The MSC GFP group showed an increase in filiform papillae and decreased ulceration, but inflammatory cell infiltration was still observed in the submucosal area. However, extensive ulceration was absent in the MSC CXCR2 group, which exhibited the lowest level of inflammatory cell infiltration and increased thickness of the epithelium layer and mucosal lining Fig.
Hence, we examined the levels of inflammatory cytokines after MSC treatment to assess the anti-inflammatory effects of MSCs. All the experiments were repeated three times. Furthermore, a flow cytometry analysis of the intracellular redox status confirmed that primary tongue epidermal or fibroblast cells co-cultured with MSCs exhibited less intracellular ROS generation than mono-cultured cells Fig.
In addition, an oxidative environment contributes to cell death, which is also responsible for ulcer healing delays 22 , 27 , Therefore, we used an H 2 O 2 -induced cell death model to determine whether MSCs protect tongue epidermal or fibroblast cells from cell death in an oxidative environment. The apoptosis of primary tongue cells subjected to co-culture with MSCs was significantly decreased compared with mono-cultured MSCs Supplementary Fig. Taken together, these findings indicate that MSCs accelerate mucositis recovery, likely by decreasing radiogenic ROS production and protecting tongue cells from cell death.
Genetic Engineering of Mesenchymal Stem Cells for Regenerative Medicine. - Semantic Scholar
Oral RIM is one of the most common complications of radiotherapy among patients with head and neck cancer. However, traditional treatments for severe mucositis are limited and cannot prevent ulcer recurrence. Here, we report an innovate and highly efficient treatment, namely, the systematic transplantation of MSCs with an enhanced targeting capacity to mucositis sites. This study sheds new light on the treatment of RIM and provides groundwork for the clinical application of MSC-based therapy for mucositis.
Due to their anti-inflammatory and immunomodulatory capabilities, MSCs have therapeutic potential for the treatment of inflammatory diseases 6 , 9. However, systemically infused MSCs rarely home to inflamed mucosa in the orofacial region. MSC targeting depends on the interactions between chemokine receptors and specific chemokines from inflamed sites 12 , Recent studies have reported that the overexpression of corresponding chemokine receptors in MSCs can enhance their targeting ability to inflamed sites. Conventional treatments, such as pain management and nutrition support therapy, have limited healing benefits in mucositis 1 , These factors play a marked role in the proliferation of various cell types, including endothelial cells, fibroblasts, vascular smooth muscle cells, and keratinocytes, thus promoting mucosa recovery However, because growth factors also promote angiogenesis in tumors 35 , 36 , the administration of growth factors might increase the risk of head and neck cancer recurrence, which hinders their application.
In addition, prolonged pro-inflammatory cytokine production greatly impacts the development, metastasis, and recurrence of cancer However, growth factors can hardly alleviate mucosal inflammation. ROS generation is widely acknowledged to be the primary event in most mucositis-causing pathways 4 , For example, ROS lead to DNA damage, cell death, and upregulation of various inflammation-related signaling pathways 4 , Hence, the effects of MSCs on alleviating inflammation in mucositis are partially due to their anti-oxidant capability.
The mechanism of how transplanted MSCs promote tissue repair have long been debated 38 , 39 , MSCs accelerate tissue repair probably in three ways: by direct differentiation 38 , direct cellular interactions 39 , or secretion of soluble factors Our BLI analysis showed that MSCs were not detected in the newly formed epithelium layer and mucosal lining 10 days after injection in the RIM mouse models, suggesting that MSCs may not accelerate ulcer healing via directly generating tongue epithelial cells or fibroblast cells.
In addition, we found that MSCs significantly decreased the ROS levels of tongue epithelial cell or those of the fibroblast cell in both transwell and direct co-culture systems with no significant difference between the two co-culture systems data not show. Because MSCs can secrete factors in both co-culture systems while exhibited similar anti-oxidant effects in the two systems, MSCs might decrease the cellular ROS levels not via direct cellular interactions.
Growing evidences indicate that MSCs can produce soluble factors, especially antioxidant factors, to promote tissue repair via their secretome 40 , Our study showed that tongue epithelial cells or fibroblast cells exhibited reduced cellular ROS levels when co-cultured with MSCs via a transwell system. We deduced that the anti-oxidative capability of MSCs mainly lies in their secretion of factors.
However, these anti-oxidative secretion factors of MSCs needs to be further identified. Because radiotherapy is one of the most widely used anti-neoplastic treatments for head and neck cancer, reducing the harmful side effects of radiation will greatly impact patient quality of life. Thus, this innovative strategy to reduce the side effects of radiotherapy and improve patient tolerance to treatment is promising for clinical application. In addition, MSCs are attractive candidates for the delivery of anti-tumor reagents or cytokines in human malignancies. MSC treatment will lead to a safer and more effective clinical therapy for tumors and reduce radiotherapy or chemotherapy complications.
However, further investigations are needed to establish the long-term safety and effects of MSC transplantation therapy. Animal models were established as previously described The results are representative of three independent experiments six animals per group. Detailed information is shown in Supplementary Fig. MSCs were obtained from healthy donors who provided informed consent.
MSCs were isolated from the bone marrow using our previously reported method Briefly, bone marrow cells were separated by Ficoll-Paque 1. After the fourth passage, the capacity of MSCs to differentiate into multiple lineages was confirmed by their differentiation to osteoblasts, chondrocytes, or adipocytes, which was performed using previously described methods Three independent experiments were performed.
Bands were detected with a chemiluminescence kit Millipore. For lentiviral infection, the optimal multiplicity of infection MOI was 50 50 viral particles for each cell , which was determined in our previous experiments. Each analysis was performed with at least three independent experiments. The data were analyzed with FlowJo7. BLI of luc is a reliable noninvasive imaging tool used to quantitatively monitor MSC survival and distribution with high sensitivity.
The plate was then incubated for an appropriate length of time in the incubator. The relative growth rate RGR was calculated using the following formula:. Cells that migrated to the lower surface were counted under a microscope. Cryosections were prepared and counterstained with 1. Tongue epithelial cells and fibroblasts were prepared and maintained as previously described 44 , Briefly, both cell types were isolated from mouse tongue tissue specimens. The tongue epithelial tissues could then easily be separated from whole tissues with forceps.
Tongue epithelial cells were derived from epithelial tissues, and fibroblasts were derived from connective tissues. Third-passage cells were used in the experiments. The tissue sections were visualized under a fluorescence microscope with a nm wavelength filter. The apoptotic population was assessed by flow cytometry.
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All the statistical analyses were performed with SPSS version Lalla, R. Cancer , — Rodriguez-Caballero, A. Cancer treatment-induced oral mucositis: a critical review. Sciubba, J. Oral complications of radiotherapy. Lancet Oncol. Sonis, S. Perspectives on cancer therapy-induced mucosal injury: pathogenesis, measurement, epidemiology, and consequences for patients. Nicolatou-Galitis, O. Systematic review of anti-inflammatory agents for the management of oral mucositis in cancer patients.
Support Care Cancer 21 , — Griffin, M. Concise review: adult mesenchymal stromal cell therapy for inflammatory diseases: how well are we joining the dots? Stem Cells 31 , — Phinney, D. Stem Cells 25 , — Moon, H. MSC-based VEGF gene therapy in rat myocardial infarction model using facial amphipathic bile acid-conjugated polyethyleneimine.
Biomaterials 35 , — Chang, P. Certain suicide genes encode enzymes that convert nontoxic prodrugs into highly toxic metabolites, thereby precisely eliminating cells expressing these enzymes [ 6 ]. Suicide genes have been studied for the treatment of cancer through the introduction of these genes into cancer cells or neighboring cells.
The expression of suicide genes in cancer cells results in the generation of cytotoxic products, killing the cancer cell; nearby cancer cells that do not express the suicide gene can also be killed by exposure to cytotoxic products by a process known as the bystander effect [ 7 — 12 ]. The combination of the suicide gene HSV1-TK with a tightly controlled system, such as Tet-On, which simply regulated by presence, or absence of doxycycline, could reduce the side effects associated with gene-based therapy. Mesenchymal stem cells MSCs are multipotent, and are derived from a variety of tissues or organs, including adipose tissue, umbilical cord, placenta, and bone marrow [ 14 ].
MSCs are attractive vehicles for the delivery of therapeutic agents to cancer cells, based on their tumor-specific tropism, and have been used for suicide gene therapy [ 15 — 17 ]. MSCs engineered with apoptosis inducers e. Imaging technologies have been utilized for the diagnosis and monitoring of therapeutic effects and these tools are indispensable for developing new drugs before their clinical translation [ 22 ]. Globally, thyroid cancer incidence has been continuously increasing over the past few decades, and has become the fifth most common malignancy in females [ 23 ].
The most aggressive form of thyroid cancer, anaplastic thyroid cancer ATC , is a highly aggressive form of the disease with poor prognosis [ 24 ]. Unfortunately, resistance to radio- and chemotherapy is a common feature; thus, the development of novel therapeutics is essential to improve the treatment of ATC patients.
The aim of this study was to develop therapeutic MSCs expressing an inducible HSV1-TK suicide gene, and to validate therapeutic gene expression in vitro using optical molecular imaging and a model of ATC. Tet-responsive regulatory systems are normally comprised of two different expression cassettes, one containing the regulatory protein tTA and the second containing the Tet regulatory element fused to a minimal promoter, which regulates the expression of the reporter or therapeutic transgene.
Based on previously described transcriptional regulators [ 25 — 27 ], the Tet-On 3G is a modified form of the Tet-On advanced transactivator protein that has been modified for markedly increased sensitivity to DOX [ 28 ]. The inducible promoter pTRE3G yields very low basal expression and maximal expression after induction [ 29 ].
It consists of seven repeats of a 19 bp tet operator sequence located upstream of a minimal CMV promoter to give higher performance for retroviruses. After overnight transfection, the medium was changed, and the virus was collected daily for 2 days, pooled, passed through a 0. The sorted cells were screened and characterized. After 24 h, 0. After BLI imaging we carefully drew a separate region of interest ROI in each well and the signal was quantified using living image software.
We further analyzed the dose- and time-dependent Fluc activity. In addition, after 24 h of DOX induction, the medium was removed and fresh medium was added for another 24 or 48 h to assess residual Fluc activity. For functional assessment of the transfected cells, we performed a 3 H-Penciclovir uptake assays as described in Sekar et al.
Next, we assessed the bystander effect. Subsequently, cells were treated with GCV for 48 h.
Further, to confirm bystander-mediated cytotoxicity of GCV treatment, we performed an analysis of morphological changes using fluorescence microscopy. For IVIS imaging, we used a controlled amount of isoflurane anesthesia and oxygen system to the imaging chamber for animal imaging. After four days, treatment we analyzed the Rluc activity and quantified. Fluc signal was normalized with DOX - Fluc activity which were increased with increased 11, The expression of mCherry was also visualized by fluorescence microscopy S1C Fig.
Overall, DOX increased Flux activity in a dose-dependent manner at 24, 48, and 72 h of culture.
NS: denotes no significant change. GCV 0.
Furthermore, we also confirmed the bystander effect by examining morphological changes. Although some cells remained attached to the plate, many of them had altered morphology with evidence of cell shrinkage and many cells had the appearance of thin and elongated cytoplasm, both of which are morphological characteristics of non-viable cells S3B and S3C Fig.
From these results, we confirmed that the therapeutic suicidal gene, either with or without the Tet-On system, in engineered MSC cells, has a significant bystander effect. Therefore, suicide gene based therapy may be useful for anaplastic thyroid cancer. The efficacy of MSCs from various sources, that have been engineered with genes appropriate for an enzyme-prodrug approach for cancer therapy, has been reported in several preclinical studies [ 12 , 31 , 32 ].
In the current study, we developed MSCs with a tetracycline inducible therapeutic moiety HSV1-TK along with a reporter system that could be monitored using bioluminescent imaging. In order to develop successful cell-based cancer therapies, non-invasive imaging methods to monitor both therapeutic cells and target cells are essential. For example, molecular imaging has been successfully used for monitoring of administered cells and tumors in living animals [ 7 , 22 , 33 ].
Furthermore, an anaplastic thyroid cancer cell line, CAL62, S1 Fig was successfully transduced with a double-fusion reporter gene mCherry-Rluc ; therefore, tumor progression could potentially be visualized with BLI using coelenterazine as a luciferase substrate [ 7 , 34 ]. Both infinite proliferation and decreased cell death are characteristics of cancer cells. ATC demonstrates local invasion and metastasis to the lung, lymph nodes, and other tissues, and therefore, the mortality rate is very high [ 35 ]. In this study, we reported that the Tet-On suicide gene system in MSCs successfully exhibited a bystander effect after induction with DOX and with GCV treatment, which could potentially be used for the treatment of thyroid cancer.
Antitumor drugs are able to kill some types of cancer cells; however, antitumor drugs generally produce significant toxicity in normal organs, generate host morbidity, and induce treatment related mortality [ 36 — 38 ]. Suicide gene therapy also generates systemic toxicity and byproducts.
Therefore, controlling suicide gene expression might be a valuable tool to prevent side effects. Manipulating the expression of a suicide gene using an inducible system can block the generation of the suicide protein before its arrival at the target tissue for example a tumor ; therefore, suicide gene therapy utilizing an inducible system represent a safe choice among the diverse treatment options for cancer treatment. The Tet-On system uses an E. DOX induces gene expression in a dose-dependent manner through the activation of the Tet-response element in cells harboring the Tet-On system.
Thus, the expression of a gene could be turned on after the arrival of therapeutic cells to the cancer site, potentially avoiding systemic toxicity of the therapeutic agent. These results indicate that HSV1-TK expression might selectively enhance the sensitivity of ATC cells to GCV in vitro , in agreement with other reports suggesting that the Tet-On system exhibits high activity after induction, but low basal transcriptional activity [ 41 ].
The benefits of MSCs for cancer treatment are controversial as MSCs can also play a positive role in cancer progression depending on the cellular context [ 42 , 43 ]. Thus, our system might be a safe alternative strategy for the treatment of human ATC. Genetically engineered MSCs expressing HSV1-TK have the ability to inhibit the growth of cancer cells in both in vitro and in vivo tumor models [ 7 , 44 — 46 ].
The advantages of the Tet-On reporter system will be helpful for developing MSC-based therapeutic gene expression systems that could reduce the adverse effects of gene-based therapies. The limitation of this system is that the in vitr o effects seen here might not be well mimicked in vivo. In the cell culture medium, DOX directly affects every cell and therefore efficiently up regulates transgene expression.
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However, in the in vivo situation responses response may not be so reproducible and robust. There may be inherent animal-to-animal variation; the DOX concentration may also vary owing to pharmacokinetic differences, such as biological half-life, across individuals, and the factors affecting tumor penetration by DOX may be complex and perhaps vary from tumor to tumor.
Another limitation is that usually DOX is given through water, and mice have to drink sufficient amount of water for optimal transgene expression. This may introduce another source of complication. Immunogenicity against the transactivator protein of the tet-on inducible system has been observed after adminstration of tet-on encoding vectors and it raises concerns about the clinical value of the system [ 47 , 48 ]. We concluded that overexpression of CXCR4 and CXCR7 receptors in murine MSCs cannot improve the homing and therapeutic potentials of these cells and it can be due to severe chromosomal abnormalities that these cells bear during ex vivo expansion.
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