Endotoxin Tolerance Creates Favourable Conditions for Cancer Development

Abstract

Endotoxin tolerance (ET) is an adaptive phenomenon of the immune system that protects the host from clinical complications due to repeated exposure of the body to endotoxins such as lipopolysaccharide (LPS). Since ET is an immunosuppressive mechanism in which a significant reprogramming of macrophages is observed, we hypothesized that it could influence cancer development by modifying the tumour environment. This study aimed to explore whether ET influences cancer progression by altering the tumour microenvironment. Endotoxin-tolerant macrophages (Mo_ET) were examined for their impact on breast and colon cancer cells via direct interaction and conditioned media exposure. We characterized cancer cell behaviour by viability, clonogenic potential, motility, scratch assays, and 3D spheroidal assays. Mo_ET-derived factors increased cancer cell viability, motility, and clonogenicity, suggesting a conducive environment for cancer development. Remarkably, despite reduced TNFα and IL-6 levels, Mo_ET exhibited M1 polarization. These findings uncover an ET-associated macrophage reprogramming that fosters a favourable context for cancer progression across diverse tumours. Targeting ET could emerge as a promising avenue for cancer therapy and prevention.

Keywords: cancer; cytokines; endotoxin tolerance; immunosuppression; macrophage polarization M1/M2; pro-cancerogenic conditions; tumour microenvironment.

Figure 1

The concentration of TNFα and IL-6 produced by the following groups of RAW 264.7 cells: non-treated macrophages (Mo_NT), LPS-tolerated macrophages (Mo_ET), and macrophages treated with LPS only once (Mo_LPS). Cells were stimulated either once for 24 h, Mo_LPS (24 h) or twice for 24 h, Mo_ET (48 h) with LPS at a concentration of 100 ng/mL. The amount of TNFα (A) and IL-6 (B) was assessed by ELISA. The data are shown as the means ± SEM of three independent experiments with three wells for each condition. Asterisks denote a significant difference between Mo_NT and Mo_LPS (*** p < 0.001).

Figure 2

Cell viability of breast cancer 4T1 cells (A,B) and colon cancer CT26 (C,D) cells stimulated with different concentrations (10–75%) of conditioned media (CM) derived from endotoxin-tolerant macrophages (CM_ET) and non-treated macrophages (CM_NT) for 24 and 48 h. Cell viability was assessed by the MTT colourimetric method. The results are expressed as a percentage of control non-stimulated cells (which is represented as 100%). The data are shown as the mean ± SEM of three independent experiments with six wells for each condition. Asterisks denote significant differences between the respective concentrations of CM_ET and CM_NT (*** p < 0.001, ** p < 0.01).

Figure 3

The number of colonies of 4T1 (A,B) and CT26 (C,D) cancer cells counted after 7 and 5 days of culture, respectively. Before seeding, the cells were cultivated in the conditioned media (CM) derived from endotoxin-tolerant macrophages CM_{ET 10–50%} and non-treated macrophages CM_{NT 10–50%} for 48 h. The data are shown as the mean ± SEM of three independent experiments with 3 wells for each condition. Asterisks denote a significant difference between the cells cultured in CM derived from non-treated cells (Mo_NT) and LPS-tolerated cells (Mo_ET) (*** p < 0.001; ** p < 0.01; * p < 0.05). Hashes denote the significant difference between CMET and the control cells (## p < 0.01).

Figure 4

The motility of 4T1 (A,B) and CT26 (C,D) cancer cells cultured in the conditioned media (CM) derived from endotoxin-tolerant macrophages (CM_ET) and non-treated macrophages (CM_NT) at a concentration of 10–50% for 20 h and 24 h, respectively. Cell migration was assessed with a scratch assay. (A,C) present the representative images of the cells treated with CM at a concentration of 25%. (B,D) show the quantitative scratch closure measured between 0 h and 20 or 24 h (%) using ImageJ software. (*** p < 0.001). The data are shown as the mean ± SEM of three independent experiments with 3 wells for each condition. Asterisks denote a significant difference between the cells cultured in CM_NT and CM_ET (*** p < 0.001). Hashes denote the significant difference between MoET and the control cells (### p < 0.001).

Figure 5

The area of 3D spheroids formed by the breast cancer 4T1 cells (A,B) and colon cancer CT26 cells (C,D) in the conditioned media (CM) derived from endotoxin-tolerant macrophages (CM_ET), non-treated macrophages (CM_NT), and normal media at a concentration of 10–50% after 48 h. Asterisks show a significant difference between the cells cultured in CM_ET and CM_NT (*** p < 0.001). Hashes denote the significant difference between Mo_ET and the control cells (### p < 0.001; ## p < 0.01).

Figure 6

Protein concentration of IL-6 (A,C) and TNFα (B,D) produced in a monoculture of Mo_NT, Mo_ET, 4T1 or CT26, and co-culture of 4T1 or CT26 cells with Mo_ET, Mo_NT, and Mo_LPS that was determined by ELISA assays. Asterisks denote significant differences between the co-culture conditions of 4T1 or CT26 cells with Mo_ET and Mo_NT (*** p < 0.001). The data are shown as the mean ± SEM of three independent experiments with three wells for each condition.

Figure 7

Characterization of the macrophage phenotype in LPS-tolerated RAW264.7 cell. The percentage of M1 cells is plotted on the left Y-axis and M2 cells on the right Y-axis, respectively. To induce endotoxin tolerance, the cells were stimulated twice with 100 ng/mL of LPS. The data are shown as the mean ± SEM of three independent experiments. Asterisks denote a significant difference in individual groups of cells (*** p < 0.001, ** p < 0.01, * p < 0.05).

Figure 8

Effect of endotoxin tolerance on cancer cell growth in vitro. Endotoxin-tolerant macrophages release low levels of TNF-α and IL-6 into the medium. Conditioned medium collected from these macrophages enhances cancer aggressiveness measured by cell viability, clonogenic potential, cell motility, and spheroid size. Up and down arrows indicate the direction of effect in response to each conditioned medium. (Partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license.)