Improved Functionality of the Vasculature during Conventionally Fractionated Radiation Therapy of Prostate Cancer

<div><p>Although endothelial cell apoptosis participates in the tumor shrinkage after single high-dose radiotherapy, little is known regarding the vascular response after conventionally fractionated radiation therapy. Therefore, we evaluated hypoxia, perfusion and vascular microenvironment changes in an orthotopic prostate cancer model of conventionally fractionated radiation therapy at clinically relevant doses (2 Gy fractions, 5 fractions/week). First, conventionally fractionated radiation therapy decreased tumor cell proliferation and increased cell death with kinetics comparable to human prostate cancer radiotherapy. Secondly, the injection of Hoechst 33342 or fluorescent-dextrans showed an increased tumor perfusion within 14 days in irradiated tumors, which was correlated with a clear reduction of hypoxia. Improved perfusion and decreased hypoxia were not explained by increased blood vessel density, size or network morphology. However, a tumor vascular maturation defined by perivascular desmin+/SMA+ cells coverage was clearly observed along with an increase in endothelial, zonula occludens (ZO)-1 positive, intercellular junctions. Our results show that, in addition to tumor cell killing, vascular maturation plays an uncovered role in tumor reoxygenation during fractionated radiation therapy.</p></div

Fractionated irradiation induces vascular remodeling.

<p>(<b>A</b>) Pseudo-confocal images of tumor blood vessels during CFRT and stained for ZO-1/CD31 (top) or SMA/CD31 (bottom). (<b>B</b>,<b>C</b>). Image quantification of ZO-1+/CD31+ (<b>B</b>) and peri-CD31+ SMA surfaces (<b>C</b>). Values represent the average of n≥13 per point ± sem. (<b>D</b>) Image quantification of peri-CD31+ desmin surface and frequency of desmin+/SMA+ vessels. (<b>B</b>,<b>E</b>,<b>D</b>) Statistical comparisons vs. t0. (<b>E</b>) Representative confocal images of a blood vessel from a 14-day treated tumor stained for CD31/desmin/SMA. (<b>F</b>) histogram analysis of CD31/desmin/SMA pseudocolor profile of confocal image cross-section.</p

Maintenance of vascular density and distribution during fractionated irradiation.

<p>(<b>A</b>) Microvessel density in tumors during CFRT. Values represent the average of n≥13 per point ± sem. (<b>B</b>) Distance profile between cells and the closest blood vessel, from tumors during CFRT. Profiles are based on n≥13. Statistical comparisons vs. t0. (<b>C</b>) Pseudo-confocal images of tumor-associated blood vessels (CD31+) stained for TUNEL during CFRT. Arrows: TUNEL+/CD31+ cells. (<b>D</b>) Image quantification of CD31+/TUNEL+ surface. Values represent the average of n≥13 per point ± sem. (<b>E</b>) Representative Z-stack images of 100 µm-thick tumor sections before (t0) or after 2 weeks of CFRT (t14) and stained for blood vessels (CD31+/Fli-1+). (<b>F</b>) Image analysis of blood vessel network from 100 µm-thick tumor sections. Values represent the average of n  =  9 per point ± sem.</p

Fractionated irradiation reduces hypoxia and increases tumor perfusion.

<p>(<b>A</b>) Pseudo-confocal images of tumors during CFRT, stained for hypoxia (EF5) and endothelial cells (CD31). (<b>B</b>) Image quantification of EF5+ surface in tumors during CFRT. Values represent the average of n≥13 per point ± sem. (<b>C</b>) Pseudo-confocal images of tumors perfused with Hoechst 33342 and 10 kDa/2 MDa dextrans before (t0) or after 2 weeks of CFRT (t14). SYBR green was used as a counterstain of total cell nuclei. (<b>D,E,F</b>) Image quantification of Hoechst+ (<b>D</b>), and medium (<b>E</b>) and large (<b>F</b>) dextran+ surfaces in tumors during CFRT (n  =  6). (<b>B</b>,<b>D</b>,<b>E</b>,<b>F</b>). Statistical comparisons vs. t0.</p

3E2 targets Gb3 on the endothelial cell membrane.

<p>A) Gb3 immunolocalization in HMEC-1 by FITC-conjugated 3E2 (green; upper panel) <i>vs.</i> control isotypic 11E10 IgM (lower panel) and counterstaining with Draq5. Scale bar represent 20 µm. B) Percentage of Gb3-positive cells by Facs analysis after hybridization of HMEC-1, RAJI and NXS2 cells with using 3E2 or isotypic control. C) GSLs profiles by orcinol staining after HPTLC (panel a) and Gb3 expression by immunohybridization using 3E2 (panel b) or commercially available 38.13 (panel c). Lane 1: rat brain gangliosides; lane 2: neutral GSLs mixture: lane 3, purified Gb3; lane 4: HMEC-1 glycolipids extract.</p

3E2 antibody inhibits <i>ex vivo</i> angiogenesis.

<p>A) Viability measured by MTT of HMEC-1 treated for 24 h with increasing concentration of 3E2 (▾) or isotypic control (▪) (n = 3; mean±SEM; *p<0.05). B) Western blot of phosphor-ERK and phosphor-AKT from cells treated with either 40 µg/ml 3E2 or IgM-control antibody (n = 3). C) Photographs of sprouting vessels from aorta 5 d post-treatment by an increasing dose of 3E2 (n = 3). D) Sprouting index from aorta rings 5 d post-treatment with 3E2 or isotypic control (n = 3, mean±SEM; *p<0.05).</p

3E2 inhibits in <i>vivo</i> metastases spreading.

<p>A) Pictures of NXS2 hepatic metastases by confocal microscopy stained with Alexa488-conjuguated CD31 mAb (green), Alexa568-conjuguated 3E2 (red) or isotypic IgM control and counterstained with Draq5. Colocalization of the endothelial marker CD31 and Gb3 is shown by the yellow staining on the merge image. B) Number of liver metastases per animal (n = 6; mean±SEM; *p<0.05). C) Representative photography of liver 28 days after NXS2 injection and the different immunotherapies (scale bar represents 1 cm).</p

3E2 antibody inhibits endothelial cell proliferation.

<p>HMEC-1 cells are tracked by videomicroscopy up to 24 h after treatment by 3E2 or isotypic control. A) Cell number per field in function of time. Histograms show the mean doubling time (n = 6 for IgM and n = 9 for 3E2, mean±SEM; *p<0.05). B) Microphotographs of representative fields of HMEC-1 in function of time. Magnification 10×. C) Number of mitosis summed every 6 h for 24 h (n = 5, mean±SEM; *p<0.05). D) Duration of the mitosis (n = 5, mean±SEM; *p<0.05). E) Cell death quantification detected by sub-G1 and hoechst assays from 3E2- or IgM-treated HMEC-1 (n = 3; mean±SD; ns: p>0.1).</p

Gb3 is not modulated by 3E2 antibody treatment.

<p>HMEC-1 cells were treated with either 40 µg/ml 3E2- or IgM control-antibody. A) RT-Q PCR 24 h after treatment (n = 6; mean±SEM; ns: p>0.1). B) Gb3-positive HMEC-1 cells determined by Facs 24 h after treatment (n = 3). C) Table analysis (% of positive cells, means) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045423#pone-0045423-g003" target="_blank">Fig. 3 B</a> (ns>0.1).</p

Distribution of Gb3 in organs from C57Bl/6 mouse obtained after staining with Alexa568-conjuguated 3E2.

<p>Frozen healthy organs sections of 5 µm were hybridized with biotinylated 3E2 or its isotypic control, revealed by an Alexa 568-conjugated streptavidin and counterstained with DAPI. Pictures were observed under a confocal microscope (n = 3).</p>#<p>: High background.</p>*<p>: Lipofuscin autofluorescence.</p