Global and single-cell proteomics view of the co-evolution between neural progenitors and breast cancer cells in a co-culture model.

BACKGROUND: Presence of nerves in tumours, by axonogenesis and neurogenesis, is gaining increased attention for its impact on cancer initiation and development, and the new field of cancer neuroscience is emerging. A recent study in prostate cancer suggested that the tumour microenvironment may influence cancer progression by recruitment of Doublecortin (DCX)-expressing neural progenitor cells (NPCs). However, the presence of such cells in human breast tumours has not been comprehensively explored. METHODS: Here, we investigate the presence of DCX-expressing cells in breast cancer stromal tissue from patients using Imaging Mass Cytometry. Single-cell analysis of 372,468 cells across histopathological images of 107 breast cancers enabled spatial resolution of neural elements in the stromal compartment in correlation with clinicopathological features of these tumours. In parallel, we established a 3D in vitro model mimicking breast cancer neural progenitor-innervation and examined the two cell types as they co-evolved in co-culture by using mass spectrometry-based global proteomics. FINDINGS: Stromal presence of DCX + cells is associated with tumours of higher histological grade, a basal-like phenotype, and shorter patient survival in tumour tissue from patients with breast cancer. Global proteomics analysis revealed significant changes in the proteomic landscape of both breast cancer cells and neural progenitors in co-culture. INTERPRETATION: These results support that neural involvement plays an active role in breast cancer and warrants further studies on the relevance of nerve elements for tumour progression. FUNDING: This work was supported by the Research Council of Norway through its Centre of Excellence funding scheme, project number 223250 (to L.A.A), the Norwegian Cancer Society (to L.A.A. and H.V.), the Regional Health Trust Western Norway (Helse Vest) (to L.A.A.), the Meltzer Research Fund (to H.V.) and the National Institutes of Health (NIH)/NIGMS grant R01 GM132129 (to J.A.P.).

PanIN and CAF transitions in pancreatic carcinogenesis revealed with spatial data integration.

This study introduces a new imaging, spatial transcriptomics (ST), and single-cell RNA-sequencing integration pipeline to characterize neoplastic cell state transitions during tumorigenesis. We applied a semi-supervised analysis pipeline to examine premalignant pancreatic intraepithelial neoplasias (PanINs) that can develop into pancreatic ductal adenocarcinoma (PDAC). Their strict diagnosis on formalin-fixed and paraffin-embedded (FFPE) samples limited the single-cell characterization of human PanINs within their microenvironment. We leverage whole transcriptome FFPE ST to enable the study of a rare cohort of matched low-grade (LG) and high-grade (HG) PanIN lesions to track progression and map cellular phenotypes relative to single-cell PDAC datasets. We demonstrate that cancer-associated fibroblasts (CAFs), including antigen-presenting CAFs, are located close to PanINs. We further observed a transition from CAF-related inflammatory signaling to cellular proliferation during PanIN progression. We validate these findings with single-cell high-dimensional imaging proteomics and transcriptomics technologies. Altogether, our semi-supervised learning framework for spatial multi-omics has broad applicability across cancer types to decipher the spatiotemporal dynamics of carcinogenesis.

Endocannabinoids and their receptors modulate endometriosis pathogenesis and immune response.

Endometriosis (EM), characterized by the presence of endometrial-like tissue outside the uterus, is the leading cause of chronic pelvic pain and infertility in females of reproductive age. Despite its high prevalence, the molecular mechanisms underlying EM pathogenesis remain poorly understood. The endocannabinoid system (ECS) is known to influence several cardinal features of this complex disease including pain, vascularization, and overall lesion survival, but the exact mechanisms are not known. Utilizing CNR1 knockout (k/o), CNR2 k/o, and wild-type (WT) mouse models of EM, we reveal contributions of ECS and these receptors in disease initiation, progression, and immune modulation. Particularly, we identified EM-specific T cell dysfunction in the CNR2 k/o mouse model of EM. We also demonstrate the impact of decidualization-induced changes on ECS components, and the unique disease-associated transcriptional landscape of ECS components in EM. Imaging mass cytometry (IMC) analysis revealed distinct features of the microenvironment between CNR1, CNR2, and WT genotypes in the presence or absence of decidualization. This study, for the first time, provides an in-depth analysis of the involvement of the ECS in EM pathogenesis and lays the foundation for the development of novel therapeutic interventions to alleviate the burden of this debilitating condition.

Interleukin-2 immunotherapy reveals human regulatory T cell subsets with distinct functional and tissue-homing characteristics.

Due to its stimulatory potential for immunomodulatory CD4+ regulatory T (Treg) cells, low-dose interleukin-2 (IL-2) immunotherapy has gained considerable attention for the treatment of autoimmune diseases. In this investigator-initiated single-arm non-placebo-controlled phase-2 clinical trial of low-dose IL-2 immunotherapy in systemic lupus erythematosus (SLE) patients, we generated a comprehensive atlas of in vivo human immune responses to low-dose IL-2. We performed an in-depth study of circulating and cutaneous immune cells by imaging mass cytometry, high-parameter flow cytometry, transcriptomics, and targeted serum proteomics. Low-dose IL-2 stimulated various circulating immune cells, including Treg cells with a skin-homing phenotype that appeared in the skin of SLE patients in close interaction with endothelial cells. Analysis of surface proteins and transcriptomes revealed different IL-2-driven Treg cell activation programs, including gut-homing CD38+, skin-homing HLA-DR+, and highly proliferative inflammation-homing CD38+ HLA-DR+ Treg cells. Collectively, these data define the distinct human Treg cell subsets that are responsive to IL-2 immunotherapy.

Single-cell mass cytometry in immunological skin diseases.

Immune-related skin diseases represent a collective of dermatological disorders intricately linked to dysfunctional immune system processes. These conditions are primarily characterized by an immoderate activation of the immune system or deviant immune responses, involving diverse immune components including immune cells, antibodies, and inflammatory mediators. However, the precise molecular dysregulation underlying numerous individual cases of these diseases and unique subsets respond under disease conditions remains elusive. Comprehending the mechanisms and determinants governing the homeostasis and functionality of diseases could offer potential therapeutic opportunities for intervention. Mass cytometry enables precise and high-throughput quantitative measurement of proteins within individual cells by utilizing antibodies labeled with rare heavy metal isotopes. Imaging mass cytometry employs mass spectrometry to obtain spatial information on cell-to-cell interactions within tissue sections, simultaneously utilizing more than 40 markers. The application of single-cell mass cytometry presents a unique opportunity to conduct highly multiplexed analysis at the single-cell level, thereby revolutionizing our understanding of cell population heterogeneity and hierarchy, cellular states, multiplexed signaling pathways, proteolysis products, and mRNA transcripts specifically in the context of many autoimmune diseases. This information holds the potential to offer novel approaches for the diagnosis, prognostic assessment, and monitoring responses to treatment, thereby enriching our strategies in managing the respective conditions. This review summarizes the present-day utilization of single-cell mass cytometry in studying immune-related skin diseases, highlighting its advantages and limitations. This technique will become increasingly prevalent in conducting extensive investigations into these disorders, ultimately yielding significant contributions to their accurate diagnosis and efficacious therapeutic interventions.

Multiparameter quantitative analyses of diagnostic cells in brain tissues from tuberous sclerosis complex.

The advent of high-dimensional imaging offers new opportunities to molecularly characterize diagnostic cells in disorders that have previously relied on histopathological definitions. One example case is found in tuberous sclerosis complex (TSC), a developmental disorder characterized by systemic growth of benign tumors. Within resected brain tissues from patients with TSC, detection of abnormally enlarged balloon cells (BCs) is pathognomonic for this disorder. Though BCs can be identified by an expert neuropathologist, little is known about the specificity and broad applicability of protein markers for these cells, complicating classification of proposed BCs identified in experimental models of this disorder. Here, we report the development of a customized machine learning pipeline (BAlloon IDENtifier; BAIDEN) that was trained to prospectively identify BCs in tissue sections using a histological stain compatible with high-dimensional cytometry. This approach was coupled to a custom 36-antibody panel and imaging mass cytometry (IMC) to explore the expression of multiple previously proposed BC marker proteins and develop a descriptor of BC features conserved across multiple tissue samples from patients with TSC. Here, we present a modular workflow encompassing BAIDEN, a custom antibody panel, a control sample microarray, and analysis pipelines-both open-source and in-house-and apply this workflow to understand the abundance, structure, and signaling activity of BCs as an example case of how high-dimensional imaging can be applied within human tissues.

Associations of amyloid-ß oligomers and plaques with neuropathology in the App NL-G-F mouse.

Amyloid-ß pathology and neurofibrillary tangles lead to glial activation and neurodegeneration in Alzheimer's disease. In this study, we investigated the relationships between the levels of amyloid-ß oligomers, amyloid-ß plaques, glial activation and markers related to neurodegeneration in the App NL-G-F triple mutation mouse line and in a knock-in line homozygous for the common human amyloid precursor protein (App hu mouse). The relationships between neuropathological features were characterized with immunohistochemistry and imaging mass cytometry. Markers assessing human amyloid-ß proteins, microglial and astrocytic activation and neuronal and synaptic densities were used in mice between 2.5 and 12 months of age. We found that amyloid-ß oligomers were abundant in the brains of App hu mice in the absence of classical amyloid-ß plaques. These brains showed morphological changes consistent with astrocyte activation but no evidence of microglial activation or synaptic or neuronal pathology. In contrast, both high levels of amyloid-ß oligomers and numerous plaques accumulated in App NL-G-F mice in association with substantial astrocytic and microglial activation. The increase in amyloid-ß oligomers over time was more strongly correlated with astrocytic than with microglia activation. Spatial analyses suggested that activated microglia were more closely associated with amyloid-ß oligomers than with amyloid-ß plaques in App NL-G-F mice, which also showed age-dependent decreases in neuronal and synaptic density markers. A comparative study of the two models highlighted the dependence of glial and neuronal pathology on the nature and aggregation state of the amyloid-ß peptide. Astrocyte activation and neuronal pathology appeared to be more strongly associated with amyloid-ß oligomers than with amyloid-ß plaques, although amyloid-ß plaques were associated with microglia activation.

High throughput spatial immune mapping reveals an innate immune scar in post-COVID-19 brains.

The underlying pathogenesis of neurological sequelae in post-COVID-19 patients remains unclear. Here, we used multidimensional spatial immune phenotyping and machine learning methods on brains from initial COVID-19 survivors to identify the biological correlate associated with previous SARS-CoV-2 challenge. Compared to healthy controls, individuals with post-COVID-19 revealed a high percentage of TMEM119+P2RY12+CD68+Iba1+HLA-DR+CD11c+SCAMP2+ microglia assembled in prototypical cellular nodules. In contrast to acute SARS-CoV-2 cases, the frequency of CD8+ parenchymal T cells was reduced, suggesting an immune shift toward innate immune activation that may contribute to neurological alterations in post-COVID-19 patients.

Multimodal Mass Spectrometry Imaging of an Osteosarcoma Multicellular Tumour Spheroid Model to Investigate Drug-Induced Response.

A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.

Development of a high dimensional imaging mass cytometry panel to investigate spatial organization of tissue microenvironment in formalin-fixed archival clinical tissues.

To decipher the interactions between various components of the tumor microenvironment (TME) and tumor cells in a preserved spatial context, a multiparametric approach is essential. In this pursuit, imaging mass cytometry (IMC) emerges as a valuable tool, capable of concurrently analyzing up to 40 parameters at subcellular resolution. In this study, a set of antibodies was selected to spatially resolve multiple cell types and TME elements, including a comprehensive panel targeted at dissecting the heterogeneity of cancer-associated fibroblasts (CAF), a pivotal TME component. This antibody panel was standardized and optimized using formalin-fixed paraffin-embedded tissue (FFPE) samples from different organs/lesions known to express the markers of interest. The final composition of the antibody panel was determined based on the performance of conjugated antibodies in both immunohistochemistry (IHC) and IMC. Tissue images were segmented employing the Steinbock framework. Unsupervised clustering of single-cell data was carried out using a bioinformatics pipeline developed in R program. This paper provides a detailed description of the staining procedure and analysis workflow. Subsequently, the panel underwent validation on clinical FFPE samples from head and neck squamous cell carcinoma (HNSCC). The panel and bioinformatics pipeline established here proved to be robust in characterizing different TME components of HNSCC while maintaining a high degree of spatial detail. The platform we describe shows promise for understanding the clinical implications of TMA heterogeneity in large patient cohorts with FFPE tissues available in diagnostic biobanks worldwide.

OMIP-103: A 35-marker imaging mass cytometry panel for the co-detection of HIV and immune cell populations in human formalin fixed paraffin embedded intestinal tissue.

We introduce a 35-marker imaging mass cytometry (IMC) panel for a detailed examination of immune cell populations and HIV RNA in formalin fixed paraffin embedded (FFPE) human intestinal tissue. The panel has broad cell type coverage and particularly excels in delineating subsets of mononuclear phagocytes and T cells. Markers for key tissue structures are included, enabling identification of epithelium, blood vessels, lymphatics, and musculature. The described method for HIV RNA detection can be generalized to other low abundance RNA targets, whether endogenous or pathogen derived. As such, the panel presented here is useful for high parameter spatial mapping of intestinal immune cells and their interactions with pathogens such as HIV.

Coupling imaging mass cytometry with Alcian blue histochemical staining for a single-slide approach.

Imaging mass cytometry (IMC) is a metal mass spectrometry-based method allowing highly multiplex immunophenotyping of cells within tissue samples. However, some limitations of IMC are its 1-µm resolution and its time and costs of analysis limiting respectively the detailed histopathological analysis of IMC-produced images and its application to small selected tissue regions of interest (ROI) of one to few square millimeters. Coupling on a single-tissue section, IMC and histopathological analyses could permit a better selection of the ROI for IMC analysis as well as co-analysis of immunophenotyping and histopathological data until the single-cell level. The development of this method is the aim of the present study in which we point to the feasibility of applying the IMC process to tissue sections previously Alcian blue-stained and digitalized before IMC tissue destructive analyses. This method could help to improve the process of IMC in terms of ROI selection, time of analysis, and the confrontation between histopathological and immunophenotypic data of cells.

Development of 42 marker panel for in-depth study of cancer associated fibroblast niches in breast cancer using imaging mass cytometry.

Imaging Mass Cytometry (IMC) is a novel, and formidable high multiplexing imaging method emerging as a promising tool for in-depth studying of tissue architecture and intercellular communications. Several studies have reported various IMC antibody panels mainly focused on studying the immunological landscape of the tumor microenvironment (TME). With this paper, we wanted to address cancer associated fibroblasts (CAFs), a component of the TME very often underrepresented and not emphasized enough in present IMC studies. Therefore, we focused on the development of a comprehensive IMC panel that can be used for a thorough description of the CAF composition of breast cancer TME and for an in-depth study of different CAF niches in relation to both immune and breast cancer cell communication. We established and validated a 42 marker panel using a variety of control tissues and rigorous quantification methods. The final panel contained 6 CAF-associated markers (aSMA, FAP, PDGFRa, PDGFRb, YAP1, pSMAD2). Breast cancer tissues (4 cases of luminal, 5 cases of triple negative breast cancer) and a modified CELESTA pipeline were used to demonstrate the utility of our IMC panel for detailed profiling of different CAF, immune and cancer cell phenotypes.

Spatial Single Cell Profiling Using Imaging Mass Cytometry: Inflammatory Versus Penetrating Crohn's Disease.

BACKGROUND AND AIMS: Fistula formation is a major complication in Crohn's disease [CD] and the role of the immune cell compartment remains to be elucidated. Thus, we compared the immune cell compartment of CD fistula to inflammatory CD colitis using imaging mass cytometry [IMC] and immunofluorescence. METHODS: A 36-marker panel including structural, functional, and lineage markers for use in IMC was established. This panel was applied to analyse paraffin-embedded CD fistula tract [n = 11], CD colitis [n = 10], and colon samples from non-inflamed controls [n = 12]. Computational methods for cell segmentation, dimensionality reduction, and cell type clustering were used to define cell populations for cell frequency, marker distribution, and spatial neighbourhood analysis. Multiplex immunofluorescence was used for higher resolution spatial analysis. RESULTS: Analysis of cell frequencies in CD fistulas compared to CD colitis and control colonic samples revealed a significant increase in neutrophils, effector cytotoxic T cells, and inflammatory macrophages in CD fistula samples, whereas regulatory T cells were decreased. Neutrophils in CD fistula expressed significantly more matrix metalloproteinase 9 [MMP9], correlating with extracellular matrix remodelling. Neighbourhood analysis revealed a strong association between MMP9+ neutrophils and effector cytotoxic T cells in both CD fistulas and colitis. CONCLUSIONS: This study presents the first highly multiplexed single cell analysis of the immune cell compartment of CD fistulas and their spatial context. It links immune cell dynamics, particularly MMP9+ neutrophils, to extracellular matrix remodelling in CD fistulas, offering insights into the complex network of cellular interactions and potential therapeutic targets for CD complications.

Multi-omics and imaging mass cytometry characterization of human kidneys to identify pathways and phenotypes associated with impaired kidney function.

Despite the recent advances in our understanding of the role of lipids, metabolites, and related enzymes in mediating kidney injury, there is limited integrated multi-omics data identifying potential metabolic pathways driving impaired kidney function. The limited availability of kidney biopsies from living donors with acute kidney injury has remained a major constraint. Here, we validated the use of deceased transplant donor kidneys as a good model to study acute kidney injury in humans and characterized these kidneys using imaging and multi-omics approaches. We noted consistent changes in kidney injury and inflammatory markers in donors with reduced kidney function. Neighborhood and correlation analyses of imaging mass cytometry data showed that subsets of kidney cells (proximal tubular cells and fibroblasts) are associated with the expression profile of kidney immune cells, potentially linking these cells to kidney inflammation. Integrated transcriptomic and metabolomic analysis of human kidneys showed that kidney arachidonic acid metabolism and seven other metabolic pathways were upregulated following diminished kidney function. To validate the arachidonic acid pathway in impaired kidney function we demonstrated increased levels of cytosolic phospholipase A2 protein and related lipid mediators (prostaglandin E2) in the injured kidneys. Further, inhibition of cytosolic phospholipase A2 reduced injury and inflammation in human kidney proximal tubular epithelial cells in vitro. Thus, our study identified cell types and metabolic pathways that may be critical for controlling inflammation associated with impaired kidney function in humans.

Spatial Mass Cytometry-Based Single-Cell Imaging Reveals a Disrupted Epithelial-Immune Axis in Prurigo Nodularis.

Prurigo nodularis (PN) is a chronic, inflammatory skin condition that disproportionately affects African Americans and features intensely pruritic, hyperkeratotic nodules on the extremities and trunk. PN is understudied compared with other inflammatory skin diseases, with the spatial organization of the cutaneous infiltrate in PN yet to be characterized. In this work, we employ spatial imaging mass cytometry to visualize PN lesional skin inflammation and architecture with single-cell resolution through an unbiased machine learning approach. PN lesional skin has increased expression of caspase 3, NF-kB, and phosphorylated signal transducer and activator of transcription 3 compared with healthy skin. Keratinocytes in lesional skin are subdivided into CD14+CD33+, CD11c+, CD63+, and caspase 3-positive innate subpopulations. CD14+ macrophage populations expressing phosphorylated extracellular signal-regulated kinase 1/2 correlate positively with patient-reported itch (P = .006). Hierarchical clustering reveals a cluster of patients with PN with greater atopy, increased NF-kB+ signal transducer and activator of transcription 3-positive phosphorylated extracellular signal-regulated kinase 1/2-positive monocyte-derived myeloid dendritic cells, and increased vimentin expression (P < .05). Neighborhood analysis finds interactions between CD14+ macrophages, CD3+ T cells, monocyte-derived myeloid dendritic cells, and keratinocytes expressing innate immune markers. These findings highlight phosphorylated extracellular signal-regulated kinase-positive CD14+ macrophages as contributors to itch and suggest an epithelial-immune axis in PN pathogenesis.

Imaging Mass Cytometry for In Situ Immune Profiling.

The complexities and cellular heterogeneity associated with tissues necessitate the concurrent detection of markers beyond the limitations of conventional imaging approaches in order to spatially resolve the relationships between immune cell populations and their environments. This is a necessary complement to single-cell suspension-based methods to inform a better understanding of the events that may underlie pathological conditions. Imaging mass cytometry is a high-dimensional imaging modality that allows for the concurrent detection of up to 40 protein markers of interest across tissues at subcellular resolution. Here, we present an optimized staining protocol for imaging mass cytometry with modifications that integrate RNAscope. This unique addition enables combined protein and single-molecule RNA detection, thereby expanding the utility of imaging mass cytometry to researchers investigating low abundance or noncoding targets. In general, the procedure described is broadly applicable for comprehensive immune profiling of host-pathogen interactions, tumor microenvironments and inflammatory conditions, all within the tissue contexture.

High-Dimensional Mass Cytometry Reveals Emphysema-associated Changes in the Pulmonary Immune System.

RATIONALE: Chronic inflammation plays an important role in alveolar tissue damage in emphysema, but the underlying immune alterations and cellular interactions are incompletely understood. OBJECTIVE: To explore disease-specific pulmonary immune cell alterations and cellular interactions in emphysema. METHODS: We used single-cell mass cytometry to compare the immune compartment in alveolar tissue from 15 patients with severe emphysema and 5 controls. Imaging mass cytometry (IMC) was applied to identify altered cell-cell interactions in alveolar tissue from emphysema patients (n=12) compared to controls (n=8). MEASUREMENTS AND MAIN RESULTS: We observed higher percentages of central memory CD4 T cells in combination with lower proportions of effector memory CD4 T cells in emphysema. In addition, proportions of cytotoxic central memory CD8 T cells and CD127+CD27+CD69- T cells were higher in emphysema, the latter potentially reflecting an influx of circulating lymphocytes into the lungs. Central memory CD8 T cells, isolated from alveolar tissue from emphysema patients exhibited an IFN-γ-response upon anti-CD3/anti-CD28 activation. Proportions of CD1c+ dendritic cells (DC), expressing migratory and costimulatory markers, were higher in emphysema. Importantly, IMC enabled us to visualize increased spatial colocalization of CD1c+ DC and CD8 T cells in emphysema in situ. CONCLUSION: Using single-cell CyTOF, we characterized the alterations of the immune cell signature in alveolar tissue from patients with COPD stage III/IV emphysema versus control lung tissue. These data contribute to a better understanding of the pathogenesis of emphysema and highlight the feasibility of interrogating the immune cell signature using single-cell and IMC in human lung tissue.

High-multiplex single-cell imaging analysis reveals tumor immune contexture associated with clinical outcomes after CAR T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has made great progress in treating lymphoma, yet patient outcomes still vary greatly. The lymphoma microenvironment may be an important factor in the efficacy of CAR T therapy. In this study, we designed a highly multiplexed imaging mass cytometry (IMC) panel to simultaneously quantify 31 biomarkers from 13 patients with relapsed/refractory diffuse large B cell lymphoma (DLBCL) who received CAR19/22 T cell therapy. A total of 20 sections were sampled before CAR T cell infusion or after infusion when relapse occurred. A total of 35 cell clusters were identified, annotated, and subsequently redefined into 10 metaclusters. The CD4+ T cell fraction was positively associated with remission duration. Significantly higher Ki67, CD57, and TIM3 levels and lower CD69 levels in T cells, especially the CD8+/CD4+ Tem and Te cell subsets, were seen in patients with poor outcomes. Cellular neighborhood containing more immune cells was associated with longer remission. Fibroblasts and vascular endothelial cells resided much closer to tumor cells in patients with poor response and short remission after CAR T therapy. Our work comprehensively and systematically dissects the relationship between cell composition, state, and spatial arrangement in the DLBCL microenvironment and the outcomes of CAR T cell therapy, which is beneficial to predict CAR T therapy efficacy.

Multiplexed Imaging Mass Cytometry Analysis in Preclinical Models of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. PDAC is characterized by a complex tumor microenvironment (TME), that plays a pivotal role in disease progression and resistance to therapy. Investigating the spatial distribution and interaction of TME cells with the tumor is the basis for understanding the mechanisms underlying disease progression and represents a current challenge in PDAC research. Imaging mass cytometry (IMC) is the major multiplex imaging technology for the spatial analysis of tumor heterogeneity. However, there is a dearth of reports of multiplexed IMC panels for different preclinical mouse models, including pancreatic cancer. We addressed this gap by utilizing two preclinical models of PDAC: the genetically engineered, bearing KRAS-TP53 mutations in pancreatic cells, and the orthotopic, and developed a 28-marker panel for single-cell IMC analysis to assess the abundance, distribution and phenotypes of cells involved in PDAC progression and their reciprocal functional interactions. Herein, we provide an unprecedented definition of the distribution of TME cells in PDAC and compare the diversity between transplanted and genetic disease models. The results obtained represent an important and customizable tool for unraveling the complexities of PDAC and deciphering the mechanisms behind therapy resistance.