Abstract
Background: The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) affects disease progression and provides resistance to therapeutic agents. Very-late-antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) is a noncovalent, heterodimeric transmembrane receptor that is strongly implicated in the pathogenesis of MM via altering cell trafficking, proliferation and drug resistance. LLP2A is a high-affinity peptidomimetic ligand for activated VLA-4. We recently reported (Soodgupta et al. J. Nucl. Med 2016) the sensitive and specific molecular imaging of activated VLA-4 in mouse MM tumors using 64Cu-LLP2A and LLP2A-Cy5. Here we extended these studies by further characterizing VLA-4 expression in primary human MM samples and malignant plasma cells in mouse models of MM.
Methods: We evaluated VLA-4 expression in 5 human MM cell lines (U266, OPM2, H929, RPMI-8226 and MM1.S), one mouse MM cell line (5TGM1) and seventeen primary human MM bone marrow samples by flow cytometry using LLP2A-Cy5, soluble VCAM-1/Fc recombinant protein and CD49d (α4) and CD29 (β1) antibodies. The relative mean fluorescence intensity (RMFI) of LLP2A-Cy5 binding was calculated by dividing the MFI of LLP2A-Cy5 binding in the absence of BIO5192 (small molecule VLA-4 inhibitor) by the MFI of LLP2A-Cy5 binding in the presence of excess BIO5192. The 5TGM1/KaLwRij immunocompetent mouse model of MM was used for in vivo study.
Results: The expression of activated VLA-4 on MM cell lines as measured by LLP2A-Cy5+ mean fluorescent intensity (MFI) varied 10-fold as follows (LLP2A-Cy5 MFI in parentheses): 5TGM1 (23.7) > U266 (16.1) > OPM2 (4.6) > H929 (3.4) > RPMI-8226 (3.2) > MM1.S (2.1). We observed similar variable expression of LLP2A-Cy5 binding to primary human CD138+CD38+ MM plasma cells (PCs), with 76.47% (13/17) of MM patients exhibiting greater than 20% LLP2A-Cy5+ PCs. expressing VLA-4 on CD138+CD38+ cells. Overall, the mean percentage of positive cells and LLP2A-Cy5 relative MFI (RMFI) on malignant CD138+ PCs from these 13 patients were 78.2% (43.8-98.3%) and 4.3 (1.7-10.8), respectively. Other hematopoietic cells within the BM samples expressed less VLA-4 in descending order as follows; monocytes (58.2%, RMFI 3.0), T-lymphocytes (34.4%, RMFI 2.1) and B-lymphocytes (21.6%, RMFI 1.6). These levels of VLA-4 expression on normal cell subsets within MM patients were comparable to normal blood donors. In general, there was good correlation between LLP2A-Cy5 binding and expression of CD49d and CD29 on CD138+ PCs in MM patients. To our surprise, the four MM patients with <20% LLP2A-Cy5 binding demonstrated high expression of CD49d (92.1%) but very low percentages of CD29 positive cells (17.3%). Using BIO5192 (VLA-4 inhibitor), we found that the LLP2A-Cy5 reagent allowed more accurate detection of activated VLA-4 than the soluble VCAM-1 binding assay as determined by the magnitude of inhibition of binding in the presence of inhibitor. We next evaluated targeting VLA-4 molecule in murine MM model. Preliminary mouse mobilization studies demonstrated that VLA-4 inhibitors effectively and rapidly mobilized murine 5TGM1 MM cells from the bone marrow to the blood (2.49-fold increase in circulating GFP+CD138+ cells) within 1 hour of injection.
Summary:This study is the first demonstration that activated VLA4 can be detected on primary human MM cells using LLP2A. These data support the continued development of LLP2A as a molecular diagnostic imaging reagent for MM and as a potential therapeutic target of VLA-4 in MM. Ongoing studies are testing whether small molecule VLA-4 inhibitors can sensitize MM cells to cytotoxic therapy in vivo.
No relevant conflicts of interest to declare.
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