We report here on a first study using synchrotron radiation-based Fourier transform infrared microspectroscopy and imaging to investigate HT1080 human fibrosarcoma cells grown onto different-aged type I collagen networks. Spectral images were analyzed with k-means and fuzzy C-means (FCM) clustering algorithms. K-means delineated tumor cells from their surrounding collagen networks and the latter as a function of age mainly due to specific changes in the sugar absorption region. The FCM analysis gave a better nuance of the spectral images. A progression of the biochemical information was observed upon going from the cellular compartments to the pericellular contact regions and to the intact collagens of the different age groups. Two spectral markers based on sugar and protein bands via the intensity ratio (I1032/I1655) and band area ratio (Asugar/Aamide II), showed an increase in advanced glycation endproducts (AGEs) with age. A clear-separation of the three age groups was obtained for spectra originating from the peripheral contact areas mainly due to changes in protein band intensities. The above-described markers decreased to constant levels for the three conditions indicating a masking of the biochemical information. These results hold promises to better understand the impact of age on tumor progression processes while highlighting new markers of the tumor cell invasion front.
Collagen is the most important component in extracellular matrix (ECM) and plays a pivotal role in individual
tissue function in mammals. During ageing, collagen structure changes, which can detrimentally affect its biophysical
and biomechanical properties due to an accumulation of advanced glycation end-products (AGEs). AGEs have been
linked to non-enzymatic cross-linking of proteins resulting in the alteration of mechanical properties of the tissue. In this
study we investigate the influence of different aged collagens on the mechanical and contractile properties of
reconstituted hydrogel constructs seeded with corneal stromal fibroblasts. A non-destructive indentation technique and
optical coherence tomography (OCT) are used to determine the elastic modulus and dimensional changes respectively. It
is revealed that the youngest collagen constructs have a higher elastic modulus and increased contraction compared to the
older collagen. These results provide new insights into the relationship between collagen molecular structures and their
The lung is an organ heavily involved in cancer as both the origin of lung cancer and where metastases from tumours
originating in other organs develop. Therefore, it is obvious that the interaction between cancer cells and lung tissue
plays a role in cancer development, invasion, and/or growth. Out of all the components of the lung's extracellular matrix,
elastin fragmentation products, the so-called elastin peptides, have been associated with tumour growth and invasion.
Therefore, we studied using a 3D model whether elastin peptides could increase the proliferative activity of lung cancer
cells. To this purpose, we grew lung cancer cells in collagen type I 3D models and used Optical Coherence Tomography
to study lung cancer cells growth in the absence and presence of elastin peptides in real-time and at different time-points
for each specimen. Our work shows that the addition of elastin peptides to lung cancer cells increased not only the size
of the cancer cell clusters in 3D models but also the number of these clusters. This work demonstrates that, using OCT,
the effects of extracellular matrix components on cancer cells can be characterised in 3D models. The biomedical
applications of this methodology can be extended to other systems.
Compared to other forms of skin cancer, a malignant melanoma has a high risk of spreading to other parts of the
body. Melanoma invasion is a complex process involving changes in cell-extracellular matrix (ECM) interaction and
cell-cell interactions. To fully understand the factors which control the invasion process, a human skin model system
was reconstructed. HBL (a commercially available cell line) melanoma cells were seeded on a skin model with and
without the presence of keratinocytes and/or fibroblasts. After 14 days culture, the skin specimens were fixed,
parafin embedded and cut into 7 µm sections. The de-parafinised sections were investigated by synchrotron Fourier
transformed infrared (FTIR) microspectroscopy to study skin cell invasion behaviour. The advantage of using FTIR
is its ability to obtain the fingerprint information of the invading cells in terms of protein secondary structure in
comparison to non-invading cells and the concentration of the enzyme (matrix-metalloproteinase) which digests
protein matrix, near the invading cells. With aid of the spectral mapping images, it is possible to pinpoint the cells in
non-invasion and invasion area and analyse the respective spectra. It has been observed that the protein bands in
cells and matrix shifted between non-invasive and invasive cells in the reconstructed skin model. We hypothesise
that by careful analysis of the FTIR data and validation by other models, FTIR studies can reveal information on
which type of cells and proteins are involved in melanoma invasion. Thus, it is possible to trace the cell invasion
path by mapping the spectra along the interface of cell layer and matrix body by FTIR spectroscopy.
Cancer is a world-wide health problem associated with an increasing death rate. The mechanisms of how normal cells transform into cancer cells are not fully understood. Intensive investigations have been undertaken to identify genes whose unregulated expression are involved in this process. In this study, we have grown, on collagen gel, adherent mouse embryo fibroblasts (MEFs) knocked out for Cyl-1 (MEFCyl1-/-) which have been transfected with the human proto-oncogene cyclin D1 (CCND1) under the control of an inducible expression system. CCND1 expression can be regulated in the fibroblasts via the presence of an inducer, isopropyl β-D-Thiogalactopyranoside (IPTG). In the absence of IPTG, CCND1 expression is silenced. The migration ability of the resultant cells on the collagen gel has been monitored by complementary optical techniques: the conventional light microscopy; optical coherence tomography and Fourier Transform Infrared Microspcopic Spectroscopy (FTIR) using Synchrotron beam source. It is found that the cells expressing CCND1 exhibited cell invasion morphology and had different matrix compositions near the cell layer in comparison to the cells not expressing CCND1. The results from this study are consistent with published findings that expression of CCND1 has oncogenic potential and is involved in cell invasion in vitro. Application of complementary optical techniques proves to be an efficient way obtaining morphological and composition information of cell invasion.
Mesothelioma is a tumour, usually fatal, which arises from and invades the pleural membrane. Investigation of the tumour cell-membrane interaction will greatly increase our understanding of the invasion mechanisms, which have the potential to improve the management of the disease. In this study, a new imaging technique, optical coherence tomography (OCT), was used to monitor tumour cell invasion in artificial membranes composed of either collagen type I or Matrigel. In parallel, standard histological section analysis was performed to validate the accuracy of the monitoring by OCT. Cross-sectional images from OCT revealed that lung tumour cells invaded only when low cell seeding density (5 x 105) and low collagen concentration (1.5 mg/ml) were combined. The cells could be easily differentiated from the artificial membranes and appeared as either a brighter layer on the top of the membrane or brighter spots embedded within the darker membrane. These cell-membrane morphologies matched remarkably to the standard histological section images. Our results suggest that OCT has a great potential to become a useful tool for fast and robust assessment of cell invasion.