CD13-positive bone marrow-derived myeloid cells promote angiogenesis, tumor growth, and metastasis

Abstract

Angiogenesis is fundamental to tumorigenesis and an attractive target for therapeutic intervention against cancer. We have recently demonstrated that CD13 (aminopeptidase N) expressed by nonmalignant host cells of unspecified types regulate tumor blood vessel development. Here, we compare CD13 wild-type and null bone marrow-transplanted tumor-bearing mice to show that host CD13(+) bone marrow-derived cells promote cancer progression via their effect on angiogenesis. Furthermore, we have identified CD11b(+)CD13(+) myeloid cells as the immune subpopulation directly regulating tumor blood vessel development. Finally, we show that these cells are specifically localized within the tumor microenvironment and produce proangiogenic soluble factors. Thus, CD11b(+)CD13(+) myeloid cells constitute a population of bone marrow-derived cells that promote tumor progression and metastasis and are potential candidates for the development of targeted antiangiogenic drugs.

Keywords: mouse models; protease; stromal cells; vascular pericytes.

Fig. 1.

Characterization of CD13-expressing stromal cells. (A) TSA, LLC, and B16-F10 tumor cells (shown and labeled in the vertically positioned graphs) were analyzed by FACS for expression of several cell type-specific markers: αSMA for pericytes/CAFs, CD31 (endothelial cells), and CD45 (immune cells), separated into CD11b⁻ and CD11b⁺ groups to distinguish nonmyeloid and myeloid cells. Pericytes and endothelial, myeloid, and nonmyeloid stromal cells are represented as percentage of total tumor cells. Means ± SEM are shown. (B) CD13-expressing cells in the three tumor cell types represented as a percentage of each stromal cell component. Means ± SEM are shown. (C) Distribution of CD13-expressing cells among stromal cells in the three tumor cell types. Means ± SEM are shown. In each case, three to five tumors per experiment were analyzed, and each experiment was performed at least three times. Data from one representative experiment are shown.

Fig. 2.

Experimental tumor growth in BMT mice. (A and B) LLC, TSA, and B16-F10 tumor growth (mean ± SEM), tumor weight (mean ± SEM), and representative photographs of tumors recovered from BMT mice administered s.c. with tumor cells. (C) Lung weight (LLC) or number of lung colonies (TSA, B16-F10) (mean ± SEM) and representative photographs of lungs recovered from BMT mice administered with tumor cells i.v. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student t test.

Fig. 3.

Evaluation of tumor angiogenesis in LLC tumor-bearing BMT mice. (A) Immunohistochemical detection of CD31 (endothelial cells, red) and NG2 (pericytes, green) in LLC tumors recovered from tumor-bearing BMT mice. DAPI, blue. (B) Higher magnification of NG2⁺ and CD31⁺ blood vessels is shown. (C) The number of CD31⁺ blood vessels per field and the number of NG2⁺ blood vessels (mean ± SEM) averaged from 10 random fields are shown. Three tumors for each of the BMT group were analyzed. *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student t test. (Scale bar, 20 µm.)

Fig. 4.

Effect of sorted BMDCs on angiogenesis. (A) Immunohistochemical detection of CD31 (endothelial cells, red) and NG2 (pericytes, green) in TSA tumors. The number of CD31⁺ blood vessels per field and the number of NG2⁺ blood vessels (mean ± SE) averaged from 10 random fields are shown. Three tumors per each group were analyzed. (B) In vitro tube-formation assay. CFSE-labeled HDMECs were incubated with sorted CD13-expressing myeloid cells (CD11b⁺CD13⁺), CD13-expressing nonmyeloid immune cells (CD11b⁻CD13⁺), and CD13-negative myeloid cells (CD11b⁺CD13⁻). Images were taken after 8 h, and tube length was analyzed by WimTube software. Representative images are shown (mean ± SEM). Each experiment was performed three times in quadruplicate. Data from one representative experiment are shown. (C) Analysis of angiogenic proteins secreted by CD11b⁺CD13⁺, CD11b⁺CD13⁻ and CD11b⁻CD13⁺ cells. Cells (1.5 × 10⁵) were isolated by FACS from LLC tumors grown in CD13 WT mice and cultured for 5 d in DMEM, 2% FBS. The supernatants were then analyzed with the Mouse Angiogenesis Antibody Array Kit (R&D). Immunoreactive spots (in duplicate) are shown. Immunoreactive control spots are also shown at the corners. **P < 0.01; ***P < 0.001 by two-tailed Student t test. (Scale bar, 20 µm.)