Neutrophils are first-responders to sites of infection and tissue damage including the inflamed t... more Neutrophils are first-responders to sites of infection and tissue damage including the inflamed tumor microenvironment. Increasing evidence suggests that crosstalk between tumors and neutrophils can affect the progression of established tumors. However, there is a gap in our understanding of the early events that lead to neutrophil recruitment to oncogene-transformed cells and how these pathways alter tumor progression. Here, we use optically transparent zebrafish larvae to probe the early signals that mediate neutrophil recruitment to Kras-transformed astrocytes. We show that zebrafish larvae with impaired neutrophil function exhibit reduced proliferation of transformed astrocytes supporting a critical role for tumor-associated neutrophils in the early progression of tumorigenesis. Moreover, using mutants and pharmacological inhibition, we show that the chemokine receptor Cxcr1 promotes neutrophil recruitment, proliferation of tumor-initiating cells, and neoplastic mass formation. These findings highlight the power of the larval zebrafish system to image and probe early events in the tumor-initiating microenvironment and demonstrate the potential for neutrophil recruitment signaling pathways such as Cxcl8-Cxcr1 as targets for anti-cancer therapies. Neutrophils are critical first-responders to sites of infection and tissue damage and play an important role in host defense. Neutrophils also localize to the tumor microenvironment (TME) in a variety of human cancers and increased neutrophil infiltration and neutrophil-to-lymphocyte ratios in tumors often correlate with poor patient prognosis 1. Therefore, there is increasing interest in understanding the role of neutrophils in the developing tumor microenvironment and how their presence alters adaptive immune responses. With recent advances in T-cell mediated immunotherapies, there is a need to better understand the role of innate leukocytes within the tumor and tumor-initiating niche, as well as the signals that mediate their recruitment to improve the efficacy of immunotherapy and patient outcomes. Several recent studies have investigated the role of tumor-associated neutrophils (TANs) and how they influence the TME and cancer development (reviewed in 2-5). For instance, there is evidence that neutrophils can modulate T cell tumoricidal activity by enhancing regulatory T cell recruitment and activity at the TME 6,7. Neutrophils also secrete several factors including neutrophil elastase (NE), matrix metalloproteinases (MMPs), reactive oxygen species (ROS), vascular endothelial growth factor (VEGF), and others, which can modify the TME in favor of cancer cell growth and spread. On the other hand, an anti-tumor role for TANs has been reported in some models, and it is important to note that neutrophils exhibit phenotypic plasticity and heterogeneity which may contribute to a variety of effects in the TME 8-10. Importantly, due to the constraints of available animal and cell culture models, few studies have investigated the role of neutrophils in early tumorigenesis or have identified signals that contribute to recruitment of neutrophils to tumor-initiating cells. Neutrophils are highly migratory cells with the capacity to move within the blood stream and migrate interstitially to rapidly localize to sites of infection or injury. Several signaling pathways are involved in neutrophil chemotaxis and recruitment, many of which are also upregulated in the TME. One of the primary chemotactic pathways that regulates neutrophil migration is the CXCL8 (IL8)-CXCR1/2 pathway. In humans, CXCL8 is a chemokine which can be produced by many different cell types in response to various cytokines, ROS, pathogens,
Abstract A117: CXCR1 is required for neutrophil recruitment to wounds and Kras-transformed cells in zebrafish
Neutrophils are the first responder cells to sites of infection and tissue damage. Evidence from ... more Neutrophils are the first responder cells to sites of infection and tissue damage. Evidence from patient tumors and animal models of cancer also suggest that they are highly recruited to the tumor microenvironment, though their role there is still poorly understood. The larval zebrafish is an excellent model to study neutrophil migration and chemotaxis in vivo and is an emerging model for cancer development and progression. Here, we use the zebrafish to model Kras-driven oncogenesis in keratinocytes and glial cells. Beginning at 3 days post fertilization (dpf), we observe invasive EMT-like cell shape changes in oncogenic mutant Kras (KrasG12V) expressing cells as well as enhanced cell proliferation as marked by phospho-histone H3. Using fluorescently labeled transgenic lines, we demonstrate that neutrophils are recruited to transformed cells and that blocking this recruitment results in decreased proliferation of transformed cells, suggesting that neutrophils may play a tumor-promotional role in our model. To determine the method of neutrophil chemotaxis to transformed cells, we used Transcription Activator-like Effector Nuclease (TALEN) mutagenesis to generate zebrafish mutants for the chemokine receptor CXCR1, which is known regulate neutrophil chemotaxis. We demonstrate that CXCR1 is required for neutrophil chemotaxis to a tail-transection wound and to Kras-transformed cells in zebrafish. Together these data suggest that blocking CXCR1 activity may reduce neutrophilic inflammation in the tumor microenvironment which may in turn reduce tumor cell proliferation and slow tumor progression. Citation Format: Davalyn Powell, Qing Deng, Anna Huttenlocher. CXCR1 is required for neutrophil recruitment to wounds and Kras-transformed cells in zebrafish [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A117.
Neural crest cells (NCCs) are essential embryonic progenitor cells that are unique to vertebrates... more Neural crest cells (NCCs) are essential embryonic progenitor cells that are unique to vertebrates and form a remarkably complex and coordinated system of highly motile cells. Migration of NCCs occurs along specific pathways within the embryo in response to both environmental cues and cell-cell interactions within the neural crest population. Here, we demonstrate a novel role for the putative Sonic hedgehog (Shh) receptor and cell adhesion regulator, cdon, in zebrafish neural crest migration. cdon is expressed in developing premigratory NCCs but is downregulated once the cells become migratory. Knockdown of cdon results in aberrant migration of trunk NCCs: crestin positive cells can emigrate out of the neural tube but stall shortly after the initiation of migration. Live cell imaging analysis demonstrates reduced directedness of migration, increased velocity and mispositioned cell protrusions. In addition, transplantation analysis suggests that cdon is required cell-autonomously for directed NCC migration in the trunk. Interestingly, N-cadherin is mislocalized following cdon knockdown suggesting that the role of cdon in NCCs is to regulate N-cadherin localization. Our results reveal a novel role for cdon in zebrafish neural crest migration, and suggest a mechanism by which Cdon is required to localize N-cadherin to the cell membrane in migratory NCCs for directed migration.
Wiley Interdisciplinary Reviews: Systems Biology and Medicine, Apr 10, 2013
The neural crest (NC) is first induced as an epithelial population of cells at the neural plate b... more The neural crest (NC) is first induced as an epithelial population of cells at the neural plate border requiring complex signaling between bone morphogenetic protein, Wnt, and fibroblast growth factors to differentiate the neural and NC fate from the epidermis. Remarkably, following induction, these cells undergo an epithelial-to-mesenchymal transition (EMT), delaminate from the neural tube, and migrate through various tissue types and microenvironments before reaching their final destination where they undergo terminal differentiation. This process is mirrored in cancer metastasis, where a primary tumor will undergo an EMT before migrating and invading other cell populations to create a secondary tumor site. In recent years, as our understanding of NC EMT and migration has deepened, important new insights into tumorigenesis and metastasis have also been achieved. These discoveries have been driven by the observation that many cancers misregulate developmental genes to reacquire proliferative and migratory states. In this review, we examine how the NC provides an excellent model for studying EMT and migration. These data are discussed from the perspective of the gene regulatory networks that control both NC and cancer cell EMT and migration. Deciphering these processes in a comparative manner will expand our knowledge of the underlying etiology and pathogenesis of cancer and promote the development of novel targeted therapeutic strategies for cancer patients.
Understanding how cell fate decisions are regulated by signaling pathways and transcription facto... more Understanding how cell fate decisions are regulated by signaling pathways and transcription factors is key to understanding how embryonic development takes place. Neural crest cells are an embryonic cell type that must undergo several cell fate choices and changes in gene expression before they are able to finally differentiate and contribute to adult tissues. Neural crest cells are formed at the border between developing neural and non-neural ectoderm in a region called the neural plate border. They are induced by several signaling factors and specified by the expression of transcription factors which will form a gene regulatory network for their development and control subsequent programs within the neural crest cells such as epithelial-to-mesenchymal transition, migration, and differentiation, which are required for proper development of the embryo. One transcription factor that is required for neural crest specification is the PR/SET domain containing transcription factor Prdm1a. The goal of this thesis is to explore the mechanisms by which Prdm1a regulates genes required for neural crest specification and migration. Prdm1a is expressed in the early neural plate border, and when its expression is abrogated, neural crest cells are significantly reduced. Here, I have demonstrated that Prdm1a is downstream of known signaling pathways that induce neural crest cells, specifically Wnt and Notch signaling. Prdm1a directly activates the expression of the neural crest specification genes foxd3 and tfap2a, which are also required for neural crest formation. In addition to its role as a transcriptional activator, Prdm1a is also required as iv a transcriptional repressor of yet unknown targets and this role is required for specified neural crest cells to continue development to migratory stages. Using whole-genome and transcriptome approaches, I was able to identify several novel targets of Prdm1a regulation, demonstrating its role as a master regulator of several genetic programs required for the formation of the neural crest and possibly other tissues as well including the neural plate and sensory placodes. Interestingly, one of the downstream targets of the Prdm1a regulatory network is a cell adhesion gene, cdon. I have demonstrated a novel role for cdon as a cell-autonomous regulator of neural crest motility and migration. Altogether, this work demonstrates the importance of the Prdm1a transcription factor and how Prdm1a and its downstream gene regulatory network influences and controls neural crest cell fate. The format and content of this abstract are approved. I recommend its publication.
Riding the crest of the wave: parallels between the neural crest and cancer in epithelial-to-mese... more Riding the crest of the wave: parallels between the neural crest and cancer in epithelial-to-mesenchymal transition and migration
Understanding how cell fate decisions are regulated by signaling pathways and transcription facto... more Understanding how cell fate decisions are regulated by signaling pathways and transcription factors is key to understanding how embryonic development takes place. Neural crest cells are an embryonic cell type that must undergo several cell fate choices and changes in gene expression before they are able to finally differentiate and contribute to adult tissues. Neural crest cells are formed at the border between developing neural and non-neural ectoderm in a region called the neural plate border. They are induced by several signaling factors and specified by the expression of transcription factors which will form a gene regulatory network for their development and control subsequent programs within the neural crest cells such as epithelial-to-mesenchymal transition, migration, and differentiation, which are required for proper development of the embryo. One transcription factor that is required for neural crest specification is the PR/SET domain containing transcription factor Prdm1a. The goal of this thesis is to explore the mechanisms by which Prdm1a regulates genes required for neural crest specification and migration. Prdm1a is expressed in the early neural plate border, and when its expression is abrogated, neural crest cells are significantly reduced. Here, I have demonstrated that Prdm1a is downstream of known signaling pathways that induce neural crest cells, specifically Wnt and Notch signaling. Prdm1a directly activates the expression of the neural crest specification genes foxd3 and tfap2a, which are also required for neural crest formation. In addition to its role as a transcriptional activator, Prdm1a is also required as iv a transcriptional repressor of yet unknown targets and this role is required for specified neural crest cells to continue development to migratory stages. Using whole-genome and transcriptome approaches, I was able to identify several novel targets of Prdm1a regulation, demonstrating its role as a master regulator of several genetic programs required for the formation of the neural crest and possibly other tissues as well including the neural plate and sensory placodes. Interestingly, one of the downstream targets of the Prdm1a regulatory network is a cell adhesion gene, cdon. I have demonstrated a novel role for cdon as a cell-autonomous regulator of neural crest motility and migration. Altogether, this work demonstrates the importance of the Prdm1a transcription factor and how Prdm1a and its downstream gene regulatory network influences and controls neural crest cell fate. The format and content of this abstract are approved. I recommend its publication.
Neutrophils are first-responders to sites of infection and tissue damage including the inflamed t... more Neutrophils are first-responders to sites of infection and tissue damage including the inflamed tumor microenvironment. Increasing evidence suggests that crosstalk between tumors and neutrophils can affect the progression of established tumors. However, there is a gap in our understanding of the early events that lead to neutrophil recruitment to oncogene-transformed cells and how these pathways alter tumor progression. Here, we use optically transparent zebrafish larvae to probe the early signals that mediate neutrophil recruitment to Kras-transformed astrocytes. We show that zebrafish larvae with impaired neutrophil function exhibit reduced proliferation of transformed astrocytes supporting a critical role for tumor-associated neutrophils in the early progression of tumorigenesis. Moreover, using mutants and pharmacological inhibition, we show that the chemokine receptor Cxcr1 promotes neutrophil recruitment, proliferation of tumor-initiating cells, and neoplastic mass formation. ...
Motile cells navigate through complex tissue environments that include both attractive and repuls... more Motile cells navigate through complex tissue environments that include both attractive and repulsive cues. In response to tissue wounding, neutrophils, primary cells of the innate immune response, exhibit bidirectional migration that is orchestrated by chemokines and their receptors. Although progress has been made in identifying signals that mediate the recruitment phase, the mechanisms that regulate neutrophil reverse migration remain largely unknown. Here, we visualize bidirectional neutrophil migration to sterile wounds in zebrafish larvae and identify specific roles for the chemokine receptors Cxcr1 and Cxcr2 in neutrophil recruitment to sterile injury and infection. Notably, we also identify Cxcl8a/Cxcr2 as a specific ligand-receptor pair that orchestrates neutrophil chemokinesis in interstitial tissues during neutrophil reverse migration and resolution of inflammation. Taken together, our findings identify distinct receptors that mediate bidirectional leukocyte motility during interstitial migration depending on the context and type of tissue damage in vivo.
Neutrophils are the first responders to sites of acute tissue damage and infection. Recent studie... more Neutrophils are the first responders to sites of acute tissue damage and infection. Recent studies suggest that in addition to neutrophil apoptosis, resolution of neutrophil inflammation at wounds can be mediated by reverse migration from tissues and transmigration back into the vasculature. In settings of chronic inflammation, neutrophils persist in tissues, and this persistence has been associated with cancer progression. However, the role of neutrophils in the tumor microenvironment remains controversial, with evidence for both pro-and anti-tumor roles. Here we review the mechanisms that regulate neutrophil recruitment and resolution at sites of tissue damage, with a specific focus on the tumor microenvironment. We discuss the current understanding as to how neutrophils alter the tumor microenvironment to support or hinder cancer progression, and in this context outline gaps in understanding and important areas of inquiry.
Neural crest cells (NCCs) are essential embryonic progenitor cells that are unique to vertebrates... more Neural crest cells (NCCs) are essential embryonic progenitor cells that are unique to vertebrates and form a remarkably complex and coordinated system of highly motile cells. Migration of NCCs occurs along specific pathways within the embryo in response to both environmental cues and cell-cell interactions within the neural crest population. Here, we demonstrate a novel role for the putative Sonic hedgehog (Shh) receptor and cell adhesion regulator, cdon, in zebrafish neural crest migration. cdon is expressed in developing premigratory NCCs but is downregulated once the cells become migratory. Knockdown of cdon results in aberrant migration of trunk NCCs: crestin positive cells can emigrate out of the neural tube but stall shortly after the initiation of migration. Live cell imaging analysis demonstrates reduced directedness of migration, increased velocity and mispositioned cell protrusions. In addition, transplantation analysis suggests that cdon is required cell-autonomously for directed NCC migration in the trunk. Interestingly, N-cadherin is mislocalized following cdon knockdown suggesting that the role of cdon in NCCs is to regulate N-cadherin localization. Our results reveal a novel role for cdon in zebrafish neural crest migration, and suggest a mechanism by which Cdon is required to localize N-cadherin to the cell membrane in migratory NCCs for directed migration.
Neural crest cells (NCC) and tumor cells undergo remarkably similar cellular and molecular change... more Neural crest cells (NCC) and tumor cells undergo remarkably similar cellular and molecular changes during the progression from an epithelial cell to a migratory mesenchymal-like cell in the course of embryonic development and metastatic disease, respectively. Many of the same genes and genetic pathways are expressed and required for delamination, migration, survival, and proliferation to occur in both neural crest and cancer. Additionally, tumors that originate from NCC and their derivatives exhibit re-activation and/or misregulation of genes that were expressed during normal development and differentiation. These parallels make neural crest development an excellent model for understanding the pathways tumor cells may employ during cancer progression and may provide clues to improve cancer diagnosis, treatment, and therapies.
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Papers by Davalyn Powell