Exosome-mediated transfer of MIF confers temozolomide resistance by regulating TIMP3/PI3K/AKT axis in gliomas

Temozolomide (TMZ) resistance is an important cause of clinical treatment failure and poor prognosis in gliomas. Increasing evidence indicates that cancer-derived exosomes contribute to chemoresistance; however, the specific contribution of glioma-derived exosomes remains unclear. The aim of this study was to explore the role and underlying mechanisms of exosomal macrophage migration inhibitory factor (MIF) on TMZ resistance in gliomas. We first demonstrated that MIF was upregulated in the exosomes of TMZ-resistant cells, engendering the transfer of TMZ resistance to sensitive cells. Our results indicated that exosomal MIF conferred TMZ resistance to sensitive cells through the enhancement of cell proliferation and the repression of cell apoptosis upon TMZ exposure. MIF knockdown enhanced TMZ sensitivity in resistant glioma cells by upregulating Metalloproteinase Inhibitor 3 (TIMP3) and subsequently suppressing the PI3K/AKT signaling pathway. Additionally, exosomal MIF promoted tumor growth and TMZ resistance of glioma cells in vivo, while IOS-1 (MIF inhibitor) promotes glioma TMZ sensitive in vivo. Taken together, our study demonstrated that exosome-mediated transfer of MIF enhanced TMZ resistance in glioma through downregulating TIMP3 and further activating the PI3K/AKT signaling pathway, highlighting a prognostic biomarker and promising therapeutic target for TMZ treatment in gliomas. Temozolomide (TMZ) resistance is an important cause of clinical treatment failure and poor prognosis in gliomas. Increasing evidence indicates that cancer-derived exosomes contribute to chemoresistance; however, the specific contribution of glioma-derived exosomes remains unclear. The aim of this study was to explore the role and underlying mechanisms of exosomal macrophage migration inhibitory factor (MIF) on TMZ resistance in gliomas. We first demonstrated that MIF was upregulated in the exosomes of TMZ-resistant cells, engendering the transfer of TMZ resistance to sensitive cells. Our results indicated that exosomal MIF conferred TMZ resistance to sensitive cells through the enhancement of cell proliferation and the repression of cell apoptosis upon TMZ exposure. MIF knockdown enhanced TMZ sensitivity in resistant glioma cells by upregulating Metalloproteinase Inhibitor 3 (TIMP3) and subsequently suppressing the PI3K/AKT signaling pathway. Additionally, exosomal MIF promoted tumor growth and TMZ resistance of glioma cells in vivo, while IOS-1 (MIF inhibitor) promotes glioma TMZ sensitive in vivo. Taken together, our study demonstrated that exosome-mediated transfer of MIF enhanced TMZ resistance in glioma through downregulating TIMP3 and further activating the PI3K/AKT signaling pathway, highlighting a prognostic biomarker and promising therapeutic target for TMZ treatment in gliomas. Graphical AbstractView Large Image Figure ViewerDownload Hi-res image Download (PPT) Glioma is the most common intracerebral tumor with high morbidity and mortality.1Ostrom Q.T. Cioffi G. Gittleman H. Patil N. Waite K. Kruchko C. Barnholtz-Sloan J.S. CBTRUS Statistical Report: Primary Brain and Other Central Nervous System Tumors Diagnosed in the United States in 2012-2016.Neuro-oncol. 2019; 21: v1-v100Crossref PubMed Scopus (713) Google Scholar The current standard glioma treatments include: surgical resection, radiotherapy, and chemotherapy.2Tan A.C. Ashley D.M. López G.Y. Malinzak M. Friedman H.S. Khasraw M. Management of glioblastoma: State of the art and future directions.CA Cancer J. Clin. 2020; 70: 299-312Crossref PubMed Scopus (178) Google Scholar Despite these approaches, most of the high-grade glioma patients suffered tumor recurrence, and the majority of them showed temozolomide (TMZ) resistance.3Munoz J.L. Rodriguez-Cruz V. Walker N.D. Greco S.J. Rameshwar P. Temozolomide resistance and tumor recurrence: Halting the Hedgehog.Cancer Cell Microenviron. 2015; 2: e747PubMed Google Scholar Although TMZ is still the standard first-line chemotherapy, drug resistance significantly limits the overall prognosis of glioma patients.4Lefranc F. Sadeghi N. Camby I. Metens T. Dewitte O. Kiss R. Present and potential future issues in glioblastoma treatment.Expert Rev. Anticancer Ther. 2006; 6: 719-732Crossref PubMed Scopus (114) Google Scholar,5Saito T. Sugiyama K. Takeshima Y. Amatya V.J. Yamasaki F. Takayasu T. Nosaka R. Muragaki Y. Kawamata T. Kurisu K. Prognostic implications of the subcellular localization of survivin in glioblastomas treated with radiotherapy plus concomitant and adjuvant temozolomide.J. Neurosurg. 2018; 128: 679-684Crossref PubMed Scopus (16) Google Scholar Therefore, it is crucial to research the molecular mechanisms of TMZ resistance and further explore feasible therapy targets. Exosomes, which can be secreted by a variety of cells, are the 40–150 nm size extracellular vesicle (EVS) with lipid bilayer membranes.6Couzin J. Cell biology: The ins and outs of exosomes.Science. 2005; 308: 1862-1863Crossref PubMed Scopus (95) Google Scholar, 7Jiang L. Gu Y. Du Y. Liu J. Exosomes: Diagnostic Biomarkers and Therapeutic Delivery Vehicles for Cancer.Mol. Pharm. 2019; 16: 3333-3349Crossref PubMed Scopus (48) Google Scholar, 8van Niel G. D’Angelo G. Raposo G. Shedding light on the cell biology of extracellular vesicles.Nat. Rev. Mol. Cell Biol. 2018; 19: 213-228Crossref PubMed Scopus (2211) Google Scholar Tumor-derived exosomes are rich in bioactive molecules, including proteins, nucleic acids, lipids, and various molecules.9Nawaz M. Shah N. Zanetti B.R. Maugeri M. Silvestre R.N. Fatima F. Neder L. Valadi H. Extracellular Vesicles and Matrix Remodeling Enzymes: The Emerging Roles in Extracellular Matrix Remodeling, Progression of Diseases and Tissue Repair.Cells. 2018; 7: 167Crossref Scopus (75) Google Scholar Accumulating evidence has shown that exosomes released by highly malignant cancer cells can be transferred to low grade cancer cells and regulate their growth, metastasis, and chemoresistance.10Cheng J. Meng J. Zhu L. Peng Y. Exosomal noncoding RNAs in Glioma: biological functions and potential clinical applications.Mol. Cancer. 2020; 19: 66Crossref PubMed Scopus (85) Google Scholar, 11Shao H. Chung J. Lee K. Balaj L. Min C. Carter B.S. Hochberg F.H. Breakefield X.O. Lee H. Weissleder R. Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma.Nat. Commun. 2015; 6: 6999Crossref PubMed Scopus (346) Google Scholar, 12Wang B. Mao J.H. Wang B.Y. Wang L.X. Wen H.Y. Xu L.J. Fu J.X. Yang H. Exosomal miR-1910-3p promotes proliferation, metastasis, and autophagy of breast cancer cells by targeting MTMR3 and activating the NF-κB signaling pathway.Cancer Lett. 2020; 489: 87-99Crossref PubMed Scopus (30) Google Scholar, 13Ge X. Liu W. Zhao W. Feng S. Duan A. Ji C. Shen K. Liu W. Zhou J. Jiang D. et al.Exosomal Transfer of LCP1 Promotes Osteosarcoma Cell Tumorigenesis and Metastasis by Activating the JAK2/STAT3 Signaling Pathway.Mol. Ther. Nucleic Acids. 2020; 21: 900-915Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 14Guo Z. Wang X. Yang Y. Chen W. Zhang K. Teng B. Huang C. Zhao Q. Qiu Z. Hypoxic Tumor-Derived Exosomal Long Noncoding RNA UCA1 Promotes Angiogenesis via miR-96-5p/AMOTL2 in Pancreatic Cancer.Mol. Ther. Nucleic Acids. 2020; 22: 179-195Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 15Zhang Y. Wang S. Lai Q. Fang Y. Wu C. Liu Y. Li Q. Wang X. Gu C. Chen J. et al.Cancer-associated fibroblasts-derived exosomal miR-17-5p promotes colorectal cancer aggressive phenotype by initiating a RUNX3/MYC/TGF-β1 positive feedback loop.Cancer Lett. 2020; 491: 22-35Crossref PubMed Scopus (14) Google Scholar, 16Tong Y. Yang L. Yu C. Zhu W. Zhou X. Xiong Y. Wang W. Ji F. He D. Cao X. Tumor-Secreted Exosomal lncRNA POU3F3 Promotes Cisplatin Resistance in ESCC by Inducing Fibroblast Differentiation into CAFs.Mol. Ther. Oncolytics. 2020; 18: 1-13Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar Recently, ever more attention has been paid to their potential mechanisms in cancer chemoresistance. For example, exosomes of glioblastoma cells containing long noncoding RNA SBF2-AS1 can induce TMZ resistance in recipient cells.17Zhang Z. Yin J. Lu C. Wei Y. Zeng A. You Y. Exosomal transfer of long non-coding RNA SBF2-AS1 enhances chemoresistance to temozolomide in glioblastoma.J. Exp. Clin. Cancer Res. 2019; 38: 166Crossref PubMed Scopus (84) Google Scholar Additionally, reactive astrocyte-derived exosomes confer TMZ resistance phenotype to glioma cells via transfer of O6-methylguanine-DNA methyltransferase (MGMT) mRNA.18Yu T. Wang X. Zhi T. Zhang J. Wang Y. Nie E. Zhou F. You Y. Liu N. Delivery of MGMT mRNA to glioma cells by reactive astrocyte-derived exosomes confers a temozolomide resistance phenotype.Cancer Lett. 2018; 433: 210-220Crossref PubMed Scopus (39) Google Scholar Hence, exosomes have emerged as a new model for research into tumor chemoresistance. Macrophage migration inhibitory factor (MIF; also known as L-dopachrome tautomerase) is a proinflammatory multifunctional cytokine that was originally discovered in 1966.19Bloom B.R. Bennett B. Mechanism of a reaction in vitro associated with delayed-type hypersensitivity.Science. 1966; 153: 80-82Crossref PubMed Scopus (1239) Google Scholar Previous studies have shown that MIF overexpressed in many tumors.20Fukaya R. Ohta S. Yaguchi T. Matsuzaki Y. Sugihara E. Okano H. Saya H. Kawakami Y. Kawase T. Yoshida K. Toda M. MIF Maintains the Tumorigenic Capacity of Brain Tumor-Initiating Cells by Directly Inhibiting p53.Cancer Res. 2016; 76: 2813-2823Crossref PubMed Scopus (35) Google Scholar,21Mittelbronn M. Platten M. Zeiner P. Dombrowski Y. Frank B. Zachskorn C. Harter P.N. Weller M. Wischhusen J. Macrophage migration inhibitory factor (MIF) expression in human malignant gliomas contributes to immune escape and tumour progression.Acta Neuropathol. 2011; 122: 353-365Crossref PubMed Scopus (55) Google Scholar Recent evidence shows that MIF is involved in tumor chemoresistance.22Parekh A. Das S. Parida S. Das C.K. Dutta D. Mallick S.K. Wu P.H. Kumar B.N.P. Bharti R. Dey G. et al.Multi-nucleated cells use ROS to induce breast cancer chemo-resistance in vitro and in vivo.Oncogene. 2018; 37: 4546-4561Crossref PubMed Scopus (33) Google Scholar,23Wu M.Y. Fu J. Xu J. O’Malley B.W. Wu R.C. Steroid receptor coactivator 3 regulates autophagy in breast cancer cells through macrophage migration inhibitory factor.Cell Res. 2012; 22: 1003-1021Crossref PubMed Scopus (39) Google Scholar As a secretory cytokine, exosomal MIF was highly expressed in lung cancer patients’ plasma.24Pan D. Chen J. Feng C. Wu W. Wang Y. Tong J. Zhou D. Preferential Localization of MUC1 Glycoprotein in Exosomes Secreted by Non-Small Cell Lung Carcinoma Cells.Int. J. Mol. Sci. 2019; 20: 323Crossref Scopus (34) Google Scholar Another study found that MIF was highly expressed in pancreatic ductal adenocarcinomas (PDAC) derived exosomes, and MIF blockade prevented liver pre-metastatic niche formation and metastasis.24Pan D. Chen J. Feng C. Wu W. Wang Y. Tong J. Zhou D. Preferential Localization of MUC1 Glycoprotein in Exosomes Secreted by Non-Small Cell Lung Carcinoma Cells.Int. J. Mol. Sci. 2019; 20: 323Crossref Scopus (34) Google Scholar,25Costa-Silva B. Aiello N.M. Ocean A.J. Singh S. Zhang H. Thakur B.K. Becker A. Hoshino A. Mark M.T. Molina H. et al.Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.Nat. Cell Biol. 2015; 17: 816-826Crossref PubMed Scopus (1371) Google Scholar However, whether exosomal MIF contributes to TMZ resistance in glioma remains to be elucidated. Here, we aim to explore whether MIF could be transmitted by exosomes and involved in TMZ resistance of glioma. We have dedicated this study to explore the molecular therapy targets of chemoresistance in glioma in our previous studies.26Liu B. Zhou J. Wang C. Chi Y. Wei Q. Fu Z. Lian C. Huang Q. Liao C. Yang Z. et al.LncRNA SOX2OT promotes temozolomide resistance by elevating SOX2 expression via ALKBH5-mediated epigenetic regulation in glioblastoma.Cell Death Dis. 2020; 11: 384Crossref PubMed Scopus (21) Google Scholar, 27Xu N. Liu B. Lian C. Doycheva D.M. Fu Z. Liu Y. Zhou J. He Z. Yang Z. Huang Q. et al.Long noncoding RNA AC003092.1 promotes temozolomide chemosensitivity through miR-195/TFPI-2 signaling modulation in glioblastoma.Cell Death Dis. 2018; 9: 1139Crossref PubMed Scopus (41) Google Scholar, 28Zeng H. Yang Z. Xu N. Liu B. Fu Z. Lian C. Guo H. Connective tissue growth factor promotes temozolomide resistance in glioblastoma through TGF-β1-dependent activation of Smad/ERK signaling.Cell Death Dis. 2017; 8: e2885Crossref PubMed Scopus (20) Google Scholar In this study, we found that MIF was upregulated in U87TR and U251TR (TMZ-resistant) cells, and our in vitro and in vivo results showed that exosomal MIF derived from TMZ-resistant cells can transfer chemoresistance character to sensitive glioma cells by downregulating TIMP3, further activating the PI3K/AKT signaling pathway. Applying ISO-1 (MIF inhibitor) is verified to be a promising treatment strategy for TMZ resistance in glioma animal models, because it could enhance the TMZ sensitivity by inhibiting MIF. In this research, we demonstrated that MIF was upregulated in higher grade gliomas and recurrent patients by immunohistochemistry (IHC) staining (Figure 1A). Next, we analyzed the mRNA expression data of gliomas in The Cancer Genome Atlas (TCGA) and The Chinese Glioma Genome Atlas (CGGA) cohorts, revealing that MIF was highly expressed in high grade gliomas and recurrent patients. These findings were in accord with our study (Figures 1B and 1C). Survival curves of glioma samples from TCGA and CGGA analyses showed that patients with high MIF expression levels had a worse prognosis (Figure 1D). These results indicated that high expression of MIF was associated with poor prognosis in glioma patients.Figure 1MIF upregulation is associated with poor prognosis in gliomasShow full caption(A) MIF was upregulated in 4 typical recurrent glioma patients by IHC staining (scale bars, 50 μm). (B–D) TCGA and CGGA database analysis showed that MIF expression level was positively correlated with glioma grade (B) and recurrence (C), but negatively correlated with prognosis (D). ∗∗∗p＜0.001, ∗∗∗∗p＜0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) MIF was upregulated in 4 typical recurrent glioma patients by IHC staining (scale bars, 50 μm). (B–D) TCGA and CGGA database analysis showed that MIF expression level was positively correlated with glioma grade (B) and recurrence (C), but negatively correlated with prognosis (D). ∗∗∗p＜0.001, ∗∗∗∗p＜0.0001. Our group had successfully established two TMZ-resistant glioma cell lines through repetitive exposure to increasing TMZ concentrations in vitro,29Zeng H. Xu N. Liu Y. Liu B. Yang Z. Fu Z. Lian C. Guo H. Genomic profiling of long non-coding RNA and mRNA expression associated with acquired temozolomide resistance in glioblastoma cells.Int. J. Oncol. 2017; 51: 445-455Crossref PubMed Scopus (19) Google Scholar named U87TR and U251TR (TMZ-resistant) cells, which had a higher IC50 (half maximal inhibitory concentration) value than U87TS and U251TS (TMZ-sensitive) cells (Figure S1A).26Liu B. Zhou J. Wang C. Chi Y. Wei Q. Fu Z. Lian C. Huang Q. Liao C. Yang Z. et al.LncRNA SOX2OT promotes temozolomide resistance by elevating SOX2 expression via ALKBH5-mediated epigenetic regulation in glioblastoma.Cell Death Dis. 2020; 11: 384Crossref PubMed Scopus (21) Google Scholar, 27Xu N. Liu B. Lian C. Doycheva D.M. Fu Z. Liu Y. Zhou J. He Z. Yang Z. Huang Q. et al.Long noncoding RNA AC003092.1 promotes temozolomide chemosensitivity through miR-195/TFPI-2 signaling modulation in glioblastoma.Cell Death Dis. 2018; 9: 1139Crossref PubMed Scopus (41) Google Scholar, 28Zeng H. Yang Z. Xu N. Liu B. Fu Z. Lian C. Guo H. Connective tissue growth factor promotes temozolomide resistance in glioblastoma through TGF-β1-dependent activation of Smad/ERK signaling.Cell Death Dis. 2017; 8: e2885Crossref PubMed Scopus (20) Google Scholar, 29Zeng H. Xu N. Liu Y. Liu B. Yang Z. Fu Z. Lian C. Guo H. Genomic profiling of long non-coding RNA and mRNA expression associated with acquired temozolomide resistance in glioblastoma cells.Int. J. Oncol. 2017; 51: 445-455Crossref PubMed Scopus (19) Google Scholar qRT-PCR and western blot results found that TR cells had a higher MIF expression level than TS cells (Figures 2A and 2B ). To assess the role of MIF in TMZ resistance of glioma, we transfected TR cells with MIF short hairpin RNA (shRNA) lentiviral vectors to knock down MIF expression and confirmed its efficiency by qRT-PCR (Figure S1B). Meanwhile, MIF overexpressed (OE) lentiviral vectors were transfected into TS cells and its overexpression efficiency was confirmed by qRT-PCR assay (Figure S1C). Cell counting kit-8 (CCK-8) assay further showed that knockdown of MIF significantly decreased IC50 values in TR cells (Figure 2C). In contrast, overexpressed MIF significantly increased IC50 values in TS cells (Figure 2D). These results demonstrated that highly expressed MIF promotes TMZ chemoresistance in glioma cells. Furthermore, the flow cytometry (FCM) showed that loss of MIF significantly increased cell apoptosis upon TMZ (50 μg/mL) treatment in TR cells (Figures 2E and 2F). Whereas TUNEL assay showed that overexpression of MIF decreased apoptosis in TS cells (Figures 2G and 2H). The percentage of the EdU-positive cells were significantly decreased in TR-MIF-sh groups than in TR-NC (negative control) groups. On the other hand, the percentage of the EdU-positive cells was significantly increased in TS-MIF-OE groups than in TS-NC groups (Figures 2I–2L). These data suggest that MIF confers TMZ chemoresistance, enhances cell proliferation, and inhibits cell apoptosis in TMZ-resistant cells.Figure 2Highly expressed MIF promotes TMZ resistance in glioma cellsShow full caption(A and B) qRT-PCR and western blot showed that the expression of MIF in U87TR/251TR cells was significantly upregulated compared with U87/251 cells. (C and D) CCK-8 showed that the IC50 values were significantly deceased after MIF knockdown in TR cells. In contrast, overexpressed MIF significantly increased the IC50 values in TS cells. (E and F) The flow cytometry (FCM) showed that loss of MIF in TR cells significantly increased cell apoptosis upon TMZ treatment for 48 h (50 μg/mL). (G and H) On the contrary, TUNEL assay showed that overexpression of MIF may cause apoptosis rates to decrease in TS cells. (I–L) The percentage of the EdU-positive cells were significantly decreased in TR-MIF-sh groups than in TR-NC groups. On the other hand, the percentage of the EdU-positive cells were significantly increased in TS-MIF-OE groups than in TS-NC groups. ∗∗p＜0.01, ∗∗∗p＜0.001, ∗∗∗∗p＜0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) qRT-PCR and western blot showed that the expression of MIF in U87TR/251TR cells was significantly upregulated compared with U87/251 cells. (C and D) CCK-8 showed that the IC50 values were significantly deceased after MIF knockdown in TR cells. In contrast, overexpressed MIF significantly increased the IC50 values in TS cells. (E and F) The flow cytometry (FCM) showed that loss of MIF in TR cells significantly increased cell apoptosis upon TMZ treatment for 48 h (50 μg/mL). (G and H) On the contrary, TUNEL assay showed that overexpression of MIF may cause apoptosis rates to decrease in TS cells. (I–L) The percentage of the EdU-positive cells were significantly decreased in TR-MIF-sh groups than in TR-NC groups. On the other hand, the percentage of the EdU-positive cells were significantly increased in TS-MIF-OE groups than in TS-NC groups. ∗∗p＜0.01, ∗∗∗p＜0.001, ∗∗∗∗p＜0.0001. We performed the differential expressed genes (DEGs) analysis of MIF in TCGA database, the cellular component (CC) analysis indicated that the DEGs were enriched in extracellular exosome (Figure S1A). In addition, enzyme-linked immunosorbent assay showed that TR cells secrete more MIF than TS cells (Figure S1B). Then, exosomes were isolated from TR and TS cells’ supernatant. The exosomes derived from glioma cells presented as 40–150 nm round shape vesicles with bilayer membranes, which were confirmed by high-resolution transmission electron microscopy (TEM; Figure 3A). The nano-flow cytometer (nFCM) further identified that the similar predominant size of these vesicles. In addition, TR cells secreted more exosomes than TS cells (Figure 3B). The exosome markers like ALIX, CD81, and TSG101 were detected in these vesicles (Figure 3C). qRT-PCR and western blot showed that the exosomes secreted by TR cells (TR EXO) expressed more MIF than those from TS cells (Figures 3C and 3D).Figure 3Exosomal MIF transfers from TR cells to TMZ-sensitive cellsShow full caption(A) The representative micrograph of round-shaped vesicles derived from TMZ-resistant and TMZ-sensitive cells by TEM (scale bars, 100 nm). (B) The nFCM further identified that the similar predominant size of these vesicles. (C) Western blot analysis showed the presence of MIF, ALIX, TSG101, and CD81 in exosomes. (D) qRT-PCR showed that the exosomes secreted by TR cells (TR EXO) expressed more MIF than those from TS cells. (E and F) Schematic diagram: TR exosomes were labeled with PKH26 and co-incubated with TS-GFP cells. Confocal photography found that exosomes could be successfully ingested by TS cells (scale bars, 10 μm). (G and H) Schematic diagram: MIF in TR cells was labeled with mCherry, and then exosomes were extracted and added into the supernatant of TS-GFP cells. After 24 h of coculture, we observed strong red fluorescence in the cytoplasm of TS-GFP cells (scale bars, 50 μm). (I and J) qRT-PCR and western blot were used to verify the expression level of MIF in TS cells after they were co-incubated with PBS, TR EXO, TR-NC EXO, or TR-MIF-sh EXO separately. ∗∗∗∗p＜0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) The representative micrograph of round-shaped vesicles derived from TMZ-resistant and TMZ-sensitive cells by TEM (scale bars, 100 nm). (B) The nFCM further identified that the similar predominant size of these vesicles. (C) Western blot analysis showed the presence of MIF, ALIX, TSG101, and CD81 in exosomes. (D) qRT-PCR showed that the exosomes secreted by TR cells (TR EXO) expressed more MIF than those from TS cells. (E and F) Schematic diagram: TR exosomes were labeled with PKH26 and co-incubated with TS-GFP cells. Confocal photography found that exosomes could be successfully ingested by TS cells (scale bars, 10 μm). (G and H) Schematic diagram: MIF in TR cells was labeled with mCherry, and then exosomes were extracted and added into the supernatant of TS-GFP cells. After 24 h of coculture, we observed strong red fluorescence in the cytoplasm of TS-GFP cells (scale bars, 50 μm). (I and J) qRT-PCR and western blot were used to verify the expression level of MIF in TS cells after they were co-incubated with PBS, TR EXO, TR-NC EXO, or TR-MIF-sh EXO separately. ∗∗∗∗p＜0.0001. Accumulating evidence has shown that MIF can be packaged into exosomes derived from tumor cells and promotes tumor progression.24Pan D. Chen J. Feng C. Wu W. Wang Y. Tong J. Zhou D. Preferential Localization of MUC1 Glycoprotein in Exosomes Secreted by Non-Small Cell Lung Carcinoma Cells.Int. J. Mol. Sci. 2019; 20: 323Crossref Scopus (34) Google Scholar,25Costa-Silva B. Aiello N.M. Ocean A.J. Singh S. Zhang H. Thakur B.K. Becker A. Hoshino A. Mark M.T. Molina H. et al.Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.Nat. Cell Biol. 2015; 17: 816-826Crossref PubMed Scopus (1371) Google Scholar Besides, several studies have demonstrated that exosomal RNA derived from resistant cells transfer chemoresistance characteristics to sensitive cells.30Xu X. Liu Y. Li Y. Chen H. Zhang Y. Liu J. Deng S. Zheng Y. Sun X. Wang J. et al.Selective exosome exclusion of miR-375 by glioma cells promotes glioma progression by activating the CTGF-EGFR pathway.J. Exp. Clin. Cancer Res. 2021; 40: 16Crossref PubMed Scopus (3) Google Scholar, 31Yin J. Zeng A. Zhang Z. Shi Z. Yan W. You Y. Exosomal transfer of miR-1238 contributes to temozolomide-resistance in glioblastoma.EBioMedicine. 2019; 42: 238-251Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 32Zeng A. Wei Z. Yan W. Yin J. Huang X. Zhou X. Li R. Shen F. Wu W. Wang X. You Y. Exosomal transfer of miR-151a enhances chemosensitivity to temozolomide in drug-resistant glioblastoma.Cancer Lett. 2018; 436: 10-21Crossref PubMed Scopus (78) Google Scholar Combining literature and our results, we speculated that MIF could transfer TMZ-resistant character via exosomes. To visualize exosome transfer, we labeled TR EXO with PKH26 (TR EXO-PKH26). TS cells transfected with lentivirus vectors carrying green fluorescent protein (TS-GFP) were incubated with the TR EXO-PKH26 for 3 h (Figure 3E). The confocal microscope captured the PKH26-labeled exosomes that were internalized by the recipient TS cells (Figure 3F). Then we validated whether the transfer of MIF occurs via exosomes. Exosomes, which were isolated from TR cells that transfected with the MIF-mCherry lentiviral activation vectors (TR MIF-mCherry), were added into culture medium of TS-GFP cells (Figure 3G). After 24 h of coculture, we observed strong red fluorescence in the cytoplasm of TS-GFP cells (Figure 3H). Furthermore, after co-incubation with TR EXO or TR-MIF-sh EXO separately, the cellular level of MIF was corresponding upregulated or downregulated in recipient cells (Figures 3I and 3J). These data demonstrated that MIF could be directly transferred from TR cells to sensitive cells through exosomes. We next investigated whether exosomal MIF could confer TMZ resistance phenotype to recipient TS glioma cells. TS cells were incubated with PBS, TR EXO, TR-NC EXO, TR-MIF-sh EXO for 24 h, and then followed with TMZ treatment for 48 h. The FCM results showed that the apoptosis rates in TR EXO and TR-NC EXO groups were significantly lower than in PBS and TR-MIF-sh EXO groups in TS cells (Figures 4A and 4B ). Similarly, EdU assay demonstrated that TR EXO and TR-NC EXO groups had higher cell proliferation than PBS and TR-MIF-sh EXO groups (Figures 4C and 4D). Meanwhile, TR EXO and TR-NC EXO significantly enhance the TMZ-resistant character after co-culture with TS cells (Figure 4E). In addition, TR EXO and TR-NC EXO obviously decrease the apoptosis-related protein (caspase-3 and caspase-9) expression level in recipient TS cells (Figure 4F). Together, these results suggested that exosomal MIF conferred TMZ resistance to recipient TS cells by promoting cell proliferation and inhibiting cell apoptosis.Figure 4Exosomal MIF confers TMZ resistance via promotion of proliferation and inhibition of apoptosis in recipient cellsShow full caption(A and B) The FCM results showed that the apoptosis rates in TR EXO and TR-NC EXO groups were significantly lower than in PBS and TR-MIF-sh EXO groups (TMZ = 50 μg/mL). (C) EdU assay demonstrated that TR EXO and TR-NC EXO groups had a higher cell proliferation rate than PBS and TR-MIF-sh EXO groups (scale bars, 50 μm). (D) EdU statistics. (E) CCK-8 showed that TR EXO and TR-NC EXO significantly enhanced the TMZ resistant characteristic in TS cells. (F) Western blot for caspase-3 and caspase-9 expression in the different treated glioma cells. ∗∗p＜0.01, ∗∗∗p＜0.001, ∗∗∗∗p＜0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) The FCM results showed that the apoptosis rates in TR EXO and TR-NC EXO groups were significantly lower than in PBS and TR-MIF-sh EXO groups (TMZ = 50 μg/mL). (C) EdU assay demonstrated that TR EXO and TR-NC EXO groups had a higher cell proliferation rate than PBS and TR-MIF-sh EXO groups (scale bars, 50 μm). (D) EdU statistics. (E) CCK-8 showed that TR EXO and TR-NC EXO significantly enhanced the TMZ resistant characteristic in TS cells. (F) Western blot for caspase-3 and caspase-9 expression in the different treated glioma cells. ∗∗p＜0.01, ∗∗∗p＜0.001, ∗∗∗∗p＜0.0001. To ascertain whether the latent mechanism of MIF induces TMZ resistance in glioma cells, we performed RNA sequencing (RNA-seq) analysis to predict the possible target genes. Tissue inhibitors of metalloproteinases-3 (TIMP3) was the most top significantly upregulated genes in U87 MIF-sh cells, and it was predicted to be the highly possible downstream target of MIF in glioma cells (Figures 5A and 5B ). TIMP3, a member of the TIMP family, is a 24-kDa secreted glycoprotein and can suppress cell proliferation and enhance chemosensitivity by activating apoptosis-related signal pathways.33Su C.W. Lin C.W. Yang W.E. Yang S.F. TIMP-3 as a therapeutic target for cancer.Ther. Adv. Med. Oncol. 2019; 11 (1758835919864247)Crossref Scopus (17) Google Scholar, 34Bodnar M. Szylberg Ł. Kazmierczak W. Marszalek A. Tumor progression driven by pathways activating matrix metal