Fig 6. Development of a spatially resolved PK/PD model for SN30000 and testing by SABM simulation of spheroid response to SN30000 exposure.

Cellular bioreductive metabolism of SN30000 (A) and clonogenic cell killing (B) was measured by serial sampling of stirred HCT116 cell suspensions (2 × 10⁶ cells/mL) under anoxia (0% oxygen gas mixture). The rate constants for SN30000 metabolism (k_met0) and cell killing (K_c) were estimated by fitting the data in panel A and panel B simultaneously using the MABM assuming the medium was fully stirred. Lines represent MABM predictions based on the parameters which minimize the overall error sum of squares (Table 1). (C) Diffusion parameters for SN30000 were estimated from HCT116 MCL transport studies illustrated for a single experiment. Lines are model fits using the reaction-diffusion program described in methods with fitted parameters for each MCL: D_SN30000 estimated from MCL transport under hyperoxia (95% O₂, filled symbols in panel C) assuming no metabolic consumption (confirmed by no production of SN30000-1-oxide); k_met0 estimated from SN30000 diffusion under anoxia (open symbols in panel C) by fixing D_SN30000 at its mean value (Table 1) determined in hyperoxic experiments. (D) To quantitate cytotoxicity, spheroids seeded with 1000 cells were exposed on day 4 to a range of SN30000 concentrations for 2 hr under 20% or 5% O₂ and clonogenic cell survival was measured (points in Panel D) and compared to predictions of SABM incorporating the optimized reaction-diffusion and cell kill parameters estimated from monolayer and MCL experiments from Table 1 (lines in Panel D). (E) To measure spheroid growth delay induced by SN30000, spheroids were exposed to 25 μM SN30000 under 5% O₂ or to 100 μM SN30000 under 20% O₂ for 2 hr and spheroid diameters were monitored (points in Panel E) and compared to the SABM predictions (lines in Panel E) using parameters from Table 1. Values are mean ± SD (n = 16 spheroids).