Journal: Neoplasia (New York, N.Y.)

Article Title: Circulating Fibronectin Controls Tumor Growth 1 2

doi:

Figure Lengend Snippet: Deletion of fibronectin in Mx-cKO and Alb-cKO mice affects tumor development. (A) Circulating fibronectin in Alb-cKO mice is decreased by 40% at 4 weeks and 87% at 8 weeks of age, while levels in Mx-cKO were decreased by 87% and 95% compared to controls (CT) as determined by ELISA (N = 9–11/time point per group). (B) Deletion of fibronectin in the bone marrow in Mx-cKO is successful and stable for at least 2 weeks after the last pIpC injection, while deletion of circulating fibronectin did not affect fibronectin amount in the bone marrow of Alb-cKO mice. Bone marrow was flushed with 100 µl of protein lysis buffer/femur. Fibronectin was measured by ELISA and corrected to protein content measured by BCA (N = 3/group). (C) Use of the Mx promoter leads to deletion of fibronectin in leukocytes (CD45+), endothelial cells (CD31+), sinusoidal cells (CD31+/PLVAP+), and pericytes (desmin+). Bone marrow was flushed from mice that express eGFP in cells in which the Mx promoter was activated and, hence, are green, stained, and examined by flow cytometry. (D) The total bioluminescence signal (tumor burden/mouse) was similar between CT and Alb-cKO but was smaller in Mx-cKO after intracardiac injection (N = 11–12/group). (E) Median survival was increased by 2 weeks in Mx-cKO. (F) Intratibial injection is associated with a decrease in bioluminescence signal in both Alb- and Mx-cKO starting at day 14 post-injection until the end of the experiment at day 40 (N = 9–11/group). The values on the y-axis represent mean bioluminescence signal of the bars shown. *P < .05, **P < .01, ***P < .0001.

Article Snippet: For mouse plasma, bone marrow lysates, conditioned media of endothelial cells, and tumor samples, the standard used was murine plasma fibronectin (#IMFBN; Dunn, Asbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Lysis, Staining, Flow Cytometry

Journal: Neoplasia (New York, N.Y.)

Article Title: Circulating Fibronectin Controls Tumor Growth 1 2

doi:

Figure Lengend Snippet: The role of circulating fibronectin. (A) Labeled fibronectin can be detected within the tumor after injection. Masson-Goldner stain confirms the presence of cancer. Bars represent 500 µm. (B) Fibronectin staining was diminished in Alb- and Mx-cKO tumors. Bars represent 100 µm. (C) Quantifying the area of staining showed a decrease by more than 60% in Alb- and Mx-cKO tumors compared to controls (N = 3–4 mice/group). Similarly, quantifying the amount of fibronectin in tumors by ELISA reveals that CT tumors had more than 10-fold higher fibronectin content compared to Alb-cKO and Mx-cKO tumors. Fibronectin was examined by ELISA and values adjusted to protein (N = 3–6/group). (D) Both cancer cell fibronectin mRNA expression corrected to human HPRT and stromal fibronectin mRNA expression corrected to murine α-actin are decreased in Alb- and Mx-cKO (N = 4/group). *P < .05, **P < .001.

Article Snippet: For mouse plasma, bone marrow lysates, conditioned media of endothelial cells, and tumor samples, the standard used was murine plasma fibronectin (#IMFBN; Dunn, Asbach, Germany).

Techniques: Labeling, Injection, Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Circulating Fibronectin Controls Tumor Growth 1 2

doi:

Figure Lengend Snippet: Effects of fibronectin on angiogenesis. (A) Conditioned media from endothelial cells originating from Mx-cKO tumors do not produce fibronectin compared to endothelial cells originating from Alb-cKO or CT tumors (N = 3–5 replicates/group). (B) Fibronectin production in conditioned media is decreased in pericytes from Mx-cKO mice. Isolated cells were cultured for 24 hours. (C) Sections from CT, Alb-, and Mx-cKO intratibial tumors show a difference in the number of CD31-stained blood vessels. Bars represent 100 µm. (D) The number of CD31-stained blood vessels as well as the area covered by these vessels was decreased in Alb- and Mx-cKO tumors at 40 days post-injection (N = 6 tumors/group, six sections/tumor). (E) The percentage of blood vessels co-stained for CD31 and α-SMA was evaluated. The decrease in the percentage of α-SMA+ blood vessels was more pronounced in Mx-than in Alb-cKO tumors. For the purpose of this graph, the number of CT, Alb-cKO, and Mx-cKO CD31+ vessels was set at 100% (N = 3, 3, 6 mice, four sections/mouse). *P < .05, **P < .01, ***P < .0001.

Article Snippet: For mouse plasma, bone marrow lysates, conditioned media of endothelial cells, and tumor samples, the standard used was murine plasma fibronectin (#IMFBN; Dunn, Asbach, Germany).

Techniques: Isolation, Cell Culture, Staining, Injection

Journal: Neoplasia (New York, N.Y.)

Article Title: Circulating Fibronectin Controls Tumor Growth 1 2

doi:

Figure Lengend Snippet: Mechanistic insights on the reason for decreased angiogenesis. (A) Cancer cell VEGF (by human-specific ELISA) was significantly diminished in tumors originating from cKO mice (N = 4–6/group). (B) A change in matrix fibronectin is associated with a concordant change in VEGF content in the matrix. Cancer cells were cultured in the presence of a peptide (FUD) that inhibits fibronectin deposition or in the presence of 200 µg/ml fibronectin (FN). Matrix was isolated on the basis of deoxycholate (DOC) insolubility, tested for fibronectin and VEGF by ELISA, and adjusted to total protein (N = 4–6/group). (C) Fibronectin retains both endogenous (CT vs FN) and exogenous VEGF (VEGF vs FN/VEGF) in the matrix. MDA cells were cultured for 72 hours in six-well plates without additives, in the presence of 400 µg of fibronectin, 12 ng of VEGF, or both. Matrix VEGF was quantified and corrected to protein in the DOC insoluble fraction as in B (N = 3–4/group). (D) Murine VEGFR-2 mRNA expression was diminished in cKO tumors, but human (Figure W6C) was not (N = 4/group). (E) The amount of phosphorylated VEGFR-2 was also diminished in cKO tumors in densitometry measurements. An example of a Western blot is shown on the right (N = 4/group). (F) Cell proliferation was significantly increased when endothelial cells were treated with either fibronectin or VEGF compared to untreated cells. The addition of both simultaneously resulted in a more pronounced increase in proliferation (N = 3 experiments with three to five replicates/condition per experiment). (G) Both phospho-ERK and phospho-AKT increased 15 minutes after the addition of both fibronectin and VEGF, while phospho-FAK and phospho-JNK did not. (H and I) Densitometries of phospho-AKT and phospho-ERK corrected to total AKT and ERK, respectively. *P < .05, **P < .01, ***P < .0001.

Article Snippet: For mouse plasma, bone marrow lysates, conditioned media of endothelial cells, and tumor samples, the standard used was murine plasma fibronectin (#IMFBN; Dunn, Asbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Isolation, Expressing, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Circulating Fibronectin Controls Tumor Growth 1 2

doi:

Figure Lengend Snippet: Effects of fibronectin content on proliferation and apoptosis. (A) The number of proliferating cells stained for BrdU in cKO tumors is diminished. Bars represent 100 µm. (B) Quantitative analysis of BrdU+ cells in tumors (N = 6 CT, 5 Alb-, and 5 Mx-cKO tumors). (C) Proliferation of tumor cells is improved by the addition of fibronectin. Fibronectin (500 µg/ml) and 10% fibronectin-depleted FCS increase proliferation to a similar extent. (D) TUNEL staining in tumor tissue from CT, Alb-, and Mx-cKO mice. Bars represent 200 µm. (E) The number of TUNEL-stained cells is increased in cKO tumors (N = 3–4/group). (F) Western blot analysis for Bcl-2 and BAX shows an increase in BAX in the absence of circulating fibronectin. A graph of densitometry of values adjusted to β-tubulin is shown on the left. On the right, an example is presented (N = 3/group).

Article Snippet: For mouse plasma, bone marrow lysates, conditioned media of endothelial cells, and tumor samples, the standard used was murine plasma fibronectin (#IMFBN; Dunn, Asbach, Germany).

Techniques: Staining, TUNEL Assay, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Circulating Fibronectin Controls Tumor Growth 1 2

doi:

Figure Lengend Snippet: Implications and relevance in human cancer. (A) Fibronectin content in tumors correlates with blood vessel numbers in the whole group including CT, Alb-cKO, and Mx-cKO mice (N = 4 CT, 3 Alb-cKO, and 3 Mx-cKO). Fibronectin content was determined by ELISA and corrected to protein content. (B) Fibronectin content in tumors determined by ELISA correlates with bioluminescence signal in the same groups as A. (C) Grading of staining intensity for breast and prostate cancer tissue arrays. (D) Fibronectin staining intensity is stronger in tumors that are ER negative (ER- = 42, ER+ = 39 samples). Kaplan-Meier survival curves for different fibronectin staining intensities in (E) breast cancer (N = 81) and (F) prostate cancer (N = 89). *P < .05, **P < .01, ***P < .001.

Article Snippet: For mouse plasma, bone marrow lysates, conditioned media of endothelial cells, and tumor samples, the standard used was murine plasma fibronectin (#IMFBN; Dunn, Asbach, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Circulating Fibronectin Controls Tumor Growth 1 2

doi:

Figure Lengend Snippet: Clinical and Histopathologic Characteristics of the Patients in the Breast Cancer Tissue Microarray.

Article Snippet: For mouse plasma, bone marrow lysates, conditioned media of endothelial cells, and tumor samples, the standard used was murine plasma fibronectin (#IMFBN; Dunn, Asbach, Germany).

Techniques: Microarray, Biomarker Discovery

Journal: Neoplasia (New York, N.Y.)

Article Title: Circulating Fibronectin Controls Tumor Growth 1 2

doi:

Figure Lengend Snippet: Clinical and Histopathologic Characteristics of the Patients in the Second Prostate Tissue Microarray.

Article Snippet: For mouse plasma, bone marrow lysates, conditioned media of endothelial cells, and tumor samples, the standard used was murine plasma fibronectin (#IMFBN; Dunn, Asbach, Germany).

Techniques: Microarray