Journal: Therapeutic Advances in Medical Oncology

Article Title: S100A4 characterize antigen-presenting cancer-associated fibroblasts and predicts surgical outcomes in relapsed ovarian cancer

doi: 10.1177/17588359261436959

Figure Lengend Snippet: Immune spatial interactions and prognostic significance of CD74 + S100A4 + antigen-presenting CAFs in ROC. (a, b) Spatial proximity analysis between CAF subpopulations and CD4 + T cells using mIHC and computational phenotyping. (a) Representative mIHC images showing spatial relationships between αSMA + , S100A4 + , CD74 + S100A4 + CAFs, and CD4 + T cells. Lines indicate nearest neighbor distances between cells. Scale bar, 50 µm. (b) Boxplot quantification of mean number of CD4 + T cells within 20 µm radius of each CAF subtype. CD74 + S100A4 + CAFs displayed significantly closer proximity to CD4 + T cells. ( p < 0.05) as shown in representative image (a). (c–e) Differences in CD74 + S100A4 + CAFs distribution and their spatial relationship with CD4 + T cells between patients achieving R0 versus Non-R0. (c) Representative images of mIHC staining illustrating differences in spatial cell arrangement. Scale bar, 200 µm. (d) Quantification of CD74 + S100A4 + CAFs densities (cells/mm²) and (e) mean count of CD4 + T cells within 20 µm of CD74 + S100A4 + CAFs between R0 and Non-R0 groups. (f, g) Prognostic significance of S100A4 + apCAFs based on multi-dataset transcriptomic analysis. (f) Forest plot showing HR of S100A4 + apCAFs-associated gene signature across 11 ovarian cancer datasets. Each horizontal black square represents the HR estimate from an individual dataset, and the horizontal line indicates the 95% CI. The overall HR for S100A4 + apCAFs is shown at the bottom, with the dashed vertical line indicating the reference value HR = 1. (g) In the TCGA ovarian cancer cohort, patients were stratified into a high-expression group (top 30%, n = 68, shown in blue) and a low-expression group (bottom 30%, n = 68, shown in red) based on the expression levels of the top 100 S100A4 + apCAFs signature genes. The Kaplan–Meier survival curves compare overall survival between these groups. The x -axis represents time since diagnosis (in months), and the y -axis indicates overall survival probability. CAF, cancer-associated fibroblasts; CI, confidence interval; HR, hazard ratio; mIHC, multiplex immunohistochemistry; ROC, relapsed ovarian cancer; S100A4, S100 calcium-binding protein A4; TCGA, The Cancer Genome Atlas; αSMA, α-smooth muscle actin.

Article Snippet: Sections underwent antigen retrieval in citrate or EDTA buffer, followed by endogenous peroxidase blocking with 3% H 2 O 2 , serum blocking, and overnight incubation at 4°C with primary antibodies: αSMA (#19245, Cell Signaling Technology, Danvers, MA, USA), FAP (#ab207178, Abcam, Cambridge, UK), S100A4 (#13018, Cell Signaling Technology, Danvers, MA, USA), PDPN (#26981, Cell Signaling Technology, Danvers, MA, USA), PAX8 (#1F8-3A8, Thermo Fisher Scientific, Waltham, MA, USA), and CD74 (#77274, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Staining, Expressing, Biomarker Discovery, Multiplex Assay, Immunohistochemistry, Binding Assay

Journal: bioRxiv

Article Title: Macrophage Migration Inhibitory Factor (MIF)-CD74 Signaling Pathway Mediates Trabecular Meshwork Dysfunction in Glaucoma

doi: 10.64898/2026.03.18.712673

Figure Lengend Snippet: The Tg.CreMYOC Y437H mice (n=12/group) were injected intravitreally with either HAd5-Cre or HAd5-empty vectors. ( A ) Schematic of the knock-in cassette showing DsRed-tagged human Y437H-mutant MYOC inserted at the transcriptionally active H11 locus. A transcriptional stop cassette prevents expression of the MYOC-DsRed fusion protein until Cre recombinase excises the stop sequence, enabling TM-specific expression of mutant MYOC. ( B-C ) Fluorescence imaging of whole-mount anterior segments ( B ) and ocular cross-sections ( C ) demonstrates robust DsRed-MYOC Y437H expression in Tg.CreMYOC Y437H mice (indicated with yellow arrows), compared with Ad5-empty controls. Scale bars: 100 μm (B), 50 μm (C). ( D ) Weekly IOP measurements show significant and sustained IOP elevation in Cre-injected Tg.CreMYOC Y437H mice compared with empty vector controls. ( E ) Immunofluorescence staining of anterior segment cross-sections shows MIF expression co-localizing with DsRed-MYOC Y437H in the TM of Tg.CreMYOC Y437H mice. ( F-H ) qPCR ( F ) and western blot ( G, H ) analyses of anterior segment tissues demonstrate upregulation of MIF and CD74 with concurrent suppression of Blimp-1 in Tg.CreMYOC Y437H mice. Western blot bands were quantified by densitometric analysis using ImageJ and normalized to β-actin, and data are presented as relative protein expression (AU) ( H ). Data represent mean ± SD. **p<0.005, and ****p<0.0001. Two-way ANOVA with Tukey’s multiple-comparison test (D), unpaired t-test (G, H).

Article Snippet: Primary antibodies used in this study were purchased from the following sources: β-actin (Millipore Sigma, #A2228), pNFκB (#3033), NFκB (#6956), pSTAT3 (#9145), STAT3 (#9139), RIP3K (#10188), pMLKL (#18640), MLKL (#14993), RhoA (#2117), ROCK1 (#4035), pMLC (#3674), MLC (#3672), MIF (#75038), CD74 (#77274), Blimp-1 (#9115) (Cell Signaling Technology, Danvers, MA).

Techniques: Injection, Knock-In, Mutagenesis, Expressing, Sequencing, Fluorescence, Imaging, Plasmid Preparation, Immunofluorescence, Staining, Western Blot, Comparison

Journal: bioRxiv

Article Title: Macrophage Migration Inhibitory Factor (MIF)-CD74 Signaling Pathway Mediates Trabecular Meshwork Dysfunction in Glaucoma

doi: 10.64898/2026.03.18.712673

Figure Lengend Snippet: Wild-type C57BL/6 mice (n=12/group) were injected intravitreally with either LV-TGF-β2 or LV-null vectors. ( A ) Weekly IOP measurements demonstrate significant and sustained IOP elevation in LV-TGF-β2-injected mice compared with LV-null controls. ( B ) Immunofluorescence staining of anterior segment cross-sections for α-SMA and MIF shows co-localization of MIF with the TM marker α-SMA, indicating increased MIF expression in the TM of LV-TGF-β2 injected mice. ( C-E ) qPCR ( C ) and western blot ( D, E ) analyses of anterior segment tissues show upregulation of MIF and CD74 accompanied by suppression of Blimp-1 in LV-TGF-β2-injected mice. Increased expression of profibrotic markers, including α-SMA, Fn-1, and MYOC, was also evident in LV-TGF-β2-injected mice. Western blot bands were quantified by densitometric analysis using ImageJ and normalized to the housekeeping control β-actin, and protein levels are presented as relative expression (AU) ( E ). Data represent mean ± SD. **p<0.005, ***p<0.0005, and ****p<0.0001. Two-way ANOVA with Tukey’s multiple-comparison test (A), unpaired t-test (C, E).

Article Snippet: Primary antibodies used in this study were purchased from the following sources: β-actin (Millipore Sigma, #A2228), pNFκB (#3033), NFκB (#6956), pSTAT3 (#9145), STAT3 (#9139), RIP3K (#10188), pMLKL (#18640), MLKL (#14993), RhoA (#2117), ROCK1 (#4035), pMLC (#3674), MLC (#3672), MIF (#75038), CD74 (#77274), Blimp-1 (#9115) (Cell Signaling Technology, Danvers, MA).

Techniques: Injection, Immunofluorescence, Staining, Marker, Expressing, Western Blot, Control, Comparison

Journal: bioRxiv

Article Title: Macrophage Migration Inhibitory Factor (MIF)-CD74 Signaling Pathway Mediates Trabecular Meshwork Dysfunction in Glaucoma

doi: 10.64898/2026.03.18.712673

Figure Lengend Snippet: Primary HTMCs (n=3) were challenged with TGF-β2 (10ng/mL), rMIF (100ng/mL), or pro-inflammatory cytokine pool (CP; TNF-α, IL-1β, IL-6, and IL-17 100ng/mL) for 24h. (A) qPCR analysis shows significant upregulation of MIF and CD74 transcripts accompanied by reduced Blimp-1 expression in response to glaucomatous stressors. ( B ) Immunofluorescence staining demonstrates increased MIF expression following TGF-β2 or CP treatment. (C) Western blot analysis confirms induction of MIF and CD74 with concurrent suppression of Blimp-1 at the protein level. Data represent mean ± SD. **p<0.005, ***p<0.0005, and ***p<0.0001, One-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: Primary antibodies used in this study were purchased from the following sources: β-actin (Millipore Sigma, #A2228), pNFκB (#3033), NFκB (#6956), pSTAT3 (#9145), STAT3 (#9139), RIP3K (#10188), pMLKL (#18640), MLKL (#14993), RhoA (#2117), ROCK1 (#4035), pMLC (#3674), MLC (#3672), MIF (#75038), CD74 (#77274), Blimp-1 (#9115) (Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Comparison

Journal: bioRxiv

Article Title: Macrophage Migration Inhibitory Factor (MIF)-CD74 Signaling Pathway Mediates Trabecular Meshwork Dysfunction in Glaucoma

doi: 10.64898/2026.03.18.712673

Figure Lengend Snippet: Primary HTMCs (n=3) were challenged with rMIF (100ng/mL) in the presence or absence of the MIF inhibitor 4-IPP (100µM) or the metabolites Agm or Thia (100 ng/mL) for 24h. (A) Western blot analysis shows that MIF suppresses Blimp-1 expression, whereas treatment with 4-IPP, Thia, or Agm restores Blimp-1 protein levels. (B) qPCR analysis reveals that MIF significantly induces mRNA expression of MIF, CD74, and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), while reducing Blimp-1 expression. Inhibition of MIF significantly restores Blimp-1 expression and suppresses MIF-CD74 signaling and pro-inflammatory cytokine expression. (C) ELISA analysis of treated and untreated TM culture supernatants demonstrates that MIF suppresses IL-10 secretion, which is restored following MIF inhibition. (D) Western blot analysis shows activation of inflammatory (STAT3, NFκB) and cell death pathways (RIP3K, MLKL, and cleaved caspase-3) following MIF treatment, which is markedly attenuated by MIF inhibition. Data represent mean ± SD. *p<0.05, and ****p<0.0001, One-way ANOVA with Dunnett’s multiple-comparison test.

Article Snippet: Primary antibodies used in this study were purchased from the following sources: β-actin (Millipore Sigma, #A2228), pNFκB (#3033), NFκB (#6956), pSTAT3 (#9145), STAT3 (#9139), RIP3K (#10188), pMLKL (#18640), MLKL (#14993), RhoA (#2117), ROCK1 (#4035), pMLC (#3674), MLC (#3672), MIF (#75038), CD74 (#77274), Blimp-1 (#9115) (Cell Signaling Technology, Danvers, MA).

Techniques: Western Blot, Expressing, Inhibition, Enzyme-linked Immunosorbent Assay, Activation Assay, Comparison

Journal: bioRxiv

Article Title: Macrophage Migration Inhibitory Factor (MIF)-CD74 Signaling Pathway Mediates Trabecular Meshwork Dysfunction in Glaucoma

doi: 10.64898/2026.03.18.712673

Figure Lengend Snippet: OHT was induced in WT C57BL/6 mice (n=12) by intravitreal injection of LV-TGF-β2. LV-null-injected mcie served as controls. Following OHT induction, extracellular vesicle-loaded agmatine (EV-Agm; 0.1 µg/eye) was applied topically once daily. (A) Weekly IOP measurements over 9 weeks show that EV-Agm treatment significantly reduces IOP compared with untreated LV-TGF-β2-injected mice. (B) qPCR analysis of anterior segment tissue demonstrates that EV-Agm significantly reduces expression of Mif, Cd74, pro-inflammatory cytokines ( Il-1 β , Il-6 ), and cytoskeletal/ECM markers (α -Sma, Myoc, Fn-1 ), while restoring Blimp-1 expression compared with untreated LV-TGF-β2-injected mice. *p<0.05, **p<0.005, ###,***p<0.0005, and ####,****p<0.0001. Two-way ANOVA with Tukey’s multiple comparison (A), one-way ANOVA with Tukey’s multiple comparison (B). In panel A, * denotes LV-null vs. LV-TGF-β2, and # denotes LV-TGF-β2 vs. LV-TGF-β2+EV-Agm comparisons.

Article Snippet: Primary antibodies used in this study were purchased from the following sources: β-actin (Millipore Sigma, #A2228), pNFκB (#3033), NFκB (#6956), pSTAT3 (#9145), STAT3 (#9139), RIP3K (#10188), pMLKL (#18640), MLKL (#14993), RhoA (#2117), ROCK1 (#4035), pMLC (#3674), MLC (#3672), MIF (#75038), CD74 (#77274), Blimp-1 (#9115) (Cell Signaling Technology, Danvers, MA).

Techniques: Injection, Expressing, Comparison