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. 2014 Oct;17(4):897-907.
doi: 10.1007/s10456-014-9437-2. Epub 2014 Jul 2.

TM4SF1: A New Vascular Therapeutic Target in Cancer

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Free PMC article

TM4SF1: A New Vascular Therapeutic Target in Cancer

Chi-Iou Lin et al. Angiogenesis. .
Free PMC article

Abstract

Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane glycoprotein that regulates cell motility and proliferation. TM4SF1 is an attractive cancer target because of its high expression in both tumor cells and on the vascular endothelial cells lining tumor blood vessels. We generated mouse monoclonal antibodies against human TM4SF1 in order to evaluate their therapeutic potential; 13 of the antibodies we generated reacted with extracellular loop-2 (EL2), TM4SF1's larger extracellular, lumen-facing domain. However, none of these antibodies reacted with mouse TM4SF1, likely because the EL2 of mouse TM4SF1 differs significantly from that of its human counterpart. Therefore, to test our antibodies in vivo, we employed an established model of engineered human vessels in which human endothelial colony-forming cells (ECFC) and human mesenchymal stem cells (MSC) are incorporated into Matrigel plugs that are implanted subcutaneously in immunodeficient nude mice. We modified the original protocol by (1) preculturing human ECFC on laminin, fibronectin, and collagen-coated plates, and (2) increasing the ECFC/MSC ratio. These modifications significantly increased the human vascular network in Matrigel implants. Two injections of one of our anti-TM4SF1 EL2 monoclonal antibodies, 8G4, effectively eliminated the human vascular component present in these plugs; they also abrogated human PC3 prostate cancer cells that were incorporated into the ECFC/MSC Matrigel mix. Together, these studies provide a mouse model for assessing tumor xenografts that are supplied by a human vascular network and demonstrate that anti-TM4SF1 antibodies such as 8G4 hold promise for cancer therapy.

Figures

Fig. 1
Fig. 1. Monoclonal antibodies reactive with human TM4SF1
(A) Structure of human TM4SF1. (B) 8G4 staining of (a) Hu TM4SF1-OE HUVEC (b) HDF (human dermal fibroblasts) that express sub-detectable levels of TM4SF1 and (c) HDF transduced to overexpress human TM4SF1. Staining is representative of that obtained with the 15 monoclonal antibodies that reacted with TM4SF1 in fixed endothelial cells. (C) Flow cytometry demonstrates population shift of HUVEC and ECFC, but not mouse endothelial cell line MS1, with 8G4 antibody. Control mouse IgG (mIgG) did not evoke a population shift in HUVEC. (D) 8G4 immunostaining of HEK293 cells that were transfected to express (a) empty vector control, (b) murine TM4SF1 (Mu-TM4SF1), or (c) Mu-Hu TM4SF1 chimera. 8G4 recognized the Mu-Hu TM4SF1 chimera but not full length murine TM4SF1. (E) Human and mouse TM4SF1 EL2 sequence alignment. Red font indicates differences between human and mouse amino acid residues. Blue underline indicates N-glycosylation sites; N (asparagine), X (any amino acid), and S/T (serine/threonine). Both human and mouse TM4SF1 contain two potential N-glycosylation sites. The first has an identical sequence and alignment in human and mouse. However, the second differs both in sequence (NVS in human, NSS in mouse) and location (159 in human, 142 in mouse). (F) 8G4 immunofluorescence staining of ECFC localized TM4SF1 to plasma membrane, to nanopodia in an intermittent pattern (i, white arrows), and to perinuclear vesicles (ii, pink arrows). F-actin (phalloidin-staining; i red arrows) extended only into the most proximal portions of nanopodia.
Fig. 2
Fig. 2. Engineered human blood vessels in Matrigel plugs implanted in mice
Passage 4 ECFC were: (A) pre-cultured 48h on either CG- or CG/FN/LN-coated plates and mixed with MSC at a ratio of 2:3, or (B) pre-cultured 48h on CG/FN/LN-coated plates and mixed with MSC at ratios of either 2:3 or 3:2, suspended in Matrigel, and then the mixture was implanted subcutaneously in the flanks of nude mice. Matrigel plugs were harvested on days -7 or -14, 30 min. after tail vein injection of 2000kd FITC-dextran. RNA was extracted for measurement of CD31 by MGTP, using human- and mouse-specific primers and FITC-dextran content was used to measure vascular volume. (A) Day-7 Matrigel plugs demonstrate (a) increased human CD31 expression levels, (b) increased vascular volumes, and (c) increased overall vascularity when ECFC were pre-cultured on CG/FN/LN versus CG alone. (B) Further increases in human CD31 expression and in the human CD31/mouse CD31 expression ratio (a) and vascular volume (b) occurred when the ratio of ECFC was increased from 2:3 to 3:2. (B,c) Matrigel plug images show increased vascularity at day-14 over day-7. (C) Confocal images show (a) Numerous UEA-1-positive human endothelial cells in close relation to FITC-dextran-filled vascular lumens in day-7 Matrigel plugs. Inset i, Z-stacked confocal image (42 stacks with 220 nm/stack), and (b) extensive FITC-dextran staining of day-14 Matrigel plugs. Results are representative of at least three independent experiments.
Fig. 3
Fig. 3. Targeting human endothelial cells in Matrigel plugs with 8G4 or control mIgG
Matrigel plugs were prepared with ECFC pre-cultured on CG/FN/LN and introduced into Matrigel plugs at a ratio of 3-ECFC:2-MSC cells (total of 2×106 cells/plug). 100 ul of HBSS containing 3mg/kg (a) mIgG or (b) 8G4 was injected i.p. on days -10 and -14 after implantation. Matrigel plugs were harvested on day-18, 30 min after tail vein injection of FITC-dextran. (A) Day-18 Matrigel images (outlined) show extensive blood vessel network following mIgG treatment (a), that was largely disrupted by 8G4 treatment (b). (B) Quantification of human and mouse CD31 content (by MGTP expression) and vascular volumes (by FITC-dextran content). (C) Hematoxylin and eosin staining of Matrigel plug sections show numerous blood vessels following mIgG injection, that are absent following 8G4 injection. Results are representative of at least three separate experiments with three mice per group in each.
Fig. 4
Fig. 4. Targeting PC3 tumor cells in Matrigel plugs with 8G4 or control mIgG
Matrigel plugs were prepared as in Fig. 3, except that PC3 tumor cells were included. Larger numbers of PC3 cells, 106, were required to induce robust tumor growth in Matrigel plugs in the absence of ECFC/MSC cells, whereas 105 PC3 cells were sufficient when they were implanted along with ECFC/MSC cells. (A) Representative macroscopic appearance of Matrigel plugs (dotted outline) in mice treated with either 3 mg/kg 8G4 or control mIgG. (B–D) Quantification of human and mouse CD31 content (by MGTP expression) and vascular volume (by FITC-dextran content) in Matrigel plugs that included PC3 cells alone or PC3 cells supplemented with ECFC/MSC cells. Results are representative of three separate experiments with three mice per group in each experiment.

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