slc1a5 fitc  (Alomone Labs)

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: Upregulation of metabolic pathway-related proteins in EVs derived from TKI-resistant cell lines and patient plasma. (A) Characterization of EVs isolated from the supernatant of HCC827 and plasma of a patient with TKI-resistant tumor by EM. Scale bar: 0.1 µM. (B) Size distribution of EVs isolated from cell culture supernatant analyzed by NanoFC. (C) Flow cytometry analysis of CD9 surface expression (MFI: Mean fluorescence intensity) on isolated EVs from plasma of a patient with TKI-resistant tumor. (D) Western blot showing expression of CD61, CD81, CD44, TSG101, and calnexin in whole cell lysates and isolated EVs. (E) Venn diagram showing the number of upregulated proteins in three different proteomics datasets from EVs of HCC827-GR/HCC827, plasma 1 (TKI-resistant NSCLC/healthy donor), and plasma 2 (pooled plasma of 5 different tumor/healthy donors). (F, G, and H) Protein expression of SLC1A5, SLC25A5, and ALDH1A1 in EVs isolated from supernatant of HCC827 and HCC827-GR cells using ELISA as described in Materials and Methods. (I, J, and K) Protein expression of SLC1A5, SLC25A5, and ALDH1A1 in the EVs isolated from the plasma of healthy donors (n = 16 for ALDH1A1 and n = 28 for SLC25A5 and n = 58 for SLC1A5) and plasma of TKI-resistant NSCLC patients (n = 111) by ELISA as described in Materials and Methods. (L) Protein expression of SLC1A5 in the EVs isolated from the plasma of healthy donors (n = 58), plasma of early-stage NSCLC (n = 20), plasma of EGFT mutant and treatment naïve NSCLC (n = 21), and late-stage and treatment-resistant NSCLC (N = 62) by ELISA as described in Materials and Methods. (M) Progression-free survival curve was generated using NSCLC patient's survival details with SLC1A5 level in fold difference (high SLC1A5 level ≥ 2, n = 44 and low SLC1A5 ≤ 2, n = 26 Log-rank Hazard Ratio: 2.632, 95% CI, 1.441-4.807, p = 0.0063). Unpaired T-test and two-way Anova were used in GraphPad Prism, version 9 for statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significance).

Article Snippet: For staining, cells were incubated with SLC1A5-FITC 1mg/ml (Alomone labs, Israel) and FAP-PE (R&D systems, USA) 10 µl/10 6 cells for 1h.

Techniques: Derivative Assay, Isolation, Cell Culture, Flow Cytometry, Expressing, Fluorescence, Western Blot, Enzyme-linked Immunosorbent Assay, Mutagenesis, Generated

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: REVs upregulate metabolic transporters. (A) Western blot showing increased SLC1A5, SLC25A5, ALDH1A1, STAT3pY705, and STAT3pS727 levels in total lysates of HCC827 cells exposed for 48 hours to SEVs or increasing concentrations of REVs. Total STAT3 and β-actin were used as loading controls. (B) Image viewed at 40X magnification by confocal microscopy showing increased surface expression of SLC1A5 in HCC827 cells after 48 hours exposure to REVs (Scale bar: 20 µM). (C) Samples from B (individual wells) were scanned using Tissue Fax and mean SLC1A5 intensity was derived and plotted using ImageJ and GraphPad prism, respectively. (D) Flow cytometry data showing surface expression of SLC1A5-FITC on HCC827 cells following incubation with 10 µg of REVs for the indicated duration. For flow analysis, at least 10,000 cells were analyzed by flow cytometry as described in Materials and Methods. (E) REVs were stained with SLC1A5-FITC and staining was analysed using NanoFC. (F) SLC1A5-FITC stained REVs were added to HCC827 cells and live imaging was done using a confocal microscope from 4 to 36 hours for every 20 minutes. A cell mask (red) was used to stain the cell membrane. (G) Increase in OCR in HCC827 cells upon exposure to REVs, measured using seahorse and plotted using GraphPad Prism software. (H) Increased intracellular glutamine levels in HCC827 cells after 48 hours exposure to REVs. Glutamine was measured as described in materials and methods and plotted using GraphPad prism software. Data are representative of at least 2-3 independent experiments and shown as mean ± SD of biological triplicates. Two-way ANOVA was employed for statistical significance (*p < 0.05, **p < 0.01).

Article Snippet: For staining, cells were incubated with SLC1A5-FITC 1mg/ml (Alomone labs, Israel) and FAP-PE (R&D systems, USA) 10 µl/10 6 cells for 1h.

Techniques: Western Blot, Confocal Microscopy, Expressing, Derivative Assay, Flow Cytometry, Incubation, Staining, Imaging, Microscopy, Membrane, Software

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: REV-induced TKI resistance involves crosstalk between the glutamine pathway and STAT3 activation. (A) HCC827 cells were pre-exposed with REVs for 48 hours before 24 hours of treatment with gefitinib (2µM) in the presence or absence of glutamine. Cell viability was measured using CCK-8 assay. (B) Effect on spheroid formation was assessed in the samples from A; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 100 µM). (C) REVs pre-exposed HCC827 cells were pre-treated with glutamine transport inhibitors, GPNA (10 µM), V9302(10 µM), and STAT3 inhibitor, STATIC (1 µM), for 1 h before exposure to gefitinib (2 µM) for 24 hours. Cell viability was measured using CCK-8 assay. (D) Effect on spheroid formation was assessed in the samples from C; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 100 µM). (E) Western blot showing expression of SLC1A5, and SLC25A5 in lysates of EVs isolated from HCC827-GR cells transiently transfected (48 hours) with Si SLC1A5 and Si SLC25A5 . CD81 and Alix were used as EV markers. (F) HCC827 cells were pre-exposed with REVs isolated from the Si SLC1A5 and Si SLC25A5 transfected cells for 48 hours before treatment with gefitinib for 24 hours. Cell viability was measured by crystal violet staining. (G) Effect on spheroid formation was assessed in the samples from F; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 100 µM). (H) HCC827 cells were stably transfected with pcmv6 vector or SLC1A5-GFP plasmid. Western blot showing over-expression of SLC1A5 and increased STAT3pY705 and STAT3pS727 levels. Total STAT3 and β-actin were used as loading controls. (I) Cell viability of HCC827 cells transfected with pcmv6 and SLC1A5 -OE following treatment with gefitinib for 24 hours. Cell viability was measured using CCK-8 assay. (J) Effect on spheroid formation was assessed in the samples from I; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 100 µM). (K) Pcmv6 transfected or SLC1A5 overexpressing cells were treated with gefitinib for 24 hours and 75,000 cells were re-seeded into ThinCert® cell culture inserts for 48 hours, stained with crystal violet, and viewed under a microscope (Scale bar: 100 µm) and (L) quantified by dissolving with 33% (v:v) acetic acid and measuring absorbance at 590 nm, as described in Materials and Methods. Migration rates are plotted in percentages with respect to control cells. (M) Increased OCR in HCC827- SLC1A5 -OE cells was inhibited upon exposure to glutamine transport inhibitors, GPNA and V9302, measured using seahorse and plotted using GraphPad Prism software. Data are representative of at least 3 independent experiments and shown as mean ± SD of biological triplicates. Two-way ANOVA was employed for statistical significance (*p<0.05, **p < 0.01, ***p < 0.0001, ****p < 0.0001, ns: not significant).

Article Snippet: For staining, cells were incubated with SLC1A5-FITC 1mg/ml (Alomone labs, Israel) and FAP-PE (R&D systems, USA) 10 µl/10 6 cells for 1h.

Techniques: Activation Assay, CCK-8 Assay, Microscopy, Western Blot, Expressing, Isolation, Transfection, Staining, Stable Transfection, Plasmid Preparation, Over Expression, Cell Culture, Migration, Control, Software

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: REVs induce resistance in different tumor models. (A) HCT116 cells were pre-exposed with SEVs, REVs, and SLC1A5 -OE-EVs for 48 hours before treatment with 5 FU for 24 hours. Cell viability was measured using CCK-8 assay. (B) Effect on spheroid formation was assessed in the samples from A; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar:10 µm). (C) A375 cells were pre-exposed to SEVs, REVs, and SLC1A5 -OE-EVs for 48 hours before treatment with Vemurafinib for 24 hours. Cell viability was measured using CCK-8 assay. (D) Effect on spheroid formation was assessed in the samples from C; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar:10 µm). (E) H1975 cells were pre-exposed to 10 µg EVs isolated from patient-derived cell lines (PDCEVs) for 48 hours before treatment with Osimertinib for 24 hours. Cell viability was measured using CCK-8 assay. (F, G) Spheroid formation in matrigel was assessed in the samples from E using the hanging drop technique (seeded in Matrigel) and left for 7-10 days before viewing under the microscope using 10X magnification or analyzed by confocal microscopy, respectively (Scale bar: 50 µM or 20 µM, respectively). (H) Western blot showing increased SLC1A5, SLC25A5, ALDH1A1, STAT3pY705, and STAT3pS727 levels in total lysates of H1975 cells exposed for 48 hours with increasing concentration of PDCEVs. Total STAT3 and B-actin were used as loading controls. (I) H1975 cells were pre-exposed with PDCEVs for 48 hours before treatment with 2 µM Gefitinib, Osimertinib, Crizotinib, Selumetinib, Afatinib, Bosutinib, and Debrafenib for 24 hours. Cell viability was measured using CCK-8 assay. Data are representative of at least 3 independent experiments and shown as mean ± SD of biological triplicates. Two-way ANOVA was employed for statistical significance (** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: For staining, cells were incubated with SLC1A5-FITC 1mg/ml (Alomone labs, Israel) and FAP-PE (R&D systems, USA) 10 µl/10 6 cells for 1h.

Techniques: CCK-8 Assay, Microscopy, Isolation, Derivative Assay, Confocal Microscopy, Western Blot, Concentration Assay

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: REVs regulate tumor microenvironment. (A) Western blot showing increased SLC1A5, TGFβ, αSMA, CD95, and Vimentin levels in total lysates of NL-20 cells exposed for 48 hours with SEVs and REVs. GAPDH was used as a loading control. (B) The scratch assay shows an increase in the migratory capacity of NL-20 cells exposed to SEVs and REVs for 48 hours (Scale bar: 10 cm). (C) Effect on spheroid formation was assessed in the co-culture (NL-20, NL-20+SEVs, and NL-20+REVs with HCC827) cells after treatment with gefitinib for 24 hours; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 10 mM). (D) From the same setup, 75,000 cells were re-seeded in ThinCert® cell culture inserts for 48 hours, stained with crystal violet, and viewed under a microscope (Scale bar: 100 µm) and (E) quantified by dissolving with 33% (v:v) acetic acid and read at an absorbance of 590 nm as described in Materials and Methods. Migration rates are plotted in percentages with respect to control cells. (F) Western blot showing increased SLC1A5, αSMA, CD95, Vimentin, STAT3pY705, and STAT3pS727 levels in total lysates MRC-5 cells exposed for 48 hours with SEVs and REVs. Total STAT3 and GAPDH was used as loading controls. (G) Increase expression of FAP in MRC-5 cells exposed to REVs and analyzed using Flow cytometry. For flow analysis, at least 10,000 cells were analyzed as described in Materials and Methods. (H) Scratch assay shows an increase in the migratory capacity of NL-20 cells after 48 hours of exposure to SEVs and REVs. (I) Effect on spheroid formation was assessed in the co-culture (NL-20, NL-20+SEVs, and NL-20+REVs with HCC827) cells after treatment with gefitinib for 24 hours; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 10 mM). (J) From the same setup, 75,000 cells were reseeded in ThinCert® cell culture inserts for 48 hours stained with crystal violet and viewed under a microscope (Scale bar: 50 µm) and (K) quantified by dissolving with 33% (v:v) acetic acid and measuring absorbance at 590 nm as described in Materials and Methods. Migration rates are plotted in percentages with respect to control cells. Data are representative of at least 3 independent experiments and shown as mean ± SD of biological triplicates. Two-way ANOVA was employed for statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (L) Western blot showing increased CD163, C-Mat, and HLA DR+DP+DQ level in naïve macrophages (M0) after being exposed to SEVs and REVs for 48 hours.

Article Snippet: For staining, cells were incubated with SLC1A5-FITC 1mg/ml (Alomone labs, Israel) and FAP-PE (R&D systems, USA) 10 µl/10 6 cells for 1h.

Techniques: Western Blot, Control, Wound Healing Assay, Co-Culture Assay, Microscopy, Cell Culture, Staining, Migration, Expressing, Flow Cytometry

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: REVs regulate tumor microenvironment in vivo . (A, B) Flow cytometry data showing increased expression of SLC1A5 on HCC827 xenograft slices 48 hours after co-culture with HCC827-GR slices. For flow analysis, at least 10,000 cells were analyzed by flow cytometry as described in Materials and Methods (MFI: Median fluorescence intensity). Data are representative of at least 3 independent experiments and shown as mean ± SD of biological triplicates. An unpaired T-test was employed for statistical significance (*p < 0.05). (C) Surface expression of SLC1A5, CD163, and FAP was assessed following co-culture of HCC827 xenograft slices with HCC827-GR xenograft slices for 48 hours. Image viewed at 10X and 20X magnification by confocal microscopy (Scale bar: 100 µM).

Article Snippet: For staining, cells were incubated with SLC1A5-FITC 1mg/ml (Alomone labs, Israel) and FAP-PE (R&D systems, USA) 10 µl/10 6 cells for 1h.

Techniques: In Vivo, Flow Cytometry, Expressing, Co-Culture Assay, Fluorescence, Confocal Microscopy

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: Upregulation of metabolic pathway-related proteins in EVs derived from TKI-resistant cell lines and patient plasma. (A) Characterization of EVs isolated from the supernatant of HCC827 and plasma of a patient with TKI-resistant tumor by EM. Scale bar: 0.1 µM. (B) Size distribution of EVs isolated from cell culture supernatant analyzed by NanoFC. (C) Flow cytometry analysis of CD9 surface expression (MFI: Mean fluorescence intensity) on isolated EVs from plasma of a patient with TKI-resistant tumor. (D) Western blot showing expression of CD61, CD81, CD44, TSG101, and calnexin in whole cell lysates and isolated EVs. (E) Venn diagram showing the number of upregulated proteins in three different proteomics datasets from EVs of HCC827-GR/HCC827, plasma 1 (TKI-resistant NSCLC/healthy donor), and plasma 2 (pooled plasma of 5 different tumor/healthy donors). (F, G, and H) Protein expression of SLC1A5, SLC25A5, and ALDH1A1 in EVs isolated from supernatant of HCC827 and HCC827-GR cells using ELISA as described in Materials and Methods. (I, J, and K) Protein expression of SLC1A5, SLC25A5, and ALDH1A1 in the EVs isolated from the plasma of healthy donors (n = 16 for ALDH1A1 and n = 28 for SLC25A5 and n = 58 for SLC1A5) and plasma of TKI-resistant NSCLC patients (n = 111) by ELISA as described in Materials and Methods. (L) Protein expression of SLC1A5 in the EVs isolated from the plasma of healthy donors (n = 58), plasma of early-stage NSCLC (n = 20), plasma of EGFT mutant and treatment naïve NSCLC (n = 21), and late-stage and treatment-resistant NSCLC (N = 62) by ELISA as described in Materials and Methods. (M) Progression-free survival curve was generated using NSCLC patient's survival details with SLC1A5 level in fold difference (high SLC1A5 level ≥ 2, n = 44 and low SLC1A5 ≤ 2, n = 26 Log-rank Hazard Ratio: 2.632, 95% CI, 1.441-4.807, p = 0.0063). Unpaired T-test and two-way Anova were used in GraphPad Prism, version 9 for statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significance).

Article Snippet: The antibodies used for immunoblotting: SLC1A5, SLC25A5, S727STAT3, and Total STAT3 (Cell signaling, USA), β-actin and c-Met (Santa-Cruz, Dallas, TX), ALDH1A1, Y705STAT3, CD63, CD81, TSG101, CD9, Anti-HLA DR + DP + DQ, and Calnexin (Abcam, Cambridge, UK), CD44, CD24, CD133 (BD Bioscience), CD163 (ThermoFisher Scientific, USA), Anti-SLC1A5-FITC antibody were purchased from Alomone labs and Anti-FAP from R&D systems.

Techniques: Derivative Assay, Isolation, Cell Culture, Flow Cytometry, Expressing, Fluorescence, Western Blot, Enzyme-linked Immunosorbent Assay, Mutagenesis, Generated

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: REVs upregulate metabolic transporters. (A) Western blot showing increased SLC1A5, SLC25A5, ALDH1A1, STAT3pY705, and STAT3pS727 levels in total lysates of HCC827 cells exposed for 48 hours to SEVs or increasing concentrations of REVs. Total STAT3 and β-actin were used as loading controls. (B) Image viewed at 40X magnification by confocal microscopy showing increased surface expression of SLC1A5 in HCC827 cells after 48 hours exposure to REVs (Scale bar: 20 µM). (C) Samples from B (individual wells) were scanned using Tissue Fax and mean SLC1A5 intensity was derived and plotted using ImageJ and GraphPad prism, respectively. (D) Flow cytometry data showing surface expression of SLC1A5-FITC on HCC827 cells following incubation with 10 µg of REVs for the indicated duration. For flow analysis, at least 10,000 cells were analyzed by flow cytometry as described in Materials and Methods. (E) REVs were stained with SLC1A5-FITC and staining was analysed using NanoFC. (F) SLC1A5-FITC stained REVs were added to HCC827 cells and live imaging was done using a confocal microscope from 4 to 36 hours for every 20 minutes. A cell mask (red) was used to stain the cell membrane. (G) Increase in OCR in HCC827 cells upon exposure to REVs, measured using seahorse and plotted using GraphPad Prism software. (H) Increased intracellular glutamine levels in HCC827 cells after 48 hours exposure to REVs. Glutamine was measured as described in materials and methods and plotted using GraphPad prism software. Data are representative of at least 2-3 independent experiments and shown as mean ± SD of biological triplicates. Two-way ANOVA was employed for statistical significance (*p < 0.05, **p < 0.01).

Article Snippet: The antibodies used for immunoblotting: SLC1A5, SLC25A5, S727STAT3, and Total STAT3 (Cell signaling, USA), β-actin and c-Met (Santa-Cruz, Dallas, TX), ALDH1A1, Y705STAT3, CD63, CD81, TSG101, CD9, Anti-HLA DR + DP + DQ, and Calnexin (Abcam, Cambridge, UK), CD44, CD24, CD133 (BD Bioscience), CD163 (ThermoFisher Scientific, USA), Anti-SLC1A5-FITC antibody were purchased from Alomone labs and Anti-FAP from R&D systems.

Techniques: Western Blot, Confocal Microscopy, Expressing, Derivative Assay, Flow Cytometry, Incubation, Staining, Imaging, Microscopy, Membrane, Software

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: REV-induced TKI resistance involves crosstalk between the glutamine pathway and STAT3 activation. (A) HCC827 cells were pre-exposed with REVs for 48 hours before 24 hours of treatment with gefitinib (2µM) in the presence or absence of glutamine. Cell viability was measured using CCK-8 assay. (B) Effect on spheroid formation was assessed in the samples from A; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 100 µM). (C) REVs pre-exposed HCC827 cells were pre-treated with glutamine transport inhibitors, GPNA (10 µM), V9302(10 µM), and STAT3 inhibitor, STATIC (1 µM), for 1 h before exposure to gefitinib (2 µM) for 24 hours. Cell viability was measured using CCK-8 assay. (D) Effect on spheroid formation was assessed in the samples from C; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 100 µM). (E) Western blot showing expression of SLC1A5, and SLC25A5 in lysates of EVs isolated from HCC827-GR cells transiently transfected (48 hours) with Si SLC1A5 and Si SLC25A5 . CD81 and Alix were used as EV markers. (F) HCC827 cells were pre-exposed with REVs isolated from the Si SLC1A5 and Si SLC25A5 transfected cells for 48 hours before treatment with gefitinib for 24 hours. Cell viability was measured by crystal violet staining. (G) Effect on spheroid formation was assessed in the samples from F; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 100 µM). (H) HCC827 cells were stably transfected with pcmv6 vector or SLC1A5-GFP plasmid. Western blot showing over-expression of SLC1A5 and increased STAT3pY705 and STAT3pS727 levels. Total STAT3 and β-actin were used as loading controls. (I) Cell viability of HCC827 cells transfected with pcmv6 and SLC1A5 -OE following treatment with gefitinib for 24 hours. Cell viability was measured using CCK-8 assay. (J) Effect on spheroid formation was assessed in the samples from I; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 100 µM). (K) Pcmv6 transfected or SLC1A5 overexpressing cells were treated with gefitinib for 24 hours and 75,000 cells were re-seeded into ThinCert® cell culture inserts for 48 hours, stained with crystal violet, and viewed under a microscope (Scale bar: 100 µm) and (L) quantified by dissolving with 33% (v:v) acetic acid and measuring absorbance at 590 nm, as described in Materials and Methods. Migration rates are plotted in percentages with respect to control cells. (M) Increased OCR in HCC827- SLC1A5 -OE cells was inhibited upon exposure to glutamine transport inhibitors, GPNA and V9302, measured using seahorse and plotted using GraphPad Prism software. Data are representative of at least 3 independent experiments and shown as mean ± SD of biological triplicates. Two-way ANOVA was employed for statistical significance (*p<0.05, **p < 0.01, ***p < 0.0001, ****p < 0.0001, ns: not significant).

Article Snippet: The antibodies used for immunoblotting: SLC1A5, SLC25A5, S727STAT3, and Total STAT3 (Cell signaling, USA), β-actin and c-Met (Santa-Cruz, Dallas, TX), ALDH1A1, Y705STAT3, CD63, CD81, TSG101, CD9, Anti-HLA DR + DP + DQ, and Calnexin (Abcam, Cambridge, UK), CD44, CD24, CD133 (BD Bioscience), CD163 (ThermoFisher Scientific, USA), Anti-SLC1A5-FITC antibody were purchased from Alomone labs and Anti-FAP from R&D systems.

Techniques: Activation Assay, CCK-8 Assay, Microscopy, Western Blot, Expressing, Isolation, Transfection, Staining, Stable Transfection, Plasmid Preparation, Over Expression, Cell Culture, Migration, Control, Software

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: REVs induce resistance in different tumor models. (A) HCT116 cells were pre-exposed with SEVs, REVs, and SLC1A5 -OE-EVs for 48 hours before treatment with 5 FU for 24 hours. Cell viability was measured using CCK-8 assay. (B) Effect on spheroid formation was assessed in the samples from A; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar:10 µm). (C) A375 cells were pre-exposed to SEVs, REVs, and SLC1A5 -OE-EVs for 48 hours before treatment with Vemurafinib for 24 hours. Cell viability was measured using CCK-8 assay. (D) Effect on spheroid formation was assessed in the samples from C; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar:10 µm). (E) H1975 cells were pre-exposed to 10 µg EVs isolated from patient-derived cell lines (PDCEVs) for 48 hours before treatment with Osimertinib for 24 hours. Cell viability was measured using CCK-8 assay. (F, G) Spheroid formation in matrigel was assessed in the samples from E using the hanging drop technique (seeded in Matrigel) and left for 7-10 days before viewing under the microscope using 10X magnification or analyzed by confocal microscopy, respectively (Scale bar: 50 µM or 20 µM, respectively). (H) Western blot showing increased SLC1A5, SLC25A5, ALDH1A1, STAT3pY705, and STAT3pS727 levels in total lysates of H1975 cells exposed for 48 hours with increasing concentration of PDCEVs. Total STAT3 and B-actin were used as loading controls. (I) H1975 cells were pre-exposed with PDCEVs for 48 hours before treatment with 2 µM Gefitinib, Osimertinib, Crizotinib, Selumetinib, Afatinib, Bosutinib, and Debrafenib for 24 hours. Cell viability was measured using CCK-8 assay. Data are representative of at least 3 independent experiments and shown as mean ± SD of biological triplicates. Two-way ANOVA was employed for statistical significance (** p < 0.01, *** p < 0.001, ns: not significant).

Article Snippet: The antibodies used for immunoblotting: SLC1A5, SLC25A5, S727STAT3, and Total STAT3 (Cell signaling, USA), β-actin and c-Met (Santa-Cruz, Dallas, TX), ALDH1A1, Y705STAT3, CD63, CD81, TSG101, CD9, Anti-HLA DR + DP + DQ, and Calnexin (Abcam, Cambridge, UK), CD44, CD24, CD133 (BD Bioscience), CD163 (ThermoFisher Scientific, USA), Anti-SLC1A5-FITC antibody were purchased from Alomone labs and Anti-FAP from R&D systems.

Techniques: CCK-8 Assay, Microscopy, Isolation, Derivative Assay, Confocal Microscopy, Western Blot, Concentration Assay

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: REVs regulate tumor microenvironment. (A) Western blot showing increased SLC1A5, TGFβ, αSMA, CD95, and Vimentin levels in total lysates of NL-20 cells exposed for 48 hours with SEVs and REVs. GAPDH was used as a loading control. (B) The scratch assay shows an increase in the migratory capacity of NL-20 cells exposed to SEVs and REVs for 48 hours (Scale bar: 10 cm). (C) Effect on spheroid formation was assessed in the co-culture (NL-20, NL-20+SEVs, and NL-20+REVs with HCC827) cells after treatment with gefitinib for 24 hours; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 10 mM). (D) From the same setup, 75,000 cells were re-seeded in ThinCert® cell culture inserts for 48 hours, stained with crystal violet, and viewed under a microscope (Scale bar: 100 µm) and (E) quantified by dissolving with 33% (v:v) acetic acid and read at an absorbance of 590 nm as described in Materials and Methods. Migration rates are plotted in percentages with respect to control cells. (F) Western blot showing increased SLC1A5, αSMA, CD95, Vimentin, STAT3pY705, and STAT3pS727 levels in total lysates MRC-5 cells exposed for 48 hours with SEVs and REVs. Total STAT3 and GAPDH was used as loading controls. (G) Increase expression of FAP in MRC-5 cells exposed to REVs and analyzed using Flow cytometry. For flow analysis, at least 10,000 cells were analyzed as described in Materials and Methods. (H) Scratch assay shows an increase in the migratory capacity of NL-20 cells after 48 hours of exposure to SEVs and REVs. (I) Effect on spheroid formation was assessed in the co-culture (NL-20, NL-20+SEVs, and NL-20+REVs with HCC827) cells after treatment with gefitinib for 24 hours; 3000 cells were re-seeded on low attachment spheroid plates and left for 7-10 days before viewing under the microscope using 10X magnification (Scale bar: 10 mM). (J) From the same setup, 75,000 cells were reseeded in ThinCert® cell culture inserts for 48 hours stained with crystal violet and viewed under a microscope (Scale bar: 50 µm) and (K) quantified by dissolving with 33% (v:v) acetic acid and measuring absorbance at 590 nm as described in Materials and Methods. Migration rates are plotted in percentages with respect to control cells. Data are representative of at least 3 independent experiments and shown as mean ± SD of biological triplicates. Two-way ANOVA was employed for statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (L) Western blot showing increased CD163, C-Mat, and HLA DR+DP+DQ level in naïve macrophages (M0) after being exposed to SEVs and REVs for 48 hours.

Article Snippet: The antibodies used for immunoblotting: SLC1A5, SLC25A5, S727STAT3, and Total STAT3 (Cell signaling, USA), β-actin and c-Met (Santa-Cruz, Dallas, TX), ALDH1A1, Y705STAT3, CD63, CD81, TSG101, CD9, Anti-HLA DR + DP + DQ, and Calnexin (Abcam, Cambridge, UK), CD44, CD24, CD133 (BD Bioscience), CD163 (ThermoFisher Scientific, USA), Anti-SLC1A5-FITC antibody were purchased from Alomone labs and Anti-FAP from R&D systems.

Techniques: Western Blot, Control, Wound Healing Assay, Co-Culture Assay, Microscopy, Cell Culture, Staining, Migration, Expressing, Flow Cytometry

Journal: Theranostics

Article Title: Tumor-derived extracellular vesicles convey solute transporters to induce bioenergetic dependence shift contributing to treatment resistance

doi: 10.7150/thno.100374

Figure Lengend Snippet: REVs regulate tumor microenvironment in vivo . (A, B) Flow cytometry data showing increased expression of SLC1A5 on HCC827 xenograft slices 48 hours after co-culture with HCC827-GR slices. For flow analysis, at least 10,000 cells were analyzed by flow cytometry as described in Materials and Methods (MFI: Median fluorescence intensity). Data are representative of at least 3 independent experiments and shown as mean ± SD of biological triplicates. An unpaired T-test was employed for statistical significance (*p < 0.05). (C) Surface expression of SLC1A5, CD163, and FAP was assessed following co-culture of HCC827 xenograft slices with HCC827-GR xenograft slices for 48 hours. Image viewed at 10X and 20X magnification by confocal microscopy (Scale bar: 100 µM).

Article Snippet: The antibodies used for immunoblotting: SLC1A5, SLC25A5, S727STAT3, and Total STAT3 (Cell signaling, USA), β-actin and c-Met (Santa-Cruz, Dallas, TX), ALDH1A1, Y705STAT3, CD63, CD81, TSG101, CD9, Anti-HLA DR + DP + DQ, and Calnexin (Abcam, Cambridge, UK), CD44, CD24, CD133 (BD Bioscience), CD163 (ThermoFisher Scientific, USA), Anti-SLC1A5-FITC antibody were purchased from Alomone labs and Anti-FAP from R&D systems.

Techniques: In Vivo, Flow Cytometry, Expressing, Co-Culture Assay, Fluorescence, Confocal Microscopy