Conditioned media of pancreatic cancer cells and pancreatic stellate cells induce myeloid-derived suppressor cells differentiation and lymphocytes suppression

Abstract

As pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs) are the two major cell types that comprise the immunosuppressive tumor microenvironment of pancreatic cancer, we aimed to investigate the role of conditioned medium derived from PCCs and PSCs co-culture on the viability of lymphocytes. The conditioned medium (CM) collected from PCCs and/or PSCs was used to treat peripheral blood mononuclear cells (PBMCs) to determine CM ability in reducing lymphocytes population. A proteomic analysis has been done on the CM to investigate the differentially expressed protein (DEP) expressed by two PCC lines established from different stages of tumor. Subsequently, we investigated if the reduction of lymphocytes was directly caused by CM or indirectly via CM-induced MDSCs. This was achieved by isolating lymphocyte subtypes and treating them with CM and CM-induced MDSCs. Both PCCs and PSCs were important in suppressing lymphocytes, and the PCCs derived from a metastatic tumor appeared to have a stronger suppressive effect than the PCCs derived from a primary tumor. According to the proteomic profiles of CM, 416 secreted proteins were detected, and 13 DEPs were identified between PANC10.05 and SW1990. However, CM was found unable to reduce lymphocytes viability through a direct pathway. In contrast, CM that contains proteins secreted by PCC and/or PSC appear immunogenic as they increase the viability of lymphocytes subtypes. Lymphocyte subtype treated with CM-induced MDSCs showed reduced viability in T helper 1 (Th1), T helper 2 (Th2), and T regulatory (Treg) cells, but not in CD8⁺ T cells, and B cells. As a conclusion, the interplay between PCCs and PSCs is important as their co-culture displays a different trend in lymphocytes suppression, hence, their co-culture should be included in future studies to better mimic the tumor microenvironment.

Figure 1

The percentage of lymphocytes in CM treated PBMCs. PBMCs were isolated and treated with medium conditioned by PCCs and/or PSCs for 7 days. Percentage of lymphocytes was determined to verify the lymphocytes suppression of CM on lymphocytes. Statistical significance is indicated by the letters above each column, in which the columns that do not share a common letter have a significance of p ≤ 0.05.

Figure 2

The proteomic profiling of PANC10.05 and SW1990. (a) Venn diagram for the number of proteins expressed by each cell line. Number in the intersection represents the number of expressed proteins shared by PANC10.05 and SW1990. (b) Volcano plot of the DEPs between PANC10.05 and SW1990. (c–e) Gene Ontology enrichment analysis of differentially expressed proteins (DEPs). X-axis represents the gene ratio and Y-axis represents the corresponding GO term. (c) Ontology = Biological Process; (d) Ontology = Molecular Function; (e) Ontology = Cellular Component.

Figure 3

The viability of lymphocyte subtypes treated with medium conditioned by PCCs and PSCs for 48 h. (a) Th1, (b) Th2, (c) Treg, (d) CD8⁺ T cells, and (e) B cells. Statistical significance is indicated by the letters above each column, in which the columns that do not share a common letter have a significance of p ≤ 0.05.

Figure 4

The ratio of Th1 against Th2. The ratio has been calculated based on the viability of Th1 and Th2 after treated with (a) CM and (b) CM-induced MDSCs for 48 h. Statistical significance is indicated by the letters above each column, in which the columns that do not share a common letter have a significance of p ≤ 0.05.

Figure 5

The viability of lymphocyte subtypes treated with MDSCs induced by PCCs and PSCs CM for 48 h. (a) Th1, (b) Th2, (c) Treg, (d) CD8⁺ T cells, and (e) B cells. Statistical significance is indicated by the asterisk above each column, in which the columns that have an asterisk represent that it has a significance of p ≤ 0.05 as compared to the untreated group.

Figure 6

The summary of phase I and phase II. The study has been divided into two phases. Lymphocyte suppression was observed in the first phase. Whereas in the second phase, lymphocyte suppression was observed only in the indirect pathway.

Figure 7

The effects of CM-induced MDSCs on each T cell subtype.