Regenerative therapy and cancer: in vitro and in vivo studies of the interaction between adipose-derived stem cells and breast cancer cells from clinical isolates

Abstract

Adipose-derived stem cells (ASCs) have been proposed to stabilize autologous fat grafts for regenerative therapy, but their safety is unknown in the setting of reconstructive surgery after mastectomy. Both bone marrow mesenchymal stem cells (MSCs) and ASC have been shown to enhance tumorigenesis of established breast cancer cell lines, but primary patient material has not been tested. Here, we ask whether ASC promote the in vitro growth and in vivo tumorigenesis of metastatic breast cancer clinical isolates. Metastatic pleural effusion (MPE) cells were used for coculture experiments. ASC enhanced the proliferation of MPE cells in vitro (5.1-fold). For xenograft experiments (100 sorted cells/injection site), nonhematopoietic MPE cells were sorted into resting and active populations: CD90+ resting (low scatter, 2.1%≥2N DNA), CD90+ active (high scatter, 10.6%≥2N DNA), and CD90-. Resting CD90+ MPE cells were tumorigenic in 4/40 sites but growth was not augmented by ASC. Active CD90+ MPE cells were tumorigenic (17/40 sites) only when coinjected with ASC (p=0.0005, χ2 test). The multilineage potentiality and MSC-like immunophenotype of ASC were confirmed by flow cytometry, differentiation cultures, and immunostaining. The secretome profile of ASC resembled that reported for MSC, but included adipose-associated adipsin and the hormone leptin, shown to promote breast cancer growth. Our data indicate that ASC enhance the growth of active, but not resting tumor cells. Thus, reconstructive therapy utilizing ASC-augmented whole fat should be postponed until there is no evidence of active disease.

FIG. 1.

Freshly isolated unsorted breast malignant pleural effusion (MPE) cells were cocultured for 1 (a, c) or 2 (b) weeks in Labtek culture chambers with low passage carboxyfluorescein succinimidyl ester (CFSE)-labeled adipose-derived stem cells (ASCs). Cells were fixed with 2% formaldehyde and stained for immunohistochemistry or immunofluorescence. MPE cells were cultured alone as control (d). ASC were observed using an antifluorescein antibody and revealed by 3,3′-diaminobenzidine staining (brown, a, b). Nests of CFSE− tumor cells were surrounded by CFSE+ ASC (a, b). MPE cells were further identified as cytokeratin+ using immunofluorescence (c, d, red). Tumor cell mitotic activity was estimated by counting the number of Ki67+ mitotic figures, excluding CFSE+ feeder cells (a, b, pink). Color images available online at www.liebertonline.com/ten.

FIG. 2.

Detection and characterization of CD90+ breast cancer cells in a pleural effusion. (a) Elimination of sources of artifact. A representative sample (10 million events) is presented. After confirming the absence of fluidic disturbances (side scatter vs. event count), cell clusters were eliminated based on pulse analysis (region A), before selecting nucleated DAPI+ events with DNA content ≥2N (region B) and removing subcellular particles and early apoptotic cells. Region C was adjusted based on the localization of CD14−CD33−CD235a−CD45+ resting lymphocytes (H, color evented red in the SS vs. FS panel). Nonhematopoietic cells and cell-binding antibody nonspecifically were selected and removed from further analysis using CD45 and a lineage cocktail. (b) Identification of large, active (high light scatter) and small resting (low light scatter) CD90+ breast cancer cells. Nonhematopoietic CD90+ cells were selected (region E) and subdivided into two distinct populations based on their light scatter profile (regions E1 and E2). (c) Both small and large CD90+ subpopulations and CD90− cells were further analyzed for expression of cytokeratin, CD44, CD133, CD117, and DNA profile (DAPI). Color images available online at www.liebertonline.com/ten.

FIG. 3.

In vivo tumorigenesis mouse xenograft model. Breast MPE cells were sorted into three distinct nonhematopoietic nonendothelial (CD31− Lin− CD45−) fractions based on their CD90 expression and light scatter profiles (small CD90+ vs. large CD90+ vs. CD90− cells) and were injected (100 cells per injection) into the mammary fat pads of NOD-SCID or NOD/Shi− scid interleukin-2rγ null mice (4 injection sites/animal). Sorted MPE cells were either injected alone or coinjected with either 10,000 ASC or with heavily irradiated MPE cells. Animals were sacrificed up to 6 months postinjection. Representative photographs of successful tumorigenesis are shown. Upon tissue harvesting, the presence of tumors at the site of injection was documented (a, and enlargement b). All injected sites were analyzed by immunohistochemistry and tumorigenesis was confirmed by detection of a cellular mass expressing the epithelial marker cytokeratin (c, q, brown). Both cross species-specific (AE1/AE3) and human-specific (MNF116) antibody clones were routinely used. Tumors were further characterized by immunofluorescence (d–o, 40 × objective) and immunohistochemistry (p–u, 100 × objective). Human CD90 expression was observed on small groups of cells distributed throughout the tumor and was largely restricted to nonepithelial cells (d–f) and small vessels (r, arrow). In contrast, CD44 was expressed at the membrane of all cytokeratin+ tumor cells (g–i). Tumors expressed both chemokine (C-C motif) receptor 5 (CCR5) and MMP9, markers of tumor invasion and metastasis. CCR5+ cells were detected as isolated groups of cells, whereas MMP9 was homogenously expressed throughout the tumor. A representative tumor was documented at higher magnification through serial immunostaining (p–u). The majority of tumor epithelial cells expressed estrogen receptor alpha (u). Despite the slow kinetics of tumor growth, a significant number of tumor cells were actively proliferating as shown by expression of Ki67 (t).

FIG. 4.

Immunophenotype and functional characterization of mesenchymal stem cell-like ASC. Immunophenotype: ASC were briefly expanded (passage 1 to passage 3) and analyzed by flow cytometry. A representative sample is shown including quantitative results from five independent samples (mean ± standard deviation; box A). ASC do not express the mature endothelial marker CD31. Persistence of CD45+ cells (primarily macrophages) during early passages is inconsistent, as reflected in the large standard deviation. ASC strongly and homogeneously express the mesenchymal markers CD105, CD73, CD90, and CD44. Small subpopulations of CD146+ and CD34+ cells can still be detected during early passages. These markers are associated with distinct perivascular fat populations (adipose pericytes and SA-ASC respectively), from which ASC are derived. ASC are able to differentiate in vitro toward mesenchymal lineages. (a) Oil red O staining (red) reveals the presence of lipid vesicles in differentiated ASC under adipogenic culture conditions (box B). (b) The presence of proteoglycans after in vitro differentiation reflects the chondrogenic potential of ASC as shown by Alcian blue staining (blue). (c, d) ASC osteogenic differentiation is assessed by alkaline phosphatase activity staining (blue, c) and calcium staining (alizarin red staining, red). (e–h) Freshly isolated stromal vascular cells (stromal vascular fraction) exhibit strong angiogenic potential in vitro (coexpression of mature endothelial markers CD31, red; von Willebrand factor [vWF], green); (e) ASC retain a limited in vitro angiogenic potential after short expansion (tubule formation, CD31 expression in red). (f–h) In addition to CD31 and vWF, other endothelial/perivascular marker (CD34, CD146, and alpha smooth muscle actin [α-SMA]) expressing cells are observed in the endothelial tubules. Five independent experiments were performed.