Fibronectin Protects from Excessive Liver Fibrosis by Modulating the Availability of and Responsiveness of Stellate Cells to Active TGF-β

Fibrotic tissue in the liver is mainly composed of collagen. Fibronectin, which is also present in fibrotic matrices, is required for collagen matrix assembly in vitro. It also modulates the amount of growth factors and their release from the matrix. We therefore examined the effects of the absence of fibronectin on the development of fibrosis in mice

Blood clot formation does not affect metastasis formation or tumor growth in a murine model of breast cancer.

Cancer is associated with increased fracture risk, due either to metastasis or associated osteoporosis. After a fracture, blood clots form. Because proteins of the coagulation cascade and activated platelets promote cancer development, a fracture in patients with cancer often raises the question whether it is a pathologic fracture or whether the fracture itself might promote the formation of metastatic lesions. We therefore examined whether blood clot formation results in increased metastasis in a murine model of experimental breast cancer metastasis. For this purpose, a clot was surgically induced in the bone marrow of the left tibia of immundeficient mice. Either one minute prior to or five minutes after clot induction, human cancer cells were introduced in the circulation by intracardiac injection. The number of cancer cells that homed to the intervention site was determined by quantitative real-time PCR and flow cytometry. Metastasis formation and longitudinal growth were evaluated by bioluminescence imaging. The number of cancer cells that homed to the intervention site after 24 hours was similar to the number of cells in the opposite tibia that did not undergo clot induction. This effect was confirmed using two more cancer cell lines. Furthermore, no difference in the number of macroscopic lesions or their growth could be detected. In the control group 72% developed a lesion in the left tibia. In the experimental groups with clot formation 79% and 65% developed lesions in the left tibia (p = ns when comparing each experimental group with the controls). Survival was similar too. In summary, the growth factors accumulating in a clot/hematoma are neither enough to promote cancer cell homing nor support growth in an experimental model of breast cancer bone metastasis. This suggests that blood clot formation, as occurs in traumatic fractures, surgical interventions, and bruises, does not increase the risk of metastasis formation

Induction of hematoma and clot formation in the bone marrow.

<p>(A) Induction of bleeding results in clot formation within 5 minutes as evidenced by Masson-Goldner staining. <i>Bar represents 500</i> µ<i>m</i>. The enlarged inset below shows the blood clot. <i>Bar represents 100</i> µ<i>m</i>. (B) The clot is still present, albeit partially reorganized at 24 hours after bleeding induction. <i>Bar represents 500</i> µ<i>m</i>. Enlarged inset is shown below. <i>Bar represents 100</i> µ<i>m</i>. (C) After 72 hours the clot resolved. (D) Thrombin staining of the bone shown in B confirms the presence of thrombin (in red) in the clot area. (E) Quantification of the change in hematoma size at different time points. After induction of bleeding in the bone marrow, mice were euthanized at the times mentioned, and tibiae examined. n = 3–4 mice/time point.</p

Comparison of control and experimental groups.

<p>The number of macroscopic lesions detectable by bioluminescence imaging in the left tibia was similar between control animals that did not undergo clot induction and experimental animals with a blood clot in which cancer cells were injected 1 minute prior to (−1 min), or 5 minutes after clot induction (+5 min). Data from all mice (including those that already died) 7 weeks after cancer cell injection are shown. Data were analyzed using Fisher's exact test and no significant differences were detected in the CT/experimental group pairs.</p><p>*The percentage presented is calculated as follows: number of lesions at a specific site/total number of lesions in the group.</p

Bone histomorphometry after clot induction.

<p>(A–B) After three days from the time of induction of a blood clot Masson-Goldner staining (on the left) and calcein labeling (on the right) showed no difference between control (CT) in panel A and hematoma induction in panel B in the same mouse. Neither osteoblast number (C), nor bone formation rate (D), nor osteoclast number (E), nor erosion surface (F) were different between the two tibiae. CT represents the right tibia without clot induction and hematoma represents the left tibia in which bone marrow was flushed and a blood clot was induced. Tibiae were obtained three days after clot induction in the left tibia, embedded in polymethylmethacrylate and stained using Masson-Goldner. n = 4 mice.</p

Bioluminescence and x-ray imaging <i>in vivo</i>.

<p>Representative bioluminescence images from the last measurements before death and x-ray images at the time of death from 5 CT and 5 experimental mice in the group injected with cancer cells 5 minutes after clot induction. Upper panel represents the paired pictures from the CT group and the lower panel represents the paired pictures from the experimental group.</p

Infiltration of blood clots by cancer cells.

<p>(A) Induction of a blood clot in the left tibia does not result in an increase in the number of infiltrating cancer cells compared to the right control tibia (CT) in the same mouse when cancer cells are injected 1 minute before blood clot induction using three cell lines (Breast cancer selected to home to the bone marrow: MDA-MB-231B/luc+; prostate cancer able to form bone metastases: PC3/luc+; and hepatoma cells not reported to form bone metastases: Huh-7). Cancer cells were injected 1 minute before clot induction. 24 hours later the bone marrow was isolated from the upper third of both tibiae and the number of cancer cells was evaluated by quantitative PCR of a cancer cell specific sequence and corrected to the total number of murine cells in the sample. n = 4–5/group. (B) The number of MDA cancer cells evaluated by flow cytometry was similar between the CT and hematoma group. MDA cancer cells were injected 1 minute before clot induction. 24 hours later the bone marrow was isolated from the upper third of both tibiae, red cells lysed, stained with a labeled human-specific CD49e (integrin α5) antibody and at least 3 million bone marrow cells were counted. n = 10 mice. (C) The use of surgical wax in the tibia following clot induction does not affect homing of cancer cells. 1 minute before clot induction MDA cancer cells were injected. The hole performed in the tibia in order to induce the blood clot in the bone marrow was either closed with surgical wax or left until bleeding stopped spontaneously (3–5 minutes) before closing the wound. n = 4 pairs. (D) Injection of MDA cancer cells 15 minutes before, 1 minute before and 5 minutes after clot induction did not affect the number of cancer cells in the bone marrow detected after 24 hours. Samples were prepared as in A. p = ns for each time point. (E) Evaluation of cancer cell numbers when injected 1 minute before clot induction did not reveal a difference in the number of cancer cells detected in the bone marrow at different time points (1, 4, 24 and 48 hours after clot induction). p = ns and n = 4–5 per time point. (F) Evaluation of cancer cell numbers when injected 5 minutes after clot induction showed a significant decrease in the number of cancer cells detected in the bone marrow at 1 hour after clot induction (p<0.05) but not at later time points (4, 24 and 48 hours after clot induction) (p = ns). n = 4–8 per time point.</p

Circulating Fibronectin Controls Tumor Growth

Fibronectin is ubiquitously expressed in the extracellular matrix, and experimental evidence has shown that it modulates blood vessel formation. The relative contribution of local and circulating fibronectin to blood vessel formation in vivo remains unknown despite evidence for unexpected roles of circulating fibronectin in various diseases. Using transgenic mouse models, we established that circulating fibronectin facilitates the growth of bone metastases by enhancing blood vessel formation and maturation. This effect is more relevant than that of fibronectin produced by endothelial cells and pericytes, which only exert a small additive effect on vessel maturation. Circulating fibronectin enhances its local production in tumors through a positive feedback loop and increases the amount of vascular endothelial growth factor (VEGF) retained in the matrix. Both fibronectin and VEGF then cooperate to stimulate blood vessel formation. Fibronectin content in the tumor correlates with the number of blood vessels and tumor growth in the mouse models. Consistent with these results, examination of three separate arrays from patients with breast and prostate cancers revealed that a high staining intensity for fibronectin in tumors is associated with increased mortality. These results establish that circulating fibronectin modulates blood vessel formation and tumor growth by modifying the amount of and the response to VEGF. Furthermore, determination of the fibronectin content can serve as a prognostic biomarker for breast and prostate cancers and possibly other cancers