human pca cell line c4 2b  (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