Anticancer Drugs Paclitaxel, Carboplatin, Doxorubicin, and Cyclophosphamide Alter the Biophysical Characteristics of Red Blood Cells, In Vitro

Abstract

Red blood cells (RBCs) are the most numerous cells in the body and perform gas exchange between all tissues. During the infusion of cancer chemotherapeutic (CT) agents, blood cells are the first ones to encounter aggressive cytostatics. Erythrocyte dysfunction caused by direct cytotoxic damage might be a part of the problem of chemotherapy-induced anemia-one of the most frequent side effects. The aim of the current study is to evaluate the functional status of RBCs exposed to mono and combinations of widely used commercial pharmaceutical CT drugs with different action mechanisms: paclitaxel, carboplatin, cyclophosphamide, and doxorubicin, in vitro. Using laser diffraction, flow cytometry, and confocal microscopy, we show that paclitaxel, having a directed effect on cytoskeleton proteins, by itself and in combination with carboplatin, caused the most marked abnormalities-loss of control of volume regulation, resistance to osmotic load, and stomatocytosis. Direct simulations of RBCs' microcirculation in microfluidic channels showed both the appearance of a subpopulation of cells with impaired velocity (slow damaged cells) and an increased number of cases of occlusions. In contrast to paclitaxel, such drugs as carboplatin, cyclophosphamide, and doxorubicin, whose main target in cancer cells is DNA, showed significantly less cytotoxicity to erythrocytes in short-term exposure. However, the combination of drugs had an additive effect. While the obtained results should be confirmed in in vivo models, one can envisioned that such data could be used for minimizing anemia side effects during cancer chemotherapy.

Keywords: anemia; chemotherapy; laser diffraction; microcirculation; microfluidics; osmotic fragility test; red blood cells.

Figure A1

Statistics of RBCs forms according to the confocal microscopy images obtained after the incubation with CT drugs.

Figure A2

The action of CT drugs has contributed to the accumulation of oxidised forms of hemoglobin: the formation of reversible metHb and non reversible hemichrome. Spectral scans of Hb from hypoosmotically lysed RBCs after 3 h treatment with the CT drugs showed the elevated levels of oxidised Hb. Spectral range was from 450 to 700 nm. Data are presented as Mean ± SD, n = 7; one way ANOVA, Tukey HSD post hoc, *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001 compared to control.

Figure A3

CT drugs induced RBCs transformation and microparticle formation. Representative SSC/FSC dot plots of control RBCs and RBCs under the action of TAX. The data shows the microparticle formation (left, green gate). The statistics of the formation of microparticles by CT drugs is shown on the right panel. Template and gating are corresponded to the left scan. Data are presented as Mean ± SD, n = 7 donors; one way ANOVA, Tukey HSD post hoc, ***, p ≤ 0.001 compared to control.

Figure A4

Representative image of a microchannel occlusion by RBCs treated with TAX drug.

Figure A5

Percentage of microchannels occlusion cases. The number of cases of microchannel occlusions was counted manually; an occlusion was recorded if erythrocyte retention was at least 10 s. Data presented as mean ± SE, n = 3 donors, 2-tailed non-paired t-test; *, p ≤ 0.05 compared to control.

Figure 1

OFT demonstrates that CT drugs alter RBCs’ osmotic resistance. (a) The osmotic lysis curves. TAX-treated cells started lysing earlier than the control ones, but were more rigid at 120 and 100 mOsmol; cells incubated with RUBI_PHOS were more sensitive to hemolysis at low osmolality 120 mOsmol; (b) H50 is the osmolality of the buffer at which 50% of the RBCs were lysed; CT drugs affect RBCs’ membranes differently: TAX and TAX_PLAT caused increased osmotic stiffness of RBCs, and RUBI_PHOS caused osmotic fragility of RBCs; (c) Quantification of MCV during osmotic fragility test: MCV increased for TAX and TAX_PLAT earlier than for other drugs and control cells; (d) OFT revealed increased osmotic heterogeneity in the population of RBCs exposed to CT drugs. Data are presented as mean ± SD, n = 15 donors, one way ANOVA, Tukey HSD post-hoc; *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001, ****, and p ≤ 0.0001 compared to control. Pink #, p ≤ 0.05 refers to RUBI_PHOS compared to control, blue asterisks refer to TAX and TAX_PLAT compared to control.

Figure 2

Disruption of native RBC shape under the action of CT drugs obtained by measuring the changes in amplitude of scattering light intensity (asphericity index) on laser diffractometer LaSca-TM. (a) At 300 mOsmol, discoid or flattened cells demonstrate a highly oscillated signal (left), while spherical cells demonstrate lower oscillation amplitude (right); (b) Representative amplitudes of light-scattering intensity oscillations demonstrate the shape changes of RBCs exposed to CT drugs. Three horizontal lines display the area of registration of the asphericity index (AI₁, AI₂). Above the graph, a schematic top and side view of RBCs are shown; (c) Asphericity index of RBCs, indicating TAX and its combination led to cell spherization. Data are presented as mean ± SD, n = 15 donors, one way ANOVA, Tukey HSD post-hoc; ****, p ≤ 0.0001 compared to control.

Figure 3

The action of CT drugs impaired the volume regulation of RBCs, resulting in increased MCV and volume heterogeneity. (a) MCV, (b) RDW-SD, and (c) RDW% calculated by Medonic-M hematology analyser. Data are presented as mean ± SD, n = 15 donors, one way ANOVA, Tukey HSD post-hoc; *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001 compared to control.

Figure 4

Disturbance of erythrocyte morphology under the action of CT drugs. Representative pseudocoloured confocal images (Leica TCS SP5 MP) of non-fixed RBCs show the appearance of anisocytosis and the disruption of RBCs’ native morphology (red circled RBCs as an example): control cells had the normal shape of discocytes, TAX—discocytes and stomatocytes I–II, PLAT—discocytes and echinocytes I, PHOS—discocytes, echinocytes I (1) and schistocytes (2), RUBI—discocytes and echinocytes I; the combinations of drugs intensified the change in morphology of RBCs: TAX_PLAT demonstrated discocytes and stomatocytes at different stages (3, 4) while RUBI_PHOS—discocytes and echinocytes II.

Figure 5

Effects of the anticancer drugs on RBCs’ cytology parameters. (a) Annexin V test: all drugs induced the externalization of PS in RBCs (n = 10 donors); (b) EMA test revealed no membrane changes associated with Band3 translocation (n = 10 donors); (c) Calcein-AM test: CT drugs reduced RBCs’ vitality. RUBI vs. control p = 0.052 (n = 13 donors); (d) Free Hb content in RBCs’ incubation medium (n = 7 donors). Data are presented as mean ± SD, one way ANOVA, Tukey HSD post-hoc (C-AM, EMA, Hemolysis) and Dunn’s post-hoc (AnnexinV); *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001 compared to control; #, p = 0.0128, compared to TAX, &, p ≤ 0.05, compared to PHOS.

Figure 6

Distributions of RBCs’ relative velocities in the microchannels of the microfluidic device. The action of CT drugs did not cause a significant shift in the main peak of cells’ velocities but led to the appearance of a subpopulation of slow cells for all used chemotherapy drugs. The data are presented as mean ± SE, and the error bars are shaded. n = 10 donors.