Abstract
CD44 is a cell-surface glycoprotein involved in cell–cell interaction, adhesion, and migration. CD44 is found on colon cancer cells and on immune cells. Previous studies of 89Zr PET imaging of CD44 have relied on an anti-human antibody (Ab), which can influence biodistribution in murine models. In this study, we used an Ab that cross-reacts with both human and mouse origin CD44 of all isoforms to unveil the type of leukocyte responsible for high splenic anti-CD44 uptake and investigate how its regulation can influence tumor immuno-PET. The Ab was site-specifically labeled with 89Zr-deferoxamine on cysteine residues. 89Zr-anti-CD44 demonstrated high-specific binding to HT29 human colon cancer cells and monocytic cells that showed CD44 expression. When 89Zr-anti-CD44 was administered to Balb/C nude mice, there was remarkably high splenic uptake but low SNU-C5 tumor uptake (1.2 ± 0.7%ID/g). Among cells isolated from Balb/C mouse spleen, there was greater CD44 expression on CD11b positive myeloid cells than lymphocytes. In cultured monocytic and macrophage cells, LPS stimulation upregulated CD44 expression and increased 89Zr-anti-CD44 binding. Similarly, normal Balb/C mice that underwent lipopolysaccharide (LPS) stimulation showed a significant upregulation of CD44 expression on splenic myeloid cells. Furthermore, LPS treatment stimulated a 2.44-fold increase of 89Zr-anti-CD44 accumulation in the spleen, which was attributable to splenic myeloid cells. Finally, in Balb/C nude mice bearing HT29 tumors, we injected 89Zr-anti-CD44 with greater Ab doses to reduce binding to splenic cells. The results showed lower spleen uptake and improved tumor uptake (2.9 ± 1.3%ID/g) with a total of 300 μg of Ab dose, and further reduction of spleen uptake and greater tumor uptake (5.7 ± 0.0%ID/g) with 700 μg Ab dose. Thus, using an 89Zr labeled Ab that cross-reacts with both human and mouse CD44, we demonstrate that CD44 immuno-PET has the capacity to monitor CD44 regulation on splenic myeloid cells and may also be useful for imaging colon tumors.
Highlights
CD44 is a multifunctional, non-kinase, single-pass transmembrane glycoprotein involved in cell–cell and cellextracellular matrix interactions[1]
Non-reduced sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis demonstrated complete reduction of target disulfide bonds of the Ab by TCEP, which remained reduced after DFO-conjugation (Fig. 1A). 89Zr radiolabeling of deferoxamine-conjugated anti-CD44 was reproducible, with a labeling efficiency of 80%
Radiochemical stability by radioinstant thin layer chromatography analysis showed that the radiolabel was more than 95% intact after up to 96 h incubation in 50% fetal bovine serum (FBS) or phosphate buffered saline (PBS) (Fig. 1A)
Summary
CD44 is a multifunctional, non-kinase, single-pass transmembrane glycoprotein involved in cell–cell and cellextracellular matrix interactions[1]. Malignant cells expressing CD44 are associated with tumor initiation and tumorsphere formation capacity that leads to cancer progression and poor patient outcome. Other tissues such as splenic leukocytes in addition to tumor cells. Myeloid cells that include monocytes/macrophages and neutrophils have discrete immune functions with central roles in cellular stress, foreign material removal, and immune response regulation[15]. Activation response to LPS is one of the best characterized pathogen-associated molecular patterns that cause phagocytic cells to switch to an inflammatory phenotype without leaving the tissue. A previous study using an 89Zr-labelled anti-human CD44 Ab observed high uptake in human cancer xenografts with low spleen uptake in mice but found remarkably high splenic uptake in non-human p rimates[7]. In vivo biodistribution and imaging findings in human cancer-bearing mice that better predicts the results in human subjects will be benefited from the use of an Ab that cross-reacts with both murine and human CD44
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