Introduction
Mesenchymal stromal cells (MSC) are found in a variety of tissues where they constitute a stem cell pool [
1]. They can be mobilized to sites of inflammation and angiogenesis, such as wounded tissue and, importantly, various solid cancers [
2‐
4]. No single MSC-specific marker exists for in vivo detection of this cell type and it has been shown that the marker expression of MSCs greatly depends on the tissue in which they reside [
5,
6]. Human MSCs are defined according to in vitro consensus criteria developed by The International Society for Cellular Therapy (ISCT); in culture, MSC should adhere to plastic, they should express CD73, CD90, CD105 but lack expression of CD11b or CD14, CD19 or CD79α, CD34, CD45 and HLA-DR, and they should be able to differentiate into adipocytes, osteoblasts and chondrocytes [
7].
MSC constitute a promising vector system for cell mediated gene therapy [
8] as they display inherent tumor-tropic migratory capabilities [
9,
10] are found in several adult tissues [
1] and are easy to isolate and expand in vitro [
11]. However the use of MSCs as a tumor-tropic cellular vector system in cancer including brain tumor gene therapy also raise several concerns as it is not clarified how the cells affect the tumor and its microenvironment [
12]. Consequently the presence and role of endogenous in human brain tumors has now gained attention [
13]. Isolation of glioma-associated human MSCs was recently performed. Analysis of bulk cultures of primary dissociated tissue from human glioma revealed that glioma-associated MSCs were nontumorigenic stromal cells with phenotypical similarities to bone marrow-derived human MSCs [
13]. Genomic sequencing of the glioma-associated MSCs suggested that most MSCs were normal non-transformed cells recruited into the glioma.
The present investigation was undertaken in order to expand on these previous recent findings. For this, a robust protocol for the detection of MSCs using 9 phenotypic markers according to the full ISCT consensus criteria has been developed. Following single cell sorting, we here report the findings of two distinct populations of glioma-derived MSC-like cells that differ in their CD90 expression, their gene expression pattern and their production of pro-angiogenic VEGF and immune-suppressive PGE2.
Materials and methods
Tumor samples
Fourteen primary brain tumor samples were obtained from the neurosurgery department at Skåne University Hospital in Lund, Sweden. Ethical permit H15 642/2008. The tissue was washed in phosphate buffered saline (PBS, Life Technologies, Carlsbad, CA, USA), placed in a Petri dish with StemMACS MSC Expansion Media (Miltenyi Biotec, Bergisch Gladbach, Germany) and minced with a scalpel. Afterwards, cells were washed in PBS and then incubated in Accutase (Sigma-Aldrich, Stockholm, Sweden) at 37 °C for 20 min. They were rotated every 5 min to prevent sedimentation. Finally, cells were passed 2–3 times through an 18 G needle before being filtered through a 100 µm nylon mesh. The single cell suspension was put in cell culture flasks with MSC Expansion Media.
Cell culturing
Tumor-derived cells were grown adherently in MSC Expansion Media supplemented with Antibiotic–Antimycotic solution (AAS, Sigma-Aldrich) in cell culturing flasks at 37 °C and 5 % CO2. Half of the medium was changed 1–3 times per week. Cells at 70–100 % confluency were passaged using Accutase.
Flow cytometry
Culture-derived cells at passage 2–4 were stained with the following monoclonal antibodies; Brilliant Violet 421-CD73 (AD2), Alexa Fluor 700-CD90 (5E10, both from Nordic BioSite, Täby, Sweden), PE-CD105 (266), PE-Cy5-HLA-ABC (G46-2.6), FITC-CD14 (M5E2), FITC-CD19 (HIB19), FITC-CD34 (581), FITC-CD45 (HI30) and FITC-HLA-DR (G46-6, all from BD Biosciences) and isotype matched controls for 30 min at 4 °C. TO-PRO-1 (Life Technologies) was used as viability marker and cells were analyzed and sorted on a FACSAria III cell sorter (BD Biosciences, Heidelberg, Germany).
Cell sorting
Culture-derived cells analyzed on the FACSAria III cell sorter were simultaneously sorted based on surface marker expression. Two populations were sorted from each tumor, one expressing the defined MSC phenotype and one expressing the defined MSC phenotype except CD90. Cells were sorted at passage 2–4 and immediately put back in culture.
“In vitro” differentiation
Sorted tumor-derived stromal cells at passage 8–11 were used for in vitro differentiation assays. Bone marrow-derived (BM) MSCs from healthy donors were used as positive control and the experiments were performed as described elsewhere [
14].
For adipocyte differentiation, cells were cultured in StemMACS AdipoDiff Media (Miltenyi Biotec) for 14 days. They were fixed with 4 % formaldehyde solution (APL, Stockholm, Sweden) for 60 min at and then incubated in 60 % isopropanol (VWR International, Stockholm, Sweden) for 5 min before being stained with 0.18 % Oil Red O (Sigma-Aldrich) for 5 min, all at room temperature (RT).
For osteoblast differentiation, cells were cultured in osteoblast induction medium for 21 days. They were incubated in ice-cold 70 % ethanol for 1 h at 4 °C before being stained with 40 mM Alizarin Red solution (Sigma-Aldrich) for 10 min at RT.
For chondrocyte differentiation, cell pellets were grown in chondrogenesis induction medium for 28 days. They were fixed in Stefanini’s fixative [
15] and then incubated in 20 % sucrose solution (Merck KGaA, Darmstadt, Germany), both at 4 °C overnight. The pellets were frozen in Tissue-Tek O.C.T. Compound (Sakura Finetek Sweden AB, Göteborg, Sweden). 5 µm thick cryosections were permeabilized and blocked with PBS supplemented with 0.3 % Triton X-100, 0.1 % sodium azide, 0.1 % fish skin gelatin (all from Sigma-Aldrich) and 10 % normal donkey serum (Jackson ImmunoResearch Europe Ltd., Suffolk, United Kingdom) for 45 min at RT. They were stained with polyclonal goat anti-human aggrecan antibody (5 µg/ml, R&D Systems) at 4 °C overnight and then Alexa Fluor 594 donkey anti-goat IgG (5 µg/ml, Life Technologies) for 60 min at RT. Nuclei were visualized by incubation with Hoechst 33342 (8.1 µM, Life Technologies) for 15 min at RT.
Stained adipocytes and osteoblasts were analyzed with a Nikon Diaphot 300 and a Nikon D70 (both from Nikon, Yurakucho, Tokyo, Japan). Chondrocyte pellets were analyzed with an Olympus BX61 and an Olympus DP72 (both from Olympus, Shinjuku, Tokyo, Japan).
RNA-sequencing extraction and gene analysis measurements
Total RNA from GBM-47 (passage number 2); GBM-48 (passage number 2); GBM-47-derived MSC-like CD90
− cells (passage number 8); GBM-48-derived MSC-like CD90
− cells (passage number 5); GBM-47-derived MSC-like CD90
+(passage number 10); GBM-48-derived MSC-like CD90
+(passage number 6); U87 primary GBM cell line (passage number 30) and human bone-marrow derived MSC (hBM-MSC, passage number 1, from a 61 years old healthy male donor) was isolated by using RNAesay (Qiagen) with DNase treatment. RNA was extracted according to manufacturer instructions and their concentration was determined spectrophotometrically by Nanodrop-ND 1000 spectophotometer (Nanodrop). RNA quality was analyzed using a Bioanalyzer (Agilent) and samples with a RNA integrity greater than seven were further analyzed. Subsequent RNA amplification and analysis were largely performed as previously reported in [
16,
17] except that libraries were prepared using TruSeq Stranded mRNA Kit for NeoPrep from Illumina.
ELISA
Sorted tumor-derived stromal cells at passage 10–13 were analyzed for VEGF and PGE2 production using ELISA. 1 × 105 cells/well were plated in a 24 well plate and grown in 1 ml MSC Expansion Media for 24 h. Supernatants were collected, centrifuged and stored at −80 °C prior to analysis. VEGF levels were determined using the Human VEGF DuoSet kit (R&D Systems). PGE2 levels were determined using the Prostaglandin E2 EIA kit (Cayman Chemical Company, Larodan Fine Chemicals, Malmö, Sweden). Experiments were performed in duplicate on three separate occasions.
Statistical analysis
The RNA sequencing data processing was performed using the analysis pipe line method described in SCAN-B article [
16]. Thus, after the first filtering processes, only the mRNAs showing differential expression between different groups are taken for further analysis. To compare the mRNA expression profile between the 2 GBM-derived MSC groups, CD90
− and CD90
+ cells, simple
t test was used and a p value <0.01 was considered significant. The VEGF and PGE
2 production analysis was performed using Two-way ANOVA, where p < 0.05 was considered statistically significant. Linear regression analysis was performed correlating the survival time of patients to % total MSC in bulk culture or CD90
+ cells in bulk culture or CD90
− cells in bulk culture in all possible combinatorial forms. A p value lower than 0.05 was considered to be significant.
Discussion
Malignant gliomas with a mesenchymal gene expression profile are associated with an invasive growth pattern and short survival and recurring tumors often display a mesenchymal phenotype [
18,
19]. In this study, we analyzed 14 primary human malignant gliomas and conclude that all of them harbor cells expressing surface markers defined by ISCT [
7].
By employing a stricter flow cytometric analysis, adhering to the ISCT criteria, and by, for the first time for this particular purpose, sorting cells, we expand on previous reports of MSC-like cells in malignant glioma [
13]. We found that malignant gliomas harbor two distinct MSC-like cell populations, differing in their CD90 expression. Although a different tri-mesenchymal differentiation capacity was noticed between CD90
+ and CD90
− populations, as indicated by adipocyte differentiation only in cells lacking CD90, this data is preliminary and further experiments, optimizing the expansion of MSC clones from a larger number of tumor samples, is warranted in order to confirm if the absence of adipocyte differentiation is a distinctive feature of CD90
+ cell population.
In most tumor samples, the CD90
− population was larger than the CD90
+ population. Both populations were expandable and retained their proliferative capacity in vitro. Unfortunately, these cell populations were difficult to detach from the plastic surface during the first passages and we could only perform complete studies in cells derived from 2 patients. The number of MSC-like cells compared to the total number of cells (MSC to total cell number in culture in Table
1) following 2–4 passages in vitro showed a very wide variation. Yet, the present study was not designed in order to quantify the ratio of MSC-like cells within glioma stroma. Interestingly though, the tumor with the highest proportion of MSC-like cells was a gliosarcoma, a GBM subtype with a clear mesenchymal component [
14].
To confirm that the two MSC-like cell populations CD90
+ and CD90
− constitute two distinct cell populations, we analyzed their gene expression profile. Although we were limited in the number of samples, the mRNA expression profiles clearly support the notion that these are, in fact, two different populations. Intriguingly, although the cell surface expression of CD90 separates these populations, their CD90 mRNA expression profile did not differ, suggesting that cell surface glycoprotein CD90 could be either engulfed from the surface due to a cell reaction to some environmental stimuli, thus becoming undetectable on the surface or, that CD90
− cells react to some stimuli and overexpress CD90 as a response to the stimuli. In vitro angiogenic stimuli, for example, generate hM-MSC lacking CD90 expression [
20] whilst overexpression of surface cell markers upon inflammatory stimuli has been already reported for neurotrophic receptors [
21]. In addition, mechanical stress has been reported to decrease CD90 cell surface glycoprotein in MSC [
22]. Since little mechanical stress was performed during culturing, and the number of passages were low when mRNA expression analysis was performed, this hypothesis is quite unlikely. Alternatively, the CD90 mRNA could suffer any type of posttranslational preventing the receptor from being visible on the surface [
23,
24]. Future experiments for a further deep characterization of CD90
+ and CD90
− populations are envisaged.
Likewise, the functional significance of CD90 expression, or lack thereof, on glioma-derived MSC-like cells needs further investigations. Previous studies have suggested that CD90
+ pericytes within gliomas promote vascularization and immunosuppression [
15]. However, CD90 has also been reported to be a tumor suppressor [
25]. The present study might lend some support to the latter, as we found that the MSC-like CD90
− cells could be producing elevated levels of VEGF compared to the CD90
+ cells from the same tumors. If confirmed, this could indicate that the CD90
− population of glioma-derived MSC-like cells might be more actively involved in tumor angiogenesis than the CD90
+ subpopulation. In addition to its critical role in tumor angiogenesis, VEGF has been reported to attract MSCs themselves to glioma cells [
3]. Furthermore, VEGF has been reported to stimulate MSC proliferation [
26], indicating that autocrine mechanisms might function to stimulate MSC recruitment and proliferation within glioma.
The immunosuppressive function of BM-MSCs is well characterized and utilized clinically [
27]. Among the key immunoactive factors expressed by MSCs is prostaglandin E
2 (PGE
2) [
28], which has been shown to induce immunosuppression within tumors [
15,
29]. Because of technical reasons, we were only able to expand FACS sorted cells from two glioblastoma, which severely restricted their further analysis. However, interestingly, the CD90
− MSC-like cells secreted markedly higher amounts of PGE
2 compared to the CD90
+ cells. PGE
2 is a molecule known to be important for BM-MSC-induced immunosuppression [
30], suggesting that glioma-derived MSC-like cells, and the CD90
− subpopulation in particular, may actively contribute to the glioma immunosuppression.
Even though the source of the glioma-derived MSC-like cells is unknown, the most probable explanation is that the tumor recruits them from normal tissue. Inflammatory factors, such as IL-8 [
31], monocyte chemotactic protein-1 [
32] and stromal cell-derived factor 1α [
33] are known to be expressed in gliomas and have been reported as attractants of MSCs in other cancers [
34,
35]. Further on, the extensive angiogenesis within malignant gliomas is believed to play a major part in MSC migration. In addition to VEGF, angiogenic factors such as platelet-derived growth factor-BB [
36] and transforming growth factor-β1 [
3], known to be involved in glioma angiogenesis, have been shown to mediate MSC recruitment [
3,
34]. Moreover, we have previously shown that drug-mediated angiogenesis inhibition markedly decreased the migration of BM-MSCs transplanted into experimental rat gliomas [
10].
In conclusion, we show that two distinct MSC-like cell populations, differing in their expression of the CD90 surface marker expression, can be isolated from primary human malignant gliomas. Further analysis revealed important differences between these two populations with regards to mRNA expression patterns and, present results show that CD90− cells produced higher amounts of VEGF and PGE2 compared to cells with the true MSC phenotype, implying that CD90− cells may be more active in tumor vascularization and immunosuppression than their CD90+ counterpart. Taken together, our results highlight the CD90− MSC-like subpopulation as an important tumor component, however, its functional effects in glioma remains to be resolved. Using the protocols presented here, it will be possible to isolate, characterize and analyze brain tumor-derived MSC-like cells in more detail and to further test their functions in vitro and in in vivo xenograft models of glioma.