Mean deformation metrics for quantifying 3D cell–matrix interactions without requiring information about matrix material properties

Mechanobiology relates cellular processes to mechanical signals, such as determining the effect of variations in matrix stiffness with cell tractions. Cell traction recorded via traction force microscopy (TFM) commonly takes place on materials such as polyacrylamide- and polyethylene glycol-based gels. Such experiments remain limited in physiological relevance because cells natively migrate within complex tissue microenvironments that are spatially heterogeneous and hierarchical. Yet, TFM requires determination of the matrix constitutive law (stress–strain relationship), which is not always readily available. In addition, the currently achievable displacement resolution limits the accuracy of TFM for relatively small cells. To overcome these limitations, and increase the physiological relevance of in vitro experimental design, we present a new approach and a set of associated biomechanical signatures that are based purely on measurements of the matrix''s displacements without requiring any knowledge of its constitutive laws. We show that our mean deformation metrics (MDM) approach can provide significant biophysical information without the need to explicitly determine cell tractions. In the process of demonstrating the use of our MDM approach, we succeeded in expanding the capability of our displacement measurement technique such that it can now measure the 3D deformations around relatively small cells (∼10 micrometers), such as neutrophils. Furthermore, we also report previously unseen deformation patterns generated by motile neutrophils in 3D collagen gels.Mechanical cues within the cellular microenvironment regulate numerous fundamental functions including cell adhesion, deformation, and generation of traction (1–6). Analysis of cellular force generation, and its role in regulating homeostasis across a variety of cellular phenotypes and experimental platforms, has received much attention over the last three decades (7–13). Experimental quantification of cellular forces has produced several cell traction measurement techniques, ranging from surface wrinkle detection and flexure of micropillars to traction force microscopy (TFM) (12, 14–20). In TFM, measured cell-induced displacements are converted into tractions using various mathematical frameworks (14, 15, 17, 18, 21, 22). Both two- and 3D TFM techniques have steadily increased in sophistication and now feature high-spatial displacement resolution and advanced computational formalisms to connect this displacement information to complex material constitutive laws (17, 23, 24).To successfully perform TFM, it is critical to know the stress–strain constitutive behavior of the matrix surrounding the cell. Although many TFM substrates feature relatively simple artificial gel constructs, such as polyacrylamide and polyethylene glycol, these constructs are impenetrable by cells and obviate measures obtained while cells are in a 3D setting (as would be the case within a bodily tissue). Most physiologically important matrices have complicated, hierarchical microstructures and tend to be spatially heterogeneous. In addition, many cells apply significant forces leading to large matrix deformations that require more complicated, nonlinear constitutive laws for accurate calculation (17, 24–28). Moreover, to generate a quantitative map of tractions exerted by a moving cell, the mechanical properties of the surrounding matrix must be resolved at the cellular level. Taken together, significant experimental challenges exist for accurately deducing the microconstitutive laws for many physiologically realistic matrices and, by extension, the ability to map tractions onto small cells is lacking. Furthermore, and perhaps most importantly, many cells are known to actively remodel the matrix as they move through it. As a result, the constitutive laws are constantly evolving, making a one-time measurement of the matrix material properties insufficient for accurate determination of cell tractions in real time.To circumvent this fundamental hurdle of requiring a material’s constitutive law, we present an alternate, kinematics-based quantification method that correlates cellular deformations to biological function. Based solely on the displacement field in the surrounding matrix, we define tensor-valued mean deformation metrics (MDM) that quantify the overall shape change of the cell (e.g., contractility, mean volume change, and rotation). Because of its kinematic nature, the mean deformation metric approach does not aim to provide any information about the cell tractions. Rather, the approach should be viewed as a distinct, immediately applicable methodology complimentary to TFM for investigations in which the material properties of the extracellular matrix are unknown.Thus, the mean deformation metric approach provides a significant advantage to laboratories and investigators who may not have the means to conduct sophisticated material characterization measurements at cellular length scales. The method is also well-suited for studies using primary cells for which fluorescent transfection approaches, such as in the use of FRET-based force sensors (29), are not applicable.We present the formulation of the MDM and validate our technique using well-established analytical solutions, demonstrating our technique is accurate to within 1%. Next, we apply our MDM approach to analyze neutrophil-generated collagen matrix displacement fields obtained using confocal microscopy and fast iterative digital volume correlation (FIDVC) (30). In the process of demonstrating the MDM approach, we solved the challenging problem of resolving local displacements around neutrophils (which are only 10 μm in size) with submicron resolution, and subsequently revealed previously unknown deformation patterns during the migration of neutrophils through a 3D collagen matrix.     

Comparison of vascularity and angiogenesis in primary invasive mammary carcinomas and in their respective axillary lymph node metastases

It is well established that the ability of a neoplasm to induce a blood supply from a pre-existing circulation (angiogenesis) is a major factor in tumour growth, invasion and metastasis. However, the angiogenic potential of metastases and their subsequent growth have not been extensively studied. The question arises: can metastatic clones induce the same level of angiogenesis as in the primary neoplasm they emanated from? In this study it is hypothesised that in the same patient the level of vascularity and angiogenesis is the same in both the primary invasive ductal carcinoma and in the axillary lymph node metastasis at the time of surgery, according to Kerbels theory of clonal-dominance. To directly address the hypothesis, morphological measures of the established blood/lymphatic circulation (vascularity) as well as estimates of angiogenesis (endothelial cell proliferation) were measured in primary tumours and directly compared to the same parameters in the corresponding lymph node metastasis in a case by case basis (n=17). The results demonstrate varying associations between the level of vascularity and angiogenesis between matched individual tumours and their metastatic lymph nodal deposits. It is possible that either variations in the angiogenic characteristics of the metastasising clone or local or systemic promoters or inhibitors of angiogenesis influence tumour angiogenesis at the different sites.     

Reflex sympathetic dystrophy syndrome in a child

Reflex sympathetic dystrophy syndrome (RSDS) is a painful condition that usually follows regional trauma. We report the case of a 13-year-old girl that was seen for a painful swelling of the right hand associated with palmar hyperhidrosis, which occurred after a trauma to the hand. Bone scan images showed early tissue abnormality, which was more significant on the right hand and wrist, as well as moderate bone uptake on the right side. Nonsteroidal anti-inflammatory drugs and alternating hot and cold baths led to a marked improvement. RSDS occurs following trauma or subsequent to various diseases or drug intake. This syndrome is related to impaired tissue microvasculature under the influence of abnormal sympathetic reflex hyperactivity. Bone scan is the diagnostic procedure of choice in RSDS, but it may be normal. Physiotherapy should be preferred in pediatric cases.     

Assessing the effectiveness of a mammography screening service

BACKGROUND: Trials have shown that mammography screening reduces mortality and probably decreases morbidity related to breast cancer. METHODS: We assessed whether the major mammography service in Western Australia (BreastScreen WA) is likely to reduce mortality by comparing prognostic variables between screen-detected and other cases of breast cancer diagnosed in 1999. We assessed likely reductions in morbidity by comparing treatments received by these two groups. To confirm mortality and morbidity reduction, we also compared prognostic variables and treatments with targets. Information on demographic variables, tumour characteristics at presentation and treatments were collected from medical records for all incident cases of breast cancer in Western Australia in 1999. We matched cases with the Western Australian Cancer Registry records to determine which cases had been detected by BreastScreen WA. RESULTS: BreastScreen WA achieved the targets for mortality reduction. Tumours detected by BreastScreen WA were smaller in size, less likely to have vascular invasion, of lower histological grade and were more likely to be ductal carcinoma in situ alone without invasive carcinoma. Oestrogen receptor status was more likely to be positive, the difference in progesterone status was not significant, and lymph node involvement tended to be lower. BreastScreen WA patients were treated more often with local therapy and less often with systemic therapy, and the proportion of patients treated with breast-conserving surgery was close to the target for minimizing morbidity in breast cancer. CONCLUSION: Mammographic detection of breast cancer by BreastScreen WA is associated with reduced breast cancer morbidity and a more favourable prognosis.     

Expression of the pS2 gene in breast tissues assessed by pS2-mRNA analysis and pS2-protein radioimmunoassay

Summary The expression of the pS2 gene in breast tissues was assessed by measuring pS2-protein using a radioimmunoassay, and by determining pS2-mRNA using Northern blotting. There was a good correlation between the two measurements, indicating that expression of the pS2 gene in breast tissues may be assessed by either method. Since radioimmunoassay is technically easier and more efficient than Northern blotting, radioimmunoassay will be the method of choice in routine applications.     

It's Not Planned,But Is It Okay? The Acceptability of Unplanned Pregnancy Among Young People

Background

It is well-established that current measures of pregnancy intentions fail to capture the complexity of couples' lived experiences and decisions regarding reproductive decision making. Despite limitations, these measures guide programs, policy, and clinical practice. Herein, we explore prospective pregnancy acceptability, which captures whether individuals anticipate considering an unexpected pregnancy welcomed, manageable, or okay.