Immune cell-based screening assay for response to anticancer agents: applications in pharmacogenomics

Figures & data

Figure 1 Assay methodology overview.

Notes: Spleens were isolated from inbred mice (A), and a single-cell splenocyte suspension was generated (B). Splenocytes were sequentially incubated with drugs (ie, doxorubicin, idarubicin, BEZ-235, and selumetinib) cellular health indicator dyes (ie, detecting viability, caspase activation, and mitochondrial health), and cell surface marker antibodies (ie, detecting CD-19+ B-cells, CD-3e+ T-cells, CD-11b+ monocytes, and Ly-6G+ granulocytes) (C). The cells were fixed and analyzed using flow cytometry (D), and dose-response curves were generated from flow cytometry data (E).
Abbreviations: FSC, forward scatter; SSC, side scatter.

Figure 2 Flow cytometry gating strategy.

Notes: The population of splenocytes (A) was derived from SSC and FSC. Subpopulations of interest derived from (A) included CD-19+ B-cells (B), CD-3e+ T-cells (C), CD-11b+ monocytes (D), and Ly-6G+ granulocytes (E). Viability (F), caspase-3/7 activation (G), mitochondrial health (H), and, when applicable, anthracycline uptake (I) were subsequently gated from (B–E) subpopulations.
Abbreviations: AmCyan, Anemonia majano cyan fluorescent protein; APC, allophycocyanin; Cy, cyanine; FITC, fluorescein isothiocyanate; FSC, forward scatter; PE, phycoerythrin; PerCP, peridinin-chlorophyll proteins; SSC, side scatter.

Table 1 Multiparameter flow cytometry assay

Excitation wavelength (nm)	Emission wavelength (nm)	Fluorochrome channel	Cell surface marker or dye	Parameter investigated
405	450	PacBlue	V450 rat anti-mouse CD11b	Monocytes
405	500	AmCyan	V500 Syrian hamster anti-mouse CD3e	T-cells
488	530	FITC	CellEvent™ caspase-3/7 green detection reagent	Caspase-3/7 activation
488	570	PE	Anthracycline autofluorescence	Anthracycline uptake
488	695	PerCPCy5.5	7-AAD	Viability
488	785	PE-Cy7	PE-Cy7 rat anti-mouse Ly-6G	Granulocytes
635	660	APC	Mitotracker^® Deep Red	Mitochondrial health
635	785	APC-Cy7	APC-H7 rat anti-mouse CD19	B-cells

Abbreviations: AmCyan, Anemonia majano cyan fluorescent protein; APC, allophycocyanin; Cy, cyanine; FITC, fluorescein isothiocyanate; PacBlue, Pacific blue; PE, phycoerythrin; PerCP, peridinin-chlorophyll proteins.

Figure 3 Interstrain variation of viability across immune cell types and anticancer drugs.

Notes: Dose-response curves depict cell populations (columns, ie, T-cells [A, E, I, and M], B-cells [B, F, J, and N], monocytes [C, G, K, and O], and granulocytes [D, H, L, and P]) exposed to anticancer drugs (rows, ie, doxorubicin [A-D], idarubicin [E-H], BEZ-235 [I-L], and selumetinib [M-P]). Nine strains are included: A/J

, C57BLKS/J

, CBA/J

, DBA/2J

, FVB/NJ

, LP/J

, NOD/LtJ

, NZB/BINJ

, and PWK/PhJ

.

Figure 4 Replicability of dose-response curves.

Notes: Four sensitive and four less sensitive strains were chosen for replication in a repeat experiment. The results for the most heritable viability phenotypes are presented: T-cells exposed to idarubicin (A) and B-cells exposed to doxorubicin (B). Eight strains are included: 129S1SvlmJ

, BALB/cByJ

, C57BLKS/J

, CBA/J

, KK/HiJ

, LG/J

, NZO/HiLtJ

, and SEA/GnJ

.

Figure 5 Interstrain phenotype comparisons for log₁₀ (IC₅₀ [nM]).

Notes: Log₁₀ (IC₅₀ [nM]) values across strains and heritability are displayed for T-cells exposed to idarubicin (A), B-cells exposed to doxorubicin (B), B-cells exposed to idarubicin (C), monocytes exposed to doxorubicin (D), and monocytes exposed to idarubicin (E). Strains are arranged from least sensitive to most sensitive along the X-axis.
Abbreviation: IC₅₀, half maximal inhibitory concentration.

Table 2 Phenotype correlations for log₁₀ (IC₅₀ [nM]) values across strains

	T-cells exposed to idarubicin	B-cells exposed to doxorubicin	B-cells exposed to idarubicin	Monocytes exposed to doxorubicin	Monocytes exposed to idarubicin
T-cells exposed to idarubicin	–	0.573 P=0.0004	0.698 P=3.2×10^−6	0.468 P=0.0053	0.835 P=1.5×10^−9
B-cells exposed to doxorubicin	–	–	0.771 P=9.6×10^−8	0.703 P=5.2×10^−5	0.534 P=0.0016
B-cells exposed to idarubicin	–	–	–	0.585 P=0.00028	0.630 P=8.4×10^−5
Monocytes exposed to doxorubicin	–	–	–	–	0.672 P=2.5×10^−5
Monocytes exposed to idarubicin	–	–	–	–	–

Abbreviation: IC₅₀, half maximal inhibitory concentration.

Figure S1 Cellular subpopulations of freshly isolated splenocytes.

Notes: Splenocytes were isolated from male C57BL/6J (n=4) mice. Our populations were comparable to murine spleen cell composition when available in the Mouse Phenome Database (B-cells, granulocytes, and monocytes P<0.05 using t-tests).

Figure S2 Interstrain variation of viability across immune cell types and anticancer drugs.

Notes: Dose-response curves depict cell populations (columns, ie, T-cells [A, E, I, and M], B-cells [B, F, J, and N], monocytes [C, G, K, and O], and granulocytes [D, H, L, and P]) exposed to anticancer drugs (rows, ie, doxorubicin [A-D], idarubicin [E-H], BEZ-235 [I-L], and selumetinib [M-P]). Thirty-six strains are included: 129S1/SvImJ, 129X1/SvJ, A/J, AKR/J, BALB/cByJ, BTBR T⁺ Itpr3^tf/J, BUB/BnJ, C3H/HeJ, C57BLKS/J, C57BL/6J, C57BR/cdJ, C58/J, CBA/J, CZECHII/EiJ, DBA/2J, FVB/NJ, I/LnJ, KK/HiJ, LG/J, LP/J, MA/MyJ, NOD/LtJ, NON/LtJ, NZB/BINJ, NZO/HiLtJ, NZW/LacJ, PERA/EiJ, PL/J, PWD/PhJ, PWK/PhJ, RIIIS/J, SEA/GnJ, SJL/J, SM/J, SWR/J, and WSB/EiJ.

Abbreviations: AUC, area under the curve; IC₅₀, half maximal inhibitory concentration.

Figure S3 Interstrain phenotype comparisons for area under the curve.

Notes: Area under the curve values across strains, heritability, and correlation to log₁₀ (IC₅₀ [nM]) values, when relevant, are displayed for T-cells exposed to selumetinib (A), T-cells exposed to BEZ-235 (B), T-cells exposed to doxorubicin (C), T-cells exposed to idarubicin (D), B-cells exposed to selumetinib (E), B-cells exposed to BEZ-235 (F), B-cells exposed to doxorubicin (G), B-cells exposed to idarubicin (H), monocytes exposed to selumetinib (I), monocytes exposed to BEZ-235 (J), monocytes exposed to doxorubicin (K), and monocytes exposed to idarubicin (L). Strains are arranged from largest to smallest area under the curve (±SEM) along the X-axis. Abbreviations: AUC, area under the curve; IC₅₀, half maximal inhibitory concentration; SEM, standard error of the mean.

Figure S4 Interstrain phenotype comparisons for slope coefficients.

Notes: Slope coefficients across strains and correlation to log₁₀ (IC₅₀ [nM]) values are displayed for T-cells exposed to idarubicin (A), B-cells exposed to doxorubicin (B), B-cells exposed to idarubicin (C), monocytes exposed to doxorubicin (D), and monocytes exposed to idarubicin (E). Strains are arranged from largest to smallest slope coefficient (±SEM) along the X-axis.

Abbreviations: IC₅₀, half maximal inhibitory concentration; SEM, standard error of the mean.

Table S1 Heritability (%) of viability (%) at specific drug doses (μM) among different phenotypes

Phenotype	100 μM	33.3 μM	11.1 μM	3.7 μM	1.2 μM	0.4 μM	0.1 μM	0.05 μM	0.02 μM
B-cells exposed to selumetinib	58.3	47.6	43.5	47.9	51.7	41.6	35.2	30.6	27.5
B-cells exposed to BEZ-235	56.9	46.3	52	45	46.9	41.5	44	36.1	58.6
B-cells exposed to doxorubicin	73.5	79.2	87.8	83.4	78.8	70.6	58.2	38.1	38.9
B-cells exposed to idarubicin	35.2	45.4	60.7	85	69.3	51.1	49.5	36.2	51.5
T-cells exposed to selumetinib	53.2	47	46.7	43.5	51.5	54.4	50.7	44.5	50.9
T-cells exposed to BEZ-235	69.1	61.8	52	55.6	40.9	42.5	32	31.7	20.4
T-cells exposed to doxorubicin	89.3	86.8	85.8	81.6	77.9	79.2	81.6	75.8	63
T-cells exposed to idarubicin	44.5	42.7	83.6	90.6	87.5	72.5	45.3	37.8	22.8
Monocytes exposed to selumetinib	64.4	53.2	37.5	47	50.1	43.2	27.9	27.5	26.2
Monocytes exposed to BEZ-235	83.9	87.5	83.4	79.4	74.1	49.9	52.3	45	41.9
Monocytes exposed to doxorubicin	76.8	59.5	81.3	77.6	71.7	75.6	66.2	44.8	40.6
Monocytes exposed to idarubicin	58.6	47.9	80.5	75.1	73.6	74.9	55	46.8	32

Table S2 Phenotype correlations for area under the curve values across strains

	T-cells exposed to selumetinib	T-cells exposed to BEZ-235	T-cells exposed to doxorubicin	T-cells exposed to idarubicin	B-cells exposed to selumetinib	B-cells exposed to BEZ-235	B-cells exposed to doxorubicin	B-cells exposed to idarubicin	Monocytes exposed to selumetinib	Monocytes exposed to BEZ-235	Monocytes exposed to doxorubicin	Monocytes exposed to idarubicin
T-cells exposed to selumetinib	–	0.49 P=0.003	0.058 P=0.75	0.14 P=0.43	0.23 P=0.19	0.053 P=0.76	0.2 P=0.26	0.24 P=0.18	0.51 P=0.002	0.26 P=0.12	0.19 P=0.28	0.15 P=0.41
T-cells exposed to BEZ-235	–	–	0.41 P=0.017	0.12 P=0.51	−0.0069 P=0.97	0.22 P=0.2	0.48P=0.004	0.4 P=0.018	0.097 P=0.58	0.74 P=3.5×10^−7	0.27 P=0.12	0.08 P=0.65
T-cells exposed to doxorubicin	–	–	–	0.49 P=0.002	0.28 P=0.1	0.44 P=0.009	0.38 P=0.021	0.28 P=0.1	0.11 P=0.54	0.48 P=0.004	0.21 P=0.21	0.32 P=0.054
T-cels exposed to idarubicin	–	–	–	–	0.49 P=0.003	0.18 P=0.31	0.61 P=9.1×10^−5	0.75 P=1.5×10^−7	0.3 P=0.087	0.35 P=0.04	0.53 P=0.001	0.91 P=3×10^−14
B-cells exposed to selumetinib	–	–	–	–	–	0.47 P=0.004	0.17 P=0.33	0.54 P=0.001	0.52 P=0.001	0.19 P=0.28	0.06 P=0.74	0.41 P=0.016
B-cells exposed to BEZ-235	–	–	–	–	–	–	0.11 P=0.51	0.42 P=0.014	0.41 P=0.02	0.38 P=0.023	−0.14 P=0.44	0.037 P=0.84
B-cells exposed to doxorubicin	–	–	–	–	–	–	–	0.75 P=1.4×10^−7	0.067 P=0.7	0.63 P=6×10^−5	0.6 P=1×10^−4	0.55 P=4.6×10^−4
B-cells exposed to idarubicin	–	–	–	–	–	–	–	–	0.32 P=0.61	0.65 P=2.9×10^−5	0.42 P=0.011	0.68 P=5.3×10^−6
Monocytes exposed to selumetinib	–	–	–	–	–	–	–	–	–	0.12 P=0.48	0.2 P=0.27	0.31 P=0.075
Monocytes exposed to BEZ-235	–	–	–	–	–	–	–	–	–	–	0.39 P=0.023	0.28 P=0.11
Monocytes exposed to doxorubicin	–	–	–	–	–	–	–	–	–	–	–	0.69 P=4×10^−6
Monocytes exposed to idarubicin	–	–	–	–	–	–	–	–	–	–	–	–

Table S3 Phenotype correlations for slope coefficient values across strains

	T-cells exposed to idarubicin	B-cells exposed to doxorubicin	B-cells exposed to idarubicin	Monocytes exposed to doxorubicin	Monocytes exposed to idarubicin
T-cells exposed to idarubicin	–	0.16 P=0.37	0.11 P=0.53	0.093 P=0.6	0.11 P=0.55
B-cells exposed to doxorubicin	–	–	0.3 P=0.085	0.16 P=0.37	0.48 P=0.006
B-cells exposed to idarubicin	–	–	–	−0.066 P=0.7	0.33 P=0.055
Monocytes exposed to doxorubicin	–	–	–	–	0.57 P=0.001
Monocytes exposed to idarubicin	–	–	–	–	–