Carolyn Pehlke

Aligned Collagen Is a Prognostic Signature for Survival in Human Breast Carcinoma

Evidence for the potent influence of stromal organization and function on invasion and metastasis of breast tumors is ever growing. We have performed a rigorous examination of the relationship of a tumor-associated collagen signature-3 (TACS-3) to the long-term survival rate of human patients. TACS-3 is characterized by bundles of straightened and aligned collagen fibers that are oriented perpendicular to the tumor boundary. An evaluation of TACS-3 was performed in biopsied tissue sections from 196 patients by second harmonic generation imaging of the backscattered signal generated by collagen. Univariate analysis of a Cox proportional hazard model demonstrated that the presence of TACS-3 was associated with poor disease-specific and disease-free survival, resulting in hazard ratios between 3.0 and 3.9. Furthermore, TACS-3 was confirmed to be an independent prognostic indicator regardless of tumor grade and size, estrogen or progesterone receptor status, human epidermal growth factor receptor-2 status, node status, and tumor subtype. Interestingly, TACS-3 was positively correlated to expression of stromal syndecan-1, a receptor for several extracellular matrix proteins including collagens. Because of the strong statistical evidence for poor survival in patients with TACS, and because the assessment can be performed in routine histopathological samples imaged via second harmonic generation or using picrosirius, we propose that quantifying collagen alignment is a viable, novel paradigm for the prediction of human breast cancer survival.

3D Collagen Alignment Limits Protrusions to Enhance Breast Cancer Cell Persistence

Patients with mammographically dense breast tissue have a greatly increased risk of developing breast cancer. Dense breast tissue contains more stromal collagen, which contributes to increased matrix stiffness and alters normal cellular responses. Stromal collagen within and surrounding mammary tumors is frequently aligned and reoriented perpendicular to the tumor boundary. We have shown that aligned collagen predicts poor outcome in breast cancer patients, and postulate this is because it facilitates invasion by providing tracks on which cells migrate out of the tumor. However, the mechanisms by which alignment may promote migration are not understood. Here, we investigated the contribution of matrix stiffness and alignment to cell migration speed and persistence. Mechanical measurements of the stiffness of collagen matrices with varying density and alignment were compared with the results of a 3D microchannel alignment assay to quantify cell migration. We further interpreted the experimental results using a computational model of cell migration. We find that collagen alignment confers an increase in stiffness, but does not increase the speed of migrating cells. Instead, alignment enhances the efficiency of migration by increasing directional persistence and restricting protrusions along aligned fibers, resulting in a greater distance traveled. These results suggest that matrix topography, rather than stiffness, is the dominant feature by which an aligned matrix can enhance invasion through 3D collagen matrices.

Control of 3-dimensional collagen matrix polymerization for reproducible Human Mammary Fibroblast cell culture in microfluidic devices

Interest in constructing a reliable 3-dimensional (3D) collagen culture platform in microfabricated systems is increasing as researchers strive to investigate reciprocal interaction between extracellular matrix (ECM) and cells under various conditions. However, in comparison to conventional 2-dimensional (2D) cell culture research, relatively little work has been reported about the polymerization of collagen type I matrix in microsystems. We, thus, present a study of 3D collagen polymerization to achieve reproducible 3D cell culture in microfluidic devices. Array-based microchannels are employed to efficiently examine various polymerization conditions, providing more replicates with less sample volume than conventional means. Collagen fibers assembled in microchannels were almost two-times thinner than those in conventional gels prepared under similar conditions, and the fiber thickness difference influenced viability and morphology of embedded human mammary fibroblast (HMF) cells. HMF cells contained more actin stress fibers and showed increased viability in 3D collagen matrix composed of thicker collagen fibers. Relatively low pH of the collagen solution within a physiological pH range (6.5-8.5) and pre-incubation at low temperature (approximately 4 degrees C) before polymerization at 37 degrees C allow sufficient time for molecular assembly, generating thicker collagen fibers and enhancing HMF cell viability. The results provide the basis for improved process control and reproducibility of 3D collagen matrix culture in microchannels, allowing predictable modifications to provide optimum conditions for specific cell types. In addition, the presented method lays the foundation for high throughput 3D cellular screening.

Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer

Abstract. Second-harmonic generation (SHG) imaging can help reveal interactions between collagen fibers and cancer cells. Quantitative analysis of SHG images of collagen fibers is challenged by the heterogeneity of collagen structures and low signal-to-noise ratio often found while imaging collagen in tissue. The role of collagen in breast cancer progression can be assessed post acquisition via enhanced computation. To facilitate this, we have implemented and evaluated four algorithms for extracting fiber information, such as number, length, and curvature, from a variety of SHG images of collagen in breast tissue. The image-processing algorithms included a Gaussian filter, SPIRAL-TV filter, Tubeness filter, and curvelet-denoising filter. Fibers are then extracted using an automated tracking algorithm called fiber extraction (FIRE). We evaluated the algorithm performance by comparing length, angle and position of the automatically extracted fibers with those of manually extracted fibers in twenty-five SHG images of breast cancer. We found that the curvelet-denoising filter followed by FIRE, a process we call CT-FIRE, outperforms the other algorithms under investigation. CT-FIRE was then successfully applied to track collagen fiber shape changes over time in an in vivo mouse model for breast cancer.

Understanding the impact of 2D and 3D fibroblast cultures on in vitro breast cancer models.

The utilization of 3D, physiologically relevant in vitro cancer models to investigate complex interactions between tumor and stroma has been increasing. Prior work has generally focused on the cancer cells and, the role of fibroblast culture conditions on tumor-stromal cell interactions is still largely unknown. Here, we focus on the stroma by comparing functional behaviors of human mammary fibroblasts (HMFs) cultured in 2D and 3D and their effects on the invasive progression of breast cancer cells (MCF10DCIS.com). We identified increased levels of several paracrine factors from HMFs cultured in 3D conditions that drive the invasive transition. Using a microscale co-culture model with improved compartmentalization and sensitivity, we demonstrated that HMFs cultured in 3D intensify the promotion of the invasive progression through the HGF/c-Met interaction. This study highlights the importance of the 3D stromal microenvironment in the development of multiple cell type in vitro cancer models.

Microtubules regulate GEF-H1 in response to extracellular matrix stiffness

Rho GTPase plays a role in mechanosensing, and breast epithelial cells sense the stiffness of the extracellular matrix through Rho-mediated contractility. Microtubule stability is reduced by a stiff matrix, which leads to the activation of the Rho exchange factor GEF-H1.

Automation of Three-Dimensional Cell Culture in Arrayed Microfluidic Devices

The increasing interest in studying the interactions between cells and the extracellular matrix (ECM) has created a need for high throughput low-cost three-dimensional (3D) culture systems. The recent development of tubeless microfluidics via passive pumping provides a high throughput microchannel culture platform compatible with existing high throughput infrastructures (e.g., automated liquid handlers). Here, we build on a previously reported high throughput two-dimensional system to create a robust automated system for 3D culture. Operational controls including temperature and sample handling have been characterized and automated. Human mammary fibroblasts (HMFs) suspended in type I collagen are loaded and cultured in microchannel arrays and used to optimize the system operational parameters. A Peltier cooler maintains the collagen as a liquid at 4 °C during cell seeding, followed by polymerization at 37 °C. Optimization of this platform is discussed (e.g., controlling collagen contraction, increasing cell viability, preventing the removal of microchannel contents), and 3D distribution of HMFs is examined by fluorescent microscopy. Finally, we validate the platform by automating a previously developed 3D breast carcinoma coculture assay. The platform allows more efficient 3D culture experiments and lays the foundation for high throughput studies of cell—ECM interactions.

A nondenatured, noncrosslinked collagen matrix to deliver stem cells to the heart

AIMS Stem cell transplantation holds promise as a therapeutic approach for the repair of damaged myocardial tissue. One challenge of this approach is efficient delivery and long-term retention of the stem cells. Although several synthetic and natural biomaterials have been developed for this purpose, the ideal formulation has yet to be identified. MATERIALS & METHODS Here we investigate the utility of a nondenatured, noncrosslinked, commercially available natural biomaterial (TissueMend(®) [TEI Biosciences, Boston, MA, USA]) for delivery of human mesenchymal stem cells (MSCs) to the murine heart. RESULTS We found that MSCs attached, proliferated and migrated within and out of the TissueMend matrix in vitro. Human MSCs delivered to damaged murine myocardium via the matrix (2.3 × 10(4) ± 0.8 × 10(4) CD73(+) cells/matrix) were maintained in vivo for 3 weeks and underwent at least three population doublings during that period (21.9 × 10(4) ± 14.4 × 10(4) CD73(+) cells/matrix). In addition, collagen within the TissueMend matrix could be remodeled by MSCs in vivo, resulting in a significant decrease in the coefficient of alignment of fibers (0.12 ± 0.12) compared with the matrix alone (0.28 ± 0.07), and the MSCs were capable of migrating out of the matrix and into the host tissue. CONCLUSION Thus, TissueMend matrix offers a commercially available, biocompatible and malleable vehicle for the delivery and retention of stem cells to the heart.

Abstract B17: A microscale 3-D in vitro model of the breast cancer progression from DCIS to IDC: Deciphering the role of the stromal fibroblasts

Breast cancer progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is a critical step in breast cancer. This invasive transition of DCIS is defined by stromal invasion and is a life-threatening step accompanied by a dramatic drop in prognosis. In addition, the invasive transition of DCIS is largely driven by stromal alteration; thus it is critical to understand how DCIS alters the surrounding microenvironment, thus causing the cancer cells to become invasive. This study investigates the role of stromal fibroblasts in the vicinity of DCIS during the invasive transition using an innovative multidisciplinary approach that combines 3D cancer biology, microfluidics, and high-resolution imaging. We recently developed an efficient 3D microfluidic system that supports the transition from DCIS to IDC. The in vitro system employs microchannels with two inputs and one output enabling MCF10-DCIS.com cells (MCF-DCIS) and human mammary fibroblasts (HMF) to be loaded in two adjacent (side-by-side) compartments. This platform allows investigations of effects of spatial organization on the transition by independently analyzing their morphology and the modifications to the surrounding collagen architecture. Importantly, the compartmentalized platform enables monitoring of both MCF-DCIS and HMF independently including quantitative measures of the collagen architecture associated with each cell type. We observed that the HMF near MCF-DCIS became more protrusive versus HMF relatively far from MCF-DCIS. We have also begun to identify how the HMF become activated and protrusive when co-cultured with MCF-DCIS and to understand the biological function and impact of protrusive HMF during DCIS progression to IDC. We verified that the signaling based on Cathepsin D produced from MCF-DCIS and low-density lipoprotein receptor-related protein-1 (LRP1) from HMF is involved in regulation of the protrusive activity of HMF. Additionally, knocking down LRP1 in HMF inhibited the invasive transition of MCF-DCIS. This study demonstrates one possible route through which MCF-DCIS activate pre-existing fibroblasts and subsequently, leads to the modification of the ECM and the progression to IDC. Citation Format: Kyung Eun Sung, Carolyn Pehlke, Erwin Berthier, Kevin W. Eliceiri, Andreas Friedl, David J. Beebe. A microscale 3-D in vitro model of the breast cancer progression from DCIS to IDC: Deciphering the role of the stromal fibroblasts. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B17.

Abstract A35: Aligned collagen is a prognostic signature for survival in human breast carcinoma

Evidence for the potent influence of stromal organization and function on invasion and metastasis of breast tumors is ever growing. Here we have performed a rigorous examination of the relationship of a tumor-associated collagen signature (TACS-3), to the long term survival rate of human patients diagnosed with invasive breast cancer. TACS-3 is characterized by bundles of straightened and aligned collagen fibers that are oriented perpendicular to the tumor boundary. An evaluation of TACS-3 was performed in biopsied tissue sections from 196 patients by second harmonic generation (SHG) imaging of the backscattered signal generated by collagen. Univariate analysis of a Cox proportional hazard model demonstrated that the presence of TACS-3 was associated with poor disease-specific and disease free survival, resulting in hazard ratios between 3.0-3.9. Furthermore, TACS-3 was confirmed to be an independent prognostic indicator regardless of tumor grade and size, ER or PR status, HER-2 status, node status, and tumor subtype. Interestingly, TACS-3 was positively correlated to expression of stromal syndecan-1, a receptor for several extracellular matrix proteins including collagens. Ongoing research is currently investigating both the biochemical signaling pathways that give rise to the TACS-3 phenotype as well as the extent of the influence the mechanical properties of an aligned matrix have on invasion. Because of the strong statistical evidence for poor survival in patients with TACS, and since the assessment can be performed in routine histopathologic samples imaged via SHG or using picrosirius, we propose that quantifying collagen alignment is a viable, novel paradigm for the prediction of human breast cancer survival. Citation Format: Matthew W. Conklin, Jens Eickhoff, Kristin Riching, Carolyn Pehlke, Kevin Eliceiri, Paolo Provenzano, Andreas Friedl, Patricia J. Keely. Aligned collagen is a prognostic signature for survival in human breast carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A35.