Clinical challenges of tissue preparation for spatial transcriptome

Abstract

Spatial transcriptomics is considered as an important part of spatiotemporal molecular images to bridge molecular information with clinical images. Of those potentials and opportunities, the excellent quality of human sample preparation and handling will ensure the precise and reliable information generated from clinical spatial transcriptome. The present study aims at defining potential factors that might influence the quality of spatial transcriptomics in lung cancer, para‐cancer, or normal tissues, pathological images of sections and the RNA integrity before spatial transcriptome sequencing. We categorised potential influencing factors from clinical aspects, including patient selection, pathological definition, surgical types, sample harvest, temporary preservation conditions and solutions, frozen approaches, transport and storage conditions and duration. We emphasis on the relationship between the combination of histological scores with RNA integrity number (RIN) and the unique molecular identifier (UMI), which is determines the quality of of spatial transcriptomics; however, we did not find significantly relevance between them. Our results showed that isolated times and dry conditions of sample are critical for the UMI and the quality of spatial transcriptomic samples. Thus, clinical procedures of sample preparation should be furthermore optimised and standardised as new standards of operation performance for clinical spatial transcriptome. Our data suggested that the temporary preservation time and condition of samples at operation room should be within 30 min and in ‘dry’ status. The direct cryo‐preservation within OCT media for human lung sample is recommended. Thus, we believe that clinical spatial transcriptome will be a decisive approach and bridge in the development of spatiotemporal molecular images and provide new insights for understanding molecular mechanisms of diseases at multi‐orientations.

Regulatory roles of external cholesterol in human airway epithelial mitochondrial function through STARD3 signalling

“…For example, spatial transcriptomics has been recently applied to identify cell–cell communication, define in situ expression of transcriptomic profiles and provide new insights of understanding molecular mechanisms in lung cancers, although there are still challenges to be overcome. 53 The molecular mechanisms by which those multi‐factors and multi‐cells interact need to be furthermore defined. It should be clarified whether the mitochondrial dysfunction is a center to regulate airway epithelial sensitivity in response to pathogens or pollutions, whether the re‐distribution of intracellular cholesterol among organelles contributes to the inflammation, and whether cholesterol or cholesterol‐driven metabolites directly deteriorate epithelial capacities of inflammatory regulations.…”

“…With rapid development of biotechnology, the interaction and intercommunication among those genes, proteins and cells will be furthermore clarified and understood. For example, spatial transcriptomics has been recently applied to identify cell–cell communication, define in situ expression of transcriptomic profiles and provide new insights of understanding molecular mechanisms in lung cancers, although there are still challenges to be overcome 53 . The molecular mechanisms by which those multi‐factors and multi‐cells interact need to be furthermore defined.…”

Spatially resolved transcriptomic profiling of degraded and challenging fresh frozen samples

“…Within these few days, the SOC contains many different cell-types, including osteoblasts, hematopoietic cells, mesenchymal stromal cells and endothelial cells, which are suddenly located within a few cell-diameters of the resting-zone chondrocytes, potentially influencing these cells 31 . To investigate potential effectors that derive from the newly forming SOC, we applied RRST to mouse growth plate specimens before SOC formation (postnatal day 4, P4) and immediately after SOC formation (postnatal day 11, P11) 10 .…”

“…The Visium protocol is optimized for fresh frozen (FF) tissue specimens and recommends a RIN (RNA Integrity Number) score higher than or equal to 7. RIN is a critical metric to assess the quality and level of RNA degradation before starting an SRT experiment 10,11 . FF samples are the preferred choice for unbiased polyA-based SRT technologies due their high preservation of polyadenylated transcripts.…”

Forward single‐cell sequencing into clinical application: Understanding of ageing and rejuvenation from clinical observation to single‐cell solution

“…One of Clinical and Translational Medicine's missions is to advance spatiotemporal molecular medicine by advancing single‐cell sequencing into clinical application, translating single‐cell measurements into routine practice, and identifying disease‐specific biomarkers and therapies 4–6 . Advanced technologies such as single‐cell RNA sequencing (scRNA‐seq) and spatial transcriptomics have broad applications in clinical tissue or fluid samples to understand intra‐ and intercellular communications and network interactions within the tissue or within the circulation 7–10 . The current Editorial extends our understanding of ageing and rejuvenation from clinical observation in clinical and translational medicine to molecular mechanisms of heterochronic parabiosis at single‐cell solution (Figure 1).…”