Journal: Cancer letters

Article Title: Rigosertib promotes anti-tumor immunity via autophagic degradation of PD-L1 in colorectal cancer cells.

doi: 10.1016/j.canlet.2023.216422

Figure Lengend Snippet: Fig. 2. RGS down-regulated PD-L1 in colorectal cancer cell lines both in vivo and in vitro. (A) Immunostaining of PD-L1 in the CT26 isograft tumors based on BALB/c mice. Scale bar, 100 μm. (B) Following incubation of CT26 cells with IFN-γ for 48 h, CT26 cells were treated with 5 μM RGS for various durations prior to lysis; the levels of inducible PD-L1 and p-STAT1 were examined by western blotting. Levels of PD-L1 and GAPDH were quantitated using the Image J software. Data shown are mean ± SEM. **P < 0.01. (C) RKO cells were treated with 0.5 or 1 μM RGS for 12 h or 1 μM for various durations prior to lysis. PD-L1 and apoptosis related protein markers were examined as indicated. (D) RKO cells were treated with RGS, with or without pretreatment with Z-VAD-FMK, for 12 h prior to lysis. PD-L1 and apoptosis-related protein markers were measured by western blot analysis. (E) After intraperitoneal injection of RGS, the levels of PD-L1 in the subcutaneous RKO xenograft tumors were evaluated by immunohistochemical staining, and the immunoreactive scores were calculated. Data are presented as the mean ± SEM from three different experiments performed in triplicate. *P < 0.05. (F) The level of PD-L1 in subcutaneous RKO xenograft tumors were examined by western blotting. (G) After incubation with IFN-γ for 48 h, DLD1, LOVO, and HCT116 cells were treated with 1 or 2 μM RGS for various time periods prior to lysis. The levels of inducible PD-L1 and those of two other membrane proteins (HGFR and E-cadherin) were examined by western blotting.

Article Snippet: Primary antibody against murine PD-L1 (AF1019) was purchased from R&D systems.

Techniques: In Vivo, In Vitro, Immunostaining, Incubation, Lysis, Western Blot, Software, Injection, Immunohistochemical staining, Staining, Membrane

Journal: Cancer letters

Article Title: Rigosertib promotes anti-tumor immunity via autophagic degradation of PD-L1 in colorectal cancer cells.

doi: 10.1016/j.canlet.2023.216422

Figure Lengend Snippet: Fig. 3. RGS accelerated the degradation of PD-L1 via the lysosomal pathway. (A) RKO cells were exposed to 1 μM RGS for 0–12 h. Total RNA was extracted and the expression of PD-L1 mRNA was analyzed by RT-PCR, using Gapdh as control. (B) RKO cells were treated with CHX in the presence or absence of RGS (1 μM) for different durations, and the levels of PD-L1 were then measured by western blotting. (C and D) RKO cells were pretreated with MG132, CQ, or BafA1 for 30 min to inhibit the function of proteasomes and lysosomes. Cells were then treated with or without RGS (1 μM) for 12–24 h, after which the levels of PD-L1 were determined. (E) In PD-L1-EGFP-RKO cells, the levels of PD-L1-EGFP and PD-L1 were determined after treatment with 1 μM RGS for 12–24 h; PD-L1 fragments are indicated by stars. (F) In PD-L1-EGFP-RKO cells, the lysosomes were stained using Lyso-tracker. After treatment with RGS (1 μM) for 12 h, the distribution of PD-L1-EGFP and lysosomes was determined under a confocal microscope. Scale bar, 10 μm.

Article Snippet: Primary antibody against murine PD-L1 (AF1019) was purchased from R&D systems.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Staining, Microscopy

Journal: Cancer letters

Article Title: Rigosertib promotes anti-tumor immunity via autophagic degradation of PD-L1 in colorectal cancer cells.

doi: 10.1016/j.canlet.2023.216422

Figure Lengend Snippet: Fig. 6. RGS-induced PD-L1 degradation is an autophagy-dependent process. (A) RKO cells were pretreated with Compound C for 30 min to inhibit the activity of AMPK. Cells were then treated with or without RGS (1 μM) for 12–24 h, after which the levels of PD-L1 were determined by western blotting. (B) RKO cells were pretreated with indicated concentrations of MRT68921, SBI0206965 for 30 min to inhibit the activity of ULK1. Cells were then treated with or without RGS (1 μM) for 12–24 h, after which the levels of PD-L1 were determined. (C and D) Following transfection with specifically targeted siRNA (ATG5#1-#2 or LC3#1-#2) for 24 h, RKO cells were treated with RGS (1 μM) for 0–24 h, after which the levels of PD-L1, ATG5, and LC3 were measured by western blotting. (E and F) RKO cells were pretreated with BafA1 for 30 min to inhibit lysosome function. Cells were then treated with or without RGS (1 μM) for 0–24 h, after which the levels of PD-L1 and P62 were determined by western blotting. Data shown are mean ± SEM. ***P < 0.001. Levels of PD-L1 and GAPDH were quantitated using the Image J software. (G) RKO cells were exposed to RGS (1 μM) for 9 h, and then the distribution of PD-L1 and P62 was analyzed by confocal microscopy. Scale bar, 10 μm.

Article Snippet: Primary antibody against murine PD-L1 (AF1019) was purchased from R&D systems.

Techniques: Activity Assay, Western Blot, Transfection, Software, Confocal Microscopy

Journal: Cancer letters

Article Title: Rigosertib promotes anti-tumor immunity via autophagic degradation of PD-L1 in colorectal cancer cells.

doi: 10.1016/j.canlet.2023.216422

Figure Lengend Snippet: Fig. 7. The combination of RGS and CTLA-4 blockade effectively suppressed tumor growth in vivo. (A) Anti-tumor effects of RGS, CTLA-4 mAb, and the combi natorial treatment (RGS + CTLA-4 mAb) were assessed in CT26 isograft models based on BALB/c mice. Tumor volumes were measured every 3 d after palpable tumors reached volumes of 50–100 mm3. Tumor growth was evidently inhibited in the combinatorial treated group (N = 5). (B) CT26 tumor weight was measured at day 13 (N = 5). (C) CDI was calculated to test for synergy between RGS and CTLA-4 mAb using mean tumor weight measurements. The CDI value of 0.522 indicates synergy (defined as CDI <1, with CDI <0.7 indicating a significantly synergistic effect). (D) Representative images of CT26 tumors after RGS and/or CTLA-4 mAb treatment at the end of time point. (E–H) Immunostaining, followed by quantification of PD-L1, cleaved caspase 3, granzyme B, and CD8 in the CT26 isograft tumor based on BALB/c mice using Image J (N = 5). Data shown are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. (I) Schematic diagram depicting the mechanism by which RGS exerts its anti-tumor effects. RGS triggers the mitochondria-related apoptosis in CRC cells. Concomitantly, RGS activates AMPK-ULK1 axis dependent autophagy, which plays an essential role in the degradation of PD-L1.

Article Snippet: Primary antibody against murine PD-L1 (AF1019) was purchased from R&D systems.

Techniques: In Vivo, Immunostaining

Journal: Microbiome

Article Title: Tumor-colonized Streptococcus mutans metabolically reprograms tumor microenvironment and promotes oral squamous cell carcinoma.

doi: 10.1186/s40168-024-01907-9

Figure Lengend Snippet: Fig. 5 Single-cell transcriptomic landscape reveals the shift of the cellular types during oral carcinogenesis, and KYNA is a predominant driving force for expansions and infiltrations of neutrophils in the TME of OSCC. A Overview of the workflow for scRNA-seq analyses of OLK and OSCC rat tongue tissues. B UMAP plot of the clustering results for 13 major cell types from OLK and OSCC tissues. C Stacked histogram of the percentages of different cells from OLK and OSCC tissues. D Quantitative analysis of neutrophils, macrophages, and T cells in non-epithelial cells in OLK and OSCC tissues. E H&E and IHC staining for CD11b in OLK and OSCC tissues of experimental rats. Scale bar: 600 µm for 4 × magnification and 300 µm for 10 × magnification. F Representative IHC staining images and statistical analysis of CD16 in saline and KYNA-treated 4NQO rats. Scale bars: 200 µm for 10 × magnification and 50 µm for 40 × magnification. G Representative flow cytometry dot plots and statistical analysis of CD11b + CD16 + neutrophils from the peripheral blood of OSCC patients after KYNA treatment. H Relative SLC7A8 expression in neutrophils from OSCC patients after KYNA treatment detected by RT-qPCR. I Representative flow cytometry dot plots and statistical analysis of AHR + neutrophils from OSCC patients after treatments with KYNA in indicated concentrations. In D, unpaired Student’s t-test; E and F, Mann– Whitney U test; G, H, and I, one-way ANOVA

Article Snippet: At week 20, the rats were sacrificed, and the tongues were dissected, and a longitudinal mid-lingual incision was made. (4) IL-1β/PD-L1monoclonal antibody (mAb) intervention (8 rats/group): (A) 4NQO + IL-1β mAb (200 μg/rat, BE0246, cloneB122, Bioxcell, USA); (B) 4NQO + KYNA + IL-1β mAb; (C) 4NQO + PD-L1 mAb (200 μg/rat, BE0383, clone 368A.4H1, Bioxcell, USA); (D) 4NQO + KYNA + PD-L1 mAb; (E) 4NQO + control IgG (200 μg/rat, BE0091, Bioxcell, USA).

Techniques: Immunohistochemistry, Saline, Flow Cytometry, Expressing, Quantitative RT-PCR, MANN-WHITNEY