Brian Burkel

Anthocyanin Vacuolar Inclusions Form by a Microautophagy Mechanism

Densely packed vacuolar anthocyanin bodies form by aggregation of anthocyanins in the cytoplasm and subsequent direct engulfment by the vacuolar membrane. Anthocyanins are flavonoid pigments synthesized in the cytoplasm and stored inside vacuoles. Many plant species accumulate densely packed, 3- to 10-μm diameter anthocyanin deposits called anthocyanin vacuolar inclusions (AVIs). Despite their conspicuousness and importance in organ coloration, the origin and nature of AVIs have remained controversial for decades. We analyzed AVI formation in cotyledons of different Arabidopsis thaliana genotypes grown under anthocyanin inductive conditions and in purple petals of lisianthus (Eustoma grandiorum). We found that cytoplasmic anthocyanin aggregates in close contact with the vacuolar surface are directly engulfed by the vacuolar membrane in a process reminiscent of microautophagy. The engulfed anthocyanin aggregates are surrounded by a single membrane derived from the tonoplast and eventually become free in the vacuolar lumen like an autophagic body. Neither endosomal/prevacuolar trafficking nor the autophagy ATG5 protein is involved in the formation of AVIs. In Arabidopsis, formation of AVIs is promoted by both an increase in cyanidin 3-O-glucoside derivatives and by depletion of the glutathione S-transferase TT19. We hypothesize that this novel microautophagy mechanism also mediates the transport of other flavonoid aggregates into the vacuole.

Collagen Matrix Density Drives the Metabolic Shift in Breast Cancer Cells

Increased breast density attributed to collagen I deposition is associated with a 4–6 fold increased risk of developing breast cancer. Here, we assessed cellular metabolic reprogramming of mammary carcinoma cells in response to increased collagen matrix density using an in vitro 3D model. Our initial observations demonstrated changes in functional metabolism in both normal mammary epithelial cells and mammary carcinoma cells in response to changes in matrix density. Further, mammary carcinoma cells grown in high density collagen matrices displayed decreased oxygen consumption and glucose metabolism via the tricarboxylic acid (TCA) cycle compared to cells cultured in low density matrices. Despite decreased glucose entry into the TCA cycle, levels of glucose uptake, cell viability, and ROS were not different between high and low density matrices. Interestingly, under high density conditions the contribution of glutamine as a fuel source to drive the TCA cycle was significantly enhanced. These alterations in functional metabolism mirrored significant changes in the expression of metabolic genes involved in glycolysis, oxidative phosphorylation, and the serine synthesis pathway. This study highlights the broad importance of the collagen microenvironment to cellular expression profiles, and shows that changes in density of the collagen microenvironment can modulate metabolic shifts of cancer cells.

Radiation Promptly Alters Cancer Live Cell Metabolic Fluxes: An In Vitro Demonstration

Quantitative data is presented that shows significant changes in cellular metabolism in a head and neck cancer cell line 30 min after irradiation. A head and neck cancer cell line (UM-SCC-22B) and a comparable normal cell line, normal oral keratinocyte (NOK) were each separately exposed to 10 Gy and treated with a control drug for disrupting metabolism (potassium cyanide; KCN). The metabolic changes were measured live by fluorescence lifetime imaging of the intrinsically fluorescent intermediate metabolite nicotinamide adenosine dinucleotide (NADH) fluorescence; this method is sensitive to the ratio of bound to free NADH. The results indicated a prompt shift in metabolic signature in the cancer cell line, but not in the normal cell line. Control KCN treatment demonstrated expected metabolic fluxes due to mitochondrial disruption. The detected radiation shift in the cancer cells was blunted in the presence of both a radical scavenger and a HIF-1α inhibitor. The HIF-1α abundance as detected by immunohistochemical staining also increased substantially for these cancer cells, but not for the normal cells. This type of live-cell metabolic monitoring could be helpful for future real-time studies and in designing adaptive radiotherapy approaches.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis(1). Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration(2,3). The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Abstract B02: Matrix stiffness regulates local metabolism of breast carcinoma cells

Dense breast tissue is one of the single largest risk factors for the development of breast cancer, and one of the primary proteins responsible for increased breast density is the core extracellular matrix (ECM) component, collagen. Similar to other ECM proteins, collagen plays a structural role underlying tissue organization and increased collagen deposition correlates to a stiffer ECM and cellular microenvironment. Interestingly, changes to the stiffness of the ECM or microenvironment have profound and poorly-understood effects on cell migration, cell proliferation, and cancer progression. Consistent with an expanding role of ECM stiffness in cell signaling, we report that increased matrix stiffness also affects cellular metabolism and respiration. Using specific pharmacological inhibitors and quantitative imaging modalities like fluorescence lifetime microscopy, we are able to show that changes in collagen stiffness can cause a metabolic shift towards a more glycolytic, Warburg-like equilibrium in breast carcinoma cells. Matrix stiffness regulates the expression of several metabolic enzymes, including PDHK-1, which is poised to regulate this shift. Note: This abstract was not presented at the conference. Citation Format: Brian Burkel, Suzanne Ponik, Brett Morris, Kevin Eliceiri, Patricia Keely. Matrix stiffness regulates local metabolism of breast carcinoma cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B02. doi:10.1158/1538-7445.CHTME14-B02

Imaging Vacuolar Anthocyanins with Fluorescence Lifetime Microscopy (FLIM)

Anthocyanins are intrinsically fluorescent pigments that accumulate in plant vacuoles. We have developed a platform to analyze the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), under in vitro and in vivo conditions. Fluorescence lifetime of a fluorophore can be influenced by temperature, pH, oxygen concentration, and other environmental conditions. Within plant cells, the anthocyanin fluorescence lifetime correlates with distinct subcellular compartments. Vacuolar anthocyanins exhibit shorter fluorescence lifetime than the cytoplasmic pool. Consistent with these observations, lower pH of anthocyanins solutions correlated with shorter fluorescence lifetimes. We discuss here the use of FLIM as a tool for analyzing the subcellular distribution of anthocyanins and estimating variation in vacuolar pH in intact cells.

Mechanical Response of Fibrous Materials to Local Contractile Loads

When cells contract or migrate within a three-dimensional fibrous matrix, they pull on the fibers surrounding them, generating displacements in the fibrous network. As there is no clear constitutive relationship for fibrous materials, the connections between force, displacement, and the fibrous structure are unclear. This is especially true for nonuniform forces such as those applied by a contractile cell. This talk will describe experiments to simulate the contraction of a cell using particles made of poly(N-isopropylacrylamide), a hydrogel that undergoes a phase transition when heated, resulting in a dramatic decrease in volume. By embedding particles made of this gel into networks of fibrous collagen I and controlling the temperature, we can generate well-controlled, repeatable contractile forces within the network. The data show two properties that differ from homogeneous, linear materials. Firstly, displacements propagate over a longer range than predicted by classical linear theory. We provide evidence that the long-range propagation results from nonlinearity caused by weakening of the fiber network under compression. Secondly, the random fibrous structure induces heterogeneity in both the displacement field and the modulus at different positions within the same material. Experiments are ongoing to connect these findings to how cells sense and deform the surrounding fibrous matrix. Bio Jacob Notbohm is an Assistant Professor in the Department of Engineering Physics at the University of Wisconsin-Madison. After receiving his Ph.D. from the California Institute of Technology in Mechanical Engineering in 2013, he worked as a postdoctoral researcher at the Harvard Chan School of Public Health. Notbohm studies mechanical properties of biological materials and how physical interactions between cells and their surroundings control cell adhesion, contraction, and migration. In all cases, the focus of this research is on mechanics with an emphasis on experiments. Notbohm has received multiple awards, including a 3M Non-Tenured Faculty Award and an NSF CAREER Award

Microbuckling of Fibrous Matrices Enables Long Range Cell Mechanosensing

When biological cells migrate, divide, and invade, they push and pull on individual fibers of the matrix surrounding them. The resulting fiber displacements are neither uniform nor smooth; rather, displacements localize to form dense fibrous bands that span from one cell to another. It is thought that these bands may be a mechanism by which cells can sense their neighbors, but this hypothesis remains untested, because the mechanism for band formation remains unknown. Using digital volume correlation, we measure the displacements induced by contractile cells embedded in a fibrous matrix. We find that cell-induced displacements propagate over a longer range than predicted by linear elasticity. To explain the long-range propagation of displacements, we consider the effect of buckling of individual matrix fibers, which generates a nonlinear stress-strain relationship. We show that fiber buckling is the mechanism that causes the displacements to propagate over a long range and the bands to form between nearby cells. The results thus show that buckling of individual fibers provides a mechanism by which cells may sense their distant neighbors mechanically.

Quantitative image analysis for investigating cell-matrix interactions

The extracellular matrix provides both chemical and physical cues that control cellular processes such as migration, division, differentiation, and cancer progression. Cells can mechanically alter the matrix by applying forces that result in matrix displacements, which in turn may localize to form dense bands along which cells may migrate. To quantify the displacements, we use confocal microscopy and fluorescent labeling to acquire high-contrast images of the fibrous material. Using a technique for quantitative image analysis called digital volume correlation, we then compute the matrix displacements. Our experimental technology offers a means to quantify matrix mechanics and cell-matrix interactions. We are now using these experimental tools to modulate mechanical properties of the matrix to study cell contraction and migration.

SU-G-TeP3-10: Radiation Induces Prompt Live-Cell Metabolic Fluxes

PURPOSE To compare metabolic dynamics and HIF-1α expression following radiation between a cancerous cell line (UM-SCC-22B) and a normal, immortalized cell line, NOK (Normal Oral Keratinocyte). HIF-1 is a key factor in metabolism and radiosensitivity. A better understanding of how radiation affects the interplay of metabolism and HIF-1 might give a better understanding of the mechanisms responsible for radiosensitivity. METHODS Changes in cellular metabolism in response to radiation are tracked by fluorescence lifetime of NADH. Expression of HIF-1α was measured by immunofluorescence for both cell lines with and without irradiation. Radiation response is also monitored with additional treatment of a HIF-1α inhibitor (chrysin) as well as a radical scavenger (glutathione). Changes in oxygen consumption and respiratory capacity are also monitored using the Seahorse XF analyzer. RESULTS An increase in HIF-1α was found to be in response to radiation for the cancer cell line, but not the normal cell line. Radiation was found to shift metabolism toward glycolytic pathways in cancer cells as measured by oxygen consumption and respiratory capacity. Radiation response was found to be muted by addition of glutathione to cell media. HIF-1α inhibition similarly muted radiation response in cancer. CONCLUSION The HIF-1 protein complex is a key regulator cellular metabolism through the regulation of glycolysis and glucose transport enzymes. Moreover, HIF-1 has shown radio-protective effects in tumor vascular endothelia, and has been implicated in metastatic aggression. Monitoring interplay between metabolism and the HIF-1 protein complex can give a more fundamental understanding of radiotherapy response.