Luke Cassereau

The cancer glycocalyx mechanically primes integrin-mediated growth and survival

Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.

Rapid disorganization of mechanically interacting systems of mammary acini

Significance Tissue mechanics are important in differentiation and development but also in diseases like breast cancer. Most breast cancers start in mammary acini, which are basic anatomical units of the mammary gland. We found in a model system that mammary acini can coordinate their disorganization toward a malignant phenotype through long-range mechanical interactions. When two or more contractile acini are sufficiently close together, they can interact via collagen lines that form between them due to acinar contractility and the nonlinearity of collagen mechanics. Disorganization of interacting acini is more probable, rapid, and extensive than that of noninteracting acini. The results may help to better understand how extrinsic factors such as tissue architecture and mechanics contribute to tumor initiation and progression. Cells and multicellular structures can mechanically align and concentrate fibers in their ECM environment and can sense and respond to mechanical cues by differentiating, branching, or disorganizing. Here we show that mammary acini with compromised structural integrity can interconnect by forming long collagen lines. These collagen lines then coordinate and accelerate transition to an invasive phenotype. Interacting acini begin to disorganize within 12.5 ± 4.7 h in a spatially coordinated manner, whereas acini that do not interact mechanically with other acini disorganize more slowly (in 21.8 ± 4.1 h) and to a lesser extent (P < 0.0001). When the directed mechanical connections between acini were cut with a laser, the acini reverted to a slowly disorganizing phenotype. When acini were fully mechanically isolated from other acini and also from the bulk gel by box-cuts with a side length <900 μm, transition to an invasive phenotype was blocked in 20 of 20 experiments, regardless of waiting time. Thus, pairs or groups of mammary acini can interact mechanically over long distances through the collagen matrix, and these directed mechanical interactions facilitate transition to an invasive phenotype.

A 3D tension bioreactor platform to study the interplay between ECM stiffness and tumor phenotype.

Extracellular matrix (ECM) structure, composition, and stiffness have profound effects on tissue development and pathologies such as cardiovascular disease and cancer. Accordingly, a variety of synthetic hydrogel systems have been designed to study the impact of ECM composition, density, mechanics, and topography on cell and tissue phenotype. However, these synthetic systems fail to accurately recapitulate the biological properties and structure of the native tissue ECM. Natural three dimensional (3D) ECM hydrogels, such as collagen or hyaluronic acid, feature many of the chemical and physical properties of tissue, yet, these systems have limitations including the inability to independently control biophysical properties such as stiffness and pore size. Here, we present a 3D tension bioreactor system that permits precise mechanical tuning of collagen hydrogel stiffness, while maintaining consistent composition and pore size. We achieve this by mechanically loading collagen hydrogels covalently-conjugated to a polydimethylsiloxane (PDMS) membrane to induce hydrogel stiffening. We validated the biological application of this system with oncogenically transformed mammary epithelial cell organoids embedded in a 3D collagen I hydrogel, either uniformly stiffened or calibrated to create a gradient of ECM stiffening, to visually demonstrate the impact of ECM stiffening on transformation and tumor cell invasion. As such, this bioreactor presents the first tunable 3D natural hydrogel system that is capable of independently assessing the role of ECM stiffness on tissue phenotype.

Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix

Mammary tumor cells adopt a basal-like phenotype when invading through a dense, stiffened, 3D matrix. These cells exert higher integrin-mediated traction forces, consistent with a physical motor-clutch model, display an altered molecular organization at the nanoscale, and recruit a suite of paxillin-associated proteins implicated in metastasis.

α5β1-Integrin promotes tension-dependent mammary epithelial cell invasion by engaging the fibronectin synergy site

Fibronectin-ligated α5β1 integrin promotes malignancy by inducing tissue tension.

Morphogenesis: Laying down the tracks

To drive the formation of tubular structures, cells remodel their extracellular microenvironment to induce coordinated migration. It is now found that a mechanical feedback loop, involving the interaction of cell traction forces with collagen fibres, facilitates the formation of long epithelial tubules.

Abstract A098: A role for fibrosis in promoting pro-tumor immune response in breast cancer

We established a positive correlation between a fibrotic phenotype in human breast tumors — especially the Her2 and basal-like breast cancer subtypes — and CD45 and CD68 positive immune cell infiltration. We were interested in elucidating how this fibrotic phenotype may influence the immune response. To address this question, we examined if matrix stiffness alters the function of STAT3, a central regulator of tumor inflammation. We hypothesize that tissue fibrosis promotes STAT3 signaling in mammary tumor cells and alter the cytokine milieu to induce a pro-tumor immune response. We found that ECM stiffness directly enhanced STAT3 phosphorylation in tumor cells both in vitro and in vivo. Our data suggest the fibrotic phenotype promotes STAT3 activity, enhancement of which may drive a pro-tumor immune response. Indeed, we observed several alterations in cytokines and immune cell populations upon STAT3 ablation consistent with anti-tumor immune response. Interestingly, our data also suggest STAT3 knockout in tumor cells doesn9t necessary influence immune cell infiltration, but rather their differentiation in mammary tumors. Finally, we investigated if matrix stiffness has potentiated macrophage differentiation when cultured with specific immunosuppressive cytokines. Overall, our work reveals a novel mechanistic insight into how a pro-tumor immune response stems from the interplay between fibrosis and STAT3 signaling in tumor cells. As such, our findings may stimulate an interest in exploring combinational treatment options with anti-fibrotic agents and immunotherapy. Citation Format: Ori Maller, Luke Cassereau, Allison Drain, Brian Ruffell, Irene Acerbi, Miranda Broz, Jennifer Munson, Melody Swartz, Matthew Krummel, Lisa Coussens, Valerie Weaver. A role for fibrosis in promoting pro-tumor immune response in breast cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A098.

Abstract 5122: Characterization of the effects of hypoxia and hydrostatic pressure on the expression of immunotherapeutic targets on cell lines used for translational research

INTRODUCTION The characterization of the tumor microenvironment includes mechanotransduction, hypoxia, acidosis, and tissue remodeling. These factors can influence the gene and protein expression of various cell types that make up the tumor as well as influence the selection of cells that can thrive in a given microenvironment; however, typical cancer cell culture rarely uses hypoxia and pressure nor utilizes substrates similar to the native extracellular matrix (ECM). We designed a system to study the influence of hypoxia, pressure, and native ECM conditions on cancer cell lines and primary cells, with the goal of creating culturing environments relevant for translational studies involving key immunotherapeutic targets. METHODS First, we studied how hypoxia with or without pressure influences cell biology and gene expression, using transcriptome profiling across a range of physiologically-relevant culturing conditions to mimic various tumor microenvironments found within the body. To do this, we utilized Xcell9s primary cell culture platform that allows for the precise control of oxygen concentration (0.1%-20% O2) and hydrostatic pressure levels (26 to 260 mmHg / 0.5 psig to 5 psig). Additionally, we studied the influence of biomimetic substrates by ECM composition and organization (aligned or unaligned collagen at concentrations from 1-2.5 mg/ml +/- fibronectin 0.1-10 microgram/ml). Cell lines studied include models for brain (U-87, A172), pancreatic (PANC10.05), and prostate cancer (DU-145, PC-3, 22Rv1, LNCaP). We performed high-resolution immunofluorescence imaging and western blot protein expression analysis of key targets, including immunotherapeutic targets CTLA-4, PD-1, and PD-L1. Finally, we applied these culturing conditions to characterize primary tissues obtained from cancer patients for studies focused on biomarker discovery, patient monitoring and treatment decision-making. CONCLUSIONS We identified both common and unique gene expression signatures across different cells lines, with hypoxic conditions activating HIF1 signaling, whereas hydrostatic pressure resulted in restricted signatures of high clinical value. Cancer cell lines and PBMCs differentially express immunotherapeutic targets, at low oxygen and high pressure culturing conditions, resulting in reduced expression of key drug targets. Analysis of mRNA-seq data revealed alterations in gene expression profiles of immunotherapeutic and drug-target pathways involving CTLA-4 and AR signaling. In contrast, we observed increased CD47 and CD44 expression at low oxygen and high pressure culturing conditions in cancer cell lines and immune cells. Therefore, these results support the presence of physiologically-relevant drug targets in tumors and immune cells characterized by low oxygen and high interstitial fluid pressure. Citation Format: Bruce A. Adams, James Lim, Luke Cassereau, Tianna Chow, Susan Bernstein. Characterization of the effects of hypoxia and hydrostatic pressure on the expression of immunotherapeutic targets on cell lines used for translational research. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5122.