Figure 1.
MSC enhances 4T1 cell proliferation and tumor growth.
(a,b) To assess cell proliferation, 4T1 or JC cells were cultured in RPMI-1640 medium or MSC-CM with the medium changed every 2 days. Cells were counted from 6 wells at each time point after culture at the indicated time points. Cells cultured in CM proliferated more than those cultured in regular medium. (c, d) To carry out the colony forming assay, 500 4T1 or JC cells were seeded and cultured using RPMI-1640 medium or MSC-CM. The colonies were stained with crystal violet after being cultured for 6 days. The cells grown using MSC-CM showed greater potential to form colonies. (c) 5×105 GFP-expressing 4T1 cells alone or premixed with an equal amount of RFP-expressing MSCs were injected subcutaneously into the mammary fat pad of nude mice. Each group consisted of ten mice. Tumor growth was measured twice a week and the data are shown as the mean ± SD. Tumors derived from a mixture of 4T1 cells and MSCs had significantly enhanced growth compared to tumors derived from 4T1 cells alone. All data are shown as the mean ± SD. “*” represents a significant difference of p<0.05.
Figure 2.
Tumor immunostaining for proliferation, apoptosis and angiogenesis.
5×105 GFP-expressing 4T1 cells alone or premixed with an equal amount of RFP-expressing MSCs were injected subcutaneously into the mammary fat pad of nude mice. Tumors were excised for subsequent sectioning and immunostaining. Tumor sections from a 4T1 tumor or a 4T1+MSCs tumor were stained with antibody raised against Ki67 (a), CD31 (e) or subjected to TUNEL assay (c). (b, d and f) Quantification of Ki67, CD31 and TUNEL staining of tumor sections from a 4T1 tumor or a 4T1+MSCs tumor. Data represent mean values ±SD (n = 3). “*” represents a significant difference of p<0.001.
Figure 3.
MSC-CM enhances 4T1 cell migration and lung metastasis.
(a,b) Representative photomicrographs of the gaps of 4T1 and JC cells for each culture condition at different time points after scratching in a wound healing assay (upper). Quantification of wound-healing was assessed by measuring gap distance (lower). The cells showed a higher migratory ability when cultured in MSC-CM compared to cells cultured in RPMI-1640 medium. Data are presented as the percentage change in gap distance relative to 0 hr. Each bar shows mean ± SD of six measurements of each gap. Asterisk indicates a significant difference using the Student’s t test. (*p<0.05) (c) For the in vivo lung metastasis assay, 5×105 GFP-expressing 4T1 cells alone, or premixed with an equal number of MSCs, were injected into the mammary fat pad. Each group contained at least nine mice. Three weeks later, the mice were sacrificed and their lungs excised and then imaged using the Olympus OV100 Imaging System with a constant exposure time and offset. Representative fluorescence images of the various lobes derived from the same lung. Lung tissue was excised from mice bearing 4T1 (right panel, n = 10) or 4T1+MSCs (left panel, n = 9) tumors. (d) Calculated number of GFP-fluorescent lung colonies. The number of colonies present in five lobes per lung, from both lungs was counted. MSCs promote spontaneous lung metastasis of orthotopic 4T1 tumors. Data are presented as mean ± SD with a significant difference (p = 0.03).
Figure 4.
MSCs enhance 4T1 mammosphere formation and tumorigenicity.
For the mammosphere formation assay, 4T1 cells were cultured in regular medium or MSC-CM for 1 week. After typsinization, a single-cell suspension was confirmed by microscopic observation. The cells were then seeded at 2000 cells/100 µl/well into 96 well ultra-low-attachment plates and cultured in mammosphere-forming medium. The number of spheres formed was counted after 1 week incubation. (a) Number of spheres formed from 2000 cells pre-cultured with RPMI-1640 or MSC-CM and counted from five wells. Cells pre-cultured in MSC-CM formed mammospheres more efficiently than those cultured in RPMI-1640 medium (p = 0.03). Data are presented as the mean ± SD. To evaluate the in vivo tumorigenic potential of 4T1 cell, a total of either 5, 10 or 100 RFP-expressing 4T1 cells alone or premixed with 1×105 GFP-expressing MSCs, were injected into the mammary fat pad. Tumor incidence was observed and imaged. (b) Representative image of tumorigenesis. The white and black arrows indicate the sites where 4T1 cells mixed with MSCs or 4T1 cells alone were injected, respectively. Tumor growth was recorded and is shown in (b). (c) Summary of tumor formation when analyzed using limiting dilution.
Figure 5.
In vivo imaging of MSC-4T1 cell-cell interaction.
GFP-expressing MSCs and RFP-expressing 4T1 cells (5×105 each) were co-implanted into the mouse thoracic mammary fat pad. (a) 5 days after implantation, an arc shape incision was made in the abdominal skin. A skin flap was lifted above the mammary fat pad and imaged with the OV-100. White arrow indicates the location of tumor. (b–d). GFP and RFP signals were detected from the tumor which was surrounded by a dense vascular network. (e) The excised tumor was cross-sectioned and imaged using the Olympus IV100. GFP and RFP-expressing cells were observed within the tumor. (f) On day 11, GFP-MSCs were rarely observed within the tumor, suggesting MSCs were not proliferating during the growing of tumor.
Figure 6.
In vivo imaging of 4T1 tumorigenesis.
Ten RFP-expressing 4T1 cells were injected alone or co-injected with 1×105 GFP-expressing MSCs. An arc-shaped incision was made in the thoracic and abdominal skin and imaged with the OV-100 over time. (a∼k) Tumor initiation by ten 4T1 cells in the presence of MSCs was monitored. (l) No tumor incidence was found when 10 4T1 cells without MSCs were injected.
Table 1.
Cluster of genes involved in the positive and negative regulation of apoptosis.
Table 2.
Cluster of genes involved in the positive and negative regulation of proliferation.
Table 3.
Genes classified into oncogenes, tumor markers and tumor promoters.