Applications of single‐cell sequencing for the field of otolaryngology: A contemporary review
Abstract Objectives Single‐cell RNA sequencing (scRNA‐Seq) is a new technique used to interrogate the transcriptome of individual cells within native tissues that have already resulted in key discoveries in auditory basic science research. Rapid advances in scRNA‐Seq make it likely that it will soon be translated into clinical medicine. The goal of this review is to inspire the use of scRNA‐Seq in otolaryngology by giving examples of how it can be applied to patient samples and how this information can be used clinically. Methods Studies were selected based on the scientific quality and relevance to scRNA‐Seq. In addition to mouse auditory system (inner ear including hair cells and supporting cells, spiral ganglion neurons, and inner ear organoids), recent studies using human primary cell samples are discussed. We also perform our own analysis on publicly available, published scRNA‐Seq data from oral head and neck squamous cell carcinoma (HNSCC) samples to serve as an example of a clinically relevant application of scRNA‐Seq. Results Studies focusing on patient tissues show that scRNA‐Seq reveals tissue heterogeneity and rare‐cell types responsible for disease pathogenesis. The heterogeneity detected by scRNA‐Seq can result in both the identification of known or novel disease biomarkers and drug targets. Our analysis of HNSCC data gives an example for how otolaryngologists can use scRNA‐Seq for clinical use. Conclusions Although there are limitations to the translation of scRNA‐Seq to the clinic, we show that its use in otolaryngology can give physicians insight into the tissue heterogeneity within their patient's diseased tissue giving them information on disease pathogenesis, novel disease biomarkers or druggable targets, and aid in selecting patient‐specific drug cocktails. Uses of single‐cell RNA sequencing (scRNA‐Seq) in clinical medicine. scRNA‐Seq characterizes tissue heterogeneity including diseased and rare cell types allowing for identification of unique genes expressed by these cells that might have been obscured by traditional RNA‐Seq and that may be used as potential drug targets. Interrogation of drug target expression within each cell cluster can aid in creating a patient‐specific drug cocktail. scRNA‐Seq can also be performed before or after treatment to get a sensitive picture of how a patient responded with elimination of diseased cells, continued presence of diseased cells unaffected by treatment, or emergence of new drug‐resistant clones. 
INTRODUCTION
Currently in otolaryngology, histology, microarray, and bulk RNA‐sequencing (RNA‐Seq) methodologies are used on patient biopsy samples to diagnose disease including microbial infections, human papilloma virus, genetic disorders such as neurofibromatosis type II, and various head and neck cancers like head and neck squamous cell carcinoma (HNSCC). These methodologies can be biased in their investigation of tissues and are limited by extremely low sensitivity, antibody or probe availability, and averaging of gene expression across the tissue of interest, ignoring tissue and cellular heterogeneity. For example, the use of bulk RNA‐Seq in tumor diagnostics can obscure the importance of tumor heterogeneity and rare cell types to tumor biology and disease prognosis. 
Single‐cell RNA‐Seq (scRNA‐Seq) is a powerful new method that allows users to obtain the transcriptome of individual cells. This technology avoids the cell averaging seen in bulk RNA‐Seq allowing for novel discoveries in tissue heterogeneity and biology at single‐cell resolution.3, 4, 5, 6, 7 The use of scRNA‐Seq in auditory basic science research has proven important in deciphering cellular heterogeneity of inner ear tissues and is just beginning to provide answers to questions regarding development of diverse cell types in this organ, how this heterogeneity relates to auditory function, and how we can use this information to understand auditory disease and mechanisms for hearing restoration. The explosive use of scRNA‐Seq in auditory research has resulted in several new discoveries in the field of otolaryngology.8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 In endolymphatic sac, scRNA‐Seq showed that mitochondrial‐rich cells were likely responsible for the pathogenesis of enlarged vestibular aqueducts. Two recent studies used scRNA‐Seq on spiral ganglion neurons (SGN) to detect four subpopulations, including three novel SGN subtypes, differing in their expression of novel marker genes and in sound intensity‐specific activation19, 20 that underlie the clinical phenomenon of “hidden hearing loss.” Another study used scRNA‐Seq of cells from the organ of Corti to prove that cochlear hair cells not only differentiate from progenitor supporting cells but also from nonsensory cell types. We provide a selected list of scRNA‐Seq studies done in the mouse that are relevant to hearing for further review (Table 1). Although most scRNA‐Seq experiments related to otolaryngology are currently conducted on mice in basic science laboratories (Table 1), with the increasingly low‐cost and new high‐throughput scRNA‐Seq technologies, this exciting tool can be readily applied to human tissue to generate new discoveries in human biology and may soon be translated to clinical otolaryngology. Human vestibular and cochlear tissues, while both difficult and unethical to obtain from healthy patients, are frequently discarded as a result of standard transcranial surgical approaches to resections of acoustic neuromas, for example.21, 22 With collaborations between treating surgeons and surgeon‐scientists, sequencing of human inner ear tissue and other more easily obtained pathologic tissue, including acoustic neuromas, HNSCC, vocal cord polyps, and recurrent respiratory papillomas, could deliver valuable information on human cell types responsible for disease and possible druggable targets within these cell types. 

Selected list of scRNA‐Seq studies from the mouse inner ear
Abbreviations: ES, endolymphatic sac; HC, hair cells; IHC, inner hair cells; mESC, mouse embryonic stem cells; OHC, outer hair cells; SC, cochlear supporting cells; scRNA‐Seq, single‐cell RNA sequencing; SE, sensory epithelium; SGN, spiral ganglion neurons, SV, stria vascularis.
All the above studies were conducted using mouse tissue.
INTRODUCTION
A few studies have used scRNA‐Seq on human otolaryngologic patient samples including HNSCC,23, 24, 25, 26 sinus mucosa, and melanoma. These, as well as studies in other human tissues,29, 30 have shown the utility of this method in human clinical samples. These studies have generated excitement for the use of scRNA‐Seq in unraveling tissue heterogeneity with the hope of using this information to provide care that is tailored to each patient, but for this goal to be attained physicians need be aware of how this technology may be applied to their clinical practice and the potential novel diagnostic information and therapeutic options it might provide in the future.32, 33 Here we give a brief overview of scRNA‐Seq methodology, suggest potential clinical applications for scRNA‐Seq in otolaryngology, and comment on potential limitations for its clinical use.
To make this review as clinically relevant as possible, we will focus on reviewing studies that have been conducted in human samples relevant to otolaryngology. While many laboratories are focusing on optimizing sequencing for human samples such as cochlear and vestibular tissues, currently it is much easier to obtain human tumor samples and the only published scRNA‐Seq studies of human tissue relevant to otolaryngology use HNSCC,23, 25, 26 melanoma, or chronic rhinosinusitis samples. We use these studies as examples of the use of scRNA‐Seq as a research tool in otolaryngology and its potential clinical applications. We perform our own scRNA‐Seq analysis (methods available in Data S2) of a publicly available HNSCC data set as an example of how this technique may impact clinical care in the future (Data S1).
scRNA‐Seq WORKFLOW
To give clinicians a general understanding of scRNA‐Seq concepts, we provide a brief overview of the scRNA‐Seq experimental workflow to inform the discussion of potential applications of this technology in otolaryngology. While a comprehensive review of this complex process is not provided here, the experimental design and analysis of scRNA‐Seq data have been extensively reviewed elsewhere and we point interested readers that would like to learn more about the details of designing scRNA‐Seq experiments to the reviews mentioned within our discussion.34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 Most scRNA‐Seq protocols follow a similar sequence of reactions (Figure 1) including tissue dissociation to generate a single‐cell suspension, cell isolation (Figure 1A), mRNA capture and barcoding (Figure 1B), library preparation (Figure 1C), followed by next‐generation sequencing (NGS) (Figure 1D). Single‐nucleus RNA‐Seq (sNuc‐Seq) protocols have also recently been developed to isolate single nuclei from difficult to dissociate or archived patient samples. From a single‐cell or nuclei suspension, most methodologies require each cell or nucleus to be captured and isolated for barcoding and pooling. Cell isolation methods have been reviewed previously including a review by Qi et al on designing scRNA‐Seq experiments for head and neck cancer.34, 35 Briefly, isolation of cells into wells or microwells of a plate can involve limiting dilution, laser capture microdissection, micropipette isolation, fluorescence‐activated cell sorting (FACS), or in situ barcoding including split‐pool barcoding methods.49, 50 Isolation of cells can also be accomplished using microfluidics that either use circuits to distribute cells into nanowells (ie, Fluidigm C1 ) or isolate cells into nanoliter oil‐based droplets (ie, 10x Genomics Chromium ). Most methods, after isolating cells into either plates (ie, STRT‐Seq, CEL‐Seq2, and SMART‐Seq2 ) or droplets (ie, inDrops, Drop‐seq, 10x Genomics Chromium ), require cell lysis to release mRNA molecules. Oligonucleotide primers with barcodes and oligo‐dT sequences are added to capture the 3′‐end mRNA poly‐A tail, give each mRNA a cell‐type‐specific barcode, and amplify mRNA. Within these primers, some methods also contain a unique molecular identifier (UMI) that labels each mRNA as a unique molecule. Recent developments in scRNA‐Seq methodologies have eliminated the need to isolate single cells or nuclei by using split‐pool barcoding.49, 50 This method allows multiple cells to be placed in individual wells containing unique barcodes and through multiple mixing, well distributions, and barcoding reactions each mRNA within each cell receives a unique cell‐specific barcode. While this technique allows for sequencing of hundreds of thousands of cells, including multiple tissue samples and fixed cells or nuclei, the large number of mixing reactions and pipetting required consumes valuable time and may perturb the native transcriptome, so precious tissue may not be amenable to this technique.

Diagram of scRNA‐Seq, single‐cell RNA sequencing (scRNA‐Seq) workflow. A, Single‐cell isolation involves generation of a single‐cell suspension. Cells are isolated by microdissection and micropipette, microfluidic circuits, droplets, or split pool barcoding. After single‐cell isolation, cells are compartmentalized into wells or droplet for library preparation depending on the scRNA‐Seq platform that is used. B, mRNA capture and barcoding involves cell lysis releasing mRNA which is barcoded and reverse transcribed into cDNA. Polymerase chain reaction or in situ transcription is used to amplify cDNA, C, library preparation involves pooling and fragmentation of cDNA and addition of adaptors used for, D, next‐generation sequencing. mRNA reads are aligned to known genes and genes are mapped back to their cell of origin. To quantify gene expression, a matrix with cells on the x‐axis and genes on the y‐axis is generated with read counts for each gene. This matrix is then used for, E, bioinformatic analysis including: quality control, filtering of unhealthy cells, normalization and scaling of mRNA read counts, principal component analysis to determine genes responsible for the most variation between cells, dimensionality reduction, unbiased clustering, followed by data visualization
scRNA‐Seq WORKFLOW
After RNA is barcoded, it is reverse transcribed into cDNA and cDNA is amplified by polymerase chain reaction or in vitro transcription. In most cases, amplified cDNA from each cell is then pooled for preparation of sequencing libraries where cDNA molecules are fragmented and adaptor sequences are added for further amplification and sequencing. Methods utilized for library preparation from amplified cDNA are varied and reviewed elsewhere including a review dedicated to discussing the details of current methods for library preparation by Head et al.39, 40, 41, 42, 43 One clinically important distinction between library preparation methods is that various methodologies result in 3′‐end, 5′‐end, or full‐length sequencing data.34, 39 Most droplet‐based protocols are biased to sequencing of the 3′‐end of mRNA resulting in loss of genetic information from the 5′‐end37, 38; however, some plate‐based techniques, like SMART‐Seq2, use template switching during reverse transcription to amplify the full length strand of mRNA allowing for the detection of different mRNA isoforms, splicing variants, single‐nucleotide polymorphisms, and variant mutations that would go undetected with 3′‐end enriched sequencing. 
Plate‐based techniques, due to lower cell numbers, allow for extremely high sensitivity and read depth (number of genes detected per cell) and this incredible depth of sequencing makes these techniques clinically useful for precious patient samples of low cell number; however, the low throughput and high cost per cell make them less clinically feasible. In contrast, due to the microscopic size of the droplets created by microfluidics, the reagent cost drops significantly and with no limit on the number of droplets created, the throughput is much higher; however, the read depth is usually lower than plate‐based methodologies because a larger number of single‐cell libraries can be pooled and loaded on the same sequencing lane. The Chromium (10x Genomics) is becoming the gold‐standard microfluidic device because of its unprecedented throughput, speed, and low cost per cell. This parallels our group's experience in transitioning from circuit‐based to nanoliter droplet‐based microfluidic single‐cell RNA‐Seq systems.11, 15 The ability to sequence a greater number of cells allows for a more comprehensive picture of the transcriptomic profile as typically only 5% to 15% of the mRNA within a given single cell are captured, making high‐throughput technologies an attractive platform for clinical use. Table 2 compares different single cell capture platforms. For a more in‐depth review of the intricacies of designing a single‐cell experiment and selecting the appropriate platforms readers are pointed to excellent reviews by Nguyen et al and Lafzi et al. 

Brief comparison of single‐cell RNA sequencing platforms
scRNA‐Seq WORKFLOW
To analyze scRNA‐Seq data, computational bioinformatic analysis is necessary to convert gene expression matrices (Figure 1E) into a more interpretable graphic format. Normalization, quality control measures, dimensionality reduction, and unbiased clustering algorithms are applied to cluster cells by their potential cellular identity. Seurat (https://satijalab.org/seurat/) is a commonly used bioinformatics pipeline for scRNA‐Seq data analysis and visualization that outputs plots depicting clusters of cells grouped together based on similarities in their gene expression58, 59 (Figure 1E). These plots aid in characterizing tissue heterogeneity by identifying novel and known cell types as well as detecting expression of genes that are unique to a specific cell type or unknown to be previously expressed by a certain cell type within a given tissue. For a thorough description of computational analysis for complex scRNA‐Seq data, we encourage readers to see reviews by Hwang et al, Shafer, and Chen et al. 
CLINICAL APPLICATIONS OF scRNA‐Seq FOR OTOLARYNGOLOGY
scRNA‐Seq techniques have already advanced the field of auditory research by identifying heterogeneity of cell types within the inner ear, including those involved in hair cell regeneration, hidden hearing loss, and the pathogenesis of enlarged vestibular aqueducts8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and are becoming more widespread in their utilization in human tissues.23, 25, 26, 60, 61 Fostering awareness of this technique and its applications for basic biological discovery in diseases managed by practicing otolaryngologists may assist with diagnosis, prognosis, and potentially personalized treatment recommendations in future clinical practice. To demonstrate these clinical applications, we will use three examples of diseases related to otolaryngology where scRNA‐Seq has already been applied to human samples including chronic sinusitis, oral HNSCC, and melanoma; but we encourage otolaryngologists to think about how scRNA‐Seq can be applied to their disease of interest as we walk through its potential applications.
Detecting tissue heterogeneity to understand disease pathogenesis
Diseases, like head and neck squamous cell carcinoma, are composed of a complex group of heterogenous cell types, consisting of both native and disease causing, in this case cancer causing, cells as well as cells within the microenvironment, including immune cells, extracellular matrix, fibroblasts, stem cells, and vasculature, which all communicate to play a role in tumorogenesis.62, 63, 64 Cellular heterogeneity in the setting of cancers of the head and neck and in the setting of chronic inflammation such as chronic rhinosinusitis has been implicated in disease maintenance or recurrence, patient variability in drug efficacy, and the failure of clinical drug trials.65, 66 As an example, understanding the interplay between diseased and native cell types, such as the case between tumor cells and native cell types (ie, fibroblasts, immune cells) in the tumor microenvironment (TME), will improve the knowledge base surrounding cancerous cell types and their interplay with the microenvironment. Using unbiased clustering of each individual cell followed by cell type identification of each cluster by expression of known marker genes, scRNA‐Seq data can distinguish disease‐causing cells from native tissue and reveal complex heterogeneity within diseased tissue samples.24, 25 
Heterogeneity of malignant tumor cells
Several scRNA‐Seq studies on human tumor samples have identified tumor heterogeneity by demonstrating subpopulations within malignant tumor cells.67, 68, 69, 70 By interrogating the gene expression programs within malignant cell populations of 18 metastatic melanomas, a landmark scRNA‐Seq study by Tirosh et al identified the existence of a subpopulation of malignant cells with a high level of AXL cell surface receptor tyrosine kinase program expression, a set of genes related to drug resistance and melanoma cancer stem cell (CSC) maintenance. The AXL‐high subpopulation signature could not be detected in bulk RNA‐Seq data from melanoma tumors which proves the utility of scRNA‐Seq in identifying heterogenous cells responsible for the pathogenesis of metastasis and intrinsic drug resistance. With the availability of AXL inhibitors such as BGB324 that show efficacy in decreasing human soluble AXL levels and treating human‐derived spheroid melanoma tumors,71, 72 detection of this signature by scRNA‐Seq in tumors of the head and neck may help identify which patients would respond best in human clinical trials. The influence of tumor heterogeneity on disease pathogenesis has also been demonstrated in oral HNSCC by Puram et al in the first scRNA‐Seq study relevant to otolaryngology. Clustering of malignant cells from 10 HNSCC showed that malignant cells clustered based on patient, as we confirmed in our own analysis of their published data set using Seurat58, 59 (Figure 2A‐C). This suggests that clonal evolution of HNSCC malignant cells is unique to each patient and each tumor should be treated therapeutically as such. As in melanoma, malignant HNSCC cells did contain groups of cells with shared gene expression profiles or subpopulations, including one expressing high levels of extracellular matrix genes that also expressed markers related to a partial epithelial‐to‐mesenchymal transition (EMT). The authors suggest that it is possible this partial‐EMT expressing subpopulation is responsible for causing invasion and lymph node metastasis in HNSCC. To confirm the presence of malignant intratumoral heterogeneity in HNSCC, we performed our own scRNA‐Seq unbiased clustering analysis of cells from one HNSCC patient, T25, which displayed three subpopulations of malignant cells within one tumor that varied in their gene expression (Figure 2D).

Example of our new analysis of scRNA‐Seq, single‐cell RNA sequencing (scRNA‐Seq) data from Puram et al head and neck squamous cell carcinoma (HNSCC) using Seurat. A,B, Unbiased clustering of cells from 10 HNSCC patients. Dotted circles represent clusters of cells from the same cell type. Cell types were determined by cluster‐specific expression of known cell‐type markers. Seurat identified 25 independent clusters of similar cells, A. Each cell was colored by the patient of origin with malignant cell clusters consisting of cells from the same patient while tumor microenvironment (TME) cells consisted of cells from multiple HNSCC patients, B. TME cells are demarcated by the dotted outlines while the malignant cells are denoted by the absence of the dotted outlines, B. C, Unbiased clustering of only the malignant cells from the HNSCC data. Cells were colored by the patient of origin. This analysis shows that malignant cells cluster by patient. D, Unbiased clustering of all cells from HNSCC patient T25 showing that malignant cells are distributed in multiple clusters or subpopulations (malignant cells 1‐3 outlined in solid grey). Cancer associated fibroblasts (CAF) are outlined in dotted black. E, Unbiased clustering of only the TME cells from the HNSCC data. Cells are colored based on cell‐type identity demonstrating TME heterogeneity
Heterogeneity of cells in the microenvironment
Single‐cell profiles obtained from tumor tissue will consist of not only individual malignant tumor cells but also cells from the TME, which consists of blood vessels, immune cells, fibroblasts, signaling molecules, and extracellular matrix that surrounds malignant cells, allowing for identification of heterogeneity within these cell types. Detection and interrogation of TME subpopulations within multiple tumor types allows for insight into potential stroma‐tumor communication programs that may be playing a role in disease pathogenesis.25, 74, 75 In contrast to malignant HNSCC cells that clustered by patient, the study by Puram et al found that HNSCC TME cells were not patient‐specific and clustered based on cell type as we also confirmed in our own analysis showing that each TME cluster (Figure 2E) consisted of cells from multiple patients (Figure 2B). Interestingly, HNSCC fibroblasts displayed vast heterogeneity including two subpopulations that expressed genes affiliated with cancer‐associated fibroblasts (CAF) (Figure 2E). Genes that were highly differentially expressed in CAF included mesenchymal and transforming growth factor‐beta (TGF‐β) signaling genes whose expression was shared by the malignant HNSCC partial‐EMT subpopulation, suggesting that CAF‐malignant cell signaling may be involved in promoting invasion and metastasis in HNSCC. Since most HNSCC tumors contain similar TME subpopulations, these cells could represent shared disease pathogenesis between all HNSCC patients that could be targeted by a similar drug program.
The sensitivity of scRNA‐Seq to detect TME heterogeneity also allows for the detection of rare cell types such as stem‐ or progenitor‐like cells responsible for disease maintenance70, 76, 77 or rare immune cell subpopulations involved in evasion of the immune response.74, 75, 78, 79 Ordovas‐Montanes et al performed scRNA‐Seq on ethmoid sinus tissue samples from patients with chronic rhinosinusitis with (CRwNP) or without (CRsNP) nasal polyps and performed trajectory analysis on individual basal epithelial cells to computationally determine a potential pathogenic developmental cell state in CRwNP. Trajectory analysis examines gene expression and places cells along a developmental timeline based on unique gene expression. Their analysis showed that most basal cells in CRwNP are stuck in a progenitor state expressing, among other genes, increased Wnt‐signaling genes that could potentially be responsible for polyp development. Another rare cell type that can be detected by scRNA‐Seq are CSC, which have been shown to play a role in the creation of HNSCC heterogeneity, resistance to drug therapy, disease recurrence, and metastasis making the elimination of these cells critical to eradicating residual disease in HNSCC. In support of this, Puram et al were able to detect varying levels of epithelial gene expression within malignant HNSCC subpopulations reflecting variable levels of epithelial differentiation. Various biomarkers to identify CSC have been suggested and we demonstrate how CSC may be identified within scRNA‐Seq data using these known markers in one HNSCC patient, T25 (Figure 3). Other clinically relevant rare TME subpopulations that were detected by Puram et al in HNSCC and Tirosh et al in melanoma were CD4+ T‐regulatory cells and exhausted T cells (Figure 2E), which likely inhibit the antitumor immune response and prevent the efficacy of immune‐modulating therapy.

Cancer stem cell (CSC) markers present in head and neck squamous cell carcinoma (HNSCC) patient T25. The level of expression of different CSC markers are indicated on the t‐distributed stochastic neighborhood embedding (tSNE) plot of all HNSCC cells from patient T25 (Figure 2D). Cells that express CSC genes are colored in gradations of purple depending on their expression level, with blue representing the highest expression level. Malignant clusters (solid grey outline in Figure 2D) vary in their expression of CSC markers and some markers are also expressed in cancer‐associated fibroblasts (CAF) (dotted black outline in Figure 2D)
Identification of disease biomarkers
Obtaining single‐cell profiles from patient biopsy samples, for example HNSCC tumors or sinus mucosa, may aid in a more sensitive diagnosis by allowing physicians to identify unique cell‐type‐specific signatures or biomarkers for disease subtyping. Disease subtyping is crucial in tumor diagnostic and prognostic decision making in HNSCC and a few studies have shown that scRNA‐Seq‐derived signatures that account for tumor heterogeneity can more accurately classify tumor subtypes.67, 74 Conventional bulk RNA‐Seq analysis has classified HNSCC into four subtypes based on molecular gene signatures: basal, classical, atypical, or mesenchymal. By scoring each HNSCC cell in their scRNA‐Seq data based on expression levels of previously defined HNSCC subtype signatures, Puram et al showed that malignant HNSCC cells only mapped to basal, classical, or atypical signatures and the mesenchymal subtype was not present in any HNSCC malignant cells. However, the mesenchymal subtype scored highest in fibroblasts of the TME suggesting that this subtype truly represents HNSCC with high levels of TME gene expression highlighting the need for tumor classification systems, potentially based on compiled scRNA‐Seq data that account for tissue heterogeneity.
Investigating gene expression within heterogenous cell clusters detected by scRNA‐Seq can be used to identify cell‐type‐specific known or novel biomarkers associated with tumor metastasis, prognosis, and survival.76, 77, 83, 84 Puram et al showed the novel partial‐EMT signature detected in a subset of malignant cells was present in existing bulk RNA‐Seq HNSCC tumor data and the levels of expression of this signature were associated with invasion, metastasis, and poor prognosis. Currently, most head and neck cancer patients undergo aggressive nodal dissection, a procedure associated with significant morbidity, based on unreliable markers such as tumor size or grade.85, 86 Determining the presence and extent of the partial‐EMT signature in HNSCC patients could aid in weighing the risks of nodal dissection in patients with lower risk of metastasis. Here we also show that recently published HNSCC prognostic signatures associated with decreased survival, metastasis, and lack of response to radiotherapy can be detected in single cells from HNSCC patient T25 (Figure 4). Further characterization of all head and neck tumors using scRNA‐Seq could reveal other novel signatures associated with prognosis, drug response, or survival.

Known prognostic markers present in head and neck squamous cell carcinoma (HNSCC) patient T25. Feature plots of the original clustering from patient T25 (Figure 2D). Cells that express known metastatic, A, poor survival, B, or radiation sensitivity, C, marker genes are colored in gradations of purple depending on their expression level, with blue representing the highest expression level
Identification of disease biomarkers
scRNA‐Seq may also detect cell‐type‐specific gene expression associated with the severity of disease. For example, Ordovas‐Montanes et al was also able to detect a novel cell‐type‐specific change distinguishing between severe CRwNP and less severe CRsNP. They found high interleukin (IL)‐4/IL‐13 expression within secretory epithelial cells from patients with severe disease and interferon (IFN)‐alpha/IFN‐gamma expression in less severe disease. Not only does this represent a cell‐type‐specific drug target, but also detection of the severe disease program by scRNA‐Seq in chronic rhinosinusitis (CR) patient biopsies could influence treatment decisions including surgical intervention. Ordovas‐Montanes et al were also able to track changes in basal‐cell transcriptomes before and after treatment with an IL‐4Rα inhibitor. While many polyp‐specific genes were downregulated in disease‐causing basal cells, they also identified a basal‐cell polyp‐specific program that did not change with treatment revealing a drug‐resistant signature that could be interrogated for new druggable targets. This demonstrates how scRNA‐Seq can also be conducted before and after treatment to monitor drug response, emergence of resistance, and identify markers of these events.
Patient‐specific drug selection
Heterogeneity detected by scRNA‐Seq can also be used to generate novel or look for known biomarkers for drug sensitivity or resistance that will aid in selecting which patients should be treated with specific drugs.83, 90, 91 While drug targets can been analyzed in cohorts of scRNA‐Seq samples, there is a need for analysis of heterogeneity at the level of individual tumors to aid in patient‐specific drug selection, enrollment of patients in clinical trials, and analyzing ablation or alteration of specific cell types before and after drug treatment.92, 93, 94 Immunotherapy with immune checkpoint inhibitors or small‐molecule immune modulators is currently an active area of research in head and neck cancer and many clinical trials are aimed at using these drugs to subvert tumor‐induced immunosuppression.63, 95 As mentioned, both melanoma and HNSCC scRNA‐Seq studies revealed the presence of rare exhausted CD8+ T cells and developed an exhausted T‐cell signature; however, both authors found that each patient varied in their number of exhausted T cells and their expression of known exhaustion markers including receptors that inhibit the T‐cell immune response.23, 28 For example, in one melanoma tumor expression of coinhibitory CTLA4 receptor was absent from the exhausted T‐cell population; however, this patient had previously been treated with CTLA4 inhibitor, ipilimumab, and subsequently became resistant. Identification of T‐regulatory and T‐exhausted subpopulations through scRNA‐Seq can lead to the creation of novel drug‐response biomarkers or potential new drug targets within these cell types. Detecting biomarkers from single‐cell TME profiles of head and neck tumor patients may aid in determining which patients will respond best to immune checkpoint inhibitors or should be considered for various immunotherapy clinical trials.
Cell clusters generated from scRNA‐Seq data can also be analyzed for expression of known drug targets to determine if or which cell types express certain drug targets and how effective the drug might be in targeting all diseased subpopulations and/or pathogenic TME cells. We show how this can theoretically be done on a patient‐specific basis by using the HNSCC data from patient T25 and displaying the cells that express the targets of current drugs used to treat HNSCC (Figure 5).86, 96 For example, epidermal growth factor receptor (EGFR) is the target of EGFR inhibitors such as cetuximab, and this gene is expressed in malignant cells from patient T25 suggesting that these cells are likely susceptible to this drug. Ideally, if a given drug does not target all subpopulations of malignant cells or a particularly pathogenic cell type of the TME such as CAF or CSC, then other drug targets could be identified within these populations and these drugs could be added to the drug cocktail until all cells are targeted.

Drug targets for commonly used and new head and neck squamous cell carcinoma (HNSCC) drugs used to treat HNSCC in patient T25. Feature plots of the original clustering from patient T25 (Figure 2D). Cells that express drug target genes are colored in gradations of purple depending on their expression level, with blue representing the highest expression level. Drug target gene is written in the black in the plot title and the drug that targets it is written at the bottom of the plot in red. A, Drugs that show strong cell‐type‐specific target expression in patient T25. B, Drugs that show nonspecific or weak target expression in patient T25
Patient‐specific drug selection
One potential agnostic approach to finding new druggable targets in malignant subpopulations or TME cells is to look for the presence of genetic targets of FDA‐approved drugs or small molecules within clusters derived from scRNA‐Seq data that could be repurposed for use in head and neck cancer or other otolaryngologic disease. A new database called Pharos describes 20 000 gene/protein targets and the availability of FDA‐approved drugs or small‐molecule ligands for each target. To demonstrate a possible use of this resource, we used the batch search option to search for druggable targets within the top marker genes from HNSCC patient T28's one malignant and two CAF clusters (Figure 6). We found 67 genes that could be targeted with FDA‐approved drugs in the malignant population, 28 in the first CAF subpopulation, and 19 in the second CAF subpopulation (full data in Table 3). Interestingly, some drug targets overlapped between these populations suggesting shared signaling between malignant and TME cells that could be targeted by a single drug. Of note, while we only analyzed a single patient's tumor sample, this methodology could be applied across all 18 HNSCC and 5 lymph node samples in this cohort to make a more informed judgment on effective drug combinations for HNSCC and effect on patient outcomes. However, these data demonstrate the potential use of scRNA‐Seq cluster expression profiles to identify previously approved drugs and available small molecules that could have activity against specific cells for the treatment of refractory disease or generating data for new clinical drug trials.

Potential drug targets and drugs that inhibit them for each malignant cell and cancer‐associated fibroblast (CAF) cluster in head and neck squamous cell carcinoma (HNSCC) patient T28. A, Unbiased clustering of HNSCC cells from patient T28. Potential cell‐type specific drug targets are shown under each cluster. Example antagonistic drugs that target each malignant and CAF cluster are shown. B, Feature plots of the original clustering of HNSCC patient T28 showing cells that cluster specific drug target genes colored in gradations of purple depending on their expression level, with blue representing the highest expression level. The drugs that target these genes are written in color under each plot and are shown in Figure 6A. Full drug target data are available in Table 3 
Pharos cell‐type‐specific druggable targets from HNSCC patient T28
Limitations of scRNA‐Seq in clinical medicine
While scRNA‐Seq will help in the transition towards personalized medicine, challenges with its translation to the clinic remain including lack of large cohorts of scRNA‐Seq human patient samples, cost, user‐friendliness, and tissue preservation. The use of scRNA‐Seq on individual patient tumors to inform drug selection is currently possible as we described above and as reported in a recent case study using scRNA‐Seq of skin and blood samples from a patient with refractory drug‐induced hypersensitivity syndrome to identify patient‐specific drug targets and successfully repurpose tofacitinib to treat this difficult case. While both our findings and the findings from case reports would need to be extended by performing studies on larger cohorts to make conclusions about prognostic outcomes from this type of drug selection, they demonstrate that individualized drug selection based on sequencing from a single patient sample is feasible and has been successful. More of these types of studies are needed to conclude that personalized drug selection and drug repurposing using scRNA‐Seq results in better patient outcomes. 
The cost of scRNA‐Seq varies based on the chosen methodology and depends on the cost of equipment, reagents, and sequencing. For any platform, a higher number of cells results in higher costs. The costs of isolation and sequencing per cell have dropped significantly, but the throughput of sequencing machines has also increased, so the cost per run with more cells remains high. Most isolation platforms are also only available at basic science laboratories and would require a large upfront investment to purchase for hospital use.
In addition to cost, analysis of scRNA‐Seq data requires users to have basic bioinformatic knowledge and coding skills; however, user‐friendly bioinformatic pipelines such as the gene expression analysis resource (gEAR) (https://umgear.org/), and scRNA‐Seq workbench created to facilitate data sharing and visualization, are currently available. Further standardization of these pipelines will be necessary for clinical use. Tissue preservation is also an issue due to fragility and the small amount of time that cells remain viable. Procurement of tumor biopsies and surgical specimens may not always be predictable or fast and, currently, the use of frozen tissue samples or methanol tissue fixation on scRNA‐Seq platforms is in its infancy; however, a few options to aid in tissue preservation are available. Temporary tissue stabilization buffers, PrepProtect (Miltenyi Biotec) or RNAlater (Thermo Fisher Scientific), can preserve cells for sequencing for 48 hours. While these methods may not be compatible with scRNA‐Seq if used in combination with sNuc‐Seq experiments, which have the advantage of lysing cells and only sequencing mRNA from the nucleus, RNALater tissue preservation of fresh or frozen patient samples can result in useful data. Cell lysis used in sNuc‐Seq allows for potentially more efficient cell type delineation including even the most interdigitated cell types and minimizes the skewing effect of degraded mRNA or cell‐stress response genes on the data.11, 60, 61, 100, 102 These advantages potentially make sNuc‐Seq an excellent option for precious patient samples including human vestibular and cochlear tissues.
CONCLUSIONS
Despite these limitations and the lack of widespread clinical availability, the rapid advances in scRNA‐Seq throughput and bioinformatic pipelines are making the ability for clinicians to perform scRNA‐Seq on patient tissue a reality in the next few years. We have described how this technology applied to three different otolaryngologic diseases, CR and head and neck cancers HNSCC and melanoma, can make otolaryngologists more aware of the cellular heterogeneity within their patients' diseased tissue. Further analysis of tissue heterogeneity across human patient samples can help otolaryngologist understand basic human biology and disease pathogenesis, make diagnostic and prognostic decisions, and select personalized drug therapy combinations.
We demonstrate the feasibility and importance of conducting scRNA‐Seq on human patient samples to generate new insight into healthy and diseased tissue biology and the possibility for this technique to move patient care further into the era of precision medicine where treatment can be tailored to a patient's unique transcriptome. Currently, a human single‐cell atlas project is underway with the goal of creating single‐cell profiles for every normal and pathologic cell in the human body. It is not difficult to imagine a time where physicians could conduct scRNA‐Seq on each pathologic patient sample and compare it to the normal transcriptome within this atlas to determine which cells are diseased and how other patients with similar diseased‐cell transcriptomes responded to various treatments.
We hope to inform otolaryngologists of the potential of this technique to further our practice in the future and inspire ideas for how they may use it now to gain insight into both healthy and diseased tissues that may have dramatic impacts on clinical care. Because there have been so few scRNA‐Seq studies of human samples in our field, we argue for immediate use of scRNA‐Seq on more otolaryngologic patient samples to not only aid in more sensitive diagnosis and drug selection, but also to contribute single‐cell profiles to a larger database like the single‐cell atlas which will help identify new disease biomarkers associated with prognosis, subtypes, or drug resistance. This technique can be applied immediately to human auditory hair cells and spiral ganglion neurons to characterize human deafness genes and the cell types that express them. scRNA‐Seq can also be readily applied to characterize benign and malignant tumors of the head and neck, such as HNSCC and vestibular schwannomas, to identify tumor heterogeneity among cohorts of patients as these pathologies stand to benefit the most from identification of new druggable targets, selection of more effective drugs, or selection of patients for clinical trials to optimize the balance between medical and surgical treatments. As this technique becomes more readily available, other otolaryngologic problems may also be answered. These uses will not only increase our understanding of the cellular diversity involved in these diseases, but also have the potential to change the way we diagnose, monitor, and treat patients in the field of otolaryngology.
CONFLICT OF INTEREST
The authors declare no potential conflict of interest.

 Data S1. Example R script for processing data from Puram et al's data set.
Click here for additional data file.
 Data S2. Supplementary descriptive methods detailing bioinformatic analyses utilized to analyze data from Puram et al's data set.
Click here for additional data file.
