Integrin VLA-4 as a PET imaging biomarker of hyper-adhesion in transgenic sickle mice

Abstract

In sickle cell disease (SCD), very late antigen-4 (VLA-4 or integrin α4β1) mediates the adhesion of reticulocytes to inflamed, proinflammatory endothelium, a key process in promoting vaso-occlusive episodes (VOEs). We hypothesized that a radionuclide tracer targeting VLA-4 could be harnessed as a positron emission tomography (PET) imaging biomarker of VOEs. We tested the VLA-4 peptidomimetic PET tracer 64Cu-CB-TE1A1P-PEG4-LLP2A (64Cu-LLP2A) for imaging hyper-adhesion-associated VOEs in the SCD Townes mouse model. With lipopolysaccharide (LPS)-induced VOEs, 64Cu-LLP2A uptake was increased in the bone marrow of the humeri and femurs, common sites of VOEs in SCD mice compared with non-SCD mice. Treatment with a proven inhibitor of VOEs (the anti-mouse anti-P-selectin monoclonal antibody [mAb] RB40.34) during LPS stimulation led to a reduction in the uptake of 64Cu-LLP2A in the humeri and femurs to baseline levels, implying blockade of VOE hyper-adhesion. Flow cytometry with Cy3-LLP2A demonstrated an increased percentage of VLA-4-positive reticulocytes in SCD vs non-SCD mice in the bone and peripheral blood after treatment with LPS, which was abrogated by anti-P-selectin mAb treatment. These data, for the first time, show in vivo imaging of VLA-4-mediated hyper-adhesion, primarily of SCD reticulocytes, during VOEs. PET imaging with 64Cu-LLP2A may serve as a valuable, noninvasive method for identifying sites of vaso-occlusion and may provide an objective biomarker of disease severity and anti-P-selectin treatment efficacy in patients with SCD.

Graphical abstract

Figure 1.

Rationale for PET imaging of VLA-4 as a biomarker of hyper-adhesion. The PET tracer ⁶⁴Cu-CB-TE1A1P-PEG₄-LLP2A binds specifically to the active conformation of VLA-4. VLA-4 is expressed on leukocytes and reticulocytes and contributes to vaso-occlusion.^-,,

Figure 2.

Experimental design and representative PET/CT scans. (A) SCD and non-SCD mice were imaged at baseline, and after 1 week they were injected intravenously with LPS and ⁶⁴Cu-LLP2A. (B) In a separate set of experiments, mice were imaged at baseline, and after 1 week they were injected intravenously with LPS and either IgG isotype control or anti-P-selectin antibody plus ⁶⁴Cu-LLP2A. (C) Representative PET/CT images of SCD and non-SCD mice 24 hours after injection of ⁶⁴Cu-LLP2A at baseline and post-LPS challenge. There are distinct differences between VLA-4 expression in SCD and non-SCD mice post-LPS in the humeri and femurs (blue arrows). (D) Representative PET/CT images of SCD mice 24 hours after injection of ⁶⁴Cu-LLP2A at baseline and post-LPS with IgG control treatment or anti-P-selectin antibody treatment. Anti-P-selectin treatment mitigates signal in the humeri and femurs (blue arrows) post-LPS.

Figure 3.

Mean SUVr for humeri and femurs at baseline and post-LPS challenge in non-SCD, SCD, and SCD mice co-treated with LPS and IgG isotype control or anti-P-selectin mAb. (A-D) Mean SUV for humeri divided by mean SUV for muscle for an SUV ratio normalized to background (humeri SUVr) at baseline and post-LPS for non-SCD (A), SCD (B), SCD + IgG isotype control (C), and SCD + anti-P-selectin mAb (D). *Denotes outlier identified by robust regression and outlier removal (ROUT, Q = 1%). (E-H) Mean SUV for femurs divided by mean SUV for muscle for an SUV ratio normalized to background (femurs SUVr) at baseline and post-LPS for non-SCD (E), SCD (F), SCD + IgG isotype control (G), and SCD + anti-P-selectin mAb (H) mice. *Denotes outlier identified by ROUT, Q = 1%. (A-H) Baseline and post-LPS means are shown as a red hollow circle with standard deviation (SD) error bars. Baseline and post-LPS groups were compared by using a paired 2-tailed Student t test. P < .05 was considered statistically significant. (I-J) SUVr fold-change from baseline to post-LPS (post-LPS/baseline) for humeri and femurs, respectively. The solid line depicts the mean of the non-SCD fold-change (no change, ∼1) and is shaded below the threshold. The data were compared using 1-way analysis of variance (ANOVA) with multiple comparisons by controlling false discovery rate (FDR) by a 2-stage linear step-up procedure from Benjamini, Krieger, and Yekutieli. FDR-corrected P < .05 was considered statistically significant. ANOVA for humeri and femurs were corrected for unequal variance. (I) Non-depicted P values for femurs fold-change: non-SCD vs SCD + IgG (P = .22), non-SCD vs SCD + anti-P-selectin mAb (P = .43), and SCD vs SCD + IgG (P = .53). (J) Non-depicted P values for femurs fold-change: non-SCD vs SCD + IgG (P = .07), non-SCD vs SCD + anti-P-selectin mAb (P = .52), and SCD vs SCD + IgG (P = .59).

Figure 4.

WBC and neutrophil counts for the non-SCD (control), SCD, and SCD mice treated with either IgG isotype control or anti-P-selectin mAb. (A-D) WBC counts (× 10⁹/L) at baseline and post-LPS challenge for non-SCD (A), SCD (B), SCD + IgG isotype control (C), and SCD + anti-P-selectin mAb (D). (E-H) Neutrophil (NEU) counts (× 10⁹/L) at baseline and post-LPS for non-SCD (E), SCD (F), SCD + IgG isotype control (G), and SCD + anti-P-selectin mAb (H). (A-H) Baseline and post-LPS means are shown as a black hollow circle with SD error bars. Baseline and post-LPS groups were compared using a paired 2-tailed Student t test. P < .05 was considered statistically significant. (I) Baseline and post-LPS WBC (× 10⁹/L) measurements for all conditions. Non-depicted P values: post-LPS non-SCD was significantly different from post-LPS SCD (P ≤ .001), SCD + IgG (P = .002), and SCD + anti-P-selectin mAb (P ≤ .001). (J) Baseline and post-LPS neutrophil (× 10⁹/L) measurements for all conditions. Non-depicted P values: post-LPS non-SCD was significantly different from post-LPS SCD (P ≤ .001), SCD + IgG (P = .002), and SCD + anti-P-selectin mAb (P ≤ .001). (I-J) Bars represent mean ± SD. Baseline analysis between SCD and non-SCD used a 2-tailed Student t test. One-way ANOVA with multiple comparisons by controlling FDR by a 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli was used for the post-LPS analysis. FDR-corrected P < .05 was considered statistically significant.

Figure 5.

Flow cytometry of isolated bone marrow and blood cells labeled with Cy3-LLP2A post-LPS challenge. (A-B) Reticulocyte-gated measurements. (A) Percent of reticulocytes that express active VLA-4 in the bone marrow and blood. (B) The MFI of Cy3-LLP2A-bound VLA-4 on VLA-4⁺ reticulocytes. (C-D) WBC-gated measurements. (C) Percent of WBCs that express active VLA-4 in the bone marrow and blood. (D) The MFI of Cy3-LLP2A-bound VLA-4 on VLA-4⁺ reticulocytes. (A-D) Bars represent mean ± SD. Data were analyzed by 1-way ANOVA with multiple comparisons by controlling FDR by a 2-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. FDR-corrected P < .05 was considered statistically significant.