Abstract The complex interplay between genetic alterations, cellular environments, and immune responses in ovarian cancer demands advanced analytical approaches to inform personalized therapies. Conventional two-dimensional analyses fail to capture the tumor’s complex three-dimensional (3D) architecture and cellular organization, limiting insight into immune dynamics and the tumor microenvironment. To overcome this, we developed a fully automated spatial biology workflow integrating 3D multiomics. This workflow combines RNAsky® technology for precise RNA detection with multiplexed protein profiling using recombinant REAfinity™ and REAdye_lease™ antibody conjugates, analyzed via the MACS® iQ View - Spatial Biology Image Analysis Software for accurate 3D segmentation. The inclusion of multiple layers in the z-dimension allows precise annotation of individual cells, and thus precise RNA and protein localization at the single-cell level. By analyzing ovarian cancer tissues with an immuno-oncology antibody panel and a complementary RNA panel, we characterized primary, untreated ovarian cancer tissues and their microenvironment. This enabled spatial mapping of diverse lymphoid cell populations including T-cells, B-cells, and NK cells, as well as cells of myeloid lineage such as dendritic cells and macrophages. Additionally, cancer cells were characterized and the stromal compartment encompassing fibroblasts, endothelial and neuronal cells were identified. These aspects revealed structural and immunological features which may influence tumor progression and treatment response. The 3D analysis uncovered spatial relationships among various cell types, in particular, 3D analysis resolved key anatomical features of tissue architecture, which play crucial roles in tumor growth and metastasis. By correlating these 3D spatial data with clinical outcomes, the workflow may provide insights into resistance mechanisms, potential biomarkers, and therapeutic targets. This integrated 3D multiomics workflow offers a holistic, high-resolution view of tumor organization and immune contexture, advancing the understanding of ovarian cancer biology and supporting precision medicine across cancer types. Citation Format: Lena Nolte, Diogo Bessa-Neto, Salpy Baghdo, Bernadett Szabó, Fabio El Yassouri, Emily Neil, Robert Pinard, Werner Müller, Dominik Eckardt, Christoph Herbel, Andreas Bosio. 3D spatial multiomics characterization of ovarian cancer tissue samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1225.

In neurosurgery procedures, cerebrospinal fluid leakage is a commonly encountered complication. Reconstructing skull base defects with patch materials can reduce the risk of cerebrospinal fluid leakage which can lead to serious issues such as infection, meningitis, arachnoiditis, and delayed wound healing. An ideal skull base reconstruction material should not only serve as a leak-proof barrier but also promote skull base bone regeneration. To fulfill this challenge, this research designed and fabricated a Janus orthogonal bilayer nanofiber membrane (OPCL/PG-PCPP). The aligned PCL (APCL) nanofibers were constituted as the top layer to resist cerebrospinal fluid leakage, while the perpendicular PCL/gelatin (APG) fibers with calcium polyphosphate encapsulated polydopamine nanoparticles (CPP@PDA, labeled as PCPP) were designed as the bottom layer (APG-PCPP) to facilitate osteoblast migration and osteogenic differentiation. Among these, APG-1%PCPP nanofibers demonstrated the most effective induction of osteogenic differentiation in bone marrow mesenchymal stem cells (rBMSCs). Subsequent in vivo animal experiments revealed that the bone surface area (BS), bone volume fraction (BV/TV), and number of trabeculae (Tb.N) in the APG-1%PCPP group were twice as high as those in the control group, which confirmed the good osteogenic potentials. Therefore, due to its unique leak-proof and osteoinductive properties, the OPCL/PG-PCPP membrane holds promise as an applicable skull base reconstruction material in the field of neurosurgery.

There is a need for novel nanomaterials with properties not yet exploited in regenerative nanomedicine. Based on lessons learned from the oldest metazoan phylum, sponges, it has been recognized that two previously ignored or insufficiently recognized principles play an essential role in tissue regeneration, including biomineral formation/repair and wound healing. Firstly, the dependence on enzymes as a driving force and secondly, the availability of metabolic energy. The discovery of enzymatic synthesis and regenerative activity of amorphous biosilica that builds the mineral skeleton of siliceous sponges formed the basis for the development of successful strategies for the treatment of osteochondral impairments in humans. In addition, the elucidation of the functional significance of a second regeneratively active inorganic material, namely inorganic polyphosphate (polyP) and its amorphous nanoparticles, present from sponges to humans, has pushed forward the development of innovative materials for both soft (skin, cartilage) and hard tissue (bone) repair. This energy-rich molecule exhibits a property not shown by any other biopolymer: the delivery of metabolic energy, even extracellularly, necessary for the ATP-dependent tissue regeneration. This review summarizes the latest developments in nanobiomaterials based on these two evolutionarily old, regeneratively active materials, amorphous silica and amorphous polyP, highlighting their specific, partly unique properties and mode of action, and discussing their possible applications in human therapy. The results of initial proof-of-concept studies on patients demonstrating complete healing of chronic wounds are outlined

Rationale: Tissue regeneration of skin and bone is an energy-intensive, ATP-consuming process that, if impaired, can lead to the development of chronic clinical pictures. ATP levels in the extracellular space including the exudate of wounds, especially chronic wounds, are low. This deficiency can be compensated by inorganic polyphosphate (polyP) supplied via the blood platelets to the regenerating site. Methods: The contribution of the different forms of energy derived from polyP (metabolic energy, mechanical energy and heat) to regeneration processes was dissected and studied both in vitro and in patients. ATP is generated metabolically during the enzymatic cleavage of the energy-rich anhydride bonds between the phosphate units of polyP, involving the two enzymes alkaline phosphatase (ALP) and adenylate kinase (ADK). Exogenous polyP was administered after incorporation into compressed collagen or hydrogel wound coverages to evaluate its regenerative activity for chronic wound healing. Results: In a proof-of-concept study, fast healing of chronic wounds was achieved with the embedded polyP, supporting the crucial regeneration-promoting activity of ATP. In the presence of Ca2+ in the wound exudate, polyP undergoes a coacervation process leading to a conversion of fibroblasts into myofibroblasts, a crucial step supporting cell migration during regenerative tissue repair. During coacervation, a switch from an endothermic to an exothermic, heat-generating process occurs, reflecting a shift from an entropically- to an enthalpically-driven thermodynamic reaction. In addition, mechanical forces cause the appearance of turbulent flows and vortices during liquid-liquid phase separation. These mechanical forces orient the cellular and mineralic (hydroxyapatite crystallite) components, as shown using mineralizing SaOS-2 cells as a model. Conclusion: Here we introduce the energetic triad: metabolic energy (ATP), thermal energy and mechanical energy as a novel theranostic biomarker, which contributes essentially to a successful application of polyP for regeneration processes.

Obesity is a major cause of metabolic dysfunction-associated steatohepatitis (MASH) and is characterized by inflammation and insulin resistance. Interferon-γ (IFNγ) is a pro-inflammatory cytokine elevated in obesity and modulating macrophage functions. Here, we show that male mice with loss of IFNγ signaling in myeloid cells (Lyz-IFNγR2−/−) are protected from diet-induced insulin resistance despite fatty liver. Obesity-mediated liver inflammation is also attenuated with reduced interleukin (IL)−12, a cytokine primarily released by macrophages, and IL-12 treatment in vivo causes insulin resistance by impairing hepatic insulin signaling. Following MASH diets, Lyz-IFNγR2−/− mice are rescued from developing liver fibrosis, which is associated with reduced fibroblast growth factor (FGF) 21 levels. These results indicate critical roles for IFNγ signaling in macrophages and their release of IL-12 in modulating obesity-mediated insulin resistance and fatty liver progression to MASH. In this work, we identify the IFNγ-IL12 axis in regulating intercellular crosstalk in the liver and as potential therapeutic targets to treat MASH.

Abstract The HubMap consortium has developed a new standard to report on normal histological samples using multicolor immunofluorescence imaging. An Organ Mapping Antibody Panel (OMAP) currently describes in a table the antibodies used, the cycle number assigned to a given reagent, and provides the rationale for using a particular antibody to better understand features of the tissue. This table is accompanied by antibody validation templates that show example images from the tissue described for the OMAP table. In addition, links to existing databases, gene symbols, the antibody features and colors are included. For each tissue, a further table called "Anatomical Structures, Cell Types, plus Biomarkers" (ASCT+B), which contains structures of the tissue, cell types in a given tissue structure, key RNA transcripts for a given cell type, and antibody stains as used in the OMAP table, is created. So far, this process has not been established for spatial multiomic datasets including protein and RNA detected on the same tissue section. We have extended spatial biology methodology to combine cyclic RNA transcript detection with cyclic antibody-based protein detection using the MACSima Imaging Cyclic Staining (MICS) technology as well as H&E-staining on the same tissue section. For such datasets, the current OMAP data table needs to be extended by adding information on the RNA detection, cycle IDs and RNA validation templates, demonstrating the correct performance of a given RNA probe detection. This poster will demonstrate a proposal to extend the OMAP table structure on an extended version of the “tonsil OMAP (OMAP-10)”, which was published on the Zenodo data repository page (https://zenodo.org/records/7875938). In addition, we will apply this extended OMAP to a colorectal cancer sample, to highlight the applicability and relevance of the OMAP structure for tumor tissue. Citation Format: Werner Müller, Julia Femel, Emily Neil, Dongju Park, Rebecca C. Hennessey, Eric C. DiBiasio, Michael DiBuono, Hanna Lafayette, Erica Lloyd, Hsinyi Lo, Alex Makrigiorgos, Sameh Soliman, Dominic Mangiardi, Paurush Praveen, Silvia Rüberg, Fabian Staubach, Ryan Hindman, Thomas Rothmann, Hansueli Meyer, Tanya Wantenaar, Jinling Wang, Robert Pinard, Andreas Bosio. A proposal to extend standardized organ mapping antibody panels (OMAPs) to integrate protein and RNA analysis in spatial biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4953.

To combat infections, silver was used extensively in biomedical field but there was a need for a capping agent to eliminate its cytotoxic effects. In this study, polymeric calcium polyphosphate was doped by silver with three concentrations 1, 3 or 5 mol.% and were characterized by TEM, XRD, FTIR, TGA. Moreover, cytotoxicity, antibacterial, cell migration and DNA fragmentation assays were done to assure its safety. The results showed that the increase in silver percentage caused an increase in particle size. XRD showed the silver peaks, which indicated that it is present in its metallic form. The TGA showed that thermal stability was increased by increasing silver content. The antibacterial tests showed that the prepared nanoparticles have an antibacterial activity against tested pathogens. In addition, the cytotoxicity results showed that the samples exhibited non-cytotoxic behavior even with the highest doping concentration (5% Ag-CaPp). The cell migration assay showed that the increase in the silver concentration enhances cell migration up to 3% Ag-CaPp. The DNA fragmentation test revealed that all the prepared nanoparticles caused no fragmentation. From the results we can deduce that 3% Ag-CaPp was the optimum silver doped calcium polyphosphate concentration that could be used safely for medical applications.

Nanoparticles of a particular, evolutionarily old inorganic polymer found across the biological kingdoms have attracted increasing interest in recent years not only because of their crucial role in metabolism but also their potential medical applicability: it is inorganic polyphosphate (polyP). This ubiquitous linear polymer is composed of 10-1000 phosphate residues linked by high-energy anhydride bonds. PolyP causes induction of gene activity, provides phosphate for bone mineralization, and serves as an energy supplier through enzymatic cleavage of its acid anhydride bonds and subsequent ATP formation. The biomedical breakthrough of polyP came with the development of a successful fabrication process, in depot form, as Ca- or Mg-polyP nanoparticles, or as the directly effective polymer, as soluble Na-polyP, for regenerative repair and healing processes, especially in tissue areas with insufficient blood supply. Physiologically, the platelets are the main vehicles for polyP nanoparticles in the circulating blood. To be biomedically active, these particles undergo coacervation. This review provides an overview of the properties of polyP and polyP nanoparticles for applications in the regeneration and repair of bone, cartilage, and skin. In addition to studies on animal models, the first successful proof-of-concept studies on humans for the healing of chronic wounds are outlined.

Abstract In solid tumors, the tumor microenvironment (TME) is composed of diverse cell types including cancer cells, immune cells, stromal cells, and other tissue specific cell types. The complex intercellular interactions that occur between these various cell populations determine cancer development and progression. Cancer-associated fibroblasts (CAFs) have been identified as key players in the TME, capable of promoting tumor cell growth and invasion, as well as manipulating immune responses. To better resolve potential subpopulations, spatial relationships, and signaling occurring between cell types within the TME, we performed same-section multiomic profiling of colorectal cancer (CRC) using the MACSima™ Imaging Cyclic Staining (MICS) technology. We combined a custom 40plex RNA panel with a panel of antibodies to characterize the drivers of tumorigenesis and the activation state of immune cells. FFPE CRC specimens were reviewed by a pathologist for clinical assessment and region of interest selection. Then, gene expression profiles were generated using RNAsky™ technology with each gene being detected via cyclic rounds of detection probe hybridization, image acquisition, and signal erasure. Subsequently, fluorescently labeled antibodies were applied to the same section following an equivalent acquisition process. Finally, multiomic clustering and cell population analyses were performed using MACS® iQ View software. The analysis revealed spatially separated subpopulations of CAFs. Functional characterization of cellular neighborhoods and the cell-to-cell interactions occurring within the TME showed that one of the CAF populations potentially promoted cancer cell growth while another subpopulation, resembling antigen-presenting CAFs (apCAFs), closely interacted with T cells in the TME. These findings will deepen our understanding of tumor progression in colorectal cancer and potentially other solid tumors. Citation Format: Emily Neil, Rebecca C. Hennessey, David Agorku, Dongju Park, Julia Femel, Michael DiBuono, Hanna Lafayette, Erica Lloyd, Hsinyi Lo, Alex Makrigiorgos, Shaina Lu, John Lee, Sameh Soliman, Dominic Mangiardi, Paurush Praveen, Philip Ströbel, Silvia Rüberg, Fabian Staubach, Ryan Hindman, Thomas Rothmann, Olaf Hardt, Hansueli Meyer, Tanya Wantenaar, Jinling Wang, Werner Müller, Robert Pinard, Andreas Bosio. Multiomic characterization of colorectal cancer using MICS technology reveals interaction of antigen presenting cancer associated fibroblasts and T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5565.

Inorganic materials are of increasing interest not only for bone repair but also for other applications in regenerative medicine. In this study, the combined effects of energy-providing, regeneratively active inorganic polyphosphate (polyP) and also morphogenetically active pearl powder on wound healing were investigated. Aragonite, the mineralic constituent of pearl nacre and thermodynamically unstable form of crystalline calcium carbonate, was found to be converted into a soluble state in the presence of a Ca2+-containing wound exudate, particularly upon addition of sodium polyP (Na-polyP), driven by the transfer of Ca2+ ions from aragonite to polyP, leading to liquid-liquid phase separation to form an aqueous Ca-polyP coacervate. This process is further enhanced in the presence of Ca-polyP nanoparticles (Ca-polyP-NP). Kinetic studies revealed that the coacervation of polyP and nacre aragonite in wound exudate is a very rapid process that results in the formation of a stronger gel with a porous structure compared to polyP alone. Coacervate formation, enabled by phase transition of crystalline aragonite in the presence of Na-polyP/Ca-polyP-NP and wound exudate, could also be demonstrated in a hydroxyethyl cellulose-based hydrogel used for wound treatment. Furthermore, it is shown that Na-polyP/Ca-polyP-NP together with nacre aragonite strongly enhances the proliferation of mesenchymal stem cells and promotes microtube formation in the in vitro angiogenesis assay with HUVEC endothelial cells. The latter effect was confirmed by gene expression studies, applying real-time polymerase chain reaction, using the biomarker genes VEGF (vascular endothelial growth factor) and hypoxia-inducible factor-1 alpha (HIF-1 alpha). Division of Escherichia coli is suppressed when suspended in a matrix containing Na-polyP/Ca-polyP-NP and aragonite. The potential medical relevance of these findings is supported by an animal study on genetically engineered diabetic mice (db/db), which demonstrated a marked increase in granulation tissue and microvessel formation in regenerating experimental wounds treated with Ca-polyP-NP compared to controls. Co-administration of aragonite significantly accelerated the wound healing-promoting effect of polyP in db/db mice. Based on these results, we propose that the ability of polyP to form a mixed coacervate with aragonite, in addition to its energy (ATP)-generating function, can decisively contribute to the regenerative activity of this polymer in wound repair. Polyphosphate (polyP) is a physiologically significant polymer with regenerative properties, crucial for supplying the metabolic fuel (ATP) essential for various regeneration processes in humans, including wound healing.

Abstract The recent increase in image-based, spatially-resolved technologies enables researchers to profile the tumor microenvironment (TME) by capturing gene expression profiles within tissue sections. However, a significant limitation of these technologies is the lack of ability to resolve protein and RNA information in the same section, as well as conveniently analyze multimodal data sets. Here, we report a spatial RNA detection method, RNAsky, using Miltenyi Biotec’s MACSima™ Platform as an automated, multiomic approach. Our method integrates spatial proteomics and transcriptomics data to provide in-depth profiling with single-cell resolution on the same tissue section. We demonstrate these capabilities by characterizing key immune-oncology markers across normal and diseased tissues using our specialized MACS® iQ View analysis software. MACS iQ View provides fast segmentation and intuitive gating and clustering strategies to simultaneously assess protein and RNA data. We investigated the impact of clustering using protein, RNA, or the combination of both to evaluate the contribution of different information modalities on TME spatial dynamics. This cutting-edge approach will enable the identification of valuable parameters and new cell types, furthering the discovery and development of predictive and prognostic biomarkers. Citation Format: Dongju Park, Emily Neil, Rebecca C. Hennessey, Michael DiBuono, Hanna Lafayette, Erica Lloyd, Hsinyi Lo, Julia Femel, Alex Makrigiorgos, Shaina Lu, John Lee, Sameh Soliman, Dominic Mangiardi, Paurush Praveen, Fabian Staubach, Ryan Hindman, Thomas Rothmann, Telma Santos, Stefan Borbe, Hansueli Meyer, Tanya Wantenaar, Jinling Wang, Werner Müller, Robert Pinard, Andreas Bosio. Same-section spatial multiomic analyses using MICS technology for investigating the dynamics of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4308.

Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.

Efficient inactivation of RNase H2 in the epidermis, and skin phenotype of Rnaseh2EKO mice.

Influence of p53-deficiency on phenotype of Rnaseh2EKO mice.

This file contains transcripts induced by type I interferon (S1) or regulated by p53 in response to DNA damage (S2).

Understanding the optimal conditions required for bone healing can have a substantial impact to target the problem of non-unions and large bone defects. The combination of bioactive factors, regenerative progenitor cells and biomaterials to form a tissue engineered (TE) complex is a promising solution but translation to the clinic has been slow. We hypothesized the typical material testing algorithm used is insufficient and leads to materials being mischaracterized as promising. In the first part of this study, hu-man bone marrow - derived mesenchymal stromal cells (hBM-MSCs) were embedded in three commonly used biomaterials (hyaluronic acid methacrylate, gelatin methacrylate and fibrin) and combined with rel-evant bioactive osteogenesis factors (dexamethasone microparticles and polyphosphate nanoparticles) to form a TE construct that underwent in vitro osteogenic differentiation for 28 days. Gene expression of relevant transcription factors and osteogenic markers, and von Kossa staining were performed. In the sec -ond and third part of this study, the same combination of TE constructs were implanted subcutaneously (cell containing) in T cell-deficient athymic Crl:NIH-Foxn1rnu rats for 8 weeks or cell free in an immuno-competent New Zealand white rabbit calvarial model for 6 weeks, respectively. Osteogenic performance was investigated via MicroCT imaging and histology staining. The in vitro study showed enhanced up-regulation of relevant genes and significant mineral deposition within the three biomaterials, generally considered as a positive result. Subcutaneous implantation indicates none to minor ectopic bone forma-tion. No enhanced calvarial bone healing was detected in implanted biomaterials compared to the empty defect. The reasons for the poor correlation of in vitro and in vivo outcomes are unclear and needs further investigation. This study highlights the discrepancy between in vitro and in vivo outcomes, demonstrating that in vitro data should be interpreted with extreme caution. In vitro models with higher complexity are necessary to increase value for translational studies.Statement of significancePreclinical testing of newly developed biomaterials is a crucial element of the development cycle. Despite this, there is still significant discrepancy between in vitro and in vivo test results. Within this study we investigate multiple combinations of materials and osteogenic stimulants and demonstrate a poor cor-relation between the in vitro and in vivo data. We propose rationale for why this may be the case and suggest a modified testing algorithm.(c) 2022 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

Inorganic polyphosphates (polyP) consist of linear chains of orthophosphate units linked together by high-energy phosphoanhydride bonds. The family of polyP molecules are evolutionarily old biopolymers and found from bacteria to man. PolyP is exceptional, no other molecule concentrates as much (bio)chemically usable energy as polyP in animals, including humans. Before this discovery, we found that the long-neglected polymer provides orthophosphate units required for bone (hydroxyapatite) synthesis. Hence, polyP is a cornerstone for bone synthesis and repair, especially in higher animals. Besides its importance for regenerative medicine, especially for the reconstitution of osteoarticular impairments/defects, a further imperative property could be attributed the polyP. This polymer is the only extracellular generator of metabolic energy in the form of ATP. While the mitochondria synthesize ATP in large amounts intracellularly, it is polyP, which functions as the storage for extracellular ATP. After enzymatic hydrolysis of polyP by alkaline phosphatase (ALP) the released free energy is partially stored in ADP (formed from AMP), which in the second step is up-phosphorylated to ATP by adenylate kinase (ADK). In turn, the two enzymes ALP and ADK are the biocatalytic proteins that conserve the released free energy and store it in ATP, especially in the extracellular space. In a proof-of-concept, we could demonstrate that polyP is an essential component for human regeneration processes, especially in those regions, which are poorly vascularised, like in bone, cartilage and wounds (including chronic wounds).

Spontaneous DNA damage response and neoplastic ulcerations in Rnaseh2EKO mice.

This file contains histology of skin and skin lesions of RNaseH2EKO mice with intact or defective Trp53 alleles.

Spatial Biology has evolved from the molecular characterization of microdissected cells to high throughput spatial RNA and protein expression analysis at scale. The main limitation of spatial technologies so far is the inability to resolve protein and RNA information in the same histological section. Here, we report for the first time the integration of highly multiplexed RNA and protein detection on the same tissue section. We developed a new, automated, spatial RNA detection method (RNAsky™), which is based on targeted rolling circle amplification and iterative staining. We combine RNAsky with MACSima™ Imaging Cyclic Staining (MICS) based protein analysis and show compatibility with subsequent standard hematoxylin and eosin (H&E) staining. Using both, open-source tools and our recently developed software suite MACS® iQ View, we demonstrate our multiomics MICS workflow by characterizing key immune-oncology markers at subcellular resolution across normal and diseased tissues.