Robert Stickels

All Publications

• Transcript-specific enrichment enables profiling of rare cell states via single-cell RNA sequencing. Nature genetics Abay, T., Stickels, R. R., Takizawa, M. T., Nalbant, B. N., Hsieh, Y. H., Hwang, S., Snopkowski, C., Yu, K. K., Abou-Mrad, Z., Tabar, V., Howitt, B. E., Ludwig, L. S., Chaligné, R., Satpathy, A. T., Lareau, C. A. 2025

  Abstract

  Single-cell genomics technologies have accelerated our understanding of cell-state heterogeneity in diverse contexts. Although single-cell RNA sequencing identifies rare populations that express specific marker transcript combinations, traditional flow sorting requires cell surface markers with high-fidelity antibodies, limiting our ability to interrogate these populations. In addition, many single-cell studies require the isolation of nuclei from tissue, eliminating the ability to enrich learned rare cell states based on extranuclear protein markers. In the present report, we addressed these limitations by developing Programmable Enrichment via RNA FlowFISH by sequencing (PERFF-seq), a scalable assay that enables scRNA-seq profiling of subpopulations defined by the abundance of specific RNA transcripts. Across immune populations (n = 184,126 cells) and fresh-frozen and formalin-fixed, paraffin-embedded brain tissue (n = 33,145 nuclei), we demonstrated that programmable sorting logic via RNA-based cytometry can isolate rare cell populations and uncover phenotypic heterogeneity via downstream, high-throughput, single-cell genomics analyses.

  View details for DOI 10.1038/s41588-024-02036-7

  View details for PubMedID 39779958

  View details for PubMedCentralID 6295155

• Spatiotemporal lineage tracing reveals the dynamic spatial architecture of tumor growth and metastasis. bioRxiv : the preprint server for biology Jones, M. G., Sun, D., Min, K. H., Colgan, W. N., Tian, L., Weir, J. A., Chen, V. Z., Koblan, L. W., Yost, K. E., Mathey-Andrews, N., Russell, A. J., Stickels, R. R., Balderrama, K. S., Rideout, W. M., Chang, H. Y., Jacks, T., Chen, F., Weissman, J. S., Yosef, N., Yang, D. 2024

  Abstract

  Tumor progression is driven by dynamic interactions between cancer cells and their surrounding microenvironment. Investigating the spatiotemporal evolution of tumors can provide crucial insights into how intrinsic changes within cancer cells and extrinsic alterations in the microenvironment cooperate to drive different stages of tumor progression. Here, we integrate high-resolution spatial transcriptomics and evolving lineage tracing technologies to elucidate how tumor expansion, plasticity, and metastasis co-evolve with microenvironmental remodeling in a Kras;p53-driven mouse model of lung adenocarcinoma. We find that rapid tumor expansion contributes to a hypoxic, immunosuppressive, and fibrotic microenvironment that is associated with the emergence of pro-metastatic cancer cell states. Furthermore, metastases arise from spatially-confined subclones of primary tumors and remodel the distant metastatic niche into a fibrotic, collagen-rich microenvironment. Together, we present a comprehensive dataset integrating spatial assays and lineage tracing to elucidate how sequential changes in cancer cell state and microenvironmental structures cooperate to promote tumor progression.

  View details for DOI 10.1101/2024.10.21.619529

  View details for PubMedID 39484491

  View details for PubMedCentralID PMC11526908

• Mechanism-guided engineering of a minimal biological particle for genome editing. bioRxiv : the preprint server for biology Ngo, W., Peukes, J. T., Baldwin, A., Xue, Z. W., Hwang, S., Stickels, R. R., Lin, Z., Satpathy, A. T., Wells, J. A., Schekman, R., Nogales, E., Doudna, J. A. 2024

  Abstract

  The widespread application of genome editing to treat or even cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped Delivery Vehicles (EDVs) are engineered virally-derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication components in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs, and demonstrated increased editing in cell lines and therapeutically-relevant primary human T cells. These results show that virally-derived particles can be streamlined to create efficacious genome editing delivery vehicles that could simplify production and manufacturing.

  View details for DOI 10.1101/2024.07.23.604809

  View details for PubMedID 39091760

  View details for PubMedCentralID PMC11291128

• Transcript-specific enrichment enables profiling rare cell states via scRNA-seq. bioRxiv : the preprint server for biology Abay, T., Stickels, R. R., Takizawa, M. T., Nalbant, B. N., Hsieh, Y. H., Hwang, S., Snopkowski, C., Yu, K. K., Abou-Mrad, Z., Tabar, V., Ludwig, L. S., Chaligné, R., Satpathy, A. T., Lareau, C. A. 2024

  Abstract

  Single-cell genomics technologies have accelerated our understanding of cell-state heterogeneity in diverse contexts. Although single-cell RNA sequencing (scRNA-seq) identifies many rare populations of interest that express specific marker transcript combinations, traditional flow sorting limits our ability to enrich these populations for further profiling, including requiring cell surface markers with high-fidelity antibodies. Additionally, many single-cell studies require the isolation of nuclei from tissue, eliminating the ability to enrich learned rare cell states based on extranuclear protein markers. To address these limitations, we describe Programmable Enrichment via RNA Flow-FISH by sequencing (PERFF-seq), a scalable assay that enables scRNA-seq profiling of subpopulations from complex cellular mixtures defined by the presence or absence of specific RNA transcripts. Across immune populations (n = 141,227 cells) and fresh-frozen and formalin-fixed paraffin-embedded brain tissue (n = 29,522 nuclei), we demonstrate the sorting logic that can be used to enrich for cell populations via RNA-based cytometry followed by high-throughput scRNA-seq. Our approach provides a rational, programmable method for studying rare populations identified by one or more marker transcripts.

  View details for DOI 10.1101/2024.03.27.587039

  View details for PubMedID 38586040

  View details for PubMedCentralID PMC10996707

• Latent human herpesvirus 6 is reactivated in CAR T cells. Nature Lareau, C. A., Yin, Y., Maurer, K., Sandor, K. D., Daniel, B., Yagnik, G., Peña, J., Crawford, J. C., Spanjaart, A. M., Gutierrez, J. C., Haradhvala, N. J., Riberdy, J. M., Abay, T., Stickels, R. R., Verboon, J. M., Liu, V., Buquicchio, F. A., Wang, F., Southard, J., Song, R., Li, W., Shrestha, A., Parida, L., Getz, G., Maus, M. V., Li, S., Moore, A., Roberts, Z. J., Ludwig, L. S., Talleur, A. C., Thomas, P. G., Dehghani, H., Pertel, T., Kundaje, A., Gottschalk, S., Roth, T. L., Kersten, M. J., Wu, C. J., Majzner, R. G., Satpathy, A. T. 2023

  Abstract

  Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are understudied. For example, we lack a comprehensive understanding of the mechanisms of toxicities observed in patients receiving T cell therapies, including recent reports of encephalitis caused by reactivation of human herpesvirus 6 (HHV-6)1. Here, through petabase-scale viral genomics mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in cultures of human CD4+ T cells. Using single-cell sequencing, we identify a rare population of HHV-6 'super-expressors' (about 1 in 300-10,000 cells) that possess high viral transcriptional activity, among research-grade allogeneic chimeric antigen receptor (CAR) T cells. By analysing single-cell sequencing data from patients receiving cell therapy products that are approved by the US Food and Drug Administration2 or are in clinical studies3-5, we identify the presence of HHV-6-super-expressor CAR T cells in patients in vivo. Together, the findings of our study demonstrate the utility of comprehensive genomics analyses in implicating cell therapy products as a potential source contributing to the lytic HHV-6 infection that has been reported in clinical trials1,6-8 and may influence the design and production of autologous and allogeneic cell therapies.

  View details for DOI 10.1038/s41586-023-06704-2

  View details for PubMedID 37938768

  View details for PubMedCentralID 9827115