Journal: Research

Article Title: Tumor-Derived Exosomal Fatty Acids Reprogram Neutrophils to Drive Neutrophil Extracellular Traps Formation and Lung Cancer Progression

doi: 10.34133/research.1278

Figure Lengend Snippet: Exposure to lung cancer cells led to lipid accumulation of neutrophils. (A) Volcano plots showing differentially expressed genes following A549-conditioned medium (CM) stimulation. (B) Statistics of reactome enrichment analysis in A549-CM-treated neutrophils compared with control neutrophils. (C) Nile Red staining for mouse bone marrow neutrophils (BMNs) and tumor-infiltrated neutrophils (TINs). (D) BMNs were cultured with LLC-CM for 4 h. Lipid levels were measured by Nile Red staining with Cytation 3 Cell Reader. (E) Human differentiated HL-60 (dHL-60) neutrophils were cultured with A549-CM for 4 h. Lipid levels were measured by Nile Red staining with flow cytometry. (F and G) Lipid levels in BMNs (F) and dHL-60 cells (G) were measured by BODIPY 493/503 staining with a confocal microscope. All data are shown as means ± standard error of the mean (SEM) ( n = 3 to 4, * P < 0.05, ** P < 0.01, and *** P < 0.001). MFI, mean fluorescence intensity.

Article Snippet: The following cell lines were used: LLC (CL-0140; Procell), A549 (CL-0016; Procell), and HL-60 (CL-0110; Procell), cultured in Dulbecco’s modified Eagle’s medium, F12, and RPMI 1640 media (KeyGEN, China), respectively; Beas2B (FH0319; Fuheng) and MLE (FH1103; Fuheng) cells, both cultured in RPMI 1640.

Techniques: Control, Staining, Cell Culture, Flow Cytometry, Microscopy, Fluorescence

Journal: Research

Article Title: Tumor-Derived Exosomal Fatty Acids Reprogram Neutrophils to Drive Neutrophil Extracellular Traps Formation and Lung Cancer Progression

doi: 10.34133/research.1278

Figure Lengend Snippet: Lung cancer cell-derived exosomes potentiated lipid accumulation in neutrophils, thereby boosting their protumoral function. (A) Mouse bone marrow neutrophils (BMNs) were cultured with LLC-conditioned medium (CM), exosomes (EXOs)-free LLC-CM or LLC-EXOs for 4 h. Lipid levels were quantified using Nile Red staining with Cytation 3 Cell Reader. (B) Human differentiated HL-60 (dHL-60) neutrophils were cultured with A549-CM, EXOs-free A549-CM or A549-EXOs for 4 h. Lipid levels were quantified using Nile Red staining with Cytation 3 Cell Reader. (C) BMNs were cultured with LLC-CM or GW4869 (GW, 10 μM, 48 h)-treated LLC-CM. Lipid levels were quantified using Nile Red staining with Cytation 3 Cell Reader. (D) Human dHL-60 neutrophils were cultured A549-CM or GW-treated A549-CM. Lipid levels were quantified using Nile Red staining with Cytation 3 Cell Reader. (E and F) BMNs were cultured with CD9-GFP-CM or control-CM. (E) Green fluorescent protein (GFP) intensity of BMNs was detected by Cytation 3 Cell Reader at indicated time. (F) Lipid levels were quantified using Nile Red staining at indicated time. (G) LLC-GFP-exosomes taken up by BMNs were visualized via fluorescence microscopy. The arrow: GFP-exosome. (H) BMNs lipid levels were quantified using Nile Red staining after culturing with LLC-EXOs or murine lung epithelial cell line (MLE)-EXOs for 4 h. (I) dHL-60 cell lipid levels were measured by Nile Red staining after culturing with A549-EXOs or Beas2b-EXOs for 4 h. (J) The proliferation of LLC cells cultured in CM from BMNs (pretreated with or without LLC-exosomes) was determined by CCK-8 assay. (K) The proliferation of A549 cells cultured in CM from dHL-60 cells (pretreated with or without A549-exosomes) was determined by CCK-8 assay. (L) Tumor growth of LLC tumors receiving BMNs treated with or without exosomes. Representative tumor pictures are shown. (M) The tumor volumes were monitored. (N) Tumor weight analysis. All data are shown as means ± standard error of the mean (SEM) ( n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant). OD.450, optical density 450.

Article Snippet: The following cell lines were used: LLC (CL-0140; Procell), A549 (CL-0016; Procell), and HL-60 (CL-0110; Procell), cultured in Dulbecco’s modified Eagle’s medium, F12, and RPMI 1640 media (KeyGEN, China), respectively; Beas2B (FH0319; Fuheng) and MLE (FH1103; Fuheng) cells, both cultured in RPMI 1640.

Techniques: Derivative Assay, Cell Culture, Staining, Control, Fluorescence, Microscopy, CCK-8 Assay

Journal: Research

Article Title: Tumor-Derived Exosomal Fatty Acids Reprogram Neutrophils to Drive Neutrophil Extracellular Traps Formation and Lung Cancer Progression

doi: 10.34133/research.1278

Figure Lengend Snippet: Lung cancer cell-derived exosomes deliver free fatty acids (FFA) into neutrophils to induce lipid accumulation. (A) PCA score plot and (B) variable importance in projection (VIP) plot of lipid metabolites from bone marrow neutrophils (BMNs) treated with or without LLC-EXOs. The VIP plot highlights the top 10 key metabolites contributing to group separation. (C) Heat map illustrating the fatty acids metabolic profiles. (D) FFA levels were measured in exosomes derived from murine lung epithelial (MLE) and LLC cells. (E) FFA levels were measured in BMNs cultured with exosomes. (F) Schematic diagram of fatty acid transfer experiment. (G) C16 fluorescence intensity was measured in LLC cultured with C16 for 24 h. (H) C16 fluorescence intensity was measured in exosomes from LLC. (I) C16 fluorescence intensity was measured in BMNs cultured with exosomes from LLC. (J) C16 fluorescence intensity was measured in A549 cultured with C16 for 24 h. (K) C16 fluorescence intensity was measured in exosomes from A549. (L) C16 fluorescence intensity was measured in differentiated HL-60 (dHL-60) cultured with exosomes from A549. (M) C16-LLC cells were cultured with or without GW4869. C16 fluorescence intensity was measured in BMNs cultured with LLC-conditioned medium (CM). (N) C16-A549 cells were cultured with or without GW4869. C16 fluorescence intensity was measured in dHL-60 cultured with LLC-CM. (O) BMNs were cultured with LLC-CM with or without C75 for 4 h. Lipid levels were quantified using Nile Red staining. (P) dHL-60 were cultured with A549-CM with or without C75 for 4 h. Lipid levels were quantified using Nile Red staining. All data are shown as means ± standard error of the mean (SEM) ( n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant).

Article Snippet: The following cell lines were used: LLC (CL-0140; Procell), A549 (CL-0016; Procell), and HL-60 (CL-0110; Procell), cultured in Dulbecco’s modified Eagle’s medium, F12, and RPMI 1640 media (KeyGEN, China), respectively; Beas2B (FH0319; Fuheng) and MLE (FH1103; Fuheng) cells, both cultured in RPMI 1640.

Techniques: Derivative Assay, Cell Culture, Fluorescence, Staining

Journal: Research

Article Title: Tumor-Derived Exosomal Fatty Acids Reprogram Neutrophils to Drive Neutrophil Extracellular Traps Formation and Lung Cancer Progression

doi: 10.34133/research.1278

Figure Lengend Snippet: Lung cancer cell-derived exosomes promote neutrophil-induced tumor progression in a neutrophil extracellular traps (NETs)-dependent manner. (A) Mouse bone marrow neutrophils (BMNs) were cultured with LLC-EXOs for 4 h. Supernatant myeloperoxidase (MPO)-DNA levels were assessed by enzyme-linked immunosorbent assay (ELISA). (B) Cell-free DNA (cfDNA) levels in the supernatant were quantified using Sytox green. (C) Citrullinated histone H3 (CitH3) expressions were measured by western blot. (D) Human differentiated HL-60 (dHL-60) neutrophils were cultured with A549- EXOs for 4 h. Supernatant MPO–DNA levels were measured by ELISA. (E) Supernatant cfDNA levels were measured by Sytox green staining. (F) CitH3 expressions were measured by western blot. (G) LLC were injected subcutaneously with phosphate-buffered saline (PBS), BMNs, or BMNs and LLC-EXOs. Deoxyribonuclease I (DNase I) was injected intraperitoneally daily. Representative tumor pictures are shown. (H) The tumor volumes were monitored. (I) Tumor weights analysis. (J) Immunofluorescence staining for MPO and CitH3 in tumor tissues. All data are shown as means ± standard error of the mean (SEM) ( n = 3 to 4, * P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant).

Article Snippet: The following cell lines were used: LLC (CL-0140; Procell), A549 (CL-0016; Procell), and HL-60 (CL-0110; Procell), cultured in Dulbecco’s modified Eagle’s medium, F12, and RPMI 1640 media (KeyGEN, China), respectively; Beas2B (FH0319; Fuheng) and MLE (FH1103; Fuheng) cells, both cultured in RPMI 1640.

Techniques: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Injection, Saline, Immunofluorescence

Journal: Research

Article Title: Tumor-Derived Exosomal Fatty Acids Reprogram Neutrophils to Drive Neutrophil Extracellular Traps Formation and Lung Cancer Progression

doi: 10.34133/research.1278

Figure Lengend Snippet: Lung cancer-derived exosomal fatty acids promote NETs formation through fatty acid β-oxidation (FAO) pathways activation in neutrophils. (A) Human differentiated HL-60 (dHL-60) neutrophils were cultured with A549-EXOs or free fatty acids for 4 h. Supernatant myeloperoxidase (MPO)-DNA levels were detected by enzyme-linked immunosorbent assay (ELISA). (B) Supernatant cell-free DNA (cfDNA) levels were measured by Sytox green staining. (C) Citrullinated histone H3 (CitH3) expressions were measured by western blot. (D) Mouse bone marrow neutrophils (BMNs) were cultured with LLC-EXOs or free fatty acids for 4 h. Supernatant MPO–DNA levels were measured by ELISA. (E) Supernatant cfDNA levels were measured by Sytox green staining. (F) CitH3 expressions were measured by western blot. (G) Gene expression analysis of FAO/oxidative phosphorylation pathways and (H) acylcarnitine heatmap in BMNs with or without LLC-EXOs treatment. (I and J) Analysis of CPT1a protein expression by western blot. (K) Quantification of mitochondrial mass in mouse BMNs cultured with LLC-EXOs, using MitoTracker staining and Cytation 3 Cell Reader. (L) Human dHL-60 neutrophils were cultured with A549-EXOs. Mitochondria mass was measured by MitoTracker staining with Cytation 3 Cell Reader. (M) Mouse BMNs were cultured with LLC-EXOs. mtROS was measured by mitoSOX staining with Cytation 3 Cell Reader. (N) Human dHL-60 neutrophils were cultured with A549-EXOs. mtROS was measured by mitoSOX staining. (O) Mouse BMNs were cultured with LLC-EXOs with 40 μM etomoxir (Eto) for 4 h. MPO–DNA levels were measured by ELISA. (P) cfDNA levels were measured by Sytox green staining. (Q) CitH3 expressions were measured by western blot. (R) dHL-60 neutrophils were cultured with A549-EXOs with 40 μM etomoxir for 4 h. MPO–DNA levels were measured by ELISA. (S) cfDNA levels were measured by Sytox green staining. (T) CitH3 protein expression was assessed by western blot. All data are shown as means ± standard error of the mean (SEM) ( n = 3 to 4, ** P < 0.01, *** P < 0.001).

Article Snippet: The following cell lines were used: LLC (CL-0140; Procell), A549 (CL-0016; Procell), and HL-60 (CL-0110; Procell), cultured in Dulbecco’s modified Eagle’s medium, F12, and RPMI 1640 media (KeyGEN, China), respectively; Beas2B (FH0319; Fuheng) and MLE (FH1103; Fuheng) cells, both cultured in RPMI 1640.

Techniques: Derivative Assay, Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Gene Expression, Phospho-proteomics, Expressing

Journal: Research

Article Title: Tumor-Derived Exosomal Fatty Acids Reprogram Neutrophils to Drive Neutrophil Extracellular Traps Formation and Lung Cancer Progression

doi: 10.34133/research.1278

Figure Lengend Snippet: Rab34 mediates neutrophil uptake of lung cancer-derived exosomes to promote NETs formation and tumor progression. (A) Volcano plots showing differentially expressed genes associated lung the prognosis of cancer patients. (B) Rab34 expression in bone marrow neutrophils (BMNs) was analyzed by western blot following a 4-h incubation with LLC-EXOs. (C) Human differentiated HL-60 (dHL-60) neutrophils were cultured with A549-EXOs for 4 h. Rab34 expression was measured by western blot. (D and E) Human dHL-60 neutrophils with or without Rab34 knockdown were cultured with fluorescein isothiocyanate (FITC)-labeled EXOs for 4 h. (D) FITC intensity was detected by Cytation 3 Cell Reader. (E) Lipid levels were measured by Nile Red staining. (F) Human dHL-60 neutrophils with or without Rab34 knockdown were cultured with A549-exosomes for 4 h. Mitochondrial mass was detected by MitoTracker staining. (G) mtROS was detected by mitoSOX staining. (H) Myeloperoxidase (MPO)-DNA levels were measured by enzyme-linked immunosorbent assay (ELISA). (I) Cell-free DNA (cfDNA) levels were measured by Sytox green staining in supernatant. (J) Citrullinated histone H3 (CitH3) and Rab34 expressions were measured by western blot. (K) Tumor growth of LLC tumors receiving BMNs treated with or without Rab34-small interfering RNA (siRNA) liposomes. Representative tumor pictures are shown. (L) The tumor volumes were monitored. (M) Tumor weight analysis. (N) Tumor-infiltrating neutrophil lipid content was quantified by flow cytometry using Nile Red staining. (O) MPO–DNA levels in tumor tissues were assessed via ELISA. (P) Following orthotopic implantation of LLC cells into C57 mice, the animals received phosphate-buffered saline (PBS) or exosome-pretreated neutrophils 7 d later, with or without prior Rab34 knockdown. Representative macroscopic and histological images of hematoxylin and eosin (H&E)-stained lung tissue are presented. (Q) Lung weights and (R) body weights were recorded. (S) MPO–DNA levels in lung tissues were determined by ELISA. Data are presented as the means ± standard error of the mean (SEM) ( n = 3 to 5, * P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: The following cell lines were used: LLC (CL-0140; Procell), A549 (CL-0016; Procell), and HL-60 (CL-0110; Procell), cultured in Dulbecco’s modified Eagle’s medium, F12, and RPMI 1640 media (KeyGEN, China), respectively; Beas2B (FH0319; Fuheng) and MLE (FH1103; Fuheng) cells, both cultured in RPMI 1640.

Techniques: Derivative Assay, Expressing, Western Blot, Incubation, Cell Culture, Knockdown, Labeling, Staining, Enzyme-linked Immunosorbent Assay, Small Interfering RNA, Liposomes, Flow Cytometry, Saline

Journal: Cancer Discovery

Article Title: PD-1 Blockade–Induced DKK1 Expression by CD8 + T Cells Promotes Blood–Brain Barrier Permeabilization

doi: 10.1158/2159-8290.CD-25-1222

Figure Lengend Snippet: Induction with anti–PD-1 antibody prior to cisplatin chemotherapy increases the intracranial therapeutic efficacy. A, A schematic illustration depicting various treatment groups and their therapeutic regimens, evaluating the efficacy of anti–PD-1 and cisplatin-based combination therapy sequences on experimental BrM of anti-PD-1–resistant LLC cells. B, An experimental BrM assay was performed on 8-week-old C57BL/6 mice treated with anti–PD-1 and cisplatin chemotherapy in opposing sequences. A Kaplan–Meier survival curve is plotted ( n = 6–7 mice/group). C–E, Eight-week-old C57BL/6 mice harboring subcutaneous LLC xenografts (∼100 mm 3 ) were treated with IgG, anti–PD-1, IgG + cisplatin, anti–PD-1 + cisplatin, and the opposing sequence, cisplatin + anti–PD-1, and the therapeutic response was monitored. Tumor growth across various treatment groups is plotted (Arrow indicates start of the treatment; n = 4–5 mice/group; C ). A bar graph showing the activation status of CD8 + T cells isolated from the spleens of these mice is plotted, as assessed by flow cytometry ( D ). A bar graph showing the percentage of dead cells [propidium iodide (PI)–positive] in a tumor cell killing assay, using CD8 + T cells isolated from the spleens of mice from the different conditions cocultured with LLC cells, as assessed by flow cytometry ( E ). F and G, Adoptive transfer of wild-type (Vector) and DKK1 KD [guide RNA (gRNA)] CD8 + T cells obtained from BALB/c mice was performed on 8-week-old SCID mice subjected to an experimental BrM assay using the 4T1 cancer cells. These mice were subsequently treated with a combination sequence of anti–PD-1 and cisplatin. An illustration depicting the experimental methodology for testing the effect of DKK1 KD on the therapeutic efficacy of the anti–PD-1 + cisplatin combination sequence is provided. Kaplan–Meier survival curve is plotted ( n = 6 mice/group; G ). H, Eight-week-old BALB/c mice, preconditioned with a single dose of IgG/anti–PD-1 or untreated (naïve), received cisplatin monotherapy 72 hours later. After 6 hours, the brains were harvested and prepared for the assessment of cisplatin concentration using LC/MS. A bar graph showing absolute quantification of cisplatin in the brains of BALB/c mice from various treatment groups is plotted ( n = 3–4 mice/group). Significance was assessed by means of the log-rank test for B and G , two-way ANOVA for C , and one-way ANOVA for D , E , and H . *, P < 0.05; **, P < 0.01; ***, P < 0.001. [ A, Created in BioRender. Raviv, Z. (2026) https://BioRender.com/rezdwrc ; F, Created in BioRender. Raviv, Z. (2026) https://BioRender.com/oo7uvma .]

Article Snippet: Murine EMT6 (cat. #CRL-2755; RRID:CVCL_1923), 4T1 (cat. #CRL-2539; RRID:CVCL_0125) mammary carcinoma, and LLC (cat. #CRL-1642; RRID:CVCL_4358) cell lines were purchased from the ATCC.

Techniques: Drug discovery, Sequencing, Clinical Proteomics, Activation Assay, Isolation, Flow Cytometry, Adoptive Transfer Assay, Plasmid Preparation, Concentration Assay, Liquid Chromatography with Mass Spectroscopy, Quantitative Proteomics