Metabolic profiling of single cells by exploiting NADH and FAD fluorescence via flow cytometry.

OBJECTIVE: The metabolism of different cells within the same microenvironment can differ and dictate physiological or pathological adaptions. Current single-cell analysis methods of metabolism are not label-free. METHODS: The study introduces a label-free, live-cell analysis method assessing endogenous fluorescence of NAD(P)H and FAD in surface-stained cells by flow cytometry. RESULTS: OxPhos inhibition, mitochondrial uncoupling, glucose exposure, genetic inactivation of glucose uptake and mitochondrial respiration alter the optical redox ratios of FAD and NAD(P)H as measured by flow cytometry. Those alterations correlate strongly with measurements obtained by extracellular flux analysis. Consequently, metabolically distinct live B-cell populations can be resolved, showing that human memory B-cells from peripheral blood exhibit a higher glycolytic flexibility than naïve B cells. Moreover, the comparison of blood-derived B- and T-lymphocytes from healthy donors and rheumatoid arthritis patients unleashes rheumatoid arthritis-associated metabolic traits in human naïve and memory B-lymphocytes. CONCLUSIONS: Taken together, these data show that the optical redox ratio can depict metabolic differences in distinct cell populations by flow cytometry.

Radiotherapy combined with docetaxel alters the immune phenotype of HNSCC cells and results in increased surface expression of CD137 and release of HMGB1 of specifically HPV-positive tumor cells.

PURPOSE: Human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC) tumors respond significantly better to anticancer treatments. It is assumed to be due to a better response to radiotherapy (RT), and presumably to an increased immunogenicity. However, little is known how the immune phenotype of HNSCC tumor cells is modulated by standard treatment, namely by radiochemotherapy (RCT). METHODS: Therefore, we aimed to examine the impact of the HPV status on the immune phenotype of HNSCC cell lines following RCT with 5 × 3Gy or 1 × 19.3Gy and/or docetaxel, by analyzing cell death, release of damage-associated molecular patterns (DAMPs), surface expression of immune checkpoint molecules (ICMs) and the impact on activation of human monocyte-derived dendritic cells (hmDCs). RESULTS: Cell death induction and Hsp70 release following RCT was independent of the HPV status, and RCT significantly increased the expression of the immune suppressive ICMs PD-L1, PD-L2 and HVEM. An immune stimulatory ICM, CD137, was significantly increased following RCT only on HPV-positive cell lines, as well as the release of HMGB1. Although the treatment increased cell death and modulated ICM expression in HNSCC, the hmDCs were not activated after co-incubation with treated tumor cells. CONCLUSION: Our data with the HPV-dependent release of HMGB1 and increased expression of CD137 following RCT provide a hint for increased immunogenicity underlining the better prognosis for HPV positive tumors following RCT.

Clonogenicity-based radioresistance determines the expression of immune suppressive immune checkpoint molecules after hypofractionated irradiation of MDA-MB-231 triple-negative breast cancer cells.

Only a subset of patients with triple-negative breast cancer (TNBC) benefits from a combination of radio- (RT) and immunotherapy. Therefore, we aimed to examine the impact of radioresistance and brain metastasizing potential on the immunological phenotype of TNBC cells following hypofractionated RT by analyzing cell death, immune checkpoint molecule (ICM) expression and activation of human monocyte-derived dendritic cells (DCs). MDA-MB-231 triple-negative breast cancer tumor cells were used as model system. Apoptosis was the dominant cell death form of brain metastasizing tumor cells, while Hsp70 release was generally significantly increased following RT and went along with necrosis induction. The ICMs PD-L1, PD-L2, HVEM, ICOS-L, CD137-L and OX40-L were found on the tumor cell surfaces and were significantly upregulated by RT with 5 x 5.2 Gy. Strikingly, the expression of immune suppressive ICMs was significantly higher on radioresistant clones compared to their respective non-radioresistant ones. Although hypofractionated RT led to significant cell death induction and release of Hsp70 in all tumor cell lines, human monocyte-derived DCs were not activated after co-incubation with RT-treated tumor cells. We conclude that radioresistance is a potent driver of immune suppressive ICM expression on the surface of TNBC MDA-MB-231 cells. This mechanism is generally known to predominantly influence the effector phase, rather than the priming phase, of anti-tumor immune responses.