Fibrillar collagen plays an important role in maintaining soft tissue integrity and providing chemical and physical cues for cell fate decisions. Collagen remodeling, which alternates the amount,... Show moreFibrillar collagen plays an important role in maintaining soft tissue integrity and providing chemical and physical cues for cell fate decisions. Collagen remodeling, which alternates the amount, distribution, and biomechanics of collagen, primarily type I (COLI) and type III (COLIII), can change tissue properties. This process is essential not only in biological developments but also in pathological processes. Thus, it is meaningful to understand the correlation between collagen remodeling and tissue dysfunction and investigate the cells' response to fibrous protein matrices. However, current studies in biochemical analysis of collagen and biomechanical study of tissues were carried out at different scales. So it is hard to correlate the data to draw solid conclusions. In this thesis research, we used two collagen disorder associated pathological conditions, pelvic organ prolapse (POP) and micropapillary serous carcinoma (MPSC) of the fallopian tube, as models to unravel the correlation between tissue dysfunctions and the impaired microenvironment relevant to the composition, nanostructure, and biomechanics of a collagen fibril. In the case of POP, we found the collagen fibers in tissues of POP patients were less abundant but stiffer than those of non-POP individuals, implying a loose and fragile matrix that is weakly integrated with other components of the connective tissue to provide adequate support of the pelvic organs. On the other hand, the collagen D-period, the characteristic banding feature which signals the proper assembly of collagen molecules, decreased in POP tissues. We surmised that the molecular level changes of collagen in POP were accountable for the weak matrix mechanics, verified by a systematic in vitro study. We also examined the collagen matrix alternation in MPSC of the fallopian tube, which is thought to cause ovarian cancer via metastasis. Since cancer metastasis is often related to collagen remodeling, we examined the collagen matrix alternation in this disease. We observed the heterogeneous distribution of COLI and COLIII in the papillae of the tumor tissue. Noticeably, COLI was accumulated at the papillae tip, whereas COLIII was dominant at the papillae base. We also observed the absence of collagen matrix between the micropapillary tip and the fibrosis base. Such an uneven collagen distribution implies that the matrix exerted distinctive forces on the tumor cells to regulate their behaviors, including cell migration, directional growth, and shedding from the primary tumor to initiate metastasis. These conclusions have been supported by the results of our in vitro experiments. In investigating the effect of the microenvironment on cell behavior, we established and validated an AFM-based method to collect and quantitatively analyze the mRNA samples from targeted live cells at the single-cell level. This method overcomes issues, such as severe cell damage or even cell death, the capability of time-dependent and in situ analyses, in current methods. The application of the method in studying heterogeneous gene expression in single cells and the interaction between cancer cells and cancer-associated fibroblasts was demonstrated. We also demonstrated that this method can be potentially used to quantitatively analyze the gene expression level changes in a targeted cell in response to the microenvironment. Show less
