**6. In vivo homing of CD34-TRAIL+ cells**

Homing properties of transduced cells in healthy tissues as well as tumor nodules were extensively investigated in tumor-bearing NOD/SCID mice who received a single

<sup>1</sup> Six- to eight-week-old female NOD/SCID mice with body weight of 20 to 25 g were purchased from Charles River (Milano, Italy, EU). Mice were housed under standard laboratory conditions according to our institutional guidelines. Animal experiments were performed according to the Italian laws (D.L. 116/92 and following additions), and were approved by the institutional Ethical Committee for Animal Experimentation. KMS-11 cells (5 × 106 cells/mouse) were inoculated subcutaneosuly in the left flank of each mouse. When tumor reached approximately 7 - 10 mm in diameter (usually 10–12 days after tumor inoculation), mice were randomly assigned to planned treatments consisting of daily injections of either CD34-TRAIL+ cells or mock-transduced CD34+ cells (1 × 106 cells/mouse/injection/day, intravenous, days 12–15), or a 4-day course of recombinant soluble TRAIL (30 mg/kg/day, intraperitoneal, days 12– 15). Mice were checked twice weekly for tumor appearance, tumor dimensions, body weight, and toxicity. Tumor volumes were measured with calipers and their weights calculated using the formula: (a × b2)/2, where a and b represented the longest and shortest diameters, respectively. Mice were followed up for 3 weeks after the end of the treatments. The endpoint of the subcutaneous model was tumor weight. Each experiment was performed on at least two separate occasions, using five mice per treatment group.

<sup>2</sup> KMS-11 (0.5 × 106 cells/mouse) cell line was inoculated intravenously. CD34-mock or CD34-TRAIL+ cells (1 × 106 cells/mouse/injection) were inoculated intravenously weekly for 4 weeks starting either on day 7 (early-stage tumor model), or 14 (advanced-stage tumor model) after tumor cell injection.

than 80% (range 70% - 96%), a high level expression of mTRAIL, and a cell viability ≥85%. Flow cytometry analysis of CD34-TRAIL+ cells shows significant levels of transgene expression for at least 96 hours after transduction, and Western blot analysis reveals the presence of 32- and 55-kDa proteins, which are the expected products for full-length

The antitumor activity of CD34-TRAIL+ cells has been investigated in a variety of localized and disseminated tumor models in NOD/SCID mice. Using a localized, subcutaneous multiple myeloma model (KMS-11 cell line), intravenously-injected mTRAIL-expressing cells significantly reduced tumor growth over controls as well as soluble TRAIL.1 In fact, compared with untreated controls, both CD34-TRAIL+ cells and soluble TRAIL significantly inhibited tumor growth by day 28 after tumor injection, when tumor volumes were reduced by 38% (P < .05) and 31% (P < .05), respectively. However, on day 35, CD34-TRAIL+ cells induced a 40% reduction in tumor growth over controls (4.2 ± 1.2 vs 7.0 ± 2.0 g, P < .001), whereas a 29% reduction of tumor growth was detected in mice receiving soluble TRAIL (5.0 ± 1.7 g vs 7.0 ± 2.0 g, P < .001) (Lavazza et al., 2010). Even more importantly, an efficient antitumor activity of intravenously injected mTRAIL-expressing CD34+ cells was also detected in NOD/SCID mice bearing disseminated, systemic multiple myeloma and non-Hodgkin lymphoma xenografts (Carlo-Stella et al., 2006; Carlo-Stella et al., 2007; Carlo-Stella et al., 2008). Using KMS-11 as model system, treatment of advanced-stage disease with CD34-TRAIL+ cells resulted in a significant increase of median survival over controls (83 vs 55 days, P ≤ 0.0001), with 28% of NOD/SCID mice alive and disease-free at the end of the

Homing properties of transduced cells in healthy tissues as well as tumor nodules were extensively investigated in tumor-bearing NOD/SCID mice who received a single

1 Six- to eight-week-old female NOD/SCID mice with body weight of 20 to 25 g were purchased from Charles River (Milano, Italy, EU). Mice were housed under standard laboratory conditions according to our institutional guidelines. Animal experiments were performed according to the Italian laws (D.L. 116/92 and following additions), and were approved by the institutional Ethical Committee for Animal Experimentation. KMS-11 cells (5 × 106 cells/mouse) were inoculated subcutaneosuly in the left flank of each mouse. When tumor reached approximately 7 - 10 mm in diameter (usually 10–12 days after tumor inoculation), mice were randomly assigned to planned treatments consisting of daily injections of either CD34-TRAIL+ cells or mock-transduced CD34+ cells (1 × 106 cells/mouse/injection/day, intravenous, days 12–15), or a 4-day course of recombinant soluble TRAIL (30 mg/kg/day, intraperitoneal, days 12– 15). Mice were checked twice weekly for tumor appearance, tumor dimensions, body weight, and toxicity. Tumor volumes were measured with calipers and their weights calculated using the formula: (a × b2)/2, where a and b represented the longest and shortest diameters, respectively. Mice were followed up for 3 weeks after the end of the treatments. The endpoint of the subcutaneous model was tumor weight. Each experiment was performed on at least two separate occasions, using five mice per

2 KMS-11 (0.5 × 106 cells/mouse) cell line was inoculated intravenously. CD34-mock or CD34-TRAIL+ cells (1 × 106 cells/mouse/injection) were inoculated intravenously weekly for 4 weeks starting either on day 7 (early-stage tumor model), or 14 (advanced-stage tumor model) after tumor cell injection.

monomer and dimer TRAIL, respectively (Carlo-Stella et al., 2006).

**5. Antitumor activity of mTRAIL-expressing cells** 

150-day observation period (Carlo-Stella et al., 2006).2

**6. In vivo homing of CD34-TRAIL+ cells** 

treatment group.

intravenous injection of CD34-TRAIL+ cells (3 × 106 cells/mouse) (Lavazza et al., 2010). Tumor and healthy tissue sections were immunostained with an anti-human CD45 antibody and digitally recorded to count transduced cells on entire tissue sections.3 Early following injection, transduced cells were detected at high frequencies in the lung, liver and spleen (**Figure 1**). CD34-TRAIL+ cells progressively decreased and were no longer detectable in these tissues 24 hours after injection. Bone marrow CD34-TRAIL+ cells peaked 5 hours after injection and were detectable up to 24 hours. Low frequencies of transduced cells were detected within tumors as early as 30 minutes following injection. They progressively increased and peaked 48 hours post-injection when on average 188 ± 25 CD45+ cells per 105 tumor cells (i.e., 0.2 ± 0.03%) were recorded (**Figure 1**). Overall, kinetics data suggest that transduced cells transiently circulate through healthy tissues, whereas they are preferentially recruited within tumor nodules, allowing to hypothesize that homing signals by tumor endothelial cells actively promote intratumor homing of transduced cells.

Fig. 1. Tissue kinetics of CD34-TRAIL+ cells. Lung, liver, spleen, femur, and tumor nodules were harvested from tumor-bearing NOD/SCID mice 0.5 (), 5 (), 24 (), and 48 () hours after a single intravenous injection of CD34-TRAIL+ cells (3 × 106 cells/mouse). Shown is the quantification of CD34-TRAIL+ cells on digitally acquired tissue sections stained with anti-CD45. Frequency of CD34-TRAIL+ cells is expressed as the mean (± SD) number of CD45+ cells per tissue section.

<sup>3</sup> Images of tissue sections were acquired at 20× magnification with an automatic high-resolution scanner (dotSlide System, Olympus, Tokyo, Japan) and subdivided into a collection of non-overlapping red, green, and blue (RGB) images in TIFF format (final resolution 3.125 pixels/µm). Image analysis was carried out using the open-source ImageJ software (http://rsb.info.nih.gov/ij/). Routines for image analysis were coded in ImageJ macro language and executed on RGB images without further treatment. Per each experimental condition, at least three sections from different tumor nodules or healthy tissues were analyzed. Intratumor frequency of CD34-TRAIL+ cells was expressed as the number of CD45+ cells per total cells per tisue section. Total cells were counted by the ImageJ internal function for particle analysis, whereas CD45+ cells were manually counted in all images from whole scanning of histochemically stained tissue sections.

Targeting TRAIL Receptors with Genetically-Engineered CD34+ Hematopoietic Stem Cells 667

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

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-

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

from in vivo biotinylated tumors. Cryosections were stained with Alexa Fluor 488-

intratumor recruitment of CD34-TRAIL+ cells specifically triggered tumor necrosis.

R2 (*i*) expression by endothelial cells. Objective lens, 40×.
