Mario Gorenjak1, Larisa Goričan1, Boris Gole1, Uršula Prosenc*2, Erik Melén3, Michael Kabesch4, Anke H Maitland-van der Zee5,6, Susanne Reinartz7, Susanne J H Vijverberg5,6, Uroš Potočnik1,8,9 and the PERMEABLE consortium
1University of Maribor, Faculty of Medicine, Centre for Human Molecular Genetics and Pharmacogenomics, Slovenia
2BIA Separations CRO – Labena d.o.o., Teslova 30, 1000 Ljubljana, Slovenia
3Karolinska Institutet, Department of Clinical Sciences and Education Södersjukhuset, Stockholm, Sweden
4University Children’s Hospital Regensburg (KUNO), Department of Pediatric Pneumology and Allergy, Regensburg, Germany
5University of Amsterdam, Amsterdam University Medical Centers, Department of Respiratory Medicine, Amsterdam, The Netherlands
6University of Amsterdam, Amsterdam University Medical Centers, Department of Pediatric Pulmonology, Amsterdam, The Netherlands
7Tergooi Medical Center, Department of Otorhinolaryngology, Hilversum, The Netherlands
8University of Maribor, Faculty for Chemistry and Chemical Engineering, Laboratory for Biochemistry, Molecular Biology and Genomics, Maribor, Slovenia
9Maribor University Medical Centre, Department for Science and Research, Maribor, Slovenia
ursula.prosenc [at] biaseparationscro.com
Abstract
Biological therapies have revolutionized management of the severe cases of Chronic Immune Diseases refractory to the standard therapies. However, many patients do not respond to the selected biological therapy, loose response over time, or develop adverse effects. A personalized approach to treatment of these patients, based on reliable biomarkers is thus clearly needed.
Non-invasive approaches, such as use of the peripheral blood immune cells, are favored for novel biomarker discovery. However, the attention has shifted away from the bulk immune cells and towards specific immune cell sub-populations. Thus, the single-cell RNA sequencing (scRNA-seq) can prove highly valuable. By simultaneously capturing and profiling all the cells in a sample, scRNA-seq allows the analysis of cellular heterogeneity and gene expression in all immune cell sub-populations, targeted or adversely affected by the biological treatment.
In our ongoing research, scRNA-seq was utilized to analyze samples from Inflammatory Bowel Disease and Childhood Asthma patients with varied response to the biological therapy. Confounding effects of disease conditions and (biological) therapies on marker genes were eliminated using computational integration in order to identify conserved marker genes across all states. It turned out, that a reliable identification of the different immune cell sub-populations in this setting is quite challenging due to subjective cell-landscape clustering resolution. Several resolutions and automated annotation approaches were subsequently tested and validated.
A reference-based approach (Seurat-Azimuth) combined with manual cluster validation proved superior. Alas, manual cluster validation is time consuming. Annotation validation is important, especially to provide additional insights into unidentified clusters, which are essential for the identification of predictive biomarkers for personalized therapies in the vast heterogeneity of immune cell landscapes residing behind pathophysiology of chronic immune diseases.
Keywords: precision medicine, chronic immune diseases, biological therapy, Single-Cell RNA Sequencing, identification of cell sub-populations
Acknowledgments: This research was funded by the Slovenian Research Agency- Research core funding P3-0427; Research grant J3-9258; the Labena company with Grant challenge program and the PERMEABLE consortium.
The PERMEABLE consortium is supported by the ZonMW (project number: 456008004), the Swedish Research Council (proj.nr. 2018-05619), the Ministry of Education, Science and Sport of the Republic of Slovenia (proj.nr. C3330-19-252012), and the German Ministry of Education and Research (BMBF) (proj.nr. FKZ01KU1909A), under the frame of the ERA PerMed JTC 2018 Call.
