Figure 5.

Multiplex Drug Assay

(A) Experimental design for short-term Entinostat assays. CD45.1- and CD45.2-derived color-coded H9M cells were treated with Entinostat for 24 hr before assessment of cell growth by Vi-Cell counts. Treated cells from a CD45.1- and a CD45.2-derived H9M line were mixed in equal ratios to yield the “group 1” and “group 2” 12xFGB mixes for longitudinal in vitro tracking and injection into four CD45.1 × CD45.2 F1 mice per group. For comparison, non-treated 12xFGB cell mixes were prepared in parallel. (B) AlamarBlue-dependent assessment of dose-dependent cell growth of four different H9M lines after 24 hr of Entinostat exposure (n = 3–4 per data point). (C) Each color-coded population was exposed to a specific Entinostat concentration for 24 hr before the preparation of cell mixes and subsequent longitudinal assessment of growth properties. (D) Assessment of color code distribution within non-treated 12xFGB H9M cell mixes at the time of transplantation (0 hr) and after 24 hr of in vitro cultivation. Cells were stained for CD45.1 and CD45.2 to distinguish all six color codes within the parental CD45 (CD45.x = CD45.1 and CD45.2) populations (n = 4 per color code). (E) Exemplified flow cytometry profiles of non-treated 12xFGB cell mixes recovered from the BM of lethally irradiated mice 24 hr after transplantation. The BM cells were first analyzed for expression of CD45.1 and CD45.2 markers before detecting color codes within these populations. (F) Assessment of color code distribution within Entinostat-treated 12xFGB H9M cell mixes at the time of transplantation (0 hr) and after 24 hr of in vitro cultivation (combined data from group 1 and group 2 cells). Cells were stained for CD45.1 and CD45.2 to distinguish all six color codes within the parental CD45 (CD45.x = CD45.1 and CD45.2) populations and normalized to the size of the DMSO control-treated samples (n = 4 per color code). (G) Exemplified flow cytometry profiles of Entinostat-treated 12xFGB H9M cell mixes 24 hr after transplantation into lethally irradiated CD45.1 × CD45.2 F1-derived mice. The gating strategy is as in (E). (H) Entinostat-dependent color code sizes normalized to DMSO control-treated cells from 12xFGB cell mixes recovered from the BM of recipient mice 24 hr after transplantation. M1–M4 indicate the individual mice transplanted for each group. (I) IC₅₀ (nM) determination for Entinostat-dependent homing potential from 96 data points from all 8 mice depicted in (H). The error bars represent mean values ± SD.