A Perspective Review: Analyzing Collagen Alterations in Ovarian Cancer by High-Resolution Optical Microscopy.

High-grade serous ovarian cancer (HGSOC) is the predominant subtype of ovarian cancer (OC), occurring in more than 80% of patients diagnosed with this malignancy. Histological and genetic analysis have confirmed the secretory epithelial of the fallopian tube (FT) as a major site of origin of HGSOC. Although there have been significant strides in our understanding of this disease, early stage detection and diagnosis are still rare. Current clinical imaging modalities lack the ability to detect early stage pathogenesis in the fallopian tubes and the ovaries. However, there are several microscopic imaging techniques used to analyze the structural modifications in the extracellular matrix (ECM) protein collagen in ex vivo FT and ovarian tissues that potentially can be modified to fit the clinical setting. In this perspective, we evaluate and compare the myriad of optical tools available to visualize these alterations and the invaluable insights these data provide on HGSOC initiation. We also discuss the clinical implications of these findings and how these data may help novel tools for early diagnosis of HGSOC.

Collagen is More Abundant and Structurally Altered in Lichen Sclerosus.

OBJECTIVE: To test the hypothesis that genital skin and male urethra affected by lichen sclerosus (LS) has increased collagen content and altered collagen structure. METHODS: We used picrosirius red to stain and image collagen in human urethral, vulvar, and foreskin specimens with and without LS. Using Image J software, we quantified and compared (1) collagen content (using 2o metrics: collagen proportionate area [CPA] and collagen fiber count), (2) collagen fiber length and width, and (3) collagen structure using the texture analysis technique gray level co-localization matrix (GLCM) with respect to LS status and tissue type. RESULTS: We analyzed 23 LS specimens (vulva n=9, urethra n=7, foreskin n=7) and 29 non-LS specimens (vulva n=9, urethra n=7, foreskin n=13). Fiber count and CPA were significantly higher in all LS specimens compared to non-LS specimens (CPA: mean±SD 0.971±0.03 vs 0.948±0.02, P < .007; fiber count: mean±SD = 2906±127 vs 2509±78 fibers; P = .003). Collagen fiber width and length were similar with respect to LS status. GLCM analysis showed decreased inverse difference moment and increased entropy in LS tissues indicative of less homogeneous and more disorganized tissue structure (P<.001). CONCLUSION: LS tissues have greater collagen content compared to non-LS tissues. Quantitative assessment of collagen organization, using GLCM, revealed less homogeneity and more disorganization of collagen in LS compared to non-LS tissues. Taken together, our findings suggest that alterations in physical tissue properties seen in LS may be due to both increased collagen abundance and altered structure.

Reduced SPAG17 Expression in Systemic Sclerosis Triggers Myofibroblast Transition and Drives Fibrosis.

Systemic sclerosis (SSc) is a clinically heterogeneous fibrotic disease with no effective treatment. Myofibroblasts are responsible for unresolving synchronous skin and internal organ fibrosis in SSc, but the drivers of sustained myofibroblast activation remain poorly understood. Using unbiased transcriptome analysis of skin biopsies, we identified the downregulation of SPAG17 in multiple independent cohorts of patients with SSc, and by orthogonal approaches, we observed a significant negative correlation between SPAG17 and fibrotic gene expression. Fibroblasts and endothelial cells explanted from SSc skin biopsies showed reduced chromatin accessibility at the SPAG17 locus. Remarkably, mice lacking Spag17 showed spontaneous skin fibrosis with increased dermal thickness, collagen deposition and stiffness, and altered collagen fiber alignment. Knockdown of SPAG17 in human and mouse fibroblasts and microvascular endothelial cells was accompanied by spontaneous myofibroblast transformation and markedly heightened sensitivity to profibrotic stimuli. These responses were accompanied by constitutive TGF-ß pathway activation. Thus, we discovered impaired expression of SPAG17 in SSc and identified, to our knowledge, a previously unreported cell-intrinsic role for SPAG17 in the negative regulation of fibrotic responses. These findings shed fresh light on the pathogenesis of SSc and may inform the search for innovative therapies for SSc and other fibrotic conditions through SPAG17 signaling.

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.

Evaluation of Collagen Alterations in Early Precursor Lesions of High Grade Serous Ovarian Cancer by Second Harmonic Generation Microscopy and Mass Spectrometry.

Background: The collagen architecture in high grade serous ovarian cancer (HGSOC) is highly remodeled compared to the normal ovary and the fallopian tubes (FT). We previously used Second Harmonic Generation (SHG) microscopy and machine learning to classify the changes in collagen fiber morphology occurring in serous tubal intraepithelial carcinoma (STIC) lesions that are concurrent with HGSOC. We now extend these studies to examine collagen remodeling in pure p53 signatures, STICs and normal regions in tissues that have no concurrent HGSOC. This is an important distinction as high-grade disease can result in distant collagen changes through a field effect mechanism. Methods: We trained a linear discriminant model based on SHG texture and image features as a classifier to discriminate the tissue groups. We additionally performed mass spectrometry analysis of normal and HGSOC tissues to associate the differential expression of collagen isoforms with collagen fiber morphology alterations. Results: We quantified the differences in the collagen architecture between normal tissue and the precursors with good classification accuracy. Through proteomic analysis, we identified the downregulation of single α-chains including those for Col I and III, where these results are consistent with our previous SHG-based supramolecular analyses. Conclusion: This work provides new insights into ECM remodeling in early ovarian cancer and suggests the combined use of SHG microscopy and mass spectrometry as a new diagnostic/prognostic approach.

Examining lysyl oxidase-like modulation of collagen architecture in 3D spheroid models of idiopathic pulmonary fibrosis via second-harmonic generation microscopy.

SIGNIFICANCE: Idiopathic pulmonary fibrosis (IPF) patients have a poor prognosis with short lifespan following diagnosis as there are limited effective treatment options. Despite matrix stiffening being the hallmark of the disease there remains a lack of knowledge surrounding the underlying collagen alterations in the disease. Specifically, while increased collagen crosslinking has been implicated, the resulting effects on collagen macro/supramolecular changes have not been explored. AIM: We sought to determine if second-harmonic generation (SHG) microscopy could characterize differences in the collagen architecture in 3D spheroid models of IPF grown under different crosslinking modulation conditions (promotion and inhibition). APPROACH: We used SHG metrics based on the fiber morphology, relative SHG brightness, and macro/supramolecular structure by SHG polarization analyses to compare the structure of the IPF spheroids. RESULTS: Comparison of the fiber morphology of the spheroids showed that the control group had the longest, straightest, and thickest fibers. The spheroids with crosslink enhancement and inhibition had the highest and lowest SHG conversion efficiencies, respectively, consistent with the resulting harmonophore density. SHG polarization analyses showed that the peptide pitch angle, alignment of collagen molecules, and overall chirality were altered upon crosslink modulation and were also consistent with reduced organization relative to the control group. CONCLUSIONS: While no single SHG signature is associated with crosslinking, we show that the suite of metrics used here is effective in delineating alterations across the collagen architecture sizescales. The results largely mirror those of human tissues and demonstrate that the combination of 3D spheroid models and SHG analysis is a powerful approach for hypothesis testing the roles of operative cellular and molecular factors in IPF.

Recent Advancements in Optical Harmonic Generation Microscopy: Applications and Perspectives.

Second harmonic generation (SHG) and third harmonic generation (THG) microscopies have emerged as powerful imaging modalities to examine structural properties of a wide range of biological tissues. Although SHG and THG arise from very different contrast mechanisms, the two are complimentary and can often be collected simultaneously using a modified multiphoton microscope. In this review, we discuss the needed instrumentation for these modalities as well as the underlying theoretical principles of SHG and THG in tissue and describe how these can be leveraged to extract unique structural information. We provide an overview of recent advances showing how SHG microscopy has been used to evaluate collagen alterations in the extracellular matrix and how this has been used to advance our knowledge of cancers, fibroses, and the cornea, as well as in tissue engineering applications. Specific examples using polarization-resolved approaches and machine learning algorithms are highlighted. Similarly, we review how THG has enabled developmental biology and skin cancer studies due to its sensitivity to changes in refractive index, which are ubiquitous in all cell and tissue assemblies. Lastly, we offer perspectives and outlooks on future directions of SHG and THG microscopies and present unresolved questions, especially in terms of overall miniaturization and the development of microendoscopy instrumentation.

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.

Platform for quantitative multiscale imaging of tissue composition.

Changes in the multi-level physical structure of biological features going from cellular to tissue level composition is a key factor in many major diseases. However, we are only beginning to understand the role of these structural changes because there are few dedicated multiscale imaging platforms with sensitivity at both the cellular and macrostructural spatial scale. A single platform reduces bias and complications from multiple sample preparation methods and can ease image registration. In order to address these needs, we have developed a multiscale imaging system using a range of imaging modalities sensitive to tissue composition: Ultrasound, Second Harmonic Generation Microscopy, Multiphoton Microscopy, Optical Coherence Tomography, and Enhanced Backscattering. This paper details the system design, the calibration for each modality, and a demonstration experiment imaging a rabbit eye.

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.

Probing ECM remodeling in idiopathic pulmonary fibrosis via second harmonic generation microscopy analysis of macro/supramolecular collagen structure.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis with short lifespan following diagnosis as patients have limited effective treatment options. A fundamental limitation is a lack of knowledge of the underlying collagen alterations in the disease, as this could lead to better diagnostics, prognostics, and measures of treatment efficacy. While the fibroses is the primary presentation of the disease, the collagen architecture has not been well studied beyond standard histology. Here, we used several metrics based on second harmonic generation (SHG) microscopy and optical scattering measurements to characterize the subresolution collagen assembly in human IPF and normal lung tissues. Using SHG directional analysis, we found that while collagen synthesis is increased in IPF, the resulting average fibril architecture is more disordered than in normal tissue. Wavelength-dependent optical scattering measurements lead to the same conclusion, and both optical approaches are consistent with ultrastructural analysis. SHG circular dichroism revealed significant differences in the net chirality between the fibrotic and normal collagen, where the former has a more randomized helical structure. Collectively, the measurements reveal significant changes in the collagen macro/supramolecular structure in the abnormal fibrotic collagen, and we suggest these alterations can serve as biomarkers for IPF diagnosis and progression.

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.

Second-harmonic generation microscopy analysis reveals proteoglycan decorin is necessary for proper collagen organization in prostate.

Collagen remodeling occurs in many prostate pathologies; however, the underlying structural architecture in both normal and diseased prostatic tissues is largely unexplored. Here, we use second-harmonic generation (SHG) microscopy to specifically probe the role of the proteoglycan decorin (Dcn) on collagen assembly in a wild type (wt) and Dcn null mouse (Dcn - / - ). Dcn is required for proper organization of collagen fibrils as it regulates size by forming an arch-like structure at the end of the fibril. We have utilized SHG metrics based on emission directionality (forward-backward ratio) and relative conversion efficiency, which are both related to the SHG coherence length, and found more disordered fibril organization in the Dcn - / - . We have also used image analysis readouts based on entropy, multifractal dimension, and wavelet transforms to compare the collagen fibril/fiber architecture in the two models, where all these showed that the Dcn - / - prostate comprised smaller and more disorganized collagen structures. All these SHG metrics are consistent with decreased SHG phase matching in the Dcn - / - and are further consistent with ultrastructural analysis of collagen in this model in other tissues, which show a more random distribution of fibril sizes and their packing into fibers. As Dcn is a known tumor suppressor, this work forms the basis for future studies of collagen remodeling in both malignant and benign prostate disease.

The Extracellular Matrix of Ovarian Cortical Inclusion Cysts Modulates Invasion of Fallopian Tube Epithelial Cells.

A growing body of research supports the idea that the fallopian tube epithelium (FTE) is the precursor for most high-grade serous ovarian canacers (HGSOC) but that the ovary plays a critical role in tumor metastasis. Cortical inclusion cysts (CICs) in the ovarian cortex have been hypothesized to create a niche environment that plays a role in HGSOC progression. Through histological analysis of pathology samples from human ovaries, we determined that collagen I and III were elevated near CICs and that the collagen fibers in this dense region were oriented parallel to the cyst boundary. Using this information from human samples as design parameters, we engineered an in vitro model that recreates the size, shape, and extracellular matrix (ECM) properties of CICs. We found that FTE cells within our model underwent robust invasion that was responsive to stimulation with follicular fluid, while ovarian surface epithelial (OSE) cells, the native cells of the ovary, were not invasive. We provide experimental evidence to support a role of the extracellular matrix in modulating FTE cell invasion, as decreased collagen I concentration or the addition of collagen III to the matrix surrounding FTE cells increased FTE cell invasion. Taken together, we show that an in vitro model of CICs informed by the analysis of human tissue can act as an important tool for understanding FTE cell interactions with their environment.

Fast and improved bioimaging via temporal focusing multiphoton excitation microscopy with binary digital-micromirror-device holography.

Conventional temporal focusing-based multiphoton excitation microscopy (TFMPEM) can offer widefield optical sectioning with an axial excitation confinement of a few microns. To improve the axial confinement of TFMPEM, a binary computer-generated Fourier hologram (CGFH) via a digital-micromirror-device (DMD) was implemented to intrinsically improve the axial confinement by filling the back-focal aperture of the objective lens. Experimental results show that the excitation focal volume can be condensed and the axial confinement improved about 24% according to the DMD holography. In addition, pseudouniform MPE can be achieved using two complementary CGFHs with rapid pulse-width modulation switching via the DMD. Furthermore, bioimaging of CV-1 in origin with SV40 genes-7 cells demonstrates that the TFMPEM with binary DMD holography can improve image quality by enhancing axial excitation confinement and rejecting out-of-focus excitation.

Polarization-resolved second harmonic generation imaging of human ovarian cancer.

Remodeling of the extracellular matrix in human ovarian cancer can be manifested in increased collagen concentration, changes in alignment within fibrils/fibers and/or up-regulation of different collagen isoforms. We used pixel-based second harmonic generation (SHG) polarization microscopy analyses to probe these molecular changes in human ovarian tissues [normal stroma, benign tumors, and high-grade serous (HGS) tumors] by: (i) determination of the α-helical pitch angle via the single-axis molecular model, (ii) collagen alignment within fibrils via SHG anisotropy, and (iii) chirality via SHG circular dichroism (SHG-CD). Pixel approaches are required due to the complex structure of the matrix that lacks a high degree of fiber alignment. The largest differences in the helical pitch angle were between normal stroma and benign tumors, consistent with gene expression showing the Col III isoform is up-regulated in the latter. The data were not consistent with up-regulation of Col III in HGS tumors as previous reports have suggested. The different tissues also displayed differing SHG anisotropies and SHG-CD responses, consistent with either Col III incorporation or randomization of Col I alignment within benign and malignant tumors. Additionally, the high-grade tumors displayed higher collagen concentration, where this desmoplasia is consistent with the higher fiber density in these tissues. These results collectively indicate that the fibril assemblies are distinct in all tissues, where these differences likely result from the synthesis of collagen rather than remodeling of existing collagen. Importantly, these analyses are label-free and interrogate subresolution collagen structure on intact tissues, without the need for conventional structural biology tools.

Developmental Pathways Pervade Stem Cell Responses to Evolving Extracellular Matrices of 3D Bioprinted Microenvironments.

Developmental studies and 3D in vitro model systems show that the production and engagement of extracellular matrix (ECM) often precede stem cell differentiation. Yet, unclear is how the ECM triggers signaling events in sequence to accommodate multistep process characteristic of differentiation. Here, we employ transcriptome profiling and advanced imaging to delineate the specificity of ECM engagement to particular differentiation pathways and to determine whether specificity in this context is a function of long-term ECM remodeling. To this end, human mesenchymal stem cells (hMSCs) were cultured in 3D bioprinted prisms created from ECM proteins and associated controls. We found that exogenous ECM provided in 3D microenvironments at early time points impacts on the composition of microenvironments at later time points and that each evolving 3D microenvironment is uniquely poised to promote stem cell differentiation. Moreover, 2D cultures undergo minimal ECM remodeling and are ill-equipped to stimulate pathways associated with development.

Assessing local stromal alterations in human ovarian cancer subtypes via second harmonic generation microscopy and analysis.

The collagen architecture in all human ovarian cancers is substantially remodeled, where these alterations are manifested in different fiber widths, fiber patterns, and fibril size and packing. Second harmonic generation (SHG) microscopy has differentiated normal tissues from high-grade serous (HGS) tumors with high accuracy; however, the classification between low-grade serous, endometrioid, and benign tumors was less successful. We postulate this is due to known higher genetic variation in these tissues relative to HGS tumors, which are genetically similar, and this results in more heterogeneous collagen remodeling in the respective matrix. Here, we examine fiber widths and SHG emission intensity and directionality locally within images (e.g., 10×10 microns) and show that normal tissues and HGS tumors are more uniform in fiber properties as well as in fibril size and packing than the other tissues. Moreover, these distributions are in good agreement with phase matching considerations relating SHG emission directionality and intensity. The findings show that in addition to average collagen assembly properties the intrinsic heterogeneity must also be considered as another aspect of characterization. These local analyses showed differences not shown in pure intensity-based image analyses and may provide further insight into disease etiology of the different tumor subtypes.

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.