Elliot C. Woods

Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis.

Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase CD45 is excluded from contact sites, using single-molecule tracking. The mobility of CD45 increased markedly upon engagement of Fcγ receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. By micropatterning IgG, the ligand of Fcγ receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fcγ receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the zippering of Fcγ receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup.

Precision glycocalyx editing as a strategy for cancer immunotherapy

Significance Successful tumors are able to evade the immune system, which is otherwise capable of killing transformed cells. Therapies that prevent this evasion have become revolutionary treatments for incurable cancers. One mechanism of evasion is the presentation of sugars, called sialic acids, within the cell surface’s sugar coating, or glycocalyx. Here, we designed biotherapeutic molecules, termed “antibody–enzyme conjugates,” that selectively remove sialic acids from tumor cells. The antibody directs the enzyme to the cancer cells, the enzyme cleaves the sugars, and then the antibody directs immune cells to kill the desialylated cancer cells. The conjugate increased tumor cell killing compared with the antibody alone. Editing the cancer cell glycocalyx with an antibody–enzyme conjugate represents a promising approach to cancer immune therapy. Cell surface sialosides constitute a central axis of immune modulation that is exploited by tumors to evade both innate and adaptive immune destruction. Therapeutic strategies that target tumor-associated sialosides may therefore potentiate antitumor immunity. Here, we report the development of antibody–sialidase conjugates that enhance tumor cell susceptibility to antibody-dependent cell-mediated cytotoxicity (ADCC) by selective desialylation of the tumor cell glycocalyx. We chemically fused a recombinant sialidase to the human epidermal growth factor receptor 2 (HER2)-specific antibody trastuzumab through a C-terminal aldehyde tag. The antibody–sialidase conjugate desialylated tumor cells in a HER2-dependent manner, reduced binding by natural killer (NK) cell inhibitory sialic acid-binding Ig-like lectin (Siglec) receptors, and enhanced binding to the NK-activating receptor natural killer group 2D (NKG2D). Sialidase conjugation to trastuzumab enhanced ADCC against tumor cells expressing moderate levels of HER2, suggesting a therapeutic strategy for cancer patients with lower HER2 levels or inherent trastuzumab resistance. Precision glycocalyx editing with antibody–enzyme conjugates is therefore a promising avenue for cancer immune therapy.

Biomaterials-based modulation of the immune system.

The immune system is traditionally considered from the perspective of defending against bacterial or viral infections. However, foreign materials like implants can also illicit immune responses. These immune responses are mediated by a large number of molecular signals, including cytokines, antibodies and reactive radical species, and cell types, including macrophages, neutrophils, natural killer cells, T-cells, B-cells, and dendritic cells. Most often, these molecular signals lead to the generation of fibrous encapsulation of the biomaterials, thereby shielding the body from these biomaterials. In this review we will focus on two different types of biomaterials: those that actively modulate the immune response, as seen in antigen delivery vehicles for vaccines, and those that illicit relatively small immune response, which are important for implantable materials. The first serves to actively influence the immune response by co-opting certain immune pathways, while the second tries to mimic the properties of the host in an attempt to remain undetected by the immune system. As these are two very different end points, each type of biomaterial has been studied and developed separately and in recent years, many advances have been made in each respective area, which will be highlighted in this review.

Glycocalyx Engineering with a Recycling Glycopolymer that Increases Cell Survival In Vivo.

Synthetic glycopolymers that emulate cell-surface mucins have been used to elucidate the role of mucin overexpression in cancer. However, because they are internalized within hours, these glycopolymers could not be employed to probe processes that occur on longer time scales. In this work, we tested a panel of glycopolymers bearing a variety of lipids to identify those that persist on cell membranes. Strikingly, we found that cholesterylamine (CholA) anchored glycopolymers are internalized into vesicles that serve as depots for delivery back to the cell surface, allowing for the display of cell-surface glycopolymers for at least ten days, even while the cells are dividing. As with native mucins, the cell-surface display of CholA-anchored glycopolymers influenced the focal adhesion distribution. Furthermore, we show that these mimetics enhance the survival of nonmalignant cells in a zebrafish model of metastasis. CholA-anchored glycopolymers therefore expand the application of glycocalyx engineering in glycobiology.

A bulky glycocalyx fosters metastasis formation by promoting G1 cell cycle progression

Metastasis depends upon cancer cell growth and survival within the metastatic niche. Tumors which remodel their glycocalyces, by overexpressing bulky glycoproteins like mucins, exhibit a higher predisposition to metastasize, but the role of mucins in oncogenesis remains poorly understood. Here we report that a bulky glycocalyx promotes the expansion of disseminated tumor cells in vivo by fostering integrin adhesion assembly to permit G1 cell cycle progression. We engineered tumor cells to display glycocalyces of various thicknesses by coating them with synthetic mucin-mimetic glycopolymers. Cells adorned with longer glycopolymers showed increased metastatic potential, enhanced cell cycle progression, and greater levels of integrin-FAK mechanosignaling and Akt signaling in a syngeneic mouse model of metastasis. These effects were mirrored by expression of the ectodomain of cancer-associated mucin MUC1. These findings functionally link mucinous proteins with tumor aggression, and offer a new view of the cancer glycocalyx as a major driver of disease progression.

Metabolic engineering of lactate dehydrogenase rescues mice from acidosis

Acidosis causes millions of deaths each year and strategies for normalizing the blood pH in acidosis patients are greatly needed. The lactate dehydrogenase (LDH) pathway has great potential for treating acidosis due to its ability to convert protons and pyruvate into lactate and thereby raise blood pH, but has been challenging to develop into a therapy because there are no pharmaceutical-based approaches for engineering metabolic pathways in vivo. In this report we demonstrate that the metabolic flux of the LDH pathway can be engineered with the compound 5-amino-2-hydroxymethylphenyl boronic acid (ABA), which binds lactate and accelerates the consumption of protons by converting pyruvate to lactate and increasing the NAD+/NADH ratio. We demonstrate here that ABA can rescue mice from metformin induced acidosis, by binding lactate, and increasing the blood pH from 6.7 to 7.2 and the blood NAD+/NADH ratio by 5 fold. ABA is the first class of molecule that can metabolically engineer the LDH pathway and has the potential to have a significant impact on medicine, given the large number of patients that suffer from acidosis.

A tension-mediated glycocalyx–integrin feedback loop promotes mesenchymal-like glioblastoma

Glioblastoma multiforme (GBMs) are recurrent lethal brain tumours. Recurrent GBMs often exhibit mesenchymal, stem-like phenotypes that could explain their resistance to therapy. Analyses revealed that recurrent GBMs have increased tension and express high levels of glycoproteins that increase the bulkiness of the glycocalyx. Studies showed that a bulky glycocalyx potentiates integrin mechanosignalling and tissue tension and promotes a mesenchymal, stem-like phenotype in GBMs. Gain- and loss-of-function studies implicated integrin mechanosignalling as an inducer of GBM growth, survival, invasion and treatment resistance, and a mesenchymal, stem-like phenotype. Mesenchymal-like GBMs were highly contractile and expressed elevated levels of glycoproteins that expanded their glycocalyx, and they were surrounded by a stiff extracellular matrix that potentiated integrin mechanosignalling. Our findings suggest that there is a dynamic and reciprocal link between integrin mechanosignalling and a bulky glycocalyx, implying a causal link towards a mesenchymal, stem-like phenotype in GBMs. Strategies to ameliorate GBM tissue tension offer a therapeutic approach to reduce mortality due to GBM.Barnes et al. report a dynamic and reciprocal crosstalk between tissue tension and glycocalyx bulkiness that promotes a mesenchymal, stem-like phenotype in GBM.

Abstract PR05: Glycoprotein-mediated tissue mechanics regulate glioblastoma aggression

Glioblastoma multiforme (GBM) is a malignant glioma whose progression is associated with rampant extracellular matrix (ECM) remodeling. We recently found that GBM ECM stiffness predicts reduced survival in human patients. Instead of collagen fibrosis, which is common in many solid tumors, we showed that GBM stiffening involves increased production of extracellular glycoproteins, glycosaminoglycans, and sugar-binding proteins. Using bioinformatics, we revealed that genes of the glycocalyx (transmembrane glycoproteins and their interacting partners) are disproportionately upregulated in GBM relative to lower grade gliomas. Further, these genes are overexpressed within GBM in the mesenchymal (MES) relative to the proneural (PRO) subtype, the former of which is associated with treatment resistance and relapse. Using mouse models of human GBM, we showed that MES tumors are more lethal than PRO, and present with elevated ECM stiffness and mechanical signaling. To test our hypothesis that mechanical signaling can drive the MES phenotype, we engineered PRO GBM cells with constitutively-elevated integrin signaling. Compared to control PRO cells, these undergo a robust MES-like transition, upregulate bulky glycoprotein expression, and result in stiffer and more lethal tumors. This phenotype was reversed by the inhibition of focal adhesion kinase in MES cells. To test whether an enhanced glycocalyx can directly elevate mechanical signaling, we decorated GBM cells with synthetic glycoprotein polymers. Indeed, this resulted in enhanced integrin-focal adhesion signaling and more aggressive tumor progression. The invasive properties and therapy resistance observed in mesenchymal tumor cells are often associated with elevated stem cell-like features. To investigate a link between the glycocalyx, tissue mechanics, and the mesenchymal-stem cell phenotype, we interfered with components of the gylcocalyx or mechanical signaling machinery and found a reduction in stem cell genes and surface proteins, as well as increased sensitivity to chemotherapy. These data support a model in which glycoprotein-mediated tissue stiffening drives GBM aggression through promotion of a mesenchymal phenotype. This abstract is also being presented as Poster A39. Citation Format: J. Matthew Barnes, Elliot C. Woods, Russell O. Bainer, Yekaterina A. Miroshnikova, Kan Lu, Gabriele Bergers, Carolyn Bertozzi, Valerie M. Weaver. Glycoprotein-mediated tissue mechanics regulate glioblastoma aggression. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr PR05.