RESUMEN
Paclitaxel is a standard of care neoadjuvant therapy for patients with triple negative breast cancer (TNBC); however, it shows limited benefit for locally advanced or metastatic disease. Here we used a coordinated experimental-computational approach to explore the influence of paclitaxel on the cellular and molecular responses of TNBC cells. We found that escalating doses of paclitaxel resulted in multinucleation, promotion of senescence, and initiation of DNA damage induced apoptosis. Single-cell RNA sequencing (scRNA-seq) of TNBC cells after paclitaxel treatment revealed upregulation of innate immune programs canonically associated with interferon response and downregulation of cell cycle progression programs. Systematic exploration of transcriptional responses to paclitaxel and cancer-associated microenvironmental factors revealed common gene programs induced by paclitaxel, IFNB, and IFNG. Transcription factor (TF) enrichment analysis identified 13 TFs that were both enriched based on activity of downstream targets and also significantly upregulated after paclitaxel treatment. Functional assessment with siRNA knockdown confirmed that the TFs FOSL1, NFE2L2 and ELF3 mediate cellular proliferation and also regulate nuclear structure. We further explored the influence of these TFs on paclitaxel-induced cell cycle behavior via live cell imaging, which revealed altered progression rates through G1, S/G2 and M phases. We found that ELF3 knockdown synergized with paclitaxel treatment to lock cells in a G1 state and prevent cell cycle progression. Analysis of publicly available breast cancer patient data showed that high ELF3 expression was associated with poor prognosis and enrichment programs associated with cell cycle progression. Together these analyses disentangle the diverse aspects of paclitaxel response and identify ELF3 upregulation as a putative biomarker of paclitaxel resistance in TNBC.
RESUMEN
Peripheral sensory neurons are a critical part of the nervous system that transmit a multitude of sensory stimuli to the central nervous system. During larval and juvenile stages in zebrafish, this function is mediated by Rohon-Beard somatosensory neurons (RBs). RBs are optically accessible and amenable to experimental manipulation, making them a powerful system for mechanistic investigation of sensory neurons. Previous studies provided evidence that RBs fall into multiple subclasses; however, the number and molecular makeup of these potential RB subtypes have not been well defined. Using a single-cell RNA sequencing (scRNA-seq) approach, we demonstrate that larval RBs in zebrafish fall into three, largely nonoverlapping classes of neurons. We also show that RBs are molecularly distinct from trigeminal neurons in zebrafish. Cross-species transcriptional analysis indicates that one RB subclass is similar to a mammalian group of A-fiber sensory neurons. Another RB subclass is predicted to sense multiple modalities, including mechanical stimulation and chemical irritants. We leveraged our scRNA-seq data to determine that the fibroblast growth factor (Fgf) pathway is active in RBs. Pharmacological and genetic inhibition of this pathway led to defects in axon maintenance and RB cell death. Moreover, this can be phenocopied by treatment with dovitinib, an FDA-approved Fgf inhibitor with a common side effect of peripheral neuropathy. Importantly, dovitinib-mediated axon loss can be suppressed by loss of Sarm1, a positive regulator of neuronal cell death and axonal injury. This offers a molecular target for future clinical intervention to fight neurotoxic effects of this drug.
Asunto(s)
Células Receptoras Sensoriales , Pez Cebra , Animales , Pez Cebra/metabolismo , Animales Modificados Genéticamente , Supervivencia Celular , Células Receptoras Sensoriales/fisiología , Axones/fisiología , Análisis de la Célula Individual , MamíferosRESUMEN
Multicellular rosettes are transient epithelial structures that serve as important cellular intermediates in the formation of diverse organs. Using the zebrafish posterior lateral line primordium (pLLP) as a model system, we investigated the role of the RhoA GEF Mcf2lb in rosette morphogenesis. The pLLP is a group of â¼150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA-sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This resulted in an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are properly polarized. In contrast, RhoA activity, as well as signaling components downstream of RhoA, Rock2a and non-muscle Myosin II, were diminished apically. Thus, Mcf2lb-dependent RhoA activation maintains the integrity of epithelial rosettes.
Asunto(s)
Sistema de la Línea Lateral , Pez Cebra , Animales , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo , Transducción de Señal/fisiología , Movimiento Celular/genética , Morfogénesis/fisiologíaRESUMEN
Peripheral sensory neurons are a critical part of the nervous system that transmit a multitude of sensory stimuli to the central nervous system. During larval and juvenile stages in zebrafish, this function is mediated by Rohon-Beard somatosensory neurons (RBs). RBs are optically accessible and amenable to experimental manipulation, making them a powerful system for mechanistic investigation of sensory neurons. Previous studies provided evidence that RBs fall into multiple subclasses; however, the number and molecular make up of these potential RB subtypes have not been well defined. Using a single-cell RNA sequencing (scRNA-seq) approach, we demonstrate that larval RBs in zebrafish fall into three, largely non-overlapping classes of neurons. We also show that RBs are molecularly distinct from trigeminal neurons in zebrafish. Cross-species transcriptional analysis indicates that one RB subclass is similar to a mammalian group of A-fiber sensory neurons. Another RB subclass is predicted to sense multiple modalities, including mechanical stimulation and chemical irritants. We leveraged our scRNA-seq data to determine that the fibroblast growth factor (Fgf) pathway is active in RBs. Pharmacological and genetic inhibition of this pathway led to defects in axon maintenance and RB cell death. Moreover, this can be phenocopied by treatment with dovitinib, an FDA-approved Fgf inhibitor with a common side effect of peripheral neuropathy. Importantly, dovitinib-mediated axon loss can be suppressed by loss of Sarm1, a positive regulator of neuronal cell death and axonal injury. This offers a molecular target for future clinical intervention to fight neurotoxic effects of this drug.
RESUMEN
Triple-negative breast cancer (TNBC) patients have a poor prognosis and few treatment options. Mouse models of TNBC are important for development of new therapies, however, few mouse models represent the complexity of TNBC. Here, we develop a female TNBC murine model by mimicking two common TNBC mutations with high co-occurrence: amplification of the oncogene MYC and deletion of the tumor suppressor PTEN. This Myc;Ptenfl model develops heterogeneous triple-negative mammary tumors that display histological and molecular features commonly found in human TNBC. Our research involves deep molecular and spatial analyses on Myc;Ptenfl tumors including bulk and single-cell RNA-sequencing, and multiplex tissue-imaging. Through comparison with human TNBC, we demonstrate that this genetic mouse model develops mammary tumors with differential survival and therapeutic responses that closely resemble the inter- and intra-tumoral and microenvironmental heterogeneity of human TNBC, providing a pre-clinical tool for assessing the spectrum of patient TNBC biology and drug response.
Asunto(s)
Neoplasias Mamarias Animales , Neoplasias de la Mama Triple Negativas , Animales , Femenino , Humanos , Ratones , Agresión , Modelos Animales de Enfermedad , Mutación , Fosfohidrolasa PTEN/genética , Neoplasias de la Mama Triple Negativas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
During development, multicellular rosettes serve as important cellular intermediates in the formation of diverse organ systems. Multicellular rosettes are transient epithelial structures that are defined by the apical constriction of cells towards the rosette center. Due to the important role these structures play during development, understanding the molecular mechanisms by which rosettes are formed and maintained is of high interest. Utilizing the zebrafish posterior lateral line primordium (pLLP) as a model system, we identify the RhoA GEF Mcf2lb as a regulator of rosette integrity. The pLLP is a group of ~150 cells that migrates along the zebrafish trunk and is organized into epithelial rosettes; these are deposited along the trunk and will differentiate into sensory organs called neuromasts (NMs). Using single-cell RNA sequencing and whole-mount in situ hybridization, we showed that mcf2lb is expressed in the pLLP during migration. Given the known role of RhoA in rosette formation, we asked whether Mcf2lb plays a role in regulating apical constriction of cells within rosettes. Live imaging and subsequent 3D analysis of mcf2lb mutant pLLP cells showed disrupted apical constriction and subsequent rosette organization. This in turn resulted in a unique posterior Lateral Line phenotype: an excess number of deposited NMs along the trunk of the zebrafish. Cell polarity markers ZO-1 and Par-3 were apically localized, indicating that pLLP cells are normally polarized. In contrast, signaling components that mediate apical constriction downstream of RhoA, Rock-2a and non-muscle Myosin II were diminished apically. Altogether our results suggest a model whereby Mcf2lb activates RhoA, which in turn activates downstream signaling machinery to induce and maintain apical constriction in cells incorporated into rosettes.
RESUMEN
Existing preclinical methods for acquiring dissemination kinetics of rare circulating tumor cells (CTCs) en route to forming metastases have not been capable of providing a direct measure of CTC intravasation rate and subsequent half-life in the circulation. Here, we demonstrate an approach for measuring endogenous CTC kinetics by continuously exchanging CTC-containing blood over several hours between un-anesthetized, tumor-bearing mice and healthy, tumor-free counterparts. By tracking CTC transfer rates, we extrapolated half-life times in the circulation of between 40 and 260 s and intravasation rates between 60 and 107,000 CTCs/hour in mouse models of small-cell lung cancer (SCLC), pancreatic ductal adenocarcinoma (PDAC), and non-small cell lung cancer (NSCLC). Additionally, direct transfer of only 1-2% of daily-shed CTCs using our blood-exchange technique from late-stage, SCLC-bearing mice generated macrometastases in healthy recipient mice. We envision that our technique will help further elucidate the role of CTCs and the rate-limiting steps in metastasis.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma Ductal Pancreático/patología , Neoplasias Pulmonares/patología , Células Neoplásicas Circulantes/patología , Neoplasias Pancreáticas/patología , Carcinoma Pulmonar de Células Pequeñas/patología , Animales , Transfusión Sanguínea/métodos , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma Ductal Pancreático/sangre , Línea Celular Tumoral , Humanos , Cinética , Neoplasias Pulmonares/sangre , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Metástasis de la Neoplasia , Neoplasias Pancreáticas/sangre , Puntaje de Propensión , RNA-Seq/métodos , Análisis de la Célula Individual/métodos , Carcinoma Pulmonar de Células Pequeñas/sangre , Neoplasias PancreáticasRESUMEN
Cell size is believed to influence cell growth and metabolism. Consistently, several studies have revealed that large cells have lower mass accumulation rates per unit mass (i.e., growth efficiency) than intermediate-sized cells in the same population. Size-dependent growth is commonly attributed to transport limitations, such as increased diffusion timescales and decreased surface-to-volume ratio. However, separating cell size- and cell cycle-dependent growth is challenging. To address this, we monitored growth efficiency of pseudodiploid mouse lymphocytic leukemia cells during normal proliferation and polyploidization. This was enabled by the development of large-channel suspended microchannel resonators that allow us to monitor buoyant mass of single cells ranging from 40 pg (small pseudodiploid cell) to over 4,000 pg, with a resolution ranging from â¼1% to â¼0.05%. We find that cell growth efficiency increases, plateaus, and then decreases as cell cycle proceeds. This growth behavior repeats with every endomitotic cycle as cells grow into polyploidy. Overall, growth efficiency changes 33% throughout the cell cycle. In contrast, increasing cell mass by over 100-fold during polyploidization did not change growth efficiency, indicating exponential growth. Consistently, growth efficiency remained constant when cell cycle was arrested in G2 Thus, cell cycle is a primary determinant of growth efficiency. As growth remains exponential over large size scales, our work finds no evidence for transport limitations that would decrease growth efficiency.
Asunto(s)
Técnicas Biosensibles , Aumento de la Célula , Proliferación Celular/genética , Leucemia Linfoide/genética , Animales , Ciclo Celular/genética , División Celular/genética , Línea Celular Tumoral , Humanos , Leucemia Linfoide/patología , Ratones , Técnicas Analíticas Microfluídicas , PoliploidíaRESUMEN
Mass and growth rate are highly integrative measures of cell physiology not discernable via genomic measurements. Here, we introduce a microfluidic platform enabling direct measurement of single-cell mass and growth rate upstream of highly multiplexed single-cell profiling such as single-cell RNA sequencing. We resolve transcriptional signatures associated with single-cell mass and growth rate in L1210 and FL5.12 cell lines and activated CD8+ T cells. Further, we demonstrate a framework using these linked measurements to characterize biophysical heterogeneity in a patient-derived glioblastoma cell line with and without drug treatment. Our results highlight the value of coupled phenotypic metrics in guiding single-cell genomics.
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Aumento de la Célula , Genómica/métodos , Técnicas Analíticas Microfluídicas , Análisis de la Célula Individual/métodos , Animales , Linfocitos T CD8-positivos/metabolismo , Línea Celular Tumoral , Humanos , Activación de Linfocitos , RatonesRESUMEN
A fundamental trade-off between flow rate and measurement precision limits performance of many single-cell detection strategies, especially for applications that require biophysical measurements from living cells within complex and low-input samples. To address this, we introduce 'active loading', an automated, optically-triggered fluidic system that improves measurement throughput and robustness by controlling entry of individual cells into a measurement channel. We apply active loading to samples over a range of concentrations (1-1000 particles µL-1), demonstrate that measurement time can be decreased by up to 20-fold, and show theoretically that performance of some types of existing single-cell microfluidic devices can be improved by implementing active loading. Finally, we demonstrate how active loading improves clinical feasibility for acute, single-cell drug sensitivity measurements by deploying it to a preclinical setting where we assess patient samples from normal brain, primary and metastatic brain cancers containing a complex, difficult-to-measure mixture of confounding biological debris.
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Análisis de la Célula Individual/instrumentación , Análisis de la Célula Individual/métodos , Animales , Línea Celular , Células Cultivadas , Diseño de Equipo , Humanos , Ratones , Reproducibilidad de los Resultados , Células Tumorales CultivadasRESUMEN
Multiple myeloma (MM) has benefited from significant advancements in treatment that have improved outcomes and reduced morbidity. However, the disease remains incurable and is characterized by high rates of drug resistance and relapse. Consequently, methods to select the most efficacious therapy are of great interest. Here we utilize a functional assay to assess the ex vivo drug sensitivity of single multiple myeloma cells based on measuring their mass accumulation rate (MAR). We show that MAR accurately and rapidly defines therapeutic susceptibility across human multiple myeloma cell lines to a gamut of standard-of-care therapies. Finally, we demonstrate that our MAR assay, without the need for extended culture ex vivo, correctly defines the response of nine patients to standard-of-care drugs according to their clinical diagnoses. This data highlights the MAR assay in both research and clinical applications as a promising tool for predicting therapeutic response using clinical samples.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Análisis de la Célula Individual/métodos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Humanos , CinéticaRESUMEN
Assays that can determine the response of tumor cells to cancer therapeutics could greatly aid the selection of drug regimens for individual patients. However, the utility of current functional assays is limited, and predictive genetic biomarkers are available for only a small fraction of cancer therapies. We found that the single-cell mass accumulation rate (MAR), profiled over many hours with a suspended microchannel resonator, accurately defined the drug sensitivity or resistance of glioblastoma and B-cell acute lymphocytic leukemia cells. MAR revealed heterogeneity in drug sensitivity not only between different tumors, but also within individual tumors and tumor-derived cell lines. MAR measurement predicted drug response using samples as small as 25 µl of peripheral blood while maintaining cell viability and compatibility with downstream characterization. MAR measurement is a promising approach for directly assaying single-cell therapeutic responses and for identifying cellular subpopulations with phenotypic resistance in heterogeneous tumors.