A vaccine targeting angiomotin induces an antibody response which alters tumor vessel permeability and hampers the growth of established tumors

Abstract

Angiomotin (Amot) is one of several identified angiostatin receptors expressed by the endothelia of angiogenic tissues. We have shown that a DNA vaccine targeting Amot overcome immune tolerance and induce an antibody response that hampers the progression of incipient tumors. Following our observation of increased Amot expression on tumor endothelia concomitant with the progression from pre-neoplastic lesions to full-fledged carcinoma, we evaluated the effect of anti-Amot vaccination on clinically evident tumors. Electroporation of plasmid coding for the human Amot (pAmot) significantly delayed the progression both of autochthonous tumors in cancer prone BALB-neuT and PyMT genetically engineered mice and transplantable TUBO tumor in wild-type BALB/c mice. The intensity of the inhibition directly correlated with the titer of anti-Amot antibodies induced by the vaccine. Tumor inhibition was associated with an increase of vessels diameter with the formation of lacunar spaces, increase in vessel permeability, massive tumor perivascular necrosis and an effective epitope spreading that induces an immune response against other tumor associated antigens. Greater tumor vessel permeability also markedly enhances the antitumor effect of doxorubicin. These data provide a rationale for the development of novel anticancer treatments based on anti-Amot vaccination in conjunction with chemotherapy regimens.

Fig. 1

Amot expression on tumor endothelial cells and in vivo tumors. Western blot of protein extracts of: a mammary glands from BALB-neuT mice bearing foci of hyperplasia (week 6), in situ carcinomas (week 10) and microscopic invasive cancer (week 16, 22) and from TUBO tumors of progressive sizes (2–10 mm mean diameter); b MAEp80 and TUBO cells cultured in vitro; c TUBO tumors of progressive sizes. Immunoblots were probed with antibodies to p80 mouse Amot (upper band, ~80 kDa) and vinculin (lower band, ~100 kDa). Faint bands visible in the samples from extracts at 6, 10 and 16 weeks of age should be considered as a cross-reacting contaminant. For each determination 3 samples were analyzed. Immunofluorescence of cryosections of 5 mm mean diameter: d TUBO tumors growing in BALB/c mice and e autochthonous clinically evident mammary carcinomas from BALB-neuT mice stained with anti-CD31 (as marker of endothelial cells) and anti-Amot antibodies

Fig. 2

Effect of anti-Amot vaccination on mammary carcinogenesis in BALB-neuT. a, b BALB-neuT and d, e PyMT mice electroporated twice (arrows) with the pAmot (black lines, n = 12 BALB-neuT and n = 19 PyMT mice) or pcDNA3 (dotted lines, n = 11 BALB-neuT and n = 13 PyMT mice) plasmids. a, b P < 0.0001; d P = 0.0002; e P = 0.004 (Mantel-Cox test). Titers of anti-Amot antibodies in BALB-neuT (c) and PyMT mice (f) vaccinated with pAmot (black bars) or with pcDNA3 (white bars). c P = 0.008; f P < 0.0001 (Student’s t test)

Fig. 3

Effect of anti-Amot vaccination on the growth of transplantable TUBO tumors. BALB/c mice were challenged with TUBO cells and electroporated with pAmot or pcDNA3 both when the TUBO tumor reached 4 mm mean diameter and 7 days later. a Progression of TUBO tumors: each line depicts the growth of a single tumor in pAmot vaccinated mice (n = 29). Grey area: tumors growing in the controls electroporated with pcDNA3 plasmid (n = 28). Data were cumulated from three independent and concordant experiments. b Titer of antibodies against Amot (white bars) and against neu (black bars) in the sera of fast and low progressor mice electroporated with pAmot and in controls electroporated with the pcDNA3 plasmid. The titers of anti-Amot antibodies are significantly higher in slow progressor mice, P = 0.03 (Student’s t test)

Fig. 4

Vessel architecture, necrosis and microsphere extravasation in TUBO tumors from pAmot vaccinated mice. Architecture of the vascular system stained in red with anti-CD31 antibody in tumor sections from mice electroporated with: a pcDNA3 and b pAmot plasmids. c Typical areas of necrosis in a tumor from pcDNA3 electroporated mice and d large perivascular necrotic areas in a tumor from pAmot electroporated mice. Extravasation of fluorescent microspheres (green spots) extravasated in the payload of perivascular space of tumors from mice: e electroporated with pcDNA3 and f pAmot plasmids. Magnification ×200

Fig. 5

DCE–MRI of tumor vasculature on BALB/c mice bearing large vascularized TUBO tumors. a Representative T2-w (upper panels) or T1-w (lower panels) of MRI-POST images of mice vaccinated with pAmot (left panels) or pcDNA3 plasmid (right panels). b Mean curves of MRI-PRE (dotted lines) and MRI-POST (continuous lines) tumor signal intensity enhancement from mice electroporated with pAmot (red, n = 6) and pcDNA3 (black, n = 3). c Relative changes of slope (sec⁻¹), d peak enhancement, and e washout (sec⁻¹), parameters after CA administration in mice electroporated with pAmot (black bars) or pcDNA3 (white bars). ∆ slope P = 0.009; ∆ peak P = 0.007; ∆ washout P = 0.03 (Student’s t-test)

Fig. 6

Therapeutic effect of doxorubicin in mice vaccinated against Amot. BALB/c mice challenged with TUBO cells were electroporated with pcDNA3 or pAmot when their TUBO tumor reached 4 mm mean diameter and then again 9 days later. Two days after the second electroporation, a few mice received an i.v. injection of doxorubicin. Tumor growth kinetics in mice electroporated with: pcDNA3 alone (grey area, n = 7); pcDNA3 and injected with doxorubicin (single black lines, n = 11); pAmot alone (orange area, n = 9); pAmot and injected with doxorubicin (single red lines, n = 7). A marked tumor regression was evident 4/7 (57%) of mice of the latter group. P ≤ 0.04 versus any other treatment group (Yates’s χ ² test)