These considerations regarding induction and maintenance therapies highlight the importance of evaluating the cellular impact of these treatments to ensure the success of HSCT. Prior studies have largely focused on the anti-cancer mechanisms of these agents but have not adequately addressed their broader impact on hematopoietic integrity or immune modulation. This gap is particularly relevant given the increased incidence of secondary health complications in cancer survivors, including immune system dysfunction and hematological disorders. Here, we aimed to understand the effects of topotecan and 13cisRA on CB-derived HSPCs and monocytes. We sought to elucidate the mechanistic basis of drug-induced alterations in these cell populations and their potential contribution to impaired hematopoiesis and immune dysregulation. These findings may inform strategies to mitigate adverse outcomes by adjusting therapeutic regimens or timing in HSCT protocols, particularly for auto-HSCT in neuroblastoma patients.
The concentration used in this study was 25 nM for Topotecan Hydrochloride (USP, Rockville, MD, USA) and 10 μM for 13-cis-retinoic acid (13cisRA; Sigma Aldrich, MERCK KGaA, Darmstadt, Germany). The concentrations used are based on the kinetics and maximum concentration of the drugs measured in the plasma of pediatric oncology patients, 13cisRA 0,4-11,2 μM and Topotecan Hydrochloride 2,6-11,2 ng/ml.
Monocytes from healthy donors were isolated from fresh buffy-coats (Department of Transfusion & Tissue Medicine of the Brno University Hospital, Brno, Czech Republic) using gradient centrifugation with Lymphoprep (density 1.077 g/ml; STEMCELL Technologies) and the Pan Monocyte Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturers' recommendations. Untouched monocytes were resuspended in X-VIVO 15 medium (Lonza, Basel, Switzerland) without supplementation and seeded at a concentration of 1 × 10 cells/ml into cell culture plates (Thermo Fisher Scientific, Waltham, MA, USA). Monocytes were treated with 25 nM Topotecan Hydrochloride or 10 μM 13cisRA for 24 h (RNA extraction and RT-qPCR, phagocytosis, reactive oxygen species (ROS) detection, intracellular staining, LDH Cytotoxicity assay, ELISA, and multiplex bead-based assay) or 48 h (β-galactosidase activity assay).
After 24 h of monocyte treatment with Topotecan Hydrochloride (25 nM) and 13cisRA (10 μM), supernatant was carefully collected and stored in microcentrifuge tubes at -80 °C. The cells were harvested, transferred to microcentrifuge tubes, and washed in phosphate-buffered saline (PBS; Lonza). The pellets were lysed using Trizol LS reagent (Thermo Fisher Scientific) following the manufacturer's recommendations, snap-frozen on dry ice, and stored at -80 °C. Total cellular RNA was extracted from monocytes using Trizol LS reagent (Thermo Fisher Scientific) following the manufacturer's protocol. Samples were centrifuged at 12,000 × g for 15 min at 4 °C, and the upper aqueous phase was mixed with an equal volume of 70% ethanol before transfer to an RNeasy spin column (Qiagen, Hilden, Germany). RNA purification was completed using the RNeasy Mini Kit. RNA concentration and quality were determined spectrophotometrically using a Nanodrop (Agilent, Santa Clara, CA, USA).
After 24 h of monocyte treatment with Topotecan Hydrochloride (25 nM) and 13cisRA (10 μM), 5 μL of pHrodo Green Staphylococcus aureus Bioparticles Conjugate for Phagocytosis (Invitrogen) were added directly to the wells of cell culture plates and incubated for 60 min (37 °C, 5% CO). Following the incubation, the cells were harvested, transferred to a 96-well round-bottom plate (Falcon), and washed with PBS (Lonza) supplemented with 0.5 M EDTA (Gibco) and 0.5% FBS (Sigma Aldrich). The cells were stained with biotinylated lineage antibodies (CD3, CD19, CD20, CD66b, CD235α, Thermo Fisher Scientific; and CD56, BioLegend, San Diego, CA, USA) for 30 min on ice, followed by washing and staining with CD45 PerCP-eF710, Live/Dead NIR, and Streptavidin eFluor 450 Conjugate (Thermo Fisher Scientific). After the final staining, the cells were washed, resuspended in PBS supplemented with 0.5 M EDTA (Gibco) and 0.5% FBS (Sigma Aldrich), and acquisition was performed using a FACS Canto II (BD Biosciences). Data were analyzed using FlowJo (BD Biosciences).
After 24 h of monocyte treatment with Topotecan Hydrochloride (25 nM) and 13cisRA (10 μM), CellROX Deep Red Reagent (Invitrogen) was added directly to the wells of cell culture plates at a final concentration of 1 μM and incubated for 30 min (37 °C, 5% CO). Tert-butyl hydroperoxide pre-treatment at a final concentration of 200 μM incubated for 30 min (37 °C, 5% CO) was used as a positive control. Following the incubation, the cells were harvested, transferred to a 96-well round-bottom plate (Falcon), and washed with PBS (Lonza) supplemented with 0.5 M EDTA (Gibco) and 0.5% FBS (Sigma Aldrich). The cells were stained with biotinylated lineage antibodies (CD3, CD19, CD20, CD66b, CD235α, Thermo Fisher Scientific; and CD56, BioLegend) for 30 min on ice, followed by washing and additional staining with CD45 PerCP-eF710, Live/Dead NIR, and Streptavidin eFluor 450 Conjugate (Thermo Fisher Scientific). After washing, the cells were resuspended in PBS supplemented with 0.5 M EDTA (Gibco) and 0.5% FBS (Sigma Aldrich), and acquisition was performed using a FACS Canto II. Data were analyzed using FlowJo (BD Biosciences).
After 24 h of monocyte treatment with Topotecan Hydrochloride (25 nM) and 13cisRA (10 μM), the cells were harvested and transferred to a 96-well round-bottom plate (Falcon). Cells were stained with the LIVE/DEAD Fixable Violet Dead Cell Stain Kit (Invitrogen) for 30 min on ice. For cytoplasmic and total protein expression, the cells were permeabilized using either the Intracellular Fixation & Permeabilization Buffer Set (eBioscience) or the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturers' recommendations. Staining was performed using anti-HMGB1 (ab79823, Abcam) and anti-γH2A.X (phospho S139; ab26350, Abcam), followed by secondary antibody labeling with Alexa Fluor 488 (donkey anti-rabbit, A-21206, Invitrogen) and Alexa Fluor 647 (goat anti-mouse, A-21236, Invitrogen). Total protein expression was assessed using Foxp3/Transcription Factor Staining Buffer Set and stained with p53 conjugated to PE (Cell Signaling, R-Phycoerythrin Conjugation Kit, ab102918, Abcam), P-p53 (Ser15) Alexa Fluor 647 (Cell Signaling), and p21 Alexa Fluor 488 (Cell Signaling). Acquisition was performed using a FACS Canto II, and data were analyzed using FlowJo (BD Biosciences).
The Pierce LDH Cytotoxicity Assay (Thermo Fisher Scientific) was performed according to the manufacturer's recommendations. Briefly, 0.5 × 10^6 monocytes were plated in a 48-well plate and treated with Topotecan Hydrochloride (25 nM), 13cisRA (10 μM) or ultrapure dH₂O for spontaneous LDH activity for 24 h, and lysis buffer for maximum LDH activity control for 45 min. Supernatants were collected and snap-frozen on dry ice. The LDH assay was carried out in duplicate for all samples, mixing 50 μL of supernatant with 50 μL of reaction mixture, incubated for 30 min protected from light, and stopped with 50 μL of stop solution. Absorbance was measured at 490 nm and 680 nm using a Multiskan GO Microplate Reader (Thermo Scientific).
After 48 h of monocyte treatment with Topotecan Hydrochloride (25 nM) and 13cisRA (10 μM), the cells were harvested and transferred to a 96-well round-bottom plate (Falcon). Cells were stained with a Senescence Assay Kit (β-Galactosidase, Fluorescence; Abcam) dye (334 × diluted) in X-VIVO media for 2 h. For the remaining 30 min, LIVE/DEAD Fixable nearIR Dead Cell Stain Kit (Invitrogen) was added. The cells were washed in Senescence Assay Kit wash buffer (Abcam). Acquisition was performed using a FACS Canto II, and data were analyzed using FlowJo (BD Biosciences).
After 24 h of monocyte treatment with Topotecan Hydrochloride (25 nM) and 13cisRA (10 μM), the cells were harvested, washed, and 1 × 10^5 cells were spun onto glass slides using a cytospin (Centurion Scientific K3 Series) in 3% BSA/PBS. The cells were fixed with paraformaldehyde, washed, permeabilized with 0.1% Triton X-100/PBS, and blocked with 3% BSA/PBS-Tween 20. The cells were stained with anti-HMGB1 (ab79823, Abcam) and anti-γH2A.X (phospho S139; ab26350, Abcam), followed by secondary antibody labeling with Alexa Fluor 488 (donkey anti-rabbit, A-21206, Invitrogen) and Alexa Fluor 555 (donkey anti-mouse, A-31570, Invitrogen), and nuclear counterstaining with DAPI. Samples were mounted in Mowiol 40-88 (Sigma Aldrich), and images were captured using a Zeiss LSM 780 confocal microscope and processed in FIJI ImageJ.
Cord blood was collected after written informed consent. Typically, 10-20 ml of cord blood was collected in sterile heparinized tubes (10 U/ml) for further analyses. CB-HSPCs were isolated using the RosetteSep Human Hematopoietic Progenitor Cell Enrichment Cocktail (STEMCELL Technologies) followed by gradient centrifugation with Lymphoprep (density 1.077 g/ml; STEMCELL Technologies) according to the manufacturers' recommendations. The study was approved by the Ethical Committee of the Institute for the Care of Mother and Child (approval date: 31st March 2014, approval code: 31,032,014).
For the differentiation assay, 5 × 10 cells/mL of semi-solid methylcellulose media (MethoCult™ GF + H4435, STEMCELL Technologies) were seeded into 35 mm Petri dishes (Thermo Fisher Scientific, Waltham, MA, USA). The HSPCs were treated with 25 nM Topotecan Hydrochloride (USP, Rockville, MD, USA) or 10 μM 13-cis-retinoic acid (13cisRA; Sigma Aldrich, MERCK KGaA, Darmstadt, Germany). The dishes were incubated at 37 °C with 5% CO₂ for 14 days. After the incubation, the cells were harvested, washed, and stained for flow cytometry analysis.
For the expansion assay, HSPCs were resuspended in X-VIVO 15 medium (Lonza, Basel, Switzerland) supplemented with SCF (50 ng/mL), Flt3-L (50 ng/mL), TPO (20 ng/mL), and IL-3 (10 ng/mL), then left overnight to recover. After recovery, the cells were harvested, counted, and seeded at a concentration of 4 × 10 cells/mL into 48-well cell culture plates (Thermo Fisher Scientific, Waltham, MA, USA). The HSPCs were treated with 25 nM Topotecan Hydrochloride or 10 μM 13cisRA for 5 days (CFSE staining and flow cytometry phenotyping) or 7 days (β-galactosidase activity assay, RNA extraction, flow cytometry phenotyping, CYTO-ID staining, ROS detection, intracellular staining), with half media changes every 3 days.
Following differentiation, the cells were harvested, washed, and stained on ice for 30 min using conjugated antibodies: CD11b PE-Cy7, CD13 APC, CD15 eF450, CD34 APC-eF780, CD45 PerCP-eF710 (all eBioscience), CD14 BV510, and CD235a PE (both Sony), along with the LIVE/DEAD Fixable Green Dead Cell Stain Kit (Invitrogen). Data acquisition was performed on a FACS Canto II, and the results were analyzed using FlowJo v10 (BD Biosciences).
For the CFSE assay, HSPCs were resuspended in X-VIVO 15 medium supplemented with SCF (50 ng/mL), Flt3-L (50 ng/mL), TPO (20 ng/mL), and IL-3 (10 ng/mL), then left overnight to recover. The cells were harvested and stained in 50 µl of 10 μM CFSE (BioLegend) for 20 min at 37 °C. Staining was quenched in 250 µl of X-VIVO 15 medium supplemented with 10% FBS. The cells were then counted and seeded at 4 × 10 cells/mL into 48-well cell culture plates. The HSPCs were treated with 25 nM Topotecan Hydrochloride or 10 μM 13cisRA for 5 days with half media changes on day 3. After 5 days, the cells were harvested and stained with biotinylated antibodies CD11b and CD235a (eBioscience), followed by incubation with CD34 PE-Cy7, CD123 BV421, CD45RA BV711 (Sony), CD38 PE-Cy5, CD90 PE (BioLegend), Streptavidin eFluor 450 Conjugate (eBioscience), and CD45 Amcyan (BD Biosciences). Propidium iodide (0.1 mg/mL; Sigma Aldrich) was added before acquisition. Data acquisition was performed on an SA3800 Spectral Analyzer (Sony), and analysis was conducted using FlowJo v10 (BD Biosciences).
After 7 days of HSPC culture, the cells were harvested, washed, and resuspended in 100 μL X-VIVO medium supplemented with cytokines and treatments, followed by addition of 0.3 μL Senescence Dye (ab228562, Senescence Assay Kit; Abcam). The cells were incubated for 2 h at 37 °C, 5% CO₂. Biotinylated lineage depletion antibodies CD11b and CD235a (eBioscience) were added during the final 20 min of incubation. The cells were then washed and stained with CD34 PE-Cy7, CD123 BV421, CD45RA BV711 (Sony), CD38 PE-Cy5, CD90 PE (BioLegend), Streptavidin eFluor 450 Conjugate (eBioscience), CD45 Amcyan (BD Biosciences), and propidium iodide (0.1 mg/mL). Data acquisition was performed on an SA3800 Spectral Analyzer (Sony), and analysis was conducted using FlowJo v10 (BD Biosciences).
After 7 days of HSPC culture, positive control cells were treated with 100 nM rapamycin and negative control cells with DMSO for 2 h. All cells were washed with assay buffer provided in the CYTO-ID Autophagy Detection Kit (Enzo Life Sciences) and stained with CYTO-ID dye (1:1000 dilution) in assay buffer supplemented with 5% FBS for 30 min at room temperature in the dark. Cells were subsequently stained with lineage antibodies CD235a and CD11b (eBioscience), followed by Streptavidin eF450 (eBioscience), CD45 Amcyan (BD Biosciences), and LIVE/DEAD Near IR cell stain (Invitrogen). Data acquisition was performed on a BD FACS Canto II, and analysis was conducted using FlowJo v10 (BD Biosciences).
Positive control cells were pre-treated with 200 μM tert-butyl hydroperoxide, while negative control cells were pre-treated with 5 mM N-acetylcysteine for 30 min at 37 °C. ROS quantification was performed using the CellROX Deep Red Flow Cytometry Assay Kit (Life Technologies) at a final concentration of 1 μM, with staining for 30 min at 37 °C. Tert-butyl hydroperoxide pre-treatment at a final concentration of 200 μM incubated for 30 min (37 °C, 5%CO) was used as a positive control. Following ROS staining, cells were stained with lineage antibodies CD235a and CD11b (eBioscience), Streptavidin eF450 (eBioscience), CD45 Amcyan (BD Biosciences), and LIVE/DEAD Near IR Cell Stain (Invitrogen). Data acquisition was performed on a FACS Canto II, and analysis was conducted with FlowJo v10 (BD Biosciences).
After 7 days of HSPC cultivation, cells were harvested and stained with lineage antibodies (CD235a and CD11b; eBioscience), followed by Streptavidin eF450 (eBioscience), CD45 AmCyan (BD Biosciences), and LIVE/DEAD Near IR Cell Stain (Invitrogen). Following staining, cells were fixed and permeabilized using the FoxP3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's recommendations. Intracellular staining was performed using anti-HMGB1 (ab79823, Abcam) and anti-γH2A.X (phospho S139; ab26350, Abcam), followed by secondary antibody labeling with Alexa Fluor 488 (donkey anti-rabbit, A-21206, Invitrogen) and Alexa Fluor 647 (goat anti-mouse, A-21236, Invitrogen). Data acquisition was performed on a FACS Canto II, and analysis was conducted using FlowJo v10 (BD Biosciences).
After 7 days of HSPC cultivation, cells in culture plates were spun at 300 × g for 10 min, and the supernatant was carefully removed without disturbing the cell pellet. Cells were harvested, washed with PBS, and resuspended in RLT buffer. RNA extraction was performed using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA concentration and quality were determined spectrophotometrically using a Nanodrop (Agilent, Santa Clara, CA, USA).
RNA from monocytes and CB-HSPCs was transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Real-time PCR (qPCR) was conducted using TaqMan probes and TaqMan Gene Expression Master Mix (Thermo Fisher Scientific). qPCR was performed on a LightCycler II (Roche, Basel, Switzerland). The Ct values of target genes were normalized to the housekeeping gene GAPDH (ΔCt), and relative expression was calculated as 2^ - ΔCt. The following TaqMan probes were used: CDKN1A (Hs00355782_m1), CDKN2A (p16 splicing isoform, Hs02902543_mH), CDKN2A (p14 splicing isoform, Hs99999189_m1), TP53 (Hs01034249_m1), GATA1 (Hs01085823_m1), CEBPA (Hs00269972_s1), SPI1 (Hs02786711_m1), POT1 (Hs00209984_m1), TERF1 (Hs00819517_mH), TERF2 (Hs01030567_m1), TINF2 (Hs01554307_g1), RARA (Hs00940446_m1), RARB (Hs00977140_m1), RARG (Hs01559234_m1), ATG5 (Hs00169468_m1), ATG7 (Hs00893766_m1), SIRT1 (Hs01009006_m1), SIRT3 (Hs00953477_m1) and GAPDH (Hs02758991_g1).
The DuoSet ELISA (R&D Systems, Minneapolis, MN, USA) was used to detect CD163 and S100A8/A9 in supernatants, following the manufacturer's instructions. HMGB1 concentrations in supernatants were measured using the HMGB1 ELISA Kit (IBL International, Hamburg, Germany). Data acquisition was performed using Multiskan GO Microplate Spectrophotometer (Thermo Scientific).
Simultaneous quantification of 13 human inflammatory cytokines/chemokines, including IL-1, IFN-α2, IFN-γ, TNF-α, MCP-1 (CCL2), IL-6, IL-8 (CXCL8), IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33, was performed using the LEGENDplex Human Inflammation Panel (BioLegend) according to the manufacturer's protocol. Sample acquisition was performed using a FACS Canto II (BD Biosciences), and data were analyzed using LEGENDplex Data Analysis Software v8.0 (VigeneTech).
Human monocytic THP-1 cells were seeded in RPMI 1640 medium (Lonza) without antibiotics at a density of 5 × 10 cells/mL. Cells were transfected with the Cignal Lenti NFκB Reporter (luc; Qiagen) and incubated overnight at 37 °C in 5% CO₂. The medium was replaced with complete low-glucose RPMI 1640 (Lonza, glucose 1 g/L) for 2 days. Puromycin selection (0.5 μg/mL; Santa Cruz Biotechnology) was applied to ensure a homogeneous population carrying the reporter. The THP-1 NFκB reporter line was maintained in RPMI 1640 before stimulation. Cells were treated with 25 nM Topotecan Hydrochloride, 10 μM 13cisRA, or 20 ng/ml TNF-α (recombinant, Sino Biological) as a positive control. Luciferase activity was measured 24 h post-treatment using the ONE-Glo Luciferase Assay System (Promega, Madison, WI, USA).
Statistical analyses were conducted using GraphPad Prism v6. Data were tested for normality, and parametric or nonparametric tests were applied as appropriate. Statistical tests used are specified in the figure legends. P values < 0.05 were considered statistically significant and marked with an asterisk.