Exploring heterogeneous expression of beta-actin (ACTB) in bladder cancer by producing a monoclonal antibody 6D6.

BACKGROUND: To predict outcomes and identify potential therapeutic targets for cancers, it is critical to find novel specific biomarkers. The objective of this study was to search for and explore novel bladder cancer-associated protein biomarkers. METHODS: A library of monoclonal antibodies (mAbs) against the JAM-ICR cell line was first generated, and clones with high affinity were selected. Hybridomas were screened using bladder cancer (BLCA) cell lines and normal cells. The target of the selected mAb was then characterized through immunoaffinity purification, western blotting, and mass spectrometry analysis. Expression of the target antigen was assessed by flow cytometry and IHC methods. Several databases were also used to evaluate the target antigen in BLCA and other types of cancers. RESULTS: Based on screenings, a 6D6 clone was selected that recognized an isoform of beta-actin (ACTB). Our data showed that ACTB expression on different cell lines was heterogeneous and varied significantly from low to high intensity. 6D6 bound strongly to epithelial cells while showing weak to no reactivity to stromal, endothelial, and smooth muscle cells. There was no association between ACTB intensity and related prognostic factors in BLCA. In silico evaluations revealed a significant correlation between ACTB and overexpressed genes and biomarkers in BLCA. Additionally, the differential expression of ACTB in tumor and healthy tissue as well as its correlation with survival time in a number of cancers were shown. CONCLUSIONS: The heterogeneous expression of ACTB may suggest the potential value of this marker in the diagnosis or prognosis of cancer.

Enhancing cancer immunotherapy with Anti-NKG2D/IL-15(N72D)/Sushi fusion protein: Targeting cytotoxic immune cells and boosting IL-15 efficacy.

BACKGROUND: There are a number of distinct challenges and complexities associated with administering IL-15 for cancer immunotherapy that must be taken into consideration. OBJECTIVE: The purpose of this study was to design a fusion protein for targeting cytotoxic immune cells and enhance IL-15 efficiency. METHODS: A fusokine that contains IL-15(N72D), a Sushi domain, and anti-NKG2D scFv was designed. The fusion protein was in-silico modeled using the Swiss model server, followed by docking and molecular dynamics simulations. The in-vitro purified fusokine was evaluated using dot blot and Western blot. Then, flow cytometry was employed to evaluate biological properties such as proliferation, cytotoxicity, and degranulation. RESULTS: Fusokine and IL-15(N72D)/Sushi, which had molecular weights of about 52 kDa and 26 kDa, respectively, were expressed in CHO-K1 cells. The fusokine binds 69.6 % of the CHO-NKG2D+ cells that express 83.1 % NKG2D. Both the fusokine and the IL-15(N72D)/Sushi significantly stimulate the proliferation of lymphocytes. After 14 days of growth, the vitality of untreated cells decreased to about 17.5 %, but 82.2 % and 56.6 % of cells were still alive when fusokine and IL-15(N72D)/Sushi were present. Furthermore, administration of fusokine was associated with the highest rates of target tumor cell cytotoxicity. Additionally, although it was not statistically significant, fusokine increased the expression of CD107a and granzyme B by 1.25 times and 2.4 times, respectively. CONCLUSION: The fusokine possesses the capability to stimulate the survival and multiplication of lymphocytes, as well as their ability to eliminate tumors. These characteristics have led to its consideration as a potential treatment for immunotherapy.

Anti-CD19/CD8 bispecific T cell engager for the potential treatment of B cell malignancies.

The administration of blinatumomab was accompanied by several adverse effects, including activation of regulatory T-cells and cytokine storm. The objective of this study was to produce and evaluate a novel αCD8/CD19 BiTE (αCD8/CD19) with the potency to directly target CD8+T-cells. In-silico studies were utilized for determining proper folding, receptor binding, and structural stability of αCD8/CD19 protein. Western blotting and indirect surface staining were used to evaluate the size accuracy and binding potency of the purified protein. Functionality was assessed for granzyme B production, cytotoxicity, and proliferation. TheαCD8/CD19recombinant protein was produced in the CHO-K1 cell line with a final concentration of 1.94 mg/l. The αCD8/CD19 bound to CD8+and CD19+cell lines and induced significant granzyme B production, cytotoxic activity and proliferation potential in the presence of IL-2 and tumor target cells. The maximum CD8+T-cell biological activity was observed on the 10th day with 10:1 effector-to-target ratio.

Dual Functions of T Lymphocytes in Breast Carcinoma: From Immune Protection to Orchestrating Tumor Progression and Metastasis.

Breast cancer (BC) is the most common cancer type in women and the second leading cause of death. Despite recent advances, the mortality rate of BC is still high, highlighting a need to develop new treatment strategies including the modulation of the immune system and immunotherapies. In this regard, understanding the complex function of the involved immune cells and their crosstalk with tumor cells is of great importance. T-cells are recognized as the most important cells in the tumor microenvironment and are divided into several subtypes including helper, cytotoxic, and regulatory T-cells according to their transcription factors, markers, and functions. This article attempts to provide a comprehensive review of the role of T-cell subsets in the prognosis and treatment of patients with BC, and crosstalk between tumor cells and T-cells. The literature overwhelmingly contains controversial findings mainly due to the plasticity of T-cell subsets within the inflammatory conditions and the use of different panels for their phenotyping. However, investigating the role of T-cells in BC immunity depends on a variety of factors including tumor types or subtypes, the stage of the disease, the localization of the cells in the tumor tissue and the presence of different cells or cytokines.

Production of a biosimilar version of aflibercept to improve VEGF blocker cytotoxicity on endothelial cells.

This project aimed to produce a biosimilar version of aflibercept (AFL) and evaluate the effect of the co-treatment of AFL with other vascular endothelial growth factor (VEGF) blocker drugs. For this purpose, the optimized gene was inserted into the pCHO1.0 plasmid and transfected into the CHO-S cell line. The final concentration of biosimilar-AFL for the selected clone was 782 mg/L. Results revealed that the inhibition potential of the biosimilar-AFL on HUVEC cells was significant at 10 and 100 nM concentrations and in a dose-dependent manner. Furthermore, co-treatment of biosimilar-AFL with Everolimus (EVR), Lenvatinib (LEN), and Sorafenib (SOR) could reduce HUVEC cell viability/proliferation, more than when used alone. When LEN and SOR were co-treated with biosimilar-AFL, their cytotoxicity increased 10-fold. The most and least efficient combination was seen when biosimilar-AFL combined with LEN and EVR, respectively. Finally, biosimilar-AFL may improve the efficiency of LEN, EVR, and SOR in reducing the VEGF effect on endothelial cells.

GM-CSF-producing lymphocytes in tumor-draining lymph nodes of patients with bladder cancer.

Bladder cancer (BC) is the tenth common cancer worldwide. Despite progress in treatment and the use of chemotherapoeutic drugs, the survival rate of BC patients is still low. Manipulation of the immune system was recently introduced as an interesting alternative treatment for this immunogenic cancer with fewer side effects. Accordingly, in the present study, we assessed the frequency of GM-CSF-producing lymphocytes in tumor-draining lymph nodes (TDLNs) of BC patients and evaluated their relationship with clinicopathological factors and survival rate. Fifty-four patients with BC who had received no treatment were recruited. Mononuclear cells were isolated from fresh homogenized lymph nodes by centrifugation over Ficoll-Hypaque, activated and subsequently analyzed by flow cytometry for the cell surface expression of CD4 and CD8 and the intracellular production of GM-CSF. Flow cytometric analysis revealed that 4.97 ± 2.7% of lymphocytes in TDLNs of patients with BC produced GM-CSF. The mean frequency of GM-CSF-producing cells was 5.5% among CD4+ lymphocytes and 11.7% in the CD8+ population. Elevated frequencies of GM-CSF-producing lymphocytes, as well as a higher production of GM-CSF by CD4+ lymphocytes was observed in the patients with tumor-free lymph nodes, as compared to those with at least one tumor-infiltrated lymph node (p < 0.05). On the other hand, the lower frequency of GM-CSF-producing CD4+ lymphocytes (ThGM) was associated with improved overall, but not one-year, survival. No other significant relationship was observed between clinicopathological parameters and the frequency of GM-CSF-producing subsets. Collectively, our findings suggest a protective role for GM-CSF in the early stages of BC; however, the unfavorable association of ThGM frequency with survival rate may imply a more complex role for this cytokine in BC.

Establishment of a bladder cancer cell line expressing both mesenchymal and epithelial lineage-associated markers.

Several experimental models including patient biopsies, animal models, and cell lines have been recommended to study the mechanism of bladder cancer development. After several passages in culture, cell lines lose some original features, and no longer resemble the cells of their original tumor. This makes it necessary to establish various cell lines. In an attempt to establish a new cell line for bladder cancer, JAM-ICR (RRID: CVCL_A9QB) was derived from a 64-year-old man diagnosed with a high-grade tumor. This cell line was characterized in multiple experiments involving morphological studies, immunophenotyping (by immunohistochemistry and flow cytometry), karyotyping, short tandem repeat analysis, colony-forming assays, migration and invasion assays, and chemosensitivity to anti-cancer drugs. JAM-ICR cells are pale with an irregular polygonal shape, and show some similarities to mesenchymal stem cells but with a wider shape and shorter arms. Phenotypic assessment demonstrated the simultaneous expression of mesenchymal-(vimentin, desmin, CD29, CD90, and CD106) and epithelial lineage (pan-cytokeratin) markers, which supports a phenotype similar to epithelial-mesenchymal transition for this cell line. JAM-ICR displayed high metastatic potential and stem-like properties, i.e., self-renewal, colony forming, and the coexpression of TRA-1 with CD44 and CD166. Furthermore, this cell line was significantly more resistant to doxorubicin in comparison to the 5637 cell line. These features make JAM-ICR a new bladder cancer cell line with metastatic potential and stem-like properties, which may be potentially useful as a model to elucidate the molecular and cellular mechanisms of bladder cancer pathogenesis or evaluate new drugs.

IL-35 and IL-18 Serum Levels in Children With Acute Lymphoblastic Leukemia: The Relationship With Prognostic Factors.

Acute lymphoblastic leukemia (ALL) is the most common type of cancer among children. In this study, we investigated the serum levels of interleukin (IL)-35 and IL-18 in children with ALL to compare with healthy subjects and find their relationship with prognostic factors and response to therapy. IL-35 and IL-18 serum concentrations in 40 children diagnosed with ALL and 35 age-matched and sex-matched healthy children were measured using ELISA. The association between cytokine levels and patients' clinical and laboratory data were determined. A significant difference was found in IL-35 serum levels between the patients (3.6±1.5 ng/mL) and controls (2.5±1.8 ng/mL) (P=0.007). No significant difference in IL-18 serum levels between these groups was observed. A positive correlation between IL-35 and IL-18 levels was detected (P=0.001). The authors found that patients with lower platelet count had higher IL-35 concentration (P=0.003). By considering a cut-off value of 6.21 ng/mL (mean±2SD of controls) for IL-35, it was found that white blood cell (WBC) count was higher in patients with IL-35 >6.21 ng/mL (P=0.016), and the majority of these patients had T-ALL (P=0.01). Although the mean overall survival in patients with IL-35 >6.21 ng/mL was shorter (937±381 d) than in those with IL-35 ≤6.21 ng/mL (1567±103 d), but the result was not significant (P=0.1, log-rank test). The IL-18 level was associated with a lower hemoglobin level (P=0.027). These data suggested a role for IL-35 in ALL development. The significant relation of IL-35 to white blood cells and platelet counts may imply a possible influence of IL-35 on ALL prognosis.

Interleukin-23 Receptor Gene Variants in Acute Lymphoblastic Leukemia and Their Relation to Prognostic Factors.

BACKGROUND: Interleukin (IL)-23 has an important role in tumor immune regulation. OBJECTIVE: To investigate the possible association of interleukin-23 receptor (IL23R) gene variants rs1884444, rs10889677 and rs11209026 with development of acute lymphoblastic leukemia (ALL). METHODS: The IL23R variants were studied in 164 ALL patients and compared to 175 healthy controls by polymerase chain reaction-restriction fragment length polymorphism. The relationship between these variants and clinical and laboratory features of the patients and response to therapy were evaluated. RESULTS: No significant differences in genotype and allele frequencies existed between patients and controls. The rs1884444TG genotype was significantly lower in patients who relapsed (24.2%) compared to those without relapse (55.9%, p=0.006). Fewer patients who relapsed had evidence of the G allele (p=0.034). The TG genotype was associated with a longer complete remission at 1804±116 days compared to other genotypes (<1217 days, p=0.028), however, this result was not significant in multivariate analysis. The rs10889677 AA genotype and A allele were associated with age (p<0.041) and platelet number (p=0.03) in precursor-B cell ALL (B-ALL) patients. Both occurred more frequently in patients aged 2-10 years (63.6% and 66%, respectively) and in those with platelets >100×10ˆ3 µL (68.4% and 52.4%, respectively). CONCLUSION: Our findings showed a lack of association of the studied polymorphisms with the risk of ALL. The influence of the rs1884444 polymorphism on relapse rate and association of rs10889677 AA genotype with favorable prognostic factors suggest the effect of the studied polymorphisms on ALL response to therapy and prognosis.