Spatially Resolved and Highly Multiplexed Protein and RNA In Situ Detection by Combining CODEX With RNAscope In Situ Hybridization

Cheng, Yiran; Burrack, Rachel Kubik; Li, Qingsheng

doi:10.1369/00221554221114174

Cited by 10 publications

(6 citation statements)

References 59 publications

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“…Five µm FFPE tissue sections of selected samples sectioned 5 were cut and mounted on poly-L-lysine coated coverslips. Akoya Biosciences CODEX multiplex immunostaining (CODEX) and ACDbio RNAscope HiPlex v2 (12-plex) multiplex FISH (RNAscope) were each performed according to their respective manufacturer's instructions (kits used are listed in Supplemental Table S8), and combined in sequence as previously described, with slight modifications 82 .…”

Section: Multiplex Immunostaining Followed By Multiplex Fishmentioning

confidence: 99%

A TCF4/BRD4-dependent regulatory network confers cross-resistance to targeted and immune checkpoint therapy in melanoma

Poźniak

Pedri

Landeloos

et al. 2022

Preprint

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Primary resistance drastically limits the clinical success of immune checkpoint blockade (ICB) in melanoma. Resistance to ICB may also develop when tumours relapse after targeted therapy. To identify cancer cell-intrinsic mechanisms driving resistance to ICB, we generated single-cell RNA-sequencing (scRNA-seq) data from a prospective longitudinal cohort of patients on ICB therapy, including an early time point obtained after only one cycle of treatment. Comparing these data with murine scRNA-seq datasets, we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem, and defined 6 evolutionarily conserved melanoma transcriptional metaprograms (Melanocytic or MEL, Mesenchymal-like or MES, Neural Crest-like, Antigen Presentation, Stress (hypoxia response) and Stress (p53 response)). Spatial multi-omics revealed a non-random geographic distribution of cell states that is, at least partly, driven by the tumour microenvironment. The single-cell data allowed unambiguous discrimination between melanoma MES cells and cancer-associated fibroblasts both in silico and in situ, a long-standing challenge in the field. Importantly, two of the melanoma transcriptional metaprograms were associated with divergent clinical responses to ICB. While the Antigen Presentation cell population was more abundant in tumours from patients who exhibited a clinical response to ICB, MES cells were significantly enriched in early on-treatment biopsies from non-responders, and their presence significantly predicted lack of response. Critically, we identified TCF4 (E2-2) as a master regulator of the MES program and suppressor of both MEL and Antigen Presentation programs. Targeting TCF4 expression in MES cells either genetically or pharmacologically using a bromodomain inhibitor increased immunogenicity and sensitivity to targeted therapy. This study describes an increasingly complex melanoma transcriptional landscape and its rapid evolution under ICB. It also identifies a putative biomarker of early response to ICB and an epigenetic therapeutic strategy that increases both immunogenicity of ICB-refractory melanoma and their sensitivity to targeted therapy.

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Section: Multiplex Immunostaining Followed By Multiplex Fishmentioning

confidence: 99%

A TCF4/BRD4-dependent regulatory network confers cross-resistance to targeted and immune checkpoint therapy in melanoma

Poźniak

Pedri

Landeloos

et al. 2022

Preprint

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“…Formalin-fixed paraffin-embedded MesLNs tissues collected from the necropsied animals LC31, LB66, DJ01, and EM77 were studied using the combination of CODEX and RNAscope in situ hybridization by following our reported method ( 10 ) in a single slide from each animal. Briefly, a 6-μm tissue section on a coverslip was deparaffinized, hydrated, and pretreated with 3% hydrogen peroxide for 10 min at room temperature, boiled in citrate buffer (pH 6, Sigma-Aldrich, catalog no.…”

Section: Methodsmentioning

confidence: 99%

“…ATI-1 was intended to evaluate the timing and extent of viral rebound in plasma and composition of viral clonotypes in peripheral blood after long-term ART and to examine the potential influence of a prior ATI on viral barcodes detected after a subsequent ATI. One week after the second ATI (post–ATI-2), when all macaques still had plasma viral RNA loads below 22 copies/ml, necropsies were performed, and multiple tissues, including the mesenteric LNs (MesLNs), inguinal LNs (IngLNs), spleen, ileum, colon, lung, liver, and brain and blood, were examined by viral barcode sequencing, analysis of cell-associated DNA (CA-DNA) and cell-associated RNA (CA-RNA), intact proviral DNA assay (IPDA), combined co-detection by indexing (CODEX) and RNAscope (Comb-CODEX-RNAscope) in situ hybridization ( 10 ), single-cell RNA sequencing (scRNA-seq), and other approaches. The results suggested that, among the tissues studied, MesLNs, IngLNs, and spleen showed overlap with viral barcodes detectable by sensitive sequencing of plasma at necropsy, suggesting that these tissues may contribute to viral reactivation and eventual rebound after ATI.…”

Section: Introductionmentioning

confidence: 99%

Lymphoid tissues contribute to plasma viral clonotypes early after antiretroviral therapy interruption in SIV-infected rhesus macaques

Solis-Leal,

Boby,

Mallick

et al. 2023

Sci. Transl. Med.

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The rebound-competent viral reservoir, composed of a virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after ART interruption (ATI), remains the biggest obstacle to treating HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop therapeutic strategies for reducing the rebound-competent viral reservoir. In this study, barcoded simian immunodeficiency virus (SIV), SIVmac239M, was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood and tissues from secondary lymphoid organs (spleen, mesenteric lymph nodes, and inguinal lymph nodes) and from the colon, ileum, lung, liver, and brain were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX and RNAscope in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy, although plasma viral RNA remained below 22 copies per milliliter. Among the tissues studied, mesenteric lymph nodes, inguinal lymph nodes, and spleen contained viral barcodes detected in plasma. CD4 + T cells were the main cell type harboring viral RNA after ATI. Furthermore, T cell zones in lymphoid tissues showed higher viral RNA abundance than B cell zones for most animals. These findings are consistent with lymphoid tissues contributing to the virus present in plasma early after ATI.

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“…In general, the respective HMTI methods are not necessarily confined to the detection of a single type of target molecule. Recent concepts and protocols have describe simultaneous or successive imaging of both proteins and RNA in tissue, e.g., by including RNAscope®-derived branched DNA constructs with metal-tagged label probes in IMC panels [ 23 ], or combining CODEX and RNAscope® [ 28 ]. The SM-omics platform achieves further integration of synergistic readouts, as it combines antigen detection with DNA-barcoded antibodies with spatial transcriptomics in one analytic system [ 29 ].…”

Section: Synergistic Potential With Spatial Transcriptomics and Spati...mentioning

confidence: 99%

High-multiplex tissue imaging in routine pathology—are we there yet?

Einhaus

Rochwarger²,

Mattern³

et al. 2023

Virchows Arch

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High-multiplex tissue imaging (HMTI) approaches comprise several novel immunohistological methods that enable in-depth, spatial single-cell analysis. Over recent years, studies in tumor biology, infectious diseases, and autoimmune conditions have demonstrated the information gain accessible when mapping complex tissues with HMTI. Tumor biology has been a focus of innovative multiparametric approaches, as the tumor microenvironment (TME) contains great informative value for accurate diagnosis and targeted therapeutic approaches: unraveling the cellular composition and structural organization of the TME using sophisticated computational tools for spatial analysis has produced histopathologic biomarkers for outcomes in breast cancer, predictors of positive immunotherapy response in melanoma, and histological subgroups of colorectal carcinoma. Integration of HMTI technologies into existing clinical workflows such as molecular tumor boards will contribute to improve patient outcomes through personalized treatments tailored to the specific heterogeneous pathological fingerprint of cancer, autoimmunity, or infection. Here, we review the advantages and limitations of existing HMTI technologies and outline how spatial single-cell data can improve our understanding of pathological disease mechanisms and determinants of treatment success. We provide an overview of the analytic processing and interpretation and discuss how HMTI can improve future routine clinical diagnostic and therapeutic processes.

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Spatially Resolved and Highly Multiplexed Protein and RNA In Situ Detection by Combining CODEX With RNAscope In Situ Hybridization

Cited by 10 publications

References 59 publications

A TCF4/BRD4-dependent regulatory network confers cross-resistance to targeted and immune checkpoint therapy in melanoma

A TCF4/BRD4-dependent regulatory network confers cross-resistance to targeted and immune checkpoint therapy in melanoma

Lymphoid tissues contribute to plasma viral clonotypes early after antiretroviral therapy interruption in SIV-infected rhesus macaques

High-multiplex tissue imaging in routine pathology—are we there yet?

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