a-label-free-segmentation-approach-for-intravital-imaging-mammary

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

University of Wisconsin-Madison

Cellular damage triggers rapid resealing of the plasma membrane and repair of the cortical cytoskeleton. Plasma membrane resealing results from calcium-dependent fusion of membranous organelles and the plasma membrane at the site of the damage. Cortical cytoskeletal repair results from local assembly of actin filaments (F-actin), myosin-2 and microtubules into an array that closes around the original wound site. Control of the cytoskeletal response is exerted by local activation of the small GTPases, Rho and Cdc42. Recent work has given insight into both the membrane fusion and cytoskeletal responses to plasma membrane damage and we propose that Rho GTPase activation results at least in part from the events that drive membrane repair.

Rehabilitation and the single cell.

Actin filaments (F-actin) are protein polymers that undergo rapid assembly and disassembly and control an enormous variety of cellular processes ranging from force production to regulation of signal transduction. Consequently, imaging of F-actin has become an increasingly important goal for biologists seeking to understand how cells and tissues function. However, most of the available means for imaging F-actin in living cells suffer from one or more biological or experimental shortcomings. Here we describe fluorescent F-actin probes based on the calponin homology domain of utrophin (Utr-CH), which binds F-actin without stabilizing it in vitro. We show that these probes faithfully report the distribution of F-actin in living and fixed cells, distinguish between stable and dynamic F-actin, and have no obvious effects on processes that depend critically on the balance of actin assembly and disassembly.

Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.

Contractile arrays of actin filaments (F-actin) and myosin-2 power diverse biological processes. Contractile array formation is stimulated by the Rho GTPases Rho and Cdc42; after assembly, array movement is thought to result from contraction itself. Contractile array movement and GTPase activity were analyzed during cellular wound repair, in which arrays close in association with zones of Rho and Cdc42 activity. Remarkably, contraction suppression prevents translocation of F-actin and myosin-2 without preventing array or zone closure. Closure is driven by an underlying "signal treadmill" in which the GTPases are preferentially activated at the leading edges and preferentially lost from the trailing edges of their zones. Treadmill organization requires myosin-2-powered contraction and F-actin turnover. Thus, directional gradients in Rho GTPase turnover impart directional information to contractile arrays, and proper functioning of these gradients is dependent on both contraction and F-actin turnover.

A Rho GTPase signal treadmill backs a contractile array.

It is well established that collagen alignment in the breast tumor microenvironment provides biophysical cues to drive disease progression. Numerous mechanistic studies have demonstrated that tumor cell behavior is driven by the architecture and stiffness of the collagen matrix. However, the mechanical properties within a 3D collagen microenvironment, particularly at the scale of the cell, remain poorly defined. To investigate cell-scale mechanical cues with respect to local collagen architecture, we employed a combination of intravital imaging of the mammary tumor microenvironment and a 3D collagen gel system with both acellular pNIPAAm microspheres and MDA-MB-231 breast carcinoma cells. Within the in vivo tumor microenvironment, the displacement of collagen fiber was identified in response to tumor cells migrating through the stromal matrix. To further investigate cell-scale stiffness in aligned fiber architectures and the propagation of cell-induced fiber deformations, precise control of collagen architecture was coupled with innovative methodology to measure mechanical properties of the collagen fiber network. This method revealed up to a 35-fold difference in directional cell-scale stiffness resulting from contraction against aligned fibers. Furthermore, the local anisotropy of the matrix dramatically altered the rate at which contractility-induced fiber displacements decayed over distance. Together, our results reveal mechanical properties in aligned matrices that provide dramatically different cues to the cell in perpendicular directions. These findings are supported by the mechanosensing behavior of tumor cells and have important implications for cell-cell communication within the tissue microenvironment. STATEMENT OF SIGNIFICANCE: It is widely appreciated that the architecture of the extracellular matrix impacts cellular behavior in normal and disease states. Numerous studies have determined the fundamental role of collagen matrix architecture on cellular mechanosensing, but effectively quantifying anisotropic mechanical properties of the collagen matrix at the cell-scale remains challenging. Here, we developed innovative methodology to discover that collagen alignment results in a 35-fold difference in cell-scale stiffness and alters contractile force transmission through the fiber network. Furthermore, we identified bias in cell response along the axis of alignment, where local stiffness is highest. Overall, our results define cell-scale stiffness and fiber deformations due to collagen architecture that may instruct cell communication within a broad range of tissue microenvironments.

Directional cues in the tumor microenvironment due to cell contraction against aligned collagen fibers.

Cells sense mechanical signals within the extracellular matrix, the most familiar being stiffness, but matrix stiffness cannot be simply described by a single value. Randomness in matrix structure causes stiffness at the scale of a cell to vary by more than an order of magnitude. Additionally, the extracellular matrix contains ducts, blood vessels, and, in cancer or fibrosis, regions with abnormally high stiffness. These different features could alter the stiffness sensed by a cell, but it is unclear whether the change in stiffness is large enough to overcome the noise caused by heterogeneity due to the random fibrous structure. Here we used a combination of experiments and modeling to determine the extent to which matrix heterogeneity disrupts the potential for cell sensing of a locally stiff feature in the matrix. Results showed that, at the scale of a single cell, spatial heterogeneity in local stiffness was larger than the increase in stiffness due to a stiff feature. The heterogeneity was reduced only for large length scales compared to the fiber length. Experiments verified this conclusion, showing spheroids of cells, which were large compared to the average fiber length, spreading preferentially toward stiff inclusions. Hence, the propagation of mechanical cues through the matrix depends on length scale, with single cells being able to sense only the stiffness of the nearby fibers and multicellular structures, such as tumors, also sensing the stiffness of distant matrix features.

Effect of matrix heterogeneity on cell mechanosensing.

Previous therapeutic attempts to deplete cancer-associated fibroblasts (CAFs) or inhibit their proliferation in pancreatic ductal adenocarcinoma (PDAC) were not successful in mice or patients. Thus, CAFs may be tumor suppressive or heterogeneous, with distinct cancer-restraining and -promoting CAFs (rCAFs and pCAFs, respectively). Here, we showed that induced expression of the glycosylphosphatidylinositol-anchored protein Meflin, a rCAF-specific marker, in CAFs by genetic and pharmacological approaches improved the chemosensitivity of mouse PDAC. A chemical library screen identified Am80, a synthetic, nonnatural retinoid, as a reagent that effectively induced Meflin expression in CAFs. Am80 administration improved the sensitivity of PDAC to chemotherapeutics, accompanied by increases in tumor vessel area and intratumoral drug delivery. Mechanistically, Meflin was involved in the suppression of tissue stiffening by interacting with lysyl oxidase to inhibit its collagen crosslinking activity. These data suggested that modulation of CAF heterogeneity may represent a strategy for PDAC treatment.

Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to chemotherapeutics.

Correction: Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to chemotherapeutics.

Article	Year
Rehabilitation and the single cell. Current opinion in cell biology\| PubMed ID: 17174083	2007
Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin. Cell motility and the cytoskeleton\| PubMed ID: 17685442	2007
A Rho GTPase signal treadmill backs a contractile array. Developmental cell\| PubMed ID: 22819338	2012
Directional cues in the tumor microenvironment due to cell contraction against aligned collagen fibers. Acta biomaterialia\| PubMed ID: 33965625	2021
Effect of matrix heterogeneity on cell mechanosensing. Soft matter\| PubMed ID: 34125129	2021
Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to chemotherapeutics. Oncogene\| PubMed ID: 35414659	2022
Correction: Pharmacologic conversion of cancer-associated fibroblasts from a protumor phenotype to an antitumor phenotype improves the sensitivity of pancreatic cancer to chemotherapeutics. Oncogene\| PubMed ID: 35508544	2022

Brian M. Burkel

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