Journal: bioRxiv

Article Title: Selenoprotein P Deficiency Drives Hepatocellular Carcinoma Progression via Induction of Neutrophil Senescence and Immunosuppressive Microenvironment

doi: 10.1101/2025.06.24.661430

Figure Lengend Snippet: (a) Representative images of livers from S100a8-Cre and Lrp8 f/f ; S100a8-Cre ( Lrp8 ΔNeu ) mice at endpoint, demonstrating increased tumor burden in Lrp8 ΔNeu mice. (b, c) Quantification of liver-to-body weight ratio (b) and tumor counts (c) in Lrp8 ΔNeu versus control groups, showing a significantly higher tumor burden in the Lrp8 ΔNeu group (n = 9 per group). (d) Flow cytometry analysis showing an increased percentage of neutrophils in the tumor microenvironment of Lrp8 ΔNeu mice compared to controls (n = 6 per group). (e, f) Flow cytometric analysis of senescence markers in tumor-infiltrating neutrophils from represent mice. Senescence-associated β-galactosidase (SA-β-Gal) positive neutrophils (e) and p21 + neutrophils (f) were significantly increased in Lrp8 ΔNeu mice compared to control group (n = 5-6 per group). (g, h) Flow cytometric analysis of neutrophils isolated from Lrp8 ΔNeu and control tumors. Representative histogram showing NIR-H 2 SE fluorescence, a hydrogen selenide probe, in neutrophils. Quantification of the percentage of NIR-H 2 SE + neutrophils (g) and mean fluorescence intensity (MFI) of NIR-H 2 SE in neutrophils (h), showing significantly reduced hydrogen selenide levels in neutrophils after Lrp8 knockout (n = 5–6 per group). (i) Quantitative RT-PCR analysis of senescence-associated markers Cdkn1a in tumor-infiltrating neutrophils (n = 5–6 per group). (j, k) mRNA expression levels of S100a8 and S100a9 significantly elevated in tumor-infiltrating neutrophils from Lrp8 ΔNeu mice (n = 5-6 per group). (l) Schematic representation of hydrodynamic tail vein injection (HDTVi) constructs. Mice were injected with constructs encoding oncogenic NRas G12V and myr-AKT along with a plasmid that encodes a green fluorescent protein (GFP) -Sepp1 fusion protein for tracking Sepp1 localization. (m) Representative immunofluorescence images of liver tissues showing GFP (green) indicating Sepp1 localization, S100A9 (red) indicating neutrophils, lymphocyte antigen 6 family member G (Ly6G; white) as another neutrophil marker, and 4′,6-diamidino-2-phenylindole (DAPI; blue) for nuclei. Images demonstrate the colocalization of GFP with neutrophils, indicating that neutrophils uptake Sepp1. The lower row of images shows magnified views of the boxed regions, highlighting Sepp1 uptake by neutrophils. Data are presented as mean ± SEM; statistical significance was determined using two-tailed unpaired Student’s t-tests.

Article Snippet: Tumor sections were blocked with 1% bovine serum albumin (BSA) for 30 min at room temperature, after which sections were incubated (overnight at 4 °C) in PBS-1% BSA containing a primary antibody against green fluorescent protein (GFP; 1:500, Abmart, Cat. M20004), Ly6G (1:200, Servicebio, Cat. GB11229), or S100A9 (1:500, R&D Systems, Cat. AF2065).

Techniques: Control, Flow Cytometry, Isolation, Fluorescence, Knock-Out, Quantitative RT-PCR, Expressing, Injection, Construct, Plasmid Preparation, Immunofluorescence, Marker, Two Tailed Test