Evaluation of the Interplay Between the Complement Protein C1q and Hyaluronic Acid in Promoting Cell Adhesion

Summary

The complement component C1q is a pro-inflammatory molecule highly expressed in the tissue microenvironment that can interact with the extracellular matrix. Here, we describe a method to test how C1q bound to hyaluronic acid impacts cell adhesion.

Abstract

It has been increasingly demonstrated that the tumor microenvironment plays an active role in neoplasia growth and metastasis. Through different pathways, tumor cells can efficiently recruit stromal, immune and endothelial cells by secreting stimulatory factors, chemokines and cytokines. In turn, these cells can alter the signaling properties of the microenvironment by releasing growth-promoting signals, metabolites and extracellular matrix components to sustain high proliferation and metastatic competence. In this context, we identify that the complement component C1q, highly expressed locally by a range of human malignant tumors, upon interacting with the extracellular matrix hyaluronic acid, strongly affects the behavior of primary cells isolated from human tumor specimens. Here, we describe a method to test how C1q bound to hyaluronic acid (HA) impacts tumor cell adhesion, underlying the fact that the biological properties of key components of the extracellular matrix (in this case HA) can be shaped by bioactive signals toward tumor progression.

Introduction

The tumor microenvironment (TME) influences cancer development and progression since it can provide a permissive niche for cell survival, growth and invasion. The identification of new key players in TME may be useful for the discovery of new molecular tools for target therapy. TME includes a complex and dynamic network of non-malignant cells, such as endothelial cells, fibroblasts and cells of the immune system, embedded in the surrounding extracellular matrix (ECM) components including collagens, laminins, fibronectins, proteoglycans and hyaluronans. Both tumor and non-tumor cells synthesize and secrete ECM components together with cytokines, chemokines, growth fact....

Protocol

Tissue samples from patients were collected after informed consent following approval of the ethical considerations by the Institutional Board of the University Hospital of Trieste, Italy.

1. Tumor cell isolation and culture (Day 1)

1. Isolate human mesothelioma cells from MPM solid specimens. Finely mince the tissue with a cutter to obtain fragments of about 2-3 mm² in size and incubate in 5 mL of digestion solution composed of Hanks' Balanced Salt Solution (HBSS) supplemented with 0.5 mM Ca²⁺/Mg²⁺, 0.5% trypsin and 50 µg/mL DNase I, overnight at 4 °C.
2. Tumor cell isolation a....

Results

HA is a negatively charged high-molecular-weight polysaccharide, which is made up of repeating (β,1-4)-D-glucuronic acid-(β,1-3)-N-acetyl-D-glucosamine disaccharide units (Figure 1B)⁷. The occurrence of the binding of HA on the 96-well plate as well as the efficiency of its immobilization were tested taking advantage of biotinylated HA (bio-HA). Different concentrations of Bio-HA, ranging from 10 µg/mL to 1 mg/mL, were re-suspended in .......

Discussion

We describe an easy method to investigate how the complement component C1q, interacting with hyaluronic acid, is able to modulate the behavior of primary cells isolated from human tumor tissues. Both HA and C1q are abundantly present in the tissue microenvironment both under physiological and pathological conditions, participating to several cell biological processes. For instance, C1q has been shown to be present in the microenvironment of the placenta where it favors extravillous trophoblast invasion of the matern.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 µm pore filter	BD Falcon	352360
Amphotericin B solution (fungizone)	Sigma-Aldrich	1397-89-3
basic FGF	Immunological Sciences	GRF-15595
Calcium Chloride	Sigma-Aldrich	C-4901
Collagenase type I	Worthington Biochemical Corporation, DBA	MX1D12644
D-Glucose	Sigma-Aldrich	50-99-7
DNase I	Roche	10 104 159 001
EDTA	Sigma-Aldrich	60-00-4
EGF	Immunological Sciences	GRF-10544
FAST DiI	Molecular probes, Invitrogen, Thermo Fisher Scientific	D7756
Fetal bovine serum	Gibco,  Thermo Fisher Scientific	10270-106
Fibronectin	Roche	11051407001
Flask for cell culture	Corning	430639	Sterile, vented
Gentamicin solution	Sigma-Aldrich	G1397-10ML
Hank’s Balanced Salt Solution (HBBS)	Sigma-Aldrich	H6648	Supplemented with EDTA, Glucose, penicillin-streptamicin, gentamicin and fungizone
High molecular weight hyaluronic acid	Kind gift by Prof. Ivan Donati
Human endothelial serum free medium	Gibco,  Thermo Fisher Scientific	11111-044	Supplemented with EGF (5 ng/mL), basic FGF (10 ng/mL), and  1% penicillin–streptomycin (Sigma-Aldrich)
Magnesium Chloride	Carlo Erba	13446-18-9
Medium 199 with Hank’s salt	Sigma-Aldrich	M7653
Penicillin-Streptomycin	Sigma-Aldrich	P0781
Time-lapse microscopy	Nikon	Imaging System BioStation IM-Q
Titertek Multiskan ELISA Reader	Flow Labs
Trypsin	Sigma-Aldrich	T4674