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VEGFR1 activity modulates myeloid cell infiltration in growing lung metastases but is not required for spontaneous metastasis formation.

Dawson MR, Duda DG, Chae SS, Fukumura D, Jain RK - PLoS ONE (2009)

Bottom Line: Recent reports suggested that blocking VEGFR1 activity or the interaction with its ligands (VEGF and PlGF) has anti-tumor effects.All these effects may be exerted indirectly by recruitment of bone marrow-derived cells (BMDCs), such as myeloid cells.Moreover, in line with emerging clinical observations, we show that blockade of VEGFR1 activity neither prevents nor changes the rate of spontaneous metastasis formation after primary tumor removal.

View Article: PubMed Central - PubMed

Affiliation: Steele Laboratory for Tumor Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
The role of vascular endothelial growth factor receptor 1 (VEGFR1/Flt1) in tumor metastasis remains incompletely characterized. Recent reports suggested that blocking VEGFR1 activity or the interaction with its ligands (VEGF and PlGF) has anti-tumor effects. Moreover, several studies showed that VEGFR1 mediates tumor progression to distant metastasis. All these effects may be exerted indirectly by recruitment of bone marrow-derived cells (BMDCs), such as myeloid cells. We investigated the role of VEGFR1 activity in BMDCs during the pre-metastatic phase, i.e., prior to metastatic nodule formation in mice after surgical removal of the primary tumor. Using pharmacologic blockade or genetic deletion of the tyrosine kinase domain of VEGFR1, we demonstrate that VEGFR1 activity is not required for the infiltration of de novo myeloid BMDCs in the pre-metastatic lungs in two tumor models and in two mouse models. Moreover, in line with emerging clinical observations, we show that blockade of VEGFR1 activity neither prevents nor changes the rate of spontaneous metastasis formation after primary tumor removal. Prevention of metastasis will require further identification and exploration of cellular and molecular pathways that mediate the priming of the metastatic soil.

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Representative images and analysis of CD11b and MF1 staining of normal lung tissue.(A–H) Confocal microscopy images of lung tissue from C57BL (A–D, non-irradiated, non-GFP control) and BMT-Actb-GFP/C57BL (E–H, irradiated mice, GFP+ BMDCs shown in green) mice stained with CD11b-AF546 (red) and MF1-AF647 (yellow) and counterstained with DAPI nuclear dye (blue). The number of GFP, CD11b, or MF1 positive cells was calculated as a ratio of green, red, or yellow surface area to DAPI surface area, respectively (I). All images are 512 µm across.
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pone-0006525-g006: Representative images and analysis of CD11b and MF1 staining of normal lung tissue.(A–H) Confocal microscopy images of lung tissue from C57BL (A–D, non-irradiated, non-GFP control) and BMT-Actb-GFP/C57BL (E–H, irradiated mice, GFP+ BMDCs shown in green) mice stained with CD11b-AF546 (red) and MF1-AF647 (yellow) and counterstained with DAPI nuclear dye (blue). The number of GFP, CD11b, or MF1 positive cells was calculated as a ratio of green, red, or yellow surface area to DAPI surface area, respectively (I). All images are 512 µm across.

Mentions: At the time of the primary tumor resection, as well as 10 days after that, evaluation of lungs showed no macroscopic metastatic tumor nodules. Nevertheless, BMDC infiltration at the time of resection was small but detectable in both LLC1 and B16 tumor models, and comparable with BMDC accumulation in tumor-free BMT-Actb-GFP/C57BL mice (Figure 5). Thus, VEGFR1 blockade by MF1 treatment did not reduce BMDC infiltration in the lungs prior to macroscopic metastases formation (i.e., at days 0 and 10) in mice that had primary tumors removed [17]. These BMDCs were likely pulmonary alveolar macrophages, which reside in the normal, non-irradiated lung in comparable numbers in tumor-free non-BMT C57BL mice (Figure 6). To directly address the issue of BMDC phenotype (Figure 7A,D), and to exclude the possibility that inflammatory BMDCs infiltration in lungs was an artifact due to prior whole body irradiation and BMT, we performed CD11b (Mac1) and VEGFR1 immunostaining in normal, non-irradiated C57BL and flt-1TK–/–/C57BL mice. In the lungs of these mice, we detected myeloid (CD11b+) cells in numbers comparable to those of lung infiltrating BMDCs in BMT-Actb-GFP/C57BL mice. Moreover, the number of VEGFR1+ cells in the lung tissue was not significantly different between C57BL and flt-1TK–/–/C57BL mice (Figure 7B,E). Next, we measured the number of CD11b+ cells in spontaneous metastatic nodules in flt-1TK–/–/C57BL and C57BL mice formed after primary tumor resection. Consistent with the effect of antibody blockade of VEGFR1, we detected a significant decrease in the number of CD11b+ cells but not VEGFR1+ cells in the peri-tumor areas in LLC1 lung metastases from (non-irradiated) flt-1TK–/–/C57BL and C57BL mice (Figure 7C,F,G).


VEGFR1 activity modulates myeloid cell infiltration in growing lung metastases but is not required for spontaneous metastasis formation.

Dawson MR, Duda DG, Chae SS, Fukumura D, Jain RK - PLoS ONE (2009)

Representative images and analysis of CD11b and MF1 staining of normal lung tissue.(A–H) Confocal microscopy images of lung tissue from C57BL (A–D, non-irradiated, non-GFP control) and BMT-Actb-GFP/C57BL (E–H, irradiated mice, GFP+ BMDCs shown in green) mice stained with CD11b-AF546 (red) and MF1-AF647 (yellow) and counterstained with DAPI nuclear dye (blue). The number of GFP, CD11b, or MF1 positive cells was calculated as a ratio of green, red, or yellow surface area to DAPI surface area, respectively (I). All images are 512 µm across.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2744279&req=5

pone-0006525-g006: Representative images and analysis of CD11b and MF1 staining of normal lung tissue.(A–H) Confocal microscopy images of lung tissue from C57BL (A–D, non-irradiated, non-GFP control) and BMT-Actb-GFP/C57BL (E–H, irradiated mice, GFP+ BMDCs shown in green) mice stained with CD11b-AF546 (red) and MF1-AF647 (yellow) and counterstained with DAPI nuclear dye (blue). The number of GFP, CD11b, or MF1 positive cells was calculated as a ratio of green, red, or yellow surface area to DAPI surface area, respectively (I). All images are 512 µm across.
Mentions: At the time of the primary tumor resection, as well as 10 days after that, evaluation of lungs showed no macroscopic metastatic tumor nodules. Nevertheless, BMDC infiltration at the time of resection was small but detectable in both LLC1 and B16 tumor models, and comparable with BMDC accumulation in tumor-free BMT-Actb-GFP/C57BL mice (Figure 5). Thus, VEGFR1 blockade by MF1 treatment did not reduce BMDC infiltration in the lungs prior to macroscopic metastases formation (i.e., at days 0 and 10) in mice that had primary tumors removed [17]. These BMDCs were likely pulmonary alveolar macrophages, which reside in the normal, non-irradiated lung in comparable numbers in tumor-free non-BMT C57BL mice (Figure 6). To directly address the issue of BMDC phenotype (Figure 7A,D), and to exclude the possibility that inflammatory BMDCs infiltration in lungs was an artifact due to prior whole body irradiation and BMT, we performed CD11b (Mac1) and VEGFR1 immunostaining in normal, non-irradiated C57BL and flt-1TK–/–/C57BL mice. In the lungs of these mice, we detected myeloid (CD11b+) cells in numbers comparable to those of lung infiltrating BMDCs in BMT-Actb-GFP/C57BL mice. Moreover, the number of VEGFR1+ cells in the lung tissue was not significantly different between C57BL and flt-1TK–/–/C57BL mice (Figure 7B,E). Next, we measured the number of CD11b+ cells in spontaneous metastatic nodules in flt-1TK–/–/C57BL and C57BL mice formed after primary tumor resection. Consistent with the effect of antibody blockade of VEGFR1, we detected a significant decrease in the number of CD11b+ cells but not VEGFR1+ cells in the peri-tumor areas in LLC1 lung metastases from (non-irradiated) flt-1TK–/–/C57BL and C57BL mice (Figure 7C,F,G).

Bottom Line: Recent reports suggested that blocking VEGFR1 activity or the interaction with its ligands (VEGF and PlGF) has anti-tumor effects.All these effects may be exerted indirectly by recruitment of bone marrow-derived cells (BMDCs), such as myeloid cells.Moreover, in line with emerging clinical observations, we show that blockade of VEGFR1 activity neither prevents nor changes the rate of spontaneous metastasis formation after primary tumor removal.

View Article: PubMed Central - PubMed

Affiliation: Steele Laboratory for Tumor Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
The role of vascular endothelial growth factor receptor 1 (VEGFR1/Flt1) in tumor metastasis remains incompletely characterized. Recent reports suggested that blocking VEGFR1 activity or the interaction with its ligands (VEGF and PlGF) has anti-tumor effects. Moreover, several studies showed that VEGFR1 mediates tumor progression to distant metastasis. All these effects may be exerted indirectly by recruitment of bone marrow-derived cells (BMDCs), such as myeloid cells. We investigated the role of VEGFR1 activity in BMDCs during the pre-metastatic phase, i.e., prior to metastatic nodule formation in mice after surgical removal of the primary tumor. Using pharmacologic blockade or genetic deletion of the tyrosine kinase domain of VEGFR1, we demonstrate that VEGFR1 activity is not required for the infiltration of de novo myeloid BMDCs in the pre-metastatic lungs in two tumor models and in two mouse models. Moreover, in line with emerging clinical observations, we show that blockade of VEGFR1 activity neither prevents nor changes the rate of spontaneous metastasis formation after primary tumor removal. Prevention of metastasis will require further identification and exploration of cellular and molecular pathways that mediate the priming of the metastatic soil.

Show MeSH
Related in: MedlinePlus