A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

Current prophylactic human immunodeficiency virus 1 (HIV-1) vaccine research aims to elicit broadly neutralizing antibodies (bnAbs). Membrane-proximal external region (MPER)-targeting bnAbs, such as 10E8, provide exceptionally broad neutralization, but some are autoreactive. Here, we generated humanized B cell antigen receptor knock-in mouse models to test whether a series of germline-targeting immunogens could drive MPER-specific precursors toward bnAbs. We found that recruitment of 10E8 precursors to germinal centers (GCs) required a minimum affinity for germline-targeting immunogens, but the GC residency of MPER precursors was brief due to displacement by higher-affinity endogenous B cell competitors. Higher-affinity germline-targeting immunogens extended the GC residency of MPER precursors, but robust long-term GC residency and maturation were only observed for MPER-HuGL18, an MPER precursor clonotype able to close the affinity gap with endogenous B cell competitors in the GC. Thus, germline-targeting immunogens could induce MPER-targeting antibodies, and B cell residency in the GC may be regulated by a precursor-competitor affinity gap.

Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4-binding site of the HIV envelope (Env) have shown promise in clinical trials. Here, we preclinically validated a lipid nanoparticle-encapsulated nucleoside mRNA (mRNA-LNP) encoding eOD-GT8 60mer as a soluble self-assembling nanoparticle in mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation without exclusionary competition. Boosts drove precursor B cell participation in germinal centers; the accumulation of somatic hypermutations, including in key VRC01-class positions; and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles encoded by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversified along the bnAb pathway.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

Broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem of influenza A viruses (IAVs) tend to be effective against either group 1 or group 2 viral diversity. In rarer cases, intergroup protective bnAbs can be generated by human antibody paratopes that accommodate the conserved glycan differences between the group 1 and group 2 stems. We applied germline-engaging nanoparticle immunogens to elicit a class of cross-group bnAbs from physiological precursor frequency within a humanized mouse model. Cross-group protection depended on the presence of the human bnAb precursors within the B cell repertoire, and the vaccine-expanded antibodies enriched for an N55T substitution in the CDRH2 loop, a hallmark of the bnAb class. Structurally, this single mutation introduced a flexible fulcrum to accommodate glycosylation differences and could alone enable cross-group protection. Thus, broad IAV immunity can be expanded from the germline repertoire via minimal antigenic input and an exceptionally simple antibody development pathway.

The skeletal muscle system is the major organ associated with movement of the body. Myogenesis and regeneration induced post-injury contribute to muscle formation and maintenance. Here, we provide detailed protocol for the accelerated repair of injured skeletal muscles and generation of hypertrophic muscle fibers. This protocol includes cardiotoxin induced muscle injury and also describes isolation of satellite cells from skeletal muscle tissues of mice. This protocol can be used to study the mechanisms associated with accelerated muscle repair and hypertrophy. For complete details on the use and execution of this protocol, please refer to Ray et al. (2021).

Melanoma originates from melanin-producing cells called melanocytes. Melanoma poses a great risk because of its rapid ability to spread and invade new organs. Cellular metastasis involves alteration in the gene expression profile and their transformation from epithelial to mesenchymal state. Despite of several advances, metastatic melanoma being a key cause of therapy failure and mortality remains poorly understood. p32 has been found to be involved in various physiological and pathophysiological conditions. However, the role of p32 in melanoma progression and metastasis remains underexplored. Here, we identify the role of p32 in the malignancy of both murine and human melanoma. p32 knockdown leads to reduced cell proliferation, migration, and invasion in murine and human melanoma cells. Furthermore, p32 promotes in vitro tumorigenesis, inducing oncogenes and EMT markers. Mechanistically, we show p32 regulates tumorigenic and metastatic properties through the Akt/PKB signaling pathway in both murine and human melanoma. Furthermore, p32 silencing attenuates melanoma tumor progression and lung metastasis in vivo, modulating the tumor microenvironment by inhibiting the angiogenesis, infiltration of macrophages, and leukocytes in mice. Taken together, our findings identify that p32 drives melanoma progression, metastasis, and regulates the tumor microenvironment. p32 can be a target of a novel therapeutic approach in the regulation of melanoma progression and metastasis.

Muscle differentiation is a multifaceted and tightly controlled process required for the formation of skeletal muscle fibers. Satellite cells are the direct cellular contributors to muscle repair in injuries or disorders. Here, we show that autotaxin (Atx) expression and activity is required for satellite cell differentiation. Conditional ablation of Atx or its pharmacological inhibition impairs muscle repair. Mechanistically, we identify LPAR1 as the key receptor in Atx-LPA signaling. Myogenic gene array and pathway analysis identified that Atx-LPA signaling activates ribosomal protein S6 kinase (S6K), an mTOR-dependent master regulator of muscle cell growth via LPAR1. Furthermore, Atx transgenic mice show muscle hypertrophic effects and accelerated regeneration. Intramuscular injections of Atx/LPA show muscle hypertrophy. In addition, the regulatory effects of Atx on differentiation are conserved in human myoblasts. This study identifies Atx as a critical master regulator in murine and human muscles, identifying a promising extracellular ligand in muscle formation, regeneration, and hypertrophy.

Receptor for advanced glycation end products (RAGE) is a multi-ligand transmembrane receptor of the immunoglobulin superfamily. Lysophosphatidic acid (LPA) is a ligand for RAGE and is involved in physiological and pathophysiological conditions including cancer. However, RAGE-LPA axis is unexplored in lung and mammary cancer. RAGE was silenced in A549, MDA MB-231 and MCF7 using RAGE shRNA. For in vitro tumorigenesis, we performed wound healing, colony formation, cell proliferation and invasion assays. Evaluation of expression of oncogenes, EMT markers and downstream signaling molecules was done by using western blot and immunohistochemistry. For subcellular expression of RAGE, immunofluorescence was done. In vivo tumorigenesis was assessed by intraperitoneal injection of cancer cells in nude mice. Here we show RAGE mediated profound increase in proliferation, migration and invasion of lung and mammary cancer cells via LPA in Protein kinase B (PKB) dependent manner. LPA mediated EMT transition is regulated by RAGE. In vivo xenograft results show significance of RAGE in LPA mediated lung and mammary tumor progression, angiogenesis and immune cell infiltration to tumor microenvironment. Our results establish the significance and involvement of RAGE in LPA mediated lung and mammary tumor progression and EMT transition via RAGE. RAGE-LPA axis may be a therapeutic target in lung and mammary cancer treatment strategies.

Receptor for advanced glycation end products (RAGE) is an immunoglobulin-like receptor present on cell surface. RAGE binds to an array of structurally diverse ligands, acts as a pattern recognition receptor (PRR) and is expressed on cells of different origin performing different functions. RAGE ligation leads to the initiation of a cascade of signaling events and is implicated in diseases, such as inflammation, cancer, diabetes, vascular dysfunctions, retinopathy, and neurodegenerative diseases. Because of the significant involvement of RAGE in the progression of numerous diseases, RAGE signaling has been targeted through use of inhibitors and anti-RAGE antibodies as a treatment strategy and therapy. Here in this review, we have summarized the physical and physiological aspects of RAGE biology in mammalian system and the importance of targeting this molecule in the treatment of various RAGE mediated pathologies.HighlightsReceptor for advanced glycation end products (RAGE) is a member of immunoglobulin superfamily of receptors and involved in many pathophysiological conditions.RAGE ligation with its ligands leads to initiation of distinct signaling cascades and activation of numerous transcription factors.Targeting RAGE signaling through inhibitors and anti-RAGE antibodies can be promising treatment strategy.

B-cell receptor (BCR) knock-in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one-step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD-GT8 60mer, a germline-targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class-switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases.

Non-muscle myosin IIA heavy chain (MYH9) has been implicated in many physiological and pathological functions including cell adhesion, polarity, motility to cancer. However, its role in melanoma remains unexplored. The aim of our study was to evaluate the role of MYH9 in melanoma tumor development and metastasis and further to find out the potential underlying mechanisms. In this study, we evaluated the in vitro migratory and invasive properties and in vivo tumor development and metastasis in C57BL/6 mice by silencing MYH9 in B16F10 melanoma cells. Knocking down MYH9 enhanced migration and invasiveness of B16F10 cells in vitro. Furthermore, MYH9 silencing accelerated tumor growth and metastasis in melanoma subcutaneous and intravenous mouse models. Next, oncogenes analysis revealed epithelial–mesenchymal transition and Erk signaling pathway are being regulated with MYH9 expression. Finally, MYH9 silencing in B16F10 cells modulates the tumor microenvironment by manipulating the leukocytes and macrophages infiltration in tumors. These findings established the opposing role of MYH9 as a tumor suppressor in melanoma suggesting specific MYH9 based approaches in therapeutics.

Lung and breast cancer signaling are very complex; receptor for advanced glycation end products (RAGE) is highly expressed in various cancers and is correlated with poorer outcome in lung and other cancers. It is an immunoglobulin like membrane receptor shows binding with various ligands sharing structural likeness such as amyloid beta, advanced glycation end products (AGEs), S100B proteins, amphoterin and HMGB1 and mediates numerous inflammatory and cellular events including diseases including various cancers. LPA a RAGE ligand is a biologically active phospholipid involved in cell proliferation, migration and survival via its different G protein couple receptors (GPCRs). LPA and its signalling pathways have been implicated in different pathological conditions of lung tissue, including inflammation, fibrosis and cancer activating various signalling pathways. Here, we have identified RAGE-LPA signaling mechanisms showing AKT, STAT3 and ERK as downstream signaling pathways gets activated upon LPA treatment via RAGE receptor in both lung and breast cancer cells. RAGE knockdown with multiple independent shRNAs in lung and breast cancer cells led to decreased transwell invasion and soft agar colony formation, and proliferation upon LPA stimulation. Our RAGE silencing study in lung and breast cancer cells upon LPA treatment showed upregulation of specific oncogenes Cyclin D1, c-Myc and VEGF via RAGE receptor. RAGE association with LPA induced epithelial to mesenchymal transition (EMT) in both lung and breast cancer cells have also been observed by the RAGE attenuation studies by examining the expression of some EMT markers. The physiological significance of LPA-RAGE axis was exposed by our xenograft study by nude mice intraperitoneal injections of control and RAGE inhibited lung and mammary tumors generated by LPA doses in alternative days, results the formation of tumor foci on peritoneum wall via RAGE receptor. This study provides novel therapeutic insights as the use of RAGE blocking antibodies and antagonists or competitive inhibitors of LPA against RAGE might be useful of breast cancer treatment. This study also suggests that RAGE and LPA can be used as biomarkers for the early detection of lung and breast cancers. Citation Format: Nitish Jangde, Rashmi Ray, Vivek Rai. RAGE is the key receptor for LPA in lung and mammary cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4307.

Tumor microenvironment consist of dynamic interactions between tumor cells and the surrounding non-transformed cells. Inflammatory cells constitute a major population of the non-transformed cells. Tumor associated macrophages (TAMs), the predominant population of inflammatory cells have major roles in cancer progression and metastasis but the exact stimulus and triggers for the tumor cells-macrophage interaction remains unclear. Autotaxin or lysophospholipaseD (LysoPLD) catalyses the synthesis of lyophosphatidic acid - the smallest phospholipid from lysophosphatidylcholine by its enzymatic action. Autotaxin is implicated in breast cancer, ovarian cancer and many other cancers. Lysophosphatidic acid (LPA) is involved in numerous biological processes encompassing cell growth, cell proliferation, cell migration, cancer and metastasis. LPA effects are mediated on different cell types via its cognate G-protein coupled receptors (GPCRs) or non-receptor pathways. Our recent study has shown that LPA converts monocytes into macrophages both in mice and humans and has an important role to play with immune cells. Here, we show that cancer cells in tumor growth are associated with tumor associated macrophages via a signature paracrine link. Our studies identify previously unknown signaling link between tumor cells and macrophages. Furthermore, we identify that suppression of this paracrine network can suppress tumor growth. This study suggests that inhibition of this paracrine network may act as a new therapeutic approach to control cancer and metastasis

*Correspondence: Lynne L Johnson, Department of Medicine, Columbia University Medical Center, 630 W. 168th St, PH 10, room 203, New York NY 10032, USA, Tel: 212-305-4275; Fax: 212-305-4648; E-mail: lj2129@columbia.edu Received Date: 13 Apr 2019 Accepted Date: 06 May 2019 Published Date: 09 May 2019 Citation: Tekabe Y, Johnson J, Li Q, Ray R, Rai V, Kokoshka J, et al. Molecular Imaging of RAGE Expression in Human Glioblastoma. Clin Oncol. 2019; 4: 1609.

Inflammatory cells and their mediators are an essential component of the tumor microenvironment. Tumor associated macrophages (TAMs) represent a predominant population of inflammatory cells present in the solid tumor and tumor microenvironment. TAMs are associated with tumor progression and metastasis and the infiltration of TAMs or the enrichment of TAM-associated genes relates to poor prognosis and disease outcome in most human tumor types. However, the stimulus and triggers for the tumor cells-macrophage interaction remain unclear. Autotaxin, an ectoenzyme generates smallest phospholipid, lyophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Autotaxin is also known to play important roles in breast cancer, ovarian cancer and many other cancers. LPA is involved in numerous biological processes encompassing cell growth, cell proliferation, cell migration, cancer and metastasis. LPA exerts its functions on different cell types via its different G-protein coupled receptors (GPCRs). More progress has been made in recent years in dissecting the mechanisms of LPA generation and how it directly acts on its target cells. Recently we have shown that LPA converts monocytes into macrophages both in mice and humans. Here, we show that cancer cells in tumor growth are associated with tumor associated macrophages via a signature paracrine link. Our studies identify an unknown signaling link between tumor cells and macrophages. Furthermore, we have functionally characterized these macrophages and the suppression of this paracrine network leads to decrease in tumor growth and its progression. Our preliminary data also shows that blocking of this paracrine link decreases the secretion of proangiogenic factor such as TGF-b, TNF-a, MMP-9, and VEGF. This study suggests that suppression of this paracrine network can act as a new therapeutic approach to control cancer and metastasis. New insights into the role of LPA in regulating immune responses via cancer cells should be explored in the near future. Citation Format: Rashmi Ray, Vivek Rai. Regulation of a novel cell surface protein in macrophage activity in tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B67.

Receptor for Advanced Glycation End product (RAGE) is a multiligand receptor implicated in diverse pathological conditions such as diabetes, atherosclerosis, cancer and neural diseases. Extracellular, RAGE consists of V, C1 and C2 domains. Here, we show RAGE exists as a monomer in equilibrium with a fraction of a covalently linked dimer of monomers via its V domain through cysteine. In order to understand the functional implication of this dimer, we examined the binding capacity and functional potential of RAGE dimer via advanced glycation end products (AGEs) which shows enhanced binding capacity towards V domain, ERK phosphorylation, cytokine release and actin polymerization ability of the dimeric form for AGEs compared with the reduced monomeric form. Our data, suggests that the dimeric state of RAGE controls its function and ligand mediated signaling which may play important role in RAGE mediated various diseases.