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

Diet-independent effect of obesity on tumor growth

First, we wished to uncouple the direct effect of obesity on cancer progression from indirect effect of the diet. We set up cohorts by rendering mice obese (body mass > 45 g) or lean (body mass < 30 g) by prefeeding with HFD or LFD. To exclude diet as a variable, lean and obese mice have been placed on regular chow for 1 month before cancer initiation. HFD prefed mice slightly lost weight upon diet normalization, however, still had higher body mass due to increased body fat (Supplementary Fig. S1A and S1B). After diet normalization, mice were isografted with tumor cells. Obese mice displayed accelerated kinetics of Lewis lung carcinoma (LLC) growth, as compared with lean mice (Fig. 1A). As tumors grew in size, obese mice gradually lost weight, whereas lean mice maintained it (Fig. 1A). Fat, but not lean, body mass decreased in response to cancer (Supplementary Fig. S1B), indicating that weight loss was specifically due to the reduction in WAT amount. Human breast MDA-231 adenocarcinoma orthotopically xenografted into the mammary fat pad of RAG-1 mice also grew faster in DIO mice, although the effect was subtle, coincidentally with obesity being less pronounced in this strain (Supplementary Fig. S1C). Using an ovarian ID8 adenocarcinoma model, we segregated animals into obese, lean, and DIO-resistant mice that had become only moderately overweight on HFD before tumor implantation. Despite HFD prefeeding, the kinetics of tumor growth in DIO-resistant mice was not significantly different from that in the lean LFD controls (Supplementary Fig. S1C), indicating that prolonged positive energy balance does not fully account for the effect of obesity. Combined, these findings show that tumor growth can be promoted by obesity irrespective of diet at the time of tumor implantation and growth. Our data indicate excess WAT as an important component in the obesity–cancer relationship. However, the contribution of systemic physiologic obesity consequences, remaining post-diet normalization (34), cannot be excluded.

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Figure 1.

ASC expansion and promoted tumor growth irrespective of diet in obesity. A, tumor growth in lean and obese (DIO) C57BL/6 mice grafted with LLC cells at week 0 upon diet normalization (left) and concomitant changes in body mass (right). B, flow cytometric enumeration of ASC (% of viable cells) among cells from i.p. WAT by gating SVF on viable CD31⁻CD45⁻ cells and then on CD34⁺ cells. SSC-A, side scatter. C, relative net numbers of ASCs in i.p. WAT of lean and obese mice quantified on the basis of flow cytometric ASC frequency and total i.p. WAT mass. D, phase contrast micrograph of adherent India ink–stained cells from i.p. WAT after 1 day in culture featuring ASCs (arrowheads) and monocytes (arrows). E, adherent ASC recovery from i.p. WAT of obese mice normalized to lean mice (from equal WAT amount). F, enumeration of ASC as CD34⁺CD31⁻CD45⁻ cells (% of viable) in PBMCs of lean and obese mice bearing tumors. G, micrographs of adherent CD34⁺CD31⁻CD45⁻ cells sorted from WAT or from PBMCs of an obese mouse (left). H, oil red O staining of lipid droplets (arrows) in a colony formed by obese mouse PBMC–derived CD34⁺CD31⁻CD45⁻ cell upon adipogenesis induction. Scale bar, 50 μm. Error bars, SEM. *, P < 0.05.

Obesity results in ASC expansion

We next set out to test whether recruitment of cells from excess WAT by tumors is implicated in obesity effect on cancer. Obese mice displayed a 7-fold increase in body fat amount (Supplementary Fig. S1B). To investigate accompanying ASC expansion, we compared ASC quantities in lean and obese animals. Unlike bone marrow MSCs, ASCs express CD34 (15), which enables their enumeration by flow cytometry as CD34⁺CD31⁻CD45⁻ cells (7, 14). For intraperitoneal (i.p.) WAT, the major ASC reservoir (26), the ASC frequency was found to be 30.4% in lean and 32.6% in obese mice, respectively (Fig. 1B; Supplementary Fig. S2). On the basis of the total amounts of i.p. WAT recovered (1.0 ± 0.23 g from lean and 6.19 ± 0.25 g from obese mice), we calculated that the net number of i.p. ASCs is 6 times higher in obese mice (Fig. 1C). Frequencies of CD31⁻CD45⁻CD34⁺ cells were very low in the bone marrow, lungs, and liver of both lean and obese animals (Supplementary Fig. S2). Upon plating WAT stromal/vascular cells fraction, adherent ASCs appear as large fibroblasts with well-defined nuclei and nucleoli and can be clearly distinguished from smaller myelomonocytic cells internalizing India ink (Fig. 1D). Quantification of adherent ASCs from i.p. WAT upon enzymatic tissue digestion showed increased efficiency in their recovery from obese mice (Fig. 1E). Combined, these data indicate that WAT expanded in obesity serves as a reserve of extra ASCs.

Obesity-associated cell mobilization and tumor infiltration

To test whether ASCs traffic from WAT through the systemic circulation, we conducted a comparative analysis of PBMCs from lean and obese mice bearing tumors. Analysis of PBMCs (Supplementary Fig. S3A and S3B) showed that while circulating CD34⁺CD45⁻ cells were rare in lean animals (0.06%), their frequency increased 6-fold (to 0.37%) in obesity (Fig. 1F). The majority of these cells had the CD34⁺CD31⁻CD45⁻ ASC phenotype: the endothelial marker CD31 was expressed only by 5.9% of CD34⁺CD45⁻ cells in obese mice (Supplementary Fig. S3A). Analysis of individual CD34⁺CD31⁻CD45⁻ cells isolated by FACS showed morphology undistinguishable from that of ASCs sorted from WAT in parallel (Fig. 1G). Blood-derived CD34⁺CD31⁻CD45⁻ cells formed colonies and accumulated lipid droplets upon adipogenic induction (Fig. 1H). The obesity-associated egress of CD34⁺CD31⁻CD45⁻ adipocyte progenitors strongly suggests their ASC identity. To obtain evidence that ASCs may be recruited by tumors, we subjected tumor cell suspension to flow cytometric analysis. Indeed, tumors contained CD34⁺CD31⁻CD45⁻ cells (Supplementary Fig. S2), and the increase in their frequency associated with obesity was consistent with their possible WAT origin.

A bone marrow transplantation model for tracking WAT-derived cells

To enable tracking of hematopoietic tumor stroma (17, 18, 35, 36) in parallel with ASCs, we designed an in vivo competitive repopulation assay. This obesity/cancer model is based on 2 syngeneic mouse strains: host ubiquitously expressing GFP and donor expressing RFP. In chimeric GFP/RFP mice generated through bone marrow transplantation (Fig. 2A), it is possible to distinguish hematopoietic (RFP⁺) from the host (GFP⁺) cells. We reasoned that, if ASC traffic in cancer as hypothesized, obesity should be associated with increased frequency of GFP⁺ cells in tumors. Upon DIO induction and RFP bone marrow transplantation followed by 1-month recovery on LFD and HFD, GFP/RFP chimeras were subjected to diet normalization and then orthotopically grafted into the mammary pad with E0771 cells, a line derived from syngeneic breast adenocarcinoma.

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Figure 2.

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.

As expected, RFP⁺ and GFP⁺ cells were observed in all mouse tissues (Supplementary Fig. S4A and S4B). RFP⁺ cells were confirmed as CD45⁺ leukocytes, and less than 0.5% of bone marrow cells were GFP⁺ (Supplementary Fig. S4A). Femur sections revealed hematopoietic RFP⁺ cells relative to GFP⁺ vascular, bone, muscle, and WAT cells (Fig. 2B). Microscopic analysis of adherent WAT-derived cells showed the typical ASC morphology of GFP⁺ cells and RFP fluorescence of adherent leukocytes (Fig. 2B). In contrast, in peripheral blood, only rare adherent GFP⁺ cells were observed among RFP⁺ leukocytes (Fig. 2B). The majority of small adherent fibroblastoid RFP⁺ monocytes, also observed in no-color mice (Fig. 1D), were macrophages, as evident from India ink staining and F4/80 expression (Supplementary Fig. S3B).

Consistent with the data in no-color mice (Fig. 1F), we observed the frequency of circulating GFP⁺ cells being higher in obese mice (1.2%) than in lean mice (0.3%) by flow cytometry (Fig. 2C). In PBMCs of obese mice, 0.3% of GFP⁺ cells had the ASC CD34⁺CD31⁻CD45⁻ immunophenotype, whereas the majority were CD45⁺ leukocytes (Fig. 2C). Adherent cell analysis confirmed that while in lean mice, virtually all circulating GFP⁺ cells had the monocytic appearance, larger GFP⁺ fibroblasts were present in PBMCs of obese mice (Fig. 2D). Their typical ASC appearance has become pronounced after 4 days in culture (Fig. 2E). Collagen-I, alpha-smooth muscle actin (α-SMA), and decorin, proteins expressed by ASCs (7, 14, 23) were expressed in colonies of GFP⁺CD34⁺CD31⁻CD45⁻ cells (Supplementary Fig. S5). Accumulation of lipid droplets upon adipogenesis induction of the expanded GFP⁺CD34⁺CD31⁻CD45⁻ cells as adipocyte progenitors (Fig. 2F) confirmed them as ASCs.

Engraftment of hematopoietic and WAT-derived cells in tumors

Tumors grew significantly faster in obese GFP/RFP chimeras concomitantly with fat mass loss (Fig. 3A). Confocal immunofluorescence on tumor sections (Fig. 3B) revealed that large GFP⁺ cells disseminated throughout both the peripheral and central tumor zones (Fig. 3B). They were observed at a higher frequency in obese mice, whereas RFP⁺ cell frequency showed the opposite trend (Supplementary Fig. S4B). Analysis of tumor cell suspensions in culture revealed adherent GFP⁺ cells with the ASC morphology (Fig. 3C). As expected, we also observed GFP⁺ and RFP⁺ monocytes (Fig. 3C). More adherent GFP⁺ cells were recovered from tumors grown in obese mice (Fig. 3D). Approximately 44% of adherent GFP⁺ tumor–derived cells had the ASC morphology in obese mice, compared with 23% in lean (Fig. 3E). To exclude tumor size as a confounder in assessing the effect of WAT excess on ASC numbers in tumors, we have taken advantage of gender-specific differences in tumor growth rate. Upon grafting E0771 tumors into chimeric GFP/RFP lean females and age-matched obese (HFD prefed) males, we observed comparable tumor growth curves in the 2 groups. Flow cytometry indicated that tumor GFP⁺ cell frequency was still higher in obese mice (9.8%) than in lean mice (3.0%), despite similar tumor size (Fig. 3F). Importantly, 20.0% of tumor GFP⁺ cells in lean mice and 30.1% in obese mice had the CD34⁺CD31⁻CD45⁻ phenotype (Fig. 3F) indicating them as ASCs.

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Figure 3.

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.

We tested whether a pericyte marker desmin could be used to further validate GFP⁺ cells as ASCs. Our data show that 91.3% of ASCs are indeed desmin-positive in WAT (Supplementary Fig. S6A). All PBMC CD34⁺CD31⁻CD45⁻ cells and the majority (52.9%) of CD34⁺CD31⁻CD45⁻ cells recovered from tumors also were desmin-positive (Supplementary Fig. S6A), suggesting that mobilized ASCs retain their pericyte identity. Four-color confocal immunofluorescence on tissue sections from GFP/RFP chimeras showed co-expression of CD34 and desmin on perivascular GFP⁺ cells in both WAT and in tumors (Supplementary Fig. S6B). Finally, immunoblotting revealed the presence of delta-decorin (ΔDCN), a recently identified marker of ASC (14), in tumor protein extracts (Supplementary Fig. S6C), confirming ASC recruitment by tumors.

Tumor vascularization and cell proliferation associated with ASC recruitment

Masson's trichrome staining revealed comparable deposition of collagen in tumors from lean and obese animals (Supplementary Fig. S7A). This analysis also revealed less extensive areas of hemorrhage and necrosis in obese mice. Immunofluorescence analysis revealed comparable fibronectin deposits typically devoid of GFP⁺ cells in tumors for each group (Supplementary Fig. S7B). These data show that the desmoplastic reorganization of the internal tumor matrix is not significantly influenced by obesity in the models used.

Blood vessels are essential for oxygen and nutrient delivery, and vascular patency predetermines tumor growth (35, 37). We therefore investigated whether ASC recruitment is associated with vascular remodeling. In obese mice, a higher proportion of endothelial cells were from the host, as revealed by luminal co-localization of CD31 with GFP (Fig. 4A and B). In lean mice, blood vessels were sparse in certain tumor areas, whereas in obese mice, they were abundant throughout the tumor mass (Fig. 4A). Quantification revealed a 2-fold higher vascular density in tumors from obese mice (Fig. 4C). Tumor vessels in lean mice were compressed and slim, whereas in obese mice, they were more hyperdilated (Fig. 4C) and filled with circulating blood cells (Fig. 4A and B). Enumeration of vessels positive for desmin⁺GFP⁺ cells (Fig. 4D) indicated a trend for increased vessels maturation in tumors from obese mice (Fig. 4C). In addition, expression of α-SMA, a perivascular marker expressed on ASCs (7, 14, 23), was more abundant on perivascular GFP⁺ cells in obese mice (Fig. 4E). Combined, these results reinforce the notion that ASCs contribute to the pool of perivascular cells in tumors in an obesity-dependent manner.

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Figure 4.

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.

An observation consistently made for tumors grown in obese animals was that tumor capsules were notably thicker than in lean mice (Figs. 3B and 5A and B). Tumor capsule composition also appeared different in obese animals (Supplementary Fig. S8A). Visually obvious increased adiposity was confirmed by immunofluorescence identifying perilipin, a marker of mature lipid droplets (Fig. 5A). Numerous large GFP⁺ cells were also observed dispersed throughout the tumor and contained unilocular perilipin-positive lipid droplets indicating them as adipocytes. While the presence of adipocytes at the tumor periphery could potentially be explainable by ingrowth of the surrounding connective tissue, the presence of separate adipocytes abundant within the tumor core indicates their differentiation from engrafting progenitors. Interestingly, the average size of adipocytes was notably larger in tumors grown in obese animals (Figs. 3 and 5), despite diet excluded as a variable.

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Figure 5.

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.

To test whether recruitment of ASCs is associated with changes in malignant cell proliferation, we conducted immunofluorescence analysis to detect Ki-67 (Supplementary Fig. S8B). Tumors in obese mice were found to contain comparatively more widespread areas populated with proliferating cells (Fig. 5B and C). Importantly, up to 10% of tumor cells were positive for Ki-67 in the vicinity of intratumoral adipocytes, as opposed to only 2% in areas devoid of adipocytes for obese mice (Fig. 5D). Cell proliferation was even more strikingly associated with the vasculature containing GFP⁺ cells (Fig. 5C). In obese mice, up to 30% of tumor cells were Ki-67⁺ next to large GFP⁺ blood vessels and less than 5% in poorly vascularized areas devoid of GFP⁺ cells (Fig. 5D). This trend was also observed for tumors in lean mice, although the frequency of tumor Ki-67⁺ cells was overall 4-fold lower (Fig. 5D). Because adipocytes themselves are found in association with blood vessels in tumors, at this point, it is unclear whether adipocytes support malignant cell proliferation and survival independently of the vasculature. Combined, our data from independent mouse models suggest that WAT excess leads to functional ASC engraftment in tumor stroma.