Journal: iScience

Article Title: FAP promotes metastasis and chemoresistance via regulating YAP1 and macrophages in mucinous colorectal adenocarcinoma

doi: 10.1016/j.isci.2023.106600

Figure Lengend Snippet: FAP promotes CRC cell growth in vivo and in vitro (A) LS174T and HCT8 CRC cells were infected with FAP-overexpressing lentivirus; the length of scale bar is 100 μm. The expression level of FAP was assayed by quantitative RT-qPCR and western blot. Data are represented as mean ± SEM. (B) Cell growth stimulated by FAP in LS174T and HCT8 CRC cells was detected by CCK-8 assay. Data are represented as mean ± SEM, using two-way ANOVA. (C) The effects of FAP on the growth of LS174T cells were evaluated in BALB/c-nu mice. (D) Tumor growth curves. Points and bars represent the mean tumor volume ±SD (six mice in each group, using two-way ANOVA, p < 0.05). (E) Tumor weight derived from LS174T overexpressing FAP and control cells. Data are represented as mean ± SD, using Student’s t test, ∗∗p < 0.01. See also Figure S2 .

Article Snippet: Human CRC cell lines HCT116, HCT8, and LS174T were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, NY, USA) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: In Vivo, In Vitro, Infection, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Derivative Assay, Control

Journal: iScience

Article Title: FAP promotes metastasis and chemoresistance via regulating YAP1 and macrophages in mucinous colorectal adenocarcinoma

doi: 10.1016/j.isci.2023.106600

Figure Lengend Snippet: FAP promotes tumor invasion and liver metastasis (A) Wound-healing assay showed that cell motility was enhanced following FAP overexpression in LS174T cells. Representative images from wounds made using a confluent monolayer of LS174T overexpressing FAP and control cells taken at 0 h and 24 h post wounding. The length of scale bar is 500 μm. (B) The Boyden chamber assay was performed to investigate the effect of FAP on LS174T cells motility. The length of scale bar is 100 μm. (C) FAP enhanced liver metastasis of LS174T cells. The tumor burden on liver was quantified by in vivo bioluminescence imaging using an IVIS Spectrum-CT. (D) Tumor burden quantified weekly for LS174T overexpressing FAP, controls by in vivo bioluminescent imaging. Data are expressed as mean ± SD (n = 6). A mixed-effect linear model was used to determine significance of differences in tumor growth, p <0.05. (E) In vivo 3D bioluminescence imaging. See also Figure S2 .

Article Snippet: Human CRC cell lines HCT116, HCT8, and LS174T were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, NY, USA) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Wound Healing Assay, Over Expression, Control, Boyden Chamber Assay, In Vivo, Imaging

Journal: iScience

Article Title: FAP promotes metastasis and chemoresistance via regulating YAP1 and macrophages in mucinous colorectal adenocarcinoma

doi: 10.1016/j.isci.2023.106600

Figure Lengend Snippet: FAP induces chemotherapy resistance of CRC cells and drives the TIC phenotype (A) The IC50 of 5-FU, CPT-11, and doxorubicin in control or FAP-overexpressing CRC cells, HCT8, HCT116, and LS174T. Data are represented as mean ± SEM. (B) The expression level of FAP in HCT8, HCT8-5-Fu, and HCT8-CPT-11 cell lines was assayed by RT-qPCR and western blotting. Data are represented as mean ± SEM. (C) The expression level of FAP in CRC spheroids was assayed using RT-qPCR and western blotting. Data are represented as mean ± SEM. (D) (a) Phase-contrast micrographs illustrate that FAP-overexpressing HCT116 cells can form primary (first) and serially passaged (second) spheroids easily. The length of scale bar is 200 μm. (b) Histograms show spheroid-formation efficiency of the FAP-overexpressing cells and control cells. Bars are mean ± SD of three independent experiments (n = 3). Student’s t test, ∗p <0.05. (E) The expression levels of several stem cell markers in FAP-overexpressing HCT116 cells and control cells were assayed by RT-qPCR and western blotting. Data are represented as mean ± SEM.

Article Snippet: Human CRC cell lines HCT116, HCT8, and LS174T were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, NY, USA) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: iScience

Article Title: FAP promotes metastasis and chemoresistance via regulating YAP1 and macrophages in mucinous colorectal adenocarcinoma

doi: 10.1016/j.isci.2023.106600

Figure Lengend Snippet: FAP activates RhoA-Hippo signaling in CRC cells. Knockdown of FAP reverses tumorigenicity and chemoresistance in CRC cells (A) Analysis of active RhoA using RhoA-GTP pull-down assay in FAP-overexpressing HCT116 cells and control cells. (B) RhoA expression in input samples was used as control for quantification and GAPDH as loading control (n = 2, two-sided Student’s t test, ∗∗ p <0.01). (C) Protein analysis of phosphorylated (S109, S397) and total YAP1 in FAP-overexpressing HCT116 cells and control cells. (D) Immunofluorescence staining of YAP1 (green) in FAP-overexpressing HCT116 cells and control cells. DAPI was used to stain the nuclei (blue). The length of scale bar is 10 μm. (E) Analysis of active RhoA using RhoA-GTP pull-down assay in FAP-overexpressing LS174T cells and control cells. (F) The effect of siMPRIP on the activity of RhoA in FAP-overexpressing LS174T cells. (G) Protein analysis of phosphorylated (S109, S397) and total YAP1 in MPRIP knock downed FAP-overexpressing LS174T cells and control cells. (H) MPRIP siRNA inhibited cell proliferation in FAP-overexpressing LS174T cells assayed by the colony formation assay. (I) MPRIP siRNA inhibited cell motility in FAP-overexpressing LS174T cells assayed by wound-healing test. The length of scale bar is 500 μm. (J) MPRIP siRNA rescued the sensitivity to 5-Fu in FAP-overexpressing LS174T cells. Data are represented as mean ± SEM. (K) Fractions of immune cell subsets in tumor samples inferred from TCGA MCs data using CIBERSORT. Šídák’s multiple comparisons test was used to compare the difference of immune cell subsets between FAP high- and low-expression groups, ∗∗ p <0.01, ∗∗∗ p <0.001, ∗∗∗∗ p <0.0001. (L) Proportion of F4/80+ cells after gating on CD45 + cell in tumor tissues from FAP-overexpressed and control groups. (M) Proportion of CD206+CD86 − macrophages in tumor. (N) Proportion of CD206+CD86 + macrophages in tumor. (O) Proportion of CD206-CD86 + macrophages in tumor. (P) Proportion of F4/80+ cells after gating on CD45 + cell in blood. (Q) Proportion of CD206+CD86 − monocytes in blood. (R) Proportion of CD206+CD86 + monocytes in blood. (S) Proportion of CD206-CD86 + monocytes in blood. Student’s t test was used to compare the difference of monocytes between FAP-overexpressed and control groups. ∗p < 0.05, ∗∗p < 0.01. (T) Chemokine levels in conditioned medium (CM) from control or FAP-overexpressing LS174T cells measured by protein array. See also Figure S4 .

Article Snippet: Human CRC cell lines HCT116, HCT8, and LS174T were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, NY, USA) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Knockdown, Pull Down Assay, Control, Expressing, Immunofluorescence, Staining, Activity Assay, Colony Assay, Protein Array

Journal: iScience

Article Title: FAP promotes metastasis and chemoresistance via regulating YAP1 and macrophages in mucinous colorectal adenocarcinoma

doi: 10.1016/j.isci.2023.106600

Figure Lengend Snippet: Knockdown of FAP reversed tumorigenicity and chemoresistance in CRC cells (A) LS174T cells were transfected with FAP siRNA and control. The expression level of FAP was assayed by western blot. (B) FAP siRNA inhibited cell growth in LS174T cells using CCK-8 assay. The results are expressed as mean ± SD of three independent experiments, using two-way ANOVA (p < 0.05). (C) FAP siRNA inhibited cell motility. Wound-healing test was used to assay cell motility. The length of scale bar is 500 μm. (D) The Boyden chamber assay was performed to assay cell migration and cell invasion. The length of scale bar is 100 μm. (E) The IC50 of CPT-11 in FAP-knockdown LS174T cells and control cells. Data are represented as mean ± SEM, using two-way ANOVA. (F) The IC50 of 5-FU in FAP-knockdown LS174T cells and control cells. Data are represented as mean ± SEM, using two-way ANOVA.

Article Snippet: Human CRC cell lines HCT116, HCT8, and LS174T were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, NY, USA) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Knockdown, Transfection, Control, Expressing, Western Blot, CCK-8 Assay, Boyden Chamber Assay, Migration

Journal: iScience

Article Title: FAP promotes metastasis and chemoresistance via regulating YAP1 and macrophages in mucinous colorectal adenocarcinoma

doi: 10.1016/j.isci.2023.106600

Figure Lengend Snippet:

Article Snippet: Human CRC cell lines HCT116, HCT8, and LS174T were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, NY, USA) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere with 5% CO 2 at 37°C.

Techniques: Virus, Magnetic Beads, Activation Assay, Software