Collagen I but not Matrigel matrices provide an MMP-dependent barrier to ovarian cancer cell penetration

Abstract Background The invasive potential of cancer cells is usually assessed in vitro using Matrigel as a surrogate basement membrane. Yet cancer cell interaction with collagen I matrices is critical, particularly for the peritoneal metastatic route undertaken by several cancer types including ovarian. Matrix metalloprotease (MMP) activity is important to enable cells to overcome the barrier constraints imposed by basement membranes and stromal matrices in vivo. Our objective was to compare matrices reconstituted from collagen I and Matrigel as representative barriers for ovarian cancer cell invasion. Methods The requirement of MMP activity for ovarian cancer cell penetration of Matrigel and collagen matrices was assessed in 2D transwell and 3D spheroid culture systems. Results The broad range MMP inhibitor GM6001 completely prevented cell perforation of polymerised collagen I-coated transwell membranes. In contrast, GM6001 decreased ES-2 cell penetration of Matrigel by only ~30% and had no effect on HEY cell Matrigel penetration. In 3D culture, ovarian cancer cells grown as spheroids also migrated into surrounding Matrigel matrices despite MMP blockade. In contrast, MMP activity was required for invasion into 3D matrices of collagen I reconstituted from acid-soluble rat-tail collagen I, but not from pepsin-extracted collagen I (Vitrogen/Purecol), which lacks telopeptide regions. Conclusion Matrigel does not form representative barriers to ovarian cancer cells in either 2D or 3D culture systems. Our findings support the use of collagen I rather than Matrigel as a matrix barrier for invasion studies to better approximate critical interactions and events associated with peritoneal metastasis

Cell–cell and cell–matrix dynamics in intraperitoneal cancer metastasis

The peritoneal metastatic route of cancer dissemination is shared by cancers of the ovary and gastrointestinal tract. Once initiated, peritoneal metastasis typically proceeds rapidly in a feed-forward manner. Several factors contribute to this efficient progression. In peritoneal metastasis, cancer cells exfoliate into the peritoneal fluid and spread locally, transported by peritoneal fluid. Inflammatory cytokines released by tumor and immune cells compromise the protective, anti-adhesive mesothelial cell layer that lines the peritoneal cavity, exposing the underlying extracellular matrix to which cancer cells readily attach. The peritoneum is further rendered receptive to metastatic implantation and growth by myofibroblastic cell behaviors also stimulated by inflammatory cytokines. Individual cancer cells suspended in peritoneal fluid can aggregate to form multicellular spheroids. This cellular arrangement imparts resistance to anoikis, apoptosis, and chemotherapeutics. Emerging evidence indicates that compact spheroid formation is preferentially accomplished by cancer cells with high invasive capacity and contractile behaviors. This review focuses on the pathological alterations to the peritoneum and the properties of cancer cells that in combination drive peritoneal metastasis

Collagen I but not Matrigel matrices provide an MMP-dependent barrier to ovarian cancer cell penetration

Abstract Background The invasive potential of cancer cells is usually assessed in vitro using Matrigel as a surrogate basement membrane. Yet cancer cell interaction with collagen I matrices is critical, particularly for the peritoneal metastatic route undertaken by several cancer types including ovarian. Matrix metalloprotease (MMP) activity is important to enable cells to overcome the barrier constraints imposed by basement membranes and stromal matrices in vivo. Our objective was to compare matrices reconstituted from collagen I and Matrigel as representative barriers for ovarian cancer cell invasion. Methods The requirement of MMP activity for ovarian cancer cell penetration of Matrigel and collagen matrices was assessed in 2D transwell and 3D spheroid culture systems. Results The broad range MMP inhibitor GM6001 completely prevented cell perforation of polymerised collagen I-coated transwell membranes. In contrast, GM6001 decreased ES-2 cell penetration of Matrigel by only ~30% and had no effect on HEY cell Matrigel penetration. In 3D culture, ovarian cancer cells grown as spheroids also migrated into surrounding Matrigel matrices despite MMP blockade. In contrast, MMP activity was required for invasion into 3D matrices of collagen I reconstituted from acid-soluble rat-tail collagen I, but not from pepsin-extracted collagen I (Vitrogen/Purecol), which lacks telopeptide regions. Conclusion Matrigel does not form representative barriers to ovarian cancer cells in either 2D or 3D culture systems. Our findings support the use of collagen I rather than Matrigel as a matrix barrier for invasion studies to better approximate critical interactions and events associated with peritoneal metastasis.</p

A novel function for lysyl oxidase in pluripotent mesenchymal cell proliferation and relevance to inflammation-associated osteopenia.

Lysyl oxidase is a multifunctional enzyme required for collagen biosynthesis. Various growth factors regulate lysyl oxidase during osteoblast differentiation, subject to modulation by cytokines such as TNF-α in inflammatory osteopenic disorders including diabetic bone disease. Canonical Wnt signaling promotes osteoblast development. Here we investigated the effect of Wnt3a and TNF-α on lysyl oxidase expression in pluripotent C3H10T1/2 cells, bone marrow stromal cells, and committed osteoblasts. Lysyl oxidase was up-regulated by a transcriptional mechanism 3-fold in C3H10T1/2 cells, and 2.5-fold in bone marrow stromal cells. A putative functional TCF/LEF element was identified in the lysyl oxidase promoter. Interestingly, lysyl oxidase was not up-regulated in committed primary rat calvarial- or MC3T3-E1 osteoblasts. TNF-α down-regulated lysyl oxidase both in Wnt3a-treated and in non-treated C3H10T1/2 cells by a post-transcriptional mechanism mediated by miR203. Non-differentiated cells do not produce a collagen matrix; thus, a novel biological role for lysyl oxidase in pluripotent cells was investigated. Lysyl oxidase shRNAs effectively silenced lysyl oxidase expression, and suppressed the growth of C3H10T1/2 cells by 50%, and blocked osteoblast differentiation. We propose that interference with lysyl oxidase expression under excess inflammatory conditions such as those that occur in diabetes, osteoporosis, or rheumatoid arthritis can result in a diminished pool of pluripotent cells which ultimately contributes to osteopenia

Wnt3a up-regulates lysyl oxidase through the canonical Wnt signaling pathway.

<p>DKK1 and SOST are inhibitors of the canonical Wnt pathway. DKK1 or SOST were over-expressed in C3H10T1/2 cells. The empty vector (pcDNA3.1⁺) used as a control. Transfected cells were serum starved prior to treatment with Wnt3a- or control conditioned medium for 24 hours. Total RNA was extracted and subjected to real time PCR analysis to measure mRNA levels of lysyl oxidase, alkaline phosphatase (AKLP), lysyl oxidase-like 1 (LOXL1). These mRNA levels were normalized to HPRT1 mRNA levels. Data are presented as means ± SD (n = 3; *, p<0.05, N.S, not significant; Student's t-test) and are representative of two independent experiments with the same outcome.</p

LOX shRNA inhibits C3H10T1/2 cell proliferation and does not stimulate cell death.

<p>(<b>A</b>) DNA accumulation over a 24-hour period as an index of proliferation was determined in sub-confluent C3H10T1/2 cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100669#s2" target="_blank">Materials and Methods</a>. Data shown are means +/− SD; n = 4. (B) DNA fragmentation was assayed after isolation of genomic DNA and 2% agarose gel electrophoresis stained with ethidium bromide. 24 Micrograms of DNA was loaded per sample. Lane 1, scrambled shRNA; lane 2, LOX shRNA 1850; lane 3, LOX shRNA 1852, lane 4, non-transduced C3H10T1/2 cells. This experiment was performed 3 times with the same outcome. (C) Western blot of cell extracts for active caspase-3 shows no difference as a function of LOX knockdown, while the positive control of C3H10T1/2 cells subjected to 20 Gy radiation exhibited activated caspase 3 as expected.</p

Primers for Site-directed Mutagenesis of the Lysyl Oxidase Promoter.

<p>Primers for Site-directed Mutagenesis of the Lysyl Oxidase Promoter.</p

A potential cis-element for Wnt3a regulation of lysyl oxidase is located at −1321 to −1328 bp upstream of lysyl oxidase translation start site.

<p>Three putative TCF/LEF cis-elements within the first 1.5 kbp of the murine lysyl oxidase promoter (pLOXFL) were mutated by site-directed mutagenesis individually, in pairs, and all three together. C3H10T1/2 cells were transfected with a Renilla luciferase thymidine kinase (pRL-TK) and either wild type pLOXFFL or mutant pLOXFFL reporter constructs. After 24 hours, transfected cells were serum starved and treated with Wnt3a- or control-conditioned media for 24 hours. Luciferase activity was assessed as explained in Experimental Procedures. The fold change of lysyl oxidase transcriptional activity of wild-type and mutated reporter constructs in response to Wn3a are presented as means ± SD. Data are pooled from three independent experiments (n = 9; *, p<0.05; Student's t-test).</p

TNF-α post-transcriptionally attenuates Wnt3a-stimulated lysyl oxidase mRNA levels.

<p><b>A)</b> The effect of TNF-α on Wnt3a-stimulated lysyl oxidase mRNA levels in C3H10T1/2 cells was examined by treating cells with Wnt3a- or control-conditioned media in the presence or absence of increasing concentrations of TNF-α. Lysyl oxidase mRNA analysis of total RNA extracted from these cells was performed by real time PCR. Lysyl oxidase mRNA levels were normalized to the levels of GAPDH mRNA. Data are means ± SD (n = 3; *, p<0.05; Student's t-test). <b>B)</b> Luciferase reporter was used to functionally assess lysyl oxidase transcriptional activity in response to TNF-α. Data are means ± SD (n = 6, N.S, not significant; Student's t-test). Data shown were pooled from two independent experiments.</p

Lysyl oxidase (LOX) regulates C3H10T1/2 cell growth.

<p>C3H10T1/2 cells were transduced with lentiviral particles containing LOX shRNA1850, LOX shRNA 1852, or control shRNA. Non-transduced cells were eliminated using puromycin, a selective reagent. Transduced cells were seeded at 20,000 cells per well in 12-well plates for the growth curve analysis. Total RNA was also collected from these cells to measure lysyl oxidase mRNA levels by real-time PCR. <b>A)</b> The growth curves were plotted for cells transduced with either LOX shRNA 1852 or control shRNA. Data are presented as means ± SD (n = 4). <b>B)</b> The growth curves were plotted for cells transduced with either LOX shRNA 1850 or control shRNA. Data are presented as means ± SD (n = 4). <b>C)</b> The chart shows lysyl oxidase mRNA levels in LOX knockdown and control cells. Two independent LOX shRNA with different shRNA sequences were used in this experiment. Data are presented as means ± SD (n = 3*, p<0.05; Student's t-test).</p