Identification of Ras suppressor-1 (RSU-1) as a potential breast cancer metastasis biomarker using a three-dimensionalin vitroapproach

Abstract

Breast cancer (BC) is the most common malignant disease in women, with most patients dying from metastasis to distant organs, making discovery of novel metastasis biomarkers and therapeutic targets imperative. Extracellular matrix (ECM)-related adhesion proteins as well as tumor matrix stiffness are important determinants for metastasis. As traditional two-dimensional culture does not take into account ECM stiffness, we employed 3-dimensional collagen I gels of increasing concentration and stiffness to embed BC cells of different invasiveness (MCF-7, MDA-MB-231 and MDA-MB-231-LM2) or tumor spheroids. We tested the expression of cell-ECM adhesion proteins and found that Ras Suppressor-1 (RSU-1) is significantly upregulated in increased stiffness conditions. Interestingly, RSU-1 siRNA-mediated silencing inhibited Urokinase Plasminogen Activator, and metalloproteinase-13, whereas tumor spheroids formed from RSU-1-depleted cells lost their invasive capacity in all cell lines and stiffness conditions. Kaplan-Meier survival plot analysis corroborated our findings showing that high RSU-1 expression is associated with poor prognosis for distant metastasis-free and remission-free survival in BC patients. Taken together, our results indicate the important role of RSU-1 in BC metastasis and set the foundations for its validation as potential BC metastasis marker.

Cell Adhesion and Matrix Stiffness: Coordinating Cancer Cell Invasion and Metastasis

“…For the study of ECM stiffness, cancer cells usually grow in three dimensions (3D) within a collagen, hyaluronan, or similar gel that mimics the ECM and parameters that most often vary to modulate stiffness are either the gel’s concentration or the degree of collagen crosslinking for gels that contain collagen. Cancer cell spheroids are also employed for the study of cell invasion through the matrix ( 65 – 70 ). Increasing ECM stiffness has been shown to induce malignant phenotype ( 71 – 73 ) characterized by Rho-dependent cytoskeletal tension that leads to enhanced cell–ECM adhesions, disruption of cell–cell junctions and increased growth ( 69 ) (Figure 1 ) and is actually associated with activated FAK and extracellular signal-regulated kinase signaling ( 69 ).…”

Defining the Role of Solid Stress and Matrix Stiffness in Cancer Cell Proliferation and Metastasis

“…Furthermore, in a 3D model consisting of breast tumor spheroids growing in collagen matrix, the Ras suppressor-1 (RSU-1), a cell-ECM adhesion protein, was shown to be upregulated as a response to increasing stiffness. Interestingly, tumor spheroids knockdown for RSU-1 or actin polymerization regulator (VASP) lost their invasiveness through the 3D matrix ( 46 , 47 ). Matrix stiffening is also shown to induce fibroblast activation and migration, which leads to a fibrotic response setting a positive feedback to matrix stiffness ( 13 , 15 , 35 , 48 , 49 ).…”

Collagen content and extracellular matrix cause cytoskeletal remodelling in pancreatic fibroblasts

“…In order to form 3D collagen I gels of desired concentration and consequently of different stiffness we used a modification of previously published protocols [21,22] [23] software. All images were acquired at a fixed resolution (512 Â 512 data points) with scan rate between 0.5 and 1 Hz.…”

“…For the formation of FB and CAF spheroids the 'hanging drop' technique was used as described previously [21,22]. Briefly, after a 2 day incubation period in Petri dishes with pure medium or medium containing TGF-b (5 mg ml 21 ), FBs and CAFs were collected by trypsinization and put in suspension at a concentration of 2.5 Â 10 4 cells ml 21 . Hanging drops containing 500 cells each were formed on the inside of the cover of a culture dish.…”

“…As tumour stiffening is an important consequence of desmoplasia, we investigated the effect of matrix stiffness on basic cellular characteristics of pancreatic FBs and CAFs, such as cell stiffness, a-SMA expression (the most common FB activation marker), cell spreading and stress fibre formation and orientation. Our approach involved the preparation of collagen substrates of different concentrations which evidently exhibit different stiffness properties [21,28].…”