Research ArticleBIOMEDICAL ENGINEERING

A versatile 3D tissue matrix scaffold system for tumor modeling and drug screening

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Science Advances  13 Sep 2017:
Vol. 3, no. 9, e1700764
DOI: 10.1126/sciadv.1700764
  • Fig. 1 TMS fabrication and structural property characterization.

    (A) The workflow of the porous TMS fabrication. (1) Collection of breast tissues from 8- to 12-week-old mice. (2) Decellularization of the native tissues to produce ECM. (3) Lyophilization of the ECM at −50°C. (4) Enzymatic digestion of the ground ECM in acidic solution. (5) Neutralization of the acidic ECM solution–generated hydrogel. (6) Loading the hydrogel into the spherical molds. (7) Formation of the pre-scaffolds in the molds at −80°C. (8) Lyophilization of the pre-scaffolds. (9) Formation of the porous scaffolds in the molds. (10) Treatment of the scaffolds with absolute ethanol and cross-linking the ECM proteins under UV light. (11) Lyophilization of the scaffolds to remove the ethanol. (12) Characterization of the finished TMS scaffolds. A microscopic view of the TMS cross sections after H&E staining is shown. Scale bars, 100 μm. (B) Comparison of the composition of the decellularized tissues with that of the native tissues at DNA and major ECM protein levels. Error bars represent the SD of the measurements of three independent batches of the ECM samples. (C) Characterization of the TMS porosity under SEM. Different amounts of the lyophilized ECM powder were used to generate TMSs at different pore sizes. (D) Histological comparison of the cross sections of the blank and the cell-laden DBT-TMS with the decellularized and the native mouse breast tissue. (E) Comparison of the occupancies of the cells grown inside the DBT-TMS with that of the native cells that lived in mouse breast tissues. Left: The closeup views of the H&E-stained cross sections of the fibroblast-laden TMS and the mammary fat pad tissues. Top right: An SEM image showing the occupancies of the MM231 cells on the surface and within the porous TMS. Bottom right: Distribution patterns of the MM231 cells and stromal cells immunostained with Ki-67 (green) and HER2 (red), respectively, on the cross sections of mouse breast tumors that originated from the MM231 cell–laden TMS. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nuclei of the cells. The red and the yellow arrows indicate stromal and MM231 cells, respectively. Scale bars, 100 μm (C to E).

  • Fig. 2 Cell survival and proliferation in TMS.

    (A) Macroscopic and microscopic views of the blank and cell-laden porous DBT-TMSs. Scale bars, 1 mm (for the macroscopic views and the microscopic views of the H&E-stained cross sections) and 200 μm (for the regional blowups of the H&E-stained cross sections). (B) Proliferation of MCF10A and MM231 cells grown on DBT-TMSs over a period of 14 days. Error bars represent the SD of the means of the values from three independent experiments. *P < 0.01; **P < 0.001, compared to the first-day culture. (C to F) The proliferation and distribution of the MM231 cells on the DBT-TMSs were examined on the cross sections of the scaffolds using H&E staining coupled with light microscopy. Scale bars, 100 μm. (G to J) Live/Dead Cell assays showing robust survival and proliferation of the MM231 cells on the DBT-TMSs over time. Scale bars, 100 μm. The images (C to J) are top (surface) to bottom (center) views of the cross sections of the scaffolds. (K to N) Comparison of MCF10A and MM231 cell proliferation profiles on different 3D scaffolds within the defined time frame. Error bars represent the SD of the means of three independent experiments. **P < 0.01, compared to the proliferation profiles on the PCL/PLGA scaffolds; #P < 0.05, compared to the proliferation profiles on the collagen scaffolds.

  • Fig. 3 Compartmental 3D tissue culture using the TMS system.

    (A) Generation of the multilayered/compartmentalized TMS culture system. MM231 cells were cultured on the porous DBT-TMS followed by either covering them with a layer of blank TMS hydrogel or directly placing them into culture for in vitro or in vivo experiments. Hydrogel premixed with another type of cells different from those coated on the porous TMS was applied outside the first layer and enzymatically cross-linked, forming a second gel layer. The multilayered TMS assembly was then subjected to culture and/or implantation into animals for further analysis or applications. (B) H&E staining of the cross sections of a TMS coated with MM231 cells and a layer of hydrogel. (C) H&E staining of the cross sections of a multilayered TMS containing the porous TMS core coated with MM231 cells and two hydrogel layers with the second gel layer containing the human GM637 fibroblasts. The middle region outlined by dotted lines was a blank hydrogel layer. (D) DAPI staining of the cross sections of the compartmentally cultured cells grown in the multilayered TMS after 3 days of culture, as shown in (C). (E) IF microscopic view of Ki-67 (green, MM231 cells) and HER2 (red, GM637 cells) staining on the cross sections of the compartmental TMS samples. Selected regional blowups of the Ki-67 and HER2 staining are shown as insets. (F to I) Live/Dead Cell staining of the cross sections of the compartmentally cultured MM231 cells (on the porous TMS, right side of the blank hydrogel layer) and the human GM637 fibroblasts (within the second hydrogel layer, left side of the blank hydrogel layer) at different time points of the cultures. Scale bars, 100 μm.

  • Fig. 4 Characterization of TMS support of tumor formation in animals.

    (A) Evaluation of the biodegradability of the scaffolds and their supports on the MM231 cell–originated tumor development (dissection microscopy images). Scale bars, 4 mm. (B) Quantification of the sizes of the tumors formed from the different MM231 cell–laden scaffolds. The plotted values reflect the ex vivo measurements of the tumors. The error bars represent the SD of the sizes of three individual tumors of the same implantation background. *P < 0.05; **P < 0.01, significance of the comparison between the indicated sample groups. (C) (i to iv) H&E staining of the cross sections of the tumors that originated from the MM231 cell–laden DBT-TMS and DMM231 scaffolds with or without hydrogel coverage. The tumor ECM structure, cell distribution, and capillaries (containing the stained red blood cells) are demonstrated. (v to viii) IF staining of Ki-67 (green) and HER2 (red) on the tumor cross sections. The cell nuclei were stained with DAPI (blue). Scale bars, 100 μm (C, i to viii).

  • Fig. 5 Comparison of the sensitivities of the cancer cells grown on the different scaffolds to anticancer drugs.

    (A) The impact of the anticancer drugs on cell growth and proliferation supported by the DBT-TMS, collagen, lrECM, and PLGA scaffolds was analyzed and compared. The drug administration pattern and cell proliferation measurements are detailed in Materials and Methods. The error bars represent the SD of three independent experiments. The black and green lines within the plot area indicate the comparison of the average T47D or BT474 cell proliferation (day 8 to day 14) between the drug-treated groups and the nontreated control groups. The red lines indicate the comparison of the average cell proliferation between the collagen or PLGA scaffold groups and the DBT-TMS groups. **P < 0.01; ✪ , posttreatment recovery measurement. (B) Evaluation of the cell proliferation in response to Taxol or HT treatment in 2D cultures. Error bars represent the SD of the means of three independent experiments. **P < 0.01, comparison of the average cell proliferation (day 8 to day 14) between the drug-treated groups and the nontreated control groups. ✪ , posttreatment recovery measurement. (C) Proliferation/inhibition curve plots. The means (from three independent experiments) of the cell proliferation status on the different scaffolds on day 1 (the start date of cell proliferation), day 8 (1 day after the first treatment), day 14 (1 day after the last treatment), and day 21 (the end of recovery) as shown in (A) were plotted.

  • Table 1 The major proteins identified in mouse mammary tissue ECM that were preserved in TMS.

    The proteins were grouped according to their similarities in a family or their functions within ECM and were listed from high to low spectrum counts. FACIT, fibril-associated collagens with interrupted triple helices.

    #ProteinGeneAccession
    number
    Protein
    molecular
    mass (kDa)
    Spectrum
    count
    Functions
    Collagen1Collagen
    type I
    alpha 1 chain
    Col1a1P11087138899Strengthens tissue structure; participates in ECM organization;
    interacts with metal ions and other proteins; regulates cell
    mobility
    2Collagen
    type III
    alpha 1 chain
    Col3a1P08121139812Strengthens tissue structure; associates with and facilitates
    collagen I fibrillogenesis; participates in ECM organization;
    interacts with metal ions and other proteins; regulates
    cell mobility
    3Collagen
    type I alpha
    2 chain
    Col1a2Q01149130769Strengthens tissue structure; participates in ECM organization;
    interacts with metal ions and other proteins; regulates
    cell mobility
    4Collagen
    type V
    alpha 2 chain
    Col5a2Q3U962145143Mediates the assembly of other collagen fibrils; participates
    in ECM organization; interacts with metal ions and other
    proteins
    5Collagen
    type VI alpha
    3 chain
    Col6a3E9PWQ3354123A major structural component of microfibrils; links BMs to
    nearby cells; participates in ECM organization; interacts with
    other proteins
    6Collagen
    type II
    alpha 1 chain
    Col2a1P2848114262Adds structure and strength to connective tissues that resist
    compression; participates in ECM organization; interacts
    with metal ions and other proteins
    7Collagen
    type V
    alpha 1 chain
    Col5a1O8820718460Participates in heterotypic assembly with other collagen
    fibrils and organization of ECM; interacts with metal ions
    and other proteins; regulates cell mobility
    8Collagen
    type VII alpha
    1 chain
    Col7a1Q6387029556A major component of anchoring fibrils that contributes to
    epithelial BM organization and adherence by interacting with
    other ECM proteins; participates in ECM organization;
    regulates cell mobility
    9Collagen
    type IV
    alpha 2 chain
    Col4a2P0812216747A major structural component of BM that forms a meshwork
    together with laminins, proteoglycans, and nidogen/
    entactin; participates in ECM organization; regulates cell
    adhesion; its cleaved product canstatin inhibits
    angiogenesis and tumor growth
    10Collagen
    type V
    alpha 3 chain
    Col5a3Q9JLI217233Participates in heterotypic assembly with other collagen fibrils
    and organization of ECM; interacts with metal ions and other
    proteins; regulates cell mobility
    11Collagen
    type IV
    alpha 1 chain
    Col4a1P0246316127A major structural component of BM that forms a meshwork
    together with laminins, proteoglycans, and nidogen/
    entactin; participates in ECM organization; regulates cell
    adhesion; its cleaved product arresten inhibits
    angiogenesis and tumor growth
    12Collagen
    type VI
    alpha 1 chain
    Col6a1Q0485710820A major structural component of microfibrils; links BM to
    nearby cells; participates in ECM organization; interacts with
    other proteins
    13Collagen
    type VI
    alpha 2 chain
    Col6a2Q0278811015A major structural component of microfibrils; links BM to
    nearby cells; participates in ECM organization; interacts with
    other proteins
    14Collagen
    type XI
    alpha 2 chain
    Col11a2Q647391727Mediates the spacing and width of type II collagen; its
    proteolytic product poly(ADP-ribose) polymerase is
    involved in cellular stress response; interacts with calcium
    and metal ions
    15Collagen
    type XVI
    alpha 1 chain
    Col16a1A3KFV71224Maintains the integrity of ECM; regulates cell attachment
    and integrin-mediated cell spreading and morphology
    changes
    16Collagen
    type XIV
    alpha 1 chain
    Col14a1B7ZNH7(+2)1932Interacts with the interstitial collagen fibrils via type I
    collagen and mediates fibrillogenesis; plays an adhesive
    role by integrating collagen bundles; participates in ECM
    organization; regulates cell adhesion
    17Collagen
    type XXII
    alpha 1 chain
    Col22a1E9Q7P11602A member of the FACIT subgroup of the collagen family;
    specifically localizes to tissue junctions; acts as a cell
    adhesion ligand
    18Collagen
    type XV
    alpha 1 chain
    Col15a1A2AJY21381A member of the FACIT collagen family; its BM expression
    adheres the BM to the underlying connective tissue
    stroma; its cleaved product restin inhibits angiogenesis
    19Collagen
    type IV
    alpha 3 chain
    Col4a3Q9QZS01621A major structural component of BM that forms a meshwork
    together with laminins, proteoglycans, and nidogen/
    entactin; its cleaved fragment tumstatin has
    antiangiogenic and antitumor activities; participates in
    ECM organization; regulates cell adhesion
    20Collagen
    type IV
    alpha 5 chain
    Col4a5Q63ZW61621A major structural component of BM that forms a meshwork
    together with laminins, proteoglycans, and nidogen/
    entactin; participates in ECM organization; regulates cell
    adhesion
    Glycoprotein and
    proteoglycan/
    GAG
    21PeriostinPostnQ620099366Functions in tissue development and regeneration; binds to
    integrins to support adhesion and migration of epithelial
    cells; plays a role in cancer stem cell maintenance and
    metastasis
    22Laminin
    subunit
    gamma 1
    Lamc1F8VQJ317726The major noncollagenous constituent of BM; regulates cell
    adhesion, differentiation, migration, signaling, neurite
    outgrowth, and cancer metastasis; interacts with other
    ECM components; participates in ECM organization
    23Laminin
    subunit
    beta 1
    Lamb1E9QN70(+1)20213The major noncollagenous constituent of BM; regulates cell
    adhesion, differentiation, migration, signaling, neurite
    outgrowth, and cancer metastasis; interacts with other
    ECM components; participates in ECM organization
    24Laminin
    subunit
    alpha 1
    Lama1P191373389The major noncollagenous constituent of BM; regulates cell
    adhesion, differentiation, migration, signaling, neurite
    outgrowth, and cancer metastasis; interacts with other
    ECM components; participates in ECM organization
    25Laminin
    subunit
    beta 2
    Lamb2Q612921978The major noncollagenous constituent of BM; regulates cell
    adhesion, differentiation, migration, signaling, neurite
    outgrowth, and cancer metastasis; interacts with other
    ECM components; participates in ECM organization
    26Laminin
    subunit
    alpha 5
    Lama5Q610014045The major noncollagenous constituent of BM; regulates cell
    adhesion, differentiation, migration, signaling, neurite
    outgrowth, and cancer metastasis; interacts with other ECM
    components; participates in ECM organization
    27FibronectinFn1P1127627323Binds cell surface and collagen, fibrin, heparin, DNA, and
    actin; regulates type I collagen deposition, cell
    morphology, adhesion, migration, and opsonization;
    mediates angiogenesis and tumor metastasis; its cleaved
    product anastellin binds fibronectin and induces fibril
    formation
    28Fibrillin 1Fbn1A2AQ5331216Fibrillin is secreted into ECM by fibroblasts and incorporated
    into the insoluble microfibrils, which appear to provide a
    scaffold for deposition of elastin
    29Fibrinogen
    alpha chain
    FgaE9PV24(+1)8714Forms fibrinogen with fibrinogen beta and gamma chains;
    thrombin converts fibrinogen to fibrin, which mediates
    blood clotting; various cleaved products of fibrinogen
    and fibrin regulate cell adhesion and spreading, display
    vasoconstrictor and chemotactic activities, and act as
    mitogens
    30Fibrinogen
    gamma chain
    FggQ8VCM74913Forms fibrinogen with fibrinogen alpha and beta chains;
    thrombin converts fibrinogen to fibrin, which mediates
    blood clotting; various cleaved products of fibrinogen
    and fibrin regulate cell adhesion and spreading, display
    vasoconstrictor and chemotactic activities, and act as
    mitogens
    31Fibrinogen beta
    chain
    FgbQ8K0E8559Forms fibrinogen with fibrinogen alpha and gamma chains;
    thrombin converts fibrinogen to fibrin, which mediates
    blood clotting; various cleaved products of fibrinogen
    and fibrin regulate cell adhesion and spreading, display
    vasoconstrictor and chemotactic activities, and act as
    mitogens
    32Nidogen 1/
    entactin
    Nid1P104931379Essential component of BM; connects the networks formed
    by collagens and laminins to each other; plays a role in
    cell-ECM interactions
    33Tenascin-XTnxbO354524352The tenascins have antiadhesive effects, as opposed to
    fibronectin, which is adhesive; it is thought to function in
    matrix maturation
    34EMILIN 1Emilin1Q99K411081Associates with elastic fibers at the interface between
    elastin and microfibrils; may play a role in the
    development of elastic tissues including large blood
    vessels, dermis, heart, and lung
    35PerlecanHspg2E9PZ1647030BM-specific; serves as an attachment substrate for cells;
    plays essential roles in vascularization; its cleaved
    products (endorepellin and LG3) have antiangiogenic and
    antitumor properties; binds to calcium and metal ions;
    maintains the integrity of BM
    36LumicanLumP518853813A proteoglycan class II member of the small leucine-rich
    proteoglycan family; binds to other extracellular
    components and mediates collagen fibril organization;
    ubiquitously distributed in most mesenchymal tissues;
    regulates epithelial cell migration and tissue repair
    Other ECM
    proteins
    37TitinTtnA2ASS639069Essential component of sarcomeres in striated muscles;
    provides structural support, flexibility, and stability to cell
    structures; mediates chemical signaling; serves as
    connections between microfilaments; contributes to the
    fine balance of forces between the two halves of
    sarcomere
    38Perilipin 1Plin1Q8CGN5565Coats lipid storage droplets in adipocytes, thereby
    protecting them until they can be broken down by
    hormone-sensitive lipase; phosphorylation of perilipin is
    essential for the mobilization of fats in adipose tissues
    39Perilipin 4Plin4O884921393Highly expressed in white adipose tissues, with lower
    expression in heart, skeletal muscle, and brown adipose
    tissues; coats lipid droplets in adipocytes to protect them
    from lipases
    40ElastinElnP54320723The major component of elastic fibers for structural support of
    tissues; participates in ECM organization; allows tissues to
    resume their shape after stretching or contracting; a major
    contributor to tissue stiffness
    41DermatopontinDptQ9QZZ6243A proteoglycan-binding cell adhesion protein that
    potentially functions in cell-matrix interactions through
    integrins; may enhance TGFB1 activity, inhibit cell
    proliferation, accelerate collagen fibril formation, and
    stabilize collagen fibrils against low-temperature
    dissociation

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/9/e1700764/DC1

    fig. S1. Comparision of the differences of the major proteins identified from TMS/mouse mammary tissue ECM versus those from lrECM hydrogel.

    fig. S2. Monitor tumor growth with x-ray–based imaging.

    fig. S3. Tumor development from the cancer cell–laden scaffolds in mice mammary tissues.

    fig. S4. Quantification of the capillary areas on the cross sections of the tumors.

    fig. S5. Histological examination of the tumors developed from the cancer cells grown on the PLGA scaffolds.

    fig. S6. Analysis of T47D and BT474 cell proliferation on the different scaffolds and the potential of using the systems in drug screening.

    fig. S7. Cancer cell survival and growth status on TMS after drug treatment.

    fig. S8. Cancer cell survival and growth status on PLGA scaffolds after drug treatment.

    table S1. The major proteins identified in the lrECM.

    movie S1. Testing the sponginess of the TMS scaffolds.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Comparision of the differences of the major proteins identified from TMS/mouse mammary tissue ECM versus those from lrECM hydrogel.
    • fig. S2. Monitor tumor growth with x-ray–based imaging.
    • fig. S3. Tumor development from the cancer cell–laden scaffolds in mice mammary tissues.
    • fig. S4. Quantification of the capillary areas on the cross sections of the tumors.
    • fig. S5. Histological examination of the tumors developed from the cancer cells grown on the PLGA scaffolds.
    • fig. S6. Analysis of T47D and BT474 cell proliferation on the different scaffolds and the potential of using the systems in drug screening.
    • fig. S7. Cancer cell survival and growth status on TMS after drug treatment.
    • fig. S8. Cancer cell survival and growth status on PLGA scaffolds after drug treatment.
    • table S1. The major proteins identified in the lrECM.
    • Legend for movie S1

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1. (.mp4 format). Testing the sponginess of the TMS scaffolds.

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