Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. However, the duration of clinical response is often limited, and patients ultimately develop drug resistance. Here, we use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors. These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Recurrently overrepresented ontologies in genes that are differentially expressed between drug tolerant cell populations and drug sensitive cells include epithelial-to-mesenchymal transition, epithelium development, vesicle mediated transport, drug metabolism and cholesterol homeostasis. We show analysis of identified markers using the LINCS database to predict and functionally validate small molecules that target selected drug tolerant cell populations. In combination with EGFR inhibitors, crizotinib inhibits the emergence of a defined subset of EGFR inhibitor-tolerant clones. In this study, we describe the spectrum of changes associated with drug tolerance and inhibition of specific tolerant cell subpopulations with combination agents.

The function of Retinoblastoma tumor suppressor (pRB) is greatly influenced by the cellular context, therefore the consequences of pRB inactivation are cell-type-specific. Here we employ single cell RNA-sequencing (scRNA-seq) to profile the impact of an Rbf mutation during Drosophila eye development. First, we build a catalogue of 11,500 wild type eye disc cells containing major known cell types. We find a transcriptional switch occurring in differentiating photoreceptors at the time of axonogenesis. Next, we map a cell landscape of Rbf mutant and identify a mutant-specific cell population that shows intracellular acidification due to increase in glycolytic activity. Genetic experiments demonstrate that such metabolic changes, restricted to this unique Rbf mutant population, sensitize cells to apoptosis and define the pattern of cell death in Rbf mutant eye disc. Thus, these results illustrate how scRNA-seq can be applied to dissect mutant phenotypes.

In Drosophila , the wing disc‐associated muscle precursor cells give rise to the fibrillar indirect flight muscles (IFM) and the tubular direct flight muscles (DFM). To understand early transcriptional events underlying this muscle diversification, we performed single‐cell RNA‐sequencing experiments and built a cell atlas of myoblasts associated with third instar larval wing disc. Our analysis identified distinct transcriptional signatures for IFM and DFM myoblasts that underlie the molecular basis of their divergence. The atlas further revealed various states of differentiation of myoblasts, thus illustrating previously unappreciated spatial and temporal heterogeneity among them. We identified and validated novel markers for both IFM and DFM myoblasts at various states of differentiation by immunofluorescence and genetic cell‐tracing experiments. Finally, we performed a systematic genetic screen using a panel of markers from the reference cell atlas as an entry point and found a novel gene, Amalgam which is functionally important in muscle development. Our work provides a framework for leveraging scRNA‐seq for gene discovery and details a strategy that can be applied to other scRNA‐seq datasets.

The receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. Here, we profile the Drosophila larval brain by single cell RNA-sequencing and identify Amalgam (Ama), encoding a cell adhesion protein of the immunoglobulin IgLON family, that regulates the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood-brain barrier (BBB) that leads to hemocyte infiltration and neuronal death. We show that Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPOUTY2 in glioblastoma cell lines suggesting that the relationship between Ama and Sty is conserved.

Single-cell RNA sequencing (scRNA-seq) has revolutionized cell biology by enabling the profiling of transcriptomes at a single-cell resolution, leading to important discoveries that have advanced our understanding of cellular and tissue heterogeneity, developmental trajectories, and disease progression. Despite these important advances, scRNA-seq is limited to measuring the transcriptome providing a partial view of cellular function. To address this limitation, multimodal scRNA-seq assays have emerged, allowing for the simultaneous measurement of RNA expression and protein. Intracellular Transcriptomic and Protein Sequencing (InTraSeq), a novel multimodal scRNA-seq technology described here, enables the concurrent measurement of mRNA, surface markers, cytoplasmic proteins, and nuclear proteins within individual cells through oligo-barcoded antibodies. This method offers a comprehensive approach to studying cellular function by combining RNA and protein profiling from the same sample and utilizing a relatively simple protocol. The InTraSeq method enables researchers to expand their view of critical intracellular protein expression including post-translational modifications (PTMs) and transcription factors, allowing for the identification of novel cellular subtypes and states that may be obscured by RNA-based analyses alone. This is particularly valuable in understanding the heterogeneity of cell populations and identifying distinct functional states. In this report, we used InTraSeq to characterize the complex cellular states and regulatory mechanisms during Th17 cell differentiation. We simultaneously profiled RNA and protein expression in over 85,000 cells, capturing transcriptional changes, changes in protein expression and the dynamics of signaling pathways at a high resolution. Our results revealed novel insights into Th17 cell differentiation, including the identification of key regulatory factors and their target genes. By simultaneously measuring mRNA, extra and intra-cellular proteins, signaling proteins, and PTMs, InTraSeq offers a comprehensive understanding of cellular processes and enables the identification of novel regulatory mechanisms.