Exosomes from tumor-associated macrophages promote glycolysis and malignant NSCLC progression by delivery of hsa_circ_0000896

Jiao Liu; Qianqian Cai; Gang Huang; Mingming Jin; Qingqing Huang

doi:10.1186/s13062-026-00791-7

. 2026 Apr 20;21:87. doi: 10.1186/s13062-026-00791-7

Exosomes from tumor-associated macrophages promote glycolysis and malignant NSCLC progression by delivery of hsa_circ_0000896

Jiao Liu ^1,^#, Qianqian Cai ^1,^#, Gang Huang ^1,^✉, Mingming Jin ^1,^✉, Qingqing Huang ^1,^✉

PMCID: PMC13224439 PMID: 42010627

Abstract

Non-small-cell lung cancer (NSCLC) is amongst the most common tumors, which is responsible for most cancer-related mortality worldwide. Tumor-associated macrophages (TAMs) can regulate tumor microenvironment (TME) and malignant progression of NSCLC; however, the regulatory mechanisms of TAMs exosome (M2-Exo) in the context of NSCLC progression are still unclear. In this study, exosomes isolated from TAMs (M2-Exo) were subjected to FISH and RT-qPCR to explore hsa_circ_0000896 expressions in NSCLC specimens. Dual-luciferase reporter data were utilized to investigate hsa_circ_0000896 downstream targets. Transwell migration, 5-ethynyl-2′-deoxyuridine incorporation, cell counting, and wound hearing experiments were performed to assess NSCLC cell migration and proliferation. Hippocampus experiment was used to detect cellular metabolism. Mouse tumor xenograft model was constructed to assess the hsa_circ_0000896 roles in NSCLC metastasis and progressions. The data revealed that M2-Exo treatment promoted NSCLC cell proliferation and migrations. Hsa_circ_0000896 in M2-Exos mediated NSCLC malignant progression, which was ameliorated by downregulation of hsa_circ_0000896 expression. Both SLC2A3 and miR-503-5p were identified as hsa_circ_0000896 downstream targets. SLC2A3 overexpression or miR-503-5p suppression can reverse hsa_circ_0000896 silence inhibit effects to NSCLC malignant progression. The hippocampus experiment confirmed that downregulation of hsa_circ_0000896 inhibited aerobic glycolysis by regulating miR-503-5p/SLC2A3. A549-DDP were used to construct a subcutaneous tumor model, outcomes of which showed that downregulation of hsa_circ_0000896 increased NSCLC chemosensitivity. Together, our results revealed that exosomes from TAMs promoted glycolysis and malignant progression of NSCLC by delivering hsa_circ_0000896. And hsa_circ_0000896 can promote glycolysis by regulation miR-503-5p/SLC2A3.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13062-026-00791-7.

Keywords: Exosome, Tumor-associated macrophages, Glycolysis, Non-small cell lung cancer, Hsa_circ_0000896

Introduction

Non-small-cell lung cancer (NSCLC) is amongst the most common cancers, with the highest rate of cancer-related mortality worldwide. The NSCLC management has been significantly improved, especially in the last decade [1]. Alterations to the function of the tumor microenvironment (TME) can regulate malignant progression [2–4]. However, the specific regulatory mechanisms are unclear.

Previous studies have confirmed that tumor-associated macrophages (TAMs) are a class of highly plastic immune cells in TME, which are grouped into two phenotypes: classically activated M1 and alternatively activated M2 macrophages. M2-polarized TAMs function to promote NSCLC progression via secreting exosomes [5, 6]. Exosomes are small extracellular vesicles [7], and those from TAMs comprise a good amount of the blood, providing novel clinical biomarkers for minimally invasive liquid biopsies in NSCLC patients [5, 8]. TAM-derived exosomes transport ncRNAs, lipids, and proteins, etc [9–11]. However, M2-Exo role and regulatory mechanisms in context of NSCLC progression are still unknown.

Current investigation discovered that exosomes from TAMs promoted glycolysis and NSCLC malignant progression by delivery of hsa_circ_0000896.

Materials and methods

Ethics statement

Nude BALB/c female mice (4-weeks-old, 15 ~ 20 g) were gained in Shanghai Ji Hui Experimental Animal Breeding Co., Ltd. (Shanghai, China). The Animal Research Committee of Shanghai University of Medicine and Health Sciences approved the protocols (2023-GZR-18-340406198707142817).

Exosome isolation and collection

After inducing THP-1 cells into M2 macrophages, cells were washed with phosphate-buffered saline (PBS), before adding serum-free medium for 2d. Subsequently, M2 macrophage culture fluid was obtained and exosomes were isolated as follows: centrifugation at 1000 × g for 10 min; centrifugation at 2000 × g for 10 min to erase the cells; centrifugation at 10,000 × g for 30 min to eliminate the cell debris; and ultracentrifugation at 120,000 × g for 1.5 h to obtain exosomes. Exosomes were resuspended in PBS and ultracentrifuged again at 120,000 × g for 1.5 h, before resuspending in PBS and collecting in 1.5-mL EP tubes. Exosome size and concentration were quantified by nanoparticle tracking analyzer.

Patient samples

Samples were collected from lung cancer and paracancerous tissues from 57 NSCLC patients. Tissues were collected from 2016 to 2020 in the Department of Cardiothoracic Surgery, Xinhua Hospital, Affiliated with Shanghai Jiao Tong University School of Medicine. All patients signed the consent form and did not undergo radiotherapy or radiotherapy. The study was approved by the Ethics Committee of the Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine, all methods were performed in accordance with the Declarations of Helsinki. Written informed consents were signed by all participants before the study.

Strand-specific NGS RNA-Seq library

Total RNA from M0-Exo and M2-Exo were extracted utilizing TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). We used 3 µg of total RNA and eliminated ribosomal RNA and retained RNA classes, including ncRNAs. We treated RNA with 40 U RNase R (Epicenter) for 3 h at 37 °C following TRIzol purification. We generated the RNA-seq library using KAPA Stranded RNA-Seq Library Prep kits (Roche, Basel, Switzerland) for NGS. Differential expression analysis of circRNAs was performed using the edgeR R package. The fold change was log2 transformed, and we used a log2 (fold change) > 2 (or < − 2) and a p value < 0.01 to sort the differently expressed circRNAs. Subsequently, to generate an overview of circRNA expression profiles between the two groups, hierarchical clustering analysis was performed.

Fluorescence in situ hybridization (FISH)

Hsa_circ_0000896 expression in samples was determined by specific probes for hsa_circ_0000896 (Dig-5′-ACACCTTCTTCCTTCGAGGACTCTGAAGACCTAC–3′-Dig) obtained from Geneseed Biotech (Guangzhou, China). For FISH analysis, samples that stained were counterstained with DAPI (Yeasen Biotechnology, Shanghai, China) for 15 min to visualize the nuclei. Images were obtained using a Zeiss LSM 700 confocal microscope (Carl Zeiss GmbH, Oberkochen, Germany).

Cell culture

Human NSCLC cell lines and a normal lung cell line were obtained from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM (Gibco, Thermo Fisher Scientific, Inc., Billings, MT, USA).

Cell counting kit-8 (CCK-8) assay

CCK-8 assay was employed to assess cell proliferation. Briefly, HCC1833 & A549 cells were incubated with 10% CCK-8. The proliferation rates were tracked 0, 1, 2, and 3 d post transfections.

RT-qPCR

RNA was obtained from tumor samples. cDNA was synthesized and amplified by a TaqMan miRNA Reverse Transcription Kit (Thermo Fisher Scientific). 2^−ΔΔCT method was utilized to obtain relative expression fold changes. Following primers were utilized (forward/reverse): hsa_circ_0000896: 5′-GCTCCTTCTGGAACTTTGAC-3′/5′-TTGCTCACCCAGTAGGTCTT-3′; miR-503-5p: 5′-CCTATTTCCCATGATTCCTTCATA-3′ / 5′-GTAATACGGTTATCCACGCG-3ʹ; SLC2A3 (GLUT3): 5´-GGTGAATTGGAGAAAGTGTAT-3´/5´-CCACTCTTCTCTCTCCCCGAT-3´; U6: 5´-CTCGCTTCGGCAGCACA-3´/5´-AACGCTTCACGAATTTGCGT-3´; and GAPDH: 5′-CTTTGGTATCGTGGAAGGACTC-3′ / 5′-GTAGAGGCAGGGATGATGTTCT-3′.

Dual-luciferase reporter assay

Putative SLC2A3 3′-UTR and hsa_circ_0000896 miR-503-5p-binding sites were cloned into a psi-CHECK vector (Promega, Madison, WI, USA). Firefly luciferase 3′-UTR or hsa_circ_0000896 was utilized as the primary luciferase signal. Clones were termed SLC2A3-WT/hsa_circ_0000896-WT and SLC2A3-MUT/hsa_circ_0000896-MUT. A psi-CHECK vector containing Renilla luciferase signal was utilized for Firefly reporter signal normalization. A549 cells were transfected by applying Lipofectamine 3000. Firefly and Renilla luciferase functions were determined 1 day post-transfection.

Western blotting

Different cell groups were inoculated into 6-well plates at 5 × 10⁵ cells/well. If the cell fusion rate was ≥ 80%, proteins were collected with 200 µL of RIPA lysate per well. The protein concentrations of samples were investigated by BCA protein concentration assay kit (Biyun Tian). Proteins were isolated using SDS-PAGE gels. After blocking for 1 h, the membranes were cleaned and incubated with the primary antibody overnight. ECL chemiluminescence detection solution (Vazyme) was added onto PVDF membranes. Images were acquired by exposing and developing blots with a chemiluminescence image analysis system (Tanon 5200). ImageJ was employed for semi-quantitative analyses. Antibodies applied in present study were: anti-Calnexin, anti-CD63, anti-CD81, anti-SLC2A3, anti-HK2, anti-GAPDH.

In vivo tumor xenograft model

Viable WT or sh-hsa_circ_0000896-transfected A549 or A549-DDP cells (1 × 10⁶) were injected into the mouse right flanks. The tumor volume was detected every 5 days via Vernier calipers, which was computed by formula: width² × length/2. Relative Ki-67 expressions in tumor samples were detected.

Metastasis was characterized utilizing stably transfected luminescence-labeled A549 (Luc-A549) cells and sh-hsa_circ_0000896-Luc-A549 (2 × 10⁵) was injected into mouse tail vein. After 4 weeks, lung tissue metastatic foci were calculated in samples stained with hematoxylin and eosin (HE).

Cell migration assay

Cell migration assay was performed in 24-well Transwell chamber (BD Biosciences, San Jose, CA, USA). A549 and HCC1833 cells were plated in the upper chamber, and 500 µL DMEM was placed in the lower chamber. Cells were cultured for 1 day, and those that adhered to the bottom chamber were fixed in 4% paraformaldehyde and stained.

5-ethynyl-2ʹ-deoxyuridine (EdU) assay

Cell proliferation was detected by a EdU Assay Kit (Thermo Fisher Scientific). HCC1833 & A549 cells were seeded in 6-well plates for 2 d and cells were incubated with EdU for 2 h.

Extracellular acid ratio (ECAR) and oxygen consumption rate (OCR) analyses

A549 and HCC1833 cells from different treatments were incubated overnight at 15,000 cells/well. Probe plates (Seahorse XFe96 Flux Assay Kit) were hydrated through overnight incubation. Medium was replaced before experiments, and incubated in a non-CO₂ incubator. Assays were performed via Seahorse XF Glycolytic Rate assay and Cell Mito Stress Test Kits (Agilent). Rotenone/antimycin A, 2-DG, oligomycin (1 µM), FCCP, and rotenone/antimycin A were added to specific dosing wells. ECAR & OCR data were detected.

Statistical analysis

Data analysis was performed via GraphPad Prism (San Diego, CA, USA). Overall survival analyses were made via Kaplan-Meier curves and log-rank tests. Two-tailed Student’s t-tests were employed to capture significance between two groups, and one-way analysis of variance was utilized for ≥ 3 groups. Correlations were explored by Pearson’s correlation test. Data are denoted by mean ± SD. P-values < 0.05 were assumed to be statistically significant.

Results

M2-Exo treatment promotes NSCLC cell proliferation and migration

After isolating exosomes from M0 and M2 macrophages using the monocytic leukemia cell line THP-1, flow cytometry and RT-qPCR revealed that THP-1 were induced into M2 type via IL-4 (Fig. 1A and B). Nanoparticle tracking data and transmission electron microscope (TEM) detection revealed that the exosomes isolated from macrophages had a diameter of 100 nM (Fig. 1C–D). Western blotting showed that M2-Exos expressed CD63 and CD81, but not calnexin (Fig. 1E). Results from the immunofluorescence assay showed that PKH26-labeled M2-Exos were taken-up by A549 and HCC1833 cells (Fig. 1F). CCK8 (Fig. 1G–H) and EdU (Fig. 1I–K) detection revealed that M0-Exo treatment did not promote A549 and HCC1833 cell proliferations. M2-Exo treatment significantly enhanced the A549 and HCC1833 cell proliferations. Transwell (Fig. 1L–N) and wound healing (Fig. 1O–R) assays showcased that M2-Exo treatment significantly increased A549 & HCC1833 cell migration abilities.

Fig. 1 — Identification of M2-Exo and the promotional effect of M2-Exo on NSCLC cell proliferation and migration. (A–B) Expression of the M2 macrophage marker CD206 analyzed by RT-qPCR and flow cytometry. Data are expressed as the mean ± SD. ^***P < 0.001 vs. M0-Exo. (C) Quantification of exosomes by nanoparticle tracking analysis. (D) Exosomes were examined by TEM. (E) The expression levels of CD63, CD81, and calnexin were detected by WB. (F) Fluorescence microscopy confirmed that both A549 and HCC1833 cells internalized M2-Exo. (G and H) CCK8 assays showed the proliferation ability of both A549 and HCC1833 cells with or without M2-Exo treatment. Data are expressed as the mean ± SD. ^***P < 0.001 vs. NC; ^###P < 0.001 vs. M0-Exo. (I–K) EdU detection showed the proliferation ability of both A549 and HCC1833 cells with or without M2-Exo treatment. Data are expressed as the mean ± SD. ^**P < 0.01 vs. NC; ^##P < 0.01 vs. M0-Exo. (L–N) Transwell assays were used to study the migration ability of both A549 and HCC1833 cells after treatment with M0-Exo and M2-Exo. Data are expressed as the mean ± SD. ^**P < 0.01, ^***P < 0.001 vs. NC; ^##P < 0.01, ^###P < 0.001 vs. M0-Exo. (O–R) Wound healing assays in both A549 and HCC1833 cells after treatment with M0-Exo and M2-Exo. Data are expressed as the mean ± SD. ^**P < 0.01, ^***P < 0.001 vs. NC; ^##P < 0.01, ^###P < 0.001 vs. M0-Exo

M2-Exo treatment promotes tumor growth and metastasis of NSCLC

Examination of nude mouse subcutaneous tumors showed that M2-Exo treatment promoted tumor growth in terms of increasing both the tumor volume and weight (Fig. 2A–C). Immunohistochemical detection unraveled that M2-Exo treatment significantly promoted Ki67 expression (Fig. 2D and E). Living imaging detection showed that M2-Exo treatment enhanced luc-A549 cell pulmonary metastasis, as evidenced by increased fluorescence intensity (Fig. 2F and G). HE staining showcased that M2-Exo treatment promoted metastatic nodule numbers in lung tissues (Fig. 2H and I).

Fig. 2 — M2-Exo treatment promoted tumor growth and metastasis of NSCLC. (A) Representative mouse subcutaneous tumors (n = 5 for each group). (B–C) Curves of tumor volumes and weights in the M2-Exo treatment and control groups. Data are expressed as the mean ± SD. ^***P < 0.001 vs. NC. (D and E) Ki67 expression in tumor tissues was detected using immunohistochemical staining. Data are expressed as the mean ± SD. ^**P < 0.01 vs. NC. (F and G) Living imaging detection showed the pulmonary metastasis ability of luc-A549 with or without M2-Exo treatment. Data are expressed as the mean ± SD. ^***P < 0.001 vs. NC. (H and I) HE staining showed the number of metastatic nodules in the lung tissues. Data are expressed as the mean ± SD. ^***P < 0.001 vs. NC

Hsa_circ_0000896 expression was increased in M2-Exos compared with M0-Exo cells

A volcano plot (Fig. 3A) and heat map (Fig. 3B) showed that there was a significant amount of differentially expressed circRNAs between M0-Exo and M2-Exo using RNA-seq. RT-qPCR outputs revealed that hsa_circ_0000896 expression increased in M2-Exo cells compared with M0-Exo cells (Fig. 3C), suggesting that hsa_circ_0000896 may play a role in M2-Exo-mediated regulation progress of NSCLC. Analysis of 57 pairs of clinical lung cancer samples revealed that hsa_circ_0000896 correlated positively with both tumor grade and tumor size (Table 1). Overall survival analysis showcased that high hsa_circ_0000896 expression was negative to patient survival (Fig. 3D). FISH data illustrated that hsa_circ_0000896 was located in cytoplasm, and that its expression increased with an increase in clinical grading (Fig. 3E and F). Sanger sequencing (Fig. 3H) and AGE (agarose gel electrophoresis) (Fig. 3I) showed that isolated hsa_circ_0000896 were circRNA and with correct sequence. We performed bioinformatics analysis and found that hsa_circ_0000896 (338 bp) originated from FARSA gene exon (506 bp), which was located on chr19:13039155–13039661; thus, hsa_circ_0000896 was named circ-0000896 or circ-FARSA (Fig. 3G). RT-qPCR detection revealed that hsa_circ_0000896 was upregulated in NSCLC cell lines (Fig. 3J). Circ-FARSA was expressed at the highest levels in A549 & HCC1833 cells, and the cell lines were thus used for subsequent experiments. Immunofluorescence detection confirmed that circ-FARSA was located mainly in A549 and HCC1833 cell cytoplasms (Fig. 3K).

Table 1.

The high expression of hsa_circ_0000896 indicates a poor prognosis for NSCLC patients

Characteristics	Number	hsa_circ_0000896 expression (n = 57)		P Value
Characteristics	Number	Low expression (n = 28)	High expression (n = 29)	P Value
Gender
Male	21	9	12	0.4937
Female	36	19	17	0.4937
Age
≤ 60	23	12	11	0.7570
> 60	34	16	18	0.7570
TNM
Ⅰ+Ⅱ	32	19	13	0.0033
Ⅲ+Ⅳ	25	9	16	0.0033
lymphatic metastasis
Negative	39	21	18	0.0062
Positive	18	7	11	0.0062
Tumor Diameter (cm)
<3	18	13	5	0.0458
≥ 3	39	15	24	0.0458

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Downregulation of hsa_circ_0000896 inhibits M2-Exo-promoting effects on malignant progression of NSCLC

EdU data demonstrated that downregulation of circ-FARSA inhibited the promoting effect of M2-Exo to proliferation ability. Experiments confirmed that circ-FARSA overexpression increased A549 and HCC1833 cell proliferation abilities. Circ-FARSA downregulation reduced A549 and HCC1833 cell proliferation (Fig. 4A and D). Transwell (Fig. 4E and H) and wound hearing (Fig. 4I and L) assays revealed that circ-FARSA downregulation inhibited the promoting effects of M2-Exo to the migration ability. Experiments confirmed that circ-FARSA overexpression increased A549 and HCC1833 cell migration abilities, while downregulation of circ-FARSA reduced A549 & HCC1833 cell migration abilities.

Fig. 4 — Downregulation of hsa_circ_0000896 inhibited the M2-Exo-mediated promotion of malignant NSCLC progression. (A–D) EdU detection showed the proliferation ability of both A549 and HCC1833 cells. Data are expressed as the mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. NC. ^##P < 0.01, ^###P < 0.001 vs. sh-circ-FARSA-M2-Exo. (E–H) Transwell assay to detect the migration ability of both A549 and HCC1833 cells. Data are expressed as the mean ± SEM. ^**P < 0.01, ^***P < 0.001 vs. NC. ^#P < 0.05, ^###P < 0.001 vs. sh-circ-FARSA-M2-Exo. (I–L) Detection of wound healing shows the migration ability of both A549 and HCC1833 cells. Data are expressed as the mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. NC. ^#P < 0.05, ^###P < 0.001 vs. sh-circ-FARSA-M2-Exo

The miR-503-5p is a downstream target of hsa_circ_0000896

A Venn diagram showed the differentially expressed miRNA using miRNA sequencing and Starbase analysis. The results showed that miR-4731-5p and miR-28-5p, among others, were downstream targets of hsa_circ_0000896 (Fig. 5A). Dual luciferase reporter data showcased that fluorescence intensity decreased significantly post miR-503-5p transfection (Fig. 5B), and that miR-503-5p was a hsa_circ_0000896 downstream target (Fig. 5C–D). Venn diagram detection showed the shared miRNA with miRNA sequencing, miRbase, circbank, ENCORI analysis further confirmed that miR-503-5p was a hsa_circ_0000896 downstream target (Fig. 5E). RT-qPCR outputs verified that hsa_circ_0000896 downregulation increased miR-503-5p levels, while inhibitory miR-503-5p reversed si-circ-FARSA-induced miR-503-5p expression (Fig. 5F–I). EdU detection showed that a miR-503-5p inhibitor reversed the si-circ-FARSA suppressive effects on A549 and HCC1833 cell proliferations (Fig. 5J–M). Transwell assays showed that miR-503-5p inhibitors reversed inhibitory effects regarding si-circ-FARSA on the A549 and HCC1833 cell migration abilities (Fig. 5N–Q).

Fig. 5 — The miR-503-5p was the downstream target of hsa_circ_0000896. (A) Venn diagram showing the differentially expressed miRNAs with miRNA sequencing and Starbase analysis. (B) Dual luciferase reporter assay showing the fluorescence intensity of hsa_circ_0000896 as a downstream target of miRNA. (C and D) Dual luciferase reporter assay show the relationship between hsa_circ_0000896 and miR-503-5p. Data are expressed as the mean ± SEM. ***P < 0.001 vs. NC. (E) Venn diagram detection showing the shared miRNAs with miRNA sequencing, miRbase, circbank, and ENCORI analyses. (F–J) RT-qPCR showing the expression of hsa_circ_0000896 and miR-503-5p in both A549 and HCC1833 cells following transfection with siRNA against hsa_circ_0000896 and miR-503-5p inhibitor, both single and combined. Data are expressed as the mean ± SEM. ^***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circ-FARSA. (J–M) EdU showing the proliferation ability of both A549 and HCC1833 cells. Data are expressed as the mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. NC. ^###P < 0.001 vs. si-circ-FARSA. (N–Q) Transwell assay showing the migration ability of both A549 and HCC1833 cells. Data are expressed as the mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. NC. ^##P < 0.01, ^###P < 0.001 vs. si-circ-FARSA. (R–U) A hippocampus experiment was used to detect the cellular metabolism in both A549 and HCC1833 cells

Bioenergetic profiles of both A549 and HCC1833 cells were tested with the Seahorse XFe96 Analyzer. ECAR values of glycoPER in both A549 and HCC1833 cells with sh-circ-FARSA adjuvant decreased significantly compared to the controls, but inhibitory miR-503-5p reversed inhibitory effects regarding sh-circ-FARSA on glycolytic capability of NSCLC cells (Fig. 5R and S). The basal OCR values, as well as the ATP production and maximal respiration values, increased significantly in sh-circ-FARSA groups compared with the control group, yet miR-503-5p inhibitions reversed promoting effects of sh-circ-FARSA upon mitochondrial respiration in NSCLC cells, and facilitated oxidative phosphorylation procedures (Fig. 5T and U).

SLC2A3 is a miR-503-5p downstream target

KEGG data showed that M2-Exo treatment regulated HIF-1α and aerobic glycolysis in A549 cells (Fig. 6A). The Venn diagram showed the differentially expressed of mRNA with different database analysis showed that SLC2A3 (GLUT3), EIF4G1, NUTF2 and TNPO1 were miR-503-5p downstream targets (Fig. 6B). RT-qPCR detection showed that downregulation of circ-FARSA significantly suppressed SLC2A3 expression in A549 cells (Fig. 6C). The dual luciferase reporter data showcased SLC2A3 and miR-503-5p correlations, demonstrating that SLC2A3 was a miR-503-5p downstream target (Fig. 5D–E). EdU detection demonstrated that SLC2A3 overexpressions reversed inhibitory effects of miR-503-5p upon A549 and HCC1833 cell proliferations (Fig. 6F–H). Transwell assay unraveled that SLC2A3 overexpression inverted inhibitory effects of miR-503-5p on A549 and HCC1833 cell migrations (Fig. 6I–K).

The miR-503-5p regulates SLC2A3-mediated aerobic glycolysis

Western blot data demonstrated that miR-503-5p overexpression suppressed SLC2A3 and HK2, while SLC2A3 overexpression promoted SLC2A3 and HK2 expression (Fig. 7A and B). The ECAR values of glycoPER in A549 and HCC1833 cells with miR-503-5p mimic adjuvant significantly decreased, when compared to controls, but SLC2A3 overexpression reversed inhibitory effects of miR-503-5p to NSCLC cell glycolytic capabilities (Fig. 7C and D). The basal OCR values, as well as ATP production and maximal respiration values, increased significantly in miR-503-5p mimic groups compared to control group, while SLC2A3 overexpression reversed the mitochondrial respiration-promoting effects regarding miR-503-5p upon NSCLC cells, and facilitated oxidative phosphorylation procedures (Fig. 7E and F).

Fig. 7 — The miR-503-5p regulated SLC2A3-mediated aerobic glycolysis. (A and B) WB revealed the expression of SLC2A3 and Hk2 in both A549 and HCC1833 cells. (C–F) A hippocampus experiment was used to detect the cellular metabolism in both A549 and HCC1833 cells

M2-Exo treatment increased the chemotherapy resistance of NSCLC by regulating SLC2A3

Nude mouse subcutaneous tumors revealed that M2-Exo treatment increased the chemotherapy resistance of A549-DDP to cis-platinum (6 mg/kg). However, the downregulation of SLC2A3 inhibited the chemotherapy resistance of A549-DDP to cis-platinum (Fig. 8A–C) Immunohistochemistry for Ki67 expression showed that downregulation of SLC2A3 inhibited Ki67 expression in tumor tissues (Fig. 8D–E). Apoptosis of tumor tissues was detected using immunohistochemistry and showed that downregulation of SLC2A3 increased apoptosis after treatment with 6 mg/kg of cis-platinum (Fig. 8F and G).

Fig. 8 — M2-Exo treatment increased the chemotherapy resistance of NSCLC by regulating SLC2A3. (A) Representative mouse subcutaneous tumor (n = 5 for each group). (B–C) Curves of tumor volumes and weights in the M2-Exo treatment and control groups. Data are expressed as the mean ± SD. ^***P < 0.001 vs. NC. (D and E) Ki67 expression in tumor tissues was detected using immunohistochemical staining. Data are expressed as the mean ± SD. ^**P < 0.01 vs. NC. (F and G) Tumor tissue apoptosis was detected using immunohistochemical staining. Data are expressed as the mean ± SD. ^**P < 0.01 vs. NC

Discussion

Numerous studies have shown that TAMs are the most abundant immune cells in TME [12, 13]. Reprogramming TAMs toward a proinflammatory M1 phenotype is a new method to induce antitumor immunity. Exosomes can be engineered to provide diverse therapeutic payloads to target cells, and represent an efficient delivery system that can mediate communications between macrophages and tumor cells [14–16]. Current investigation isolated the exosomes from M2-Exo and discovered that they increased malignant progression of NSCLC.

CircRNAs are a type of ncRNA that are resistant to exonuclease degradation [17, 18]. Former reports have unraveled that circRNAs function importantly in cancers, including serving as cancer biomarkers [19, 20]. Former investigation discovered that hsa_circ_0000896 has important functions in M2-Exo-mediated malignant progression of NSCLC. Hsa_circ_0000896 originates from FARSA gene exon (506 bp), which is located on chr19:13039155–13039661. Previous studies have confirmed that serum exosomal hsa_circ_0000896 can be utilized as diagnostic biomarker for lung adenocarcinomas [21]. Hsa_circ_0000896 promotes NSCLC tumorigenesis by elevating B7H3 [22]. Current research discovered that reducing hsa_circ_0000896 expression in M2-Exos significantly promoted the malignant progression of NSCLC. Thus, hsa_circ_0000896 was suggested to play an important role in M2-Exo-mediated NSCLC malignant progression.

High-throughput sequencing and luciferase reporter analyses confirmed that miR-503-5p was a hsa_circ_0000896 downstream target. Former investigations have confirmed that miR-503-5p can suppress different cancer malignant progressions, including tumorigenesis, angiogenesis, lymphangiogenesis, and chemotherapy resistance [23–27]. In the present study, we discovered that downregulation of hsa_circ_0000896 enhanced miR-503-5p expression. The miR-503-5p inhibitory levels reversed sh-circ-FARSA inhibitions of A549 and HCC1833 cell proliferation and migration. The results of aerobic glycolysis test verified that inhibiting miR-503-5p restored aerobic glycolysis after sh-circ-FARSA, by recovering ECAR and OCR, suggesting that circ-FARSA promoted aerobic glycolysis in NSCLC by miR-503-5p adsorption.

KEGG analysis from high-throughput sequencing results showed that M2-Exo treatment regulated HIF-1α and aerobic glycolysis in NSCLC. Bioinformatics and luciferase reporter data validated that SLC2A3 (GLUT3) was a miR-503-5p downstream target. In mammalian cells, glucose transport across the plasma membrane is mediated by SLC2A family [28]. SLC2A3 can enhance macrophage infiltration by glycolysis reprogramming [17]. Previous studies have confirmed that SLC2A3 expression is up-regulated in cancer tissues. SLC2A3 activates TGF-β signaling pathways via lactic acid [29, 30]. Present research revealed that circ-FARSA downregulation or miR-503-5p overexpression inhibited SLC2A3 expression. Moreover, SLC2A3 overexpression reversed the miR-503-5p-mediated inhibitory effects on A549 and HCC1833 cell proliferation and migration. Data of aerobic glycolysis test validated that SLC2A3 overexpression restored aerobic glycolysis post miR-503-5p overexpression, by recovering ECAR and OCR, suggesting that miR-503-5p decreased aerobic glycolysis of NSCLC by the downregulation of SLC2A3.

Previous research has confirmed that aerobic glycolysis is positively correlated with chemotherapy resistance [31–33]. In this study, using A549-DDP cells, we found that cis-platinum (6 mg/kg) treatment did not inhibit tumor growth. M2-Exo treatment further increased chemotherapy resistance and promoted tumor growth, while downregulation of SLC2A3 increased chemosensitivity of A549-DDP tumors by inhibiting tumor growth and promoting apoptosis.

Our data verified that TAM-derived exosomes promoted glycolysis and malignant progression of NSCLC by hsa_circ_0000896 delivery (Fig. 9), thus providing possible biomarkers and therapy targets for NSCLC.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1^{(204.7KB, pdf)}

Acknowledgements

Not applicable.

Author contributions

Gang Huang, Qingqing Huang and Mingming Jin mainly participated in literature search, study design, writing and critical revision, prepared Figs. 1, 2, 3, 4, 5, 6, 7, 8 and 9. Jiao Liu and Qianqian Cai mainly participated in data collection, data analysis and data interpretation. All authors read and approved the final manuscript.

Funding

This research was funded by the Shanghai 2024 “Science and Technology Innovation Action Plan” Natural Science Fund Project (24ZR1429400), Medical new technology research and transformation seed program of Shanghai Municipal Health Commission (2024ZZ2056), National Key Research and Development Program of China (2020YFA0909000), Shanghai Science and Technology program (23010502600), National Natural Science Foundation of China (82127807, 52103171, 82003142) and Shanghai Key Laboratory of Molecular Imaging (18DZ2260400).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Medical Ethics Committee of Shanghai University of Medicine and Health Sciences (2016-GZR-16-340406198707142817) and were conducted in strict accordance with relevant laboratory animal regulations. The study was carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org). The study was approved by the Ethics Committee of the Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine, all methods were performed in accordance with the Declarations of Helsinki. Written informed consents were signed by all participants before the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jiao Liu and Qianqian Cai are co-first authors.

Contributor Information

Gang Huang, Email: huanggang502@sumhs.edu.cn.

Mingming Jin, Email: asdjinmingming@126.com.

Qingqing Huang, Email: qingqinghuang80@163.com.

References

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8.Farzam OR, Eslami S, Jafarizadeh A, Alamdari SG, Dabbaghipour R, Nobari SA, Baradaran B. The significance of exosomal non-coding RNAs (ncRNAs) in the metastasis of colorectal cancer and development of therapy resistance. Gene. 2025;937:149141. [DOI] [PubMed] [Google Scholar]
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12.Bai R, Li Y, Jian L, Yang Y, Zhao L, Wei M. The hypoxia-driven crosstalk between tumor and tumor-associated macrophages: mechanisms and clinical treatment strategies. Mol Cancer. 2022;21(1):177. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Friedman-DeLuca M, Karagiannis GS, Condeelis JS, Oktay MH, Entenberg D. Macrophages in tumor cell migration and metastasis. Front Immunol. 2024;15:1494462. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Kamerkar S, Leng C, Burenkova O, Jang SC, McCoy C, Zhang K, Dooley K, Kasera S, Zi T, Siso S, et al. Exosome-mediated genetic reprogramming of tumor-associated macrophages by exoASO-STAT6 leads to potent monotherapy antitumor activity. Sci Adv. 2022;8(7):eabj7002. [DOI] [PMC free article] [PubMed] [Google Scholar]
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18.Liang Y, Ye F, Luo D, Long L, Wang Y, Jin Y, Wang L, Li Y, Han D, Chen B, et al. Exosomal circSIPA1L3-mediated intercellular communication contributes to glucose metabolic reprogramming and progression of triple negative breast cancer. Mol Cancer. 2024;23(1):125. [DOI] [PMC free article] [PubMed] [Google Scholar]
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21.Kang Y, You J, Gan Y, Chen Q, Huang C, Chen F, Xu X, Chen L. Serum and Serum Exosomal CircRNAs hsa_circ_0001492, hsa_circ_0001439, and hsa_circ_0000896 as Diagnostic Biomarkers for Lung Adenocarcinoma. Front Oncol. 2022;12:912246. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Nie J, Yang R, Zhou R, Deng Y, Li D, Gou D, Zhang Y. Circular RNA circFARSA promotes the tumorigenesis of non-small cell lung cancer by elevating B7H3 via sponging miR-15a-5p. Cell Cycle. 2022;21(24):2575–89. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Guo Y, Zeng J, Zhuang Y, Jiang C, Xie W. MiR-503-5p alleviates peripheral neuropathy-induced neuropathic pain in T2DM mice by regulating SEPT9 to inhibit astrocyte activation. Sci Rep. 2024;14(1):14361. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Li J, Zhang F, Li H, Peng F, Wang Z, Peng H, He J, Li Y, He L, Wei L. Circ_0010220-mediated miR-503-5p/CDCA4 axis contributes to osteosarcoma progression tumorigenesis. Gene. 2020;763:145068. [DOI] [PubMed] [Google Scholar]
25.Peng Q, Wang S, Huang S, Deng Y, Li Z, Liu C, Hong Y, Duan R, Xue X. Ge P: FTO/miR-503-5p/USP10 axis regulates neuronal endoplasmic reticulum stress-mediated apoptosis in ischemic stroke. Int Immunopharmacol. 2025;149:114150. [DOI] [PubMed] [Google Scholar]
26.Fei Y, Shan W, Chen X. MiR-503-5p functions as an oncogene in oral squamous cell carcinoma by targeting Smad7. Histol Histopathol. 2020;35(8):893–901. [DOI] [PubMed] [Google Scholar]
27.Zhang J, Wang M, Wang J, Wang W. JMJD2C-mediated long non-coding RNA MALAT1/microRNA-503-5p/SEPT2 axis worsens non-small cell lung cancer. Cell Death Dis. 2022;13(1):65. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Yao X, He Z, Qin C, Deng X, Bai L, Li G, Shi J. SLC2A3 promotes macrophage infiltration by glycolysis reprogramming in gastric cancer. Cancer Cell Int. 2020;20:503. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Jiang W, Xu S, Li P. SLC2A3 promotes tumor progression through lactic acid-promoted TGF-beta signaling pathway in oral squamous cell carcinoma. PLoS ONE. 2024;19(4):e0301724. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Li Y, Lei H, Zhang M, Wu G, Guo C, Yang Z, Zhang J, Wang Y, Zhu J, Du Y. The Effect of SLC2A3 Expression on Cisplatin Resistance of Colorectal Cancer Cells. Iran J Public Health. 2021;50(12):2576–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Wang S, Zhou L, Ji N, Sun C, Sun L, Sun J, Du Y, Zhang N, Li Y, Liu W, et al. Targeting ACYP1-mediated glycolysis reverses lenvatinib resistance and restricts hepatocellular carcinoma progression. Drug Resist Updat. 2023;69:100976. [DOI] [PubMed] [Google Scholar]
32.Xu X, Chen Y, Shao S, Wang J, Shan J, Wang Y, Chang J, Zhou T, Chen R, Liu S, et al. USP21 deubiquitinates and stabilizes HSP90 and ENO1 to promote aerobic glycolysis and proliferation in cholangiocarcinoma. Int J Biol Sci. 2024;20(4):1492–508. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Jiang X, Guo S, Wang S, Zhang Y, Chen H, Wang Y, Liu R, Niu Y, Xu Y. EIF4A3-Induced circARHGAP29 Promotes Aerobic Glycolysis in Docetaxel-Resistant Prostate Cancer through IGF2BP2/c-Myc/LDHA Signaling. Cancer Res. 2022;82(5):831–45. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1^{(204.7KB, pdf)}

Data Availability Statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

[CR1] 1.Hendriks LEL, Remon J, Faivre-Finn C, Garassino MC, Heymach JV, Kerr KM, Tan DSW, Veronesi G, Reck M. Author Correction: Non-small-cell lung cancer. Nat Rev Dis Primers. 2025;11(1):3. [DOI] [PubMed] [Google Scholar]

[CR2] 2.Cui X, Liu S, Song H, Xu J, Sun Y. Single-cell and spatial transcriptomic analyses revealing tumor microenvironment remodeling after neoadjuvant chemoimmunotherapy in non-small cell lung cancer. Mol Cancer. 2025;24(1):111. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR3] 3.Chen H, Deng C, Gao J, Wang J, Fu F, Wang Y, Wang Q, Zhang M, Zhang S, Fan F, et al. Integrative spatial analysis reveals tumor heterogeneity and immune colony niche related to clinical outcomes in small cell lung cancer. Cancer Cell. 2025;43(3):519–36. e515. [DOI] [PubMed] [Google Scholar]

[CR4] 4.Song KJ, Choi S, Kim K, Hwang HS, Chang E, Park JS, Shim SB, Heo YJ, An WJ, Yang DY, et al. Proteogenomic analysis reveals non-small cell lung cancer subtypes predicting chromosome instability, and tumor microenvironment. Nat Commun. 2024;15(1):10164. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR5] 5.Fang H, Chi X, Wang M, Liu J, Sun M, Zhang J, Zhang W. M2 macrophage-derived exosomes promote cell proliferation, migration and EMT of non-small cell lung cancer by secreting miR-155-5p. Mol Cell Biochem. 2025;480(5):3019–32. [DOI] [PubMed] [Google Scholar]

[CR6] 6.Cheng D, Ge K, Yao X, Wang B, Chen R, Zhao W, Fang C, Ji M. Tumor-associated macrophages mediate resistance of EGFR-TKIs in non-small cell lung cancer: mechanisms and prospects. Front Immunol. 2023;14:1209947. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020;367(6478). [DOI] [PMC free article] [PubMed]

[CR8] 8.Farzam OR, Eslami S, Jafarizadeh A, Alamdari SG, Dabbaghipour R, Nobari SA, Baradaran B. The significance of exosomal non-coding RNAs (ncRNAs) in the metastasis of colorectal cancer and development of therapy resistance. Gene. 2025;937:149141. [DOI] [PubMed] [Google Scholar]

[CR9] 9.Zhu R, Huang J, Qian F. The role of tumor-associated macrophages in lung cancer. Front Immunol. 2025;16:1556209. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR10] 10.Zhang J, Liu J, Liu Z, Guo L, Liu X. M2 macrophages-derived exosomal MDH1 drives lung adenocarcinoma progression via the Hippo/YAP signaling. Pathol Res Pract. 2025;269:155902. [DOI] [PubMed] [Google Scholar]

[CR11] 11.Zhou J, Ma W, Zhou R, Ma D, Liu C, Wang F, Yuan L, Xu L, Wang C, Li Q. Exosome-derived SLC12A2-DT promotes macrophage M2 polarization-mediated hepatocellular carcinoma progression. Int Immunopharmacol. 2025;160:114955. [DOI] [PubMed] [Google Scholar]

[CR12] 12.Bai R, Li Y, Jian L, Yang Y, Zhao L, Wei M. The hypoxia-driven crosstalk between tumor and tumor-associated macrophages: mechanisms and clinical treatment strategies. Mol Cancer. 2022;21(1):177. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.Friedman-DeLuca M, Karagiannis GS, Condeelis JS, Oktay MH, Entenberg D. Macrophages in tumor cell migration and metastasis. Front Immunol. 2024;15:1494462. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Kamerkar S, Leng C, Burenkova O, Jang SC, McCoy C, Zhang K, Dooley K, Kasera S, Zi T, Siso S, et al. Exosome-mediated genetic reprogramming of tumor-associated macrophages by exoASO-STAT6 leads to potent monotherapy antitumor activity. Sci Adv. 2022;8(7):eabj7002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR15] 15.Zhong W, Lu Y, Han X, Yang J, Qin Z, Zhang W, Yu Z, Wu B, Liu S, Xu W, et al. Upregulation of exosome secretion from tumor-associated macrophages plays a key role in the suppression of anti-tumor immunity. Cell Rep. 2023;42(10):113224. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR16] 16.Xu Z, Chen Y, Ma L, Liu J, Guo Y, Yu T, Zhang L, Zhu L, Shu Y. Role of exosomal non-coding RNAs from tumor cells and tumor-associated macrophages in the tumor microenvironment. Mol Ther. 2022;30(10):3133–54. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Chen S, Chen Z, Li Z, Li S, Wen Z, Cao L, Chen Y, Xue P, Li H, Zhang D. Tumor-associated macrophages promote cholangiocarcinoma progression via exosomal Circ_0020256. Cell Death Dis. 2022;13(1):94. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR18] 18.Liang Y, Ye F, Luo D, Long L, Wang Y, Jin Y, Wang L, Li Y, Han D, Chen B, et al. Exosomal circSIPA1L3-mediated intercellular communication contributes to glucose metabolic reprogramming and progression of triple negative breast cancer. Mol Cancer. 2024;23(1):125. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR19] 19.Zhao Y, Jia Y, Wang J, Chen X, Han J, Zhen S, Yin S, Lv W, Yu F, Xu F, et al. circNOX4 activates an inflammatory fibroblast niche to promote tumor growth and metastasis in NSCLC via FAP/IL-6 axis. Mol Cancer. 2024;23(1):47. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR20] 20.Zhong Y, Lin H, Li Q, Liu C, Shen J. CircRNA_100565 contributes to cisplatin resistance of NSCLC cells by regulating proliferation, apoptosis and autophagy via miR-337-3p/ADAM28 axis. Cancer Biomark. 2021;30(2):261–73. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.Kang Y, You J, Gan Y, Chen Q, Huang C, Chen F, Xu X, Chen L. Serum and Serum Exosomal CircRNAs hsa_circ_0001492, hsa_circ_0001439, and hsa_circ_0000896 as Diagnostic Biomarkers for Lung Adenocarcinoma. Front Oncol. 2022;12:912246. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR22] 22.Nie J, Yang R, Zhou R, Deng Y, Li D, Gou D, Zhang Y. Circular RNA circFARSA promotes the tumorigenesis of non-small cell lung cancer by elevating B7H3 via sponging miR-15a-5p. Cell Cycle. 2022;21(24):2575–89. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR23] 23.Guo Y, Zeng J, Zhuang Y, Jiang C, Xie W. MiR-503-5p alleviates peripheral neuropathy-induced neuropathic pain in T2DM mice by regulating SEPT9 to inhibit astrocyte activation. Sci Rep. 2024;14(1):14361. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Li J, Zhang F, Li H, Peng F, Wang Z, Peng H, He J, Li Y, He L, Wei L. Circ_0010220-mediated miR-503-5p/CDCA4 axis contributes to osteosarcoma progression tumorigenesis. Gene. 2020;763:145068. [DOI] [PubMed] [Google Scholar]

[CR25] 25.Peng Q, Wang S, Huang S, Deng Y, Li Z, Liu C, Hong Y, Duan R, Xue X. Ge P: FTO/miR-503-5p/USP10 axis regulates neuronal endoplasmic reticulum stress-mediated apoptosis in ischemic stroke. Int Immunopharmacol. 2025;149:114150. [DOI] [PubMed] [Google Scholar]

[CR26] 26.Fei Y, Shan W, Chen X. MiR-503-5p functions as an oncogene in oral squamous cell carcinoma by targeting Smad7. Histol Histopathol. 2020;35(8):893–901. [DOI] [PubMed] [Google Scholar]

[CR27] 27.Zhang J, Wang M, Wang J, Wang W. JMJD2C-mediated long non-coding RNA MALAT1/microRNA-503-5p/SEPT2 axis worsens non-small cell lung cancer. Cell Death Dis. 2022;13(1):65. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR28] 28.Yao X, He Z, Qin C, Deng X, Bai L, Li G, Shi J. SLC2A3 promotes macrophage infiltration by glycolysis reprogramming in gastric cancer. Cancer Cell Int. 2020;20:503. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR29] 29.Jiang W, Xu S, Li P. SLC2A3 promotes tumor progression through lactic acid-promoted TGF-beta signaling pathway in oral squamous cell carcinoma. PLoS ONE. 2024;19(4):e0301724. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR30] 30.Li Y, Lei H, Zhang M, Wu G, Guo C, Yang Z, Zhang J, Wang Y, Zhu J, Du Y. The Effect of SLC2A3 Expression on Cisplatin Resistance of Colorectal Cancer Cells. Iran J Public Health. 2021;50(12):2576–83. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR31] 31.Wang S, Zhou L, Ji N, Sun C, Sun L, Sun J, Du Y, Zhang N, Li Y, Liu W, et al. Targeting ACYP1-mediated glycolysis reverses lenvatinib resistance and restricts hepatocellular carcinoma progression. Drug Resist Updat. 2023;69:100976. [DOI] [PubMed] [Google Scholar]

[CR32] 32.Xu X, Chen Y, Shao S, Wang J, Shan J, Wang Y, Chang J, Zhou T, Chen R, Liu S, et al. USP21 deubiquitinates and stabilizes HSP90 and ENO1 to promote aerobic glycolysis and proliferation in cholangiocarcinoma. Int J Biol Sci. 2024;20(4):1492–508. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR33] 33.Jiang X, Guo S, Wang S, Zhang Y, Chen H, Wang Y, Liu R, Niu Y, Xu Y. EIF4A3-Induced circARHGAP29 Promotes Aerobic Glycolysis in Docetaxel-Resistant Prostate Cancer through IGF2BP2/c-Myc/LDHA Signaling. Cancer Res. 2022;82(5):831–45. [DOI] [PubMed] [Google Scholar]

PERMALINK

Exosomes from tumor-associated macrophages promote glycolysis and malignant NSCLC progression by delivery of hsa_circ_0000896

Jiao Liu

Qianqian Cai

Gang Huang

Mingming Jin

Qingqing Huang

Abstract

Supplementary Information

Introduction

Materials and methods

Ethics statement

Exosome isolation and collection

Patient samples

Strand-specific NGS RNA-Seq library

Fluorescence in situ hybridization (FISH)

Cell culture

Cell counting kit-8 (CCK-8) assay

RT-qPCR

Dual-luciferase reporter assay

Western blotting

In vivo tumor xenograft model

Cell migration assay

5-ethynyl-2ʹ-deoxyuridine (EdU) assay

Extracellular acid ratio (ECAR) and oxygen consumption rate (OCR) analyses

Statistical analysis

Results

M2-Exo treatment promotes NSCLC cell proliferation and migration

Fig. 1.

M2-Exo treatment promotes tumor growth and metastasis of NSCLC

Fig. 2.

Hsa_circ_0000896 expression was increased in M2-Exos compared with M0-Exo cells

Fig. 3.

Table 1.

Downregulation of hsa_circ_0000896 inhibits M2-Exo-promoting effects on malignant progression of NSCLC

Fig. 4.

The miR-503-5p is a downstream target of hsa_circ_0000896

Fig. 5.

SLC2A3 is a miR-503-5p downstream target

Fig. 6.

The miR-503-5p regulates SLC2A3-mediated aerobic glycolysis

Fig. 7.

M2-Exo treatment increased the chemotherapy resistance of NSCLC by regulating SLC2A3

Fig. 8.

Discussion

Fig. 9.

Supplementary Information

Acknowledgements

Author contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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