Fig. 1Fibrin hydrogel-supported 3-dimensional organ culture of the gastric submucosa (GS). (A) Phase-contrast microphotographs show robust cell outgrowth into the hydrogel from the embedded GS. High-power observation discloses outgrown cells with the fibroblastoid feature in the hydrogel (a) and in situ renewed cells inside of the GS (b). (B) After two weeks of organ culture, the number of cells increases in the fibrin hydrogel (FG) and the GS. Most cells express or incorporate proliferating cell nuclear antigen (PCNA) or bromodeoxyuridine (BrdU). (C) The limited dilution assay indicates the highly clonogenic potential of recovered outgrown cells from the hydrogel with fibroblastoid morphology. H&E, hematoxylin and eosin.
Fig. 2The immunophenotype, localization, and differentiation potential of in situ renewed and outgrown cells. (A) Immunohistochemical staining reveals that outgrown cells express mesenchymal stem cell markers and pericyte markers but do not express endothelial cell markers. (B) Hematoxylin and eosin staining shows increased cell density around the microvessels at day 14 of culture compared to day 0. Immunofluorescent staining shows perivascular localization of in situ renewed cells with a mesenchymal stem cell-and pericyte-like phenotype. (C, D) Nuclei are counterstained with propidium iodide (red). Subcultured outgrown cells show differentiation potential into adipogenic (C, Oil red O), osteogenic (C, Alizarin red), and endothelial cells (D). Cells induced with vascular endothelial growth factor form a capillary-like network at 12 hours. Then, cells aggregate with each other to form a spheroid structure at day 3 and express FIk-1, CD31, and CD34. SMA, smooth muscle actin; GS, gastric submucosa; FG, fibrin hydrogel.
Fig. 3Paracrine effects of gastric cancer cells on gastric submucosa-derived mesenchymal stem cells (GS-MSCs). Conditioned gastric cancer cell line media (AGS, NCI-N87, and SNU-1), adipose-derived stem cells (ADSC), and HEK293 are used. (A) GS-MSCs migration do not significantly increased in cells incubated with conditioned gastric cancer cell media. **p<.01 compared to vehicle. (B) Cell proliferation increases meaningfully in GS-MSCs incubated with the conditioned gastric cancer cell media. *p<.05 compared to vehicle. (C) The tube length and branch point of capillary-like network significantly increase in GS-MSCs induced by conditioned gastric cancer cell media. *p<.05 compared to HEK293. (D) Endothelial cell-specific mRNAs measured with quantitative real-time reverse transcriptase polymerase chain reaction are highly expressed in GS-MSCs induced by conditioned gastric cancer cell media. vWF, von Willebrand factor. **p<.01 compared to HEK293. (E) Quantitative analysis of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) contents with enzyme-linked immunosorbent assay in conditioned gastric cancer cell line media.
Fig. 4The effect of gastric submucosa-derived mesenchymal stem cells (GS-MSCs) on the growth of gastric cancer cells. (A) Conditioned GS-MSCs media (CM) enhances in vitro tumor spheroid formation of NCI-N87 in a dose-dependent manner. *p<.05 and **p<.01 compared to vehicle. (B) The weight of the tumor measured at three weeks post-injection increases in mice injected with NCI-N87 and GS-MSCs. *p<.05 compared to NCI-N87 (2×106 cells). (C) Tumors formed by injection of NCI-N87 and GS-MSCs show decreased coagulative necrosis (asterisk) but increased CD34+ microvascular density compared to tumors formed by injection of NCI-N87 only. (D) Tumors formed by injection of NCI-N87 and GS-MSCs show increased cellular and vascular stroma (arrow, hematoxylin and eosin [H&E]) compared to tumors formed by injection of NCI-N87 only. Human-specific mitochondrial antigen (hMt-Ag) is uniformly expressed in all cancer cells, but hMt-Ag+ stromal cells (arrows) are detected in tumors formed by NCI-N87 and GS-MSC injections.
Fig. 5The contribution of gastric submucosa-derived mesenchymal stem cells (GS-MSCs) to the formation of cancer stroma. Immunofluorescent staining reveals that human-specific mitochondrial antigen (hMT-Ag)+ stromal cells coexpress CD105, CD140b, and smooth muscle actin (SMA) and are easily observed in the cancer stroma formed by co-injection of NCI-N87 and GS-MSCs, but hMT-Ag+/CD34+ endothelial cells are rarely observed. Nuclei are counterstained with ToPro.
Fig. 6Representative microphotographs of double immunostaining against human-specific mitochondrial antigen and stromal cell markers (CD105, CD140b, and smooth muscle actin [SMA]) and endothelial cells (CD34) in the cancer stroma formed by injection of NCI-N87 only. hMT-Ag, human-specific mitochondrial antigen. Nuclei are counterstained with ToPro.
Table 1.Antibodies used in the study
Antibody |
Application |
Dilution |
Company |
CD29 |
IHC, IF |
1:200 |
Novus Biologicals |
CD31 |
IHC, IF |
1:40 |
Dako |
CD34 |
IHC, IF |
1:800 |
Neomarker |
CD44 |
IHC |
1:40 |
Leica |
CD105 |
IHC, IF |
1:500 |
BD Bioscicence |
CD140b |
IHC, IF |
1:400 |
Abcam |
CD146 |
IHC |
1:200 |
Abcam |
SMA |
IHC, IF |
1:400 |
Dako |
Vimentin |
IHC |
1:200 |
Zymed |
Human-specific mitochondrial antigen |
IHC, IF |
1:200 |
Millipore |
Delight 488-goat anti-mouse IgG |
IF, FCM |
1:800 |
Jackson Immunoresearch |
Delight 488-goat anti-rabbit IgG |
IF, FCM |
1:800 |
Jackson Immunoresearch |
Delight 594-goat anti-mouse IgG |
IF, FCM |
1:800 |
Jackson Immunoresearch |
Delight 591-goat anti-rabbit IgG |
IF, FCM |
1:800 |
Jackson Immunoresearch |
HRP-conjugated anti-rabbit/mouse IgG |
IHC |
|
Dako |
CD14 (PE conjugated) |
FCM |
1:100 |
BD Bioscience |
CD29 (PE conjugated) |
FCM |
1:100 |
BD Bioscience |
CD31 (PE conjugated) |
FCM |
1:100 |
BD Bioscience |
CD34 (FITC conjugated) |
FCM |
1:100 |
BD Bioscience |
CD44 (PE conjugated) |
FCM |
1:100 |
BD Bioscience |
CD45 (PE conjugated) |
FCM |
1:100 |
BD Bioscience |
CD73 (PE conjugated) |
FCM |
1:100 |
BD Bioscience |
CD90 (PE conjugated) |
FCM |
1:100 |
BD Bioscience |
CD105 (unconjugated) |
FCM |
1:100 |
R&D Systems |
CD133 (PE conjugated) |
FCM |
1:100 |
Mitenyl Biotech |
CD140b (unconjugated) |
FCM |
1:100 |
Abcam |
CD146 (unconjugated) |
FCM |
1:100 |
Abcam |
c-Kit (unconjugated) |
FCM |
1:100 |
eBioscience |
Flk-1 (unconjugated) |
FCM |
1:100 |
R&D Systems |
SMA (unconjugated) |
FCM |
1:100 |
Dako |
MHC-1 (PE conjugated) |
FCM |
1:100 |
BD Bioscience |
MHC-II (PE conjugated) |
FCM |
1:100 |
BD Bioscience |
Table 2.Primers used in the study
Gene |
Primer sequence (5’-3’) |
β-Actin |
F: AGCAAGCAGGAGTATGACGA |
|
R: TGTGAACTTTGGGGGATG |
CD31
|
F: GTGGTGGAGTCTGGAGAGGA |
|
R: TCCGATGATAACCACTGCAA |
CD34
|
F: AAGTGAAATTGACTCAGGGC |
|
R: CCAGTGCAATCAGGGTCTTT |
CD144
|
F: GGATGACCAAGTACAGC |
|
R: ACACACTTTGGGCTGGTAGG |
VWF
|
F: TGGATGAGCTTTTGCAGACC |
|
R: GTGGGAGCCGTCGTGGTACT |
Table 3.Expression rate of the surface markers analyzed with flow cytometry
Antibody |
Expression rate (%) |
CD29 |
97.63 ± 5.21 |
CD44 |
97.30 ± 3.25 |
CD73 |
94.33 ± 3.47 |
CD90 |
95.83 ± 4.87 |
CD105 |
93.93 ± 5.28 |
HLA-ABC |
95.57 ± 3.14 |
HLA-DR |
0.84 ± 0.02 |
CD14 |
0.54 ± 0.01 |
CD34 |
0.87 ± 0.05 |
CD45 |
0.71 ± 0.04 |
CD133 |
0.21 ± 0.01 |
SMA |
81.13 ± 9.78 |
CD140b |
94.90 ± 6.57 |
STRO-1 |
47.90 ± 7.98 |
c-Kit |
0.01 ± 0.01 |
Flk-1 |
0.01 ± 0.01 |