VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche

Abstract

The cellular and molecular mechanisms by which a tumour cell undergoes metastasis to a predetermined location are largely unknown. Here we demonstrate that bone marrow-derived haematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 (VEGFR1; also known as Flt1) home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells. Preventing VEGFR1 function using antibodies or by the removal of VEGFR1(+) cells from the bone marrow of wild-type mice abrogates the formation of these pre-metastatic clusters and prevents tumour metastasis, whereas reconstitution with selected Id3 (inhibitor of differentiation 3)-competent VEGFR1+ cells establishes cluster formation and tumour metastasis in Id3 knockout mice. We also show that VEGFR1+ cells express VLA-4 (also known as integrin alpha4beta1), and that tumour-specific growth factors upregulate fibronectin--a VLA-4 ligand--in resident fibroblasts, providing a permissive niche for incoming tumour cells. Conditioned media obtained from distinct tumour types with unique patterns of metastatic spread redirected fibronectin expression and cluster formation, thereby transforming the metastatic profile. These findings demonstrate a requirement for VEGFR1+ haematopoietic progenitors in the regulation of metastasis, and suggest that expression patterns of fibronectin and VEGFR1+VLA-4+ clusters dictate organ-specific tumour spread.

Figure 1. Bone marrow-derived cells form the pre-metastatic niche

a, β-gal⁺ bone marrow cells (left panel) are rarely observed in lungs after irradiation and before LLC cell implantation (n = 6). By day 14, β-gal⁺ bone marrow-derived clusters appear in the lung parenchyma (left middle panel and magnified inset of the region arrowed; n = 25) and are associated with micrometastases by day 23 (right panel, arrows) and in gross metastases (right panel, inset; n = 12). Also shown is a cluster with associated stroma between a terminal bronchiole and bronchial vein, a common metastatic site (right middle panel). B, terminal bronchiole; V, bronchial vein. b, GFP⁺ bone marrow in the lungs after irradiation and before DsRed-tagged B16 cell implantation (left panel; n = 6). On day 14, GFP⁺ (green) BMDCs are seen with no DsRed⁺ (red) tumour cells (left middle panel and inset; n = 12). Beginning on day 18, a few single DsRed⁺ B16 cells adhere to GFP⁺ bone marrow clusters (right middle panel), and by day 23, DsRed⁺ tumour cells proliferate at cluster sites (right panel; n = 8). DAPI stain (blue) shows cell nuclei. c, A graph showing flow cytometric data of bone marrow-derived GFP⁺ BMDCs and DsRed⁺ B16 cells in the lung, and two flow diagrams on day 14 (left panel) and day 18 (right panel) (n = 30; error bars show s.e.m.). d, GFP⁺ BMDCs mobilized with B16 conditioned media, then DsRed-tagged tumour cells injected through the tail vein adhere 24 h later (right panel, arrows) compared with animals receiving media alone (left panel; P < 0.01). Inset shows proliferating tumour cells in a cluster after four days (right panel inset; n = 6). e, Number of clusters per ×100 objective field in animals with intradermal LLC or B16 tumours (n = 12). Scale bar on top left panel applies to panels a (left, left middle, right middle, 80 µm; left middle inset, 8 µm; right, 20 µm; right inset, 47 µm), b (left, left middle, 80 µm; left middle inset, 8 µm; right middle, right, 40 µm) and d (40 µm; right inset, 20 µm).

Figure 2. Pre-metastatic clusters are comprised of VEGFR1⁺ haematopoietic progenitors

a, VEGFR1 staining in irradiated lung before tumour implantation (left panel and inset; n = 10) and 14 days after LLC cell implantation showing clusters in the lung (right panel, arrows; n = 18, 3.9 ± 0.2% cells with VEGFR1 staining per ×100 objective field, P < 0.05). b, c, Double immunofluorescence in the lung of an animal with day 14 LLC tumour. b, VEGFR1⁺ (red) and GFP⁺ (green) bone marrow cells (left panel), VEGFR1⁺ (red) and CD133⁺ (green) (right panel). c, VEGFR1⁺ (red) and CD117⁺ (green). d, VEGFR1⁺ clusters in c-Myc transgenic lymph node at day 40 of life and before tumorigenesis (middle panel and inset showing VEGFR1⁺ cells (red)) as compared with wild-type littermate lymph node without the transgene (left panel), and day 120 c-Myc transgenic node with lymphoma (right panel). In the inset of the right panel, arrows indicate the VEGFR1⁺ clusters (red) surrounded by lymphoma (green) (n = 6). Scale bar at bottom right applies to panels a (80 µm; left inset, 40 µm), b (20 µm), c (20 µm) and d (80 µm; insets, 8 µm).

Figure 3. Expression of VEGFR1 in pre-metastatic human tissue

a–f, Cellular clusters stained with VEGFR1 in malignant and non-malignant tissues in individuals with breast (n = 15), lung (n = 15) and gastrointestinal (n = 3) cancers. Lymph node with evidence of breast adenocarcinoma metastasis (a, red arrows indicate tumour) and lymph node without malignancy from same patient (b). Primary lung adenocarcinoma (c) and adjacent ‘normal’ lung without neoplasm (d, red arrows indicate VEGFR1⁺ cells). No VEGFR1⁺ clusters were seen in lymph node (b, inset; n = 6) and lung tissue (d, inset; n = 3) from individuals without cancer. Also shown is a primary adenosquamous carcinoma of the gastrooesophageal junction (e), and a hepatic lymph node without carcinoma (f). Insets in e, f, show co-immunofluorescence of VEGFR1 (red) and c-Kit (green). Scale bar at bottom right applies to all panels (40 µm; insets, 40 µm).

Figure 4. Inhibition of homing of bone marrow cells prevents metastasis

a, VEGFR1⁺-selected bone marrow (R1-pos) permits micrometastasis (red arrows, middle panel) but prevents well-vascularized large metastases as seen in wild types (left panel), 24 days after LLC implantation. Insets show CD31 (endothelial marker) expression. Bone marrow depleted of VEGFR1⁺ cells (non-R1) abrogates both clusters and metastases (right panel) (P < 0.01 by ANOVA). The table shows the number of clusters and micrometastases per ×100 objective field. *denotes that the metastasis filled the lung. (R1-pos, n = 4; non-R1, n = 4; wild type, n = 6; non-R1 plus wild type, n = 4). b, Treatment with antibodies to VEGFR1 (anti-R1) and VEGFR2 (anti-R2) in mice with LLC tumours prevents both clusters and metastases (P < 0.01 by ANOVA; for all groups, n = 5). Arrows in the lung of the wild type denote a large LLC metastasis. Arrows in anti-R2 show a cluster, inset shows a micrometastasis within a cluster. T, tumour cells. The table shows the number of clusters and LLC micrometastases in lung per ×100 objective field. *denotes that the metastasis filled the tissue. Scale bar at bottom right applies to panels a (20 µm; wild type inset, 26 µm; R1-pos inset, 32 µm) and b (40 µm; anti-R2 inset, 20 µm).

Figure 5. The VLA-4/fibronectin pathway mediates cluster formation

a–c, Wild-type mice 14 days after tumour implantation develop clusters expressing VLA-4 (inset, VEGFR1 (red) and VLA-4 (green)), MMP9 and Id3 (inset, VEGFR1 (red) and Id3 (green)). d, Lung tissue in Id3 knockout (KO) mice with LLC tumours given VEGFR1⁺GFP⁺ BMDCs (P < 0.01 by ANOVA; n = 6). Green arrows show region in upper inset. Red arrows (lower inset) show the site of metastasis with GFP⁺VEGFR1⁺ cells. e, Baseline fibronectin expression in the wild-type lung (n = 6) (left panel). Increased stromal fibronectin in the peribronchial region of the pre-metastatic lung at day three (middle panel, arrows), with maximal expression on day 14 (right panel). Insets, PDGRFα expression indicates resident fibroblasts laying down fibronectin. f, Quantitative RT–PCR reveals increased fibronectin expression in the lungs of mice with LLC tumours compared with wild type (*P < 0.05 by ANOVA; n = 6), and a similar earlier trend in lungs from animals with B16 melanoma. Scale bar at top right applies to panels a, b, c (40 µm; insets, 8 µm), d (80 µm; top right inset, 20 µm; bottom right inset 80 µm) and e (40 µm; insets, 20 µm).

Figure 6. Redirection of LLC metastases to atypical sites

a, b, By quantitative RT–PCR analysis, increased fibronectin expression was seen in the oviduct (a) and intestine (b) in mice given MCM compared with wild-type and LCM-treatment. For oviduct, *P < 0.05 at days 3–5 and **P < 0.001 for days 7–9 compared with wild type, and for intestine, *P < 0.001 at days 7–9 compared with wild type by ANOVA (n = 6). c, ELISA assay (in triplicate) for VEGF and PlGF levels in the conditioned media (*P < 0.05 when compared with L-LCM, **P < 0.01 when compared with media alone, by ANOVA). d, Transwell migration assays (in triplicate) demonstrate enhanced migration of VEGFR1⁺ cells to LCM and MCM (**P < 0.001 by ANOVA). e, Treatment with MCM redirects the metastatic spread of LLC to B16 melanoma metastatic sites, such as the spleen (left panel), kidney (left middle panel), intestine (right middle panel) and oviduct (right panel). Arrows denote the regions of metastatic borders, which are shown in the insets (n = 6). T, LLC tumour cells. Scale bar at bottom right applies to panel e (200 µm; insets, 20 µm).