To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging ...from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.
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•Profiling of 10 human skin SCCs and matched normals via scRNA-seq, ST, and MIBI•Tumor-specific keratinocytes (TSKs) reside within a fibrovascular niche at leading edges•Distinct ligand-receptor and spatial niche associations for tumor and stromal cells.•Subpopulation essential tumorigenic gene networks defined by in vivo CRISPR screening
Integration of high-dimensional multi-omics approaches to characterize human cutaneous squamous cell carcinoma identifies a tumor-specific keratinocyte population as well as the immune infiltrates and heterogeneity at tumor leading edges.
Abstract
Simultaneous visualization of the relationship between multiple biomolecules and their ligands or small molecules at the nanometer scale in cells will enable greater understanding of how ...biological processes operate. We present here high-definition multiplex ion beam imaging (HD-MIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added structurally-unmodified small molecules. With this technology, the atomic constituents of the biomolecules themselves can be used in our system as the “tag” and we demonstrate measurements down to ~30 nm lateral resolution. We correlated the subcellular localization of the chemotherapy drug cisplatin simultaneously with five subnuclear structures. Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. Unexpectedly, cells surviving multi-drug treatment with cisplatin and the BET inhibitor JQ1 demonstrated near total cisplatin exclusion from the nucleus, suggesting that selective subcellular drug relocalization may modulate resistance to this important chemotherapeutic treatment. Multiplexed high-resolution imaging techniques, such as HD-MIBI, will enable studies of biomolecules and drug distributions in biologically relevant subcellular microenvironments by visualizing the processes themselves in concert, rather than inferring mechanism through surrogate analyses.
Multiplexed ion beam imaging (MIBI) has been previously used to profile multiple parameters in two dimensions in single cells within tissue slices. Here, a mathematical and technical framework for ...three-dimensional (3D) subcellular MIBI is presented. Ion-beam tomography (IBT) compiles ion beam images that are acquired iteratively across successive, multiple scans, and later assembled into a 3D format without loss of depth resolution. Algorithmic deconvolution, tailored for ion beams, is then applied to the transformed ion image series, yielding 4-fold enhanced ion beam data cubes. To further generate 3D sub-ion-beam-width precision visuals, isolated ion molecules are localized in the raw ion beam images, creating an approach coined as SILM, secondary ion beam localization microscopy, providing sub-25 nm accuracy in original ion images. Using deep learning, a parameter-free reconstruction method for ion beam tomograms with high accuracy is developed for low-density targets. In cultured cancer cells and tissues, IBT enables accessible visualization of 3D volumetric distributions of genomic regions, RNA transcripts, and protein factors with 5 nm axial resolution using isotope-enrichments and label-free elemental analyses. Multiparameter imaging of subcellular features at near macromolecular resolution is implemented by the IBT tools as a general biocomputation pipeline for imaging mass spectrometry.
Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to ...dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.
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•Development of a method (PANINI) for multiplexed nucleic acid and protein imaging•Identification of orchestrated immune events in response to SIV infection•IL-10 levels in B cells and macrophages correlate with immunosuppression•Tissue microenvironmental cues can influence SIV viral transcription state
Our understanding of persistent HIV tissue reservoirs is constrained by our limited ability to visualize the cellular components of these viral reservoirs. Jiang et al. develop a robust method that combines nucleic acid and protein imaging, termed PANINI, and use it to uncover the orchestrated immune events of retroviral lymphoid tissue reservoirs.
Cellular organization and functions encompass multiple scales in vivo. Emerging high-plex imaging technologies are limited in resolving subcellular biomolecular features. Expansion Microscopy (ExM) ...and related techniques physically expand samples for enhanced spatial resolution, but are challenging to be combined with high-plex imaging technologies to enable integrative multiscaled tissue biology insights. Here, we introduce Expand and comPRESS hydrOgels (ExPRESSO), an ExM framework that allows high-plex protein staining, physical expansion, and removal of water, while retaining the lateral tissue expansion. We demonstrate ExPRESSO imaging of archival clinical tissue samples on Multiplexed Ion Beam Imaging and Imaging Mass Cytometry platforms, with detection capabilities of > 40 markers. Application of ExPRESSO on archival human lymphoid and brain tissues resolved tissue architecture at the subcellular level, particularly that of the blood-brain barrier. ExPRESSO hence provides a platform for extending the analysis compatibility of hydrogel-expanded biospecimens to mass spectrometry, with minimal modifications to protocols and instrumentation.
Antigen-specific T cells traffic to, are influenced by, and create unique cellular microenvironments. Here we characterize these microenvironments over time with multiplexed imaging in a melanoma ...model of adoptive T cell therapy and human patients with melanoma treated with checkpoint inhibitor therapy. Multicellular neighborhood analysis reveals dynamic immune cell infiltration and inflamed tumor cell neighborhoods associated with CD8+ T cells. T cell-focused analysis indicates T cells are found along a continuum of neighborhoods that reflect the progressive steps coordinating the anti-tumor immune response. More effective anti-tumor immune responses are characterized by inflamed tumor-T cell neighborhoods, flanked by dense immune infiltration neighborhoods. Conversely, ineffective T cell therapies express anti-inflammatory cytokines, resulting in regulatory neighborhoods, spatially disrupting productive T cell-immune and -tumor interactions. Our study provides in situ mechanistic insights into temporal tumor microenvironment changes, cell interactions critical for response, and spatial correlates of immunotherapy outcomes, informing cellular therapy evaluation and engineering.
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•CODEX multiplexed imaging of human and mouse, immunotherapy-treated melanomas•Tumor-specific T cells cause inflamed tumor phenotype and unique cell neighborhoods•T cell phenotype influences capacity to make effective tumor-killing neighborhoods•Potent therapy creates larger, linked areas of inflamed tumor and immune infiltrate
Hickey et al. discovered that tumor-specific T cells induce tumor inflammation and establish progressive inflammatory environments. Effective therapies exhibit compartmentalized immune infiltration, co-located with T cell and inflamed tumor zones, emphasizing the need for therapeutic T cells to be designed to curate local and distal microenvironments.
Intratumoral heterogeneity is a seminal feature of human tumors contributing to tumor progression and response to treatment. Current technologies are still largely unsuitable to accurately track ...phenotypes and clonal evolution within tumors, especially in response to genetic manipulations. Here, we developed epitopes for imaging using combinatorial tagging (EpicTags), which we coupled to multiplexed ion beam imaging (EpicMIBI) for in situ tracking of barcodes within tissue microenvironments. Using EpicMIBI, we dissected the spatial component of cell lineages and phenotypes in xenograft models of small cell lung cancer. We observed emergent properties from mixed clones leading to the preferential expansion of clonal patches for both neuroendocrine and non-neuroendocrine cancer cell states in these models. In a tumor model harboring a fraction of PTEN-deficient cancer cells, we observed a non-autonomous increase of clonal patch size in PTEN wild-type cancer cells. EpicMIBI facilitates in situ interrogation of cell-intrinsic and cell-extrinsic processes involved in intratumoral heterogeneity.
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•EpicTags enable in situ tracking of barcoded cells•EpicMIBI provides spatial, cell-type, and cell-state depiction of clonal tumor patches•Non-neuroendocrine cells display increased local clonal growth•PTEN loss can promote a non-cell autonomous increase of clonal wild-type cell growth
Rovira-Clave et al. employ a barcoding system and subcellular multiplex protein imaging to identify clonal behaviors in small cell lung cancer models. Non-neuroendocrine cells display increased local clonal growth compared with neuroendocrine cells. Loss of the PTEN tumor suppressor shifts tumor evolution through cell-intrinsic mechanisms and modifications on neighboring wild-type cells.
Ataxia-telangiectasia mutated (ATM) is a cell cycle checkpoint kinase that upon activation by DNA damage leads to cell cycle arrest and DNA repair or apoptosis. The absence of Atm or the occurrence ...of loss-of-function mutations in Atm predisposes to tumorigenesis. MAPK7 has been implicated in numerous types of cancer with pro-survival and pro-growth roles in tumor cells, but its functional relation with tumor suppressors is not clear. In this study, we show that absence of MAPK7 delays death due to spontaneous tumor development in Atm-/- mice. Compared with Atm-/- thymocytes, Mapk7-/-Atm-/- thymocytes exhibited an improved response to DNA damage (increased phosphorylation of H2AX) and a restored apoptotic response after treatment of mice with ionizing radiation. These findings define an antagonistic function of ATM and MAPK7 in the thymocyte response to DNA damage, and suggest that the lack of MAPK7 inhibits thymic lymphoma growth in Atm-/- mice by partially restoring the DNA damage response in thymocytes.
ERK5 is required for the optimal modulation of TCR/CD3 complex expression at the cell surface.
Regulation of the levels of the TCR/CD3 complex at the cell surface is critical to proper T cell ...development and mature T cell activation. We provide evidence that the MAPK ERK5 regulates the surface expression of the TCR/CD3 complex by controlling the degradation of the CD3ζ chain and the recovery of the complex after anti‐CD3ɛ stimulation. ERK5 knockdown led to TCR/CD3 up‐regulation at the cell surface and increased amounts of the CD3ζ chain. Inhibition of the MEK5‐dependent phosphorylation status of the kinase domain of ERK5 in human T CD4+ cells reduced CD3ζ ubiquitination and degradation, limiting TCR/CD3 down‐regulation in anti‐CD3‐stimulated cells. Moreover, TCR/CD3 recovery at the cell surface, after anti‐CD3ɛ treatment, is impaired by ERK5 knockdown or pharmacological inhibition of autophosphorylation in the ERK5 C‐terminal region. ERK5 loss in thymocytes augmented cellular CD3ζ and increased cell surface levels of TCR/CD3 on CD4+CD8+ thymocytes. This correlated with enhanced generation of CD4+CD8−CD25+ thymocytes. Our findings define ERK5 as a novel kinase that modulates the levels of TCR/CD3 at the cell surface by promoting CD3ζ degradation and TCR/CD3 recovery after TCR stimulation.
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