Structural changes in mixed Col I/Col V collagen gels probed by SHG microscopy: implications for probing stromal alterations in human breast cancer

Abstract

Second Harmonic Generation (SHG) microscopy has been previously used to describe the morphology of collagen in the extracellular matrix (ECM) in different stages of invasion in breast cancer. Here this concept is extended by using SHG to provide quantitative discrimination of self-assembled collagen gels, consisting of mixtures of type I (Col I) and type V (Col V) isoforms which serve as models of changes in the ECM during invasion in vivo. To investigate if SHG is sensitive to changes due to Col V incorporation into Col I fibrils, gels were prepared with 0-20% Col V with the balance consisting of Col I. Using the metrics of SHG intensity, fiber length, emission directionality, and depth-dependent intensities, we found similar responses for gels comprised of 100% Col I, and 95% Col I/5% Col V, where these metrics were all significantly different from those of the 80% Col I/20% Col V gels. Specifically, the gels of lower Col V content produce brighter SHG, are characterized by longer fibers, and have a higher forward/backward emission ratio. These attributes are all consistent with more highly organized collagen fibrils/fibers and are in agreement with previous TEM characterization as well as predictions based on phase matching considerations. These results suggest that SHG can be developed to discriminate Col I/Col V composition in tissues to characterize and follow breast cancer invasion.

Optical clearing of biological tissues: prospects of application in medical diagnostics and phototherapy

“…At present SHG is one of the new high-resolution nonlinear-optical imaging methods for the study of intact tissues and cell structures [276][277][278][279][280][281][282][283][284]. Similar to multiphoton fluorimetry, the SHG method has a few important advantages compared to the methods of linear spectroscopy and imaging, since the exciting near infrared light is weakly scattered by the tissue, while the second harmonic generation occurs already inside the tissue, in the small focal region of the focused laser beam.…”

Second-harmonic generation scattering directionality predicts tumor cell motility in collagen gels

“…In addition to the simplified environmental cues, the alteration of known factors of fibrillogenesis allows for the controlled manipulation of collagen fiber microstructure, [12][13][14] allowing one to manipulate F/B and hence to explore the relationship between F/B ratio and tumor cell motility. In this work, we change the F/B ratio of collagen gels by three different methods: via alterations in pH, ionic strength [KCl], and collagen I/collagen III ratio.…”

Imaging of biological tissues with pixel-level analysis of second-order susceptibility

“…[38][39][40] A mixture of type I and V collagen emulating the state of human breast cancer has been quantifiably diagnosed with SHG intensity measurement, which deceases in parallel with an increase in the content of Col V; mixture with lower Col V is also characterized by longer fibers and a higher emission ratio of Forward SHG to Backward SHG FSHG/BSHG. 41 A similar investigation on breast cancer using SHG microscopy also clarified the relationship between collagen density and carcinogenesis. In that investigation, three tumor-associated collagen signatures were observed and defined based on density, shape, and orientation of collagen fibers, which may provide novel markers for locating and characterizing tumors.…”