**8. CD34-TRAIL+ cells induce tumor cell apoptosis and hemorrhagic necrosis**

Tumor-homing of CD34-TRAIL+ cells is associated with significant levels of tumor cell apoptosis (Carlo-Stella et al., 2006). To obtain an objective quantification of apoptosis, a computer-aided image analysis using ImageJ software was performed.6 As compared to controls, TUNEL+ cells were increased by 8- (2.4 ± 1.4% vs 0.3 ± 0.3%, P < .0001) and 4-fold (1.2 ± 0.7% vs 0.3 ± 0.3%, P < .0001) following treatment with CD34-TRAIL+ cells and soluble TRAIL, respectively (**Figure 3A**). Interestingly, apoptotic effects of CD34-TRAIL+ cells resulted significantly more potent than those exerted by soluble TRAIL (P < .0001). Additionally, TUNEL staining of tumor sections from untreated, mock- and soluble TRAIL-

 4 Cryosections were fixed with cold acetone, rinsed with PBS, and then blocked with 2% BSA. Sections were first incubated with the appropriate primary antibody, including mouse anti-human stromal cellderived factor-1 (SDF-1) (R&D Systems), rat anti-mouse VCAM-1 (Southern Biotech), or hamster antimouse TRAIL-R2 (BD Pharmingen). After washing, sections were incubated with the appropriate Alexa Fluor 568-conjugated secondary antibody (Invitrogen). Biotinylated tumor vessels were revealed with Alexa Fluor 488-conjugated streptavidin (Invitrogen). Sections were examined under an epifluorescent microscope equipped with a laser confocal system (MRC-1024, Bio-Rad Laboratories). Image processing was carried out using LaserSharp computer software (Bio-Rad Laboratories). 5 To inhibit intratumor homing of CD34-TRAIL+ cells, mice received either one single intraperitoneal

dose of anti–VCAM-1 (vascular cell adhesion molecule-1) antibody (clone M/K-2; Southern Biotech, Birmingham, AL, USA) at 0.5 mg/mouse, 3 hours before cell administration, or two doses of AMD3100 (5 mg/kg, subcutaneous, 1 hour prior to and 3 hours after cell administration).

<sup>6</sup> The number of total and TUNEL+ cells per section was counted as follows. Briefly, the dynamic range of images was expanded to full by contrast enhancement, and cells were identified by appropriate filtering in the red, green, and blue (RGB) channels. Resulting black and white images were combined to represent only pixels selected in every color channel. For each image, both total and TUNEL+ cells were counted by the ImageJ internal function for particle analysis.

This issue was investigated by evaluating the expression of homing receptors on tumor vasculature. Confocal microscopy4 revealed that 30% of tumor vessels expressed high levels of VCAM-1 on the luminal surface (**Figure 2b-c**) (Jin et al., 2006), whereas SFD-1 was ubiquitously expressed on tumor vessels and tumor cells (**Figure 2e-f**). Thus, α4β1 integrins and the CXCR4 chemokine (De Raeve et al., 2004; Peled et al., 1999) seem to play a critical role in regulating intratumor homing of mTRAIL-expressing cells. To further investigate the functional relevance of SDF-1/CXCR4 and VCAM-1/VLA-4 pathways in mediating tumor homing of transduced cells, inhibitory experiments with an anti–VCAM-1 antibody and the CXCR4 antagonist AMD3100 were performed.5 As compared to controls, tumor homing of CD34-TRAIL+ cells was significantly reduced in mice administered with anti–VCAM-1 antibody [0.2 ± 0.03% vs 0.09 ± 0.01% (P = .001)] or the CXCR4 antagonist AMD3100 (Fricker et al., 2006) [0.2 ± 0.03% vs 0.05 ± 0.006% (P = .0003)]. Tumor vasculature was also analyzed for the expression of TRAIL-R2 receptor. Indeed, confocal microscopy revealed that approximately 8 - 12% of tumor endothelial cells expressed TRAIL-R2 receptor on their luminal surface (**Figure 2h-i**), suggesting that mechanisms other than SDF-1/CXCR4 and VCAM-1/VLA-4, such as the mTRAIL/TRAIL-R2 interactions, might be involved in regulating intratumor homing as well as functional activity of CD34-TRAIL+ cells (Lavazza

**8. CD34-TRAIL+ cells induce tumor cell apoptosis and hemorrhagic necrosis**  Tumor-homing of CD34-TRAIL+ cells is associated with significant levels of tumor cell apoptosis (Carlo-Stella et al., 2006). To obtain an objective quantification of apoptosis, a computer-aided image analysis using ImageJ software was performed.6 As compared to controls, TUNEL+ cells were increased by 8- (2.4 ± 1.4% vs 0.3 ± 0.3%, P < .0001) and 4-fold (1.2 ± 0.7% vs 0.3 ± 0.3%, P < .0001) following treatment with CD34-TRAIL+ cells and soluble TRAIL, respectively (**Figure 3A**). Interestingly, apoptotic effects of CD34-TRAIL+ cells resulted significantly more potent than those exerted by soluble TRAIL (P < .0001). Additionally, TUNEL staining of tumor sections from untreated, mock- and soluble TRAIL-

4 Cryosections were fixed with cold acetone, rinsed with PBS, and then blocked with 2% BSA. Sections were first incubated with the appropriate primary antibody, including mouse anti-human stromal cellderived factor-1 (SDF-1) (R&D Systems), rat anti-mouse VCAM-1 (Southern Biotech), or hamster antimouse TRAIL-R2 (BD Pharmingen). After washing, sections were incubated with the appropriate Alexa Fluor 568-conjugated secondary antibody (Invitrogen). Biotinylated tumor vessels were revealed with Alexa Fluor 488-conjugated streptavidin (Invitrogen). Sections were examined under an epifluorescent microscope equipped with a laser confocal system (MRC-1024, Bio-Rad Laboratories). Image processing was carried out using LaserSharp computer software (Bio-Rad Laboratories). 5 To inhibit intratumor homing of CD34-TRAIL+ cells, mice received either one single intraperitoneal dose of anti–VCAM-1 (vascular cell adhesion molecule-1) antibody (clone M/K-2; Southern Biotech, Birmingham, AL, USA) at 0.5 mg/mouse, 3 hours before cell administration, or two doses of AMD3100

6 The number of total and TUNEL+ cells per section was counted as follows. Briefly, the dynamic range of images was expanded to full by contrast enhancement, and cells were identified by appropriate filtering in the red, green, and blue (RGB) channels. Resulting black and white images were combined to represent only pixels selected in every color channel. For each image, both total and TUNEL+ cells

(5 mg/kg, subcutaneous, 1 hour prior to and 3 hours after cell administration).

were counted by the ImageJ internal function for particle analysis.

**7. Vascular signals involved in tumor homing** 

et al., 2010).

Fig. 2. Vascular molecules involved in intratumor homing of CD34-TRAIL+ cells. Confocal microscopy analysis of intratumor recruiting signals was carried out on 4-µm cryosections from in vivo biotinylated tumors. Cryosections were stained with Alexa Fluor 488 conjugated streptavidin (*green*) to detect tumor vasculature (*a, d, g*). Cryosections were also stained with anti–VCAM-1 (*b*), anti–SDF-1 (*e*), or anti–TRAIL-R2 (*h*) followed by the appropriate Alexa Fluor 568-conjugated secondary antibody for indirect detection of the corresponding antigen (*red*). Merged images demonstrate VCAM-1 (*c*), SDF-1 (*f*), or TRAIL-R2 (*i*) expression by endothelial cells. Objective lens, 40×.

treated mice revealed a homogeneous mass of viable cells with necrotic areas accounting only for 1.4 ± 1.0%, 1.8 ± 1%, and 2.9 ± 1% of total tissue, respectively (**Figure 3B**). In contrast, tumors from CD34-TRAIL-treated mice displayed a significant increase of necrotic areas as compared to controls, with percentages of necrotic areas per tissue section ranging from 6% to 18%, and a mean 8-fold increase over controls (11 ± 3.8% vs 1.4 ± 1.0%, P < .0001), and 4-fold increase over soluble TRAIL-treated mice (11 ± 3.8% vs 2.9 ± 1%, P = .0001) (**Figure 3B**). Pharmacological inhibition of intratumor recruitment of CD34- TRAIL+ cells using AMD3100, or anti-VCAM-1 antibody significantly reduced necrotic areas by 37% (P = .02) and 56% (P = .002), respectively (**Figure 3C**), suggesting that intratumor recruitment of CD34-TRAIL+ cells specifically triggered tumor necrosis.

Targeting TRAIL Receptors with Genetically-Engineered CD34+ Hematopoietic Stem Cells 669

histological sections from tumors treated with transduced cells and subsequently in vivo biotinylated to detect tumor vasculature (Lavazza et al., 2010; Rybak et al., 2005). In untreated mice, tumor vasculature was abundant, tortuous, and evenly distributed throughout the tumor (**Figure 6A**). In striking contrast, in NOD/SCID mice treated with CD34-TRAIL+ cells, viable tumor cells surrounding necrotic areas appeared deficient in capillaries and small-caliber blood vessels, which were less tortuous and had fewer branches and sprouts (**Figure 6A**). Globally, mean percentages of endothelial areas from control and mock-treated tumors were 8.8 ± 5.6% and 8.2 ± 3.3%, respectively (**Figure 6B**). Administration of soluble TRAIL did not affect endothelial area compared to controls (8.1 ± 2.9% vs 8.8 ± 5.6%, P = ns). In contrast, a single intravenous injection of 3 × 106 CD34- TRAIL+ cells caused a 37% decrease of endothelial area compared to control (5.6 ± 3.2% vs 8.8 ± 5.6%, P < .0001) (**Figure 6B**). Additionally, blood vessels from tumors treated with CD34-TRAIL+ cells were thicker than those observed in untreated or soluble TRAIL-treated animals (**Figure 6A**). Based on these findings, we isolated images of transversally oriented vessels in streptavidin-HPR stained sections and calculated vessel wall thickness by processing images with ImageJ and specifically written macros. As shown in **Figure 6C**, wall thickness was 1.7-fold increased compared to control (5.5 ± 1.4 vs 3.2 ± 0.8 µm, P < .0001), whereas no increases emerged after soluble TRAIL administration (3.3 ± 0.7 vs 3.2 ± 0.8 µm).

Experimental data obtained in a variety of preclinical models of both localized and disseminated disease strongly suggest that TRAIL-expressing CD34+ cells can efficiently vehiculate mTRAIL within the tumors where they exert potent antivascular and antitumor activities resulting in a significant reduction of tumor growth. Analysis of tumor nodules obtained 48 hours after a single administration of transduced cells showed that TRAILexpressing cells were 2-fold more effective than soluble TRAIL in inducing apoptosis of tumor cells. Broad necrotic events, involving up to 18% of tumor tissue, were detected only after administration of CD34-TRAIL+ cells and were associated with a hemorrhagic component which was not detectable after soluble TRAIL administration. Hemorrhagic

distribution. Then, the lumen of each vessel in the image was identified as a non-endothelial area, ringed by endothelial tissue and greater than an arbitrary threshold. This procedure rejected smaller artifacts and allowed for recognition of hollow vessels even when erythrocytes or other cells occupied the lumen. Subsequently, we identified the endothelium surrounding a given lumen by an iterative procedure. At first, we subdivided the binary representation of stained tissue into areas by means of a watershed algorithm. Then we selected only those regions adjacent to the lumen, obtaining a minimal image of the vessel wall. This minimal image was used to compute a working thickness according to the previously stated formula. Then, to avoid arbitrary removal of bona fide portions of the walls, we calculated a theoretical vessel contour expanding the lumen outline by a number of pixels equivalent to the working thickness. We next turned back to the watershedded image of endothelium distribution, selecting only those areas connected with the new, theoretical contour. Inclusion of the new regions in the minimal image produced the final vessel image. Both images were saved to allow for manual appreciation of proper vessel identification. At last, the final vessel thickness was calculated after assessment of the final vessel area and external perimeter. Per each parameter, the accuracy and appropriate cut-off levels were determined by comparing processed images to the RGB originals. In all instances, automatic routines were validated by comparing results with those obtained by visual

counting of up to 10% of the total images by two independent pathologists.

**10. Conclusions** 

Fig. 3. Pro-apoptotic and necrotic effects of CD34-TRAIL+ cells. NOD/SCID mice bearing subcutaneous tumor nodules 10 mm in diameter were randomly assigned to receive CD34- TRAIL+ cells, mock-transduced CD34+ cells (3 × 106 cells/mouse, intravenous), recombinant soluble TRAIL (500 µg/mouse, IP), or control vehicle. (A) Percentages of apoptotic cells in tumors from untreated or treated animals were computationally calculated on digitally acquired images (objective lens, 20×) using ImageJ. At least three sections from different animals were analyzed. The boxes extend from the 25th to the 75th percentiles, the lines indicate the median values, and the whiskers indicate the range of values. \* P < .0001, compared to controls. # P < .0001, compared to soluble TRAIL. (B) Quantification of necrotic areas by ImageJ analysis on tissue sections stained with TUNEL. At least six sections from different animals were analyzed per treatment group. \* P < .0001, compared to controls. # P = .0001, compared to soluble TRAIL. (C) Anti–VCAM-1 and AMD3100 reduced tumor necrosis in mice treated with CD34-TRAIL+ cells. # P < .0001, compared to control. \* P = .002, compared with CD34-TRAIL+ cells. \*\* P = .02, compared with CD34-TRAIL+ cells.

A distinctive and prominent feature of tumors treated with CD34-TRAIL+ cells was represented by hemorrhagic phenomena within necrotic areas close to damaged vessels which were detected by immunohistochemical staining with glycophorin A (**Figure 4A**). Hemorrhagic phenomena exactly matched TUNEL+ necrotic areas and closely associated with apoptotic endothelial cells (**Figure 4BL**). In striking contrast, apoptotic vessels and hemorrhagic phenomena could not be detected neither in tumors from mice treated with soluble TRAIL (**Figure 4BK** and **4A**), nor in healthy tissues (**Figure 5**), suggesting a tumorrestricted antivascular activity by CD34-TRAIL+ cells.

#### **9. Antivascular effects of CD34-TRAIL+ cells**

To better understand the relationship between the antitumor effects of CD34-TRAIL+ cells and apoptosis of endothelial cells, an extensive vascular analysis7 was performed on

<sup>7</sup> Tumor vasculature was analyzed on cryosections using the open-source ImageJ software (http://rsb.info.nih.gov/ij/) from in vivo biotinylated mice stained with HRP-conjugated streptavidin. To calculate endothelial area, i.e., the percentage of tissue section occupied by endothelium, endothelial cells were identified by contrast enhancement and appropriate filtering. Background signal was removed considering only structures larger than an arbitrary minimal value. To analyze vessel wall thickness, we manually selected rectangular regions of RGB input images containing at least a hollow vessel. An automatic routine computed vessel thickness according to the formula: Thickness = 2 × (vessel area)/[(vessel perimeter) + (lumen perimeter)]. At first, endothelial tissue was identified applying a threshold on the blue channel and obtaining a binary image representative of its

histological sections from tumors treated with transduced cells and subsequently in vivo biotinylated to detect tumor vasculature (Lavazza et al., 2010; Rybak et al., 2005). In untreated mice, tumor vasculature was abundant, tortuous, and evenly distributed throughout the tumor (**Figure 6A**). In striking contrast, in NOD/SCID mice treated with CD34-TRAIL+ cells, viable tumor cells surrounding necrotic areas appeared deficient in capillaries and small-caliber blood vessels, which were less tortuous and had fewer branches and sprouts (**Figure 6A**). Globally, mean percentages of endothelial areas from control and mock-treated tumors were 8.8 ± 5.6% and 8.2 ± 3.3%, respectively (**Figure 6B**). Administration of soluble TRAIL did not affect endothelial area compared to controls (8.1 ± 2.9% vs 8.8 ± 5.6%, P = ns). In contrast, a single intravenous injection of 3 × 106 CD34- TRAIL+ cells caused a 37% decrease of endothelial area compared to control (5.6 ± 3.2% vs 8.8 ± 5.6%, P < .0001) (**Figure 6B**). Additionally, blood vessels from tumors treated with CD34-TRAIL+ cells were thicker than those observed in untreated or soluble TRAIL-treated animals (**Figure 6A**). Based on these findings, we isolated images of transversally oriented vessels in streptavidin-HPR stained sections and calculated vessel wall thickness by processing images with ImageJ and specifically written macros. As shown in **Figure 6C**, wall thickness was 1.7-fold increased compared to control (5.5 ± 1.4 vs 3.2 ± 0.8 µm, P < .0001), whereas no increases emerged after soluble TRAIL administration (3.3 ± 0.7 vs 3.2 ± 0.8 µm).
