Lipopolysaccharide (LPS) stimulation of Pancreatic Ductal Adenocarcinoma (PDAC) and macrophages activates the NLRP3 inflammasome that influences the levels of pro-inflammatory cytokines in a co-culture model

Figures & data

Table 1. The different treatment/control groups included for each designated co-culture setup. All treatments were conducted for 24 h.

Group	Description of PDAC treatment
L	Stimulated with 1 μg/mL LPS
A	Treated with 5 μM ATP
M	Treated with 10 μM MCC950
LA	Stimulated with 1 μg/mL LPS in the presence of 5 μM ATP
LM	Stimulated with 1 μg/mL LPS in the presence of 10 μM MCC950
AM	Stimulated with 5 μM ATP in the presence of 10 μM MCC950
LAM	Stimulated with 1 μg/mL LPS in the presence of 5 μM ATP and 10 μM MCC950
C	RPMI SFM only (control)

Figure 1. Cell proliferation of Panc 10.05 and SW 1990 cells co-cultured with macrophages under LPS-stimulated inflammation with or without ATP and MCC950.

PDAC cell lines were co-cultured with macrophages and the proliferation of the PDAC cells was determined by performing the MTT Assay.

PDAC cell lines were co-cultured with macrophages and the proliferation of the PDAC cells was determined by performing the MTT Assay. Data are presented as mean ± SEM of three individual experiments. Means with the same letter(s) above the bar are not significantly different from each other (p>.05), whereas means with different letters are significantly different from each other.

Figure 2. Levels of cytokine a) IL-1β and b) TNF-α production by i) Panc 10.05 PDAC cells and ii) SW 1990 PDAC cells co-cultured with macrophages under LPS-stimulated inflammation, with or without ATP and MCC950.

Panc 10.05 PDAC cells and SW 1990 PDAC cells were co-cultured with macrophages and the levels of cytokine IL-1β and TNF-α production by PDAC cells, in their respective co-cultures, were determined by performing ELISA and Bradford Assays.

Data are presented as mean ± SEM of three individual experiments. Means with the same letter(s) above the bar are not significantly different from each other (p>.05), whereas means with different letters are significantly different from each other.

Figure 3. Levels of cytokines i) IL-1β and ii) TNF-α production by RAW 264.7 macrophages in the co-cultures A) Panc 10.05 PDAC cells/RAW 264.7 macrophages and B) SW 1990 PDAC cells/RAW 264.7 macrophages, under LPS- and ATP-induced inflammation with or without NLRP3 inhibition by MCC950.

Panc 10.05 PDAC cells and SW 1990 PDAC cells were co-cultured with RAW 264.7 macrophages and the levels of cytokine IL-1β and TNF-α production by RAW 264.7 macrophages, in their respective co-cultures, were determined by performing ELISA and Bradford Assays.

Data are presented as mean ± SEM of three individual experiments. Means with the same letter(s) above the bar are not significantly different from each other (p>.05), whereas means with different letters are significantly different from each other.

Figure 4. Relative protein expression levels of NLRP3 inflammasome by a) Panc 10.05 and b) SW 1990 PDAC cells in their respective co-cultures with RAW 264.7 macrophages under LPS and ATP-induced inflammation with or without MCC950 inhibition were analysed using Western blot.

Panc 10.05 and SW 1990 PDAC cells were co-cultured with RAW 264.7 macrophages and the relative protein expression levels of NLRP3 inflammasome by the PDAC cells, in their respective co-cultures, was determined by performing and imaging Western Blots.

β-actin expression serves as the control. Data are presented as mean ± SEM of three individual experiments. Means with the same letter(s) above the bar are not significantly different from each other (p>.05), whereas means with different letters are significantly different from each other.

Figure 5. Relative levels of NLRP3 inflammasome by macrophages when co-cultured with a) Panc 10.05 cells and b) SW 1990 cells under LPS-stimulated inflammation with or without ATP and MCC950. Proteins in cell lysates of each treatment were subjected to the Western blot analysis.

Panc 10.05 and SW 1990 PDAC cells were co-cultured with RAW 264.7 macrophages and the relative protein expression levels of NLRP3 inflammasome by the RAW 264.7 macrophages, in their respective co-cultures, was determined by performing and imaging Western Blots.

Levels of NLRP3 were normalized with the levels of β-actin, which served as the endogenous control. Data presented as mean ± SEM of three individual experiments. Means with the same letter(s) above the bar are not significantly different from each other (p>.05), whereas means with different letters are significantly different from each other.

Figure 6. The proposed pathways of interaction between Panc 10.05 PDAC cells and RAW 264.7 macrophages.

This system emulates the interaction between primary adenocarcinoma cells (Panc 10.05) and TAMs in an isolated microenvironment in the absence of other immune cells and stromal cells.

This system emulates the interaction between primary adenocarcinoma cells (Panc 10.05) and TAMs in an isolated microenvironment in the absence of other immune cells and stromal cells. Central to the co-culture model would be the role of macrophage-conditioned medium (MCM) in inhibiting PP2A repression, leading to increased activity of kinases, including IKK, JNK, PKC, and ERK. The roles of LPS, ATP, MCC950, TRIM 31, and GFI1 on the negative regulation of NLRP3 inflammasome were also appreciated. MCM: Macrophage Conditioned Medium; PP2A: Protein Phosphatase 2A; IKK: IκB kinase; JNK: c-Jun N-terminal Kinases; PKC: Protein Kinase C; ERK: Extracellular signal Related Kinase; LPS: Lipopolysaccharide; ATP: Adenosine Triphosphate; TRIM31: Tripartite Motif Containing 31; GFI1: Growth Factor Independence 1; p50-p65: NF-κB.

Figure 7. The proposed pathways of interaction between SW 1990 PDAC cells and RAW 264.7 macrophages.

This system emulates the interaction between primary adenocarcinoma cells (SW 1990) and TAMs in an isolated microenvironment in the absence of other immune cells and stromal cells.

This system emulates the interaction between primary adenocarcinoma cells (SW 1990) and TAMs in an isolated microenvironment in the absence of other immune cells and stromal cells. Central to the co-culture model would be the role of macrophage-conditioned medium (MCM) in inhibiting PP2A repression, leading to increased activity of kinases, including IKK, JNK, PKC, and ERK. The roles of LPS, ATP, MCC950, TRIM 31, and GFI1 on the negative regulation of NLRP3 inflammasome were also appreciated. MCM: macrophage conditioned medium; PP2A: protein phosphatase 2A; IKK: IκB kinase; JNK: c-Jun N-terminal kinases; PKC: protein kinase C; ERK: extracellular signal-related kinase; LPS: lipopolysaccharide; ATP: adenosine triphosphate; TRIM31: tripartite motif containing 31; GFI1: growth factor independence 1; p50-p65: NF-κB.