Figure 1.
The effect of ASCs on the growth of MDA-MB 231 cells.
A. MDA-MB-231 were cultured in the bottom well of a Boyden Chamber and ASCs were cultured in the insert. Growth of MDA-MB-231 cells was assessed using the MTT assay. B. 2.5×104ASCs were cultured in 6 well plates for 24 hrs. prior to addition of MDA-MB-231-GFP breast cancer cells at a 1∶1 ratio. Bright field and fluorescent microscopy photographs were taken on days 1–4 after addition of the MDA-MB-231 cells. Data are representative of experiments using three different ASC donors.
Figure 2.
ASC effect on migration of MDA-MB-231 cells.
A. ASCs were cultured in the bottom well of a Boyden Chamber and MDA-MB-231 cells were cultured in the insert. Migration of MDA-MB-231 cells was assessed by using crystal violet staining of the insert membrane and quantification of color development. †P<0.02, *P<0.04. B. MDA-MB-231 cells were cultured 24 h followed by replacement with medium containing 0%, 20% or 50% growth conditioned media (GCM) or adipocyte-differentiated conditioned medium (ADCM) from ASCs. A horizontal scratch was made using a P200 pipette tip and bright field pictures were taken at 0 and 6 h (Figure S1) following the scratch wound. Graphical representation of % gap closure quantitated using ImageJ software (NIH, Bethesda, MD). **P<0.01, ***P<0.0001. Data are representative of experiments using three different ASC donors.
Figure 3.
ASC effect on primary MDA-MB-231 xenografts.
3×106 human MDA-MB-231/GFP breast cancer cells were bilaterally injected subcutaneously into the mammary fat pads of 5 female NUDE mice (n = 10 tumors/group) with or without 3×106 human ASC/RFP cells from donor with BMI 25.0 (A) or donor with BMI 18.3 (B).Tumor volume was monitored for 40 days by caliper measurement. Tumors were removed at day 40 and fluorescence of the intact, fresh tumors from the MDA-MB-231/GFP alone group (C) or MDA-MB-231/GFP+ASC/RFP group (D) were visualized for GFP and RFP within 10 minutes of removal using a dissecting fluorescent microscope. The white arrow indicates a region of RFP fluorescence only in the MDA-MB-231/GFP+ASC/RFP group tumors. E. 5 µM paraffin embedded section of MDA-MB-231/GFP and MDA-MB-231/GFP+ASC/RFP tumors were prepared for Hematoxylin and Eosin (H&E) staining. F. 10 µM frozen sections of tumors were stained with DAPI (blue) and prepared for fluorescence microscopy for GFP and RFP. DAPI+GFP (DG); DAPI+RFP (DR); DAPI+GFP+RFP (DGR).
Figure 4.
Metastasis of MDA-MB-231/GFP and MDA-MB-231/GFP+ASC/RFP tumors.
40 days after subcutaneous injection of either human MDA-MB-231/GFP cells, ASC/RFP cells or MDA-MB-231/GFP+ASC/RFP cells, mouse organs were collected. A. Visual macrometastatic lesions were observed in the liver, lungs only in mice co-injected with MDA-MB-231/GFP and ASC/RFP (arrows). B. H&E sections of the liver and lungs of mice bearing MDA-MB-231/GFP+ASC/RFP tumors showing metastatic MDA-MB-231 cancer cells (insets). C. To quantitate micrometastases, DNA was prepared from mouse organs from two separate experiments (n = 10 mice/group) for detection of human chromosome 17 by real time RT-PCR. A significant increase in micrometastasis for MDA-MB-231/GFP+ASC/RFP tumors was detected in liver, lung and spleen. * P<0.05.
Figure 5.
Detection of metastasis in whole organs by fluorescence quantitation.
Metastases in fresh, whole organs were quantitated by detection of green fluorescence protein in mouse liver, lung and spleen. Image J software was used to quantitate the area of the fluorescent signal on the image as described in the Materials and Methods.
Figure 6.
Metastatic lesions in lung and liver from the MDA-MB-231/GFP+ASC/RFP group tumors.
40 days after subcutaneous injection of MDA-MB-231/GFP+ASC/RFP cells, mouse organs were collected and 10 µM frozen sections were prepared for immunofluorescence of the lung and liver. A representative section of lung demonstrating multifocal metastatic lesions expressing GFP. A representative section of the liver demonstrated a small region expressing GFP. RFP was not detected above background level in any frozen tissue sections.
Figure 7.
IHC was conducted as described in Materials and Methods.Paraffin-embedded tumor sections from MDA-MB-231/GFP and the MDA-MB-231/GFP+ASC/RFP groups were stained for vimentin, MMP9, IL-8, CD-31, and VEGF. Bright-field photomicrographs were taken and representative images are presented. Quantitative representation of the staining is indicated.