Journal: bioRxiv

Article Title: ASPARAGINE-RICH METASTATIC NICHES DRIVE PROSTATE CANCER ORGANOTROPISM BY ENABLING TRANSLATIONAL REWIRING TOWARD N-GLYCOSYLATED PROTEINS

doi: 10.64898/2026.02.27.708521

Figure Lengend Snippet: (A) Western blot analysis of ASNS in metastatic cells isolated from bone (B-M-1, B-M-2) and lung (L-M-1, L-M-2) lesions. Vinculin was used as loading control. The image is representative of three independent experiments. (B) Schematic representation of asparaginyl-tRNA synthetase 1 (NARS1) mechanism of action. (C) NARS1 mRNA levels in PC3 cells following NARS1 silencing. Cells were transfected with NARS1-targeting small interfering RNA (siRNA) or negative control, and mRNA levels were evaluated after 5 days of incubation in 3D cultures (3D-C) by quantitative RT-PCR, using scramble-transfected cells as reference. (D) Relative cell number of PC3 cells silenced for NARS1 and cultured under standard 2D conditions for 5 days in the presence or absence of Asn (0.1 mM). One-way ANOVA with Sidak’s correction. (E-G) Number of putative N-glycosylation sites in proteins encoded by genes up- or down-regulated in metastatic cells derived from bone (E), lung (F), and liver (G) relative to primary tumor (PT). RNA-seq analysis was conducted as described in . Values are expressed relative to total protein number. (H-I) Fractional enrichment of UDP-GlcNAc isotopologues. PC3 cells were grown in 2D or 3D-C for 5 days and subsequently incubated in a medium containing U- 13 C-glucose for 24h. Labeling enrichment was evaluated by LC-MS analysis, and isotopologue abundance is reported as relative to total UDP-GlcNAc amount. Welch’s t test. (J) Labeling (m+5) enrichment of penotose phosphates from U- 13 C-glucose in PC3 cells growing in 2D or 3D-C for 5 days and subsequently incubated in a medium containing U- 13 C-glucose for 24h. Labeling enrichment was evaluated by LC-MS analysis. (K, L, P) Western blot analysis of GFPT1 (K), STT3a (L), and CD44 (P) expression in PC3 cells silenced for GFPT1, STT3a/b, and CD44 respectively after 48h of gene silencing. Vinculin was used as a loading control. The image is representative of three independent experiments. (M) Concanavalin A lectin binding assay performed on lysates from PC3 cells silenced or not for GFPT1 or STT3a/b and cultured in 3D-C for 5 days. Immunoblot for vinculin was used to confirm equal protein loading across samples. The image is representative of three independent experiments. (N-O) Adhesion of PC3 3D-C to collagen type I (L) and hyaluronic acid (M). Cells were cultured with Asn (0,1 mM) for 5 days and allowed to adhere for 15 min to plates coated with matrix components as reported. Adherent cells were quantified and data are shown relative to untreated cells. Welch’s t-test.

Article Snippet: Cells were permeabilized with 0.25% Triton X-100 in PBS for 5 minutes, washed twice with PBS, and incubated overnight at 4°C with primary antibody against CD44 (Cell Signaling Technology #3570) diluted 1:100 in IFF.

Techniques: Western Blot, Isolation, Control, Transfection, Small Interfering RNA, Negative Control, Incubation, Quantitative RT-PCR, Cell Culture, Glycoproteomics, Derivative Assay, RNA Sequencing, Labeling, Liquid Chromatography with Mass Spectroscopy, Expressing, Binding Assay

Journal: bioRxiv

Article Title: ASPARAGINE-RICH METASTATIC NICHES DRIVE PROSTATE CANCER ORGANOTROPISM BY ENABLING TRANSLATIONAL REWIRING TOWARD N-GLYCOSYLATED PROTEINS

doi: 10.64898/2026.02.27.708521

Figure Lengend Snippet: (A) Volcano plot showing differential protein abundance in PC3 cells grown in 3D-C in the absence or presence of Asn (0.1 mM). The x-axis reports the difference (log 2 fold change) fold change between 3D-C and 3D-C+Asn; the y-axis shows –log 10 (p value). Proteins specific to 3D-C were identified by excluding proteins also expressed in 2D conditions. Each dot represents a quantified protein; red dots denote proteins exceeding the ≥2-fold change threshold. (B) Western blot analysis of CD44 expression in metastatic cells isolated from bone (B-M-1, B-M-2) and lung (L-M-1, L-M-2) lesions isolated as described in . Vinculin was used as a loading control. Representative of three independent experiments. (C) Western blot analysis of CD44 in PC3 cells cultured under standard 2D conditions or in 3D-C. Vinculin was used as a loading control. Representative of three independent experiments. (D) Time-course Western blot analysis of CD44 expression in PC3 cells cultured in 3D-C during spheroid formation (5 h, 1 day, 2 days, and 5 days after plating). Vinculin was used as a loading control. The image is representative of three independent experiments. (E) Representative immunohistochemical staining for CD44 in primary prostate carcinoma tissues (PT) and bone metastatic (BM) lesions from patients with PC. Boxed areas indicate regions shown at higher magnification. Brown staining denotes CD44-positive cells; nuclei are stained with hematoxylin. (F) H-score quantification of CD44 immunohistochemical staining shown in (E). H-scores were calculated by integrating staining intensity and the percentage of positive cells. Welch’s t test. (G) Total spheroid area of CD44-silenced PC3 cells grown in 3D-C compared to control silencing condition. Welch’s t-test. (H) Hyaluronic acid adhesion assay on CD44-silenced PC3 cells grown in 3D-C for 5days before plating on hyaluronic acid-coated plate. Data are shown relative to untreated cells. Welch’s t test. (I) Western blot analysis of CD44 expression in PC3 cells cultured in 3D-C in the presence or absence of Asn (0.1 mM and 1 mM). Vinculin was used as a loading control. The image is representative of three independent experiments. (J) Western blot analysis of CD44 in PC3 cells in 3D-C with or without Asn (0.1 mM) in the presence or absence of L-asparaginase (ASNase, 0.25 U/ml). Vinculin was used as loading control. The image is representative of three independent experiments. (K) CD44 mRNA expression levels in PC3 cells cultured in 3D-C with or without Asn (0.1 mM), measured by quantitative RT-PCR and normalized to the non-treated condition. Student’s t test. (L) Confocal fluorescence microscopy images showing CD44 membrane localization in PC3 cells grown in standard 2D conditions, 3D-C, and 3D-C supplemented with Asn (0,1 mM). The image is representative of three independent experiments. (M, N) Western blot analysis of CD44 expression in PC3 cells grown in 3D-C and silenced for NARS1 (M) or GFPT1 (N), cultured with or without Asn (0.1 mM). Vinculin was used as a loading control. The images are representative of three independent experiments. (O) Total spheroid area of 3D-C PC3 cells silenced for the CD44 with or without Asn (0.1 mM). One-way ANOVA with Dunnett’s correction. (P) Hyaluronic acid adhesion assay on CD44-silenced PC3 cells grown in 3D-C with or without Asn (0.1 mM). One-way ANOVA with Dunnett’s correction. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data represent mean ± s.e.m. from at least three independent experiments.

Article Snippet: Cells were permeabilized with 0.25% Triton X-100 in PBS for 5 minutes, washed twice with PBS, and incubated overnight at 4°C with primary antibody against CD44 (Cell Signaling Technology #3570) diluted 1:100 in IFF.

Techniques: Quantitative Proteomics, Western Blot, Expressing, Isolation, Control, Cell Culture, Immunohistochemical staining, Staining, Cell Adhesion Assay, Quantitative RT-PCR, Fluorescence, Microscopy, Membrane

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Immunoreactivity for CD44 was detected in vascular endothelial cells (arrows) and stromal cells (arrowheads) (A, B) . Stromal cells were leukocytes co-expressing CD45 (arrows) (C) , monocytes/macrophages co-expressing CD68 (arrows) (D) and spindle-shaped cells (arrowheads) (B) . Staining for receptor for hyaluronan-mediated motility (RHAMM) showed immunoreactivity in monocytes/macrophages (arrows) (E, F) (black scale bar 10 µm).

Article Snippet: Subsequently, the sections were incubated for 60 minutes with mouse monoclonal anti-CD31 (ready-to-use; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11+PD7/26; Dako), mouse monoclonal anti-CD68 antibody (ready-to-use; clone KP1; Dako), mouse monoclonal antibody against α-SMA (ready-to-use; clone 1A4; Dako), mouse monoclonal antibody against HAS2 (1:1000; ab140671; Abcam, Cambridge, UK), rabbit polyclonal antibody against Hyal-2 (1:250; ab68608; Abcam), mouse monoclonal anti-CD44 antibody (1:150; Cat No 3570; Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal antibody against RHAMM (CD168) (1:250; ab124729; Abcam).

Techniques: Expressing, Staining

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Immunohistochemical analysis of epiretinal fibrovascular membranes that were obtained from 14 patients with proliferative diabetic retinopathy during pars plana vitrectomy. Significant positive correlations between the numbers of blood vessels expressing CD31, reflecting angiogenic activity, and the numbers of blood vessels expressing hyaluronan synthase (HAS)2 (A) , the numbers of stromal cells expressing HAS2 (B) , the numbers of blood vessels expressing hyaluronidase (Hyal)-2 (C) and the numbers of blood vessels expressing CD44 (D) (Pearson’s correlation coefficient). Significant positive correlations between vitreous fluid levels of hyaluronan and the levels of hyaluronidase (Hyal)-1 (E) , heparan sulphate (F) and soluble syndecan-1 (G) (Pearson’s correlation coefficient). Samples from patients with proliferative diabetic retinopathy (PDR) are indicated by open circles, whereas samples from non-diabetic patients with rhegmatogenous retinal detachment (RD) are indicated by closed circles, illustrating sample clustering according to disease entity.

Article Snippet: Subsequently, the sections were incubated for 60 minutes with mouse monoclonal anti-CD31 (ready-to-use; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11+PD7/26; Dako), mouse monoclonal anti-CD68 antibody (ready-to-use; clone KP1; Dako), mouse monoclonal antibody against α-SMA (ready-to-use; clone 1A4; Dako), mouse monoclonal antibody against HAS2 (1:1000; ab140671; Abcam, Cambridge, UK), rabbit polyclonal antibody against Hyal-2 (1:250; ab68608; Abcam), mouse monoclonal anti-CD44 antibody (1:150; Cat No 3570; Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal antibody against RHAMM (CD168) (1:250; ab124729; Abcam).

Techniques: Immunohistochemical staining, Expressing, Activity Assay

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Determination of hyaluronan synthase (HAS)2 (A) , hyaluronidase (Hyal)-1 (B) , Hyal-2 (C) , soluble CD44 (D) , syndecan-1 (E) and heparan sulphate (F) levels in vitreous fluid samples. Equal volumes (15 µL) of vitreous fluid from patients with proliferative diabetic retinopathy (PDR; n=10) and from non-diabetic patients with rhegmatogenous retinal detachment (RD; n=10) were subjected to gel electrophoresis and the presence of HAS2, Hyal-1, Hyal-2, soluble CD44, syndecan-1, and heparan sulphate were detected by Western blot analysis. Representative sets of samples are shown. The intensity of the protein bands was determined in all samples and band intensities were compared between RD and PDR patients. Results are expressed as mean ± standard deviation or standard error of mean (*p < 0.05, independent t-test).

Article Snippet: Subsequently, the sections were incubated for 60 minutes with mouse monoclonal anti-CD31 (ready-to-use; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11+PD7/26; Dako), mouse monoclonal anti-CD68 antibody (ready-to-use; clone KP1; Dako), mouse monoclonal antibody against α-SMA (ready-to-use; clone 1A4; Dako), mouse monoclonal antibody against HAS2 (1:1000; ab140671; Abcam, Cambridge, UK), rabbit polyclonal antibody against Hyal-2 (1:250; ab68608; Abcam), mouse monoclonal anti-CD44 antibody (1:150; Cat No 3570; Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal antibody against RHAMM (CD168) (1:250; ab124729; Abcam).

Techniques: Nucleic Acid Electrophoresis, Western Blot, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Expression levels of hyaluronidase (Hyal)-1 (A) , CD44 (B) and receptor for hyaluronan-mediated motility (RHAMM) (C) in the retinal lysates of non-diabetic control rats (C) (n=12) and diabetic rats (D) (n=12) were determined by Western blot analysis. After determination of the intensity of the protein bands, intensities were adjusted to those of β-actin in the samples. Oxidative stress was monitored with the use of 2’,7’-Dichlorofluorescein (DCF) fluorescence intensity analysis (D) . Results are expressed as mean ± standard deviation. Ultra-Low molecular weight hyaluronan (ULMW-HA) induces breakdown of blood-retinal barrier (E) . ULMW-HA was injected intravitreally at the dose of 50 ng in 5 µL in one eye and the same volume of phosphate-buffered saline (PBS) was injected in the contralateral eye of normal rats. The BRB was quantified with the fluorescein isothiocyanate-conjugated dextran technique. Results are expressed as mean ± standard deviation of 12 rats. *p < 0.05 compared to the values obtained from PBS-injected eyes. (independent t-test). Western blot analysis of retinas demonstrated that intravitreal injection of ULMW-HA induced significant upregulation of the expression of phospho-NF-κB (F) , phospho-ERK1/2 (G) , vascular endothelial growth factor (VEGF) (H) , intercellular adhesion molecule-1 (ICAM-1) (I) , vascular cell adhesion molecule-1 (VCAM-1) (J) and high-mobility group box-1 (HMGB1) (K) . Results are expressed as mean ± standard deviation or standard error of mean of 8–10 rats in each group (*p < 0.05; independent t-test).

Article Snippet: Subsequently, the sections were incubated for 60 minutes with mouse monoclonal anti-CD31 (ready-to-use; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11+PD7/26; Dako), mouse monoclonal anti-CD68 antibody (ready-to-use; clone KP1; Dako), mouse monoclonal antibody against α-SMA (ready-to-use; clone 1A4; Dako), mouse monoclonal antibody against HAS2 (1:1000; ab140671; Abcam, Cambridge, UK), rabbit polyclonal antibody against Hyal-2 (1:250; ab68608; Abcam), mouse monoclonal anti-CD44 antibody (1:150; Cat No 3570; Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal antibody against RHAMM (CD168) (1:250; ab124729; Abcam).

Techniques: Expressing, Control, Western Blot, Fluorescence, Standard Deviation, Molecular Weight, Injection, Saline

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Human retinal Müller glial cells were left untreated or treated with high-glucose (HG) (25 mM) for 24 (h). For HG treatment, cultures treated with mannitol (25 mM) were used as a control. Protein expression of hyaluronan synthase (HAS)2 (A) , hyaluronidase (Hyal)-1 (B) , Hyal-2 (C) , CD44 (D) and receptor for hyaluronan-mediated motility (RHAMM) (E) in cell lysate was determined by Western blot analysis. Human retinal Müller glial cells were left untreated or treated with HG (25 mM), cobalt chloride (CoCl 2 ) (300 µM), tumor necrosis factor-α (TNFα) (5 ng/mL) or hydrogen peroxide (H 2 O 2 ) (10 mM) for 24 (h) Levels of hyaluronidase (Hyal)-1 were quantified in the culture media by ELISA. (F) Results are expressed as mean ± standard deviation or standard error of mean from three different experiments each performed in triplicate (*p < 0.05; independent t-test).

Article Snippet: Subsequently, the sections were incubated for 60 minutes with mouse monoclonal anti-CD31 (ready-to-use; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11+PD7/26; Dako), mouse monoclonal anti-CD68 antibody (ready-to-use; clone KP1; Dako), mouse monoclonal antibody against α-SMA (ready-to-use; clone 1A4; Dako), mouse monoclonal antibody against HAS2 (1:1000; ab140671; Abcam, Cambridge, UK), rabbit polyclonal antibody against Hyal-2 (1:250; ab68608; Abcam), mouse monoclonal anti-CD44 antibody (1:150; Cat No 3570; Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal antibody against RHAMM (CD168) (1:250; ab124729; Abcam).

Techniques: Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Dysregulated hyaluronan metabolism drives inflammation and angiogenesis in proliferative diabetic retinopathy

doi: 10.3389/fimmu.2026.1724199

Figure Lengend Snippet: Human retinal microvascular endothelial cells (HRMECs) were left untreated or treated with high-glucose (HG) (25 mM) for 24h. For HG treatment, cultures treated with mannitol (25 mM) were used as a control. Protein expression of hyaluronan synthase (HAS)2 (A) , hyaluronidase (Hyal)-1 (B) , Hyal-2 (C) and CD44 (D) in cell lysate was determined by Western blot analysis. (E) HRMECs were left untreated or treated with tumor necrosis factor–α (TNF-α) (5ng/ml) or hydrogen peroxide (H 2 O 2 ) (10mM) for 24h. Levels of Hyal-1 were quantified in the culture media by ELISA. (F) HRMECs and human retinal Müller glial cells were compared after treatment with mannitol (25 mM) or HG (25 mM) for 24h. Levels of hyaluronan (HA) were quantified in the culture media by ELISA. Results are expressed as mean ± standard deviation from three different experiments each performed in triplicate (*p < 0.05; independent t-test).

Article Snippet: Subsequently, the sections were incubated for 60 minutes with mouse monoclonal anti-CD31 (ready-to-use; clone JC70A; Dako, Glostrup, Denmark), mouse monoclonal anti-CD45 (ready-to-use; clones 2B11+PD7/26; Dako), mouse monoclonal anti-CD68 antibody (ready-to-use; clone KP1; Dako), mouse monoclonal antibody against α-SMA (ready-to-use; clone 1A4; Dako), mouse monoclonal antibody against HAS2 (1:1000; ab140671; Abcam, Cambridge, UK), rabbit polyclonal antibody against Hyal-2 (1:250; ab68608; Abcam), mouse monoclonal anti-CD44 antibody (1:150; Cat No 3570; Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal antibody against RHAMM (CD168) (1:250; ab124729; Abcam).

Techniques: Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation