Background
Gliomas are the most common primary malignancies in the central nervous system (CNS), accounting for approximately 80% of all primary brain tumors [
1]. Glioblastoma (GBM, grade IV) is the most common and malignant type of glioma in adults, presenting a high mortality rate and very poor patient outcomes. In fact, despite multimodal therapeutic approaches consisting of surgery, chemotherapy and radiotherapy, virtually all GBMs recur and lead to death, presenting a median overall survival of ~ 15 months [
2]. This poor outcome has not changed significantly in the last decades, stressing the need for novel therapeutic strategies that may, more efficiently, overcome the highly resistance nature of these tumors. A novel therapeutic approach currently being investigated for a variety of cancer types is based on the use of tumor-trophic stem cells, such as mesenchymal stem cells (MSCs) [
3‐
17]. These are multipotent progenitor cells that are defined according to 3 main characteristics: (i) expression of CD105, CD73 and CD90 (MSCs markers), and lack of expression of CD45, CD34, CD14 (hematopoietic markers); (ii) ability to adhere to plastic surfaces; and (iii) differentiation capacity into adipocytes, osteoblasts and chondrocytes (multipotency) [
18,
19]. Additionally, MSCs are also characterized by their proliferative and self-renewal abilities, and can be isolated from bone marrow (BM-MSCs) [
20], adipose tissue (ASCs) [
21], umbilical cord (e.g., human umbilical cord perivascular cells; HUCPVCs) [
22‐
24], among other sources [
25‐
30]. The use of MSCs is relatively promising since these cells: (i) can be easily isolated and subsequently expanded in vitro; (ii) show multi-lineage differentiation ability; (iii) have an immune privileged nature; (iv) present capacity to home for site of injury, including tumors; and (v) are amenable to genetic modification [
31,
32]. In fact, it was already demonstrated that MSCs present an intrinsic capacity to migrate towards gliomas and present low immunogenicity at autologous transplantation [
3,
10‐
12,
33‐
42]. However, whether this selective MSC tumor-tropism is associated with cancer suppression or promotion functions is still controversial [
3,
10,
11,
13,
34,
35,
43]. Several studies, using MSCs engineered to express anti-glioma agents, demonstrated that these cells are highly effective as anti-tumor delivery agents [
10,
11,
36,
38,
42,
44,
45]. However, few studies have evaluated the impact of non-engineered MSCs on glioma behavior [
3,
12,
13,
46], so it is still unclear if MSCs either promote or repress tumor features. Akimoto and colleagues showed that umbilical cord blood-derived MSCs induced apoptosis in glioma cells; however, in the same study, adipose-derived MSCs enhanced the growth of GBM cells [
3]. In another study, co-culturing of adipose-derived MSCs with human glioma cells led to higher survival and proliferation of glioma cells [
12], whereas in another study, bone marrow-derived MSCs co-cultured with human glioma cells inhibited tumor cell proliferation [
46]. Therefore, further studies focusing on the crosstalk between tumor cells and MSCs should be performed to strengthen the evidence that MSCs-based therapies could be efficiently and safely translated into clinical settings.
This study evaluates how the secretome of a population of MSCs isolated from Wharton Jelly of the umbilical cord (HUCPVCs) modulates critical hallmark features of GBM. In particular, using in vitro and in vivo models, we investigated the effect of HUCPVCs conditioned media (CM) on GBM cells viability, growth, migration, proliferation, angiogenesis, and response to chemotherapy. Proteomic analysis of HUCPVCs CM was performed to identify molecular players that can influence the behavior of GBM cells, which may identify novel targets for therapy.
Discussion
One of the major concerns in stem cell-based therapies is the impact that modified stem cells may have on tumor behavior. MSCs have been proposed as a new therapeutic approach for glioma treatment, because these cells have been found to have tumor chemotactic capabilities and migrate towards tumor sites through the blood-brain-barrier, where continuous bilateral molecular crosstalks occur between stromal and cancer cells [
60,
61]. Although some studies suggested that MSCs inhibit tumor growth, others demonstrated that MSCs have a pro-tumoral function by stimulating tumor growth, migration, invasion, and anticancer-drug resistance [
3,
12,
13,
62]. The pro-tumoral effects are mediated by secreted molecules and/or via direct cellular interactions [
10,
15,
33‐
35,
63‐
70]. Therefore, the clinical validity of MSCs as a potential therapeutic approach for glioma is still a matter of debate, deserving further clarification. In this study, we evaluated the influence of HUCPVCs CM on GBM aggressiveness and highlighted proteins from HUCPVCs CM potentially involved in the observed effects.
Our data shows that GBM cells exposed to HUCPVCs CM exhibit increased viability, migration and proliferation in vitro (Figs.
1,
2,
3a, c). Interestingly, the in vivo CAM model also showed an increase in tumor growth when GBM cells were exposed to HUCPVCs CM (Fig.
4). To the best of our knowledge, this is the first study on the influence of HUCPVCs CM in critical hallmark features of GBM.
Previous studies in different tumor types, including gliomas, are in agreement with our results, showing that MSCs may contribute to tumor growth/proliferation [
3,
4,
14,
39]. Additionally, it was also demonstrated that factors released by MSCs increased the migration ability of several types of cancer cells, including breast [
71], colon [
72], and gastric [
73] cancers. Regarding gliomas, Onzi and colleagues demonstrated that ASCs CM treatment was able to increase the migration capacity of U87 GBM cells, which is in line with our results [
74]. Interestingly, despite the prominent effects of HUCPVCs CM in multiple dimensions of GBM cell biology, the sensitivity of these tumor cells to TMZ chemotherapy was not significantly affected by HUCPVCs CM (Fig.
3b, d). These results are in agreement with the work of Onzi and colleagues, where they demonstrated that ASCs CM treatment did not alter the response of U87 GBM cells to TMZ [
74]. This absence of effect on the response of an anti-tumor drug (doxorubicin) was also recently observed in lung cancer cell lines when exposed to CM from Wharton’s jelly derived MSCs by Hendijani and co-workers [
16]. Our study is the first to evaluate the influence of HUCPVCs CM on glioma growth and angiogenesis in a CAM assay with formation of 3D microtumors (Fig.
4). It was previously demonstrated that MSCs can induce angiogenesis in breast and colorectal cancer [
75,
76], while in GBM these cells were associated with decreased angiogenesis [
46]. In our work, we observed that exposure to HUCPVCs CM increased tumor blood vessels density, particularly in SNB-19 GBM tumors, suggesting that MSCs can induce angiogenesis, which may also contribute to higher tumor growth and tumor aggressiveness. Interestingly, GBM-associated stromal cells (GASCs), which are endogenously present in the tumor microenvironment, were previously shown to present properties of MSCs and have tumor-promoting effects on glioma [
77‐
80], similarly to what we observed with HUCPVCs CM.
Taking into consideration that our study and others showed that MSCs can potentiate tumor aggressiveness, while others demonstrated that MSCs can be safely used as drug delivery agents [
10,
39‐
41], it is crucial to standardize the methods used in different studies to more accurately understand if MSCs are definitely a valid and safe therapeutic approach to tackle cancer. Future studies should have into account several aspects, such as, tissue source and in vitro culture conditions of MSCs; type of tumor cells; variability of experimental methodology; and studies using modified MSCs should include unmodified MSCs as control. In addition, it will also be important to study low-passage primary GBM cell lines, instead of long-term established GBM lines, as these cells resemble more closely the original tumor characteristics, and are thus considered better models.
It is widely accepted that the major mechanism by which MSCs influence cancer pathophysiology is mediated by paracrine events [
81‐
83]. In order to identify which factors secreted by HUCPVCs could be modulating the viability, proliferation, and migration of GBM cells, we performed proteomic analyses of HUCPVCs CM identifying 699 proteins in the secretome. The functional clustering annotation and integration analyses (Fig.
5) revealed that HUCPVCs secretome had a significant enrichment in pathways that have been consistently found dysregulated in cancer (e.g. Wnt, PDGF and VEGF signaling pathways). In fact, these signaling pathways are known to mediate the phenotypes we observed in GBM cells exposed to HUCPVCs CM (namely the increases in proliferation, migration, and invasion), further supporting our experimental findings [
84‐
86]. For example, VEGF is one of the most important regulators of angiogenesis and subsequent tumor growth in GBMs [
87‐
89]. In addition, the expression levels of VEGF in gliomas is correlated with poor prognosis and higher malignancy grades [
90,
91]. Regarding PDGF, it was shown that this ligand and its receptors are involved in the proliferation, differentiation and apoptosis of GBM cells, and are commonly overexpressed in GBMs [
92,
93]. Finally, overactivation of the Wnt signaling pathway has been associated with several tumor types, including GBM (reviewed in [
94]), promoting tumor growth, migration and invasion [
95‐
97]. Additionally, the expression levels of some genes of the Wnt pathway were found to be associated with poor prognosis in glioma patients [
97,
98].
Similarly, particular proteins present in the secretome of HUCPVCs (Table
1), such as C-C motif chemokine 2 (CCL2), platelet-derived growth factor C (PDGF-C), semaphorin-7A (Sema-7A), periostin, and interleukin 6 (IL-6) are known to be important regulators of homeostasis in a variety of physiological conditions, but have also been described to influence tumor cell behavior, as is the case of a classic proto-oncogene [
99‐
121]. Interestingly, IL-6, CCL2, and periostin were recently demonstrated to promote M2 macrophage polarization, and thus contribute to tumor growth [
122‐
124].
Table 1
Examples of proteins secreted by HUCPVCs that have been described to influence tumor cells’ behavior
C-C motif chemokine 2 (CCL2) | Regulates migration and invasion in several cancer types, including gliomas | |
Actin-related protein 2/3 complex subunit 5 (ARPC5) | Contributes to cell migration and invasion in head and neck squamous cell carcinoma | |
Translationally-controlled tumor protein (TPT1) | Overexpressed in glioma tissue and is associated with tumor progression and poor clinical outcome of glioma patients. TCTP promotes glioma cell viability and proliferation, in vitro | |
Platelet-derived growth factor C (PDGFC) | Plays an important role in glioma vessel maturation and stabilization and in the progression of brain tumors, such as glioblastoma and medulloblastoma; and promotes tumor growth by recruitment of cancer-associated fibroblasts | |
Alpha-actinin-4 (ACTN4) | Enhances the motility and invasion potential of various carcinoma cell lines | |
Testican-1 (SPOCK1) | Promotes the proliferation, migration and invasion and inhibits apoptosis in glioma cells | |
Neuropilin-2 (NRP2) | Essential for breast cancer tumor initiation being involved in the formation of focal adhesions and is associated with metastasis and poor prognosis; and promotes the invasion and migration of thyroid cancer cells | |
Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) | Correlated with the grade of malignancy in human glioma; increases the migration capacity of glioma stem cells, and is implicated in U87 cell invasiveness | |
Transforming growth factor-beta-induced protein (TGFβI) | Promotes cell adhesion of human astrocytoma cells, in vitro | |
Plasminogen activator inhibitor 1 (SERPINE1) | Essential in processes related to tumor development, like angiogenesis, adhesion, migration, invasion and metastasis | |
Semaphorin-7A (SEMA7A) | Contributes to the increases motility and decreases adhesion necessary for U87 cell invasion | |
Periostin (POSTN) | Secreted periostin promotes glioma cell invasion and adhesion | |
Interleukin 6 (IL6) | Secreted IL6 promotes glioma cell invasion and angiogenesis | |
Authors’ contributions
JVdC and EDG contributed equally to the work, BMC and AJS share senior authorship. JVdC participated in the design of the study, carried out the experiments, analyzed the data and wrote the manuscript. EDG participated in the design of the study, carried out the experiments and analyzed the data. SG and FB performed the CAM assay and analyzed the results related to this assay. SIA and BM performed the proteomic analysis. AJS and BMC conceived the study, participated in its design and coordination, discussed data and contributed to the manuscript. All authors read and approved the final manuscript.