Acute inflammation induces immunomodulatory effects on myeloid cells associated with anti-tumor responses in a tumor mouse model

Abstract

Given the self nature of cancer, anti-tumor immune response is weak. As such, acute inflammation induced by microbial products can induce signals that result in initiation of an inflammatory cascade that helps activation of immune cells. We aimed to compare the nature and magnitude of acute inflammation induced by toll-like receptor ligands (TLRLs) on the tumor growth and the associated inflammatory immune responses. To induce acute inflammation in tumor-bearing host, CD1 mice were inoculated with intraperitoneal (i.p.) injection of Ehrlich ascites carcinoma (EAC) (5 × 10(5) cells/mouse), and then treated with i.p. injection on day 1, day 7 or days 1 + 7 with: (1) polyinosinic:polycytidylic (poly(I:C)) (TLR3L); (2) Poly-ICLC (clinical grade of TLR3L); (3) Bacillus Calmette Guerin (BCG) (coding for TLR9L); (4) Complete Freund's adjuvant (CFA) (coding for TLR9L); and (5) Incomplete Freund's Adjuvant (IFA). Treatment with poly(I:C), Poly-ICLC, BCG, CFA, or IFA induced anti-tumor activities as measured by 79.1%, 75.94%, 73.94%, 71.88% and 47.75% decreases, respectively in the total number of tumor cells collected 7 days after tumor challenge. Among the tested TLRLs, both poly(I:C) (TLR3L) and BCG (contain TLR9L) showed the highest anti-tumor effects as reflected by the decrease in the number of EAc cells. These effects were associated with a 2-fold increase in the numbers of inflammatory cells expressing the myeloid markers CD11b(+)Ly6G(+), CD11b(+)Ly6G(-), and CD11b(+)Ly6G(-). We concluded that Provision of the proper inflammatory signal with optimally defined magnitude and duration during tumor growth can induce inflammatory immune cells with potent anti-tumor responses without vaccination.

Keywords: Anti-tumor; BCG; Ehrlich ascite carcinoma; Inflammation; Poly(I:C); TLR.

Fig. 1

The anti-tumor effects of the inflammatory signals on tumor growth. (A) Shows the total number of EAC cells harvested in each group. (B) Shows the percentage of EAC cells. ^∗P value ⩽0.01 as compared to control.

Fig. 2

Effects of the inflammatory signals on myeloid cells infiltrate in EAC ascites. (A) Shows a representative control in tumor. (B) Shows the number of cell expressing myeloid (Ly6G⁺ CD11b⁺) or (Ly6G⁺ CD11b⁻) or (Ly6G⁻ CD11b⁺) were estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry. (C) Table shows the percentage of myeloid cells in quadrates.

Fig. 3

Impact of the timing of administration of the inflammatory signals on their anti-tumor effects, (A) shows the total number of EAC cells harvested in each group and (B) shows the percentage of EAC cells. ^∗P value ⩽0.01 as compared to control.

Fig. 4

Effects of the timing of the inflammatory signals on myeloid cells in tumor site. (A) Shows the number of cell expressing myeloid (Ly6G⁺ CD11b⁺) was estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry. (B) Shows the number of cell expressing myeloid (Ly6G⁻ CD11b⁺) was estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry. (C) Shows the number of cell expressing myeloid (Ly6G⁺ CD11b⁻) was estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry.

Fig. 5

Effects of inflammation on myeloid cells in spleen. (A) Shows representative control. (B) Shows analysis of the number of expressing cells of myeloid (Ly6G⁺ CD11b⁺) or (Ly6G⁺ CD11b⁻) or (Ly6G⁻ CD11b⁺) were estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry. (C) Table shows the percentage of myeloid cells in quadrates.

Fig. 6

Effects of inflammation on myeloid cells in blood. (A) Shows the number of cell expressing myeloid (Ly6G⁺ CD11b⁺) was estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry. (B) Shows the number of cell expressing myeloid (Ly6G⁻ CD11b⁺) was estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry. (C) Shows the number of cell expressing myeloid (Ly6G⁺ CD11b⁻) was estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry.

Fig. 7

The anti-tumor effects on tumor growth according to magnitude. (A) Shows the total number of EAC cells harvested in each group. (B) Shows the percentage of EAC cells. ^∗P value ⩽0.01 as compared to control.

Fig. 8

Effects of inflammation on myeloid cells in tumor bearing mice. (A) Shows the number of cell expressing myeloid (Ly6G⁺ CD11b⁺) was estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry. (B) Shows the number of cell expressing myeloid (Ly6G⁻ CD11b⁺) was estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry. (C) Shows the number of cell expressing myeloid (Ly6G⁺ CD11b⁻) was estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry.

Fig. 9

Effects of inflammation on myeloid cells In spleen. (A) Shows representative control. (B) Shows in the analysis of the number of expressing cells of myeloid (Ly6G⁺ CD11b⁺) or (Ly6G⁺ CD11b⁻) or (Ly6G⁻ CD11b⁺), were estimated after staining with anti-Ly6G and anti-CD11b using flow cytometry. (C) Table shows the percentage of myeloid cells in quadrates.

Fig. 10

Effects of inflammation on myeloid cells in blood. The number of cells expressing myeloid (Ly6G⁺ CD11b⁺) or (Ly6G⁺ CD11b⁻) or (Ly6G⁻ CD11b⁺) after staining with anti-Ly6G and anti-CD11b using flow cytometry in blood were analyzed after 4 h of the 1st (A) and the 2nd (B) injection of poly(I:C) and BCG, (C) shows a representative data for control blood, (D) table is shown the percentage of myeloid cells in quadrates.