scDrugPrio: a framework for the analysis of single-cell transcriptomics to address multiple problems in precision medicine in immune-mediated inflammatory diseases

Background Ineffective drug treatment is a major problem for many patients with immune-mediated inflammatory diseases (IMIDs). Important reasons are the lack of systematic solutions for drug prioritisation and repurposing based on characterisation of the complex and heterogeneous cellular and molecular changes in IMIDs. Methods Here, we propose a computational framework, scDrugPrio, which constructs network models of inflammatory disease based on single-cell RNA sequencing (scRNA-seq) data. scDrugPrio constructs detailed network models of inflammatory diseases that integrate information on cell type-specific expression changes, altered cellular crosstalk and pharmacological properties for the selection and ranking of thousands of drugs. Results scDrugPrio was developed using a mouse model of antigen-induced arthritis and validated by improved precision/recall for approved drugs, as well as extensive in vitro, in vivo, and in silico studies of drugs that were predicted, but not approved, for the studied diseases. Next, scDrugPrio was applied to multiple sclerosis, Crohn’s disease, and psoriatic arthritis, further supporting scDrugPrio through prioritisation of relevant and approved drugs. However, in contrast to the mouse model of arthritis, great interindividual cellular and gene expression differences were found in patients with the same diagnosis. Such differences could explain why some patients did or did not respond to treatment. This explanation was supported by the application of scDrugPrio to scRNA-seq data from eleven individual Crohn’s disease patients. The analysis showed great variations in drug predictions between patients, for example, assigning a high rank to anti-TNF treatment in a responder and a low rank in a nonresponder to that treatment. Conclusions We propose a computational framework, scDrugPrio, for drug prioritisation based on scRNA-seq of IMID disease. Application to individual patients indicates scDrugPrio’s potential for personalised network-based drug screening on cellulome-, genome-, and drugome-wide scales. For this purpose, we made scDrugPrio into an easy-to-use R package (https://github.com/SDTC-CPMed/scDrugPrio). Supplementary Information The online version contains supplementary material available at 10.1186/s13073-024-01314-7.


Background
Immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis, Crohn's disease, and psoriatic arthritis, affect millions of people worldwide and can cause chronic pain, disability, and reduced quality of life [1].While new classes of therapies are transforming the management of IMIDs, it is still a general problem that many patients do not achieve remission with mono- [2,3] or combinatorial therapy [3].This may be due to drug development involving testing drugs on large groups of patients, with the assumption that the drug will work similarly on all patients.Such an approach does not take into account the fact that each individual's genetic makeup and environment are unique, leading to significant variations in drug efficacy and side effects.
Given that IMIDs are known to involve thousands of genes that are variably expressed in different cell types and show temporal and interindividual differences [4,5], singlecell RNA sequencing (scRNA-seq) provides a promising foundation for the identification of suitable drug treatments [6].Indeed, one pioneering case report described scRNAguided therapy of one patient with an inflammatory disease [7].The case report described successful outcomes in a patient who did not respond to standard treatment.A limitation was that drug selection was empirical rather than based on systems-level understanding of the relative importance of disease-associated cell types, pathways, and genes.
Several systematic prediction models for drug selection in cancer exist, in which omics data are leveraged to determine the chemotherapies' "killing potential" of tumour cells [8][9][10][11].However, these models are not immediately translatable to IMIDs as they are (1) trained on large public drug-response data (e.g.GDSC database [12] and PRISM [13]), which are thus far unavailable for IMIDs, and (2) pursuing the eradication of disease-associated cell types.Rather few methodologies are applicable to IMIDs, including (1) identification of all druggable targets [14,15], (2) targeting enriched pathways [7,15], (3) network-based proximity calculations [6,16] or (4) matching of transcriptomic signatures as by Connectivity Map (CMap) [17].A limitation of these approaches is that they are developed using bulk transcriptomics or genetic variants and hence do not possess inherent solutions for rank aggregation for parallel analyses of several cell types, which limits their applicability to scRNA-seq.
Aiming to create a systematic framework for scRNAseq-based drug prioritisation and repositioning in inflammatory diseases, we hypothesised that the limitations of previous methodologies could be overcome by transposing network-based approaches [6,16] to a systematic and scalable strategy for network-based virtual drug screening of multicellular disease models (MCDMs).Therefore, we composed a computational framework henceforth referred to as scDrugPrio (Fig. 1).Using scRNA-seq-derived differentially expressed genes (DEGs) of either (1) one individual or (2) a group comparison between patients and controls, scDrugPrio starts by identifying cell type-specific drug candidates by considering both proximity in a protein-protein interaction network and biopharmacological criteria.To rank drug candidates, scDrugPrio calculates two measures, intracellular and extracellular centrality.We used these two measures to capture two important drug properties, namely, (1) proficiency in targeting key disease-associated expression changes in a cell type and (2) the relative importance of the targeted cell type.These measures are then aggregated over all cell types to provide a final drug ranking.
Because of the complexity and heterogeneity of IMIDs, we started by developing scDrugPrio using scRNA-seq data from a mouse model of antigen-induced arthritis.This reduced heterogeneity since the mice are inbred and the disease induced in a standardised way.Moreover, the mouse model allowed extensive in vitro and in vivo validation studies.To illustrate some potential case-of-use scenarios, we next applied scDrugPrio to cerebrospinal fluid from multiple sclerosis patients and intestinal biopsies from Crohn's disease (CD) patients.Our analyses demonstrated drug selection and ranking capabilities through (1) the prioritisation of known drugs.For antigen-induced arthritis, drug selection was also supported by (2) experimental validation of repurposed drugs and (3) favourable comparison with previous methods.Next, we applied scDrugPrio to paired biopsies from individual CD patients, revealing its ability to capture the significant heterogeneity in individualised therapeutic prioritisations.

scDrugPrio's computational framework
As indicated in Additional file 1: Fig. S1, scDrugPrio requires (1) an adjusted scRNA-seq matrix, (2) diseaseassociated differentially expressed genes (DEGs) for each cell type from either group-based comparison of healthy Fig. 1 Overview of the scDrugPrio workflow.Samples were derived from inflamed tissue in patients (red) and healthy tissues in controls (blue) or, for individual predictions, sampling included inflamed and uninflamed tissue taken from the same patient.Single-cell RNA-sequencing (scRNA-seq) data were preprocessed by undergoing quality control, denoising, clustering, cell typing and differentially expressed gene (DEG) calculation.DEGs for each cell type were calculated between healthy and sick samples.Using DEGs alongside information on drugs, scDrugPrio selects drug candidates (for each cell type; CT) whose gene targets are (1) in network proximity to DEGs (network proximity based selection) and (2) who counteract disease-associated expression changes (pharmacological action filtering).These cell type-specific drug candidates are next ranked using intracellular and intercellular centrality.(3) Intracellular centrality is computed based on the centrality of drug targets in the largest connected component (LCC) formed by DEGs and functions as a proxy for drug target importance.(4) Intercellular centrality measures centrality in disease-associated cellular crosstalk networks called multicellular disease models (MCDMs).(5) To derive a final ranking that aggregated cell type-specific drug selection and ranking into one list, drug candidates were ranked using a composite score of intra-and intercellular centralities (Additional file 1: Fig. S1).Drugs with identical targets and mechanism of action were given identical rankings and sick samples or from inflamed and noninflamed samples of one individual, (3) a protein-protein interaction network (PPIN) and (4) drug-target information.scDrug-Prio then utilises this information for cell type-specific drug selection, calculation of drug ranking measures and finally rank aggregation.Unless otherwise stated, analysis was performed in R 3.6.3.
For drug selection, scDrugPrio first computes the mean closest network distance (d c ) between cell type-specific DEGs and drug targets in the PPIN for each cell typedrug combination [16].To calculate z-scores (z c ) for network distance, permutation tests (1000 iterations) were performed in which both cell type-specific DEGs and drug targets were randomised in a bin-adjusted manner [16] before the mean closest distance was calculated.The minimal bin size for randomisation was set at 100 genes.Drugs that did not have any target in the interactome were removed from the analysis (n = 4 for literature-curated PPIN).Based on network distance, we selected only drugs that targets were significantly close (z c < − 1.64 corresponding to one-sided P < 0.05) to DEGs and that frequently targeted DEGs directly (d c < 1).These cut-offs were chosen based on our empirical observations (e.g.Additional file 1: Fig. S2h, j; Additional file 2) and previous knowledge [18].As the significance of network proximity can depend on the number of DEGs relative to the size of the network, cut-offs allowing only the top significant DEGs to enter analysis were implemented when needed.Cell type-specific drug candidates were selected further by requiring drugs to counteract the fold change of at least one targeted DEG.This criterion intuitively removes drugs that likely will not help to restore transcriptomic homeostasis.For this purpose, the pharmacological action of the drugs on their targets was determined.Binary drug action (activating/enhancing or inhibiting) on the drug target was recorded for each drug (Additional files 3, 4, 5, 6 and 7).If the pharmacological effect of the drug on the target had not been specified explicitly in DrugBank [19], a literature search was performed using the drug name and gene symbol of the targeted DEG as search terms in PubMed and Google Scholar.Additional information gathered from the literature can be found in Additional files 3, 4, 5, 6 and 7.In case the pharmacological effect of the drug on a target, despite a literature search, could not be classified as enhancing/activating or inhibiting, the drug target was assumed to not counteract fold-change.
Drug ranking by intra-and intercellular centralities was motivated by empirical observations (Fig. 3e; Additional file 1: Fig. S3) in our study as well as previous indications of the biological importance of disease modules [6,20] and central cell types in MCDMs [6].For calculation of intracellular centrality, disease modules for each cell type were defined as the largest connected component (LCC) formed by a cell type's DEGs in the PPIN.For LCC identification, the igraph R package [21] was utilised.To avoid overparameterisation, the eigenvector centrality [22] of DEGs in the LCC was calculated using the CINNA R package [23].For each drug, the intracellular centrality was calculated as the geometric mean of its differentially expressed target centrality scores in the cell type-specific LCCs.If a drug did not target any DEG included in the LCC, intracellular centrality was set to zero.
Intercellular centrality was calculated using MCDMs that modelled disease-associated cellular crosstalk.For the creation of MCDMs, first, cell type interactions were predicted using NicheNet [24].Briefly, NicheNet predicts and ranks ligand-target links between interacting cells by combining their expression data with prior knowledge on signalling and gene regulatory networks.As suggested by Browaeys et al. [24], Pearson correlation was used to measure each ligand's ability to predict the gene expression of genes in the gene set of interest compared to background genes in the receiving cell type.This means that a ligand has a strong positive correlation coefficient if its cognate receptor and the downstream genes of that receptor are all differentially expressed in the downstream cell type.We downloaded the human ligand-target model as well as the human ligand-receptor network (downloaded from https:// zenodo.org/ record/ 32607 58 April 2020).Cell type-specific DEGs constituted the gene set of interest.A set of potentially active ligands was defined as the intersection of ligands included in the downloaded human ligand-target model and ligands among respective cell type DEGs.Background genes for each cell type were defined as genes (i) in the denoised single-cell expression matrix D of k cell type-associated cells that showed a mean aggregate expression, Ea(i), over Ea(i) ≥ 0.2.This definition of background genes was similar to definitions by Browaeys et al. [24] and Puram et al. [25].At the chosen cut-off, we identified ca.10,000 background genes that corresponded to the recommended amount for NicheNet calculations [24].
All genes were translated to human Entrez gene symbols using human-mouse orthologues downloaded from NCBI (August 2019).Ligand activity analysis in NicheNet was performed for all possible cell type pairs, including self-interactions, excluding cell types that did not express DEGs.In the next step of MCDM construction, directed cell type interactions were derived from ligand activity results and weighted by NicheNet-derived Pearson coefficients.Only ligand interactions with a positive Pearson correlation coefficient were considered negative Pearson coefficients that reflected the association of a ligand with background genes and therefore were not biologically relevant.The resulting MCDM was visualised using Cytoscape 3.6.1 [26], and for visualisation purposes, the sum of Pearson coefficients that described the directed interaction between two cell types was used.Supplemental analysis supported the relevance of identified ligand interactions (Additional file 2).Eigenvector centrality was calculated for each cell type based on the weighted, directed interactions in the MCDM using the igraph and CINNA R package [21,23].The intercellular centrality of each drug was computed as the sum of MCDM centralities of the cell types that had selected the drug as a candidate.While eigenvector centrality is well tailored to capture central disease-associated cell types in the MCDM, considering both direct and indirect node connections [22], multiple centrality measures are available.We evaluated several of them, finding them to yield similar results to eigenvector centrality (as described in Additional file 2 and listed in Additional files 3, 4, 5, 6 and 7).
Final rank aggregation involved the calculation of a drug's compounded intra-and intercellular centrality.For this, we calculated combined intracellular centrality for each drug as the sum of drug-specific intracellular centralities in all cell types.The centrality compound score consisted of a drug intercellular centrality + 0.1 × combined intracellular centrality and thereby emphasised the importance of intercellular centrality over intracellular centrality.Intracellular centrality effectively worked as a tiebreaker.Drugs were ranked based on centrality compound scores, using the average position for ties.

Drug data
We retrieved data on 13,339 drugs from DrugBank [19] (downloaded July 2019) and selected only drugs that had been or currently FDA approved (n = 4021) were indicated for use in humans (n = 1964) and had at least one human protein target (n = 1864).Of those, drug targets could be translated to human Entrez IDs for 1844 drugs.The drug-target interactions used are provided in Additional file 3. Sets of drugs that are approved for each disease were identified according to DrugBank's [19] "Indication" category (as described in Additional file 2 and listed in Additional files 3, 4, 5 & 7).Unless otherwise specified, precision is calculated using these diseasespecific sets of FDA-approved drugs as relevant drugs or true positives.
For validation of ranked drug candidates, we also downloaded data from www. clini caltr ials.gov (September 2023).Data included information on 465,269 clinical trials registered from September 17th, 1999, to September 7th, 2023.Clinical trials (n = 70,396) for 1085 of the included 1844 drugs were found.To derive information on the disease relevance of drug candidates, we filtered clinical trials further by MESH terms, resulting in sets of 724, 494, 532, and 140 drugs that had been tried for rheumatoid arthritis, multiple sclerosis, Crohn's disease and psoriatic arthritis, respectively (Additional files 3, 4, 5, 6 and 7).Even though the outcome of such trials is largely unknown, using drugs registered for clinical trials alongside approved drugs for calculation of precision tests scDrugPrio's ability to capture the pharmacological consensus of the medical community on drugs with an expected effect.
For further validation of drug ranking, we also performed a literature search for the top 100 ranked drug candidates of each data set.We systematically searched PubMed and Google Scholar between June 2020 and December 2022 using the specific disease denotation and the drug name as search terms.No restrictions or filters were applied.The relevance of the identified articles was screened by title and abstract.When no relevant articles were identified, the drug name was replaced by the substance name, and another search was conducted.To be eligible, studies had to (1) include a control group, (2) be a human clinical study or rodent experiment, (3) measure inflammatory activity and (4) be accessible.When several studies were identified that reported contradictory results, the drug was labelled as having a previously reported effect, reasoning that it would be impossible to determine the evidence and accuracy level in every such instance.In Additional files 3, 4, 5 and 6, a summary on the nature of the identified article and a full reference is provided, listed by drug.

Precision and recall
When referring to precision among the top 100 candidates, we refer to all candidates with rank ≤ 100.

Protein-protein interaction network
The human interactome was derived from do Valle et al. [27]

Antigen-induced arthritis mouse model
Antigen-induced arthritis (AIA) was triggered in six 8-week-old, anaesthetised female 129/Sve mice by intraarticular injection of methylated bovine serum (mBSA) in the left knee joint after having presensitised mice to mBSA.The left knee joints of four naïve mice were injected with phosphate-buffered saline (PBS, 20 μL) and used as a negative control.One week after intra-articular triggering of AIA, mice were euthanized with carbon dioxide, and joints were either used for immunohistochemistry or scRNA-seq.Histochemical preparation was performed as previously described [6], and specimens were examined in a blinded manner for pannus formation, cartilage and subchondral bone destruction, and synovial hypertrophy on an arbitrary scale, 0-3, as described by Magnusson et al. [28].For the scRNA-seq experiment, joint tissue was minced to ~ 1-mm 3 pieces, which were digested by collagenase IV (1.5 mg/mL) and DNase I (100 µg/mL) at 37 °C.Dissociated cells were passed through a 70-µm cell strainer.Single-cell suspensions were resuspended in RPMI-1640 at a density of 1 × 10 5 cells/mL for cell loading.One mouse in which AIA had been triggered developed only mild arthritis (arthritis score 0.5) and was therefore excluded from further analysis.All mice were housed with a 12-h light/dark cycle and fed standard chow diets in a specific pathogenfree animal facility at Linköping University.scRNA sequencing was performed using the Seq-Well technique [29] following a described protocol [6].Briefly, prepared single-cell suspensions were counted and coloaded with barcoded and functionalised oligo-dT beads (Chemgenes, Wilmington, MA, USA, cat.No. MACOSKO-2011-10) on microwell arrays synthesised as described by Gierahn et al. [29].For each sample, 20,000 live cells were loaded per array to bind with oligo-dT beads.Beads were collected for capturing mRNA and preparing the library following cell lysis, hybridisation, reverse transcription and transcriptome amplification.Except for one library, which was sequenced alone, libraries from three samples were pooled for sequencing (Additional file 2: Table S1), resulting in a coverage of 6.6 reads per base.Four libraries were prepared for each sample using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA; cat.No. FC-131-1096) according to the manufacturer's instructions.Each library was sequenced once, except for one library, which was sequenced twice using the NextSeq 500/550 system.
The single-cell data were processed into digital gene expression matrices following James Nemesh, McCarrol's lab Drop-seq Core Computational Protocol (version 1.0.1, http:// mccar rolll ab.com) using bcl2fastq Conversion [30] and Picard software [31].To increase the read depth for the cells, each sample was sequenced multiple times (Additional file 2: Table S1), and the fastq files for each sample were merged before further alignment steps.The indexed reference for alignment of reads was generated from GRCm38 (June 2017, Ensembl) using STAR software (2.5.3) [32].Only primary alignments towards the reference genome were considered during downstream analyses, according to the mapping quality using STAR software.

Sampling and sequencing of psoriatic arthritis patients Sampling
Psoriatic arthritis (PsA) patients and controls were recruited by the Immune-Mediated Inflammatory Diseases Consortium (IMIDC) [33].PsA patients were recruited from different rheumatology departments from university hospitals belonging to the IMIDC.All PsA patients were diagnosed according to the CASPAR diagnostic criteria for PsA [34] with > 1 year of disease evolution and > 18 years old at the time of recruitment.Exclusion criteria for PsA included the presence of any other form of inflammatory arthritis, rheumatoid factor levels greater than twice the normality threshold or confirmed presence of an inflammatory bowel disease.PBMCs were sampled prior to treatment with anti-TNF or anti-IL17 and cryopreserved.Treatment response was classified at week 12 according to the EULAR response [35] (Additional file 7).For the anti-TNF study, 6 males and 10 females were included.The corresponding figures for anti-IL-17 treatment were 3 males (2 responders) and 13 females (6 responders).Simultaneously, healthy ageand sex-matched control subjects (Additional file 7) were recruited from healthy volunteers recruited through the Vall d'Hebron University Hospital in Barcelona (Spain).All the controls were screened for the presence of any autoimmune disorder, as well as for first-degree family occurrence of autoimmune diseases.None were found to be positive.Four males and four females were included.

Cell thawing
PBMCs cryopreserved at − 80 °C were thawed in a 37 °C water bath and transferred with a bored tip to a 15-ml Falcon tube containing 14 ml of 37 °C prewarmed RPMI medium supplemented with 10% FBS (Thermo Fisher Scientific).Samples were centrifuged at 300 × g for 10 min at (room temperature) RT, the supernatant was removed, and pellets were resuspended in 1 ml of 1 × PBS (Thermo Fisher Scientific) supplemented with 1% BSA (PN 130-091-376, Miltenyi Biotec) and 10 µL of DNase I (PN LS002007, Worthington-Biochem).After incubation at RT for 10 min with periodic shaking, the cells were filtered with a 20-µm strainer (PN 43-10,020-70, Cell Strainer) into a new 15-ml falcon on ice, and the filter was washed by adding 9 mL of cold 1 × PBS.Samples were concentrated afterwards by centrifugation at 300 × g for 10 min at 4°C and resuspended in 1 × PBS with 0.05% BSA for further assessment of cell numbers and viability with the TC20 ™ Automated Cell Counter (Bio-Rad).Samples balanced by responders and nonresponders for each treatment were mixed in pools of 8 patients at a 50:50 ratio and concentrated by centrifugation in an appropriate volume of 1 × PBS-0.05%BSA to obtain a final cell concentration > 4000 cells/µL, suitable for 10 × Genomics scRNA-sequencing.The suspension was filtered again with a 20-µm strainer, and the cell concentration was verified by counting with the TC20 ™ Automated Cell Counter.

Cell encapsulation and library preparation
Cells were partitioned into Gel BeadInEmulsions (GEMs) by using the Chromium Controller system (10 × Genomics).Each pooled sample was loaded into two channels with a target recovery of 35,000 cells per channel to ensure a minimum final recovery of 2000 cells per sample condition.After GEM-RT incubation, the resulting cDNAs were purified with SPRI beads.To ensure maximal cDNA recovery, a second Sylane bead purification was performed on the supernatant from the first purification, and both products were eluted together and preamplified for 13 cycles, following the 10 × Genomics protocol.cDNA was quantified on an Agilent Bioanalyzer High Sensitivity chip (Agilent Technologies), and 100 ng was used for library preparation.Gene Expression (GEX) libraries were indexed with 13 cycles of amplification using the Dual Index Plate TT Set A (10 × Genomics; PN-3000431).The size distribution and concentration of full-length GEX libraries were verified on an Agilent Bioanalyzer High Sensitivity chip.Finally, sequencing of GEX libraries was carried out on a NovaSeq 6000 sequencer (Illumina) using the following sequencing conditions: 28 bp (Read 1) + 10 bp (i7 index) + 10 bp (i5 index) + 90 bp (Read 2) to obtain approximately > 20,000 paired-end reads per cell.

3' single-cell RNA sequencing (scRNA-seq)
PBMC samples from 32 patients and 8 healthy controls were evenly mixed in pools of 8 donors per library following a multiplexing approach based on donor genotype, as in Kang et al. [36], for a more cost-and time-efficient strategy.Importantly, libraries were designed to pool samples together from the same treatment (anti-TNF or anti-IL17) but mixing patients with a different response to treatment.With this approach, we aimed to avoid technical artefacts that could mask subtle biological differences between responders and nonresponders.To profile the cellular transcriptome, we processed the sequencing reads with 10X Genomics Inc. software package CellRanger v6.1.1 and mapped them against the human GRCh38 reference genome.

Library demultiplexing
The donor's genotypes (VCF format) were simplified by removing SNPs that were unannotated or located in the sexual Y, pseudoautosomal XY or mitochondrial chromosomes (chr 0, 24, 25 and 26, respectively).As genotypes were assembled using the human GRCh19 reference genome, we converted them to the same genome assembly used to map the sequencing reads, the human GRCh38 reference genome, using the USCS LiftOver (https:// genome.ucsc.edu/ cgi-bin/ hgLif tOver) command line executable.To meet the LiftOver required format (BED format), we used an available wrapper script (lift-Over_vcf.py)to support input/output from VCF format [37].The library demultiplexing by donor was performed with cellsnp-lite v1.2.2 in Mode 1a [37], which allows genotyping single-cell GEX libraries by piling up the expressed alleles based on a list of given SNPs.To do so, we used a list of 7.4 million common SNPs in the human population (MAF > 5%) published by the 10,000 Genome Project consortium and compiled by Huang et al. [37].Importantly, we used the default parameters, setting the MAF > 5% (-minMAF 0.05) and requesting genotyping in addition to counting (-genotype).Then, we performed donor deconvolution with vireo v0.5.6 [38], which assigns the deconvoluted samples to their donor identity using known genotypes while detecting doublets and unassigned cells.Finally, we discarded detected doublets and unassigned cells before moving on to the downstream processing steps.

scRNA-seq data sets and preprocessing
Below, we describe all of the scRNA-seq data sets and the preprocessing steps of the current work as outlined in Additional file 1: Fig. S1.As most preprocessing steps were applied to scRNA-seq expression matrices of all data sets, we will describe them jointly.

Quality cut-offs
Starting from a raw scRNA-seq gene expression matrix, the quality of cells was assured by application of quality cut-offs that aimed to filter out low-quality cells (few genes, low read depth), dying cells (high expression of mitochondrial genes) and doublets (unexpectedly high reads and large number of genes).While arbitrary, these specific cut-offs were adapted to the corresponding data set and reported in Additional files 3, 4, 5, 6 and 7 [39].Genes that were expressed in less than three cells were excluded from further analysis.

Batch correction
In case a high degree of interindividual expression differences existed, batch correction was performed according to a previously established pipeline [40].In short, we used Seurat's function findIntegrationAnchors() [41] for the list of objects that corresponded to each individual.These anchors were later used by IntegrateData() [41] to integrate the data from individuals to correct for patientspecific differences as suggested in [42].

Denoising
Next, data for all cells were processed by a deep count autoencoder (DCA) model [43], which is a neural network performing a nonlinear principal component analysis (PCA).The DCA method is initiated by computing a library size, log-and z-score normalised expression matrix, which is taken as an input to the neural network, and the output of the neural network log10 transformed), and denoised single-cell expression matrix D, which has the same features as the original data but is corrected for various sources of noise in the data.The DCA method also outputs a representation of the original single-cell data in a latent space.This representation has many fewer features than the original data, which is particularly important for performing accurate cluster analyses.The intercellular expression differences are generally better represented in this latent space than in purely linear PCA models, and the latent space representation is also corrected for single-cell data artefacts such as dropouts and varying library sizes.
Clustering analysis was performed using the Seurat v3.1 package [41] on the DCA-derived latent representation.A shared nearest neighbour graph was constructed, and neighbourhood overlap between every cell and its k-nearest neighbours was calculated based on the Jaccard index using the FindNeighbors() function on all supplied latent features.Next, clusters were identified through application of the Louvain algorithm to the shared nearest neighbour graph using the FindClusters() function along with a specified resolution setting.The resolution parameter and k for k-nearest neighbour analysis were tailored to each data set and are reported below.Clusters were visualised through RunTSNE ().

Analyses of interindividual molecular heterogeneity
After denoising and clustering, heterogeneity among samples was determined.For this, we trained a flexible machine learning model that attempts to find a decision boundary between the given groups of cells.If this model results in a high misclassification rate for test data, it indicates that groups are highly mixed.More specifically, the data were randomly divided in half and used for training and testing the model.A random forest classifier [44] was used to classify cells from sick samples based on what patient the sample was derived from.Cross-validation with tenfold and grid search [45] was used to find the most appropriate hyperparameters of the random forest.The bootstrap [46] percentile method [46] was used to construct the 95% confidence intervals for training and test misclassification rates.The Scikit package [47] from Python (3.7.9) was used to perform the analysis.
Furthermore, patient heterogeneity was explored through comparison of cell type proportions and examination of latent features of the non-batch-corrected data.Interindividual differences in cell type proportions were explored by the application of the chi-square test to the proportions of cell types in sick samples.The contingency table for the chi-square test included individuals in columns and cell types in rows.Latent feature comparison was conducted visually through tSNE visualisation of the latent features of each patient.The results for these analyses are found in Additional file 2.

Cell typing
While cell typing is not crucial for scDrugPrio (which might be performed on unlabelled clusters), we cell typed clusters to enhance biological interpretation.Cell types were assigned to each cluster based on the relative coexpression of several known cell type marker genes (Additional file 3).For AIA data, each gene's expression was expressed as a fraction of a cell's total gene expression score.For visualisation of gene expression differences between clusters, z-scores were calculated.Z-scores for single cells were derived by comparison of one cell's gene fraction to all other cells' gene fractions.Z-scores for gene expression of clusters were derived by comparison of the average gene fraction in a cluster to the cluster-averaged gene fractions of cells in other clusters.Murine cell type-specific marker genes for the RA data were derived from the online resources of the R&D systems (www.rndsy stems.com/ resea rch-area; accessed July 2020).Cell typing of the human data sets was performed using DCA denoised gene fractions and utilised combinations of marker genes (Additional files 4 & 5).

Differentially expressed genes
For each cell type separately, differentially expressed genes (DEGs) were calculated by comparing denoised gene expression of cells derived from healthy samples vs. cells from sick samples.For this purpose, the Find-Markers() function in Seurat [41] was used to deploy a scRNA-seq-tailored hurdle model supplied by the MAST package [48].Genes were considered significantly differentially expressed when they showed an absolute log fold change greater than or equal to 1.5 and a Bonferroniadjusted P < 0.05.The fold change cut-off was motivated by previous studies [49] and aimed to decrease the number of DEGs for later network calculations.

Antigen-induced arthritis
Quality cut-offs resulted in a total of 16,751 cells (Additional file 3).Genes were annotated as murine NCBI Gene Symbols.Following denoising, clustering using k = 20 and a resolution of 0.6 resulted in the identification of 20 clusters that were cell typed.Heterogeneity analysis used n_estimators = 1500, max_depth = 15, min_sam-ples_split = 50, and min_samples_leaf = 50 and showed no significant heterogeneity.DEGs and the denoised expression matrix were translated to human Entrez gene IDs using human-mouse orthologues downloaded from NCBI (August 2019).

Multiple sclerosis
A unique molecular identifier (UMI) matrix [50] for cerebrospinal fluid (CSF) of five human multiple sclerosis (MS) patients and five human patients with idiopathic intracerebral hypertension (IIH) were downloaded from Gene Expression Omnibus (GEO), GSE138266 [51].Gene annotation was translated from human Ensembl gene IDs to human Entrez gene IDs and symbols using the HUGO Gene Nomenclature Committee (HGNC) database [52] (downloaded November 2020).After the application of quality cut-offs (Additional file 4), we derived 33,848 cells.Initial preprocessing was performed without batch correction using cluster parameters k = 10 and resolution = 0.2 after DCA denoising to derive 17 clusters.Interindividual heterogeneity was assessed as described below using the following hyperparameters: n_estimators = 500, max_depth = 30, min_sam-ples_split = 50, min_samples_leaf = 25.Since we noticed substantial patient-related heterogeneity, preprocessing was repeated, including batch correction, DCA denoising, and clustering using k = 15 and resolution = 0.35 to derive 21 clusters that were cell typed using known marker genes derived from the original publication [50] (Additional file 4).The number of DEGs ranged from 0 to 10,076.

Crohn's disease
A unique molecular identifier (UMI) matrix [53] for eleven human Crohn's disease (CD) patients was downloaded from GEO (GSE134809) [54].Data for each patient included intestinal biopsies from one inflamed site and one uninflamed site.After application of quality cut-offs (Additional file 5), we derived 77,416 cells.Gene annotation was translated from human Ensembl gene IDs to human Entrez gene IDs and symbols using the HGNC database (2020-11-08) [52].Initially, data were DCA denoised without applying batch correction.Clustering was performed using k = 15 and resolution = 0.8.Interindividual molecular heterogeneity was assessed as described below using the following hyperparameters: n_estimators = 1000, max_depth = 20, min_sam-ples_split = 50, min_samples_leaf = 25.Since there was substantial interindividual heterogeneity, preprocessing was repeated now batch-correcting before DCA denoising.Clustering was again performed using k = 15 and resolution = 0.8.

Individual Crohn's patients
For individual patient predictions, we used the same quality cut-offs as for the pooled analysis of CD patients.As interindividual heterogeneity does not affect the predictions made for individual patients, these calculations were performed on non-batch-corrected data.DCA denoising was applied to the joint data, and gene annotation was translated to Entrez gene IDs.Thereafter, scRNA-seq data were separated by patient, and cells from each patient were clustered individually.An individual patient cluster was assigned a cell type based on which cluster it most resembled in the joined CD analysis, as measured by the number of shared cell identifiers.DEGs were then calculated between cells from sick and healthy samples.Visualisations of data were in part created using BioRender.com.

Psoriatic arthritis
Data sets were divided into one anti-TNF and one anti-IL17 data set, including responders (R), nonresponders (NR) and healthy controls.We filtered out the doublet and unassigned cells as well as those that did not meet the quality cut-off criteria (Additional file 7) and derived 78,610 cells with 5088 mean reads for the anti-TNF data set and 72,472 cells with 5343 mean reads for the anti-IL17 data set.In both data sets, 19,415 cells were derived from healthy controls.Data sets were batch-corrected, and DCA was performed.For anti-TNF, clustering was performed using k = 25 and resolution = 0.25.The corresponding parameters for anti-IL17 were k = 15 and resolution = 0.45.The remaining downstream analysis was performed for responders and nonresponders separately, which meant that DEGs were calculated between responders and healthy controls and between nonresponders and healthy controls.For the anti-TNF data set, the number of DEGs ranged from 0 to 5989 for R and from 0 to 5877 for NR.The corresponding figures for anti-IL17 were 0 to 3097 and 0 to 3284.Next, scDrugPrio was applied to DEGS from anti-TNF R and NR as well as anti-IL17 R and NR separately.

In vitro validation of potential novel drugs
To validate the predicted novel drugs, in vitro culture of murine and human B-cells upon activation with the indicated stimuli was employed to assess the effects of the predicted drugs on B-cell survival, activation, proliferation, and antibody production.Three doses for each predicted drug were used to challenge in vitro cultured B-cells (Additional file 2: Table S2).For the assessment of potential novel drugs on murine B-cell survival and activation, 300,000 murine naïve B-cells (Lin − B220 + CD43 − ) were enriched by flow cytometric sorting and cultured in the presence of AffiniPure F(ab') 2 Fragment goat antimouse anti-IgM (10 μg/mL, CAT: 115-006-075, Jackson ImmunoResearch), anti-mouse CD40 (10 μg/mL, Clone:1C10, Biolegend), or LPS (10 μg/mL) for 24 h.B-cell survival was determined by flow cytometric analysis of propidium iodide (PI) + cells.Surface CD69, CD86, and MHC-II were used as readouts for assaying B-cell activation.For the analysis of B-cell proliferation, purified B-cells were stained with carboxyfluorescein succinimidyl ester (CFSE) (1 μM) before in vitro culture for 3 days.To determine the effects of the predicted drugs on antibody production, 200,000 purified murine naïve B-cells were stimulated with anti-CD40 (10 μg/mL) + IL-4 (10 ng/mL), LPS (10 μg/mL) + IL-4 (10 ng/mL), or LPS (10 μg/mL) + IFN-γ (10 ng/mL) for 6 days.
B-cell survival was determined by flow cytometric analysis of propidium iodide (PI) +-cells.Surface CD69, CD86, and MHC-II were used as readouts for assaying murine B-cell activation.Surface CD69 was assayed for the measurement of human B-cell activation.For the analysis of B-cell proliferation, purified B-cells were prestained with carboxyfluorescein succinimidyl ester (CFSE) (1 μM) before in vitro culture for 3 days.Murine IgG2a and IgG1, as well as human IgG in the supernatant, were determined by enzyme-linked immunosorbent assay (ELISA) using goat anti-mouse Ig, goat-anti-mouse IgG1-HRP and goat-anti-human IgG2a-HRP, goat antihuman Ig, and goat-anti-human IgG-HRP (SouthernBiotech) as previously described [57].

In vivo validation of predicted drugs
Amrinone was tested for treating collagen-induced arthritis (CIA).For this, male DBA1/J mice purchased from GemPharmatech (China) were immunised intradermally with 100 μg of chicken type II collagen (2 mg/ mL, Chondrex, USA) emulsified with complete Freund's adjuvant (CFA, 1 mg/mL) and boosted on day 21 with 100 μg of chicken type II collagen emulsified with incomplete Freund's adjuvant (IFA).Mice were i.g.given with diluent (n = 5) or amrinone (30 mg/kg, n = 5) daily from day 21 for 3 weeks.The rear paw thickness and the clinical arthritis score for each limb were recorded every other day from 0 to 4 with a maximal score of 16 for each mouse according to the previous protocol [58].Mice were housed with a 12-h light/dark cycle and fed standard chow diets in a specific pathogen-free animal facility at Xuzhou Medical University.
Mice were euthanized on day 21 post drug intervention with carbon dioxide.Serum was collected for the analysis of collagen-specific autoantibodies by enzyme-linked immunosorbent assay (ELISA) as previously described [59].Briefly, diluted serum was incubated in a 96-well ELISA plate precoated with chicken type II collagen (5 μg/mL).Goat-anti-mouse IgG1-HRP, goat-anti-mouse IgG2a-HRP, and goat-anti-mouse IgG-HRP (Southern-Biotech) were used as detection antibodies.Knee joints were fixed in 4% formaldehyde and subsequently decalcified with decalcification solution (ServiceBio, China) for 1 week.The specimens were next embedded in paraffin, and sagittal Sects.(4 µm) were cut.The sections were stained with haematoxylin and eosin (H&E) for the histological analysis of immune cell infiltration and Safranin-O for the analysis of bone erosion as previously described [57,59].

The scDrugPrio framework
Aiming to create an analytical framework for scRNA-seqdriven drug prioritisation, we constructed scDrugPrio, which consists of three main modules: (1) drug candidate selection based on cell type-specific DEGs, (2) drug candidate ranking and (3) aggregated ranking of drug candidates from all cell types.For this scDrugPrio requires two components (Fig. 1): (a) differentially expressed genes (DEGs) between sick and healthy samples for each cell type and (b) drug data, including information on gene drug targets and pharmacological effects.
Preprocessing includes the calculation of DEGs based on scRNA expression from one or more healthy and one or more sick samples (Additional file 1: Fig. S1).Preprocessing starts with quality control, batch correction (if needed) and data denoising of all scRNA-seq data, followed by clustering and cell typing.DEGs are computed per cell type by using sick versus healthy expressions.In the analysis below, we computed DEGs in the two modes.In the first mode, we use data from groups of sick and healthy individuals, while in the second mode, we use scRNA-seq data from paired samples of sick and adjacent healthy tissue for personalised drug prioritisations for one patient of interest.
scDrugPrio starts by computing the mean closest distance between DEGs and gene targets of drugs (henceforth referred to as drug targets) in the protein-protein interaction network (PPIN) for each cell type and each drug candidate.Intuitively, the "closer" the drug targets are to DEGs, the better is the chance for the drug to affect the disease-associated genes [16].Specifically, a relative proximity measure (z c ) capturing the statistical significance of the observed closest distance (d c ) was calculated based on a comparison of d c to the random expectation.Furthermore, scDrugPrio assumed that drugs that counteract the fold-change of at least one DEG will have a better chance to reverse disease-associated expression changes in the targeted cell type (following a similar idea as CMap [17]).To determine whether a drug counteracted a DEG fold change, we considered (1) the direction of the fold change (upregulated or downregulated) and (2) pharmacological action (e.g.agonistic, antagonistic) on the targeted DEG.For each cell type, a list of drug candidates was derived by filtering out drugs with low network proximity (d c ≥ 1, z c ≥ − 1.64 corresponding to one-sided P < 0.05) and drugs that did not exert counteracting pharmacological action against at least one targeted DEG.
In the next step, scDrugPrio computed intra-and intercellular centrality measures that were later used to aggregate the prediction and rank drugs.To compute intracellular centralities per cell type, scDrugPrio determines the largest connected component formed by DEGs in the PPIN and next computes a drug's centrality score based on the centrality of the drug targets within this component.Intracellular centrality hence presents an approximation of a drug's target relevance for diseaseassociated expression changes in a cell type.For intercellular centrality, scDrugPrio constructs a multicellular disease model (MCDM).In short, MCDMs were based on predicted molecular interactions between differentially expressed upstream regulatory genes in any cell type and their downstream genes in any other cell type using NicheNet [24].The resulting MCDM is a network in which cell types were nodes connected by directed, weighted ligand-target interactions.Intercellular centrality refers to the centrality of cell types in the MCDM.
Finally, scDrugPrio aggregates drug predictions through aggregation of cell type-specific drug predictions and intracellular and intercellular centralities into a compound score (Fig. 1).The resulting scores were used to rank drugs.Hence, overall drug ranking prioritised drugs that targeted key disease-associated expression changes in the most important cell types.

scDrugPrio development and evaluation in antigen-induced arthritis
scRNA-seq data were generated from whole joints of five inbred AIA mice and four naïve mice.After application of quality criteria (Additional file 3), data included 16,751 cells with 132,459 mean reads per cell.Data for all mice were denoised jointly using a deep count autoencoder network (DCA) [43], and 20 clusters were cell typed using marker gene expression (Fig. 2a; Additional file 1: Fig. S2).Comparison of cells from AIA and naïve mice identified DEGs in 17 cell types (Fig. 2; Additional file 2).
We retrieved drug target information from DrugBank [19] for 13,339 drugs (Additional file 3).From those drugs, we selected all of which had been FDA approved for human use and had at least one target in the human interactome (n = 1840).According to the indication information in DrugBank, 57 drugs were FDA approved for RA and hence treated as true positives for the calculation of precision and recall.
Applying scDrugPrio, the final list of candidates included 334 out of 1840 drugs; 32 drugs were established RA drugs, of which 22 ranked among the top 100 candidates (Fisher exact P < 10 −6 ; Fig. 3 ) and control (H1 to H4) mice.c Heatmap of z-scores of the cluster-based means of normalised, denoised gene expression for cell type markers.d MCDM, in which cell types were represented by nodes connected by the predicted interactions between upstream regulatory genes in any cell type and their downstream target genes in any other cell type [24].Edge width corresponds to the sum of Pearson coefficients for all interactions between two cell types, with arrows directed from upstream to downstream cell types.Edge colour corresponds to the upstream cell type.As indicated by more central positions in the MCDM, the four B-cell clusters were the most central cell types.e Bar plot depicting eigenvector cell type centrality in MCDM.Colours in all plots correspond to cell type colours in (a) file 3).To further evaluate the candidates' relevance, we collected clinical trial data from clinicaltrials.gov to capture the medical community's interest in the identified candidates as RA medications.We also performed a literature review of the top 100 drugs to evaluate whether candidates had shown promise when tested in human RA or murine/rat RA models.Through a literature review, evidence for the relevance of 40 additional drugs was identified, whereas three of the top 100 ranking drugs had not shown effects in previous trials.Hence, 62.0% of the top 100 ranking candidates were either approved or had successful experimental validation in prior literature (Fig. 3e), and 95.4% of previously studied candidates (among the top 100 candidates) had shown promise.

; Additional
Describing scDrugPrio results in more detail, network proximity selection (d c < 1, z c < − 1.64) yielded no drug candidates for six cell types and an average of 67 drug candidates (min = 1, max = 226) for the remaining In most cell types, precision increased with decreasing z c.Too stringent z c cut-offs, by contrast, led to the exclusion of almost all candidates, including approved RA drugs.c, d Precision and recall after stepwise application of drug selection criteria.Bars with no pattern represent a z c cut-off that Guney et al. 10  had previously found to offer good coverage of known drug-disease pairs.The dotted pattern represents precision at z c < − 1.64 (corresponding to one-sided P < 0.05).The striped pattern represents precision among candidates with z c < − 1.64 and d c < 1 (in other words, candidates passing network proximity-based selection).The crosshatch pattern represents candidates passing network proximity and pharmacological action selection.The application of selection criteria substantially increased the precision among central cell types.e Precision for approved RA drugs for which clinical trials had been registered for RA, and literature evidence (only for the top 100 ranking drugs) among candidates.Drug ranking included rank ties.Literature evidence was gathered for the top 100 ranked candidates and is presented as triangles with a dashed line.The colour legend for (a-d) is depicted in (f) fourteen cell types.Precision among cell type-specific candidates ranged from 0.0 to 12.8%.However, precision of cell types that were central in the MCDM, such as activated, immature, and plasma B-cells (12.8, 11.4, and 6.3%, respectively), outperformed random selection from the 1840 included drugs (57/1,840 = 3.1%; Fisher exact P < 10 −12 , P < 10 −8 , P = 0.4, respectively).Following network proximity calculations that were performed in the absence of information on a drug's effect on the target, we found tasonermin, a synthetic version of TNF, among the top-ranking candidates for several B-cell subtypes.Since overexpression of TNF has a crucial role in RA pathogenesis [69], this ranking supported the network proximity criterion.However, because tasonermin mimics the effects of TNF, it could worsen the disease.This finding exemplified the importance of pharmacological action selection.Pharmacological action selection resulted in removal of all drug candidates in two cell types and an average of 43 drug candidates per cell type (min = 2, max = 137) in the remaining twelve cell types.Overall precision decreased to a median [range] of 1.56% [0.00-20.63],although it increased in activated, immature, and plasma B-cells (20.6, 16.1, and 9.1%, respectively) as well as in T cells (Fig. 3c).
Having identified drug candidates for every cell type individually, intra-and intercellular centrality were calculated.The use of intra-and intercellular centrality measures for composite ranking was motivated by our findings that (1) intercellular centrality correlated with the significance of GWAS enrichment among cell type-specific DEGs (Pearson's r [95% CI] = 0.62 [0.21-0.85];P < 0.01), (2) intercellular centrality correlated with the precision among cell type-specific drug candidates (Pearson's r [95% CI] = 0.77 [0.46-0.91];P < 10 −3 ; Additional file 1: Fig. S2j), (3) drugs that targeted more than one cell type were more likely to be known RA drugs (Additional file 1: Fig. S2k) and (4) intracellular centrality could improve the mean rank of known drugs more than expected by chance (Additional file 2).
Using the AIA and human RA data, we benchmarked scDrugPrio against previous methodologies (Additional file 2) and performed extensive additional testing, demonstrating the advantage of scRNA-seq-driven analysis over genetic variations or bulk transcriptomics.Furthermore, we evaluated drug selection criteria and performed robustness analysis (Additional file 2).

Experimental validation of scDrugPrio
To further validate scDrugPrio, five high-ranking drugs that were not found to have any prior literature support for their efficacy in RA were chosen alongside the highest-ranking RA drug (auranofin #6) serving as a positive control.We first evaluated the five drugs by in vitro studies of B-cells.This cell type was selected because of its crucial role in the pathogenesis of RA and its central position in MCDM [70].We used previously described in vitro models of B-cell functions [70] measuring murine B-cell survival, activation, proliferation, and antibody production upon in vitro stimulation with selected drugs at various concentrations.Auranofin dramatically suppressed B-cell functions, including cell viability, proliferation, and immunoglobulin production (Fig. 4).Additionally, two of the five novel drugs (#114 amrinone and #100 adapalene) showed concentration-dependent in vitro effects on B-cell viability, proliferation, and immunoglobulin production (Fig. 4).Adapalene also greatly inhibited murine B-cell activation (Fig. 4b).The other three drugs (#91 irbesartan, #98 isosorbide, and #134 dimethyl furamate) showed little to no effect on murine B-cell functions (Additional file 1: Fig. S4).Next, we examined the effects of drug candidates on the function of human B-cells.Similarly, auranofin, adapalene, and amrinone inhibited human B-cell viability, activation, proliferation, and IgG production (Additional file 1: Fig. S5).Thus, two out of five candidate drugs prioritised by scDrugPrio, namely, adapalene and amrinone, were successfully validated by in vitro studies.
Valid drug candidates should arguably be transposable and replicable.As drug prediction was performed using data from the AIA model of arthritis, we deployed the collagen-induced arthritis (CIA) model for further in vivo study.We selected only amrinone for further study, as adapalene was designed for topical skin use and not for systemic delivery.CIA mice were administered 30 mg/kg amrinone i.g. for 3 weeks.This treatment significantly reduced the paw thickness (Fig. 4e) and clinical scores of CIA mice (Fig. 4f ).Analysis of collagen-specific serum autoantibodies revealed a significant inhibitory effect (Fig. 4g).Considering that it even reduced immune cell infiltration (Fig. 4h) and bone erosion (Fig. 4i), in vivo studies confirmed the relevance of amrinone treatment and thereby further supported scDrugPrio.

Application of scDrugPrio to multiple sclerosis
We next applied scDrugPrio to human IMIDs using scRNA-seq data of cerebrospinal fluid (CSF) from multiple sclerosis (MS) patients and idiopathic intracranial hypertension that served as controls [50].After application of quality cut-offs (Additional file 4), the data included 33,848 cells with 47,332 mean reads per cell.Comparing MS samples with controls, DEGs were calculated from batch-corrected, normalised expression scores.DEGs were found in 19 of 20 cell types.scDrug-Prio identified on average 59 (min = 1, max = 270) drugs in these 19 cell types (Fig. 5; Additional file 1: Fig. S6).Aggregated ranking of 417 drug candidates (Additional file 1: Fig. S7; Additional file 4) included ten out of 17 approved MS drugs.Among the top 100 ranking candidates, four approved MS drugs, as well as biosimilars rituximab and obinutuzumab, were identified, which outperformed random expectation (precision 0.9%).A literature search revealed that an additional 22 of the top 100 ranking drugs had shown effects in previous studies, resulting in a precision of 28.3% (Fig. 5d).Seventy-one drugs had not yet been validated in previous studies, and one evaluated drug had not shown promise in previous studies.Hence, 96.7% of the studied candidates (among the top 100 candidates) showed efficacy.Taken together, these data supported the potential of scDrugPrio to predict the response to drugs approved for that disease, as well as for repurposing other drugs.S2.Having successfully validated adapalene and amrinone in we conducted in vivo experiments for amrinone.with collagen-induced arthritis (CIA) were treated with diluent (n 5) or (30 mg/kg, n = 5) for 3 weeks.The (e) rear paw thickness, (f) clinical scores, and (g) collagen-specific serum autoantibodies were measured.drug efficacy was assessed by analysis of joint immune cell infiltration H&E staining and (i) bone erosion using Safranin-O staining.*P < 0.05, **P < 0.01, < 0.001.Dimethyl furamate, DMF The value of above rank aggregation was supported as precision among cell type-specific drug candidates only ranging from 0.0 to 12.5%.The MCDM (Fig. 5c; Additional file 4) indicated plasma cells, mononuclear phagocytes, natural killer cells, and activated CD8 + T cells to be of central importance, which is consistent with the current pathophysiological understanding [50,71].Notably, the MS MCDM was more complex than the AIA MCDM regarding the number of cell types and interactions.

Application of scDrugPrio to Crohn's disease
Next, scDrugPrio was applied to patients with Crohn's disease (CD) from whom scRNA-seq data [53] from paired inflamed and uninflamed intestinal tissue biopsies were available.After application of quality criteria (Additional file 5), 77,416 cells were included with 3591 mean unique molecular identifiers (UMIs) per cell.Following preprocessing, analysis was performed on batchcorrected, pooled data from all patients in which DEGs were calculated through comparison of inflamed and uninflamed samples (Additional file 1: Fig. S8 & S9; Additional file 5).The aggregated ranking included 343 drug candidates, of which five were known CD drugs.These five consisted of sulfasalazine (#91), mesalazine (#169.5),rifaximin (#238), and two anti-TNF drugs (adalimumab and infliximab, tied rank #292.5).Apart from sulfasalazine, literature evidence suggested the effectiveness of 13 additional top-ranking 100 drugs, resulting in a precision of 14%.
5 scDrugPrio applied to scRNA-seq data from cerebrospinal fluid samples of MS patients and controls.a, b Clustering and cell type proportions (%) for CSF samples of idiopathic intracranial hypertension (IIH) and multiple sclerosis patients (MS).Cell type proportions showed significant interindividual differences among MS patients.c The MCDM for MS was more complex than that of AIA mice.The most central cells included mononuclear phagocyte 3, natural killer cells, mononuclear phagocyte 2, early activated CD8 + T cells and plasma B-cells (in order of eigenvector centrality).d Precision among ranked candidates for approved MS drugs, drugs that had been registered for clinical trials for MS in the ClinicalTrial.database, and literature evidence (only for top 100 ranking drugs).Abbreviations: Tm, T memory; Tem, T effector memory

Patient heterogeneity in human disease
An important difference between human data and mouse data was substantial interindividual heterogeneity in human patients.To illuminate such differences, we (1) applied chi-square test to compare cell type proportions among sick samples, (2) trained a flexible machine learning model (random forest) with the aim to separate cells from different individuals based on gene expression profiles, and (3) examined latent features of the non-batchcorrected data.Mice, as expected, showed no differences in cell type proportions (chi-square P = 0.9919; Fig. 2c), gene expression (random forest, CI of misclassification rate 0.504 ~ 0.532 in the training data and 0.638-0.655 in the test data), or latent features (Additional file 1: Fig. S2c).However, both MS and CD patients showed great interindividual heterogeneity in cell type composition (chi-square P < 10 −15 , respectively).Heterogeneity in cell type composition, although decreased, can still be observed in the batch-corrected data (Fig. 5b; Additional file 1: S7i).A patient effect was also observed in the nonbatch-corrected gene expression for MS (random forest, 95% CI of misclassification rate 0.075-0.084 in training data, and 0.124-0.132 in testing data) and CD patients (random forest; 95% CI of misclassification rate 0.270-0.283 in training data, and 0.378 ~ 0.385 in testing data) as well as latent features derived from non-batch-corrected data (Additional file 1: Fig. S6 & S9).Taken together, such patient effects necessitated batch correction for pooled prediction using the human data sets above.

Potential for individualised predictions
Even though batch correction had been applied appropriately, a potential limitation of the above MS and CD analyses was that scDrugPrio was applied to pooled data derived from heterogeneous patients and controls.Patient effects might form the bases for the responder/ nonresponder dichotomy, and we therefore evaluated scDrugPrios potential for individualised drug prioritisation.For this, we applied scDrugPrio to individual CD patients, using similar preprocessing as for CD data above with the following exceptions: (1) data were not batch-corrected and (2) following denoising, cells from inflamed and uninflamed samples of each patient were clustered separately.DEGs were derived through comparison of individual patient inflamed and uninflamed cells in each cluster.DEGs showed that the eleven patients expressed important CD drug targets differently (Additional file 1: Fig. S10a, b).To investigate whether such molecular differences could affect drug prediction outcomes, scDrugPrio was applied to all patients separately (Additional file 1: Fig. S10-12; Additional file 6).
Strikingly, individualised drug predictions of nine out of eleven patients (such as patient 1: 19.0%; patient 10: 20.5%, Fig. 6; Additional file 1: Fig. S13) outperformed the precision of pooled patient analysis (14.0%).Among the top 10 candidates, precisions for individualised predictions (20-70%) outperformed precision of pooled patient analysis in seven patients and equalled that of pooled patient analysis in four patients (10%).All predictions outperformed random chance (1.5%).More detailed analysis revealed interindividual differences in cell type proportions and network properties in the MCDM (Additional file 1: Fig. S12) as well as different drug rankings (Additional file 1: Fig. S14).Taken together, these findings supported that scDrugPrio presents a valid strategy for personalised drug prioritisation.

Application of scDrugPrio to nonresponder/responder data from patients with psoriatic arthritis highlighted the importance of local tissue samples
Since the previous analyses supported scDrugPrio's potential for another case-of-use scenario, namely, to distinguish drug responders from nonresponders.To explore this potential, we collected peripheral mononuclear blood cells (PBMCs) from patients with psoriatic arthritis (PsA) as well as healthy controls.PBMCs were chosen because the analysis of blood samples is clinically more tractable than the analysis of biopsies.Samples were cryopreserved before treatment with either anti-TNF or anti-IL17.Treatment response was later assessed by a rheumatologist according to EULAR response criteria [35] (Additional file 7).We next selected 32 patients, of whom eight were classified as responders (R) and eight as nonresponders (NR) to either of the two drugs, as well as eight healthy controls.Cryopreserved PBMCs from these patients were analysed with scRNA-seq.For preprocessing, data were divided into two data sets by treatment regimen, each data set containing the corresponding eight R and NR along with the eight healthy Fig. 6 scDrugPrio for individual drug prediction.a, b Cell type proportions differed greatly between two CD patients, as shown in the horizontally stacked bar plots representing paired biopsies from inflamed (infl) and uninflamed (uninfl) lesions that were taken from each patient.c, d Patients also show differences in the composition and interconnectivity (representing ligand interactions) of the MCDMs.Patient 1 had a cell type for which no ligand-target interactions could be found with any other cell types in the MCDM.e, f Precision among ranked candidates for approved CD drugs, drugs for which clinical trials had been registered for CD and literature evidence (only for top 100 ranking drugs).The precision for the ranked drug candidates for patient 1 was low for approved CD drugs, while literature evidence supported the top-ranking drugs, of which many are anti-inflammatory.In contrast, patient 10 had several approved CD drugs among the top 100 candidates, and a curated literature search confirmed the validity of many more candidates.g Precision for prediction based on pooled patient data was poor.h Venn diagram presenting the overlap of considered drug candidates for patients 1 and 10. i Interindividual differences between patients 1 and 10 were reflected in the prediction outcome, as no correlation existed between the drug rank of drugs that were candidates in both patients controls.After application of quality cut-offs, the data included 78,610 cells with 5088 mean reads for anti-TNF analysis and 72,472 cells with 5343 mean reads for anti-IL17 analysis.Data were batch-corrected and DCA denoised before clusters were identified and cell-typed using marker genes (Additional file 1: Fig. S15 & S16; Additional file 7).
For each data set, DEGs were calculated through comparison of cells from healthy controls to either R or NR.The precision for approved PsA drugs among the top 100 candidates in the respective aggregated ranking was 0% for anti-IL17 NR, 4% for anti-IL17 R, 1% for anti-TNF NR and 1% for anti-TNF R (Additional file 1: Fig. S15 & S16).Unexpectedly, anti-TNF treatment received a low rank in anti-TNF R but not in NR (#333 and #88, respectively), while anti-IL-17 was not considered a candidate in either R or NR.
Further analyses of our PBMC data from all R and NR patients showed that the TNF signalling pathway was significant in only 8 and 8% of clusters, respectively.The corresponding figures for the IL-17 signalling pathway were 17 and 13% in the anti-IL17 R and NR groups, respectively.In those cell types, most pathways were downregulated, including those regulated by TNF and IL17 (Additional file 1: Fig. S17).This result contrasted with previous studies of skin and synovium from PsA, which showed increased mRNA expression of TNF and IL17, as well as their pathways [72,73].A similar dichotomy between local inflamed tissues and cells in the blood in autoimmune diseases has been previously described [74].This dichotomy can be explained by the physiological need to localise inflammatory responses to inhibit systemic and possibly fatal responses.The general clinical implication may be that drug predictions should ideally be based on local tissue samples [35].

Discussion
The main problem in therapeutics, which serves as the basis for this study, is the large number of IMID patients who do not respond to treatment [2,3,75].Previous virtual drug screening methods for inflammatory diseases are based on genetic variance or bulk RNA sequencing [16,76,77] and hence do not consider variations in gene expression across different cell types, biopharmacological properties, or individual variations between patients with the same diagnosis.While harnessing the daunting complexity and heterogeneity for personalised treatment may seem impossible by health care standards today, this challenge should be put in the context of the suffering and costs resulting from ineffective drug treatment.Many IMIDs cause life-long morbidity and increased mortality.The yearly cost of treating an individual IMID patient may be hundreds of thousands of dollars for drugs and hospital care [1].
Recent efforts for drug toxicity screening [8,78] support the feasibility of scRNA-seq to capture relevant cellular information.However, systematic solutions for drug prioritisation for IMIDs based on scRNA-seq remain to be devised.We therefore propose a computational framework, scDrugPrio, that extends on existing bioinformatic tools [6,16,24] by providing a framework for data integration, enabling drug ranking based on a multifaceted understanding of cell type-specific disease mechanisms, altered cellular crosstalk and pharmacological effects.We demonstrate that scDrugPrio yields relevant and robust drug prioritisations, outperforms previous methods [7,67], and holds potential for individualised as well as pooled drug prioritisation and repurposing.
An important advance of scDrugPrio is that it can be applied to scRNA-seq data.The importance lies in the fact that complex diseases each involve differential expression of thousands of genes across multiple cell types [6].A previous case report [7] described one successful example of treating an individual patient with immunological diseases based on scRNA-seq data.However, the drug choices were empirical rather than systematic.Because scRNA-seq allows transcriptome-wide analyses in each of thousands of cells, it is possible to infer disease-associated changes in individual patients preferably by comparisons with noninflamed samples from the same individual or to groups of healthy individuals.Thus, scDrugPrio has the potential to personalise the treatment of individual patients.The importance of this advance is highlighted by our results and previous findings [53,79] showing great interindividual differences in the molecular and cellular composition of human diseases.For example, we showed that scDrugPrio ranked anti-TNF treatment high in a CD patient who was classified as a responder but not in a nonresponding patient.In the latter patient, other immunomodulatory drugs, such as natalizumab, received high ranks.Natalizumab is mainly used in MS but has, in previous studies, shown positive effects in CD [80], making it a viable recommendation.These examples emphasise that successful drug screening will need to consider variations between patients with the same diagnosis.
There are several limitations of scRNA-seq-based drug predictions in IMIDs.Many of these depend on the challenges involved in harnessing complex and heterogeneous disease-associated changes with an emerging technology such as scRNA-seq.An analogy to a historical example may illustrate how such limitations may drive scientific progress.In 1970, Needleman-Wunsch [81] and 1981, Smith-Waterman [82] published algorithms for global and local sequence alignment, which were widely used.The limitations of those algorithms were that they were mainly useful for nucleotide but not protein sequence analyses because of limited protein sequence data and no scoring system that modelled protein evolution.During the next two decades, these problems were resolved by increasingly accurate data and methods [83].Importantly, 42 years after publication of the Smith-Waterman algorithms for proteins, these algorithms can generate very accurate results when combined with scoring systems that were later developed [83].We propose that the limitations of scRNA-seq that we face today will lead to a similar development of increasingly accurate technologies.One obvious limitation of scRNA-seq is that mRNA and protein levels may be poorly correlated, which can limit biological interpretability.From this perspective, the use of DEGs for scDrugPrio's pharmacological predictions is a relative strength, as DEGs have been shown to correlate significantly better with protein levels [84] compared to mRNA levels alone and hence increase the biological relevance of our predictions.Inherent limitations of scDrugPrio also derive from the use of current interactomes, which are not comprehensive in terms of proteins, interactions, and variations across cell types [85] and are prone to investigative biases.While it is impossible to address all these concerns, we explored whether network proximity-based drug selection was influenced by investigative bias through replication of key results in a smaller yet unbiased interactome.We found that precision among candidates was slightly lower, partly due to missing drug targets in the interactome, but that results were comparable.Additionally, scDrugPrio might benefit from systematic parameter optimisation, which is currently not possible due to the limited amount of suitable scRNA-seq data sets.
Predictions were based on drug-target effects which are complex and potentially incompletely characterised [86] which could explain why some of the drug predictions could not be validated experimentally.The need for better characterised drug effects and the relative importance of drug targets is exemplified by etanercept, which inhibits TNF and its receptors but may activate IgG receptors.The TNF-inhibitory effects are beneficial in PsA, while those on IgG receptors are not clearly defined.However, because all these targets were downregulated in PBMCs from nonresponding PsA patients, etanercept (counteracting IgG downregulation) received a higher rank than in patients responding to anti-TNF treatment.While unexpected, this highlights the need for systematic information about the relative importance of drug targets.Future efforts aiming to address these limitations might find that the predictive capability of scDrugPrio can be further enhanced by integration of multiple other factors, such as binding affinity (e.g.BindingDB) or bioactivity (e.g.ChEMBL), especially if data become more comprehensive.However, it should be emphasised that treatment decisions ultimately depend on informed discussions between patients and physicians.Future clinical studies are warranted to determine which variables should be included in a drug prediction tool like scDrug-Prio, and which variables should be analysed separately depending on the specific disease-context or questions arising from patient-physician discussions.
The above example of etanercept in PsA highlights an important clinical concern, which to our knowledge has not been recognised in the context of drug prediction methods.While analyses of blood samples are often more tractable in routine clinical practice, diseaseassociated mechanisms may vary greatly between cells in blood and inflamed tissues.Our scRNA-seq analyses of PBMCs from PsA patients who did or did not respond to treatment with either anti-TNF or anti-IL-17 showed that predicted activation of TNF and IL-17 induced pathways was found only in a small portion of the PBMC cell types and, in fact, was downregulated in both responders and nonresponders.In contrast, previous studies [72,73] of synovium from PsA patients have shown consistent upregulation of both signalling pathways.Additionally, one previous study also showed differences between synovium and skin from the same patients [72].Thus, scDrugPrio should ideally be applied to local, inflamed tissue samples of the relevant tissue.
Despite these limitations, the translational relevance of scDrugPrio was supported by analyses of precision/recall for drugs that were approved for the studied diseases, as well as by in vitro and in vivo experiments.Those experiments implied two drugs, namely, adapalene (used for acne vulgaris) and amrinone (used for congestive heart failure), that had not been previously described as candidates for RA treatment.However, both have anti-inflammatory effects and could, therefore, be effective [87,88].This potential was supported by in vivo experiments in which CIA mice were treated with amrinone (adapalene is a topical skin drug and hence is not suitable for systemic treatment in this experimental system).This example also suggests a potentially important pharmacological application of scDrugPrio, namely, virtual drug repurposing by systematic screening of thousands of drugs across several inflammatory diseases, as well as in patients who do not respond to standard treatment.

Conclusions
Here, we show that scDrugPrio has the potential for individualised drug predictions.We have made data and tools freely available for this purpose.However, further parameter optimisation and controlled, prospective clinical studies are needed for clinical translation.If successful, this approach could lead to a radical change in health care, which today is largely based on treating groups of patients with the same diagnosis with a limited number of drugs based on a limited understanding of the underlying molecular complexity and heterogeneity with limited population-based efficacy [89].

Fig. 2
Fig.2Construction and analysis of a multicellular disease model (MCDM) based on scRNA-seq analysis of a mouse model of antigen-induced arthritis (AIA).a tSNE of cells (n = 16,751) pooled from all samples.b Cell type proportions (%) for individual AIA (S1 to S6) and control (H1 to H4) mice.c Heatmap of z-scores of the cluster-based means of normalised, denoised gene expression for cell type markers.d MCDM, in which cell types were represented by nodes connected by the predicted interactions between upstream regulatory genes in any cell type and their downstream target genes in any other cell type[24].Edge width corresponds to the sum of Pearson coefficients for all interactions between two cell types, with arrows directed from upstream to downstream cell types.Edge colour corresponds to the upstream cell type.As indicated by more central positions in the MCDM, the four B-cell clusters were the most central cell types.e Bar plot depicting eigenvector cell type centrality in MCDM.Colours in all plots correspond to cell type colours in (a)

Fig. 3
Fig.3 Precision and recall in relation to drug selection and ranking criteria.a, b Precision and recall at different z c cut-offs.In most cell types, precision increased with decreasing z c.Too stringent z c cut-offs, by contrast, led to the exclusion of almost all candidates, including approved RA drugs.c, d Precision and recall after stepwise application of drug selection criteria.Bars with no pattern represent a z c cut-off that Guney et al.10  had previously found to offer good coverage of known drug-disease pairs.The dotted pattern represents precision at z c < − 1.64 (corresponding to one-sided P < 0.05).The striped pattern represents precision among candidates with z c < − 1.64 and d c < 1 (in other words, candidates passing network proximity-based selection).The crosshatch pattern represents candidates passing network proximity and pharmacological action selection.The application of selection criteria substantially increased the precision among central cell types.e Precision for approved RA drugs for which clinical trials had been registered for RA, and literature evidence (only for the top 100 ranking drugs) among candidates.Drug ranking included rank ties.Literature evidence was gathered for the top 100 ranked candidates and is presented as triangles with a dashed line.The colour legend for (a-d) is depicted in (f)

Fig. 4
Fig. 4 validation of amrinone in murine function and the pathogenesis of the CIA model.Drug effect of the selected drugs and the positive on in vitro murine (a) B-cell survival, (b) activation, (c) proliferation, and (d) immunoglobulin production.Colours represent the responses to diluent and different drug concentrations (low, medium, high); for specific concentrations, see Additional file 2: Table S2.Having successfully validated adapalene and amrinone in we conducted in vivo experiments for amrinone.with collagen-induced arthritis (CIA) were treated with diluent (n 5) or (30 mg/kg, n = 5) for 3 weeks.The (e) rear paw thickness, (f) clinical scores, and (g) collagen-specific serum autoantibodies were measured.drug efficacy was assessed by analysis of joint immune cell infiltration H&E staining and (i) bone erosion using Safranin-O staining.*P < 0.05, **P < 0.01, < 0.001.Dimethyl furamate, DMF