Cell shape and motility are primarily controlled by cellular mechanics. The attachment of the plasma membrane to the underlying actomyosin cortex has been proposed to be important for cellular ...processes involving membrane deformation. However, little is known about the actual function of membrane-to-cortex attachment (MCA) in cell protrusion formation and migration, in particular in the context of the developing embryo. Here, we use a multidisciplinary approach to study MCA in zebrafish mesoderm and endoderm (mesendoderm) germ layer progenitor cells, which migrate using a combination of different protrusion types, namely, lamellipodia, filopodia, and blebs, during zebrafish gastrulation. By interfering with the activity of molecules linking the cortex to the membrane and measuring resulting changes in MCA by atomic force microscopy, we show that reducing MCA in mesendoderm progenitors increases the proportion of cellular blebs and reduces the directionality of cell migration. We propose that MCA is a key parameter controlling the relative proportions of different cell protrusion types in mesendoderm progenitors, and thus is key in controlling directed migration during gastrulation.
Mitotic spindle disassembly after chromosome separation is as important as spindle assembly, yet the molecular mechanisms for spindle disassembly are unclear. In this study, we investigated how the ...chromosomal passenger complex (CPC), which contains the Aurora B kinase Ipl1, swiftly concentrates at the spindle midzone in late anaphase, and we researched the role of this dramatic relocalization during spindle disassembly. We showed that the kinesins Kip1 and Kip3 are essential for CPC relocalization. In cells lacking Kip1 and Kip3, spindle disassembly is severely delayed until after contraction of the cytokinetic ring. Purified Kip1 and Kip3 interact directly with the CPC and recruit it to microtubules in vitro, and single-molecule experiments showed that the CPC diffuses dynamically on microtubules but that diffusion stops when the CPC encounters a Kip1 molecule. We propose that Kip1 and Kip3 trap the CPC at the spindle midzone in late anaphase to ensure timely spindle disassembly.
Given the role of myeloid cells in T cell activation and in the antitumor response, targeting checkpoint molecules expressed on this population represents a promising strategy to augment antitumor ...immunity. However, myeloid checkpoints that can be effectively used as immunotherapy targets are still lacking. Here, we demonstrate the therapeutic potential of targeting the myeloid receptors Siglec-7 and Siglec-9 in vivo. By using a humanized immunocompetent murine model, we demonstrate that human Siglec-7 and Siglec-9, in addition to the murine homolog Siglec-E, inhibit the endogenous antitumor immune response, as well as the response to tumor-targeting and immune checkpoint inhibiting antibodies in vivo. The impact of these Siglecs on tumor progression is highly dependent on the anatomical distribution of the tumor and, as a consequence, the local tumor microenvironment, as tumors with a more immune-suppressive tumor microenvironment are less sensitive to Siglec perturbation. Finally, to assess the potential of these two receptors as targets for immunotherapy, we developed Fc engineered blocking antibodies to Siglec-7 and Siglec-9 and demonstrate that Siglec-7 and Siglec-9 blockade can significantly reduce tumor burden in vivo, demonstrating the therapeutic potential of targeting these two receptors.
BackgroundDespite the curative potential of checkpoint blockade immunotherapy, a majority of patients remain unresponsive to existing treatments. Glyco-immune checkpoints – interactions of ...cell-surface glycans with lectin, or glycan binding, immunoreceptors – have emerged as prominent mechanisms of immune evasion and therapeutic resistance in cancer.MethodsHere, we describe antibody-lectin chimeras (AbLecs), a modular platform for glyco-immune checkpoint blockade. AbLecs are bispecific antibody-like molecules comprising a tumor-targeting arm as well as a lectin ‘decoy receptor’ domain that directly binds tumor glycans and blocks their ability to engage lectin receptors on immune cells (figure 1).ResultsAbLecs elicited tumor killing in vitro via macrophage phagocytosis and NK cell and granulocyte cytotoxicity, matching or outperforming combinations of monospecific antibodies with lectin-blocking or glycan-disrupting therapies. Furthermore, AbLecs synergized with blockade of the ‘don’t eat me’ signal CD47 for enhanced tumor killing.ConclusionsAbLecs can be readily designed to target numerous tumor-associated antigens and glyco-immune checkpoint ligands, and therefore represent a new modality for cancer immune therapy.Abstract 1187 Figure 1Antibody-lectin chimeras for glyco-immune checkpoint blockade
Immunotherapy for cancer that aims to promote T cell anti-tumor activity has changed current clinical practice, where some previously lethal cancers have now become treatable. However, clinical ...trials with low response rates have been disappointing for pancreatic ductal adenocarcinoma (PDAC). One suggested explanation is the accumulation of dominantly immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells in the tumor microenvironment (TME). Using retrospectively collected tumor specimens and transcriptomic data from PDAC, we demonstrate that expression of the scavenger receptor MARCO correlates with poor prognosis and a lymphocyte-excluding tumor phenotype. PDAC cell lines produce IL-10 and induce high expression of MARCO in myeloid cells, and this was further enhanced during hypoxic conditions. These myeloid cells suppressed effector T and natural killer (NK) cells and blocked NK cell tumor infiltration and tumor killing in a PDAC 3D-spheroid model. Anti-human MARCO (anti-hMARCO) antibody targeting triggered the repolarization of tumor-associated macrophages and activated the inflammasome machinery, resulting in IL-18 production. This in turn enhanced T cell and NK cell functions. The targeting of MARCO thus remodels the TME and represents a rational approach to make immunotherapy more efficient in PDAC patients.
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•Pancreatic tumors drive an immunosuppressive phenotype of myeloid cells•Antibodies activate MARCO-expressing myeloid cells to release cytotoxic cells•Expression of MARCO is connected to decreasing survival in human pancreatic cancer
Immunology; Components of the immune system; Cancer
Tumor-associated macrophages (TAMs) can have protumor properties, including suppressing immune responses, promoting vascularization and, consequently, augmenting tumor progression. To stop ...TAM-mediated immunosuppression, we use a novel treatment by injecting antibodies specific for scavenger receptor MARCO, which is expressed on a specific subpopulation of TAMs in the tumor. We now report the location of this TAM as well as the pleiotropic mechanism of action of anti-MARCO antibody treatment on tumor progression and further show that this is potentially relevant to humans. Using specific targeting, we observed decreased tumor vascularization, a switch in the metabolic program of MARCO-expressing macrophages, and activation of natural killer (NK) cell killing through TNF-related apoptosis-inducing ligand (TRAIL). This latter activity reverses the effect of melanoma cell-conditioned macrophages in blocking NK activation and synergizes with T cell-directed immunotherapy, such as antibodies to PD-1 or PD-L1, to enhance tumor killing. Our study thus reveals an approach to targeting the immunosuppressive tumor microenvironment with monoclonal antibodies to enhance NK cell activation and NK cell-mediated killing. This can complement existing T cell-directed immunotherapy, providing a promising approach to combinatorial immunotherapy for cancer.
Coordination of multiple kinesin and myosin motors is required for intracellular transport, cell motility and mitosis. However, comprehensive resources that allow systems analysis of the localization ...and interplay between motors in living cells do not exist. Here, we generated a library of 243 amino- and carboxy-terminally tagged mouse and human bacterial artificial chromosome transgenes to establish 227 stably transfected HeLa cell lines, 15 mouse embryonic stem cell lines and 1 transgenic mouse line. The cells were characterized by expression and localization analyses and further investigated by affinity-purification mass spectrometry, identifying 191 candidate protein-protein interactions. We illustrate the power of this resource in two ways. First, by characterizing a network of interactions that targets CEP170 to centrosomes, and second, by showing that kinesin light-chain heterodimers bind conventional kinesin in cells. Our work provides a set of validated resources and candidate molecular pathways to investigate motor protein function across cell lineages.
Cell division is an essential process for multicellular organisms. The fundamental goal of mitosis is to duplicate and segregate the genetic material of one cell, to ultimately generate two cells ...genetically identical to each other and to the parent cell. The mitotic spindle is a microtubule-based structure that attaches to, and segregates replicated chromosomes to the two daughter cells. To accomplish such a crucial, yet intricate task, the mitotic spindle must be correctly assembled, positioned, and disassembled. Historically, research has mostly focused on addressing the first problem. However, recent studies have highlighted the importance of proper spindle positioning and disassembly. In this dissertation we attempt to address these last two problems. We focused on understanding how the activities of a group of microtubule-associated proteins are coordinated to regulate microtubule function during spindle positioning and disassembly, to finally ensure successful cell division. The work presented in Chapters 2 and 3 of this dissertation attempts to shed light on the process of spindle disassembly. The Aurora B protein kinase, or Ipl1 in budding yeast, is one of the main regulators of spindle disassembly. Once chromosome segregation is completed, and just before the onset of spindle disassembly, the Ipl1/Aurora B kinase concentrates at the spindle midzone, the region of spindle breakage. However, we do not fully understand how it is targeted to the midzone, or its precise role there. Chapter 2 addresses these two questions. Combining live cell microscopy with yeast genetics and biochemistry we identified a kinesin-5 (Kip1) as the main kinesin responsible for the midzone localization of Ipl1/Aurora B. Kip1 alone is sufficient to recruit Ipl1/Aurora B to microtubules, and it is able to transport Ipl1/Aurora B to the microtubule end. We also found that cells rely on cytokinesis to physically break the spindle if the Ipl1/Aurora B kinase cannot concentrate at the midzone. Once at the midzone, we know that Ipl1/Aurora B phosphorylates targets to destabilize the spindle and allow its breakage. She1 is a microtubule-associated protein that is activated during late anaphase by Ipl1/Aurora B-mediated phosphorylation. However, we do not understand its precise role during spindle disassembly. Chapter 3 investigates this question, and addresses whether She1 promotes spindle disassembly by directly destabilizing spindle microtubules. The mitotic spindle needs to be positioned perpendicular to the division plane for proper chromosome segregation. Chapter 4 attempts to gain some insight into this process, and focuses on understanding how the activity of two key antagonistic kinesins, present at the same time, on the same set of microtubules, is coordinated to achieve precise spindle positioning. Together, these studies have shed light on the mechanistic nuances of how protein function is coordinated to position and disassemble the mitotic spindle, and they present a novel model for how the Ipl1/Aurora B kinase is recruited to the spindle midzone in late anaphase, a process conserved across eukaryotes.
Cell shape and motility are primarily controlled by cellular mechanics. The attachment of the plasma membrane to the underlying actomyosin cortex has been proposed to be important for cellular ...processes involving membrane deformation. However, little is known about the actual function of membrane-to-cortex attachment (MCA) in cell protrusion formation and migration, in particular in the context of the developing embryo. Here, we use a multidisciplinary approach to study MCA in zebrafish mesoderm and endoderm (mesendoderm) germ layer progenitor cells, which migrate using a combination of different protrusion types, namely, lamellipodia, filopodia, and blebs, during zebrafish gastrulation. By interfering with the activity of molecules linking the cortex to the membrane and measuring resulting changes in MCA by atomic force microscopy, we show that reducing MCA in mesendoderm progenitors increases the proportion of cellular blebs and reduces the directionality of cell migration. We propose that MCA is a key parameter controlling the relative proportions of different cell protrusion types in mesendoderm progenitors, and thus is key in controlling directed migration during gastrulation.
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