Clinical and prognostic significance of follicular helper and regulatory T cells in bladder cancer draining lymph nodes.

Follicular helper and regulatory T cells (Tfh/TFR) cells are distinct subsets of CD4+ cells that have been recognized for their critical role in regulating cellular reactions within the germinal centers of lymphoid follicles. In the present study, we aimed to determine the presence and the frequency of these cells in draining lymph nodes of patients with bladder cancer (BC). Forty-six patients with BC who had undergone radical cystectomy and pelvic lymph node dissection were enrolled. Following routine pathological examination, a portion of the dissected lymph nodes was minced to obtain a single-cell suspension. Mononuclear cells were then separated using Ficoll-Hypaque gradient centrifugation, and the samples with proper viability (> 95%) were subjected to further analysis. To phenotype the follicular subsets, cells were stained with appropriate fluorochrome-conjugated antibodies specific for CD4, CXCR5, BCL6, and FOXP3. The cells were then acquired on a four-color flow cytometer. The data were analyzed with the FlowJo software version 10.8.1 package. Our analysis indicated that, on average 37.89 ± 16.36% of CD4+ lymphocytes in draining lymph nodes of patients with BC expressed CXCR5. The majority of them were negative for FOXP3, representing helper subsets (28.73 ± 13.66). A small percent simultaneously expressed BCL6 transcription factor (1.65% ± 1.35), designated as Tfh (CD4+BCL6+CXCR5+FOXP3-). While less than 10% of CD4+ lymphocytes expressed CXCR5 and FOXP3, 1.78 ± 2.54 were also positive for BCL6, known as TFR. Statistical analysis revealed that the frequency of both Tfh and TFR cells was higher in draining lymph nodes of patients with tumor-infiltrated nodes (P = 0.035 and P = 0.079, respectively) compared to those with negative ones. The percentage of these cells was also higher in high-grade tumors compared to low-grade ones (P = 0.031 for both). Our data collectively indicated that however approximately one third of CD4+ lymphocytes expressed CXCR5 and accordingly had the capacity to enter the follicles, less than 2% of them represented Tfh and TFR phenotypes. The percentage of these cells increased in progressed tumors and showed an association with negative prognostic factors.

Diversity of Memory CD8+ T Cells in Tumor-Draining Lymph Nodes from Patients with Bladder Cancer.

The role of memory T cells in orchestrating memory responses to previously known tumor antigens is well documented. The aim of this study was to assess the frequency of different memory T cell subsets in tumor-draining lymph nodes of patients with bladder cancer (BC) and their prognostic significance. Mononuclear cells were isolated from 50 tumor-draining lymph nodes of untreated patients with BC and stained with antibodies against the markers CD8, CD95, CD45RO and CCR7. Data were collected using the FACSCalibur flow cytometer and analyzed using FlowJo software. Among the CD8+ cytotoxic lymphocytes, the frequency of different subsets was determined including total memory cells (CD8+CD45RO+CD95+), T central memory (TCM: CD8+CCR7+CD45RO+CD95+), T effector memory (TEM: CD8+CCR7-CD45RO+CD95+), T stem cell memory (TSCM: CD8+CCR7+CD45RO-CD95+) and naïve T cells (CD8+CCR7+CD45RO-CD95-). The analysis revealed that on average 49.32±20.15 (between 1.62% and 87.20%) percent of CD8+ lymphocytes in draining lymph nodes of BC had a memory phenotype. TCM cells showed the highest frequency (34.71±17.04), while TSCM cells (7.51±8.53) demonstrated the lowest. The total frequency of memory cells tended to be higher in patients with tumor invasion to muscle layer (P=0.052) and stage III (P=0.042) than in patients without invasion and stage I. The TCM subset was more frequent in patients with necrotic tumors than in patients without necrosis (P=0.048). TSCM significantly increased in patients with N2 compared to N0 (P=0.042). Conversely, the ratio of TSCM cells to total memory cells was higher in lower tumor stages (P=0.059), tumors without muscle invasion (P=0.026) and low T grouping (P=0.043). Overall the data indicated an increase in the frequency of memory T cells and their TSCM and TCM cells with tumor progression. In contrast, the ratio of TSCM to total memory cells was higher in less advanced tumors. These results suggest that the immune system is frequently exposed to tumor antigens and strives to create a memory T cell reservoir, but this is suppressed by inhibitory factors provided by the tumor. These findings emphasize the importance of understanding the dynamic interplay between memory T cell subsets and BC progression.

Expression of major histocompatibility complex class I polypeptide-related sequence B in adipose-derived stem cells from breast cancer patients and normal individuals.

CONTEXT: Through the expression of different immunomodulatory molecules, mesenchymal stem cells (MSCs) play a significant role in the regulation of immune responses against tumor cells. Herein, the expression of major histocompatibility complex class I polypeptide-related sequence B (MIC B) as an immunomodulatory molecule was investigated on adipose-derived stem cells (ASCs) isolated from breast cancer patients (Stage II and III) and healthy individuals. MATERIALS AND METHODS: ASCs were isolated enzymatically, and the expression of MIC B was measured using quantitative real-time polymerase chain reaction method before and after treatment with interferon γ (IFN-γ). The concentration of MIC B in the supernatant of ASCs and also sera of breast cancer and normal individuals were determined using ELISA method. RESULTS: The expression of MIC B in normal ASCs and Stage II ASCs was higher than Stage III ASCs. However, after treatment with IFN-γ expression of MIC B in ASCs was conversely changed as cancer ASCs showed approximately 3.5 fold higher expression of MIC B compared to normal ASCs. The mRNA expression of MIC B in Stage III, Stage II, and normal ASCs showed 61 (P = 0.02), 13 (P = 0.01) and 3 (P > 0.05) fold higher expression after stimulation with IFN-γ compared to cells with no stimulation. CONCLUSION: Expression of MIC B and upregulation of this molecule in response to IFN-γ in cancer ASCs draw attention to the effective role of MSCs in the tumor microenvironment. However, more studies will be needed to further elucidate Natural-killer Group 2, member D (NKG2D) ligands-dependent immunomodulatory roles of ASCs in the tumor progression.

Knockdown of microRNA-29a regulates the expression of apoptosis-related genes in MCF-7 breast carcinoma cells.

MicroRNA (miR), as non-coding small RNA, are key regulators of cancer-related biological cell processes and contribute to tumor growth through regulation of groups of pro- and anti-apoptotic genes. The present study aimed to investigate the effects of miR-29a on the expression of genes involved in apoptosis, including p21, B-cell lymphoma 2 (BCL-2), p53 and survivin. The MCF-7 breast cancer cell line was transfected with anti-miR-29a and treated with Taxol in subdivided treatment groups including: Scramble; anti-miR-29a; anti-miR-29a + Taxol; Taxol; and control. Expression levels of p21, BCL-2, p53 and survivin were evaluated using reverse transcription-quantitative polymerase chain reaction. miR-29a knockdown resulted in p21 and p53 upregulation and a decrease in survivin expression. These results indicated that miR-29a inhibition regulates apoptosis. The present data suggested that miR-29a inhibition may be a promising strategy for the induction of apoptosis of tumor cells.

Cancer and normal adipose-derived mesenchymal stem cells (ASCs): Do they have differential effects on tumor and immune cells?

Adipose-derived mesenchymal stem cells (ASCs) are known to have immunomodulatory properties through soluble factors or by direct cell-to-cell contact. This study aimed to assess the expression of HLA-G and IDO activity in breast cancer and normal ASCs and to see whether ASC is capable of modulating both tumor cells and immune system cells in vitro. ASCs were enzymatically isolated from 15 breast cancer patients and 10 normal individuals. Then they were cultured, and the impact of their conditioned media on the movement of the MDA-MB-231 breast cancer cell line was studied in wound healing scratch assay. Next, PBLs from the peripheral blood of normal individuals were separated and co-cultured with breast cancer and normal ASCs. PBLs proliferation and apoptosis were assessed using CFSE labeling dye and annexin V/7AAD staining, respectively. IDO activity and HLA-G protein expression in ASCs were examined using kynurenine assay and Western blotting, respectively. Tumor-derived ASCs, especially those from higher stages of breast cancer, have stronger effects on the proliferation and movement of MDA-MB-231 cells than normal ASCs (P-value < 0.05). Apoptosis in PBLs increased in the presence of ASCs compared to PBLs cultured alone (P-value < 0.05). In contrast, necrosis of PBLs decreased in the presence of ASCs compared to apoptosis in these cells (P-value < 0.001). Collectively, ASCs may have strategic effects on both tumor cells and cells of the immune system in the tumor microenvironment, resulting in tumor development, growth, and metastasis.