Inflammatory Human Umbilical Cord-Derived Mesenchymal Stem Cells Promote Stem Cell-Like Characteristics of Cancer Cells in an IL-1 β-Dependent Manner

Abstract

To ensure the safety of clinical applications of MSCs, thorough understanding of their impacts on tumor initiation and progression is essential. Here, to further explore the complex dialog between MSCs and tumor cells, umbilical cord-derived mesenchymal stem cells (UC-MSCs) were employed to be cocultured with either breast or ovarian cancer cells. Though having no obvious influence on proliferation or apoptosis, UC-MSCs exerted intense stem cell-like properties promoting effects on both cancer models. Cocultured cancer cells showed enriched side population, enhanced sphere formation ability, and upregulated pluripotency-associated stem cell markers. Human cytokine array and real-time PCR revealed a panel of MSC-derived prostemness cytokines CCL2, CXCL1, IL-8, and IL-6 which were induced upon coculturing. We further revealed IL-1β, a well-characterized proinflammatory cytokine, to be the inducer of these prostemness cytokines, which was generated from inflammatory UC-MSCs in an autocrine manner. Additionally, with introduction of IL-1RA (an IL-1 receptor antagonist) into the coculturing system, the stem cell-like characteristics promoting effects of inflammatory UC-MSCs were partially blocked. Taken together, these findings suggest that transduced inflammatory MSCs work as a major source of IL-1β in tumor microenvironment and initiate the formation of prostemness niche via regulating their secretome in an IL-1β-dependent manner.

Figure 1

Characteristics of UC-MSCs. (a) Immunofluorescent staining of CD29, CD44, CD90, and CD105 in human umbilical cord-derived MSCs (UC-MSCs). (b) Flow cytometry analysis of CD44, CD90, and CD105 expression in UC-MSCs. (c) Differentiation of UC-MSCs into 3 distinct lineages, namely, adipocytes, chondrocytes, and osteoblasts.

Figure 2

Effects of UC-MSCs on proliferation and apoptosis of cancer cells. (a-b) UC-MSC conditioned medium (MSC-CM) 1 : 1 mixed with fresh basic medium was supplemented with 10% FBS and used for culturing. CCK8 assay was performed in MDA-MB-231 (a) or IGROV1 cells (b) at the indicated time points, and OD value was measured at 450 nm. (c-d) MDA-MB-231 (c) or IGROV1 cells (d) were indirectly cocultured with UC-MSCs and harvested at the indicated time points, and the percentage of Ki67 positive cells was shown here. n = 3; ns stands for nonsignificance. (e-f) Annexin V-PI staining was performed in MDA-MB-231 (e) or IGROV1 cells (f) cocultured with UC-MSCs, and apoptotic cells were analyzed by FACS. Representative dot plots and statistical results were shown here. n = 3; ns stands for nonsignificance.

Figure 3

UC-MSCs enhance stem cell-like characteristics in cancer cells. (a-b) Hoechst staining was performed in MDA-MB-231 cells (a) and IGROV1 cells (b) indirectly cocultured with UC-MSCs, and statistical results of side population (SP) by FACS were shown here. n = 3 for MDA-MB-231 cells, and n = 2 for IGROV1 cells, ^∗p < 0.05. (c) After being indirectly cocultured with UC-MSCs, MDA-MB-231 cells were harvested and seeded into low attachment plates for sphere formation assay. Representative images and statistical results were shown here. n = 4, ^∗p < 0.05. (d) Expression levels of pluripotency markers Sox2 and Oct4 in MDA-MB-231 and IGROV1 cells indirectly cocultured with UC-MSCs by Western blots.

Figure 4

Cytokines derived from cocultured UC-MSCs enhance stem cell-like characteristics in cancer cells. (a) Detection of increased cytokines in 231-MSC coculture compared to MDA-MB-231 cell culture and IGROV1-MSC coculture compared to IGROV1 cell culture by human cytokine array. (b) Relative expression levels of CCL2, CXCL1, and IL-8 in MDA-MB-231 cells and UC-MSCs in 231-MSC coculture and relative expression of CXCL1 and IL-8 in IGROV1 cells and UC-MSCs in IGROV1-MSC coculture. ^∗∗p < 0.01. (c) Relative expression levels of CCL2, CXCL1, IL-8, and IL-6 in UC-MSCs cocultured with MDA-MB-231 cells compared to UC-MSCs cultured alone. ^∗∗p < 0.01. (d) Effects of CCL2 and CXCL1 on pluripotency markers Sox2, Oct4, and KLF4 expression in MDA-MB-231 cells. (e) Effects of CCL2 and CXCL1 on migration ability of MDA-MB-231 cells by Transwell migration assay. n = 3, ^∗∗p < 0.01.

Figure 5

Inflammatory UC-MSCs secrete IL-1β in an autocrine manner and generate a prostemness niche via upregulating CCL2, CXCL1, IL-8, and IL-6. (a) Comparison of IL-1β concentration between MDA-MB-231 cell culture and 231-MSC coculture, and between IGROV1 cell culture and IGROV1-MSC coculture by ELISA assay. ^∗∗p < 0.01. (b) Relative expression level of IL-1β in MDA-MB-231 cells and UC-MSCs in 231-MSC coculture and in IGROV1 cells and UC-MSCs in IGROV1-MSC coculture. ^∗∗p < 0.01. (c) Expression levels of CCL2, CXCL1, IL-8, and IL-6 in UC-MSCs upon IL-1β treatment. ^∗∗p < 0.01. (d) Expression levels of pluripotency markers Sox2 and Oct4 in MDA-MB-231 and IGROV1 cells upon IL-1β treatment. (e) Relative expression level of IL-1β in UC-MSCs and MDA-MB-231 cells under coculturing compared to cells cultured alone. ^∗∗p < 0.01; ^∗p < 0.05. (f) mRNA level of IL-1β upon IL-1β treatment in UC-MSCs and MDA-MB-231 cells. ^∗∗p < 0.01; ns stands for nonsignificance. (g) Protein level of IL-1β upon IL-1β treatment in UC-MSCs. (h) Effects of IL-1RA on the expressions of Sox2 and KLF4 in MDA-MB-231 and IGROV1 cells under coculturing. ns, nonsignificance.

Figure 6

A putative model for prostemness characteristics of inflammatory UC-MSCs cocultured with cancer cells. Upon coculturing with cancer cells, UC-MSCs possess inflammatory phenotype with enhanced IL-1β level. IL-1β derived from inflammatory UC-MSCs creates a prostemness niche by upregulating CCL2, CXCL1, IL-8, and IL-6 secretion in UC-MSCs in an autocrine manner, which reciprocally function on cancer cells, enhance side population (SP) ratio, and upregulate pluripotency markers.