Fig 1: Label-free sensitive detection of MCT1 and CD147 in serum-derived exosomes from a mouse model of glioma.(A to C) Representative MRI images for the brain of sham-operated mice and U251 and U87 mouse models of glioma. (D and E) Representative phase responses of the LSPR biosensor with the functionalized SAM-AuNIs sensing chip with anti-MCT1 AB or anti-CD147 AB and (F and G) representative separation force curves of the AFM biosensor with the functionalized silicon nitride cantilever tip with anti-MCT1 AB or anti-CD147 AB toward serum-derived exosomes from sham-operated mice and U251 and U87 mouse models of glioma. (H to K) Bar graph summarizing the relative strength of LSPR responses (n = 3) or AFM forces (n = 3) toward exosomal MCT1 [e.g., (D) and (E)] and CD147 [e.g., (F) and (G)]. Detailed processes of LSPR and AFM biosensing were described in Materials and Methods. All data were expressed as the means ± SD. Significance level: **P < 0.01, *P < 0.05, U251 or U87 mouse model of glioma versus sham-operated severe combined immunodeficient mouse. WT, wild type.
Fig 2: Hypoxia promotes U251 GMs’ glycolytic reprogramming, including enhancing MCT1 and CD147 expression.(A to E) Change in the mRNA expression of HIF-1?, HK-2, LDH, MCT1, and CD147 in GMs in response to hypoxia (1% O2) (n = 3), as determined by quantitative real-time polymerase chain reaction (qRT-PCR). (F to I) Protein-level change of HIF-1?, MCT1, and CD147 in GMs in response to hypoxia (1% O2) (n = 3), as determined by Western blot (WB). (J to Z and A1) Immunofluorescent staining for HIF-1?, MCT1, and CD147 in GMs under normoxia and hypoxia. (B1) A representative graph of ECAR outputs from the XF24 analyzer for normoxic and hypoxic GMs and their response to glucose, oligomycin, and 2-deoxyglucose (2-DG) in the measurement of the status of glycolytic metabolism. (C1) Comparison of glycolysis, glycolytic capacity, and glycolytic reserve between normoxic and hypoxic GMs (n = 3). Immunofluorescent staining for MCT1 in GMs treated with (D1 to G1) empty backbone lentivirus (control 1) and (H1 to K1) MCT1 OE lentivirus for 24 hours. Immunofluorescent staining for CD147 in GMs treated with (L1 to O1) empty backbone lentivirus (control 1) and (P1 to S1) CD147 OE lentivirus for 24 hours. All data were shown as the means ± SD. Significance level: **P < 0.01, *P < 0.05, hypoxia versus normoxia.
Fig 3: Noninvasive sensitive detection of increased MCT1 and CD147 in hypoxic GM-derived exosomes by LSPR and AFM biosensors.(A and B) Baseline phase response of the LSPR biosensor with the functionalized SAM-AuNIs sensing chip with anti-MCT1 AB or anti-CD147 AB after sequential treatment with 11-mercaptoundecanoic acid (MUA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS). (C and D) Phase response of the LSPR biosensor toward three different concentrations (serial dilution of 1300 µg/ml exosome solution: 1000×, 100×, and 10×) of U251 GM-derived exosomes. Standard curve fitting for phase responses toward anti-MCT1 AB (R2 = 0.9871) or anti-CD147 AB (R2 = 0.9969). (E and F) Representative phase response of the LSPR biosensor toward equal amount of normoxic and hypoxic GM-derived exosomes (50 µg/ml). (G and H) Bar graph showing the relative strength of LSPR responses toward exosomal MCT1 (E) and CD147 (F) from normoxic or hypoxic GMs (n = 3). (I to K) Two-dimensional, three-dimensional, and high resolution of three-dimensional AFM topographic images for U251 GM-derived exosomes immobilized on the SAM-AuNIs sensing chip. (L) Height profile of single U251 GM-derived exosome by AFM scanning. (M and N) Representative separation force responses of the AFM biosensor with the functionalized cantilever sensing tip with anti-MCT1 AB, or anti-CD147 AB toward equal amount (50 µg/ml) of normoxic and hypoxic GM-derived exosomes captured on the SAM-AuNIs sample discs. (O and P) Bar graph showing the relative strength of AFM separation force responses toward exosomal MCT1 (M) and CD147 (N) from normoxic or hypoxic GMs (n = 12). All data were shown as the means ± SD. Significance level: **P < 0.01, *P < 0.05, hypoxia versus normoxia.
Fig 4: Label-free quantitative detection of exosomal MCT1 and CD147 to monitor their expression levels in parent GMs, as novel surrogate biomarkers.(A and B) Representative phase responses of the LSPR biosensor with the functionalized SAM-AuNIs sensing chip with anti-MCT1 AB or anti-CD147 AB and (C and D) separation force responses of the AFM biosensor with the functionalized silicon nitride tip with anti-MCT1 AB or anti-CD147 AB toward equal amount of daughter exosomes (50 µg/ml) from parent U251 GMs with no treatment (control), MCT1 OE, MCT1 KD, CD147 OE, and CD147 KD. (E to H) Bar graph showing the relative strength of LSPR responses (n = 3) or AFM forces (n = 12) toward exosomal MCT1 [from (A) and (C)] and CD147 [from (B) and (D)]. (I and J) Correlation curve between MCT1 or CD147 levels in parent GMs and the strength of LSPR responses toward exosomal MCT1 or CD147, respectively [for MCT1, coefficient of determination (R2) = 0.9247, and for CD147, R2 = 0.9654], or the strength of AFM forces toward exosomal MCT1 or CD147, respectively (for MCT1, R2 = 0.9996, and for CD147, R2 = 0.9952). The correlation analysis was performed based on the data obtained from (A) to (D). All data were shown as the means ± SD. Significance level: **P < 0.01, *P < 0.05, MCT1 OE and MCT1 KD group versus control. CD147 OE and CD147 KD group versus control.
Fig 5: Enhanced MCT1 and CD147 in hypoxic U251 GMs promote intracellular Ca2+-dependent exosome release.(A and B) Size distribution and quantity of exosomes released from normoxic and hypoxic GMs for 24 hours (NTA analysis). (C) Enhanced release of exosomes from hypoxic GMs (versus normoxic GMs). (D) Analysis of exosome release from GMs treated with control 1, MCT1 OE, MCT1 KD, CD147 OE (all lentivirus), and control 2 and CD147 KD (antisense oligonucleotides) constructs. (E to P) Representative images of Fura Red calcium dye- loaded- hypoxic (versus normoxic), MCT1 OE- or MCT1 KD- (versus control 1) induced, CD147 OE- or CD147 KD- (versus control 1 & 2) induced, and BAPTA-AM (20 µM)-treated GMs. (Q) Enhanced exosome release from MCT1 OE– and CD147 OE–induced (versus control 1) GMs, followed by a marked decline in exosome release by treatment with BAPTA-AM (20 µM, 100 µl). (R) Enhanced intracellular Ca2+ levels in GMs by treatment with sodium-l-lactate (20 mM, 100 µl), followed by distinctive decline in intracellular Ca2+ level by treatment with BAPTA-AM (20 µM, 100 µl). (S) NTA exosome release assay from GMs exposed to four different conditions for 10 min described in Materials and Methods. Briefly, a, Exo–fetal bovine serum (FBS) medium; b, sodium-l-lactate (20 mM), c, BAPTA-AM; d, BAPTA-AM with the pretreatment of sodium-l-lactate (20 mM). All chemicals were dissolved in the Exo-FBS medium containing 1% dimethyl sulfoxide. All data were shown as the means ± SD. Significance level: **P < 0.01, *P < 0.05, hypoxia versus normoxia, BAPTA-AM versus control, MCT1 KD lentivirus versus empty backbone lentivirus (control 1), and CD147 antisense oligonucleotides versus antisense control oligonucleotides (control 2).
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