Journal: Advanced Science

Article Title: FGF19‐Activated Hepatic Stellate Cells Release ANGPTL4 that Promotes Colorectal Cancer Liver Metastasis

doi: 10.1002/advs.202413525

Figure Lengend Snippet: Activated HSCs secrete ANGPTL4 to promote CRC cells migration. A) Venn diagram showing the intersection between two datasets including GSE215882 and GSE224235, with ANGPTL4 was highlighted in the overlapping section. B) The mRNA levels of ANGPTL4 in tissues of the colorectal primary site and liver metastasis site were determined by spatially resolved transcriptomics (colorectal primary site, n = 9; Liver metastasis site, n = 8). C) The ANGPTL4 and FAP expression were simultaneously stained in the clinical sample tissues by using the microarray assay (n = 42). DAPI: blue color; FAP: green color; ANGPTL4: yellow color. D) The protein levels of ANGPTL4 in SW620 CM‐treated LX‐2 were determined by Western blotting (n = 3). E) Protein levels of ANGPTL4 after treated by the recombinant protein FGF19 were determined by Western blotting (n = 3). F) Levels of ANGPTL4 in the recombinant protein FGF19‐treated LX‐2 cell system (n = 4). G) Effect of the recombinant ANGPTL4 protein on the migration of SW620 cells (n = 3). H) Effect of coculture LX‐2 cells overexpressing ANGPTL4 with CRC cells on the migration ability of SW620 cells (n = 3). Data are shown as Mean ±SD from three independent experiments. For B, ** p < 0.01 versus the colorectal primary group. For D, * p < 0.05 versus HSCs CM. For E‐G: * p < 0.05, ** p < 0.01 versus the corresponding CTL. For H: ** p < 0.01 versus the corresponding CTL; ## p < 0.01 versus SW620‐LX‐2 coculture group.

Article Snippet: Infigratinib, human angiopoietin‐related 4 protein (ANGPTL4, HEK293, His), and mouse FGF15 Protein (His‐SUMO) were purchased from MedChemExpress (MCE, NJ, USA).

Techniques: Migration, Expressing, Staining, Microarray, Western Blot, Recombinant

Journal: Advanced Science

Article Title: FGF19‐Activated Hepatic Stellate Cells Release ANGPTL4 that Promotes Colorectal Cancer Liver Metastasis

doi: 10.1002/advs.202413525

Figure Lengend Snippet: Effects of FGF15 and ANGPTL4 in mouse models of different degrees of CRCLM. A) Expression levels of MMP2 and MMP9 in the liver tissues of each group were detected by IHC staining. B,C) Protein levels of FGF15 in the liver tissues (B) and tumor sites (C) were determined by Western blotting. D) The serum level of FGF15 was determined by the ELISA assay. E) Expression of FGFR4, a FGF15 receptor in the liver tissues of each group was detected by IHC staining. F) Expression levels of α‐SMA and FAP in the liver tissues of each group were detected by IHC staining. G,H) Protein levels of α‐SMA in the liver tissues with tumors were determined by using Western blotting (G), and quantitative results were analyzed using Image J software (H). I) mRNA levels of FAP in the liver tissues with tumors were determined using RT‐qPCR analysis. J) Protein levels of ANGPTL4 in the liver tissues were determined by Western blotting (left panel), and the quantitative data were analyzed using Image J software (right panel). K,L) The liver tissue homogenates (K) and the serum (L) levels of ANGPTL4 were determined by the ELISA assay. M) Localization of FGF15 in CTCs within the liver of CRCLM mice by using the mIF analysis. N) Localization of ANGPTL4 in the liver tissue CAFs in CRCLM mouse model determined using the mIF analysis. Data are shown as Mean ± SD, n = 6. For B, D, H‐L, * p < 0.05, ** p < 0.01 versus CTL. For C, ** p < 0.01 versus 5.0 × 10 5 group.

Article Snippet: Infigratinib, human angiopoietin‐related 4 protein (ANGPTL4, HEK293, His), and mouse FGF15 Protein (His‐SUMO) were purchased from MedChemExpress (MCE, NJ, USA).

Techniques: Expressing, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay, Software, Quantitative RT-PCR

Journal: Advanced Science

Article Title: FGF19‐Activated Hepatic Stellate Cells Release ANGPTL4 that Promotes Colorectal Cancer Liver Metastasis

doi: 10.1002/advs.202413525

Figure Lengend Snippet: The effects of FGF15 and ANGPTL4 in CRCLM mouse models with different time points. A) Timeline for the establishment of the mouse CRCLM model. CT26‐luc cells were injected into the spleens of BALB/c mice, then the mice were sacrificed at different time points. B) Representative images of live tumor‐bearing mice with tumors. C) Representative fluorescence signal imaging of the liver tissues. D,E) Quantitative results of the mice fluorescence intensity (D) and the liver fluorescence intensity (E) were analyzed using the Living Image software 4.4. F–H) Representative IHC staining of FGF15 and FGFR4 (F) MMP2 and MMP9 (G), α‐SMA, and FAP (H) in the liver tissues of mice in each group. I) Localization of ANGPTL4 in CAFs of liver tissues in CRCLM mouse model by using the mIF analysis. J) Protein levels of ANGPTL4 in the liver tissues with tumors were determined by using Western blotting (upper panel); and quantitative results were quantified and analyzed using Image J software (lower panel). K,L) Liver tissue homogenates (K) and the serum (L) levels of ANGPTL4 were determined by the ELISA assay. Data are shown as Mean ± SD. n = 6. * p < 0.05, ** p < 0.01 versus CTL.

Article Snippet: Infigratinib, human angiopoietin‐related 4 protein (ANGPTL4, HEK293, His), and mouse FGF15 Protein (His‐SUMO) were purchased from MedChemExpress (MCE, NJ, USA).

Techniques: Injection, Fluorescence, Imaging, Software, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: FGF19‐Activated Hepatic Stellate Cells Release ANGPTL4 that Promotes Colorectal Cancer Liver Metastasis

doi: 10.1002/advs.202413525

Figure Lengend Snippet: The FGF15/ANGPTL4 axis is involved in the progression of CRCLM. A,B) Representative images of IHC staining for FGF15 and FGFR4 (A), and for α‐SMA and FAP (B) in the liver tissues of mice in each group. C) The protein levels of ANGPTL4 in tumor‐bearing liver tissues were determined by using Western blotting (upper panel); and quantitative results were analyzed using Image J software (lower panel). D) Representative live‐animal imaging images of mice with tumors. CT26‐luc‐shNC cells and CT26‐luc‐shFGF15 cells were inoculated into the hepatic portal vein of BALB/c mice, respectively. Mice were randomly divided into 3 groups, including the sham, shNC, and shFGF15 groups. E) Quantitative results of the fluorescence intensity were analyzed using Living Image software 4.4. F) Representative H&E‐stained images of liver tissues in each group of mice. G) Representative images of IHC for MMP2 and MMP9 in mouse liver tissues from each group. H) Diagram showing that the FGF19/ANGPTL4 axis mediates the interaction between CRC cells and HSCs, and promotes CRC liver metastasis. Data are shown as Mean ± SD, n = 6. ** p < 0.01 versus Sham; # p < 0.05, ## p < 0.01 versus shNC.

Article Snippet: Infigratinib, human angiopoietin‐related 4 protein (ANGPTL4, HEK293, His), and mouse FGF15 Protein (His‐SUMO) were purchased from MedChemExpress (MCE, NJ, USA).

Techniques: Immunohistochemistry, Western Blot, Software, Imaging, Fluorescence, Staining

Journal: Cancer Medicine

Article Title: ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer

doi: 10.1002/cam4.418

Figure Lengend Snippet: Evaluation of ANGPTL3 expression in OSCC-derived cell lines. (A) Quantification of ANGPTL3 mRNA expression in OSCC-derived cell lines by qRT-PCR analysis. Significant (* P < 0.05, Mann–Whitney U -test) upregulation of ANGPTL3 mRNA is seen in seven OSCC-derived cell lines compared with the HNOKs. Data are expressed as the mean ± SEM of triplicate results. (B) Immunoblotting analysis of ANGPTL3 protein in the OSCC-derived cell lines and HNOKs. ANGPTL3 protein expression is upregulated in the OSCC-derived cell lines compared with the HNOKs. Densitometric ANGPTL3 protein data are normalized to GAPDH protein levels. The values are expressed as a percentage of the HNOKs ( P < 0.05, Mann–Whitney U -test).

Article Snippet: ANGPTL3 shRNA (shANGPTL3) and the control shRNA (shMock) (Santa Cruz) vectors were transfected into HSC-3 and Sa3.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, MANN-WHITNEY, Western Blot

Journal: Cancer Medicine

Article Title: ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer

doi: 10.1002/cam4.418

Figure Lengend Snippet: Evaluation of ANGPTL3 protein expression in primary OSCCs. (A and B) Representative IHC results of ANGPTL3 in primary OSCCs and normal oral tissues. Original magnification, ×100. Scale bars, 50 μ m. ANGPTL3 is highly overexpressed in OSCCs compared to normal oral tissues. (C) The status of ANGPTL3 protein expression in primary OSCCs ( n = 109) and normal counterparts based on an IHC scoring system. IHC scores are calculated as follows: IHC score = 1 × (number of weakly stained cells in the field) + 2 × (number of moderately stained cells in the field) + 3 × (number of intensely stained cells in the field). The ANGPTL3 IHC scores for normal oral tissues and OSCCs range from 30.5 to 105.2 (median, 65.5) and 55.5 to 200.0 (median, 138.6), respectively. ANGPTL3 protein expression levels in OSCCs are significantly higher than in normal oral tissues (* P = 0.003; Mann–Whitney U test). (D) Kaplan–Meier curve for overall survival. The ANGPTL3 expression level is not correlated significantly ( P = 0.076, log-rank test) with the overall survival. The overall survival rates in the ANGPTL3-positive OSCCs ( n = 65) and the ANGPTL3-negative OSCCs ( n = 44) were 78.7% and 89.9%, respectively. (E) Kaplan–Meier overall survival curves of patients with OSCC with a primary tumor size of T3/T4. A significant ( P = 0.047, log-rank test) difference is seen in the overall survival rates between the ANGPTL3-positive OSCCs ( n = 31, 58.6%) and the ANGPTL3-negative OSCCs ( n = 12, 91.6%). (F) To evaluate the diagnostic relevance of the identified IHC scores, we used the ROC curve by plotting sensitivity versus specificity. The ANGPTL3 IHC scores of patients with primary T3/T4 OSCC tumors in primary OSCCs normalized to those in normal tissues. The optimal threshold value was 2.25 (sensitivity, 75.8%; specificity, 68.2%). When the cutoff values for the ANGPTL3 IHC scores were set at 2.25, the AUC was 0.7293 (95% CI, 0.5682–0.8903, P < 0.05).

Article Snippet: ANGPTL3 shRNA (shANGPTL3) and the control shRNA (shMock) (Santa Cruz) vectors were transfected into HSC-3 and Sa3.

Techniques: Expressing, Staining, MANN-WHITNEY, Diagnostic Assay

Journal: Cancer Medicine

Article Title: ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer

doi: 10.1002/cam4.418

Figure Lengend Snippet: Correlation between ANGPTL3 expression and clinical classification in OSCCs

Article Snippet: ANGPTL3 shRNA (shANGPTL3) and the control shRNA (shMock) (Santa Cruz) vectors were transfected into HSC-3 and Sa3.

Techniques: Expressing, Immunostaining

Journal: Cancer Medicine

Article Title: ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer

doi: 10.1002/cam4.418

Figure Lengend Snippet: Expression of ANGPTL3 and ANGPTL3 knockdown inhibits ERK activation and promotes G1 arrest. (A) qRT-PCR shows that ANGPTL3 mRNA expression in the shANGPTL3-transfected cells (HSC-3- and Sa3-derived transfectants) is significantly (* P < 0.05, Mann–Whitney U -test) lower than in the shMock-transfected cells. (B) Immunoblotting analysis shows that the ANGPTL3 protein levels in the shANGPTL3-transfected cells (HSC-3- and Sa3-derived transfectants) also are decreased markedly compared with the shMock-transfected cells. (C) To determine the effect of shANGPTL3 on cellular proliferation, shANGPTL3-, and shMock-transfected cells were seeded in six-well plates at a density of 1 × 10 4 viable cells/well. Both transfectants were counted on seven consecutive days. The cellular growth of shANGPTL3-transfected cells (HSC-3- and Sa3-derived transfectants) is significantly inhibited compared with the shMock-transfected cells after 7 days (168 h). The results are expressed as the means ± SEM of values from three assays. The asterisks indicate significant (* P < 0.05, Mann–Whitney U -test) differences between the shANGPTL3 and shMock cells. (D) Immunoblotting analysis shows that ANGPTL3 knockdown results in decreased levels of pERK compared with the shMock-transfected cells (HSC-3- and Sa3-derived transfectants). Densitometric pERK/ERK protein data are normalized to GAPDH protein levels. (E) Immunoblotting analysis shows upregulation of p21 Cip1 and p27 Kip1 and downregulation of cyclin D1, cyclin E, CDK2, CDK4, and CDK6 in the shANGPTL3-transfected cells (HSC-3- and Sa3-derived transfectants) compared with the shMock-transfected cells. (F) Flow cytometric analysis was performed to investigate cell-cycle progression in the shANGPTL3- and shMock-transfected cells after synchronization at the G2/M phase to treatment with nocodazole. The percentage of cells at the G1 phase in the shANGPTL3-transfected cells (HSC-3- and Sa3-derived transfectants) is increased markedly compared with the shMock- transfected cells.

Article Snippet: ANGPTL3 shRNA (shANGPTL3) and the control shRNA (shMock) (Santa Cruz) vectors were transfected into HSC-3 and Sa3.

Techniques: Expressing, Knockdown, Activation Assay, Quantitative RT-PCR, Transfection, Derivative Assay, MANN-WHITNEY, Western Blot

Journal: Cancer Medicine

Article Title: ANGPTL3 is a novel biomarker as it activates ERK/MAPK pathway in oral cancer

doi: 10.1002/cam4.418

Figure Lengend Snippet: ANGPTL3 promotes tumoral growth in vivo. (A) ShANGPTL3- and shMock-transfected cells (HSC-3 and Sa3) were injected subcutaneously into the backs of female nude mice ( n = 3). Tumoral growth in the shANGTPL3-injected mice is inhibited significantly (* P < 0.05; Mann–Whitney U -test) compared to the shMock-injected mice. (B) IHC of the xenografted tumors clearly shows more decreased immunostaining for ANGPTL3 and pERK in the xenografted tumors from shANGPTL3 transfectants than shMock transfectants. H&E staining confirmed the presence of tumoral cells. Original magnification, ×400. Scale bars, 50 μ m.

Article Snippet: ANGPTL3 shRNA (shANGPTL3) and the control shRNA (shMock) (Santa Cruz) vectors were transfected into HSC-3 and Sa3.

Techniques: In Vivo, Transfection, Injection, MANN-WHITNEY, Immunostaining, Staining