Cell surface marker profiles of EAEASCs and WtASCs.

<p>A) Cells were analyzed by flow cytometry for MSC surface markers CD29, Sca1; hematopoietic markers CD34 and CD45; phagocytic lineage marker CD11b; and endothelial marker CD31. Gray filled: isotype control; gray line: WtASCs; black line: EAEASCs. B) Cell size based on forward scatter signal of flow cytometry (<i>P</i> = 0.26, t-test, n = 7).</p

Clinical scoring for disease onset and progression.

<p>The clinical scores for the HBSS-treated (n = 20), EAEASC-treated (n = 20) and WtASC-treated (n = 20) EAE mouse groups were recorded daily for the duration of the study. WtASCs significantly reduced the clinical symptoms from PDI13 where the EAEASCs failed to mediate any therapeutic efficacy or improvement. # means <i>P</i><0.01 vs HBSS-treated EAE group (post-hoc, n = 20); • means <i>P</i><0.01 vs EAEASCs-treated EAE group (post-hoc, n = 20).</p

Lesion and cell infiltration analysis on the spinal cord.

<p>Spinal cords were collected at sacrifice from 3 mice per group. Each spinal cord was sectioned, mounted, and stained with anti-CD3, CD11b, and CD45 followed by Hematoxylin counterstain for lesion size, number and cell infiltration analysis. # means P<0.05 vs EAEASC-treated EAE group (post-hoc, n = 9); & means P<0.05 vs HBSS-treated EAE group (post-hoc, n = 9).</p

Real-time PCR analysis of cytokine and chemokine profiles of EAEASCs and WtASCs.

<p>Both cell types were cultured overnight, trypsinized, and analyzed by real-time PCR to determine the expression levels of various cytokines and chemokines TNFα, IL-6, MCP-1, MIP-1α, RANTES, KC, MIP-2α and VEGF. # means <i>P</i><0.05 EAEASCs vs WtASCs (t-test, n = 3).</p

Pro-inflammatory cytokine protein levels in mouse serum.

<p>Sera from 5 mice per group at sacrifice were pooled and analyzed by ELISA to detect levels of TNF-α, IL-12, and IL-17. # means <i>P</i><0.05 vs HBSS-treated EAE group (post-hoc, n = 5); • means <i>P</i><0.05 vs EAEASCs-treated EAE group (post-hoc, n = 5); & means <i>P</i><0.05 vs normal naïve mouse group (post-hoc, n = 5).</p

Colony forming unit assays for EAEASCs and WtASCs.

<p>A total of 100 cells were plated on 56.7² Nunc cell culture plates and incubated for 14 days. Cells were stained with 3% crystal violet, and colonies 2 mm or larger in diameter were counted. # means <i>P</i><0.05 vs WtASCs (t-test, n = 5).</p

Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Triple Negative Breast Cancer Xenografts

<div><p>Background</p><p>Fat grafting is used to restore breast defects after surgical resection of breast tumors. Supplementing fat grafts with adipose tissue-derived stromal/stem cells (ASCs) is proposed to improve the regenerative/restorative ability of the graft and retention. However, long term safety for ASC grafting in proximity of residual breast cancer cells is unknown. The objective of this study was to determine the impact of human ASCs derived from abdominal lipoaspirates of three donors, on a human breast cancer model that exhibits early metastasis.</p><p>Methodology/Principal Findings</p><p>Human MDA-MB-231 breast cancer cells represents “triple negative” breast cancer that exhibits early micrometastasis to multiple mouse organs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089595#pone.0089595-Anbalagan1" target="_blank">[1]</a>. Human ASCs were derived from abdominal adipose tissue from three healthy female donors. Indirect co-culture of MDA-MB-231 cells with ASCs, as well as direct co-culture demonstrated that ASCs had no effect on MDA-MB-231 growth. Indirect co-culture, and ASC conditioned medium (CM) stimulated migration of MDA-MB-231 cells. ASC/RFP cells from two donors co-injected with MDA-MB-231/GFP cells exhibited a donor effect for stimulation of primary tumor xenografts. Both ASC donors stimulated metastasis. ASC/RFP cells were viable, and integrated with MDA-MB-231/GFP cells in the tumor. Tumors from the co-injection group of one ASC donor exhibited elevated vimentin, matrix metalloproteinase-9 (MMP-9), IL-8, VEGF and microvessel density. The co-injection group exhibited visible metastases to the lung/liver and enlarged spleen not evident in mice injected with MDA-MB-231/GFP alone. Quantitation of the total area of GFP fluorescence and human chromosome 17 DNA in mouse organs, H&E stained paraffin sections and fluorescent microscopy confirmed multi-focal metastases to lung/liver/spleen in the co-injection group without evidence of ASC/RFP cells.</p><p>Conclusions</p><p>Human ASCs derived from abdominal lipoaspirates of two donors stimulated metastasis of MDA-MB-231 breast tumor xenografts to multiple mouse organs. MDA-MB-231 tumors co-injected with ASCs from one donor exhibited partial EMT, expression of MMP-9, and increased angiogenesis.</p></div

ASC effect on migration of MDA-MB-231 cells.

<p>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>B.</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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089595#pone.0089595.s001" target="_blank">Figure S1</a>) 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.</p

The effect of ASCs on the growth of MDA-MB 231 cells.

<p>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>B.</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.</p

ASC effect on primary MDA-MB-231 xenografts.

<p> 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 (<b>C</b>) or MDA-MB-231/GFP+ASC/RFP group (<b>D</b>) 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. <b>E.</b> 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. <b>F.</b> 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).</p