Antitumor activity of targeting SRC kinases in endothelial and myeloid cell compartments of the tumor microenvironment - PubMed (original) (raw)

Antitumor activity of targeting SRC kinases in endothelial and myeloid cell compartments of the tumor microenvironment

Wei Liang et al. Clin Cancer Res. 2010.

Abstract

Purpose: Several Src family kinase (SFK) inhibitors have entered clinical trials based on their direct effects against tumor cells. Here, we characterize the effects of targeting Src kinases on the tumor microenvironment and how these effects influence tumor growth.

Experimental design: Human cancer cells grown in cell culture or in mice were treated with dasatinib, a small-molecule inhibitor of SFKs. Tumor cell, endothelial cell, and myeloid cell compartments within the tumor microenvironment were analyzed. Primary human endothelial cells and freshly isolated CD11b+/CD11c- myeloid cells from mice were treated with dasatinib in cell culture. Cellular functions and signaling pathways affected by dasatinib were evaluated.

Results: Dasatinib was not cytotoxic in cell culture against the human cancer cell lines investigated here. However, dasatinib administration in human tumor-bearing mice suppressed tumor growth associated with increased tumor cell apoptosis, decreased microvessel density, and reduced intratumoral CD11b+ myeloid cells. Dasatinib directly inhibited motility and other functions of endothelial and myeloid cells, accompanied by the inhibition of phosphorylation of SFKs and downstream signaling. Tumor-infiltrating myeloid cells were identified as the major source of matrix metalloproteinase (MMP)-9 in the tumor microenvironment. Dasatinib treatment reduced MMP-9 levels in the tumor microenvironment through the simultaneous inhibition of recruitment of MMP9+ myeloid cells and MMP-9 gene expression in tumor-infiltrating myeloid cells.

Conclusions: These findings suggest that Src kinase inhibitors such as dasatinib possess a previously unrecognized anticancer mechanism of action by targeting both host-derived endothelial and myeloid cell compartments within the tumor microenvironment.

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Figures

Fig. 1

Fig. 1

Dasatinib inhibited endothelial cell functions. A, DU145, Colo205 and HUVEC cells were treated with DMSO (control) or dasatinib for 48 h. HUVECs were serum-starved and treated with VEGF (HUVEC+VEGF) or bFGF (HUVEC+bFGF) to stimulate cell growth. Cell viability was measured by MTS assay. Average values in control (DMSO) wells were set as 100% viability. Results are expressed as mean ± SEM % of viability relative to control (n=3). SS: serum starvation. B, HUVECs were treated with DMSO or dasatinib for 48 h with or without VEGF. Cell apoptosis was measured by TUNEL assay. Results are shown as mean ± SEM % apoptotic cells (n=3). CM: complete medium. C, serum-starved HUVECs were treated with DMSO or dasatinib and allowed to migrate towards VEGF or bFGF for 6 h in a modified Boyden chamber assay. Average number of migrated cells in control (DMSO) wells was set as 100% migration. Results are expressed as mean ± SEM % of migration relative to control (n=4). D, Tube formation was quantified by counting the cord junctions of branches formed by endothelial cells. Average numbers of cord junctions in control wells (VEGF only or bFGF only) were set as 100% tube formation. Results are expressed as mean ± SEM % of tube formation relative to control (n=3).

Fig. 2

Fig. 2

Dasatinib selectively inhibited Src downstream signaling in HUVECs. A, B and C, Serum-starved Confluent HUVECs were pretreated with DMSO or dasatinib for 1 h prior to stimulation with 50 ng/ml VEGF for another 30 min (for SFKs, FAK, p130CAS, paxillin, VE-cadherin and Stat3) or 10 min (for VEGFR2, Erk1/2, Akt and p38). Cells were lysed and cell lysate was probed with indicated antibodies. β-actin was used to demonstrate protein loading.

Fig. 3

Fig. 3

Dasatinib inhibited human tumor growth in mouse models. Mice bearing Colo205 tumors were treated with vehicle solution or dasatinib (15 mg/kg, B.I.D., p.o.) for 21 d. A, Tumor volume was shown as mean ± SEM. (*p<0.001, n=10) B, Whole tumor lysate was isolated from 4 vehicle- or dasatinib-treated tumors and immunoblots were probed with indicated antibodies. C, Tumor sections were immunostained for p-SFKs (left and middle) and total SFKs (right). Representative images were obtained with a x40 objective. Regions surrounded by white lines were further amplified to show p-SFKs staining in endothelial cells. (Scales bars, left and right: 20 µm; middle: 5 µm.) D, Tumor sections were immunostained for CD31, TUNEL (green)/Hoechst 33342 (blue) and Ki67. Scale bars, 50 µm.

Fig. 4

Fig. 4

Dasatinib directly inhibited tumor-associated myeloid cells. A, Upper, Colo205 tumor sections were immunostained for CD11b (red) or F4/80 (red). Representative images were obtained with a x20 objective. Scale bars, 50 µm. Lower, quantification of CD11b+ cells (*p<0.01, n=10) or F4/80+ cells per field (*p<0.01, n=10). Results are shown as mean ± SEM. B, Quantification of CD11b+ cells (*p<0.01, n=3) or F4/80+ cells (*p<0.05, n=3) by flow cytometry analyses. Results are shown as mean ± SEM % infiltrating cells. C, CD11b+/CD11c− cells were isolated from the spleens of Colo205 tumor-bearing mice treated with vehicle or dasatinib and subjected to Western blot analysis. Cell lysate was probed with indicated antibodies. D, CD11b+/CD11c− cells were isolated from the spleens of tumor-bearing mice prior to dasatinib treatment, treated with DMSO or dasatinib in vitro and allowed to migrate towards 33% tumor cell conditioned medium (CM) for 18 h in a modified Boyden Chamber assay. Results are shown as mean ± SEM × 103 migrated cells per ml medium (*p<0.001, n=8).

Fig. 5

Fig. 5

c-Src and FAK as targets for inhibition of macrophage migration. RAW264.7 cells were transiently transfected with control, c-Src or FAK siRNAs. A, lysate was prepared from RAW264.7 cells 72 h post-transfection and probed with indicated antibodies to measure the knock-down efficiency by siRNAs. B, RAW264.7 cells were harvested 72 h post-transfection, resuspended in basal medium and pretreated with DMSO or 30 nM dasatinib for 1 h before loading on the collagen I-coated inserts. Cell motility was evaluated by a modified Boyden chamber assay using 33% Colo205 tumor cell conditioned medium (TCCM) as a chemoattractant. Results were shown as mean ± SEM cells per field (x20) (*p<0.01, n=3). C, RAW264.7 cells were serum-starved for 6 h, pretreated with DMSO or dasatinib for 1 h prior to stimulation with or without 33% TCCM for another 2 h. Cell lysate was extracted and probed with indicated antibodies.

Fig. 6

Fig. 6

Dasatinib reduced MMP-9 levels in the tumor microenvironment. A, Upper, MMP-9 immunostaining in Colo205 xenograft tumors treated with vehicle or dasatinib. Representative images were obtained with a x40 objective. Regions surrounded by white lines were further amplified to show different MMP-9 expression pattern. (Scales bars, left and right: 20 µm; middle: 5 µm.) Lower left, negative control for MMP-9 staining. Lower right, quantification of MMP-9+ cells per field (*_p_=0.025, n=10). Results are shown as mean ± SEM. B, Upper, immunostaining of tumor sections for CD11b (red) and MMP-9 (green). Confocal fluorescence images were obtained with a x20 objective. Scale bars, 50 µm. Lower, quantification of CD11b+/MMP-9+ cells per field (*_p_=0.032, n=10). Results are shown as mean ± SEM. C, Normalized MMP-9 mRNA expression in human tumor cells, mouse stromal cells and CD11b+/CD11c− tumor-associated myeloid cells. Results are shown as mean ± SEM (n=3).

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