Elucidating Spatially-Resolved Changes in Host Signaling During Plasmodium Liver-Stage Infection

Upon transmission to the human host, Plasmodium sporozoites exit the skin, are taken up by the blood stream, and then travel to the liver where they infect and significantly modify a single hepatocyte. Low infection rates within the liver have made proteomic studies of infected hepatocytes challenging, particularly in vivo, and existing studies have been largely unable to consider how protein and phosphoprotein differences are altered at different spatial locations within the heterogeneous liver. Using digital spatial profiling, we characterized changes in host signaling during Plasmodium yoelii infection in vivo without disrupting the liver tissue. Moreover, we measured alterations in protein expression around infected hepatocytes and identified a subset of CD163+ Kupffer cells that migrate towards infected cells during infection. These data offer the first insight into the heterogeneous microenvironment that surrounds the infected hepatocyte and provide insights into how the parasite may alter its milieu to influence its survival and modulate immunity.

Ratiometric Mass Spectrometry Imaging for Stain-Free Delineation of Ischemic Tissue and Spatial Profiling of Ischemia-Related Molecular Signatures

Mass spectrometry imaging (MSI) serves as an emerging tool for spatial profiling of metabolic dysfunction in ischemic tissue. Prior to MSI data analysis, commonly used staining methods, e.g., triphenyltetrazole chloride (TTC) staining, need to be implemented on the adjacent tissue for delineating lesion area and evaluating infarction, resulting in extra consumption of the tissue sample as well as morphological mismatch. Here, we propose an in situ ratiometric MSI method for simultaneous demarcation of lesion border and spatial annotation of metabolic and enzymatic signatures in ischemic tissue on identical tissue sections. In this method, the ion abundance ratio of a reactant pair in the TCA cycle, e.g., fumarate to malate, is extracted pixel-by-pixel from an ambient MSI dataset of ischemic tissue and used as a surrogate indicator for metabolic activity of mitochondria to delineate lesion area as if the tissue has been chemically stained. This method is shown to be precise and robust in identifying lesions in brain tissues and tissue samples from different ischemic models including heart, liver, and kidney. Furthermore, the proposed method allows screening and predicting metabolic and enzymatic alterations which are related to mitochondrial dysfunction. Being capable of concurrent lesion identification, in situ metabolomics analysis, and screening of enzymatic alterations, the ratiometric MSI method bears great potential to explore ischemic damages at both metabolic and enzymatic levels in biological research.

HTAPP_Acquisition and allocation of fresh metastatic breast cancer samples v1

This standard operating procedure was established by the Center for Cancer Genomics at Dana-Farber Cancer Institute, the Brigham and Women's Hospital and the Klarman Cell Observatory at the Broad Institute, to standardize the collection of fresh metastatic breast cancer biopsies and their allocation to various bulk and single cell assays, including whole exome and bulk RNA-sequencing, single-cell RNA sequencing, and spatial profiling of RNA and protein. The use of a well defined workflow has allowed us to generate high quality data from these different assays, by implementing efficient modes of communication, minimizing the time elapsed from sample collection to preservation or processing, and ensuring optimal transportation conditions. Visual Abstract

HTAPP_Acquisition and allocation of fresh metastatic breast cancer sample. v1

This standard operating procedure was established by the Center for Cancer Genomics at Dana-Farber Cancer Institute, the Brigham and Women's Hospital and the Klarman Cell Observatory at the Broad Institute, to standardize the collection of fresh metastatic breast cancer biopsies and their allocation to various bulk and single cell assays, including whole exome and bulk RNA-sequencing, single-cell RNA sequencing, and spatial profiling of RNA and protein. The use of a well defined workflow has allowed us to generate high quality data from these different assays, by implementing efficient modes of communication, minimizing the time elapsed from sample collection to preservation or processing, and ensuring optimal transportation conditions. Visual Abstract

527 Digital spatial profiling of paired tumor biopsies reveals indoleamine 2,3-dioxygenase (IDO)1 as a potential resistance mechanism for a tumor-targeted 4–1BB agonist in patients with solid tumors

BackgroundWe previously described the capacity of RO7122290 (RO) - a FAP-targeted 4-1BB bispecific antibody - to induce CD8+ T cell infiltration and activation in the tumor (Moreno V. et al, SITC 2020). Aiming to compare pharmacodynamic (PD) changes in tumor nests and stroma, paired tumor biopsies from patients treated with RO (Part A) and RO + atezolizumab (Part B) were analysed by digital spatial profiling (DSP, Nanostring).MethodsSeven paired (baseline and on-treatment) FFPE tumor tissue biopsies (three from Part A, four from Part B) obtained from an ongoing Phase 1/1b trial (EUDRACT 2017-003961-83) were assessed for mRNA and protein expression. Biopsies were taken from six different tumor types at different RO doses. Up to twelve Regions of Interest (ROIs) were collected per slide and the morphology markers PanCK, CD8, CD3 and DAPI were applied. The ROIs were further annotated in tumor nests and stroma segments based on PanCK staining. The immune-oncology 58-plex protein and 78-plex mRNA expression panels (Nanostring) were used to profile all samples. Data were normalized according to Nanostring guidelines and filtered based on relevance (absolute log2 fold change > 1) and significance (FDR < 0.05, p-value).ResultsThe level of CD8+ T cell infiltration measured by spatial profiling correlated with the level measured by IHC, in both tumor nests and stroma. The activation markers 4-1BB and PD-1 were upregulated, confirming the PD effect already measured by mRNA sequencing. We also identified novel protein markers - CD40, PD-L1 and IDO1 - being upregulated after treatment. Spatial regulation differed among the markers with 4-1BB, PD-1 and CD40 upregulated only in the stroma, PD-L1 and IDO1 upregulated in the tumor nests and in the stroma. IDO1 induction is particularly relevant, since this protein is known to attenuate 4-1BB-mediated effector responses. Conventional IHC analysis performed on 14 paired biopsies confirmed IDO1 being upregulated in 11 out of 14 cases and revealed dendritic cells, macrophages and stromal cells to express IDO1. Importantly, IDO1 upregulation was observed in both Part A (3 out of 3) and Part B (8 out of 11).ConclusionsSpatial profiling allowed us to identify key markers that are spatially regulated after treatment and to gain new insights on the MoA of RO. The induction of IDO1 by RO confirms the dual immunoregulatory nature of 4-1BB signaling and highlights IDO1 as a potential resistance mechanism for RO in the clinical setting, both as single agent and in combination with atezolizumab.Trial RegistrationEUDRACT Number: 2017-003961-83; Protocol Number: BP40087ReferencesMoreno V. et al, Pharmacodynamic assessment of a novel FAP-targeted 4–1BB agonist, administered as single agent and in combination with atezolizumab to patients with advanced solid tumors, Nov 1 2020, Journal for ImmunoTherapy of Cancer, presented at SITC 2020

673 (Re-) Solving the biology of colorectal cancer onset and progression to improve treatment and prevention

BackgroundColorectal cancer (CRC) development is accompanied by the gradual accumulation of genetic alterations in epithelial cells of the colon and rectum.1 2 The paradigm of the adenoma-carcinoma sequence was originally centered around cancer cells; however, it is now clear that the tumor microenvironment plays a substantial role in cancer progression and patient outcome.3 In recent years, technologies have evolved rapidly, allowing the multiplexed quantification of gene expression while preserving spatial context.4 Furthermore, some spatial transcriptomic technologies also allow the parallel interrogation of different cell populations in the tumor microenvironment. Here, we performed digital spatial profiling on early-stage CRC samples to elucidate the biological processes that are at the basis of malignant transformation and to identify novel therapeutic targets and (immune) biomarkers.MethodsEndoscopically resected early-stage CRC samples were obtained at Leiden University Medical Center. In total, 144 areas of illumination were interrogated with GeoMx digital spatial profiling using the Cancer Transcriptome Atlas (>1,800 genes). In each of eight samples, nine regions of interest with different levels of cancer progression were selected, including normal epithelium, transition areas, low-, and high-grade dysplasia, and invasive carcinoma (figure 1A). We segmented each region based on cytokeratin and vimentin protein expression (figure 1B). Immunohistochemical detection was performed on these samples and 26 additional samples to validate targets associated with disease progression.ResultsDigital spatial profiling allowed us to dissect transcriptional alterations in epithelial and stromal fractions between different regions from healthy tissue, different degrees of dysplasia, and cancer. Gene expression data revealed a clear separation of profiled areas by histologic category. Interestingly, gene expression features in the stromal compartment provided a better data-driven separation of histologic categories than the epithelial fraction (figure 1C). Substantial changes in immune-related pathways were identified, including differential expression of specific immunomodulators. We validated the expression of several candidate biomarkers/targets that demonstrated consistent alterations from normal tissue to cancer by immunohistochemistry. Several proteins were identified that could clearly discriminate benign from malignant tissue.ConclusionsWe here demonstrated the unique biological insights that are provided by spatial examination of early-stage CRC by digital spatial profiling. We identified specific genes that were altered during CRC tumorigenesis, in epithelial and stromal/immune fractions. Furthermore, our results indicate an essential role for innate immunity in colorectal cancer onset and progression. The genes identified by this approach could potentially serve as novel biomarkers and targets for early interception or prevention of CRC development.AcknowledgementsThis work was supported by the European Research Council (ERC) Starting grant awarded to Dr. Noel F. de Miranda and the Stichting Management Apothekers en de Gezondheidszorg (STIMAG) Research grant awarded to Jessica Roelands.Trial RegistrationN/AReferencesFearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61(5):759–767. doi: 10.1016/0092-8674(90)90186-I.Nowell PC. The clonal evolution of tumor cell populations. Science 1976;194(4260):23–28. doi: 10.1126/science.959840.Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144(5):646–674. doi: 10.1016/j.cell.2011.02.013.Merritt CR, et al. Multiplex digital spatial profiling of proteins and RNA in fixed tissue. Nat Biotechnol 2020;38(5):586–599. doi: 10.1038/s41587-020-0472-9.Ethics ApprovalThis study was approved by the METC Leiden-Den Haag-Delft (protocol B20.039). Patient samples were anonymised and handled according to the medical ethical guidelines described in the Code of Conduct for Proper Secondary Use of Human Tissue of the Dutch Federation of Biomedical Scientific Societies.Abstract 673 Figure 1Transcriptional alterations in early-stage colorectal cancer. Digital spatial profiling defines transcriptional alterations in early-stage colorectal cancer. (A) Schematic representation of an early-stage CRC sample containing regions with different levels of cancer progression, including normal epithelium, transition areas, low-, and high-grade dysplasia, and invasive carcinoma. (B) Segmentation based immunofluorescent labelling with antibodies directed against PanCK and Vimentin in one of the early-stage CRC samples. Artificial overlay of implemented segmentation is indicated for each ROI, visualizing Vimentin+ (pink) and PanCK+ (orange) segments. Inset: higher magnification of an individual ROI. (C) Dimension reduction of expression of all quantified genes by t-Distributed Stochastic Neighbor Embedding (tSNE). tSNE plots are annotated by segment (left), and histological region (right).

Tumor Microenvironment Profiles Reveal Distinct Therapy-Oriented Proteogenomic Characteristics in Colorectal Cancer

Advances in immunotherapy have made an unprecedented leap in treating colorectal cancer (CRC). However, more effective therapeutic regimes need a deeper understanding of molecular architectures for precise patient stratification and therapeutic improvement. We profiled patients receiving neoadjuvant chemotherapy alone or in combination with immunotherapy (PD-1 checkpoint inhibitor) using Digital Spatial Profiler (DSP), a high-plex spatial proteogenomic technology. Compartmentalization-based high-plex profiling of both proteins and mRNAs revealed pronounced immune infiltration at tumor regions associated with immunotherapy treatment. The protein and the corresponding mRNA levels within the same selected regions of those patient samples correlate, indicating an overall concordance between the transcriptional and translational levels. An elevated expression of PD-L1 at both protein and the mRNA levels was discovered in the tumor compartment of immunotherapy-treated patients compared with chemo-treated patients, indicating potential prognostic biomarkers are explorable in a spatial manner at the local tumor microenvironment (TME). An elevated expression of PD-L1 was verified by immunohistochemistry. Other compartment-specific biomarkers were also differentially expressed between the tumor and stromal regions, indicating a dynamic interplay that can potentiate novel biomarker discovery from the TME perspectives. Simultaneously, a high-plex spatial profiling of protein and mRNA in the tumor microenvironment of colorectal cancer was performed.

Spatial heterogeneity of the immune compartment within the lungs of critical COVID-19 patients

Cross AR, Sansom S, Roberts I, Cerundolo L, Melero I, De Andrea C, Landecho MF, Klenerman P,Hester J, Issa F Acute respiratory distress syndrome (ARDS) is a defining feature of severe infection with theSARS-CoV-2 virus. Approaches to understand the immune response during COVID-19 are largelyconfined to characterisation of circulating leukocytes, however this approach excludes the mostrelevant cells that are active at the site of infection and injury. The aim of this study was to characterise the immune landscape across the lungs of COVID-19patients. Lung samples from three critical COVID-19 patients were assessed for histopathology,viral load, and distribution using qPCR, in situ hybridisation and immunohistochemistry.Leukocyte distribution was then assessed, and the transcript profile of selected areas examinedagainst the >1800 genes in the Cancer Transcriptome Atlas panel on the NanoString GeoMxDigital Spatial Profiling platform. Lung samples exhibited a spectrum of typical COVID-19 pathology with diffuse alveolar damageconsistent with hyaline membrane and type II pneumocyte hyperplasia, interstitialinflammation, organising pneumonia and thrombi. All tissues tested positive for SARS-CoV-2RNA using qPCR, whilst spatially resolved techniques revealed only few and sparsely distributedcells carrying the viral nucleocapsid protein. Multiplexed immunofluorescence for lymphocytes(CD3+) and macrophages (CD68+) was used to select areas of immune enrichment for spatialtranscriptomic profiling. These targeted analyses highlighted functional pathways involved inthe interferon gamma response, TCR activation and antigen presentation. Comparison acrossimmune-enriched areas identified a heterogeneity in lung infiltrates with spatial separation ofchemokine and complement production. Our data identify pathological immune pathways thatare amenable to therapeutic intervention in critical disease.

Spatially resolved whole transcriptome profiling in human and mouse tissue using Digital Spatial Profiling

Emerging spatial profiling technology has enabled high-plex molecular profiling in biological tissues, preserving the spatial and morphological context of gene or protein expression. Here we describe expanded chemistry for the Digital Spatial Profiling platform to quantify whole transcriptomes in human and mouse tissues using a wide range of spatial profiling strategies and sample types. We designed multiplexed in situ hybridization probe pools targeting the protein-coding genes in the human and mouse transcriptomes, hereafter referred to as the human or mouse Whole Transcriptome Atlas (WTA). We validated the human and mouse WTA assays using cell lines to demonstrate concordance with orthogonal gene expression profiling methods in profiled region sizes ranging from ~10-500 cells. By benchmarking against bulk RNAseq and single-molecule fluorescence in situ hybridization, we demonstrate robust transcript detection possible down to ~100 transcripts per region. To assess the performance of WTA across tissue and sample types, we applied WTA to biological questions in cancer, molecular pathology, and developmental biology. We show that spatial profiling with WTA can detect expected spatial gene expression differences between tumor and tumor microenvironment, identify spatial disease-specific heterogeneity in gene expression in histological structures of the human kidney, and comprehensively map transcriptional programs in anatomical substructures of nine organs in the developing mouse embryo. Digital Spatial Profiling technology with the WTA assays provides a flexible method for spatial whole transcriptome profiling applicable to diverse tissue types and biological contexts.

Dissection of the bone marrow microenvironment in hairy cell leukaemia identifies prognostic tumour and immune related biomarkers

Hairy cell leukaemia (HCL) is a rare CD20+ B cell malignancy characterised by rare “hairy” B cells and extensive bone marrow (BM) infiltration. Frontline treatment with the purine analogue cladribine (CDA) results in a highly variable response duration. We hypothesised that analysis of the BM tumour microenvironment would identify prognostic biomarkers of response to CDA. HCL BM immunology pre and post CDA treatment and healthy controls were analysed using Digital Spatial Profiling to assess the expression of 57 proteins using an immunology panel. A bioinformatics pipeline was developed to accommodate the more complex experimental design of a spatially resolved study. Treatment with CDA was associated with the reduction in expression of HCL tumour markers (CD20, CD11c) and increased expression of myeloid markers (CD14, CD68, CD66b, ARG1). Expression of HLA-DR, STING, CTLA4, VISTA, OX40L were dysregulated pre- and post-CDA. Duration of response to treatment was associated with greater reduction in tumour burden and infiltration by CD8 T cells into the BM post-CDA. This is the first study to provide a high multiplex analysis of HCL BM microenvironment demonstrating significant immune dysregulation and identify biomarkers of response to CDA. With validation in future studies, prospective application of these biomarkers could allow early identification and increased monitoring in patients at increased relapse risk post CDA.