The work presented here describes the recruitment of a putative stem cell population to malignant mesothelioma spheroids in a transplantable murine tumor model. Stem cells are slowly proliferating cells whose proliferation status is niche-dependent (22
). We found that the increasing number of Sca-1-positive cells found in tumor spheroids over time is due to cell recruitment rather than cell proliferation, also confirmed by BrdU incorporation studies It is possible there is a number of proliferative Sca1-positive cells cycling but not in S-phase during this BrdU pulse labeling. Alternatively, recruited Sca-1-positive cells may undergo cell proliferation at a slow rate.
The Sca-1-positive cells consist of adherent mesenchymal stem cells. Recruitment of mesenchymal stem cells to malignant mesothelioma tumors is a novel finding. Mesenchymal stem cells comprised 82% of the total adherent Sca-1-positive cell population. We hypothesized that the malignant mesothelioma tumor spheroid microenvironment is conducive to recruitment of mesenchymal stem cells. While many chemokines and growth factors are reported to be involved in recruitment of mesenchymal stem cells (11
) the highest expression levels were found for the chemokine SDF1/CXCL12, at both the mRNA and protein levels. Trends in mRNA expression were not paralleled by protein expression in these studies; this could be explained by the fact that mRNA expression was measured using tumor spheroid cells alone while protein expression was measured using cell culture media samples containing secreted SDF1 protein. However, there is slightly higher expression of SDF1 after 7 days compared to 21 days after injection of malignant mesothelioma cells. This supports the hypothesis that the malignant mesothelioma microenvironment is conducive to ongoing recruitment of mesenchymal stem cells.
RT-PCR studies showed no expression of CXCR4 by malignant mesothelioma cells grown in monolayer in vitro
, but significant CXCR4 expression was found in ex vivo
malignant mesothelioma spheroids. These findings suggest that malignant mesothelioma cells do not express CXCR4, rather, malignant mesothelioma cells injected in vivo
recruit CXCR4-expressing host cells that are integrated into tumor spheroids. In addition to a CXCR4-expressing mesenchymal stem population, there may be recruitment of CXCR4+ lymphocyte population similar to the lymphocyte population found in human malignant pleural and peritoneal mesothelioma (23
CXCR4 expression could be dependent on cell culture conditions. Cells grown in monolayer in culture have different gene expression profiles than cells grown as spheroids in culture (24
). It is possible that the mesenchymal stem cells and adherent Sca-1-positive cells used in our studies would upregulate CXCR4 expression if grown as spheroids in vitro
or immediately used ex vivo
Transwell migration studies showed increased mesenchymal stem cell recruitment to lavage fluid collected from mice 21 days after malignant mesothelioma cell injection compared to lavage fluid from mice after 7 days, and there was greater mesenchymal stem cell recruitment at both time points compared to lavage fluid from PBS-injected mice. This trend of increasing Sca-1-positive cell recruitment over time is consistent with the ex vivo experiments described earlier.
Mesenchymal stem cell migration to lavage fluid from PBS-injected mice and to lavage fluid collected 7 days after injection of malignant mesothelioma cells was unaffected by AMD3100 pretreatment. It is possible that other chemotactic factors play a more significant role for mesenchymal stem cell recruitment at this early time point in tumor growth and progression. However, at 21 days following injection of mesothelioma cells, there is significant inhibition of mesenchymal stem cell recruitment. This result suggests that the SDF1/CXCR4 chemotactic axis plays a significant role in recruitment of mesenchymal stem cells at the later stages of malignant mesothelioma growth and progression.
The peritoneal fluid can be a depot for secreted molecules from multiple cell types found in the peritoneal cavity, including tumor cells, recruited host cells, and resident cells of the intestinal mesentery including milky spots. In experiments where the only source of SDF1 would be from cells of the tumor spheroids, pretreatment of mesenchymal stem cells with CXCR4 inhibitors significantly abrogated mesenchymal stem cell recruitment. Taken together, these findings demonstrate the dependence of MSC transmigration on the SDF1/CXCR4 chemotactic axis in this murine tumor model, and that tumor spheroids are a significant source of SDF1.
CXCR4 inhibition in vivo decreased tumor burden in our murine tumor model. Interestingly, the percent of total tumor consisting of free-floating tumor spheroids was significantly different between the two treatment groups. By 21 days after tumor cell injection, the percentage of tumor spheroids significantly decreased in AMD3100-injected mice but not in PBS-injected mice. This decrease may be attributed to tumor cell death due to apoptosis in AMD3100-injected mice during this time frame resulting in a decreased percentage of tumor spheroids. When total tumor area is divided into solid tumor area versus tumor spheroid area, it is clear that AMD3100-injected mice have less tumor area than PBS-injected mice, and that tumor area in AMD3100-injected mice is comprised mostly of tumor spheroids and not solid tumor. Taken together, there is a trend towards decreased tumor burden in mice treated with AMD3100. This may correlate with decreased Sca1-positive cell recruitment and incorporation into tumor spheroids. It is also possible that AMD3100 inhibits other stages in malignant mesothelioma progression, such as angiogenesis.
In conclusion, tumor spheroids from this orthotopic murine mesothelioma model represent a dynamic state of mesenchymal stem cell recruitment during tumor growth and progression. The SDF1/CXCR4 chemotactic axis plays a significant role in mesenchymal stem cell recruitment to malignant mesothelioma spheroids and could be a novel therapeutic target for this aggressive tumor, especially as part of a multimodal therapeutic approach following surgical debulking.