Journal: Advanced Science

Article Title: The Beneficial Role of Sunitinib in Tumor Immune Surveillance by Regulating Tumor PD‐L1

doi: 10.1002/advs.202001596

Figure Lengend Snippet: Sunitinib downregulates PD‐L1 via autophagic degradation. A) Summary of the distribution of differentially expressed genes (DE genes) identified from RNA sequencing data, which was generated from the A375 cells with or without Sunitinib treatment. The average logCPM (count per million) represented the gene expression level. The color represents differentially expressed genes (fold change >1.3, FDR < 0.05, see the Experimental Section; red: upregulation, blue: downregulation, gray: nonsignificant change). B) Top eight enriched GO biological processes pathways for genes upregulated or downregulated in A375 melanoma cell lines treated with Sunitinib. C) Representative image of the detection of autophagy using a transmission electron microscope. A375 or SK‐MEL‐28 cells were treated with 5 µmol Sunitinib for 24 h or dimethyl sulfoxide (DMSO) treated. The right side image indicates: autolysosome‐like structures. Bar, 2 µm. D) Representative image and quantification of Western blot analysis of p62 and LC3 in A375 and SK‐MEL‐28 cells after treatment with indicated doses of Sunitinib. GAPDH was used as loading control (left). Bar graphs are quantified results of three independent experiments and showed as means ± SD, * p < 0.05, ** p < 0.01 (right). E,F) Representative image and colocalization coefficiency of immunofluorescence staining in A375 and SK‐MEL‐28 cells for immunostaining of LC3 (green FITC) with red (Cy3) staining of E) p62 or F) LAMP1, nuclear was indicated by DAPI (blue). Scale bar, 20 µm in inset. Data are representatives of three independent assays. The LC3 and p62 or LAMP1 colocalization coefficiency is expressed as mean ± standard error of mean (SEM), n = ≈30–50 cells of three independent experiments. G) Representative image and quantification of western blot analysis of PD‐L1 in A375 and SK‐MEL‐28 cells after treatment with indicated doses of Sunitinib with or without BafA1 (the inhibitor of autophagy) under the IFN‐ γ exposure for 24 h. GAPDH was used as loading control (left). Bar graphs was quantified results of three independent experiments expressed as the mean ± SEM, * p < 0.05, ** p < 0.01 compared to the control (right).

Article Snippet: Anti‐SQSTM1/p62‐Rb (88588), LC3A/B (D3U4C) XP Rabbit mAb (12741) from Cell Signaling Technology; human anti‐PD‐L1 (ab213524 or ab213480); anti‐granzyme B antibody (ab4059) from Abcam; GAPDH (60004‐1‐Ig) from Proteintech; and anti‐CD8a (GB11068) from Servicebio were used.

Techniques: RNA Sequencing, Generated, Gene Expression, Transmission Assay, Microscopy, Western Blot, Control, Immunofluorescence, Staining, Immunostaining

Journal: Advanced Science

Article Title: The Beneficial Role of Sunitinib in Tumor Immune Surveillance by Regulating Tumor PD‐L1

doi: 10.1002/advs.202001596

Figure Lengend Snippet: Sunitinib regulates PD‐L1 via p62 dependent selective autophagy. A,B) Representative image (left) and colocalization coefficiency (right) of immunofluorescence staining in A) A375 and B) SK‐MEL‐28 cells for immunostaining of PD‐L1 (green FITC) with red (Cy3) staining of LC3, p62 or LAMP1, nuclear was indicated by DAPI (blue). Scale bar, 20 µm in inset. Data are representatives of three independent assays. The PD‐L1 and LC3, p62 or LAMP1 colocalization coefficiency is expressed as mean ± SEM, n = ≈30–50 cells of three independent experiments. C) Representative Western blot analysis of p62 after immunoprecipitation of endogenous PD‐L1 as indicated treatment from A375 cells. D) Representative Western blot analysis of PD‐L1 after immunoprecipitation of endogenous p62 as indicated treatment from A375 cells. E) Representative Western blot analysis of knockdown p62 in A375 cells treated with or without Sunitinib (2.5 µmol) for 24 h under IFN‐ γ exposure (left). Bar graphs were quantified results of three independent experiments expressed as the mean ± SEM, * p < 0.05, ** p < 0.01 compared to the control (right).

Article Snippet: Anti‐SQSTM1/p62‐Rb (88588), LC3A/B (D3U4C) XP Rabbit mAb (12741) from Cell Signaling Technology; human anti‐PD‐L1 (ab213524 or ab213480); anti‐granzyme B antibody (ab4059) from Abcam; GAPDH (60004‐1‐Ig) from Proteintech; and anti‐CD8a (GB11068) from Servicebio were used.

Techniques: Immunofluorescence, Staining, Immunostaining, Western Blot, Immunoprecipitation, Knockdown, Control

Journal: Advanced Science

Article Title: The Beneficial Role of Sunitinib in Tumor Immune Surveillance by Regulating Tumor PD‐L1

doi: 10.1002/advs.202001596

Figure Lengend Snippet: The reduction of PD‐L1 by Sunitinib induced p62 is clinically relevant. A) Representative image (left) and the quantification of normalized fluorescence intensity (right) of immunofluorescence staining for immunostaining of p62 and PD‐L1 in tumor mass from before and after Sunitinib treatment patients. The fluorescence of green (FITC) indicated the p62 and red (Cy3) indicated the PD‐L1. Blue (DAPI) indicated the nucleus. Scale bar, 50/100 µm in inset. The quantification of normalized fluorescence intensity was expressed as mean ± SEM, n = 6 views per sample, *** p < 0.001. B) Docetaxel‐ and sunitinib + docetaxel‐induced changes from baseline in the p62 expression to 14 d after treatment in cancer patients ( n = 14). C) Representative image (left) and the quantification of normalized fluorescence intensity (right) of immunofluorescence staining for immunostaining of p62 and PD‐L1 in tumor mass from anti‐PD‐1 treatment response or nonresponse patients. The fluorescence of green (FITC) indicated the p62 and red (Cy3) indicated the PD‐L1. Blue (DAPI) indicated the nucleus. Scale bar, 50/100 µm in inset. The quantification of normalized fluorescence intensity was expressed as mean ± SEM, n = 10 samples per group, *** p < 0.001. D) Clinicopathologic characteristics of anti‐PD‐1 monotherapy cohorts. Anti‐PD‐1, antiprogrammed death‐1; CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; PFS, progression‐free survival; OS, overall survival. Patients were stratified into response groups based on RECIST (Response Evaluation Criteria in Solid Tumors) 1.1 criteria. Patients with CR, PR, and SD > 3 months were classified as responders, while patients with SD ≤ 3 months and PD were classified as nonresponders. E) Kaplan–Meier estimates for progression‐free survival and overall survival; patients ( n = 19) were stratified in two groups: high p62 expression and low p62 expression. Significance was determined by the Gehan–Breslow–Wilcoxon test. Data represent mean ± SD, * p < 0.05,** p < 0.01. F) Kaplan–Meier estimates for progression‐free survival and overall survival; patients ( n = 19) were stratified in two groups: high PD‐L1 expression and low PD‐L1 expression. Significance was determined by the log rank test. Data represent mean ± SD, * p < 0.05,** p < 0.01. G) A proposed model illustrating the regulation of PD‐L1 stability was p62‐mediated selective autophagy degradation.

Article Snippet: Anti‐SQSTM1/p62‐Rb (88588), LC3A/B (D3U4C) XP Rabbit mAb (12741) from Cell Signaling Technology; human anti‐PD‐L1 (ab213524 or ab213480); anti‐granzyme B antibody (ab4059) from Abcam; GAPDH (60004‐1‐Ig) from Proteintech; and anti‐CD8a (GB11068) from Servicebio were used.

Techniques: Fluorescence, Immunofluorescence, Staining, Immunostaining, Expressing

Journal: Advanced Science

Article Title: The Beneficial Role of Sunitinib in Tumor Immune Surveillance by Regulating Tumor PD‐L1

doi: 10.1002/advs.202001596

Figure Lengend Snippet: Correlations between p62 and PD‐L1 expression levels in NSCLC patients with anti‐PD‐1 mAb treatment

Article Snippet: Anti‐SQSTM1/p62‐Rb (88588), LC3A/B (D3U4C) XP Rabbit mAb (12741) from Cell Signaling Technology; human anti‐PD‐L1 (ab213524 or ab213480); anti‐granzyme B antibody (ab4059) from Abcam; GAPDH (60004‐1‐Ig) from Proteintech; and anti‐CD8a (GB11068) from Servicebio were used.

Techniques: Expressing

Journal: Advanced Science

Article Title: The Beneficial Role of Sunitinib in Tumor Immune Surveillance by Regulating Tumor PD‐L1

doi: 10.1002/advs.202001596

Figure Lengend Snippet: Sunitinib downregulates PD‐L1 via autophagic degradation. A) Summary of the distribution of differentially expressed genes (DE genes) identified from RNA sequencing data, which was generated from the A375 cells with or without Sunitinib treatment. The average logCPM (count per million) represented the gene expression level. The color represents differentially expressed genes (fold change >1.3, FDR < 0.05, see the Experimental Section; red: upregulation, blue: downregulation, gray: nonsignificant change). B) Top eight enriched GO biological processes pathways for genes upregulated or downregulated in A375 melanoma cell lines treated with Sunitinib. C) Representative image of the detection of autophagy using a transmission electron microscope. A375 or SK‐MEL‐28 cells were treated with 5 µmol Sunitinib for 24 h or dimethyl sulfoxide (DMSO) treated. The right side image indicates: autolysosome‐like structures. Bar, 2 µm. D) Representative image and quantification of Western blot analysis of p62 and LC3 in A375 and SK‐MEL‐28 cells after treatment with indicated doses of Sunitinib. GAPDH was used as loading control (left). Bar graphs are quantified results of three independent experiments and showed as means ± SD, * p < 0.05, ** p < 0.01 (right). E,F) Representative image and colocalization coefficiency of immunofluorescence staining in A375 and SK‐MEL‐28 cells for immunostaining of LC3 (green FITC) with red (Cy3) staining of E) p62 or F) LAMP1, nuclear was indicated by DAPI (blue). Scale bar, 20 µm in inset. Data are representatives of three independent assays. The LC3 and p62 or LAMP1 colocalization coefficiency is expressed as mean ± standard error of mean (SEM), n = ≈30–50 cells of three independent experiments. G) Representative image and quantification of western blot analysis of PD‐L1 in A375 and SK‐MEL‐28 cells after treatment with indicated doses of Sunitinib with or without BafA1 (the inhibitor of autophagy) under the IFN‐ γ exposure for 24 h. GAPDH was used as loading control (left). Bar graphs was quantified results of three independent experiments expressed as the mean ± SEM, * p < 0.05, ** p < 0.01 compared to the control (right).

Article Snippet: Anti‐SQSTM1/p62‐Rb (88588), LC3A/B (D3U4C) XP Rabbit mAb (12741) from Cell Signaling Technology; human anti‐PD‐L1 (ab213524 or ab213480); anti‐granzyme B antibody (ab4059) from Abcam; GAPDH (60004‐1‐Ig) from Proteintech; and anti‐CD8a (GB11068) from Servicebio were used.

Techniques: RNA Sequencing, Generated, Gene Expression, Transmission Assay, Microscopy, Western Blot, Control, Immunofluorescence, Staining, Immunostaining

Journal: Cell reports. Medicine

Article Title: Dietary vitamin B3 supplementation induces the antitumor immunity against liver cancer via biased GPR109A signaling in myeloid cell.

doi: 10.1016/j.xcrm.2024.101718

Figure Lengend Snippet: Figure 6. Biased b-Arrestin/GPR109A axis activation in myeloid cells is responsible for antitumor effect of VB3 (A) Myeloid cells from WT mice or RAW264.7 were treated with/without LPC-H12 CM and VB3 for 12 h. The GPR109A mRNA expression in cells was detected (n = 3). (B) Myeloid cells from WT mice or RAW264.7 were treated with LPC-H12 CM and 50 mM VB3 for 0, 3, 6, 12, and 24 h. The protein expression of GPR109A, P-P65, and P65 was detected. (C and E) Myeloid cells from WT or GPR109A/ mice were treated with/without Hepa1-6 CM and VB3 (50 mM) for 12 h. The mRNA expression of GPR109A, Arg-1, IL-10, VEGF, CCL2, CSF-1, CXCL1, CXCL2, and CXCL5 was explored (C, n = 3). The protein expression of GPR109A, P-P65, and P65 was detected (E). (D and F) Myeloid cells from WT mice were treated with MPN (50 mM) previously for 4 h and treated with Hepa1-6 CM and VB3 (50 mM) for another 12 h. The mRNA expression of GPR109A, Arg-1, IL-10, VEGF, CCL2, CSF-1, CXCL1, CXCL2, and CXCL5 was detected (D, n = 3). The protein expression of GPR109A, P-P65, and P65 was evaluated (F). (G–I) Scheme of myeloid cells treatment and coincubation. Myeloid cells from WT mice were previously stimulated with CM for 12 h before treatment with VB3 (50 mM), ACTD (50 mM), and CHX (50 mM) for another 12 h (G). The mRNA expression (H, n = 3) and protein expression (I) of GPR109A were detected. (J) Myeloid cells from WT mice were treated with LPC-H12 CM and VB3 for 12 h. The binding between GPR109A and b-Arrestin1or b-Arrestin2 was determined. (K and L) b-Arrestin1/2-knockdown RAW264.7 cells were treated with LPC-H12 CM and VB3 (50 mM) for 12 h. The mRNA expression of GPR109A, Arg-1, IL-10, VEGF, CCL2, CSF-1, CXCL1, CXCL2, and CXCL5 was detected (K). The protein expression of GPR109A, P-P65, and P65 was explored (L). Data are presented as means ± SEM. Experiments were conducted independently in triplicates or more. Statistical analysis was performed by one-way ANOVA. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Anti-b-arrestin2 antibody Santa Cruz Cat#sc-365445; RRID: AB_10841589 Anti-b-arrestin2 antibody Santa Cruz Cat#sc-13140; RRID: AB_626701 Anti-CD8a antibody Servicebio Cat# GB11068; RRID: AB_2905511 Anti-INOS antibody Abcam Cat# ab15323; RRID: AB_301857 Anti-AFP antibody Proteintech Cat#14550-1-AP; RRID: AB_2223933 Anti-GPR109A antibody Novus Cat# NBP1-92180; RRID: AB_11039155 Anti-GPR109A antibody Cohesion Bio Cat# CPA6195; RRID: AB_3096221 Anti-Ly6G antibody CST Cat# 87048; RRID: AB_2909808 Anti-Arg-1 antibody CST Cat# 93668; RRID: AB_2800207 Anti-Ki67 antibody CST Cat#12202; RRID: AB_2620142 Anti-Cleaved Caspase-3 Antibody CST Cat#9661; RRID: AB_2341188 Anti-Phospho-ERK1/2 antibody CST Cat# 9101S; RRID: AB_331646 Anti-ERK1/2 antibody CST Cat# 9102S; RRID: AB_330744 Anti-b-arrestin1 antibody CST Cat#12697S; RRID: AB_2797996 Anti-NF-kB p65 antibody CST Cat# 4764; RRID: AB_823578 Anti-Phospho-NF-kB p65 antibody CST Cat# 3033; RRID: AB_331284 Anti-Phospho-PKA C antibody CST Cat#4781; RRID: AB_2300165 PKA C-a Antibody antibody CST Cat#4782; RRID: AB_2170170 Anti-GAPDH antibody CST Cat# 2118L; RRID: AB_561053 Anti-rabbit IgG HRP-linked CST Cat# 7074S; RRID: AB_2099233 Anti-mouse IgG HRP-linked CST Cat# 7076S; RRID: AB_330924 Chemicals, peptides, and recombinant proteins Mouse GM-CSF Novoprotein Cat# CK02 Mouse G-CSF Novoprotein Cat# CB53 Mouse M-CSF Novoprotein Cat# CB34 Mouse IL-2 Novoprotein Cat# CK24 Vitamin B3 Sigma Cat#N4126-100G Lenvatinib Psaitong Cat# L10232 Mepenzolate bromide Sigma Cat# M5651 Actinomycin D MCE Cat#HY-17559 MK-0354 MCE Cat#HY-13008 (Continued on next page) Cell Reports Medicine 5, 101718, September 17, 2024 e3

Techniques: Activation Assay, Expressing, Binding Assay, Knockdown