Journal: bioRxiv

Article Title: Neutrophils orchestrate pre-metastatic immunosuppressive bone marrow remodeling during prostate cancer metastasis

doi: 10.64898/2026.10.08.757800

Figure Lengend Snippet: (a) Experimental design of the conditioned-medium (CM) model. Mice received daily intraperitoneal injections of TRAMP-C1 CM or matched blank medium (Control) for 2 weeks; BM was collected after 1 or 2 weeks. (b) Flow-cytometric quantification of CCR3-positive neutrophils, CD8Temra cells, intermediate monocytes (intMono) and non-classical monocytes (ncMono) during niche establishment. Each dot represents one mouse; for CCR3 neutrophils, intMono and ncMono measurements, n = 6, 7 and 7 mice for Control, 1 week and 2 weeks groups, respectively. For CD8 T cells, n = 3 mice per group. (c) Representative multiplex immunofluorescence images of BM stained for DAPI, CCR3 and Ly6G. Scale bars, 50 µm in the main images and 5 µm in the insets. (d) Frequency of CCR3-positive Ly6G-positive neutrophils in BM, liver, lung, peripheral blood (PB) and spleen from sham-operated and orthotopic prostate-tumor-bearing mice. Each dot represents one mouse; n = 3 mice per group. (e) Differential expression in epithelial cells from primary prostate cancer versus normal prostate datasets; selected tumor-secreted factors are labeled. (f) Activity of IGF1-, CXCL8-and ERBB-family downstream gene modules across neutrophil states. (g) Representative immunohistochemical staining of IGF1 in normal prostate and prostate cancer tissues from patients with low/medium risk, high risk or established bone metastasis. Scale bar, 200 µm. (h) Quantification of prostate-tissue IGF1 staining and plasma IGF1 bioavailability across normal, low/medium-risk, high-risk and bone-metastatic prostate cancer groups. For tissue IGF1 staining, normal prostate, n = 15 patients; low/medium-risk PCa, n = 16; high-risk PCa, n = 21; bone-metastatic PCa, n = 11. For plasma IGF1 bioavailability, normal prostate, n = 6 patients; low/medium-risk PCa, n = 13; high-risk PCa, n = 14; bone-metastatic PCa, n = 10. (i) Paired plasma IGF1 bioavailability before and after radical prostatectomy (RP); n = 9 matched patient pairs. (j) IGF1 staining in primary prostate cancer tissues and plasma IGF1 bioavailability in patients with advanced localized PCa, stratified according to subsequent bone metastasis development during follow-up (non-BMets, remained free of bone metastasis; BMets, subsequently developed bone metastases). For tissue IGF1 staining, non-BMets, n = 36 patients; BMets, n = 33. For plasma IGF1 bioavailability, non-BMets, n = 18 patients; BMets, n = 8. (k) Flow-cytometric quantification of IGF1R mean fluorescence intensity (MFI) on neutrophils from PB, spleen and BM. Each dot represents one mouse; n = 4, 4 and 3 mice for PB, spleen and BM, respectively. (l) UMAP of epithelial cells in metastatic BM samples. Early and late subtypes were labeled. (m) Diffusion-map and RNA-velocity analysis of epithelial-subtype progression. (n) UMAP feature plots of IGF1 , COL5A2 , ACAD8 , FOS , FOSB and JUN . (o) Malignant epithelial cells from paired primary-prostate cancer and BM samples from patients M8 and M9, shown by cluster identity and tissue-specific density. (p) EMP scores across paired-sample epithelial clusters. (q) Patient-specific diffusion maps with RNA-velocity vectors, colored by cluster identity. Unless otherwise indicated, bar plots with error bars show mean ± s.d. Statistical significance was assessed using two-sided unpaired Student’s t-tests (b,d,h,j,k), a paired Student’s t-test (i), Fisher’s exact tests with multiple-testing correction (e). Student’s t-test P values were adjusted using the Benjamini-Hochberg method where multiple comparisons were performed.

Article Snippet: Purified BM neutrophils were treated with saline vehicle (Control) or recombinant IGF1 (100 ng/ml; MCE, HY-P70698A).

Techniques: Control, Multiplex Assay, Immunofluorescence, Staining, Quantitative Proteomics, Labeling, Activity Assay, Immunohistochemical staining, Tissue, Clinical Proteomics, Fluorescence, Diffusion-based Assay, Analysis

Journal: bioRxiv

Article Title: Neutrophils orchestrate pre-metastatic immunosuppressive bone marrow remodeling during prostate cancer metastasis

doi: 10.64898/2026.10.08.757800

Figure Lengend Snippet: (a) Heatmap of genes differentially expressed in control and IGF1-treated mouse BM neutrophils; values are scaled expressions. Two mice were analysed per group, with each mouse serving as an independent biological replicate. (b) Gene-set enrichment analysis of the TAN signature in IGF1-treated versus control neutrophils using the RNA-seq data shown in a (n = 2 mice per group). (c) Representative immunofluorescence images of peripheral-blood and BM neutrophils from control- or IGF1-treated mice stained for DAPI, CD274, Ly6G and CCR3. Scale bars, 10 µm. (d) Frequencies of CCR3-positive and CD274-positive neutrophils in peripheral blood and BM; n = 3 mice per group. (e) Transcription-factor motifs enriched in accessible chromatin after IGF1 stimulation. n = 2 biologically independent neutrophil preparations per group. (f) Intersection of IGF1-upregulated genes (RNA-seq), IGF1-regulated transcription factors (ATAC-seq) and TAN-upregulated genes (scRNA-seq), identifying KLF4 as a candidate regulator. (g) KLF4 MFI in neutrophils after IGF1 stimulation with or without MAPK/ERK inhibition; n = 3 biologically independent samples per group. (h) ChIP-qPCR analysis of c-Myc occupancy at the KLF4 promoter after IGF1 stimulation; normal IgG served as the negative control. n = 3 biologically independent samples per group. (i) Experimental design for IGF1-induced BM preconditioning in mice receiving matched vehicle (Control), IGF1 or IGF1 plus the IGF1R inhibitor picropodophyllin (PPP). (j) CCR3 and ICAM1 MFIs in BM neutrophils after the indicated treatments; n = 6 mice per group. (k) Representative BM immunofluorescence images stained for DAPI, CCR3 and Ly6G. Scale bars, 50 µm in the main images and 5 µm in the insets. (l) Experimental design of the CM-induced BM-preconditioning model using matched blank medium (Control), wild-type TRAMP-C1 conditioned-medium (WT CM), Igf1-knockdown CM (Igf1-KD CM) or wild-type CM combined with an IGF1R-blocking antibody. (m) Frequencies of CCR3-positive neutrophils, PD-1-positive CD8 T cells and Tregs after the indicated CM treatments; n = 3 mice per group. (n) Experimental design for analysis of IGF1-induced BM remodeling in neutrophil-specific Igf1r conditional-knockout (cKO) mice and littermate controls. (o) CD274, CCR3 and ICAM1 MFIs in neutrophils, intermediate-monocyte frequency and CD8Temra frequency in control and cKO mice after IGF1 treatment; n = 3 mice per group. (p) Design of the mouse BM scRNA-seq experiment comprising saline-control, IGF1-induced HMP-like, and IGF1-preconditioned TRAMP-C1 metastatic groups. For each experimental group, BM cells from three mice were pooled to generate one scRNA-seq sample. (q) UMAP of all mouse BM cells, colored by major cell type. (r) UMAP of mouse neutrophil states with group-specific embeddings. (s) Human-derived TAN signature scores across mouse neutrophil states. (t) Differential abundance of mouse neutrophil states across experimental groups. Color indicates the log odds ratio, and “+” or “-” denotes significant enrichment or depletion, respectively. (u) UMAP of mouse BM CD8 T cell subtypes with group-specific embeddings. (v) Differential abundance of CD8 T cell subtypes across experimental groups. (w) IL-1β-induced signature scores in CD8Temra cells across saline-control, IGF1-induced HMP and TRAMP-C1 metastatic groups. Unless otherwise indicated, bar plots with error bars show mean ± s.d. Statistical comparisons in panels d, g, h, j, m and o were performed using two-sided Student’s t-tests. P values were adjusted using the Benjamini–Hochberg method where multiple comparisons were performed. For the GSEA in panel b, nominal P values and normalized enrichment scores are shown.

Article Snippet: Purified BM neutrophils were treated with saline vehicle (Control) or recombinant IGF1 (100 ng/ml; MCE, HY-P70698A).

Techniques: Control, Analysis, RNA Sequencing, Immunofluorescence, Staining, Inhibition, ChIP-qPCR, Negative Control, Knockdown, Blocking Assay, Gene Knockout, Saline, Derivative Assay, Oligomer Restriction

Journal: bioRxiv

Article Title: Neutrophils orchestrate pre-metastatic immunosuppressive bone marrow remodeling during prostate cancer metastasis

doi: 10.64898/2026.10.08.757800

Figure Lengend Snippet: (a) Experimental design for testing whether conditioned-medium (CM)-induced BM preconditioning promotes bone metastasis and whether PPP prevents this effect. Mice received matched blank medium (Control), wild-type TRAMP-C1 CM or CM plus PPP before intracardiac TRAMP-C1 injection. (b) Experimental design for testing whether recombinant IGF1-induced BM preconditioning promotes bone metastasis and whether PPP prevents this effect. Control mice received matched IGF1 and PPP vehicles. (c, d) Bone-metastasis-free curves and longitudinal bone bioluminescence imaging (BLI) signals after CM (c) or IGF1 (d) preconditioning, with or without PPP. For longitudinal BLI analyses, the area under the curve (AUC) was calculated for each mouse from ln(signal + 1)-transformed serial BLI measurements, and per-mouse AUC values were compared between groups; n = 10 mice per group. P 1 denotes Control versus CM in c and Control versus IGF1 in d; P 2 denotes CM versus CM plus PPP in c and IGF1 versus IGF1 plus PPP in d. (e) Experimental design using neutrophil-specific Igf1r conditional-knockout (cKO) mice and littermate controls to evaluate IGF1-induced BM preconditioning and subsequent bone metastasis. (f) Bone-metastasis-free curves and longitudinal bone BLI signals in control and cKO mice. BLI signals were natural-log transformed as ln(signal + 1); n = 10 mice per group. (g) Experimental design of the metastatic model evaluating Control, anti-IGF1R, anti-PD-1 or combined anti-IGF1R plus anti-PD-1 treatment. Control mice received matched saline. (h) Bone-metastasis-free curves and longitudinal bone BLI signals across treatment groups. BLI signals were natural-log transformed as ln(signal + 1); n = 10 mice per group. P values for the prespecified pairwise comparisons are shown in the panel: P 1, P 2 and P 3 denote Control versus Combination, Saline versus anti-IGF1R and Saline versus anti-PD-1, respectively. (i) Representative haematoxylin-and-eosin-stained bone sections from control, anti-IGF1R-, anti-PD-1- and combination-treated mice. Dashed lines outline tumor regions (T); B denotes bone. Scale bar, 400 µm. (j) Quantification of tumor area in the indicated treatment groups; each dot represents one mouse. (k) Frequencies of CCR3-positive neutrophils, IFNG-positive and PD1-positive CD8 T cells, and Tregs, together with BM IL-1β concentrations, across treatment groups; n = 3 mice per group. (l) Flowchart of the investigator-initiated trial of teprotumumab plus tislelizumab in metastatic castration-resistant prostate cancer; seven patients were enrolled and received treatment; one subsequently withdrew from the study. (m) Best percentage change in serum prostate-specific antigen (PSA) from baseline. (n) PSA progression-free survival from the first dose. (o) Representative axial PET/CT images before treatment and at day 84. (p) Best percentage change in serum alkaline phosphatase (ALP) from baseline. (q) Patient-reported pain outcomes at days 28, 56 and 84 after the first dose, categorized as improved, stable or worse relative to the immediately preceding assessment (day 28 versus baseline, day 56 versus day 28 and day 84 versus day 56); n = 6 evaluable patients at each time point. Unless otherwise indicated, bar plots with error bars show mean ± s.d. Bone-metastasis-free curves were compared using log-rank tests. Longitudinal BLI data were compared using a two-sided Welch’s t-test. Pairwise cellular or histological comparisons were analyzed using two-sided Student’s t-tests, with Benjamini-Hochberg adjustment where multiple comparisons were performed. Exact P values are shown in the panels.

Article Snippet: Purified BM neutrophils were treated with saline vehicle (Control) or recombinant IGF1 (100 ng/ml; MCE, HY-P70698A).

Techniques: Control, Injection, Recombinant, Imaging, Transformation Assay, Gene Knockout, Saline, Staining, Positron Emission Tomography-Computed Tomography

Journal: Aging Cell

Article Title: IGF ‐1/ SLC25A33 Maintains Blood‐Labyrinth Barrier Integrity by Suppressing TGF ‐β/Smad2‐Mediated Pericyte Activation in Age‐Related Hearing Loss

doi: 10.1111/acel.70712

Figure Lengend Snippet: Transcriptomic analysis identifies IGF‐1/SLC25A33 dysregulation and TGF‐β pathway activation in aged pericytes, which may be associated with ARHL. (A) Representative click‐evoked ABR waveforms showing a threshold of 30 dB SPL (red trace, left) in a young mouse versus 60 dB SPL (red trace, right) in an aged mouse. (B) Click‐evoked ABR thresholds are significantly elevated in old mice compared to young controls. (C) Frequency‐specific ABR thresholds are markedly increased across all tested frequencies (4–32 kHz) in aged mice. (D) DPOAE thresholds are significantly higher in old mice at F 2 = 17.44 kHz ( n = 6 mice). (E) RNA sequencing of FACS‐purified pericytes from young and aged stria vascularis reveals significant downregulation of Igf1 and Slc25a33 mRNA in aged mice ( n = 6 mice). (F) The most enriched DO terms for the differentially expressed genes when old vs. young. (G) The most enriched GO terms for the downregulated genes when old versus young. (H) The most enriched Reactome terms for the downregulated genes when old versus young. (I) KEGG pathway enrichment analysis highlights the TGF‐β signaling pathway as one of the most differentially activated pathways. (J) Gene–gene interaction network for the downregulated genes when old versus young.

Article Snippet: Sections were blocked in 10% goat serum + 0.3% Triton X‐100 in PBS for 1 h. Primary antibodies were applied overnight at 4°C: IGF‐1 (NOVUS, SPM406, 1:200), SLC25A33 (Bioss, bs‐21231R, 1:200), PDGFRβ (CST, #3169, 1:200), E‐cadherin (CST, #14472, 1:200), α‐SMA (Bioss, bs‐10196R, 1:200), TGFβ1 (Bioworld, BS80030, 1:50), p‐Smad2 (Bioworld, bs‐3419R, 1:500).

Techniques: Analysis, Activation Assay, RNA Sequencing, Purification

Journal: Aging Cell

Article Title: IGF ‐1/ SLC25A33 Maintains Blood‐Labyrinth Barrier Integrity by Suppressing TGF ‐β/Smad2‐Mediated Pericyte Activation in Age‐Related Hearing Loss

doi: 10.1111/acel.70712

Figure Lengend Snippet: Downregulation of IGF‐1 and SLC25A33 are confirmed in the aged stria vascularis. In vitro studies reveal IGF‐1 regulates SLC25A33 expression. (A, B) qPCR shows downregulation of Igf1 and Slc25a33 mRNA expression in aged pericytes ( n = 3 mice); (C) Immunofluorescence staining confirms colocalization of IGF‐1 (green), SLC25A33 (red) and PDGFRβ (purple, pericyte marker) in the stria vascularis. Age‐dependent alteration of IGF‐1 and SLC25A33 were markedly occurred in pericytes, as indicated by arrows. (D‐E) Quantitative analysis reveals significant downregulation of both IGF‐1 and SLC25A33 fluorescence intensity in aged mice relative to young controls ( n = 5, five typical sections from five individual mice were analyzed per group). (F) qPCR shows that inhibition of IGF‐1 signaling (via NVP‐AEW541) significantly reduces Slc25a33 mRNA levels. (G) Representative Western blot of SLC25A33 protein expression. (H) Densitometric quantification demonstrates reduced SLC25A33 protein upon IGF‐1 inhibition. Control: HBVPs without treatment; IGF‐1 (−): HBVPs treated with NVP‐AEW541; IGF‐1 (+): Cells treated with recombinant IGF‐1. Data are mean ± SEM; n = 3 mice.

Article Snippet: Sections were blocked in 10% goat serum + 0.3% Triton X‐100 in PBS for 1 h. Primary antibodies were applied overnight at 4°C: IGF‐1 (NOVUS, SPM406, 1:200), SLC25A33 (Bioss, bs‐21231R, 1:200), PDGFRβ (CST, #3169, 1:200), E‐cadherin (CST, #14472, 1:200), α‐SMA (Bioss, bs‐10196R, 1:200), TGFβ1 (Bioworld, BS80030, 1:50), p‐Smad2 (Bioworld, bs‐3419R, 1:500).

Techniques: In Vitro, Expressing, Immunofluorescence, Staining, Marker, Analysis, Fluorescence, Inhibition, Western Blot, Control, Recombinant

Journal: Aging Cell

Article Title: IGF ‐1/ SLC25A33 Maintains Blood‐Labyrinth Barrier Integrity by Suppressing TGF ‐β/Smad2‐Mediated Pericyte Activation in Age‐Related Hearing Loss

doi: 10.1111/acel.70712

Figure Lengend Snippet: IGF‐1 upregulation promotes SLC25A33 expression in the stria vascularis of aged mice. (A) Immunofluorescence images show IGF‐1 (green) and SLC25A33 (red) expression in the stria vascularis. (B, C) Quantitative analysis reveals that exogenous IGF‐1 supplementation increases both IGF‐1 and SLC25A33 fluorescence intensity, while IGF‐1 inhibition further reduces their expression ( n = 5, five typical sections from five individual mice were analyzed per group). (D) qPCR confirms exogenous IGF‐1 supplementation upregulates Slc25a33 mRNA levels, whereas IGF‐1 inhibition decreases its expression ( n = 3 mice). Control: wild‐type C57BL/6J mice without treatment; IGF‐1 (−): mice treated with NVP‐AEW541; IGF‐1 (+): mice treated with recombinant IGF‐1. Data are mean ± SEM.

Article Snippet: Sections were blocked in 10% goat serum + 0.3% Triton X‐100 in PBS for 1 h. Primary antibodies were applied overnight at 4°C: IGF‐1 (NOVUS, SPM406, 1:200), SLC25A33 (Bioss, bs‐21231R, 1:200), PDGFRβ (CST, #3169, 1:200), E‐cadherin (CST, #14472, 1:200), α‐SMA (Bioss, bs‐10196R, 1:200), TGFβ1 (Bioworld, BS80030, 1:50), p‐Smad2 (Bioworld, bs‐3419R, 1:500).

Techniques: Expressing, Immunofluorescence, Analysis, Fluorescence, Inhibition, Control, Recombinant

Journal: Aging Cell

Article Title: IGF ‐1/ SLC25A33 Maintains Blood‐Labyrinth Barrier Integrity by Suppressing TGF ‐β/Smad2‐Mediated Pericyte Activation in Age‐Related Hearing Loss

doi: 10.1111/acel.70712

Figure Lengend Snippet: Exogenous IGF‐1 rescues blood‐labyrinth barrier integrity and improves hearing function in aged mice. (A) TEM images reveal ultrastructural changes in the stria vascularis. In the IGF‐1‐inhibited group, pericytes exhibit abnormal morphology, loose association with endothelial cells, and widened tight junctions (yellow arrows). These defects are ameliorated by exogenous IGF‐1 administration. (B) Click‐evoked ABR thresholds are significantly elevated in IGF‐1‐suppressed mice but partially rescued by IGF‐1 supplementation. (C–E) At 12 kHz, ABR thresholds are significantly lower in the IGF‐1‐treated group compared to control and suppressed groups, indicating improved auditory sensitivity. (F) Proposed model of IGF‐1/SLC25A33‐mediated regulation of pericyte phenotype and BLB integrity in age‐related hearing loss. Under physiological conditions, pericytes maintain a quiescent state characterized by low α‐SMA and high E‐cadherin expression, contributing to BLB stability. With aging, declining IGF‐1 levels lead to reduced SLC25A33 expression, resulting in mtROS accumulation and activation of the TGF‐β/Smad2 signaling pathway. This drives pericyte transition toward a contractile, pro‐fibrotic phenotype (high α‐SMA, low E‐cadherin), disrupting BLB integrity and promoting cochlear dysfunction. Targeting this axis may offer therapeutic potential for ARHL. Control: wild‐type C57BL/6J mice without treatment; IGF‐1 (−): mice treated with NVP‐AEW541; IGF‐1 (+): mice treated with recombinant IGF‐1. Data are mean ± SEM; n = 6 mice.

Article Snippet: Sections were blocked in 10% goat serum + 0.3% Triton X‐100 in PBS for 1 h. Primary antibodies were applied overnight at 4°C: IGF‐1 (NOVUS, SPM406, 1:200), SLC25A33 (Bioss, bs‐21231R, 1:200), PDGFRβ (CST, #3169, 1:200), E‐cadherin (CST, #14472, 1:200), α‐SMA (Bioss, bs‐10196R, 1:200), TGFβ1 (Bioworld, BS80030, 1:50), p‐Smad2 (Bioworld, bs‐3419R, 1:500).

Techniques: Morphology, Control, Sensitivity, Expressing, Stability, Activation Assay, Recombinant