Exosomal NOX1 promotes tumor-associated macrophage M2 polarization-mediated cancer progression by stimulating ROS production in cervical cancer: a preliminary study

Background Cervical cancer the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death in women, with an estimated 604,000 new cases and 342,000 deaths worldwide in 2020 for high rates of recurrence and metastasis. Identification of novel targets could aid in the prediction and treatment of cervical cancer. NADPH oxidase 1 (NOX1) gene-mediated production of reactive oxygen species (ROS) could induce migration and invasion of cervical cancer cells. Tumor-associated macrophages (TAMs) play important roles in cervical cancer. Tumor cell-derived exosomes mediate signal transduction between the tumor and tumor microenvironment. Elucidation of the mechanisms of NOX1-carrying exosomes involved in the regulation of TAMs may provide valuable insights into the progression of cervical cancer. Methods Uniformly standardized mRNA data of pan-carcinoma from the UCSC database were downloaded. Expression of NOX1 in tumor and adjacent normal tissues for each tumor type was calculated using R language software and significant differences were analyzed. SNP data set were downloaded for all TCGA samples processed using MuTect2 software from GDC. Cell experiment and animal tumor formation experiment were used to evaluate whether exosomal NOX1 stimulating ROS production to promote M2 polarization of TAM in cervical cancer. Results NOX1 is highly expressed with a low mutational frequency in pan-carcinoma. Upregulation of NOX1 may be associated with infiltration of M2-type macrophages in cervical cancer tissues, and NOX1 promotes malignant features of cervical cancer cells by stimulating ROS production. Exosomal NOX1 promotes M2 polarization of by stimulating ROS production. Exosomal NOX1 enhances progression of cervical cancer and M2 polarization in vivo by stimulating ROS production. Conclusion Exosomal NOX1 promotes TAM M2 polarization-mediated cancer progression through stimulating ROS production in cervical cancer. Supplementary Information The online version contains supplementary material available at 10.1186/s40001-023-01246-9.


Introduction
Cervical cancer, a heterogeneous tumor type, is the third most commonly diagnosed cancer in women worldwide.While screening programs and HPV vaccinations have had a successful impact in reducing the incidence of cervical cancer, over 13,000 cases continue to be diagnosed each year in the United States.[1].From Global Cancer Statistics 2020, the data showed in 2020, there were 604,127 new cases of cervical cancer worldwide, of which 109,741 were in China.The number of deaths from cervical cancer worldwide in 2020 was 341,831, of which 59,060 were in China [2].Surgery, chemotherapy, radiotherapy, and immunotherapy are the common modalities of treatment, but high rates of recurrence and metastasis remain the main causes of death [3,4].Identification of novel targets that could aid in the prediction and treatment of cervical cancer thus remains an urgent unmet clinical need.
NADPH oxidase 1 (NOX1), located on the X chromosome, is reported to be involved in a variety of pathogenic mechanisms including vascular disease, tumor angiogenesis, and fibrosis [5].Recently, overexpression of DEAD-box helicase 19A (DDX19A) was reported to increase NOX-1-mediated production of reactive oxygen species (ROS) to induce migration and invasion of cervical squamous cell carcinoma cells.Additionally, high expression of NOX1 was associated with significantly decreased overall patient survival [6].However, expression patterns of the NOX1 gene in cervical cancer and adjacent tissues and its tumor-promoting mechanisms remain to be established.
Exosomes, a type of extracellular vesicles, are released by almost all eukaryotic cells, including tumor cells.Tumor cell-derived exosomes mediate signal transduction between the tumor and surrounding nontumor cells.This intercellular communication actively contributes to remodeling of the tumor microenvironment (TME) to facilitate tumor growth, invasion, and metastasis [7].The importance of the TME in dynamic regulation of cancer progression is widely accepted [8].Tumor-associated macrophages (TAMs) in the TME play important roles in the genesis, development, and prognosis of cervical cancer [9].Earlier studies indicate that TAMs are redefined into anti-tumor M1-like and pro-tumor M2-like TAMs during tumor development [10].The relationship between NOX1 and exosomes has also been reported in the published literature, Vytautas Žėkas et al. found Oxidative Properties of Blood-Derived Extracellular Vesicles in 15 Patients After Myocardial Infarction [11].Renato et al. found platelet-derived extracellular vesicles express NADPH oxidase-1 (NOX1), generate superoxide, and modulate platelet function [12].These studies demonstrate that NOX1 can indeed act through the exosome pathway.
Elucidation of whether NOX1-carrying exosomes are involved in the regulation of TAMs may therefore provide valuable insights into the mechanisms associated with progression of cervical cancer.
In this study, we investigated the expression patterns of NOX1 in cervical cancer.Our collective findings suggest that NOX1-carrying exosomes promote the malignant features of cervical cancer by regulating TAMs via ROS production, providing a theoretical basis and novel targets for effective clinical treatment of cervical cancer.

Bioinformatics analysis
In terms of data sources for bioinformatics analysis, we downloaded uniformly standardized mRNA data of pan-carcinoma from the UCSC database (https:// xenab rowser.net/).Expression of NOX1 in tumor and adjacent normal tissues for each tumor type (Additional file 2: Table S1) was calculated using R language software (version 3.6.4)and significant differences analyzed using unpaired Wilcoxon Rank Sum and Signed Rank Tests.We downloaded the simple nucleotide variation data set for all TCGA samples processed using MuTect2 software from GDC (https:// portal.gdc.cancer.gov/).The R language software package "maftools" (version 2.2.10) was used for mapping.

Clinical samples
Ten pairs of cervical cancer and adjacent tissues were collected between January 2020 and June 2020 from patients in the Department of Obstetrics and Gynecology, Renji Hospital, Shanghai Jiao Tong University School of Medicine(Additional file 2: Table S2).None of the patients had undergone chemotherapy or radiotherapy before surgery.Histologic examinations were performed by two pathologists from the Department of Pathology at our hospital based on WHO criteria.This study was approved by the Institutional Review Board of Shanghai Renji Hospital (Approval number: KY2020-018).Written informed consent was obtained from all patients.

Real-time fluorescence quantitative PCR (RT-PCR)
Total RNA from tissues or cells was extracted using TRIzol (1559601; Ambion, USA) and reverse transcribed into cDNA using a PrimeScript ™ RT reagent Kit (RR037Q, Takara, Japan).qPCR was further performed using TB Green ® Fast qPCR Mix (RR430A, Takara) and 2 -ΔΔCT was applied to calculate relative gene expression using GAPDH as the internal reference.All primer sequences are presented in Additional file 2: Table S3.

Transmission electron microscopy (TEM)
Exosomes were re-suspended in PBS, immobilized in 30 μL of 2% paraformaldehyde, and adsorbed on a lightdischarging copper grid.The copper grid attached to exosomes was suspended in 3% glutaraldehyde droplets, fixed in 0.1 M phosphate buffer of 1% glutaraldehyde (pH 7.4), and suspended on 4% uranyl acetate droplets for exosomal staining.Transmission electron microscopy (Tecnai G2 Spirit BioTwin, FEI, USA) was employed for image acquisition.

Immunofluorescence analysis of exosome uptake
Following induction of THP-1 cells into M0 macrophages, co-culture was performed with 10 µg/mL HeLa-NC (blank exosomes) or HeLa-OE (NOX1-overexpressing exosomes) for 0.5, 2, and 12 h, following which cells were fixed with 4% paraformaldehyde at room temperature for 30 min.FITC-labeled phalloidin working solution (250 µL) prepared with 1% BSA (100 nM, 40735ES75; Yeasen, China) was added to each well and incubated at room temperature in the dark for 30 min.Following the addition of anti-fluorescence quenching agent (E675011; Sangon Biotech, China), cells were visualized under a microscope (ECLIPSE Ts2, Nikon, Japan).

Cell counting kit-8 (CCK-8) assay
Cells (2 × 10 4 cells/mL) were plated on a 96-well plate (100 μL/well) and incubated at 37 °C under 5% CO 2 for 24 h with different concentrations of Ace.After 24 h of culture, CCK-8 solution was added to each well and cell viability was estimated by measuring absorbance at 450 nm using a microplate reader (VICTOR Nivo, Perkin Elmer, USA).

Transwell assay
Cell invasion ability was evaluated with the Transwell chamber assay (8 μm, Corning Costar, USA).Cells were implanted into the upper compartment and the lower compartment filled with 20% serum.The filter was fixed and cultured for 48 h prior to staining with methanol and 0.1% crystal violet.A cotton swab was utilized to wipe the cells in the upper compartment.Cells below the filter were counted and images obtained under a microscope (IX83, OLYMPUS, JAPAN).All experiments were repeated three times.

Wound healing assay
Aliquots of cells were inoculated on 6-well plates (1 × 10 6 /well).A single layer of cells was scratched with the tip of a pipette to make a notch in the dish.Images of the wound were captured at 0, 6, 24, and 48 h under a light microscope (ECLIPSE NI, Nikon, Japan).Wound closure was evaluated as follows: wound width (0 h)wound width (6, 24, or 48 h)/wound width (0 h).

ROS detection
Cells were incubated with H2DCFDA (HY-D0940, MCE, USA) for 30 min.After washing with cold PBS three times, images of cells were acquired via confocal scanning laser microscopy (ECLIPSE Ts2, Nikon, Japan).

In vivo experiments
HeLa, HeLa-NC, and HeLa-OE cells were collected and the cell density adjusted to 1 × 10 8 /mL.Mice were captured and left forelimb was fixed.The left underarm skin was wiped with an alcohol cotton ball.A syringe was subcutaneously inserted into the left armpit in parallel and the tip moved from side to side twice to inject 100 µL of the cell suspension.Subsequently, the syringe was slowly removed and mice transferred into cages to continue feeding.The drug was administered 10 days later and samples collected after 4 weeks of intervention.Mice were euthanized via spinal dislocation.All animal experiments were approved by the Ethics Committee of our hospital.The method of administration is depicted in Additional file 2: Table S3.GW4869 is an inhibitor of exosome biogenesis/release [13].

Statistical analysis
Cell experiments were performed at least three times and animal experiments at least six times.Statistical analysis was conducted using SPSS 22.0 software and GraphPad Prism 7.0.Numerical data are expressed as the means ± SD.The Student's t-test and one-way ANOVA were applied to compare differences between groups.Differences were considered statistically significant at P < 0.05.

NOX1 is highly expressed with a mutational frequency of < 3% in pan-carcinoma
Based on the TCGA database, we systematically evaluated the expression patterns and mutational frequency of NOX1 in pan-carcinoma.Within 26 tumor types, NOX1 was highly expressed in tumor compared to adjacent normal tissues (Fig. 1A).In addition, NOX1 displayed low mutational frequency in 25 tumor tissues (Fig. 1B).In particular, NOX1 was highly expressed in cervical cancer tissues relative to the non-tumor counterparts (Fig. 1A) with a mutational frequency of 2.1% (Fig. 1B).Univariate analysis revealed mRNA expression of NOX1 as a risk factor for poor overall and disease-specific survival (Fig. 1C).We further evaluated the expression patterns of NOX1 in cervical cancer.RT-PCR (Fig. 1D) and IHC (Fig. 1E) results consistently showed elevated expression of NOX1 in cervical cancer tissues compared with adjacent normal tissues, prompting further investigation of the potential functions and mechanisms of action of NOX1 in cervical cancer.

Elevated expression of NOX1 may be associated with infiltration of M2-type macrophages in cervical cancer tissues
Based on QUANTISEQ analysis, a significant positive correlation between NOX1 expression and M2-type, but not M1-type macrophage infiltration was observed in cervical cancer tissues (Fig. 2A).Confocal fluorescence analyses showed that M1-type macrophages were substantially reduced, while M2-type macrophages were substantially increased in cervical cancer compared with adjacent normal tissues (Fig. 2B).In Kaplan-Meier survival analysis, a higher proportion of M2 macrophages was associated with poor prognosis of patients with cervical cancer (Fig. 2C).The collective results suggest that poor prognosis associated with the overexpression of NOX1 is attributed to its activity in promoting M2 polarization of macrophages.

NOX1 promotes malignant features of cervical cancer cell by stimulating ROS production
Plasmids overexpressing NOX1 were generated and their transfection efficiencies examined using RT-PCR (Fig. 3A).The CCK-8 assay was used to determine the optimal concentrations of ROS inhibitors (Acetylcysteine, Ace).The experiments showed that 50 µM Ace induced a substantial reduction in proliferation of HeLa cells (Additional file 1: Fig. S1).Overexpression of NOX1 led to a significant increase in the level of ROS.Treatment with the ROS inhibitor, Ace, induced a significant decrease in the ability of cells overexpressing NOX1 to generate ROS (Fig. 3B).WB analysis disclosed higher expression of NOX1 in the NOX1-OE compared with NOX1-NC group but no significant differences between the NOX1-OE and NOX1-OE + Ace groups (Fig. 3C).Notably, Transwell (Fig. 3D) and scratch (Fig. 3E) experiments demonstrated that invasion and migration abilities of HeLa cell were substantially reduced in the NOX1-OE + Ace compared to NOX1-OE group.Our findings clearly suggest that NOX1 promotes malignant features of cervical cancer cells via stimulation of ROS production.

Exosomal NOX1 promotes M2 polarization of macrophages by stimulating ROS production
We utilized nanoparticle tracking analysis (NTA) to detect particle size and transmission electron microscopy (TEM) analysis to determine the morphology of NC-exo and OE-exo (Fig. 4A).WB was employed to detect the exosomal markers HSP70 and TSG101 (Fig. 4B) and further revealed substantially higher expression of NOX1 in the OE-exo compared to NC-exo group (Fig. 4C).
Electron microscopy (Fig. 5A) and flow cytometry (Fig. 5B) results showed that PMA successfully induced the conversion of THP-1 to M0 macrophages.Data obtained from fluorescence confocal microscopy analysis showed that M0 macrophages ingest exosomes secreted by HeLa cells (Fig. 5C).The ROS concentration in each group of cells was further determined using H2DCFDA (Fig. 5D).Flow cytometry experiments showed that OEexo promoted M2-type polarization of M0 macrophages compared to the NC-exo group.However, polarization of M0-to M2-type macrophages was substantially reduced in OE-exo + Ace relative to the OE-exo group (Fig. 5E).The collective results suggest that exosomal NOX1 promotes M2 polarization of macrophages through stimulating ROS production.

Exosomal NOX1 enhances progression of cervical cancer and M2 polarization of macrophages in vivo by stimulating ROS production
Compared with the HeLa-NC treatment group, the weight and volume of tumor tissues in the HeLa-OE group were significantly increased.However, relative to the HeLa-OE group, the weight and volume of tumor tissues in the HeLa-OE + Ace and HeLa-OE + GW4869 treatment groups were markedly decreased (Fig. 6A).IHC results showed that expression of Ki67 in tumor tissues was substantially increased in HeLa-OE compared with the HeLa-NC group.However, Ki67 expression in tumor tissues was significantly reduced in HeLa-OE + Ace and HeLa-OE + GW4869 groups relative to the HeLa-OE group (Fig. 6B).Fluorescence confocal microscopy revealed a significant increase in the number of M2-type macrophages and, conversely, decrease in M1-type macrophages in the HeLa-OE group compared with the HeLa-NC group.Relative to the HeLa-NC group, the M1-type macrophage content was substantially increased, while that of M2-type macrophages was decreased in HeLa-OE + Ace and HeLa-OE + GW4869 treatment groups (Fig. 6C, D).Based on these results, we propose that exosomal NOX1 promotes tumor growth and M2 macrophage infiltration by stimulating ROS production.

Discussion
Bioinformatics analysis revealed a < 6% mutational frequency of NOX1 across pan-cancer tumor types.However, expression of NOX1 was significantly higher in cancer tissues relative to the corresponding adjacent normal counterparts, including cervical cancer.This differential expression of NOX1 may be associated with tumor occurrence and progression.
RT-PCR and IHC analyses confirmed the significant overexpression of NOX1 in cervical cancer compared with adjacent normal tissues.Similar results based on tissue microarray immunohistochemical staining were reported by Jiang et al. [6].Data from Transwell  Macrophages are one of the major cell types in the innate immune system [14].Macrophages in the tumor microenvironment (designated TAMs) account for 30-50% of cells in the tumor microenvironment.TAMs are involved in invasion, migration, and several other biological processes of tumors [15].Depending on the phenotype, TAMs are divided into M1 and M2 macrophages [16].M1 macrophages display the characteristic property of releasing proinflammatory cytokines and are considered anti-tumor factors.However, M2-type macrophages have low antigen presenting ability and low anti-tumor activity, which are beneficial for tumor growth and invasion [17].Based on QUANTISEQ analysis, we observed an almost significant positive correlation between NOX1 expression and M2 macrophages.High expression of NOX1 is reported to be associated with poor prognosis of cervical cancer patients [7,8].Consistently, our results showed that the higher proportion of M2 macrophages was associated with poor prognosis of patients with cervical cancer.Both in vitro and in vivo experiments collectively demonstrate that overexpression of NOX1 promotes the polarization of macrophages to M2 type, suggesting a carcinogenic role of NOX1 via this pathway.
Combined with the findings of Jiang et al. [7,8], we infer from these experiments that NOX1 promotes M2 polarization of macrophages, which contributes to poor prognosis of patients with cervical cancer.
Exosomes play an important role in intercellular communication among not only cancer cells but also between cancer cells and immune cells [18].Diverse roles of exosomes in cervical cancer have been reported, including exosomal lncRNA-or microRNA-based promotion of cell proliferation [19], angiogenesis  In summary, exosomal NOX1 promotes tumor-associated macrophage M2 polarization-mediated cancer progression through stimulating ROS production in cervical cancer.

Fig. 1
Fig. 1 NOX1 is highly expressed and maintained at a low mutational frequency in pan-carcinoma.A NOX1 is highly expressed in tissues of many tumor types compared with adjacent normal tissues.B Low mutational frequency of NOX1 in tissues of multiple tumor types.C Univariate analysis showing that NOX1 is a potential risk factor for patient prognosis.D RT-PCR and E IHC detection of NOX1 expression in paired adjacent normal and tumor tissues

Fig. 2
Fig. 2 NOX1 promotes infiltration of M2-type macrophages into cervical cancer tissues.A QUANTISEQ-based prediction of the correlation between NOX1 expression and macrophages.B Fluorescence confocal microscopy detection of the infiltration of M1(CD68 + iNOS) and M2(CD68 + CD163) macrophages in adjacent normal tissues and tumor tissues (20x).C Kaplan-Meier survival analysis showing significant association of a higher proportion of M2 macrophages with poor prognosis.H, high, L, low.Scale bar, 100 µm for HE, 50 µm for IHC

Fig. 3 Fig. 4
Fig. 3 NOX1 promotes malignant characteristics of HeLa cells by stimulating ROS production.A RT-PCR analysis of the effects of NOX1-overexpressing plasmids.B Detection of ROS levels for evaluation of NOX1 overexpression and the inhibitory effect of Acetylcysteine.C WB analysis of NOX1 expression in individual groups of cells.D Transwell and E scratch experiments for detecting the invasion and migration of HeLa cells in each group.Con, control; NC, negative control; OE, NOX1 overexpression; Ace, Acetylcysteine.Scale bar, 50 µm [20], and immune escape[21].In the present study, exosomal NOX1 promoted tumor growth and M2 macrophage infiltration in vivo by stimulating ROS production.In both in vitro and in vivo assays, compared with the NOX1 overexpression group, treatment with the exosome inhibitor, GW4869, induced a significant decrease in NOX1-mediated invasion, migration, and growth of HeLa cells and inhibited M2 polarization of macrophages.These results indicate that HeLa cells promote M2 polarization of macrophages by secreting NOX1-carrying exosomes, in turn, stimulating M2 macrophage-mediated carcinogenesis.The collective findings provide a valuable insight into the potential mechanisms by which oncogenic NOX1 promotes cervical cancer progression.The present study had some limitations that should be taken into consideration.The main research results were based on in vitro cell line experiments, and further in vivo animal experiments and clinical sample verification are needed to elucidate the specific mechanisms by which exosome NOX1-derived ROS induce M2 macrophage polarization.

Fig. 5 Fig. 6
Fig. 5 NOX1 promotes polarization of M0-to M2-type macrophages by stimulating ROS production.A Electron microscopy analysis of cell morphology.B Flow cytometry detection of CD11b and CD14 expression.C Fluorescence confocal microscopy for evaluation of exosomal uptake in M0 macrophages.D Fluorescence microscopy for evaluation of ROS production.E Flow cytometry detection of polarization of macrophages.Scale bar, 50 µm.Con, control; NC, negative control; OE, NOX1 overexpression; Ace, Acetylcysteine; exo, exosome