Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 1. Involvement of FGF2-FGFR1 axisin Akt activation. (A) The effect of CAF-CM on proliferation of breast cancer (MCF-7, MDA-MB-231, and MDA-MB-468) cells was determined by the MTT assay. Cells were incubated with or without CAF-CM for 72 hours. ***Significantly different be- tween the groups compared (P < 0.001). (B) MDA-MB-231 cells were incubated with CAF-CM for the indicated time periods. Phosphorylation of Akt and STAT3 were detected by Western blot analysis. (C) MDA-MB-231 cells were exposed to CAF-CM with or without FGF-2-neutralizing antibody for 3 hours. Phosphorylation of Akt was detected by Western blot analysis. *,***Significantly different between the groups compared (*P < 0.05; ***P < 0.001). (D) MDA-MB-231 cells were treated with 20 ng/mL of FGF2 for the indicated time periods. The phosphorylation of FRS2α as well as Akt was analyzed by Western blot. (E) RNA-seq data set of TCGA breast invasive carcinoma was downloaded from XenaBrower (https://xenabrowser.net). mRNA expression levels of total 1,097 samples (Illumina HiSeq log [normalized counts + 1]) were prepared by quantile normalization. Pearson cor- relation coefficient was calculated to assess the relationship between FGF2 and FGFR1. (F, G) Correlation of FGFR1 protein expression with FGF2 (F) and Akt (G), based on 105 breast invasive carcinoma protein specimens (TCGA, Pan-Cancer Atlas) from the cBioportal database (www.cbiopor- tal.org). FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; CAFs, cancer-associated fibroblasts; NFs, normal fibroblasts; CM, conditioned medium; ns, not significantly different; FRS2, FGFR substrate 2; TCGA, The Cancer Genome Atlas; CPTAC, the Clinical Proteomic Tumor Analysis Consortium.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Activation Assay, MTT Assay, Incubation, Phospho-proteomics, Western Blot, RNA Sequencing, Expressing

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 2. Role of FGFR1 in Akt phosphorylation and breast cancer cell growth and progression. (A) MDA-MB-231 cells were transfected with scrambled or FGFR1 si-RNA for 24 hours. Cells were then incubated with 20 ng/mL of FGF2 for 15 minutes to measure phosphorylated FRS2α. (B) Mice were subjected to xenograft co-injecting with fibroblasts and MDA-MB-231 breast cancer cells. A complex collagen network was detected in H&E-stained tumors by an intense pink and in Masson’s trichrome stain by a blue stain (arrows). Stromal compartment was also detected by α-SMA immunostaining. Magnification, x100. Bars, 100 μm. (C) Phosphorylated Akt in the xenograft tumors was determined by Western blot analysis. *Sig- nificantly different between the groups compared (P < 0.05). (D) Enrichment plots of hallmark gene sets in the high FGFR1-expressing group. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; FRS2, FGFR substrate 2; α-SMA, alpha-smooth muscle actin; CONT, control; EMT, epithelial- mesenchymal transition.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Phospho-proteomics, Transfection, Incubation, Staining, Immunostaining, Western Blot, Expressing, Control

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 3. The involvement of FGF2-induced ROS generation in nuclear localization of FGFR1. (A) MDA-MB-231 cells were co-cultured with NFs or CAFs for 24 hours. MDA-MB-231 (5 x 10 3 cells) and NFs or CAFs (5 x 10 3 cells) were mixed prior to seeding and incubated for 24 hours. Immunocytochemical analysis was performed using anti-FGFR1 antibody. Cells were then stained with DAPI for detection of nuclei. Magnification, x100. Bars, 200 μm. (B) MDA-MB-231 cells were incubated with FGF2 for 1 hour. Immunocytochemical analysis was performed using anti-FGFR1 antibody. Cells were then stained with PI for detection of nuclei. Magnification, x100. Bars, 200 μm. (C) MDA-MB-231 cells were treated with 20 ng/ mL of FGF2 for 1 hour, followed by Western blot analysis of FGFR1 in cytosolic and nuclear extracts. Lamin B was used as a nuclear marker. *Sig- nificantly different between the groups compared (P < 0.05). (D, E) MDA-MD-231 cells were incubated with CAF-CM or FGF2 for 3 hours and 1 hour, respectively. After staining with DCF-DA for 30 minutes, fluorescent microscopic (D) or flow cytometric (E) analysis was performed to detect intracellu- lar ROS accumulation. Magnification, x40. (F) After pretreatment with NAC for 3 hours, cells were exposed to FGF2 for additional 1 hour. Nuclear ex- tracts were subjected to Western blot analysis to detect the presence of FGFR1 and Nrf2 in the nucleus. **Significantly different between the groups compared (P < 0.01). (G) MDA-MB-231 cells were exposed to FGF2 (20 ng/mL) for 1 hour. Cell lysates were subjected to immunoprecipitation using CBP antibody for 16 hours followed by immunoblotting with. FGFR1 or Nrf2 antibody. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; ROS, reactive oxygen species; CAFs, cancer-associated fibroblasts; CM, conditioned medium; NFs, normal fibroblasts; DAPI, 4′,6-diamidino-2-phenylindole; PI, propidium iodide; CONT, cotrol; DCF-DA, 2’,7’-dichlorodihydrofluorescein diacetate; NAC, N-acetylcysteine; CBP, CREB-binding protein.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Cell Culture, Incubation, Staining, Western Blot, Marker, Immunoprecipitation, Binding Assay

Journal: Journal of Cancer Prevention

Article Title: Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts

doi: 10.15430/jcp.2022.27.1.68

Figure Lengend Snippet: Figure 4. Possible association between nuclear FGFR1 and Nrf2. (A) TNBC patient cohorts were validated based on the mean expression value of the indicated single genes (FGFR1 or NFE2L2) or as a signature of two genes together and patient survival was analyzed (n = 255). (B, C) MDA- MB-231 cells were transfected with scrambled or Nrf2 si-RNA for 24 hours. Cells were then incubated with 20 ng/mL of FGF2 for 3 hours. The mRNA (B) and protein (C) expression of cyclin D1 was assessed by RT-PCR and Western blot analyses, respectively. The expression of cyclin D1 was mea- sured by RT-PCR (B) and Western blot (C) analyses. (D) In tumor microenvironment, fibroblasts are activated to form CAFs, which secrete FGF2. CAF-derived FGF2 could induces nuclear translocation as well as de novo synthesis of FGFR1, ultimately contributing to cancer cell proliferation, mi- gration and tumor growth. While membrane bound FGFR1 may translocate to nucleus as a complex with FGF2 which has nuclear localization signal (NLS), the complex is likely rather to stimulate the intracellular signaling via FRS2α, which induces transcription of FGFR-1 gene. On the other hand, newly synthesized FGFR-1 is speculated to enter the nucleus as a complex with a cargo protein harboring NLS. FGFR-1 is translocated to the inner nuclear membrane through the nuclear pore complexes (NPCs), which is regulated by importin β. FGF2, fibroblast growth factor 2; FGFR1, FGF receptor 1; TNBC, triple negative breast cancer; HR, hazard ratio; CAFs, cancer-associated fibroblasts; ER, endoplasmic reticulum; FRS2, FGFR substrate 2; CBP, CREB-binding protein.

Article Snippet: For neutralization of FGF2 in the CM of CAFs, CM was pre-incubated with 25 μg/mL of human FGF2 antibody or its IgG control (R&D Systems, Inc., Minneapolis, MN, USA) for 1 hour at room temperature prior to use.

Techniques: Expressing, Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay, Translocation Assay, Membrane, Synthesized, Binding Assay

Journal: Brain : a journal of neurology

Article Title: Improvement in disability after alemtuzumab treatment of multiple sclerosis is associated with neuroprotective autoimmunity.

doi: 10.1093/brain/awq176

Figure Lengend Snippet: Figure 3 Neurotrophin secretion from PBMC cultures from 15 patients before and 12 months after alemtuzumab treatment. Cultures were either unstimulated (unstim) or stimulated with the myelin antigen, MBP or polyclonally stimulated with anti-CD3/anti-CD28 antibodies (CD3/28). Supernatants were harvested after 72 h and assayed for: (A) brain-derived neurotrophic factor, (B) CNTF, (C) platelet-derived neurotrophic factor, (D) FGF, and (E) insulin-like growth factor-1. Error bars represent 95% confidence intervals. (*P50.05, **P50.01, ***P50.001).

Article Snippet: For blocking experiments, neutralizing antibodies to BDNF (2 mg/ml—Sigma B5050), CNTF (0.2 mg/ml—Peprotech 500-P140), FGF (2 mg/ml RnD systems AB-233-NA) and PDGF (20 mg/ml RnD systems AN-20-NA) were added at the time of resuspension in PBMC derived conditioning medium.

Techniques: Derivative Assay

Journal: Brain : a journal of neurology

Article Title: Improvement in disability after alemtuzumab treatment of multiple sclerosis is associated with neuroprotective autoimmunity.

doi: 10.1093/brain/awq176

Figure Lengend Snippet: Figure 4 Peripheral blood mononuclear cell neurotrophin secretion from three patients 12 months after alemtuzumab in response to a wide variety of antigens. PBMCs were cultured either unstimulated (unstim) or stimulated with; MBP, tetanus toxoid (TT), myelin oligodendrocyte glycoprotein (MOG), collagen type II fragment aa245–270 (Collagen), myelin basic peptide aa87–99 (MBP-P), keyhole limpet haemocyanin (KLH) or recombinant human insulin (Insulin). Supernatants were harvested after 72 h and assayed for: (A) brain-derived neurotrophic factor, (B) CNTF, (C) platelet-derived neurotrophic factor, (D) insulin-like growth factor-1 and (E) FGF (*P50.05).

Article Snippet: For blocking experiments, neutralizing antibodies to BDNF (2 mg/ml—Sigma B5050), CNTF (0.2 mg/ml—Peprotech 500-P140), FGF (2 mg/ml RnD systems AB-233-NA) and PDGF (20 mg/ml RnD systems AN-20-NA) were added at the time of resuspension in PBMC derived conditioning medium.

Techniques: Cell Culture, Recombinant, Derivative Assay

Journal: Brain : a journal of neurology

Article Title: Improvement in disability after alemtuzumab treatment of multiple sclerosis is associated with neuroprotective autoimmunity.

doi: 10.1093/brain/awq176

Figure Lengend Snippet: Figure 5 Peripheral blood mononuclear cell neurotrophin secretion, induced by MBP stimulation. PBMCs from 15 healthy controls (HC), 15 patients before (pre) and at three time points (6, 9 and 12 months) after alemtuzumab and from 10 patients treated with interferon b-1a (rebif), were cultured with MBP. Supernatants were harvested after 72 h and assayed for: (A) brain-derived neurotrophic factor, (B) CNTF, (C) platelet-derived neurotrophic factor, (D) insulin-like growth factor-1 and (E) FGF. Error bars represent 95% confidence intervals (**P50.01, ***P50.001).

Article Snippet: For blocking experiments, neutralizing antibodies to BDNF (2 mg/ml—Sigma B5050), CNTF (0.2 mg/ml—Peprotech 500-P140), FGF (2 mg/ml RnD systems AB-233-NA) and PDGF (20 mg/ml RnD systems AN-20-NA) were added at the time of resuspension in PBMC derived conditioning medium.

Techniques: Cell Culture, Derivative Assay

Journal: Nature Communications

Article Title: Malignant subclone drives metastasis of genetically and phenotypically heterogenous cell clusters through fibrotic niche generation

doi: 10.1038/s41467-021-21160-0

Figure Lengend Snippet: a Representative photographs of AKTP mono-culture and co-culture with hepatic stellate cells (HSCs). Images are representative of n = 5 independent cultures. The inset shows an enlarged image. Arrowheads indicate ES cell-like colonies. Bars, 100 μm. b Results of PCR array analysis (average Log10 of 3 independent samples). Magenta and blue, upregulated and downregulated genes, respectively, in AKTP cells co-cultured with HSCs. c Fluorescent immunohistochemistry for AKTP cells (green, left), FGF2 (red, center), and merged images with DAPI nuclear staining (right) of the AKTP cell-arrested liver vessels at days 3 (top) and 14 (bottom) after spleen transplantation. Bars, 100 μm. The photographs in c are representative images from n = 3 biologically independent animals. Bars, 100 μm. d Growth rates of AP cells stimulated with AKTP conditioned medium (CM) or FGF2 ( n = 3 biologically independent samples for each culture condition). e Cloning efficiency of AP cells cultured with AKTP-CM or FGF2 ( n = 5 plates of independent 96-well plate for each culture condition). f Representative photographs of fluorescent immunohistochemistry of AP mono-culture (left) and co-culture with HSCs (right) in the presence or absence of AKTP-CM or FGF2. tdTomato-labeled AP cells (red)/Venus-labeled HSCs (green) (top) and tdTomato-labeled AP cells (red)/Ki67 (green) (bottom) with DAPI nuclear staining. Bars, 100 μm (top) and 50 μm (bottom). Arrowheads, AP cells; H, HSCs in chimeric spheroids. g Ki67-labeling indices of AP cells in monoculture or co-culture with HSCs in the presence or absence of AKTP-CM or FGF2 (experiments in f , g : n = 3 and 6 biologically independent samples for AP mono-culture in the absence and presence of AKTP-CM, respectively; n = 6 and 7 biologically independent samples for control and FGF2- or AKTP-CM-treated AP cell co-culture, respectively). The data in d , e , and g are presented as the mean ± s.d. The data in d were analyzed by one-way ANOVA test, ns not significant. The data in e and g were analyzed by a two-sided unpaired t -test, and p values are provided. h Schematic illustration of possible polyclonal metastasis mechanism. Source data are provided as a Source Data File.

Article Snippet: Antibodies against Ki67 (1:1000, Abcam; 1:50, BD Biosciences), GFP (1:500, MBL), RFP (1:1000, Rockland Immunochemicals), DsRed (1:200, Clonetech), αSMA (1:800, Sigma), CD31 (1:20, Clone SZ31; Dianova, Hamburg, Germany), and FGF2 (1:100, OriGene) were used as primary antibodies for immunohistochemistry.

Techniques: Co-Culture Assay, Cell Culture, Immunohistochemistry, Staining, Transplantation Assay, Cloning, Labeling, Control