Journal: Cell reports

Article Title: Alterations of ceramide synthesis induce PD-L1 internalization and signaling to regulate tumor metastasis and immunotherapy response

doi: 10.1016/j.celrep.2024.114532

Figure Lengend Snippet: (A) Shh protein abundance in SCR or CerS4 shRNA-transfected 4T1 cells was detected in western blots. Data quantification is shown in right panel ( n = 3). (B) ELISA detected shh protein in the cell-culture supernatant from SCR or CerS4 shRNA-transfected 4T1 cells ( n = 4). (C) Representative images of migration assay measured in fibronectin-coated Boyden chambers ( n = 3). Scale bars, 10 μm. (D) Interaction of Shh and PTCH1 was detected by a co-immunoprecipitation with a Shh-targeting antibody and blotting for Shh and PTCH1 in 4T1 cells stably transfected with SCR or CerS4 shRNA ( n = 2). Data quantification is shown in right panel. (E) Effect of exogenous shh ligand (100–200 μg/mL) on cellular migration in 4T1 cells stably expressing SCR or CerS4 shRNA ( n = 3). (F) Effect of a neutralizing SMO and pSMO targeting antibody on cellular migration in 4T1 cells stably expressing SCR or CerS4 shRNA. Data quantification isshown in right panel ( n = 3). Scale bars, 10 μm. (G) Effect of exogenous shh ligand (200 μg/mL) on cellular migration in 4T1 cells stably expressing SCR or CerS4 shRNA with or without neutralizing pSMO antibody treatment ( n = 2). (H) TGFBR1 protein abundance in SCR or CerS4 shRNA-transfected 4T1 cells was detected by western blot. Data quantification is shown in lower panel ( n = 4). (I) Interaction of pSmo and TGFBR1 proteins was measured using a PLA in 4T1 cells stably expressing SCR or CerS4 shRNA with or without neutralizing pSMO antibody treatment. Data quantification is shown in right panel ( n = 3). Scale bars, 5 μm. (J) Cell-surface expression of SMO and TGFBR1 were determined using flow cytometry in 4T1 cells stably expressing SCR or CerS4 shRNA ( n = 3). Data represent means ± SD; Student’s t test or two-way ANOVA was used to determine significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Mouse TGFBR1 Taqman ® Probe , Thermo-Fisher , Assay ID: Mm00436964_m1.

Techniques: Quantitative Proteomics, shRNA, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Migration, Immunoprecipitation, Stable Transfection, Expressing, Flow Cytometry

Journal: Cell reports

Article Title: Alterations of ceramide synthesis induce PD-L1 internalization and signaling to regulate tumor metastasis and immunotherapy response

doi: 10.1016/j.celrep.2024.114532

Figure Lengend Snippet: (A) CerS4 protein abundance in MDA-MB-231 and LM2-4175 cells was detected by western blotting. Data quantification is shown in right panel ( n = 3). (B) The abundance of CerS4-generated ceramides in MDA-MB-231 and LM2-4175 cells was determined using LC-MS/MS-based lipidomics analysis ( n = 3). (C) Interaction between PD-L1 and caprin-1 was detected by PLA using anti-PD-L1 and anti-caprin-1 antibodies in MDA-MB-231 and LM2-4175 cells. Data quantification is shown in left panel ( n = 3). Scale bars, 20 μm. (D–G) CerS4 (D and E), Shh (F), and TGFBR1 (G) abundance was determined in doxycycline-inducible CerS4 expression LM2-4175 cells after 48 h in 5 μg/mL doxycycline detected by western blotting using an anti-CerS4, anti-TGFBR1, and anti-Shh antibodies. Data quantification is shown in right panels ( n = 3). (H) The abundance of CerS4-generated ceramides in doxycycline-inducible CerS4 expression LM2-4175 cells was determined using LC-MS/MS-based lipidomics analysis. The raw values were normalized to the control conditions for each ceramide species ( n = 3). (I) Cell-surface expression of PD-L1 was determined by flow cytometry in doxycycline-inducible CerS4 expression LM2-4175 cells after 48 h in 5 μg/mL doxycycline ( n = 3). (J) Interaction between PD-L1 and caprin-1 was detected by PLA using anti-PD-L1 and anti-caprin-1 antibodies in doxycycline-inducible CerS4 expression LM2-4175 cells after 48 h in 5 μg/mL doxycycline. Data quantification is shown in left panel ( n = 3). Scale bars, 20 μm. (K) Cellular migration was measured in doxycycline-inducible CerS4 expression LM2-4175 cells after 48 h in 5 μg/mL doxycycline. Data quantification is shown in left panel ( n = 3). Scale bars, 10 μm. Data represent means ± SD; Student’s t test was used to determine significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Mouse TGFBR1 Taqman ® Probe , Thermo-Fisher , Assay ID: Mm00436964_m1.

Techniques: Quantitative Proteomics, Western Blot, Generated, Liquid Chromatography with Mass Spectroscopy, Expressing, Control, Flow Cytometry, Migration

Journal: Cell reports

Article Title: Alterations of ceramide synthesis induce PD-L1 internalization and signaling to regulate tumor metastasis and immunotherapy response

doi: 10.1016/j.celrep.2024.114532

Figure Lengend Snippet: (A) Interaction between caprin-1 and Shh mRNA was quantified using RNA-immunoprecipitation coupled RT-qPCR in 4T1 cells stably expressing SCR or CerS4 shRNA with empty vector, WT-PD-L1-FLAG, or Mut-PD-L1-FLAG overexpressed ( n = 3). (B–D) Stability of Shh mRNA was determined in 4T1 cells stably expressing SCR or CerS4 shRNA with empty vector, WT-PD-L1-FLAG, or Mut-PD-L1-FLAG overexpressed along with actinomycin D (5 μg/mL) treatment ( n = 3). (E) Interaction between caprin-1 and TGFBR1 mRNA was quantified using RNA-immunoprecipitation coupled RT-qPCR with an anti-caprin-1 antibody in 4T1 cells stably expressing SCR or CerS4 shRNA with empty vector, WT-PD-L1-FLAG, or Mut-PD-L1-FLAG overexpressed ( n = 3). (F–H) Stability of TGFBR1 mRNA was determined in 4T1 cells stably expressing SCR or CerS4 shRNA with empty vector, WT-PD-L1-FLAG, or Mut-PD-L1-FLAG overexpressed along with actinomycin D (5 μg/mL) treatment ( n = 3). (I) Abundance and immunoprecipitation of EXOSC10 were determined by western blotting in 4T1 cells stably expressing SCR or CerS4 shRNA ( n = 2). (J and K) Interaction between EXOSC10 and Shh (J) or TGFBR1 (K) mRNA was quantified using RNA-immunoprecipitation coupled RT-qPCR with an anti-EXOSC10 antibody in 4T1 cells stably expressing SCR or CerS4 shRNA with empty vector, WT-PD-L1-FLAG, or Mut-PD-L1-FLAG overexpressed ( n = 3). (L and M) The interaction between EXOSC10 and Shh (L) or TGFBR1 (M) mRNA was quantified using RNA-immunoprecipitation coupled RT-qPCR with an anti-EXOSC10 antibody in 4T1 cells stably expressing SCR or CerS4 shRNA and SCR or caprin-1 shRNA ( n = 3). (N) Expression of CerS4, Shh, and TGFBR1 was determined by RT-qPCR in doxycycline-inducible CerS4 expression LM2-4175 cells after 48 h in 5 μg/mL doxycycline ( n = 3). (O and P) The interaction between EXOSC10 and Shh (O) or TGFBR1 (P) mRNA was quantified using RNA-immunoprecipitation coupled RT-qPCR with an anti-EXOSC10 antibody in doxycycline-inducible CerS4 expression LM2-4175 cells after 48 h in 5 μg/mL doxycycline ( n = 3). Data represent means ± SD; one-way or two-way ANOVA was used to determine significance: * p < 0.05, ** p < 0.01, *** p < 0.001, ****p < 0.0001; ns, not significant.

Article Snippet: Mouse TGFBR1 Taqman ® Probe , Thermo-Fisher , Assay ID: Mm00436964_m1.

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Stable Transfection, Expressing, shRNA, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Cell reports

Article Title: Alterations of ceramide synthesis induce PD-L1 internalization and signaling to regulate tumor metastasis and immunotherapy response

doi: 10.1016/j.celrep.2024.114532

Figure Lengend Snippet: (A–D) Primary mammary tumors and lungs were collected from MMTV-PyMT-CerS4 +/+ , -CerS4 +/− , and -CerS4 −/− mice after 150 days. (A) The number of metastatic lung nodules from MMTV-PyMT-CerS4 +/+ ( n = 12), -CerS4 +/− ( n = 10), and -CerS4 −/− ( n = 8) mice were quantified by an independent pathologist after H&E staining of lungs collected after 150 days. (B) The percentage of mice that developed lung metastasis was calculated. (C) Interaction between shh and PTCH1 was detected by PLA assay using anti-Shh and anti-PTCH1 antibodies in metastatic lung nodules from MMTV-PyMT-CerS4 +/+ and MMTV-PyMT-CerS4 −/− mice ( n = 3). (D) Interaction between PD-L1 and caprin-1 was detected by PLA assay using anti-PD-L1 and anti-caprin-1 antibodies in metastatic lung nodules from MMTV-PyMT-CerS4 +/+ and MMTV-PyMT-CerS4 −/− mice. Quantification of data is shown in the left panel ( n = 3). Scale bars, 10 μm. (E–O) 4T1-derived tumors stably expressing SCR or CerS4 shRNA were established in BALB/c mice unilaterally. After 1 week, the mice were treated with vehicle ( n = 6 SCR and n = 5 CerS4), sonidegib ( n = 4 SCR and n = 4 CerS4), anti-PD-L1 therapy ( n = 5 SCR and n = 6 CerS4), or a combination ( n = 5 SCR and n = 6 CerS4) for 21 days. (E) The total area of lung metastasis was determined using Akoya Inform analysis software. (F) Endpoint primary tumors were collected and weighed. (G and H) The interaction between PD-L1 and caprin-1 (G) and Shh and PTCH1 (H) was detected by PLA assay using anti-PD-L1 and anti-caprin-1 or anti-Shh and anti-PTCH-1 antibodies, respectively, in primary tumors from the vehicle, anti-PD-L1, or combination groups ( n = 4). (I) The expression of Shh and TGFBR1 in primary tumors from the vehicle or combination groups was determined by multiplexed immunofluorescence using anti-Shh and anti-TGFBR1 antibodies. Data quantification is shown in right panels ( n = 4). Scale bars, 20 μm. (J and K) The expression of Wnt5b (J) and TCF7L2 (K) in primary tumors was determined by RT-qPCR on tumors from vehicle ( n = 6 SCR and n = 6 CerS4), sonidegib ( n = 5 SCR and n = 5 CerS4), anti-PD-L1 therapy ( n = 4 SCR and n = 6 CerS4), or a combination ( n = 5 SCR and n = 6 CerS4). (L) 4T1 allografts stably expressing SCR ( n = 8) or CerS4 ( n = 8) shRNA were established in BALB/c mice unilaterally. Tumors were collected after 21 days. Immune cells were characterized in the tumor microenvironment using spectral flow cytometry. (M) Tumor-infiltrating lymphocytes (TILs) were isolated from shSCR ( n = 6) or shCerS4-transfected ( n = 6) tumors from (L). TILs were cultured with anti-CD3 and anti-CD28 for 24 h before being treated with Golgi-stop for 3 h and processed for spectral flow cytometry. (N) Tumor-infiltrating lymphocytes (TILs) were analyzed in primary tumors from the vehicle or combination groups by performing multiplexed immunofluorescence using anti-CD3, anti-CD8, anti-FoxP3, and anti-PanCK antibodies. Scale bars, 20 μm. (O) Quantification of TIL populations counted from mulitplexed immunofluoresence shown in (N) is shown in lower panels ( n = 4). Data represent means ± SD; tumor growth data and metastasis quantification, means ± SEM; data from (L) and (M), means ± interquartile range. Student’s t test, two-way ANOVA, or one-way ANOVA were used to determine significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Mouse TGFBR1 Taqman ® Probe , Thermo-Fisher , Assay ID: Mm00436964_m1.

Techniques: Staining, Derivative Assay, Stable Transfection, Expressing, shRNA, Software, Immunofluorescence, Quantitative RT-PCR, Flow Cytometry, Isolation, Transfection, Cell Culture

Journal: Cell reports

Article Title: Alterations of ceramide synthesis induce PD-L1 internalization and signaling to regulate tumor metastasis and immunotherapy response

doi: 10.1016/j.celrep.2024.114532

Figure Lengend Snippet:

Article Snippet: Mouse TGFBR1 Taqman ® Probe , Thermo-Fisher , Assay ID: Mm00436964_m1.

Techniques: Recombinant, Red Blood Cell Lysis, Lysis, Clinical Proteomics, Staining, Transfection, In Situ, Proximity Ligation Assay, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Sequencing, shRNA, Software