October 19th, 2021

New Advances in Sampling Methods


Chairmen : Lance Liotta & Claudia Bevilacqua





Claudia Bevilacqua & Lance Liotta

Lance Liotta



Tissue spatially resolved molecular profiling for personalized oncology

15.45-16.15Daniel RosenbergAnalysis of immune and inflammatory signaling in colonic microadenomas
16.15-16.20 Break
16.20-16.50Amanda KedaigleCerebral Organoids: Creating a Solid Foundation to Study Human Disease
16.50-17.20Joe FoleyProfiling the tissue microenvironment with laser-capture microdissection and Smart-3SEQ
   A round-table discussion will conclude the webinar



Lance Liotta (George Mason University, USA): "Tissue spatially resolved molecular profiling for personalized oncology"

The future of molecular oncology is personalized therapies guided by molecular profiling of the patient’s tumor. Ten years ago oncology diagnostics faced the reality of tumor heterogeneity at the level of genomics, proteomics, and the tissue cellular microenvironment.  At the molecular and cellular level each patient’s tumor is unique. Addressing this challenge, over the past five years, there has been an explosion in specialized technology for spatially resolved molecular profiling of tumor tissue.

Recognition of the limitations of whole-tissue extraction profiling has driven a rapidly expanding field of investigators who are creating technology, software, and analytical tools to explore the tissue cellular spatial interactome. Consequently, a rich set of new research tools now exists to histologically localize proteomic and genomic markers, or to physically procure subpopulations of cells, or individual cells, within a formalin fixed or frozen section. Within this cadre of technologies Laser Capture Microdissection (LCM), CyTOF, NanoString, Geo-Seq, tomo-Seq, TIVA, smFISH, RNAscope, SeqFISH, VISIUM, LCseq, Scanning MALDI, and a host of others, has proven to be a reliable means of procuring tissue cellular populations, achieving high sensitivity and high precision for multiplex genomic and proteomic profiling. Post translational modifications such as protein phosphorylation provide key functional information that cannot be obtained by genomic and transcriptomic profiling. Now we are seeing the transition of tumor tissue spatial profiling from the research lab to clinical trial validation of tissue cellular genomic (mRNA, NGS), proteomic, and phosphoprotein signal pathway quantitation, as strategies to guide patient therapy.

Genomic profiling of whole tissue specimens has shown considerable clinical potential for the classification of patient responses to chemotherapy. Nevertheless extracting a whole tissue specimen is associated with a very high degree of preanalytical variability due to cellular heterogeneity, the unknown proportion of tumor or host subpopulations that all contribute to the extract, and the destruction of information about the spatial location of cell types within the tissue. For a variety of molecular targeted therapies, an RNA transcript related to the molecular drug target can be expressed in tumor cells as well as non-tumor host stroma and immune cells. The need for spatial molecular mapping is particularly important in tumor immunology, and tumor sub-clonal analysis, where the spatial proximity (and class) of tumor cell and immune cell populations provides critical information relevant to immunotherapy and drug resistance phenotypes. Thus, the ideal tissue spatial profiling technology should permit genomic, and proteomic functional analysis of the same cells or cellular subpopulations while retaining their spatial context. The major area of emphasis for this Special Session is the exciting rapidly emerging frontier of tissue single cell molecular analysis that combines proteomics with genomic, and transcriptomic analysis.

We  envisage near-term future workflows for the technology in which a diagnostic biopsy pathologic tissue section histologic image is digitally captured at high resolution, sent to the cloud, and then marked up remotely on a scientist or clinician’s computer screen. Once the specific histopathology regions are marked for interrogation on-screen (including groups of cells, single cells, immune cells, stromal cells etc.), these regions are automatically and remotely microdissected by LCM, followed by proteomic, genome sequencing and mRNA transcriptomic evaluation. The data from the molecular analysis of each selected region is then ported back through the cloud and can be viewed on-screen as a separate laboratory report for each individual histologic region that is highlighted, for integrated clinical and molecular data analysis.

Daniel Rosenberg (Uconn School of Medicine, USA): "Analysis of immune and inflammatory signaling in colonic microadenomas"

Stromal cells play an important role in promoting colorectal cancer progression. In this study, we have analyzed molecular changes associated with the epithelial and stromal compartments of lesions associated with early colonic neoplasia found in normal screening colonoscopy subjects. Our focus is on microadenomas (less than 5-mm) that are formed in the proximal colon, a region where rapidly developing ‘interval’ cancers often occur. In order to study these small lesions, we have combined laser-capture microdissection with high-sensitivity targeted gene expression profiling.

We report strong activation of a panel of neutrophil/monocyte chemokines, a finding that is consistent with ongoing localized inflammation. Our data also indicate reduced interferon signaling and cell-based immunity. The immune checkpoint and T cell exhaustion gene, PDCD1, was one of the most significantly up-regulated genes, a finding accompanied by decreased cytotoxic T cell effector gene expression. Additionally, CDKN2A expression was strongly up-regulated in the stroma and down-regulated in the epithelium, consistent with a set of alterations often found in senescence-associated signaling pathways. These findings have several important implications, including reduced CD8 T cell infiltration and increased T cell PD1 expression occurring within microadenomas found in the ascending colon. These changes occur within the context of a robust inflammatory response and potential stromal cell senescence, thus providing new insight into potential promotional drivers that may underlie rapid cancer development in the ascending colon.

Amanda Kedaigle (Broad Institute, USA): "Cerebral Organoids: Creating a Solid Foundation to Study Human Disease"

Neurodevelopmental disorders are difficult to study due to practical and ethical concerns in human studies, and the vast differences between developmental processes in human brain compared to common model organisms. Cerebral organoids are three-dimensional in vitro models of human brain organogenesis, which have been shown to produce a large diversity of cell types resembling those in the human developing cortex. However, realizing the full potential of these tools requires a deep understanding of the model: the cell types produced, their developmental trajectories, the reproducibility between batches and organoids, and any differences from endogenous cells. Here, using a large single-cell atlas of organoid development, we evaluate their reproducibility and fidelity over time. Finally, we demonstrate the utility of these highly reproducible organoids by evaluating the molecular and cellular effects of mutations in genes which have been associated with Autism Spectrum Disorder. The work provides a comprehensive, single-cell molecular map of human corticogenesis in organoids, identifying consistent molecular programs of cellular diversification, and elucidating novel functions for genes important to human neurodevelopment.

Joe Foley (Stanford  University School of Medicine, USA): "Profiling the tissue microenvironment with laser-capture microdissection and Smart-3SEQ"

In recent decades the biological sciences have applied a series of new techniques to quantify genome-wide gene activity as a molecular phenotype, and in recent years these phenotypes have been profiled at the level of individual cells within heterogeneous tissue. However, most techniques for single-cell genomics currently rely on homogenizing a large sample of cells from fresh tissue and retroactively categorizing them by molecular phenotype. Instead we have developed a complementary approach combining laser-capture microdissection (LCM) and Smart-3SEQ, a streamlined version of RNA-seq that is robust with small and degraded samples, which allows us to choose cells by their histological phenotype prior to molecular profiling. By identifying and profiling small regions within a complex tissue sample, we can compare the gene expression of distinct clusters of cells, such as different stages of tumor progression in clinical biopsies preserved as FFPE blocks, or even individual cells within a single heterogeneous lesion. The combination of LCM and Smart-3SEQ unlocks the vast archives of existing FFPE tissue samples and opens new directions in molecular histology and pathology.

A round table discussion will close the session.

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