Recent studies have demonstrated that the cellular contribution of the bone marrow to tumor neovascularization is highly complex. In this context, the extent to which bone marrow-derived cells incorporate as bona fide endothelial (nonhematopoietic) cells into perfused tumor vessels, or any new vessels formed postnatally (vasculogenesis), is unclear. To this end, we developed models to characterize local vesselderived and bone marrow-derived endothelial cells (BMD-ECs). Then, we characterized the BMD-ECs based on a set of endothelial markers and morphology. Finally, we quantified their contribution to perfused blood vessels in tumors using transplanted as well as spontaneous primary and metastatic tumor models. We demonstrate that BMD-ECs incorporate in perfused tumor vessels, and that this contribution varies with organ site and mouse strain. IntroductionThe contribution of bone marrow-derived endothelial cells (BMD-ECs) to blood vessels in postnatal life (adult vasculogenesis) remains controversial. For example, BMD-EC involvement in tumor neovascularization has been reported to be significant [1][2][3][4][5] to undetectable [6][7][8] in some of the same tumor models. 9 Other studies have identified hematopoietic cells incorporated into the endothelial lining of tumor vessels. [10][11][12] We hypothesized that the reasons for these discordant findings may be the inability of some of the reported models to detect and distinguish both bone marrow-and local vessel-derived endothelial cells and the fact that most studies relied on ambigous markers to identify the endothelialcell phenotype. 9 Other potential confounding factors may result from the genetic deficiency or transgenic models used, tumor type and organ site. 2,13 To address these issues, we quantified BMD-ECs in perfused blood vessels in tumors using bone marrow transplantation (BMT) in 2 strains of mice and 2 tumor models: isograft and spontaneous metastasis. Study designWe quantified vasculogenesis with a set of markers for bone marrow-derived cells incorporated in perfused tumor blood vessels. To detect and analyze donor-derived cells, we used flow cytometry and fluorescence microscopy. In one transgenic mouse line, green fluorescence protein (GFP) was constitutively expressed in all cells (under ␤-actin promoter activation, Actb-Gfp/C57BL6 14 ); in another, GFP was expressed in cells upon Tie2 promoter activation (Tie2-Gfp/ FVB 15 ). We employed restorative BMT from these mice to wild-type (WT) nontransgenic syngeneic counterparts after lethal irradiation. Using the resulting chimeric mice (WT/Tie2-Gfp-BMT and WT/Actb-Gfp-BMT), we analyzed isografted and spontaneous primary tumors, as well as distant metastases. Then, using fluorescence confocal microscopy, we directly compared the incorporation of BMD-ECs and locally derived endothelial cells into lectin-perfused vessels (see supplemental methods, available on the Blood website; see the Supplemental Materials link at the top of the online article). We identified BMD-ECs by simultaneous cytoplasmic G...

Functional roles for the cancer cell-associated membrane type I matrix metalloproteinase (MT1-MMP) during early steps of the metastatic cascade in primary tumors remain unresolved. In an effort to determine its significance, we determined the in vivo effects of RNAi-mediated downregulation in mammary cancer cells on the migration, blood and lymphatic vessel invasion (LVI), and lymph node and lung metastasis. We also correlated the expression of cancer cell MT1-MMP with blood vessel invasion (BVI) in 102 breast cancer biopsies. MT1-MMP downregulation in cancer cells decreased lung metastasis without affecting primary tumor growth. The inhibition of lung metastasis correlated with reduced cancer cell migration and BVI. Furthermore, cancer cell-expressed MT1-MMP upregulated the expression of MT1-MMP in vascular endothelial cells, but did not affect MT1-MMP expression in lymphatic endothelial cells, LVI, or lymph node metastasis. Of clinical importance, we observed that elevated MT1-MMP expression correlated with BVI in biopsies from triplenegative breast cancers (TNBC), which have a poor prognosis and high incidence of distant metastasis, relative to other breast cancer subtypes. Together, our findings established that MT1-MMP activity in breast tumors is essential for BVI, but not LVI, and that MT1-MMP should be further explored as a predictor and therapeutic target of hematogenous metastasis in TNBC patients. Cancer Res; 71(13); 4527-38. Ó2011 AACR.

Here we integrated multiphoton laser scanning microscopy and the registration of second harmonic generation images of collagen fibers to overcome difficulties in tracking stromal cell-matrix interactions for several days in live mice. We show that the matrix-modifying hormone relaxin increased tumor-associated fibroblast (TAF) interaction with collagen fibers by stimulating β 1 -integrin activity, which is necessary for fiber remodeling by matrix metalloproteinases.Our current understanding of extracellular matrix remodeling derives from in vitro experiments 1,2 , which are difficult to interpret and relate to in vivo physiology. Multiphoton laser scanning microscopy and second harmonic generation (SHG) of fibrillar collagen allow visualization of the matrix of normal and tumor tissues in vivo 3 . Using these technologies, it is possible to study the real-time movement of cells through the collagen network on time scales of minutes to hours 4,5 . However, the slow rate of extracellular matrix remodeling makes it difficult to monitor matrix reorganization by stromal cells.To track stromal cell collagen fiber interactions in vivo, we grew tumors (human sarcoma HSTS26T) in transparent dorsal skinfold chambers of immunodeficient mice expressing GFP under the control of the Vegfa promoter (VEGF-GFP mice). The transparent tumor chamber facilitated the tracking of the same cells and fibers for several days (Supplementary Methods online). Also, previous data in VEGF-GFP mice 6 and our immunostaining results suggest that peritumor GFP-positive cells are tumor-associated fibroblasts (TAFs) ( Supplementary Fig. 1 online).To induce collagen remodeling in a reliable fashion, we treated tumors with the small hormone relaxin, which causes matrix remodeling and increases tumor invasion and progression 7 . In vitro, relaxin enhanced the invasion of fibroblasts through increased collagen I degradation but did not affect the HSTS26T tumor cells ( Supplementary Fig. 2 Temporal changes in tumors and the inability to place mice under the microscope in the same orientation between imaging sessions necessitated the development of a registration method to align image sequences. The SHG signal arising from fibrillar collagen provided a convenient registration landmark because of the relative stability and the wide distribution of collagen fibers in comparison with other potential landmarks, such as blood vessels or exogenous fluorescent beads. Blood vessels within tumors are porous to injected tracer particles, can have time-dependent perfusion fluctuations and are more sparsely distributed than the extracellular matrix, which forms a distinct network distributed throughout the tumor. Likewise, fluorescent beads pose the problem of potentially being unevenly distributed in the region of interest or being unstable because of photobleaching or cellular phagocytosis. We used an intensity-based registration approach (Turboreg) 8 to align subsequent imaged volumes with those at the initial time point (Supplementary Methods).After ima...