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 Wnt5b Taqman ® Probe , Thermo-Fisher , Assay ID: Mm00437350_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: (A) Kaplan-Meier survival curve representing the percentage of distant relapse-free survival in breast cancer patients from GSE25066 ( n = 507) separated based on low vs. high CerS4 mRNA expression. (B) Kaplan-Meier survival curve representing the percentage of disease-free survival in breast cancer patients from GSE21653 ( n = 248). (C) Kaplan-Meier survival curve representing the percentage relapse-free survival in breast cancer patients from KM-Plotter ( n = 4,890). (D and E) Interaction between PD-L1 and caprin-1 was detected by PLA using anti-PD-L1 and anti-caprin-1 antibodies in a commercially available tissue microarray containing normal breast tissue ( n = 4 patients) and TNBC primary tumors ( n = 62 patients). Two images were taken per tissue core, and two cores were present per patient sample. (D) Interaction between PD-L1 and caprin-1 was quantified in normal breast tissue ( n = 4), lymph-node-negative primary tumors (N0, n = 42), and lymph-node-positive primary tumors (N1–N3, n = 20). Scale bars, 10 μm. (E) Interaction between PD-L1 and caprin-1 was quantified in normal breast tissue ( n = 4), stage 2 primary tumors (T2, n = 42), stage 3 primary tumors (T3, n = 12), and stage 4 primary tumors (T4, n = 8). Scale bars, 10 μm. (F and G) Correlation analysis of caprin-1 mRNA expression and Shh score in TNBC patients from GSE58812 ( n = 53 for PD-L1 + [F], and n = 54 for PD-L1 [G]). (H) Kaplan-Meier survival curve representing the percentage metastasis-free survival in PD-L1 + TNBC patients from GSE58812 ( n = 24) separated based on low vs. high CerS4 mRNA and Shh score. (I–K) Kaplan-Meier survival curves representing the percentage metastasis-free survival in PD-L1 + TNBC patients from GSE58812 ( n = 20) separated based on low vs. high Cers4 and CTNNB1 (I), Wnt5b (J), or TCF7L2 (K) mRNA levels. (L and M) Kaplan-Meier survival curves representing the percentage overall survival (L) and progression-free survival (M) in cancer patients treated with PD-L1 immunotherapy ( n = 459 for overall survival and n = 138 for progression-free survival) separated based on low vs. high CerS4 mRNA expression. One-way ANOVA or log-rank test was used to determine significance: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Mouse Wnt5b Taqman ® Probe , Thermo-Fisher , Assay ID: Mm00437350_m1.

Techniques: Expressing, Microarray

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 Wnt5b Taqman ® Probe , Thermo-Fisher , Assay ID: Mm00437350_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