Assessing the roles of collagen fiber morphology and matrix stiffness on ovarian cancer cell migration dynamics using multiphoton fabricated orthogonal image-based models.

Ovarian cancer remains the deadliest of the gynecological cancers, where this arises from poor screening and imaging tools that can detect early disease, and also limited understanding of the structural and functional aspects of the tumor microenvironment. To gain insight into the underlying cellular dynamics, we have used multiphoton excited fabrication to create Second Harmonic Generation (SHG) image-based orthogonal models from collagen/GelMA that represent both the collagen matrix morphology and stiffness (∼2-8 kPa) of normal ovarian stroma and high grade serous ovarian cancers (HGSOC). These scaffolds are used to study migration/cytoskeletal dynamics of normal (IOSE) and ovarian cancer (OVCA433) cell lines. We found that the highly aligned fiber morphology of HGSOC promotes aspects of motility (motility coefficient, motility, and focal adhesion expression) through a contact guidance mechanism and that stiffer matrix further promotes these same processes through a mechanosensitive mechanism, where these trends were similar for both normal and cancer cells. However, cell specific differences were found on these orthogonal models relative to those providing only morphology, showing the importance of presenting both morphology and stiffness cues. Moreover, we found increased cadherin expression and decreased cell alignment only for cancer cells on scaffolds of intermediate modulus suggesting different stiffness-dependent mechanotransduction mechanisms are engaged. This overall approach affords decoupling the roles of matrix morphology, stiffness and cell genotype and affords hypothesis testing of the factors giving rise to disease progression and metastasis. Further, more established fabrication techniques cannot simultaneously reproduce both the 3D collagen fiber morphology and stiffness. STATEMENT OF SIGNIFICANCE: Ovarian cancer metastasizes when lesions are small, where cells exfoliate from the surface of the ovary and reattach at distal sites in the peritoneum. The adhesion/migration dynamics are not well understood and there is a need for new 3D in vitro models of the extracellular matrix to study the biology. Here we use multiphoton excited crosslinking to fabricate ECM orthogonal models that represent the collagen morphology and stiffness in human ovarian tissues. These are then used to study ovarian cancer cell migration dynamics and we found that contact guidance and a mechanosensitive response and cell genotype all combine to affect the behavior. These models provide insight into disease etiology and progression not readily possible by other fabrication methods.

Role of Collagen Fiber Morphology on Ovarian Cancer Cell Migration Using Image-Based Models of the Extracellular Matrix.

Remodeling of the extracellular matrix (ECM) is an important part in the development and progression of many epithelial cancers. However, the biological significance of collagen alterations in ovarian cancer has not been well established. Here we investigated the role of collagen fiber morphology on cancer cell migration using tissue engineered scaffolds based on high-resolution Second-Harmonic Generation (SHG) images of ovarian tumors. The collagen-based scaffolds are fabricated by multiphoton excited (MPE) polymerization, which is a freeform 3D method affording submicron resolution feature sizes (~0.5 µm). This capability allows the replication of the collagen fiber architecture, where we constructed models representing normal stroma, high-risk tissue, benign tumors, and high-grade tumors. These were seeded with normal and ovarian cancer cell lines to investigate the separate roles of the cell type and matrix morphology on migration dynamics. The primary finding is that key cell-matrix interactions such as motility, cell spreading, f-actin alignment, focal adhesion, and cadherin expression are mainly determined by the collagen fiber morphology to a larger extent than the initial cell type. Moreover, we found these aspects were all enhanced for cells on the highly aligned, high-grade tumor model. Conversely, the weakest corresponding responses were observed on the more random mesh-like normal stromal matrix, with the partially aligned benign tumor and high-risk models demonstrating intermediate behavior. These results are all consistent with a contact guidance mechanism. These models cannot be synthesized by other conventional fabrication methods, and we suggest this approach will enable a variety of studies in cancer biology.

Parallel multiphoton excited fabrication of tissue engineering scaffolds using a diffractive optical element.

Multiphoton excited photochemistry is a powerful technique for freeform nano/microfabrication. However, the construction of large and complex structures using single point scanning is slow, where this is a significant limitation for biological investigations. We demonstrate increased throughput via parallel fabrication using a diffractive optical element. To implement an approach with large field of view and near-theoretical resolution, a scan lens was designed that is optimized for using low-magnification high NA objective lenses. We demonstrate that with this approach it is possible to synthesize large scaffolds at speeds several times faster than by single point scanning.

Analysis of fibroblast migration dynamics in idiopathic pulmonary fibrosis using image-based scaffolds of the lung extracellular matrix.

Idiopathic pulmonary fibrosis (IPF) is characterized by a profound remodeling of the collagen in the extracellular matrix (ECM), where the fibers become both denser and more highly aligned. However, it is unknown how this reconfiguration of the collagen matrix affects disease progression. Here, we investigate the role of specific alterations in collagen fiber organization on cell migration dynamics by using biomimetic image-based collagen scaffolds representing normal and fibrotic lung, where the designs are derived directly from high-resolution second harmonic generation microscopy images. The scaffolds are fabricated by multiphoton-excited (MPE) polymerization, where the process is akin to three-dimensional printing, except that it is performed at much greater resolution (∼0.5 microns) and with collagen and collagen analogs. These scaffolds were seeded with early passaged primary human normal and IPF fibroblasts to enable the decoupling of the effect of cell-intrinsic characteristics (normal vs. IPF) versus ECM structure (normal vs. IPF) on migration dynamics. We found that the highly aligned IPF collagen structure promoted enhanced cell elongation and F-actin alignment along with increased cell migration speed and straightness relative to the normal tissues. Collectively, the data are consistent with an enhanced contact guidance mechanism on the aligned IPF matrix. Although cell intrinsic effects were observed, the aligned collagen matrix morphology had a larger effect on these metrics. Importantly, these biomimetic models of the lung cannot be synthesized by conventional fabrication methods. We suggest that the MPE image-based fabrication method will enable additional hypothesis-based testing studies of cell-matrix interactions in the context of tissue fibrosis.

Migration dynamics of ovarian epithelial cells on micro-fabricated image-based models of normal and malignant stroma.

A profound remodeling of the collagen in the extracellular matrix (ECM) occurs in human ovarian cancer but it is unknown how this affects migration dynamics and ultimately tumor growth. Here, we investigate the influence of collagen morphology on ovarian cell migration through the use of second harmonic generation (SHG) image-based models of ovarian tumors. The scaffolds are fabricated by multiphoton excited (MPE) polymerization, where the process is akin to 3D printing except it achieves much greater resolution (∼0.5 µm) and utilizes collagen and collagen analogs. We used this technique to create scaffolds with complex 3D submicron features representing the collagen fiber morphology in normal stroma, high risk stroma, benign tumors, and high grade ovarian tumors. We found the highly aligned malignant stromal structure promoted enhanced motility and also increased cell and f-Actin alignment relative to the other tissues. However, using models based on fiber crimping characteristics, we found cells seeded on linear fibers based on normal stromal models yielded the highest degree of alignment but least motility. These results show that both the fiber properties themselves and as well as their overall alignment govern the resulting migration dynamics. These models cannot be synthesized by other conventional fabrication methods and we suggest the MPE image-based fabrication method will enable a variety of studies in cancer biology. STATEMENT OF SIGNIFICANCE: The extracellular matrix collagen in ovarian cancer is highly remodeled but the consequences on cell function remain unknown. It is important to understand the operative cell matrix interactions, as this could lead to better prognostics and better prediction of therapeutic efficacy. We probe migration dynamics using high resolution (∼0.5 µm) multiphoton excited fabrication to synthesize scaffolds whose designs are derived directly from Second Harmonic Generation microscope images of the collagen in normal ovarian tissues as well as benign and malignant tumors. Collectively our results show the importance of the matrix morphology (fiber shape and alignment) on driving cell motility, cell shape and f-Actin alignment. These collagen-based models have complex fiber morphology and cannot be created by conventional fabrication technologies.

Ovarian and Breast Cancer Migration Dynamics on Laminin and Fibronectin Bidirectional Gradient Fibers Fabricated via Multiphoton Excited Photochemistry.

INTRODUCTION: Migration mis-regulation is a hallmark of cancer, and remains an important problem in cancer biology. We postulate the needs for better in vitro models to understand the details of cell-matrix interactions. Here, we utilized multiphoton excited (MPE) photochemistry to fabricate models to systematically study migration dynamics operative in breast and ovarian cancer. Gradients are a convenient means to modulate concentration and also have been implicated in metastases. METHODS: We specifically pattern sub-micron structured gradients from laminin and fibronectin whose up-regulation is associated with increased metastasis and poor prognosis. We developed a new continuous linear bi-directional gradient design, permitting exploration of the underlying cell-matrix interactions of migration, including speed, directness, and f-actin cytoskeleton alignment as a function of concentration. These new models provide both contact guidance and ECM binding cues, and provide a more relevant environment than possible with existing technologies such as flow chambers or 2D printed surfaces. RESULTS: We found an overall increase in these processes with increasing concentration on both laminin and fibronectin gradients for a series of ovarian and breast cancer lines. Moreover, directness was higher for more metastatic cells, indicating that epithelial or mesenchymal state of the cell type governs the dynamics. However, the specifics of the speed and directedness depend on both the cell type and protein, thus we found that we must consider these processes collectively to obtain a self-consistent picture of the migration. For this purpose, we performed a linear discriminate analysis (LDA) and successfully classified the different cell types on the two protein gradients without molecular biology analysis. CONCLUSIONS: The bi-gradient structures are versatile tools to performing detailed studies of cell migration, specifically haptotxis. We further suggest the can be used in assessing efficacy of drug treatments targeted at specific matrix components.