Multiplex Imaging Research Articles

Activity-based trapping for multiplex imaging illuminates the hidden role of endogenous formaldehyde in proinflammatory signaling

Activity-based trapping for multiplex imaging illuminates the hidden role of endogenous formaldehyde in proinflammatory signaling

Abstract A045: Imaging ferroptosis-mediated TME remodeling that sensitize cold tumors for immunotherapy

Abstract Background: Immune checkpoint inhibitors (ICIs) harness the power of the immune system to fight against systemic cancers. Despite being one of the most promising current therapeutic strategies, response rates remain heterogeneous. Immunologically cold tumors, such as triple-negative breast cancer (TNBC), are among those that do not respond well to checkpoint inhibition. Ferroptosis-related therapies are emerging as a new class of therapeutics for chemo-resistant and metastatic cancers, as they exploit the antioxidant dependence of cancer cells, circumventing most resistance mechanisms. However, the relationship between ferroptosis and the tumor microenvironment (TME) is not well characterized. Here, we leverage ferroptosis-inducing therapies as an adjuvant for immunotherapy and monitor the myeloid dynamics in response with immuno-Positron Emission Tomography (immuno-PET). Methods: A myeloid-heavy TNBC model was used to study myeloid dynamics in response to ferroptosis therapy. The immunogenicity of ferroptotic cancer cells was investigated through in vitro quantification of damage-associated molecular patterns (DAMPs). The impact of ferroptosis therapies on macrophages was also evaluated. Furthermore, the synergy of ferroptosis-inducing therapy (using IKE) and ICB (with aPD-1) was assessed in vivo, and immune infiltration changes were quantified via flow cytometry and tissue staining. To facilitate this investigation, a pan-myeloid radioimmunoconjugate, 89Zr-DFO-CD11b, was developed as an immunoPET agent to quantitatively monitor spatiotemporal dynamics of tumor myeloid cells during ferroptosis-based therapy. Results: TNBC cells undergoing ferroptosis exhibit an immunogenic phenotype, characterized by increased DAMP production, which can contribute to TME reprogramming. Macrophages exposed to cells undergoing ferroptosis were polarized to an anti-tumor phenotype, exhibited increased tumor spheroid infiltration, and increased phagocytosis signaling. In vivo combination of IKE with aPD-1 suppressed tumor growth. The combination therapy increased tumor infiltration of lymphocyte populations, particularly CD8+ T cells, as well as anti-tumor macrophages, into the tumor core. After confirming 89Zr-DFO-CD11b showed high radiochemical conversion, stability in serum, and specific binding to myeloid cells both in vitro and in vivo, 89Zr-DFO-CD11b was injected IV into tumor-bearing mice receiving therapy. Tumor uptake was significantly less in the combination therapy group, with a distinct pocket of myeloid cells in the center of treated tumors, compared to control tumors with myeloid cells around the tumor periphery. Conclusions: This study underscores the potential of ferroptosis as an adjuvant to immunotherapy, with spatiotemporal immune cell dynamics highlighted by 89Zr-DFO-CD11b myeloid imaging. Future studies will multiplex PET imaging with T cell and ferroptosis markers to further elucidate TME dynamics. Citation Format: Elana Apfelbaum, Nermin Mostafa, Edwin C Pratt, Jan Grimm. Imaging ferroptosis-mediated TME remodeling that sensitize cold tumors for immunotherapy [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A045.

Abstract B048: Spatiotemporal analysis during tumor rejection reveals coordinated cellular dynamics underlying successful immune-checkpoint therapy

Abstract There is a growing appreciation for the role that the spatial organization of immune cells within the tumor microenvironment (TME) plays in shaping cellular function and response to therapy. However, the majority of studies have been limited to analyzing immune responses at a single time point, and so, little is known about the spatiotemporal changes that underlie successful tumor rejection after cancer immunotherapy. In this study, we used the CODEX multiplex imaging system with a 32-plex antibody panel to longitudinally characterize the spatial changes in the lymphoid and myeloid cell populations that were previously identified by scRNA-Seq and CyTOF to be associated with complete tumor rejection in mice treated with the combination of α-PD-1 and α-CTLA-4 (combo ICT) in our well-characterized mouse T3 MCA sarcoma model. T3 tumor-bearing mice were treated with control antibody or combo ICT, tumors were harvested at multiple time points (day 7, 9, 10, 11 and 13), fresh frozen, sectioned, and subjected to CODEX multiplex imaging. A total of 42 tumors were imaged with the full range of the tumor-immune boundary. A hierarchical cell clustering approach developed in-house was used to profile 13,051,156 cells resulting in the identification of 13 distinct cell types. Our results show that after ICT the decrease in T3 cells in the tumor tissue starts on day 10 and culminates in the complete elimination of tumor cells by day 13. This immune-mediated response is driven by an increase in CD4+ T cells, which allows them to more frequently co-localize with CD8+ T cells and type 1 conventional dendritic cells (cDC1), forming three-cell clusters with increased cell contacts. This change in cell-to-cell interactions is followed by a broader reorganization of the TME as noted by the expansion of a lymphoid-rich cellular neighborhood (CN) that harbors the majority of CD4+ and CD8+ T cells. Interestingly, this CN becomes a hub for T cell effector functions, characterized by an enrichment of GZM-B+ and KI67+ T cells, and exhibits spatial dynamics throughout tumor regression. While both GZM-B+ and KI67+ T cells are observed in proximity to blood vessels on days 7 and 9, they begin to spatially separate into distinct regions after day 10, indicating that cytotoxic T cells move towards areas with tumor cells, while proliferating T cells are more frequently observed at the periphery. This dynamic behavior was further confirmed by assessing the communication rules between CNs, which showed that the lymphoid-rich CN acts by eliminating layers of tumor cells at the tumor boundary and by modulating nearby myeloid populations. Additionally, integration of CODEX dataset with scRNA-seq revealed CD4+ T cells produce Interferon-gamma and a change in the molecular chemotactic pathways orchestrating lymphoid and myeloid communication. Overall, this study provides a detailed spatial view of the cellular events that result in successful ICT and allows us to propose a computational model of the actions of T cells in coordinating the antitumor response after α-CTLA-4/α-PD1. Citation Format: Ruan FV Medrano, Vladimir Sukhov, Maxim Artyomov, Robert Schreiber. Spatiotemporal analysis during tumor rejection reveals coordinated cellular dynamics underlying successful immune-checkpoint therapy [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B048.

Sample-specific learning of lymphovascular invasion with heterogeneous spatial patterns

Lymphovascular invasion (LVI) represents one of the earliest stages of metastasis. This article is motivated by using the multiplex immunofluorescence imaging data for identification of LVI that can improve diagnosis of early breast cancer prior to metastasis. One unique aspect for this type of data is that individual-level imaging are taken at various locations depending on where the biopsy was located on the breast. Thus, there exists substantial spatial heterogeneity between the images. We present a novel sample-specific learning framework for l 1 -penalized logistic regression to aid accurate LVI classification in real time. We derive finite sample guarantees for our proposed estimator in this paper and present a computationally efficient algorithm for translation. The finite sample performance for the proposed method is assessed via intensive simulation studies. Using images generated from the Stanford cohort, we rigorously assess the LVI classification performance with both internal and external validation and demonstrate high concordance with pathologist labeling.

Usage of time multiplexing super resolution for imaging through scattering medium

This paper presents an extension of time multiplexing super-resolution imaging concept to allow imaging through scattering medium. The presented concept includes laser illuminating an object through a diffuser. The technique performs time multiplexing super-resolved imaging through this diffuser while using the unknown speckle pattern the diffuser projects on the imaged object to enhance the resolution at which the object is imaged. Thus, unlike in conventional case where imaging through a diffuser or other scattering medium destroys the imaging resolution, here the speckle pattern the diffuser generates assists in performing super-resolved imaging. Experimental results demonstrate the efficacy of the approach, showing significant improvement in image quality and resolution.

CD163 detection in immune check-point inhibitors-related acute interstitial nephritis.

Acute interstitial nephritis (AIN) is the most common renal immune-related adverse event after immune check-point inhibitors (ICI). We hypothesized that alternatively activated macrophages (CD163-M) could be involved in ICI-AIN and wished to evaluate the use of their soluble urinary form (us)CD163 as a non-invasive diagnostic marker. CD163-M infiltrates were evaluated by both immune-histochemistry and multiplex immunofluorescence and imaging. usCD163 was detected with ELLA technology and evaluated together with urinary creatinine to be expressed as a ratio to creatinuria in ng/mmol. Clinical data were collected to perform correlations with renal function assessed by estimated glomerular filtration rate (eGFR). A retrospective cohort of 63 ICI-exposed patients with tubular acute kidney injury profile requiring a biopsy were selected. AIN patients (n=44) were compared to acute tubular necrosis (ATN) patients (n=19). CD163-M staining was detectable in all ICI-AIN patients, which was significantly higher than in ATN patients (18.4%vs 3.6% of area, P =.005). CD163-M staining was restricted to the interstitial compartment. CD163-M infiltrate inversely correlated with initial eGFR (r=-0.6, P =.003), and was positively correlated with delta eGFR, reflecting a renal improvement outcome (r=0.48; P =.02). usCD163 was well detected in urines of patients, but did not allow us to distinguish ATN from AIN patients at diagnosis. No correlation was observed, neither between usCD163 and CD163-M staining nor with renal response after 3 months of glucocorticoid tapering. CD163-M are detected in ICI-AIN and correlate both with severity at diagnosis and better prognosis at 3months. CD163-M may help us to distinguish AIN from ATN but, it does not allow us to assess ICI imputability. Although detected in urine, usCD163 is clearly not a surrogate biomarker for AIN diagnosis.

Claudin18.2 expression in gallbladder cancer correlates with immune activation and a favourable prognosis

AimsGallbladder carcinoma (GBC) is frequently diagnosed and treated in advanced stages and has a poor prognosis. Recent studies have identified claudin18.2 (CLDN18.2) as a promising target in digestive system cancer....

Cellular and molecular determinants mediating the dysregulated germinal center immune dynamics in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by dysregulated humoral immunity, leading to the generation of autoreactive B cells that can differentiate both within and outside of lymph node (LN) follicles. Here, we employed spatial transcriptomics and multiplex imaging to investigate the follicular immune landscaping and the in situ transcriptomic profile in LNs from SLE individuals. Our spatial transcriptomic analysis revealed robust type I IFN and plasma cell signatures in SLE compared to reactive, control follicles. Cell deconvolution revealed that follicular T cell subsets are mainly affected by the type I IFN fingerprint of SLE follicles. Dysregulation of TFH differentiation was documented by i) the significant reduction of Bcl6hi TFH cells, ii) the reduced cell density of potential IL-4 producing TFH cell subsets associated with the impaired transcriptomic signature of follicular IL-4 signaling and iii) the loss of their correlation with GC-B cells. This profile was accompanied by a marked reduction of Bcl6hi B cells and an enrichment of extrafollicular CD19hiCD11chiTbethi, age-associated B cells (ABCs), known for their autoreactive potential. The increased prevalence of follicular IL-21hi cells further reveals a hyperactive microenvironment in SLE compared to control. Taken together, our findings highlight the altered immunological landscape of SLE follicles, likely fueled by potent inflammatory signals such as sustained type I IFN and/or IL-21 signaling. Our work provides novel insights into the spatial molecular and cellular signatures of SLE follicular B and TFH cell dynamics, and points to druggable targets to restore immune tolerance and enhance vaccine responses in SLE patients.

Perivascular glial reactivity is a feature of phosphorylated tau lesions in chronic traumatic encephalopathy

Chronic traumatic encephalopathy (CTE), a neurodegenerative disease associated with repetitive head injuries, is characterised by perivascular hyperphosphorylated tau (p-tau) accumulations within the depths of cortical sulci. Although the majority of CTE literature focuses on p-tau pathology, other pathological features such as glial reactivity, vascular damage, and axonal damage are relatively unexplored. In this study, we aimed to characterise these other pathological features, specifically in CTE p-tau lesion areas, to better understand the microenvironment surrounding the lesion. We utilised multiplex immunohistochemistry to investigate the distribution of 32 different markers of cytoarchitecture and pathology that are relevant to both traumatic brain injury and neurodegeneration. We qualitatively assessed the multiplex images and measured the percentage area of labelling for each marker in the lesion and non-lesion areas of CTE cases. We identified perivascular glial reactivity as a prominent feature of CTE p-tau lesions, largely driven by increases in astrocyte reactivity compared to non-lesion areas. Furthermore, we identified astrocytes labelled for both NAD(P)H quinone dehydrogenase 1 (NQO1) and L-ferritin, indicating that lesion-associated glial reactivity may be a compensatory response to iron-induced oxidative stress. Our findings demonstrate that perivascular inflammation is a consistent feature of the CTE pathognomonic lesion and may contribute to the pathogenesis of brain injury-related neurodegeneration.

PRAME Expression in Melanoma is Negatively Regulated by TET2-Mediated DNA Hydroxymethylation.

PRAME Expression in Melanoma is Negatively Regulated by TET2-Mediated DNA Hydroxymethylation.

Assessing spatial sequencing and imaging approaches to capture the molecular and pathological heterogeneity of archived cancer tissues.

Spatial transcriptomics (ST) offers enormous potential to decipher the biological and pathological heterogeneity in precious archival cancer tissues. Traditionally, these tissues have rarely been used and only examined at a low throughput, most commonly by histopathological staining. ST adds thousands of times as many molecular features to histopathological images, but critical technical issues and limitations require more assessment of how ST performs on fixed archival tissues. In this work, we addressed this in a cancer-heterogeneity pipeline, starting with an exploration of the whole transcriptome by two sequencing-based ST protocols capable of measuring coding and non-coding RNAs. We optimised the two protocols to work with challenging formalin-fixed paraffin-embedded (FFPE) tissues, derived from skin. We then assessed alternative imaging methods, including multiplex RNAScope single-molecule imaging and multiplex protein imaging (CODEX). We evaluated the methods' performance for tissues stored from 4 to 14 years ago, covering a range of RNA qualities, allowing us to assess variation. In addition to technical performance metrics, we determined the ability of these methods to quantify tumour heterogeneity. We integrated gene expression profiles with pathological information, charting a new molecular landscape on the pathologically defined tissue regions. Together, this work provides important and comprehensive experimental technical perspectives to consider the applications of ST in deciphering the cancer heterogeneity in archived tissues. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

New methods at the transition from research to routine diagnostics

Pathology, traditionally focused on classification and diagnosis, is continuously evolving through new technologies. Advances in proteomics, epigenetics, tissue staining, and 3D imaging expand the possibilities of classical morphology. The aim of this study was to investigate how modern technologies can improve diagnostic accuracy and therapy selection and how they can be integrated into pathologic routine diagnostics. Recent studies in proteomics, epigenetics, multiplex tissue staining, and 3D tissue imaging were analyzed to assess their application and the challenges of clinical implementation. The analysis shows significant potential for pathologic diagnostics. Proteomics provides adeeper understanding of the molecular architecture of tumors, while epigenetics and 3D genome architecture offer new insights into genetic regulation and tumor heterogeneity. Multiplex tissue staining and 3D tissue imaging improve spatial tissue analysis. Despite the potential to improve diagnostics, high costs, technical complexity, and lack of standardization hinder integration into clinical practice. Nevertheless, these technologies offer promising approaches for optimizing diagnostics and therapy selection. Research and interdisciplinary collaboration are crucial to successfully integrating these innovations into routine clinical practice.

Benchmarking Spatial Co-Localization Methods for Single-Cell Multiplex Imaging Data with Applications to High-Grade Serous Ovarian and Triple Negative Breast Cancer.

Single-cell multiplex imaging (scMI) measures cell locations and phenotypes within a tissue and can be used to understand the tumor microenvironment. In scMI studies, it is often of interest to quantify spatial co-localization of immune cells and its association with clinical outcomes; however, it remains unknown which of the many available spatial indices have adequate power to detect spatial within-sample co-localization and its association with patient outcomes, such as survival. In this study, the performance of six frequentist metrics of spatial co-localization used in scMI studies were evaluated. Simulated data was used to assess the power and type I error of these spatial metrics to detect signficant co-localization. Furthermore, these spatial co-localization methods were applied to two scMI studies - a high-grade serous ovarian cancer (HGSOC) study and triple negative breast cancer (TNBC) study - to detect within-sample co-localization between cell types and their sensitivity to detect differences in survival across samples. In the simulation study, Ripley's K had the greatest power to identify co-localization followed closely by pair correlation g; all other statistics showed little power across all simulation scenarios. In the application of the methods to cancer studies, the results consistently point to pair correlation g and Ripley's K as indices with the most power for detecting significant co-localization in scMI data. Furthermore, pair correlation g, Ripley's K, and the scLMM index were most effective for estimating between-sample associations between level of co-localization and survival.

Highly multiplexed imaging reveals prognostic immune and stromal spatial biomarkers in breast cancer.

Spatial profiling of tissues promises to elucidate tumor-microenvironment interactions and generate prognostic and predictive biomarkers. We analyzed single-cell spatial data from 3 multiplex imaging technologies: cyclic immunofluorescence (CycIF) data we generated from 102 patients with breast cancer with clinical follow-up as well as publicly available mass cytometry and multiplex ion-beam imaging datasets. Similar single-cell phenotyping results across imaging platforms enabled combined analysis of epithelial phenotypes to delineate prognostic subtypes among patients who are estrogen-receptor+ (ER+). We utilized discovery and validation cohorts to identify biomarkers with prognostic value. Increased lymphocyte infiltration was independently associated with longer survival in triple-negative (TN) and high-proliferation ER+ breast tumors. An assessment of 10 spatial analysis methods revealed robust spatial biomarkers. In ER+ disease, quiescent stromal cells close to tumor were abundant in tumors with good prognoses, while tumor cell neighborhoods containing mixed fibroblast phenotypes were enriched in poor-prognosis tumors. In TN disease, macrophage/tumor and B/T lymphocyte neighbors were enriched, and lymphocytes were dispersed in good-prognosis tumors, while tumor cell neighborhoods containing vimentin+ fibroblasts were enriched in poor-prognosis tumors. In conclusion, we generated comparable single-cell spatial proteomic data from several clinical cohorts to enable prognostic spatial biomarker identification and validation.

Data-driven synapse classification reveals a logic of glutamate receptor diversity.

The rich diversity of synapses facilitates the capacity of neural circuits to transmit, process and store information. We used multiplex super-resolution proteometric imaging through array tomography to define features of single synapses in mouse neocortex. We find that glutamatergic synapses cluster into subclasses that parallel the distinct biochemical and functional categories of receptor subunits: GluA1/4, GluA2/3 and GluN1/GluN2B. Two of these subclasses align with physiological expectations based on synaptic plasticity: large AMPAR-rich synapses may represent potentiated synapses, whereas small NMDAR-rich synapses suggest "silent" synapses. The NMDA receptor content of large synapses correlates with spine neck diameter, and thus the potential for coupling to the parent dendrite. Overall, ultrastructural features predict receptor content of synapses better than parent neuron identity does, suggesting synapse subclasses act as fundamental elements of neuronal circuits. No barriers prevent future generalization of this approach to other species, or to study of human disorders and therapeutics.

A Single Bioorthogonal Reaction for Multiplex Cell Surface Protein Labeling.

Small-molecule fluorophores are invaluable tools for fluorescence imaging. However, means for their covalent conjugation to the target proteins limit applications in multicolor imaging. Here, we identify 2-[(alkylthio)(aryl)methylene]malononitrile (TAMM) molecules reacting with 1,2-aminothiol at a labeling rate over 104 M-1 s-1 through detailed mechanistic investigation. The fast TAMM molecules and mild reaction conditions enable site-specific labeling of surface proteins in not only cell lines but also primary neurons and living mice. The combination of genetic code expansion and sequence-specific proteolytic cleavage enables selective modification of three different cell surface proteins through iterative TAMM condensation. TAMM condensation is also compatible with Cu-catalyzed azide-alkyne cycloaddition and tetrazine ligation for four-color fluorescent labeling, reaching the maximum available colors of conventional confocal microscopes. Thus, bioconjugation chemistry is no longer the limiting factor for multiplex cell surface protein imaging.

Divergent paths of mammary gland involution: unveiling the cellular dynamics in abruptly and gradually involuted mouse models

BackgroundEpidemiological studies associate an increase in breast cancer risk, particularly triple-negative breast cancer (TNBC), with lack of breastfeeding. This is more prevalent in African American women, with significantly lower rate of breastfeeding compared to Caucasian women. Prolonged breastfeeding leads to gradual involution (GI), whereas short-term or lack of breastfeeding leads to abrupt involution (AI) of the breast. Our previous study utilizing a murine model demonstrated precancerous changes, specifically hyperplasia, a non-obligate precursor of breast cancer in the mammary glands of AI mice. Here we investigated mechanisms during early events of AI that prompts precancerous changes in mouse mammary glands.MethodsUniparous FVB/N mice were randomized to AI and GI on postpartum day 7 when all pups were removed from AI dams. GI dams were allowed to nurse the pups till day 31. Cell death kinetics and gene expression were assessed by TUNEL assay and qPCR respectively. Immune cell changes were investigated by flow cytometry, cytokine array and multiplex immunofluorescence. 3D-organoid cultures were used for in vitro assay of luminal progenitor cells.ResultsAI results in rapid cell death, DNA repair response, and immunosuppressive myeloid cells infiltration, leading to a chronically inflamed microenvironment. GI elicits a more controlled immune response and extended cell death. At the peak of cell death, AI glands harbored more immunosuppressive myeloid-derived suppressor cells (MDSCs) and CD206 + M2-like macrophages, known to promote oncogenic events, compared to GI glands. AI glands exhibit an enrichment of CCL9-producing MDSCs and CD206 + M2-like macrophages that promote expansion of ELF5 + /ERα- luminal cells, both in vitro and in vivo. Multiplex imaging of AI glands demonstrated an increase in ELF5 + /WNT5a + luminal cells alongside a reduction in the ELF5 + /ERα + population when involution appeared histologically complete. A significantly higher number of CD206 + cells in post involution AI gland attests to a chronically inflamed state induced by AI.ConclusionsOur findings reveal significant disparities between AI and GI gland dynamics at the early phase of involution. CCL9, secreted by immune cells at the peak of cell death promotes expansion of Elf5 + /ERα- luminal progenitor cells, the putative precursors of TNBC connecting early events of AI with increased breast cancer risk.

Establishment of a National Surgical Tissue Biobank for Pediatric Crohn's Disease: An Implementation Feasibility Study.

Establishment of a National Surgical Tissue Biobank for Pediatric Crohn's Disease: An Implementation Feasibility Study.

Unveiling Cellular Heterogeneity and Liver Pathophysiology in Liver Transplant Recipients through Multiplex Image Analysis

Unveiling Cellular Heterogeneity and Liver Pathophysiology in Liver Transplant Recipients through Multiplex Image Analysis

Highly-Multiplexed Immunofluorescence PhenoCycler Panel for Murine FFPE Yields Insight into Tumor Microenvironment Immunoengineering

Highly-Multiplexed Immunofluorescence PhenoCycler Panel for Murine FFPE Yields Insight into Tumor Microenvironment Immunoengineering