Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: F. nucleatum aggregates at the invasive margin of OSCC and correlates with tumor invasion. (A) F. nucleatum abundance for adjacent normal tissues (right) and OSCC cancerous tissues (left) is plotted. A total of 70 matched OSCC tissue pairs were tested. Each symbol represents data from one sample (OSCC cancerous tissue and adjacent normal tissue) (one-way-ANOVA), P = 0.0006. (B–D) F. nucleatum abundance for OSCC tissues with or without cervical lymph node metastasis (B), at high, middle and low pathological stages (C), as well as with flange, spike and skipping invasive phenotypes (D). (one-way-ANOVA), (B), P = 0.0231; (C), ANOVA P = 5.0E-16; (D), ANOVA P = 7.9E-05. (E) Representative images of H&E staining for tissue structure (up) and F. nucleatum spatial distribution by FISH staining (down) from clinical samples of OSCC lesions with flange, spike and skipping invasive phenotypes, respectively. Black and white curves indicate the borders between tumor tissues and adjacent normal tissues. (F) Representative data of xenograft tumors in C3H mice bearing SCC-7 cells in different groups. Local injection of F. nucleatum was applied to establish the F. nucleatum -associated tumor model. (G and H) Statistical analysis of mouse tumor weights (I) and volumes (J) in different groups. n = 5/group (two-tailed t test), Weight, P = 3.5E-05; Volume, P = 0.0052. (I) Representative FISH images of clinical OSCC samples using F. nucleatum -specific 16S rDNA-directed probe (left) and fluorescent images stained with 488-anti-SOX2, 488-anti-N-cad and 488-anti-ZEB1 (right) for measurement of tumor invasive levels by successive sections. (J) Representative fluorescent images of xenograft tumor samples with or without F. nucleatum infection stained with SOX2 (green), N-cad (green) and DAPI (blue).

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Staining, Injection, Two Tailed Test, Infection

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: Colonized F. nucleatum drives M2-like tumor-associated macrophages formation. (A and B) Representative fluorescent images of F. nucleatum spatial distribution, total macrophages and TAMs enrichment status in successive sections from clinical OSCC samples. Cy3-labeled F. nucleatum -specific probe was used for the detection of F. nucleatum . 488-anti-CD68 and 488-anti-CD206 were applied to visualize total macrophages and TAMs, respectively. A total of 40 paired successive sections on OSCC primary locus were tested to explore the relationship between F. nucleatum colonization and macrophage accumulation. (C) The Pearson linear correlation analysis was conducted based on F. nucleatum counts, total macrophages and TAMs in OSCC. (D) Representative fluorescent images of xenograft tumor samples (left) or normal oral epithelial samples (right) from C3H mice with or without F. nucleatum colonization stained with Cy3-anti-CD206 for identification of TAMs. (E) Representative data of xenograft tumors in C3H mice bearing SCC-7 cells in different groups. Local injection of F. nucleatum was used for in vivo F. nucleatum -associated tumor model. Injection of clodronate liposomes was applied for the eradication of local macrophages. (F and G) Statistical analysis of mouse tumor weights (I) and volumes (J) in different groups. n = 5/group (two-tailed t test), Weight, P = 0.0001; Volume, P = 4.9E-07. (H) The fluorescent assay showed a significant reversal of F. nucleatum -induced EMT markers (SOX2 and ZEB1) upregulation by eradicating local macrophages using clodronate liposomes. (I) schematic graph of indirect co-culture system including two crucial steps: extraction of oral tumor/epithelial cell culturing supernatant with or without F. nucleatum infection, and stimulation of M0 macrophages by extracted cell supernatant. (J and K) Assessment of macrophage polarization status in the indirect co-culture system mentioned above by immunofluorescent assay for CD86+ (green) M1-like macrophages and CD206+ (red) M2-like macrophages derived from M0 macrophages (J) as well as measuring mRNA expression of M2 markers ( CCL2 and IL12 ) in macrophages (K) indirectly co-cultured with oral tumor/epithelial cells with or without F. nucleatum infection (two-tailed t test), CCL2 , P = 0.0165; IL12 , P = 6.5E-05.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Labeling, Staining, Injection, In Vivo, Two Tailed Test, Fluorescence, Co-Culture Assay, Cell Culture, Infection, Derivative Assay, Expressing

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: F. nucleatum -induced lactate production of OSCC cells is required for M2-like tumor-associated macrophages formation. (A) Representative Giemsa staining images of OSCC invasive margins. The red square indicates the acidic border between tumor tissue and adjacent normal tissue, and the orange square indicates the non-acidic central regions. (B) Volcano plot for metabolite changes in filtered cell culturing supernatant with or without F. nucleatum infection. The red and green symbols indicate significantly upregulated and downregulated metabolites between the two groups. (C) Metabolic classification analysis of the top 5 metabolic terms based on differential metabolites in different groups (ranked by the number of enriched metabolites). (D) Pathway enrichment analysis of the top 5 metabolic pathways based on differential metabolites between the above two groups. (E) Alteration of intermediate metabolites of glycolysis in CAL27 culturing supernatant with or without infection of F. nucleatum (two-tailed t test), G6P, P = 0.0006; F-1,6-bisP, P = 1.2E-06; G3P, P = 0.0003; PEP, P = 0.0022; Pyruvate, P = 0.0004; Lactate, P = 0.0037. (F) Infection of F. nucleatum significantly enhanced the production of lactate, pyruvate, glucose consumption, and production of ATP in CAL27 cells. (two-tailed t test), Lactate, P = 0.0001; Pyruvate, P = 2.4E-06; Glucose consumption, P = 2.3E-05; ATP production, P = 0.0018. (G) Schematic graph of indirect co-culture system includes two crucial steps: extraction of CAL27 cell culturing supernatant with or without F. nucleatum infection, and stimulation of M0 macrophages by extracted cell supernatant with or without addition of 7ACC2 (10 μM) for inhibition of lactate adsorption. (H and I) Assessment of macrophage polarization status by measuring mRNA expression of M2 markers ( CCL2 and IL12 ) (I) as well as immunofluorescent assay of CD206 (J) in CAL27 cells treated with DMSO, F. nucleatum suspension or 7ACC2. (one-way-ANOVA), CCL2 , ANOVA P = 0.0048; IL12 , ANOVA P = 5.3E-05. (J) Schematic graph of indirect co-culture system including two crucial steps: extraction of oral tumor cell culturing supernatant with or without F. nucleatum infection plus 2-DG (20 mM) for inhibition of glycolysis, and stimulation of M0 macrophages by extracted cell supernatant. (K and L) Assessment of macrophage polarization status by measuring mRNA expression of M2 markers ( IL10 and IL12 ) (I) as well as immunofluorescent assay of CD206 (J) in CAL27 cells treated with DMSO, F. nucleatum suspension or 2-DG. (one-way-ANOVA), CCL2 , ANOVA P = 0.0141; IL12 , ANOVA P = 1.7E-06. (M) Schematic graph depicting indirect co-culture system with F. nucleatum -mediated TAM failed to promote tumor invasive behavior by inhibiting lactate adsorption of macrophages using 7ACC2. (N and O) Fluorescence assay of CAL27 cells for expression levels of ZEB1 and VIMENTIN (left) and Transwell assay depicting OSCC invasive potential under the above conditions. (one-way-ANOVA), ANOVA P = 0.0005.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Staining, Cell Culture, Infection, Two Tailed Test, Co-Culture Assay, Inhibition, Adsorption, Expressing, Fluorescence, Transwell Assay

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: F. nucleatum -induced accumulation of GLUT1 increases lactate production of OSCC cells. (A) Western blot analysis of glycolysis-related rate-limiting components (HK2, PFKP, PKM2, ENO1, LDHA, GLUT1) and GAPDH (for loading controls) in CAL27 cells co-cultured with or without F. nucleatum . All experiments were performed repeatedly three times. (two-tailed t test), PKM2, P = 0.048; LDHA, P = 0.0419; GLUT1, P = 0.0148. (B) Representative fluorescent images of successive sections from OSCC tissues for localization of F. nucleatum (red) and GLUT1 (green). (C) Representative images of GLUT1 (green) expression pattern in OSCC. (D) Clinical OSCC sections stained with 488-anti-KRT19 (green) and Cy3-anti-GLUT1 (red). High expression levels of GLUT1 could be detected mainly in KRT19+ tumor cells. (E) Representative images of xenograft tumor sections for measurement of GLUT1 expression (green) after injection of F. nucleatum suspension or PBS vehicle. (F and G) Glucose uptake and lactate production in CAL27 cells treated with F. nucleatum infection, BAY-876 (50 nM) or GLUT1 knockdown operations. (one-way-ANOVA), (F), Glucose, ANOVA P = 1.7E-06; Lactate, ANOVA P = 0.0142. (G), Glucose, ANOVA P = 0.0025; Lactate, ANOVA P = 0.0049. (H and I) Evaluation of macrophage polarization status by confocal fluorescence assay for CD206 (H) and mRNA expression levels of CCL2 and IL-12 (I). Macrophages were indirectly co-cultured with CAL27 cells treated by PBS, F. nucleatum infection or BAY-876. (one-way-ANOVA), CCL2 , ANOVA P = 0.0001; IL12 , ANOVA P = 0.0216.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Western Blot, Cell Culture, Two Tailed Test, Expressing, Staining, Injection, Infection, Fluorescence

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: F. nucleatum promotes GLUT1 localization on the cell surface through autophagy-dependent TBC1D5 decrease. (A) Representative confocal images of GLUT1 (green) and DIL-labelled cellular membrane (red) in CAL27 cells with or without F. nucleatum co-culture. (B) Western blot analysis of GLUT1 in the cytoplasm and cellular membrane of CAL27 cells with or without F. nucleatum infection. GAPDH was applied as the loading control for cytoplasm protein, and ATP1A was used as the loading control for membrane protein (two-tailed t test), P = 0.0002. (C) Western blot analysis of GLUT1 in CAL27 cells with or without F. nucleatum infection after pretreatment of CHX for 6 h at the proper concentration of 50 μg/mL. GAPDH was applied as the loading control. (one-way-ANOVA), ANOVA P = 7.4E-09. (D) Fluorescence assay for colocalization of GLUT1 (green) and LAMP1 (red) in CAL27 cells with or without F. nucleatum infection. (E) Western blot analysis of TBC1D5 and GLUT1 in CAL27 and CAL27/TBC1D5 treated by F. nucleatum infection or not. (one-way-ANOVA), TBC1D5, ANOVA P = 1.2E-05; GLUT1, ANOVA P = 0.0076. (F) The functional enrichment analysis of differentially expressed genes between CAL27 cells with or without F. nucleatum infection. (G) Confocal images of BECLIN1 (green) and DIL-labelled cellular membrane (red) in CAL27 cells with or without F. nucleatum infection. (H) Indicated proteins were detected by Western blot in CAL27 cells. The cells were co-cultured with F. nucleatum or treated by 3-MA (12 mM) for inhibition of autophagy. (one-way-ANOVA), TBC1D5, ANOVA P = 0.0040; GLUT1, ANOVA P = 2.3E-05; BECLIN1, ANOVA P = 1.1E-05; ATG5, ANOVA P = 0.0008. (I) Measurement of glucose uptake in CAL27 cells treated by PBS, F. nucleatum suspension or 3-MA. (one-way-ANOVA), ANOVA P = 0.0005.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Co-Culture Assay, Western Blot, Infection, Two Tailed Test, Concentration Assay, Fluorescence, Functional Assay, Cell Culture, Inhibition

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: F. nucleatum binds to GalNAc on OSCC cell surface to induce cell autophagy. (A) Fluorescence assay of KRT19 (red) and GalNAc (green) in clinical OSCC sections. (B) Representative fluorescent images of F. nucleatum (red) and FITC-PNA (green) in clinical OSCC sections. (C) Confocal images of SYTO9-labelled F. nucleatum (green) in CAL27 cells treated by PBS or PNA solutions at the proper concentration of 80 μg/mL. (D and E) Western blot analysis of indicated proteins in CAL27 cells treated with PBS, F. nucleatum suspension or PNA solution (one-way-ANOVA), ATG5, ANOVA P = 0.0004; BECLIN1, ANOVA P = 0.0016. (F) Glucose consumption (left) and lactate production (right) in CAL27 cells under F. nucleatum infection with or without PNA treatment. (one-way-ANOVA), Glucose, ANOVA P = 1.9E-07; Lactate, ANOVA P = 3.8E-08. (G and H) Confocal images of GLUT1 (green), LAMP1 (red), DIL-labelled cellular membrane (red) and cellular nuclei (blue) in CAL27 cells under F. nucleatum infection for 12 h and 24 h pretreated by PNA solution for localization of cellular GLUT1. (I) Western blot analysis of key components in AKT/mTOR pathway in CAL27 cells treated by F. nucleatum suspension or PNA solution. (one-way-ANOVA), P-AKT, ANOVA P = 2.3E-06; p-mTOR, ANOVA P = 0.0003. (J) Western blot analysis of GLUT1, BECLIN1 and ATG5 in CAL27 cells under F. nucleatum , SC79 or 3BDO treatment. GAPDH was used as the loading control. GLUT1, (one-way-ANOVA), ANOVA P = 0.0002; BECLIN1, ANOVA P = 2.0E-09; ATG5, ANOVA P = 0.0001.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Fluorescence, Concentration Assay, Western Blot, Infection

Journal: eBioMedicine

Article Title: F. nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production

doi: 10.1016/j.ebiom.2023.104444

Figure Lengend Snippet: Double targeting at GalNAc and GLUT1 inhibits OSCC progression. (A) Workflow diagram for translational medical research using BAY-876 and PNA for treatment of OSCC. (B) Representative images of xenograft tumors in C3H mice bearing SCC-7 cells in different groups. Local injection of F. nucleatum was used for in vivo F. nucleatum -associated tumor model, and injection of PNA, BAY-876 and a combination of them were applied as a different therapeutic strategy. (C and D) Statistical analysis of mouse tumor weights (C) and volumes (D) in different groups. n = 5/group. (one-way-ANOVA), Weight, ANOVA P = 1.7E-06; Volume, ANOVA P = 2.3E-08. (E) Representative images of CD68+ CD206+ M2-like TAMs (above) and CD86+ M1-like anti-tumor macrophages (below) in xenograft tumor samples from different groups. (F) Representative images of SOX2 (above) and ZEB1 (below) in xenograft tumor samples from different groups.

Article Snippet: Human primary oral keratinocytes (OKCs) derived from fresh oral mucosa tissues during operative resection, human OSCC cell lines CAL27 (ATCC Cat# CRL-2095, RRID: CVCL_1107), SCC15 (ATCC Cat# CRL-1623, RRID: CVCL_1681) and SCC25 (ATCC Cat# CRL-1628, RRID: CVCL_1682), and human monocyte cell line THP-1 (ATCC Cat# TIB-202, RRID: CVCL_0006) were cultured in air-humidified cell incubator with 5% CO2 at 37 °C.

Techniques: Injection, In Vivo