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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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Experimental design.(A) Timeline for spontaneous metastasis studies: C57BL mice were lethally irradiated and transplanted 24 hours later with bone marrow cells isolated from Actb-GFP/C57BL mice (BMT). BMT mice were allowed to recover for 8 weeks prior to study. LLC1 or B16 tumor cells (TCs) were subcutaneously (s.c.) implanted in the left leg. Three days after LLC1 or B16 injection and until sacrifice, mice were treated 3 times per week by intraperitoneal (i.p.) injection of rat IgG (control) or VEGFR1-blocking antibody (MF1, 20 mg/kg). Primary tumors were resected when they reached a maximum length of 10 mm. Lung tissue was isolated from mice at three time points: at time of primary tumor resection and 10 and 14 days after removal of the primary tumor. Note: Tumor growth varied from 13–17 days; no macroscopic metastases are detectable at days 0 or 10 after tumor resection. (B) Bone marrow chimerism (each blue line represents GFP+ BMDCs in one BMT mouse, 30 mice prepared in one BMT procedure). Blood flow cytometric analysis was used to enumerate the percentage of GFP+ BMDCs among nucleated blood cells. (C) Mouse brain endothelial cells treated with rat IgG or MF1 were stimulated with 100 ng/ml of recombinant mouse PlGF-2 and incubated with anti-VEGFR1 antibody (MF1). VEGFR1 was immunoprecipitated and the ratio of phosphorylated-VEGFR1 to total VEGFR1 was compared to the ratio calculated for non-stimulated endothelial cells (assigned the value of 1). MF1 inhibited VEGFR1 phosphorylation in endothelial cells after PlGF2 stimulation in a dose-dependent manner.
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pone-0006525-g001: Experimental design.(A) Timeline for spontaneous metastasis studies: C57BL mice were lethally irradiated and transplanted 24 hours later with bone marrow cells isolated from Actb-GFP/C57BL mice (BMT). BMT mice were allowed to recover for 8 weeks prior to study. LLC1 or B16 tumor cells (TCs) were subcutaneously (s.c.) implanted in the left leg. Three days after LLC1 or B16 injection and until sacrifice, mice were treated 3 times per week by intraperitoneal (i.p.) injection of rat IgG (control) or VEGFR1-blocking antibody (MF1, 20 mg/kg). Primary tumors were resected when they reached a maximum length of 10 mm. Lung tissue was isolated from mice at three time points: at time of primary tumor resection and 10 and 14 days after removal of the primary tumor. Note: Tumor growth varied from 13–17 days; no macroscopic metastases are detectable at days 0 or 10 after tumor resection. (B) Bone marrow chimerism (each blue line represents GFP+ BMDCs in one BMT mouse, 30 mice prepared in one BMT procedure). Blood flow cytometric analysis was used to enumerate the percentage of GFP+ BMDCs among nucleated blood cells. (C) Mouse brain endothelial cells treated with rat IgG or MF1 were stimulated with 100 ng/ml of recombinant mouse PlGF-2 and incubated with anti-VEGFR1 antibody (MF1). VEGFR1 was immunoprecipitated and the ratio of phosphorylated-VEGFR1 to total VEGFR1 was compared to the ratio calculated for non-stimulated endothelial cells (assigned the value of 1). MF1 inhibited VEGFR1 phosphorylation in endothelial cells after PlGF2 stimulation in a dose-dependent manner.

Mentions: C57BL mice (6–7 weeks old, male) were lethally irradiated (137Cs Irradiator; Atomic Energy of Canada Ltd, Mississauga, Canada) using two 6 Gy fractions (with less than 12 hour time interval between dosing) delivered to the whole body. Irradiated mice were rescued 24 hours later by a bone marrow transplant isolated from Actb-GFP/C57BL, as previously described [13] (Figure 1A). We used the model after confirming that no weight loss or fibrosis, inflammation or any other sign of damage was detectable in the lungs two months after BMT. The BMT protocol was also optimized to ensure reproducible levels of GFP+ BMDC chimerism in BMT-Actb-GFP/C57BL mice. Blood was collected at four and eight weeks after BMT for flow cytometry analysis. To limit the variability in GFP-BMDC chimerism, BMT mice were prepared in large groups (32 recipient mice were injected with pooled BM from 11 donor mice). One group was prepared for each tumor model (see Figure 1B).


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)

Experimental design.(A) Timeline for spontaneous metastasis studies: C57BL mice were lethally irradiated and transplanted 24 hours later with bone marrow cells isolated from Actb-GFP/C57BL mice (BMT). BMT mice were allowed to recover for 8 weeks prior to study. LLC1 or B16 tumor cells (TCs) were subcutaneously (s.c.) implanted in the left leg. Three days after LLC1 or B16 injection and until sacrifice, mice were treated 3 times per week by intraperitoneal (i.p.) injection of rat IgG (control) or VEGFR1-blocking antibody (MF1, 20 mg/kg). Primary tumors were resected when they reached a maximum length of 10 mm. Lung tissue was isolated from mice at three time points: at time of primary tumor resection and 10 and 14 days after removal of the primary tumor. Note: Tumor growth varied from 13–17 days; no macroscopic metastases are detectable at days 0 or 10 after tumor resection. (B) Bone marrow chimerism (each blue line represents GFP+ BMDCs in one BMT mouse, 30 mice prepared in one BMT procedure). Blood flow cytometric analysis was used to enumerate the percentage of GFP+ BMDCs among nucleated blood cells. (C) Mouse brain endothelial cells treated with rat IgG or MF1 were stimulated with 100 ng/ml of recombinant mouse PlGF-2 and incubated with anti-VEGFR1 antibody (MF1). VEGFR1 was immunoprecipitated and the ratio of phosphorylated-VEGFR1 to total VEGFR1 was compared to the ratio calculated for non-stimulated endothelial cells (assigned the value of 1). MF1 inhibited VEGFR1 phosphorylation in endothelial cells after PlGF2 stimulation in a dose-dependent manner.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2744279&req=5

pone-0006525-g001: Experimental design.(A) Timeline for spontaneous metastasis studies: C57BL mice were lethally irradiated and transplanted 24 hours later with bone marrow cells isolated from Actb-GFP/C57BL mice (BMT). BMT mice were allowed to recover for 8 weeks prior to study. LLC1 or B16 tumor cells (TCs) were subcutaneously (s.c.) implanted in the left leg. Three days after LLC1 or B16 injection and until sacrifice, mice were treated 3 times per week by intraperitoneal (i.p.) injection of rat IgG (control) or VEGFR1-blocking antibody (MF1, 20 mg/kg). Primary tumors were resected when they reached a maximum length of 10 mm. Lung tissue was isolated from mice at three time points: at time of primary tumor resection and 10 and 14 days after removal of the primary tumor. Note: Tumor growth varied from 13–17 days; no macroscopic metastases are detectable at days 0 or 10 after tumor resection. (B) Bone marrow chimerism (each blue line represents GFP+ BMDCs in one BMT mouse, 30 mice prepared in one BMT procedure). Blood flow cytometric analysis was used to enumerate the percentage of GFP+ BMDCs among nucleated blood cells. (C) Mouse brain endothelial cells treated with rat IgG or MF1 were stimulated with 100 ng/ml of recombinant mouse PlGF-2 and incubated with anti-VEGFR1 antibody (MF1). VEGFR1 was immunoprecipitated and the ratio of phosphorylated-VEGFR1 to total VEGFR1 was compared to the ratio calculated for non-stimulated endothelial cells (assigned the value of 1). MF1 inhibited VEGFR1 phosphorylation in endothelial cells after PlGF2 stimulation in a dose-dependent manner.
Mentions: C57BL mice (6–7 weeks old, male) were lethally irradiated (137Cs Irradiator; Atomic Energy of Canada Ltd, Mississauga, Canada) using two 6 Gy fractions (with less than 12 hour time interval between dosing) delivered to the whole body. Irradiated mice were rescued 24 hours later by a bone marrow transplant isolated from Actb-GFP/C57BL, as previously described [13] (Figure 1A). We used the model after confirming that no weight loss or fibrosis, inflammation or any other sign of damage was detectable in the lungs two months after BMT. The BMT protocol was also optimized to ensure reproducible levels of GFP+ BMDC chimerism in BMT-Actb-GFP/C57BL mice. Blood was collected at four and eight weeks after BMT for flow cytometry analysis. To limit the variability in GFP-BMDC chimerism, BMT mice were prepared in large groups (32 recipient mice were injected with pooled BM from 11 donor mice). One group was prepared for each tumor model (see Figure 1B).

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