Stromal Progenitor Cells from Endogenous Adipose Tissue Contribute to Pericytes and Adipocytes That Populate the Tumor Microenvironment

In-parallel tracking of WAT and medullary cells. A, transplant scheme. Bone marrow from RFP (red) mice is transplanted into lethally irradiated lean or obese (DIO) GFP mice, which are grafted with tumors after diet normalization. In obese mice, trafficking of GFP⁺ ASCs (green) from WAT (yellow) is increased. B, GFP (green) and RFP (red) fluorescence of tissues and cells (1 day post-plating) isolated from an obese GFP/RFP chimera. Leukocytes (arrows) of host (green) and donor (red) origin and host ASCs (arrowheads) are indicated. C, flow cytometric enumeration of circulating GFP⁺ (FITC channel) cells among viable PBMCs from representative lean and obese E0771 tumor–grafted mice (left) and gating of the GFP⁺ cells from PBMCs of the obese mouse to enumerate CD34⁺ ASCs as a percentage of GFP⁺ cells (right). SSC-A, side scatter. D, total GFP⁺ cells from PBMCs of a lean and an obese mouse (C) were plated in culture for 1 day. GFP-fluorescent (green) adherent monocytes (arrows) and cells with the ASC morphology (arrowheads) are indicated. E, high magnification of obese mouse PBMC–derived GFP⁺ cells 4 days post-plating. F, lipid droplet formation (*) in a colony formed by an obese mouse PBMC–derived GFP⁺ cell ASCs 7 days after adipogenesis induction. Scale bar, 50 μm.

Recruitment of ASCs by tumors increased in obesity. A, tumor growth in lean and obese GFP/RFP chimeras grafted with E0771 cells at week 0 upon diet normalization (left) and concomitant changes in fat body mass measured by EchoMRI (right). B, confocal immunofluorescence on sections from E0771 tumors with anti-GFP (green) and anti-RFP (red) antibodies identifies host and donor cells, respectively. Shown for lean and obese mice are peripheral and internal tumor areas as indicated. RFP⁺ cells within the stroma (red arrows), GFP⁺ cells within the stroma (green arrows), GFP⁺ vasculature-associated cells (green arrowheads), and tumor capsule (brackets) are indicated. Nuclei are stained with TO-PRO-3 (blue). C, adherent GFP⁺ cells with ASC morphology (arrowheads) and RFP⁺ monocytes (arrows) observed in culture 1 day post-plating of E0771 tumor cell suspension from an obese GFP/RFP chimera. D, adherent GFP+ cells FACS-sorted from E0771 tumors grown in lean and obese mice 1 day post-plating. Cells with ASC morphology (arrowheads) and other cell types (arrows) are indicated. E, percentages of cells with ASC morphology among GFP⁺ cells from D. F, flow cytometric analysis of cell suspensions from size-matched E0771 tumors grown in lean and obese mice. Viable cells were gated to separate RFP⁺ (Texas Red channel) and GFP⁺ (FITC channel) cells from malignant cells (blue). The combined GFP⁺ and RFP⁺ cells were then gated to visualize CD31⁺ and CD45⁺ cells. Finally, GFP⁺CD31⁻CD45⁻ cells were gated to enumerate GFP⁺ cells with the ASC CD31⁻CD45⁻CD34⁺ phenotype (as a percentage of total GFP⁺ cells). *, P < 0.001. Error bar, SEM. Scale bar, 50 μm.

Pericyte recruitment in obesity and tumor vascularization. A and B, confocal immunofluorescence analysis of sections from E0771 tumors with anti-GFP (green) and anti-CD31 (red) antibodies. Shown for lean and obese GFP/RFP chimeras are internal tumor areas at low (A) and high (B) magnification. Blood vessels (red) contain luminal GFP⁺CD31⁺ endothelial cells (yellow arrows) and perivascular/stromal GFP⁺CD31⁻ cells (green arrows). Note increased pericyte coverage and dilation of tumor vessels in obese mice. C, quantitative vasculature analysis in E0771 tumors from lean and obese mice. Vascular density was assessed as mean number of vessels per ×100 view field. Blood vessel size was assessed as mean lumen width for all vessels scored. Blood vessel maturity was assessed as mean percentage of vessels associated with desmin-positive pericytes among all vessels scored. Error bar, SEM. D, confocal immunofluorescence tumor analysis with anti-GFP (green) and anti-desmin (red) antibodies. Yellow signal upon digital channel merging indicates GFP⁺ pericytes, which is confirmed by Z-stack projections of median series for individual cells in the indicated magnified area (bottom). E, confocal immunofluorescence analysis of internal areas from E0771 tumors grown in lean and obese GFP/RFP chimeras with anti-GFP (green) and anti-α-SMA (red) antibodies. Nuclei are stained blue (TO-PRO-3). Scale bar, 100 μm.

Adipogenesis and proliferation associated with tumor vascularization in obesity. A, analysis of sections from tumors by immunofluorescence with antibodies against GFP (green) and perilipin (red). a, GFP⁺ adipocytes containing perilipin⁺ unilocular lipid droplets; b, GFP⁺perilipin⁻ blood vessels. B and C, analysis of sections from tumors with antibodies against GFP (green) and Ki-67 (red). Shown are proliferating tumor cells (arrowheads) and rare proliferating GFP⁺ cells (arrow). Shown are peripheral (A and B) and internal (C) E0771 tumor areas. Tumor capsule indicated with brackets. Scale bar, 100 μm. D, effect of proximity to clusters of intratumoral adipocytes (left) and to clusters of dilated blood vessels (right) on tumor cell proliferation. From 15 representative ×100 view fields, Ki-67+ nuclei were counted among 100 Hoechst 33258–stained nuclei. Shown are mean ± SEM. *, P < 0.05; ***, P < 0.001.