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. 2013 May 10;332(1):120-9.
doi: 10.1016/j.canlet.2012.11.016. Epub 2012 Nov 27.

Development of an anti-angiogenic therapeutic model combining scAAV2-delivered siRNAs and noninvasive photoacoustic imaging of tumor vasculature development

Qing Ruan  1 , Lei Xi, Sanford L Boye, Song Han, Zhi J Chen, William W Hauswirth, Alfed S Lewin, Michael E Boulton, Brian K Law, Wen G Jiang, Huabei Jiang, Jun Cai
Affiliations

Development of an anti-angiogenic therapeutic model combining scAAV2-delivered siRNAs and noninvasive photoacoustic imaging of tumor vasculature development

Qing Ruan et al. Cancer Lett. .

Abstract

We aimed to develop an anti-angiogenic model for breast cancer by combining (1) siRNA-based therapy delivered by self-complementary adeno-associated virus serotype 2 (scAAV2) vectors to _target tumor vasculature, and (2) noninvasive monitoring to tumor response to anti-angiogenesis by photoacoustic (PA) imaging. scAAV2 vector containing 7 surface exposed tyrosine to phenylanine capsid mutations was able to transduce microvascular endothelial cells with high efficiency. siRNAs against UPR (unfolded protein response)-IRE1α, XBP-1, ATF6 significantly inhibited breast cancer-induced angiogenesis in vitro by inhibiting endothelial cell survival. PA imaging showed that knockdown of UPR proteins greatly reduced tumor angiogenesis in vivo in breast cancer models.

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Conflict of interest statement

Conflict of Interest

W. W. H. and the University of Florida have a financial interest in the use of AAV therapies and own equity in a company (AGTC Inc.) that might, in the future, commercialize some aspects of this work.

Figures

Figure. 1
Figure. 1
Comparative analysis of scAAV2-mediated transduction of MMECs. A, cells were infected with the WT or the triple-, quadruple- or septuplete-tyrosine mutant scAAV2-hGFP vectors at a of multiplicity infection (MOI) of 10,000 vgs/cell. Transgene expression was detected by fluorescence microscopy, 72 hr post-infection. Quantitative analysis of AAV2 transduction efficiency in MMECs is shown in arbitrary units calculated by multiplying the percentage of positive cells by the mean fluorescence intensity in each sample. B, shows a scAAV2 construct with smCBA driving expression of siRNAs against mice UPR proteins. C, transduction efficiency of scAAV2 septuplet-tyrosine mutant vectors in MMECs. D, a comparison of mCherry expression at different MOIs as indicated shown in arbitrary units calculated by multiplying the percentage of positive cells by the mean fluorescence intensity. Each value represents the average of three samples (eight pooled wells of a 24-well plates/sample), based on 10,000 counted cells.
Figure. 2
Figure. 2
Pro-angiognic role of the UPR proteins on endothelial cells. Mice microvascular endothelial cells (MMECs) were co-cultured with NeuT EMTCL2 for 48 hr followed by treated with scAAV2 encoding siRNAs against UPR proteins. A, MMECs were cultured between two layers of Matrigel for 48 hr. Morphometric analysis of the degree of tubule formation was then performed. B, representative photomicrographs of microscope fields showing tubule formation.
Figure. 3
Figure. 3
Survival role of the UPR proteins on endothelial cells. A, anexin V-propidium iodide-positive cells were shown by flow cytometric analysis (top right, late stage apoptosis; bottom right, early apoptosis). B, cell apoptosis was expressed as a percentage of apoptotic cells in the total cell population. C, the cell proliferation was assessed by crystal violet staining.
Figure. 4
Figure. 4
Serial noninvasive photoacoustic (PA) imaging of the developing tumor vasculature and quantitative analysis for mice breast cancer xenograft models. Mice breast cancer xenografts were established by subcutaneous injection of 1×105 NeuT or NeuT EMTCL2 cells into the mammary fat pads of the mice. Serial PA imaging was performed on the same tumor inoculation site on day 3, 5, 7 and 9 post tumor inoculations. A, representative serial PA images of NeuT model (first and second panels) and NeuT EMTCL2 model (third and forth panels). B, Entropy extraction for change in the vessel density over different time points as indicated. C, shows comparative analysis of normalized vessel volumes at different time point as indicated. D, tumor volume was calculated from daily caliper measurements of the large (a) and smallest (b) diameters of each tumor using formula a×b2×0.4. Representative images of H&E staining for breast cancer tissue sections of NeuT (top) and NeuT EMTCL2 (bottom) xenograft models.
Figure. 5
Figure. 5
Knockdown of the siRNAs against the UPR protein resulted in decreased tumor growth and tumor vasculature development in mice breast cancer xenografts. Mice breast cancer xenografts received intratumoral scAAV2 sept-mutant vector-delivered siRNAs against IRE1α or XBP-1 or ATF6. PA imaging was performed on the same tumor inoculation site on day 3, 5, 7 and 9 post tumor inoculations. A, representative PA images of different treatments: the scrambled siRNA (top panel), IRE1α siRNA (second panel), XBP-1 siRNA (third panel) and ATF6 siRNA (bottom panel). B, Entropy extraction for change in the vessel density over different time points as indicated. C, shows comparative analysis of normalized vessel volumes at different time point as indicated. D, tumor volume was calculated from daily caliper measurements of the large (a) and smallest (b) diameters of each tumor using formula a×b2×0.4.
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