Volume 11, Issue 6, Pages 812-824 (December 2012) CCR2-Dependent Recruitment of Macrophages by Tumor-Educated Mesenchymal Stromal Cells Promotes Tumor Development and Is Mimicked by TNFα  Guangwen Ren, Xin Zhao, Ying Wang, Xin Zhang, Xiaodong Chen, Chunliang Xu, Zeng-rong Yuan, Arthur I. Roberts, Liying Zhang, Betty Zheng, Ting Wen, Yanyan Han, Arnold B. Rabson, Jay A. Tischfield, Changshun Shao, Yufang Shi  Cell Stem Cell  Volume 11, Issue 6, Pages 812-824 (December 2012) DOI: 10.1016/j.stem.2012.08.013 Copyright © 2012 Elsevier Inc. Terms and Conditions

Cell Stem Cell 2012 11, 812-824DOI: (10.1016/j.stem.2012.08.013) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 L-MSCs Promoted Lymphoma Growth to a Greater Extent than Did BM-MSCs (A) Morphology of cultured L-MSCs from lymphoma-bearing p53+/− mice at passage 1 and passage 5. (B) L-MSCs are phenotypically similar to BM-MSCs. After isolation from BM and lymphoma tumors, respectively, BM-MSCs and L-MSCs, each from passage 5, were analyzed for the indicated markers by immunofluorescence staining and flow cytometry. (C) Effect of coadministration of small number of MSCs on growth of EL4 lymphoma. EL4 cells were coadministered with BM-MSCs or L-MSCs from p53+/−, gld/gld, or mlh1−/− mice or BM-MSCs from the respective wild-type mice at a 1:5 ratio (1 × 105 MSCs and 5 × 105 EL4 cells) into WT C57BL/6 mice by intramuscular injection. After 12 days, tumors were excised and weighed. (D) Impact of coadministration of large number of MSCs on lymphoma progression. A representative L-MSC line established from a lymphoma that developed spontaneously in a p53+/− mouse, or BM-MSCs derived from corresponding WT mice, were coinjected with EL4 cells at a 2:5 ratio (2 × 105 MSCs and 5 × 105 EL4 cells) into C57BL/6 mice. On days 6, 9, and 12, tumor size was measured (mean ± SD), and tumors from day 12 were sectioned and imaged microscopically. Four mice per treatment group were used and results are representative of three independent experiments. Cell Stem Cell 2012 11, 812-824DOI: (10.1016/j.stem.2012.08.013) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 L-MSCs and BM-MSCs Inhibited Adaptive Immune Cells to a Similar Degree (A) Influence of MSC coadministration on host adaptive immune cells. L-MSCs or BM-MSCs were coadministered with EL4 cells at a 2:5 ratio (MSCs: EL4 cells) into C57BL/6 mice. At the indicated time points after tumor inoculation, tumors were excised and single-cell suspensions prepared. These tumor cells and peripheral blood cells were stained for CD3, CD19, CD4, and CD8 and analyzed by flow cytometry. (B) Serum levels of IFNγ and IL-12 on day 12 in each group described in (A) were assayed by multiplexed bead-based immunoassay. Four mice per treatment group were used and results (means ± SD) are representative of four independent experiments. (C) BM-MSCs or L-MSCs were cocultured with splenocytes at graded ratios in the presence of anti-CD3 (1 μg/ml). After 2 days, cell proliferation was assayed by 3H-thymidine incorporation, and IFNγ in the culture supernatants was measured by multiplexed bead immunoassay. Values are means ± SD of four replicate wells from a representative of three experiments. Cell Stem Cell 2012 11, 812-824DOI: (10.1016/j.stem.2012.08.013) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Recruitment of Monocytes, Macrophages, and Neutrophils by L-MSCs and BM-MSCs In Vivo BM-MSCs or L-MSCs were coadministered with EL4 cells into C57BL/6 mice, as in Figure 1D. After 6, 9, or 12 days, single-cell suspensions prepared from tumor tissues or from peripheral blood were analyzed for the frequency of CD11b+Ly6G+ neutrophils, CD11b+Ly6C+ monocytes, and F4/80+ macrophages. Flow cytometry data of tumor-infiltrated myeloid cells from day 6 are shown (A–C), along with means ± SD of both tumor tissues and peripheral blood for all time points (D). Four mice per treatment group were used and results are representative of four independent experiments. Cell Stem Cell 2012 11, 812-824DOI: (10.1016/j.stem.2012.08.013) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Monocytes/Macrophages, but Not Neutrophils, Were Required for Tumor-Promotion by L-MSCs (A–C) Depletion of CD11b+Ly6G+ neutrophils did not alter the tumor-promoting activity of MSCs. BM-MSCs or L-MSCs were coadministered with EL4 cells into C57BL/6 mice, as in Figure 1D, and Ly6G neutralizing Ab (1A8;150 μg per injection/mouse, intraperitoneally [i.p.]) was administered on days 4, 7, and 10 after cell inoculation. On day 6, tumors were excised and single-cell preparations were analyzed by flow cytometry for the extent of depletion of different myeloid cell types in tumors. Shown are representative flow cytometry data for tumor-bearing mice injected with EL4 without MSC coadministration (A) and averaged results for all treatment groups (B). To determine the effect on tumor growth, we excised tumors from identically treated mice and weighed them on day 12 (C). (D–F) Ablation of monocytes/macrophages completely abolished the tumor-promoting activity of MSCs. After coadministration of BM-MSCs or L-MSCs with EL4 cells, as in Figure 1D, into CD11b-DTR mice, diphtheria toxin (DT, 5 ng/g/injection) or vehicle (1% BSA in sterile water) was administered i.p. on days 4, 7, and 10, and extent of myeloid cell depletion in tumor tissues was measured on day 6 (D and E). Tumors were weighed on day 12 (F). Four mice per treatment group were used and results (means ± SD) are representative of three independent experiments. Cell Stem Cell 2012 11, 812-824DOI: (10.1016/j.stem.2012.08.013) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Tumor-Promoting Effect of L-MSCs Requires CCR2 (A) Overexpression of CCL-2 ligands by L-MSCs. Supernatants from cultures of BM-MSCs and L-MSCs from the same passage number were assayed for the indicated cytokines by multiplexed bead array immunoassay. Values are means ± SD of four replicate wells from a representative of five different MSC lines. (B) Chemotaxis by BM-derived monocytes in response to culture supernatants from L-MSCs or BM-MSCs was tested in a Transwell system, with or without the addition of anti-CCR2 (10 μg/ml). After 3 hr incubation, cells that had migrated through membrane pores and into bottom wells were quantitated under microscope. Values are means ± SD of three replicate wells from a representative of three experiments. (C–E) Impact of CCR2 deficiency on myeloid cells in tumor-bearing mice. BM-MSCs or L-MSCs were coadministered with EL4 cells, as in Figure 1D, into WT C57BL/6 mice or CCR2−/− mice, and myeloid cell frequency was examined on day 7. Representative flow cytometry data show the frequency of CD11b+Ly6G+ neutrophils, CD11b+Ly6C+ monocytes, and F4/80+ macrophages from tumor and peripheral blood of EL4-bearing control mice (C). Means and statistical differences among all treatment groups in tumors of WT and CCR2-KO mice are given (D). Tumors were weighed on day 12 (E). Four mice per group were used and results (means ± SD) are representative of three independent experiments. Cell Stem Cell 2012 11, 812-824DOI: (10.1016/j.stem.2012.08.013) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 TNFα-Stimulated BM-MSCs Mimic the Characteristics of L-MSCs in Promoting Tumor Growth (A) Lethally irradiated WT C57BL/6 mice were transplanted with bone marrow cells from GFP-transgenic mice (C57 background) via intrabone injection. Three months later, these mice were injected with EL4 lymphoma cells (5 × 105 cells), and MSCs were isolated from the resultant tumors after 12 days. The phenotype of EL4-derived MSCs was assayed by flow cytometry, as in Figure 1B, by gating on GFP+ cells. Results are representative of two independent experiments. (B) Treatment with TNFα altered the cytokine production profile of BM-MSCs. Supernatants from cultured BM-MSCs treated with or without TNFα (20 ng/ml for 24 hr) were assayed for the indicated cytokines by multiplexed bead immunoassay. Results are representative of three different BM-MSC lines. (C and D) TNFα-pretreated BM-MSCs enhanced the growth of EL4 tumors more significantly than did control BM-MSCs. BM-MSCs (2 × 105 cells), with or without TNFα treatment (20 ng/ml for 24 hr), were coadministered with EL4 cells (5 × 105 cells) into C57BL/6 mice. Tumor sizes were measured at the indicated time points after inoculation (C). Extent of F4/80+ macrophage infiltration in tumors from each group was examined by flow cytometry on day 6 (D). (E) Depletion of monocytes/macrophages or deficiency in CCR2 prevented tumor promotion by TNFα-treated MSCs. In experiments conducted as in Figure 4F and Figure 5E, the tumor-promoting capacity of TNFα-treated BM-MSCs was examined and compared to control BM-MSCs. Tumors were weighed on day 12. Five mice per group were used and results (means ± SD) are representative of three independent experiments. Cell Stem Cell 2012 11, 812-824DOI: (10.1016/j.stem.2012.08.013) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 The Interaction between MSCs and Macrophages in Spontaneous Lymphomas and Transplanted Solid Tumors (A) Numbers of MSCs and macrophages were positively correlated in spontaneous lymphomas. Spontaneous T cell lymphomas and B cell lymphomas were harvested from p53−/− and gld mice, respectively. The frequencies of LinlowSca-1+CD140a+ cells (representing tissue MSCs) and F4/80+ macrophages were analyzed by flow cytometry. (B–D) Effects of BM-MSCs on tumor growth in a B16-F0 melanoma model. Control BM-MSCs or TNFα-treated BM-MSCs were coadministered with B16-F0 cells into C57BL/6 mice subcutaneously, as in Figure 1D. Tumor size was measured at the indicated time points postinoculation (B). Deficiency in CCR2 eliminated the tumor-promoting activity of TNFα-treated MSCs, as indicated by tumor weights on day 12 (C). Similar to the lymphoma model, F4/80+ macrophage infiltration into tumors was much greater in mice coadministered TNFα-pretreated MSCs, as revealed by flow cytometry on day 8 (D). Four mice per group were used and results (means ± SD) are representative of three independent experiments. (E and F) Breast carcinoma 4T1 model. Control BM-MSCs or TNFα-treated BM-MSCs were coadministered with 4T1 cells into the mammary gland fat pad of Balb/c mice. Tumor size was measured at the indicated time points postinoculation (E). Extent of F4/80+ macrophage infiltration on day 7 was detected by immunohistochemical staining (F). Five mice per group were used and results (means ± SD) are representative of two independent experiments. (G) Proposed model depicting the mechanism of tumor growth promotion by MSCs. TNFα, and possibly other proinflammatory cytokines, induce tumor-resident MSCs to express CCR2-chemokines, which recruit monocytes/macrophages that are directly responsible for augmenting tumor growth, by previously described mechanisms. Cell Stem Cell 2012 11, 812-824DOI: (10.1016/j.stem.2012.08.013) Copyright © 2012 Elsevier Inc. Terms and Conditions