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

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

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 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

Vedolizumab staining of colonic biopsies detected by CLE at inclusion and response to vedolizumab at week 22.

Vedolizumab staining of colonic biopsies detected by CLE at inclusion and response to vedolizumab at week 22.</p

S2 Fig -

Absolute counts of T cells (panel A), B cells (panel B) and NK cells (panel D) as determined by flow cytometry in responders (R) and non-responders (NR) to vedolizumab at week 0 (W0) and week 22 (W22). The monocyte blood count (panel C) was measured using the Sysmex XS-800i analyzer. There was no difference of absolute counts of the different cells between responders and non-responders to vedolizumab. In responder patients, the absolute count of monocytes (panel C) decreased significantly between week 0 and 22 and monocytes were also significantly fewer at week 22 in responders compared to non-responders. α4β7 expression was analyzed by flow cytometry using a FITC-conjugated vedolizumab antibody in T cells (panel E), B cells (panel F), NK cells, monocytes (panel G) and NK cells (panel H). There was no difference between responders and non-responders to vedolizumab at week 0 before the initiation of the treatment. At week 22, the number of vedolizumab positive B cells decreased significantly in both responders and non-responders (panel F). (TIF)</p

Comparison of FITC-labelled vedolizumab and Alexa fluor- labelled adalimumab staining detected by CLE in inflamed and non-inflamed mucosa.

Comparison of FITC-labelled vedolizumab and Alexa fluor- labelled adalimumab staining detected by CLE in inflamed and non-inflamed mucosa.</p

Fig 2 -

Representative CLE pictures obtained from the same colonic biopsy after staining with vedolizumab-FITC (panel A, green) and adalimumab-Alexa fluor (panel B, red). Arrows identified positive vedolizumab -FITC positive cells and solid arrows, adalimumab-Alexa fluor positive cells in each panel. CLE images were merged (panel C) and showed an overlay of 80%.</p

Adalimumab staining of colonic biopsies detected by CLE at inclusion and response to vedolizumab at week 22.

Adalimumab staining of colonic biopsies detected by CLE at inclusion and response to vedolizumab at week 22.</p

Adalimumab staining of colonic biopsies detected by CLE and response to adalimumab at week 30.

Adalimumab staining of colonic biopsies detected by CLE and response to adalimumab at week 30.</p