Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma

Abstract

Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-α, have been shown to decrease tumor size in hematological and epithelial tumor models by interfering with the protection from phagocytosis by macrophages that intact CD47 bestows upon tumor cells. Leiomyosarcoma (LMS) is a tumor of smooth muscle that can express varying levels of colony-stimulating factor-1 (CSF1), the expression of which correlates with the numbers of tumor-associated macrophages (TAMs) that are found in these tumors. We have previously shown that the presence of TAMs in LMS is associated with poor clinical outcome and the overall effect of TAMs in LMS therefore appears to be protumorigenic. However, the use of inhibitory antibodies against CD47 offers an opportunity to turn TAMs against LMS cells by allowing the phagocytic behavior of resident macrophages to predominate. Here we show that interference with CD47 increases phagocytosis of two human LMS cell lines, LMS04 and LMS05, in vitro. In addition, treatment of mice bearing subcutaneous LMS04 and LMS05 tumors with a novel, humanized anti-CD47 antibody resulted in significant reductions in tumor size. Mice bearing LMS04 tumors develop large numbers of lymph node and lung metastases. In a unique model for neoadjuvant treatment, mice were treated with anti-CD47 antibody starting 1 wk before resection of established primary tumors and subsequently showed a striking decrease in the size and number of metastases. These data suggest that treatment with anti-CD47 antibodies not only reduces primary tumor size but can also be used to inhibit the development of, or to eliminate, metastatic disease.

Fig. 1.

Evaluation of CD47 expression in LMS versus benign leiomyoma. (A) Gene-expression profiling on 51 LMS and on 19 benign leiomyoma samples revealed that the transcript levels of CD47, as well as TAM markers CD68 and CD163, were significantly higher on LMS versus their benign counterparts. P values were calculated using Student t test. (B) Immunofluorescence staining with anti-CD47 (Upper) or isotype control (Lower) antibodies on 16 LMS, three leiomyoma, and three normal muscle samples revealed that CD47 protein was more highly expressed on LMS than on benign or normal tissues. Representative images are shown and additional images are available Fig. S1 (see also Table S1). Magnification, 200×.

Fig. 2.

In vitro system to study interaction between human macrophages and LMS cell lines. (A) CD47 protein expression was confirmed in human LMS cell lines, LMS04 and LMS05, by flow cytometry. (B) PBMC-derived macrophages were assessed by IHC for a panel of immune markers. Scale bar, 50 μm. (C) Representative images showing that anti-CD47 mAb treatment resulted in robust phagocytosis of green fluorescently labeled LMS05 cells by human macrophages (arrows), whereas treatment of LMS05 cells with control antibodies resulted in no phagocytosis. Magnification, 200×.

Fig. 3.

Real-time monitoring of LMS cell phagocytosis by macrophages. RFP-positive mouse macrophages (red) and fluorescently labeled LMS05 cells (green) were cocultured in the presence of anti-CD47 (A) or IgG (B) antibodies and were imaged using video microscopy. (Movies S1, S2, and S3).

Fig. 4.

Effect of anti-CD47 treatment on primary tumor growth in xenotransplantation models of LMS. Representative tumors (A) and comparison of primary tumor volumes (B) of LMS05 xenografts treated with anti-CD47 or IgG antibodies. Representative tumors (C), comparison of primary tumor volumes (D), and comparison of the presence of lung metastases (E) of LMS04 xenografts treated with anti-CD47 or IgG antibodies. P values were calculated using Student t test. (F) Vimentin IHC on representative lungs from anti-CD47 or IgG control-treated mice. Scale bar, 40 μm. (G) Example of multicell metastatic focus from IgG control-treated mouse lung. Scale bar, 25 μm.

Fig. 5.

Characterization of metastatic model of LMS. (A) Mice were injected subcutaneously with 100,000 LMS04 cells and individual mice were killed at weekly intervals until the appearance of single-cell lung metastases. Thereafter, primary tumors were resected and the remaining mice were serially killed to assess the progression of lung metastases and secondary tumor outgrowth in the axilla. (B) Representative Vimentin IHC stains on mouse lungs 2 wk before primary tumor resection, 1 wk after primary tumor resection, and 6 wk after primary tumor resection. Magnification, 40×. (C) Autopsy image (Left) and Vimentin IHC (Right) of axillary region of mouse that had developed two lymph-node tumors after resection of primary tumor.

Fig. 6.

Effect of anti-CD47 therapy in neoadjuvant treatment model. (A) Mice were implanted subcutaneously with 100,000 LMS04 cells. Tumor volumes were measured at week 6, and mice with a similar range of tumor sizes were assigned to either anti-CD47 or IgG treatment groups (as detailed in Table S3). Effect of anti-CD47 antibody treatment on the volume (B) and incidence (C) of secondary lymph-node tumors. P values were calculated using Student t test. (D) Effect of anti-CD47 antibody treatment on the presence of lung metastases, as measured using computerized morphometric analysis. P value calculated using Wilcoxon rank-sum test. (E) Sample lungs from IgG control-treated or anti-CD47–treated lungs. (F) Presence of fibrin deposits, inflammatory cells, and degraded tumor cells in lung metastases of mouse treated with anti-CD47 antibodies. Magnification, 400×. (G) Comparison of multicell clusters and single-cell growths in IgG control or anti-CD47–treated mouse lungs. Magnification, 200×.