human nsclc cell lines  (ATCC)

Journal: Journal of Cellular and Molecular Medicine

Article Title: Exosomal HMGB1 Orchestrates NSCLC Progression and Immunosuppressive Macrophage Polarisation Through the TLR4 / NF ‐ κB / IL ‐6/ STAT3 Signalling Cascade

doi: 10.1111/jcmm.71050

Figure Lengend Snippet: Exosomal HMGB1 promotes NSCLC progression. (A) Western blot analysis of HMGB1 expression in vector control and HMGB1‐overexpressing (OE) A549 and PC9 cells. (B) Measurement of HMGB1 levels in exosomes derived from vector control and HMGB1 OE A549 and PC9 cells. (C) Cell proliferation assays of vector and HMGB1 OE A549 and PC9 cells. (D) Cell migration assays in vector and HMGB1 OE A549 and PC9 cells. (E) Colony formation assays for vector and HMGB1 OE A549 and PC9 cells. (F) Cell proliferation of A549 and PC9 cells treated with PBS, recombinant HMGB1 (10 or 100 ng) or exosomes derived from vector or HMGB1 OE cells (cell‐to‐exosome ratio = 1:10). (G) Cell migration of A549 and PC9 cells under the same treatment conditions as in (F). (H) Colony formation capacity of A549 and PC9 cells under the same treatment conditions as in (F). (I) Tumour volume in A549‐bearing mice treated with PBS, HMGB1 (10 ng or 100 ng per mouse) or exosomes from vector or HMGB1 OE cells (1 × 10 10 exosomes per mouse). (J) A549 and PC9 cells were seeded at densities of 1 × 10 5 , 1 × 10 6 and 5 × 10 6 cells per six‐well plate and cultured for 3 days. Exosome production was then analysed.

Article Snippet: Human NSCLC cell lines (A549, PC9), human embryonic kidney cells (HEK293T) and the human monocytic leukaemia cell line THP‐1 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Western Blot, Expressing, Plasmid Preparation, Control, Derivative Assay, Migration, Recombinant, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Exosomal HMGB1 Orchestrates NSCLC Progression and Immunosuppressive Macrophage Polarisation Through the TLR4 / NF ‐ κB / IL ‐6/ STAT3 Signalling Cascade

doi: 10.1111/jcmm.71050

Figure Lengend Snippet: Exosomal HMGB1 activates JAK/STAT3 signalling to promote NSCLC progression. (A) Protein–protein interaction (PPI) network analysis of HMGB1 using the STRING database. (B) Western blot analysis of NF‐κB in A549 and PC9 cells treated with PBS, recombinant HMGB1 (100 ng), exosomes from vector cells or exosomes from HMGB1 OE cells (cell‐to‐exosome ratio = 1:10). (C) ELISA quantification of IL‐6 in the supernatant of A549 and PC9 cells under the same treatment conditions as in (B). (D) Immunofluorescence staining of p‐STAT3 of A549 and PC9 cells under the same treatments, including an additional group co‐treated with exosomes from HMGB1 OE cells and NF‐κB inhibitor (50 μM). (E) Cell proliferation of A549 and PC9 cells treated with HMGB1 OE‐derived exosomes alone or in combination with NF‐κB inhibitor (50 μM) or STAT3 inhibitor (20 μM). (F) Cell migration under the same treatment conditions as in (E). (G) Colony formation assays of A549 and PC9 cells under the same treatment conditions as in (E).

Article Snippet: Human NSCLC cell lines (A549, PC9), human embryonic kidney cells (HEK293T) and the human monocytic leukaemia cell line THP‐1 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Western Blot, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Derivative Assay, Migration

Journal: Journal of Cellular and Molecular Medicine

Article Title: Exosomal HMGB1 Orchestrates NSCLC Progression and Immunosuppressive Macrophage Polarisation Through the TLR4 / NF ‐ κB / IL ‐6/ STAT3 Signalling Cascade

doi: 10.1111/jcmm.71050

Figure Lengend Snippet: Targeting HMGB1 signalling improves therapeutic outcomes in NSCLC. (A) Correlation analysis between immune infiltration scores and HMGB1 expression in 491 LUAD and 500 LUSC patients from the TCGA database. (B) Correlation between HMGB1 expression and the distribution of various immune cell subsets in LUAD and LUSC patients. (C, D) THP‐1–derived M0 macrophages were treated with PBS, HMGB1 (10 or 100 ng) or exosomes derived from vector or HMGB1 OE cells (cell‐to‐exosome ratio = 1:10). M1 macrophage markers (CD86, CD80, iNOS) and M2 markers (CD206, IL‐10, Arg1) were quantified by PCR. (E) Lewis tumour‐bearing mice were treated with PBS, HMGB1 OE‐derived exosomes (1 × 10 10 exosomes per mouse, twice per week), anti‐PD‐1 antibody (RMP1‐14, 200 μg per mouse, twice per week) or combination therapy ( n = 5 per group). Tumour volumes and apoptosis levels in tumour tissues (day 25) were assessed. (F) PC9 cells were treated with PBS or exosomes from HMGB1 OE cells (cell‐to‐exosome ratio = 1:10), followed by Osimertinib (50 nM, 48 h), and apoptosis was measured. (G) A549 and PC9 cells were similarly treated with PBS or HMGB1 OE‐derived exosomes, followed by Cisplatin (5 μM, 48 h), and apoptosis was analysed. (H) A549 and PC9 cells were similarly treated with paclitaxel (10 μM, 48 h) under the same conditions, and cell apoptosis was determined. (I) A549‐bearing mice were treated with HMGB1 OE‐derived exosomes (1 × 10 10 exosomes per mouse), followed by PBS, paclitaxel (PTX, 10 mg/kg, twice per week), STAT3 inhibitor (5 mg/kg, twice per week) or combination therapy. (J) Schematic diagram illustrating the proposed mechanism: HMGB1 upregulates TLR4, thereby activating the NF‐κB–IL‐6 axis and stimulating JAK2/STAT3 signalling to promote tumour progression. Concurrently, HMGB1 facilitates M2 macrophage polarisation.

Article Snippet: Human NSCLC cell lines (A549, PC9), human embryonic kidney cells (HEK293T) and the human monocytic leukaemia cell line THP‐1 were obtained from the American Type Culture Collection (ATCC, USA).

Techniques: Expressing, Derivative Assay, Plasmid Preparation