An Animal Model of Local Breast Cancer Recurrence in the Setting of Autologous Fat Grafting for Breast Reconstruction

Abstract

Autologous fat grafting after breast cancer surgery is commonly performed, but concerns about oncologic risk remain. To model the interaction between fat grafting and breast cancer cells, two approaches were employed. In the first approach, graded numbers of viable MDA-MB-231 or BT-474 cells were admixed directly into human fat grafts and injected subcutaneously into immune-deficient mice to determine if the healing graft is a supportive environment for the tumor. In the second approach, graded doses of MDA-MB-231 cells were suspended in Matrigel and injected into the mammary fat pads of mice. Two weeks after the tumor cells engrafted, 100 μL of human adipose tissue was grafted into the same site. Histologically, MDA-MB-231 cells seeded within fat grafts were observed and stained positive for human-specific pan-cytokeratin and Ki67. The BT-474 cells failed to survive when seeded within fat grafts at any dose. In the second approach, MDA-MB-231 cells had a strong trend toward lower Ki67 staining at all doses. Regression analysis on all groups with fat grafts and MDA-MB-231 revealed fat tissue was associated with lower cancer cell Ki67 staining. Healing fat grafts do not support the epithelial BT-474 cell growth, and support the mesenchymal MDA-MB-231 cell growth only at doses ten times greater than in Matrigel controls. Moreover, fat grafts in association with MDA-MB-231 cancer cells already present in the wound resulted in decreased tumor proliferation and increased fibrosis. These findings suggest that clinical fat grafting does not induce breast cancer cell growth, and may even have a suppressive effect. Stem Cells Translational Medicine 2018;7:125-134.

Keywords: Animal model; Autologous fat grafting; BT-474; Breast cancer local recurrence; MDA-MB-231; NOD scid gamma mice.

Figure 1

(A): Microscopic imaging of BT‐474 and MDA‐MB‐231 cells in culture. Each scale bar indicates 200 µm. (B): Phenotypic characterization of BT474 (left column) and MDA‐MB231 (right column) cell lines. The top panels show identification of cytokeratin+ epithelial cells and DAPI+ nucleated cells. These were further phenotyped (green box) for CD44, CD90, CD73, and E‐cadherin expression (bottom panels). Direct comparison of BT474 and MDAMB231 phenotypes confirmed that BT474 is a purely epithelial breast cancer cell line (Cytokeratin+, CD44−, CD90−, E‐cadherin+, and CD73−), while the MDA‐MB‐231 cell line co‐expresses mesenchymal markers CD44 and CD73 (Cytokeratin+, CD44+, CD90−, E‐cadherin−, and CD73+). A small subset of the MDA‐MB‐231 cell line also co‐expresses CD90 (<0.5%). Abbreviation: DAPI, 4′,6‐diamino‐2‐phenylindole.

Figure 2

Macroscopic appearance of MDA‐MB‐231 plus lipo (A), 10 × 10³ and 1 × 10³ MDA‐MB‐231 cells injected with 300 µl of lipo on right and left side. Macroscopic appearance of MDA‐MB‐231 cells plus Matrigel; 10 × 10³, 1 × 10³, 100, and 10 MDA‐MB‐231 cells injected with Matrigel on upper left, upper right, lower left, and lower right (B). (C): Regression analysis on graft mass and weight of tumor and fibrosis (g) between the MDA‐MB‐231 plus lipo group and the MDA‐MB‐231 plus Matrigel group. Regression analysis showed no effect on tumor mass when lipo was co‐injected (p = .231). As more MDA‐MB‐231 cancer cells were co‐injected, bigger masses of tumor plus fibrosis were obtained (p < .001). When cancer cells were not injected, no significant tumor mass was seen (p = .263). Histological pictures of MDA‐MB‐231 plus lipo grafts (D) and MDA‐MB‐231 plus Matrigel grafts (E). H&E (I), human specific pan‐cytokeratin (II), Ki67 (III), and negative control (IV) are shown from left to right. Each scale bar depicts 200 µm. (F): Regression analysis of the effect of lipo on pan‐cytokeratin/Ki67 positive MDA‐MB‐231 cells. Lipoaspirate in association with MDA‐MB‐231 tumor cells resulted in decreased tumor cell proliferation index (% positive Ki67 stained tumor cells) (p = .001).

Figure 3

Macroscopic appearance of BT‐474 plus lipo; 1 × 10⁶ and 100 × 10³ BT‐474 cancer cells were co‐injected subcutaneously with 300 µl of lipo on right and left side of back (A). Macroscopic appearance of BT‐474 plus Matrigel; 1 × 10⁶ and 100 × 10³ BT‐474 cells were subcutaneously injected with 50 µl of Matrigel and media on the right and left abdominal side (B), 10 × 10³, 1 × 10³, 100, and 10 BT‐474 cancer cells were co‐injected with 50 µl of Matrigel plus media on upper left, upper right, lower left, and lower right of the back (C). (D): Regression analysis on graft mass and weight of tumor and fibrosis (g) between BT‐474 plus lipo group and BT‐474 plus Matrigel group. Regression analysis depicts the significant effect on tumor mass when lipo was co‐injected (p < .001). As more tumor cells were injected, bigger tumor plus fibrosis masses were obtained (p = .000). Histological observations of BT‐474 plus lipo (E): and BT‐474 plus Matrigel (F): grafts. H&E (I), human specific pan‐cytokeratin (II), Ki67 (II), and negative control (IV) are shown from left to right. Each scale bar depicts 200 µm. (G): Regression analysis of the effect of lipo on pan‐cytokeratin/Ki67 positive BT‐474 cancer cells. In the BT‐474 plus Matrigel group, pan‐cytokeratin/Ki67 positive tumors were obtained dose‐dependently. No BT‐474 plus 300 µl human lipo grafts showed pan‐cytokeratin/Ki67 positivity (p = .033).

Figure 4

Macroscopic appearance of MDA‐MB‐231 plus Matrigel followed by lipo; 100, 30, 10, and 3 MDA‐MB‐231 cells were subcutaneously injected with 50 µl of media and Matrigel on the upper right, upper left, lower right, and lower left abdominal sides followed by 100 µl lipo injection on each site after 2 weeks (A): As a positive control, 100, 30, 10, and 3 MDA‐MB‐231 cells were subcutaneously injected with 50 µl of Matrigel and media on upper right, upper left, lower right, and lower left abdominal sides (B). (C): Regression analysis on graft mass and mass of tumor and fibrosis (g) between the MDA‐MB‐231 plus Matrigel followed by lipo injection group and the MDA‐MB‐231 plus Matrigel group. Regression analysis showed a significant effect on tumor mass when lipo was co‐injected (p = .000). As more tumor cells were co‐injected, bigger tumor plus fibrosis masses were obtained (p < .001). Histological observations of MDA‐MB‐231 plus Matrigel followed by lipo grafts (D) and MDA‐MB‐231 plus Matrigel grafts (E). H&E (I), human specific pan‐cytokeratin (II), Ki67 (III), and negative control (IV) are shown from left to right. Each scale bar depicts 200 µm.