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Background. Despite therapeutic advancements, metastasis remains a major cause in breast cancer-specific mortality. Breast cancer cells are susceptible to oxidative damage and exhibit high levels of oxidative stress, including protein damage, DNA damage, and lipid peroxidation. Some breast cancer risk factors may change the level of endogenous oxidative stress. Circulating exosomes play critical roles in tumorigenesis, distant metastasis, and poor prognosis in patients with breast cancer. Methods. We used an online database to analyze the expression and prognostic value of core binding factor subunit β (CBFB) and oxidative stress–related targets in patients with breast cancer. Serum from healthy controls and patients with primary breast cancer or bone metastatic breast cancer in the bone was collected. Exosomes were isolated from the sera or cell culture media. We used an MDA-MB-436-innoculated tumor xenograft mouse model for silencing CBFB. Results. Circulating exosomes from patients with breast cancer metastasis to the bone were rich in CBFB. The human mammary fibroblast cells HMF3A and fibroblasts derived from patient samples cocultured with exosomes had increased α-SMA and vimentin expression and IL-6 and OPN secretion. Similarly, nonmetastatic breast cancer cells cocultured with exosomes exhibited increased levels of certain markers, including vimentin, snail1, CXCR4, and Runx2, and the exosomes had high CBFB expression. Silencing CBFB in metastatic MDA-MB-436 and MDA-MB-157 cells resulted in suppressed migration and invasion and downregulation of vimentin, CXCR4, snail1, Runx2, CD44, and OPN. Conversely, CBFB overexpression resulted in upregulation of Runx2, vimentin, snail1, CD44, and OPN in nonmetastatic T47D and MCF12A cells. The CBFB-rich exosomes derived from MDA-MB-436 cells induced enhanced metastatic phenotypes in the low-metastatic T47D and MCF12A cell lines. Conclusion. Our results revealed that CBFB may promote bone metastasis in patients with breast cancer. Of therapeutic relevance, targeting CBFB resulted in decreased tumor burden and bone metastasis, downregulation of bone metastasis markers, and impaired regulation of oxidative stress–related proteins NAE1 and NOS1.

Background. Treating advanced colon cancer remains challenging in clinical settings because of the development of drug resistance and distant metastasis. Mechanisms underlying the metastasis of colon cancer are complex and unclear. Methods. Computational analysis was performed to determine genes associated with the exosomal long noncoding (lncRNA) plasmacytoma variant translocation 1 (PVT1)/vascular endothelial growth factor A (VEGFA) axis in patients with colon cancer. The biological importance of the exosomal lncRNA PVT1/VEGFA axis was examined in vitro by using HCT116 and LoVo cell lines and in vivo by using a patient-derived xenograft (PDX) mouse model through knockdown (by silencing of PVT1) and overexpression (by adding serum exosomes isolated from patients with distant metastasis (M-exo)). Results. The in silico analysis demonstrated that PVT1 overexpression was associated with poor prognosis and increased expression of metastatic markers such as VEGFA and epidermal growth factor receptor (EGFR). This finding was further validated in a small cohort of patients with colon cancer in whom increased PVT1 expression was correlated with colon cancer incidence, disease recurrence, and distant metastasis. M-exo were enriched with PVT1 and VEGFA, and both migratory and invasive abilities of colon cancer cell lines increased when they were cocultured with M-exo. The metastasis-promoting effect was accompanied by increased expression of Twist1, vimentin, and MMP2. M-exo promoted metastasis in PDX mice. In vitro silencing of PVT1 reduced colon tumorigenic properties including migratory, invasive, colony forming, and tumorsphere generation abilities. Further analysis revealed that PVT1, VEGFA, and EGFR interact with and are regulated by miR-152-3p. Increased miR-152-3p expression reduced tumorigenesis, where increased tumorigenesis was observed when miR-152-3p expression was downregulated. Conclusion. Exosomal PVT1 promotes colon cancer metastasis through its association with EGFR and VEGFA expression. miR-152-3p targets both PVT1 and VEGFA, and this regulatory pathway can be explored for drug development and as a prognostic biomarker.

Locally advanced oral squamous cell carcinoma (OSCC) requires multimodal therapy, including surgery and concurrent chemoradiotherapy (CCRT). CCRT-resistant and recurrent cancer has a poor prognosis. We investigated the effects of Bruton’s tyrosine kinase (BTK) on CCRT-resistant OSCC tissues. The effect of ibrutinib, a first-in-class BTK inhibitor, was tested on stem cell-like OSCC tumorspheres. A tissue array was constructed using tissue samples from 70 patients with OSCC. Human OSCC cell lines, SAS, TW2.6 and HSC-3, were examined. Wound healing, Matrigel invasion, and tumorsphere formation assays, as well as immunofluorescence analysis and flow cytometry, were used to investigate the effects of BTK knockdown (shBTK), ibrutinib, cisplatin, and ibrutinib/cisplatin combination on OSCC cells. We demonstrated that BTK was aberrantly highly expressed in the clinical CCRT-resistant OSCC tissue array, which resulted in poor overall survival in our local Tri-Service General Hospital and freely accessible TCGA OSCC cohorts. shBTK significantly downregulated the stemness markers Nanog, CD133, T cell immunoglobulin-3 (TIM-3), and Krüppel-like factor 4 (KLF4) in SAS tumorspheres and attenuated OSCC cell migration and colony formation. Ibrutinib reduced the number of aldehyde dehydrogenase (ALDH)-rich OSCC cells and reduced tumorsphere formation, migration, and invasion in a dose-dependent manner. Compared with ibrutinib or cisplatin monotherapy, the ibrutinib/cisplatin combination significantly reduced the formation of ALDH + OSCC tumorspheres and enhanced apoptosis. These results demonstrate that ibrutinib effectively inhibits the CSCs-like phenotype of OSCC cells through dysregulation of BTK/CD133 signaling. The ibrutinib/cisplatin combination may be considered for future clinical use.

The exact mechanisms of Head and neck squamous carcinoma (HNSCC) chemoresistance and metastatic transformation remain unclear. In recent decades, members of the transient receptor potential (TRP) channel family have been proposed as potential biomarkers and/or drug targets in cancer treatment. First, in a TCGA cohort of HNSCC, TRPM7 is highly expressed in cancer tissues, especially the expression in invasive cancer tissues is statistically significant (p0.001). In GEO and TCGA cohort, patients with high expression of TRPM7 and NFATC2 have poor overall survival rates. The expression of TRPM7 and NFATC2 showed a positive correlation. Compared to human normal oral keratinocytes (hNOK), TRPM7 is overexpressed in FaDU, SAS, and TW2.6 cell lines. Similarly, patients with HNSCC exhibited higher TRPM7 expression than non-HNSCC subjects, and this high TRPM7 expression was associated with worse 5-year overall survival. Furthermore, TRPM7 inversely correlated with E-cadherin, but positively correlated with Vimentin, NANOG, and BMI-1 mRNA levels. Consistent with this, we demonstrated the overexpression of TRPM7 in cisplatin-resistant subjects, compared to the cisplatin-sensitive counterparts. Moreover, shRNA-mediated silencing of TRPM7 significantly suppressed the migration, invasion, colony formation, and tumorsphere formation of SAS cells, with associated downregulation of Snail, c-Myc, cyclin D1, SOX2, OCT4, and NANOG proteins expression. Finally, compared with the untreated wild-type SAS cells or cisplatin-treated cells, shTRPM7 alone or in combination with cisplatin significantly inhibited tumorsphere and colony formation. These findings serving as the basis for development of novel therapeutic strategies against metastasis and chemoresistance, while providing new insights into TRPM7 biology and activity in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is a major contributor to cancer incidence globally and is currently managed by surgical resection followed by adjuvant chemoradiotherapy. However, local recurrence is the major cause of mortality, indicating the emergence of drug-tolerant persister cells. A specific histone demethylase, namely lysine-specific demethylase 5D (KDM5D), is overexpressed in diverse types of cancers and involved in cancer cell cycle regulation. However, the role of KDM5D in the development of cisplatin-tolerant persister cells remains unexplored. Here, we demonstrated that KDM5D contributes to the development of persister cells. Aurora Kinase B (AURKB) disruption affected the vulnerability of persister cells in a mitotic catastrophe–dependent manner. Comprehensive in silico, in vitro, and in vivo experiments were performed. KDM5D expression was upregulated in HNSCC tumor cells, cancer stem cells, and cisplatin-resistant cells with biologically distinct signaling alterations. In an HNSCC cohort, high KDM5D expression was associated with a poor response to platinum treatment and early disease recurrence. KDM5D knockdown reduced the tolerance of persister cells to platinum agents and caused marked cell cycle deregulation, including the loss of DNA damage prevention, and abnormal mitosis-enhanced cell cycle arrest. By modulating mRNA levels of AURKB, KDM5D promoted the generation of platinum-tolerant persister cells in vitro, leading to the identification of the KDM5D/AURKB axis, which regulates cancer stemness and drug tolerance of HNSCC. Treatment with an AURKB inhibitor, namely barasertib, resulted in a lethal consequence of mitotic catastrophe in HNSCC persister cells. The cotreatment of cisplatin and barasertib suppressed tumor growth in the tumor mouse model. Thus, KDM5D might be involved in the development of persister cells, and AURKB disruption can overcome tolerance to platinum treatment in HNSCC.

Activation of mitogen-activated protein kinase (MAPK) and PI3K signaling confers resistance against sorafenib, a mainstay treatment for advanced hepatocellular carcinoma (HCC). Antrocin and ovatodiolide constitute as the most potent secondary metabolites isolated from Antrodia camphorata and Anisomeles indica, respectively. Both natural compounds have recently gained a lot of attention due to their putative inhibition of MAPK and PI3K signaling in various solid cancers. However, whether their combination is effective in HCC remains unknown. Here, we investigated their effect, alone or in various combinations, on MAPK and PI3K signaling pathways in HCC cells. An array of in vitro study were used to investigate anticancer and stemness effects to treat HCC, such as cytotoxicity, drug combination index, migration, invasion, colony formation, and tumor sphere formation. Drug effect in vivo was evaluated using mouse xenograft models. In this study, antrocin and ovatodiolide synergistically inhibited the SNU387, Hep3B, Mahlavu, and Huh7 cell lines. Sequential combination treatment of Huh7 and Mahlavu with ovatodiolide followed by antrocin resulted stronger cytotoxic effect than did treatment with antrocin followed by ovatodiolide, their simultaneous administration, antrocin alone, or ovatodiolide alone. In the Huh7 and Mahlavu cell lines, ovatodiolide→antrocin significantly suppressed colony formation and proliferation as well as markedly downregulated ERK1/2, Akt, and mTOR expression. Inhibition of ERK1/2 and Akt/mTOR signaling by ovatodiolide→antrocin suppressed ribosomal biogenesis, autophagy, and cancer stem cell-like phenotypes and promoted apoptosis in Huh7 and Mahlavu cells. The sorafenib-resistant clone of Huh7 was effectively inhibited by synergistic combination of both compound in vitro. Eventually, the ovatodiolide→antrocin combination synergistically suppressed the growth of HCC xenografts. Taken together, our findings suggested that ovatodiolide→antrocin combination may represent potential therapeutic approach for patients with advanced HCC.

Simple Summary Our study provided data that the inhibition of MEK/ERK signaling could overcome Osimertinib resistance both in vitro and in vivo. Mechanistically, MEK inhibitor Trametinib suppressed the tumorigenic properties of NSCLC cells by reducing the generation of CAFs. The trametinib-mediated anti-cancer function was also associated with the significantly suppressed level of miR-21, of which primary targets included PDCD4, as shown in this study and MEK inhibitor Trametinib significantly suppressed Osimertinib-resistant NSCLC tumor growth by abolishing both processes. Abstract Background: The third-generation epidermal growth factor receptor (EGFR) inhibitor, Osimertinib, is used to treat non-small cell lung cancer (NSCLC) patients with tyrosine kinase inhibitor (TKI) resistance caused by acquired EGFR T790M mutation. However, patients eventually develop resistance against Osimertinib with mechanisms not yet fully clarified. Activated alternative survival pathways within the tumor cells and cancer-associated fibroblasts (CAFs) have been proposed to contribute to Osimertinib resistance. MET and MEK inhibitors may overcome EGFR-independent resistance. Another acquired resistance mechanism of EGFR-TKI is the up-regulation of the RAS/RAF/MEK/ERK signaling pathway, which is the key to cell survival and proliferation; this may occur downstream of various other signaling pathways. In this report, we reveal the possible regulatory mechanism and inhibitory effect of the MEK inhibitor trametinib applied to MEK/ERK/miR-21 axis and PDCD4 in Osimertinib resistance. We found a possible regulatory role of PDCD4 in ERK signaling. PDCD4 is a new type of tumor suppressor that has multiple functions of inhibiting cell growth, tumor invasion, metastasis, and inducing apoptosis. Previous bioinformatics analysis has confirmed that PDCD4 contains the binding site of miR-21 and acts as a tumor suppressor in the regulation of various processes associated with the development of cancer, including cell proliferation, invasion, metastasis, and neoplastic transformation. Based on the above analysis, we hypothesized that the tumor suppressor PDCD4 is one of the effective inhibitory targets of miR-21-5p. Methods: The expression between EGFR and ERK2 in lung adenocarcinoma was evaluated from the TCGA database. Osimertinib-sensitive and resistant NSCLC cells obtained from patients were used to co-culture with human lung fibroblasts (HLFs) to generate CAF cells (termed CAF_R1 and CAF_S1), and the functional roles of these CAF cells plus the regulatory mechanisms were further explored. Then, MEK inhibitor Trametinib with or without Osimertinib was applied in xenograft model derived from patients to validate the effects on growth inhibition of Osimertinib-resistant NSCLC tumors. Result: ERK2 expression correlated with EGFR expression and higher ERK2 level was associated with worse prognosis of patients and Osimertinib resistance. CAFs derived from Osimertinib-resistant cells secreted more IL-6, IL-8, and hepatocyte growth factor (HGF), expressed stronger CAF markers including α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP) plus platelet-derived growth factor receptor (PDGFR), and enhanced stemness and Osimertinib resistance in NSCLC cells. Meanwhile, increased MEK/ERK/miR-21 expressions were found in both CAFs and NSCLC cells. MEK inhibitor Trametinib significantly abrogated the abovementioned effects by modulating β-catenin, STAT3, and ERK. The xenograft model showed combining Osimertinib and Trametinib resulted in the most prominent growth inhibition of Osimertinib-resistant NSCLC tumors. Conclusions: Our results suggested that MEK/ERK/miR-21 signaling is critical in Osimertinib resistance and CAF transformation of NSCLC cells, and MEK inhibitor Trametinib significantly suppressed Osimertinib-resistant NSCLC tumor growth by abolishing both processes.

In our previous study, we produced a monoclonal antibody EB2 that recognized an epitope in the HA1 domain on the hemagglutinin (HA) of H6N1 influenza virus (A/chicken/Taiwan/2838 V/00). The residue Arg-201 (R201) on this epitope was protected by the glycan at Asn-167 (N167) from tryptic digestion; therefore, the infectivity of the virus was retained. R201 was extremely conserved in various subtypes of the influenza virus. To explore the role of R201 and the protecting glycan, we developed a bi-cistronic baculovirus expression system for the production of H6HA1 and H6HA0 (nearly full-length HA), which were glycosylated in insect cells. The expressed H6HA1 was mostly found in the trimeric form, and the H6HA0 protein was only found in the monomeric form. The trimeric H6HA1 was resistant to tryptic digestion; however, it could not bind to fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans. By contrast, the monomeric H6HA0 could bind to fetuin but was sensitive to tryptic digestion. We found that the positive charge on R201 was critical for binding HA to the negatively charged surface of host cells because the mutant R201A of H6HA0 lost its binding capacity substantially. Moreover, this binding capacity was dependent on the pH value and inhibited by free electrically charged amino acids. We propose a two-step model for binding the influenza virus with a host cell. The first step involved the specific recognition of the receptor binding site on HA to the sialylated glycan on the host cell. After the virus is engulfed by the acidic endosome, R201 could bind to the cell surface with stronger interactions and trigger the fusion process.

Simple Summary Hepatitis virus is a major risk factor for liver cancer. We analyzed possible synergism between momelotinib and sorafenib in hepatitis virus-associated liver cancer. The combined effect of momelotinib and sorafenib both at in vitro and in vivo synergistically sup-presses the proliferation of vHCC cells and effectively reduces the tumor burden. Our results showed that momelotinib effectively suppressed the expression of the IFNGR-JAK-STAT-PARP1 pathway, which results in the downregulation of cancer stem cell genes and enhances the antitumor efficacy of sorafenib by initiating the expression of apoptosis-related genes and inhibiting the DNA repair gene in vHCC cells, thus maximizing its therapeutic potential for patients with HCC. Abstract Background: Hepatitis virus is a major risk factor for liver cancer. The mitochondrial dysfunction IFN gamma-related pathways are activated after virus infection. Jak family-related protein is involved in the downstream of IFN gamma-related pathways. However, the effect of the IFNGR-JAK-STAT pathway acting as functional regulators of their related protein expression on virus infection and hepatocellular carcinoma (HCC) remains unclear. Interestingly, the role of the DNA repair gene (PARP1) in therapy resistant cancers also has not been studied and explored well. In this study, we hypothesized that momelotinib could suppress the progression of HCC by targeting Jak family related and PARP1 DNA repair protein. Based on this observation, we link the relevant targets of the JAK family and the potential applications of targeted therapy inhibitors. Methods: We analyzed possible synergism between momelotinib and sorafenib in hepatitis virus-associated liver cancer. Immunostaining, colony formation assay, cell invasion, migration, and tumorsphere-formation assay were used for drug cytotoxicity, cell viability, and possible molecular mechanism. Result: We first demonstrated that the expression of Jak1 and 2 is significantly upregulated in vHCC than in nvHCC/normal liver tissues. In addition, the gene expression of IFN gamma-related pathways is activated after virus infection. Additionally, we found that momelotinib significantly inhibited the growth of HCC cells and reduces the expression of Jak2, which showed the importance of momelotinib in targeting Jak2 and reducing tumorigenesis in HCC. Meanwhile, momelotinib effectively inhibited the IFNGR-JAK-STAT pathway and reduced the migratory/invasive ability of vHCC cells through down-regulating EMT biomarkers (E-cadherin and vimentin), transcription factor (Slug), and significantly inhibits the DNA damage repair enzyme PARP1. It also induced cell apoptosis of vHCC cells. Furthermore, the combined effect of momelotinib and sorafenib both at in vitro and in vivo synergistically suppresses the proliferation of vHCC cells and effectively reduces the tumor burden. Conclusions: Our results showed that momelotinib effectively suppressed the expression of the IFNGR-JAK-STAT-PARP1 pathway, which results in the downregulation of cancer stem cell genes and enhances the antitumor efficacy of sorafenib by initiating the expression of apoptosis-related genes and inhibiting the DNA repair gene in vHCC cells, thus maximizing its therapeutic potential for patients with HCC.

The current standard therapy for metastatic pancreatic cancer is ineffective, necessitating a new treatment approach for prognosis improvement. The urokinase-plasmin activator (uPA) is a critical factor in epithelial-mesenchymal transition (EMT) and cancer metastasis, but its underlying mechanisms in pancreatic cancer remains elusive.We investigated uPA expression in our pancreatic cancer cohort. A bioinformatics approach was used to further determine the role of uPA in pancreatic cancer. We employed MiaPaCa-2 and PANC-1 cell lines to investigate how uPA regulates EMT and metastasis in pancreatic cancer and present a novel approach aimed at inhibiting uPA in pancreatic cancer.We observed that higher uPA mRNA expression was significantly associated with overall-poor survival and progression-free survival in pancreatic cancer. uPA was highly expressed in tumor tissue. Gene set enrichment analysis revealed a positive association between uPA mRNA expression and EMT and transforming growth factor β (TGF-β) signaling pathways. Moreover, shRNA-mediated uPA gene knockdown reduced plasmin, MMP14, and TGF-β activation, leading to the inhibition of PANC-1 cells' EMT marker expression, migration, invasion, and cell viability. Notably, 4-acetyl-antroquinonol B (4-AAQB) treatment suppressed MiaPaCa-2 and PANC-1 cell migratory and invasive abilities by inhibiting the uPA/MMP14/TGF-β axis through upregulation of miR-181d-5p. In the xenograft mouse model of orthotropic pancreatic cancer, 4-AAQB treatment has reduced tumor growth and metastasis rate by deactivating uPA and improving the survival of the mice model.Accordingly, to extent of our knowledge and previous studies, we demonstrated that 4-AAQB is an anti Pan-Cancer drug, and may inhibit pancreatic cancer EMT and metastasis and serve as a new therapeutic approach for patients with late-stage pancreatic cancer.

The authors have requested that this preprint be removed from Research Square.

The authors have requested that this preprint be removed from Research Square.

A bi-cistronic baculovirus expression vector was constructed to facilitate the expression, detection, and isolation of the hemagglutinin (HA) fragment HA1 of H6N1 avian influenza virus (AIV) in an insect and a culture of its cells. In this construct, the GP67sp signal peptide promoted the secretion of the recombinant protein into the culture medium, and improved protein expression and purification. Enhanced green fluorescent protein, co-expressed through an internal ribosome entry site, served as a visible reporter for protein expression detection. The hemolymph of Spodoptera litura larvae infected with the bi-cistronic baculovirus was collected for the purification of the recombinant HA1, which was found to be glycosylated, and monomeric and trimeric forms of the recombinant HA1 were identified. Proteins expressed in both the cell culture and larvae served as effective subunit vaccines for the production of antiserum against HA. The antiserum recognized the H6 subtype of AIV but not the H5 subtype., Highlights • HA1 of H6N1 influenza virus was expressed in insect and cell culture. • The expressed HA1 was glycosylated, and estimated as monomeric and trimeric forms. • The expressed HA1 served as an effective subunit vaccine for producing antisera. • The antisera specifically recognized influenza H6 subtype but not the H5 subtype.

Oral squamous cell carcinoma (OSCC) is among the most prevalent cancers of the head and neck. This study revealed that isoorientin attenuates OSCC cell stemness and epithelial-mesenchymal transition potential through the inhibition of JAK/signal transducer and activator of transcription 3 (STAT3) and Wnt/β-catenin signaling in cell lines. Our findings indicated that isoorientin is a potential inhibitor of β-catenin/STAT3 in vitro and in vivo. We analyzed possible synergism between isoorientin and cisplatin in OSCC. A sulforhodamine B assay, colony formation assay, tumorsphere-formation assay, and Wnt reporter activity assay were used for determining cell invasion, cell migration, drug cytotoxicity, and cell viability with potential molecular mechanisms in vitro. Isoorientin reduced the expression of p-STAT3, β-catenin, and p-GSK3 as well as downstream effectors TCF1/TCF7 and LEF1 and significantly reduced β-catenin colocalization in the nucleus. Isoorientin markedly strengthened the cytotoxic effects of cisplatin against SAS and SCC-25. Therefore, combining isoorientin and cisplatin treatments can potentially improve the anticancer effect of cisplatin. Isoorientin inhibited the tumorigenicity and growth of OSCC through the abrogation of Wnt/β-catenin/STAT3 signaling in vivo. Thus, isoorientin disrupted the β-catenin signaling pathway through the inactivation of STAT3 signaling. In conclusion, targeting OSCC-SC-mediated stemness with isoorientin to eradicate OSCC-SCs may be an effective strategy for preventing relapse and metastasis of OSCC and providing long-term survival benefits.

Neutralizing antibodies on the globular head of the hemagglutinin (HA) of avian influenza virus (AIV) are crucial for controlling this disease. However, most neutralizing antibodies lack cross reaction. This report describes the identification of a hemagglutinin epitope on the globular head near the receptor binding site of the H6N1 AIV. A monoclonal antibody named EB2 was prepared against the H6N1 AIV HA. Flow cytometry of AIV-infected chicken embryo fibroblast, DF-1 cells and specific-pathogen-free embryonated eggs were used to verify the neutralizing activity of this mAb. To narrow down the binding region, partially overlapping HA fragments and synthetic peptides were used to map the epitope by immune-blotting. The linear motif RYVRMGTESMN, located on the surface on the globular head of the HA protein, was identified as the epitope bound by EB2 mAb. Alignment of the EB2-defined epitope with other H6 AIVs showed that this epitope was conserved and specific to H6. We propose that this motif is a linear B-cell epitope of the HA protein and is near the receptor binding site. The identified epitope might be useful for clinical applications and as a tool for further study of the structure and function of the AIV HA protein.

This study established a novel method of pre-screening peptides for monoclonal antibody (mAb) production. Whole virus particles were used as antigens to produce mAbs in the first stage. However, most mAbs obtained from this method were aimed toward hemagglutinin. For this reason, synthetic peptides were used as antigens for mAb production that aimed at the AIV proteins with low abundance or poor immunogenicity in the virus particle. The peptides that showed high immunogenicity were designed using bioinformatic tools for immunization. For high-throughput, a rabbit was used to screen the immunogenicities of the synthetic peptides. Those showed high immunity were used for mAb preparation in mice. Several new mAbs against PB2, PA, M1, M2, NS1 and NS2 proteins were successfully obtained in this study. Furthermore, the epitopes of M1 and NS1 mAbs were determined using competitive western blot assay and competitive ELISA. This study might simplify the mAb preparation and serves as the basis for developing mAb against poor immunogenic proteins.