calcified-artery-preparation-processing-with-preserved-morphology-rna

Calcified Artery Preparation and Processing with Preserved Morphology and RNA for Digital Spatial Profiling

Spatial transcriptomics maps whole-transcriptome profiles directly onto tissue sections, correlating between cellular neighborhoods and gene expression ...

University of Washington

A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.

A technology platform to assess multiple cancer agents simultaneously within a patient's tumor.

A persistent issue in cancer drug development is the discordance between robust antitumor drug activity observed in laboratory models and the limited benefit frequently observed when patients are treated with the same agents in clinical trials. Difficulties in accurately modeling the complexities of human tumors may underlie this problem. To address this issue, we developed Comparative  Oncology (CIVO), which enables  investigation of multiple microdosed drugs simultaneously in a patient's tumor. This study was designed to test CIVO's safety and feasibility in patients with soft tissue sarcoma (STS).

Multiplexed Evaluation of Microdosed Antineoplastic Agents  in the Tumor Microenvironment of Patients with Soft Tissue Sarcoma.

Cancer drug development is currently limited by a paradigm of preclinical evaluation that does not adequately recapitulate the complexity of the intact human tumor microenvironment (TME). To overcome this, we combined trackable intratumor microdosing (CIVO) with spatial biology readouts to directly assess drug effects in patient tumors in situ.

Trackable Intratumor Microdosing and Spatial Profiling Provide Early Insights into Activity of Investigational Agents in the Intact Tumor Microenvironment.

Spatial transcriptomic profiling enables precise quantification of gene expression with simultaneous localization of expression profiles onto tissue structures. This new technology promises to improve our understanding of the disease mechanisms. Therefore, there is intense interest in applying these methods in clinical trials or as laboratory developed tests to aid in diagnosis of disease. Before these technologies can be more broadly deployed in clinical research and diagnostics, it is necessary to thoroughly understand their performance in real world conditions. In this study, we vet technical reproducibility, data normalization methods and assay sensitivity focusing predominantly on one widely used spatial transcriptomic methodology, digital spatial profiling. Using clinically sourced human tissue specimens, we find that digital spatial profiling exhibits high rigor and reproducibility. Our approach lays the foundation for incorporation of digital spatial profiling methods into clinical workflows.

Rigor and Reproducibility of Digital Spatial Profiling on Clinically Sourced Human Tissues.

Spatial transcriptomic profiling enables precise quantification of gene expression with simultaneous localization of expression profiles onto tissue structures. Several implementations of these approaches have been released as commercialized platforms that will allow multiple laboratories to improve our understanding of human disease mechanisms. There is also intense interest in applying these methods in clinical trials or as laboratory-developed tests to aid in the diagnosis of disease. However, before these technologies can be broadly deployed in clinical research and diagnostics, it is necessary to thoroughly understand their performance in real-world conditions. In this study, we vet the technical reproducibility, data normalization methods, and assay sensitivity focusing predominantly on one widely used spatial transcriptomics methodology, digital spatial profiling. We also compare its performance with a single molecular imager, a newer platform with single-cell resolution. Using clinically sourced human kidney tissues and biopsies as exemplars, we find that digital spatial profiling exhibits high rigor and reproducibility. We show that normalization approaches can impact the biological interpretation of spatial transcriptomics data. Although there is good concordance between multicellular and single-cell resolution methods, there are tradeoffs in cost, execution time, and sensitivity of detection, which may affect which approach is chosen. Our study lays a practical foundation for the incorporation of spatial transcriptomics methods into clinical workflows.

Rigor and Reproducibility of Spatial Transcriptomics Performed on Clinically Sourced Human Tissues.

Vaccines against emerging SARS-CoV-2 variants and sarbecoviruses with pandemic potential must elicit a robust humoral immune response in a population imprinted with the SARS-CoV-2 spike (S) protein. Here, we designed protein nanoparticle (NP) vaccines co-displaying the SARS-CoV-2 BA.5, SARS-CoV-1, and BtKY72 receptor-binding domains (RBDs) with or without the Wuhan-Hu-1 (Wu) RBD. We show that these vaccines elicit cross-reactive and broadly neutralizing plasma antibody responses against SARS-CoV-2 variants and sarbecoviruses in naive and pre-immune animals. Immunization with multivalent RBD-NPs overcomes immune imprinting and elicits neutralizing antibodies and memory B cells specific for the BA.5, SARS-CoV-1, and BtKY72 RBDs in mRNA-1273-vaccinated non-human primates. Multivalent RBD-NPs outperform a monovalent Wu RBD-NP vaccine by providing superior protection in mice and non-human primates challenged with the vaccine-mismatched SARS-CoV-2 XBB.1.5 or the pre-emergent RsSHC014. These data support the use of multivalent RBD-NP vaccines for SARS-CoV-2 variants and sarbecoviruses in naive and pre-immune populations.

Phylogeny-driven design of broadly protective sarbecovirus receptor-binding domain nanoparticle vaccines.

Effective antitumor immunity ultimately depends on the priming and activation of tumor-specific cytotoxic CD8+ T cells; however, the role of intratumoral cell-cell immune interactions remains incompletely understood. Recent work has revealed that the temporospatial colocalization of dendritic cells (DCs), helper T cells (Th), and cytotoxic T lymphocytes (CTL) within the tumor immune microenvironment following immune checkpoint blockade correlates with clinical response. Herein, we report the integration of more than one million spatially resolved single-cell profiles across six spatial proteomic and transcriptomic assays, which demonstrated that DC:Th:CTL three-cell-type clusters were common even in immunotherapy-naïve and highly desmoplastic tumors, such as fibrolamellar carcinoma and pancreatic ductal adenocarcinoma (PDAC). We found that these immune triads were enriched for functionally important type 1 conventional DC, mature DCs enriched in immunoregulatory molecules (mregDC), CXCL13+ Th, and GZMK+ effector CTL phenotypes. Subsequent multiplex immunofluorescence imaging of more than 450 primary PDAC tumors showed that the density of antigen-presenting cell (APC):Th:CTL three-cell-type clusters was correlated with intratumoral T-cell clonal expansion and improved overall survival. These findings suggest that DC:Th:CTL triads are conserved across solid tumors and highlight the importance of intratumoral spatial niches in mediating endogenous antitumor immunity.

Article	Year
A technology platform to assess multiple cancer agents simultaneously within a patient's tumor. Science translational medicine\| PubMed ID: 25904742	2015
Multiplexed Evaluation of Microdosed Antineoplastic Agents in the Tumor Microenvironment of Patients with Soft Tissue Sarcoma. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 32299817	2020
Trackable Intratumor Microdosing and Spatial Profiling Provide Early Insights into Activity of Investigational Agents in the Intact Tumor Microenvironment. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 37389981	2023
Rigor and Reproducibility of Digital Spatial Profiling on Clinically Sourced Human Tissues. bioRxiv : the preprint server for biology\| PubMed ID: 39463992	2024
Rigor and Reproducibility of Spatial Transcriptomics Performed on Clinically Sourced Human Tissues. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 40311874	2025
Phylogeny-driven design of broadly protective sarbecovirus receptor-binding domain nanoparticle vaccines. bioRxiv : the preprint server for biology\| PubMed ID: 40463102	2025
Intratumoral three-cell-type clusters are a conserved feature of endogenous antitumor immunity. Cancer immunology research\| PubMed ID: 41201469	2025

Emily Beirne

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