Collagen Fiber Orientation Research Articles

Abstract 4425: Extracellular matrix segmentation combined with cell classification as a novel method for detailed tumor tissue mapping

Abstract Tumor microenvironment (TME) represents a dynamic niche that regulates cancer cell behavior contributing directly to disease outcome. Systemic approach to analysis of TME should uncover its complexity and facilitate discovery of mechanisms orchestrating tumor development and metastasis. Multiplexed fluorescent tissue stain followed by spatial analysis of tumor tissue architecture can provide insights to pivotal interactions of cellular and acellular components of TME. Extracellular matrix (ECM), one of constitutive of TME, is represented mainly by collagen deposition. Recently it is became increasingly recognized that ECM contribution to TME dynamics not only depend upon amount of accumulated collagen but its geometrical features and spatial orientation of fibers. These characteristics of collagen contribute directly to physical and mechanical properties of tissue and can contribute to tumor growth and metastasis. Current algorithms of tissue stain-based methods include estimation of ECM deposition by measuring percent of positive area and separate to it cell classification and cell count. The goal of current work was to develop a new computational tool to perform spatial distribution analysis based on geometrical features and orientation of collagen fibers in combination with cell class assessment aimed to achieve detailed tumor tissue mapping. To pursue this goal, we generated fluorescent images of human breast tumor tissue, stained for following markers: CD3 - marker of T-lymphocytes, PanCytokeratin - marker of epithelial/tumor cells, collagen hybridizing peptide (3Helix) - marker of collagen, DAPI - nuclear counterstain. To develop image analysis pipeline, we utilized open source graphical interface analytical platform KNIME, where we generated modular workflow. For ECM analysis, we integrated Python written code into KNIME node. Segmentation of collagen fibers was performed using skeletonization with subsequent calculation of geometrical properties (length, alignment, widths) and orientation of each fiber. Data, collected from single cell analysis and ECM architecture assessment, were combined and forwarded to downstream spatial analysis, where distances from cell to cell or cell to ECM were computed and neighborhood analysis was performed. We demonstrate different patterns in tumor microenvironment organization that correlate with cancer outcome. Developed image analysis algorithm provides additional dimensionality to fluorescent tissue stains and can reveal underrecognized patterns of tumor microenvironment that can contribute to better understanding of tumorigenesis and metastasis. Citation Format: GEORGII VASIUKOV, Tatiana Novitskaya, Sergey Gutor, Anna Menshikh, Andries Zijlstra, Sergey Novitskiy. Extracellular matrix segmentation combined with cell classification as a novel method for detailed tumor tissue mapping [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4425.

Relationships among collagen fiber orientation, mineralization, mineral maturity, and crystallinity

Bone quality is a combination of the material and structural properties of bone. Reductions in bone quality parameters, such as the degree of collagen fiber orientation, mineralization, mineral maturity, and crystallinity, affect bone strength. However, the relationships among the bone quality parameters remain poorly understood. In this work, we investigated the relationships among collagen fiber orientation and other bone quality parameters in the rat femur using Fourier transform infrared spectroscopic imaging and infrared dichroism imaging. In cortical bone, collagen fibers were found to be aligned longitudinally and an increase in the degree of mineral maturity or mineralization was associated with an increase in the degree of collagen fiber orientation.

Human articular cartilage is orthotropic where microstructure, micromechanics, and chemistry vary with depth and split-line orientation

Quantitative, micrometer length scale assessment of human articular cartilage is essential to enable progress toward new functional tissue engineering approaches, including utilization of emerging 3D bioprinting technologies, and for improved computational modeling of the osteochondral unit. Thus the objective of this study was to characterize the structural organization, material properties, and chemical composition of human skeletally mature articular cartilage with respect to depth and defined morphological features: normal to the articulating surface, parallel to the split-line, and transverse to the split-line. Three samples from the lateral femoral condyles of 4 healthy adult donors (55-61 years old) were evaluated via histology, second harmonic generation, microindentation, and Raman spectroscopy. All metrics were evaluated as a function of depth and direction relative to the split-line. All donors presented with intact and healthy tissue. Collagen fiber orientation varied significantly between testing directions and with increasing depth from the articular surface. Both compressive and tensile modulus increased significantly with depth and differed across the middle and deep zones and depended on orthogonal direction relative to the split-line. Similarly, matrix components varied with both depth and direction, where chondroitin sulfate steadily increased with depth while collagen prevalence was highest in the surface layer. Microscale measurements of human articular cartilage demonstrate that properties are both depth-dependent and orthotropic and depend on the underlying tissue structure and composition. These findings improve upon existing knowledge establishing more accurate measurements, with greater degree of depth and spatial specificity, as inputs for tissue engineering and computational modeling.

Investigation of effect of collagen fiber orientation on tumor cell invasion

Collagen fibers can both inhibit and promote cellular migration, based on their orientation. Physical models such as Random walk can regenerate cellular migration. As such, we proposed a model based on (biased) random walk to study migration on collagen fibers. We took mean squared displacement as the determinant factor for metastatic risk and found that direction of fibers, spatial correlations and migration mode together regulate the risk and we proposed an approach to quantify an existing prognostic approach.

Mueller Matrix Analysis of Collagen and Gelatin Containing Samples Towards More Objective Skin Tissue Diagnostics.

Electrospun polycaprolactone:gelatin (PCL:GT) fibre scaffolds are widely employed in the field of tissue implants. Here, the orientation of fibres plays an important role in regard to implantation due to the impact on the mechanical properties. Likewise, the orientation of collagen fibres in skin tissue is relevant for dermatology. State-of-the-art fibre orientation measurement methods like electron microscopy are time consuming and destructive. In this work, we demonstrate polarimetry as a non-invasive approach and evaluate its potential by measuring the Mueller matrix (MM) of gelatin and collagen containing samples as simple skin tissue phantoms. We demonstrate that it is possible to determine the orientation of PCL:GT fibre scaffolds within one MM measurement. Furthermore, we determine the structural orientation in collagen film samples. Currently, the diagnosis of skin diseases is often performed by image analysis or histopathology respectively, which are either subjective or invasive. The method presented, here, provides an interesting alternative approach for such investigations. Our findings indicate that the orientation of collagen fibres within skin lesions might be detectable by MM measurements in the future, which is of interest for skin diagnostics, and will be further investigated during the next step.

Collagen fibre orientation in human bridging veins.

Bridging veins (BVs) drain the blood from the cerebral cortex into dural sinuses. BVs have one end attached to the brain and the other to the superior sagittal sinus (SSS), which is attached to the skull. Relative movement between these two structures can cause BV to rupture producing acute subdural haematoma, a head injury with a mortality rate between 30 and 90%. A clear understanding of the BVs microstructure is required to increase the biofidelity of BV models when simulating head impacts. Twelve fresh BV samples draining in the superior sagittal sinus (SSS) from a single human cadaver were cut open along their length and placed on an inverted multiphoton microscope. To ensure that the BVs were aligned with the axial direction an in-house built, uniaxial tension set-up was used. Two scans were performed per sample. Before the first scan, a minor displacement was applied to align the tissue; then, a second scan was taken applying 50% strain. Each BV was scanned for a length of 5 mm starting from the drainage site into the SSS. Imaging was performed on a Zeiss LSM780 microscope with an 25[Formula: see text] water immersion objective (NA 0.8), coupled to a tunable MaiTai DS (Spectraphysics) pulsed laser with the wavelength set at 850 nm. Second harmonic and fluorescence signals were captured in forward and backward direction on binary GaAsP (BiG) detectors and stored as four colour Z-stacks. Prior to the calculation of the local orientations, acquired Z-stacks were denoised and enhanced to highlight fibrillar structures from the background. Then, for each Z-plane of the stack, the ImageJ plugin OrientationJ was used to extract the local 2D orientations of the fibres based on structure tensors. Two kinds of collagen architectures were seen. The most common (8[Formula: see text]12 samples) was single layered and had a uniform distribution of collagen. The less common (4[Formula: see text]12 samples) had 2 layers and 7 to 34 times thicker collagen bundles on the outer layer. Fibre angle analysis showed that collagen was oriented mainly along the axial direction of the vessel. The von Mises fittings showed that in order to describe the fibre distribution 3 components were needed with mean angles [Formula: see text] at [Formula: see text] 0.35, 0.21, [Formula: see text] 0.02 rad or [Formula: see text] 20.2[Formula: see text], 12.1[Formula: see text], [Formula: see text] 1.2[Formula: see text] relative to the vessel's axial direction which was also the horizontal scan direction.

A 3D biophysical model for cancer spheroid cell-enhanced invasion in collagen-oriented fiber microenvironment**Project supported by the National Natural Science Foundation of China (Grant Nos. 11974066 and 11674043), the Fundamental Research Funds for the Central Universities, China (Grant No. 2019CDYGYB007), and the Natural Science Foundation of Chongqing, China (Grant No. cstc2019jcyj-msxmX0477).

The process of in situ tumors developing into malignant tumors and exhibiting invasive behavior is extremely complicated . From a biophysical point of view, it is a phase change process affected by many factors, including cell-to-cell, cell-to-chemical material, cell-to-environment interaction, etc. In this study, we constructed spheroids based on green fluorescence metastatic breast cancer cells MDA-MB-231 to simulate malignant tumors in vitro, while constructed a three-dimensional (3D) biochip to simulate a micro-environment for the growth and invasion of spheroids. In the experiment, the 3D spheroid was implanted into the chip, and the oriented collagen fibers controlled by collagen concentration and injection rate could guide the MDA-MB-231 cells in the spheroid to undergo directional invasion. The experiment showed that the oriented fibers greatly accelerated the invasion speed of MDA-MB-231 cells compared with the traditional uniform tumor micro-environment, namely obvious invasive branches appeared on the spheroids within 24 hours. In order to analyze this interesting phenomenon, we have developed a quantitative analyzing approach to explore strong angle correlation between the orientation of collagen fibers and invasive direction of cancer cell. The results showed that the oriented collagen fibers produced by the chip can greatly stimulate the invasion potential of cancer cells. This biochip is not only conducive to modeling cancer cell metastasis and studying cell invasion mechanisms, but also has the potential to build a quantitative evaluation platform that can be used in future chemical drug treatments.

Biomechanics of infarcted left ventricle: a review of modelling.

Mathematical modelling in biomechanics of infarcted left ventricle (LV) serves as an indispensable tool for remodelling mechanism exploration, LV biomechanical property estimation and therapy assessment after myocardial infarction (MI). However, a review of mathematical modelling after MI has not been seen in the literature so far. In the paper, a systematic review of mathematical models in biomechanics of infarcted LV was established. The models include comprehensive cardiovascular system model, essential LV pressure-volume and stress-stretch models, constitutive laws for passive myocardium and scars, tension models for active myocardium, collagen fibre orientation optimization models, fibroblast and collagen fibre growth/degradation models and integrated growth-electro-mechanical model after MI. The primary idea, unique characteristics and key equations of each model were identified and extracted. Discussions on the models were provided and followed research issues on them were addressed. Considerable improvements in the cardiovascular system model, LV aneurysm model, coupled agent-based models and integrated electro-mechanical-growth LV model are encouraged. Substantial attention should be paid to new constitutive laws with respect to stress-stretch curve and strain energy function for infarcted passive myocardium, collagen fibre orientation optimization in scar, cardiac rupture and tissue damage and viscoelastic effect post-MI in the future.

Adipose-Derived Stem Cells Promote Intussusceptive Lymphangiogenesis by Restricting Dermal Fibrosis in Irradiated Tissue of Mice.

Currently, there is no definitive treatment for lymphatic disorders. Adipose-derived stem cells (ADSCs) have been reported to promote lymphatic regeneration in lymphedema models, but the mechanisms underlying the therapeutic effects remain unclear. Here, we tested the therapeutic effects of ADSC transplantation on lymphedema using a secondary lymphedema mouse model. The model was established in C57BL/6J mice by x-irradiation and surgical removal of the lymphatic system in situ. The number of lymphatic vessels with anti-lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) immunoreactivity increased significantly in mice subjected to transplantation of 7.5 × 105 ADSCs. X-irradiation suppressed lymphatic vessel dilation, which ADSC transplantation could mitigate. Proliferative cell nuclear antigen staining showed increased lymphatic endothelial cell (LEC) and extracellular matrix proliferation. Picrosirius red staining revealed normal collagen fiber orientation in the dermal tissue after ADSC transplantation. These therapeutic effects were not related to vascular endothelial growth factor (VEGF)-C expression. Scanning electron microscopy revealed structures similar to the intraluminal pillar during intussusceptive angiogenesis on the inside of dilated lymphatic vessels. We predicted that intussusceptive lymphangiogenesis occurred in lymphedema. Our findings indicate that ADSC transplantation contributes to lymphedema reduction by promoting LEC proliferation, improving fibrosis and dilation capacity of lymphatic vessels, and increasing the number of lymphatic vessels via intussusceptive lymphangiogenesis.

Integrational Technologies for the Development of Three-Dimensional Scaffolds as Platforms in Cartilage Tissue Engineering.

The prevalence of osteoarthritis is on the rise, and an effective treatment for cartilage defects is still being sought. Cartilage tissue in vivo encompasses complex structures and composition, both of which influence cells and many properties of the native cartilage. The extracellular matrix structure and components provides both morphological cues and the necessary signals to promote cell functions including metabolism, proliferation, and differentiation. In the present study, cryo-printing and electrospinning were combined to produce multizone scaffolds that consist of three distinctive zones. These scaffolds successfully mimic the collagen fiber orientation of the native cartilage. Moreover, in vitro analysis of chondrocyte-seeded scaffolds demonstrated the ability of multizone scaffolds to support long-term chondrocyte attachment and survival over a 5 week culture period. Moreover, multizone scaffolds were found to regulate the expression of key genes in comparison to the controls and allowed the detection of sulfated glycosaminoglycan. Evaluation of the compressive properties revealed that the multizone scaffolds possess more suitable mechanical properties, for the native cartilage, in comparison to the electrospun and phase-separated controls. Multizone scaffolds provide viable initial platforms that capture the complex structure and compressive properties of the native cartilage. They also maintain chondrocyte phenotype and function, highlighting their potential in cartilage tissue engineering applications.

Probing the Role of Bone Lamellar Patterns through Collagen Microarchitecture Mapping, Numerical Modeling, and 3D‐Printing

The fibrillar organization is crucial for the mechanics of osteonal bone and for its anisotropy. Different collagen fiber orientation (CFO) patterns are observed in bone and correlated to the main local loading, leading to the hypothesis that the fibers are preferentially aligned to bear specific loading types. A heterogeneous distribution of osteon morphotypes (OMs) is noticed in long bones, although the relationship between the OM spatial distribution and the local predominant loading is still unclear. This study aims to shed light on the reasons why Nature uses specific OMs in diverse stress‐dominated regions. A multimodal approach is adopted, including collagen fiber orientation mapping, numerical modeling of different OMs under tensile and compressive loading, and 3D‐printing of OM‐inspired samples, to unravel the relationship between different OMs and their mechanical behavior. Simulation results suggest a better performance of the vertical OM (VOM) under tensile loading and of the twisted OM (TOM) under compression, confirming earlier hypotheses. The outcomes of mechanical testing, conducted on 3D‐printed samples, highlight a possible buckling‐induced failure of fibers with preferential vertical orientation and provide evidence that OMs are stress‐tailored, opening new venues for the design of stress‐tuned bioinspired composites.

Anisotropic properties of articular cartilage in an accelerated in vitro wear test

Many material properties of articular cartilage are anisotropic, particularly in the superficial zone where collagen fibers have a preferential direction. However, the anisotropy of cartilage wear had not been previously investigated. The objective of this study was to evaluate the anisotropy of cartilage material behavior in an in vitro wear test. The wear and coefficient of friction of bovine condylar cartilage were measured with loading in directions parallel (longitudinal) and orthogonal (transverse) to the collagen fiber orientation at the articular surface. An accelerated cartilage wear test was performed against a T316 stainless-steel plate in a solution of phosphate buffered saline with protease inhibitors. A constant load of 160 N was maintained for 14000 cycles of reciprocal sliding motion at 4 mm/s velocity and a travel distance of 18 mm in each direction. The contact pressure during the wear test was approximately 2 MPa, which is in the range of that reported in the human knee and hip joint. Wear was measured by biochemically quantifying the glycosaminoglycans (GAGs) and collagen that was released from the tissue during the wear test. Collagen damage was evaluated with collagen hybridizing peptide (CHP), while visualization of the tissue composition after the wear test was provided with histologic analysis. Results demonstrated that wear in the transverse direction released about twice as many GAGs than in the longitudinal direction, but that no significant differences were seen in the amount of collagen released from the specimens. Specimens worn in the transverse direction had a higher intensity of CHP stain than those worn in the longitudinal direction, suggesting more collagen damage from wear in the transverse direction. No anisotropy in friction was detected at any point in the wear test. Histologic and CHP images demonstrate that the GAG loss and collagen damage extended through much of the depth of the cartilage tissue, particularly for wear in the transverse direction. These results highlight distinct differences between cartilage wear and the wear of traditional engineering materials, and suggest that further study on cartilage wear is warranted. A potential clinical implication of these results is that orienting osteochondral grafts such that the direction of wear is aligned with the primary fiber direction at the articular surface may optimize the life of the graft.

Axial stretch regulates rat tail collecting lymphatic vessel contractions

Lymphatic contractions play a fundamental role in maintaining tissue and organ homeostasis. The lymphatic system relies on orchestrated contraction of collecting lymphatic vessels, via lymphatic muscle cells and one-way valves, to transport lymph from the interstitial space back to the great veins, against an adverse pressure gradient. Circumferential stretch is known to regulate contractile function in collecting lymphatic vessels; however, less is known about the role of axial stretch in regulating contraction. It is likely that collecting lymphatic vessels are under axial strain in vivo and that the opening and closing of lymphatic valves leads to significant changes in axial strain throughout the pumping cycle. The purpose of this paper is to quantify the responsiveness of lympatic pumping to altered axial stretch. In situ measurements suggest that rat tail collecting lymphatic vessels are under an axial stretch of ~1.24 under normal physiological loads. Ex vivo experiments on isolated rat tail collecting lymphatics showed that the contractile metrics such as contractile amplitude, frequency, ejection fraction, and fractional pump flow are sensitive to axial stretch. Multiphoton microscopy showed that the predominant orientation of collagen fibers is in the axial direction, while lymphatic muscle cell nuclei and actin fibers are oriented in both circumferential and longitudinal directions, suggesting an axial component to contraction. Taken together, these results demonstrate the significance of axial stretch in lymphatic contractile function, suggest that axial stretch may play an important role in regulating lymph transport, and demonstrate that changes in axial strains could be an important factor in disease progression.

Transmural Variation of Collagen Orientation and Structure Distributions in the Right Pulmonary Arterial Wall

Pulmonary arterial hypertension (PAH) is a disease characterized by elevated blood pressure (mean pulmonary arterial pressure above 25 mmHg. Remodeling of pulmonary arteries due to this elevated blood pressure compromises their normal physiological function. Our previous work showed that the right pulmonary artery significantly stiffens circumferentially in PAH and collagen fibers become engaged and significantly aligned in the medial layer (Pursell et al 2016). To better understand the changes in tissue mechanical response, here we explore the distributions of collagen orientation and tortuosity through the vessel wall.MethodsTo induce PAH, four male Sprague‐Dawley rats were injected with 60 mg/kg monocrotaline. Once PAH was established (4 weeks post‐injection), pulmonary arteries were harvested, and collagen imaged via multiphoton microscopy. A custom MATLAB code was then used to determine collagen fiber orientation and tortuosity.ResultsMPM images of collagen fibers from the intima, media, and adventitia of normotensive and hypertensive rat RPAs were traced with our custom MATLAB code. In the hypertensive animals, fiber direction and tortuosity varied more between vascular wall layers than those of normotensive animals.ConclusionsWhile our previous study only examined the medial layer, this study determined that fiber distribution varies throughout the vessel wall. This will significantly affect the mechanics or behavior of pulmonary vessels. Therefore, future studies will need to incorporate collagen structures from multiple vessel layers to better understand the relationship between vascular remodeling and mechanics.

Defining the Design Principles of Skin Epidermis Postnatal Growth

SummaryDuring embryonic and postnatal development, organs and tissues grow steadily to achieve their final size at the end of puberty. However, little is known about the cellular dynamics that mediate postnatal growth. By combining in vivo clonal lineage tracing, proliferation kinetics, single-cell transcriptomics, and in vitro micro-pattern experiments, we resolved the cellular dynamics taking place during postnatal skin epidermis expansion. Our data revealed that harmonious growth is engineered by a single population of developmental progenitors presenting a fixed fate imbalance of self-renewing divisions with an ever-decreasing proliferation rate. Single-cell RNA sequencing revealed that epidermal developmental progenitors form a more uniform population compared with adult stem and progenitor cells. Finally, we found that the spatial pattern of cell division orientation is dictated locally by the underlying collagen fiber orientation. Our results uncover a simple design principle of organ growth where progenitors and differentiated cells expand in harmony with their surrounding tissues.

Polarimetric second-harmonic generation microscopy of the hierarchical structure of collagen in stage I-III non-small cell lung carcinoma.

Polarimetric second-harmonic generation (P-SHG) microscopy is used to quantify the structural alteration of collagen in stage-I,-II and -III non-small cell lung carcinoma (NSCLC) ex vivo tissue. The achiral and chiral molecular second-order susceptibility tensor components ratios (R and C, respectively), the degree of linear polarization (DLP) and the in-plane collagen fiber orientation (δ) were extracted. Further, texture analysis was performed on the SHG intensity, R, C, DLP and δ. The distributions of R, C, DLP and δ as well as the textural features of entropy, correlation and contrast show significant differences between normal and tumor tissues.

Histopathological Evaluation of Mechanoreceptors in the Metatarsophalangeal Joint Capsule in Hallux Valgus

To date, we could find no study concerning the relationship between mechanoreceptors in the joint capsule of the first metatarsophalangeal joint and hallux valgus deformity. We aimed to investigate the presence of mechanoreceptors in samples obtained from the first metatarsophalangeal joint capsules of patients with hallux valgus deformity to improve our understanding of the clinical and histopathological features of the disease. Samples were taken from the first metatarsophalangeal joint capsules of 13 fresh-frozen cadavers with normal anatomy (controls) and 29 patients undergoing surgery for hallux valgus (cases). For light microscopy, excised specimens were fixed in 10% formaldehyde and processed for routine histopathological investigation. All samples were dehydrated in a series of ethanol, cleared in xylene, and embedded in paraffin. Orientation of collagen fibers was determined on Masson's trichrome–stained sections, and mechanoreceptors were evaluated on S-100-immunostained sections. In the sections stained with Masson's trichrome, the orientation of collagen fibers was regular in the control group. However, coarse and disoriented collagen bundles were observed in the hallux valgus cases (P ≤ .05). S-100 immunostaining was positive in the sections of both the cases and controls. Finally, free nerve endings were more abundant in the samples obtained from the capsules of hallux valgus cases than from the control group (P ≤ .05). An increase in the number of free nerve endings within the capsules of the first metatarsophalangeal joints in feet with hallux valgus deformity might have a role in the development of clinically relevant joint pain and instability.

Vertical Fibrous Morphology and Structure-Function Relationship in Natural and Biomimetic Suction-Based Adhesion Discs

Summary Recent years have seen rapid development in bio-inspired materials for adhesion. However, it remains challenging to realize robust adhesion on rough aquatic surfaces. An inspiring natural model is the remora fish, which has evolved to retain powerful adhesion to hosts using a dorsal suction disc. We find that the remora suction disc has a unique fibrous architecture of vertically oriented collagen fibers that enable anisotropic mechanical properties and enhanced adhesion performance. In the engineered prototype, vertically oriented nylon fibers are embedded into the soft silicone matrix using electrostatic flocking. The anisotropic mechanical properties are validated in both natural and biomimetic suction discs. Furthermore, the biomimetic suction disc demonstrates an enhanced adhesion function with a maximum 62.5% increase in pull-off force and a 340% increase in attachment time compared with the silicone control. This work can shed light on natural adhesion mechanisms and inspire novel designs for aquatic soft adhesives and actuators.

Abstract P5-06-16: Histomorphometric measure of disorder of collagen fiber orientation is associated with risk of recurrence in ER+ breast cancers in ECOG-ACRIN E2197 and TCGA-BRCA

Abstract Background: Recent research has shown that collagen fiber arrangement in the tumor microenvironment plays a key role determining breast cancer prognosis. In this work, we present a new quantitative histomorphometric approach to identify and evaluate whether Collagen Fiber Orientation Disorder from Tumor-associated Stroma (CFOD-TS) on digitized H&E slides are associated with disease free survival (DFS) in estrogen receptor positive (ER+) breast cancer patients. Method: 234 and 171 ER+ invasive breast cancer patient’s slides were acquired and digitized from E2197 (training set) and TCGA-BRCA (test set) respectively. Collagen fibers were first segmented out on the digitized H&E slides by a combination of Derivative of Gaussian filters. The CFOD-TS features were then extracted from tumor patches in the whole slide image. A multiple linear regression model (MLR) was then trained in conjunction with the CFOD-TS features distinguishing patients likely to recur from not (>15 year follow-up period) to predict DFS for the non-censored patients. Result: The CFOD-TS discriminated patients (p=0.01) with and without recurrence and was significantly associated with DFS on ECOG 2197. The univariate and multivariate analysis on the training set were summarized in table 1 and table 2. CFOD-TS was also independently prognostic on patients in the TCGA for stratifying patients into low- and high-risk groups with p=0.009 via a log rank test and a hazard ratio (HR) of 3.09 (95% CI =1.33~7.18, p=0.0088). HR between the two risk groups was 7.11 (n=104, 95% CI =1.53~32.94, p=0.01) for stage I / II tumors and 3.03 (n=39, 95% CI =0.79-8.67, p=0.11) for stage III / IV cancers. In a multivariate setting, CFOD-TS (HR -3.7) was more significant than T-stage (HR-2.88) in stratifying risk. (Table2) Table 1: Univariate DFS analysis on the training setP-valueHR(95% CI)Case numberCFDO-TS derived risk groups0.00272.04 (1.28 – 3.26)234low / intermediate Oncotype DX (ODx) vs High ODx0.940.96(0.36 – 2.56)68low ODx vs intermediate / High ODx0.00323.49 (1.52 – 8.01)68grade0.300.82 (0.56 – 1.19)209 Table 2: Multivariate DFS analysis on the training set (n=68) and test set (n = 171)VariableP-valueHR(95% CI)ECOG 2197low ODx vs intermediate / High ODx0.00144.00 (1.71 – 9.34)CFDO-TS derived risk groups: Low vs high-risk0.0192.53 (1.17 – 5.47)TCGAT-stage: T1/T2 vs T3/T40.032.88 (1.10 -7.50)N-stage: N0 vs N1/N2/N30.411.47 (0.59 - 3.70)CFOD-TS derived risk groups: Low vs high-risk0.0033.70 (1.57 - 8.67) Conclusion: Over-expression of CFOD-TS was found to be independently associated with lower likelihood of recurrence and could potentially serve as a prognostic marker of outcome for ER+ breast cancer. Citation Format: Haojia Li, Kaustav Bera, Hannah Gilmore, Nancy E Davidson, Lori J Goldstein, Anant Madabhushi. Histomorphometric measure of disorder of collagen fiber orientation is associated with risk of recurrence in ER+ breast cancers in ECOG-ACRIN E2197 and TCGA-BRCA [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-16.

Directional dependent variation in mechanical properties of planar anisotropic porcine skin tissue

Nonlinear and anisotropic mechanical behavior of skin is essential in various applications such as dermatology, cosmetic products, forensic science, and computational studies. The present study quantifies the mechanical anisotropy of skin using the bulge method and full-field imaging technique. In bulging, the saline solution at 37 °C mimics the in vivo body temperature and fluid conditions, and all experiments were performed in the control environment. Assumption of thin spherical shell membrane theory and imaging techniques were implemented to obtain the anisotropic stress strain relations. Further, stress strain relations at an interval of 10° were calculated to obtain the variation in modulus with direction. Histological examinations were performed to signify the role of the collagen fibers orientation on the mechanical properties. The maximum and minimum linear modulus and collagen fiber orientation intensity were found in good agreement. The angular difference between maximum and minimum linear modulus and orientation intensity was found 71° ± 7° and 76° ± 5° respectively, and the percentage difference was 43.4 ± 8.2 and 52.5 ± 6.4 respectively. Further, a significant difference in the maximum and minimum collagen orientation intensity between the untested and tested specimens indicates the realignment of the fibers. Additionally, a cubic polynomial empirical relation was established to calculate the quantitative variation in the apparent modulus with the directions, which serves for the anisotropic modeling of the skin. The experimental technique used in this study can be applied for anisotropic quantification of planar soft tissues as well as can be utilized to imitate the tissue expansion procedure used in reconstructive surgery.