High yield killing of lymphoma cells by anti-CD22 CAR-NK cell therapy.

Chimeric antigen receptors (CARs) offer a promising new approach for targeting B cell malignancies through the immune system. Despite the proven effectiveness of CAR T cells targeting CD19 and CD22 in hematological malignancies, it is imperative to note that their production remains a highly complex process. Unlike T cells, NK cells eliminate targets in a non-antigen-specific manner while avoiding graft vs. host disease (GvHD). CAR-NK cells are considered safer than CAR-T cells because they have a shorter lifespan and produce less toxic cytokines. Due to their unlimited ability to proliferate in vitro, NK-92 cells can be used as a source for CAR-engineered NK cells. We found that CARs created from the m971 anti-CD22 mAb, which specifically targets a proximal CD22 epitope, were more effective at anti-leukemic activity compared to those made with other binding domains. To further enhance the anti-leukemic capacity of NK cells, we used lentiviral transduction to generate the m971-CD28-CD3ζ NK-92. CD22 is highly expressed in B cell lymphoma. To evaluate the potential of targeting CD22, Raji cells were selected as CD22-positive cells. Our study aimed to investigate CD22 as a potential target for CAR-NK-92 therapy in the treatment of B cell lymphoma. We first generated m971-CD28-CD3ζ NK-92 that expressed a CAR for binding CD22 in vitro. Flow cytometric analysis was used to evaluate the expression of CAR. The 7AAD determined the cytotoxicity of the m971-CD28-CD3ζ NK-92 towards target lymphoma cell lines by flow cytometry assay. The ELISA assay evaluated cytokine production in CAR NK-92 cells in response to target cells. The m971-CD28-CD3ζ NK-92 cells have successfully expressed the CD22-specific CAR. m971-CD28-CD3ζ NK-92 cells efficiently lysed CD22-expressing lymphoma cell lines and produced large amounts of cytokines such as IFN-γ and GM-CSF but a lower level of IL-6 after coculturing with target cells. Based on our results, it is evident that transferring m971-CD28-CD3ζ NK-92 cells could be a promising immunotherapy for B cell lymphoma.

The effect of microvesicles derived from K562 cells on proliferation and apoptosis of human bone marrow mesenchymal stem cells.

Objectives: Microvesicles (MVs) are small membrane-bound particles that act as a vehicle to transfer their contents, such as proteins, RNAs, and miRNAs, to the target cells, making them undergo several changes. Depending on the origin and the target cell, MVs may cause cell survival or apoptosis. This study investigated the effects of MVs released from the leukemic K562 cell line on the human bone marrow mesenchymal stem cells (hBM-MSCs) to evaluate changes in the survival or apoptosis of the cells in an in vitro system. Materials and Methods: In this experimental study, we added the isolated MVs from the K562 cell line to hBM-MSCs, and after three and then seven days, subsequently cell count, cell viability, transmission electron microscopy, tracing MVs by carboxyfluorescein diacetate, succinimidyl ester (CFSE) solution, flow cytometry analysis for Annexin-V/PI staining and qPCR for the evaluation of BCL-2, KI67, and BAX expression were carried out. On the 10th day of the culture, hBM-MSCs were examined by Oil red O and Alizarin Red staining to evaluate their differentiation into adipocytes and osteoblasts. Results: There was a significant decrease in cell viability and KI67 and BCL-2 expression; however, BAX was significantly upregulated in the hBM-MSCs compared to control groups. Annexin-V/PI staining results also showed the apoptotic effects of K562-MVs on hBM-MSCs. Moreover, the differentiation of hBM-MSCs into adipocytes and osteoblasts was not observed. Conclusion: MVs from the leukemic cell line could affect the viability of normal hBM-MSCs and induce cell apoptosis.

Extracellular vesicles (EVs): What we know of the mesmerizing roles of these tiny vesicles in hematological malignancies?

Cancer is a complex disease in which a bidirectional collaboration between malignant cells and surrounding microenvironment creates an appropriate platform which ultimately facilitates the progression of the disease. The discovery of extracellular vesicles (EVs) was a turning point in the modern era of cancer biology, as their importance in human malignancies has set the stage to widen research interest in the field of cell-to-cell communication. The implication in short- and long-distance interaction via horizontally transfer of cellular components, ranging from non-coding RNAs to functional proteins, as well as stimulating target cells receptors by the means of ligands anchored on their membrane endows these "tiny vesicles with giant impacts" with incredible potential to re-educate normal tissues, and thus, to re-shape the surrounding niche. In this review, we highlight the pathogenic roles of EVs in human cancers, with an extensive focus on the recent advances in hematological malignancies.

Upregulation of Pro-inflammatory Cytokine Genes by Parvovirus B19 in Human Bone Marrow Mesenchymal Stem Cells.

Chronic inflammation plays a prominent role in cancer initiation and development. On the other hand, the Inflammation can be established by a number of factors such as viral infections. Parvovirus B19 (B19V) is a pathogen with widespread infection, which infects bone marrow erythroid progenitor cells. It has been shown that B19V can also enter human bone marrow mesenchymal stem cells (BM-MSCs). In this study, we hypothesized that BM-MSCs as the main cellular component of bone marrow niche may be induced to secret pro-inflammatory cytokines after B19V infection. BM-MSCs were cultured up to passage 3. The cells were then subjected to nucleofection to transfer a plasmid containing B19V genome. After 36 h, total RNA was extracted and the expression levels of IL-1ß, IL-6, TNF-α and NF-κB genes were examined using qRT-PCR. Data analysis showed the significant increase in expression levels of all studied genes in the B19V-transfected cells (P < 0.05). Although further researches are required, our findings for the first time suggest the importance of B19V infection to establish an inflammatory microenvironment in the bone marrow and its involvement in inflammation-related diseases. Finally, based on our results, molecular assay to diagnose B19V infection of BM-MSCs prior to stem cell therapy is strongly recommended.

The effect of simultaneous administration of arsenic trioxide and microvesicles derived from human bone marrow mesenchymal stem cells on cell proliferation and apoptosis of acute myeloid leukemia cell line.

Acute Promyelocytic Leukemia is one of the most prevalent forms of leukemia which has been treated with arsenic trioxide (ATO) and/or all-trans retinoic acid (ATRA). Although, ATRA and ATO are broadly accessible and administrated, some adverse side effects have been reported recently. Nowadays, microvesicles (MVs) are considered as a potential therapeutic agent. Their capacity to alter the behavior of the cells is one of the most controversial issues. In this study, we investigated apoptotic effects of MVs derived from human bone marrow mesenchymal stem cells (hBM-MSCs) in combination with ATO on NB4 cell line. MVs were isolated by ultra-centrifugation. After 7 days, MTT assay, Annexin-V-fluorescein staining assay and RT-qPCR for BCL-2, KI67, BAX genes expression were performed. The results showed lower cell viability rate, higher apoptosis ratio, higher BAX gene, and lower KI67 and BCL-2 genes' expression in cells exposed to MVs in combination with ATO compared to cells treated with each agent alone and non-treated control. We showed that MVs in combination with ATO had more apoptotic effect on NB4 cell line than each agent alone. MVs in combination with ATO in APL treatment might play an effective therapeutic role with fewer adverse side effects compared to any other agents.