Subcellular Detection of SARS-CoV-2 RNA in Human Tissue Reveals Distinct Localization in Alveolar Type 2 Pneumocytes and Alveolar Macrophages

Acheampong, Kofi; Schaff, Dylan L.; Emert, Benjamin; Lake, Jonathan I.; Reffsin, Sam; Shea, Emily; Comar, Courtney E; Litzky, Leslie A.; Khurram, Nigar; Linn, Rebecca L.; Feldman, Michael D.; Weiss, Susan R.; Montone, Kathleen T.; Cherry, Sara; Shaffer, Sydney M.

doi:10.1128/mbio.03751-21

Cited by 20 publications

(28 citation statements)

References 46 publications

(58 reference statements)

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“…To analyze NT5E and SOX10 expression in mouse tumors, we used HCRv3.0 with probes targeting NT5E and SOX10 (Acheampong et al 2022; Choi et al 2018). The probes and fluorescently labeled hairpins were purchased from Molecular Instruments ( NT5E lot #: PRK825, SOX10 lot #: PRK826).…”

Section: Methodsmentioning

confidence: 99%

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Disrupting cellular memory to overcome drug resistance

Harmange

Hueros

Schaff

et al. 2022

Preprint

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Cellular memory describes the length of time a particular transcriptional state exists at the single-cell level. Transcriptional states with memory can underlie important processes in biology, including therapy resistance in cancer. Here we present a new experimental and computational approach for identifying gene expression states with memory at single-cell resolution by combining single-cell RNA sequencing (scRNA-seq) with cellular barcoding. With this technique, we can systematically quantify the full expression profile of these memory states as well as their population dynamics including the relative growth rates of cells within different states and the rates of switching between states. We applied this approach to human melanoma cells and uncovered memory gene expression states that are predictive of which cells will be resistant to combination BRAF and MEK inhibition. While these cells have not been treated with targeted therapies, they already express markers of resistance, and thus are referred to here as “primed” for resistance. From the scRNA-seq data alone, the drug-susceptible and primed cells appear as two distinct and unrelated cell populations. From the lineage barcodes, we found that most cells remain within the same state, demonstrating memory of gene expression. However, we also directly observe state switching as we find that about 18% of lineages contain cells that have switched states. While the molecular drivers of state switching are not immediately apparent from scRNA-seq data alone, we specifically analyzed lineages that undergo state switching and identified TGF-β and PI3K as drivers of state switching at the single-cell level. Through functional validation and single-cell RNA-sequencing, we show mechanistic single-cell manipulation of state switching that ultimately yields changes in cellular phenotype. We leverage these mechanisms of state switching to delay resistance by demonstrating that reducing the number of primed cells with a PI3K inhibitor prior to BRAF and MEK inhibition ultimately leads to fewer resistant colonies. Our results show that modulation of cellular signaling can globally regulate gene expression programs to overcome therapy resistance.

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Section: Methodsmentioning

confidence: 99%

“…The probes and fluorescently labeled hairpins were purchased from Molecular Instruments ( NT5E lot #: PRK825, SOX10 lot #: PRK826). To perform HCR in tissue, we made slight modifications to published protocols (Choi et al 2018; Acheampong et al 2022). First, we used cryostat sectioning to generate 6µm sections of fresh frozen tumor.…”

Section: Methodsmentioning

confidence: 99%

Disrupting cellular memory to overcome drug resistance

Harmange

Hueros

Schaff

et al. 2022

Preprint

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“…The expression of well-known replication-associated proteins, including nsp7, nsp8, nsp9, nsp10, nsp12, nsp13, and nsp15, could not recover the impaired replication of the viral replicon with the corresponding nsp deletion, indicating that the viral RTC assembly likely prefers the in cis component nsps generated from the same original polyproteins. The preference is likely due to the time window and the subcellular location for the viral RTC assembly ( van Hemert et al, 2008 ; Acheampong et al, 2022 ). The exogenous expression of some nsps cannot fully satisfy the requirements, so these nsps cannot restore the assembly and the proper function of viral RTC.…”

Section: Discussionmentioning

confidence: 99%

Genome-Wide Analysis of the Indispensable Role of Non-structural Proteins in the Replication of SARS-CoV-2

Jin

Ouyang

et al. 2022

Front. Microbiol.

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Understanding the process of replication and transcription of SARS-CoV-2 is essential for antiviral strategy development. The replicase polyprotein is indispensable for viral replication. However, whether all nsps derived from the replicase polyprotein of SARS-CoV-2 are indispensable is not fully understood. In this study, we utilized the SARS-CoV-2 replicon as the system to investigate the role of each nsp in viral replication. We found that except for nsp16, all the nsp deletions drastically impair the replication of the replicon, and nsp14 could recover the replication deficiency caused by its deletion in the viral replicon. Due to the unsuccessful expressions of nsp1, nsp3, and nsp16, we could not draw a conclusion about their in trans-rescue functions. Our study provided a new angle to understand the role of each nsp in viral replication and transcription, helping the evaluation of nsps as the target for antiviral drug development.

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“…To avoid exposure to the air, the samples were sealed with a thin layer of paraffin, then stored at room temperature. Within 24 hours of beginning the clampFISH 2.0 steps, we performed a protocol adapted from (Choi et al 2018; Acheampong et al 2022) . First, we deparaffinized the sample in HistoChoice Clearing Agent (Sigma-Aldrich, H2779-1L) for 40 to 110 minutes total, then, while the sample was in LockMailer jars, in 100% ethanol for 2 x 5 minutes.…”

Section: Methodsmentioning

confidence: 99%

clampFISH 2.0 enables rapid, scalable amplified RNA detection in situ

Dardani

Emert

Goyal

et al. 2022

Preprint

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RNA labeling in situ has enormous potential to visualize transcripts and quantify their levels in single cells, but it remains challenging to produce high levels of signal while also enabling multiplexed detection of multiple RNA species simultaneously. Here, we describe clampFISH 2.0, a method that uses an inverted padlock design to efficiently detect and exponentially amplify signals from many RNA species at once, while also reducing time and cost compared to the prior clampFISH method. We leverage the increased throughput afforded by multiplexed signal amplification and sequential detection by demonstrating the ability to detect 10 different RNA species in over 1 million cells. We also show that clampFISH 2.0 works in tissue sections. We expect the advantages offered by clampFISH 2.0 will enable many applications in spatial transcriptomics.

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Subcellular Detection of SARS-CoV-2 RNA in Human Tissue Reveals Distinct Localization in Alveolar Type 2 Pneumocytes and Alveolar Macrophages

Cited by 20 publications

References 46 publications

Disrupting cellular memory to overcome drug resistance

Disrupting cellular memory to overcome drug resistance

Genome-Wide Analysis of the Indispensable Role of Non-structural Proteins in the Replication of SARS-CoV-2

clampFISH 2.0 enables rapid, scalable amplified RNA detection in situ

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