Cancer combination therapies by silencing of CTLA-4, PD-L1, and TIM3 in osteosarcoma.

Osteosarcoma (OS) is the most common orthopedic neoplasm, with a high metastasis rate and a dismal prognosis despite surgery and chemotherapy. Immunotherapies have offered cancer patients a ray of optimism, but their impact on OS has been disappointing. The objective of this study is to assess the effect of mono, dual, and triple combinations of CTLA-4, PD-L1, and TIM3 blockade on OS cell viability, apoptosis, and migration. The MG-63 and U-2 OS cell lines were transfected with mono, dual, and triple combinations of siRNAs specific for CTLA-4, PD-L1, and TIM3. After evaluation for transfection efficacy by qRT-PCR, MTT assay and flow cytometry were applied to assess cell viability and apoptosis rate in siRNA-transfected cells, respectively. Ultimately, the migration of transfected cells was measured by wound-healing assay. First, the qRT-PCR analysis revealed that in siRNA-transfected OS cells, CTLA-4, PD-L1, and TIM3 were downregulated. The MTT assay and flow cytometry results confirmed that silencing of these immune checkpoints in dual or triple combinations, but not in the single-agent blockade, significantly decreases cell viability and increases apoptosis, respectively. These effects were more significant when triple silencing was performed. Finally, the wound-healing assay revealed that dual and triple silencing of immune checkpoints significantly inhibits cell migration, with triple silencing exhibiting a greater effect. Our findings suggest that triple blockade of CTLA-4, PD-L1, and TIM3 is an effective strategy for inhibiting tumor cell progression and migration in OS, which requires large-scale clinical investigations to be translated into broad therapeutic applicability for OS patients.

Anastasis: cell recovery mechanisms and potential role in cancer.

Balanced cell death and survival are among the most important cell development and homeostasis pathways that can play a critical role in the onset or progress of malignancy steps. Anastasis is a natural cell recovery pathway that rescues cells after removing the apoptosis-inducing agent or brink of death. The cells recuperate and recover to an active and stable state. So far, minimal knowledge is available about the molecular mechanisms of anastasis. Still, several involved pathways have been explained: recovery through mitochondrial outer membrane permeabilization, caspase cascade arrest, repairing DNA damage, apoptotic bodies formation, and phosphatidylserine. Anastasis can facilitate the survival of damaged or tumor cells, promote malignancy, and increase drug resistance and metastasis. Here, we noted recently known mechanisms of the anastasis process and underlying molecular mechanisms. Additionally, we summarize the consequences of anastatic mechanisms in the initiation and progress of malignancy, cancer cell metastasis, and drug resistance. Video Abstract.

A comprehensive review about immune responses and exhaustion during coronavirus disease (COVID-19).

Coronavirus disease (COVID-19) is a viral infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The infection was reported in Wuhan, China, in late December 2019 and has become a major global concern due to severe respiratory infections and high transmission rates. Evidence suggests that the strong interaction between SARS-CoV-2 and patients' immune systems leads to various clinical symptoms of COVID-19. Although the adaptive immune responses are essential for eliminating SARS-CoV-2, the innate immune system may, in some cases, cause the infection to progress. The cytotoxic CD8+ T cells in adaptive immune responses demonstrated functional exhaustion through upregulation of exhaustion markers. In this regard, humoral immune responses play an essential role in combat SARS-CoV-2 because SARS-CoV-2 restricts antigen presentation through downregulation of MHC class I and II molecules that lead to the inhibition of T cell-mediated immune response responses. This review summarizes the exact pathogenesis of SARS-CoV-2 and the alteration of the immune response during SARS-CoV-2 infection. In addition, we've explained the exhaustion of the immune system during SARS-CoV-2 and the potential immunomodulation approach to overcome this phenomenon. Video Abstract.

miR-200c increases the sensitivity of breast cancer cells to Doxorubicin through downregulating MDR1 gene.

BACKGROUND: Breast cancer (BC) has been known as the most common type of cancer worldwide and resulted in death among women. BC is usually resistant to standard therapies that are significant problems in managing BC patients. miR-200c belongs to the miRNA family, which is considered as a tumor suppressor with reduced expression levels in various kinds of cancer like BC. Increased expression of miR-200c has been reported as a potent inhibitor of drug resistance and tumor advancement. The purpose of this paper is to examine the outcome of miR-200c restoring on enhancing the BC cells' sensitivity to Doxorubicin through downregulating the MDR1 expression. METHODS: Initially, MDA-MB-231 cells were transfected with miR-200c to perform functional analyses. After that, MTT assay was performed to investigate the viability of the cell. Finally, qRT-PCR was used to assess gene expression. RESULTS: According to the results, the miR-200c expression was downregulated in BC cells compared to control. Moreover, the cell viability was reduced in transfected cells via regulation in gene expression associated with apoptosis. Furthermore, miR-200c could increase the BC cells' sensitivity to Doxorubicin by reducing the MDR1 gene expression. CONCLUSION: Hence, this study's findings recommend that miR-200c can consider as a method of therapy for the treatment of BC.

Targeted Therapy of B7 Family Checkpoints as an Innovative Approach to Overcome Cancer Therapy Resistance: A Review from Chemotherapy to Immunotherapy.

It is estimated that there were 18.1 million cancer cases worldwide in 2018, with about 9 million deaths. Proper diagnosis of cancer is essential for its effective treatment because each type of cancer requires a specific treatment procedure. Cancer therapy includes one or more approaches such as surgery, radiotherapy, chemotherapy, and immunotherapy. In recent years, immunotherapy has received much attention and immune checkpoint molecules have been used to treat several cancers. These molecules are involved in regulating the activity of T lymphocytes. Accumulated evidence shows that targeting immune checkpoint regulators like PD-1/PD-L1 and CTLA-4 are significantly useful in treating cancers. According to studies, these molecules also have pivotal roles in the chemoresistance of cancer cells. Considering these findings, the combination of immunotherapy and chemotherapy can help to treat cancer with a more efficient approach. Among immune checkpoint molecules, the B7 family checkpoints have been studied in various cancer types such as breast cancer, myeloma, and lymphoma. In these cancers, they cause the cells to become resistant to the chemotherapeutic agents. Discovering the exact signaling pathways and selective targeting of these checkpoint molecules may provide a promising avenue to overcome cancer development and therapy resistance. Highlights: (1) The development of resistance to cancer chemotherapy or immunotherapy is the main obstacle to improving the outcome of these anti-cancer therapies. (2) Recent investigations have described the involvement of immune checkpoint molecules in the development of cancer therapy resistance. (3) In the present study, the molecular participation of the B7 immune checkpoint family in anticancer therapies has been highlighted. (4) Targeting these immune checkpoint molecules may be considered an efficient approach to overcoming this obstacle.

Immune Features of Tumor Microenvironment: A Genetic Spotlight.

A tumor represents a highly intricate tissue entity, characterized by an exceptionally complex microenvironment that starkly contrasts with the typical physiological surroundings of healthy tissues. Within this tumor microenvironment (TME), every component and factor assume paramount importance in the progression of malignancy and exerts a pivotal influence on a patient's clinical outcome. One of the remarkable aspects of the TME is its remarkable heterogeneity, not only across different types of cancers but even within the same histological category of tumors. In-depth research has illuminated the intricate interplay between specific immune cells and molecules and the dynamic characteristics of the TME. Recent investigations have yielded compelling evidence that several mutations harbored by tumor cells possess the capacity to instigate substantial alterations in the TME. These mutations, often acting as drivers of tumorigenesis, can orchestrate a cascade of events that remodel the TME, thereby influencing crucial aspects of cancer behavior, including its invasiveness, immune evasion, and response to therapies. It is within this nuanced context that the present study endeavors to provide a concise yet comprehensive summary of how specific mutations, within the genetic landscape of cancer cells, can instigate profound changes in TME features. By elucidating the intricate relationship between genetic mutations and the TME, this research aims to contribute to a deeper understanding of cancer biology. Ultimately, the knowledge gained from this study holds the potential to inform the development of more targeted and effective treatments, thereby offering new hope to patients grappling with the complexities of cancer.

Current approaches in glioblastoma multiforme immunotherapy.

Glioblastoma multiform (GBM) is the most prevalent CNS (central nervous system) tumor in adults, with an average survival length shorter than 2 years and rare metastasis to organs other than CNS. Despite extensive attempts at surgical resecting, the inherently permeable nature of this disease has rendered relapse nearly unavoidable. Thus, immunotherapy is a feasible alternative, as stimulated immune cells can enter into the remote and inaccessible tumor cells. Immunotherapy has revolutionized patient upshots in various malignancies and might introduce different effective ways for GBM patients. Currently, researchers are exploring various immunotherapeutic strategies in patients with GBM to target both the innate and acquired immune responses. These approaches include reprogrammed tumor-associated macrophages, the use of specific antibodies to inhibit tumor progression and metastasis, modifying tumor-associated macrophages with antibodies, vaccines that utilize tumor-specific dendritic cells to activate anti-tumor T cells, immune checkpoint inhibitors, and enhanced T cells that function against tumor cells. Despite these findings, there is still room for improving the response faults of the many currently tested immunotherapies. This study aims to review the currently used immunotherapy approaches with their molecular mechanisms and clinical application in GBM.

Dendritic cell-derived exosomes: A new horizon in personalized cancer immunotherapy?

Dendritic cells (DCs) release nanometer-sized membrane vesicles known as dexosomes, containing different molecules, particularly proteins, for presenting antigens, i.e., major histocompatibility complex (MHC)-I/II and CD86. Dexosomes can, directly and indirectly, stimulate antigen-reactive CD8+ and CD4+ T cell responses. Antigen-loaded dexosomes can lead to the development of potent anti-tumoral immune responses. Notably, developing dexosome-based cell-free vaccines could serve as a new vaccination platform in the era of immunotherapy for various cancers. Furthermore, combining dexosomes vaccination strategies with other treatment approaches can considerably increase tumor-specific T cell responses. Herein, we aimed to review how dexosomes interact with immune cells, e.g., CD4+ and CD8+ T cells and natural killer (NK) cells. Besides, we discussed the limitations of this approach and suggested potential strategies to improve its effectiveness for affected patients.

siRNA-Mediated B7H7 Knockdown in Gastric Cancer Lysate-Loaded Dendritic Cells Amplifies Expansion and Cytokine Secretion of Autologous T Cells.

BACKGROUND: Gastric cancer, ranked as the fifth most common cancer worldwide, presents multiple treatment challenges. These obstacles often arise due to cancer stem cells, which are associated with recurrence, metastasis, and drug resistance. While dendritic cell (DC)-based immunotherapy has shown promise as a therapeutic strategy, its efficacy can be limited by the tumor microenvironment and certain inhibitory immune checkpoint molecules, such as B7H7. SiRNA-medicated knockdown of B7H7 in tumor cell lysate-pulsed DCs can increase cytokine secretion and autologous T lymphocyte expansion. This study aimed to evaluate the impact of B7H7 suppression in gastric cancer cell lysate-pulsed DCs on the stimulatory potential of autologous CD3+ T lymphocytes. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated and monocytes were obtained; then, they were differentiated to immature DCs (iDCs) by GM-CSF and IL-4. Tumor cell lysates from human gastric cancer cell lines were harvested, and iDCs were transformed into mature DCs (mDCs) by stimulating iDCs with tumor cell lysate and lipopolysaccharide. B7H7-siRNA was delivered into mDCs using electroporation, and gene silencing efficiency was assessed. The phenotypic characteristics of iDCs, mDCs, and B7H7-silenced mDCs were evaluated using specific surface markers, an inverted light microscope, and flow cytometry. CD3+ T cells were isolated via magnetically activated cell sorting. They were labeled with CFSE dye and co-cultured with mDCs and B7H7-silenced mDCs to evaluate their ability to induce T-cell proliferation. T-cell proliferation was assessed using flow cytometry. The concentration of TGF-ß, IL-4, and IFN-γ secreted from CD3+ T cells in the co-cultured supernatant was evaluated to investigate the cytokine secretory activity of the cells. RESULTS: Transfection of B7H7 siRNA into mDCs was performed in optimal conditions, and the siRNA transfection effectively reduced B7H7 mRNA expression in a dose-dependent manner. SiRNA-mediated B7H7 knockdown in mDCs enhanced maturation and activation of the DCs, as demonstrated by an increased surface expression of CD11c, CD86, and CD40. Co-culture experiments revealed that B7H7-silenced mDCs had more capacity to induce T cell proliferation compared to non-transfected mDCs. The cytokine production patterns of T cells were also altered. Upon examining the levels of TGF-ß, IL-4, and IFN-γ released by CD3+ T cells in the co-culture supernatant, we found that silencing B7H7 in mDCs resulted in a rise in IL-4 secretion and a reduction in TGF-ß levels compared to mDCs that were not transfected. CONCLUSIONS: The study found that suppressing B7H7 expression in DCs significantly enhances their maturation and stimulatory activity when exposed to gastric cancer cell lysate. These B7H7-silenced DCs can substantially increase cytokine production and promote co-cultured T-cell expansion. Consequently, inhibiting B7H7 in DCs may offer a practical strategy to enhance the ability of DCs to initiate T lymphocyte responses and improve the effectiveness of DC-based cell therapy for cancer patients.

The Basis and Advances in Clinical Application of Cytomegalovirus-Specific Cytotoxic T Cell Immunotherapy for Glioblastoma Multiforme.

A high percentage of malignant gliomas are infected by human cytomegalovirus (HCMV), and the endogenous expression of HCMV genes and their products are found in these tumors. HCMV antigen expression and its implications in gliomagenesis have emerged as a promising target for adoptive cellular immunotherapy (ACT) strategies in glioblastoma multiforme (GB) patients. Since antigen-specific T cells in the tumor microenvironments lack efficient anti-tumor immune response due to the immunosuppressive nature of glioblastoma, CMV-specific ACT relies on in vitro expansion of CMV-specific CD8+ T cells employing immunodominant HCMV antigens. Given the fact that several hurdles remain to be conquered, recent clinical trials have outlined the feasibility of CMV-specific ACT prior to tumor recurrence with minimal adverse effects and a substantial improvement in median overall survival and progression-free survival. This review discusses the role of HCMV in gliomagenesis, disease prognosis, and recent breakthroughs in harnessing HCMV-induced immunogenicity in the GB tumor microenvironment to develop effective CMV-specific ACT.

Survivin; a novel therapeutic target that correlates with survival of autoreactive T lymphocytes obtained from patients with ankylosing spondylitis.

Ankylosing spondylitis (AS) is progressive immune-mediated arthritis. Persistent autoreactivity of T cells with an up-regulated Survivin expression is strongly implicated in AS immunopathogenesis. Besides, Survivin can inhibit proapoptotic caspase 9 activations. Moreover, microRNAs are small non-coding RNAs that are dysregulated in various diseases, in which their altered expression could modulate Survivin expression. The primary goal of this study was to assess the role of Survivin and its-targeting microRNAs in the immunopathogenesis of AS disease. For this aim, peripheral blood mononuclear cells (PBMCs) were isolated from 15 patients with AS and healthy matched controls using Ficoll-Hypaque. T cells were obtained using the magnetic-activated cell sorting (MACS) method. After that, the expression levels of Survivin, Caspase 9, and specific miRNAs were determined using qT-qPCR. Also, the expression of Survivin and Caspase 9 at protein levels was determined by western blotting. Then, the isolated T cells were co-cultured with interleukin (IL)-2 and muromonab-CD3 (OKT-3) for active-induced cell death (AICD) induction, Survivin siRNA for inhibition of Survivin expression, and their combination to assess the implication of Survivin expression in autoreactive T lymphocytes' resistance to apoptosis by determining the rate of apoptosis by Flowcytometry assay. The results showed that Survivin was up-regulated while Caspase 9 was downregulated in patients with AS. It was also revealed that microRNAs that directly or indirectly target the Survivin mRNA were dysregulated in patients with AS. It was also revealed that T cells obtained from AS patients were more resistant to apoptosis induction than those obtained from healthy people. In summary, the results obtained from this study showed that dysregulation of Survivin and Survivin-targeting miRNAs in T lymphocytes obtained from AS patients contribute to their resistance to apoptosis, suggesting the future development of targeted therapies for AS.

Expression of proliferation-related genes in BM-MSC-treated ALL cells in hypoxia condition is regulated under the influence of epigenetic factors in-vitro.

Mesenchymal stem cells affect ALL cell biology under hypoxic conditions. We studied survival, proliferation, expression, and promoter methylation levels of essential genes involved in expanding MOLT-4 cells co-cultured with BM-MSC under the hypoxic condition. Here, MOLT-4 cells were co-cultured with BMMSCs under hypoxic conditions. First, the apoptosis rate was evaluated by Flow cytometry. Then, MOLT-4 cells' proliferation rate was assessed using MTT assay, and the expressions and methylation rates of genes were determined by qRT-PCR and MS-qPCR, respectively. The results showed that although MOLT-4 cells proliferation and survival rates were reduced under hypoxic conditions, this reduction was not statistically significant. Also, we showed that hypoxic conditions caused upregulation of candidate genes and affected their methylation status. Besides, it was revealed that Pontin was downregulated, while KDM3A, SKP2, and AURKA had an upward trend in the presence of MOLT-4 cells plus BM-MSC. The co-culture of leukemia cells with BMMSCs under hypoxic conditions may be a potential therapeutic approach for ALL.

Tumor necrosis factor­α in systemic lupus erythematosus: Structure, function and therapeutic implications (Review).

Tumor necrosis factor­α (TNF­α) is a pleiotropic pro­inflammatory cytokine that contributes to the pathophysiology of several autoimmune diseases, such as multiple sclerosis, inflammatory bowel disease, rheumatoid arthritis, psoriatic arthritis and systemic lupus erythematosus (SLE). The specific role of TNF­α in autoimmunity is not yet fully understood however, partially, in a complex disease such as SLE. Through the engagement of the TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), both the two variants, soluble and transmembrane TNF­α, can exert multiple biological effects according to different settings. They can either function as immune regulators, impacting B­, T­ and dendritic cell activity, modulating the autoimmune response, or as pro­inflammatory mediators, regulating the induction and maintenance of inflammatory processes in SLE. The present study reviews the dual role of TNF­α, focusing on the different effects that TNF­α may have on the pathogenesis of SLE. In addition, the efficacy and safety of anti­TNF­α therapies in preclinical and clinical trials SLE are discussed.

Novel CAR T therapy is a ray of hope in the treatment of seriously ill AML patients.

Acute myeloid leukemia (AML) is a serious, life-threatening, and hardly curable hematological malignancy that affects the myeloid cell progenies and challenges patients of all ages but mostly occurs in adults. Although several therapies are available including chemotherapy, allogeneic hematopoietic stem cell transplantation (alloHSCT), and receptor-antagonist drugs, the 5-year survival of patients is quietly disappointing, less than 30%. alloHSCT is the major curative approach for AML with promising results but the treatment has severe adverse effects such as graft-versus-host disease (GVHD). Therefore, as an alternative, more efficient and less harmful immunotherapy-based approaches such as the adoptive transferring T cell therapy are in development for the treatment of AML. As such, chimeric antigen receptor (CAR) T cells are engineered T cells which have been developed in recent years as a breakthrough in cancer therapy. Interestingly, CAR T cells are effective against both solid tumors and hematological cancers such as AML. Gradually, CAR T cell therapy found its way into cancer therapy and was widely used for the treatment of hematologic malignancies with successful results particularly with somewhat better results in hematological cancer in comparison to solid tumors. The AML is generally fatal, therapy-resistant, and sometimes refractory disease with a disappointing low survival rate and weak prognosis. The 5-year survival rate for AML is only about 30%. However, the survival rate seems to be age-dependent. Novel CAR T cell therapy is a light at the end of the tunnel. The CD19 is an important target antigen in AML and lymphoma and the CAR T cells are engineered to target the CD19. In addition, a lot of research goes on the discovery of novel target antigens with therapeutic efficacy and utilizable for generating CAR T cells against various types of cancers. In recent years, many pieces of research on screening and identification of novel AML antigen targets with the goal of generation of effective anti-cancer CAR T cells have led to new therapies with strong cytotoxicity against cancerous cells and impressive clinical outcomes. Also, more recently, an improved version of CAR T cells which were called modified or smartly reprogrammed CAR T cells has been designed with less unwelcome effects, less toxicity against normal cells, more safety, more specificity, longer persistence, and proliferation capability. The purpose of this review is to discuss and explain the most recent advances in CAR T cell-based therapies targeting AML antigens and review the results of preclinical and clinical trials. Moreover, we will criticize the clinical challenges, side effects, and the different strategies for CAR T cell therapy.