Figure 2. In Vitro effects of pan IR700 based PIT on EGFR expressing cells.

A. Pan IR700 based PIT induces cell death in UMUC5 cells. Untreated UMUC5 cells, UMUC5 cells treated with panitumumab (10µg/ml), pan IR700 (10µg/ml) and UMUC5 cells pre-treated with panitumumab (50µg/ml) followed by pan IR700 (10µg/ml) were subjected to no NIR (NIR 0 J/cm² (upper panel)) or 4 J/cm² of NIR (lower panel). Thirty minutes post-NIR treatment, the cell death was measured on flow cytometry using green LIVE/DEAD reagent (Ex. 488nm/ Em. 535nm). The cell death is observed only in the cells treated with pan IR700 + 4 J/cm² NIR. No appreciable cell death was observed under blocking conditions indicating the specificity of pan IR700 (lower, rightmost panel) for EGFR binding.

B. IC₅₀ measurement of pan IR700 mediated PIT for bladder cancer cell lines. UMUC5 (i), 5637 (ii) and TCCSUP (iii) and UMUC-3 (iV) were treated with increasing concentrations of pan IR700 (▲), panitumumab (▼) without NIR treatment or with pan IR700 (●) and panitumumab (■) in the presence of 4 J/cm² (UMUC5), 32 J/cm² (5637) and 64 J/cm² (TCCSUP, UMUC-3) of NIR. After 24 hours, cell survival was monitored using MTS reagent. The data is represented as percent cell survival. The IC50 values for UMUC5, 5637, TCCSUP and UMUC-3 are 4.7nM, 5.8nM, 4 nM and 108nM respectively. No cell death was observed under any other conditions.

C. The amount of NIR required to achieve equivalent amount of cell death (in terms of IC50 values) in bladder cancer cell lines is plotted as a function of cell surface EGFR expression (RMFI). As the expression of EGFR increases, the NIR needed to achieve similar IC₅₀ values of pan IR700 decreases.