Peter Naredi

PD-1 expression, absolut counts and frequences of T cells in peripheral blood before and after surgery

Pancreatic cancer surgery entails reduced levels of NK cells with abridged expression of the activating NK cell receptor NKp30. Absolute numbers (A, D) and frequencies (B, E) of CD16+ (A, B) and CD56bright (D, E) NK cells in preoperative (pre-op) and postoperative (day 1, days 3–5, and day 28) samples from patients with periampullary cancer and healthy donors (HD). Frequencies of NKp30high-expressing cells among CD16+ (C) and CD56bright (F) NK cells (N = 16 for pre-op, N = 15 for day 1, N = 14 days 3–5, and N = 10 for day 28 for A and D and N = 16 for pre-op, day 1 and days 3–5, N = 10 for day 28, N = 10 for HD for B–C and E–F). Preoperative and postoperative samples were compared using mixed-effects analysis followed by Šídák multiple comparison test (P-values indicated by *). HD and cancer samples were compared using Kruskal–Wallis test followed by Dunn multiple comparison test (P-values indicated by #). #, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

NOX2 inhibition prevents surgery-induced metastasis in a murine model. A, Schematic outline of the experimental model of surgery-induced metastasis. Blood samples were collected from naïve (Ctrl) and sponge-bearing (Inf) WT and NOX2-KO mice 1 week after a surgical procedure (sponge implantation). A group of WT mice received intraperitoneal injections with histamine dihydrochloride (WT HDC) every other day starting one day before surgery and continuing until 3 days after tumor cell challenge. Frequency of CD11b+Ly6C+M-MDSC (B) and intracellular ROS levels in M-MDSC (C) were measured by flow cytometry. At 8 days postsurgery, mice were intravenously inoculated with B16F10 cells. D, Metastatic lesions in lungs were enumerated 19–21 days after tumor cell challenge. Differences in inflammatory monocytes, DCFDA levels, and tumor numbers in vivo were calculated using one-way ANOVA following Holm-Šídák multiple comparisons test. The frequency of M-MDSC and number of metastatic foci was evaluated in four independent experiments for WT mice with or without surgery-induced inflammation, and in two experiments for KO mice and HDC treated mice. DCFDA levels of M-MDSC were measured in three independent experiments for WT mice, two experiments for KO mice, and one experiment for HDC-treated mice. In each experiment, 4–5 mice per group were included. Fold change was calculated by dividing raw data with the mean of raw data in the WT mice control group in each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Expression of NK cell receptors on NK cells before and after surgery

M-MDSC induction in a non-cancerous patient undergoing pancreatic cancer surgery

M-MDSC induction in a non-cancerous patient undergoing pancreatic cancer surgery

M-MDSC suppress NK cells by generating ROS and show enhanced NK cell suppression after surgery. A–C, NK cells were co-cultured with or without PANC-1 cells and freshly isolated healthy donor (HD) monocytes in the presence or absence of 100 µmol/L HDC, 200 U/mL catalase (Cat), 10 µmol/L GSK2795039 (GSK) or 1% DMSO (control). NK cell activation and degranulation were measured by flow cytometry and are shown by frequencies of NK cells expressing CD69+CD16+ (A) or CD107a+CD16+ (B), respectively. C, NK cell cytotoxicity against PANC-1 cells was determined by frequency of LIVE/DEAD stained PANC-1 cells. NK cell cytotoxicity against PANC-1 cells was measured in four independent experiments, that each included two to three combinations of NK cells and monocyte from different donors. D, ROS formation was measured by CL from CD33+ cells isolated 3–5 days after surgery in the absence of stimulation (PDAC Ctrl, orange), in the presence of rituximab (PDAC RTX, red), in the presence of rituximab and HDC (PDAC RTX + HDC, green) and from 2 HDs stimulated with rituximab (mean ROS formation shown, HD RTX, black). NK cells were cocultured with K562 cells and CD33+ cells isolated from HDs or patients before surgery (pre-op) or 3–5 days after surgery (days 3–5) in the presence (HDC) or absence of HDC. NK cell activation and degranulation were measured by flow cytometry as frequencies of CD16+ NK cells expressing CD69 (E) or CD107a (F) in NK:K562:Myeloid cell cocultures. NK cell cytotoxicity was measured in 10 independent experiments that were normalized by setting the mean NK cell cytotoxicity against K562 in the absence of myeloid cells to 1 in each experiment. Statistics by mixed-effects analysis followed by Šídák multiple comparison test (P-values indicated by *). HD and cancer samples were compared using Kruskal–Wallis test followed by Dunn multiple comparison test (P-values indicated by #).*, P < 0.05; **/##, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Pancreatic cancer surgery triggers inflammation and expansion of M-MDSC. A, Flow chart of included IPEP study participants. B, Study design and available samples. Absolute numbers of neutrophils (C), monocytes (D), and lymphocytes (E) in patients undergoing surgery for periampullary cancer with whole blood samples (differential counts) analyzed before (pre-op) and 1, 3–5 and 28 days after pancreatic cancer surgery (day 1, days 3–5, and day 28, respectively; N = 17 for pre-op, N = 16 for day 1, N = 15 for days 3–5, N = 11 for day 28). F, Plasma levels of IL6 in healthy donors (HD) and in pre- and post-op samples from patients (N = 16 for pre-op and for day 1, N = 15 for days 3–5, N = 9 for day 28, N = 6 for HD). G–I, CD14+HLADRlow M-MDSC in peripheral blood of healthy donors and patients before and after surgery analyzed by flow cytometry. G, Representative FACS plot of the frequency of CD14+HLADRlow cells among CD14+ cells. Absolute numbers (H) and frequencies (I) of M-MDSC (N = 17 for pre-op, N = 16 for day 1 and days 3–5, N = 10 for day 28, N = 12 for HD). J, M-MDSC levels of ROS as measured by DCFDA MFI (N = 16 for pre-op, for day 1 and for days 3–5, N = 10 for day 28, N = 12 for HD). Preoperative and postoperative samples were compared using mixed-effects analysis followed by Šídák multiple comparison test (P-values indicated by *). HD and cancer samples were compared using Kruskal–Wallis test followed by Dunn multiple comparison test (P-values indicated by #). */#P < 0.05, ##P < 0.01, ***/###P < 0.001, ****/####P < 0.0001.