Activation of glucagon-like peptide-1 receptor (GLP-1R) could affect cancer treatment responses through direct action in tumor or immune cells. However, the field lacks a comprehensive assessment of GLP-1R expression and activity across human tumors. Herein, we report detection GLP-1R across multiple human tumor types and focus on triple-negative breast cancer (TNBC) for deeper analysis. In TNBC, GLP-1R is present in immune and tumor cell compartments. GLP-1 treatment of cancer cells activated survival pathways, drove proliferation, induced paclitaxel resistance and dampened cytokine secretion, effects that required expression of GLP-1R. Spatial transcriptomics of human tumors revealed that GLP-1 exposure remodeled the tumor microenvironment, promoted a mesenchymal transition in malignant cells and disrupted productive macrophage inflammation in tumor-proximate niches. Patients taking GLP-1 drugs during neoadjuvant chemotherapy experienced reduced pathological complete response rates (pCR: 30.8%) compared to controls (65%, p<0.001). Thus, GLP-1-exposure acts on tumor and immune cells to impair chemoimmunotherapy efficacy in TNBC.

The tumor microenvironment (TME) is composed of diverse heterogeneous components and plays a crucial role in immune cell infiltration, immune evasion, and dynamic interactions between tumor cells and the immune system. A precise understanding of the TME is essential for tissue immunology research and the development of effective immunotherapies. Technologies that spatially dissect the TME and analyze it at the molecular level are increasingly important. Recently, cutting-edge, high-resolution, high-multiplex molecular profiling technologies capable of high-throughput RNA and protein profiling while incorporating spatial information have been rapidly developing. This review describes a variety of cutting-edge spatial transcriptomics and proteomics technologies, including sequencing-based spatial transcriptomics, multiplex in situ hybridization imaging-based spatial proteomics, and multiomics, which are particularly useful for tissue immunology research. Technological advances in computational tools are discussed, as well as how researchers have interpreted visualized data using our own results as examples. These technologies enable simultaneous analysis of the diverse types and functional states of immune cells in cancer tissues within the tissue architecture, providing a crucial foundation for understanding immune cell organization, function, and cell-to-cell interactions of tissue immune responses. Current spatial molecular profiling technologies still face technological limitations in resolution and analytical complexity. The lack of data standardization across diverse platforms and experimental conditions remains a significant issue, hindering the reproducibility and comparability of research results. To overcome these limitations, multiomics integrated research combining spatial transcriptomics, proteomics, and genomics data is expected to become more active in the future, which will enable a more multidimensional understanding of the TME and tissue immune environment. The introduction of artificial intelligence and machine learning technologies is expected to enable more precise interpretation of the functional status and interactions of immune cells within organizations, ultimately contributing to the development of next-generation immunotherapies.

Women of African ancestry develop more aggressive breast cancer (BCa) with poorer survival outcomes, yet only 8% of available cell lines represent this population, impeding targeted treatment development. Here, we aimed to establish and characterize new cell lines from an Afro-Caribbean patient to better understand population-specific BCa biology. We developed three lines (ACRJ-BC24 parent, α, and β) from a patient with 100% African ancestry. Karyotype analysis revealed progressive chromosomal instability, with the β-clone showing X-11 translocations correlating with higher Ki-67 expression and enhanced tumorigenic capacity. Immunohistochemistry and immunoblotting demonstrated their transition from hormone-positive to triple-negative phenotypes. Transcriptional profiling identified significant enrichment in extracellular matrix organization pathways mechanistically linked to chromosomal instability, explaining their distinct drug responses. The parent line demonstrated notable sensitivity to PARP inhibitors and microtubule-targeting agents, while the β-clone showed enhanced platinum sensitivity correlating with its chromosomal abnormalities. The parent line exhibited atypical dose-response patterns to gemcitabine and docetaxel, possibly relating to ECM-mediated drug transport mechanisms. This resource provides valuable tools for studying BCa disparities in African ancestry populations, offering the first cell line models from this underrepresented population for hypothesis-driven mechanistic studies.

Normal tissues from Black individuals, regardless of their country of birth or residence, remain critically understudied. This gap in research is particularly concerning, as Black individuals disproportionately experience aggressive pathological diseases, often exhibit resistance or refractoriness to treatment, and face higher rates of premature death. In women, breast cancer is not only more common among U.S. Black women, but it also tends to develop at a younger age compared to other ancestral groups. Furthermore, U.S. Black women are approximately twice as likely as White women to be diagnosed with triple-negative breast cancer (TNBC), a subtype characterized by higher recurrence rates and poorer survival outcomes. To investigate the biological factors contributing to these disparities, we spatially profiled TNBC tumors from U.S. Black and White patients. Our analysis revealed significant differences in the tumor immune microenvironment (TiME). Tumors from Black women exhibited higher immune infiltration, particularly among macrophage and T cell populations. To explore how differences in key immune cell populations could impact immunotherapeutic outcomes, we employed a multiomic spatial proteomics approach, aimed at (1) identifying immune cell populations, (2) mapping their activation states, and (3) revealing PD-1/PD-L1 checkpoint activity. All of this was accomplished with a novel serial CellScapeTM workflow, which allows the integration multiplex immunofluorescence (mIF) with and in situ Proximity Ligation Assay (isPLA). Our mIF assay consisted of more than 30 VistaplexTM antibodies and efficiently labeled immune cell lineages, activation states and checkpoints. Cell-to-cell interactions and protein-protein interactions at immune checkpoints were then surveyed throughout the TiME using isPLA, which enables the imaging-based identification of checkpoint activation via detection of protein-protein interactions. The assay is based on a 40 nm proximity of the interacting partners and uses rolling circle amplification (RCA) to amplify the signal, ensuring high sensitivity and specificity of the detected interaction, in this case, that of PD1 and PDL1. Collectively these results affirmed heightened immune infiltration in tumors from Black woman and, crucially, allowed us to map checkpoint activation on a cell-by-cell phenotypic basis. By leveraging multiomic spatial phenotyping, we detected distinctions in the TNBC TiME from Black women compared to White women. Linking together the occurrence of certain cell phenotypes, their spatial signatures, and their checkpoint activation states. These findings underscore the importance of studying diverse patient populations to better understand tumor biology and inform more equitable therapeutic strategies. Felipe Segato Dezem, Matt Ingalls, Omotoso Ayodele, Destiny Burnett, Priscila Coelho, Carmen Gomez, Sara Bodbin, Judith Hurley, Agata Zieba Wicher, Arne Christians, Jannik Boog, Daniel Jimenez Sanchez, Oliver Braubach, Sophia George, Jasmine Plummer. Comprehensive spatial profiling of the tumor immune microenvironment in triple negative breast cancer from Black women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7106.

Single-cell RNA sequencing has revolutionized our understanding of cellular diversity but remains constrained by scalability, high costs, and the destruction of cells during analysis. To overcome these challenges, we developed STAMP (single-cell transcriptomics analysis and multimodal profiling), a highly scalable approach for the profiling of single cells. By leveraging transcriptomics and proteomics imaging platforms, STAMP eliminates sequencing costs, enabling cost-efficient single-cell genomics of millions of cells. Immobilizing (stamping) cells in suspension onto imaging slides, STAMP supports multimodal (RNA, protein, and H&E) profiling, while retaining cellular structure and morphology. We demonstrate STAMP's versatility by profiling peripheral blood mononuclear cells, cell lines, and stem cells. We highlight the capability of STAMP to identify ultra-rare cell populations, simulate clinical applications, and show its utility for large-scale perturbation studies. In total, we present data for 10,962,092 high-quality cells/nuclei and 6,030,429,954 transcripts. STAMP makes high-resolution cellular profiling more accessible, scalable, and affordable.

This Comment outlines the creation of the Global Alliance for Spatial Technologies (GESTALT), a collaborative initiative aimed at fostering the growth and standardization of spatial tissue profiling technologies. It explores the need for GESTALT, its community-driven structure and its goals, spanning from the immediate to the long term.

Formalin-fixed paraffin-embedded (FFPE) samples remain an underutilized resource in single-cell omics due to RNA degradation from formalin fixation. Here, we present snPATHO-seq, a robust and adaptable approach that enables the generation of high-quality single-nucleus (sn) transcriptomic data from FFPE tissues, utilizing advancements in single-cell genomic techniques. The snPATHO-seq workflow integrates optimized nuclei isolation with the 10× Genomics Flex assay, targeting short RNA fragments to mitigate FFPE-related RNA degradation. Benchmarking against standard 10× 3' and Flex assays for fresh/frozen tissues confirmed robust detection of transcriptomic signatures and cell types. snPATHO-seq demonstrated high performance across diverse FFPE samples, including diseased tissues like breast cancer. It seamlessly integrates with FFPE spatial transcriptomics (e.g., FFPE Visium) for multi-modal spatial and single-nucleus profiling. Compared to workflows like 10× Genomics' snFFPE, snPATHO-seq delivers superior data quality by reducing tissue debris and preserving RNA integrity via nuclei isolation. This cost-effective workflow enables high-resolution transcriptomics of archival FFPE samples, advancing single-cell omics in translational and clinical research. Key features • Optimized nuclei isolation from FFPE tissues enables high-quality single-nucleus transcriptomics by minimizing debris and maximizing intact nuclear yield. • Compatible with 10× Genomics Flex, leveraging short RNA probes to overcome FFPE RNA fragmentation challenges. • Outperforms existing FFPE workflows in cell type detection sensitivity across archival, degraded, or aged samples. • Low-cost, accessible protocol using off-the-shelf reagents, suitable for broad translational and archival tissue applications.

Certain pediatric cancers, like blood leukemia, have shown high cure rates, but effective treatments of solid tumors remain elusive and survival rates for affected children remain low. While CAR-T therapy has demonstrated success in treating acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), its effectiveness against solid tumors has been limited. Yet, pediatric patients with solid tumors may still receive CAR-T therapy with minimal prior evidence of treatment efficacy. It is imperative to understand why CAR-T treatments succeed or fail in solid pediatric tumors, as such knowledge can guide the development of future treatment options. This study focuses on a pediatric patient enrolled in a Phase 1/2 clinical trial exploring the use of CAR-T therapy for solid tumors. The patient underwent tumor resection before receiving CAR-T therapy (PRE). Following treatment, the tumor did not respond, leading to recurrence and a second resection (POST).Using advanced spatial transcriptomic profiling we compared the tumor immune microenvironment (TiME) between the PRE and POST treatment samples. Our findings revealed significant differences, including changes in immune cell populations and the spatial distribution of T cells in the TiME. However, while we observed alterations in T cell distribution, the precise localization of CAR-T cells relative to other cell types remained unclear. To address this, we developed a novel targeted spatial multi-omics assay that combines next-generation RNA fluorescence in situ hybridization (HCR™ Gold RNA-FISH) with multiplex immunofluorescence (mIF). HCR™ Gold RNA-FISH is broadly compatible with protein imaging methods, and therefore enables RNA and protein co-detection on the same tissue sample, which is needed to affirmatively detect and map the putative infiltration of CAR-T cells in the TiME. CAR-T cell detection was enabled by custom-designed HCR™ HiFi probes, targeted against B3-H7. Affirmative identification, phenotyping, and spatial profiling of these cells were enabled via VistaPlex™ mIF assay panels deployed serially on the same formalin-fixed paraffin-embedded (FFPE) tissue samples. All experiments were automated on the CellScape™ spatial biology platform and image data were processed and integrated with a custom image analysis pipeline. Our method enables conclusive follow-up investigations into the efficacy of CAR-T treatments in solid pediatric tumors. With this toolkit in hand, it is possible to (a) confirm the status of the CAR-T cell infiltration, (b) define the phenotype and activation states of these cells, and (c) map the spatial biology and cell-to-cell interactions of the CAR-T cells within the TiME. Jasmine T. Plummer, Felipe Segato-Dezem, Yutian Liu, Arjumand Wani, Hannah Chasteen, Arne Christians, Jannik Boog, Randy Chen, Harry Choi, Aneesh Acharya, Oliver Braubach, Giedre Krenciute, Jason Chiang, Christopher De Renzo, Kelsey Bertrand. Development of a multi-omic spatial phenotyping assay for affirmative detection of CAR-T cells in the tumor immune microenvironment of solid pediatric tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1372.

Spared regions of the damaged central nervous system undergo dynamic remodelling and exhibit a remarkable potential for therapeutic exploitation1. Lesion-remote astrocytes (LRAs), which interact with viable neurons and glia, undergo reactive transformations whose molecular and functional properties are poorly understood2. Here, using multiple transcriptional profiling methods, we investigated LRAs from spared regions of mouse spinal cord following traumatic spinal cord injury. We show that LRAs acquire a spectrum of molecularly distinct, neuroanatomically restricted reactivity states that evolve after spinal cord injury. We identify transcriptionally unique reactive LRAs in degenerating white matter that direct the specification and function of local microglia that clear lipid-rich myelin debris to promote tissue repair. Fuelling this LRA functional adaptation is the secreted matricellular protein CCN1. Loss of astrocyte-derived CCN1 results in excessive, aberrant activation of local microglia, characterized by abnormal molecular specification, impaired debris processing reflected by the intracellular accumulation of myelin and axon debris, and dysregulated lipid metabolism with distinctive attenuation in lipid droplet accumulation. Mechanistically, we find that CCN1 binds microglial SDC4 to augment lipid storage, linking this signalling axis to a vital repair-associated lipid buffering response in debris-clearing microglia. Accordingly, microglial deficits resulting from astrocyte CCN1 depletion culminate in blunted clearance of white matter debris and impaired neurological recovery from spinal cord injury. Ccn1-expressing white matter astrocytes are induced by local myelin damage and are generated in diverse demyelinating disorders in mice and humans, pointing to their fundamental, evolutionarily conserved role in white matter repair. Our findings show that context-specific cues shape regionally distinct LRA reactivity states with functional adaptations that orchestrate multicellular processes underlying neural repair and influence disease outcome.

Spatial transcriptomics has emerged as a transformative technology in biomedical research, offering unprecedented insights into gene and protein expression within their native tissue context. Unlike conventional bulk or single-cell sequencing approaches, spatial omics has the advantage of preserving the spatial structure of tissues, allowing researchers to directly map molecular information onto histological structures. This review provides an overview of the current state of spatial omics technologies, highlighting their application in cancer research. Spatial omics has enabled detailed characterization of the tumor microenvironment (TME), revealing spatial heterogeneity, immune cell infiltration patterns, and complex mechanisms of tumor progression and therapy resistance across various cancer types. The review covers future directions, including artificial intelligence–driven analytics, improved standardization, and cost reduction to accelerate clinical translation. Ultimately, spatial omics is poised to play a central role in precision oncology, enabling a deeper understanding of tumor biology and informing more effective individualized treatment strategies.

Phenotypic plasticity is a hallmark of cancer and is increasingly realized as a mechanism of resistance to androgen receptor- targeted (AR-targeted) therapy. Now that many prostate cancer (PCa) patients are treated upfront with AR-targeted agents, it is critical to identify actionable mechanisms that drive phenotypic plasticity, to prevent the emergence of resistance. We showed that loss of tristetraprolin (TTP; gene ZFP36) increased NF-kappa B activation, and was associated with higher rates of aggressive disease and early recurrence in primary PCa. We also examined the clinical and biological impact of ZFP36 loss with co-loss of PTEN, a known driver of PCa. Analysis of multiple independent primary PCa cohorts demonstrated that PTEN and ZFP36 co-loss was associated with increased recurrence risk. Engineering prostate-specific Zfp36 deletion in vivo induced prostatic intraepithelial neoplasia, and, with Pten codeletion, resulted in rapid progression to castration- resistant adenocarcinoma. Zfp36 loss altered the cell state driven by Pten loss, as demonstrated by enrichment of epithelial- mesenchymal transition (EMT), inflammation, TNF-alpha/NF-kappa B, and IL-6-JAK/STAT3 gene sets. Additionally, our work revealed that ZFP36 loss also induced enrichment of multiple gene sets involved in mononuclear cell migration, chemotaxis, and proliferation. Use of the NF-kappa B inhibitor dimethylaminoparthenolide (DMAPT) induced marked therapeutic responses in tumors with PTEN and ZFP36 co-loss and reversed castration resistance.

1115 Background: Glucagon-like peptide-1 receptor agonists (GLP-1RAs) have emerged as a key class of drugs for treating type 2 diabetes mellitus (DM2) and obesity. GLP-1 is rapidly degraded by DPP4, which led to the development of DPP4 inhibitors (DPP4i). Prior work has shown GLP-1R in tumor cells activates key growth signaling and GLP-1RA likely dampen inflammation. This suggests that GLP-1R activation may influence response rates to chemoimmunotherapy. This study aims to investigate the impact of GLP-1RAs and DPP4i (GLP1 drugs) exposure on pathological complete response (pCR) rates for patients with early-stage triple negative breast cancer (TNBC) receiving neoadjuvant chemoimmunotherapy. Methods: Patients with early-stage TNBC diagnosed between July 1, 2021, and December 31, 2023, who received the KEYNOTE-522 regimen were identified at three institutions. Patients using GLP-1RAs and DPP4i at breast cancer diagnosis and throughout the neoadjuvant period, alone or with other diabetes medications, were included. Those who started or discontinued GLP-1 drugs during chemoimmunotherapy were excluded. Group comparisons were made using Chi-square and two-sample t-tests. Human TNBCs were analyzed by IHC and CosMx 6000-plex spatial transcriptomics. Results: Among 343 patients, 7.5% were using GLP-1 drugs. The pCR rate among patients exposed to GLP-1 drugs was 30.8% compared to 64.4% in those not exposed (p = 0.001). For patients using other classes of DM2 medications (n = 46), the pCR rate was 65.2%, while for those not taking any DM2 medications (n = 271), the pCR rate was 64.2%. In univariate analysis, patients exposed to GLP-1 drugs were significantly older than non-exposed (median age: 60 vs. 51 years; p = 0.009), had a higher BMI (35.0 vs. 28.9 kg/m²; p = 0.002), and had higher rates of DM2, hypertension, and hyperlipidemia. In multivariate analysis, only age was associated with pCR (OR: 0.97, 95% CI: 0.96-0.99, p = 0.007). When comparing patients taking GLP-1 drugs with those using other DM2 medications, no significant differences were observed regarding age, BMI, or clinical T or N stage. To evaluate tumor-intrinsic factors that may influence treatment response, we examined TNBC specimens (n = 84) and identified GLP-1 receptor expression in tumor cells in 35.7% of cases and in the tumor microenvironment in 60.7% of cases. A spatial transcriptomics atlas of GLP-1 drug-exposed tumors (469,029 cells) provides evidence of GLP-1 pathway activity in both malignant and non-malignant cells of the tumor microenvironment. Conclusions: We observed significantly lower pCR rate among patients taking GLP1 drugs during neoadjuvant chemotherapy for TNBC. These effects were not observed with other diabetic medications. Detection of GLP1R expression in TNBC specimens indicates there may be direct and indirect effects of agonists to the GLP1 pathway on chemoimmunotherapy response rates.

The Spatial Atlas of Human Anatomy (SAHA) represents the first multimodal, subcellular-resolution reference of healthy adult human tissues across multiple organ systems. Integrating spatial transcriptomics, proteomics, and histological features across over 15 million cells from more than 100 donors, SAHA maps conserved and organ-specific cellular niches in gastrointestinal and immune tissues. High-resolution profiling using CosMx SMI, 10x Xenium, RNAscope, GeoMx DSP, and single-nucleus RNA-seq reveals spatially organized cell states, rare adaptive immune populations, and tissue-specific cell-cell interactions and ligand-receptor pairs. Comparative analyses with colorectal cancer and inflammatory bowel disease demonstrate the power of SAHA to detect disease-associated spatial disruptions, including crypt dedifferentiation, perineural invasion, and therapy-resistant immune remodeling. All data are openly accessible through a FAIR-compliant interactive portal to support exploration, benchmarking, and machine learning model training. Through SAHA, we provide a foundational framework for spatial diagnostics and next-generation precision medicine grounded in a comprehensive human tissue atlas, enabling the development of context-aware models that simulate tissue behavior, decode complex pathologies, and accelerate therapeutic innovation at unprecedented scale.

The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.

Spared regions of the damaged central nervous system undergo dynamic remodeling and exhibit a remarkable potential for therapeutic exploitation. Here, lesion-remote astrocytes (LRAs), which interact with viable neurons, glia and neural circuitry, undergo reactive transformations whose molecular and functional properties are poorly understood. Using multiple transcriptional profiling methods, we interrogated LRAs from spared regions of mouse spinal cord following traumatic spinal cord injury (SCI). We show that LRAs acquire a spectrum of molecularly distinct, neuroanatomically restricted reactivity states that evolve after SCI. We identify transcriptionally unique reactive LRAs in degenerating white matter that direct the specification and function of local microglia that clear lipid-rich myelin debris to promote tissue repair. Fueling this LRA functional adaptation is Ccn1 , which encodes for a secreted matricellular protein. Loss of astrocyte CCN1 leads to excessive, aberrant activation of local microglia with (i) abnormal molecular specification, (ii) dysfunctional myelin debris processing, and (iii) impaired lipid metabolism, culminating in blunted debris clearance and attenuated neurological recovery from SCI. Ccn1 -expressing white matter astrocytes are specifically induced by local myelin damage and generated in diverse demyelinating disorders in mouse and human, pointing to their fundamental, evolutionarily conserved role in white matter repair. Our findings show that LRAs assume regionally divergent reactivity states with functional adaptations that are induced by local context-specific triggers and influence disorder outcome. Astrocytes tile the central nervous system (CNS) where they serve vital roles that uphold healthy nervous system function, including regulation of synapse development, buffering of neurotransmitters and ions, and provision of metabolic substrates 1 . In response to diverse CNS insults, astrocytes exhibit disorder-context specific transformations that are collectively referred to as reactivity 2-5 . The characteristics of regionally and molecularly distinct reactivity states are incompletely understood. The mechanisms through which distinct reactivity states arise, how they evolve or resolve over time, and their consequences for local cell function and CNS disorder progression remain enigmatic. Immediately adjacent to CNS lesions, border-forming astrocytes (BFAs) undergo transcriptional reprogramming and proliferation to form a neuroprotective barrier that restricts inflammation and supports axon regeneration 6-9 . Beyond the lesion, spared but dynamic regions of the injured CNS exhibit varying degrees of synaptic circuit remodeling and progressive cellular responses to secondary damage that have profound consequences for neural repair and recovery 10,11 . Throughout these cytoarchitecturally intact, but injury-reactive regions, lesion-remote astrocytes (LRAs) intermingle with neurons and glia, undergo little to no proliferation, and exhibit varying degrees of cellular hypertrophy 7,12,13 . The molecular and functional properties of LRAs remain grossly undefined. Therapeutically harnessing spared regions of the injured CNS will require a clearer understanding of the accompanying cellular and molecular landscape. Here, we leveraged integrative transcriptional profiling methodologies to identify multiple spatiotemporally resolved, molecularly distinct states of LRA reactivity within the injured spinal cord. Computational modeling of LRA-mediated heterotypic cell interactions, astrocyte-specific conditional gene deletion, and multiple mouse models of acute and chronic CNS white matter degeneration were used to interrogate a newly identified white matter degeneration-reactive astrocyte subtype. We define how this reactivity state is induced and its role in governing the molecular and functional specification of local microglia that clear myelin debris from the degenerating white matter to promote repair.

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but under-utilised in single-cell omics research due to their low DNA and RNA quality. Leveraging recent single-cell genomic technology advances, we introduce snPATHO-seq: a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples.

The quality of standard single-cell experiments often depends on the immediate processing of cells or tissues post-harvest to preserve fragile and vulnerable cell populations, unless the samples are adequately fixed and stored. Despite the recent rise in popularity of probe-based and aldehyde-fixed RNA assays, these methods face limitations in species and target availability and are not suitable for immunoprofiling or assessing chromatin accessibility. Recently, a reversible fixation strategy known as FixNCut has been successfully deployed to separate sampling from downstream applications in a reproducible and robust manner, avoiding stress or necrosis-related artifacts. In this article, we present an optimized and robust practical guide to the FixNCut protocol to aid the end-to-end adaptation of this versatile method. This protocol not only decouples tissue or cell harvesting from single-cell assays but also enables a flexible and decentralized workflow that unlocks the potential for single-cell analysis as well as unconventional study designs that were previously considered unfeasible.

Understanding the composition of human immune responses to SARS-CoV-2 and vaccines is essential for predicting protection from infection and determining vaccine efficacy. Here, we explored T-cell immune responses to SARS-CoV-2 and the UK (B.1.1.7) variant of concern (VOC) in infected and vaccinated individuals. In infected patients, CD4+ T-cells demonstrated consistent, robust responses against Spike peptides, while CD8+ T-cells had heterogeneous responses to 5 SARS-CoV-2 proteins. We found 80% of infected and vaccinated individuals showed positive CD4+ T-cell immunity against SARS-CoV-2. Moreover, CD4+/CD8+ T-cell responses to SARS-CoV-2 and the United Kingdom (B.1.1.7) variant are robust and nearly identical in infected and vaccinated individuals. Thus, the UK variant did not interfere with T-cell recognition and elicited responses. These observations will be of critical importance in assessing human immune responses to emerging VOCs.

Genetic Engineering & Biotechnology NewsVol. 44, No. 3 DepartmentsSounding an Alarm over Spatial BiologyScientists from across the globe highlight spatial biology's triumphs and legal trials while issuing an urgent call for antitrust actionMiranda E. Orr, Arutha Kulasinghe, Grant R. Kolar, Holger Heyn, Jasmine Plummer, Lasse Sommer Kristensen, Jorgen Kjems, Gordon Mills, Juan J. Garcia-Vallejo, I.J. Nijman, Nicholas P. West, and Amanda CoxMiranda E. OrrMiranda E. Orr, PhD, associate professor, gerontology and geriatric medicine, Wake Forest University School of Medicine.Search for more papers by this author, Arutha KulasingheArutha Kulasinghe, PhD, group leader, Clinical-o-Mx Lab, Faculty of Medicine, University of Queensland.Search for more papers by this author, Grant R. KolarGrant R. Kolar, MD, PhD, professor of pharmacology and physiology, Saint Louis University.Search for more papers by this author, Holger HeynHolger Heyn, PhD, team leader, Single Cell Genomics Group, Spanish National Center for Genomic Analysis.Search for more papers by this author, Jasmine PlummerJasmine Plummer, PhD, associate member, St. Jude Faculty, and director, Center for Spatial OMICs.Search for more papers by this author, Lasse Sommer KristensenLasse Sommer Kristensen, PhD, associate professor, Department of Biomedicine, Aarhus University.Search for more papers by this author, Jorgen KjemsJorgen Kjems, PhD, professor, Department of Molecular Biology and Genetics, Aarhus University.Search for more papers by this author, Gordon MillsGordon Mills, MD, PhD, professor of cell, developmental and cancer biology and director of precision oncology, Knight Cancer Institute, Oregon Health & Science University.Search for more papers by this author, Juan J. Garcia-VallejoJuan J. Garcia-Vallejo, PhD, associate professor, Molecular Cell Biology and Immunology, Amsterdam University Medical CentersSearch for more papers by this author, I.J. NijmanI.J. Nijman, PhD, manager, Utrecht Sequencing Facility, Bioinformatic Facility, and High Performance Compute Facility. University Medical Center Utrecht (Center for Molecular Medicine) and the Netherlands X-omics Institute.Search for more papers by this author, Nicholas P. WestNicholas P. West, PhD, associate professor, Central Facility for Genomics and School of Pharmacy and Medical Science, Griffith University.Search for more papers by this author, and Amanda CoxAmanda Cox, PhD, senior lecturer, Central Facility for Genomics and School of Pharmacy and Medical Science, Griffith University.Search for more papers by this authorPublished Online:6 Mar 2024https://doi.org/10.1089/gen.44.03.07AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookXLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 44Issue 3Mar 2024 Information© 2024 by GEN PublishingTo cite this article:Miranda E. Orr, Arutha Kulasinghe, Grant R. Kolar, Holger Heyn, Jasmine Plummer, Lasse Sommer Kristensen, Jorgen Kjems, Gordon Mills, Juan J. Garcia-Vallejo, I.J. Nijman, Nicholas P. West, and Amanda Cox.Sounding an Alarm over Spatial Biology.Genetic Engineering & Biotechnology News.Mar 2024.16-17.http://doi.org/10.1089/gen.44.03.07Published in Volume: 44 Issue 3: March 6, 2024 PDF download

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3' and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization. A combination of an FFPE nuclei preparation protocol and a probe-based transcriptomic profiling technique enables snRNA-seq characterization of archival human FFPE tissues, holding promise for retrospective studies involving aged clinical cohorts.