November 23, 2021
Chairmen : Pamela Pinzani & Mikael Kubista
Molecular characterization of single Circulating Tumor Cells: challenges and promise for translation to the clinic
|15.35-16.05||Lukas Valihrach||Spatiotemporal dynamics of ischemic brain injury resolved at single-cell level|
|16.05-16.25||Stefan Heckmann, Invited by Stilla||Single haploid nucleus genotyping in barley - measuring meiotic recombination rates in pollen nuclei|
|16.30-17.00||Vladimir Benes||Flow cytometry and single-cell transcriptomics: much more than a marriage of convenience|
|17.00-17.30||Nicola Luchi||Single-cell analysis as a promising technique for plant disease|
|A round-table discussion will conclude the webinar|
Introduction : Single-cell analysis is attracting considerable interest in various areas of scientific research, particularly in oncology, where it is used to dissect tumor heterogeneity both at the tissue level and at the level of circulating tumor cells. Despite the continuous technological development in this field, researchers often face problems related to the application of single-cell analysis in their different and specific fields. Many challenges still need to be overcome to achieve widespread use of these new and often sophisticated technologies, especially when it comes to the management of cancer patients. This webinar aimed to highlight the most critical issues and the most important results that can be achieved by this type of research.
Pamela Pinzani (UNIFI, Italy): "Molecular characterization of single Circulating Tumor Cells: challenges and promise for translation to the clinic"
Circulating Tumor Cells (CTCs) represent a “liquid biopsy of the tumor” which might allow real-time monitoring of cancer biology and therapies in individual patients. CTCs are extremely rare in the blood stream and their analysis is technically challenging.
Considerable progress has been made towards elucidating the basic biology of primary cancers, however, the molecular characterization of metastatic disease, which generally occurs months or years after primary tumor excision, remains limited. When patients with disseminated cancer undergo re-biopsy of metastatic disease, this generally happens for a single lesion even if multiple foci are present, with the majority of metastatic lesions not biopsied due to anatomic inaccessibility and/or associated morbidity of the procedure. Thus, the biological characterization of multiple metastatic sites is rarely performed, although recent data suggest that the selection of therapies according to the alterations of the metastatic lesions, rather than primary tumors, could be beneficial for the patient.
Alternatively, CTCs may offer a readily accessible means of evaluating the biology of metastatic cells, providing a non-invasive approach that could potentially identify drug sensitivity and resistance-associated markers, guiding therapeutic decisions.
While being a promising approach, CTC analysis, as a means of assessing the biological characteristics of metastatic disease, involves challenging technical aspects and requires further clinical validation.
During this presentation the feasibility of a protocol for the molecular characterization of single CTCs will be explored by reporting the results obtained in different type of cancers and highlighting the challenges to be overcome to obtain reliable results that can find applications in the clinic.
Lucas Valihrach (Czech Academy of Sciences, Czech Republic): "Spatiotemporal dynamics of ischemic brain injury resolved at single-cell level"
Ischemic brain injury (stroke) is second leading cause of death and primary cause of long-term disability. Pathophysiology of ischemic stroke is complex and involves interaction of large number of cell types in time- and space-dependent manner. A better understanding of the cellular and spatial heterogeneity is therefore crucial for understanding of stroke pathobiology and development of effective treatment strategies. In this webinar, we will present results of analysis of ischemic brain injury in mouse model using the state-of-art methods for single-cell and spatial transcriptomic analysis. The analysis provides unique insight on the processes and cell-types involved in pathogenesis of ischemia with spatial, temporal and single-cell resolution.
Stefan Heckmann (Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Germany. Invited by our GOLD sponsor Stilla): Single haploid nucleus genotyping in barley – measuring meiotic recombination rates in pollen nuclei
Meiotic recombination assures novel allelic combinations that are harnessed during breeding. Assessing meiotic recombination outcome in gametes prior fertilization enables measuring large sample numbers from single individuals without the need of growing large segregating populations. So far, methods to genotype or sequence a single barley pollen nucleus rely on whole-genome amplification (WGA) of individual single pollen nuclei, thus restricting the number of analyzable samples.
Using Crystal Digital PCRTM (naica, STILLA Technologies) we developed a single pollen nucleus genotyping assay to measure meiotic recombination rates within defined chromosomal intervals in high-throughput without WGA. Measured rates of meiotic recombination in hybrid pollen nuclei corresponded to that in segregating populations. Initial data acquired using the 6-color naicaTM system suggest that the throughput can be further increased by multiplexing our assays.
Nuclei from several crop plants with different nuclear sizes and amounts of DNA are compatible with the set-up, suggesting a broad application of Crystal Digital PCRTM-based single nuclei genotyping assays.
Vladimir Benes (European Molecular Biology Laboratory): "Flow cytometry and single-cell transcriptomics: much more than a marriage of convenience"
Diana Ordoñez, Beata Ramasz, Nayara de Azevedo, Laura Villacorta, Jonathan Landry, Vladimir Benes
Flow Cytometry Core Facility, Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany
A methodological possibility to analyse transcriptomes of individual cells, natural principal units of form and function in living organisms, has become an important drive enabling a dramatic increase of our knowledge on functional principles governing their existence. It also provides us with an unprecedented insight into the previously under-appreciated large spectrum of cell types, their states as well as their heterogeneity. However, this on its own represents one of the largest obstacles of the methodology itself as it is becoming increasingly apparent that different cells respond differently to implemented experimental approaches. Although the principal workflow is established, its steps will vary depending upon the specimen of interest and will not be always identical among experimental systems. Therefore – in order to obtain meaningful results - it is necessary to pay an utmost attention to the performance and efficiency of all individual steps of the complex workflow. Ultimately, collectively we should be more concerned by what we are not ‘seeing’ as it inherently reduces our understanding of biological systems we are studying.
Nicola Luchi (National Research Council, Italy): "Single-cell analysis as a promising technique for plant disease"
Over the last 20 years, biosecurity protocols for plant protection have been developed in order to prevent the introduction, diffusion and eradication of invasive plant pathogens. Ceratocystis platani is an invasive fungal pathogens threatening plane trees (Platanus), causing rapid death of the plant by blocking the vascular system. Recently, in the study of blood cancer disease new innovative approaches, based on single-cell have, been developed and they can be easily and successfully transferred to plant wilt pathogens.
In the present work the DEPArray was able to recovery single C. platani conidia or small group of conidia in single microtubes. After the whole genome amplification, the identification of C. platani DNA was performed by amplification and sequencing of ITS target region by using a specific real-time PCR markers. Sequences analysis confirms the presence of C. platani target qPCR amplicon detected in a single conidia.
The use of single-cell will provide new insight in plant pathology especially in the studies of vascular disease.
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