Journal: Journal of Translational Medicine

Article Title: Colonization by Porphyromonas gingivalis in cervical squamous cell carcinomas promotes metastasis through FimA/CD151/ITGB1 signaling

doi: 10.1186/s12967-025-06928-y

Figure Lengend Snippet: Porphyromonas gingivalis promotes CSCC cell migration and invasion in vitro. A , B Porphyromonas gingivalis treatment had no significant effects on cell proliferation and viability. Confluence analysis ( A ) and a CCK-8 assay ( B ) were used to assess the proliferation and viability of SiHa cells cocultured with P. gingivalis at different MOIs (10, 50, and 100). Heat-killed P. gingivalis was generated by heating at 60 °C for 30 min. Supernatant: the culture supernatant of P. gingivalis was filtered through a 0.2 μm pore size filter. The results are shown as the means ± SEMs, n = 3; ns, not significant; * P < 0.05, two-way ANOVA. C – E Migration and invasion abilities were measured via Transwell assays in SiHa, HCC94, and HCEC cells which were cocultured with P. gingivalis (Pg) or L. crispatus (Lc) at different MOIs (10, 50, and 100). Heat-killed Pg: P. gingivalis was generated by heating at 60 °C for 30 min. Supernatant of Pg: the culture supernatant of P. gingivalis was filtered through a 0.2 μm pore size filter. F Wound healing assay was used to evaluate the migration of SiHa cells cocultured with P. gingivalis at different MOIs. n = 3, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, one-way ANOVA

Article Snippet: The human CSCC cell lines, including SiHa, HCC94, CaSki, C33A and MS751, were provided by the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Migration, In Vitro, CCK-8 Assay, Generated, Pore Size, Wound Healing Assay

Journal: Journal of Translational Medicine

Article Title: Colonization by Porphyromonas gingivalis in cervical squamous cell carcinomas promotes metastasis through FimA/CD151/ITGB1 signaling

doi: 10.1186/s12967-025-06928-y

Figure Lengend Snippet: Porphyromonas gingivalis accelerates CSCC metastatic progression in vivo. A A subcutaneous xenograft model in BALB/c-nude mice was established via SiHa cells treated with P. gingivalis (Pg) at an MOI of 50 for 12 h. n = 5 in each group. B Tumor volume and the weight of mice were measured and analyzed in the subcutaneous xenograft model. C Representative bioluminescence images of mice and lung tissues in the tail vein metastasis model. SiHa-luciferase cells cocultured with P. gingivalis at an MOI of 50 were injected intravenously through the tail vein of BALB/c-nude mice. n = 5 in each group. D Representative H&E images of lung metastasis in the tail vein metastasis model. The arrows showed the tumor cells. E Quantification of bioluminescence of lung tissues and the number of lung metastases in the tail vein metastasis model. F In vivo bioluminescence images of the footpad and popliteal lymph nodes (PLNs) in a lymph node metastasis model. SiHa-luciferase cells cocultured with P. gingivalis at an MOI of 50 were injected into the footpads of BALB/c-nude mice, n = 5 per group. H Representative images of H&E and IHC staining of CK5/6 (cytokeratin 5/6, a biomarker of squamous cell carcinoma) of PLN. G Quantification of bioluminescence of PLN and the number of metastatic PLN. The results are shown as the mean ± SEM, * P < 0.05, Student’s t test

Article Snippet: The human CSCC cell lines, including SiHa, HCC94, CaSki, C33A and MS751, were provided by the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vivo, Luciferase, Injection, Immunohistochemistry, Biomarker Discovery

Journal: Journal of Translational Medicine

Article Title: Colonization by Porphyromonas gingivalis in cervical squamous cell carcinomas promotes metastasis through FimA/CD151/ITGB1 signaling

doi: 10.1186/s12967-025-06928-y

Figure Lengend Snippet: Porphyromonas gingivalis preferentially adheres to CSCC cells. A Representative fluorescence images showing the adhesion of P. gingivalis to human cervical epithelial cells (HCECs) and five CSCC cell lines, including SiHa, HCC94, CaSki, C33A, and MS751. The cells were cocultured with CFSE-labelled P. gingivalis (green) at an infection MOI of 50 for 2 h. F-actin was stained with TRITC-phalloidin (red), and the nuclei were stained with DAPI (blue). B Quantification of P. gingivalis (Pg) adhesion in (A), expressed as the mean number of bacteria per cell. C Representative fluorescence images showing the adhesion of P. gingivalis or L. crispatus (ATCC 33820) to SiHa cells. SiHa cells were cocultured with CFSE-labelled L. crispatus or P. gingivalis at an MOI of 50 for 12 h. F-actin was stained with TRITC-phalloidin (red), and the nuclei were stained with DAPI (blue). D Quantification of the number of P. gingivalis or L. crispatus adhering to SiHa cells in (C). E Representative images showing intracellular localization of P. gingivalis in both adherent and trypsinized (suspension) SiHa cells after 12-h coculture (MOI = 50). F TEM (transmission electron microscopy) images of P. gingivalis within the cytoplasm of SiHa and HCEC cells. The red arrows indicate internalized P. gingivalis and red dashed lines indicate cell nucleus. The results are shown as the mean ± SEM. n = 3 per group, ns, not significant, * P < 0.05, **** P < 0.0001, Student’s t test (two groups) or one-way ANOVA (more than two groups)

Article Snippet: The human CSCC cell lines, including SiHa, HCC94, CaSki, C33A and MS751, were provided by the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Fluorescence, Infection, Staining, Bacteria, Suspension, Transmission Assay, Electron Microscopy

Journal: Journal of Translational Medicine

Article Title: Colonization by Porphyromonas gingivalis in cervical squamous cell carcinomas promotes metastasis through FimA/CD151/ITGB1 signaling

doi: 10.1186/s12967-025-06928-y

Figure Lengend Snippet: Porphyromonas gingivalis FimA selectively interacts with CD151/ITGB1 in CSCC cells. A Schematic diagram of the His pull-down assay. FimA, a major subunit of fimbriae of P. gingivalis , was overexpressed with a 6 × His tag to pull down its corresponding receptors in SiHa cells. B SDS-PAGE and Western blot assays showing the recombinant His-FimA protein in E. coli . C Silver staining showing the candidate proteins that interact with His-FimA. D Mass spectrometry identification of potential protein interacting with FimA, and western blot analysis for the binding of FimA, ITGB1 and CD151 in His pull-down assay. E Expanded His pull-down assays to additional members of the tetraspanin and integrin families. F Validation of the interaction between FimA and CD151/ITGB1 by co-immunoprecipitation using anti-CD151 and anti-ITGB1 antibodies. G FimA was pulled down by the biotin-pull down assay of SiHa cell membrane proteins. H The basic protein levels of CD151 and ITGB1 in CSCC lines and HCECs were detected by Western blot assay. Compared with those in other CSCC lines and HCECs, CD151 and ITGB1 were overexpressed in SiHa cells. I , J STORM images showing that P. gingivalis (green) directly binds to CD151 (red) or ITGB1 (red) during adhesion to SiHa cells

Article Snippet: The human CSCC cell lines, including SiHa, HCC94, CaSki, C33A and MS751, were provided by the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Pull Down Assay, SDS Page, Western Blot, Recombinant, Silver Staining, Mass Spectrometry, Binding Assay, Biomarker Discovery, Immunoprecipitation, Membrane

Journal: Journal of Translational Medicine

Article Title: Colonization by Porphyromonas gingivalis in cervical squamous cell carcinomas promotes metastasis through FimA/CD151/ITGB1 signaling

doi: 10.1186/s12967-025-06928-y

Figure Lengend Snippet: P. gingivalis adheres to CSCC cells through the FimA-CD151/ITGB1 interaction A Knockdown of CD151 and ITGB1 by siRNA attenuated the adhesion of P. gingivalis to SiHa cells. SiHa cells transfected with siRNA CD151, siRNA ITGB1 or nontarget siRNA (NT) were cocultured with P. gingivalis at an MOI of 50 for 4 h. B Western blot was used to confirm the knockdown efficacy of CD151 and ITGB1. C Quantification of the number of P. gingivalis adhering to SiHa cells with CD151 and ITGB1 knockdown. D Overexpression of CD151 and ITGB1 in SiHa cells facilitated the adhesion of P. gingivalis . SiHa cells transfected with lentiviruses encoding CD151, ITGB1 or an empty vector (EV) were cocultured with P. gingivalis at an MOI of 50 for 4 h. E Western blot analysis was used to detect the overexpression of CD151 and ITGB1. F Quantification of the number of P. gingivalis adhering to SiHa cells with overexpression of CD151 and ITGB1. G Knockdown of CD151 and ITGB1 in SiHa cells suppressed the promotion of migration and invasion mediated by P. gingivalis . Transwell assays were performed to detect the migration and invasion abilities of SiHa cells with CD151 and ITGB1 knockdown and cocultured with P. gingivalis . H Quantification of the Transwell assay results of SiHa cells. I , J Neutralizing antibodies against FimA and CD151/ITGB1 suppressed the adhesion of P. gingivalis in SiHa cells. SiHa cells were cocultured with CFSE-labelled P. gingivalis at an MOI of 50 for 2 h. K – M Transwell assays were performed to detect the migration and invasion of SiHa cells treated with neutralizing antibodies against FimA and CD151/ITGB1. The quantification results are shown as the mean ± SEM; ns, not significant; * P < 0.05; ** P < 0.01; one-way ANOVA for column analyses and two-way ANOVA for grouped analyses

Article Snippet: The human CSCC cell lines, including SiHa, HCC94, CaSki, C33A and MS751, were provided by the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Knockdown, Transfection, Western Blot, Over Expression, Plasmid Preparation, Migration, Transwell Assay

Journal: The Journal of Clinical Investigation

Article Title: HIC1 deletion promotes breast cancer progression by activating tumor cell/fibroblast crosstalk

doi: 10.1172/JCI99974

Figure Lengend Snippet: (A) Left: relative RT-qPCR analysis of 8 differentially expressed genes (CCL2, CXCL14, CX3CL1, IL1B, IL24, IL8, TGFA, and VEGFA) after HIC1 deletion in MCF7 and T47D cells (n = 3). Right: relative RT-qPCR analysis of Hic1, Cxcl14, and Sirt1 mRNA levels in the mammary glands of Hic1–/– or Hic1+/+ mice (n = 5). (B) ELISA analysis of CXCL14 levels in the CM of MCF7sgHIC1MCF7Ctrl and T47DsgHIC1T47DCtrl cells. The supernatants were collected after culture of the cells for 24 hours or 48 hours (n = 4). (C) Schematic of the CXCL14 promoter region. The positions of selected consensus binding sites are indicated above the diagram; the lengths of the promoter constructs used in the reporter assays are shown below. (D) CXCL14 promoter activity after transfection of the full-length construct (–2000/+136) alone or together with HIC1 expression vectors. p–GL3-Basic, control for promoter constructs; pc3.1, control for the HIC1 expression vector. The results are expressed as the ratio of firefly luciferase to Renilla luciferase (n = 3). (E) CXCL14 promoter activity after cotransfection with 100 ng of the HIC1 expression vector and each of the promoter constructs. The –41/+136 construct had a significant repressive effect, despite its lower promoter activity (n = 3). (F) ChIP analysis of HIC1 at the CXCL14 promoter region in MCF7 and T47D cells (n = 3). Data are shown as mean ± SEM. n = 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, 2-tailed Student’s t tests (A, B, and E), 1-way ANOVA followed by Bonferroni’s post hoc test (D and F).

Article Snippet: High-density TMA of human BrCa clinical samples (catalog BRC2281) were obtained from a cohort of 228 patients and constructed by Superbiotek Inc. IHC staining was performed with specific antibodies against HIC1 (catalog bs15485R; Bioss), CXCL14 (catalog ab46010; Abcam), CCL17 (catalog ab195044; Abcam), GPR85 (catalog ab140783; Abcam), and CCR4 (catalog ab1669; Abcam).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Binding Assay, Construct, Activity Assay, Transfection, Expressing, Control, Plasmid Preparation, Luciferase, Cotransfection

Journal: The Journal of Clinical Investigation

Article Title: HIC1 deletion promotes breast cancer progression by activating tumor cell/fibroblast crosstalk

doi: 10.1172/JCI99974

Figure Lengend Snippet: (A) Left: NAF2 or NAF6 cells were treated with rhCXCL14 at various concentrations (0–200 ng/ml) for 4 days. The corresponding primary CAF2 or CAF6 cells were used as positive controls. Right: immortalized NAF10 cells stably overexpressed CXCL14. Cell lysates were analyzed by Western blot with antibodies against α-SMA, FAP, PDGFRα, and GAPDH. (B) Representative immunofluorescence staining detection of α-SMA, FAP, and PDGFRα expression in NAF6 or CAF6 cells treated similarly to the cells described in A. (C) NAF6 cells were cocultured with MCF7CtrlMCF7sgHIC1 or with T47DCtrlT47DsgHIC1 luminal BrCa cells for 4 days in the presence or absence of α-CXCL14 at 1 μg/ml or an isotype-matched IgG control. Cell lysates from NAF6 cells were analyzed by Western blot with antibodies against α-SMA, FAP, PDGFRα, and GAPDH. (D) Three MCF7 cell lines (Ctrl-NC, sgHIC1-NC, sgHIC1-shCXCL14) were injected bilaterally into the fourth mammary fat pads of female BALB/c nude mice (n = 10 per group). Tumor volumes were measured with calipers at the indicated time points. Data are shown as mean ± SD. ***P < 0.001, RM ANOVA followed by post hoc LSD test. (E) Photographs and weights of the tumors obtained from the animals described in D. Data are shown as mean ± SD. ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post hoc test. (F) Representative immunohistochemical staining for Ki67 and stromal α-SMA in tumor tissues obtained from each experimental group. Dot plots show the mean value for the percentage of Ki67 or stromal α-SMA–positive cells with statistical evaluation (n = 5–6). Data are shown as mean ± SEM. n = 3 independent experiments. **P < 0.01; ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: High-density TMA of human BrCa clinical samples (catalog BRC2281) were obtained from a cohort of 228 patients and constructed by Superbiotek Inc. IHC staining was performed with specific antibodies against HIC1 (catalog bs15485R; Bioss), CXCL14 (catalog ab46010; Abcam), CCL17 (catalog ab195044; Abcam), GPR85 (catalog ab140783; Abcam), and CCR4 (catalog ab1669; Abcam).

Techniques: Stable Transfection, Western Blot, Immunofluorescence, Staining, Expressing, Control, Injection, Immunohistochemical staining

Journal: The Journal of Clinical Investigation

Article Title: HIC1 deletion promotes breast cancer progression by activating tumor cell/fibroblast crosstalk

doi: 10.1172/JCI99974

Figure Lengend Snippet: (A) Confocal microscopy of NAF6 cells treated with 100 ng/ml biotin or biotin-CXCL14 at 4°C and stained with an antibody against Cy3-streptavidin. An isotype-matched IgG was used as a control. Cell nuclei were counterstained with DAPI. (B) Schematic of the procedure used to detect biotin-CXCL14–binding proteins using HuProt human proteome microarrays containing 18,583 affinity-purified N-terminally GST-tagged proteins. (C) Representative CXCL14-binding membrane proteins in the proteome microarrays. (D) Western blot validation using a streptavidin-agarose pull-down assay of proteome microarray determination that CXCL14 binds directly to GPR85. (E) Mobilization of [Ca2+]i in NAF6 cells that were transfected with control siRNA (NC) or GPR85-3 siRNA and then treated with 100 ng/ml HBSS, rhCXCL14, or rhCXCL12. The black arrows denote the times at which stimulation was initiated. (F) 125I-CXCL14 binding properties between 293T-NC and 293T-GPR85 cells. Data are shown as mean ± SD. n = 4 repetitions. **P < 0.01; ***P < 0.001, 2-tailed Student’s t test. (G) Binding assay with 10 nM 125I-CXCL14 in the presence or absence of increasing concentrations of unlabeled rhCXCL14, rhCXCL12, and rhCXCL3 for 293T cells that were transfected with GPR85. B, specific binding; T, total binding. (H) Knockdown of GPR85 expression by GPR85-3 siRNA in NAF6 cells in the presence or absence of 100 ng/ml rhCXCL14 for the indicated times (0, 30, and 60 minutes). Cell lysates were analyzed by Western blot with antibodies against p-Akt (Ser 473), Akt, p-ERK1/2, ERK1/2, and GAPDH. (I) Knockdown of GPR85 expression by GPR85-3 siRNA in NAF6 cells treated with rhCXCL14 at various concentrations (0–100 ng/ml) for 4 days. Cell lysates were analyzed by Western blot with antibodies against α-SMA, FAP, PDGFRα, and GAPDH. (J) NAF6 cells were transfected with control siRNA (NC) or GPR85-3 siRNA and then cocultured with MCF7Ctrl or MCF7sgHIC1 cells, respectively, for 4 days. NAF6 cell lysates were analyzed by Western blot with antibodies against α-SMA, FAP, PDGFRα, p-Akt (Ser 473), Akt, p-ERK1/2, ERK1/2, and GAPDH.

Article Snippet: High-density TMA of human BrCa clinical samples (catalog BRC2281) were obtained from a cohort of 228 patients and constructed by Superbiotek Inc. IHC staining was performed with specific antibodies against HIC1 (catalog bs15485R; Bioss), CXCL14 (catalog ab46010; Abcam), CCL17 (catalog ab195044; Abcam), GPR85 (catalog ab140783; Abcam), and CCR4 (catalog ab1669; Abcam).

Techniques: Confocal Microscopy, Staining, Control, Binding Assay, Affinity Purification, Membrane, Western Blot, Biomarker Discovery, Pull Down Assay, Microarray, Transfection, Knockdown, Expressing

Journal: The Journal of Clinical Investigation

Article Title: HIC1 deletion promotes breast cancer progression by activating tumor cell/fibroblast crosstalk

doi: 10.1172/JCI99974

Figure Lengend Snippet: (A) Upper panel: schematic showing the coculture of MDA-231-LM2 BrCa cells with primary NAF10, CXCL14-activated NAF10, or primary CAF10 cells in a Transwell apparatus (0.4 μm pore size) for 4 days. Lower panel: Boyden chamber assay of MDA-231-LM2 cells that were treated as above. (B) Upper panel: Human XL Cytokine Array Kits (R&D Systems) were used to measure the levels of 102 cytokines in the CM from diverse fibroblasts. Cytokines that were upregulated in the CM of CXCL14-activated NAF10 and CAF10 cells are indicated by colored boxes; they include CCL17 (red), IL-5 (green), and angiopoietin-2 (blue). Black frames indicate the positive controls, and the dashed boxes indicate the negative controls in each membrane. Lower panel: table showing the relative signal intensities of the 3 selected cytokines noted above. The signal intensities were quantified by densitometry using ImageJ software and normalized to the intensity of the internal positive controls. (C) Boyden chamber assay of MDA-231-LM2 cells plated with rhCCL17, rhIL-5, and rh angiopoietin-2 in the lower chambers at 100 ng/ml for 20 hours. (D) Boyden chamber assay of MDA-231-LM2 cells that were cocultured with primary NAF10, CXCL14-activated NAF10, or primary CAF10 cells in a Transwell apparatus for 4 days in the presence or absence of α-CCL17 (1 μg/ml) or an isotype-matched IgG control. (E) MCF7 or MDA-231-LM2 cells were treated with various concentrations (0–100 ng/ml) of rhCCL17 for 4 days, and lysates of the cells were analyzed by Western blot using antibodies against E-cadherin, N-cadherin, and GAPDH. (F) MCF7 or MDA-231-LM2 cells were treated with rhCCL17 at 100 ng/ml for the indicated times (0, 5, 15, 30, and 60 minutes). Cell lysates were analyzed by Western blot with antibodies against p-Akt (Ser 473), Akt, p-GSK-3β (Ser9), GSK-3β, and GAPDH. (G) Knockdown of CCR4 expression by siRNA-3 in MCF7 and MDA-231-LM2 cells in the presence or absence of rhCCL17 at 100 ng/ml for 4 days. Cell lysates were analyzed by Western blot with antibodies against E-cadherin, N-cadherin, p-Akt (Ser 473), Akt, and GAPDH. Data are shown as mean ± SEM. n = 3 independent experiments. ***P < 0.001, 1-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: High-density TMA of human BrCa clinical samples (catalog BRC2281) were obtained from a cohort of 228 patients and constructed by Superbiotek Inc. IHC staining was performed with specific antibodies against HIC1 (catalog bs15485R; Bioss), CXCL14 (catalog ab46010; Abcam), CCL17 (catalog ab195044; Abcam), GPR85 (catalog ab140783; Abcam), and CCR4 (catalog ab1669; Abcam).

Techniques: Pore Size, Boyden Chamber Assay, Membrane, Software, Control, Western Blot, Knockdown, Expressing

Journal: The Journal of Clinical Investigation

Article Title: HIC1 deletion promotes breast cancer progression by activating tumor cell/fibroblast crosstalk

doi: 10.1172/JCI99974

Figure Lengend Snippet: (A) Percentages of low and high expression of epithelial HIC1 (upper), stromal CXCL14 (middle), and stromal CCL17 (bottom) in benign breast tissue and various BrCa subtypes are shown as a pie chart. P vs. benign, χ2 test. (B) Representative IHC images showing the correlation between epithelial HIC1 expression and stromal CXCL14/CCL17 expression in 228 benign and malignant breast tumor samples. Broken lines indicate the margins of the tumor. (C) Schematic model showing how HIC1-mediated crosstalk between cancer cells and mammary fibroblasts promotes BrCa progression. Conditional deletion of HIC1 in the mouse mammary gland may contribute to premalignant transformation at the early stage of breast tumor formation. Moreover, chemokine CXCL14 secreted by HIC1-deleted BrCa cells binds to its cognate receptor GPR85 on mammary fibroblasts in the microenvironment, thereby activating the fibroblasts through the ERK1/2, Akt, and neddylation pathways. The activated fibroblasts in turn promote BrCa progression through induction of EMT by activation of the CCL17/CCR4 chemokine axis.

Article Snippet: High-density TMA of human BrCa clinical samples (catalog BRC2281) were obtained from a cohort of 228 patients and constructed by Superbiotek Inc. IHC staining was performed with specific antibodies against HIC1 (catalog bs15485R; Bioss), CXCL14 (catalog ab46010; Abcam), CCL17 (catalog ab195044; Abcam), GPR85 (catalog ab140783; Abcam), and CCR4 (catalog ab1669; Abcam).

Techniques: Expressing, Transformation Assay, Activation Assay