In our present study, we utilized an orthotopic murine model of neuroblastoma to mimick the actual neuroblastoma condition in vivo. Adrenal gland is the most common primary site of neuroblastoma and implanting xenografts of human neuroblastoma cells into the adrenal gland area provide a more relevant microenvironment setting compared to the subcutaneously implanted in vivo tumor model. The orthotopic xenograft offers a more ideal in vivo condition to mimic the relationship between cancer cells and the microenvironment. This model also allows us to evaluate the metastasis potential of neuroblastoma cells in vivo. In our model, bioluminescence gene luciferase was used to label transplanted tumor cells, which can be utilized to monitor the early growth of tumor in vivo non-invasively. This model is free of apparent auto-fluorescence background since luciferase cannot be produced naturally by mice. Thus, this orthotopic model provides a better platform for investigating the underlying tumorigenic pathophysiology, and furthermore, for evaluating the efficacy of novel anti-cancer therapies.
We further explored the effects of MSCs on the initiation and progression of human neuroblastoma. Until now, the exact impact of MSCs on tumor growth and progression in vivo is still controversial. The favorable effects of MSCs have been reported in the tumorigenesis of ovarian cancer , colorectal carcinoma , breast cancer  and pancreatic cancer . The potential underlying mechanisms include the supportive effects of MSCs on cancer growth and metastasis. MSCs could enhance tumor growth via constituting the cancer microenvironment, enhancing neovascularization, producing growth factors and exerting immunosuppressive effects. In addition, MSCs enhance cancer metastasis through releasing soluble factors such as chemokine SDF-1, IL-6 and CCL5. They impact on cancer metastasis through releasing soluble factors such as chemokine SDF-1, IL-6 and CCL5 [6, 14, 15]. Moreover, MSCs protect cancer cell survival from cytotoxicity of anti-cancer reagents . However, MSCs also exerted negative effects on the growth of colon carcinoma , Kaposi’s carcinoma, glioma and hepatoma model . Conflict findings were reported even in same type of cancer or study [19–22]. To date, the exact reasons underlying these controversial effects remain largely unknown. It is potentially related to the specific histological types of cancer, particular experimental model and research design, different in vitro culture conditions and the dosage of cell inoculation. Furthermore, such effects could also be closely related with tumor-specific background and in certain scenarios even be cell-line specific. Majority of studies observed the supportive effects of co-transplanted MSCs on cancers using excessive number of MSCs than cancer cells or at least an equal number. Specially, my study validated that lower dosage of MSCs co-injection using 0.5⋅105 MSCs and 1⋅105 neuroblastoma cells was enough to generate the promoting effects on tumor growth and metastasis. However, much lower dosage of MSCs (102 MSCs to 104 cancer cells) was found to induce tumor rejection .
The data from current research focusing on the interaction between MSCs and neuroblastoma is very limited. It was revealed that MSCs could protect neuroblastoma from oxidative stress in vitro . IL-6 produced by MSCs was reported to participate in promoting survival of neuroblastoma cells and the bone metastasis . In addition, SDF-1/CXCR4 axis plays a pivotal role in growth, progression and metastasis modulation in diverse kinds of cancers including head and neck cancer, pancreatic cancer and lung cancer. It was suggested that SDF-1/CXCR4 axis could promote the dissemination of cancer cells towards sites highly secreting SDF-1 through binding to the cognate receptor CXCR4 expressed on cancer cells including neuroblastoma [26–28]. Our previous in vitro study demonstrated that MSCs could benefit the metastasis of neuroblastoma via the secretion of SDF-1. It was also reported that MSCs secretome could modulate CXCR4 expression and invasion to the bone marrow of neuroblastoma in vitro . Despite the above progress, the exact role of MSCs in neuroblastoma development has yet to be defined and majority of data require further validation by in vivo experiments. In this study, using in vivo model, we demonstrated that MSCs indeed exerted tumorigenic effects on neuroblastoma in vivo. In early period of post-inoculation, mice co-transplanted with MSCs and SK-N-LP showed stronger tumor signals compared to the mice injected with SK-N-LP alone. Such phenomenon was further verified by evaluation of gross tumor volume. The facilitative effect of MSCs on neuroblastoma’s metastasis was also studied. We observed that compared to SK-N-LP group, MSCs co-transplantation may accelerate the metastasis since all mice in this group developed metastasis in all organs studied and had stronger bioluminescent signals.
Based on the above results, we further explored whether MSCs could be a therapeutic target to eradicate tumors from the microenvironment shelter. To achieve this goal, we investigated the trafficking of MSCs in mice bearing neuroblastoma and investigated the potential modifier involved in this whole process. It has been extensively reported that SDF-1/CXCR4 axis is actively involved in the homing of MSCs to injured tissues and thereafter exert biological immunomodulatory and regeneration effects. MSCs were also found to have the propensity of being guided towards tumors. However, unlike the advanced understanding of MSCs trafficking to injured tissues, the mechanism responsible for homing of MSCs towards tumors is just starting to be unfolded and it has not been adequately explored especially in the setting of neuroblastoma. Whether SDF-1/CXCR4 axis is a vital modifier in MSCs homing towards neuroblastoma, like it is described in the trafficking towards injured tissues is still uncertain. We found that MSCs could preferentially migrate to neuroblastoma. Importantly, we also demonstrated that such trafficking was in a CXCR4-dependent manner. Pretreatment with AMD3100, the specific antagonist of CXCR4, significantly abolished the homing of MSCs towards primary tumor. The strong evidence supporting this concept came from the striking phenomenon that no hMSCs signal could be detected in liver or lung without metastatic disease. Moreover, supplementing the early studies, we observed that systemically infused MSCs could also be attracted by the metastatic loci other than the primary tumor.
To the best of our knowledge, few studies have demonstrated the preferential homing of MSCs towards metastatic loci. One study reported intravenously injected MSCs could be guided towards primary tumor and lung metastatic sites. However, they established the tumor model using subcutaneous inoculation and the lung metastasis was induced separately through intravenous injection of tumor cells. After systemically infusing the MSCs 4 days post-injection of cancer cells, they indicated higher signal intensity and longer retention of MSCs at lung than normal control and thus proposed the specific recruitment of MSCs by metastatic lesions . This can be due to trapping of cancer cells in the lung tissue via the “first-pass effect” during venous return to the right heart and then the lung. In our study, an orthotopic tumor model was established, and other than lung invasion, multiple metastatic diseases were triggered naturally by generated primary tumor. Using this clinically relevant model, we provided convincing evidence for the tumor tropism of MSCs. Firstly it was demonstrated that intravenously injected MSCs could preferentially home towards both the primary tumor site and the multiple metastatic loci. And more strikingly, through labeling the MSCs and tumor cells, we can directly observe the preferential migration of MSCs to organs invaded, whereas, no MSCs could be detected in normal tissues. Moreover, such finding was further supported by the observation that higher intensity of MSCs signal was observed in organs with higher degree of invasion indicating indeed MSCs could only be recruited by tumor cells at both primary site or metastatic loci and such homing was correlated with the invasive tumor cell number. This property of tropism could provide a promising cue for targeting the microenvironment in high risk metastatic neuroblastoma.
Interestingly, albeit significant inhibitory effects on MSCs trafficking towards primary tumor, AMD3100 pretreatment failed to show similar impact on the recruitment of MSCs by the metastasis loci. The mechanism underlying this paradoxical phenomenon warrants further detailed investigations. We hypothesize several possible mechanisms underlying this phenomenon. The first potential reason is the level of SDF-1 was much higher at metastatic loci than primary site . The inhibition of MSCs homing towards metastatic loci may require much higher dosage of AMD3100 than used in blocking the migration of MSCs towards primary site. In support of this, using high-density tissue microarrays, a large cohort study of more than 600 human prostate carcinoma specimens indicated that compared to primary tumor sites, higher SDF-1 was expressed by metastatic lesions . In addition, CXCR7, the other receptor of SDF-1, was reported to express in cancer cells and associated with tumorigenesis and metastasis [32, 34, 35]. In our previous study, both CXCR4 and CXCR7 were found to express in neuroblastoma cell lines. CXCR7 was involved in increasing neuroblastoma migration acting as alternative receptor of SDF-1 in the absence of CXCR4, but not functional in regulating cell migration and adhesion . However, the exact role of CXCR7 in guiding the homing of MSCs towards tumor metastatic loci deserves further study. Finally, the potential variation in microenvironment between primary tumors and metastatic lesions may result in different profiles of released chemokines. The metastatic lesions might trigger different tissue injury signals and involved other non-SDF-1 related pathways.
The limitations of current study could not ascertain the effects of MSCs on early metastasis of neuroblastoma, which requires further in vivo studies. The sequence in which intravenously infused MSCs migrate towards primary tumor or metastatic loci remains unknown. Moreover, one notable phenomenon that AMD3100 pre-treatment could not inhibit the homing of MSCs towards metastatic loci requires further verification. In addition, albeit useful, it was observed that the bioluminescence imaging underestimated the tumor burden at longer time period.