Cholangiopathies represent a heterogeneous group of human liver diseases comprising several conditions affecting the biliary tree. Cholangiocarcinoma (CCA) includes a heterogeneous group of cancers affecting the biliary tree. The primary target of these diseases is represented by cholangiocytes, the epithelial cells lining the bile ducts. Despite progress in the understanding of cholangiopathies and CCA, a considerable translational gap remains between putative targets and effective patient therapies. This is in part due to limited definition of the functional heterogeneity and interactome of cell lineages that contribute to these diseases.
The GeoMx Digital Spatial Profiler (DSP) and NCounter® Sprint Profiler is an integrated workstation developed by Nanostring® and represents a benchtop multiplex system that combines the advantages of high-throughput molecular (both proteomic and transcriptomic) analysis with the spatial definition of histological samples, allowing to perform molecular analysis from formalin-fixed, paraffin-embedded samples and to obtain data into a morphological context.
The general aim of this project is to unveil the complete identity, function, and molecular profile of cells involved in cholangiopathies and CCA. This could be pursued thanks to the use of full potentiality of the GeoMX DSP platform. Since cholangiopathies and CCA are characterized by cross-interaction among parenchymal cells (i.e. hepatocytes, cholangiocytes, stem/progenitor cells), myofibroblast population and inflammatory cells, the morphological distinction at single cell level will ensure a precise cell recognition and the identification of eventual modifications in the molecular profile induced by disease state. The multi-omics analyses will generate large datasets that could be processed by artificial intelligence approaches (e.g. machine learning and network medicine), that could lead to identify novel pathogenetic, diagnostic, and therapeutic targets.
The GeoMx Digital Spatial Profiler (DSP) platform represents an advanced technology allowing high-throughput, multiplex proteomic/transcriptomic analyses with highly reproducible results. This innovative platform represents a unique instrumentation that allows spatially-restricted high-throughput analyses within the tissue, which is at the basis of unveiling the cellular cross-talks in pathologic and neoplastic cell niches as in the aim of the present project. The GeoMx Digital Spatial Profiler (DSP) platform would be the only instrument in Sapienza with such potentiality, thus representing a unique tool for the University.
RNA analysis is usually performed using RT-qPCR or next generation sequencing (NGS) approaches. RT-qPCR is limited by the low throughput compared to NGS which, however, has higher costs and more complex procedures. Remarkably, RT-qPCR and NGS approaches collect RNA from the entire specimen, thus lacking any information regarding the specific cell within the tissue or organ from which the RNAs come. Novel single-cell approaches can only be performed using specific equipment and can identify cell subtypes based on their transcriptomic profile but not by their location within the tissue/organ or by their morphology. Conventional proteomics approaches also imply the lack of spatial information. Uniquely, the GeoMx DSP technology allows to obtain spatially restricted analysis by segmenting the tissue/organ, even at single-cell resolution.
Laser capture microscopy is used for molecular analysis of small fragments dissected from tissue slides. However, this technique has several disadvantages compared to GeoMx DSP; it requires the physical removal (i.e. dissection) of the tissue from the slide, and it is subjected to the risk of RNA degradation; moreover, standard protocols are not optimized for immunostaining and ideal use exclude FFPE material, requiring the development of non-standard procedures for histologic sample handling to preserve the integrity of the target molecules. The advantages of GeoMx DSP reside in the possible use of standardized immunostaining protocols and the extraction of molecular information from the slides without physically interacting with the tissue. GeoMx DSP is specifically optimized to work on FFPE specimens, thus allowing the use of FFPE specimens already collected in histology archives.
Technically, both RT-qPCR and NGS require working in extraction environment, with the risk of RNA degradation and contamination. Uniquely, Nanostring technology largely simplifies the steps for transcriptomic analysis; RNA extraction and library synthesis are substituted by RNA hybridization with the oligo-tags, and amplification and sequencing steps are substituted by nCounter system analysis. Conventional proteomic analysis requires complicated methods to fractionate complex protein or peptide mixtures from samples and needs mass spectrometry technology. Differently, Nanostring technology requires a relatively simple sample preparation procedure, and allows to obtain information from different parts of the section or different cell types without altering tissue integrity, only requiring a single slide.
The GeoMx DSP platform by Nanostring is internationally considered as an innovative resourceful tool for the characterization of human pathologies, especially in cancer research. The proponents are already developing an international collaborative project on CCA characterization, involving several European referral centers. GeoMx DSP segmentation tools would allow to thoroughly characterize cellular and microenvironment interactions in tumour tissue slides. Moreover, investigators interested in cholangiocyte pathophysiology and working on experimental models of human diseases or clinicians/surgeons with a biopsy-based population ¿ already collaborating with the proponents - will be intrigued to further start new collaborative projects based on GeoMx DSP and the possibility to spatially-resolve -omics approaches. The possibility to perform high-throughput analysis on readily available samples as FFPE sections makes it the ideal tool to carry on collaborative studies on rare diseases or large case series. In fact, FFPE samples can be easily received from collaborators from foreign Institutions, as the transportation will not alter the quality of the samples. The possibility to combine the morphological expertise with a thorough molecular characterization of cell populations, also integrating this information with clinical data, represents a novel approach to further improve ongoing fields of research. Therefore, the presence of this innovative instrument at Sapienza will further strengthen already established international collaborations and will draw new interests in collaborative studies by national and international investigators, both clinically oriented and basic scientists.