P0092 Advancing precision medicine in IBD: Systematic evaluation of single-cell transcriptomics protocols for intestinal biopsies
Authors: Bernardes J P , Schulte-Schrepping J , Tran F , Lorant A , Soumyabrata G , Taubenheim J , Volk V , Yim A L , Wang X , Aden K , Ellinghaus D , Feuerhake F , Häsler R , Kaleta C , Röcken C , Satagopam V , Schultze J L , Vetrano S , Schreiber S , Rosenstiel P 
Published: January 2025

Abstract

Background
Intestinal biopsies from inflammatory bowel disease (IBD) patients are routinely collected for histopathological examination. These samples offer a unique opportunity to study cellular heterogeneity and molecular pathways, and assist in selecting relevant features for disease prognosis in precision medicine. Single-cell transcriptomic approaches can yield insights into cellular and molecular signatures linked to disease progression and treatment response (1, 2). Nonetheless, the absence of a systematical evaluation of tissue processing, dissociation, and the availability of multiple technological methods remains a significant limitation that requires testing to maximize data quality and insights across varied biopsy profiles.

Methods
This study systematically tested tissue dissociation and fixation protocols, as well as droplet-based and plate-based single-cell processing platforms to evaluate cell yield, viability, and cell-type granularity. Furthermore, biopsies were collected from both ulcerative colitis (UC) and Crohn’s disease (CD) patients, on the least- and most-inflamed areas of the intestinal mucosa. We included intestinal biopsies cryopreserved for various time spans to assess limitations in single-cell transcriptomics processing. Finally, we compare the probe-base to total RNA-seq methods to understand the depth of the resulting profiles and how comparable these two approaches are.

Results
We capture 120,952 high-quality transcription profiles from 32 biopsies of 13 IBD patients (9 UC and 4 CD patients). Independent of biopsy characteristics, the dissociation and processing that included a priori fixation method was more easily applied due to the multiple stop points throughout the protocol and the capability of multiplex biopsies in a run. The plate-based method could capture more cell-types, including epithelium cells and could be particularly beneficial for biopsies with low cellular integrity. In contrast, droplet-based methods enabled higher cell throughput, flexibility of multiplexing samples, and additional immune profile information (CITE-seq). Furthermore, the time of sample collection did not affect the overall cell numbers. Both total and probe-based approaches were easily integrated after subsetting for the common features.

Conclusion
These findings underscore the importance of methodological standardization, stability and comparability of protocols, which are key in disease-informed single-cell analysis. Emphasizing the critical need to refine processing techniques for precision medicine applications in de-centralized single-cell base studies under clinical trial settings.

References
1. Martin JC, Chang C, Boschetti G, et al. Single-Cell Analysis of Crohn's Disease Lesions Identifies a Pathogenic Cellular Module Associated with Resistance to Anti-TNF Therapy. Cell. 2019;178(6):1493-1508.e20. doi:10.1016/j.cell.2019.08.008
2. Smillie CS, Biton M, Ordovas-Montanes J, et al. Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis. Cell. 2019;178(3):714-730.e22. doi:10.1016/j.cell.2019.06.029