Cell Density-Dependent Cytological Stage Profile and Its Application for a Screen of Cytostatic Agents Active Toward Leukemic Stem Cells

Abstract

Proliferation and expansion of leukemia is driven by leukemic stem cells (LSCs). Multidrug resistance (MDR) of LSCs is one of the main reasons of failure and relapses in acute myeloid leukemia (AML) treatment. In this study, we show that maintaining HL-60 at low cell culture density or applying a 240-day treatment with anthrapyridazone (BS-121) increased the percentage of primitive cells, which include LSCs determining the overall stage profile. This change manifested in morphology, expression of both cell surface markers and redox-state proteins, as well as mitochondrial potential. Moreover, four sublines were generated, each with unique and characteristic stage profile and cytostatic sensitivity. Cell density-induced culture alterations (affecting stage profiles) were exploited in a screen of anthrapyridazones. Among the compound tested, C-123 was the most potent against primitive cell stages while generating relatively low amounts of reactive oxygen species (ROS). Furthermore, it had low toxicity in vivo and weakly affected blood morphology of healthy mice. The cell density-dependent stage profiles could be utilized in preliminary drug screens for activity against LSCs or in construction of patient-specific platforms to find drugs effective in case of AML relapse (drug extrapolation). The correlation between ROS generation in differentiated cells and toxic effect observed in HL-60 has a potential application in myelotoxicity predictions. The discovered properties of C-123 indicate its potential application in AML treatment, specifically in conditioned myeloablation preceding allogeneic transplantation and/or ex vivo treatment preceding autologous transplantation.

Effective Drug Concentration and Selectivity Depends on Fraction of Primitive Cells

“…Crucially, low cell density makes it possible to maintain high proliferation potential of human synovium-derived stem cell cultures [ 32 ]. As previously shown by our group, keeping HL-60 at low cell density by frequent passages significantly increased the PC content, including the LSC HSC-like stages [ 33 ]. Culturing at different specified cell densities resulted in three HL-60 sublines with various PC content, named Primitive, Standard, and Mature [ 33 ].…”

“…As previously shown by our group, keeping HL-60 at low cell density by frequent passages significantly increased the PC content, including the LSC HSC-like stages [ 33 ]. Culturing at different specified cell densities resulted in three HL-60 sublines with various PC content, named Primitive, Standard, and Mature [ 33 ]. The establishment and cytological profiling of HL-60 sublines then served as a useful tool for determining the pharmacological parameters of compounds active against PCs [ 33 ].…”

“…Culturing at different specified cell densities resulted in three HL-60 sublines with various PC content, named Primitive, Standard, and Mature [ 33 ]. The establishment and cytological profiling of HL-60 sublines then served as a useful tool for determining the pharmacological parameters of compounds active against PCs [ 33 ].…”

“…Recently, it has become evident that many experiments and assays have been significantly affected by stage-dependent differences that are nontrivial to account for [ 33 , 34 ], as notoriously observed in the complex problem of the reproducibility of in vitro assays using cell models [ 35 ]. Even assuming that two laboratories remove all sources of error (such as different laboratory skills, types and volumes of multi-well plates, incubation times, cell density and viability, quality of culture media, equipment accuracies), the commonly used determination of the dose of a substance as the molar concentrations in the medium still has significant drawbacks [ 35 , 36 , 37 ].…”

Conjugates of Ciprofloxacin and Levofloxacin with Cell-Penetrating Peptide Exhibit Antifungal Activity and Mammalian Cytotoxicity

“…ROS generation activity was evaluated as described previously [37]. Briefly, HEK 293 cells were seeded on 35-mm Petri dishes in the amount of 15,000 cells per plate for 24 h in DMEM medium.…”