Functional characterization and development of novel human kinase insert domain receptor chimeric antigen receptor T-cells for immunotherapy of non-small cell lung cancer.

CAR-T cell therapy, in which T cells are transfected or transduced with a chimeric antigen receptor (CAR), is a transformative type of cancer immunotherapy. Despite outstanding success in hematological malignancies, their efficacy against solid tumors has been limited. Here, we aimed to explore whether T cells modified by a CAR targeting the vascular endothelial growth factor 2 receptor/ kinase insert domain receptor (KDR) could destroy tumors and their vasculature. A second-generation KDR-CAR was constructed and transfected into T cells using lentivirus. The 3D structure of the CAR construct and target antigen was predicted. Moreover, in silico analysis, including molecular docking and molecular dynamics (MD) simulation, were used to evaluate the minimum energy of interaction and stability of the complex. The anti-cancer effect of KDR-specific CAR-T cells was tested with KDR-expressing and KDR overexpressing A549 cell line. The in-silico study suggested that this CAR construct could be effective for lung cancer therapy. We evaluated this using both in vitro and in vivo experiments. The KDR-CAR-T cells targeted and killed KDR-A549 with high efficiency by expressing IFN-γ and releasing granzyme B. The in vivo study showed that KDR-CAR-T cells dramatically inhibited the growth of lung cancer KDR-A549 xenografts in BALB/c-nu mice at day 10. The characterization of T cells modified by KDR-CAR by computational biology and wet-lab experiments suggested its applicability as a new treatment strategy for lung cancer and, potentially, for other vascularized solid tumors.

The integrative multi-omics approach identifies the novel competing endogenous RNA (ceRNA) network in colorectal cancer.

Circular RNAs (circRNA) are known to function as competing endogenous RNA (ceRNA) in various cancers by regulating microRNAs (miRNA). However, in colorectal cancer (CRC), the precise pathological role of circ000240/miRNA/mRNA remains indeterminate. The expression level of hsa_circ_000240 was evaluated using qRT-PCR in matching pairs of CRC tumor and adjacent normal tissue samples in our laboratory. Then, to determine whether hsa_circ_000240 acted as a ceRNA in CRC, the linked miRNAs and gene targets were retrieved. Topological analysis of candidate genes using a network approach identified the most critical hub genes and subnetworks related to CRC disease. Microarray and bulk RNA sequencing analyses were utilized to comprehensively evaluate the expression levels of both miRNA and mRNA in CRC. Single-cell RNA-seq analysis was also used to evaluate the significant overall survival (OS) genes at the cellular level. ATAC-seq data provided insights into candidate genes' accessible chromatin regions. The research uncovered a considerable upregulation of hsa_circ_000240 in CRC tissues. Three miRNAs interacted with the target circRNA. One thousand six hundred eighty intersected genes regulated by three miRNAs were further identified, and the relevant functionality of identified neighbor genes highlighted their relevance to cancer. The topological analysis of the constructed network has identified 33 hub genes with notably high expression in CRC. Among these genes, eight, including CHEK1, CDC6, FANCI, GINS2, MAD2L1, ORC1, RACGAP1, and SMC4, have demonstrated a significant impact on overall survival. The utilization of single-cell RNA sequencing unequivocally corroborated the augmented expression levels of CDC6 and ORC1 in individuals with CRC, alongside their noteworthy connection with the infiltration of immune cells. ATAC-seq analyses revealed altered accessibility regions in Chr2, 4, and 12 for CDC6 and ORC1 high-expression. Correlation analysis of CDC6 and ORC1 further highlighted the association of candidate gene expression with exhaustion markers such as CTLA4, CD247, TIGIT, and CD244. The candidate genes exhibit a positive correlation with chromatin remodeling and histone acetylation. These epigenetic modifications play a significant role in influencing the cancer progression following expression of CDC6 and ORC1 in CRC. Additionally, results showed that the methylation rate of the promoter region of CDC6 was elevated in CRC disease, confirming the functional importance of CDC6 and their interaction with hsa_circ_000240 and associated ceRNA in CRC. In conclusion, this study highlights hsa_circ_000240's role as a ceRNA in CRC. It opens new avenues for further dissection of CDC6, ORC1, and underlying novel epigenetics and immunotherapy targets for CRC therapy.

Integrative and in-vitro analysis reveal hsa_circ_001787 can act as a diagnostic biomarker for colorectal cancer.

BACKGROUND: Circular RNAs (circRNAs) are a new kind of non-coding RNA(ncRNA). Throughout research, we see an increase in the number of studies demonstrating that circRNAs occupy a pivotal role in the growth and advancement of human tumors. Nevertheless, hsa_circ_001787's role in the evolution of colorectal cancer (CRC) remains unclear. This current study ascertained the expression level of circRNA001787 in CRC specimens and neighboring healthy tissues, and investigated the miRNAs associate with hsa_circ_001787, as well as the relationship between hsa_circ_001787 and pathological factors. METHOD: First, the expression level of hsa_circ_001787 was measured in 43 matched Tissues from CRC and normal tissues through using real-time quantitative reverse transcription PCR (qRT-PCR). Second, based on circular RNA-microRNA and microRNA-mRNA pairs, a circRNA-miRNA-mRNA network was created. The survival rate of mRNAs was investigated through the GEPIA in the network. Regarding the elucidated function analysis of hsa_circ_001787, The biological, molecular, cellular function (GO) and pathway (KEGG) enrichment was obtained. RESULT: We detected that hsa_circ_001787 expression level was significantly down expressed in CRC tissue versus paired CRC histological normal tissue. The area under the curve (AUC) was 0.83. The expression level of hsa_circ_001787 was significantly associated with pathological factors such as tumor grade and the primary site of the tumor. Based on the hsa_circ_001787, a novel circRNA/miRNA/mRNA network has been built up, four miRNAs, and 24 mRNA. The pathway of mRNAs analyzed in the pathogenesis of CRC. Four genes distinguished via the GEPIA database were positively linked to the overall survival of CRC patients. CONCLUSION: Our study suggested that hsa_circ_001787 was significantly down-regulated in CRC. We might be able to use this as a new biomarker in the screening of CRC. Furthermore, our finding achieves a broader understanding of the regulatory mechanisms by which hsa_circ_001787 acts as ceRNA in colorectal cancer.

Components from spider venom activate macrophages against glioblastoma cells: new potential adjuvants for anticancer immunotherapy.

Immunomodulation has been considered an important approach in the treatment of malignant tumours. However, the modulation of innate immune cells remains an underexplored tool. Studies from our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the infiltration of macrophage in glioblastoma, in addition to decreasing the tumour size in a preclinical model. The hypothesis that PnV would be modulating the innate immune system led us to the main objective of the present study: to elucidate the effects of PnV and its purified fractions on cultured macrophages. Results showed that PnV and the three fractions activated macrophages differentiated from bone marrow precursors. Further purification generated 23 subfractions named low weight (LW-1 to LW-12) and high weight (HW-1 to HW-11). LW-9 presented the best immunomodulatory effect. Treated cells were more phagocytic, migrated more, showed an activated morphological profile and induced an increased cytotoxic effect of macrophages on tumour cells. However, while M1-controls (LPS) increased IL-10, TNF-alpha and IL-6 release, PnV, fractions and subfractions did not alter any cytokine, with the exception of LW-9 that stimulated IL-10 production. These findings suggest that molecules present in LW-9 have the potential to be used as immunoadjuvants in the treatment of cancer.

In silico Analysis, Molecular Docking, Molecular Dynamic, Cloning, Expression and Purification of Chimeric Protein in Colorectal Cancer Treatment.

INTRODUCTION: Colorectal cancer (CRC) is a type of cancer in humans that leads to high mortality and morbidity. CD166 and CD326 are immunoglobulins that are associated with cell migration. These molecules are included in tumorigenesis of CRC and serve a great marker of CRC stem cells. In the present study, we devised a novel chimeric protein including the V1-domain of the CD166 and two epitopes of CD326 to use in diagnostic or therapeutic applications. METHODS: In silico techniques were launched to characterize the properties and structure of the protein. We have predicted physicochemical properties, structures, stability, MHC class I binding properties and ligand-receptor interaction of this chimeric protein by means of computational bioinformatics tools and servers. The sequence of chimeric gene was optimized for expression in prokaryotic host using online tools and cloned into pET-28a plasmid. The recombinant pET28a was transformed into the E. coli BL21DE3. Expression of recombinant protein was examined by SDS-PAGE and Western blotting. RESULTS: The designed chimeric protein retained high stability and the same immunogenicity as of the original proteins. Bioinformatics data indicated that the epitopes of the synthetic chimeric protein might induce B-cell- and T-cell-mediated immune responses. Furthermore, a gene was synthesized using the codon bias of a prokaryotic expression system. This synthetic gene expressed a bacterial expression system. The recombinant protein with molecular weights of 27kDa was expressed and confirmed by anti-his Western blot analysis. CONCLUSION: The designed recombinant protein may be useful as a CRC diagnostic tool and for developing a protective vaccine against CRC.

A New World of Biomarkers and Therapeutics for Female Reproductive System and Breast Cancers: Circular RNAs.

As one of the most recently (re)discovered types of non-coding RNAs (ncRNA), circular RNAs (circRNAs) differentiate from other ncRNAs by a specific biogenesis, high stability, and distinct functions. The biogenesis of circRNAs can be categorized into three mechanisms that permit the back-splicing reaction: exon-skipping, pairing of neighboring introns, and dimerization of RNA-binding proteins. Regarding their stability, circRNAs have no free ends, specific to linear RNA molecules, prompting a longer half-life and resistance to exonuclease-mediated activity by RNase R, bypassing the common RNA turnover process. Regarding their functions, circular transcripts can be categorized into four broad roles: miRNA sponging, protein binding, regulation of transcription, and coding for proteins and peptides. Female reproductive system (including mainly ovarian, corpus, and cervix uteri cancers) and breast cancers are the primary causes of death in women worldwide, accounting for over 1,212,772 deaths in 2018. We consider that a better understanding of the molecular pathophysiology through the study of coding and non-coding RNA regulators could improve the diagnosis and therapeutics of these cancers. Developments in the field of circRNA in regard to breast or gynecological cancers are recent, with most circRNA-related discoveries having been made in the last 2 years. Therefore, in this review we summarize the newly detected roles of circRNAs in female reproductive system (cervical cancer, ovarian cancer, and endometrial cancer) and breast cancers. We argue that circRNAs can become essential elements of the diagnostic and therapeutic tools for female reproductive system cancers in the future.

Small interfering RNAs (siRNAs) in cancer therapy: a nano-based approach.

Cancer is one of the most complex diseases that has resulted in multiple genetic disorders and cellular abnormalities. Globally, cancer is the most common health concern disease that is affecting human beings. Great efforts have been made over the past decades in biology with the aim of searching novel and more efficient tools in therapy. Thus, small interfering RNAs (siRNAs) have been considered one of the most noteworthy developments which are able to regulate gene expression following a process known as RNA interference (RNAi). RNAi is a post-transcriptional mechanism that involves the inhibition of gene expression through promoting cleavage on a specific area of a target messenger RNA (mRNA). This technology has shown promising therapeutic results for a good number of diseases, especially in cancer. However, siRNA therapeutics have to face important drawbacks in therapy including stability and successful siRNA delivery in vivo. In this regard, the development of effective siRNA delivery systems has helped addressing these issues by opening novel therapeutic windows which have allowed to build up important advances in Nanomedicine. In this review, we discuss the progress of siRNA therapy as well as its medical application via nanoparticle-mediated delivery for cancer treatment.