Intrinsic fluorescence detection with multiphoton excitation in combination with SHG facilitates three-dimensional, high resolution, imaging in unfixed, unsectioned, unstained mammary tissues. This imaging provides information commonly obtained with classical histology and EM without the need for complex and destructive sample preparation, and with additional structural information in four dimensions. With MPE/SHG, mammary gland tissue could be clearly imaged at depths of 440 nm, and changes in collagen density could be reliably detected. Furthermore, three-dimensional imaging of tumors in situ revealed three now defined TACS, which provide standard hallmarks to locate and characterize tumors: TACS-1, the presence of dense collagen, indicated by increased signal intensity at a region around the tumor as a standard hallmark for locating small tumor regions; TACS-2, the presence of taut (straightened) collagen fibers stretched around the tumor, indicating growth leading to increased tumor volume; and TACS-3, the identification of radially aligned collagen fibers facilitating invasion, which may be indicative of the invasive and metastatic growth potential of a tumor. Together these signatures may serve a mechanism to help identify and characterize breast tumors in experimental animal models as well as human cancers and fresh tumor biopsies.
The breast epithelial cell-ECM interaction is responsible for influencing cell polarity, proliferation, differentiation, adhesion, and migration [44
] and type I collagen is an important regulator of mammary ductal formation during development [4
]. Analysis of normal mammary glands reveals collagen fibers wrapping around, as well as radiating away from, the duct (Figure ). This organization is remarkably consistent with the observation that in fixed whole mounts of developing mammary gland, analyzed with multiphoton microscopy, collagen fibers are "pulled in" perpendicular to the terminal end bud [46
], similar to what we observe for radially aligned collagen fibers near tumors (TACS-3). Combined, these morphologies provide insight into the structure-function relationship in the mammary gland and imply that collagen may provide directional cues during development that also influence changes in the normal mammary gland. For instance, the crimped (wavy) collagen structure (i.e. Figure ) is consistent with numerous reports of crimped collagen fibers in connective tissue that allow normal tissue deformation with a strain-stiffening behavior [47
]. This behavior may hold true for the mammary gland as well, allowing for tissue deformation and normal ductal growth and involution without over constraining the system, yet providing adequate levels of tensile resistance to contracting cells and resisting large deformations that can damage the tissue. The less numerous taut fibers may serve a different purpose. They may act as locally constraining structures at the single cell level and may act to interconnect various ducts in the tissue together and to the nipple structure (Figure ), which may transmit mechanical signals to the ducts during activities such as nursing to elicit mechanotransductive signaling related to lactation. Furthermore, such mechanical signals acting directly on epithelial cells or transmitting stress across the basement membrane would be amplified by increased breast tissue density. Hence, increased breast tissue density in vivo may promote carcinoma formation by increased mechanical signaling events in dense tissue, consistent with in vitro work showing that increased matrix density alters breast epithelial cell signaling [49
The importance of matrix composition and morphology around the mammary epithelium is illustrated by studies showing that misregulated stromal-epithelial interactions can promote tumorigenesis [6
] and the fact that breast carcinomas often exhibit desmoplasia (excessive collagen surrounding an invasive tumor [50
]). Moreover, cancer cells can locally invade across basement membrane and collagenous stroma to spread into neighboring ECM environments, where they can migrate further to enter lymphatic and blood vessels, resulting in metastatic growth in distant tissues [40
]. Therefore, understanding the mechanisms of invasion in vivo is of great importance. Yet, to our knowledge, no study has visualized local invasion in endogenous tumors in vivo in relation to stromal organization. Consequently, it is noteworthy that we observe alignment of collagen fibers, and association of individual cells with those fibers at regions of local invasion in live tissue (TACS-3), which is similar to observations of individual cell migration along collagen fibers in a xenograft model [9
], and confirms and expands upon in vitro studies in 3D matrices that have identified collagen reorganization (alignment) at the front of invading cells [22
]. Moreover, the concept of alignment-facilitated invasion appears to be of significance in collective cell migration (e.g. tubulogenesis in the mammary gland; [41
]) as collagen alignment is noted at the terminal end bud during invasion of the mammary ductal tree [46
]. Thus, collagen alignment may facilitate motility and migration during normal development, while tumor invasion may resemble misregulated developmental processes.