Collagen Organization Does Not Influence T-Cell Distribution in Stroma of Human Pancreatic Cancer

The dominant intrastromal T-cell infiltration in pancreatic cancer is mainly caused by the contact guidance through the excessive desmoplastic reaction and could represent one of the obstacles to an effective immune response in this tumor type. This study analyzed the collagen organization in normal and malignant pancreatic tissues as well as its influence on T-cell distribution in pancreatic cancer. Human pancreatic tissue was analyzed using immunofluorescence staining and multiphoton and SHG microscopy supported by multistep image processing. The influence of collagen alignment on activated T-cells was studied using 3D matrices and time-lapse microscopy. It was found that the stroma of malignant and normal pancreatic tissues was characterized by complex individual organization. T-cells were heterogeneously distributed in pancreatic cancer and there was no relationship between T-cell distribution and collagen organization. There was a difference in the angular orientation of collagen alignment in the peritumoral and tumor-cell-distant stroma regions in the pancreatic ductal adenocarcinoma tissue, but there was no correlation in the T-cell densities between these regions. The grade of collagen alignment did not influence the directionality of T-cell migration in the 3D collagen matrix. It can be concluded that differences in collagen organization do not change the spatial orientation of T-cell migration or influence stromal T-cell distribution in human pancreatic cancer. The results of the present study do not support the rationale of remodeling of stroma collagen organization for improvement of T-cell–tumor cell contact in pancreatic ductal adenocarcinoma.

Abstract P351: Grayscale Texture Feature Angular Second Moment Correlates With Collagen Alignment in Carotid Artery Plaques

Introduction: Grayscale (GS) texture features that examine homogeneity and echogenicity have been used to identify vulnerable plaques with in vivo ultrasound imaging have been shown to correlate with plaque tissue composition. However, the relationship of collagen fiber organization to GS texture features extracted from in vivo images is a novel idea to provide additional information about plaque structure. We hypothesize that collagen fiber alignment is clinically relevant to identify vulnerable plaques. The objective of this feasibility study was to use multiscale imaging (in vivo ultrasound and high resolution optical microscopy) to determine how GS texture features are related to plaque collagen structure. Methods: Participants (n=6) scheduled for clinically indicated carotid endarterectomy underwent in vivo carotid ultrasound imaging with texture feature extraction (spatial gray level dependence matrices method for calculating angular second moment [SGLDM-ASM] and grayscale median value [GSM]). Plaque specimens were sent to histopathology and stained with H&E. The collagen fibers in the fibrous cap of the plaque histopathology slides were imaged with liquid crystal based polarization microscopy and quantified using an established software tool (CurveAlign). Correlations between collagen alignment coefficient (range 0-1, 1 represents perfectly aligned fibers) and the texture feature SGLDM-ASM (a measure of homogeneity, higher values are more homogenous) and GSM (a measure of echogenicity higher values are more echogenic) were examined. Results: Participants were mean (SD) 72.5 (6.1) years of age, had 71.67 (8.16) percent stenosis. The mean SGLDM-ASM was 0.0017 (0.0023), the mean SD GSM was 73.13 (30.98). SGLDM-ASM was significantly correlated to collagen alignment (r=0.83; p=0.028). There was no significant correlation detected between GSM and collagen alignment (r=-0.43;p=0.38). Conclusion: Results of this study indicate the potential role for using high resolution optical microscopy with ultrasound to characterize collagen fiber alignment in plaques with measures of homogeneity. Future studies are needed to see how multiscale imaging can be used to inform in vivo imaging for identification of vulnerable plaque features.

Microstructural and Mechanical Properties of the Anterolateral Ligament of the Knee

Background: The variable anatomy and controversy of the anterolateral ligament (ALL) reflect the complex relationship among the anterolateral knee structures. Purpose/Hypothesis: The purpose was to quantify the microstructural and mechanical properties of the ALL as compared with the anterolateral capsule (ALC) and lateral collateral ligament (LCL). The primary hypotheses were that (1) there is no difference in these properties between the ALL and ALC and (2) the LCL has significantly different properties from the ALL and ALC. Study Design: Descriptive laboratory study. Methods: The LCL, ALL, and ALC were harvested from 25 cadaveric knees. Mechanical testing and microstructural analyses were performed using quantitative polarized light imaging. The average degree of linear polarization (AVG DoLP; mean strength of collagen alignment) and standard deviation of the angle of polarization (STD AoP; degree of variation in collagen angle orientation) were calculated. Results: Linear region moduli were not different between the ALC and ALL (3.75 vs 3.66 MPa, respectively; P > .99). AVG DoLP values were not different between the ALC and ALL in the linear region (0.10 vs 0.10; P > .99). Similarly, STD AoP values were not different between the ALC and ALL (24.2 vs 21.7; P > .99). The LCL had larger modulus, larger AVG DoLP, and smaller STD AoP values than the ALL and ALC. Of 25 knee specimens, 3 were observed to have a distinct ALL, which exhibited larger modulus, larger AVG DoLP, and smaller STD AoP values as compared with nondistinct ALL samples. Conclusion: There were no differences in the mechanical and microstructural properties between the ALL and ALC. The ALC and ALL exhibited comparably weak and disperse collagen alignment. However, when a distinct ALL was present, the properties were suggestive of a ligamentous structure. Clinical Relevance: The properties of the ALL are similar to those of a ligament only when a distinct ALL is present, but otherwise, for the majority of specimens, ALL properties are closer to those of the capsule. Variability in the ligamentous structure of the ALL suggests that it may be more important in some patients than others and reconstruction may be considered in selective patients. Further study is needed to better understand its selective role and optimal indications for reconstruction.

Mechanics-driven mechanobiological mechanisms of arterial tortuosity

Arterial tortuosity manifests in many conditions, including hypertension, genetic mutations predisposing to thoracic aortopathy, and vascular aging. Despite evidence that tortuosity disrupts efficient blood flow and that it may be an important clinical biomarker, underlying mechanisms remain poorly understood but are widely appreciated to be largely biomechanical. Many previous studies suggested that tortuosity may arise via an elastic structural buckling instability, but the novel experimental-computational approach used here suggests that tortuosity arises from mechanosensitive, cell-mediated responses to local aberrations in the microstructural integrity of the arterial wall. In particular, computations informed by multimodality imaging show that aberrations in elastic fiber integrity, collagen alignment, and collagen turnover can lead to a progressive loss of structural stability that entrenches during the development of tortuosity. Interpreted in this way, microstructural defects or irregularities of the arterial wall initiate the condition and hypertension is a confounding factor.

Cell-Scale Biophysical Cues from Collagen Fiber Architecture Instruct Cell Behavior and the Propagation of Mechanosensory Signals

Mechanosensory cues from the extracellular matrix underpin numerous cellular behaviors including tumor cell migration yet are influenced by the local structure and organization of the matrix in unknown ways. To investigate mechanical cues with respect to local collagen organization, we used a combination of intravital imaging of the mammary tumor microenvironment and 3D collagen gel systems with both migratory MDA-MB-231 cells and acellular pNIPAAm beads. We identified that fiber organization directs a bias in cell response along the axis of alignment. Using innovative methodology, we determined that local collagen alignment resulted in a 30-fold difference in directional cell-scale stiffness and also dramatically altered the rate at which cell-induced fiber displacements decayed over distance. Our results reveal differential mechanical properties across orthogonal directions in aligned matrices that provide sizeable cues to the cell and have important implications for cellular mechanosensing and cell-cell communication within the tissue microenvironment.

Microstructural and Mechanical Properties of the Anterolateral Ligament (ALL) of the Knee

Objectives: The anterolateral ligament (ALL) of the knee has recently emerged as a potential contributor to rotational stability of the knee, with growing interest in ALL reconstruction as a supplement to anterior cruciate ligament reconstruction. The prevalence of the ALL in the knee has varied in anatomic dissection and imaging studies, raising questions about its importance as a knee stabilizer. The purpose of this study was to assess the microstructural and mechanical properties of the anterolateral knee, to better understand the ALL structure compared to the surrounding anterolateral capsule (ALC) and lateral collateral ligament (LCL). A polarized light imaging technique was used to quantify collagen fiber alignment simultaneously with measurement of tensile mechanical properties. Our primary hypothesis was that there is no difference in the microstructural and mechanical properties between the ALL and ALC. Our secondary hypothesis was that the properties of the LCL are different from the ALL and ALC. Methods: Twenty-five knee specimens from sixteen donors (five males, eleven females; mean age 45.6 +/- 6.4; age range 35-59 years; mean BMI 26.5 +/- 8.4) were obtained as determined by a priori power analysis. The anatomic technique to dissect the anterolateral knee structures was performed as described previously. Three tissue samples (LCL, ALL, and ALC) were harvested (Fig. 1). The ALL was taken as a quadrilateral piece of tissue starting posterior/proximal from the lateral femoral epicondyle and ending at the lateral border of Gerdy’s tubercle. During gross dissection, the knee was assessed for the presence or absence of a distinct visible and palpable structure within the area defined as the ALL. Harvested samples were thinned to approximately 1-mm thick using a freezing-stage sliding microtome. Cross-sectional area was measured using a 3D laser scanning system. Four 0.8-mm diameter aluminum beads were attached to the sample surface to enable strain measurement. Mechanical testing was performed with preconditioning followed by both a stress-relaxation test and a quasi-static ramp to failure. Microstructural analysis was performed using transmitted circularly-polarized incident light and a high-resolution, division-of-focal-plane polarization camera. The average degree of linear polarization (AVG DoLP; i.e., mean strength of collagen alignment) and standard deviation of the angle of polarization (STD AoP; i.e., degree of variation in collagen angle orientation) were calculated for the region of interest of each sample. Statistical analysis was performed using Kruskal-Wallis test (assuming nonparametric data) with Dunn’s correction for multiple comparisons. Results: Mechanical analysis of elastic moduli for the toe- and linear-region of the stress-strain curves showed no difference between the ALL and ALC but were significantly higher for the LCL (p<0.0001; Fig. 2). Microstructural analysis of the ALL and ALC during quasi-static ramp to failure showed no difference in AVG DoLP and STD AoP values at all strain levels (Fig. 3). Larger DoLP values (i.e., stronger collagen fiber alignment) were observed for the LCL than both the ALL and ALC (p<0.0001). Larger STD AoP values (i.e., more variation in collagen orientation) were observed for the ALL and ALC compared to the LCL (p<0.0001; Fig. 3). When looking at correlations between mechanical and microstructural properties (Fig. 4), we found clustering of the LCL data points at high linear modulus and AVG DoLP while the ALL and ALC data points were clustered together. Similarly, we found clustering of the LCL at high linear modulus and low STD AoP while the ALL and ALC were clustered together. Only three of 25 knee specimens (12%) were observed to have a distinct, ligamentous structure in the region of the ALL. Interestingly, these distinct ALL samples (outlined in black on figures) showed relatively larger elastic moduli, higher AVG DoLP, and lower STD AoP (i.e., uniform and organized collagen alignment) across the stress-strain curve compared to samples harvested from knees without a distinct ALL. The distinct ALL tissues were also seen clustered near the LCL data points in the correlation plots. Conclusions: Overall, there were no differences in the mechanical and microstructural properties between the ALL and ALC, while the LCL demonstrated different properties compared to both the ALL and ALC. Both the ALC and ALL show significantly weaker collagen fiber alignment and more variation in the direction of collagen fiber alignment compared to the LCL. These findings suggest that the ALL has similar properties to capsule (i.e., ALC). However, when a distinct ALL was present at dissection (12%), the data indicates stronger and more uniform collagen alignment suggestive of more ligament-type qualities. Further research is needed to more precisely define the prevalence and properties of distinct ALLs in the knee.

Heparanase promotes Syndecan-1 expression to mediate fibrillar collagen and mammographic density in human breast tissue cultured ex vivo

Mammographic density (MD) is a strong and independent factor for breast cancer (BC) risk and is increasingly associated with BC progression. We have previously shown in mice that high MD, which is characterised by the preponderance of a fibrous stroma, facilitates BC xenograft growth and metastasis. This stroma is rich in extracellular matrix (ECM) factors, including heparan sulfate proteoglycans (HSPGs), such as the BC-associated syndecan-1 (SDC1). These proteoglycans tether growth factors, which are released by heparanase (HPSE). MD is positively associated with estrogen exposure and, in cell models, estrogen has been implicated in the upregulation of HPSE, the activity of which promotes SDC expression. Herein we describe a novel measurement approach (single-sided NMR) using a patient-derived explant (PDE) model of normal human (female) mammary tissue cultured ex vivo to investigate the role(s) of HPSE and SDC1 on MD. Relative HSPG gene and protein analyses determined in patient-paired high versus low MD tissues identified SDC1 and SDC4 as potential mediators of MD. Using the PDE model we demonstrate that HPSE promotes SDC1 rather than SDC4 expression and cleavage, leading to increased MD. In this model system, synstatin (SSTN), an SDC1 inhibitory peptide designed to decouple SDC1-ITGαvβ3 parallel collagen alignment, reduced the abundance of fibrillar collagen as assessed by picrosirius red viewed under polarised light, and reduced MD. Our results reveal a potential role for HPSE in maintaining MD via its direct regulation of SDC1, which in turn physically tethers collagen into aligned fibres characteristic of MD. We propose that inhibitors of HPSE and/or SDC1 may afford an opportunity to reduce MD in high BC risk individuals and reduce MD-associated BC progression in conjunction with established BC therapies.