A small-molecule screen for enhanced homing of systemically infused cells

Abstract

Poor homing of systemically infused cells to disease sites may limit the success of exogenous cell-based therapy. In this study, we screened 9,000 signal-transduction modulators to identify hits that increase mesenchymal stromal cell (MSC) surface expression of homing ligands that bind to intercellular adhesion molecule 1 (ICAM-1), such as CD11a. Pretreatment of MSCs with Ro-31-8425, an identified hit from this screen, increased MSC firm adhesion to an ICAM-1-coated substrate in vitro and enabled targeted delivery of systemically administered MSCs to inflamed sites in vivo in a CD11a- (and other ICAM-1-binding domains)-dependent manner. This resulted in a heightened anti-inflammatory response. This represents a new strategy for engineering cell homing to enhance therapeutic efficacy and validates CD11a and ICAM-1 as potential targets. Altogether, this multi-step screening process may significantly improve clinical outcomes of cell-based therapies.

Figure 1. A medium throughput screen identified Ro-31-8425, a kinase inhibitor which upregulates CD11a expression on MSC surface

(a) Native MSCs lack surface expression of CD11a. Cells (HL-60 or MSCs) were incubated with PE-CY5-CD11a Ab and analyzed by flow cytometry (representative data from n=3 independent experiments). (b) Global screening data obtained from the medium-throughput screening to identify compounds that up-regulate CD11a expression on the MSC surface (9000 cpds in 112 384-well assay plates were screened. Green bars: S/B=signal/background ratio, Blue curve: Z’ values. Also see Experimental Procedures). (c) A dose-dependent increase in the percentage of CD11a⁺ MSCs in response to Ro-31-8425 pretreatment. MSCs were pretreated with DMSO vehicle control (0.1%) or Ro-31-8425 (0.1, 1, 3 and 10μM) for 24h and CD11a expression levels were assessed by CyTOF analysis (n=3. Blue dots - CD11a⁺ MSCs, black dots - CD11a⁻ MSCs. * = p < 0.05 vs. DMSO-treated control MSCs, Tukey's HSD test). (d) CD11a mRNA levels in response to Ro-31-8425 pretreatment as analyzed by RT-PCR. MSCs were pretreated with Ro-31-8425 (3μM) and CD11a mRNA levels were analyzed at indicated times post pretreatment (n=3. * = p < 0.05 vs. DMSO-treated control MSCs, Tukey's HSD test).

Figure 2. Upregulation of CD11a, in response to pretreatment with Ro-31-8425, increases MSC firm adhesion to an ICAM-1-coated surface in-vitro

(ai) MSCs firm adhesion to an ICAM-1-coated surface following pretreatment with ruboxistaurin (Rubox) or Ro-31-8425 (3μM for 24h, 10X magnification). (aii) Quantification of MSC firm adhesion to an ICAM-1 surface in response to pretreatment with ruboxistaurin and Ro-31-8425. Error bars represent standard deviation (n=3. Statistically significant difference vs. vehicle-treated control is denoted by * = p < 0.05, Tukey's HSD test). (aiii) A pie chart of the percent distribution of MSC population that express active ICAM-1 binding domains following Ro-31-8425 pretreatment (b) Ab blocking experiments demonstrate a significant involvement of CD11a in the increased firm adhesion of Ro-31-8425-treated MSCs to ICAM-1 surface. Error bars represent standard deviation (n=3. Statistically significant difference vs. no Ab control and vs. CD90 Ab control is denoted by * = p < 0.05, Tukey's HSD test).

Figure 3. Ro-31-8425-pretreated MSCs exhibit increased homing to inflamed sites and an improved anti-inflammatory impact following systemic administration

(a) Homing of systemically infused MSCs to LPS-induced inflamed mouse ears was assessed 24hr following cell infusion. An example 2D projection of a 3D image stack (scale bar = 50 μm) demonstrates homing to the inflamed ear of Ro-31-8425-pre-treated MSCs (green cells) compared to vehicle-treated MSCs (blue cells). MSCs are found in the vascularized region of the skin (left side of image), with the skin surface exhibiting autofluorescence in multiple channels and a characteristic tiled pattern (right side of image). Ro-31-8425 pretreatment significantly promoted MSC homing versus the vehicle-treated control cells (** = p < 0.01, Tukey's HSD test, n=8 mice). (b) For Ab blocking experiments, Ro-31-8425 or vehicle-pretreated MSCs were washed and incubated for 30 min with mouse anti-human CD11a (clone TS1/22) or Mouse IgG1 isotype control prior to staining with the Vybrant dyes and retro-orbital infusion as described above. Ab blocking experiments demonstrate involvement of CD11a and other ICAM-1 binding domains in the increased homing response of systemically infused Ro-31-8425-treated MSCs to the inflamed ear. CD11a-blocked or Ab isotype control-incubated Ro-31-8425-pretreted MSCs were co-injected systemically with vehicle MSCs (1:1 ratio) and the homing response to inflamed ear was assessed via intravital microscopy. Error bars represent standard deviation (statistically significant difference vs. Ab isotype control is denoted by * = p < 0.05, Tukey's HSD test, n=5 mice per group). (c) Ro-31-8425 -treated MSCs displayed a superior effect in reducing swollen ear thickness of the inflamed ear compared to native MSCs (* = p < 0.05, ** = p < 0.01, Tukey's HSD test, n=8 mice). (d) MSCs treated with Ro-31-8425 significantly reduced the TNF-α level in the inflamed ear compared to the control ear (** = p <0.01, Tukey's HSD test, n = 6 mice).