Detection of neoplastic-immune hybrid cells with metastatic properties in uveal melanoma.

BACKGROUND: Uveal melanoma is the most common non-cutaneous melanoma and is an intraocular malignancy affecting nearly 7,000 individuals per year worldwide. Of these, approximately 50% will progress to metastatic disease for which there are currently no effective curative therapies. Despite advances in molecular profiling and metastatic stratification of uveal melanoma tumors, little is known regarding their underlying biology of metastasis. Our group has identified a disseminated neoplastic cell population characterized by co-expression of immune and melanoma proteins, circulating hybrid cells (hybrids), in patients with uveal melanoma. Compared to circulating tumor cells, which lack expression of immune proteins, hybrids are detected at an increased prevalence in peripheral blood and can be used as a non-invasive biomarker to predict metastatic progression. METHODS: To ascertain mechanisms underlying enhanced hybrid cell dissemination we identified hybrid cells within primary uveal melanoma tumors using single cell RNA sequencing (n = 8) and evaluated their gene expression and predicted ligand-receptor interactions in relation to other melanoma and immune cells within the primary tumor. We then verified expression of upregulated hybrid pathways within patient-matched tumor and peripheral blood hybrids (n = 4) using cyclic immunofluorescence and quantified their protein expression relative to other non-hybrid tumor and disseminated tumor cells. RESULTS: Among the top upregulated genes and pathways in hybrid cells were those involved in enhanced cell motility and cytoskeletal rearrangement, immune evasion, and altered cellular metabolism. In patient-matched tumor and peripheral blood, we verified gene expression by examining concordant protein expression for each pathway category: TMSB10 (cell motility), CD74 (immune evasion) and GPX1 (metabolism). Both TMSB10 and GPX1 were expressed on significantly higher numbers of disseminated hybrid cells compared to circulating tumor cells, and CD74 and GPX1 were expressed on more disseminated hybrids than tumor-resident hybrids. Lastly, we identified that hybrid cells express ligand-receptor signaling pathways implicated in promoting metastasis including GAS6-AXL, CXCL12-CXCR4, LGALS9-P4HB and IGF1-IGFR1. CONCLUSION: These findings highlight the importance of TMSB10, GPX1 and CD74 for successful hybrid cell dissemination and survival in circulation. Our results contribute to the understanding of uveal melanoma tumor progression and interactions between tumor cells and immune cells in the tumor microenvironment that may promote metastasis.

In situ single-cell therapeutic response imaging facilitated by the TRIPODD fluorescence imaging platform.

Purpose: Small molecule drugs such as tyrosine kinase inhibitors (TKIs) targeting tumoral molecular dependencies have become standard of care for numerous cancer types. Notably, epidermal growth factor receptor (EGFR) TKIs (e.g., erlotinib, afatinib, osimertinib) are the current first-line treatment for non-small cell lung cancer (NSCLC) due to their improved therapeutic outcomes for EGFR mutated and overexpressing disease over traditional platinum-based chemotherapy. However, many NSCLC tumors develop resistance to EGFR TKI therapy causing disease progression. Currently, the relationship between in situ drug target availability (DTA), local protein expression and therapeutic response cannot be accurately assessed using existing analytical tools despite being crucial to understanding the mechanism of therapeutic efficacy. Procedure: We have previously reported development of our fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution) that is capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. TRIPODD combines two complementary fluorescence imaging techniques: intracellular paired agent imaging (iPAI) to measure DTA and cyclic immunofluorescence (cyCIF), which utilizes oligonucleotide conjugated antibodies (Ab-oligos) for spatial proteomic expression profiling on tissue samples. Herein, TRIPODD was modified and optimized to provide a downstream analysis of therapeutic response through single-cell DTA and proteomic response imaging. Results: We successfully performed sequential imaging of iPAI and cyCIF resulting in high dimensional imaging and biomarker assessment to quantify single-cell DTA and local protein expression on erlotinib treated NSCLC models. Pharmacodynamic and pharmacokinetic studies of the erlotinib iPAI probes revealed that administration of 2.5 mg/kg each of the targeted and untargeted probe 4 h prior to tumor collection enabled calculation of DTA values with high Pearson correlation to EGFR, the erlotinib molecular target, expression in the tumors. Analysis of single-cell biomarker expression revealed that a single erlotinib dose was insufficient to enact a measurable decrease in the EGFR signaling cascade protein expression, where only the DTA metric detected the presence of bound erlotinib. Conclusion: We demonstrated the capability of TRIPODD to evaluate therapeutic response imaging to erlotinib treatment as it relates to signaling inhibition, DTA, proliferation, and apoptosis with preserved spatial context.

Ultra high content analyses of circulating and tumor associated hybrid cells reveal phenotypic heterogeneity.

Persistently high, worldwide mortality from cancer highlights the unresolved challenges of disease surveillance and detection that impact survival. Development of a non-invasive, blood-based biomarker would transform survival from cancer. We demonstrate the functionality of ultra-high content analyses of a newly identified population of tumor cells that are hybrids between neoplastic and immune cells in patient matched tumor and peripheral blood specimens. Using oligonucleotide conjugated antibodies (Ab-oligo) permitting cyclic immunofluorescence (cyCIF), we present analyses of phenotypes among tumor and peripheral blood hybrid cells. Interestingly, the majority of circulating hybrid cell (CHC) subpopulations were not identified in tumor-associated hybrids. These results highlight the efficacy of ultra-high content phenotypic analyses using Ab-oligo based cyCIF applied to both tumor and peripheral blood specimens. The combination of a multiplex phenotypic profiling platform that is gentle enough to analyze blood to detect and evaluate disseminated tumor cells represents a novel approach to exploring novel tumor biology and potential utility for developing the population as a blood-based biomarker in cancer.

Detection of neoplastic-immune hybrid cells with metastatic properties in uveal melanoma.

Background: Uveal melanoma is the most common non-cutaneous melanoma and is an intraocular malignancy affecting nearly 7,000 individuals per year worldwide. Of these, approximately 50% will progress to metastatic disease for which there are currently no effective therapies. Despite advances in molecular profiling and metastatic stratification of uveal melanoma tumors, little is known regarding their underlying biology of metastasis. Our group has identified a disseminated neoplastic cell population characterized by co-expression of immune and melanoma proteins, circulating hybrid cells (hybrids), in patients with uveal melanoma. Compared to circulating tumor cells, which lack expression of immune proteins, hybrids are detected at an increased prevalence in peripheral blood and can be used as a non-invasive biomarker to predict metastatic progression. Methods: To ascertain mechanisms underlying enhanced hybrid cell dissemination we identified hybrid cells within primary uveal melanoma tumors using single cell RNA sequencing and evaluated their gene expression and predicted ligand-receptor interactions in relation to other melanoma and immune cells within the primary tumor. We then verified expression of upregulated hybrid pathways within patient-matched tumor and peripheral blood hybrids using cyclic immunofluorescence and quantified their protein expression relative to other non-hybrid tumor and disseminated tumor cells. Results: Among the top upregulated genes and pathways in hybrid cells were those involved in enhanced cell motility and cytoskeletal rearrangement, immune evasion, and altered cellular metabolism. In patient-matched tumor and peripheral blood, we verified gene expression by examining concordant protein expression for each pathway category: TMSB10 (cell motility), CD74 (immune evasion) and GPX1 (metabolism). Both TMSB10 and GPX1 were expressed on significantly higher numbers of disseminated hybrid cells compared to circulating tumor cells, and CD74 and GPX1 were expressed on more disseminated hybrids than tumor-resident hybrids. Lastly, we identified that hybrid cells express ligand-receptor signaling pathways implicated in promoting metastasis including GAS6-AXL, CXCL12-CXCR4, LGALS9-P4HB and IGF1-IGFR1. Conclusion: These findings highlight the importance of TMSB10, GPX1 and CD74 for successful hybrid cell dissemination and survival in circulation. Our results contribute to the understanding of uveal melanoma tumor progression and interactions between tumor cells and immune cells in the tumor microenvironment that may promote metastasis.

Single-cell and spatial analyses reveal a tradeoff between murine mammary proliferation and lineage programs associated with endocrine cues.

Although distinct epithelial cell types have been distinguished in glandular tissues such as the mammary gland, the extent of heterogeneity within each cell type and the degree of endocrine control of this diversity across development are incompletely understood. By combining mass cytometry and cyclic immunofluorescence, we define a rich array of murine mammary epithelial cell subtypes associated with puberty, the estrous cycle, and sex. These subtypes are differentially proliferative and spatially segregate distinctly in adult versus pubescent glands. Further, we identify systematic suppression of lineage programs at the protein and RNA levels as a common feature of mammary epithelial expansion during puberty, the estrous cycle, and gestation and uncover a pervasive enrichment of ribosomal protein genes in luminal cells elicited specifically during progesterone-dominant expansionary periods. Collectively, these data expand our knowledge of murine mammary epithelial heterogeneity and connect endocrine-driven epithelial expansion with lineage suppression.

Mapping human natural killer cell development in pediatric tonsil by imaging mass cytometry and high-resolution microscopy.

Natural killer (NK) cells develop from CD34+ progenitors in a stage-specific manner defined by changes in cell surface receptor expression and function. Secondary lymphoid tissues, including tonsil, are sites of human NK cell development. Here we present new insights into human NK cell development in pediatric tonsil using cyclic immunofluorescence and imaging mass cytometry. We show that NK cell subset localization and interactions are dependent on NK cell developmental stage and tissue residency. NK cell progenitors are found in the interfollicular domain in proximity to cytokine-expressing stromal cells that promote proliferation and maturation. Mature NK cells are primarily found in the T-cell rich parafollicular domain engaging in cell-cell interactions that differ depending on their stage and tissue residency. The presence of local inflammation results in changes in NK cell interactions, abundance, and localization. This study provides the first comprehensive atlas of human NK cell development in secondary lymphoid tissue.

Single-Cell and Spatial Analysis of Emergent Organoid Platforms.

Organoids have emerged as a promising advancement of the two-dimensional (2D) culture systems to improve studies in organogenesis, drug discovery, precision medicine, and regenerative medicine applications. Organoids can self-organize as three-dimensional (3D) tissues derived from stem cells and patient tissues to resemble organs. This chapter presents growth strategies, molecular screening methods, and emerging issues of the organoid platforms. Single-cell and spatial analysis resolve organoid heterogeneity to obtain information about the structural and molecular cellular states. Culture media diversity and varying lab-to-lab practices have resulted in organoid-to-organoid variability in morphology and cell compositions. An essential resource is an organoid atlas that can catalog protocols and standardize data analysis for different organoid types. Molecular profiling of individual cells in organoids and data organization of the organoid landscape will impact biomedical applications from basic science to translational use.

Flexible Cyclic Immunofluorescence (cyCIF) Using Oligonucleotide Barcoded Antibodies.

Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss-particularly in fragile samples-, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images.

An in situ analysis pipeline for initial host-pathogen interactions reveals signatures of human colorectal HIV transmission.

The initial immune response to HIV determines transmission. However, due to technical limitations we still do not have a comparative map of early mucosal transmission events. By combining RNAscope, cyclic immunofluorescence, and image analysis tools, we quantify HIV transmission signatures in intact human colorectal explants within 2 h of topical exposure. We map HIV enrichment to mucosal dendritic cells (DCs) and submucosal macrophages, but not CD4+ T cells, the primary targets of downstream infection. HIV+ DCs accumulate near and within lymphoid aggregates, which act as early sanctuaries of high viral titers while facilitating HIV passage to the submucosa. Finally, HIV entry induces recruitment and clustering of target cells, facilitating DC- and macrophage-mediated HIV transfer and enhanced infection of CD4+ T cells. These data demonstrate a rapid response to HIV structured to maximize the likelihood of mucosal infection and provide a framework for in situ studies of host-pathogen interactions and immune-mediated pathologies.

cyc-DEP: Cyclic immunofluorescence profiling of particles collected using dielectrophoresis.

Cancer is a highly heterogenous disease that requires precise detection tools and active surveillance methods. Liquid biopsy assays provide an agnostic way to follow the complex trajectory of cancer, providing better patient stratification tools for optimized treatment. Here, we present the development of a low-volume liquid biopsy assay called cyc-DEP (cyclic immunofluorescent imaging on dielectrophoretic chip) to profile biomarkers collected on a dielectrophoretic microfluidic chip platform. To enable on-chip cyclic imaging, we optimized a fluorophore quenching method and sequential rounds of on-chip staining with fluorescently conjugated primary antibodies. cyc-DEP allows for the quantification of a multiplex array of proteins using 25 µl of a patient plasma sample. We utilized nanoparticles from a prostate adenocarcinoma (LNCaP) cell line and a panel of six target proteins to develop our proof-of-concept technique. We then used cyc-DEP to quantify blood plasma levels of target proteins from healthy individuals, low-grade and high-grade prostate cancer patients (n = 3 each) in order to demonstrate that our platform is suitable for liquid biopsy analysis in its present form. To ensure accurate quantification of signal intensities and comparisons between different samples, we incorporated a signal intensity normalization method (fluorescent beads) and a custom signal intensity quantification algorithm that account for the distribution of signal across hundreds of collection regions on each chip. Our technique enabled a threefold improvement in multiplicity for detecting proteins associated with fluid samples, opening doors for early detection, and active surveillance through quantification of a multiplex array of biomarkers from low-volume liquid biopsies.

Single-Cell Proteomics Analysis of Recurrent Low-Grade Serous Ovarian Carcinoma and Associated Brain Metastases.

Between 2% and 6% of epithelial ovarian cancer (EOC) patients develop brain metastases (brain mets), which are incurable and invariably result in death. This poor outcome is associated with a lack of established guidelines for the detection and treatment of brain mets in EOC patients. In this study, we characterize an unusual case of low-grade serous ovarian carcinoma (LGSOC) that metastasized to the brain. Using a spatially oriented single-cell proteomics platform, we compared sequential biopsies of a primary tumor with a peritoneal recurrence and brain mets. We identified several targetable oncogenic pathways and immunosuppressive mechanisms that are amplified in the brain mets and could be involved in the progression of LGSOC to the brain. Furthermore, we were able to identify cell populations that are shared between the primary tumor and the brain mets, suggesting that cells that have a propensity for metastasis to the brain could be identified early during the course of disease. Taken together, our findings further a path for personalized therapeutic decisions in LGSOC.

TRIPODD: a Novel Fluorescence Imaging Platform for In Situ Quantification of Drug Distribution and Therapeutic Response.

PURPOSE: Personalized medicine has largely failed to produce curative therapies in advanced cancer patients. Evaluation of in situ drug target availability (DTA) concomitant with local protein expression is critical to an accurate assessment of therapeutic efficacy, but tools capable of both are currently lacking. PROCEDURE: We developed and optimized a fluorescence imaging platform termed TRIPODD (Therapeutic Response Imaging through Proteomic and Optical Drug Distribution), resulting in the only methodology capable of simultaneous quantification of single-cell DTA and protein expression with preserved spatial context within a tumor. Using TRIPODD, we demonstrate the feasibility of combining two complementary fluorescence imaging techniques, intracellular paired agent imaging (iPAI) and cyclic immunofluorescence (cyCIF), conducted with oligonucleotide-conjugated antibodies (Ab-oligos) on tissue samples. RESULTS: We successfully performed sequential imaging on a single tissue section of iPAI to capture single-cell DTA and local protein expression heterogeneity using Ab-oligo cyCIF. Fluorescence imaging data acquisition was followed by spatial registration resulting in high dimensional data correlating DTA to protein expression at the single-cell level where uptake of a targeted probe alone was not well correlated to protein expression. CONCLUSION: Herein, we demonstrated the utility of TRIPODD as a powerful imaging platform capable of interpreting tumor heterogeneity for a mechanistic understanding of therapeutic response and resistance through quantification of drug target availability and proteomic response with preserved spatial context at single-cell resolution.

Cyclic Multiplexed-Immunofluorescence (cmIF), a Highly Multiplexed Method for Single-Cell Analysis.

Immunotherapy harnesses the power of the adaptive immune system and has revolutionized the field of oncotherapy, as novel therapeutic strategies have been introduced into clinical use. The development of immune checkpoint inhibitors has led to durable control of disease in a subset of advanced cancer patients, such as those with melanoma and non-small cell lung cancer. However, predicting patient responses to therapy remains a major challenge, due to the remarkable genomic, epigenetic, and microenvironmental heterogeneity present in each tumor. Breast cancer (BC) is the most common cancer in women, where hormone receptor-positive (HR+; estrogen receptor and/or progesterone receptor) BC comprises the majority (>50%) and has better prognosis, while a minority (<20%) are triple negative BC (TNBC), which has an aggressive phenotype. There is a clinical need to identify predictors of late recurrence in HR+ BC and predictors of immunotherapy outcomes in advanced TNBC. Tumor-infiltrating lymphocytes (TILs) have recently been shown to predict late recurrence in HR+, counter to the findings that TILs confer good prognosis in TNBC and human epidermal growth factor receptor 2 positive (HER2+) subtypes. Furthermore, the spatial arrangement of TILs also appears to have prognostic value, with dense clusters of immune cells predicting poor prognosis in HR+ and good prognosis in TNBC. Whether TIL clusters in different breast cancer subtypes represent the same or different landscapes of TILs is unknown and may have treatment implications for a significant portion of breast cancer patients. Current histopathological staining technology is not sufficient for characterizing the ensembles of TILs and their spatial patterns, in addition to tumor and microenvironmental heterogeneity. However, recent advances in cyclic immunofluorescence enable differentiation of the subsets based on TILs, tumor heterogeneity, and microenvironment composition between good and poor responders. A computational framework for understanding the importance of the spatial relationships between TILs and tumor cells in cancer tissues, which will allow for quantitative interpretation of cyclic immunostaining, is also under development. This chapter will explore the workflow for a newly developed cyclic multiplexed-immunofluorescence (cmIF) assay, which has been optimized for formalin-fixed. paraffin-embedded tissues and developed to process digital images for quantitative single-cell based spatial analysis of tumor heterogeneity and microenvironment, including immune cell composition.

Signal removal methods for highly multiplexed immunofluorescent staining using antibody conjugated oligonucleotides.

Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and have vastly affected how cancer is diagnosed and treated. However, the translation of complex genomic analyses to pathological diagnosis remains challenging using conventional immunofluorescence (IF) staining, which is typically limited to 2-5 antigens. Numerous strategies to increase distinct antigen detection on a single sample have been investigated, but all have deleterious effects on the tissue limiting the maximum number of biomarkers that can be imaged on a single sample and none can be seamlessly integrated into routine clinical workflows. To facilitate ready integration into clinical histopathology, we have developed a novel cyclic IF (cycIF) technology based on antibody conjugated oligonucleotides (Ab-oligos). In situ hybridization of complementary oligonucleotides (oligos) facilitates biomarker labeling for imaging on any conventional fluorescent microscope. We have validated a variety of oligo configurations and their respective signal removal strategies capable of diminishing fluorescent signal to levels of autofluorescence before subsequent staining cycles. Robust signal removal is performed without the employment of harsh conditions or reagents, maintaining tissue integrity and antigenicity for higher dimensionality immunostaining of a single sample. Our platform Ab-oligo cycIF technology uses conventional fluorophores and microscopes, allowing for dissemination to a broad audience and congruent integration into clinical histopathology workflows.

Multiplexed Single-Cell Imaging: Past, Present, and Future.

Jia-Ren Lin from the Laboratory of Systems Pharmacology at Harvard Medical School was awarded best poster at the annual Society of Biomolecular Imaging and Informatics meeting held in Boston, September 2016. His work focuses on single-cell imaging, especially on developing new methods for simultaneously detecting many antigens, named cyclic immunofluorescence (CycIF). This method could be applied in different stages of drug development, from discovery phase, preclinical research to clinical research. The current works and future directions of CycIF method are summarized in the following overview.

Multiplexed imaging of intracellular protein networks.

Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. © 2016 International Society for Advancement of Cytometry.