Investigating VLA-4 as a PET imaging biomarker vaso-occlusive crisis in a mouse model of sickle cell disease

Objectives: The debilitating and unpredictable pain of vaso-occlusive crisis (VOC) in sickle cell disease (SCD) is the most common cause for patients to seek emergency medical attention. VOC evaluation is currently limited to patients’ measurement of pain intensity and reported location. There is a clinical need for quantitative imaging to improve VOC assessment and determine the best pain management strategy for each patient. VOC is a direct consequence of the adhesion of sickle erythrocytes (SSRBCs) to an inflamed, proinflammatory endothelium. Very late antigen-4 (VLA-4 or integrin α₄β₁) mediates the adhesion of SSRBCs to endothelium, and could therefore be harnessed as a neuroimaging biomarker of VOC. The ultimate goal of this project is to validate whether the VLA-4 peptidomimetic PET tracer ⁶⁴Cu-CB-TE1A1P-LLP2A can image VOC events in SCD patients. Here we investigate this tracer in a mouse model of SCD.

Methods: LLP2A was conjugated with PEG4 and a cross-bridge copper chelator (CB-TE1A1P) and radiolabeled with Cu-64, as previously described (Beaino et al., JNM 2014; 55:1856-63). ⁶⁴Cu-PEG4-CB-TE1A1P-LLP2A (⁶⁴Cu-LLP2A) was injected via tail vein in homozygous sickle Townes mice and non-sickling controls (n=5/group) at the dose of 200 µCi per animal (1mCi/µg). Baseline PET/CT images were acquired at 4 and 24 h post-injection (p.i.). After one week, all mice were challenged with i.v. injection of 0.1 µg/Kg lipopolysaccharide (LPS) to elicit inflammation and VOC, followed immediately by injection of ⁶⁴Cu-LLP2A and subsequent PET/CT imaging at 4 and 24 h p.i.. Standardized uptake values (SUVs) of liver, lung, femur, and humerus were measured using VivoQuant software (InviCRO; Boston, MA). After obtaining 24 h post-LPS images, mice were sacrificed, and organs (femur, spleen, and brain) were harvested and prepared for histology and immunofluorescence imaging. Single cell suspensions from the blood, bone marrow, and spleen were obtained from sickle and non-sickle mice with and without LPS treatment and then labeled with fluorescent markers to measure VLA-4 expression in reticulocytes and leukocytes.

Results: The sickle mice showed significant uptake of ⁶⁴Cu-LLP2A post-LPS challenge in the humerus, a common area of pain related to vaso-occlusion in humans. The ratio of SUV at 24 h p.i in humerus and entire femur compared to muscle in sickle cell mice was increased over baseline whereas the SUV ratio in the control mice did not show any significant change. In these mice, VLA-4 immunofluorescence imaging confirmed signal localization in the femur, spleen, and brain, and histology studies showed that areas of VLA immunofluorescence corresponded to regions of PET uptake. Additionally, flow-cytometry analysis of blood, bone-marrow, and spleen demonstrated a significant increase in VLA-4 signal in sickle mice treated with LPS but not in the controls.

Conclusions: Increased ⁶⁴Cu-LLP2A uptake in the humerus and femur, where VOC occurs, post LPS challenge in sickle mice but not in control mice, suggests localization of the tracer in areas of vaso-occlusion. Validation studies using histology, fluorescent imaging, and flow cytometry confirmed increased VLA-4 expression. These results suggest ⁶⁴Cu-LLP2A imaging in patients with sickle cell disease who undergo vaso-occlusive events will be clinically important to determine the extent of disease, and possibly in patient management.