human pca cell lines du145  (ATCC)

Journal: Advanced Science

Article Title: Intercellular Horizontal Transfer of TXNDC5 mRNA via Extracellular Vesicles Contributes to Tumor‐Associated Macrophage‐Mediated Prostate Cancer Metastasis

doi: 10.1002/advs.202511052

Figure Lengend Snippet: CCM of M2 macrophages promotes migration and invasion of PCa cells. (A) Illustration of the strategy used to induce M0 and M2 macrophages in human leukemia monocytic THP‐1 cells. THP‐1 cells were differentiated into M0 macrophages by incubation with 100 ng/mL phorbol‐12‐myristate‐13‐acetate (PMA) for 48 h. M0 macrophages were polarized into M2 macrophages by culturing in 20 ng/mL IL‐4 and IL‐10 for 48 h. (B) Characterization of morphological changes in the course of differentiation from THP‐1 cells to M2 macrophages under a light microscope. Scale bars, 200 µm (100×), 100 µm (200×), 50 µm (400×). (C) ELISA revealed elevated levels of secretory TGF‐β, CCL22, and VEGFA in the CCM of M2 macrophages compared with those of M0 macrophages. (D) Evaluation of M2 macrophage‐associated protein markers by flow cytometry before and after differentiation. (E) Verification of classical M2‐associated genes by qRT‐PCR in M0 and M2 macrophages. Gene expression normalized to GAPDH . (F) The proportional change of CD68 + CD163 + cells upon induction was shown by IF. Scale bar, 100 µm. (G, H) Migration and invasion assays in M2 CCM‐treated versus M0 CCM‐treated DU145 (G) and PC3 (H) cells. (I, J) The wound healing assay showed different migration rates of DU145 (I) and PC3 (J) cells upon M2 CCM treatment. All experiments were repeated three times. Data presented as the mean ± SD. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; and ns for non‐significant data.

Article Snippet: Human PCa cell lines DU145 (HTB‐81, ATCC, RRID: CVCL_0105) cells and murine RAW264.7 (TIB‐71, ATCC, RRID: CVCL_0493) were maintained in Dulbecco's modified eagle medium (11965092, Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin.

Techniques: Migration, Incubation, Light Microscopy, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Quantitative RT-PCR, Gene Expression, Wound Healing Assay

Journal: Advanced Science

Article Title: Intercellular Horizontal Transfer of TXNDC5 mRNA via Extracellular Vesicles Contributes to Tumor‐Associated Macrophage‐Mediated Prostate Cancer Metastasis

doi: 10.1002/advs.202511052

Figure Lengend Snippet: EVs are a crucial contributor to M2 macrophage‐mediated migration and invasion of PCa cells. (A,B) Migration, invasion (A), and wound healing assay (B) of DU145 and PC3 cells treated with M2 CCM, EV‐depleted M2 CCM, or M2 CCM plus GW4869. (C) Quantitative statistics for the above experiment. (D‐E) Migration, invasion (D), and wound healing assay (E) of DU145 and PC3 cells treated with 50 µg/mL M0 or M2 EVs. (F) Quantitative statistics for the above (D‐E) experiment. All experiments were repeated three times. Data presented as the mean ± SD. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; and ns for non‐significant data.

Article Snippet: Human PCa cell lines DU145 (HTB‐81, ATCC, RRID: CVCL_0105) cells and murine RAW264.7 (TIB‐71, ATCC, RRID: CVCL_0493) were maintained in Dulbecco's modified eagle medium (11965092, Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin.

Techniques: Migration, Wound Healing Assay

Journal: Advanced Science

Article Title: Intercellular Horizontal Transfer of TXNDC5 mRNA via Extracellular Vesicles Contributes to Tumor‐Associated Macrophage‐Mediated Prostate Cancer Metastasis

doi: 10.1002/advs.202511052

Figure Lengend Snippet: M2 EVs are internalized by PCa cells and mediate MLS transition in recipient cells. (A, B) The internalization of both M0 and M2 EVs by DU145 (A) and PC3 (B) cells was shown by confocal microscopy. Scale bar, 10 µm. (C) GO analysis in PC3 cells based on the difference between treatment with 50 µg/mL M0 EVs and M2 EVs. (D) Regulation of MLS‐related markers in DU145 and PC3 after treatment with EVs based on qRT‐PCR analysis. (E) Characterization of MLS‐related protein markers with western blotting following treatment with 50 µg/mL M0 or M2 EVs. (F, G) Migration and invasion of DU145 and PC3 cells treated with M0 EVs (Con), MLSi, M2 EVs, and M2 EVs + MLSi (F). Quantitative statistics for the above experiment (G). MLSi: an MLS inhibitor Apigenin, 10 µM. All experiments were repeated three times. Data presented as the mean ± SD. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; and ns for non‐significant data.

Article Snippet: Human PCa cell lines DU145 (HTB‐81, ATCC, RRID: CVCL_0105) cells and murine RAW264.7 (TIB‐71, ATCC, RRID: CVCL_0493) were maintained in Dulbecco's modified eagle medium (11965092, Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin.

Techniques: Confocal Microscopy, Quantitative RT-PCR, Western Blot, Migration

Journal: Advanced Science

Article Title: Intercellular Horizontal Transfer of TXNDC5 mRNA via Extracellular Vesicles Contributes to Tumor‐Associated Macrophage‐Mediated Prostate Cancer Metastasis

doi: 10.1002/advs.202511052

Figure Lengend Snippet: TXNDC5 is the key mediator of M2 EVs‐induced PCa cell migration and invasion. (A) Venn diagram illustrating the cross‐analysis of upregulated genes in DU145 and PC3 cells following M2 EVs treatment based on RNA sequencing. The analysis integrated two independent DU145 datasets (M2 EV vs. M0 EV; 6 vs. 3 and 3 vs. 3) and one PC3 dataset (M2 EV vs. M0 EV; 4 vs. 3). Significantly upregulated genes (Foldchange > 1.5, p < 0.05) from the two DU145 datasets were combined (union) and subsequently intersected with upregulated genes from the PC3 dataset to define the final core set of conserved targets. (B) Pan‐cancer analysis of TXNDC5 mRNA expression across multiple cancer types from the TCGA database. (C) TXNDC5 expression in PCa tissues and normal prostate tissues based on public transcriptome data using the Xiantao Academic cloud platform. (D) Kaplan‐Meier survival analysis showing the association between TXNDC5 expression and PCa patient prognosis. (E, F) Representative IHC images (E) and quantitative analysis (F) of TXNDC5 protein expression in primary lesions of mPCa and nmPCa. Scale bar, 50 µm and 25 µm (for magnification). (G) Tumor Immune Estimation Resource (TIMER) analysis of the correlation between TXNDC5 expression and the infiltration of all macrophages or M2 macrophages. (H‐N) Changes of migration, invasion (H, I) and wound healing abilities (J, K) of DU145 and PC3 cells with TXNDC5 knockdown. Quantitative statistics for the above experiment (L‐N). All experiments were repeated three times. Data presented as the mean ± SD. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; and ns for non‐significant data.

Article Snippet: Human PCa cell lines DU145 (HTB‐81, ATCC, RRID: CVCL_0105) cells and murine RAW264.7 (TIB‐71, ATCC, RRID: CVCL_0493) were maintained in Dulbecco's modified eagle medium (11965092, Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin.

Techniques: Migration, RNA Sequencing, Expressing, Knockdown

Journal: Advanced Science

Article Title: Intercellular Horizontal Transfer of TXNDC5 mRNA via Extracellular Vesicles Contributes to Tumor‐Associated Macrophage‐Mediated Prostate Cancer Metastasis

doi: 10.1002/advs.202511052

Figure Lengend Snippet: M2 macrophages promote PCa metastasis via horizontal TXNDC5 mRNA transfer. (A) Demonstration of efficient inhibition of de novo TXNDC5 mRNA synthesis in DU145 and PC3 cells with 1 µg/mL actinomycin D at 6 and 24 h. (B) TXNDC5 mRNA levels in PCa cells under transcriptional inhibition. TXNDC5 mRNA levels were measured at 0 h (Con, 0 h) as a baseline, and at 6 h post‐inhibition in cells receiving no EVs (Con, 6 h), M0 EVs (50 µg/mL), or M2 EVs (50 µg/mL). (C) Schematic depiction of the generation of PKH67‐labeled M2 EVs containing Cy5‐tagged TXNDC5 ‐ Flag mRNA. (D) Single‐particle analysis by nFCM validating the successful generation of positive (PKH67⁺/Cy5⁺) EVs groups. (E, F) Validation of the protective role of EV lipid bilayer via RNase and Triton treatment (E). Quantitative statistics of P1 (PKH67⁺/Cy5⁺), P2 (PKH67−/Cy5⁺ group), P4 (PKH67⁺/Cy5− group) subgroups for the above experiment (F). (G) Confocal microscopy images showing internalization of dual‐labeled M2 EVs (PKH67, green; Cy5‐ TXNDC5 mRNA, red) by DU145 and PC3 cells. Nuclei were stained with DAPI (blue). Scale bar, 20 µm. (H, I) Quantitative comparison of mRNA loading efficiency between M0 and M2 EVs, shown by (H) the percentage of total mRNA‐carrying EVs (P1+P2) and (I) the ratio of mRNA‐carrying to total macrophage‐derived EVs (P1/[P1+P4]). (J) Western blotting analysis of Flag‐tagged protein expression in PCa cells after treatment with M0 or M2 EVs loaded with identical amounts of TXNDC5‐Flag mRNA. All experiments were repeated three times. Data presented as the mean ± SD. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; and ns for non‐significant data.

Article Snippet: Human PCa cell lines DU145 (HTB‐81, ATCC, RRID: CVCL_0105) cells and murine RAW264.7 (TIB‐71, ATCC, RRID: CVCL_0493) were maintained in Dulbecco's modified eagle medium (11965092, Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin.

Techniques: Inhibition, Labeling, Single Particle, Biomarker Discovery, Confocal Microscopy, Staining, Comparison, Derivative Assay, Western Blot, Expressing

Journal: Advanced Science

Article Title: Intercellular Horizontal Transfer of TXNDC5 mRNA via Extracellular Vesicles Contributes to Tumor‐Associated Macrophage‐Mediated Prostate Cancer Metastasis

doi: 10.1002/advs.202511052

Figure Lengend Snippet: TXNDC5 in M2 EVs drives MLS and metastatic progression in vitro and in vivo. (A–C) Migration, invasion (A), and wound healing assay (B) of DU145 and PC3 cells treated with 50 µg/mL M2 EVs and M2 EVs shTXNDC5 . Quantitative statistics for the above experiment (C). (D) Western blotting of MLS‐related markers of PCa cells treated with 50 µg/mL M2 EVs or M2 EVs shTXNDC5 . (E) Schematic diagram of the in vivo experimental design: DU145‐Luc cells pretreated with EVs were intracardially injected, followed by repeated EV administration via the tail vein. EV‐treated PCa cells are injected into the left ventricle of nude mice (day 0). 5 mg/kg EVs of three types were routinely injected into the tail vein of the mice at day 0, day 3, and day 6. The animals were subjected to downstream analyses at day 9 ( n = 6). (F,G) In vivo metastasis assessment by bioluminescence imaging. Representative whole‐body bioluminescence images (F). and quantification of total flux (G) showed that M2 EVs enhanced metastatic burden, which was attenuated by M2 EVs shTXNDC5 . (H) Representative H&E‐stained lung sections harvested from mice treated with control, M2 EVs or M2 EVs shTXNDC5 . Scale bar, 2 mm. (I, J) IHC analysis of MLS markers in lung metastases. Representative images (I) and quantitative scoring (J) showing expression of N‐Cadherin, SLUG, and ZEB1 in the control (M0 EVs), M2 EVs, and M2 EVs shTXNDC5 group. Data presented as the mean ± SD. * , p < 0.05; ** , p < 0.01; *** , p < 0.001; **** , p < 0.0001; and ns for non‐significant data.

Article Snippet: Human PCa cell lines DU145 (HTB‐81, ATCC, RRID: CVCL_0105) cells and murine RAW264.7 (TIB‐71, ATCC, RRID: CVCL_0493) were maintained in Dulbecco's modified eagle medium (11965092, Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin.

Techniques: In Vitro, In Vivo, Migration, Wound Healing Assay, Western Blot, Injection, Imaging, Staining, Control, Expressing

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: Characterization of PCa EVs. (A) EV size distribution measured by nanoparticle tracking analysis. (B) EV morphology visualized by transmission electron microscopy. Scale bars are 300 nm. (C) Western blot analysis was performed to investigate the expression levels of TSG101, Alix, Hsc70, CD9, calnexin, and cytochrome c in PC3 and DU145 cells and in their EVs. One representative of three experiments performed is shown.

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Expressing

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: PCa EVs promote lipolysis in adipocytes. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h. Lipid accumulation was then evaluated by cytofluorimetric analysis after staining with Bodipy 1 μM for 30 min. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h. FFA release was then evaluated by colorimetric assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (C) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h. Glycerol release was then evaluated by colorimetric assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (D) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 6 h. cAMP levels were then evaluated by ELISA assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control). (E) After PCa EV treatment (30 μg/mL for 12 h), Western blot analysis was performed to investigate the expression levels of PKA substrates, HSL, G0S2, and ATGL in 3T3‐L1 adipocytes. GAPDH expression was evaluated as a loading control. One representative of three experiments performed is shown. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. C (control), *** p < 0.001 vs. C (control).

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Staining, Control, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: The secretome from EV‐treated adipocytes increases PCa cell growth. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (96 h). Cell proliferation was then evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control). (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (72 h). Clonogenic ability was then evaluated by colony formation assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control), ** p < 0.01 vs. Adipo CM (control).

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Trypan Blue Exclusion Assay, Control, Colony Assay

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: The secretome from EV‐exposed adipocytes enhances PCa cell invasive potential. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Cell migration was then evaluated by wound healing assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05. Scale bars are 200 μm. (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Cell migration was then evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control), *** p < 0.001 vs. Adipo CM (control). (C) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (48 h). Anoikis was then evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. * p < 0.05 vs. Adipo CM (control). (D) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Cell invasion was then evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. ** p < 0.01 vs. Adipo CM (control).

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Migration, Wound Healing Assay, Transwell Assay, Control, Trypan Blue Exclusion Assay

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: FFAs in EV‐conditioned adipocyte secretome are taken up by PCa cells and mediate the above pro‐tumor effects. (A) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). FFA uptake was then evaluated by colorimetric assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Dunnet's test. *** p < 0.001. (B) 3T3‐L1 adipocytes were incubated with PC3 and DU145 EVs (30 μg/mL) for 48 h, and their medium was then given to PC3 and DU145 cells (24 h). Lipid accumulation was then evaluated by cytofluorimetric analysis after staining with Bodipy 1 μM for 30 min. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by t ‐test. *** p < 0.001 vs. C (control). (C) After delipidation, EV‐Adipo CM was given to PC3 and DU145 cells (72 h). Cell proliferation was then evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05, ** p < 0.01. (D) After delipidation, EV‐Adipo CM was given to PC3 and DU145 cells (24 h). Cell migration was then evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05.

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Incubation, Colorimetric Assay, Staining, Control, Trypan Blue Exclusion Assay, Migration, Transwell Assay

Journal: Biofactors (Oxford, England)

Article Title: Extracellular Vesicles Released From Prostate Cancer Cells Confer Pro‐Tumor Properties to Adipocytes by Stimulating Lipolysis

doi: 10.1002/biof.70067

Figure Lengend Snippet: FFAs in EV‐conditioned adipocyte secretome trigger the Akt signaling in PCa cells. (A) After EV‐Adipo CM treatment (24 h), Western blot analysis was performed to investigate the expression levels of p‐Akt in PC3 and DU145 cells. GAPDH expression was evaluated as a loading control. One representative of three experiments performed is shown. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. *** p < 0.001 vs. C (control). (B) After BEZ235 treatment (100 nM, 72 h) following EV‐Adipo CM treatment (3 h), PC3 and DU145 cell proliferation was evaluated by Trypan Blue exclusion assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05, *** p < 0.001. (C) After BEZ235 treatment (100 nM, 24 h) following EV‐Adipo CM treatment (3 h), PC3 and DU145 cell migration was evaluated by transwell assay. Each experiment was repeated three times. Data represent mean values ± SEM and were analyzed by one‐way ANOVA followed by Bonferroni's test. * p < 0.05, *** p < 0.001.

Article Snippet: PC3 and DU145 PCa cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS), glutamine, and antibiotics.

Techniques: Western Blot, Expressing, Control, Trypan Blue Exclusion Assay, Migration, Transwell Assay