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.
# Protein Gene Accession
numberProtein
molecular
mass (kDa)Spectrum
countFunctions Collagen 1 Collagen
type I
alpha 1 chainCol1a1 P11087 138 899 Strengthens tissue structure; participates in ECM organization;
interacts with metal ions and other proteins; regulates cell
mobility2 Collagen
type III
alpha 1 chainCol3a1 P08121 139 812 Strengthens tissue structure; associates with and facilitates
collagen I fibrillogenesis; participates in ECM organization;
interacts with metal ions and other proteins; regulates
cell mobility3 Collagen
type I alpha
2 chainCol1a2 Q01149 130 769 Strengthens tissue structure; participates in ECM organization;
interacts with metal ions and other proteins; regulates
cell mobility4 Collagen
type V
alpha 2 chainCol5a2 Q3U962 145 143 Mediates the assembly of other collagen fibrils; participates
in ECM organization; interacts with metal ions and other
proteins5 Collagen
type VI alpha
3 chainCol6a3 E9PWQ3 354 123 A major structural component of microfibrils; links BMs to
nearby cells; participates in ECM organization; interacts with
other proteins6 Collagen
type II
alpha 1 chainCol2a1 P28481 142 62 Adds structure and strength to connective tissues that resist
compression; participates in ECM organization; interacts
with metal ions and other proteins7 Collagen
type V
alpha 1 chainCol5a1 O88207 184 60 Participates in heterotypic assembly with other collagen
fibrils and organization of ECM; interacts with metal ions
and other proteins; regulates cell mobility8 Collagen
type VII alpha
1 chainCol7a1 Q63870 295 56 A 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 mobility9 Collagen
type IV
alpha 2 chainCol4a2 P08122 167 47 A 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 growth10 Collagen
type V
alpha 3 chainCol5a3 Q9JLI2 172 33 Participates in heterotypic assembly with other collagen fibrils
and organization of ECM; interacts with metal ions and other
proteins; regulates cell mobility11 Collagen
type IV
alpha 1 chainCol4a1 P02463 161 27 A 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 growth12 Collagen
type VI
alpha 1 chainCol6a1 Q04857 108 20 A major structural component of microfibrils; links BM to
nearby cells; participates in ECM organization; interacts with
other proteins13 Collagen
type VI
alpha 2 chainCol6a2 Q02788 110 15 A major structural component of microfibrils; links BM to
nearby cells; participates in ECM organization; interacts with
other proteins14 Collagen
type XI
alpha 2 chainCol11a2 Q64739 172 7 Mediates 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 ions15 Collagen
type XVI
alpha 1 chainCol16a1 A3KFV7 122 4 Maintains the integrity of ECM; regulates cell attachment
and integrin-mediated cell spreading and morphology
changes16 Collagen
type XIV
alpha 1 chainCol14a1 B7ZNH7(+2) 193 2 Interacts 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 adhesion17 Collagen
type XXII
alpha 1 chainCol22a1 E9Q7P1 160 2 A member of the FACIT subgroup of the collagen family;
specifically localizes to tissue junctions; acts as a cell
adhesion ligand18 Collagen
type XV
alpha 1 chainCol15a1 A2AJY2 138 1 A member of the FACIT collagen family; its BM expression
adheres the BM to the underlying connective tissue
stroma; its cleaved product restin inhibits angiogenesis19 Collagen
type IV
alpha 3 chainCol4a3 Q9QZS0 162 1 A 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 adhesion20 Collagen
type IV
alpha 5 chainCol4a5 Q63ZW6 162 1 A major structural component of BM that forms a meshwork
together with laminins, proteoglycans, and nidogen/
entactin; participates in ECM organization; regulates cell
adhesionGlycoprotein and
proteoglycan/
GAG21 Periostin Postn Q62009 93 66 Functions 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
metastasis22 Laminin
subunit
gamma 1Lamc1 F8VQJ3 177 26 The major noncollagenous constituent of BM; regulates cell
adhesion, differentiation, migration, signaling, neurite
outgrowth, and cancer metastasis; interacts with other
ECM components; participates in ECM organization23 Laminin
subunit
beta 1Lamb1 E9QN70(+1) 202 13 The major noncollagenous constituent of BM; regulates cell
adhesion, differentiation, migration, signaling, neurite
outgrowth, and cancer metastasis; interacts with other
ECM components; participates in ECM organization24 Laminin
subunit
alpha 1Lama1 P19137 338 9 The major noncollagenous constituent of BM; regulates cell
adhesion, differentiation, migration, signaling, neurite
outgrowth, and cancer metastasis; interacts with other
ECM components; participates in ECM organization25 Laminin
subunit
beta 2Lamb2 Q61292 197 8 The major noncollagenous constituent of BM; regulates cell
adhesion, differentiation, migration, signaling, neurite
outgrowth, and cancer metastasis; interacts with other
ECM components; participates in ECM organization26 Laminin
subunit
alpha 5Lama5 Q61001 404 5 The major noncollagenous constituent of BM; regulates cell
adhesion, differentiation, migration, signaling, neurite
outgrowth, and cancer metastasis; interacts with other ECM
components; participates in ECM organization27 Fibronectin Fn1 P11276 273 23 Binds 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
formation28 Fibrillin 1 Fbn1 A2AQ53 312 16 Fibrillin is secreted into ECM by fibroblasts and incorporated
into the insoluble microfibrils, which appear to provide a
scaffold for deposition of elastin29 Fibrinogen
alpha chainFga E9PV24(+1) 87 14 Forms 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
mitogens30 Fibrinogen
gamma chainFgg Q8VCM7 49 13 Forms 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
mitogens31 Fibrinogen beta
chainFgb Q8K0E8 55 9 Forms 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
mitogens32 Nidogen 1/
entactinNid1 P10493 137 9 Essential component of BM; connects the networks formed
by collagens and laminins to each other; plays a role in
cell-ECM interactions33 Tenascin-X Tnxb O35452 435 2 The tenascins have antiadhesive effects, as opposed to
fibronectin, which is adhesive; it is thought to function in
matrix maturation34 EMILIN 1 Emilin1 Q99K41 108 1 Associates 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 lung35 Perlecan Hspg2 E9PZ16 470 30 BM-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 BM36 Lumican Lum P51885 38 13 A 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 repairOther ECM
proteins37 Titin Ttn A2ASS6 3906 9 Essential 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
sarcomere38 Perilipin 1 Plin1 Q8CGN5 56 5 Coats 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 tissues39 Perilipin 4 Plin4 O88492 139 3 Highly 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 lipases40 Elastin Eln P54320 72 3 The 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 stiffness41 Dermatopontin Dpt Q9QZZ6 24 3 A 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.
Additional Files
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
Other Supplementary Material for this manuscript includes the following:
- movie S1. (.mp4 format). Testing the sponginess of the TMS scaffolds.
Files in this Data Supplement:
- fig. S1. Comparision of the differences of the major proteins identified from
TMS/mouse mammary tissue ECM versus those from lrECM hydrogel.
