK pathway, and protein synthesis is regulated by the AKT/mTOR pathway.
  • mutations that constituitvely activate these signaling pathways could lead to uncoupling of the FAK/Src mechanosensory complex with its downstream effectors.
  • Protein synthesis, and perhaps other aspects of cellular metabolism are regulated by the rigidity of the microenvironment, and this contributes to slower growth in soft tissue and tumor dormancy at distant metastatic sites.
  • the rigidity of the extracellular matrix regulates cellular metabolism and protein synthesis in cancer cells.
  • the rigidity-dependent cancer cell lines A549 and MDA-MB-231 sustain the expression of cyclin Dl when cultured on
  • polyacrylamide gels that have mechanical properties similar to that of soft tissue such as lung or breast.
  • A549 cells when cultured on soft gels, show a lengthening of the Gl phase of the cell cycle, suggesting that there may be defects in the synthesis of the structural and enzymatic components necessary for the transition into S phase.
  • the rigidity-dependent cell lines show lower levels of cellular ATP levels when cultured on soft gels.
  • protein synthesis is slower under this condition, with the synthesis of specific structural proteins and glycolytic enzymes such as tubulin, actin, and phosphofructokinase especially sensitive to the decrease in extracellular rigidity.
  • Proteins that were less sensitive to the change in rigidity included enzymes such as epoxide hydrolase and nicotinamide phosphoribosyltransferase that are involved in the metabolism of cellular macromolecules.
  • Tumor dormancy is defined as a stage in cancer progression in which residual disease is present but is asymptomatic (Aguirre-Ghiso, J.A. 2007. Models, mechanisms and clinical evidence for cancer dormancy. Nat Rev Cancer. 7:834-46).
  • Dormant cancer cells can reside undetected at sites distant from the primary tumor, pending subsequent growth and clinical recurrence.
  • Matrigel is a mixture of laminin, collagen IV, and entactin, in addition to a variety of proteases and growth factors such as TGFb, FGF, EGF, PDGF, and IGF (Kleinman, H.K., and G.R. Martin. 2005. Matrigel: basement membrane matrix with biological activity.
  • Matrigel Because it is generated from a tumor, the specific composition of Matrigel is not well defined, and it may vary from batch to batch, producing variability in experimental results. While the mechanical properties of Matrigel have been analyzed, they also are subject to variability because its polymerization is affected by its composition and other experimental factors such as temperature (Soofi, S.S., J.A. Last, S.J. Liliensiek, P.F. Nealey, and C.J. Murphy. 2009. The elastic modulus of Matrigel as determined by atomic force microscopy. J Struct Biol. 167:216-9).
  • polyacrylamide gels that mimic the mechanical properties of the soft tissue where metastases commonly occur, such as the lung, liver, and bone marrow.
  • polyacrylamide is a stable, homogeneous polymer, and its mechanical properties are not sensitive to changes in temperature (Sunyer, R., X. Trepat, J.J. Fredberg, R. Farre, and D. Navajas. 2009. The temperature dependence of cell mechanics measured by atomic force microscopy. Phys Biol. 6:025009). Its rigidity is easily tunable by modulating the amount of bis crosslinker without affecting its ability to crosslink ECM molecules to its surface (Mih, J.D., A.S. Sharif, F.
  • Acrylamide encompasses a spectrum of elastic moduli that includes a range of human tissues from fat to skeletal muscle, and a variety of ECM molecules can be conjugated to its surface, including collagen, fibronectin, and unpolymerized Matrigel.
  • a high-throughput system was developed to enable the screening of small molecule inhibitors with cells cultured on polyacrylamide gels spanning a range of stiffnesses (Mih, J.D., A.S. Sharif, F. Liu, A. Marinkovic, M.M.
  • PFK phosphofructokinase-1
  • EH epoxide hydrolase
  • Nampt nicotinamide phosphoribosyltransferase
  • Nampt is the rate-limiting step in the salvage pathway to generate NAD from nicotinamide (Rongvaux, A., F. Andris, F. Van Gool, and O. Leo. 2003. Reconstructing eukaryotic NAD metabolism. Bioessays. 25:683-90).
  • the conservation of EH and Nampt in cells cultured on soft gels suggests that these cells are undergoing a form of autophagy to promote survival by metabolizing cellular components.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé permettant de déterminer un phénotype de cellule cancéreuse, réalisé par la mise en culture d'une cellule tumorale ou de sa descendance sur un premier substrat et sur un second substrat, et par la mesure de la croissance, de la morphologie, de la migration, de l'apoptose, ou de l'expression de protéine, ou de l'expression de gène. Lesdits substrats se caractérisent par des modules d'élasticité différents.
PCT/US2011/053003 2010-09-23 2011-09-23 Compositions et procédés de modification de la rigidité d'une matrice destinés à réguler la croissance de cellules cancéreuses et phénotype Ceased WO2012040587A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US38561710P 2010-09-23 2010-09-23
US61/385,617 2010-09-23

Publications (2)

Publication Number Publication Date
WO2012040587A2 true WO2012040587A2 (fr) 2012-03-29
WO2012040587A3 WO2012040587A3 (fr) 2012-07-05

Family

ID=45874407

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/053003 Ceased WO2012040587A2 (fr) 2010-09-23 2011-09-23 Compositions et procédés de modification de la rigidité d'une matrice destinés à réguler la croissance de cellules cancéreuses et phénotype

Country Status (1)

Country Link
WO (1) WO2012040587A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107828729A (zh) * 2017-11-15 2018-03-23 世翱（上海）生物医药科技有限公司 一种人肺癌a549衍生细胞株及其制备方法和应用
CN116194770A (zh) * 2020-09-16 2023-05-30 新加坡科技研究局 伤口愈合模型
CN116459391A (zh) * 2022-12-19 2023-07-21 四川大学 一种兼具肿瘤消融及骨缺损修复的生物医用凝胶及其制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9097702B2 (en) * 2008-07-29 2015-08-04 Cornell University Pathologically relevant tumor microenvironments for high-throughput drug screening

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107828729A (zh) * 2017-11-15 2018-03-23 世翱（上海）生物医药科技有限公司 一种人肺癌a549衍生细胞株及其制备方法和应用
CN107828729B (zh) * 2017-11-15 2020-05-05 世翱（上海）生物医药科技有限公司 一种人肺癌a549衍生细胞株及其制备方法和应用
CN116194770A (zh) * 2020-09-16 2023-05-30 新加坡科技研究局 伤口愈合模型
CN116459391A (zh) * 2022-12-19 2023-07-21 四川大学 一种兼具肿瘤消融及骨缺损修复的生物医用凝胶及其制备方法

Also Published As

Publication number Publication date
WO2012040587A3 (fr) 2012-07-05

Similar Documents

Publication Publication Date Title
Bell et al. Low lamin A levels enhance confined cell migration and metastatic capacity in breast cancer
Huang et al. Epithelial–mesenchymal transition: The history, regulatory mechanism, and cancer therapeutic opportunities
Tilghman et al. Matrix rigidity regulates cancer cell growth and cellular phenotype
Xu et al. Upregulated circular RNA circ_0030235 predicts unfavorable prognosis in pancreatic ductal adenocarcinoma and facilitates cell progression by sponging miR-1253 and miR-1294
Tilghman et al. Matrix rigidity regulates cancer cell growth by modulating cellular metabolism and protein synthesis
EP2989460B1 (fr) Procédé pour un test de criblage de médicaments à base de cellules et son utilisation
Zhao et al. Hypoxia-induced cell stemness leads to drug resistance and poor prognosis in lung adenocarcinoma
Lehtinen et al. 15‐Hydroxyprostaglandin dehydrogenase associates with poor prognosis in breast cancer, induces epithelial–mesenchymal transition, and promotes cell migration in cultured breast cancer cells
Akkoc et al. Tumor-derived CTF1 (cardiotrophin 1) is a critical mediator of stroma-assisted and autophagy-dependent breast cancer cell migration, invasion and metastasis
Chen et al. The marine-derived fungal metabolite, terrein, inhibits cell proliferation and induces cell cycle arrest in human ovarian cancer cells
Nolan et al. Collagen and fibronectin promote an aggressive cancer phenotype in breast cancer cells but drive autonomous gene expression patterns
JPWO2018124210A1 (ja) 環状心筋細胞塊
Zhao et al. Sequencing-free analysis of multiple methylations on gene-specific mRNAs
Eftekharjoo et al. Epithelial cell-like elasticity modulates actin-dependent E-cadherin adhesion Organization
WO2012040587A2 (fr) Compositions et procédés de modification de la rigidité d'une matrice destinés à réguler la croissance de cellules cancéreuses et phénotype
CN111172283B (zh) 检测linc00673表达量在食管癌靶向治疗预后评估试剂盒中的应用
US20220205981A1 (en) A human in vitro cardiotoxicity model
Das et al. IL-9 abrogates the metastatic potential of breast cancer by controlling extracellular matrix remodeling and cellular contractility
US9102984B2 (en) Systems and methods for individualized functional genomic profiling related to cancer cell growth
Craig et al. Involvement of the MEKK1 signaling pathway in the regulation of epicardial cell behavior by hyaluronan
CA2803677C (fr) Analyses de l'expression de genes pour caracteriser et identifier des composes genotoxiques
WO2011137302A1 (fr) Procédés d'identification de voies de signalisation intracellulaire régulées de manière aberrante dans des cellules cancéreuses
Huang et al. Roles of small GTPases in acquired tamoxifen resistance in MCF-7 cells revealed by targeted, quantitative proteomic analysis
Nakaya et al. Enrichment of stem-like cell population comprises transformation ability of Epstein–Barr virus latent membrane protein 2A for non-transformed cells
Kälvälä et al. Air-liquid interface culture of midbrain organoids improves neuronal functionality and integration of microglia

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11827620

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11827620

Country of ref document: EP

Kind code of ref document: A2