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www.nature.com/npjbcancer All rights reserved 2374-4677/15 MEETING REPORT OPEN 1 2 3 4 5 4 6 5 Maja H Oktay , Yi-Fen Lee , Allison Harney , Dorothy Farrell , Nastaran Z Kuhn , Stephanie A Morris , Emily Greenspan , Suresh Mohla , 4 7,8 Piotr Grodzinski and Larry Norton Recent advances in cancer biology and the development of new research tools have enabled interrogations of single cells and cell–cell interactions. Emerging technologies are capable of revealing data on the physical characteristics of cells, differences in the genome and proteome between cancerous and healthy cells, and variations in distinct cell subpopulations. Dynamic measurements enable studies that can reveal the evolution of cell characteristics. Cells can also be assembled in vitro or ex vivo into two- and three- dimensional cell environments, allowing for studies of cell–cell interactions and cell signaling. The Memorial Sloan Kettering Cancer Center, in collaboration with the Breast Cancer Research Foundation and the National Cancer Institute, co-organized a workshop as an opportunity for leading researchers in their respective ﬁelds to present and discuss scientiﬁc research highlights relevant to the utilization of techniques and technologies for studying cell-to-cell communications in cancer. Avenues of future development and the potential for clinical utility were primary features of these discussions. The scientiﬁc presentations and extensive ensuing discussions resulted in the identiﬁcation of a number of research opportunities, which are summarized in this report. npj Breast Cancer (2015) 1, 15022; doi:10.1038/npjbcancer.2015.22; published online 25 November 2015 INTRODUCTION presentations and ﬁndings made by participants in three key areas—improving cancer diagnosis and therapy using single-cell Advanced technologies such as nanotechnologies and micro- analyses; the effects of cell-to-cell communication within the ﬂuidics platforms as well as conventional three-dimensional (3D) tumor microenvironment on cancer progression and meta- organotypic cultures have contributed to an increased under- stasis; and methods and models for recapitulating the tumor standing of cancer biology at the single-cell level and at the level microenvironment. of communication between cells. This in turn is leading to a greater appreciation of the implications of intratumoral hetero- geneity and the role of tumor microenvironment and vasculature SINGLE-CELL ANALYSES in cancer progression and metastasis. Both, the Breast Cancer Recent technological advances have enabled the detailed genetic Research Foundation (BCRF) and the National Cancer Institute and proteomic analysis of single tumor cells, as well as the (NCI) support research in these areas. In collaboration with the isolation and analysis of either intact circulating tumor cells (CTCs) Memorial Sloan Kettering Cancer Center (MSKCC), the BCRF and or circulating DNA from the blood of cancer patients. These NCI convened a workshop titled “Cell-to-Cell Communication in techniques have already demonstrated clinical utility, such as use Cancer” that was hosted by MSKCC on 14–15 July 2014. The 2 of CTCs as prognostic markers for advanced prostate cancer. workshop consisted of plenary presentations from researchers Workshop presentations focused on the ways in which these working at the interface of biology and engineering interspersed technologies have also enabled fundamental studies of cancer cell with breakout sessions to discuss aspects of cancer biology for 3 4,5 signaling pathways, genomic heterogeneity and evolution, and 6,7 which cellular analyses can yield clinically relevant insights into physical characteristics. Both presentations and discussions tumor behavior. Breakout session discussions focused on intra- highlighted the potential for insights from these studies to spark tumoral heterogeneity, single-cell measurements, and modeling discovery and development of new therapeutic strategies and complex tumor microenvironment interactions. Discussions were associated diagnostics. aimed at identifying ways in which the characterization of single Howard Scher discussed the use of CTC analysis to provide non- cells and cell-to-cell interactions could improve the understanding invasive patient monitoring, including the possibility of capturing and the treatment of cancer. the transformation into therapy resistant disease in prostate A number of themes emerged from the presentations and cancer by tracking androgen receptor status of CTCs. Scher views discussions, including the diversity of factors that contribute to CTC analysis as more effective at capturing the heterogeneity of malignancy, heterogeneity, and the evolution of progression- cancer cells than traditional bulk analysis of the tumor, and linked mutations; the importance of intercellular signaling in therefore potentially more likely to discover drug resistance as it tumor progression and invasion; and the need to develop in vitro emerges. The evolution of disease and emergence of therapy- and ex vivo models that capture these factors and can predict resistant phenotypes has important implications for cancer tumor response to intervention. This report highlights the treatment, and Nicholas Navin presented his work revealing that, 1 2 3 Department of Pathology, Monteﬁore Medical Center, New York, NY, USA; Department of Pathology, University of Rochester Medical Center, Rochester, MN, USA; Department of Anatomy & Structural Biology, Albert Einstein College of Medicine, New York, NY, USA; Ofﬁce of Cancer Nanotechnology Research, National Cancer Institute, Bethesda, MD, 5 6 USA; Division of Cancer Biology, National Cancer Institute, Rockville, MD, USA; Center for Strategic Scientiﬁc Initiatives, National Cancer Institute, Bethesda, MD, USA; 7 8 Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA and The Breast Cancer Research Foundation, New York, NY, USA. Correspondence: L Norton (firstname.lastname@example.org) Received 21 October 2015; accepted 21 October 2015 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Cell-to-cell communication in cancer MH Oktay et al in at least some cancers, evolution occurs not gradually but rather discovery that oncogenes induce the malignant phenotype only in by stepwise clonal expansions, with different mutational types the proper context of the microenvironment began a 30-year (e.g., copy number rearrangements versus point mutations) program on the study of tissue architecture and its effect on having quite different natural histories. Wei Wei focused his talk malignancy. Bissell’s work shows that the profound effect on analyzing intracellular signaling pathways in single tumor cells microenvironment can have on cell phenotype, even dominating and how integrated genomics and functional proteomics studies over the cellular genotype, and the importance of 3D models that on single tumor cells reveal connections between genomic recapitulate the tumor microenvironment and cellular interactions information and biological function. By analyzing protein to properly understand the sequence of progression. In 3D gel networks associated with acquired drug resistance in glioblastoma culture systems, phenotypic characteristics of malignant transfor- multiforme, his team is developing strategies to predict and mation that are often considered to be a consequence of pre-empt resistance through the use of targeted combination alterations in the genome have the ability to activate oncogenic therapies. Scott Manalis demonstrated the use of his suspended signaling pathways and contribute to malignant transformation. microchannel resonator device to measure the mass of single cells For example, increased glycolysis in a 3D model can become an in real time and showed that growth measurements on genetically oncogenic event, and dormant cells residing in metastatic characterized single cells can reveal the correlation between microvascular niches could be reactivated upon loss of treatment efﬁcacy and genomic features in a way that cannot be endothelial-derived thrombospondin-1 that coincide with the replicated in bulk tumor studies. onset of endothelial sprouting. Other ﬁndings with particularly These presentations provided rich data and context for strong clinical relevance are that cell attachment to basement discussions centered on exploiting single-cell analyses to improve membrane and formation of polarized tridimensional structures cancer diagnosis and therapy. Participants discussed immediate protect from chemotherapy-induced apoptosis, and manipulation opportunities to use single-cell studies, particularly involving CTCs, of integrin signaling in 3D matrices can increase cancer-cell to monitor drug response and to identify and target the most sensitivity to ionizing radiation. aggressive phenotypes within a tumor. At the same time, Echoing Bissell’s ﬁndings on the importance of paracrine participants recognized that certain important aspects of cell signaling in malignancy, Clifford Hudis spoke on the relationship or tumor behavior cannot be deduced at single-cell level between obesity, inﬂammation, and cancer. He presented a series measurements, and for many single-cell assays there is a need of analyses on adipose tissue from different malignancies and to establish the number of measurements allowing for reliable found “crown-like structures” present in the white adipose tissue, predictions. In addition, there is currently little consensus on which correlate positively with excess weight, serum levels of what would be the most useful functional assays (e.g., viability, inﬂammatory cytokines, aromatase, and worse outcomes in some immune/cytokine assays) to characterize tumor cells or CTCs, cancers. Paul Newton and Johanna Joyce both discussed the role especially from solid tumors. of cellular communication in breast cancer metastasis. Newton Discussions led to the identiﬁcation of additional questions that employs evolutionary game theory models of single-cell and cell must be addressed before the results of these analyses could be population trafﬁcking to study cancer progression from one site to 14,15 translated into clinical strategies. For example, what is the role of another. Using a breast cancer patient data set, including intratumoral heterogeneity in treatment response and what is the outcomes and ER/Her2 status, possible anatomic pathways and clinical utility of measuring this diversity at the single cell level? progression timescales of metastases were compared and These questions immediately raise the issue of resistance-linked probability distributions of cell migration patterns were established. mutations, and whether they are pre-existing, or a result of Joyce’s work focuses on understanding the mechanisms of tumor therapy(ies) that may induce or select for speciﬁc mutations, or metastasis by investigating how the microenvironment responds both. Greater understanding of how surviving cancer cells adapt to metastatic tumor cells and how heterotypic interactions to therapy and the post-therapy environment is necessary to between tumor and stromal cells differ between secondary organ enable targeting of these cells without promoting more resistant metastasis sites. By comparing gene expression array proﬁles or aggressive disease. All of these considerations indicate that the from tumor and stroma cells in micro- and macromestases in the disease state must be analyzed over time, potentially using brain, lung, and bone, they identiﬁed a protease, cathepsin S, with mutations as markers of lineage evolution. Practical limitations on high speciﬁcity for brain metastases. These studies indicate that biopsy collection for many cancer types suggest CTCs are one of cellular signaling and trafﬁcking patterns can provide both the only avenues through which these longitudinal data can be diagnostic signatures and therapeutic targets for metastasis. collected. Each of these presentations highlighted scenarios in which Several participants emphasized that the focus on genomic and studies of cancer cells in context are necessary to predict or functional analyses should not lead to neglect of the physical understand how the phenotype, or even genotype, of those cells parameters and phenotypes of cells, which may not only indicate will progress. Workshop participants recognized numerous the level of heterogeneity and malignancy but may also inﬂuence implications of these ﬁndings, which point to the continued them. For example, it is not known how physical constraints such relevance of cancer-cell phenotype even in the era of genomic as high pressure within tumors, and the eventual release from medicine. Participants emphasized that for clinicians, the ques- these constraints through extravasation or migration, affect the tions of which phenotypes to target and how to identify them will invasiveness or metastatic potential of cancer cells. Another be crucial to determining treatment strategies. Clinicians also important open question in cellular characterization is how to need to be able to identify and target dormant cells and distinguish dormant cancer cells from normal cells, and then understand catastrophic events that lead to pan resistance in differentially target the dormant cells. This targeting will require metastatic cells and rapid patient death. determining the relative importance of cell-intrinsic and cell- Better understanding of the activation of dormant cells and extrinsic inﬂuences in dormancy. what triggers the metastatic pathway in these cells is necessary to identify targets to short circuit this pathway and improve outcomes in cancer care. The role of inﬂammation in activating TUMOR MICROENVIRONMENT dormant cells, along with the evidence of collective behavior The importance of the tumor microenvironment was understood underlying progression and metastasis, point to the need to study from the outset of the workshop, which began with a plenary cell-to-cell communication within the tumor microenvironment. address by Mina Bissell on the effect of tissue architecture on Participants were excited at the prospect of looking to the tumor gene expression and subsequently cell behavior. Bissell’s group stroma to provide clues to which lesions are likely to progress over npj Breast Cancer (2015) 15022 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Cell-to-cell communication in cancer MH Oktay et al time, potentially providing a route to limit overdiagnosis and or tissue fragments, rather than dissociated cells, and multiple overtreatment of indolent tumors, which result in few saved lives models may be necessary to study different aspects of tumor but signiﬁcant health care and quality of life costs. Participants microenvironment interactions. The important factor will be that a also raised the prospect of identifying another phenotypic model captures the relevant cell types, tissue structure, and cancer target for intervention besides mitosis, perhaps a migratory, molecular subtypes being probed in a given investigation. dissemination-linked, or inﬂammatory signature. Participants did recognize that coupling technologies to create In thinking about CTCs in the context of the tumor a master model in which multiple aspects of a tumor (physical, microenvironment, participants raised the idea of CTC “memory”, chemical, and biological) are simultaneously interrogated and or the ability of a CTC to survive outside the context of the primary multiple discrete steps in tumor progression are recapitulated tumor and its microenvironment. It was speculated that perhaps would enable major strides in understanding cancer-cell behavior. those CTC subtypes that are associated with metastasis are those There was consensus on the importance of using “humanized” that retain some “memory” of the primary tumor’s microenvironment in vitro/ex vivo models, and that these models should incorporate across an as yet to be determined timescale. Answering this information derived from other model systems to improve question depends on understanding several other not performance. Validation in human samples to ensure relevance well-deﬁned processes, such as identifying the initiating event in to human disease was also called for during the discussions. In the a cell becoming a CTC and determining what drives molecular development of models, in particular, the group called for team alterations, such as gene mutations, during circulation. In any case, science approaches, with strong engagement of clinicians, clinical it was deemed important to deﬁne the differences between researchers, basic cancer biologists, and engineers. single-cell assays in the context of the primary tumor versus CTC assays where tumor cells survive without the tumor OPPORTUNITIES, BARRIERS, AND SOLUTIONS microenvironment. Workshop participants identiﬁed numerous opportunities for both discovery oriented research and clinical applications based on 3D TUMOR MODELS technologies that characterize single cells or enable study of The ﬁnal session of the workshop focused on the development of cell-to-cell communications. New technologies are being used to accurate 3D in vitro or ex vivo models of tissues that mimic the capture and analyze tumor heterogeneity, and the associated physiological conditions in the tumor microenvironment and implications for patient outcomes. Further studies in this area are recapitulate individual aspects in the metastatic cascade. David expected to have major impact on both cancer biology and Beebe is developing microﬂuidic tools that balance the ability to patient care through improved choice of therapeutic course, isolate speciﬁc interactions between cells in vitro and the more including more informed tumor surveillance. The identiﬁcation of realistic complexity but greater cost of in vivo models. His group signatures of dormancy and reactivation in cancer cells is also has used a microscale cell-culture platform capable of compart- expected to be clinically actionable. Integrated models of tumors mentalizing adherent and non-adherent cells in controlled that enable studies of cellular behavior at multiple scales, and that environments to understand and predict patient response to the can include or isolate environmental factors, will provide insight proteasome inhibitor bortezomib. Only in cultures with both into cancer progression and disease markers that are badly multiple myeloma and stromal cells were tumor cell viabilities needed. similar to patient response to bortezomib, indicating that this Among the most clinically relevant technologies are those that platform can be used to recapitulate clinical response in vitro. can be used for in depth, longitudinal proﬁling of therapeutic Christopher Chen described the use of 3D printed networks to response, such as CTC capture and analysis devices, and recreate vascular networks for the study of angiogenesis. integrated approaches to diagnostics and therapy. Effective use Endothelial cells are seeded inside a network that mimics in vivo of these techniques to characterize tumors and guide clinical blood vessels, and via a parallel channel, angiogenic factors can be decision making will require extensive studies to determine the correspondence between single-cell measurement results and ﬂowed through to identify combinations that produce different types of endothelial cell invasion. The system allows for the bulk tumor characteristics. Patient-speciﬁc models to predict visualization of sprouting angiogenesis over time, providing therapeutic response and rationalize treatment choices also have mechanistic information on the function of different growth the potentially-to-radically alter treatment outcomes. Deployment factors and the mechanism underlying treatment with of such models depends on further development of fully anti-angiogenic drugs. Roger Kamm described his efforts to humanized in vitro and ex vivo models, which workshop understand tumor-cell migration through reconstruction of participants saw as a pressing but not fully recognized need. blood ﬂow in a microﬂuidic device to study intravasation and A central point of discussion among workshop participants was extravasation. Intravasation increased in the presence of development of effective collaborations to capitalize on the macrophages, and macrophage-induced intravasation is strengths of researchers in each ﬁeld. One item of concern is that associated with increased endothelial permeability, which can many investigators are not aware of valuable experimental models be stimulated by the addition of tumor necrosis factor-α. These and tools that are available and currently in use by other studies indicate the role of non-neoplastic cells and signaling in researchers. Another concern is the difﬁculty of bringing together migration and metastasis and demonstrate that 3D models can researchers from different disciplines and disease focus areas. illuminate the processes underlying these events, as well as Joint workshops, special sessions at larger meetings and smaller malignant transformation and progression. dedicated, Gordon Research Conference-type meetings were all Workshop participants discussed the minimum requirements of suggested to bridge these gaps. However, participants noted the multiple available in vitro or ex vivo models to be friction at the social interface between disciplines at both physiologically relevant to the in vivo environment. Although institutions and conferences, which can lead to ineffective and there was consensus that the complexity of the model depends inefﬁcient technology development. For example, clinicians are on the experimental question(s), it was agreed that issues of frequently disillusioned by seeing engineers present technologies, vascularization and the viability of endothelial cells in mixed devices, and strategies that are not clinically relevant, leading to cultures and organoids, as well as how to retain viability of stromal decreased engagement. cells, are inescapable. Capturing the clinically relevant characteristics There was some concern that the NCI’s emphasis on the R01 of the tumor microenvironment, such as oxygen levels, hypoxia, single project mechanism may make it difﬁcult to pursue the and necrosis, will probably require use of patient-derived models sort of multidisciplinary, integrated research presented at the © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15022 Cell-to-cell communication in cancer MH Oktay et al workshop. Participants saw a role for the NCI in bringing different 8. 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Published: Nov 25, 2015
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