PI3K Isoforms in CD8+ T Cell Development and Function.

CD8+ T cells are an essential part of the immune system and play a vital role in defending against tumors and infections. The phosphoinositide-3-kinase (PI3K), especially class I, is involved in numerous interrelated signaling pathways which control CD8+ T cell development, maturation, migration, activation, and differentiation. While CD8+ T lymphocytes express all class I PI3K isoforms (PI3Kα, PI3Kß, PI3Kδ, and PI3Kγ), isoform-specific functions, especially for PI3Kα and PI3Kß have not been fully elucidated. A few studies suggest the important role of p110δ and p110γ in CD8+ T cell activation, signaling, chemotaxis and function and several clinical trials are currently testing the effect of isoform-specific inhibitors in various types of cancers, including Indolent Non-Hodgkin Lymphoma, Peripheral T cell Lymphoma, Chronic Lymphocytic Leukemia, Small Lymphocytic Lymphoma, non-small cell lung carcinoma (NSCLC), head & neck cancer, and breast cancer. This chapter summarizes current knowledge of the roles of various PI3K isoforms and downstream signaling pathways in regulating CD8+ T cell fate, including cell proliferation, migration, and memory generation. We also discuss certain clinical trials employing PI3K inhibitors for cancer therapy, their limitations, and future perspectives.

CML derived exosomes promote tumor favorable functional performance in T cells.

BACKGROUND: Leukemic cells facilitate the creation of the tumor-favorable microenvironment in the bone marrow niche using their secreted factors. There are not comprehensive details about immunosuppressive properties of chronic myelogenous leukemia-derived exosomes in the bone marrow stromal and immune compartment. We explained here that K562-derived exosomes could affect the gene expression, cytokine secretion, nitric oxide (NO) production, and redox potential of human primary cord blood-derived T cells (CB T cells). METHODS: Human primary cord blood-derived T cells were treated with K562-derived exosomes. We evaluated the expression variation of some critical genes activated in suppressor T cells. The alterations of some inflammatory and anti-inflammatory cytokines levels were assessed using ELISA assay and real-time PCR. Finally, NO production and intracellular ROS level in CB T cells were evaluated using Greiss assay and flow cytometry, respectively. RESULTS: Our results showed the over-expression of the genes involved in inhibitory T cells, including NQO1, PD1, and FoxP3. In contrast, genes involved in T cell activation such as CD3d and NFATc3 have been reduced significantly. Also, the expression of interleukin 10 (IL-10) and interleukin 6 (IL-6) mRNAs were significantly up-regulated in these cells upon exosome treatment. In addition, secretion of the interleukin 10, interleukin 6, and interleukin 17 (IL-17) proteins increased in T cells exposed to K562-derived exosomes. Finally, K562-derived exosomes induce significant changes in the NO production and intracellular ROS levels in CB T cells. CONCLUSIONS: These results demonstrate that K562-derived exosomes stimulate the immunosuppressive properties in CB-derived T cells by inducing anti-inflammatory cytokines such as IL-10, reducting ROS levels, and arising of NO synthesis in these cells. Moreover, considering the elevation of FOXP3, IL-6, and IL-17 levels in these cells, exosomes secreted by CML cells may induce the fates of T cells toward tumor favorable T cells instead of conventional activated T cells.

MEK inhibition reprograms CD8+ T lymphocytes into memory stem cells with potent antitumor effects.

Regenerative stem cell-like memory (TSCM) CD8+ T cells persist longer and produce stronger effector functions. We found that MEK1/2 inhibition (MEKi) induces TSCM that have naive phenotype with self-renewability, enhanced multipotency and proliferative capacity. This is achieved by delaying cell division and enhancing mitochondrial biogenesis and fatty acid oxidation, without affecting T cell receptor-mediated activation. DNA methylation profiling revealed that MEKi-induced TSCM cells exhibited plasticity and loci-specific profiles similar to bona fide TSCM isolated from healthy donors, with intermediate characteristics compared to naive and central memory T cells. Ex vivo, antigenic rechallenge of MEKi-treated CD8+ T cells showed stronger recall responses. This strategy generated T cells with higher efficacy for adoptive cell therapy. Moreover, MEKi treatment of tumor-bearing mice also showed strong immune-mediated antitumor effects. In conclusion, we show that MEKi leads to CD8+ T cell reprogramming into TSCM that acts as a reservoir for effector T cells with potent therapeutic characteristics.

Anti-metastatic properties of a potent herbal combination in cell and mice models of triple negative breast cancer.

AIM: To determine the anti-metastatic potential of combinations of two bioactive carotenoids of saffron, crocin and crocetin, on 4T1 breast cancer and on a mice model of TNBC, and assess the effect of the most potent combination on the Wnt/ß-catenin pathway. MAIN METHODS: The effects of the carotenoid combinations on the viability of 4T1 cells were determined by MTT assay. The effects of the nontoxic doses on migration, mobility, invasion and adhesion to ECM were examined by scratch assay, Transwell/Matrigel-coated Transwell chamber and adhesion assay respectively. Tumors were inoculated by injecting mice with 4T1 cells. The weights and survival rates of the mice and tumor sizes were monitored. Histological analysis of the tissues was conducted. The expression levels of Wnt/ß-catenin pathway genes were measured by Real-time PCR and western blotting. KEY FINDINGS: Treatment of 4T1 cells with combination doses inhibited viability in a dose-dependent manner. The nontoxic combinations significantly inhibited migration, cell mobility and invasion, also attenuating adhesion to ECM. The combination therapy mice possessed more weight, higher survival rates and smaller tumors. Histological examination detected remarkably fewer metastatic foci in their livers and lungs. It was also demonstrated that the combinations exerted anti-metastatic effects by disturbing the Wnt/ß-catenin target genes in the liver and tumors. SIGNIFICANCE: Our findings propose a carotenoid combination as an alternative potent herbal treatment for TNBC, which lacks the adverse effects associated with either chemotherapeutic agents or herb-chemotherapeutic drugs.

Promotion of angiogenesis by M13 phage and RGD peptide in vitro and in vivo.

One of the most important goals of regenerative medicines is to generate alternative tissues with a developed vascular network. Endothelial cells are the most important cell type required in angiogenesis process, contributing to the blood vessels formation. The stimulation of endothelial cells to initiate angiogenesis requires appropriate extrinsic signals. The aim of this study was to evaluate the effects of M13 phage along with RGD peptide motif on in vitro and in vivo vascularization. The obtained results demonstrated the increased cellular proliferation, HUVECs migration, cells altered morphology, and cells attachment to M13 phage-RGD coated surface. In addition, the expression of Vascular Endothelial Growth Factor A (VEGF-A), VEGF Receptors 2 and 3, Matrix Metalloproteinase 9 (MMP9), and epithelial nitric oxide synthase (eNOS) transcripts were significantly upregulated due to the HUVECs culturing on M13 phage-RGD coated surface. Furthermore, VEGF protein secretion, nitric oxide, and reactive oxygen species (ROS) production were significantly increased in cells cultured on M13 phage-RGD coated surface.

Alteration of cellular and immune-related properties of bone marrow mesenchymal stem cells and macrophages by K562 chronic myeloid leukemia cell derived exosomes.

Leukemic cells can impact the bone marrow niche to create a tumor-favorable microenvironment using their secreted factors. Little knowledge is available about immunosuppressive and tumor-promoting properties of chronic myeloid leukemia derived exosomes in bone marrow stromal components. We report here that K562-derived exosomes can affect the gene expression, cytokine secretion, nitric oxide (NO) production, and redox potential of bone marrow mesenchymal stem cells (BM-MSCs) and macrophages. Human BM-MSCs and mouse macrophages were treated with K562-derived exosomes. Our results demonstrated that the expression of the genes involved in hematopoietic developmental pathways and immune responses, including C-X-C motif chemokine 12 (Cxcl12), Dickkopf-related protein 1 (DKK1), wnt5a, interleukin 6 (IL-6), transforming growth factor-beta, and tumor necrosis factor-alpha (TNF-alpha), changed with respect to time and exosome concentration in BM-MSCs. The TNF-alpha level was higher in exosome-treated BM-MSCs compared with the control. Exosome treatment of BM-MSCs led to an increased production of NO and a decreased production of reactive oxygen species (ROS) in a time- and concentration-dependent manner. We have shown that K562-derived exosomes induce overexpression of IL-10 and TNF-alpha and downregulation of iNOS transcript levels in macrophages. The enzyme-linked immunosorbent assay results showed that TNF-alpha and IL-10 secretions increased in macrophages. Treatment of macrophages with purified exosomes led to reduced NO and ROS levels. These results suggest that K562-derived exosomes may alter the local bone marrow niche toward a leukemia-reinforcing microenvironment. They can modulate the inflammatory molecules (TNF-alpha and NO) and the redox potential of BM-MSCs and macrophages and direct the polarization of macrophages toward tumor-associated macrophages.

Inhibitory Effect of Crocin on Metastasis of Triple-Negative Breast Cancer by Interfering with Wnt/ß-Catenin Pathway in Murine Model.

Triple-negative breast cancer (TNBC) is the most metastatic subtype of breast cancer and cannot be controlled with any standard-of-care therapy. However, various studies have recommended medicinal plants as complementary treatments for cancer. In particular, crocin, the main bioactive carotenoid of saffron, has exhibited anticancer effects on primary tumors. This research, for the first time, investigated the antimetastatic potency of crocin on murine model of metastatic TNBC and its effect on the Wnt/ß-catenin pathway. To induce tumors, 4T1 cells were injected to female BALB/c mice. Measurement of biochemical markers showed nontoxicity of crocin. The crocin-treated mice possessed more weight, higher survival rates, and smaller tumors. Histological examination detected no metastatic deposits in their livers and lungs. Also, downregulation of the expression of Wnt/ß-catenin target genes in tumors and lungs was observed compared to the untreated group. Our findings suggest crocin as a promising complementary antimetastatic herbal medicine for treatment of TNBC.

A Comparative Study on Anti-Invasion, Antimigration, and Antiadhesion Effects of the Bioactive Carotenoids of Saffron on 4T1 Breast Cancer Cells Through Their Effects on Wnt/ß-Catenin Pathway Genes.

Crocus sativus L. (saffron) has been used as a spice and as a medicine for the past four thousand years. Recently, saffron has been well documented to possess anticancer effects on primary tumors. However studies of its antimetastatic potential are lacking. The present study is a comparative investigation of the antimetastatic effects of saffron carotenoids, crocin and crocetin, on triple negative metastatic breast cancer cells (4T1) and their effects on the Wnt/ß-catenin pathway. It was found that treatment of 4T1 cells with crocin and crocetin resulted in the inhibition of viability in a dose-dependent manner. Scratch and Transwell chamber assays showed that the nontoxic doses of crocin and crocetin significantly inhibited migration, cell mobility, and invasion, also attenuating adhesion to extracellular matrix. Crocin downregulated mRNA expression of FZD7, NEDD9, VIM, and VEGF-α genes and upregulated E-CAD. Crocin and crocetin exhibited comparable anti-invasion properties on 4T1 cells. However, crocin and crocetin exerted more pronounced antimigration and antiadhesion potency, respectively. Furthermore, we showed that the antimetastatic effects of crocin can occur through interfering with the Wnt/ß-catenin pathway.

Atrial and ventricular specification of ADSCs is stimulated by different doses of BMP4.

To investigate the effect of BMP4 on cardiomyocyte differentiation of adipose tissue-derived stem cells (ADSCs), mouse ADSCs were treated with different concentrations of BMP4 in media containing fetal bovine serum (FBS) or Knockout™ Serum Replacement (KoSR). 3 weeks after cardiac induction, differentiated ADSCs expressed some cardiac-specific genes and proteins. BMP4 treatment upregulated the expression of cardiac transcription factors. In both FBS and KoSR-supplemented media, lower concentrations of BMP4 had a positive effect on the expression of MLC2A gene, while MLC2V was more expressed with higher concentrations of BMP4. BMP4 treatment in KoSR supplemented medium was more efficient for cardiac induction. Supplementation of culture media with insulin-transferrin-selenium improved the expression of MLC2A gene. The results of this study indicated that BMP4 is important for cardiac differentiation of the ADSCs. However, BMP4 was not enough for structural and functional maturation of the ADSC-derived cardiomyocytes.

Oxytocin improves proliferation and neural differentiation of adipose tissue-derived stem cells.

The neurohypophyseal hormone oxytocin plays a role in stimulation of neurogenesis in the adult brain. However, the exact role of oxytocin in neural development is still not well understood. In the present study, we evaluated the effect of oxytocin on neural differentiation of mouse adipose tissue-derived stem cells (ADSCs). For this purpose, ADSCs were cultured in a medium containing Knockout™ Serum Replacement (KoSR) and treated with different concentrations of oxytocin at the first or eighth day of differentiation. Two weeks after neural induction, ADSCs expressed several early and late neuron-specific genes and proteins. MTT assay and cell cycle analysis revealed a stimulatory effect of oxytocin on viability and proliferation of differentiating ADSCs. As detected by quantitative real-time PCR, treatment of the ADSCs with low concentrations of oxytocin induced neurogenesis. Oxytocin treatment also upregulated the expression of oxytocin receptor mRNA. These results demonstrated for the first time that oxytocin treatment can promote neural differentiation of the ADSCs in a dose-dependent and time-dependent manner. Oxytocin has a significant role in neurogenesis, and this may have implications in regeneration of adult neurons.