Tumor-associated macrophages (TAMs) play a role in tumor angiogenesis and are recruited into the tumor microenvironment (TME) by secreted chemokines, including Monocyte Chemoattractant Protein-1 ...(MCP-1/CCL2). Angiogenesis is required to sustain proliferation and enable metastasis of breast cancer (BCa) cells. Understanding the underlying mechanisms of TAM recruitment would allow for the identification of desperately needed novel drug targets. Sushi Domain Containing 2 (SUSD2), a transmembrane protein on BCa cells, was previously shown to promote tumor angiogenesis in a murine model. To identify the role of SUSD2 in angiogenesis, 175 human breast tumors were surveyed by immunohistochemical analysis for the presence of SUSD2 and macrophages. Tumors with high levels of SUSD2 staining contained 2-fold more TAMs, mainly of the M2 pro-angiogenic phenotype. An in vitro co-culture model system was developed by differentiating SC monocytes into SC M0 macrophages. A 2-fold increase in polarized M2 macrophages was observed when M0 macrophages were incubated with SUSD2-expressing BCa cells compared to cancer cells that do not contain SUSD2. Since MCP-1 is known to recruit macrophages, levels of MCP-1 were compared between SUSD2-expressing MDA-MB-231 and MBA-MB-231-vector control cell lines. MCP-1 RNA, intracellular protein and secreted MCP-1 were all significantly increased compared to the vector control. Knockdown of SUSD2 in SKBR3 resulted in significantly decreased levels of secreted MCP-1. Consistently, increased levels of MCP-1 were observed in Susd2-expressing tumors generated from an in vivo isogeneic mouse model compared to the vector control tumors. Because SUSD2 recruits macrophages into the TME and promotes M2 polarization, inhibiting the function of SUSD2 may be an effective therapy for breast cancer patients.
Galectin-1 (Gal-1) is a 14 kDa protein that has been well characterized for promoting cancer metastasis and tumor immune evasion. By localizing to the cancer cell surface, Gal-1 induces T cell ...apoptosis through binding T cell surface receptors. The transmembrane protein, Sushi Domain Containing 2 (SUSD2), has been previously shown to be required for Gal-1 surface presentation in breast cancer cells. Western immunoblot analysis revealed that SUSD2 is cleaved into two fragments. However, the significance of this cleavage for Gal-1 surface localization has not been investigated. To define the location of cleavage, a mutagenesis analysis of SUSD2 was performed. Our studies demonstrated that SUSD2 is cleaved at its glycine-aspartic acid-proline-histidine (GDPH) amino acid sequence. Generation of a noncleavable SUSD2 mutant (GDPH∆-SUSD2) showed that SUSD2 cleavage was required for SUSD2 and Gal-1 plasma membrane localization. Noncleavable cysteine mutants were also unable to present Gal-1 at the cell surface, further demonstrating that SUSD2 cleavage is required for Gal-1 surface presentation. Treatment with the serine protease inhibitor, Pefabloc SC, inhibited SUSD2 cleavage in a dose dependent manner, suggesting that SUSD2 is cleaved by a serine protease. Therefore, identification and inhibition of this protease may provide a new therapeutic tool for inhibiting SUSD2 and Gal-1's combined tumorigenic function in breast cancer.
Abstract
Passive antibody immunotherapeutics directed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are promising countermeasures for protection and treatment of coronavirus ...disease 2019 (COVID-19). SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) can impact the clinical efficacy of immunotherapeutics. A fully human polyclonal antibody immunotherapeutic purified from plasma of transchromosomic (Tc) bovines hyperimmunized with SARS-CoV-2 WA-1 spike (SAB-185) is being assessed for efficacy in a phase 2/3 clinical trial when different circulating SARS-CoV-2 variants predominated. We evaluated antibody binding, avidity maturation, and SARS-CoV-2 VOCs/VOIs virus-neutralizing capacity of convalescent plasma compared with different lots of SAB-185 and individual Tc bovine sera sequentially obtained after each vaccination against Alpha, Epsilon, Iota, Gamma, Beta, Kappa, and Delta variants. In contrast to convalescent plasma, sera and SAB-185 derived from hyperimmunized Tc bovines demonstrated higher antibody avidity and more potent cross-neutralizing activity of VOCs/VOIs. Thus, SAB-185 is a potential promising therapeutic candidate for the treatment of patients infected with SARS-CoV-2 variants.
Polyclonal human antibodies were derived from transchromosomic bovines hyperimmunized with SARS-CoV-2 spike. These high-affinity antibodies, which are being evaluated in clinical trials, demonstrated broad cross-neutralization activity against emerging SARS-CoV-2 variants and can be used for treatment of COVID-19.
Metastatic breast cancer (BCa) is most often diagnosed months after completion of treatment of the primary tumor when a patient reports physical symptoms. Besides a physical examination, no other ...alternative recurrence screening method is recommended for routine follow-up care. Detection of autoantibodies against tumor-associated antigens (TAAs) has demonstrated promise for distinguishing healthy women from patients diagnosed with primary BCa. However, it is unknown what changes occur to patient autoantibody levels during and after treatment.
Three serial blood draws were collected from 200 BCa patients: before treatment, 6 and 12 months after surgery. Patients were categorized according to treatment regimen, including surgery, chemotherapy, radiation, trastuzumab and hormonal therapies. The longitudinal samples were assayed for autoantibody responses against 32 conformation-carrying TAAs using a Luminex multiplex bead assay.
The treatment modality groups that had the greatest decrease in autoantibody response levels were radiation + hormonal therapy; radiation + chemotherapy; and radiation + hormonal therapy + chemotherapy. For these three treatment groups, autoantibody responses against 9 TAAs (A1AT, ANGPTL4, CAPC, CST2, DKK1, GFRA1, GRN, LGALS3 and LRP10) were significantly reduced at 12 months after surgery compared to before treatment. One TAA, GRP78, had a significantly increased autoantibody response after 12 months.
Single treatment regimens alone did not significantly alter autoantibodies levels against the studied TAAs. Radiation treatment was the common denominator of the three most affected groups for significant changes in autoantibody response levels.
Routinely used therapies are not adequate to treat the heterogeneity of breast cancer, and consequently, more therapeutic targets are desperately needed. To identify novel targets, we generated a ...breast cancer cDNA library enriched for genes that encode membrane and secreted proteins. From this library we identified SUSD2 (Sushi Domain Containing 2), which encodes an 822-amino acid protein containing a transmembrane domain and functional domains inherent to adhesion molecules. Previous studies describe the mouse homolog, Susd2, but there are no studies on the human gene associated with breast cancer. Immunohistochemical analysis of human breast tissues showed weak or no expression of SUSD2 in normal epithelial cells, with the endothelial lining of vessels staining positive for SUSD2. However, staining was observed in pathologic breast lesions and in lobular and ductal carcinomas. SUSD2 interacts with galectin-1 (Gal-1), a 14-kDa secreted protein that is synthesized by carcinoma cells and promotes tumor immune evasion, angiogenesis, and metastasis. Interestingly, we found that localization of Gal-1 on the surface of cells is dependent on the presence of SUSD2. Various phenotype assays indicate that SUSD2 increases the invasion of breast cancer cells and contributes to a potential immune evasion mechanism through induction of apoptosis of Jurkat T cells. Using a syngeneic mouse model, we observed accelerated tumor formation and decreased survival in mice with tumors expressing Susd2. We found significantly fewer CD4 tumor infiltrating lymphocytes in mice with tumors expressing Susd2. Together, our findings provide evidence that SUSD2 may represent a promising therapeutic target for breast cancer.
The cause of death among high-grade serous ovarian cancer (HGSOC) patients involves passive dissemination of cancer cells within the peritoneal cavity and subsequent implantation of cancer spheroids ...into adjacent organs.
Sushi Domain
Containing 2
(
SUSD2
) encodes a type I transmembrane protein containing several functional domains inherent to adhesion molecules. Previous studies using
in vitro
methods have indicated that
SUSD2
functions as a tumor suppressor in several cancers, including HGSOC. In this study, we generated a HGSOC xenograft mouse model to investigate
SUSD2
expression in the context of HGSOC late-stage metastasis and overall survival. OVCAR3 cells with knock-down expression of
SUSD2
(OVCAR3 SUSD2-KD) or endogenous expression of
SUSD2
(OVCAR3-Non-Targeting (NT)) were injected into the peritoneal cavity of athymic nude mice. Immunohistochemistry analysis was utilized to identify infiltrating cancer cells and metastatic tumors in mouse ovaries, pancreas, spleen, omentum and liver. OVCAR3-NT mice developed significantly less cancer cell infiltrate and tumors in their pancreas and omentum compared to OVCAR3 SUSD2-KD mice. Furthermore, OVCAR3-NT mice displayed a longer median survival when compared to OVCAR3 SUSD2-KD mice (175 days and 185.5 days, respectively;
p
-value 0.0159). Altogether, the findings generated through the preclinical mouse model suggest that increased
SUSD2
expression in HGSOC impedes
in vivo
metastasis to pancreas and omentum. These results correlate to longer median survival and prove to be consistent with previous findings showing prolonged survival of HGSOC patients with high
SUSD2
-expressing primary tumors.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with amino-acid substitutions and deletions in spike protein (S) can reduce the effectiveness of monoclonal antibodies (mAbs) and ...may compromise immunity induced by vaccines. We report a polyclonal, fully human, anti-SARS-CoV-2 immunoglobulin produced in transchromosomic bovines (Tc-hIgG-SARS-CoV-2) hyperimmunized with two doses of plasmid DNA encoding the SARS-CoV-2 Wuhan strain S gene, followed by repeated immunization with S protein purified from insect cells. The resulting Tc-hIgG-SARS-CoV-2, termed SAB-185, efficiently neutralizes SARS-CoV-2, and vesicular stomatitis virus (VSV) SARS-CoV-2 chimeras in vitro. Neutralization potency was retained for S variants including S477N, E484K, and N501Y, substitutions present in recent variants of concern. In contrast to the ease of selection of escape variants with mAbs and convalescent human plasma, we were unable to isolate VSV-SARS-CoV-2 mutants resistant to Tc-hIgG-SARS-CoV-2 neutralization. This fully human immunoglobulin that potently inhibits SARS-CoV-2 infection may provide an effective therapeutic to combat COVID-19.
Since SARS-CoV-2 emerged in late 2019, it spread from China to the rest of the world. An initial concern was the potential for vaccine- or antibody-dependent enhancement (ADE) of disease as had been ...reported with other coronaviruses. To evaluate this, we first developed a ferret model by exposing ferrets to SARS-CoV-2 by either mucosal inoculation (intranasal/oral/ocular) or inhalation using a small particle aerosol. Mucosal inoculation caused a mild fever and weight loss that resolved quickly; inoculation via either route resulted in virus shedding detected in the nares, throat, and rectum for 7–10 days post-infection. To evaluate the potential for ADE, we then inoculated groups of ferrets intravenously with 0.1, 0.5, or 1 mg/kg doses of a human polyclonal anti-SARS-CoV-2 IgG from hyper-immunized transchromosomic bovines (SAB-185). Twelve hours later, ferrets were challenged by mucosal inoculation with SARS-CoV-2. We found no significant differences in fever, weight loss, or viral shedding after infection between the three antibody groups or the controls. Signs of pathology in the lungs were noted in infected ferrets but no differences were found between control and antibody groups. The results of this study indicate that healthy, young adult ferrets of both sexes are a suitable model of mild COVID-19 and that low doses of specific IgG in SAB-185 are unlikely to enhance the disease caused by SARS-CoV-2.
Importance
Breast cancer (BC) death rates have not improved for American Indian/Alaska Native (AI/AN) women, whereas, it has significantly decreased for non-Hispanic White (White) women.
Objective
...Delineate the differences in patient and tumor characteristics among AI/AN and Whites with BC, and its impact on age and stage at diagnosis as well as overall survival (OS).
Methods
Hospital-based, cohort study using the National Cancer Database to identify female AI/AN and Whites diagnosed with BC between the years 2004 and 2016.
Results
BC in 6866 AI/AN (0.3%) and 1,987,324 Whites (99.7%) were studied. The median age at diagnosis was 58 for AI/AN and 62 for Whites. AI BC patients traveled double the distance for treatment, lived in lower median income zip codes, had a higher percentage of uninsured, higher comorbidities, lower percentage of Stage 0/I, larger tumor size, greater number of positive lymph nodes, higher proportion of triple negative and HER2-positive BC than Whites. All the above comparisons were significant,
p
<0.001. Association between patient/tumor characteristics with age and stage at diagnosis was not significantly different between AI/AN and Whites. Unadjusted OS was worse for AI/AN as compared to Whites (HR=1.07, 95% CI=1.01–1.14,
p
=0.023). After adjustment of all covariates, OS was not different (HR=1.038, 95%CI=0.902–1.195,
p
=0.601).
Conclusion
There were significant differences in patient/tumor characteristics among AI/AN and White BC which adversely impacted OS in AI/AN. However, when adjusted for various covariates, the survival was similar, suggesting that the worse survival in AI/AN is mostly the impact of known biological, socio-economic, and environmental determinants of health.
Patients with breast cancer elicit an autoantibody response against cancer proteins, which reflects and amplifies the cellular changes associated with tumorigenesis. Detection of autoantibodies in ...plasma may provide a minimally invasive mechanism for early detection of breast cancer. To identify cancer proteins that elicit a humoral response, we generated a cDNA library enriched for breast cancer genes that encode membrane and secreted proteins, which are more likely to induce an antibody response compared with intracellular proteins. To generate conformation-carrying antigens that are efficiently recognized by patients' antibodies, a eukaryotic expression strategy was established. Plasma from 200 patients with breast cancer and 200 age-matched healthy controls were measured for autoantibody activity against 20 different antigens designed to have conformational epitopes using ELISA. A conditional logistic regression model was used to select a combination of autoantibody responses against the 20 different antigens to classify patients with breast cancer from healthy controls. The best combination included ANGPTL4, DKK1, GAL1, MUC1, GFRA1, GRN, and LRRC15; however, autoantibody responses against GFRA1, GRN, and LRRC15 were inversely correlated with breast cancer. When the autoantibody responses against the 7 antigens were added to the base model, including age, BMI, race and current smoking status, the assay had the following diagnostic capabilities: c-stat (95% CI), 0.82 (0.78-0.86); sensitivity, 73%; specificity, 76%; and positive likelihood ratio (95% CI), 3.04 (2.34-3.94). The model was calibrated across risk deciles (Hosmer-Lemeshow, P = 0.13) and performed well in specific subtypes of breast cancer including estrogen receptor positive, HER-2 positive, invasive, in situ and tumor sizes >1 cm.
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