Brillouin and Raman scattering spectroscopy are established techniques for the nondestructive contactless and label-free readout of mechanical, chemical, and structural properties of condensed ...matter. Brillouin-Raman investigations currently require separate measurements and a site-matched approach to obtain complementary information from a sample. Here, we demonstrate a new concept of fully scanning multimodal microspectroscopy for simultaneous detection of Brillouin and Raman light scattering in an exceptionally wide spectral range, from fractions of GHz to hundreds of THz. It yields an unprecedented 150-dB contrast, which is especially important for the analysis of opaque or turbid media such as biomedical samples, and spatial resolution on a subcellular scale. We report the first applications of this new multimodal method to a range of systems, from a single cell to the fast reaction kinetics of a curing process, and the mechanochemical mapping of highly scattering biological samples.
Mid-infrared (MIR) imaging has emerged as a valuable tool to investigate biological samples, such as tissue histological sections and cell cultures, by providing non-destructive chemical specificity ...without recourse to labels. While feasibility studies have shown the capabilities of MIR imaging approaches to address key biological and clinical questions, these techniques are still far from being deployable by non-expert users. In this review, we discuss the current state of the art of MIR technologies and give an overview on technical innovations and developments with the potential to make MIR imaging systems more readily available to a larger community. The most promising developments over the last few years are discussed here. They include improvements in MIR light sources with the availability of quantum cascade lasers and supercontinuum IR sources as well as the recently developed upconversion scheme to improve the detection of MIR radiation. These technical advances can substantially speed up data acquisition of multispectral or hyperspectral datasets thus providing the end user with vast amounts of data when imaging whole tissue areas of many mm2. Therefore, effective data analysis is of tremendous importance, and progress in method development is discussed with respect to the specific biomedical context.
We describe the first application of confocal Brillouin and Raman microscopy to
ex vivo
human epithelial tissue - Barrett's oesophagus. This non-invasive label-free approach provides high-resolution ...mechanical mapping with chemical specificity, opening the route to a new integrated method for multiple biomedical and bioengineering applications, and potentially
in vivo
real-time diagnostics.
Confocal Brillouin and Raman microscopies provide non-contact label-free mechanochemical mapping of epithelial tissue, Barrett's oesophagus at high spatial resolution.
Bone has a sophisticated architecture characterized by a hierarchical organization, starting at the sub-micrometre level. Thus, the analysis of the mechanical and structural properties of bone at ...this scale is essential to understand the relationship between its physiology, physical properties and chemical composition. Here, we unveil the potential of Brillouin-Raman microspectroscopy (BRaMS), an emerging correlative optical approach that can simultaneously assess bone mechanics and chemistry with micrometric resolution. Correlative hyperspectral imaging, performed on a human diaphyseal ring, reveals a complex microarchitecture that is reflected in extremely rich and informative spectra. An innovative method for mechanical properties analysis is proposed, mapping the intermixing of soft and hard tissue areas and revealing the coexistence of regions involved in remodelling processes, nutrient transportation and structural support. The mineralized regions appear elastically inhomogeneous, resembling the pattern of the osteons' lamellae, while Raman and energy-dispersive X-ray images through scanning electron microscopy show an overall uniform distribution of the mineral content, suggesting that other structural factors are responsible for lamellar micromechanical heterogeneity. These results, besides giving an important insight into cortical bone tissue properties, highlight the potential of BRaMS to access the origin of anisotropic mechanical properties, which are almost ubiquitous in other biological tissues.
Small interfering RNAs (siRNAs) are emerging as promising therapeutic tools. However, the widespread clinical application of such molecules as modulators of gene expression is still dependent on ...several aspects that limit their bioavailability. One of the most promising strategies to overcome the barriers faced by gene silencing molecules involves the use of lipid-based nanoparticles (LNPs) and viral vectors, such as adenoviruses (Ads). The primary obstacle for translating gene silencing technology from an effective research tool into a feasible therapeutic strategy remains its efficient delivery to the targeted cell type in vivo. In this study, we tested the capability of LNPs and Ad to transduce and treat locally tumors in vivo. Efficient knockdown of a surrogate reporter (luciferase) and therapeutic target genes such as the kinesin spindle protein (KIF11) and polo-like kinase 1 were observed. Most importantly, this activity led to a cell cycle block as a consequence and slowed down tumor progression in tumor-bearing animals. Our data indicate that it is possible to achieve tumor transduction with si/short hairpin RNAs and further improve the delivery strategy that likely in the future will lead to the ideal non-viral particle for targeted cancer gene silencing.
Abstract Gastric cancers with mismatch repair (MMR) inactivation are characterised by microsatellite instability (MSI). In this study, the transcriptional profile of 38 gastric cancers with and ...without MSI was analysed. Unsupervised analysis showed that the immune and apoptotic gene networks efficiently discriminated these two cancer types. Hierarchical clustering analysis revealed numerous gene expression changes associated with the MSI phenotype. Amongst these, the p53-responsive genes maspin and 14-3-3 sigma were significantly more expressed in tumours with than without MSI. A tight immunosurveillance coupled with a functional p53 gene response is consistent with the better prognosis of MSI cancers. Frequent silencing of MLH1 and downregulation of MMR target genes, such as MRE11 and MBD4, characterised MSI tumours. The downregulation of SMUG1 was also a typical feature of these tumours. The DNA repair gene expression profile of gastric cancer with MSI is of relevance for therapy response.
Control of gene expression for gene therapy application requires the design of a sophisticated system embodying multiple properties. The ideal system should present the following features: (1) low or ...undetectable gene expression in the absence of inducer; (2) strong expression upon induction; and (3) fast kinetics of induction in the presence of inducers and rapid reversal of induction after its withdrawal. To evaluate these parameters, the features of the latest generation tetracycline-sensitive reverse-transactivator (rtTA2(s)-M2) alone or in combination with Tet-repressor (tTS-Kid) were explored in the context of helper-dependent adenovirus vector. Various genetic elements were assembled in a series of vectors and the ability to control secreted alkaline phosphatase expression evaluated in vitro in HeLa cells and in vivo by intramuscular injection in both C57/B6 and Balb/C nude mice. The results allow us to draw some general conclusions about the combination of transcription regulators and their relative orientation to the transgene to achieve maximal induction, while minimizing leakiness of expression.
Abstract
Background: Microcalcifications resulting from calcium deposition in the mammary gland play a central role in the early detection of breast cancer 1. However, the relationship between their ...occurrence in the breast and cancer progression remains poorly understood. Our approach is to use vibrational spectroscopy and imaging, which is non-invasive, non-destructive, label-free and chemically specific, to assess the composition and distribution of the deposits in an in vitro cancer cell model of mineralisation 2. In parallel we will utilise the same methods to measure the biochemical composition of microcalcifications found in breast biopsies from different grades of cancer. The ultimate aim of the study is to link the changes identified during the in vitro mineralisation process with the different stages of breast cancer. Vibrational spectroscopic methods can provide incredibly detailed biomolecular fingerprints enabling us to elucidate both the compositional changes with advancing pathology and the spatial distribution of those changes within the calcification and the surrounding tissue.
Methods: The breast cancer cell line MDA-MB-231 has the ability to produce mineralisation. This mineralisation was assessed over a 14-day period in the presence of different osteogenic cocktails: one composed of ascorbic acid, β-glycerophosphate (βG) and dexamethasone (Dex), and another one composed of inorganic phosphate (Pi). Fixed cells were analysed using Raman spectroscopy and micro-FTIR imaging at different time points (3, 7, 11 and 14 days). Tissue sections from patients with microcalcifications identified in histopathology will be sectioned to 3 mm and imaged with infrared (Agilent 670 FTIRinterferometer and Focal Plane Array imaging microscope) and Raman (Renishaw inVia) microspectrometers.
Results: We observed distinct and specific phosphate peak (PO43-) at 960 and 1020 cm-1 in Raman and FTIR spectra, respectively, corresponding to hydroxyapatite crystal and indicating the presence of microcalcification formation. Treatment with Pi induced a faster mineralisation (day 3) compared to cells treated with βG (day 11) and different spectral profiles during this development phase. In addition, there are changes in both the relative DNA and protein concentrations in the cells following 11 days exposure to the osteogenic agents.
It has been shown that the level of carbonate substitution in the calcium hydroxyapatite crystal correlates directly with the pathology of the tissue surrounding the microcalcification. Here we compare the mineral composition found ex vivo versus the in vitro model.
Conclusion: It could be possible to link the progressive biophysical changes associated with mineralisation to distinct stages of breast cancer pathology based on protein, lipid and carbonated apatite contents of the mineralised cells.
Support:This work was conducted as part of the Marie Curie Innovative Training Network Mid-TECH H2020-MSCA-ITN-2014-642661
References:
1 R. Baker, KD. Rogers, N. Shepherd and N. Stone. British Journal of Cancer. 103, 1034-1039 (2010)
2 RF. Cox, A. Hernandez-Santana, S. Ramdass, G. McMahon, JH. Harmey and MP. Morgan. British Journal of Cancer. 106, 525–537 (2012)
Citation Format: Bouzy P, O'Grady S, Palombo F, Morgan MP, Stone N. Exploring the relationship between an in vitro model of breast cancer cell mineralisation and the cancer grade specific composition of ex vivo microcalcifications abstract. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-01-03.
Cancer vaccines are a promising approach to treating tumors or preventing tumor relapse through induction of an immune response against tumor-associated antigens (TAA). One major obstacle to ...successful therapy is the immunological tolerance against self-antigens which limits an effective antitumor immune response. As a transient reduction of immunological tolerance may enable more effective vaccination against self-tumor antigens, we explored this hypothesis in a CEA tolerant animal model with an adenovirus expressing CEA vaccine in conjunction with inactivation of CD4(+)CD25(+) regulatory T cells. This vaccination modality resulted in increased CEA-specific CD8(+), CD4(+) T cells and antibody response. The appearance of a CD4(+) T-cell response correlated with a stronger memory response. The combined CD25(+) inactivation and genetic vaccination resulted in significant tumor protection in a metastatic tumor model. Non-invasive tumor visualization showed that not only primary tumors were reduced, but also hepatic metastases. Our results support the viability of this cancer vaccine strategy as an adjuvant treatment to prevent tumor relapse in cancer patients.
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