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. 2021 May 6;22(9):4931.
doi: 10.3390/ijms22094931.

Effective Drug Concentration and Selectivity Depends on Fraction of Primitive Cells

Jan Jakub Lica  1 , Miłosz Wieczór  2 , Grzegorz Jan Grabe  3 , Mateusz Heldt  4 , Marta Jancz  4 , Majus Misiak  4 , Katarzyna Gucwa  1 , Wioletta Brankiewicz  4 , Natalia Maciejewska  4 , Anna Stupak  5 , Maciej Bagiński  4 , Krzysztof Rolka  1 , Andrzej Hellmann  6 , Andrzej Składanowski  4
Affiliations

Effective Drug Concentration and Selectivity Depends on Fraction of Primitive Cells

Jan Jakub Lica et al. Int J Mol Sci. .

Abstract

Poor efficiency of chemotherapeutics in the eradication of Cancer Stem Cells (CSCs) has been driving the search for more active and specific compounds. In this work, we show how cell density-dependent stage culture profiles can be used in drug development workflows to achieve more robust drug activity (IC50 and EC50) results. Using flow cytometry and light microscopy, we characterized the cytological stage profiles of the HL-60-, A-549-, and HEK-293-derived sublines with a focus on their primitive cell content. We then used a range of cytotoxic substances-C-123, bortezomib, idarubicin, C-1305, doxorubicin, DMSO, and ethanol-to highlight typical density-related issues accompanying drug activity determination. We also showed that drug EC50 and selectivity indices normalized to primitive cell content are more accurate activity measurements. We tested our approach by calculating the corrected selectivity index of a novel chemotherapeutic candidate, C-123. Overall, our study highlights the usefulness of accounting for primitive cell fractions in the assessment of drug efficiency.

Keywords: C-123; cell culture density-dependent cytological stage profile; drug screening platform; effective drug concentration; primitive acute myeloid leukemia cellular stages; primitive cancer cellular stages; selectivity index.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
AML stage transformations. (A) Scheme of leukemogenesis and initiation of acute myeloid leukemia (AML). Left panel: Initiation of AML caused by the accumulation of DNA malfunctions resulting in the formation of LIC from the pathogenic primitive stage. LIC formation can occur independently in different cells and at different stages of their stage development. Right panel: Initiation of AML by LIC following the sudden and spontaneous appearance of multiple mutations in the primitive stage. Currently, LIC formation at a stage other than LSC HSC-like remains speculative. (B,C) Pattern of AML development and the effect of stage differentiation on interactions with a biologically active substance: (B) The surface level of ABC transporters decreases with the degree of differentiation. (C) As cells divide, the number of drug molecules per cell decreases. Top: hypothetical model of symmetrical divisions without maturation of the myelocyte-like stage. The model assumes that the “colony” would only be able to increase in the number of cells of the same stage: Myelocytes-like cells undergoing cell division terminally and maturing into metamyelocytes-like cells, unable to divide further and beginning to age. Bottom: Model of specific developmental stages of cell division with maturation, with a dynamic evolution of cytological stage fractions. (D) The stage transformations of AML over 5 days. The panel shows the AML stages capable of completing cell division within 120 h.
Figure 2
Figure 2
Primitive stages in HL-60. (A) Colony morphology. Micrographs show MGG-stained cells. Top panel: ×400 magnification. Bottom panel: ×1000 magnification. Marker: –20 µm. (B) Colony formation assay. Microphotographs show colonies formed by culture sublines. The values are averages ±SD of n = 3 independent experiments. (C) Expression of topoisomerase IIα isoforms in the HL-60 Standard and Primitive sublines.
Figure 3
Figure 3
Cytological stage profiles derived at different cell culture densities. (A) Morphology of the A-549 subline. Micrographs of MGG-stained cells and SSC/FSC cytometric parameters. Marker: –50 µm. (B) Cell cycle progression, ROS generation, and extracellular phosphatidylserine in the A-549 Standard and Mature sublines. (C) HEK-293 morphology (laser light scattering) and ROS effect. All values are averages ±SD of n = 3 independent experiments. LB—left bottom, RB—right bottom, LT—left top, RT—right top.
Figure 4
Figure 4
C-123 studies in vitro. Topoisomerase IIα and proteasome inhibition assays and molecular modeling of the interactions of C-123 with DNA. (A) Inhibition of topoisomerase IIα as measured by in vitro decatenation assay. (B) Quantification of proteasome inhibition by C-123 as measured by in vitro assay on A-549 cell lysates. Bortezomib was used as a positive control. The values represent averages of n = 3 independent experiments ±SEM. (C) Side and top views of an intercalated complex in which C-123 displaced an AT pair while interacting with the phosphosugar backbone via positively charged side chains. (D,E) Relative densities of the side-chain amino nitrogens of C-123 (D) or a central carbon from the fused ring core (E) integrated along the DNA axis, shown in a local coordinate system with the minor groove on the top and major groove on the bottom (see the schematic base pair for approximate location of bases and backbone). Note the nonlinear scale in the color bars.
Figure 5
Figure 5
Relative IC50 and EC50 for C-123, idarubicin, ethanol, and DMSO as a function of the relative initial number of cells or primitive cells in HL-60, shown as linear fits to the data. (A) Relative IC50 as a function of relative total initial cell number (solid line) or total initial primitive cell number (dotted line). Solid line: IC50 values obtained at initial densities (5, 25, and 100 × 103 cells per mL) were normalized relative to the value at lowest cell density (5 × 103 cells per mL) and averaged over the 3 sublines: Primitive, Standard, and Mature. These averages were plotted as a function of multiples of the lowest cell density (1, 5, and 20 corresponding to 5, 25, and 100 × 103 cells per mL). Dotted line: An analogous procedure was performed considering the fractions of primitive cells in each subline (0.04 in Mature, 0.4 in Standard, and 0.85 in Primitive; Table 1). *IC50 values were normalized to the Primitive subline, and the relative values were averaged over the 3 initial densities. The averaged values were plotted as a function of multiples of the lowest fraction of primitive cells (1, 2.1, and 21 corresponding to the Primitive/Primitive, Primitive/Standard, and Primitive/Mature ratios). (B) Normalization of EC50 as a function of relative initial cell number, calculated per total (solid line) or primitive (dotted line). Solid line: EC50 values obtained at initial densities (5, 25, and 100 × 103 cells per mL) were averaged over the 3 sublines and normalized relative to the value at lowest cell density (5 × 103 cells per mL). These averages were plotted as a function of multiples of the lowest cell density (1, 5, and 20 corresponding to 5, 25, and 100 × 103 cells per mL). Dotted line: *EC50 values were averaged over the 3 initial densities and normalized to the Primitive subline. The normalized values were plotted as a function of multiples of the lowest fraction of primitive cells (1, 2.1, and 21 corresponding to the Primitive/Primitive, Primitive/Standard, and Primitive/Mature ratios). EC50 and *EC50 averaged values are presented in panel Table 4.
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