Journal: Cell Research

Article Title: Dynamic magneto-mechanical force in lysosomes induces durable macrophage repolarization for antitumor immunity

doi: 10.1038/s41422-025-01217-1

Figure Lengend Snippet: a RAW 264.7 cells were polarized into M0, M1 (induced by IFN-γ and LPS), or M2 (induced by IL-4 and IL-13) macrophages. M0 macrophages were incubated with MNMs and then treated with 1 Hz RMF for 15 min. Macrophages were stained with ActinRed (red), and nuclei were stained with Hoechst (blue). b Polarized M0 RAW 264.7 cells were incubated with MNMs and then treated with 1 Hz RMF for 15 min. Flow cytometry analysis of CD86 and CD206 expression was performed. c , d RNA-sequencing analyses of polarized M2 RAW 264.7 cells treated with or without 1 Hz RMF for 15 min. KEGG enrichment analysis and heat map analysis were performed ( n = 3 independent biological replicates). e , f RAW 264.7 cells or BMDMs were polarized into M0, M1, or M2 macrophages. M2 macrophages were incubated with MNMs and then treated with or without 1 Hz RMF for 15 min (M2-MNM represents M2 macrophages incubated with MNMs, and M2-RMF represents M2 macrophages incubated with MNMs and treated with RMF). Concentrations of M1-associated proteins (IL-1β) and M2-associated proteins (IL-10) were examined in the conditioned medium of these cells. Data are presented as means ± SD. Statistical significance was defined as P < 0.05 ( n = 3 independent biological replicates). g RAW 264.7 cells were polarized into M0, M1, or M2 macrophages. M0 macrophages were cultured in conditioned medium derived from LLC and incubated with or without MNMs, then treated with or without 1 Hz RMF for 15 min. mRNA levels of M1-associated genes ( Cd80 , Cd86 , Il1b , Il6 ) and M2-associated genes ( Cd163 , Cd206 , Il4 , Il10 ) were examined. Data are presented as means ± SD ( n = 4 independent biological replicates).

Article Snippet: To explore the proportions of different macrophage subtypes in tumor tissues after MagLMP treatment, macrophages in the sections were stained by immunofluorescence with anti-mouse CD206 (R&D Systems, AF2535), anti-mouse CD80 (Proteintech, 14292-1-AP), anti-mouse F4/80 (R&D Systems, MAB5580), and DAPI (Beyotime Biotech, C1002).

Techniques: Incubation, Staining, Flow Cytometry, Expressing, RNA Sequencing, Cell Culture, Derivative Assay

Journal: Cancer Cell International

Article Title: Comprehensive analysis of prognostic immune-related genes and drug sensitivity in cervical cancer

doi: 10.1186/s12935-021-02333-9

Figure Lengend Snippet: The prognostic IRGPs and their risk coefficients involved in PRSM

Article Snippet: Immunohistochemical staining was performed with the antibody against CD80 (1:100, 66406-1-lg, Proteintech) and antibody against CD28 (1:100, 65099-1-lg, Proteintech).

Techniques: Activation Assay, Transduction, Inhibition

Journal: Cancer Cell International

Article Title: Comprehensive analysis of prognostic immune-related genes and drug sensitivity in cervical cancer

doi: 10.1186/s12935-021-02333-9

Figure Lengend Snippet: IHC of CD80 and CD28 in adjacent normal tissues and tumor tissues from patients with cervical cancer. A CD80 and CD28 expression is lower in cervical squamous cell carcinoma tissues compared with adjacent normal tissues. B Comparison of CD80 and CD28 protein expression in 11 pairs of matched tissue section samples by IHC

Article Snippet: Immunohistochemical staining was performed with the antibody against CD80 (1:100, 66406-1-lg, Proteintech) and antibody against CD28 (1:100, 65099-1-lg, Proteintech).

Techniques: Expressing, Comparison

Journal: Frontiers in Oncology

Article Title: CA19-9-related macrophage polarization drives poor prognosis in HCC after immune checkpoint inhibitor treatment

doi: 10.3389/fonc.2024.1528138

Figure Lengend Snippet: Results of correlation between FUT3 expression and immune cell infiltration. (A) Gene Set Enrichment Analysis (GSVA) of the expression level between FUT3 and the relative abundance of 24 types of immune cells. The bubble size represents Spearman’s rank correlation coefficient. (B) Correlation between the expression level FUT3 and the relative enrichment scores of immune cells (including NK CD56 bright cells, macrophages, Tem, Th2 cells, and Th17 cells). (C) Spearman’s correlation analysis of the immune infiltration level of immune cells (including NK CD56 bright cells, macrophages, Tem, Th2 cells, and Th17 cells) in the high and low FUT3 expression groups. (D) Representative MRI images of patients with HCC with or without elevated CA19-9 levels before/after immunotherapy (left panel). Representative immunohistochemistry images (CD80 and CD206) of patients with HCC with or without elevated CA19-9 levels after immunotherapy (right panel). CD80, a key marker for M1 macrophages; CD206, a key marker for M2 macrophages.

Article Snippet: The primary antibodies (CD80 (1:100, Proteintech #66406) and CD206 (1:200, CST #24595)) were used in this study.

Techniques: Expressing, Immunohistochemistry, Marker

Journal: Acta Pharmaceutica Sinica. B

Article Title: Converting bacteria into autologous tumor vaccine via surface biomineralization of calcium carbonate for enhanced immunotherapy

doi: 10.1016/j.apsb.2023.08.028

Figure Lengend Snippet: A23187-mediated Ca 2+ influx and the immunological effects. The cumulative release of (A) Ca 2+ from Sal @CaCO 3 and (B) A23187 from Sal @CaCO 3 /A23187 at pH 7.4/6.5. (C) The intracellular Ca 2+ quantification and (D) the CLSM images of RAW264.7 cells post different treatments after staining with Fluo-8 AM. Scale bar = 20 μm. (E) Quantification results from (D). The flow cytometry results indicating the expression of (F) M1 macrophage marker CD80 and (G) M2 macrophage marker CD206. (H) The quantification of the MHC Ⅱ and CD86 double positive cells in DC2.4 cells post different treatments by flow cytometry. (I) IL-2 and (J) IFN- γ secretion from Jurkat T cells post different treatments. Data are presented as mean ± SD ( n = 3). ns, not significant. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001.

Article Snippet: The primary antibody against connexin 43 (Cx43, 26980-1-AP), CD80 (66406-1-Ig), CD86 (13395-1-AP), and CD206 (18704-1-AP) were bought from Proteintech Group (Chicago, USA).

Techniques: Staining, Flow Cytometry, Expressing, Marker

Journal: Acta Pharmaceutica Sinica. B

Article Title: Converting bacteria into autologous tumor vaccine via surface biomineralization of calcium carbonate for enhanced immunotherapy

doi: 10.1016/j.apsb.2023.08.028

Figure Lengend Snippet: Combinatorial immunological effects of Sal and Ca 2+ influx. (A) The apoptosis of B16F10 cells post different treatments detected by Annexin V-FITC/PI double staining. (B) The flow cytometry results showing the CRT exposure for B16F10 cells post different treatments. (C) The mean fluorescence intensity quantified from (B). The release of (D) HMGB1 and (E) ATP from B16F10 cells post different treatments measured by corresponding kits. The Cx43 expression of (F) B16F10 cells and (G) DC2.4 cells post different treatments. (H) The flow cytometry results indicating the fluorescence intensity of Calcein-AM transferred from B16F10 cells to DC2.4 cells. (I) The analysis of MHC II and CD86 double positive cells in DC2.4 cells post different treatments by flow cytometry. (J) The quantified results from (I). The flow cytometry results indicating the expression of (K) M1 macrophage marker CD86 and (L) M2 macrophage marker CD206. (M,N) The quantified results from (K,L). Data are presented as mean ± SD ( n = 4). (O) IFN- γ secretion from Jurkat T cells post different treatments. Data are presented as mean ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: The primary antibody against connexin 43 (Cx43, 26980-1-AP), CD80 (66406-1-Ig), CD86 (13395-1-AP), and CD206 (18704-1-AP) were bought from Proteintech Group (Chicago, USA).

Techniques: Double Staining, Flow Cytometry, Fluorescence, Expressing, Marker

Journal: Acta Pharmaceutica Sinica. B

Article Title: Converting bacteria into autologous tumor vaccine via surface biomineralization of calcium carbonate for enhanced immunotherapy

doi: 10.1016/j.apsb.2023.08.028

Figure Lengend Snippet: The anti-tumor mechanisms of Sal @CaCO 3 /A23187. The representative flow cytometry results indicating the ratio of (A) activated DCs (CD11c + CD86 + ), (B) M1 macrophages (F4/80 + CD80 + ), (C) M2 macrophages (F4/80 + CD206 + ), (D) CD4 + T cells (CD3 + CD4 + ) and (E) CD8 + T cells (CD3 + CD8 + ). (F–J) The quantified results of (A–E). (K) The protein expression of CD86, CD80, CD206, CD4 and CD8 in tumor tissues of mice post different treatments. (L) The immunofluorescence images of CD86, CD80, CD206, CD4 and CD8 in tumor sections from mice post different treatments. Scale bar = 50 μm. (M) The content of TNF- α and IL-10 in tumor tissues of mice post different treatments. Data are presented as mean ± SD ( n = 3). ns, not significant. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: The primary antibody against connexin 43 (Cx43, 26980-1-AP), CD80 (66406-1-Ig), CD86 (13395-1-AP), and CD206 (18704-1-AP) were bought from Proteintech Group (Chicago, USA).

Techniques: Flow Cytometry, Expressing, Immunofluorescence

Journal: Theranostics

Article Title: ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation

doi: 10.7150/thno.52435

Figure Lengend Snippet: ILT4 in EGFR-activated tumor cells promoted TAM recruitment and M2-like polarization. (A-B) Patients with high ILT4 expression in tumor cells of NSCLC tissues showed markedly elevated infiltration of CD68 + TAMs by IHC analysis. (A) Representative images of ILT4 expression and TAM infiltration, brown granules represent positive staining. (B) Average results from 80 patients. ILT4-low and -high were defined by IHC score < 6 (median) and ≥ 6. Scale bar: 20 µm. (C-D) TAMs induced by PC9 and H1975 cells displayed increased migration ability compared with parental macrophages, whereas ILT4 knockdown in PC9 and H1975 cells restricted tumor-induced TAM migration. Tumor cells were first transfected with lentivirus carrying ILT4 shRNA for 48 h, and CM was collected for macrophage culture and TAM induction. The migration ability of TAMs was evaluated by the Transwell migration assay. (C) Images of migrated cells and (D) Average results from 3 independent experiments. Scale bar: 50 µm. *, p < 0.05; **, p < 0.01 compared with the medium group; ***, p < 0.001. ### , p < 0.001 compared with the LV-shNC group. (E-F) ILT4 knockdown in PC9 and H1975 cells decreased the secretion of CCL2 and CCL5. (E) showed ELISA results of CCL2, (F) showed CCL5. (G-H) Recombinant human CCL2 or CCL5 reversed ILT4 knockdown and decreased macrophage migration. 100 ng/mL recombinant human CCL2 or 200 ng/mL CCL5 were added to the CM of ILT4 knockdown PC9 and H1975 cells to induce TAM migration. The migration ability of TAMs was evaluated by the Transwell migration assay. (G) Images of migrated cells. (H) Average results from 3 independent experiments. Scale bar: 50 µm. *, p < 0.05; **, p < 0.01 compared with the medium group; ***, p < 0.001. compared with LV-shNC group. (I-J) Patients with high ILT4 expression in tumor cells displayed more frequent CD206 + but less CD86 + TAM accumulation in tumor tissues by IHC analysis. (I) Representative images of CD68 + TAMs, CD206 + M2-like TAMs and CD86+ M1-like TAMs frequencies; brown granules represent positive staining. (J) Average results from 80 patients. ILT4-low and -high were defined by IHC score < 6 and ≥ 6 respectively. Scale bar: 20 µm. (K-L) ILT4 knockdown in PC9 and H1975 cells decreased M2-like markers (CD163, CD206, IL-10 and Arg1) and increased M1-like markers (CD80, CD86, IL-12 and TNFα) in TAMs by real-time PCR (K) and flow cytometry (L). TAMs were induced by CM as in (D) for 24 h. (M-N) TAMs induced by ILT4-downregulated PC9 and H1975 cells promoted the proliferation (M) and IFN-γ expression (N) of T cells. For T cell proliferation assay, CD3 + T cells separated from fresh PBMCs were first pre-activated by anti-CD3 for 24 h and stained with CFSE (1:1000), and then co-cultured for 4 days with TAMs induced by CM of ILT4-downregulated PC9 and H1975 cells. Flow cytometry was performed to determine CFSE strength. For IFN-γ expression, pre-activated CD3 + T cells were co-cultured with TAMs as described above for 48 h and IFN-γ levels were analyzed by flow cytometry. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Arg1: arginase 1; CM, conditioned medium; LV-shILT4: lentivirus carrying ILT4 shRNA; LV-shNC: lentivirus carrying control shRNA.

Article Snippet: Following antigen retrieval, the sections were incubated with 3% hydrogen peroxide for 20 min. Tissue slides were then incubated overnight at 4 °C with following primary antibodies: anti-ILT4 antibody (1:50; Affinity; Cat No. DF9604), anti-pEGFR antibody (1:200; Abcam; Cat No. ab40815), anti-PD-L1 antibody (1:100; CST; Cat No.13684), anti-CD68 antibody (1:100; Abcam; Cat No. ab125212), anti-CD206 antibody (1:100; Abcam; Cat No. ab64693), anti-CD163 antibody (1:100; Abcam; Cat No. ab182422), anti-CD3 antibody (1:200; Abcam; Cat No. ab5690; 1:100; CST; Cat No. 85061), anti-IFN-γ antibody (1:50; Affinity; Cat No. DF6045), and anti-F4/80 antibody (1:200; Abcam; Cat No. ab111101), anti-CD80 antibody (1:500; Boster; Cat No. BM4121), anti-CD86 antibody (1:500; Proteintech; Cat No. 66406-1-Ig), anti-PIRB antibody (1:100; Immunoway; Cat No. YN1914).

Techniques: Expressing, Staining, Migration, Knockdown, Transfection, shRNA, Transwell Migration Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Real-time Polymerase Chain Reaction, Flow Cytometry, Proliferation Assay, Cell Culture, Control

Journal: Life sciences

Article Title: TPX2 influences the regulation of macrophage polarization via the NF-κB pathway in lung adenocarcinoma.

doi: 10.1016/j.lfs.2024.122437

Figure Lengend Snippet: Fig.2 Expression of TPX2 in A549 cells affects macrophage polarization. (A) Detection of macrophage surface markers CD80 and CD163 after cell supernatant induction in A549 group, Vehicle group and TPX2-shRNA group. (B) The analysis percentage of macrophage surface marker CD80, CD163; CD80/CD163 after induction by cell supernatant of the three groups. (C) CD80 and CD163 protein expression in macrophages after induction in the supernatant of the three groups cells. (D) The

Article Snippet: Antibodies: TPX2 (ab252944, Abcam, UK), CD80/B7-1 (66406-1-Ig, Proteintech, China), CD163 (16646-1-AP, Proteintech, China), M-CSF (ab233387, Abcam, UK).

Techniques: Expressing, shRNA, Marker

Journal: Life sciences

Article Title: TPX2 influences the regulation of macrophage polarization via the NF-κB pathway in lung adenocarcinoma.

doi: 10.1016/j.lfs.2024.122437

Figure Lengend Snippet: Fig.4 The expression of M-CSF and CD163 in transplant tumors after TPX2 silencing. (A-C) Comparison of tumor-forming ability of Vehicle and TPX2-shRNA groups. (D, E) IHC detection of TPX2, M-CSF, and CD163 expression in Vehicle and TPX2-shRNA groups. (F) Immunofluorescence

Article Snippet: Antibodies: TPX2 (ab252944, Abcam, UK), CD80/B7-1 (66406-1-Ig, Proteintech, China), CD163 (16646-1-AP, Proteintech, China), M-CSF (ab233387, Abcam, UK).

Techniques: Expressing, Comparison, shRNA, Immunofluorescence

Journal: Acta Pharmaceutica Sinica. B

Article Title: Converting bacteria into autologous tumor vaccine via surface biomineralization of calcium carbonate for enhanced immunotherapy

doi: 10.1016/j.apsb.2023.08.028

Figure Lengend Snippet: A23187-mediated Ca 2+ influx and the immunological effects. The cumulative release of (A) Ca 2+ from Sal @CaCO 3 and (B) A23187 from Sal @CaCO 3 /A23187 at pH 7.4/6.5. (C) The intracellular Ca 2+ quantification and (D) the CLSM images of RAW264.7 cells post different treatments after staining with Fluo-8 AM. Scale bar = 20 μm. (E) Quantification results from (D). The flow cytometry results indicating the expression of (F) M1 macrophage marker CD80 and (G) M2 macrophage marker CD206. (H) The quantification of the MHC Ⅱ and CD86 double positive cells in DC2.4 cells post different treatments by flow cytometry. (I) IL-2 and (J) IFN- γ secretion from Jurkat T cells post different treatments. Data are presented as mean ± SD ( n = 3). ns, not significant. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001.

Article Snippet: The primary antibody against connexin 43 (Cx43, 26980-1-AP), CD80 (66406-1-Ig), CD86 (13395-1-AP), and CD206 (18704-1-AP) were bought from Proteintech Group (Chicago, USA).

Techniques: Staining, Flow Cytometry, Expressing, Marker

Journal: Acta Pharmaceutica Sinica. B

Article Title: Converting bacteria into autologous tumor vaccine via surface biomineralization of calcium carbonate for enhanced immunotherapy

doi: 10.1016/j.apsb.2023.08.028

Figure Lengend Snippet: Combinatorial immunological effects of Sal and Ca 2+ influx. (A) The apoptosis of B16F10 cells post different treatments detected by Annexin V-FITC/PI double staining. (B) The flow cytometry results showing the CRT exposure for B16F10 cells post different treatments. (C) The mean fluorescence intensity quantified from (B). The release of (D) HMGB1 and (E) ATP from B16F10 cells post different treatments measured by corresponding kits. The Cx43 expression of (F) B16F10 cells and (G) DC2.4 cells post different treatments. (H) The flow cytometry results indicating the fluorescence intensity of Calcein-AM transferred from B16F10 cells to DC2.4 cells. (I) The analysis of MHC II and CD86 double positive cells in DC2.4 cells post different treatments by flow cytometry. (J) The quantified results from (I). The flow cytometry results indicating the expression of (K) M1 macrophage marker CD86 and (L) M2 macrophage marker CD206. (M,N) The quantified results from (K,L). Data are presented as mean ± SD ( n = 4). (O) IFN- γ secretion from Jurkat T cells post different treatments. Data are presented as mean ± SD ( n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: The primary antibody against connexin 43 (Cx43, 26980-1-AP), CD80 (66406-1-Ig), CD86 (13395-1-AP), and CD206 (18704-1-AP) were bought from Proteintech Group (Chicago, USA).

Techniques: Double Staining, Flow Cytometry, Fluorescence, Expressing, Marker

Journal: Acta Pharmaceutica Sinica. B

Article Title: Converting bacteria into autologous tumor vaccine via surface biomineralization of calcium carbonate for enhanced immunotherapy

doi: 10.1016/j.apsb.2023.08.028

Figure Lengend Snippet: The anti-tumor mechanisms of Sal @CaCO 3 /A23187. The representative flow cytometry results indicating the ratio of (A) activated DCs (CD11c + CD86 + ), (B) M1 macrophages (F4/80 + CD80 + ), (C) M2 macrophages (F4/80 + CD206 + ), (D) CD4 + T cells (CD3 + CD4 + ) and (E) CD8 + T cells (CD3 + CD8 + ). (F–J) The quantified results of (A–E). (K) The protein expression of CD86, CD80, CD206, CD4 and CD8 in tumor tissues of mice post different treatments. (L) The immunofluorescence images of CD86, CD80, CD206, CD4 and CD8 in tumor sections from mice post different treatments. Scale bar = 50 μm. (M) The content of TNF- α and IL-10 in tumor tissues of mice post different treatments. Data are presented as mean ± SD ( n = 3). ns, not significant. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: The primary antibody against connexin 43 (Cx43, 26980-1-AP), CD80 (66406-1-Ig), CD86 (13395-1-AP), and CD206 (18704-1-AP) were bought from Proteintech Group (Chicago, USA).

Techniques: Flow Cytometry, Expressing, Immunofluorescence