Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: FHL2 is highly expressed in lung cancer tissue and is a myofibroblast marker as demonstrated by using a previously published single‐cell RNA‐sequencing dataset. (A) Left, UMAP projection color‐coded by major cell lineages in lung tissue. Right, FHL2 expression in each subset. (B) Violin plot showing the expression level of FHL2 in fibroblasts of normal lung tissue (normal lung) and lung cancer tissue (tumor lung). (C) Upper left, UMAP projection of fibroblasts, color‐coded by subtypes identified after subclustering of the fibroblast subset in (B). Upper right, FHL2 expression of each subset. Lower, Violin plot showing the expression level of FHL2 in subtypes of fibroblasts.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Marker, RNA Sequencing, Expressing

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: Knock‐down of FHL2 in CAF reduces proliferation of CAFs and establishment of FHL2 KO CAFs and TGF‐β1 upregulate FHL2 expression on CAF and characteristics of FHL2 KO CAFs. (A) Silencing of FHL2 in CAF094 hTERTRFP by siRNA. Western blot analysis showing FHL2 expression in CAF094 hTERTRFP transfected with siRNAs. (B) WST‐8 assay for the assessing the growth of CAF094 hTERTRFP cells transfected with siFHL2 or scrambled siRNA (siScr) after a 96‐h incubation. Data represent the mean ± SEM ( n = 3). * p < .05; ** p < .01. (C) Western blot analysis of FHL2 in CAF094 YFPhTERT and CAF094 YFPhTERT FHL2KO cells. GAPDH was used as an internal control. (D) WST‐8 assay for assessing the growth of CAF094 YFPhTERT cells (WT) and CAF094 YFPhTERT FHL2KO cells (KO) after a 72‐h incubation. (E) Western blot analysis of FHL2 in CAF094 YFPhTERT cells. CAF094 YFPhTERT cells were stimulated with 5 ng/mL of recombinant human TGF‐β1 for 48 h. GAPDH was used as an internal control. (F) SRB assay for the assessing the growth of CAF094 YFPhTERT cells (WT) and CAF094 YFPhTERT FHL2KO cells (KO) cells stimulated with 5 ng/mL of recombinant human TGF‐β1 for 72‐h. Data represent the mean ± SEM (n = 3). * p < .05. (G) Results of a gel contraction assay of CAF094 YFPhTERT cells (WT) and CAF094 YFPhTERT FHL2KO cells (KO). Gel contraction was monitored after 72 h by taking photographs of the gels and the percent contraction was calculated using the formula: 100 × (well area − gel area)/well area. (H) Results of a PDGF‐BB‐induced Transwell migration assay. Human recombinant PDGF‐BB (40 ng/mL) was placed in the lower chamber and cells migrating through the membrane were counted after a 24‐h incubation. Data show the mean ± SEM ( n = 3). * p < .05.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Knockdown, Expressing, Western Blot, Transfection, Incubation, Control, Recombinant, Sulforhodamine B Assay, Collagen Gel Contraction Assay, Transwell Migration Assay, Membrane

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: FHL2 KO on CAFs decreases CAF‐induced migration and invasion of human lung adenocarcinoma cells in vitro and metastasis in an orthotopic lung cancer model. (A) Migration of lung adenocarcinoma cells induced by CAFs was analyzed. In the lower chamber of the Transwell chamber, CAF094 YFPhTERT cells (WT) or CAF094 YFPhTERT FHL2KO cells (KO) were seeded and incubated for 48 h. Normal medium (DMEM supplemented with 10% FBS) was used as a control (Cont). A110L or H23 adenocarcinoma cells were placed into the upper compartment of a Transwell chamber and the cells migrating through the membrane were counted after a 24‐h incubation. (B) Invasion of lung adenocarcinoma cells induced by CAF were analyzed. Matrigel‐coated cell culture inserts were used. In the lower compartment of the Transwell chamber, CAF094 YFPhTERT cells (WT) or CAF094 YFPhTERT FHL2KO cells (KO) were seeded and incubated for 48 h. Normal medium (DMEM supplemented with 10% FBS) was used as a control (Cont). A110L adenocarcinoma cells were placed into the upper compartment of a Transwell chamber and the cells migrating through the membrane were counted after a 24‐h incubation. Data represent the mean ± SEM ( n = 3). ** p < .01; * p < .05. (C) Assessment of the influence of FHL2 KO in CAFs on pulmonary metastasis using an orthotopic lung cancer model. A110L cells (A110L group) or A110L cells with CAF094 YFPhTERT cells (A110L + CAF group), or A110L cells with CAF094 YFPhTERT FHL2KO cells (A110L + CAFFHL2KO group) were inoculated into the left lungs of mice. All the mice in one experiment were sacrificed once significant morbidity was observed in any mouse in the experimental group. As a result, the experiments were terminated at approximately d37–d46. The formation of a primary lung tumor was confirmed and macroscopic pulmonary metastasis to the right lung was evaluated. (Left) Representative macroscopic view of the primary tumor and pulmonary metastasis in the A110L + CAF group. Arrow: Primary tumor arrowhead: Pulmonary metastasis. (Middle) Representative microscopic view of cytokeratin AE1/AE3 staining of main tumor in the left lung in the A110L group as a positive control of immunohistochemical staining to detect A110L lung cancer cells. Scale bar, 200 μm. (Right) Representative microscopic view of cytokeratin AE1/AE3 staining of micrometastasis in the right lung in the A110L + CAF group. Arrow: Primary pulmonary metastasis. Scale bar, 100 μm. (Table) Incidence of pulmonary metastasis in each group is shown. * p < .05.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Migration, In Vitro, Incubation, Control, Membrane, Cell Culture, Staining, Positive Control, Immunohistochemical staining

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: FHL2 KO in CAFs reduces CAF‐induced tube formation of MS1 cells and relationship of microvascular density and stromal FHL2 expression in clinical tumor samples. (A, B) Results of an endothelial cell tube formation assay. Murine endothelial MS1 cells were seeded in conditioned medium from CAF094 YFPhTERT cells (WT) or CAF094 YFPhTERT FHL2KO cells (KO). Photographs taken 6 h post‐seeding were analyzed. (A) Representative photographs of WT and KO groups are shown. (B) results of the quantification of tube formation are shown. Data represent the mean ± SEM. (C) Immunohistochemical analysis of CD34 in lung adenocarcinoma tissues from patients who underwent surgery. Scale bar, 50 μm. (Left) Representative image showing a tumor with high microvascular density. (Right) Representative image showing a tumor with low microvascular density. (D) Relationship of microvascular density and FHL2 expression. (Left) Relationship of microvascular density and tumoral FHL2 expression. (Right) Relationship of microvascular density and stromal FHL2 expression. **p < .01; *p < .05.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Expressing, Endothelial Cell Tube Formation Assay, Immunohistochemical staining

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: Survival analysis according to tumoral or stromal FHL2 expression.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Expressing

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: FHL2 KO in CAFs reduces OPN secretion from CAF and knock‐down of OPN in CAF or antibody blocking of OPN reduces CAF induced migration of A110L. (A) Relative concentrations of 13 proteins of which concentration was significantly different between CM of CAF094 YFPhTERT cells (WT) and CAF094 YFPhTERT FHL2KO cells (KO). Data represent the mean ± SEM ( n = 3). (B) Western blot analysis of OPN in cell lysate of CAF094 YFPhTERT and CAF094 YFPhTERT FHL2KO cells. GAPDH was used as an internal control. (C) Silencing of OPN in CAF094 hTERTRFP by siRNA. Three different siRNAs and smart pool (SP; mixture of three different siOPN RNAs) were used. Western blot analysis showing OPN expression in CAF094 hTERTRFP transfected with siRNAs. (D) Knock down effect of OPN in CAF on migration of lung adenocarcinoma cell A110L induced by CAF CM was analyzed. In the lower chamber of the Transwell chamber, CM from CAF094 YFPhTERT cells transfected with siScr (control) or siOPN‐SP (mixture of three different siOPN RNAs) were placed. A110L adenocarcinoma cells were placed into the upper compartment of a Transwell chamber and the cells migrating through the membrane were counted after a 24‐h incubation. Data of average of three experiments was shown. ** p < .01. (E) Antibody blocking effect of OPN on migration of lung adenocarcinoma cell A110L induced by CAF CM was analyzed. In the lower chamber of the Transwell chamber, CM from CAF094 YFPhTERT cells incubated with control IgG or anti OPN antibody were placed. A110L adenocarcinoma cells were placed into the upper compartment of a Transwell chamber and the cells migrating through the membrane were counted after a 24‐h incubation. Data of average of three experiments was shown. * p < .05.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Knockdown, Blocking Assay, Migration, Concentration Assay, Western Blot, Control, Expressing, Transfection, Membrane, Incubation

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: Gene expression signature analysis and comparisons to common lung adenocarcinoma driver alterations. (A) Scatter plots of FHL2 mRNA expression versus six proposed biological metagenes in lung cancer. Correlation was calculated using Pearson correlation. Dotted red line corresponds to a linear regression model of FHL2 versus respective metagene. Both FHL2 mRNA expression and metagene scores were mean‐centered as described in Karlsson et al. ¹⁸ (B) FHL2 mRNA expression stratified by gene mutation status for EGFR , KRAS , STK11 , and TP53 in the Lund lung adenocarcinoma cohort. p ‐Values were calculated using Wilcoxon's test.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Gene Expression, Expressing, Mutagenesis

Journal: International Journal of Cancer

Article Title: FHL2 expression by cancer‐associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma

doi: 10.1002/ijc.35174

Figure Lengend Snippet: Expression and significance of FHL2 in clinical tumor samples. (A) Immunohistochemical analysis of FHL2 in lung adenocarcinoma tissues from patients with lung adenocarcinoma who underwent surgery (Osaka cohort). Scale bar, 100 μm. Upper panels show representative images of tumoral and stromal FHL2‐negative, tumoral FHL2‐negative stromal FHL2‐positive, tumoral FHL2‐positive stromal FHL2 negative, and tumoral and stromal FHL2‐positive tumor tissues. Tissues were stained with an anti‐FHL2 antibody (brown) and counterstained with hematoxylin. Lower panels show schematic image of tumoral and stromal components. S, stromal component; T, tumoral component. (B) Relationship between tumoral and stromal FHL2 expression in each tumor. (C) Relationship between stromal FHL2 expression and lymph node metastasis. (D) Relationship between stromal FHL2 expression and histologic subtype. AIS, adenocarcinoma in situ; MIA, minimally invasive adenocarcinoma. (E) (Left) Association of stromal FHL2 expression with recurrence in the Osaka lung adenocarcinoma cohort. Kaplan–Meier plot showing the cumulative recurrence rate in patients with lung adenocarcinoma who underwent surgery relative to stromal FHL2 expression. Patients were divided into four groups according to tumoral and stromal FHL2 expression, that is, both tumoral and stromal FHL2‐negative group (double negative [DN]), both tumoral and stromal FHL2‐positive group (double positive [DP]), tumoral FHL2‐negative and stromal FHL2‐positive group (stromal positive [SP]), and tumoral FHL2‐positive and stromal FHL2‐negative group (tumoral positive [TP]). The 5‐year cumulative recurrence rate of DN, DP, SP, TP groups were 11.6%, 18.2%, 38.1%, and 15.0%, respectively. The 5‐year cumulative recurrence rate of SP group was significantly higher compared with that of DN group ( p < .01), however, the difference between SP and DP, TP groups were not significant. (Right) Association of stromal FHL2 expression with disease‐specific survival in the Osaka cohort. Kaplan–Meier plot of the disease‐specific survival rate in patients with lung adenocarcinoma who underwent surgery according to stromal FHL2 expression. The 5‐year disease‐specific survival rate of DN, DP, SP, TP groups were 96.2%, 88.9%, 88.0%, and 96.3%, respectively. The 5‐year disease‐specific survival rate of SP group was significantly lower compared with that of DN group ( p < .05), however, the difference between SP and DP, TP groups were not significant. DN, double negative; DP, double positive; SP, stromal positive; TP, tumoral positive.

Article Snippet: The sections were then incubated at 4°C overnight with rabbit polyclonal anti‐human FHL2 antibody (1/200) (HPA006028, Atlas antibodies, Stockholm, Sweden) or mouse monoclonal anti‐human CD34 (1/400, clone QBEnd/10, NBP2‐32932, Novus biologicals, Novus Biological, Littleton, CO, USA).

Techniques: Expressing, Immunohistochemical staining, Staining, In Situ

Journal: Journal of Clinical Medicine

Article Title: Serum and Adipose Dipeptidyl Peptidase 4 in Cardiovascular Surgery Patients: Influence of Dipeptidyl Peptidase 4 Inhibitors

doi: 10.3390/jcm11154333

Figure Lengend Snippet: Patient characteristics.

Article Snippet: Secondary antibody reactions were performed at room temperature for 1 h. The antibodies used in the assay were DPP-4 and adiponectin goat anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotech-nology, Inc., Dallas, TX, USA).

Techniques: Biomarker Discovery, Activity Assay

Journal: Journal of Clinical Medicine

Article Title: Serum and Adipose Dipeptidyl Peptidase 4 in Cardiovascular Surgery Patients: Influence of Dipeptidyl Peptidase 4 Inhibitors

doi: 10.3390/jcm11154333

Figure Lengend Snippet: Comparison of dipeptidyl peptidase 4 (DPP-4), adiponectin, and tumor necrosis factor α (TNFα) expression in subcutaneous and epicardial adipose tissue. ( a ) Expression of DPP-4 in subcutaneous and epicardial adipose tissue. ( b ) Expression of adiponectin in subcutaneous and epicardial adipose tissue. ( c ) Expression of TNFα in subcutaneous and epicardial adipose tissue.

Article Snippet: Secondary antibody reactions were performed at room temperature for 1 h. The antibodies used in the assay were DPP-4 and adiponectin goat anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotech-nology, Inc., Dallas, TX, USA).

Techniques: Comparison, Expressing

Journal: Journal of Clinical Medicine

Article Title: Serum and Adipose Dipeptidyl Peptidase 4 in Cardiovascular Surgery Patients: Influence of Dipeptidyl Peptidase 4 Inhibitors

doi: 10.3390/jcm11154333

Figure Lengend Snippet: Correlation between DPP-4 and other parameters in serum, subcutaneous adipose tissue, and epicardial adipose tissue.

Article Snippet: Secondary antibody reactions were performed at room temperature for 1 h. The antibodies used in the assay were DPP-4 and adiponectin goat anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotech-nology, Inc., Dallas, TX, USA).

Techniques: Activity Assay

Journal: Journal of Clinical Medicine

Article Title: Serum and Adipose Dipeptidyl Peptidase 4 in Cardiovascular Surgery Patients: Influence of Dipeptidyl Peptidase 4 Inhibitors

doi: 10.3390/jcm11154333

Figure Lengend Snippet: Correlations between ( a ) serum and SAT adiponectin, ( b ) serum and EAT adiponectin, and ( c ) SAT and EAT adiponectin.

Article Snippet: Secondary antibody reactions were performed at room temperature for 1 h. The antibodies used in the assay were DPP-4 and adiponectin goat anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotech-nology, Inc., Dallas, TX, USA).

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Serum and Adipose Dipeptidyl Peptidase 4 in Cardiovascular Surgery Patients: Influence of Dipeptidyl Peptidase 4 Inhibitors

doi: 10.3390/jcm11154333

Figure Lengend Snippet: Correlation between adiponectin and other parameters in serum, subcutaneous adipose tissue, and epicardial adipose tissue.

Article Snippet: Secondary antibody reactions were performed at room temperature for 1 h. The antibodies used in the assay were DPP-4 and adiponectin goat anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotech-nology, Inc., Dallas, TX, USA).

Techniques: Activity Assay

Journal: Journal of Clinical Medicine

Article Title: Serum and Adipose Dipeptidyl Peptidase 4 in Cardiovascular Surgery Patients: Influence of Dipeptidyl Peptidase 4 Inhibitors

doi: 10.3390/jcm11154333

Figure Lengend Snippet: Characteristics of patient groups taking DPP-4 inhibitors and those not taking them.

Article Snippet: Secondary antibody reactions were performed at room temperature for 1 h. The antibodies used in the assay were DPP-4 and adiponectin goat anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotech-nology, Inc., Dallas, TX, USA).

Techniques: Biomarker Discovery, Activity Assay