**10. Conclusions**

668 Non-Viral Gene Therapy

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.

restricted antivascular activity by CD34-TRAIL+ cells.

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

\* 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 tumor-

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 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.

Targeting TRAIL Receptors with Genetically-Engineered CD34+ Hematopoietic Stem Cells 671

Fig. 5. Tissue and vascular toxicity in healthy tissue after CD34-TRAIL+ cells administration. NOD/SCID mice bearing subcutaneous tumor nodules received a single intravenous injection of CD34-TRAIL+ cells (3 × 106 cells/mouse), or control vehicle. Forty-eight hours after treatment, lung, liver, spleen, and femur were harvested and analyzed. Hematoxylin and eosin staining demonstrated the absence of tissue or vascular damage. Representative

Increasing evidences suggest that recruitment of CD34+ cells in the tumor microenvironment is due to homing signals similar to those found in the bone marrow hematopoietic niches (Jin et al., 2006; Kaplan et al., 2007; Rafii et al., 2002; Wels et al., 2008). Both SDF-1/CXCR4 and VCAM-1/VLA-4 pathways play a key role in regulating bone marrow homing of transplanted hematopoietic stem cells (Aiuti et al., 1997; Peled et al., 1999) as well as intratumor recruitment of CXCR4-expressing cells and neovascularization during acute ischemia and tumor growth (Burger & Kipps, 2006; Jin et al., 2006; Petit et al., 2007). Kinetics data obtained in our models clearly show that intravenously injected transduced cells circulate in normal tissues up to 24 hours, but they progressively and preferentially home at tumor sites where they can be detected up to 48 hours after injection. Lack of intratumor detection of CD34-TRAIL+ cells beyond 48 hours after injection (data not shown) may be due to destruction of mTRAILexpressing cells in the context of antitumor activities (i.e., disruption of tumor vasculature, hemorrhagic necrosis, tumor necrosis, etc.). Pharmacological manipulation of adhesion receptor expression using either AMD3100 or anti–VCAM-1 antibodies significantly reduced both the frequency and the antitumor efficacy of CD34-TRAIL+ cells strongly suggesting that SDF-1 and VCAM-1 expressed by tumor vasculature efficiently recruit transduced CD34+ cells within tumors by challenging their trafficking and homing properties. The role of additional binding systems, such as mTRAIL/TRAIL-R2, in mediating tumor tropism of CD34-TRAIL+ cells may be hypothesized on the basis of our data. Binding of CD34-TRAIL+ cells to TRAIL-R2 expressed by tumor vasculature could significantly contribute to initiation of a cascade of events that induce early endothelial damage, leading to extensive tumor cell death (Arafat et

histological images are shown. Objective lens, 10x.

al., 2000).

Fig. 4. Tumor hemorrhagic necrosis and endothelial cell apoptosis induced by CD34- TRAIL+ cells. NOD/SCID mice bearing subcutaneous tumor nodules 10 mm in diameter were randomly assigned to receive CD34-TRAIL+ cells, recombinant TRAIL (500 µg/mouse, IP), or control vehicle. Tumors were harvested forty-eight hours after treatment. (**A**) Hematoxylin and eosin (H&E), TUNEL and glycophorin A staining were performed. Objective lens, 2×. (**B**) Just before sacrifice NOD/SCID mice were intravenously injected with sulfo-NHS-LC-biotin to biotinylate tumor vasculature. Representative confocal images of tumors from untreated and treated animals processed by triple immunofluorescence staining. (*A–C*) Cell nuclei were detected in blue by TO-PRO-3; (*D–F*) apoptotic cells were detected in green by TUNEL staining; (*G–I*) tumor endothelial cells were detected in red by Alexa 568-conjugated streptavidin. (*J–L*) After merging of single-color images, apoptotic nuclei (*green*) were detectable throughout tumor parenchyma after treatment with either soluble TRAIL or CD34-TRAIL+ cells, whereas endothelial cells with apoptotic nuclei (*yellow*) could be detected only in CD34-TRAIL+ cell–treated animals. Objective lens, 40×.

necrosis was localized near TUNEL+ blood vessels, suggesting that apoptosis of tumor endothelial cells represents an early event triggered by CD34-TRAIL+ cells. Overall, these findings support the hypothesis that CD34-TRAIL+ cells exert their cytotoxic activity not only by targeting parenchymal tumor cells but also by targeting tumor vasculature (Carlo-Stella et al., 2006; Lavazza et al., 2010). Indeed, the vascular-disrupting activity of mTRAIL might represent a major concern in view of clinical applications. Notwithstanding the intratumor vascular-disrupting activity of mTRAIL, extensive analysis of healthy tissues failed to detect any evidence of hemorrhagic necrosis, suggesting that vascular damage was tumor-restricted.

Fig. 4. Tumor hemorrhagic necrosis and endothelial cell apoptosis induced by CD34- TRAIL+ cells. NOD/SCID mice bearing subcutaneous tumor nodules 10 mm in diameter were randomly assigned to receive CD34-TRAIL+ cells, recombinant TRAIL (500 µg/mouse,

IP), or control vehicle. Tumors were harvested forty-eight hours after treatment. (**A**) Hematoxylin and eosin (H&E), TUNEL and glycophorin A staining were performed. Objective lens, 2×. (**B**) Just before sacrifice NOD/SCID mice were intravenously injected with sulfo-NHS-LC-biotin to biotinylate tumor vasculature. Representative confocal images of tumors from untreated and treated animals processed by triple immunofluorescence staining. (*A–C*) Cell nuclei were detected in blue by TO-PRO-3; (*D–F*) apoptotic cells were detected in green by TUNEL staining; (*G–I*) tumor endothelial cells were detected in red by Alexa 568-conjugated streptavidin. (*J–L*) After merging of single-color images, apoptotic nuclei (*green*) were detectable throughout tumor parenchyma after treatment with either soluble TRAIL or CD34-TRAIL+ cells, whereas endothelial cells with apoptotic nuclei (*yellow*) could be detected only in CD34-TRAIL+ cell–treated animals. Objective lens, 40×.

necrosis was localized near TUNEL+ blood vessels, suggesting that apoptosis of tumor endothelial cells represents an early event triggered by CD34-TRAIL+ cells. Overall, these findings support the hypothesis that CD34-TRAIL+ cells exert their cytotoxic activity not only by targeting parenchymal tumor cells but also by targeting tumor vasculature (Carlo-Stella et al., 2006; Lavazza et al., 2010). Indeed, the vascular-disrupting activity of mTRAIL might represent a major concern in view of clinical applications. Notwithstanding the intratumor vascular-disrupting activity of mTRAIL, extensive analysis of healthy tissues failed to detect any evidence of hemorrhagic necrosis, suggesting that vascular damage was

tumor-restricted.

Fig. 5. Tissue and vascular toxicity in healthy tissue after CD34-TRAIL+ cells administration. NOD/SCID mice bearing subcutaneous tumor nodules received a single intravenous injection of CD34-TRAIL+ cells (3 × 106 cells/mouse), or control vehicle. Forty-eight hours after treatment, lung, liver, spleen, and femur were harvested and analyzed. Hematoxylin and eosin staining demonstrated the absence of tissue or vascular damage. Representative histological images are shown. Objective lens, 10x.

Increasing evidences suggest that recruitment of CD34+ cells in the tumor microenvironment is due to homing signals similar to those found in the bone marrow hematopoietic niches (Jin et al., 2006; Kaplan et al., 2007; Rafii et al., 2002; Wels et al., 2008). Both SDF-1/CXCR4 and VCAM-1/VLA-4 pathways play a key role in regulating bone marrow homing of transplanted hematopoietic stem cells (Aiuti et al., 1997; Peled et al., 1999) as well as intratumor recruitment of CXCR4-expressing cells and neovascularization during acute ischemia and tumor growth (Burger & Kipps, 2006; Jin et al., 2006; Petit et al., 2007). Kinetics data obtained in our models clearly show that intravenously injected transduced cells circulate in normal tissues up to 24 hours, but they progressively and preferentially home at tumor sites where they can be detected up to 48 hours after injection. Lack of intratumor detection of CD34-TRAIL+ cells beyond 48 hours after injection (data not shown) may be due to destruction of mTRAILexpressing cells in the context of antitumor activities (i.e., disruption of tumor vasculature, hemorrhagic necrosis, tumor necrosis, etc.). Pharmacological manipulation of adhesion receptor expression using either AMD3100 or anti–VCAM-1 antibodies significantly reduced both the frequency and the antitumor efficacy of CD34-TRAIL+ cells strongly suggesting that SDF-1 and VCAM-1 expressed by tumor vasculature efficiently recruit transduced CD34+ cells within tumors by challenging their trafficking and homing properties. The role of additional binding systems, such as mTRAIL/TRAIL-R2, in mediating tumor tropism of CD34-TRAIL+ cells may be hypothesized on the basis of our data. Binding of CD34-TRAIL+ cells to TRAIL-R2 expressed by tumor vasculature could significantly contribute to initiation of a cascade of events that induce early endothelial damage, leading to extensive tumor cell death (Arafat et al., 2000).

Targeting TRAIL Receptors with Genetically-Engineered CD34+ Hematopoietic Stem Cells 673

approach could be exploited to develop effective autologous or allogeneic anticancer

This work was supported in part by grants from Special Program Molecular Clinical Oncology of Associazione Italiana per la Ricerca sul Cancro (Milano, Italy), Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR, Rome, Italy), Ministero della Salute (Rome, Italy), Alleanza Contro il Cancro (Rome, Italy), and the Michelangelo Foundation for

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**12. References** 

**11. Acknowledgements** 

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Fig. 6. Antivascular 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), and control vehicle. (**A**) Forty-eight hours after treatment, NOD/SCID mice were intravenously injected with 0.2 mL of sulfo-NHS-LC-biotin (5 mg/mL) to biotinylate tumor vasculature. Tumors were then excised, and biotinylated endothelium was revealed by HRP-streptavidin and 3,3'-diaminobenzidine for light microscopy analysis. Representative histological images of in vivo biotinylated mice receiving the different treatments are shown. (**B**) Sections were analyzed using ImageJ for quantification of vascular parameters. Endothelial area was calculated on whole tissue sections as (streptavidin-HRP stained area)/(total tissue area) × 100. \* P < .0001, compared to controls. # P < .0001, compared to soluble TRAIL . (**C**) Vessel wall thickness was calculated on transversally oriented vessels. \* P < .0001, compared to controls. # P < .0001, compared to soluble TRAIL .

In conclusion, under our experimental conditions the use of transduced CD34+ cells as a vehicle of mTRAIL resulted in an antitumor effect greater than that exerted by soluble TRAIL, likely because of an antivascular action. Our findings appear to be of outstanding interest in the context of the increasing need for therapeutic strategies targeting not only tumor cells but also the tumor microenvironment (De Raeve et al., 2004; Joyce, 2005; Rafii et al., 2002). Finally, the clinical feasibility of such a systemic CD34+ cell-based gene therapy approach could be exploited to develop effective autologous or allogeneic anticancer treatments.
