Multiplexed Tissue Imaging Research Articles

Abstract 482: Rapid highly multiplexed immunoprofiling of human fixed tissues by Orion imaging

Abstract The dramatic impact of immune checkpoint inhibitors (ICIs) has focused interest in investigating immune-tumor cell interactions to understand mechanisms of ICI sensitivity and resistance, to identify patients that are responsive to specific treatments, and to develop new therapies. Multiplexed tissue imaging is a highly promising approach to immunoprofile tumors; it can assess many cell types and states within the context of preserved tumor architecture. However, the promise of highly multiplexed tissue imaging remains largely unfulfilled by current methods which are not compatible with pathology workflows. Here we present the development and implementation of Orion™ technology that permits whole-slide rapid single-pass imaging of up to 21 markers from formalin fixed paraffin embedded (FFPE) tissues. This method measures spectra for specific fluorophores to optimally sample the emitted light spectrum and distinguish multiple fluorescence excitation and emission channels across the spectral range of optical microscopes (~400-900 nm). To establish the utility of the Orion™ platform for immuno-phenotyping and immune checkpoint protein detection, we created an immunoprofiling panel of 21 qualified antibodies and labelled with fluorophores to subdivide the available emission spectrum in the 438-893 nm range. The panel includes markers that define subsets of T cells and macrophages. FFPE sections of human tonsil and matched primary and brain metastatic lung adenocarcinoma were stained and imaged in a single pass demonstrating staining patterns consistent with known micro-anatomic compartments and cell types in tonsil and identifying immune cell subtypes and heterogeneous checkpoint protein expression in tumor samples. An unexpected benefit of imaging with the Orion platform is the reduction of autofluorescence which is highly advantageous for the detection of proteins, like PD-L1, that function at very low levels. Orion imaging promises to accelerate discovery of predictive and prognostic biomarkers, enable pharmacodynamics study of immuno-oncology drugs undergoing clinical trials and ultimately provide clinically actionable diagnostic tests. Citation Format: Jia-Ren Lin, Daniel E. Campton, Jeremy Cooper, Yu-An Chen, Erin F. McCarty, Keith L. Ligon, Eric P. Kaldjian, Kyla Teplitz, Steve Reese, Sandro Santagata, Peter K. Sorger. Rapid highly multiplexed immunoprofiling of human fixed tissues by Orion imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 482.

Abstract 2756: A toolbox enabling a data-driven selection of regions of interest in tumor tissue sections for imaging mass cytometry analysis

Abstract Highly multiplexed imaging technologies are increasingly applied in biomedical research to identify underlying patterns of disease. As part of an IMI-funded project, the IMMUcan (Integrated immunoprofiling of large adaptive cancer patients cohorts) consortium will generate single-cell resolved molecular profiles for thousands of cancer patients from hospitals across Europe. Patient tissues will be profiled using two imaging modalities, multispectral immunofluorescence (mIF) and Imaging Mass Cytometry (IMC), in junction with data from different omics techniques. IMC enables highly multiplexed tissue imaging, but its current throughput only enables to image a fraction of large/whole-slide tumor tissue sections. The selection of regions of interest for IMC analysis is of critical importance with respect to the cells present in an area and the antibody panel used for the analysis of such an area. To target areas of interest in tumor tissue sections, e.g. with immune cell infiltration or tertiary lymphoid structures, we have developed a data driven approach based on mIF imaging analyses from consecutive sections to select such regions of interest for analysis with IMC. A mIF panel was designed to stain T cells, B cells, macrophages, neutrophils, dendritic cells and tumor cells to have an overview of immune cell populations within the whole tissue. After quantification, mIF images and data are hosted on a webserver and regions that contain cell-types of interest are annotated with the help of a web-based tool. mIF images are used for alignment with brightfield whole slide scans of the consecutive FFPE sections which are used for IMC. After alignment, the annotated regions of interest are acquired in IMC. The mean positional offset from IF to IMC for regions of interest was roughly 120 µm and the overall cell-type numbers across both technologies were well correlated. The approach presented here has the great advantage to allow us to reproducibly target similar areas for IMC across thousands of samples based on mIF within the IMMUcan consortium and will enable single-cell analysis for whole tumor sections based on mIF and IMC to gain a deeper understanding of the underlying tumor biology. Funding: IMI2 JU grant agreement 821558, supported by EU's Horizon 2020 and EFPIA. Citation Format: Daniel Schulz, Stephanie Renaud-Tissot, Robin Liechti, Nils Eling, Michelle Daniel, Jonas Windhager, Yervand Karapetyan, Thomas Mrowiec, Sylvie Rusakiewicz, Henoch S. Hong, Marie Morfouace, Bernd Bodenmiller. A toolbox enabling a data-driven selection of regions of interest in tumor tissue sections for imaging mass cytometry analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2756.

Abstract 1816: Phenogenomic characterization of immunomodulatory purinergic signaling in glioblastoma

Abstract INTRODUCTION: Extracellular purinergic signaling plays critical roles in the regulation of tumor growth and anti-tumor immunity via autocrine/paracrine binding of metabolites to receptors on neoplastic and non-neoplastic populations. Extracellular purine concentrations are principally mediated by the ectonucleotidase enzymes CD39 and CD73, which catabolize ATP to adenosine. Within the tumor microenvironment, neoplastic, immune, and stromal cells expressing these enzymes may co-localize to generate an immunosuppressive adenosine-rich niche. However, the cellular composition, spatial architecture and phenotypic properties of these tumor ecosystems and their relationship to tumor genotype have been poorly characterized. METHODS: We quantified CD73 expression by immunohistochemistry (IHC) in a cohort of CNS tumors [meningiomas(N=222), gliomas(N=244), ependymomas(N=44), medulloblastomas(N=24), craniopharyngiomas(N=38)]. We used publicly-available single-cell RNA-seq data and 36 marker multiplexed tissue imaging (t-CyCIF) of 139 clinically and genomically annotated glioblastomas to characterize CD39 and CD73 expressing populations, define immune architecture and tumor cell states at single cell resolution, evaluate spatial correlations, and identify markers of clinical outcome. Mass spectrometry imaging (MALDI-MSI) was employed to generate spatially-resolved quantification of purine metabolite levels in glioblastoma resections (N=9). RESULTS: IHC revealed strong CD73 expression in meningiomas and gliomas. Tumor CD73 expression was associated with poor progression-free-survival in IDH-wildtype glioblastoma (p=0.04). scRNA-seq in glioblastoma revealed that CD73 is predominantly expressed by tumor cell populations, while CD39 is predominantly expressed by monocytic (macrophage, microglial) populations. t-CyCIF showed enrichment of EGFR, Ki-67, and TP53 expression in CD73-high tumor cells at a single cell level independent of genotype, as well as significant spatial correlation between CD73 expression in tumor cells and CD39 expression in macrophages. MALDI-MSI showed significantly greater adenosine concentrations in glioblastomas with high CD73 expression. CD73 expression significantly correlated with EGFR amplification or C-terminal deletion (EGFRvIII or variants), type-II interferon signaling, and PD-L1 expression in glioblastoma. CONCLUSIONS: Phenogenomic analysis of purinergic signaling in glioblastoma revealed correlations between CD73 expression and genotype, adenosine concentration, and clinical outcome. Spatial analysis revealed interaction between macrophages CD39 expression and tumor cell CD73 expression, suggesting that these populations may interact to suppress anti-tumor immunity. Anti-CD73 therapy may provide therapeutic benefits in glioblastoma by blunting immunosuppressive and oncogenic adenosine signaling. Citation Format: Shannon Coy, Jia-Ren Lin, Shu Wang, Sylwia Stopka, Rumana Rashid, Jaeho Hwang, Prasidda Khadka, Philipp Euskirchen, Pratiti Bandopadhayay, Patrick Y. Wen, Peter K. Sorger, Nathalie Agar, Keith L. Ligon, Mehdi Touat, Sandro Santagata. Phenogenomic characterization of immunomodulatory purinergic signaling in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1816.

Abstract 122: Highly multiplexed, spatially-resolved tissue imaging of genetically engineered mouse models of cancer to discover and characterize immune regulators of tumorigenesis

Abstract Despite the encouraging success of immunotherapy for certain malignancies, most patients with solid tumors do not yet benefit from such therapies. It is therefore critical that we identify factors dictating responsiveness to immunotherapy by more completely characterizing tumor-immune interactions. Even though immunotherapies are used in humans, in-depth analysis of murine models is essential for a mechanistic understanding of the crosstalk among tumor, immune, and stromal components of the tumor microenvironment (TME): only animal models have the necessary manipulability and reproducibility for causal, mechanistic studies. However, murine modelling must be combined with spatially resolved analytical methods such as highly multiplexed tissue imaging that enable accurate characterization of the TME at a single-cell level. Here we use tissue cyclic immunofluorescence (t-CyCIF) multiplexed imaging to characterize the immune microenvironment of a mouse lung adenocarcinoma model initiated via lentiviral delivery of Cre recombinase into the lungs of KrasLSL-G12D/+;p53fl/fl (KP) mice. Using this new platform, we identify tumor cells and immune cell types (dendritic cells, NK cells, macrophages, B cells, helper T cells, regulator T cells and cytotoxic T cells). We characterize the effects of expressing tumor antigen (i.e., LucOS), of CRISPRa based upregulation of the chemokines (e.g., CXCL10), and of combination immune check point inhibitor treatments (e.g., one-week treatment with anti-PD-1 and anti-CTLA-4 (PC)). Remarkably the total immune composition of the mouse lungs remains relatively unchanged following these perturbations, but there are marked changes in the immune cell localization in tumor nodules, in the number and size of immune cell networks and in the functional activation states of cytotoxic T cells. For example, one week of PC treatment did not affect tumor burden and did not change the extent of immune cell infiltration however, it did drastically change cytotoxic T cell phenotypes with increased effector phenotypes (i.e., GzmB and Perforin expression and proliferation) and decreased exhaustion-like phenotypes (i.e., PD-1 and Tim-3 expression). Lymphocyte networks decreased in number but were closer to tumors. Using high-dimensional protein expression data to characterize GEMM models following in situ genetic or therapeutic perturbation is a powerful new platform to investigate tumor-immune interactions. Citation Format: Giorgio Gaglia, Megan Burger, Claire Ritch, Danae Argyropoulou, Yang Dai, Shannon Coy, Jia-Ren Lin, Peter Sorger, Tyler Jacks, Sandro Santagata. Highly multiplexed, spatially-resolved tissue imaging of genetically engineered mouse models of cancer to discover and characterize immune regulators of tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 122.

Spatially visualized single-cell pathology of highly multiplexed protein profiles in health and disease

Deep molecular profiling of biological tissues is an indicator of health and disease. We used imaging mass cytometry (IMC) to acquire spatially resolved 20-plex protein data in tissue sections from normal and chronic tonsillitis cases. We present SpatialViz, a suite of algorithms to explore spatial relationships in multiplexed tissue images by visualizing and quantifying single-cell granularity and anatomical complexity in diverse multiplexed tissue imaging data. Single-cell and spatial maps confirmed that CD68+ cells were correlated with the enhanced Granzyme B expression and CD3+ cells exhibited enrichment of CD4+ phenotype in chronic tonsillitis. SpatialViz revealed morphological distributions of cellular organizations in distinct anatomical areas, spatially resolved single-cell associations across anatomical categories, and distance maps between the markers. Spatial topographic maps showed the unique organization of different tissue layers. The spatial reference framework generated network-based comparisons of multiplex data from healthy and diseased tonsils. SpatialViz is broadly applicable to multiplexed tissue biology.

Temporal and spatial topography of cell proliferation in cancer.

3122 Background: Tumors are complex ecosystems where exogenous and endogenous cues are integrated to either stimulate or inhibit cancer cell proliferation. However, the nature of these complex cell cycle states, their spatial organization, response to perturbation, and implications for clinical outcomes, are poorly characterized in tumor tissues. Methods: We used multiplexed tissue imaging to develop a robust classifier of proliferation, the multivariate proliferation index (MPI), using 513 unique tumors across five cancer types. Next, we used dimensionality reduction analysis to assess how the patterns of cell cycle protein expression in tumors were altered in response to perturbation. Results: The MPI outperforms single markers, like Ki67, when classifying proliferative index across diverse tumor types and reveals the proliferative architecture of tumors in situ. We find that proliferative and non-proliferative cancer cells are organized across microscopic (cell-to-cell) and macroscopic (tissue-level) scales. Both domains are reshaped by therapy, and local clusters of proliferative and non-proliferative tumor cells preferentially neighbor distinct tumor-infiltrating immune cells. We further phenotyped non-proliferating cancer cells using markers of quiescent cancer cells, cancer stem cells, and dormant cancer cells. We found that these types of non-proliferating cancer cells can occupy distinct regions within the same primary tumor. In high-dimensional marker space, populations of proliferative cancer cells express canonical patterns of cell cycle protein markers, a property we refer to as “cell cycle coherence”. Untreated tumors exist in a continuum of coherence states, ranging from optimal coherence, akin to freely cycling cells in culture, to reduced coherence characterized by either cell cycle polarization or non-canonical marker expression. Coherence can be stereotypically altered by induction and abrogation of mitogen signaling in a HER2-driven model of breast cancer. Cell cycle coherence is modulated by neoadjuvant therapy in patients with localized breast cancer, and coherence is associated with disease-free survival after adjuvant therapy in patients with colorectal cancer, mesothelioma and glioblastoma. Conclusions: The MPI robustly defines proliferating and non-proliferating cells in tissues, with immediate implications for clinical practice and research. The coherence metrics capture the diversity of post-treatment cell cycle states directly in clinical samples, a fundamental step in advancing precision medicine. More broadly, replacing binary metrics with multivariate traits provides a quantitative framework to study temporal processes from fixed static images and to investigate the rich spatial biology of human cancers.

Highly Multiplexed Phenotyping of Immunoregulatory Proteins in the Tumor Microenvironment by CODEX Tissue Imaging.

Immunotherapies are revolutionizing cancer treatment by boosting the natural ability of the immune system. In addition to antibodies against traditional checkpoint molecules or their ligands (i.e., CTLA-4, PD-1, and PD-L1), therapies targeting molecules such as ICOS, IDO-1, LAG-3, OX40, TIM-3, and VISTA are currently in clinical trials. To better inform clinical care and the design of therapeutic combination strategies, the co-expression of immunoregulatory proteins on individual immune cells within the tumor microenvironment must be robustly characterized. Highly multiplexed tissue imaging platforms, such as CO-Detection by indEXing (CODEX), are primed to meet this need by enabling >50 markers to be simultaneously analyzed in single-cells on formalin-fixed paraffin-embedded (FFPE) tissue sections. Assembly and validation of antibody panels is particularly challenging, with respect to the specificity of antigen detection and robustness of signal over background. Herein, we report the design, development, optimization, and application of a 56-marker CODEX antibody panel to eight cutaneous T cell lymphoma (CTCL) patient samples. This panel is comprised of structural, tumor, and immune cell markers, including eight immunoregulatory proteins that are approved or currently undergoing clinical trials as immunotherapy targets. Here we provide a resource to enable extensive high-dimensional, spatially resolved characterization of the tissue microenvironment across tumor types and imaging modalities. This framework provides researchers with a readily applicable blueprint to study tumor immunology, tissue architecture, and enable mechanistic insights into immunotherapeutic targets.

Using single cell spatial and transcriptional analysis to understand early host response to RNA viral infections.

Abstract The innate immune system serves as the first line of defense against pathogens. One innate defense mechanism is the RIG-I like receptor pathway (RLR) which signals through the mitochondrial anti-viral signaling protein (MAVS) which plays a role in initiating the interferon (IFN) response to RNA viruses such as vesicular stomatitis virus (VSV). To investigate the mechanisms the MAVS-dependent pathway has on early viral control following VSV infection in vivo, we used a combination of two approaches: single cell RNA sequencing and multiplex imaging. These two techniques allow us to assess the individual cellular response to VSV in whole lymph node tissue. After intradermal inoculation, skin draining lymph nodes were assessed using single cell sequencing. Macrophages and plasmacytoid dendritic cells (pDCs) showed significantly elevated IFN stimulated gene (ISG) detection in C57Bl/6J (B6) mice compared to the MAVS−/− animals at 8 hours post infection (hpi), yet there was no detectable viral reads. This early initiation of ISG expression played a role in detection of viral RNA at 24 hpi which saw significantly elevated viral RNA in the MAVS−/− animals compared to the B6 animals. Interestingly, the virus replication in both conditions was contained to the macrophage populations suggesting that without the MAVS pathway active, the virus was still contained. Combining this information along with the observed spatial organization through multiplex tissue imaging, we aim to develop a model illustrating the role of the MAVS-dependent pathway in controlling early virus replication. Animal experiments were done in accordance with the guidelines of the NIAID/NIH Institutional ACUC. This research was supported by the IRP of NIH/NIAID.

Highly multiplexed tissue imaging using repeated oligonucleotide exchange reaction.

Multiparameter tissue imaging enables analysis of cell‐cell interactions in situ, the cellular basis for tissue structure, and novel cell types that are spatially restricted, giving clues to biological mechanisms behind tissue homeostasis and disease. Here, we streamlined and simplified the multiplexed imaging method CO‐Detection by indEXing (CODEX) by validating 58 unique oligonucleotide barcodes that can be conjugated to antibodies. We showed that barcoded antibodies retained their specificity for staining cognate targets in human tissue. Antibodies were visualized one at a time by adding a fluorescently labeled oligonucleotide complementary to oligonucleotide barcode, imaging, stripping, and repeating this cycle. With this we developed a panel of 46 antibodies that was used to stain five human lymphoid tissues: three tonsils, a spleen, and a LN. To analyze the data produced, an image processing and analysis pipeline was developed that enabled single‐cell analysis on the data, including unsupervised clustering, that revealed 31 cell types across all tissues. We compared cell‐type compositions within and directly surrounding follicles from the different lymphoid organs and evaluated cell‐cell density correlations. This sequential oligonucleotide exchange technique enables a facile imaging of tissues that leverages pre‐existing imaging infrastructure to decrease the barriers to broad use of multiplexed imaging.

IBEX: A versatile multiplex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues

The diverse composition of mammalian tissues poses challenges for understanding the cell-cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only two to six molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a method that overcomes major impediments to highly multiplex tissue imaging. "Iterative bleaching extends multiplexity" (IBEX) uses an iterative staining and chemical bleaching method to enable high-resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an "open" system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, cellular indexing of transcriptomes and epitopes by sequencing, and IBEX analysis of the same tissue. To facilitate data processing, we provide an open-source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high-content imaging to reveal the complex cellular landscape of diverse organs and tissues.

TAMI-45. PHENOGENOMIC CHARACTERIZATION OF IMMUNOMODULATORY PURINERGIC SIGNALING IN GLIOBLASTOMA

Abstract INTRODUCTION Purinergic signaling plays critical roles in the regulation of tumor growth and anti-tumor immunity via autocrine/paracrine binding of metabolites to receptors on neoplastic and non-neoplastic populations. Extracellular purine concentrations are mediated by the ectonucleotidase enzymes CD39 and CD73, which catabolize ATP to adenosine. Within tumors such as glioblastoma, neoplastic, immune, and stromal cells expressing these enzymes may co-localize to generate immunosuppressive adenosine-rich environments. However, the composition, architecture, and phenotypic properties of these tumor ecosystems and their relationship to tumor genotype are poorly characterized. METHODS We quantified CD73 expression by immunohistochemistry in a cohort of CNS tumors [meningiomas(n=222), gliomas(n=244), ependymomas(n=44), medulloblastomas(n=24), and craniopharyngiomas(n=38)]. We used publicly-available single-cell RNA-seq data and 36-marker multiplexed tissue imaging (t-CyCIF) of 139 clinically and genomically annotated glioblastoma resections to characterize CD39 and CD73-expressing populations, define the immune architecture and tumor cell-states at single cell resolution, and identify markers of clinical outcome. We used mass spectrometry imaging (MALDI-MSI) to generate spatially-resolved quantification of purine metabolite levels in glioblastoma resections (n=10). RESULTS CD73 exhibited strong expression in a subset of gliomas and meningiomas but was typically not expressed in ependymomas or medulloblastomas. CD73 expression correlated with poor progression-free-survival in IDH-wildtype glioblastoma (p=0.04). scRNA-seq and t-CyCIF in glioblastoma showed CD73 expression in tumor cells, and CD39 expression in macrophages and endothelial cells. MALDI-MSI showed significantly greater adenosine concentrations (3.5-fold;p=0.04) in glioblastomas with high CD73 expression. scRNA-seq showed direct correlations between stem-like mRNA expression, proliferation, and CD73 expression in DIPG. CD73 expression significantly correlated with EGFR amplification, interferon signaling, and PD-L1 expression in glioblastoma. CONCLUSIONS Phenogenomic analysis of purinergic immunomodulatory signaling revealed significant interplay between CD73 activity and genotype, adenosine concentration, differentiation-state, clinical outcome, and possible interaction between CD39-positive macrophages and CD73-positive neoplastic cells. Anti-CD73 therapy may provide therapeutic benefits in glioblastoma by blunting immunosuppressive and oncogenic adenosine signaling.

Oncolytic vaccinia virus reinvigorates peritoneal immunity and cooperates with immune checkpoint inhibitor to suppress peritoneal carcinomatosis in colon cancer

BackgroundPeritoneal carcinomatosis (PC) is a common and devastating manifestation of colon cancer and refractory to conventional anticancer therapeutics. During the peritoneal dissemination of colon cancer, peritoneal immunity is nullified by...

Minerva: a light-weight, narrative image browser for multiplexed tissue images.

SummaryAdvances in highly multiplexed tissue imaging are transforming our understanding of human biology by enabling detection and localization of 10-100 proteins at subcellular resolution (Bodenmiller, 2016). Efforts are now underway to create public atlases of multiplexed images of normal and diseased tissues (Rozenblatt-Rosen et al., 2020). Both research and clinical applications of tissue imaging benefit from recording data from complete specimens so that data on cell state and composition can be studied in the context of overall tissue architecture. As a practical matter, specimen size is limited by the dimensions of microscopy slides (2.5 × 7.5 cm or ~2-8 cm2 of tissue depending on shape). With current microscopy technology, specimens of this size can be imaged at sub-micron resolution across ~60 spectral channels and ~106 cells, resulting in image files of terabyte size. However, the rich detail and multiscale properties of these images pose a substantial computational challenge (Rashid et al., 2020). See Rashid et al. (2020) for an comparison of existing visualization tools targeting these multiplexed tissue images.

2112-P: Sarco/Endoplasmic Reticulum ATPase (SERCA2) Deficiency in the Nonobese Diabetic (NOD) Mouse Accelerates Type 1 Diabetes Development

Recent evidence has implicated pathways intrinsic to the β cell, such as endoplasmic reticulum (ER) stress, as potential triggers of T1D. ER stress is increased by the loss of ER Ca2+, leading to decreased β cell function and increased β cell apoptosis. Maintenance of intraluminal ER Ca2+ stores is primarily regulated by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2) pump, and we have shown that β cell SERCA2 expression is reduced in islets from mouse models of T1D prior to, and after, disease onset. Thus, we hypothesized that SERCA2-mediated ER Ca2+ dyshomeostasis could be a major contributor to T1D development. To test this, we generated a mouse model haploinsufficient for SERCA2 on the NOD background (NOD-S2+/-). Compared to wild type littermates (NOD-WT), NOD-S2+/- mice had a higher incidence of diabetes (p<0.0001) and incidence was accelerated (14.5 wks vs. 19 wks, p<0.0001). Prior to diabetes onset, NOD-S2+/- islets also had increased MHC-I expression, which has been reported to follow increased ER stress and lead to increased β cell immunogenicity. To test whether SERCA2 activation may have beneficial therapeutic effects, we treated NOD-WT and NOD-S2+/- mice with CDN1163, an allosteric activator of SERCA2, from 6-8 wks of age and observed a two week delay in NOD-S2+/- average onset (p<0.05). To characterize patterns of islet immune infiltration in response to SERCA2 loss, we are utilizing the CODEX multiplexed tissue imaging platform. Our pilot study used a ten antibody panel to label and image spleen, pancreatic lymph nodes, and islets from NOD-WT. Using Indiana University-developed software, Volumetric Tissue Exploration Analysis, our preliminary analysis yields a high fidelity of nuclear segmentation and low (∼10%) false positive identification of markers. Taken together, our data suggest that loss of SERCA2 exacerbates T1D development and indicates SERCA2 may be a novel T1D therapeutic target. Disclosure R.N. Bone: None. C.A. Reissaus: None. T. Kono: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb.

Uncovering Differently Expressed Markers and Heterogeneity on Human Pancreatic Cancer

Discovery of biomarkers is critical to understand tumor heterogeneity and microenvironment. To determine differently expressed makers on cancer tissue for comprehensive profiling, the multiplexed tissue imaging mass cytometer (IMC) which uniquely combines time-of-flight mass spectrometry with metal-labeling technology to enable breakthrough discovery on single cell level was employed to investigate the expression of seven markers related to the epithelial-to-mesenchymal transition [α-smooth muscle actin (α-SMA), vimentin, collagen I, cytokeratin 7, pan-keratin], tumor proliferation (Ki-67), and human leucocyte antigen (HLA-DR) on human pancreatic cancer tissue. The difference was analyzed using bioinformatic tools. We observed the high expression of α-SMA, vimentin, collagen I, and Ki-67 on grade I but not on grade III. HLA-DR was highly expressed on grade I/III but not on grade II. Overall, the expression of markers has elucidated the heterogeneity intratumors. Additionally, to identify biomarkers on pancreatic cancer cells by blind systematic evolution of ligands by exponential enrichment (SELEX), aptamer pull-down assay and liquid chromatography–tandem mass spectrometry were used. Mortalin was identified as a potential a prognostic marker of pancreatic cancer. Our studies demonstrate that the IMC and blind SELEX might be implemented to discover biomarkers which can be used to better understand tumor biology and biomedical research applications.

HSF1 phase transition mediates stress adaptation and cell fate decisions.

Under proteotoxic stress, some cells survive whereas others die. Mechanisms governing this heterogeneity in cell fate are unknown. We report that condensation and phase transition of heat-shock factor 1 (HSF1), a transcriptional regulator of chaperones1,2, is integral to cell fate decisions underlying survival or death. During stress, HSF1 drives chaperone expression but also accumulates separately in nuclear stress bodies (foci)3–6. Foci formation has been regarded as a marker of cells actively upregulating chaperones3,6–10. Using multiplexed tissue imaging, we observed HSF1 foci in human tumors. Paradoxically, their presence inversely correlated with chaperone expression. By live-cell microscopy and single-cell analysis, we found that foci dissolution rather than formation promoted HSF1 activity and cell survival. During prolonged stress, the biophysical properties of HSF1 foci changed; small, fluid condensates enlarged into indissoluble gel-like arrangements with immobilized HSF1. Chaperone gene induction was reduced in such cells, which were prone to apoptosis. Quantitative analysis suggests that survival under stress results from competition between concurrent yet opposing mechanisms. Foci may serve as sensors that tune cytoprotective responses, balancing rapid transient responses and irreversible outcomes.

Dynamics of the Cutaneous T Cell Lymphoma Microenvironment in Patients Treated with Pembrolizumab Revealed By Highly Multiplexed Tissue Imaging

Cutaneous T cell lymphoma (CTCL) is a CD4+ T cell malignancy of the skin with heterogeneous outcomes and limited treatment options. Monoclonal antibodies directed against PD-1, such as pembrolizumab, have shown impressive efficacy in multiple advanced malignancies, and are currently tested in clinical trials in patients with CTCL. Initial data indicate that about half of the patients experience treatment response, whereas the other half are non-responders. Non-responders can be further divided into patients with stable disease versus rapid progressors. It is currently unknown why some CTCL patients respond to pembrolizumab while others rapidly progress, and no predictive biomarkers are available. Single-cell analysis approaches to identify biomarkers of response, for example quantifying the expression of PD-1 on tumor cells vs. reactive immune cells, have not enabled stratification of patients. We therefore hypothesized that more complex spatial cellular interactions within the immune tumor microenvironment (iTME) of CTCL could provide insight into the mechanisms of pembrolizumab response and enable prediction. We applied CODEX (CO-Detection by indEXing) highly multiplexed tissue imaging to study the CTCL iTME in matched biopsies before and after pembrolizumab therapy in 7 responders and 7 non-responders (see the Figure). Using 54 markers simultaneously allowed discriminating malignant CD4+ tumor cells from reactive CD4+ T cells and identified 30 different cell clusters with spatial information, including an M2 macrophage cluster that was enriched in non-responders before therapy. Unexpectedly, in pembrolizumab responders compared to non-responders, PD-1 expression levels were higher in multiple clusters of tumor cells and reactive T cells. Computational spatial analysis revealed ten distinct, conserved cellular neighborhoods in the CTCL iTME that changed in composition and frequency during therapy. Interestingly, one cellular neighborhood to be presented dramatically increased after therapy only in responders. Therefore, highly multiplexed spatial analysis of the CTCL iTME allows discovering novel, predictive biomarkers of immunotherapy response and will pave the way for future studies that functionally address the identified cell types and cellular interactions. Disclosures Khodadoust: Corvus Pharmaceuticals: Research Funding. Kim:miRagen: Research Funding; Merck: Research Funding; Medivir: Honoraria, Membership on an entity's Board of Directors or advisory committees; Innate Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Soligenix: Research Funding; Forty Seven Inc: Research Funding; Neumedicine: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trillium: Research Funding; Elorac: Research Funding; Galderma: Research Funding; Corvus: Honoraria, Membership on an entity's Board of Directors or advisory committees; Portola Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Horizon: Research Funding. Nolan:Akoya Biosciences Inc.: Consultancy, Equity Ownership, Patents & Royalties.

Qualifying antibodies for image-based immune profiling and multiplexed tissue imaging.

Multiplexed tissue imaging enables precise, spatially resolved enumeration and characterization of cell types and states in human resection specimens. A growing number of methods applicable to formalin-fixed, paraffin-embedded (FFPE) tissue sections have been described, the majority of which rely on antibodies for antigen detection and mapping. This protocol provides step-by-step procedures for confirming the selectivity and specificity of antibodies used in fluorescence-based tissue imaging and for the construction and validation of antibody panels. Although the protocol is implemented using tissue-based cyclic immunofluorescence (t-CyCIF) as an imaging platform, these antibody-testing methods are broadly applicable. We demonstrate assembly of a 16-antibody panel for enumerating and localizing T cells and B cells, macrophages, and cells expressing immune checkpoint regulators. The protocol is accessible to individuals with experience in microscopy and immunofluorescence; some experience in computation is required for data analysis. A typical 30-antibody dataset for 20 FFPE slides can be generated within 2 weeks.

The landscape of myeloid and astrocyte phenotypes in acute multiple sclerosis lesions

Activated myeloid cells and astrocytes are the predominant cell types in active multiple sclerosis (MS) lesions. Both cell types can adopt diverse functional states that play critical roles in lesion formation and resolution. In order to identify phenotypic subsets of myeloid cells and astrocytes, we profiled two active MS lesions with thirteen glial activation markers using imaging mass cytometry (IMC), a method for multiplexed labeling of histological sections. In the acutely demyelinating lesion, we found multiple distinct myeloid and astrocyte phenotypes that populated separate lesion zones. In the post-demyelinating lesion, phenotypes were less distinct and more uniformly distributed. In both lesions cell-to-cell interactions were not random, but occurred between specific glial subpopulations and lymphocytes. Finally, we demonstrated that myeloid, but not astrocyte phenotypes were activated along a lesion rim-to-center gradient, and that marker expression in glial cells at the lesion rim was driven more by cell-extrinsic factors than in cells at the center. This proof-of-concept study demonstrates that highly multiplexed tissue imaging, combined with the appropriate computational tools, is a powerful approach to study heterogeneity, spatial distribution and cellular interactions in the context of MS lesions. Identifying glial phenotypes and their interactions at different lesion stages may provide novel therapeutic targets for inhibiting acute demyelination and low-grade, chronic inflammation.

Abstract 1189: Revisiting immune exhaustion in Hodgkin’s lymphoma

Abstract Clinical successes with immune check-point blockers have demonstrated the potency of the immune system in controlling cancers, most strikingly in Hodgkin lymphoma (HL), where overall response rates to PD1/L1 inhibitors approach 90%. Complete or durable responses, however, are uncommon, therefore targeting the PD1/L1 axis alone is not sufficient. Recent work analyzing the spatial arrangement of PD1 and PDL1 expressing cells has given us new insight into the mechanism of action of PD1/L1 inhibitors, however this work limited itself to studying a single check point marker on a subset of cells. We hypothesize that comprehensive profiling of the frequency and spatial arrangement of immune cells in the Hodgkin lymphoma tumor immune microenvironment (TME) will provide new insights into the mechanism of checkpoint blockers and identify novel targets for immune therapy. Until now, multiparameter spatial analysis of the immune microenvironment was limited by technical challenges. Flow and mass cytometry are able to identify immune subsets of interest but spatial information is lost. Multiplex tissue imaging methods are limited to 6-8 simultaneous markers and cannot capture the full complexity of the immune phenotypes. The Fluidigm Hyperion imaging mass cytometry (IMC) system combines a CyTOF mass cytometer with a laser ablation system allowing for 40+ parameter simultaneous immunophenotyping on a single slide of FFPE tissue, with sub-cellular resolution. We have developed a panel of 34 antibodies that allow for comprehensive characterization of CD4, CD8 and myeloid cells components in the TME of Hodgkin lymphoma using IMC. Here we report on our spatial analysis of TIM3 and LAG3 expressing CD4+ lymphocytes. Our data suggests LAG3+CD4+ and TIM3+CD4+ lymphocytes had shorter mean nearest distance to PDL1+Hodgkin Reed-Sternberg (HRS) cells upon comparison to PDL1- HRS cells (t-test, p=1.703e-08,p=1.126e-14). Future studies should explore multiple exhausted marker models that seeks to further understand the presence of simultaneous exhaustion signals in the niche environment. These data suggest that therapies that target TIM3 and/or LAG3 should be tested in Hodgkin Lymphoma and that spatial analysis of immune subsets by IMC should be explored as selective and pharmacodynamic biomarkers. Citation Format: Anthony R. Colombo, Monirath Hav, Erik Gerdtsson, Jose Bisnesto-Villasboas, Stephen Ansell, James Hicks, Peter Kuhn, Akil Merchant. Revisiting immune exhaustion in Hodgkin’s lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1189.