Modulation of Peripheral Immune Cells Following Vitamin D₃ Supplementation in Vitamin D-Insufficient Cancer Patients

Background and aims: Low vitamin D₃ levels are common in cancer patients, and these patients might benefit from vitamin D₃ level normalization in parallel with the conventional oncology treatment. This study aimed to examine the molecular effects of moderate-high-dose vitamin D₃ supplementation in vitamin D-deficient cancer patients. Methods: Eight patients under oncological treatment (5 lung cancer, 2 colorectal cancer, and 1 urothelial carcinoma) received 30,000 IU of vitamin D₃ per week for two months. Blood samples were collected before and after supplementation, and peripheral blood mononuclear cells (PBMCs) were isolated. With the aim of assessing further potential epigenetic alterations, global DNA methylation level was estimated on the basis of LINE-1 bisulfite-sequencing experiments on cfDNA and PBMC cells. In order to explore the chromatin accessibility alterations after the treatment in PBMCs, an assay for transposase-accessible chromatin with sequencing (ATAC-Seq) was performed using the (10x Genomics, Pleasanton, CA, USA) on a NextSeq 550 instrument using High Output Sequencing kit (Illumina, San Diego, CA, USA). DNA integrity was assessed by the alkaline Comet-assay and telomere qPCR was also performed. Results: After serum 25-hydroxy-vitamin D levels were normalized, DNA integrity in mononuclear cells improved significantly (p = 0.01), while no significant changes were found in granulocytes. Vitamin D₃ supplementation also led to significant changes in telomere length in mononuclear cells (p = 0.007). No significant differences were observed in cfDNA levels or DNA methylation in PBMCs and cfDNA after supplementation. ATAC-Seq revealed changes in PBMC composition, including an increased number of NK, pDC cells, and monocytes, especially in patients treated with Pembrolizumab in parallel with vitamin D supplementation. Conclusions: These exploratory findings suggest that the observed immune cell and chromatin changes after vitamin D₃ level normalization are compatible with immunomodulatory effects and warrant confirmation in larger, controlled cohorts.