The Department has recently launched a number of top-down initiatives aimed at the set-up and dissemination of new technologies that are relevant for translational research.
Clinical Technoshot: A PET- Transcriptomic Interrogation of the Metabolic Status of Breast Cancers
Pier Paolo Di Fiore
The molecular heterogeneity of breast cancer (BC) translates into different disease courses and therapeutic outcomes and poses important challenges for patient management. Strategies to achieve patient stratification for prognostic judgment and therapy assignment have traditionally relied on the combined evaluation of clinical and pathological parameters, and more recently on molecular signatures. Together, these tools have led to the largely accepted molecular classification of BC into Luminal A, Luminal B, HER2+ and Basal-like/Triple-negative subtypes, which presently informs patient management1. Nevertheless, important unmet clinical needs remain in the management of BC patients, among which: The prediction of tumor recurrence and chemotherapy response, especially in Luminal BCs, which remain at persistent risk of recurrence for at least 15-20 years2. The scarcity of targetable alterations in Triple-negative BCs (TNBCs), which consequently lack effective targeted therapies. This technoshot aims at combining a PET-radiomics approach with transcriptomics analysis to exploit metabolic alterations in BC to meet the above clinical needs.
Clinical Technoshot: Bioenergetics Analysis Platform
Bioenergetics is the science that study the processes of energy conversion within cells. The alteration of bioenergetic phenotype is a hallmark of tumors including the most frequent breast, prostate, lung and colon cancers. In fact, there is the possibility that disturbing the particular energy processes of the cancer cell may represent a valid therapy. However, the dynamics and the regulation of bioenergetics processes during tumorigenesis remains largely unclear. Tools allowing fine analysis of mitochondrial energetics, carb and fatty acid oxidation are used in our department to associate genomic and epigenetic alteration with peculiar bioenergetics phenotypes of patient derived cancer cells. The identified reactions are then targeted by specific drugs or library of compounds to select for novel effective anticancer treatments. These researches are supported by the IEO Bioenergetics Facility equipped with the Agilent Seahorse XFe96 live cell metabolic analyzer the allows allows the measurement real time of mitochondrial, glycolytic and beta-oxidation fluxes, in living cultured cell monolayers and spheroids.
Clinical Technoshot: in situ Multiplex RNA-FISH
Mario Romolo Faretta
New Generation Sequencing technologies allowed to move the first steps towards a personalized medicine. However, they suffer an important limitation that resides in the inability to preserve single-cell resolution. The association of a genomic/transcription profile to distinct cell components in different histological locations provides an enormous amount of information. We thus decided to try to identify and quantify RNA molecules in situ starting from recently published methods.
Clinical Technoshot: in situ Proteomics
The cancer tissue is the best sample material for any molecular profiling analysis, yet tumors are characterized by high degree of heterogeneity, difficult to access by most analytical methods. Aim of this clinical technoshot it to optimize a platform based on Laser Micro Dissection coupled with shot-gun proteomics to carry out spatially-resolved measurements of protein levels and modifications.
Clinical Technoshot: in situ Tumor Cell Transcriptomes
Giuseppe Riccardo Diaferia
Analysis of gene expression profiles in cancer is informative of tumor properties and in some cases allows stratifying patients for diagnosis, prognostic assessment and therapeutic decisions. However, standard analyses of tumor transcriptomes average data obtained from tumor cells, which may be highly heterogeneous, and not-tumor cells, including infiltrating immune cells and stromal components. Single cell analyses provide detailed information on gene expression profiles of individual cells after dissociation of the tumor tissue, thus being not informative of the spatial relationship between tumor cells with different gene expression profiles and non-tumor components. For instance, the relationship between tumor cells with divergent gene expression profiles and vessels or other identifiable anatomical structures cannot be determined on the basis of the analysis. This technoshot aims at generating standardized procedures for digital gene expression analysis of defined tumor anatomical areas composed of a limited number of cells (10-100) obtained by laser capture microdissection. The activity of this unit is highly complementary to the Technoshot focusing on highly multiplexed in situ FISH.
Clinical Technoshot: Isolation of Micrometastases Alive Cells
At the end of the nineteenth century it has been observed that the tumor was initially a local disease that then expanded, gradually and in order, until it became systemic. Cancer research has shed light on much of the process of spreading (metastasis - far position) the tumor throughout the body. It is known that metastatic cancer cells often die or remain dormant for several years at a distant site before growing again and forming a secondary tumor. Furthermore, though metastatic process may involve any part of the body, different types of cancer are more likely to spread and effectively expand to certain sites than others. From these evidences, it is assumed that specific mechanisms of adaptation favor the survival and the growing of metastases in the distal site. However, in which way local tissue environment or anticancer treatments select the different metastatic phenotypes of and whether genetic abnormalities promote the phenotypic plasticity of metastasis is unclear.
Clinical Technoshot: Multiplexed Analysis of Soluble Factors in Cancer
As we are walking along the post-genome sequencing, there is an urgency for approaches to address functions and create network analysis as mean to discover new and personalized therapeutic targets. The rapid, sensitive, high-throughput and simultaneous detection of soluble factors by the Luminex® xMAP™ enable to interrogate the complexity of the tumor microenvironment in a bench-size assay.
Clinical Technoshot: Single-Cell Sequencing Program (SCS)
Starting in 2018, DEO established top-down technology development projects aimed at boosting the dissemination of the know-how required to face the newest technological challenges in cancer research. The programs are funded for 12 months and subject to evaluation with the possibility of being renewed by the DEO faculty. Program 1.0 In concomitance with the acquisition of the 10X Genomics microfluidic device and the increased power of the GU sequencing throughput (by Illumina NovaSeq 6000) that took place between 2017 and 2018, DEO decided to launch the first project in high-throughput Single Cell Transcriptomics with the goal of promoting the technological and computational know-how within the department. The program was opened to all PIs of DEO and was joined by eight different groups. It started in January 2018 and led to the generation of more than 800.000 single cell RNAseq profiles (around 220 runs) in different human and mouse model systems. A midterm (June 14th 2018) and a final (January 31st 2019) meeting were organized for the entire department with each participant group presenting and discussing their technical and computational achievements and bottlenecks. Program 2.0 Based on the success registered by this pilot program and given the strong interest that the department has shown in the development of technological approaches suitable for the study of cellular heterogeneity, the DEO has decided to continue the Single Cell programs into 2019 while also expanding its scope. The Program 2.0 aims at the implementation of new Single Cell approaches, such as 1) a Single Cell DNA Sequencing that aims at establishing the analysis of DNA copy number variations (CMV) at a single cell level; 2) a Single Cell Epigenomics with the goal to generate ATACseq analysis at Single Cell level and develop analytical pipelines that can integrate transcription and epigenomic data at single cell level; 3) Single Cell featured RNAseq, for gene expression characterization at single cell resolution coupled with the Feature Barcoding technology that could be used to identify cell-specific CRISPR-mediated perturbations or to simultaneously measure gene and cell surface protein expression in the same cell. These new programs will follow the same structure of the pilot Single Cell Transcriptomics program launched in 2018 with mid-term and final common meetings jointly taking place across the four different programs that will be active in 2019.
Clinical Technoshot: Single Molecule Program
Pier Giuseppe Pelicci
The aims of this program are: i) to favor dissemination of the single molecule technology among our scientists and ii) to further develop this technology for clinical applications. Technology Dissemination. We have previously set-up the single-molecule technology (nanopore sequencing using Oxford Nanopore Technologies), which is executed within our Genomics Unit. We have now launched eight projects, supported by the Department, each run by an individual PI, which aim at exploiting different usages of the single-molecule sequencing technologies (Sequencing of NGS-problematic regions; Structural variant characterization; Direct detection of epigenetic marks in native DNA or RNA) within specific scientific contexts. The department guarantees help in data analyses through collaborations with experts in the field. Technology development. We aim at developing a robust assay for calling somatic mutations within transcripts at single-cell level, by combining single cell and single molecule RNA and cDNA sequencing (10x Chromium and Oxford Nanopore Technologies).