Use of vascular endothelial growth factor receptor 1+ cells in treating and monitoring cancer and in screening for chemotherapeutics

#The present invention is directed to the use of vascular endothelial growth factor receptor 1+ cells in treating and monitoring cancer and in screening for chemotherapeutics.

#Cancer is the second leading cause of death next to heart attacks in the United States. There has been important progress in the development of new therapies in the treatment of this devastating disease. Much of the progress is due to better understanding of cell proliferation in both normal cells and cancerous cells.

#Normal cells proliferate as a result of the highly controlled activation of growth factor receptors by their respective ligands. Examples of such receptors are the growth factor receptor tyrosine kinases.

#FIGS. 1A-E show bone marrow-derived cells form the pre-metastatic niche. In FIG. 1A, 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 is a terminal bronchiole, and V is a bronchial vein. FIG. 1B shows GFP+ bone marrow in the lungs after irradiation and before DsRed-tagged B16 cell implantation (left panel; n=6). On day 14, GFP+ BMDCs are seen with no DsRed+ tumor 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+ tumor cells proliferate at cluster sites (right panel; n=8). DAPI stain shows cell nuclei. FIG. 1C is a graph showing flow cytometric data of bone marrow-derived GFP+ BMDCs and DsRed+ B16 cells in the lung (n=30), and two flow diagrams on day 14 (left panel) and day 18 (right panel). FIG. 1D shows GFP+ BMDCs mobilized with B16 conditioned media, then DsRed-tagged tumor 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 tumor cells in a cluster after four days (right panel inset; n=6). FIG. 1E shows a number of clusters per 100× objective field in animals with intradermal LLC or B16 tumors (n=12). Scale bar at bottom right applies for FIG. 1A (left, left middle, right middle, 80 mm; left middle inset, 8 mm; right, 20 mm; right inset, 47 mm), for FIG. 1B (left, left middle, 80 mm; left middle inset, 8 mm; right middle, right, 40 mm), and for FIG. 1D (40 mm; right inset, 20 mm). Pre-metastatic clusters are comprised of VEGFR1+ haematopoietic progenitors. FIGS. 1F-I show that pre-metastatic clusters are comprised of VEGFR1 haematopoietic progenitors. FIG. 1F shows VEGFR1 staining in irradiated lung before tumor 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). FIGS. 1G and H show double immunofluorescence in the lung of an animal with day 14 LLC tumor. VEGFR1+ and GFP+ bone marrow cells (left panel), VEGFR1+ and CD133+ (right panel) are shown in FIG. 1G. VEGFR1+ and CD117+ are shown in FIG. 1H. FIG. 1I shows VEGFR1+ clusters in c-Myc transgenic lymph node at day 40 of life and before tumorigenesis (middle panel and inset showing VEGFR1+ cells) as compared with wild-type littermate lymph node without the transgene (left panel), and day 120 c-Myc transgenic node with lymphomas (right panel). In the inset of the right panel, arrows indicate the VEGFR1+ clusters surrounded by lymphoma (n=6). Scale bar at bottom right applies to FIG. 1E (80 mm; left inset, 40 mm), FIG. 1G (20 mm), FIG. 1H (20 mm), and FIG. 1I (80 mm; insets, 8 mm).

#FIGS. 2A-B show inhibition of homing of bone marrow cells prevents metastasis. FIG. 2A shows VEGFR1+-selected bone marrow (R1-pos) permits micrometastasis (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). FIG. 2B shows treatment with antibodies to VEGFR1 (anti-R1) and VEGFR2 (anti-R2) in mice with LLC tumors 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, tumor 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 FIG. 2A (20 mm; wild type inset, 26 mm; R1-pos inset, 32 mm) and FIG. 2B (40 mm; anti-R2 inset, 20 mm).

#FIGS. 3A-F show the VLA4/fibronectin pathway mediates cluster formation. FIGS. 3A-C show wild-type mice 14 days after tumor implantation develop clusters expressing VLA-4 (inset, VEGFR1 and VLA-4), MMP9 and Id3 (inset, VEGFR1 and Id3). FIG. 3D shows lung tissue in Id3 knockout (KO) mice with LLC tumors given VEGFR1+ GFP+ BMDCs (P<0.01 by ANOVA; n=6). Arrows show region in upper inset. Arrows (lower inset) show the site of metastasis with GFP+ VEGFR1+ cells. FIG. 3E shows 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. FIG. 3F shows quantitative RT-PCR reveals increased fibronectin expression in the lungs of mice with LLC tumors 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 FIGS. 3A-C (40 mm; insets, 8 mm), FIG. 3D (80 mm; top right inset, 20 mm; bottom right inset 80 mm) and FIG. 3E (40 mm; insets, 20 mm).

#The present invention is directed to a method of inhibiting tumor formation in a cancer patient at a site remote from sites of prior tumor formation. This method involves administering to the cancer patient an inhibitor of vascular endothelial growth factor receptor 1+ bone marrow-derived cells under conditions effective to inhibit tumor formation in the cancer patient at a site remote from sites of prior tumor formation. This inhibitor can also be used to prevent tumor recurrence at the primary tumor site.

#The administering step of this method is carried out at a time period corresponding to when vascular endothelial growth factor receptor 1+ bone marrow-derived cell mobilization is stimulated. Such stimulation can occur as a result of chemotherapy, stress, surgery, bone marrow recovery in a cancer patient, inflammation, irradiation, or growth factors. In stimulating growth factors, such as granulocyte colony stimulating factors, granulocyte macrophage stimulating factors, and erythropoietin, the mobilization of hematopoietic cells in the bloodstream may be stimulated and tumor growth and metastasis promoted.

#In carrying out the present invention, the inhibitors of vascular endothelial growth factor receptor 1+ bone marrow-derived cells are selected to bind to vascular endothelial growth factor receptor 1+ bone marrow-derived cells.