Exosomes derived from imatinib-sensitive chronic myeloid leukemia cells transmit miR-711 reverses imatinib resistance by inhibiting CD44

Exosomes derived from imatinib-sensitive chronic myeloid leukemia cells transmit miR-711 reverses imatinib resistance by inhibiting CD44 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exosomes derived from imatinib-sensitive chronic myeloid leukemia cells transmit miR-711 reverses imatinib resistance by inhibiting CD44 Meiyong Li, Xuan Hou, Rong Zhang, Pei Huang, Qingting Xiong, WenQi Ma, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7873222/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Imatinib (IM) resistance is an urgent problem in the clinical treatment of chronic myeloid leukemia (CML). Accumulated evidences have identified that exosomes are recognized as an important component of tumor microenvironment and are closely related to drug resistance of tumor. However, the specific contribution of chronic myeloid leukemia-derived exosomes is poorly understood. Currently we found that exosomes derived from imatinibsensitive CML cells co-culture with drug-resistant CML cells can improve the sensitivity of imatinib resistant CML cells. We further demonstrated that miR-711 significantly decreased in exosomes derived from imatinib drug-resistant CML cells compared with exosomes derived from imatinib sensitive CML cells using microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses. Moreover, miR-711 was also downregulated in imatinib resistant cells compared with parental cells. Overexpression of miR-711 reversed chemoresistance, whereas silence of miR-711 induced imatinib resistance. In addition, enhanced miR-711 could be packaged into exosomes, incorporated into receipt cells, reversed chemoresistance by targeting CD44. To conclude, it is speculated that the exosomes from imatinib sensitive CML cells mediate the sensitivity of imatinib resistant CML by transporting miR-711 and targeting CD44. Exosomal miR-711 may be a promising therapeutic strategy and prognostic indicator for CML patients. Imatinib Drug resistance Chronic myeloid leukemia Exosomes MiR-711 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm originating from hematopoietic stem cells. The Philadelphia chromosome (Ph) and the BCR/ABL1 fusion gene are the characteristic alterations of CML (Rowley 1973 ). Imatinib (IM), a tyrosine kinase inhibitor (TKI) that target the ATP-binding site of BCR-ABL1 kinase, has become the first-line molecular targeted therapy drug for CML (Oehler et al. 2007 ). Although IM has significantly improved the survival of CML patients, secondary resistance to IM remains a major clinical challenge (Hochhaus et al. 2017 ). The mechanisms of drug resistance to IM are very complex, and the present mechanisms cannot fully explain all drug resistance phenomena. Therefore, it is essential to reveal the molecular mechanism of IM resistance and to find new markers of resistance for clinical treatment of CML. The tumor microenvironment (TME) is comprised of immune cells, endothelial cells, tumor-associated fibroblasts, adipocytes, extracellular matrix and soluble cytokines, which play a critical role in tumorigenesis, tumor metastasis, and chemosensitivity (Elhanani et al. 2023 ). Increasing evidence have shown that exosomes are an essential component of the tumor microenvironment. Exosomes are nanoscale extracellular vesicles with a diameter of about 30–150 nm (average ~ 100 nm) (Mathieu et al. 2019 ). Exosomes carry functional proteins, DNA, mRNA, metabolic products, and non-coding RNA, serve as intermediaries for communication between different cells to regulate various cellular biological functions (Kalluri et al. 2020). Our previous study found that exosomal miR-365 secreted by imatinib-resistant cells could be transmitted to imatinib-sensitive cells, resulting in acquired resistance to imatinib (Min et al. 2018 ). Therefore, exosomes as important components of the tumor microenvironment can transmit the resistance to neighboring cells. Whether exosomes derived from IM-sensitive CML cells in the tumor microenvironment could sensitise imatinib-resistant CML cells and thus reverse drug resistance is worthy of further study. In this study, we revealed that exosomes derived from IM-sensitive cells influence the drug resistance of IM-resistant cells. Moreover, exosomes derived from IM-sensitive cells could transport miR-711 to imatinib-resistant cells and increases the sensitivity of CML cells to imatinib by targeting CD44. These findings may provide new experimental evidence for the treatment of CML resistance. Materials and methods Cell culture Imatinib-sensitive (K562) and imatinib-resistant (K562/G01) human chronic myeloid leukemia cells lines were obtained from Institute of Hematology, Chinese Academy of Medical Science (Tianjin, China). All cells were cultured in RPMI 1640 media (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, South America) and 100 U/mL penicillin-streptomycin (Solarbio, China) at 37°C in a humidifed atmosphere with 5% carbon dioxide (CO2). K562/G01 cells were maintained in medium with 1.0 µM Imatinib mesylate (Sigma-Aldrich, Mo, USA) and were cultured in IM-free medium for at least 1 weeks before all experiments. Exosome isolation and identification We isolated exosomes from the cell culture medium through ultracentrifugation as previously described with slight modifications (Jiang et al. 2020 ). The culture medium was centrifuged at 400 × g for 10 min and 2000× g for 20 min at 4°C to remove cells and cell debris. Subsequently, the supernatant was collected and centrifuged at 10,000 × g for 30 min at 4°C and then was filtered through a 0.22 µm syringe membrane filter (Millipore, USA). The supernatant was ultracentrifuged at 110,000 × g for 70 min at 4°C (OptimaL-80XP, SW41 rotor, Beckman Coulter, USA) to enrich exosomes. The pellet was resuspended with cold PBS and centrifuged again at 110,000 × g for 70 min at 4°C. The final exosomes were resuspended in 50 µl PBS for subsequent analysis. Exosomes were identified as previously described. Briefly, the morphology of exosome was visualized by transmission electron microscope, the size distribution and concentration, the size distribution and concentration were measured by NTA, and exosomal protein markers were identified by Western blotting as previously described (Li et al. 2021 ). Exosome labeling and Co-culture assay Exosomes secreted from K562 cells were labeled with red fluorescent dye PKH26 (Sigma-Aldrich, USA) according to the manufacture’s instruction. The labeled exosomes were co-incubated with K562/R cells for 6 h, 12h, 24h, and 48h. Exosomes without PKH26 staining were used as negative control. The cells were stained with Hoechst 33342 and visualized under a TCS-SP5 confocal microscope (Leica, Germany). For exosome treatment, K562/R cells were co-cultured with 20 µg/mL exosomes derived from K562 cells for 48 h. Cell viability assay The sensitivity of cells to drugs was measured by cell counting kit-8 (CCK8) assays. A total of 8×10 3 cells were seeded per well into 96-well plates. Various concentrations of imatinib were added into culture media and incubated for 48 h. 10 µl CCK-8 reagent (ApexBio, USA) was added to each well and incubated for 2 h at 37°C. The optical density was read at 450 nm using a microplate reader (Thermo Fisher Scientific Inc. MA, USA). The inhibitory concentration to produce 50% cell death (IC50) of imatinib was calculated from the survival curves with GraphPad Prism® 8.0 software. Apoptosis assay Cells were collected, washed twice with cold phosphate buffer saline (PBS), and then resuspended in 1× binding buffer. The Annexin V-FITC/PI or Annexin V-PE/PI Apoptosis Detection Kit (BD Biosciences, CA, USA) was used to stain the cells according to manufacturer's instructions. Cell apoptosis rate was then measured via fow cytometry (BD FACS Calibur) and data analysis using FACSDiva software. RNA extraction and qRT-PCR analysis MiRNAs from the cells and exosomes were extracted using miRcute miRNA Isolation Kit (Tiangen, China) following the manufacturer’s instructions. Trizol reagent (Invitrogen) was used for total cellular RNA extraction. RNAs (1 µg) were reverse-transcribed to cDNA with Fast cDNA Reverse Transcriptase (Tiangen, China). qRT-PCR analysis for miR-711 and CD44 mRNA expression was performed using SYBR Green PCR Kit (Tiangen, China) with 7500 Real-Time PCR system (Applied Biosystems, USA). The results were obtained using the relative standard curve method (2-△△Ct) and the relative expression level of miR-711 and CD44 mRNA was respectively normalized with U6 and β-actin. Specific primers for hsa-miR-711 and U6 were supplied by RIBOBIO (Guangzhou, China). Western blot analysis Protein of cells or exosomes were extracted using the RIPA lysis buffer (Beyotime, China). Western blot analysis was performed according to our previous reports (Li et al. 2020 ). The primary antibodies against Alix (12422-1-AP, 1:4000), CD81(66866-1-Ig, 1:3000), TSG101(28283-1-AP,1:6000), Calnexin (66903-1-Ig,1:10000), CD44(60224-1-Ig,1:3000), and β-actin (66009-1-Ig,1:10000) were obtained from Proteintech (Wuhan, China). The bound antibodies were visualized with Pro-light HRP chemiluminescence kit (Tiangen, China) using a ChemiDoc XRS System (Bio-Rad). Cell transfection K562 cells or K562R cells were seeded into six-well plates at a density of 10 6 cells per well. The miR-711 mimic, inhibitor, and corresponding negative controls (mimic NC and inhibitor NC) were purchased from Ribobio company (Guangzhou, China). MiR-711 mimic (50 nM), miR-711 inhibitor (100 nM) and negative control were incubated with 12 µl of riboFECTTM CP reagent (RioBio) for 15 min at room temperature and transfected into cells. K562 cells or K562R cells (5 × 104 per well) were seeded into six-well plates and infected with 10 µl of lentiviral vectors containing CD44 overexpression plasmid, CD44 shRNA, and negative control plasmid (Jikai Gene, Shanghai, China) for 2 days. The stable infected cells were screened with 3.0 µg/ml puromycin (Sigma-Aldrich, MO, USA) for 2 weeks and confirmed by fluorescence microscopy, RT-qPCR and WB analyses. Statistical analysis The data were shown as mean ± standard deviation (χ ± SD). The comparison between the two groups were determined using Student's t-test, and statistical analyses were performed by SPSS22.0 software (version 22.0, NY, USA). The values of P < 0.05 was considered to be statistically significant. Exosomes characterization In order to investigate the effect of exosomes derived from K562 on the IM resistance of K562/R, we first extracted exosomes from cell supernatants by ultracentrifugation, and verified using transmission electron microscopy (TEM), Nanosight particle tracking analysis (NTA), and Western blot analysis (WB). The results showed that the size of the extracted exosomes was mainly distributed around 150 nm in diameter (Fig. 1 A). In addition, transmission electron microscopy showed that the vesicles exhibited a teacup vesicle-like structures with a particle size of about 150 nm, which was consistent with the typical exosome structure (Fig. 1 B). Finally, we proved that the extracted substances carried the exosomal characteristic marker proteins including Alix, TSG101 and CD81, whereas the marker protein of the endoplasmic reticulum Calnexin was enriched in the whole cell lysates through WB (Fig. 1 C). In summary, the above experimental results indicate that exosomes have been successfully enriched and purified from the cell supernatants by ultracentrifugation. Exosomes derived from imatinib-sensitive CML cells are internalized by IM-resistant CML cells and reduce their IM resistance. The successful uptake of exosomes is the important prerequisite by which exosomes modulate their target cells, so that exosomes can release the functional substances to target cells and then influence cellular functions. To demonstrate that K562 cell-derived exosomes were taken up by K562R cells, the exosomes were stained with PKH26 (a red florescent dye), and then co-cultured with K562/R cells. Finally, the uptake of exosomes by K562/R cells was observed by laser confocal microscopy, after PKH26-labeled exosomes was co-cultured with K562/R cells for 6h, 12h and 24h. To exclude false-positive results caused by free dyes, and PBS was also used as the negative control. Compared with the control group, different degrees of red fluorescence signals appeared in the K562/R cells (Fig. 2 A) after coincubation for 6 h, which were mainly distributed within the cytoplasm. With the increase of incubation time, the red fluorescence signal intensity was progressively strengthened. These studies indicate that K562 cell-derived exosomes can be taken up by K562/R cells. Although exosome uptake experiments found that K562/R cells were indeed able to internalize exosomes derived from K562 cells, whether K562 cell-derived exosomes would have effects on imatinib resistance of K562/R cell remains unclear. K562/R cells were respectively co-cultured with K562 cell-derived exosomes (10 µg/ml), K562R cell-derived exosomes (10 µg/ml) and equal volume PBS for 48h, and then the IM resistance and apoptotic rates of K562/R cells was respectively detected by CCK-8 assay and flow cytometry. The CCK-8 assay showed that K562 cell-derived exosomes decreased imatinib resistance in K562/R cells, compared with PBS and K562/R cell derived exosomes. No statistically significant difference was observed in IC50 values between Exo K562/R -treated and untreated groups ( P < 0.05; Fig. 2 B-C). Additionally, K562/R cells exposed to Exo K562 exhibited a markedly lower apoptosis rate relative to those treated with Exo K562R (Fig. 2 D-E). These collective findings demonstrate that exosomes originating from K562 cells can enhance IM drug sensitivity in K562/R cells. Effect of miR-711 on Cellular Drug Resistance Exosomes derived from K562 can reduce the resistance of K562/R cells to IM. However, the mechanisms K562 of cell-derived exosomes influence the resistance of K562/R cells remains unclear. Exosomes modulate drug resistance of target cells through the transfer of protein and nucleic acid molecules, particularly non-coding RNAs. The miRNA was the most abundant in exosomes and have been demonstrated to be associated with tumor drug resistance in multiple studies. In our previous study, miRNA microarray analysis of exosomes derived from K562 and K562/R cells revealed differential expression profiles: compared with K562-derived exosomes, 29 miRNAs were downregulated while 21 were upregulated in K562/R cell-derived exosomes (Fig. 3 A). MiR-711 is highly expressed in K562 cell-derived exosomes, and its dysregulation has been implicated tumorigenesis and cancer progression. The expression level of miR-711 was validated by qRT-PCR. The result indicated that miR-711 was significantly upregulated in K562 derived exosomes compared with K562/R derived exosomes, which was consistent with the miRNA array. Moreover, compared with K562/R cells, the levels of miR-711 in K562 cells was also significantly upregulated (Fig. 3 B). To further investigate the function of miR-711 in imatinib resistance, we transfected miR-711 inhibitor or mimic into K562 and K562/R cells, and were treated with imatinib at 48 h post-transfection. Transfection of K562/R cells with miR-711 mimic resulted in significant upregulation of miR-711 expression. On the contrary, after being transfected with miR-711 inhibitor in K562, the expression level of this miRNA was significantly downregulated (Fig. 3 C-D). Subsequently, CCK-8 assay showed that the IC50 of K562/R cells was lower after overexpression of miR-711 than the negative control (2.572 ± 0.212 µM vs 1.582 ± 0.105 µM) (Fig. 3 E-F). Meanwhile, the IC50 of K562 cells was increased after the expression of miR-711 was inhibited (0.109 ± 0.019 µM vs 0.225 ± 0.025 µM) (Fig. 3 E-F). Finally, we detected the apoptosis by flow cytometry. Compared with the transfected control group, the apoptosis rate of K562/R cells overexpressing miR-711 was significantly increased (17.660 ± 1.569% vs 27.850 ± 1.784%) (Fig. 3 I-J), the apoptosis rate of K562 cells inhibiting miR-711 was significantly reduced (19.400 ± 2.275% vs 12.190 ± 1.095%) (Fig. 3 K-L). The above experiments show that miR-711 may be able to influence to some extents of CML cells to IM. MiR‑711 regulates chemoresistance through CD44 MicroRNAs are small non-coding RNAs that function by binding to the 3′-untranslated regions (3′UTR) of target mRNAs to induce mRNA degradation or to inhibit translation, thereby exerting its biological functions. Thus, to clarify the target gens of miR-711-mediated drug resistance in CML cells, we performed a bioinformatics analysis combined four databases (TargetScan, miRDIP, miRDB and miRmap) and GEO datasets (GSE120932). The 9 candidate target genes (CD44、COL4A5、DUSP5、MGAT3、ALAS2、HOXC13、UBASH3A、PIK3AP1 and TMEM163) were finally screened out (Fig. 4 A). We previously identified CD44 was direct target gene of miR-711 by luciferase reporter assay. We previously identified that miR-711 can bind to the 3 'UTR region of CD44 by luciferase reporter assay (Jiang et al. 2020 ). Furthermore, western blotting and RT-qPCR analyses showed that CD44 expression was upregulated in IM-resistant cells compared with parental cells (Fig. 4 B-D). Compared with the control group, the mRNA and protein expressions of CD44 were significantly downregulated in K562/R cells by transfected with miR-711 mimic, while the mRNA and protein expressions of CD44 were significantly upregulated in K562 cells by transfected with miR-711 inhibitor (Fig. 4 E-G). These results suggest that miR-711 regulates CD44 expression by targeting the 3’ UTR of CD44 mRNA. K562 cell-derived exosomes carrying miR-711 reverses imatinib resistance in K562/R cells by downregulating CD44 Above, we verified that that miR-711 can regular imatinib resistance of K562/R cells and CD44 was the target gene of miR-711. We next investigated whether exosomal miR-711 was involved in imatinib resistance of K562/R cells by targeting CD44. First, we infected K562/R cells with lentiviral vector to construct the CD44 overexpression cell line and negative control cell line. The cell infection efficiency reached more than 90% under fluorescence microscopy after purinomycin screen (Fig. 5 A). In addition, the efficiency of overexpression was confirmed by RT-qPCR and western blot. Compared with the negative control, the expression levels of mRNA and protein of CD44 in the over-expressed cells were significantly increased (Fig. 5 B-F), indicating that we successfully constructed CD44 overexpressed stable cell line (K562/R + OV CD44) and the negative control stable cell line (K562/R + OV NC). K562 cell-derived exosome (Exo k562 ) was co-cultured with K562/R and K562/R + OV CD44 cells, respectively, and then transfected miR-711 inhibitor and inhibitor NC into K562/R cells. The equivalent amount of PBS was added to K562/R + OV NC cells as the negative control. The experimental grouping was as followed: K562/R + OV NC + PBS, K562/R + Exo k562 +inhibitor NC, K562/R + Exo k562 +miR-711 inhibitor, K562/R + OV CD44, K562/R + OV CD44 + Exo k562 . Compared with negative control (K562/R + OV NC + PBS), the expression of miR-711 was significantly increased in K562/R + Exo k562 +inhibitor NC group, while the CD44 mRNA and protein were significantly decreased. However, miR-711, CD44 mRNA and protein expression were not significantly changed in K562/R + Exo k562 +miR-711 inhibitor group, K562/R + OV CD44 + Exo k562 inhibitor group, CD44 mRNA and protein expression levels were not significantly changed. At the same time, the influence of K562 cell-derived exosome miR-711 on CD44 in K562/R cells can be reversed by overexpressed CD44 (Fig. 5 D-F). Finally, we used the CCK-8 assay and flow cytometry to verify whether K562-secreted exosomal miR-711 induces IM resistance and apoptosis of K562/R cells by targeting CD44. K562/R cells co-culture with K562-derived exosomes (K562/R + Exo k562 +inhibitor NC)exhibited lower levels of IC50 and higher levels of apoptosis (Fig. 5 I) compared with controls (K562/R + OV NC + PBS). However, transfected miR-711 inhibitor (K562/R + Exo k562 +miR-711 inhibitor) or overexpression of CD44 (K562/R + OV CD44 + Exo k562 ) failed to lead to changes in IC50 and apoptosis (Fig. 5 G-J). These results suggest that K562 cell-derived exosomes can transmit miR-711 into K562/R cells and increase IM-induced apoptosis by down-regulating CD44 expression, thereby reducing the resistance of K562/R cells to IM. Discussion The extensive application of targeted chemotherapy agents, such as tyrosine kinase inhibitors (TKIs), has marked a significant advancement in the treatment of chronic myeloid leukemia (CML). However, chemotherapy resistance has limited the clinical utility of imatinib (IM) (Jabbour et al. 2025). The resistance of chronic myeloid leukemia (CML) cells to imatinib mesylate (IM) involves multiple mechanisms. Recent research indicates that exosomes can affect the chemotherapy resistance in tumor cells by a variety of mechanisms (Wu et al. 2023 ). In this study, exosomes were successfully isolated from cell supernatants by differential centrifugation. Exosomes derived from K562 cells were co-cultured with K562R cells, where they were absorbed and partially reversed IM resistance in K562R cells. The primary mechanism involves that miR-711 is directly transferred from sensitive cells to resistant cells through exosomes, which contributes to the partial reversal of imatinib resistance by targeting CD44. Exosomes as a novel intercellular communication tool, play a pivotal role in cancer progression and chemoresistance. These exosome-encapsulated miRNAs can shuttle to recipient cells and modify their phenotypes through gene expression regulation (Bayat et al. 2024; Luo et al. 2025 ). Numerous studies have demonstrated that tumor-derived exosomes deliver miRNAs to recipient cells to mediate drug-resistant phenotypes in various malignancies, including breast cancer (Dong et al. 2020 ; Sun et al. 2024 ; Zhao et al. 2023 ), gastric cancer (Jing et al. 2022 ; Kong et al. 2025 ; Zhu et al. 2023 ), colorectal cancer (Tian et al. 2025 ; Yan et al. 2025 ; Zhang et al. 2022 ), and lung cancer (Shi et al. 2022 ; Wang et al. 2021 ; Wu et al. 2021 ). However, previous investigations primarily focused on drug-resistant cells transferring miRNAs to sensitive cells to enhance chemoresistance, while the potential impact of exosomes from sensitive cells on drug-resistant counterparts remains underexplored. To determine whether K562-derived exosomes could transfer biological information to K562/R cells and modulate their drug resistance, we co-cultured K562/R cells with K562-derived exosomes. Intriguingly, the chemoresistance of K562/R cells to IM was significantly reduced, accompanied by markedly increased IM-induced apoptosis. This observation indicates that K562-derived exosomes can partially restore IM sensitivity in K562/R cells. This phenomenon of exosome reversing tumor chemoresistance has also been reported in breast cancer and colorectal cancer. Specifically, exosomal miR-770 could significantly inhibit the DOX resistance and metastasis of triple negative breast cancer cells, which is mediated via regulation of apoptosis and EMT and modification of tumor associated macrophages (Li et al. 2018 ). Additionally, exosomes-transmitted miR-7 reverses gefitinib resistance by targeting YAP in non-small-cell lung cancer (Chen et al. 2021 ). In our preliminary investigations, microarray analysis of exosomal miRNA profiles from K562 and K562/R cells revealed several miRNAs (hsa-miR-1973, hsa-miR-652-5p, hsa-miR-1275, hsa-miR-210, hsa-miR-30c-1-3p, and hsa-miR-711) exhibiting significant enrichment in K562-derived exosomes (Min et al. 2018 ). Notably, we previously demonstrated that K562-derived exosomes could transfer miR-711 to bone marrow mesenchymal stem cells, subsequently impairing their cellular adhesion capacity (Jiang et al. 2020 ). To further investigate the role of miR-711 in imatinib (IM) resistance, we transfected K562/R cells with miR-711 mimics and K562 cells with miR-711 inhibitors. It was found that miR-711 was significantly increased in K562/R cells and the cell resistance was reduced, whereas miR-711 suppression in K562 cells enhanced drug tolerance. These findings collectively suggest the modulatory effects of miR-711 on IM resistance. The miR-711, encoded within the intronic region of collagen type VII alpha 1 chain (COL7A1), demonstrates broad tissue expression and functional relevance to tumor proliferation or apoptosis. Experimental evidence demonstrated that miR-711 overexpression in gastric cancer cells significantly inhibited malignant proliferation and suppressed epithelial-mesenchymal transition (EMT) by reducing CD44 expression (Xiao et al. 2018 ). Paradoxically, clinical analyses revealed elevated miR-711 expression in breast cancer patients undergoing radical mastectomy predicted poor prognosis and served as an independent prognostic factor. Functional assays confirmed its oncogenic potential in promoting breast cancer cell proliferation while inhibiting apoptosis (Hu et al. 2016 ). miRNAs are a class of short non-coding RNAs, approximately 19–25 nucleotides in length, that are ubiquitously present in eukaryotic organisms and participate in numerous pathophysiological processes. The primary mechanism of miRNA action involves binding to the 3'-untranslated region (3'-UTR) of target mRNAs through complementary base pairing, leading to mRNA degradation and/or translational inhibition, thereby downregulating the expression of target genes (Carthew et al. 2009).Through bioinformatic analysis and dual-luciferase reporter gene assays, we demonstrated that miR-711 specifically binds to the 3'-UTR sequence of CD44. Subsequently, we overexpressed CD44 in K562/R cells and co-cultured them with exosomes derived from K562 cells. Remarkably, no significant changes were observed in either CD44 expression or imatinib (IM) resistance in K562/R cells, indicating that the miR-711 carried by K562-derived exosomes modulates drug resistance in K562/R cells through targeted regulation of CD44. CD44 is a transmembrane glycoprotein widely expressed on the cell membrane, functioning as a critical signaling molecule that participates in the transduction of multiple intracellular signaling pathways, thereby regulating diverse pathophysiological processes (Naor et al. 2002 ). Substantial evidence has demonstrated that CD44 is overexpressed in various malignancies and closely associated with tumorigenesis, progression, drug resistance, and maintenance of cancer stemness (Morath et al. 2016 ). Evidence suggests that AGD1, USP10, and METTL13 form a functional complex that promotes prostate cancer stemness and docetaxel resistance by mediating CD44 m6A modification in castration-resistant prostate cancer (Wang et al. 2025 ). Another study demonstrated that silencing CD44 expression restored cisplatin sensitivity in cisplatin-resistant head and neck squamous cell carcinoma cells (Roy et al. 2020 ). Furthermore, CD44 plays a significant role in hematological drug resistance. In multiple myeloma, CD44 has been identified as both a biomarker for predicting lenalidomide sensitivity and a therapeutic target to overcome lenalidomide resistance (Bjorklund et al. 2014 ). Researchers discovered that CD44 may enhance doxorubicin resistance in chronic myeloid leukemia (CML) by modulating the expression of P-gp, MMP-2, MMP-9, and Bcl-2/Bax (Li et al. 2021 ). Mechanistic investigations of CML resistance to imatinib (IM) revealed that AF1q induces IM resistance through CD44 regulation (Li et al. 2018 ). This study demonstrates that exosomes derived from K562 cells deliver miR-711 to K562/R cells, which increases IM-induced apoptosis by downregulating CD44 expression, thereby reducing IM resistance in the target K562/R cells. In conclusion, our findings demonstrate that exosomes derived from K562 cells deliver miR-711 to K562/R cells, which enhances imatinib (IM)-induced apoptosis through downregulation of CD44 expression, thereby attenuating IM resistance in K562/R cells (Fig. 6 ). This study provides novel insights into chemotherapy resistance in chronic myeloid leukemia (CML) and may facilitate the development of targeted strategies to reverse IM resistance in clinical settings. Statements & Declarations Funding This study was supported by the National Natural Science Foundation of China (No. 82160038), Youth Program of the Natural Science Foundation of Jiangxi Province (No. 20232BAB216038), and Science and Technology Program of Health Commission of Jiangxi Province (No. 202410027). Competing interests The authors have no relevant financial or non-financial interests to disclose. Author contributions Bo Huang conceived and designed the study. Meiyong Li Xuan Hou, and Rong Zhang performed the experiments. Pei Huang and Qingting Xiong conducted data analysis and interpretation. WenQi Ma supervised the project and revised the manuscript. All authors contributed to the drafting and editing of the manuscript and approved the final version for submission. Acknowledgements None. Data availability No datasets were generated or analysed during the current study. References Bayat M, Sadri Nahand J. Exosomal miRNAs: the tumor's trojan horse in selective metastasis. Mol Cancer. 2024; 23:167. https://doi.org/10.1186/s12943-024-02081-0 Bjorklund CC, Baladandayuthapani V, Lin HY et al. Evidence of a role for CD44 and cell adhesion in mediating resistance to lenalidomide in multiple myeloma: therapeutic implications. Leukemia. 2014; 28:373-83. https://doi.org/10.1038/leu.2013.174 Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs. 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1","display":"","copyAsset":false,"role":"figure","size":223794,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of exosomes‌. A. Nanoparticle tracking analysis (NTA) demonstrating the size distribution profile of isolated exosomes. B. Transmission electron microscopy (TEM) image revealing the morphology of exosomes (scale bar = 200 nm). C. Western blotting analysis of exosomal markers (Alix, TSG101, CD81) and negative control endoplasmic reticulum marker Calnexin\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7873222/v1/218b36c44c34645ee3f343d0.png"},{"id":97260425,"identity":"e6f84817-236f-4c73-bf1e-4736d21af06e","added_by":"auto","created_at":"2025-12-02 13:57:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":287608,"visible":true,"origin":"","legend":"\u003cp\u003eExosomes derived from sensitive CML cells reduce chemotherapy resistant trait of drug resistant CML cells.\u003cstrong\u003e A.\u003c/strong\u003e Confocal microscopy showed K562-derived exosomes stained with PKH26 (Red fluorescence) was internalized by imatinib-resistant cells (K562/R). The nuclei of K562/R cells were counterstained with Hoechst 33342 (blue fluorescence). \u003cstrong\u003eB-C.\u003c/strong\u003e CCK-8 assay was used to measure the viability of K562/R cells that were incubated with 10 μg/ml Exo\u003csup\u003eK562\u003c/sup\u003e or Exo\u003csup\u003eK562/R \u003c/sup\u003eand treated by imatinib for 48h in 96-well plates. Experiments were performed three times, and results are expressed as mean±SD unless otherwise stated. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003eD-E.\u003c/strong\u003e FCM was used to analyze the apoptosis of K562/R cells that were incubated with 10 μg/ml Exo\u003csup\u003eK562\u003c/sup\u003e or Exo\u003csup\u003eK562/R \u003c/sup\u003eand treated by 4 μM imatinib for 48 h. Experiments were performed three times, and results are expressed as mean±SD unless otherwise stated. ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7873222/v1/8369ebf0c6abf503e5e8c43a.png"},{"id":97260421,"identity":"779e26a2-d70c-4537-8a98-2d61c8d6f873","added_by":"auto","created_at":"2025-12-02 13:57:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":255220,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of miR-711 on drug resistance in K562/R and K562 cells. \u003cstrong\u003eA.\u003c/strong\u003e The expression of miRNA in exosomes isolated from K562 cell and K562/R cells was shown by analyzing the miRNA array. \u003cstrong\u003eB.\u003c/strong\u003e Real-time qRT-PCR was performed to assess the expression level of miR-711 in K562, K562/R cells and their exosomes, ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001. \u003cstrong\u003eC-D.\u003c/strong\u003e miR-711 was overexpressed (C) or silenced (D) by transfection with miR-711 mimic and inhibitor, respectively compared with control groups, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003eE-H.\u003c/strong\u003eOverexpressed of miR-711 increased chemosensitivity of K562/R cells (E, F). Silence of miR-711 induced resistance of imatinibin K562 cells (G, H), **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. \u003cstrong\u003eI-L.\u003c/strong\u003e Flow cytometry assay revealed that miR-711 induced cell apoptosis (I, J), whereas silence of miR-711 inhibited apoptosis (K, L) compared with control groups, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7873222/v1/43dc7747ecab973222fa3ffc.png"},{"id":97260423,"identity":"654c4294-52ba-4ac8-9ed7-bca7322df70d","added_by":"auto","created_at":"2025-12-02 13:57:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":256681,"visible":true,"origin":"","legend":"\u003cp\u003ePrediction and validation of downstream target genes of miR-711. \u003cstrong\u003eA. \u003c/strong\u003eVenn diagram identifying predicted target genes of miR-711 and the schematic representation of the predicted miR-711 binding site in the 3′-UTR of CD44. \u003cstrong\u003eB-D.\u003c/strong\u003e Determination of CD44 mRNA (RT-qPCR) and protein (WB) expression levels in K562 and K562/R cells. \u003cstrong\u003eE-G.\u003c/strong\u003e CD44 mRNA and protein expression levels were detected by RT-qPCR and western blotting in K562/R cells following transfection with miR-711 mimic (E) and in K562 cells following transfection with miR-711 inhibitor (F, G)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7873222/v1/cf75af401d6ecdb803bed99d.png"},{"id":97367741,"identity":"a345459a-d7d8-47b9-807c-428c9c2b91a7","added_by":"auto","created_at":"2025-12-03 16:20:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2717583,"visible":true,"origin":"","legend":"\u003cp\u003eExosomes derived from K562 cells influence IM resistance in K562/R cells via miR-711 targeting CD44. \u003cstrong\u003eA.\u003c/strong\u003e Fluorescence images of stable CD44-overexpressing cells constructed using lentivirus. \u003cstrong\u003eB.\u003c/strong\u003eRT-qPCR analysis of CD44 mRNA expression in stable CD44-overexpressing cells. \u003cstrong\u003eC. \u003c/strong\u003eExpression levels of miR-711 in K562/R cells co-cultured with exosomes derived from K562 cells, with or without transfection of miR-711 inhibitor. \u003cstrong\u003eD-F.\u003c/strong\u003e CD44 mRNA (D) and protein (E, F) expression levels in K562/R cells co-cultured with exosomes derived from K562 cells, with or without transfection of miR-711 inhibitor, and in CD44-overexpressing K562/R cells co-cultured with exosomes derived from K562 cells. \u003cstrong\u003eG-J.\u003c/strong\u003e Changes in IC50 (G, H) and apoptosis (I, J) in K562/R cells co-cultured with exosomes derived from K562 cells, with or without transfection of miR-711 inhibitor, and in CD44-overexpressing K562/R cells co-cultured with exosomes derived from K562 cells\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7873222/v1/13368a62238456ecdcf5e836.png"},{"id":97367882,"identity":"5112a8d5-8445-460c-a927-3da6e7e9eddc","added_by":"auto","created_at":"2025-12-03 16:20:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":169973,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the potential roles of exosomal miR-711 in chronic myeloid leukemia. The imatinib-sensitive cell (K562) secreted exosomes containing miR-711 can be taken up by imatinib-resistant (K562/R). Exosomal miR-711 promote apoptosis by downregulating CD44, and reverse chemoresistance of the recipient cells\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7873222/v1/e091abed696405caf7119799.png"},{"id":98774598,"identity":"beee60ea-a786-4bc8-b632-c5b2126c2006","added_by":"auto","created_at":"2025-12-22 12:03:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4232091,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7873222/v1/81e506f6-cff8-4c62-baf2-cc6eb4792f33.pdf"},{"id":97260427,"identity":"3ec7ccbf-8c7a-492c-ad11-303c5973c441","added_by":"auto","created_at":"2025-12-02 13:57:42","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2549372,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7873222/v1/e705a05c08ea7a891fe5aaab.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosomes derived from imatinib-sensitive chronic myeloid leukemia cells transmit miR-711 reverses imatinib resistance by inhibiting CD44","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic myeloid leukemia (CML) is a myeloproliferative neoplasm originating from hematopoietic stem cells. The Philadelphia chromosome (Ph) and the BCR/ABL1 fusion gene are the characteristic alterations of CML (Rowley \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). Imatinib (IM), a tyrosine kinase inhibitor (TKI) that target the ATP-binding site of BCR-ABL1 kinase, has become the first-line molecular targeted therapy drug for CML (Oehler et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Although IM has significantly improved the survival of CML patients, secondary resistance to IM remains a major clinical challenge (Hochhaus et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The mechanisms of drug resistance to IM are very complex, and the present mechanisms cannot fully explain all drug resistance phenomena. Therefore, it is essential to reveal the molecular mechanism of IM resistance and to find new markers of resistance for clinical treatment of CML.\u003c/p\u003e\u003cp\u003eThe tumor microenvironment (TME) is comprised of immune cells, endothelial cells, tumor-associated fibroblasts, adipocytes, extracellular matrix and soluble cytokines, which play a critical role in tumorigenesis, tumor metastasis, and chemosensitivity (Elhanani et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Increasing evidence have shown that exosomes are an essential component of the tumor microenvironment. Exosomes are nanoscale extracellular vesicles with a diameter of about 30\u0026ndash;150 nm (average\u0026thinsp;~\u0026thinsp;100 nm) (Mathieu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Exosomes carry functional proteins, DNA, mRNA, metabolic products, and non-coding RNA, serve as intermediaries for communication between different cells to regulate various cellular biological functions (Kalluri et al. 2020). Our previous study found that exosomal miR-365 secreted by imatinib-resistant cells could be transmitted to imatinib-sensitive cells, resulting in acquired resistance to imatinib (Min et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, exosomes as important components of the tumor microenvironment can transmit the resistance to neighboring cells. Whether exosomes derived from IM-sensitive CML cells in the tumor microenvironment could sensitise imatinib-resistant CML cells and thus reverse drug resistance is worthy of further study.\u003c/p\u003e\u003cp\u003eIn this study, we revealed that exosomes derived from IM-sensitive cells influence the drug resistance of IM-resistant cells. Moreover, exosomes derived from IM-sensitive cells could transport miR-711 to imatinib-resistant cells and increases the sensitivity of CML cells to imatinib by targeting CD44. These findings may provide new experimental evidence for the treatment of CML resistance.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eCell culture\u003c/p\u003e\u003cp\u003eImatinib-sensitive (K562) and imatinib-resistant (K562/G01) human chronic myeloid leukemia cells lines were obtained from Institute of Hematology, Chinese Academy of Medical Science (Tianjin, China). All cells were cultured in RPMI 1640 media (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, South America) and 100 U/mL penicillin-streptomycin (Solarbio, China) at 37\u0026deg;C in a humidifed atmosphere with 5% carbon dioxide (CO2). K562/G01 cells were maintained in medium with 1.0 \u0026micro;M Imatinib mesylate (Sigma-Aldrich, Mo, USA) and were cultured in IM-free medium for at least 1 weeks before all experiments.\u003c/p\u003e\u003cp\u003eExosome isolation and identification\u003c/p\u003e\u003cp\u003eWe isolated exosomes from the cell culture medium through ultracentrifugation as previously described with slight modifications (Jiang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The culture medium was centrifuged at 400 \u0026times; g for 10 min and 2000\u0026times; g for 20 min at 4\u0026deg;C to remove cells and cell debris. Subsequently, the supernatant was collected and centrifuged at 10,000 \u0026times; g for 30 min at 4\u0026deg;C and then was filtered through a 0.22 \u0026micro;m syringe membrane filter (Millipore, USA). The supernatant was ultracentrifuged at 110,000 \u0026times; g for 70 min at 4\u0026deg;C (OptimaL-80XP, SW41 rotor, Beckman Coulter, USA) to enrich exosomes. The pellet was resuspended with cold PBS and centrifuged again at 110,000 \u0026times; g for 70 min at 4\u0026deg;C. The final exosomes were resuspended in 50 \u0026micro;l PBS for subsequent analysis.\u003c/p\u003e\u003cp\u003eExosomes were identified as previously described. Briefly, the morphology of exosome was visualized by transmission electron microscope, the size distribution and concentration, the size distribution and concentration were measured by NTA, and exosomal protein markers were identified by Western blotting as previously described (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eExosome labeling and Co-culture assay\u003c/p\u003e\u003cp\u003eExosomes secreted from K562 cells were labeled with red fluorescent dye PKH26 (Sigma-Aldrich, USA) according to the manufacture\u0026rsquo;s instruction. The labeled exosomes were co-incubated with K562/R cells for 6 h, 12h, 24h, and 48h. Exosomes without PKH26 staining were used as negative control. The cells were stained with Hoechst 33342 and visualized under a TCS-SP5 confocal microscope (Leica, Germany). For exosome treatment, K562/R cells were co-cultured with 20 \u0026micro;g/mL exosomes derived from K562 cells for 48 h.\u003c/p\u003e\u003cp\u003eCell viability assay\u003c/p\u003e\u003cp\u003eThe sensitivity of cells to drugs was measured by cell counting kit-8 (CCK8) assays. A total of 8\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells were seeded per well into 96-well plates. Various concentrations of imatinib were added into culture media and incubated for 48 h. 10 \u0026micro;l CCK-8 reagent (ApexBio, USA) was added to each well and incubated for 2 h at 37\u0026deg;C. The optical density was read at 450 nm using a microplate reader (Thermo Fisher Scientific Inc. MA, USA). The inhibitory concentration to produce 50% cell death (IC50) of imatinib was calculated from the survival curves with GraphPad Prism\u0026reg; 8.0 software.\u003c/p\u003e\u003cp\u003eApoptosis assay\u003c/p\u003e\u003cp\u003eCells were collected, washed twice with cold phosphate buffer saline (PBS), and then resuspended in 1\u0026times; binding buffer. The Annexin V-FITC/PI or Annexin V-PE/PI Apoptosis Detection Kit (BD Biosciences, CA, USA) was used to stain the cells according to manufacturer's instructions. Cell apoptosis rate was then measured via fow cytometry (BD FACS Calibur) and data analysis using FACSDiva software.\u003c/p\u003e\u003cp\u003eRNA extraction and qRT-PCR analysis\u003c/p\u003e\u003cp\u003eMiRNAs from the cells and exosomes were extracted using miRcute miRNA Isolation Kit (Tiangen, China) following the manufacturer\u0026rsquo;s instructions. Trizol reagent (Invitrogen) was used for total cellular RNA extraction. RNAs (1 \u0026micro;g) were reverse-transcribed to cDNA with Fast cDNA Reverse Transcriptase (Tiangen, China). qRT-PCR analysis for miR-711 and CD44 mRNA expression was performed using SYBR Green PCR Kit (Tiangen, China) with 7500 Real-Time PCR system (Applied Biosystems, USA). The results were obtained using the relative standard curve method (2-△△Ct) and the relative expression level of miR-711 and CD44 mRNA was respectively normalized with U6 and β-actin. Specific primers for hsa-miR-711 and U6 were supplied by RIBOBIO (Guangzhou, China).\u003c/p\u003e\u003cp\u003eWestern blot analysis\u003c/p\u003e\u003cp\u003eProtein of cells or exosomes were extracted using the RIPA lysis buffer (Beyotime, China). Western blot analysis was performed according to our previous reports (Li et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The primary antibodies against Alix (12422-1-AP, 1:4000), CD81(66866-1-Ig, 1:3000), TSG101(28283-1-AP,1:6000), Calnexin (66903-1-Ig,1:10000), CD44(60224-1-Ig,1:3000), and β-actin (66009-1-Ig,1:10000) were obtained from Proteintech (Wuhan, China). The bound antibodies were visualized with Pro-light HRP chemiluminescence kit (Tiangen, China) using a ChemiDoc XRS System (Bio-Rad).\u003c/p\u003e\u003cp\u003eCell transfection\u003c/p\u003e\u003cp\u003eK562 cells or K562R cells were seeded into six-well plates at a density of 10\u003csup\u003e6\u003c/sup\u003e cells per well. The miR-711 mimic, inhibitor, and corresponding negative controls (mimic NC and inhibitor NC) were purchased from Ribobio company (Guangzhou, China). MiR-711 mimic (50 nM), miR-711 inhibitor (100 nM) and negative control were incubated with 12 \u0026micro;l of riboFECTTM CP reagent (RioBio) for 15 min at room temperature and transfected into cells.\u003c/p\u003e\u003cp\u003eK562 cells or K562R cells (5 \u0026times; 104 per well) were seeded into six-well plates and infected with 10 \u0026micro;l of lentiviral vectors containing CD44 overexpression plasmid, CD44 shRNA, and negative control plasmid (Jikai Gene, Shanghai, China) for 2 days. The stable infected cells were screened with 3.0 \u0026micro;g/ml puromycin (Sigma-Aldrich, MO, USA) for 2 weeks and confirmed by fluorescence microscopy, RT-qPCR and WB analyses.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data were shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (χ\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). The comparison between the two groups were determined using Student's t-test, and statistical analyses were performed by SPSS22.0 software (version 22.0, NY, USA). The values of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to be statistically significant.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExosomes characterization\u003c/h3\u003e\n\u003cp\u003eIn order to investigate the effect of exosomes derived from K562 on the IM resistance of K562/R, we first extracted exosomes from cell supernatants by ultracentrifugation, and verified using transmission electron microscopy (TEM), Nanosight particle tracking analysis (NTA), and Western blot analysis (WB). The results showed that the size of the extracted exosomes was mainly distributed around 150 nm in diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In addition, transmission electron microscopy showed that the vesicles exhibited a teacup vesicle-like structures with a particle size of about 150 nm, which was consistent with the typical exosome structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Finally, we proved that the extracted substances carried the exosomal characteristic marker proteins including Alix, TSG101 and CD81, whereas the marker protein of the endoplasmic reticulum Calnexin was enriched in the whole cell lysates through WB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In summary, the above experimental results indicate that exosomes have been successfully enriched and purified from the cell supernatants by ultracentrifugation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eExosomes derived from imatinib-sensitive CML cells are internalized by IM-resistant CML cells and reduce their IM resistance.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe successful uptake of exosomes is the important prerequisite by which exosomes modulate their target cells, so that exosomes can release the functional substances to target cells and then influence cellular functions. To demonstrate that K562 cell-derived exosomes were taken up by K562R cells, the exosomes were stained with PKH26 (a red florescent dye), and then co-cultured with K562/R cells. Finally, the uptake of exosomes by K562/R cells was observed by laser confocal microscopy, after PKH26-labeled exosomes was co-cultured with K562/R cells for 6h, 12h and 24h. To exclude false-positive results caused by free dyes, and PBS was also used as the negative control. Compared with the control group, different degrees of red fluorescence signals appeared in the K562/R cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) after coincubation for 6 h, which were mainly distributed within the cytoplasm. With the increase of incubation time, the red fluorescence signal intensity was progressively strengthened. These studies indicate that K562 cell-derived exosomes can be taken up by K562/R cells.\u003c/p\u003e\u003cp\u003eAlthough exosome uptake experiments found that K562/R cells were indeed able to internalize exosomes derived from K562 cells, whether K562 cell-derived exosomes would have effects on imatinib resistance of K562/R cell remains unclear. K562/R cells were respectively co-cultured with K562 cell-derived exosomes (10 \u0026micro;g/ml), K562R cell-derived exosomes (10 \u0026micro;g/ml) and equal volume PBS for 48h, and then the IM resistance and apoptotic rates of K562/R cells was respectively detected by CCK-8 assay and flow cytometry. The CCK-8 assay showed that K562 cell-derived exosomes decreased imatinib resistance in K562/R cells, compared with PBS and K562/R cell derived exosomes. No statistically significant difference was observed in IC50 values between Exo\u003csup\u003eK562/R\u003c/sup\u003e-treated and untreated groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Additionally, K562/R cells exposed to Exo\u003csup\u003eK562\u003c/sup\u003e exhibited a markedly lower apoptosis rate relative to those treated with Exo\u003csup\u003eK562R\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E). These collective findings demonstrate that exosomes originating from K562 cells can enhance IM drug sensitivity in K562/R cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eEffect of miR-711 on Cellular Drug Resistance\u003c/h3\u003e\n\u003cp\u003eExosomes derived from K562 can reduce the resistance of K562/R cells to IM. However, the mechanisms K562 of cell-derived exosomes influence the resistance of K562/R cells remains unclear. Exosomes modulate drug resistance of target cells through the transfer of protein and nucleic acid molecules, particularly non-coding RNAs. The miRNA was the most abundant in exosomes and have been demonstrated to be associated with tumor drug resistance in multiple studies. In our previous study, miRNA microarray analysis of exosomes derived from K562 and K562/R cells revealed differential expression profiles: compared with K562-derived exosomes, 29 miRNAs were downregulated while 21 were upregulated in K562/R cell-derived exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). MiR-711 is highly expressed in K562 cell-derived exosomes, and its dysregulation has been implicated tumorigenesis and cancer progression. The expression level of miR-711 was validated by qRT-PCR. The result indicated that miR-711 was significantly upregulated in K562 derived exosomes compared with K562/R derived exosomes, which was consistent with the miRNA array. Moreover, compared with K562/R cells, the levels of miR-711 in K562 cells was also significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eTo further investigate the function of miR-711 in imatinib resistance, we transfected miR-711 inhibitor or mimic into K562 and K562/R cells, and were treated with imatinib at 48 h post-transfection. Transfection of K562/R cells with miR-711 mimic resulted in significant upregulation of miR-711 expression. On the contrary, after being transfected with miR-711 inhibitor in K562, the expression level of this miRNA was significantly downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). Subsequently, CCK-8 assay showed that the IC50 of K562/R cells was lower after overexpression of miR-711 than the negative control (2.572\u0026thinsp;\u0026plusmn;\u0026thinsp;0.212 \u0026micro;M vs 1.582\u0026thinsp;\u0026plusmn;\u0026thinsp;0.105 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). Meanwhile, the IC50 of K562 cells was increased after the expression of miR-711 was inhibited (0.109\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019 \u0026micro;M vs 0.225\u0026thinsp;\u0026plusmn;\u0026thinsp;0.025 \u0026micro;M) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). Finally, we detected the apoptosis by flow cytometry. Compared with the transfected control group, the apoptosis rate of K562/R cells overexpressing miR-711 was significantly increased (17.660\u0026thinsp;\u0026plusmn;\u0026thinsp;1.569% vs 27.850\u0026thinsp;\u0026plusmn;\u0026thinsp;1.784%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI-J), the apoptosis rate of K562 cells inhibiting miR-711 was significantly reduced (19.400\u0026thinsp;\u0026plusmn;\u0026thinsp;2.275% vs 12.190\u0026thinsp;\u0026plusmn;\u0026thinsp;1.095%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK-L). The above experiments show that miR-711 may be able to influence to some extents of CML cells to IM.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMiR‑711 regulates chemoresistance through CD44\u003c/h3\u003e\n\u003cp\u003eMicroRNAs are small non-coding RNAs that function by binding to the 3\u0026prime;-untranslated regions (3\u0026prime;UTR) of target mRNAs to induce mRNA degradation or to inhibit translation, thereby exerting its biological functions. Thus, to clarify the target gens of miR-711-mediated drug resistance in CML cells, we performed a bioinformatics analysis combined four databases (TargetScan, miRDIP, miRDB and miRmap) and GEO datasets (GSE120932). The 9 candidate target genes (CD44、COL4A5、DUSP5、MGAT3、ALAS2、HOXC13、UBASH3A、PIK3AP1 and TMEM163) were finally screened out (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We previously identified CD44 was direct target gene of miR-711 by luciferase reporter assay. We previously identified that miR-711 can bind to the 3 'UTR region of CD44 by luciferase reporter assay (Jiang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, western blotting and RT-qPCR analyses showed that CD44 expression was upregulated in IM-resistant cells compared with parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). Compared with the control group, the mRNA and protein expressions of CD44 were significantly downregulated in K562/R cells by transfected with miR-711 mimic, while the mRNA and protein expressions of CD44 were significantly upregulated in K562 cells by transfected with miR-711 inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G). These results suggest that miR-711 regulates CD44 expression by targeting the 3\u0026rsquo; UTR of CD44 mRNA.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eK562 cell-derived exosomes carrying miR-711 reverses imatinib resistance in K562/R cells by downregulating CD44\u003c/h3\u003e\n\u003cp\u003eAbove, we verified that that miR-711 can regular imatinib resistance of K562/R cells and CD44 was the target gene of miR-711. We next investigated whether exosomal miR-711 was involved in imatinib resistance of K562/R cells by targeting CD44. First, we infected K562/R cells with lentiviral vector to construct the CD44 overexpression cell line and negative control cell line. The cell infection efficiency reached more than 90% under fluorescence microscopy after purinomycin screen (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In addition, the efficiency of overexpression was confirmed by RT-qPCR and western blot. Compared with the negative control, the expression levels of mRNA and protein of CD44 in the over-expressed cells were significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-F), indicating that we successfully constructed CD44 overexpressed stable cell line (K562/R\u0026thinsp;+\u0026thinsp;OV CD44) and the negative control stable cell line (K562/R\u0026thinsp;+\u0026thinsp;OV NC).\u003c/p\u003e\u003cp\u003eK562 cell-derived exosome (Exo\u003csup\u003ek562\u003c/sup\u003e) was co-cultured with K562/R and K562/R\u0026thinsp;+\u0026thinsp;OV CD44 cells, respectively, and then transfected miR-711 inhibitor and inhibitor NC into K562/R cells. The equivalent amount of PBS was added to K562/R\u0026thinsp;+\u0026thinsp;OV NC cells as the negative control. The experimental grouping was as followed: K562/R\u0026thinsp;+\u0026thinsp;OV NC\u0026thinsp;+\u0026thinsp;PBS, K562/R\u0026thinsp;+\u0026thinsp;Exo\u003csup\u003ek562\u003c/sup\u003e+inhibitor NC, K562/R\u0026thinsp;+\u0026thinsp;Exo\u003csup\u003ek562\u003c/sup\u003e+miR-711 inhibitor, K562/R\u0026thinsp;+\u0026thinsp;OV CD44, K562/R\u0026thinsp;+\u0026thinsp;OV CD44\u0026thinsp;+\u0026thinsp;Exo\u003csup\u003ek562\u003c/sup\u003e. Compared with negative control (K562/R\u0026thinsp;+\u0026thinsp;OV NC\u0026thinsp;+\u0026thinsp;PBS), the expression of miR-711 was significantly increased in K562/R\u0026thinsp;+\u0026thinsp;Exo\u003csup\u003ek562\u003c/sup\u003e+inhibitor NC group, while the CD44 mRNA and protein were significantly decreased. However, miR-711, CD44 mRNA and protein expression were not significantly changed in K562/R\u0026thinsp;+\u0026thinsp;Exo\u003csup\u003ek562\u003c/sup\u003e+miR-711 inhibitor group, K562/R\u0026thinsp;+\u0026thinsp;OV CD44\u0026thinsp;+\u0026thinsp;Exo\u003csup\u003ek562\u003c/sup\u003e inhibitor group, CD44 mRNA and protein expression levels were not significantly changed. At the same time, the influence of K562 cell-derived exosome miR-711 on CD44 in K562/R cells can be reversed by overexpressed CD44 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F).\u003c/p\u003e\u003cp\u003eFinally, we used the CCK-8 assay and flow cytometry to verify whether K562-secreted exosomal miR-711 induces IM resistance and apoptosis of K562/R cells by targeting CD44. K562/R cells co-culture with K562-derived exosomes (K562/R\u0026thinsp;+\u0026thinsp;Exo\u003csup\u003ek562\u003c/sup\u003e+inhibitor NC)exhibited lower levels of IC50 and higher levels of apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI) compared with controls (K562/R\u0026thinsp;+\u0026thinsp;OV NC\u0026thinsp;+\u0026thinsp;PBS). However, transfected miR-711 inhibitor (K562/R\u0026thinsp;+\u0026thinsp;Exo\u003csup\u003ek562\u003c/sup\u003e+miR-711 inhibitor) or overexpression of CD44 (K562/R\u0026thinsp;+\u0026thinsp;OV CD44\u0026thinsp;+\u0026thinsp;Exo\u003csup\u003ek562\u003c/sup\u003e) failed to lead to changes in IC50 and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-J). These results suggest that K562 cell-derived exosomes can transmit miR-711 into K562/R cells and increase IM-induced apoptosis by down-regulating CD44 expression, thereby reducing the resistance of K562/R cells to IM.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe extensive application of targeted chemotherapy agents, such as tyrosine kinase inhibitors (TKIs), has marked a significant advancement in the treatment of chronic myeloid leukemia (CML). However, chemotherapy resistance has limited the clinical utility of imatinib (IM) (Jabbour et al. 2025). The resistance of chronic myeloid leukemia (CML) cells to imatinib mesylate (IM) involves multiple mechanisms. Recent research indicates that exosomes can affect the chemotherapy resistance in tumor cells by a variety of mechanisms (Wu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, exosomes were successfully isolated from cell supernatants by differential centrifugation. Exosomes derived from K562 cells were co-cultured with K562R cells, where they were absorbed and partially reversed IM resistance in K562R cells. The primary mechanism involves that miR-711 is directly transferred from sensitive cells to resistant cells through exosomes, which contributes to the partial reversal of imatinib resistance by targeting CD44.\u003c/p\u003e\u003cp\u003eExosomes as a novel intercellular communication tool, play a pivotal role in cancer progression and chemoresistance. These exosome-encapsulated miRNAs can shuttle to recipient cells and modify their phenotypes through gene expression regulation (Bayat et al. 2024; Luo et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Numerous studies have demonstrated that tumor-derived exosomes deliver miRNAs to recipient cells to mediate drug-resistant phenotypes in various malignancies, including breast cancer (Dong et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), gastric cancer (Jing et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kong et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), colorectal cancer (Tian et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Yan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and lung cancer (Shi et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, previous investigations primarily focused on drug-resistant cells transferring miRNAs to sensitive cells to enhance chemoresistance, while the potential impact of exosomes from sensitive cells on drug-resistant counterparts remains underexplored. To determine whether K562-derived exosomes could transfer biological information to K562/R cells and modulate their drug resistance, we co-cultured K562/R cells with K562-derived exosomes. Intriguingly, the chemoresistance of K562/R cells to IM was significantly reduced, accompanied by markedly increased IM-induced apoptosis. This observation indicates that K562-derived exosomes can partially restore IM sensitivity in K562/R cells. This phenomenon of exosome reversing tumor chemoresistance has also been reported in breast cancer and colorectal cancer. Specifically, exosomal miR-770 could significantly inhibit the DOX resistance and metastasis of triple negative breast cancer cells, which is mediated via regulation of apoptosis and EMT and modification of tumor associated macrophages (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, exosomes-transmitted miR-7 reverses gefitinib resistance by targeting YAP in non-small-cell lung cancer (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn our preliminary investigations, microarray analysis of exosomal miRNA profiles from K562 and K562/R cells revealed several miRNAs (hsa-miR-1973, hsa-miR-652-5p, hsa-miR-1275, hsa-miR-210, hsa-miR-30c-1-3p, and hsa-miR-711) exhibiting significant enrichment in K562-derived exosomes (Min et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Notably, we previously demonstrated that K562-derived exosomes could transfer miR-711 to bone marrow mesenchymal stem cells, subsequently impairing their cellular adhesion capacity (Jiang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To further investigate the role of miR-711 in imatinib (IM) resistance, we transfected K562/R cells with miR-711 mimics and K562 cells with miR-711 inhibitors. It was found that miR-711 was significantly increased in K562/R cells and the cell resistance was reduced, whereas miR-711 suppression in K562 cells enhanced drug tolerance. These findings collectively suggest the modulatory effects of miR-711 on IM resistance. The miR-711, encoded within the intronic region of collagen type VII alpha 1 chain (COL7A1), demonstrates broad tissue expression and functional relevance to tumor proliferation or apoptosis. Experimental evidence demonstrated that miR-711 overexpression in gastric cancer cells significantly inhibited malignant proliferation and suppressed epithelial-mesenchymal transition (EMT) by reducing CD44 expression (Xiao et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Paradoxically, clinical analyses revealed elevated miR-711 expression in breast cancer patients undergoing radical mastectomy predicted poor prognosis and served as an independent prognostic factor. Functional assays confirmed its oncogenic potential in promoting breast cancer cell proliferation while inhibiting apoptosis (Hu et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003emiRNAs are a class of short non-coding RNAs, approximately 19\u0026ndash;25 nucleotides in length, that are ubiquitously present in eukaryotic organisms and participate in numerous pathophysiological processes. The primary mechanism of miRNA action involves binding to the 3'-untranslated region (3'-UTR) of target mRNAs through complementary base pairing, leading to mRNA degradation and/or translational inhibition, thereby downregulating the expression of target genes (Carthew et al. 2009).Through bioinformatic analysis and dual-luciferase reporter gene assays, we demonstrated that miR-711 specifically binds to the 3'-UTR sequence of CD44. Subsequently, we overexpressed CD44 in K562/R cells and co-cultured them with exosomes derived from K562 cells. Remarkably, no significant changes were observed in either CD44 expression or imatinib (IM) resistance in K562/R cells, indicating that the miR-711 carried by K562-derived exosomes modulates drug resistance in K562/R cells through targeted regulation of CD44.\u003c/p\u003e\u003cp\u003eCD44 is a transmembrane glycoprotein widely expressed on the cell membrane, functioning as a critical signaling molecule that participates in the transduction of multiple intracellular signaling pathways, thereby regulating diverse pathophysiological processes (Naor et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Substantial evidence has demonstrated that CD44 is overexpressed in various malignancies and closely associated with tumorigenesis, progression, drug resistance, and maintenance of cancer stemness (Morath et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Evidence suggests that AGD1, USP10, and METTL13 form a functional complex that promotes prostate cancer stemness and docetaxel resistance by mediating CD44 m6A modification in castration-resistant prostate cancer (Wang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Another study demonstrated that silencing CD44 expression restored cisplatin sensitivity in cisplatin-resistant head and neck squamous cell carcinoma cells (Roy et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, CD44 plays a significant role in hematological drug resistance. In multiple myeloma, CD44 has been identified as both a biomarker for predicting lenalidomide sensitivity and a therapeutic target to overcome lenalidomide resistance (Bjorklund et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Researchers discovered that CD44 may enhance doxorubicin resistance in chronic myeloid leukemia (CML) by modulating the expression of P-gp, MMP-2, MMP-9, and Bcl-2/Bax (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Mechanistic investigations of CML resistance to imatinib (IM) revealed that AF1q induces IM resistance through CD44 regulation (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This study demonstrates that exosomes derived from K562 cells deliver miR-711 to K562/R cells, which increases IM-induced apoptosis by downregulating CD44 expression, thereby reducing IM resistance in the target K562/R cells.\u003c/p\u003e\u003cp\u003eIn conclusion, our findings demonstrate that exosomes derived from K562 cells deliver miR-711 to K562/R cells, which enhances imatinib (IM)-induced apoptosis through downregulation of CD44 expression, thereby attenuating IM resistance in K562/R cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This study provides novel insights into chemotherapy resistance in chronic myeloid leukemia (CML) and may facilitate the development of targeted strategies to reverse IM resistance in clinical settings.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Statements \u0026 Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This study was supported by the National Natural Science Foundation of China (No. 82160038), Youth Program of the Natural Science Foundation of Jiangxi Province (No. 20232BAB216038), and Science and Technology Program of Health Commission of Jiangxi Province (No. 202410027).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003eBo Huang conceived and designed the study. Meiyong Li Xuan Hou, and Rong Zhang performed the experiments. Pei Huang and Qingting Xiong conducted data analysis and interpretation. WenQi Ma supervised the project and revised the manuscript. All authors contributed to the drafting and editing of the manuscript and approved the final version for submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e None.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e No datasets were generated or analysed during the current study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBayat M, Sadri Nahand J. Exosomal miRNAs: the tumor\u0026apos;s trojan horse in selective metastasis. Mol Cancer. 2024; 23:167. https://doi.org/10.1186/s12943-024-02081-0\u003c/li\u003e\n \u003cli\u003eBjorklund CC, Baladandayuthapani V, Lin HY et al. Evidence of a role for CD44 and cell adhesion in mediating resistance to lenalidomide in multiple myeloma: therapeutic implications. 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Cell Biol Toxicol. 2023; 39:1923-37. https://doi.org/10.1007/s10565-021-09675-0\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Imatinib, Drug resistance, Chronic myeloid leukemia, Exosomes, MiR-711","lastPublishedDoi":"10.21203/rs.3.rs-7873222/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7873222/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImatinib (IM) resistance is an urgent problem in the clinical treatment of chronic myeloid leukemia (CML). Accumulated evidences have identified that exosomes are recognized as an important component of tumor microenvironment and are closely related to drug resistance of tumor. However, the specific contribution of chronic myeloid leukemia-derived exosomes is poorly understood. Currently we found that exosomes derived from imatinibsensitive CML cells co-culture with drug-resistant CML cells can improve the sensitivity of imatinib resistant CML cells. We further demonstrated that miR-711 significantly decreased in exosomes derived from imatinib drug-resistant CML cells compared with exosomes derived from imatinib sensitive CML cells using microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses. Moreover, miR-711 was also downregulated in imatinib resistant cells compared with parental cells. Overexpression of miR-711 reversed chemoresistance, whereas silence of miR-711 induced imatinib resistance. In addition, enhanced miR-711 could be packaged into exosomes, incorporated into receipt cells, reversed chemoresistance by targeting CD44. To conclude, it is speculated that the exosomes from imatinib sensitive CML cells mediate the sensitivity of imatinib resistant CML by transporting miR-711 and targeting CD44. Exosomal miR-711 may be a promising therapeutic strategy and prognostic indicator for CML patients.\u003c/p\u003e","manuscriptTitle":"Exosomes derived from imatinib-sensitive chronic myeloid leukemia cells transmit miR-711 reverses imatinib resistance by inhibiting CD44","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 13:57:37","doi":"10.21203/rs.3.rs-7873222/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0dd4b772-1235-4f96-bc24-a3e6b7b286a3","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-16T23:23:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 13:57:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7873222","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7873222","identity":"rs-7873222","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}