Molecular cartography uncovers evolutionary and microenvironmental dynamics in sporadic colorectal tumors.

Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.

Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

Combinatorial Transcriptional Profiling of Mouse and Human Enteric Neurons Identifies Shared and Disparate Subtypes In Situ.

BACKGROUND & AIMS: The enteric nervous system (ENS) coordinates essential intestinal functions through the concerted action of diverse enteric neurons (ENs). However, integrated molecular knowledge of EN subtypes is lacking. To compare human and mouse ENs, we transcriptionally profiled healthy ENS from adult humans and mice. We aimed to identify transcripts marking discrete neuron subtypes and visualize conserved EN subtypes for humans and mice in multiple bowel regions. METHODS: Human myenteric ganglia and adjacent smooth muscle were isolated by laser-capture microdissection for RNA sequencing. Ganglia-specific transcriptional profiles were identified by computationally subtracting muscle gene signatures. Nuclei from mouse myenteric neurons were isolated and subjected to single-nucleus RNA sequencing, totaling more than 4 billion reads and 25,208 neurons. Neuronal subtypes were defined using mouse single-nucleus RNA sequencing data. Comparative informatics between human and mouse data sets identified shared EN subtype markers, which were visualized in situ using hybridization chain reaction. RESULTS: Several EN subtypes in the duodenum, ileum, and colon are conserved between humans and mice based on orthologous gene expression. However, some EN subtype-specific genes from mice are expressed in completely distinct morphologically defined subtypes in humans. In mice, we identified several neuronal subtypes that stably express gene modules across all intestinal segments, with graded, regional expression of 1 or more marker genes. CONCLUSIONS: Our combined transcriptional profiling of human myenteric ganglia and mouse EN provides a rich foundation for developing novel intestinal therapeutics. There is congruency among some EN subtypes, but we note multiple species differences that should be carefully considered when relating findings from mouse ENS research to human gastrointestinal studies.

Succinate Produced by Intestinal Microbes Promotes Specification of Tuft Cells to Suppress Ileal Inflammation.

BACKGROUND & AIMS: Countries endemic for parasitic infestations have a lower incidence of Crohn's disease (CD) than nonendemic countries, and there have been anecdotal reports of the beneficial effects of helminths in CD patients. Tuft cells in the small intestine sense and direct the immune response against eukaryotic parasites. We investigated the activities of tuft cells in patients with CD and mouse models of intestinal inflammation. METHODS: We used microscopy to quantify tuft cells in intestinal specimens from patients with ileal CD (n = 19), healthy individuals (n = 14), and TNFΔARE/+ mice, which develop Crohn's-like ileitis. We performed single-cell RNA sequencing, mass spectrometry, and microbiome profiling of intestinal tissues from wild-type and Atoh1-knockout mice, which have expansion of tuft cells, to study interactions between microbes and tuft cell populations. We assessed microbe dependence of tuft cell populations using microbiome depletion, organoids, and microbe transplant experiments. We used multiplex imaging and cytokine assays to assess alterations in inflammatory response following expansion of tuft cells with succinate administration in TNFΔARE/+ and anti-CD3E CD mouse models. RESULTS: Inflamed ileal tissues from patients and mice had reduced numbers of tuft cells, compared with healthy individuals or wild-type mice. Expansion of tuft cells was associated with increased expression of genes that regulate the tricarboxylic acid cycle, which resulted from microbe production of the metabolite succinate. Experiments in which we manipulated the intestinal microbiota of mice revealed the existence of an ATOH1-independent population of tuft cells that was sensitive to metabolites produced by microbes. Administration of succinate to mice expanded tuft cells and reduced intestinal inflammation in TNFΔARE/+ mice and anti-CD3E-treated mice, increased GATA3+ cells and type 2 cytokines (IL22, IL25, IL13), and decreased RORGT+ cells and type 17 cytokines (IL23) in a tuft cell-dependent manner. CONCLUSIONS: We found that tuft cell expansion reduced chronic intestinal inflammation in mice. Strategies to expand tuft cells might be developed for treatment of CD.

Quantitative assessment of cell population diversity in single-cell landscapes.

Single-cell RNA sequencing (scRNA-seq) has become a powerful tool for the systematic investigation of cellular diversity. As a number of computational tools have been developed to identify and visualize cell populations within a single scRNA-seq dataset, there is a need for methods to quantitatively and statistically define proportional shifts in cell population structures across datasets, such as expansion or shrinkage or emergence or disappearance of cell populations. Here we present sc-UniFrac, a framework to statistically quantify compositional diversity in cell populations between single-cell transcriptome landscapes. sc-UniFrac enables sensitive and robust quantification in simulated and experimental datasets in terms of both population identity and quantity. We have demonstrated the utility of sc-UniFrac in multiple applications, including assessment of biological and technical replicates, classification of tissue phenotypes and regional specification, identification and definition of altered cell infiltrates in tumorigenesis, and benchmarking batch-correction tools. sc-UniFrac provides a framework for quantifying diversity or alterations in cell populations across conditions and has broad utility for gaining insight into tissue-level perturbations at the single-cell resolution.

Dual indexed library design enables compatibility of in-Drop single-cell RNA-sequencing with exAMP chemistry sequencing platforms.

BACKGROUND: The increasing demand of single-cell RNA-sequencing (scRNA-seq) experiments, such as the number of experiments and cells queried per experiment, necessitates higher sequencing depth coupled to high data quality. New high-throughput sequencers, such as the Illumina NovaSeq 6000, enables this demand to be filled in a cost-effective manner. However, current scRNA-seq library designs present compatibility challenges with newer sequencing technologies, such as index-hopping, and their ability to generate high quality data has yet to be systematically evaluated. RESULTS: Here, we engineered a dual-indexed library structure, called TruDrop, on top of the inDrop scRNA-seq platform to solve these compatibility challenges, such that TruDrop libraries and standard Illumina libraries can be sequenced alongside each other on the NovaSeq. On scRNA-seq libraries, we implemented a previously-documented countermeasure to the well-described problem of index-hopping, demonstrated significant improvements in base-calling accuracy on the NovaSeq, and provided an example of multiplexing twenty-four scRNA-seq libraries simultaneously. We showed favorable comparisons in transcriptional diversity of TruDrop compared with prior inDrop libraries. CONCLUSIONS: Our approach enables cost-effective, high throughput generation of sequencing data with high quality, which should enable more routine use of scRNA-seq technologies.

Novel kidney dissociation protocol and image-based flow cytometry facilitate improved analysis of injured proximal tubules.

Flow cytometry studies on injured kidney tubules are complicated by the low yield of nucleated single cells. Furthermore, cell-specific responses such as cell cycle dynamics in vivo have conventionally relied on indirect immunohistochemistry and proximal tubule markers that may be downregulated in injury. Here, we report a new tissue dissociation protocol for the kidney with an early fixation step that greatly enhances the yield of single cells. Genetic labeling of the proximal tubule with either mT/mG "tomato" or R26Fucci2aR (Fucci) cell cycle reporter mice allows us to follow proximal tubule-specific changes in cell cycle after renal injury. Image-based flow cytometry (FlowSight) enables gating of the cell cycle and concurrent visualization of the cells with bright field and fluorescence. We used the Fucci mouse in conjunction with FlowSight to identify a discrete polyploid population in proximal tubules after aristolochic acid injury. The tissue dissociation protocol in conjunction with genetic labeling and image-based flow cytometry is a tool that can improve our understanding of any discrete cell population after injury.

Cytometry-based single-cell analysis of intact epithelial signaling reveals MAPK activation divergent from TNF-α-induced apoptosis in vivo.

Understanding heterogeneous cellular behaviors in a complex tissue requires the evaluation of signaling networks at single-cell resolution. However, probing signaling in epithelial tissues using cytometry-based single-cell analysis has been confounded by the necessity of single-cell dissociation, where disrupting cell-to-cell connections inherently perturbs native cell signaling states. Here, we demonstrate a novel strategy (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue-DISSECT) that preserves native signaling for Cytometry Time-of-Flight (CyTOF) and fluorescent flow cytometry applications. A 21-plex CyTOF analysis encompassing core signaling and cell-identity markers was performed on the small intestinal epithelium after systemic tumor necrosis factor-alpha (TNF-α) stimulation. Unsupervised and supervised analyses robustly selected signaling features that identify a unique subset of epithelial cells that are sensitized to TNF-α-induced apoptosis in the seemingly homogeneous enterocyte population. Specifically, p-ERK and apoptosis are divergently regulated in neighboring enterocytes within the epithelium, suggesting a mechanism of contact-dependent survival. Our novel single-cell approach can broadly be applied, using both CyTOF and multi-parameter flow cytometry, for investigating normal and diseased cell states in a wide range of epithelial tissues.

Atf4 protects islet ß-cell identity and function under acute glucose-induced stress but promotes ß-cell failure in the presence of free fatty acid.

Glucolipotoxicity, caused by combined hyperglycemia and hyperlipidemia, results in ß-cell failure and type 2 diabetes (T2D) via cellular stress-related mechanisms. Activating transcription factor 4 (Atf4) is an essential effector of stress response. We show here that Atf4 expression in ß-cells is dispensable for glucose homeostasis in young mice, but it is required for ß-cell function during aging and under obesity-related metabolic stress. Henceforth, aged Atf4- deficient ß-cells display compromised secretory function under acute hyperglycemia. In contrast, they are resistant to acute free fatty acid-induced loss-of identity and dysfunction. At molecular level, Atf4 -deficient ß-cells down-regulate genes involved in protein translation, reducing ß-cell identity gene products under high glucose. They also upregulate several genes involved in lipid metabolism or signaling, likely contributing to their resistance to free fatty acid-induced dysfunction. These results suggest that Atf4 activation is required for ß-cell identity and function under high glucose, but this paradoxically induces ß-cell failure in the presence of high levels of free fatty acids. Different branches of Atf4 activity could be manipulated for protecting ß-cells from metabolic stress-induced failure. Highlights: Atf4 is dispensable in ß-cells in young miceAtf4 protects ß-cells under high glucoseAtf4 exacerbate fatty acid-induced ß-cell defectsAtf4 activates translation but depresses lipid-metabolism.

Interleukin 13 Promotes Maturation and Proliferation in Metaplastic Gastroids.

BACKGROUND & AIMS: Type 2 innate lymphoid cells (ILC2s) and interleukin-13 (IL-13) promote the onset of spasmolytic polypeptide-expressing metaplasia (SPEM) cells. However, little is known about molecular effects of IL-13 in SPEM cells. We now sought to establish a reliable organoid model, Meta1 gastroids, to model SPEM cells in vitro. We evaluated cellular and molecular effects of ILC2s and IL-13 on maturation and proliferation of SPEM cells. METHODS: We performed single-cell RNA sequencing to characterize Meta1 gastroids, which were derived from stomachs of Mist1-Kras transgenic mice that displayed pyloric metaplasia. Cell sorting was used to isolate activated ILC2s from stomachs of IL-13-tdTomato reporter mice treated with L635. Three-dimensional co-culture was used to determine the effects of ILC2s on Meta1 gastroids. Mouse normal or metaplastic (Meta1) and human metaplastic gastroids were cultured with IL-13 to evaluate cell responses. Air-Liquid Interface culture was performed to test long-term culture effects of IL-13. In silico analysis determined possible STAT6-binding sites in gene promoter regions. STAT6 inhibition was performed to corroborate STAT6 role in SPEM cells maturation. RESULTS: Meta1 gastroids showed the characteristics of SPEM cell lineages in vitro even after several passages. We demonstrated that co-culture with ILC2s or IL-13 treatment can induce phosphorylation of STAT6 in Meta1 and normal gastroids and promote the maturation and proliferation of SPEM cell lineages. IL-13 up-regulated expression of mucin-related proteins in human metaplastic gastroids. Inhibition of STAT6 blocked SPEM-related gene expression in Meta1 gastroids and maturation of SPEM in both normal and Meta1 gastroids. CONCLUSIONS: IL-13 promotes the maturation and proliferation of SPEM cells consistent with gastric mucosal regeneration.

MTGR1 is required to maintain small intestinal stem cell populations.

Undifferentiated intestinal stem cells (ISCs) are crucial for maintaining homeostasis and resolving injury. Lgr5+ cells in the crypt base constantly divide, pushing daughter cells upward along the crypt axis where they differentiate into specialized cell types. Coordinated execution of complex transcriptional programs is necessary to allow for the maintenance of undifferentiated stem cells while permitting differentiation of the wide array of intestinal cells necessary for homeostasis. Previously, members of the myeloid translocation gene (MTG) family have been identified as transcriptional co-repressors that regulate stem cell maintenance and differentiation programs in multiple organ systems, including the intestine. One MTG family member, myeloid translocation gene related 1 (MTGR1), has been recognized as a crucial regulator of secretory cell differentiation and response to injury. However, whether MTGR1 contributes to the function of ISCs has not yet been examined. Here, using Mtgr1-/- mice, we have assessed the effects of MTGR1 loss specifically in ISC biology. Interestingly, loss of MTGR1 increased the total number of cells expressing Lgr5, the canonical marker of cycling ISCs, suggesting higher overall stem cell numbers. However, expanded transcriptomic and functional analyses revealed deficiencies in Mtgr1-null ISCs, including deregulated ISC-associated transcriptional programs. Ex vivo, intestinal organoids established from Mtgr1-null mice were unable to survive and expand due to aberrant differentiation and loss of stem and proliferative cells. Together, these results indicate that the role of MTGR1 in intestinal differentiation is likely stem cell intrinsic and identify a novel role for MTGR1 in maintaining ISC function.

Identification and multimodal characterization of a specialized epithelial cell type associated with Crohn's disease.

Crohn's disease (CD) is a complex chronic inflammatory disorder with both gastrointestinal and extra-intestinal manifestations associated immune dysregulation. Analyzing 202,359 cells from 170 specimens across 83 patients, we identify a distinct epithelial cell type in both terminal ileum and ascending colon (hereon as 'LND') with high expression of LCN2, NOS2, and DUOX2 and genes related to antimicrobial response and immunoregulation. LND cells, confirmed by in-situ RNA and protein imaging, are rare in non-IBD controls but expand in active CD, and actively interact with immune cells and specifically express IBD/CD susceptibility genes, suggesting a possible function in CD immunopathogenesis. Furthermore, we discover early and late LND subpopulations with different origins and developmental potential. A higher ratio of late-to-early LND cells correlates with better response to anti-TNF treatment. Our findings thus suggest a potential pathogenic role for LND cells in both Crohn's ileitis and colitis.

Matrix stiffness enhances cancer-macrophage interactions and M2-like macrophage accumulation in the breast tumor microenvironment.

The role of intratumor heterogeneity is becoming increasingly apparent in part due to expansion in single cell technologies. Clinically, tumor heterogeneity poses several obstacles to effective cancer therapy dealing with biomarker variability and treatment responses. Matrix stiffening is known to occur during tumor progression and contribute to pathogenesis in several cancer hallmarks, including tumor angiogenesis and metastasis. However, the effects of matrix stiffening on intratumor heterogeneity have not been thoroughly studied. In this study, we applied single-cell RNA sequencing to investigate the differences in the transcriptional landscapes between stiff and compliant MMTV-PyMT mouse mammary tumors. We found similar compositions of cancer and stromal subpopulations in compliant and stiff tumors but differential intercellular communication and a significantly higher concentration of tumor-promoting, M2-like macrophages in the stiffer tumor microenvironments. Interestingly, we found that cancer cells seeded on stiffer substrates recruited more macrophages. Furthermore, elevated matrix stiffness increased Colony Stimulating Factor 1 (CSF-1) expression in breast cancer cells and reduction of CSF-1 expression on stiffer substrates reduced macrophage recruitment. Thus, our results demonstrate that tissue phenotypes were conserved between stiff and compliant tumors but matrix stiffening altered cell-cell interactions which may be responsible for shifting the phenotypic balance of macrophages residing in the tumor microenvironment towards a pro-tumor progression M2 phenotype. STATEMENT OF SIGNIFICANCE: Cells within tumors are highly heterogeneous, posing challenges with treatment and recurrence. While increased tissue stiffness can promote several hallmarks of cancer, its effects on tumor heterogeneity are unclear. We used single-cell RNA sequencing to investigate the differences in the transcriptional landscapes between stiff and compliant MMTV-PyMT mouse mammary tumors. We found similar compositions of cancer and stromal subpopulations in compliant and stiff tumors but differential intercellular communication and a significantly higher concentration of tumor-promoting, M2-like macrophages in the stiffer tumor microenvironments. Using a biomaterial-based platform, we found that cancer cells seeded on stiffer substrates recruited more macrophages, supporting our in vivo findings. Together, our results demonstrate a key role of matrix stiffness in affecting cell-cell communication and macrophage recruitment.

A contamination focused approach for optimizing the single-cell RNA-seq experiment.

Droplet-based single-cell RNA-seq (scRNA-seq) data are plagued by ambient contaminations caused by nucleic acid material released by dead and dying cells. This material is mixed into the buffer and is co-encapsulated with cells, leading to a lower signal-to-noise ratio. Although there exist computational methods to remove ambient contaminations post-hoc, the reliability of algorithms in generating high-quality data from low-quality sources remains uncertain. Here, we assess data quality before data filtering by a set of quantitative, contamination-based metrics that assess data quality more effectively than standard metrics. Through a series of controlled experiments, we report improvements that can minimize ambient contamination outside of tissue dissociation, via cell fixation, improved cell loading, microfluidic dilution, and nuclei versus cell preparation; many of these parameters are inaccessible on commercial platforms. We provide end-users with insights on factors that can guide their decision-making regarding optimizations that minimize ambient contamination, and metrics to assess data quality.

Temporal recording of mammalian development and precancer.

Key to understanding many biological phenomena is knowing the temporal ordering of cellular events, which often require continuous direct observations [1, 2]. An alternative solution involves the utilization of irreversible genetic changes, such as naturally occurring mutations, to create indelible markers that enables retrospective temporal ordering [3-8]. Using NSC-seq, a newly designed and validated multi-purpose single-cell CRISPR platform, we developed a molecular clock approach to record the timing of cellular events and clonality in vivo , while incorporating assigned cell state and lineage information. Using this approach, we uncovered precise timing of tissue-specific cell expansion during murine embryonic development and identified new intestinal epithelial progenitor states by their unique genetic histories. NSC-seq analysis of murine adenomas and single-cell multi-omic profiling of human precancers as part of the Human Tumor Atlas Network (HTAN), including 116 scRNA-seq datasets and clonal analysis of 418 human polyps, demonstrated the occurrence of polyancestral initiation in 15-30% of colonic precancers, revealing their origins from multiple normal founders. Thus, our multimodal framework augments existing single-cell analyses and lays the foundation for in vivo multimodal recording, enabling the tracking of lineage and temporal events during development and tumorigenesis.

A Specialized Epithelial Cell Type Regulating Mucosal Immunity and Driving Human Crohn's Disease.

Crohn's disease (CD) is a complex chronic inflammatory disorder that may affect any part of gastrointestinal tract with extra-intestinal manifestations and associated immune dysregulation. To characterize heterogeneity in CD, we profiled single-cell transcriptomics of 170 samples from 65 CD patients and 18 non-inflammatory bowel disease (IBD) controls in both the terminal ileum (TI) and ascending colon (AC). Analysis of 202,359 cells identified a novel epithelial cell type in both TI and AC, featuring high expression of LCN2, NOS2, and DUOX2, and thus is named LND. LND cells, confirmed by high-resolution in-situ RNA imaging, were rarely found in non-IBD controls, but expanded significantly in active CD. Compared to other epithelial cells, genes defining LND cells were enriched in antimicrobial response and immunoregulation. Moreover, multiplexed protein imaging demonstrated that LND cell abundance was associated with immune infiltration. Cross-talk between LND and immune cells was explored by ligand-receptor interactions and further evidenced by their spatial colocalization. LND cells showed significant enrichment of expression specificity of IBD/CD susceptibility genes, revealing its role in immunopathogenesis of CD. Investigating lineage relationships of epithelial cells detected two LND cell subpopulations with different origins and developmental potential, early and late LND. The ratio of the late to early LND cells was related to anti-TNF response. These findings emphasize the pathogenic role of the specialized LND cell type in both Crohn's ileitis and Crohn's colitis and identify novel biomarkers associated with disease activity and treatment response.

Dissociation and inDrops microfluidic encapsulation of human gut tissues for single-cell atlasing studies.

In droplet-based single-cell RNA-sequencing (scRNA-seq) experiments, cells, along with some of their surrounding buffer and ambient material, are encapsulated into droplets for mRNA capture and barcoding. This protocol details the steps for human gut tissue dissociation using cold active protease, and subsequent isolation of single epithelial cells, with enrichment of viability through washes. Next, the steps for encapsulation on the inDrops scRNA-seq platform are described. This procedure has been demonstrated to be applicable to polyps, cancers, and inflamed tissues. For complete details on the use and execution of this protocol, please refer to Chen et al. (2021).

Cancer-Associated Fibroblasts and Squamous Epithelial Cells Constitute a Unique Microenvironment in a Mouse Model of Inflammation-Induced Colon Cancer.

The tumor microenvironment plays a key role in the pathogenesis of colorectal tumors and contains various cell types including epithelial, immune, and mesenchymal cells. Characterization of the interactions between these cell types is necessary for revealing the complex nature of tumors. In this study, we used single-cell RNA-seq (scRNA-seq) to compare the tumor microenvironments between a mouse model of sporadic colorectal adenoma (Lrig1CreERT2/+;Apc2lox14/+) and a mouse model of inflammation-driven colorectal cancer induced by azoxymethane and dextran sodium sulfate (AOM/DSS). While both models develop tumors in the distal colon, we found that the two tumor types have distinct microenvironments. AOM/DSS tumors have an increased abundance of two populations of cancer-associated fibroblasts (CAFs) compared with APC tumors, and we revealed their divergent spatial association with tumor cells using multiplex immunofluorescence (MxIF) imaging. We also identified a unique squamous cell population in AOM/DSS tumors, whose origins were distinct from anal squamous epithelial cells. These cells were in higher proportions upon administration of a chemotherapy regimen of 5-Fluorouracil/Irinotecan. We used computational inference algorithms to predict cell-cell communication mediated by ligand-receptor interactions and downstream pathway activation, and identified potential mechanistic connections between CAFs and tumor cells, as well as CAFs and squamous epithelial cells. This study provides important preclinical insight into the microenvironment of two distinct models of colorectal tumors and reveals unique roles for CAFs and squamous epithelial cells in the AOM/DSS model of inflammation-driven cancer.

Archetype tasks link intratumoral heterogeneity to plasticity and cancer hallmarks in small cell lung cancer.

Small cell lung cancer (SCLC) tumors comprise heterogeneous mixtures of cell states, categorized into neuroendocrine (NE) and non-neuroendocrine (non-NE) transcriptional subtypes. NE to non-NE state transitions, fueled by plasticity, likely underlie adaptability to treatment and dismal survival rates. Here, we apply an archetypal analysis to model plasticity by recasting SCLC phenotypic heterogeneity through multi-task evolutionary theory. Cell line and tumor transcriptomics data fit well in a five-dimensional convex polytope whose vertices optimize tasks reminiscent of pulmonary NE cells, the SCLC normal counterparts. These tasks, supported by knowledge and experimental data, include proliferation, slithering, metabolism, secretion, and injury repair, reflecting cancer hallmarks. SCLC subtypes, either at the population or single-cell level, can be positioned in archetypal space by bulk or single-cell transcriptomics, respectively, and characterized as task specialists or multi-task generalists by the distance from archetype vertex signatures. In the archetype space, modeling single-cell plasticity as a Markovian process along an underlying state manifold indicates that task trade-offs, in response to microenvironmental perturbations or treatment, may drive cell plasticity. Stifling phenotypic transitions and plasticity may provide new targets for much-needed translational advances in SCLC. A record of this paper's Transparent Peer Review process is included in the supplemental information.