Figure 4

LLP2A-Cy5 specificity and affinity to VLA4 in vitro. (A) Strong cell surface fluorescence was observed in the WT 5TGM1-GFP cells incubated with LLP2A-Cy5 (1 µM, 15 min, at 4 °C). Blue, green and red colors represent nuclear stain, GFP, and Cy5 signal, respectively in all images (60x; scale bar: 20 µm). (B) Cell surface signal was decreased when the same was co-incubated with 100-fold excess of LLP2A-PEG4 blocking peptide (100 µM). (C) Cell surface binding was also reduced in the WT 5TGM1-GFP cells treated with scLLPA-Cy5 (1 µM, 15 min, at 4 °C). (D) Quantification of background corrected cell fluorescence showed significantly increased signal in LLP2A-Cy5 incubated cells, relative to scLLP2A-Cy5 and blocking (***p < 0.001 1-way analysis of variance (ANOVA) with Bonferroni multiple comparisons test). (E) Representative high-resolution imaging of a single cell incubated with LLP2A-Cy5 for 2.5 h at 37 °C. Cells exhibiting minimal motion at 60 × magnification were imaged in a confocal 3D z-stack. Post-deconvolution visualization of a single confocal plane (left) and maximal intensity projection (right) of the confocal 3D z-stack (100x; scale bar: 3 µm) show strong cell surface binding with minimal internalization of LLP2A-Cy5. (F) FACS of LLP2A-Cy5 incubated with WT 5TGM1-GFP cells demonstrated selective binding of LLP2A-Cy5 to VLA4. WT 5TGM1-GFP cells and varying LLP2A-Cy5 concentrations (0-1400 nM) were co-incubated for 30 min and washed with Tyrode’s buffer containing 1% BSA. BIO5192 (100 nM) was used to evaluate non-specific binding. (G) LLP2A-Cy5 showed minimal binding to VLA4 KO cells relative to VLA4⁺ WT tumor cells. (Top) 1.5 × 10⁵ WT and KO 5TGM1-GFP cells were assessed for α₄ and β₁ levels, and for binding with sVCAM-1 (Middle), and (Bottom) LLP2A-Cy5 respectively.