Altered maturation of peripheral blood dendritic cells in patients with breast cancer

Abstract

Tumours have at least two mechanisms that can alter dendritic cell (DC) maturation and function. The first affects the ability of haematopoietic progenitors to differentiate into functional DCs; the second affects their differentiation from CD14 þ monocytes, promoting an early but dysfunctional maturation. The aim of this study was to evaluate the in vivo relevance of these pathways in breast cancer patients. For this purpose, 53 patients with invasive breast cancer were compared to 68 healthy controls. To avoid isolation or culture procedures for enrichment of DCs, analyses were directly performed by flow cytometry on whole-blood samples. The expression of surface antigens and intracellular accumulation of regulatory cytokines upon LPS stimulation were evaluated. The number of DCs, and in particular of the myeloid subpopulation, was markedly reduced in cancer patients (Po0.001). Patient DCs were characterized by a more mature phenotype compared with controls (P ¼ 0.016), and had impaired production of IL-12 (Po0.001). These alterations were reverted by surgical resection of the tumour. To investigate the possible role of some tumourrelated immunoactive soluble factors, we measured the plasmatic levels of vascular endothelial growth factor, IL-10 and spermine. A significant inverse correlation between spermine concentration and the percentage of DCs expressing IL-12 was found. Evidence was also obtained that in vitro exposure of monocyte-derived DCs to spermine promoted their activation and maturation, and impaired their function. Taken together, our results suggest that both the above-described mechanisms could concomitantly act in breast cancer to affect DC differentiation, and that spermine could be a mediator of dysfunctional maturation of DCs.

A six‐color flow cytometric assay for the analysis of peripheral blood dendritic cells

“…Key advantages of this type of analysis, compared with other methods that analyze DCs from blood samples after multistep purification procedures or in vitro culture, reside in provision of results that more directly reflect the in vivo situation, need for very small blood samples and rapidity of execution. The 6-color assay presented in this study consists of a modification of a previously reported 3-color assay that allowed us to demonstrate numerical and immunophenotypic alterations of PBDCs in particular physiological and pathological conditions (6,(10)(11)(12).…”

“…Both mDC and pDC count were similarly calculated from the number of events in P3 and P4, respectively. For the dual-platform assay, samples were acquired with a Lyse/ Wash protocol, and estimates of the absolute numbers of PBDCs were calculated from the proportion of cells recorded by flow cytometry in the mononuclear gate multiplied by absolute mononuclear cell count measured using a standard hemacytometer, as previously described (6,(10)(11)(12). The expression of the activation/maturation markers was analyzed gated on mDCs and pDCs.…”

“…Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA) were added before acquisition as an internal calibrator, according to the manufacturer's instructions. For the dual-platform 3-color assay, parallel tubes were stained with the mAbs listed in Table 1, as previously described (6,(10)(11)(12). …”

“…Accordingly, possible changes in their number and function may be relevant to the comprehension of physiopathological mechanisms and the monitoring of a wide range of immune-based pathologies including infectious, allergic, neoplastic, and chronic inflammatory diseases (1,(3)(4)(5)(6)(7). Peripheral blood is the most accessible source of DCs, and this renders flow cytometric analysis of peripheral blood DCs (PBDCs) a convenient procedure that can be serially repeated in patient follow-up.…”

Tumour-released exosomes and their implications in cancer immunity

“…11,12 In particular, DCs are often described as displaying altered expression of HLA-DR and co-stimulatory molecules as well as defective cytokine release, resulting in the cross-priming of tolerogenic or anergized T cells instead of efficient antitumour effectors. [11][12][13][14] Furthermore, DCs are directly affected in their specialized ability to process and present exogenous antigens through their antigen processing machinery, thereby losing their crucial ability to prime specific T cells through cross-presentation. 15 In addition to the lack of functional APCs, cancer patients are affected by the accumulation in peripheral blood and tumour sites 16,17 of myeloid precursors arrested at an immature differentiation stage, exerting active suppression on the development of specific T cell responses, and known as MSCs.…”