Stimulating T cell responses against patient-derived breast cancer cells with neoantigen peptide-loaded peripheral blood mononuclear cells.

Breast cancer stands as a formidable global health challenge for women. While neoantigens exhibit efficacy in activating T cells specific to cancer and instigating anti-tumor immune responses, the accuracy of neoantigen prediction remains suboptimal. In this study, we identified neoantigens from the patient-derived breast cancer cells, PC-B-142CA and PC-B-148CA cells, utilizing whole-genome and RNA sequencing. The pVAC-Seq pipeline was employed, with minor modification incorporating criteria (1) binding affinity of mutant (MT) peptide with HLA (IC50 MT) ≤ 500 nm in 3 of 5 algorithms and (2) IC50 wild type (WT)/MT > 1. Sequencing results unveiled 2513 and 3490 somatic mutations, and 646 and 652 non-synonymous mutations in PC-B-142CA and PC-B-148CA, respectively. We selected the top 3 neoantigens to perform molecular dynamic simulation and synthesized 9-12 amino acid neoantigen peptides, which were then pulsed onto healthy donor peripheral blood mononuclear cells (PBMCs). Results demonstrated that T cells activated by ADGRL1E274K, PARP1E619K, and SEC14L2R43Q peptides identified from PC-B-142CA exhibited significantly increased production of interferon-gamma (IFN-γ), while PARP1E619K and SEC14L2R43Q peptides induced the expression of CD107a on T cells. The % tumor cell lysis was notably enhanced by T cells activated with MT peptides across all three healthy donors. Moreover, ALKBH6V83M and GAAI823T peptides from PC-B-148CA remarkably stimulated IFN-γ- and CD107a-positive T cells, displaying high cell-killing activity against target cancer cells. In summary, our findings underscore the successful identification of neoantigens with anti-tumor T cell functions and highlight the potential of personalized neoantigens as a promising avenue for breast cancer treatment.

Enhancement of PD-L1-attenuated CAR-T cell function through breast cancer-associated fibroblasts-derived IL-6 signaling via STAT3/AKT pathways.

BACKGROUND: Carcinoma-associated fibroblasts (CAFs) play a critical role in cancer progression and immune cell modulation. In this study, it was aimed to evaluate the roles of CAFs-derived IL-6 in doxorubicin (Dox) resistance and PD-L1-mediated chimeric antigenic receptor (CAR)-T cell resistance in breast cancer (BCA). METHODS: CAF conditioned-media (CM) were collected, and the IL-6 level was measured by ELISA. CAF-CM were treated in MDA-MB-231 and HCC70 TNBC cell lines and siIL-6 receptor (IL-6R) knocked down (KD) cells to determine the effect of CAF-derived IL-6 on Dox resistance by flow cytometry and on increased PD-L1 through STAT3, AKT and ERK1/2 pathways by Western blot analysis. After pre-treating with CM, the folate receptor alpha (FRα)-CAR T cell cytotoxicity was evaluated in 2D and 3D spheroid culture assays. RESULTS: The results showed a significant level of IL-6 in CAF-CM compared to that of normal fibroblasts (NFs). The CM with high IL-6 level significantly induced Dox resistance; and PD-L1 expression through STAT3 and AKT pathways in MDA-MB-231 and HCC70 cells. These induction effects were attenuated in siIL-6R KD cells. Moreover, the TNBC cell lines that were CM-treated with STAT3 and an AKT inhibitor had a reduced effect of IL-6 on PD-L1 expression. BCA cells with high IL-6 containing-CM treatment had resistance to cancer cell killing by FRα CAR-T cells compared to untreated cells. CONCLUSION: These results highlight CAF-derived IL-6 in the resistance of chemotherapy and T cell therapy. Using inhibitors of IL6-STAT3/AKT-PD-L1 axis may provide a potential benefit of Dox and CAR-T cell therapies in BCA patients.

Adoptive Transfer of Anti-Nucleolin T Cells Combined with PD-L1 Inhibition against Triple-Negative Breast Cancer.

Dendritic cell (DC)-based T-cell activation is an alternative immunotherapy in breast cancer. The anti-programmed death ligand 1 (PD-L1) can enhance T-cell function. Nucleolin (NCL) is overexpressed in triple-negative breast cancer (TNBC). The regulation of PD-L1 expression through autophagy and the anti-PD-L1 peptide to help sensitize T cells for NCL-positive TNBC cell killing has not been evaluated. Results showed the worst clinical outcome in patients with high NCL and PD-L1. Self-differentiated myeloid-derived antigen-presenting cells reactive against tumors presenting NCL or SmartDCs-NCL producing GM-CSF and IL-4, could activate NCL-specific T cells. SmartDCs-NCL plus recombinant human ribosomal protein substrate 3 (RPS3) successfully induced maturation and activation of DCs characterized by the reduction of CD14 and the induction of CD11c, CD40, CD80, CD83, CD86, and HLA-DR. Interestingly, SmartDCs-NCL plus RPS3 in combination with anti-PD-L1 peptide revealed significant killing activity of the effector NCL-specific T cells against NCLHigh/PD-L1High MDA-MB-231 and NCLHigh/PD-L1High HCC70 TNBC cells at the effector: a target ratio of 5:1 in 2-D and 10:1 in the 3-D culture system; and increments of IFNγ by the ELISpot assay. No killing effect was revealed in MCF-10A normal mammary cells. Mechanistically, NCL-specific T-cell-mediated TNBC cell killing was through both apoptotic and autophagic pathways. Induction of autophagy by curcumin, an autophagic stimulator, inhibited the expression of PD-L1 and enhanced cytolytic activity of NCL-specific T cells. These findings provide the potential clinical approaches targeting NCLHigh/PD-L1High TNBC cells with NCL-specific T cells in combination with a PD-L1 inhibitor or autophagic stimulator.

Vitamin C supplementation reduces expression of circulating miR-451a in subjects with poorly controlled type 2 diabetes mellitus and high oxidative stress.

BACKGROUND: Vitamin C is an essential element required for normal metabolic function. We investigated the effect of vitamin C supplementation on circulating miRNA (miR) expression in subjects with poorly controlled type 2 diabetes mellitus (T2DM). Changes in miR expression were also correlated with clinical measures of disease. METHODS: Pre- and post-vitamin C supplementation samples from five participants who had increased vitamin C levels, improved oxidative status and polymorphonuclear (PMN) function after receiving 1,000 mg of vitamin C daily for six weeks were screened for miRNA expression using the NanoString miRNA assay. Differences in miRNA expression identified from the miRNA screen were validated by qRT-PCR. RESULTS: Four miRNAs showed significantly different expression post-vitamin C supplementation relative to baseline, including the down-regulation of miR-451a (-1.72 fold change (FC), p = 0.036) and up-regulation of miR-1253 (0.62 FC, p = 0.027), miR-1290 (0.53 FC, p = 0.036) and miR-644a (0.5 FC, p = 0.042). The validation study showed only miR-451a expression was significantly different from baseline with vitamin C supplementation. MiR-451a expression was negatively correlated with vitamin C levels (r =  - 0.497, p = 0.049) but positively correlated with levels of malondialdehyde (MDA) (r = 0.584, p = 0.017), cholesterol (r = 0.564, p = 0.022) and low-density lipoproteins (LDL) (r = 0.522, p = 0.037). Bioinformatics analysis of the putative target genes of miR-451a indicated gene functions related to signaling pathways involved in cellular processes, such as the mammalian target of rapamycin (mTOR) signaling pathway. CONCLUSIONS: Vitamin C supplementation altered circulating miR-451a expression. The results from this pilot study suggest that miRNAs could be used as biomarkers to indicate oxidative status in subjects with T2DM and with poor glycemic control and could lead to a novel molecular strategy to reduce oxidative stress in T2DM.

Oral vitamin C treatment increases polymorphonuclear cell functions in type 2 diabetes mellitus patients with poor glycemic control.

This study investigated the effect of vitamin C on polymorphonuclear (PMN) cell functions in type 2 diabetes mellitus patients with poor glycemic control. We hypothesized that oral vitamin C treatment improves PMN cell functions. Patients (14) received either a vitamin C (1000 mg/d) or placebo (anhydrous calcium hydrogen phosphate) tablet for 6 weeks and were subjected to a 6-week washout period followed by a 6-week treatment crossover period. Blood samples were collected at pretreatment and posttreatment for PMN cell functions (by flow cytometry) and plasma vitamin C concentration. Phagocytosis was examined by incubating whole blood samples with fluorescein isothiocyanate-labeled Staphylococcus aureus, and oxidative burst was simultaneously evaluated by adding hydroethidine. In comparison with placebo, vitamin C increased both PMN cell phagocytosis (pretreatment: placebo, 17.8% ± 1.6% and vitamin C, 19.0% ± 3.4%, P = .70; posttreatment: placebo, 16.6% ± 1.7% and vitamin C, 27.1% ± 2.9%, P = .005) and oxidative burst (pretreatment: placebo, 6.4% ± 0.8% and vitamin C, 7.1% ± 1.2%, P = .60; posttreatment: placebo, 6.9% ± 1.3% and vitamin C, 12.1% ± 1.6%, P = .02). The plasma vitamin C concentration was elevated after vitamin C treatment as compared with that before treatment (P < .001) and was higher than that observed in the placebo treatment group (P < .01). Plasma vitamin C concentration and PMN cell functions were not significantly different before both treatments. We conclude that the 6-week 1000-mg/d vitamin C increased PMN phagocytosis and oxidative burst in type 2 diabetes mellitus patients with poor glycemic control.