Abstract

   The unique hypoxic environment in high-altitude regions is increasingly
   drawing attention for its impact on the health of residents,
   particularly in patients post-chemotherapy. This study aimed to
   investigate the effects and potential mechanisms of high-altitude
   hypoxia on myelosuppression following chemotherapy, with the goal of
   providing a theoretical basis for clinical treatment. A retrospective
   clinical study of 80 patients with breast cancer revealed that patients
   in the plateau exhibited a significantly higher incidence of grade 3 or
   higher neutropenia and any level of neutropenia post-chemotherapy than
   those in the plain, with propensity score matching (PSM) confirming
   these associations. Animal experiments revealed that high-altitude
   hypoxia reduced the white blood cell (WBC) count, granulocyte count,
   lymphocyte count, and number of bone marrow nucleated cells (BMNCs) in
   cyclophosphamide (CTX)-treated mice. Additionally, high-altitude
   hypoxia induced a significant reduction in the proliferation index and
   an elevation in apoptosis rates in BMNCs. High-altitude hypoxia also
   significantly reduced serum levels of granulocyte colony-stimulating
   factor (G-CSF) and granulocyte–macrophage colony-stimulating factor
   (GM-CSF). Transcriptomic analysis of BMNCs demonstrated that
   high-altitude hypoxia might modulate the hematopoietic function in
   CTX-induced myelosuppression mice through pathways related to
   hematopoiesis, such as porphyrin metabolism, hematopoietic cell
   lineage, ECM-receptor interaction, and PI3K-Akt signaling pathway. Our
   results suggest that high-altitude hypoxia exacerbates
   chemotherapy-induced myelosuppression, possibly through reducing the
   serum level of G-CSF/GM-CSF and regulating apoptosis and proliferation
   by PI3K-Akt signaling pathway, highlighting that cancer patients
   undergoing chemotherapy in hypoxic environments may require enhanced
   supportive care to mitigate these adverse effects.

Supplementary Information

   The online version contains supplementary material available at
   10.1007/s12672-025-02611-2.

   Keywords: High-altitude hypoxia, Chemotherapy, Myelosuppression,
   Hematopoietic function

Introduction

   High altitude is home to approximately 81.6 million people worldwide
   [[36]1–[37]3]. High altitudes are characterized by hypoxia, low
   pressure, low temperature, dry air, and strong ultraviolet radiation.
   Among these, hypoxia is the most significant factor affecting human
   physiology [[38]4, [39]5]. The effects of high-altitude hypoxia on
   human physiological functions are complex and multifaceted. Reduced
   atmospheric pressure and lower oxygen partial pressures at high
   altitudes lead to insufficient oxygen intake, triggering a series of
   physiological and pathological responses [[40]6]. Inadequate adaptation
   to high-altitude hypoxia can result in serious conditions such as
   pulmonary edema, stroke, cardiovascular dysfunction, and
   life-threatening situations [[41]7]. However, these adaptations can
   also lead to health issues such as high-altitude polycythemia and
   chronic mountain sickness [[42]8, [43]9].

   It is widely known that hypoxic environments impact the hematopoietic
   system and can rapidly and persistently alter clinical blood parameters
   in humans [[44]10–[45]13]. However, most existing research has
   primarily focused on the impact of high-altitude hypoxia on
   hematopoiesis, particularly concerning red blood cell (RBC) and
   hemoglobin (Hb), with few studies investigating its effects on
   chemotherapy-induced myelosuppression. Myelosuppression is a common and
   serious side effect of chemotherapy that can lead to life-threatening
   complications such as infections, anemia, and bleeding [[46]14–[47]17].
   In clinical practice, we have observed that the incidence of
   neutropenia, defined as an abnormally low level of neutrophil, appears
   to be higher in patients receiving chemotherapy at high altitudes
   compared to those in lowland areas. Consequently, the impact of
   high-altitude hypoxia on chemotherapy-induced myelosuppression remains
   an understudied area.

   This study investigates the effects of high-altitude hypoxia on
   chemotherapy-induced neutropenia in patients with breast cancer. It
   also examines the impact of altitude hypoxia on chemotherapy-induced
   myelosuppression in mice and explores the underlying mechanisms.

Materials and methods

Patients and methods

   The clinical portion of this study was an observational, retrospective,
   multicenter investigation involving patients treated at hospitals at
   different altitudes: Xining (2260 m), Qinghai Province, and Jining
   (37 m), Shandong Province. The inclusion criteria were female patients
   aged 18–75 years with a pathological diagnosis of breast cancer, who
   were treated with the EC regimen (epirubicin and cyclophosphamide)
   between October 2013 and December 2023, and had complete medical
   records. Patients also required no history of chemoradiotherapy,
   Karnofsky performance status (KPS) ≥ 80 points, and met the following
   laboratory parameters: Hb ≥ 95 g/L, white blood cell (WBC)
   count ≥ 3 × 10^9/L, and platelet (PLT) count ≥ 80 × 10^9/L. Exclusion
   criteria included the prophylactic use of granulocyte-stimulating
   factors after chemotherapy and the use of other chemotherapeutic
   regimens. Blood routine data were collected only after the first
   chemotherapy cycle and before the second cycle.

   The study protocol and animal experiments were approved by the Ethics
   Committee of the Affiliated Hospital of Qinghai University (approval
   number P-SL-2023-447). Written informed consent was obtained from the
   patients prior to sample collection. All patient information was
   anonymized to ensure confidentiality. This research complies with the
   ethical standards outlined in the Declaration of Helsinki.

Animal experiments and drug treatments protocol

   All procedures were conducted in strict accordance with the Guide for
   the Care and Use of Medical Laboratory Animals. Female C57BL/6
   specific-pathogen-free (SPF) inbred mice, weighing 16–18 g, were
   sourced from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co.,
   Ltd (Taizhou, China), under license number CNAS LA0021. The mice were
   housed in a controlled environment with temperatures between 24 °C and
   26 °C, humidity levels of 50–60%, and a 12-h light/dark cycle. They
   were allowed a one-week acclimatization period with access to
   irradiated standard chow and sterilized purified water.

   In this study, a total of 108 mice were used, of which 60 were used for
   blood routine tests within 21 days after chemotherapy, 24 were used for
   BMNCs counting, H&E staining, and transcriptomic sequencing, and 24
   were used for the detection of cell cycle and apoptosis in BMNCs, as
   well as ELISA testing for hematopoietic cytokines. The mice were
   randomly assigned to one of four groups: control (ctrl), normoxic
   chemotherapy (model), hypoxia plus chemotherapy (HC), and hypoxia (H).
   Mice in the ctrl and model groups were housed in Xining (altitude:
   2260 m, 16.2 kPa), Qinghai Province, while those in the HC and H groups
   were transported to Maduo County (altitude: 4300 m, PaO[2]: 12.4 kPa),
   Qinghai Province, to facilitate comparative analysis of the effects of
   hypoxic conditions on the experimental outcomes. Mice in the HC and H
   groups were exposed to the hypoxic environment for the entire duration
   of the experiment, starting from their arrival in Maduo County until
   the completion of the study. This included a one-week acclimatization
   period prior to the initiation of any experimental procedures. Mice in
   the model and HC groups received intraperitoneal (i.p.) injections of
   cyclophosphamide (CTX) at a dose of 100 mg/kg for 3 days to develop the
   myelosuppressed mouse model, while the ctrl and H groups received an
   equal volume of saline.

   All operations were conducted between 9:00 and 11:00 a.m. Blood
   samples, approximately 20 µL in volume, were collected from the
   mandibular veins on days 2, 7, 14, and 21 following the last drug
   administration. The mice used for the detection of hematopoietic
   cytokines were anesthetized with isoflurane, blood was collected via
   cardiac puncture, and then the mice were euthanized by cervical
   dislocation before they could recover from anesthesia. The other mice
   were euthanized by administering an intraperitoneal injection of
   pentobarbital at a dose of 200 mg/kg.

Cell counts in the peripheral blood

   Peripheral blood samples were obtained from myelosuppressed mice on
   days 2, 7, 14, and 21 following the last CTX injection. Blood was
   collected via the mandibular vein and transferred into EDTA-coated
   tubes (cat no. YA1460; Solarbio Science and Technology Co. Ltd.) to
   prevent coagulation. Immediately after collection, a complete blood
   count was performed using a hematology analyzer (HKANGXING; China).

Isolation of bone marrow nucleated cells (BMNCs) and count

   The experimental mice were euthanized, and the femoral bones were
   harvested immediately. Bone marrow components were flushed out using
   IMDM (cat no. PM150510; Procell Life Science and Technology Co., Ltd.)
   supplemented with 2% FBS (cat no. 10099141C; Gibco; Thermo Fisher
   Scientific, Inc.). A syringe equipped with a 21-gauge needle was used
   for this procedure. After passing through a 300-mesh strainer, BMNCs
   were isolated using red cell lysis buffer and then counted under a
   light microscope.

Cell cycle analysis

   A single suspension of BMNCs (5–10 × 10^5 cells) was collected in a
   centrifuge tube, and 1.5 mL of 70% cold ethanol was added to fix the
   cells overnight at − 20 °C. Following fixation, the cells were
   centrifuged at 300g for 5 min, and the supernatant was removed. The
   cells were then washed twice with PBS. Afterward, the cells were
   resuspended in 100 μL of RNase A Reagent from Cell Cycle Assay Kit (cat
   no. E-CK-A351, Elabscience Biotechnology Co.,Ltd.) at 37 °C for 30 min.
   Subsequently, staining was performed with 400 μL of propidium iodide
   (PI, 50 μg/mL) was performed in the dark at 2–8 °C for 30 min according
   to the manufacturer’s instructions. Prior to analysis, the stained
   cells were filtered through a 70-μm cell strainer to ensure a
   single-cell suspension. Finally, the stained cells were analyzed with a
   BD FACSCelesta Multicolor Flow Cytometer (BD Biosciences) and the data
   were analyzed with FlowJo software.

Apoptosis analysis by annexin V/PI staining

   BMNCs containing 5–10 × 10^5 cells were resuspended in 100 μL of the
   binding buffer from the apoptosis detection kit (cat no. E-CK-A211,
   Elabscience Biotechnology Co.,Ltd.). The cells were incubated with
   Annexin V-FITC and propidium iodide (PI) in the dark at room
   temperature for 15–20 min. After incubation, 400 μL of binding buffer
   was added to dilute the cells. Cell apoptosis was subsequently detected
   using a BD FACSCelesta Multicolor Flow Cytometer (BD Biosciences) and
   analyzed with FlowJo software.

Histopathological analysis

   Collected femurs were fixed in a neutral buffered 10% formalin solution
   for 48 h to preserve the original morphological structure of the cells.
   Subsequently, the tissues were embedded in paraffin and sliced to a
   thickness of 4 μm. Notably, the bones were decalcified for 7 days prior
   to embedding. All slides were analyzed using hematoxylin and eosin
   (H&E) staining. After staining, the sections were placed in a
   TissueFAXS PLUS microscope (TissueGnostics, Vienna, Austria) for
   panoramic scanning, and the TissueFAXS Viewer software was utilized for
   image acquisition.

Enzyme-linked immunosorbent assay (ELISA)

   The blood samples of mice were collected on day 2 post-chemotherapy,
   kept overnight at 4 °C, and then centrifuged at 3000g/min for 10 min to
   isolate serum. Commercial ELISA kits were utilized for the evaluation
   of Granulocyte-Colony Stimulating Factor (G-CSF) (cat no. KE10025;
   proteintech group Inc.), Granulocyte–Macrophage Colony-Stimulating
   Factor (GM-CSF) (cat no. MM-0185M1; Jiangsu Meimian industrial Co.,
   Ltd), Erythropoietin (EPO) (cat no. MM-0194M1; Jiangsu Meimian
   industrial Co., Ltd), and Thrombopoietin (TPO) (cat no. MM-0194M1;
   Jiangsu Meimian industrial Co., Ltd). The ELISA assays were performed
   according to the manufacturers’ instructions. The absorbance was
   measured at the wavelength of 450 nm using Cytation™5 (BioTek, USA).

RNA extraction and sequencing

   Total RNA from BMNCs was extracted using TRIzol reagent (cat no.
   15596026; Invitrogen; Thermo Fisher Scientific, Inc.) and subsequently
   stored at − 80 °C. Once all samples were prepared, they were sent to
   Shanghai Jiayin Biotechnology Ltd. for mRNA preparation and RNA
   sequencing (RNA-seq). Sequencing was performed using a Novaseq
   platform. The original sequencing data were deposited in the BioProject
   database (Bioproject ID PRJNA1176344).

RNA-seq data analysis

   Differentially expressed genes (DEGs) were defined as genes with a fold
   change (FC) > 1 and P values < 0.05. Xiantao tool
   ([48]www.xiantaozi.com/), an online bioinformatics analysis platform,
   was utilized to create a Venn diagram that illustrates the common and
   unique DEGs among the groups. Functional enrichment analysis was
   conducted using the Xiantao tool, generating functional processing
   categories and pathway enrichment based on the Gene Ontology (GO) and
   Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, with an
   adjusted P value < 0.05. The “ClusterProfiler” (v4.4.4) R package
   [[49]18] was used for the functional annotation and Gene Set Enrichment
   Analysis (GSEA) of the DEGs. The curated reference genesets from the
   MSigDB file: c2.cp.all.v2022.1.Hs.symbols.gmt were selected for GSEA
   [[50]19]. Significantly enriched clusters were identified based on
   false discovery rate (FDR) < 0.25 and P (adjust) < 0.05 as threshold
   parameters.

Propensity score matching (PSM)

   PSM technique represents an advanced and refined statistical approach
   for adjusting potential baseline confounders. We employed
   the MatchIt package in R 4.4.1 to execute a 1:1 match based on the
   nearest neighbor algorithm. The matched covariates included age, T
   stage, and N stage.

Statistical analysis

   Pearson's chi-square test or Fisher's exact test is used to compare the
   incidence between the plateau and plain groups. Animal experimental
   data are presented as the mean ± standard deviation (SD). The
   comparison of the means between two samples was analyzed using the
   Student’s t test, while comparisons among multiple samples were
   conducted using one-way ANOVA followed by Tukey’s post-hoc test
   (GraphPad Prism 9.5.0). Two-way ANOVA was used for repeated
   measurements followed by post-hoc analyses using the LSD test (SPSS
   28.0). P < 0.05 was considered statistically significant.

Result

Effect of high-altitude hypoxia on neutrophil counts in patients with breast
cancer undergoing chemotherapy

   The study involved 80 eligible patients: 52 (65%) from a plain hospital
   (plain group) and 28 (35%) from a plateau hospital (plateau group).
   There were no significant differences in baseline characteristics
   between the two groups, including age, menstrual status, hormone
   receptor (HR) status, HER-2 status, tumor T stage, pathological stage,
   lymph node status, pathological staging, and histological type
   (Table [51]1). The incidence of neutropenia after the first cycle of
   chemotherapy was compared between the plateau and plain groups. The
   results indicated a significant incidence of grade 3 or higher
   neutropenia, with 30 patients (57.69%) in the plain group and 25
   patients (89.29%) in the plateau group affected (P = 0.004).
   Additionally, 38 patients (73.08%) in the plain group and 27 patients
   (96.43%) in the plateau group exhibited some level of myelosuppression,
   with this difference also proving to be statistically significant
   (P = 0.011) (Table [52]2). To mitigate confounding bias and optimize
   statistical power, we employed Propensity Score Matching (PSM) to
   adjust for potential confounders and achieve balance in patient
   characteristics between the plateau and plain groups. Following PSM,
   each group consisted of 28 patients. Baseline characteristics of those
   patients are summarized in Table [53]1. There were no significant
   differences in baseline characteristics between the two groups. The
   incidence of neutropenia after the first cycle of chemotherapy was
   compared between plateau and plain groups. The results indicated a
   significant incidence of grade 3 or higher neutropenia, with 17
   patients (60.71%) in the plain group and 25 patients (89.29%) in the
   plateau group affected (P = 0.014). Additionally, 21 patients (75.00%)
   in the plain group and 27 patients (96.43%) in the plateau group
   exhibited some level of myelosuppression, with this difference also
   proving to be statistically significant (P = 0.022) (Table [54]3).
   These findings suggest that high-altitude hypoxia may play a critical
   role in influencing the incidence of neutropenia in patients with
   breast cancer undergoing chemotherapy.

Table 1.

   Baseline characteristics before and after propensity score matching
   Characteristics Before matching (n = 80) After matching (n = 56)
   Plain, n = 52 Plateau, n = 28 P Plain, n = 28 Plateau, n = 28 P
   Age 0.77 0.45
    < = 60 44 (84.62%) 23 (82.14%) 25 (89.29%) 23 (82.14%)
    > 60 8 (15.38%) 5 (17.86%) 3 (10.71%) 5 (17.86%)
   Menopause 0.06 0.28
    No 32 (61.54%) 11 (39.29%) 15 (53.57%) 11 (39.29%)
    Yes 20 (38.46%) 17 (60.71%) 13 (46.43%) 17 (60.71%)
   HR status 0.07 0.06
    Positive 35 (67.31%) 24 (85.71%) 18 (64.29%) 24 (85.71%)
    Negative 17 (32.69%) 4 (14.29%) 10 (35.71%) 4 (14.29%)
   Her-2 status 0.36 0.24
    Positive 6 (11.54%) 3 (10.71%) 5 (17.86%) 3 (10.71%)
    Negative 39 (75%) 24 (85.71%) 19 (67.86%) 24 (85.71%)
   Indeterminate 7 (13.46%) 1 (3.57%) 4 (14.29%) 1 (3.57%)
   T stage 0.4 0.75
    T1 22 (42.31%) 9 (32.14%) 11 (39.29%) 9 (32.14%)
    T2 29 (55.77%) 17 (60.71%) 16 (57.14%) 17 (60.71%)
    T3 and T4 1 (1.92%) 2 (7.14%) 1 (3.57%) 2 (7.14%)
   N stage 0.36 0.12
    N0 24 (46.15%) 10 (35.71%) 10 (35.71%) 10 (35.71%)
    N1 17 (32.69%) 8 (28.57%) 14 (50.00%) 8 (28.57%)
    N2 and N3 11 (21.15%) 10 (35.71%) 4 (14.29%) 10 (35.71%)
   Pathological staging 0.55 0.15
    I 12 (23.08%) 6 (21.43%) 4 (14.29%) 6 (21.43%)
    II 29 (55.77%) 13 (46.43%) 20 (71.43%) 13 (46.43%)
    III 11 (21.15%) 9 (32.14%) 4 (14.29%) 9 (32.14%)
   Histotype 0.18 0.28
    Ductal 40 (76.92%) 25 (89.29%) 22 (78.57%) 25 (89.29%)
    Other 12 (23.08%) 3 (10.71%) 6 (21.43%) 3 (10.71%)
   [55]Open in a new tab

Table 2.

   Incidence of neutropenia in patients with breast cancer undergoing
   chemotherapy at varying altitudes
   Plain group, n = 52 Plateau group, n = 28 P
   Grade 3 or higher neutropenia, n (%) 30 (57.69) 25 (89.29) 0.004
   Any level of neutropenia, n (%) 38 (73.08) 27 (96.43) 0.011
   [56]Open in a new tab

Table 3.

   Incidence of neutropenia in patients with breast cancer undergoing
   chemotherapy at varying altitudes after propensity score matching
   Plain group, n = 28 Plateau group, n = 28 P
   Grade 3 or higher neutropenia, n (%) 17 (60.71) 25 (89.29) 0.014
   Any level of neutropenia, n (%) 21 (75.00) 27 (96.43) 0.022
   [57]Open in a new tab

Effect of high-altitude hypoxia on peripheral blood cell counts in
CTX-induced myelosuppressive mice

   A complete blood count was performed on days 2, 7, 14, and 21 after the
   last cyclophosphamide (CTX) injection (Fig. [58]1A). The study included
   four experimental groups: control (ctrl) group, normoxic chemotherapy
   (model) group, hypoxia plus chemotherapy (HC) group, and hypoxia (H)
   group. As shown in Fig. [59]1B–G, the white blood cell (WBC),
   granulocyte and lymphocyte and red blood cells (RBC) counts in the
   model group were significantly lower than those in the ctrl group on
   day 2 after chemotherapy (P < 0.01), which suggests that
   myelosuppression took place in the mice. The WBC, granulocyte,
   lymphocyte counts decreased significantly (P < 0.05) in the HC group
   when compared with the model group on day 2, 7, 21 after chemotherapy
   and the lowest value occured on day 2 after chemotherapy. The platelet
   (PLT) count decreased significantly (P < 0.05) in the HC group when
   compared with the model group on day 2, 14, 21 after chemotherapy.
   However, RBC counts and hemoglobin (Hb) level in the HC group were
   significantly higher than those in the model group (P < 0.05) on day 2,
   7, 21 after chemotherapy. Meanwhile, RBC counts and Hb level in the H
   group were significantly higher than those in the ctrl group at any
   time point. During chemotherapy, WBC, granulocyte, and lymphocyte
   counts decreased significantly in the model group, with a more
   pronounced reduction observed in the HC group. While RBC and Hb levels
   also decreased significantly in the model group, the reduction was less
   pronounced in the HC group. The adverse effects of altitude hypoxia on
   myelosuppression mainly occurred in leukocytes, namely granulocytes and
   lymphocytes. So, we chose 2 days after chemotherapy as the time node of
   the follow-up experiment.

Fig. 1.

   [60]Fig. 1
   [61]Open in a new tab

   Effect of high-altitude hypoxia on peripheral blood cell counts in
   CTX-induced myelosuppression mice. Mice were administered CTX at a dose
   of 100 mg/(kg d) via intraperitoneal injection for 3 consecutive days,
   while the control (ctrl) group was treated with the same volume of
   saline. A Experimental design. The levels of WBC (B), granulocyte (C),
   RBC (D), Hb (E), PLT (F), and lymphocyte (G) were assessed on days 2,
   7, 14, and 21 post-chemotherapy. WBC white blood cell, Gran
   granulocyte, RBC red blood cell, Hb hemoglobin, PLT platelets, Lym
   lymphocyte. *P < 0.05, **P < 0.01, ***P < 0.001, compared to the ctrl
   group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared to the model
   group. n = 15

High-altitude hypoxia aggravates impaired hematopoiesis in CTX-induced
myelosuppressive mice

   For bone marrow nucleated cells (BMNCs) count and bone marrow
   hematoxylin and eosin (H&E) staining, mouse femurs were harvested
   2 days after chemotherapy (Fig. [62]2A). CTX significantly reduced the
   BMNCs count (P < 0.05), with an even greater reduction observed in the
   HC group (P < 0.05) (Fig. [63]2B). H&E staining of femurs demonstrated
   notable bone marrow cell depletion in CTX-treated mice (Fig. [64]2C).
   The H&E staining revealed that in the ctrl group, the bone marrow
   structure appeared normal, with all types of hematopoietic cells evenly
   distributed within rich hematopoietic zones, and mature erythrocytes
   present in the sinusoids. Conversely, in the model group, there were
   few nucleated cells in the marrow cavities of femurs, while the
   hemorrhagic changes in the HC group were more severe than those in the
   model group. These observations suggest that high-altitude hypoxia, in
   the context of CTX treatment, may result in impaired angiogenesis,
   increased hemorrhage, and further reductions in BMNC counts, which
   requires further investigation and validation.

Fig. 2.

   [65]Fig. 2
   [66]Open in a new tab

   Effects of high-altitude hypoxia on hematopoietic function in
   CTX-induced myelosuppression mice. A Experimental design. Refer to the
   "Materials and Methods" section for specific administration protocols.
   B The number of bone marrow nucleated cells (BMNCs) was counted on day
   2 post-CTX treatment (n = 6). Results are expressed as mean ± SD.
   ^###P < 0.0001 compared to the ctrl group; ***P < 0.0001 compared to
   the model group. C Representative H&E-stained sections of bone marrow
   are displayed for the indicated groups of mice. The scale bars in the
   upper and lower panels represent 100 µm and 20 µm, respectively

RNA-seq identifies transcriptomic profiles of high-altitude hypoxia in BMNCs
of CTX-induced myelosuppressive mice

   To explore the mechanism of the effect of altitude hypoxia on bone
   marrow suppression, we used CTX to establish a myelosuppression model
   by intraperitoneal injection that continued for 3 days. After
   confirming the decreased hematopoietic function resulting from CTX
   treatment through WBC count on day 2 post-chemotherapy, transcriptome
   profiling analyses of BMNCs in the ctrl group, model group, and HC
   group were conducted to elucidate the underlying mechanisms. By
   applying the cutoffs of P value < 0.05 and FC > 1, the differentially
   expressed genes (DEGs) were selected. Compared to the ctrl group, 927
   genes were upregulated, and 882 genes were downregulated in the model
   group (Fig. [67]3A, [68]B). Compared to the model group, 130 genes were
   upregulated, and 550 genes were downregulated in the HC group
   (Fig. [69]3A and [70]C). Compared to the ctrl group, 183 genes were
   upregulated, and 388 genes were downregulated in the H group
   (Fig. [71]3A and [72]D).

Fig. 3.

   [73]Fig. 3
   [74]Open in a new tab

   Transcriptomic analysis. A Comparison of the number of upregulated and
   downregulated genes among three groups: model vs ctrl, HC vs model, and
   H vs ctrl. B–D Volcano plots of differentially expressed genes (DEGs)
   for the three comparisons: model vs ctrl, HC vs model, and H vs ctrl.
   E, F Pie charts illustrating the co-regulated DEGs. G, H Heatmap of
   hierarchical clustering

   Following this, we analyzed the co-regulated DEGs between the HC group
   vs the model group and the model group vs the ctrl group. The Venn
   diagram showed that 224 co-regulated DEGs were commonly affected by
   both high-altitude hypoxia and chemotherapy treatment and
   (Fig. [75]3E). Hierarchical clustering analysis showed that of the 224
   DEGs, most of the co-regulated DEGs were consistent down-regulation
   (Fig. [76]3G). Finally, we explored the co-regulated DEGs between
   the HC group vs the model group and the H group vs the ctrl group.
   The Venn diagram showed that 146 co-regulated DEGs were related to
   hypoxia (Fig. [77]3F). Hierarchical clustering analysis showed that
   most of the co-regulated DEGs were also uniformly downregulated
   (Fig. [78]3H).

Functional enrichment analysis reveals key pathways regulated by
high-altitude hypoxia in CTX-induced myelosuppressive mice

   In order to further explore the potential mechanism of high altitude
   hypoxia affecting myelosuppression after chemotherapy, we conducted
   pathway enrichment analysis. GO analysis suggested that these DEGs
   related to high-altitude hypoxia between HC and model groups were
   classified into 961 different biological process categories, the top 10
   of which encompassing erythrocyte development, B cell receptor
   signaling pathway, phagocytosis, recognition, respiratory system
   development, complement activation, B cell activation, homeostasis of
   number of cells, respiratory tube development, complement activation
   classical pathway, and regulation of B cell activation (Fig. [79]4A).
   The results supported the assumption that high-altitude hypoxia may
   have regulatory effect on the hematopoietic process during
   chemotherapy-induced myelosuppression.

Fig. 4.

   [80]Fig. 4
   [81]Open in a new tab

   Enrichment analysis. A GO analysis related to biological processes of
   DEGs. B KEGG enrichment analysis of DEGs. C Top 10 pathways enriched in
   Gene Set Enrichment Analysis (GSEA). D Apoptosis pathway enriched in
   GSEA

   KEGG enrichment analysis of DEGs between the HC and the model groups
   enriched 10 pathways, such as PI3K-Akt signaling pathway, focal
   adhesion, hematopoietic cell lineage, and ECM-receptor interaction
   (Fig. [82]4B).

   GSEA showed that DEGs associated with hypoxia significantly enriched in
   cell cycle and DNA replication processes-related clusters, including
   cell cycle checkpoints, mitotic G1 phase and G1 S transition, DNA
   replication, cell cycle mitotic, and G2 M checkpoints, and the top 10
   terms are shown in Fig. [83]4C. DEGs also showed significant
   correlation with the apoptosis (Fig. [84]4D).

   Through enrichment analysis, we hypothesize that high-altitude hypoxia
   exacerbates chemotherapy-induced myelosuppression, possibly via
   regulation of cell cycle and apoptosis in BMNCs by multiple
   hematopoiesis-related pathways, such as PI3K-Akt signaling pathway and
   ECM-receptor interaction. To test this hypothesis, we conducted a
   follow-up experiment.

Effects of high-altitude hypoxia on the cell cycle of BMNC in CTX-induced
myelosuppressive mice

   In the model group, the proportion of cells in the G0/G1 phase
   significantly decreased compared to the ctrl group (P < 0.05), while
   the proportions of cells in the S and G2/M phases increased (P < 0.05).
   The proliferation index (PI) was significantly higher than in the ctrl
   group (P < 0.05). These results suggest that CTX-induced DNA damage
   triggered cell cycle arrest at the G2/M checkpoint. In contrast, the HC
   group showed a higher proportion of cells in the G0/G1 phase and lower
   proportions in the S and G2/M phases compared to the model group
   (P < 0.05). The PI was significantly lower in the HC group than in the
   model group (P < 0.05), indicating that hypoxia suppressed cell cycle
   progression and induced G1 phase arrest. In the H group, the proportion
   of cells in the G0/G1 phase showed no significant difference compared
   to the ctrl group (P > 0.05), while the proportions of cells in the S
   and G2/M phases were significantly increased (P < 0.05). The PI was
   also significantly higher than in the ctrl group (P < 0.05). These
   results suggest that hypoxia alone promoted cell cycle progression,
   leading to an increase in cells entering the S and G2/M phases and
   enhancing proliferative activity.

Effects of high-altitude hypoxia on the apoptosis of BMNC in CTX-induced
myelosuppressive mice

   The apoptosis of BMNCs was assessed on day 2 post-chemotherapy, with
   results shown in Fig. [85]5C, [86]D. Compared to the ctrl group, the
   model group exhibited a significant increase in apoptosis (P < 0.05),
   confirming that chemotherapy led to elevated levels of bone marrow cell
   apoptosis. Notably, the HC group, which underwent chemotherapy under
   hypoxic conditions, showed an even higher apoptosis rate, significantly
   surpassing that of the model group (P < 0.05).

Fig. 5.

   [87]Fig. 5
   [88]Open in a new tab

   Effect of hypoxia on the cell cycle and apoptosis of bone marrow
   nucleated cells (BMNCs) in CTX-induced myelosuppressed mice. A
   Propidium iodide (PI) staining was performed to assess the cell cycle
   of BMNCs using flow cytometric analysis. B Quantitative analysis of the
   durations of cell cycle phases (n = 6). C Flow cytometric analysis of
   apoptosis in BMNCs. D Quantitative analysis of apoptosis (n = 6).
   *P < 0.05, **P < 0.01, ***P < 0.001, compared to the ctrl group;
   ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, compared to the model group

Effect of high-altitude hypoxia on the hematopoiesis-related cytokines in
CTX-induced myelosuppressive mice

   As illustrated in Fig. [89]6A–C, the levels of G-CSF, GM-CSF and EPO in
   the model group were significantly elevated compared to the ctrl group
   (P < 0.05). Hypoxia resulted in a significant reduction in G-CSF and
   GM-CSF levels in the HC group compared to the model group (P < 0.001),
   while it significantly increased EPO levels (P < 0.001). No
   statistically significant differences were observed in TPO levels among
   the four groups (Fig. [90]6D).

Fig. 6.

   [91]Fig. 6
   [92]Open in a new tab

   The levels of G-CSF (A), GM-CSF (B), EPO (C), and TPO (D) in serum were
   detected on day 2 after chemotherapy. *P < 0.05, **P < 0.01,
   ***P < 0.001, compared to the ctrl group; ^#P < 0.05, ^##P < 0.01,
   ^###P < 0.001, compared to the model group

Discussion

   This study investigates the impact of high-altitude hypoxia on
   hematopoietic function through both clinical analysis and an
   experimental mouse model of CTX-induced myelosuppression. Patients with
   breast cancer residing at high altitudes exhibited a significantly
   higher incidence of grade 3 or higher neutropenia and any level of
   neutropenia after chemotherapy. Experimental results indicated that
   high-altitude hypoxia during chemotherapy further reduced WBC,
   granulocyte, lymphocyte, and PLT counts while unexpectedly increasing
   RBC counts and Hb level. Additionally, high-altitude hypoxia increased
   cell apoptosis, inhibited cell proliferation, decreased G-CSF and
   GM-CSF levels and increased EPO levels in the CTX-induced
   myelosuppressive mice. RNA-seq analysis revealed several
   hematopoiesis-related signaling pathways influenced by hypoxia,
   underscoring the complexity of its effects on bone marrow function.

   Cyclophosphamide (CTX), a classic alkylating agent, is widely used in
   the chemotherapy of various malignant tumors. Its anti-tumor mechanism
   primarily relies on its ability to damage DNA, especially affecting
   rapidly proliferating cells. The main mechanism of action of
   cyclophosphamide involves its metabolites forming cross-links with DNA,
   leading to DNA strand breaks and damage. This DNA damage activates the
   cell's repair mechanisms, but if the damage is too severe, the cell may
   enter the programmed cell death (apoptosis) pathway, resulting in cell
   death [[93]20]. The alkylating effect of cyclophosphamide is not
   limited to tumor cells; it also affects the DNA of normal cells,
   leading to myelosuppression and reproductive toxicity, which are among
   the main side effects in its clinical application [[94]21].
   Additionally, the impact of CTX on the cell cycle is mainly reflected
   in its induction of cell cycle arrest and apoptosis. Cyclophosphamide
   can cause cells to arrest in the G0/G1 or G2/M phase, preventing them
   from entering the next proliferative phase. This arrest is due to the
   DNA damage caused by cyclophosphamide, which activates cell cycle
   checkpoints, particularly the p53 signaling pathway, leading to changes
   in the expression of cell cycle regulatory proteins and thereby
   affecting the cell's proliferative capacity [[95]22]. In tumor cells
   treated with cyclophosphamide, the proportion of cells in the G2/M
   phase significantly increases, while the proportion of cells in the
   G0/G1 phase decreases, indicating that cyclophosphamide can effectively
   inhibit cell proliferation [[96]23]. Additionally, cyclophosphamide
   promotes cell death by inducing the expression of apoptosis-related
   proteins, further enhancing its anti-tumor effects [[97]20].
   Cyclophosphamide exhibits significant immunosuppressive effects,
   primarily through the inhibition of lymphocyte proliferation and
   function. Studies have reported that cyclophosphamide can lead to a
   marked decrease in the number of white blood cells in peripheral blood,
   particularly reducing the counts of T cells and B cells, thereby
   diminishing the body's immune response capacity [[98]24]. In this
   study, the pronounced decrease in WBC, granulocyte, lymphocyte, RBC,
   and BMNCs observed after CTX administration alone in the model group is
   a direct consequence of its mechanism of action. CTX induces DNA
   damage, cell cycle arrest, and apoptosis in hematopoietic stem and
   progenitor cells, leading to the suppression of erythropoiesis,
   granulopoiesis, lymphopoiesis, and overall hematopoiesis. This
   mechanistic explanation aligns with the observed data. In this study,
   the pronounced decrease in WBC, granulocyte, lymphocyte, RBC, and BMNCs
   observed after CTX administration alone in the model group is a direct
   consequence of its mechanism of action. CTX induces DNA damage, leading
   to cell cycle arrest at the G2/M checkpoint and activation of apoptosis
   in hematopoietic stem and progenitor cells. This results in the
   suppression of erythropoiesis, granulopoiesis, lymphopoiesis, and
   overall hematopoiesis. The observed increase in the proportion of cells
   in the S and G2/M phases, along with a decreased G0/G1 phase
   proportion, further supports the notion that CTX triggers DNA damage
   repair attempts and cell cycle arrest. These findings are consistent
   with the known mechanisms of CTX-induced myelosuppression and provide a
   clear explanation for the observed changes in peripheral blood and bone
   marrow cell counts.

   Chemotherapy-induced myelosuppression is a significant clinical
   challenge, characterized by the suppression of bone marrow activity,
   leading to reduced production of blood cells [[99]25, [100]26]. This
   condition severely impacts patients’ immune function, increasing the
   risk of infections, anemia, and overall morbidity [[101]27, [102]28].
   The management of myelosuppression is crucial for the continuation of
   cancer therapy, as severe cases may need dose reductions, treatment
   delays, or discontinuation, which can adversely affect overall
   treatment outcomes [[103]28, [104]29]. Our findings indicate a
   significant increase in the incidence of grade 3 or higher neutropenia
   among chemotherapy patients treated at high altitudes compared to those
   at low altitudes. This suggests that hypoxic conditions prevalent at
   higher altitudes may exacerbate the myelosuppressive effects of
   chemotherapy. Previous studies have primarily focused on the general
   effects of altitude on human physiology or the impact of chemotherapy
   on cancer patients, but none have specifically examined the interaction
   between altitude and chemotherapy-induced myelosuppression in cancer
   patients. Thus, our study fills an important gap in the literature by
   highlighting the potential risks associated with administering
   chemotherapy at high altitudes and underscores the need for tailored
   treatment strategies to mitigate these risks. This study's findings are
   particularly relevant for healthcare providers in high-altitude
   regions, as they may need to adopt more rigorous monitoring and
   supportive care protocols for patients with breast cancer undergoing
   chemotherapy. Large randomized controlled studies are needed for
   further confirmation.

   In patients with breast cancer, hormone receptor (HR)-positive breast
   cancer accounts for approximately 70%-80% of all breast cancer cases
   [[105]30]. Hormone receptor-negative breast cancers are typically more
   aggressive and have fewer treatment options compared to hormone
   receptor-positive cancers [[106]31]. Endocrine therapy, a primary
   treatment strategy for HR-positive patients, significantly improves
   disease-free survival and overall survival [[107]31, [108]32]. The
   molecular characteristics of breast cancer, such as ER status, may
   influence the occurrence and severity of neutropenia [[109]33]. In our
   study, we observed that the plateau group had a significantly higher
   incidence of grade 3 or higher neutropenia and overall neutropenia
   compared to the plain group. Interestingly, the plateau group also
   exhibited a relatively higher proportion of HR-positive patients
   compared to the plain group, although this difference did not reach
   statistical significance. This observation suggests that HR status may
   play a role in modulating the severity of chemotherapy-induced
   myelosuppression, particularly in high-altitude environments. Estrogen
   has been shown to play a protective role in bone marrow function by
   promoting the proliferation and differentiation of hematopoietic stem
   cells through its receptors [[110]34, [111]35]. In HR-positive
   patients, the presence of estrogen may help mitigate the severity of
   chemotherapy-induced myelosuppression. Conversely, estrogen deficiency,
   which is more likely in HR-negative patients, can lead to a decline in
   hematopoietic function, increasing the risk of myelosuppression. The
   high incidence of neutropenia in the plateau group, despite the higher
   proportion of HR-positive patients, may be attributed to the combined
   effects of high-altitude hypoxia and chemotherapy. This suggests that
   HR status may play an important role in aggravating the severity of
   chemotherapy-induced myelosuppression in high-altitude environments.
   These findings highlight the potential impact of HR status on patient
   treatment and prognosis, particularly in the context of high-altitude
   hypoxia. However, further validation through prospective, multicenter,
   large-sample studies is necessary to confirm these observations and to
   explore the underlying mechanisms.

   Understanding the mechanisms underlying myelosuppression is crucial for
   developing effective therapeutic strategies to mitigate these adverse
   effects and improve patient outcomes. Despite extensive research on the
   regulation of erythropoiesis, the precise effects of hypoxia on WBC
   remain indeterminate [[112]12, [113]36–[114]38]. To the best of our
   knowledge, this study is the first to investigate the impact of
   high-altitude hypoxia on hematopoietic function following chemotherapy,
   along with the underlying mechanisms involved. The present study
   specifically investigates the impact of high-altitude hypoxia on
   chemotherapy-induced myelosuppression in mice, aiming to elucidate the
   multifaceted effects of hypoxic conditions on bone marrow function and
   hematopoiesis. According to a recent study, hypobaric hypoxia enhances
   the proliferation and apoptosis of bone marrow erythroblasts in rats,
   leading to increased peripheral blood red blood cell parameters
   [[115]39]. Consistently, our research demonstrates that high-altitude
   hypoxia significantly increases apoptosis of BMNCs in mice. Further,
   the present study demonstrated that high-altitude hypoxia significantly
   increased proportion of cells in the G0/G1 phase and reduced
   proliferation index in the CTX-induced myelosuppressive mice. These
   findings provide a deeper understanding of how hypoxia modulates
   cellular responses to chemotherapy, revealing that hypoxia not only
   impairs cell proliferation but also promotes apoptosis.

   Understanding the mechanisms underlying myelosuppression is crucial for
   developing effective therapeutic strategies to mitigate these adverse
   effects. Previous studies have made some progress in understanding how
   hypoxia conditions affect bone marrow cell proliferation and
   differentiation [[116]39, [117]40]. However, these studies usually
   focus on a single factor and lack a systematic and comprehensive
   analysis. The findings of our study indicate that high-altitude hypoxia
   exacerbates myelosuppression in mice undergoing chemotherapy,
   particularly through its effects on serum hematopoietic cytokines. The
   elevated EPO levels in hypoxic conditions reflect that hypoxia can
   stimulate erythropoiesis that is crucial for maintaining Hb levels and
   enhancing RBC production, which is consistent with many previous
   studies [[118]41, [119]42]. From this side, high-altitude hypoxia seems
   as a good factor for patients undergoing chemotherapy. For cancer
   patient living in high-altitude environment, the increased levels of
   EPO and RBCs could indeed indicate enhanced oxygen transport in the
   blood, which might theoretically make tumors more susceptible to
   radiotherapy due to improved re-oxygenation. This is an intriguing
   hypothesis that could have significant clinical implications for
   personalized radiotherapy strategies, thereby pointing out a new
   direction for our future research. However, the significant reduction
   in G-CSF and GM-CSF levels observed in the HC group suggests that there
   is a relation between high-altitude hypoxia and the decrease in WBC and
   granulocyte levels. It is well known that G-CSF and GM-CSF play a
   multifaceted role in regulating hematopoiesis, which are crucial for
   the production of granulocyte [[120]43, [121]44]. WBC mainly include
   granulocyte and lymphocyte [[122]45, [123]46], therefore, the depletion
   of G-CSF and GM-CSF will induce the decline in WBC and granulocyte.
   These cytokines are essential for maintaining hematopoietic
   homeostasis, and the decline induced by hypoxia highlights the
   challenges faced by patients undergoing chemotherapy at high altitudes.
   Future studies should explore potential interventions that could
   mitigate these adverse effects, aiming to improve the management of
   chemotherapy-induced myelosuppression in hypoxic environments.

   PI3K-Akt pathway is an important biological pathway in cell signal
   transduction, which is involved in various physiological processes such
   as cell growth, proliferation, and apoptosis [[124]47, [125]48].
   Studies have shown that PI3K-Akt signaling pathway plays a key role in
   the proliferation and survival of hematopoietic stem cells (HSCs) and
   are crucial for maintaining hematopoietic stem cell (HSC) function and
   bone marrow microenvironment integrity [[126]49, [127]50]. In acute
   lymphoblastic leukemia cells, inhibition of PI3K-Akt pathway leads to
   decreased expression of Bcl-2, thus promoting apoptosis [[128]51].
   Therefore, high-altitude hypoxia is likely to regulate the
   proliferation and apoptosis of bone marrow cells through the PI3K-Akt
   pathway, and further aggravate myelosuppression after chemotherapy.
   This mechanistic understanding highlights the potential for targeting
   PI3K-Akt pathway to mitigate the adverse effects of hypoxia on bone
   marrow function during chemotherapy, thereby improving patients’
   outcomes.

   Despite the known half-life of cyclophosphamide of up to 12 h, the
   blood biochemical values in mice remained abnormal 21 days after a
   three-day treatment regimen, suggesting a complex and persistent impact
   on hemopoietic systems. The protracted recovery may be due to the
   cumulative toxic effects of cyclophosphamide on the bone marrow and
   other rapidly dividing cells, leading to prolonged hematopoietic
   suppression. Additionally, the drug’s alkylating activity could result
   in sustained damage to the liver and kidneys, which are crucial for
   drug metabolism and excretion, respectively. The slow regeneration of
   these organs, combined with potential oxidative stress and
   inflammation, may further delay the return to baseline biochemical
   parameters. Moreover, the immune system’s response to chemotherapy
   could contribute to the variability in recovery times of
   myelosuppression. These findings underscore the need for careful
   dosing, monitoring of drug metabolism, and the implementation of
   supportive care strategies to mitigate the cytotoxic effects of
   cyclophosphamide in future studies.

Conclusion

   High-altitude hypoxia significantly exacerbates chemotherapy-induced
   myelosuppression, which may be related to reducing the serum level of
   G-CSF/GM-CSF and regulating apoptosis and proliferation by PI3K-Akt
   signaling pathway. These findings imply that cancer patients undergoing
   chemotherapy in high-altitude regions necessitate enhanced supportive
   care to mitigate these adverse effects.

Supplementary Information

   [129]Additional file1 (DOCX 16 KB)^ (15.5KB, docx)

Acknowledgements