SDF1/CXCL12 is involved in recruitment of stem-like progenitor cells to orthotopic murine malignant mesothelioma spheroids

Abstract

Background/aim: Tumor progression is influenced by the microenvironment. We found stem cells are recruited to malignant mesothelioma spheroids. We aimed to determine if stem cell recruitment depends on the chemokine SDF1, and if inhibition of the cognate receptor CXCR4 affects tumor growth.

Materials and methods: The kinetics of stem cell recruitment was determined using immunofluorescence staining, BrdU incorporation and eGFP transgenic mice. Chemokines were identified using PCR array. Inhibitors of CXCR4 were used to determine the effect on cell migration and tumor progression.

Results: The increasing number of stem cells found in tumor spheroids over time is attributed to cell recruitment. Stem cell migration in vitro was enhanced by exogenous SDF1 and abrogated by CXCR4 inhibition and. CXCR4 inhibition reduced tumor burden in vivo.

Conclusion: SDF1 is a candidate chemokine for recruitment of stem cells to malignant peritoneal mesothelioma and a potential target for therapy.

Figure 1

Spatial and temporal distribution of Sca-1+ cells. (A) Confocal Z-stack (60μm) spheroid showing Sca-1+ cells (red) in the center. (B) Increasing number of Sca-1+ cells (green) in spheroids over time. (C) Flow cytometry data showing no increase in percentage of Sca-1+ cells amongst single cells but an increasing percentage in spheroids over time.

Figure 2

Sca1-positive cells are recruited to tumor spheroids and are not proliferating. (A) Schematic of experimental design. Flow cytometry shows that 69% of Sca-1-positive cells are recruited (eGFP+) while 31% are proliferative (eGFP−). (B) Day 21 tumor spheroids were pulse-labeled with BrdU and immunostained for BrdU incorporation and Sca-1. BrdU+ cells (green) were not Sca-1-positive (red).

Figure 3

SDF1 and CXCR4 expression in tumor spheroids. (A) SDF1 expression at the mRNA and protein levels. (B) left- CXCR4 expression by tumor spheroids but not by mesothelioma cells grown in vitro. β-actin is the loading control. right- No CXCR4 expression by mesenchymal stem cells grown in monolayer or by Sca-1-positive cells collected from day 21 lavage. 18S is the loading control.

Figure 4

(A) Migration of mesenchymal stem cells to lavage fluid collected from PBS-injected mice and tumor-bearing mice 7 and 21 days following injection of mesothelioma cells. (B) AMD3100 inhibits transmigration of mesenchymal stem cells to lavage fluid. (*p value<0.05 compared to no treatment control unless otherwise noted, student’s t-test)

Figure 5

Transmigration of mesenchymal stem cells to conditioned media from tumor spheroids is dependent on the SDF1/CXCR4 chemotactic axis. MSC transmigration to conditioned media is abrogated after CXCR4 inhibition with T22 or AMD3100. Transmigration is enhanced by addition of exogenous SDF1. (*p value < 0.05 compared to no treatment control, student’s t-test)

Figure 6

Tumor burden in mice after mesothelioma cell injection followed by daily injection of PBS or AMD3100. (A) Total tumor number significantly increased in PBS-injected but not in AMD3100-injected mice. (B) Initial higher percentage of tumor spheroids in AMD3100-injected mice compared to control. (C) Greater increasing trend in total tumor area in control than in AMD3100-injected mice. (*p-value ≤ 0.05, student’s t-test)