CRISPR-Cas9 technology: As an efficient genome modification tool in the cancer diagnosis and treatment.

Cancer is the second most common cause of death globally and is a major public health concern. Managing this disease is difficult due to its multiple stages and numerous genetic and epigenetic changes. Traditional cancer diagnosis and treatment methods have limitations, making it crucial to develop new modalities to combat the increasing burden of cancer. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has transformed genetic engineering due to its simplicity, specificity, low cytotoxicity, and cost-effectiveness. It has been proposed as an effective technology to enhance cancer diagnosis and treatment strategies. This article presents the most recent discoveries regarding the structure, mechanism, and delivery methods of the highly powerful genome editing tool, CRISPR-Cas9. In terms of diagnosis, the article examines the role of CRISPR-Cas9 in detecting microRNAs and DNA methylation, and discusses two popular gene detection techniques that utilize the CRISPR-Cas system: DNA endonuclease-targeted CRISPR trans reporter and specific high sensitivity enzymatic reporter unlocking. Regarding treatment, the article explores several genes that have been identified and modified by CRISPR-Cas9 for effective tumorigenesis of common cancers such as breast, lung, and colorectal cancer. The present review also addresses the challenges and ethical issues associated with using CRISPR-Cas9 as a diagnostic and therapeutic tool. Despite some limitations, CRISPR-Cas9-based cancer diagnosis has the potential to become the next generation of cancer diagnostic tools, and the continuous progress of CRISPR-Cas9 can greatly aid in cancer treatment.

Rapid Isolation of Gastric Adenocarcinoma Cancer Stem Cells as a Target for Autologous Dendritic Cell-Based Immunotherapy.

Background: Gastric cancer (GC) is a malignancy cause associated with a high death rate in the world. Cancer stem cells (CSCs) are a rare immortal subpopulation of cells within tumors with characteristics of the ability to self-renew, initiate tumor, and differentiate into defined progenies as well as and high resistance to conventional therapies. Objectives: Despite the use of surgery and chemotherapy for GC therapy, there are no efficient therapeutic protocols for it to date. Therefore, rapid isolation of CSCs in order to therapeutic targets, especially immunotherapy is very important. Materials and Methods: Cancerous cell suspension isolated from patients with GC was cultured in the serum-free medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions to generate spheres. Expression of mRNA level stemness transcription factors (OCT4, SOX2, SALL4, and Cripto-1), CD44 variable isoforms (CD44s, CD44v3, CD44v6, CD44V8-10) of spheroid-forming single cells compared with gastric normal tissue cells using real time PCR and molecules of CD44, CD54, and EpCAM as gastric CSC markers, and stemness factor Oct4 using flow cytometry, as well as tumorgenicity using subcutaneous injection of sphere-forming cells to nude mice were investigated. Results: Few cancerous cells isolated from patients with GC were able to generate three-dimensional spheroid colonies in the serum-free medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions, and form xenograft tumors in immunodeficient nude mice after subcutaneous injection. Spheroid-forming single cells upregulated stemness transcription factors OCT4, SOX2, SALL4, and Cripto-1 that are associated with pluripotency and self-renewal and CD44 isoforms (CD44s, CD44v3, CD44v6, CD44V8-10) compared with gastric normal tissue cells. Finally, molecules of CD44, CD54, and EpCAM as gastric CSC markers and stemness factor Oct4 were expressed in sphere-forming cells. Conclusion: We suggested that the sphere formation and tumorigenicity assays are two procedures, leading to the rapid isolation of cancer cells with certain stem-like properties in order to target CSCs using autologous dendritic cell therapy, especially in patients with advanced disease.

Conjugated PNC-27 peptide/PEI-superparamagnetic iron oxide nanoparticles (SPIONs) as a double targeting agent for early cancer diagnosis: In vitro study.

Objectives: Superparamagnetic iron oxide nanoparticles (SPIONs) have been considered promising non-invasive imaging tools in medicine. However, their high surface energy leads to NPs aggregation, while non-targeted SPIONs can cause cytotoxic effects on normal cells. In this work, we evaluated the in vitro potential of polyethyleneimine (PEI)-SPIONs targeted by PNC-27 peptide as a double targeting agent throughout early cancer diagnosis. Materials and Methods: Initially, PEI was conjugated to PNC-27 with HDM-2-binding domain. Then, SPIONs were loaded into PEI-PNC-27 through the ligand exchange method. The physicochemical characteristics of the synthesized NPs were evaluated. The cytotoxicity and targeting efficiency were assayed against HT-29 and CT-26 cell lines along with NIH-3t3 as normal cells by MTT method and Prussian blue staining test, respectively. Results: The mean diameter of synthesized carriers was obtained in the range of 86.6 - 116.1 nm with a positive charge. According to the cytotoxicity results, the binding and uptake abilities of the PNC-27 peptide by cancer cells were significantly higher than that of the NIH-3t3 cells. However, the results were indicative of the more toxic impacts of targeted synthesized NPs against CT-26 cancer cell line when being compared with HT-29 cells, which may be caused by the different cytotoxicity mechanisms of NPs. In addition, the targeted carriers and SPIONs were present inside and around the cells with HDM-2 expression along with only a few non-targeted vectors, while displaying no appearance throughout the normal cell. Conclusion: The results indicated the efficiency of targeted PEI-coated SPIONs for cancer diagnostic applications.

Establishment and characterization of chemotherapy-enriched sphere-forming cells with stemness phenotypes as a new cell line (BAG50) of gastric carcinoma.

Gastric cancer is a malignancy with a high mortality rate worldwide. Cancer stem cells (CSCs) are a small subpopulation of tumor cells that possess the tumor-initiating ability, self-renewal capacity, and high resistance to conventional therapies. Due to the diversity and complexity of human tumors, new cell lines are urgently needed to supply clinically and physiologically relevant cancer models. Here, we report establishing a novel cell line (BAG50) with stemness properties. Chemotherapy-enriched sphere-forming cells with CSC properties isolated from a patient with GC were cultured in a serum-containing medium and passaged for up to 51 passages. The colony-forming ability and tumor-forming capacity of BAG50 cells were evaluated in vitro and in vivo. mRNA upregulation of stemness-related transcriptional factors using real-time PCR as well as expression of CSC markers using flow cytometry was investigated. Finally, STR profiling and chromosome studies were performed. BAG50 cells formed floating spheroid colonies in a serum-free medium. Subcutaneous injection of these cells generated xenograft tumors in nude mice. Pluripotency markers (SOX-2, OCT4, and Cripto-1) in them were upregulated compared with normal gastric cells. The majority of them expressed CSC markers of CD44, CD54, and EpCAM, and stemness marker of oct-4. STR profiling showed a unique DNA fingerprint. Karyotype also demonstrated multiple aneuploidies and chromosomal translocations. We suggested that the highly tumorigenic BAG50 cell line with stem cell-like phenotypes may provide a valuable in vitro tool to support new diagnostic, prognostic, and predictive biomarkers as well as the development of more effective treatment strategies.

Elucidated tumorigenic role of MAML1 and TWIST1 in gastric cancer is associated with Helicobacter pylori infection.

BACKGROUND: Epithelial-mesenchymal transition (EMT) has a fundamental role in tumor initiation, progression, and metastasis. Helicobacter pylori (HP) induces EMT and thus causes gastric cancer (GC) by deregulating multiple signaling pathways involved in EMT. TWIST1 and MAML1 have been confirmed to be critical inducers of EMT via diverse signaling pathways such as Notch signaling. This study aimed to investigate for the first time possible associations between TWIST1/MAML1 mRNA expression levels, HP infection, and clinicopathological characteristics in GC patients. METHOD: TWIST1 and MAML1 mRNA expression levels were evaluated in tumoral and adjacent normal tissues in 73 GC patients using the quantitative reverse transcription PCR (RT-qPCR) method. PCR technique was also applied to examine the infection with HP in GC samples. RESULTS: Upregulation of TWIST1 and MAML1 expression was observed in 35 (48%) and 34 (46.6%) of 73 tumor samples, respectively. Co-overexpression of these genes was found in 26 of 73 (35.6%) tumor samples; meanwhile, there was a significant positive correlation between MAML1 and TWIST1 mRNA expression levels (P < 0.001). MAML1 overexpression exhibited meaningful associations with advanced tumor stages (P = 0.006) and nodal metastases (P ˂ 0.001). 34 of 73 (46.6%) tumors tested positive for HP, and meanwhile, MAML1 expression was positively related with T (P = 0.05) and grade (P = 0.0001) in these HP-positive samples. Increased TWIST1 expression was correlated with patient sex (P = 0.035) and advanced tumor grade (P = 0.017) in HP-infected tumors. Furthermore, TWIST1 and MAML1 expression levels were inversely linked with histologic grade in HP-negative tumor samples (P = 0.021 and P = 0.048, respectively). CONCLUSION: We propose TWIST1 and MAML1 as potential biomarkers of advanced-stage GC that determine the characteristics and aggressiveness of the disease. Based on accumulating evidence and our findings, they can be introduced as promising therapeutic targets to modify functional abnormalities in cells that promote GC progression. Moreover, HP may enhance GC growth and metastasis by disrupting TWIS1/MAML1 expression patterns and related pathways.

Expression and Prognostic Significance of Cancer/Testis Antigens, MAGE-E1, GAGE, and SOX-6, in Glioblastoma: An Immunohistochemistry Evaluation.

BACKGROUND & OBJECTIVE: Glioblastoma is the most common primary malignancy of the brain, the prognosis of which is poor. Immunotherapy with cancer/testis (CT) antigens is a novel therapeutic approach for glioblastoma. This study aimed to investigate the expression rate of MAGE-E1, GAGE, and SOX-6 in glioblastoma tumors using the method of immunohistochemistry (IHC). METHODS: Expression of MAGE-E1, GAGE, and SOX-6 were determined by IHC in 50 paraffin blocks of glioblastoma. The results were compared between variables including age, gender, tumor location, and Karnofsky performance status (Kps) score. Survival analysis was also performed. RESULTS: The expression levels of SOX-6, MAGE-E1, and GAGE were 82%, 78%, and 76%, respectively. The relationship between CT antigens and age, gender, and tumor location was not significant, while the association between MAGE-E1 expression and age was statistically significant (P=0.002). High expression levels of SOX-6 and MAGE-E1 were associated with low Kps scores (P=0.034 and P<0.001, respectively). Survival analysis showed that age >40 and Kps score <80 were associated with significant relationship with shorter survival rate. (P=0.005 and P=0.018, respectively). Expression of MAGE-E1 and GAGE was negatively associated with overall 2-year survival rate (P=0.001 and P=0.021, respectively). CONCLUSION: The expression of all the three CT antigens, especially MAGE-E1 and SOX-6, was high in patients with glioblastoma. It can be concluded that these markers could be ideal targets for immunotherapy in such patients. MAGE-E1 and SOX-6 can be considered as important markers in determining the prognosis of glioblastoma.

Correlation between the immune checkpoints and EMT genes proposes potential prognostic and therapeutic targets in ESCC.

PD-1, PD-L1, CTLA-4, TIM-3, and LAG-3, crucial immune checkpoint molecules in the tumor microenvironment, identify as key targets for cancer immunotherapy. There is a correlation between immune cells and epithelial-mesenchymal transition (EMT)-related genes expression in varies human cancers. In this study, we aimed to investigate the probable association between expression of immune checkpoints and EMT in esophageal squamous cell carcinoma (ESCC) with clinical treats for providing the new therapeutic targets and prognostic value for the disease. Quantitative real-time PCR was used to investigate the gene expression profile of immune checkpoints (PD-1, PD-L1, CTLA-4, TIM-3, and LAG-3) and EMT (TWIST1 and MMP-13) genes based on the mRNA expression levels in 51 ESCC tissues. The upregulation of CTLA-4, PD-1, PD-L1, TIM-3, LAG-3, MMP-13, and TWIST1 were observed in 31.37%, 29.41%, 21.56%, 39.21%, 25.49%, 60.78%, and 56.86% of ESCC cases at the mRNA level, respectively. Dysregulation of immune checkpoints was related to lymph node involvement, stage of tumor progression, and depth of tumor invasion (P < 0.05). While overexpression of MMP-13 and TWIST1 was associated with lymph node involvement, stage of tumor progression, and grade of tumor differentiation (P < 0.05). The mRNA expression of immune checkpoint genes was significantly correlated to each other's (P = 0.000). Of importance, the data explored the significant association between the concomitant expression of immune checkpoints and EMT-related genes with each other in a variety of clinicopathological traits (P < 0.05). Consequently, immune checkpoints were positively correlated with EMT status in ESCC. The correlation between tumor immune microenvironment with the elevation of multiple immune checkpoints and EMT status may help to identify potential biomarkers for the simultaneous clinical use of multiple immune checkpoints blockade and other immunotherapies approaches for advanced ESCC patients.

Crosstalk between MMP-13, CD44, and TWIST1 and its role in regulation of EMT in patients with esophageal squamous cell carcinoma.

Matrix metalloproteinases (MMPs) play key roles in epithelial-mesenchymal transition (EMT) for the development of cancer cell invasion and metastasis. MMP-13 is an extracellular matrix (ECM)-degrading enzyme that plays crucial roles in angiogenesis, cell cycle regulation, niche maintenance, and transforming squamous epithelial cells in various tissues. CD44, a transmembrane glycoprotein expressed on esophageal tumor cells, is required for EMT induction and invasion in esophageal squamous cell carcinoma (ESCC). The transcription factor TWIST1, as EMT and stemness marker, regulates MMPs expression and is identified as the downstream target of CD44. In this study, we aimed to investigate the probable interplay between the expression of key genes contributing to ESCC development, including MMP-13, TWIST1, and CD44 with clinical features for introducing novel diagnostic and therapeutic targets in the disease. The gene expression profiling of MMP-13, TWIST1, and CD44 was performed using quantitative real-time PCR in tumor tissues from 50 ESCC patients compared to corresponding margin non-tumoral tissues. Significant overexpression of MMP-13, CD44S, CD44V3, CD44V6, and TWIST1 were observed in 74%, 36%, 44%, 44%, and 52% of ESCC tumor samples, respectively. Overexpression of MMP-13 was associated with stage of tumor progression, metastasis, and tumor location (P < 0.05). There was a significant correlation between TWIST1 overexpression and grade (P < 0.05). Furthermore, overexpression of CD44 variants was associated with stage of tumor progression, grade, tumor invasion, and location (P < 0.05). The results indicated the significant correlation between concomitant expression of MMP-13/TWIST1, TWIST1/CD44, and CD44/MMP-13 with each other in a variety of clinicopathological traits, including depth of tumor invasion, tumor location, stage of tumor, and lymph node involvement in ESCC tissue samples (P < 0.05). Collectively, our results indicate that the TWIST1-CD44-MMP-13 axis is involved in tumor aggressiveness, proposing these genes as regulators of EMT, diagnostic markers, and therapeutic targets in ESCC.

Allogeneic tumor cell line-based vaccines: A good alternative to autologous and cancer stem cell vaccines in colorectal cancer.

OBJECTIVES: Besides the uncertainty about colorectal cancer stem cell (CCSC) markers, isolating, purifying, and enriching CCSCs to produce CCSC vaccines is highly challenging. However, allogeneic vaccines developed from CRC cell lines can provide universal, comprehensive, inexpensive, simple, and fast approach to cancer treatment. MATERIALS AND METHODS: CCSCs were isolated from human CRC tissue using the in vitro sphere formation assay and then characterized through gene expression analysis, in vivo and in vitro tumor formation assay, karyotyping, and surface marker detection. Subsequently, CCSCs and two CRC cell lines (HT-29 and SW-480) were inactivated with cisplatin (CDDP) and administrated as vaccines to the three groups of athymic C57BL/6 nude mice. Afterward, tumorigenesis was challenged with HT-29 cells. The antitumor effect of vaccines was evaluated by tumor and spleen examination and immune response analysis. The cytotoxic activity of splenocytes and serum levels of TGF-ß and IFN-γ were measured by Calcein-AM cytotoxicity assay and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: The results of gene expression analysis showed that CCSCs are CD44+CD133-LGR5-. All vaccinations resulted in decreased tumor growth, spleen enlargement, enhanced serum level of IFN-γ and TGF-ß, and increased cytotoxic activity of natural killer (NK) cells. The antitumor efficacy of the CCSC vaccine was not more than CRC cell line-based vaccines. Interestingly, the allogeneic SW-480 vaccine could effectively inhibit tumorigenesis. CONCLUSION: Despite the great challenge in developing CCSC vaccines, allogeneic vaccines based on CRC cell lines can efficiently induce antitumor immunity in CRC.

Novel Biomarkers Aim at Detecting Metastatic Sentinel Lymph Nodes in Breast Cancer

Background: Intra-operative molecular diagnostic assays are currently used for the detection of lymph node metastases. The objective of this study was to find new biomarkers to improve diagnostic accuracy in the detection of metastatic axillary lymph nodes in breast cancer patients. Methods: We applied an absolute quantitative real-time reverse transcription-PCR to quantitate the expression of CK19, KLK11, and CLEC3A mRNAs in 79 FFPE sentinel lymph nodes (SLNs) from 35 breast cancer patients. The CK19 was confirmed as a standard biomarker, and the level of expression of selected new markers, KLK11 and CLEC3A, was evaluated in pathologically negative and positive SLNs by using absolute quantitative real-time PCR. Results: The overall concordance of the CK19 gene with pathological results was 92.4% (less than 250 copies) in negative SLNs and 85% in positive SLNs (more than 250 copies). The sensitivity and specificity of CK19, which were detected by real-time PCR, was 85% and 46%, respectively. Our results revealed that lower CLEC3A was associated with more lymph node involvement. We could set a cut-off point for CLEC3A with the sensitivity of 78% and specificity of 60%. Also, the mean KLK11 had a statistically significant reverse correlation with tumor grade (p = 0.017). Higher CK19 levels were related to more tumor invasion (p < 0.0001). Conclusion: Regarding the findings, CLEC3A along with CK19 can be used as a promising marker with high sensitivity and specificity for the detection of metastatic SLN.

The Role of Interleukin-4 and 13 Gene Polymorphisms in Allergic Rhinitis: A Case Control Study.

BACKGROUND: Allergic Rhinitis (AR) is an IgE-mediated inflammatory disorder with high morbidity rates. The eitiology of this disease is understood to occur from a complex interaction between genetic and environmental factors. T helper type 2 cells have been shown to have a crucial role in atopic disease due to their production of the cytokines, intelukin (IL)-13 and IL-4, involved in inflammation. Research has shown single nucleotide polymorphisms (SNP) of the IL-13 and IL-4 genes to be associated increased levels of IgE and with allergic diseases such as, allergic rhinitis, asthma, and atopic dermatitis. Specifically, the rs2243250 SNP of IL-4 and the rs20541 SNP of IL-13 have been shown to be associated with AR. METHODS: A case-control study was designed to investigate the relationship between the two SNPs rs2243250 and rs20541 with the incidence of AR. The SNPs were examined in patients with AR and healthy controls (86 patients and 86 controls). Blood samples were collected and DNA was extracted to evaluate the SNPs by RFLP-PCR. RESULTS: Recessive analysis model of the IL-13 gene (GG vs. AA+AG) revealed that the GG genotype was more common in AR patients (P=0.36) )OR=0.8 [81% CI 0.38-1.6]). For the IL-4 gene (TC vs. TT+CC), the TC genotype was more common in AR patients (P = 0.0022)) OR=0.71 [60% CI 1.41-5.02]). Furthermore, in the IL-4 gene, the 590 T>C polymorphism had a significant association with AR. However, no association was found between AR and the IL-13 rs20541 polymorphism. CONCLUSION: Our findings suggest that the IL-13 polymorphism (rs20541, Exo 4, G>A, Arg130Gln) and IL-4 polymorphism (rs2243250= C-590T, promoter, T>C) are co-associated with AR and sensitivity to aeroallergens. However, this study used a cohort of AR patients and healthy controls from the northeast of Iran. Given the influence of ethnicity and environment on genetics, further investigation is needed to elucidate the role of SNPs in IL-4 and IL-13 in AR among different populations.

Role of MAML1 in targeted therapy against the esophageal cancer stem cells.

BACKGROUND: Esophageal cancer is the sixth-leading cause of cancer-related deaths worldwide. Cancer stem cells (CSCs) are the main reason for tumor relapse in esophageal squamous cell carcinoma (ESCC). The NOTCH pathway is important in preservation of CSCs, therefore it is possible to target such cells by targeting MAML1 as the main component of the NOTCH transcription machinery. METHODS: In present study we isolated the CD44+ ESCC CSCs and designed a MAML1-targeted therapy to inhibit the NOTCH signaling pathway. CSCs were isolated using magnetic cell sorting utilizing the CD44 cell surface marker. Several stem cell markers were analyzed in the levels of protein and mRNA expression. The isolated CSCs were characterized in vivo in NUDE mice. Biological role of MAML1 was assessed in isolated CD44+ CSCs. A drug resistance assay was also performed to assess the role of MAML1 in CD44+ CSCs with 5FU resistance. RESULTS: The CD44+ CSCs had ability to form tumors in NUDE mice. MAML1 silencing caused a significant decrease (p = 0.019) and ectopic expression caused a significant increase in migration of CD44+ CSCs (p = 0.012). Moreover, MAML1 silencing and ectopic expression significantly increased and decreased 5FU resistance, respectively (p < 0.05). MAML1 silencing significantly increased the number of cells in G1 phase (p = 0.008), and its ectopic expression significantly increased the number of CD44+ CSCS in S phase (p = 0.037). CONCLUSIONS: MAML1 may be utilized for targeted therapy with a low side effect to eliminate the CD44+ CSCs through inhibition of canonical NOTCH pathway in ESCC patients.

Correlation between expression of CatSper1,2 and sperm parameters in the gamma irradiated adult mouse testis.

Propose: CatSper protein channels are responsible for the entry of Ca2+ into sperm cells. These proteins play an important role in motility and male fertility. So it is important to find out whether or not environmental factors, such as gamma radiation, have an effect on the expression of Catsper genes. In this study, we investigated the effects of gamma radiation on the expression of CatSper1 and CatSper2 genes. Materials and methods: Twenty-one male NMRI mice were divided into three groups: a control group without gamma radiation, and two experimental groups; Group 1 treated with 1 Gy of gamma radiation, and Group 2 treated with a higher dose of 2 Gy gamma radiation. Testes were removed from all groups of animals 35 days following irradiation and the testicular tissue, processed and embedded in paraffin blocks for sectioning and histological examination. Sperm samples were also taken from the epididymis for microscopic. Sperm parameters such as sperm count, morphology, motility, and viability rates were analyzed. Expression of CatSper genes was evaluated using Real-time PCR. Data were analyzed using the SPSS software and ANOVA test. Results: Our results showed that after treatment with gamma radiation, testes morphology was changed. Epididymal sperm count, motility, and morphology rates were significantly affected in both experimental groups compared to the control group. The relative expressions of CatSper 1 and 2 genes were significantly reduced in the irradiated mice (1 Gy and 2 Gy) than non-irradiated ones. Conclusions: Gamma radiations not only change testes histology and sperm parameters, but also decrease the expression of CatSper 1 and 2 genes in male mice.

Induction of T cell-mediated immune response by dendritic cells pulsed with mRNA of sphere-forming cells isolated from patients with gastric cancer.

Gastric cancer (GC) as the third most common cause of cancer-associated mortality worldwide is one of the cancers with very high heterogeneity. Cancer stem cells (CSCs) as a small subset of cancer cells in solid tumors with the self-renewal, differentiation and tumorigenic ability are responsible for tumor initiation, progression, recurrence, metastasis, and resistance to current treatments. Therefore, eradication of CSCs is very vital to cure cancer. Here, we first isolated and identified sphere-forming cells in tumor tissue from four GC patients and then analyzed T cell responses induced by monocyte-derived dendritic cells (DCs) loaded with total mRNA of sphere-forming cells in terms of interferon-gamma (IFN-γ) gene expression and specific cytotoxicity. Spheroid colonies were formed in serum-free media. Sphere-forming cells dissociated from tumorspheres heterogeneously expressed CD44, CD54, and epithelial cell adhesion molecule (EpCAM) markers and generated one tumor in nude mice. These results demonstrated that gastric CSCs were enriched in tumorspheres. Cytokine-matured DCs loaded with mRNA of sphere-forming cells were able to induce IFN-γ gene expression in T-lymphocytes after a 12-day co-culture. mRNA level of IFN-γ gene in these lymphocytes was more highly expressed compared to stimulated T-lymphocytes by DCs transfected with normal tissue (6.4-9.39 folds). Cytotoxic activity of primed T-lymphocytes with antigens of sphere-forming cells was significantly higher than normal tissue antigens and mock DCs (P ≤ 0.0001). Taken together, DCs loaded with mRNA of sphere-forming cells that elicit effectively specific T cell-mediated immune responses in vitro, may be considered as a promising therapeutic vaccination in GC patients in future.

Ectopic Expression of Human DPPA2 Gene in ESCC Cell Line Using Retroviral System.

BACKGROUND: Cancer/Testis Antigens (CTAs) are a sub-group of tumor-associated antigens which are expressed normally in germ line cells and trophoblast, and aberrantly in a variety of malignancies. One of the most important CTAs is Developmental Pluripotency Associated-2(DPPA2) with unknown biological function. Considering the importance of DPPA2 in developmental events and cancer, preparing a suitable platform to analyze DPPA2 roles in the cells seems to be necessary. METHODS: In this study, the coding sequence of DPPA2 gene was amplified and cloned into the retroviral expression vector to produce recombinant retrovirus. The viral particles were transducted to Esophageal Squamous Cell Carcinoma (ESCC) cell line (KYSE-30 cells) and the stable transducted cells were confirmed for ectopic expression of DPPA2 gene by real-time PCR. RESULTS: According to the critical characteristics of retroviral expression system such as stable and long time expression of interested gene and also being safe due to deletion of retroviral pathogenic genes, this system was used to induce expression of DPPA2 gene and a valuable platform to analyze its biological function was prepared. Transduction results clearly showed efficient overexpression of the gene in target cells in protein level due to high level of GFP expression. CONCLUSION: Such strategies can be used to produce high levels of desired protein in target cells as a therapeutic target. The produced recombinant cells may present a valuable platform to analyze the effect of DPPA2 ectopic expression in target cells. Moreover, the introduction of its potential capacity into the mouse model to evaluate the tumorigenesis of these cancer cells in vivo leads to an understanding of the biological importance of DPPA2 in tumorigenesis. In addition, our purified protein can be used in a mouse model to produce specific antibody developing a reliable detection of DPPA2 existence in any biological fluid through ELISA system.

Isolation and identification of chemotherapy-enriched sphere-forming cells from a patient with gastric cancer.

Gastric cancer (GC) is the third and fifth cause of cancer-associated mortality for men and women throughout the world, respectively. Despite the use of surgery and chemotherapy for GC therapy, there are no efficient therapeutic protocols for it to date. Cancer stem cells (CSCs) due to their pivotal role in tumor initiation, growth, progression, invasion, distant metastasis, recurrence and resistance to anticancer drugs are very appealing targets for cancer therapies. Here, we isolated and identified CSCs from a chemotherapy-treated patient. Small subpopulation of dissociated cells after tissue digestion formed spheroid colonies in serum-free media under the non-adherent condition. These spheroid colonies differentiated into epithelial like cells in serum-containing medium. Few sphere-forming cells carried CD44 and CD54 markers overexpressed DLL4 that is responsible for tumor growth and angiogenesis. Subcutaneous injections of sphere-forming cells in different passages conferred tumorigenicity in nude mice. Sphere-forming cells upregulated CD44 polymorphisms CD44v3, -v6, and -v8 -10, stemness factors OCT4, SOX2, SALL4 and Cripto-1, self-renewal molecules IHh, Wnt, ß-catenin and BMI1, and epithelial mesenchymal transition (EMT) markers Twist1 and Snail1 in vitro and in vivo. Moreover, these cells similar to sphere-forming cells isolated from a chemotherapy-free patient expressed Oct-4 and ß-catenin proteins. However, the Twist1 protein was only expressed by sphere-forming cells derived from the chemotherapy-treated patient. Thus, these cells have all the characteristics of stationary and migratory CSCs, including tumorigenicity, self-renewal, pluripotency, invasion and metastasis. Taken together, targeting chemotherapy-enriched CSCs as chemo-resistance cells observed in GC patients can provide more effective therapeutic strategies compared to untreated patients.

Association of Two CD44 Polymorphisms with Clinical Outcomes of Gastric Cancer Patients

Objective: CD44 is an important cell adhesion molecule that plays a key role in growth, invasion, proliferation and metastasis of cancer cells. CD44 protein over-expression is associated with a poor prognosis of gastric cancer (GC) and previous studies have shown that CD44 gene polymorphisms could affect survival and recurrence. In this study, we tested the hypothesis that polymorphisms impacting on the CD44 signaling pathway may predict clinical outcomes in patients with GC. Materials and Methods: DNA was extracted from blood of 150 healthy individuals and formalin-fixed paraffin-embedded (FFPE) tumor tissue of 150 patients. The two polymorphisms rs187116 and rs7116432 were studied by RFLP-PCR and sequencing techniques. Results: There was a strong significant correlation between single nucleotide polymorphisms (SNPs) in the CD44 gene, tumor recurrence, and overall survival (p <0.0001). The existence of a significant relationship between tumor recurrence and overall survival was proved in this study, with at least one allele G for the polymorphism rs187116 and at least one allele A for polymorphism rs7116432. Conclusion: These results provide evidence of a relationship between CD44 gene polymorphisms and clinical outcomes in our GC patients. This result could help identify individuals with GC who have a high risk of tumor recurrence.

Cytokine networks and their association with Helicobacter pylori infection in gastric carcinoma.

Cytokine networks as dynamic networks are pivotal aspects of tumor immunology, especially in gastric cancer (GC), in which infection, inflammation, and antitumor immunity are key elements of disease progression. In this review, we describe functional roles of well-known GC-modulatory cytokines, highlight the functions of cytokines with more recently described roles in GC, and emphasize the therapeutic potential of targeting the complex cytokine milieu. We also focus on the role of Helicobacter pylori (HP)-induced inflammation in GC and discuss how HP-induced chronic inflammation can lead to the induction of stem cell hyperplasia, morphological changes in gastric mucosa and GC development.

Biological and Clinicopathological Significance of Cripto-1 Expression in the Progression of Human ESCC.

BACKGROUND: Human Cripto-1, a member of the EGF-CFC family, is involved in embryonic development, embryonic stem cell maintenance, and tumor progression. It also participates in multiple cell signaling pathways including Wnt, Notch, and TGF-ß. Remarkably, it is expressed in cancer stem cell (CSC) compartments, boosting tumor cell migration, invasion, and angiogenesis. Although Cripto-1 is overexpressed in a variety of human malignant tumors, its expression in esophageal squamous cell carcinoma (ESCC) remains unclear. Our aim in this study was to evaluate the possible oncogenic role of Cripto-1 in ESCC progression and elucidate its association with clinicopathological parameters in patients. METHODS: In this study, Cripto-1 expression in 50 ESCC tissue samples was analyzed and compared to corresponding margin-normal esophageal tissues using quantitative real-time PCR. RESULTS: Cripto-1 was overexpressed in nearly 40% of ESCC samples compared with normal tissue samples. Significant correlations were observed between Cripto-1 expression and tumor differentiation grade, progression stage, and location (p < 0.05). CONCLUSIONS: Our results indicate that overexpression of Cripto-1 is involved in the development of ESCC. Further assessment will be necessary to determine the role of Cripto-1 cross talk in ESCC tumorigenesis.

GATA3 Gene Polymorphisms Associated with Allergic Rhinitis in an Iranian Population.

BACKGROUND: The development of allergic rhinitis (AR) is caused by the interaction between genetic predisposition and environmental factors. In this study, the association between GATA3 single nucleotide polymorphisms and AR in an Iranian population was identified. METHODS: This case-control study was performed on 86 patients with AR and 86 healthy subjects. This study aimed to evaluate a potential association between two GATA3 SNPs, rs1269486 and rs2229360, and AR. Blood samples were collected and DNA was extracted for the evaluation of these SNPs by RFLP-PCR. RESULTS: A statistically-significant association was found between rs1269486 and AR (P<0.001). The frequencies of the A and GA genotypes were less in patients than in controls. The frequencies of the G allele and the GG genotype were greater in patients than in controls (P < 0.001). CONCLUSIONS: SNP rs1269486 of GATA3 was associated with AR and sensitivity to aeroallergens in our population. Because of the significance of this gene in AR, studying the association between GATA3 polymorphisms and AR is recommended for other populations.