Exosomal Thomsen-Friedenreich Glycoantigen: A New Liquid Biopsy Biomarker for Lung and Breast Cancer Diagnosis.

Exosomes are nanosized extracellular vesicles released by cells to transport biomolecules such as proteins and RNAs for intercellular communication. Exosomes play important roles in cancer development and metastasis; therefore, they have emerged as potential liquid biopsy biomarkers for cancer screening, diagnosis and management. Many exosome cargos, including proteins, RNAs and lipids, have been extensively investigated as biomarkers for cancer liquid biopsy. However, carbohydrates, an important type of biomolecules, have not yet been explored for this purpose. In this study, we reported a new exosomal carbohydrate biomarker, i.e., alpha-linked Thomsen-Friedenreich glycoantigen (TF-Ag-α; Galß1-3GalNAc alpha). To transform our discovery for clinical applications, we developed a surface plasmon resonance (SPR)-based assay which utilized a unique monoclonal antibody, JAA-F11, with high specificity to TF-Ag-α to measure the levels of exosomal TF-Ag-α in blood. To our knowledge, we are the first to demonstrate that exosomes carry TF-Ag-α. We detected exosomal TF-Ag-α in as low as 10 µL serum samples from cancer patients but in contrast, levels were negligible from normal controls. With a total of 233 cancer patients and normal controls, we showed that exosomal TF-Ag-α detected lung cancer (n=60) and breast cancer (n=95) from normal controls (n=78) with ≥95% and ≥97% accuracy, respectively. These results demonstrated that exosomal TF-Ag-α is a potential liquid biopsy biomarker for cancer diagnosis.

Therapeutic efficacy of the humanized JAA-F11 anti-Thomsen-Friedenreich antibody constructs H2aL2a and H3L3 in human breast and lung cancer xenograft models.

The Thomsen-Friedenreich antigen (TF-Ag-α) is found on ~85% of human carcinomas but is cryptic on normal tissue. The humanized highly specific hJAA-F11-H2aL2a and -H3L3 antibodies target TF-Ag-α without binding to TF-Ag-beta (found on surface glycolipids of some normal cells). The relative affinity of H3L3 is 17 times that of H2aL2a, which would seem to favor superior efficacy, however, increased affinity can result in less tumor penetration. To assess the potential therapeutic efficacy of these antibodies, four human cancer- mouse xenograft models were treated with H2aL2a and H3L3. The tumor xenograft models used were human non-small cell lung cancer, H520, and small cell lung cancer, HTB171 in nude mice and human triple negative breast cancer, MDA-MB-231 and HCC1806 in SCID mice. H2aL2a significantly decreased tumor growth in both breast and both lung cancer models. H2aL2a showed statistically equal or better efficacy than H3L3 and has superior production capabilities. These results suggest that H2aL2a may be superior as a naked antibody, as an antibody drug conjugate or as a radiolabeled antibody, however the higher affinity of H3L3 may lead to better efficacy in bi-specific therapies in which the binding is decreased due to the presence of only one TF-Ag-α binding site.

Responses of serum chemokines to dramatic changes of air pollution levels, a panel study.

Background: Despite the in vitro and in vivo evidence, studies are limited in evaluating whether chemokines are potential inflammatory mediators in response to air pollution exposure in humans. Methods: We conducted a panel study coinciding with the Beijing Olympics, when temporary air pollution controls were implemented. We measured a suite of serum chemokines among healthy adults before, during and after the Olympics, respectively. Linear mixed-effect models were used to evaluate changes in chemokine levels over the three time periods. Results: In response to the 50% drop in air pollution levels during the games, levels of RANTES, MCP-2, and TARC decreased by 25.8%, 20.9% and 35.3%, respectively (p < 0.001) from pre-Olympics, and then increased by 45.8%, 34.9% and 61.5%, respectively (p < 0.001) after the games when air pollution levels went up again. Similar patterns were observed in subgroup analyses by sex, age, smoking and body mass index. GRO-α and IL-8 decreased significantly during the games (22.5% and 30.4%), and increased non-significantly after the games. Eotaxin-1 only increased significantly from during- to post-games. Conclusions: The strongest associations with air pollution levels were observed among RANTES, TARC and MCP-2. Those chemokines may play important roles in the air pollution-induced inflammatory pathway.

Temporal and molecular dynamics of human metastatic breast carcinoma cell adhesive interactions with human bone marrow endothelium analyzed by single-cell force spectroscopy.

Bone is a common site of metastasis for breast cancer and the mechanisms of metastasis are not fully elucidated. The purpose of our study was to characterize temporal and molecular dynamics of adhesive interactions between human breast cancer cells (HBCC) and human bone marrow endothelium (HBME) with piconewton resolution using atomic force microscopy (AFM). In adhesion experiments, a single breast cancer cell, MDA-MB-231 (MB231) or MDA-MB-435 (MB435) was attached to the AFM cantilever and brought into contact with a confluent HBME monolayer for different time periods (0.5 to 300 sec). The forces required to rupture individual molecular interactions and completely separate interacting cells were analyzed as measures of cell-cell adhesion. Adhesive interactions between HBME and either MB231 or MB435 cells increased progressively as cell-cell contact time was prolonged from 0.5 to 300 sec due to the time-dependent increase in the number and frequency of individual adhesive events, as well as to the involvement of stronger ligand-receptor interactions over time. Studies of the individual molecule involvement revealed that Thomsen-Friedenreich antigen (TF-Ag), galectin-3, integrin-ß1, and integrin-α3 are all contributing to HBCC/HBME adhesion to various degrees in a temporally defined fashion. In conclusion, cell-cell contact time enhances adhesion of HBCC to HBME and the adhesion is mediated, in part, by TF-Ag, galectin-3, integrin-α3, and integrin-ß1.

Preclinical Analysis of JAA-F11, a Specific Anti-Thomsen-Friedenreich Antibody via Immunohistochemistry and In Vivo Imaging.

The tumor specificity of JAA-F11, a novel monoclonal antibody specific for the Thomsen-Friedenreich cancer antigen (TF-Ag-alpha linked), has been comprehensively studied by in vitro immunohistochemical (IHC) staining of human tumor and normal tissue microarrays and in vivo biodistribution and imaging by micro-positron emission tomography imaging in breast and lung tumor models in mice. The IHC analysis detailed herein is the comprehensive biological analysis of the tumor specificity of JAA-F11 antibody performed as JAA-F11 is progressing towards preclinical safety testing and clinical trials. Wide tumor reactivity of JAA-F11, relative to the matched mouse IgG3 (control), was observed in 85% of 1269 cases of breast, lung, prostate, colon, bladder, and ovarian cancer. Staining on tissues from breast cancer cases was similar regardless of hormonal or Her2 status, and this is particularly important in finding a target on the currently untargetable triple-negative breast cancer subtype. Humanization of JAA-F11 was recently carried out as explained in a companion paper "Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis" (Neoplasia 19: 716-733, 2017), and it was confirmed that humanization did not affect chemical specificity. IHC studies with humanized JAA-F11 showed similar binding to human breast tumor tissues. In vivo imaging and biodistribution studies in a mouse syngeneic breast cancer model and in a mouse-human xenograft lung cancer model with humanized 124I- JAA-F11 construct confirmed in vitro tumor reactivity and specificity. In conclusion, the tumor reactivity of JAA-F11 supports the continued development of JAA-F11 as a targeted cancer therapeutic for multiple cancers, including those with unmet need.

Cell surface Thomsen-Friedenreich proteome profiling of metastatic prostate cancer cells reveals potential link with cancer stem cell-like phenotype.

The tumor-associated Thomsen-Friedenreich glycoantigen (TF-Ag) plays an important role in hematogenous metastasis of multiple cancers. The LTQ Orbitrap LC-MS/MS mass spectrometry analysis of cell surface TF-Ag proteome of metastatic prostate cancer cells reveals that several cell surface glycoproteins expressing this carbohydrate antigen in prostate cancer (CD44, α2 integrin, ß1 integrin, CD49f, CD133, CD59, EphA2, CD138, transferrin receptor, profilin) are either known as stem cell markers or control important cancer stem-like cell functions. This outcome points to a potential link between TF-Ag expression and prostate cancer stem-like phenotype. Indeed, selecting prostate cancer cells for TF-Ag expression resulted in the enrichment of cells with stem-like properties such as enhanced clonogenic survival and growth, prostasphere formation under non-differentiating and differentiating conditions, and elevated expression of stem cell markers such as CD44 and CD133. Further, the analysis of the recent literature demonstrates that TF-Ag is a common denominator for multiple prostate cancer stem-like cell populations identified to date and otherwise characterized by distinct molecular signatures. The current paradigm suggests that dissemination of tumor cells with stem-like properties to bone marrow that occurred before surgery and/or radiation therapy is largely responsible for disease recurrence years after radical treatment causing a massive clinical problem in prostate cancer. Thus, developing means for destroying disseminated prostate cancer stem-like cells is an important goal of modern cancer research. The results presented in this study suggest that multiple subpopulation of putative prostate cancer stem-like cells characterized by distinct molecular signatures can be attacked using a single target commonly expressed on these cells, the TF-Ag.

Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and InVitro Efficacy Analysis.

JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag) which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11). Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1) suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.

Peak expiratory flow, breath rate and blood pressure in adults with changes in particulate matter air pollution during the Beijing Olympics: a panel study.

OBJECTIVES: This study aims to examine whether changes in short-term exposures to particulate matter are associated with changes in lung function, breath rate, and blood pressure among healthy adults and whether smoking status modifies the association. METHODS: We took advantage of the artificially controlled changes in air pollution levels that occurred during the 2008 Olympic Games in Beijing, China and conducted a panel study of 201 Beijing residents. Data were collected before, during, and after the Olympics, respectively. Linear mixed-effect models and generalized estimating equation models were used to compare measurements of peak expiratory flow, breath rate and blood pressure across three time points. RESULTS: The mean values of peak expiratory flow were 346.0 L/min, 399.3 L/min, and 364.1L/min over the three study periods. Peak expiratory flow levels increased in 78% of the participants when comparing the during- with pre- Olympics time points, while peak expiratory flow levels decreased in 80% of participants for the post- and during-Olympic periods comparison. In subgroup analyses comparing the during-Olympic to pre-Olympic time points, we found a larger percentage change in peak expiratory flow (+17%) among female, younger and non-smoking participants than among male, elderly and smoking participants (+12%). The percentage of participants with a fast breath rate (>20/min) changed from 9.7% to 4.9% to 30.1% among females, and from 7.9% to 2.6% to 27.3% among males over the three time points. The changes in blood pressure over the three study periods were not very clear, although there is an increase in diastolic pressure and a decrease in pulse pressure among males during the games. CONCLUSIONS: The results suggest that exposure to different air pollution levels has significant effects on respiratory function. Smoking, age and gender appear to modify participants' biological response to changes in air quality.

Endothelial integrin α3ß1 stabilizes carbohydrate-mediated tumor/endothelial cell adhesion and induces macromolecular signaling complex formation at the endothelial cell membrane.

Blood borne metastatic tumor cell adhesion to endothelial cells constitutes a critical rate-limiting step in hematogenous cancer metastasis. Interactions between cancer associated carbohydrate Thomsen-Friedenreich antigen (TF-Ag) and endothelium-expressed galectin-3 (Gal-3) have been identified as the leading molecular mechanism initiating tumor/endothelial cell adhesion in several types of cancer. However, it is unknown how these rather weak and transient carbohydrate/lectin mediated interactions are stabilized. Here, using Western blot and LC tandem mass spectrometry analyses of pull-downs utilizing TF-Ag loaded gold nanoparticles, we identified Gal-3, endothelial integrin α3ß1, Src kinase, as well as 5 additional molecules mapping onto focal adhesion pathway as parts of the macromolecular complexes formed at the endothelial cell membranes downstream of TF-Ag/Gal-3 interactions. In a modified parallel flow chamber assay, inhibiting α3ß1 integrin greatly reduced the strength of tumor/endothelial cell interactions without affecting the initial cancer cell adhesion. Further, the macromolecular complex induced by TF-Ag/Gal-3/α3ß1 interactions activates Src kinase, p38, and ERK1/2, pathways in endothelial cells in a time- and α3ß1-dependent manner. We conclude that, following the initial metastatic cell attachment to endothelial cells mediated by TF-Ag/Gal-3 interactions, endothelial integrin α3ß1 stabilizes tumor/endothelial cell adhesion and induces the formation of macromolecular signaling complex activating several major signaling pathways in endothelial cells.

Preclinical studies with JAA-F11 anti-Thomsen-Friedenreich monoclonal antibody for human breast cancer.

AIM: The Thomsen-Friedenreich antigen (TF-Ag) is a disaccharide hidden on normal cells, but selectively exposed on the surface of breast, colon, prostate and bladder cancer cells. JAA-F11, a highly specific monoclonal antibody to TF-Ag, reduces metastasis and prolongs survival in a mouse model. In addition,(124)I-JAA-F11 localizes 4T1 tumors in mice. These studies continue translation of JAA-F11 to human breast cancer. MATERIALS & METHODS & RESULTS: Of the 41 human breast cancer cell lines tested, 78% were positive for reactivity with JAA-F11 by whole-cell enzyme immunoassay and positivity occurred unrelated to estrogen, progesterone or HER2 receptor status. JAA-F11 inhibited the growth rate of the human cancer cell lines tested. At 1 h, approximately 80% of JAA-F11 internalized in the three cell lines tested. (124)I-JAA-F11 specifically imaged human triple-negative tumors in mice by microPET. CONCLUSION: The results highlight the potential that humanized JAA-F11 may have for immunotherapy and drug conjugate therapy in breast cancer patients.

Computational screening of the human TF-glycome provides a structural definition for the specificity of anti-tumor antibody JAA-F11.

Recombinant antibodies are of profound clinical significance; yet, anti-carbohydrate antibodies are prone to undesirable cross-reactivity with structurally related-glycans. Here we introduce a new technology called Computational Carbohydrate Grafting (CCG), which enables a virtual library of glycans to be assessed for protein binding specificity, and employ it to define the scope and structural origin of the binding specificity of antibody JAA-F11 for glycans containing the Thomsen-Friedenreich (TF) human tumor antigen. A virtual library of the entire human glycome (GLibrary-3D) was constructed, from which 1,182 TF-containing human glycans were identified and assessed for their ability to fit into the antibody combining site. The glycans were categorized into putative binders, or non-binders, on the basis of steric clashes with the antibody surface. The analysis employed a structure of the immune complex, generated by docking the TF-disaccharide (Galß1-3GalNAcα) into a crystal structure of the JAA-F11 antigen binding fragment, which was shown to be consistent with saturation transfer difference (STD) NMR data. The specificities predicted by CCG were fully consistent with data from experimental glycan array screening, and confirmed that the antibody is selective for the TF-antigen and certain extended core-2 type mucins. Additionally, the CCG analysis identified a limited number of related putative binding motifs, and provided a structural basis for interpreting the specificity. CCG can be utilized to facilitate clinical applications through the determination of the three-dimensional interaction of glycans with proteins, thus augmenting drug and vaccine development techniques that seek to optimize the specificity and affinity of neutralizing proteins, which target glycans associated with diseases including cancer and HIV.

Glycodendrimers: versatile tools for nanotechnology

Combining nanotechnology with glycobiology has triggered an exponential growth of research activities in the design of novel functional bionanomaterials (glyconanotechnology). More specifically, recent synthetic advances towards the tailored and versatile design of glycosylated nanoparticles namely glyconanoparticles, considered as synthetic mimetics of natural glycoconjugates, paved the way toward diverse biomedical applications. The accessibility of a wide variety of these structured nanosystems, in terms of shapes, sizes, and organized around stable nanoparticles have readily contributed to their development and applications in nanomedicine. In this context, glycosylated gold-nanoparticles (GNPs), glycosylated quantum dots (QDs), fullerenes, single-wall natotubes (SWNTs), and self-assembled glycononanoparticles using amphiphilic glycopolymers or glycodendrimers have received considerable attention to afford powerful imaging, therapeutic, and biodiagnostic devices. This review will provide an overview of the most recent syntheses and applications of glycodendrimers in glycoscience that have permitted to deepen our understanding of multivalent carbohydrate-protein interactions. Together with synthetic breast cancer vaccines, inhibitors of bacterial adhesions to host tissues including sensitive detection devices, these novel bionanomaterials are finding extensive relevance.

A combinação de nanotecnologia com glicobiologia tem desencadeado o crescimento exponencial de atividades de pesquisa em desenvolvimento de novos biomateriais funcionais (gliconanotecnologia). Mais especificamente, recentes avanços sintéticos para o planejamento sob medida e versátil de nanopartículas glicosiladas, ou seja, gliconanopartículas, consideradas como miméticos sintéticos de glicoconjugados naturais, prepararam o caminho para diversas aplicações biomédicas. A acessibilidade da grande variedade destes nanossistemas estruturados, em termos de forma, tamanho e organização, tem prontamente contribuído para seu desenvolvimento e aplicações em nanomedicina. Neste contexto, nanopartículas de ouro glicosiladas (do inglês, GNPs), pontos quânticos glicosilados (do inglês, QDs), fulerenos, nanotubos de parede simples (do inglês, SWNTs) e gliconanopartículas autoconstruídas usando glicopolímeros anfifílicos ou glicodendrímeros têm recebido considerável atenção para originar poderosos instrumentos de imagem, terapêutico e de biodiagnóstico. Esta revisão fornecerá a visão global das mais recentes sínteses e aplicações de glicodendrímeros em glicociência que têm permitindo aprofundar nosso conhecimento das interações multivalentes proteína-carboidrato. Estes novos biomateriais estão sendo considerados de grande relevância, junto com vacinas sintéticas de câncer de mama, inibidores de adesão bacteriana em tecidos hospedeiros incluindo instrumentos de detecção sensível.