Multifaceted role of mesenchymal stem cell in oral cancer: A review

Lynn Johnson; Hiroj Bagde

doi:10.4103/jpo.jpo_22_22

REVIEW ARTICLE

Year : 2022 | Volume : 2 | Issue : 2 | Page : 83-94

Multifaceted role of mesenchymal stem cell in oral cancer: A review

Lynn Johnson, Hiroj Bagde
Department of Periodontology, Rama Dental College and Research Centre, Kanpur, Uttar Pradesh, India

Date of Submission	28-Oct-2022
Date of Decision	11-Nov-2022
Date of Acceptance	23-Nov-2022
Date of Web Publication	06-Feb-2023

Correspondence Address:
Dr. Lynn Johnson
Department of Periodontology, Rama Dental College and Research Centre, Kanpur, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpo.jpo_22_22

Abstract

Oral cancer is among the top 10 most prevalent forms of cancer worldwide, characterized by a highly diverse group of tumors and the absence of specific biomarkers and poor prognosis. It is evident that oral squamous cell carcinoma (OSCC) is the most prevalent form of oral cancer in developing nations, particularly in Southeast Asia and southern Africa. Despite recent advances in the treatment modalities, including surgery, chemotherapy, and radiotherapy, the mortality rate of OSCC (mainly due to lymphatic involvement and metastasis) continues to rise, presenting both patients and healthcare systems with a challenge. It has been shown that tumors are heterogeneous due to the presence of different kinds of cancer cells. In addition to these populations of cells, cancer stem cells (CSCs) contribute substantially to the initiation and progression of cancer. The CSCs are also capable of self-renewal and differentiation, similar to their stem cell counterparts. The mesenchymal SCs (MSCs) are a specific population of CSCs which differentiate into mesodermal cells. The characteristics of MSCs include self-renewal, rapid proliferation, multipotent differentiation, and low immunogenicity. Furthermore, because MSCs are particularly prone to delivering therapeutic agents and transferring genetic material to injured tissues and tumors, they are excellent candidates for use as cell carriers. There has been a significant amount of research regarding the potential pro-or antitumorigenic effect of MSCs on the progression and initiation of tumors. The interaction between tumor cells and MSCs within the tumor microenvironment plays an important role in tumor progression. It is important to note that MSCs are recruited to the site of wound healing in order to repair damaged tissues, a process that is also related to tumorigenesis. Alternatively, resident or migrating MSCs may favor tumor angiogenesis and make the tumor more aggressive. The interaction between MSCs and cancer cells is fundamental to the development, progression, and metastasis of cancer. Therefore, an interesting topic is the relationship between cancer cells and MSCs, since contrasting reports about their respective influences have been reported. In this review, we discuss recent findings related to conflicting results on the influence of MSCs in cancer development and its management.

Keywords: Cancer stem cells, delivery vehicle, mesenchymal stem cells, oral cancer, squamous cell carcinoma, target therapy

How to cite this article:
Johnson L, Bagde H. Multifaceted role of mesenchymal stem cell in oral cancer: A review. J Precis Oncol 2022;2:83-94

How to cite this URL:
Johnson L, Bagde H. Multifaceted role of mesenchymal stem cell in oral cancer: A review. J Precis Oncol [serial online] 2022 [cited 2023 Jun 8];2:83-94. Available from: https://www.jprecisiononcology.com//text.asp?2022/2/2/83/369217

Introduction

Head-and-neck cancer (HNC) is a heterogeneous group of tumors which mainly arise in the oral cavity, oropharynx, hypopharynx, salivary glands, paranasal sinuses, and larynx.^[1] HNC is among the most common cancers worldwide, with a high prevalence in Southeast Asia, Brazil, and Southern Africa. Squamous cell carcinomas make up the majority of HNC which have an incidence of around 630,000 new cases per year worldwide. Lifestyle behaviors such as drinking alcohol, use of tobacco, and chewing betel quid are the most common risk factors associated with HNSCC.^[2] Despite recent considerable advances in the therapeutic repertoire for oral cancer, the average overall survival rate for patients with metastatic or recurrent HNC remains <1 year. Resistance to chemotherapeutic and biological agents is responsible for the failure of many current therapeutic approaches.^[3]

Tumors are composed of different types of cancer cells that contribute to tumor heterogeneity. Among these population of cells, cancer stem cells (CSCs) and its one particular population known as the mesenchymal stem cells (MSCs) play an important role in cancer initiation and progression.^[4] MSCs are a class of nonhematopoietic stem cells (SCs) belonging to the mesoderm, with the characteristics of self-renewal, high proliferation, and multi-directional differentiation potential.^[5] These cells harbor, although rare, in the bone marrow (BM) and almost all body tissues.^[6] The classical and widely used sources of human MSCs for clinical settings are the BM, the adipose tissue (AT), and the umbilical cord (UC), exhibiting peculiar in vivo biology and different native functions, which represent a promising tool for cell therapy, tissue engineering, and regenerative medicine. MSCs have been harvested also from endometrium, synovium, muscle, skin, placental, and dental pulp. Increasing evidence proposes the use of MSCs as promising therapeutic approach for the treatment of several diseases and applications in the fields of regenerative medicine, neuroscience, oncology, pharmacology, and bioengineering.^[7]

The Origin of Cancer

Several theories have been postulated in an effort to reach a compelling answer to the mystery of cancers' origin. Among these the most popular ones are as follows:

Clonal evolution model: Based on this model, cancer is the consequence of multiple mutations striking the somatic cell's gene consecutively. These mutations, interplaying with epigenetic aberrations, can alter the structure and function of normal regulatory genes; proto-oncogenes driving the cell's proliferation, tumor suppressors inhibiting the cell's growth, apoptosis-inducing genes, and genes involved in DNA repair. The result of this chain of nonlethal mutations is the formation of an “immortal” cell possessing a collection of traits, rendering an evolutionary competent cell lineage in the process of natural selection^{[8],[9],[10],[11]}
CSC model: An alternative model, i.e., gradually gaining popularity is the “cancer stem cell” hypothesis. The idea that cancers arise from a distinct group of cells, known as “germ cells” or “stem cells,” was first proposed more than a century ago. Numerous studies and the inspection of intrinsic heterogeneities among cancer cells in a single tumor have led to the CSC hypothesis. The model, also known as the hierarchical model, has four key concepts: (1) only a limited fraction of cancer cells have tumorigenic potential; (2) a distinctive profile of cell surface markers can be utilized to separate the CSC subpopulation from the rest of tumor; (3) the resulting tumor from CSC proliferation and differentiation consists of tumorigenic and non-tumorigenic cells, creating a heterogeneous environment; and (4) the CSC subpopulation can be serially transplanted through consecutive generations, suggesting its self-renewing capacity.^[12] CSCs possess two principal features: the capability to regenerate the same SC (self-renewal) and to produce a progeny that can differentiate.

^[13]

Premalignant and malignant CSC markers: The progression of normal mucosa to mild, moderate, and severe dysplasia and then to an oral SCC is a multivariate process, comprising structural and functional changes in cells. The identification of the events relevant to malignant transformation is of the utmost importance. It can be useful for the clinician to evaluate the progression risk of premalignant lesions toward cancer, and preventive strategies can be performed.^[13]

Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is the most commonly occurring oral malignancy and one of the most widely occurring cancers throughout the world.^[14] It is ranked 6^th place among all common cancers worldwide, and the overall survival rate has not improved in decades.^{[15],[16],[17],[18],[19]} OSCC is a malignancy that arises in the squamous epithelium lining the oral cavity. The risk factors for the development of OSCC include tobacco exposure, alcohol consumption, and infection with oncogenic viruses such as HPV. The tumor can invade deeply into adjacent tissues of the tongue and floor of the mouth, as well as into the bones, primarily of the alveolar crest.^[16] Its morbidity varies depending on patient age, gender, geographical region, and risk factors associated with tumor progression.^[15],[17]

Microscopically, OSCC usually shows variable degrees of keratinization, cellular and nuclear pleomorphism, and mitotic activity. They are graded as well-, moderately-, or poorly differentiated (grades 1-3) according to the WHO criteria. The tumor's features, including size and site, histologic malignant grade, perineural spread at the invasive front, lymphovascular invasion, and tumor thickness, can act as major risk factors influencing the prognosis for OSCC patients; however, the main negative prognostic factor is the presence of lymph node metastasis, which occurs in 25%–65% of cases. OSCC currently adopts a comprehensive treatment that combines surgery, radiotherapy (RT), chemotherapy (ChT), and target therapy. However, this approach does not come with a satisfactory outcome.^[18]

Normal Stem Cells

Human SCs are unspecialized cells responsible for the formation and maintenance of tissues in the body. They are broadly categorized into two types: embryonic SCs (ESCs) and adult SCs (ASCs). ESCs are the cells of the inner layer of the blastocyst formed at 3^rd or 4^th day of fertilization. These are a transient class of cells, meaning that they require a special microenvironment and intercellular signaling to remain in an “undifferentiated” state. Thus, the ESCs cannot be normally found following the completion of body development. During the entire lifetime, there are clusters of “organspecific” resident SCs in tissues called ASCs. ASCs are more likely to be “multipotent,” meaning they can only give rise to specific cell types of their tissue of origin. There are several sites in the oral cavity with the identified populations of SCs, including oral epithelium, connective tissue, and tooth structures.^[13]

The general term “stem cells” includes several different types of cells and the first distinction to be made is between (a) normal SCs, which are responsible for the development and maintenance of all of the tissues of the body, and (b) their diseased counterpart, called CSCs, that have lost the close growth control that is a property of normal SCs.

The general property that characterizes adult (somatic) SCs is that they can be divided indefinitely, normally producing one cell that remains an SC and one cell that differentiates itself into a functional tissue cell. This normal “asymmetrical” division pattern is important as it results in the maintenance of the same number of SCs while also providing another cell for tissue function. However, when it is necessary to replace SCs, such as those lost after wounding, SCs can be divided “symmetrically” to form two SCs and thus increase their number.^[16]

Cancer Stem Cells and Oral Cancer Stem Cells: History and Isolation

The idea that cancers arise from a distinct group of cells, known as “germ cells” or “stem cells,” was first proposed about 150 years ago.^[19] Observations dating back more than 50 years have evidenced similarities between cancer and embryonic development and that led to the hypothesis of the existence of CSCs. Recent growing evidences suggest that the tumor is composed of heterogeneous populations of cells with different levels of malignity and the tumor development is driven by a specialized cell subset, characterized by self-renewing, multi-potent, and tumor-initiating properties. These malignant cells are called CSCs and their maintenance is tightly ensured by the microenvironment and the stroma.^[4] They are probably generated from normal stem or precursor cells within tissues after mutations occur and are typically resistant to conventional treatments.^[20],[21]

CSCs possess two principal features: the capability to regenerate the same SC (self-renewal) and to produce a progeny that can differentiate. Both traits are achieved by the CSC's asymmetric division potential, as the daughter cell may follow either pathway of retaining the original identity or undergoing differentiation into various types of cells called “transit-amplifying (TA) cells,” which will eventually transform into the “more mature” cells.^[13]

The first reports of the presence of a CSC population have been related to the leukemic cells.^[22] Later, many evidences that CSCs also play a central role in the pathogenesis and progression of carcinomas of the head and neck (HNSCC), including OSCC, have been found.^[16] The existence of subpopulations of oral CSCs has been primarily proposed by the study, which showed just a subpopulation of OSCC cells is able to create a developing tumor mass.^[23] A study showed that a subpopulation of OSCC cells extracted from the cultivated OSCC cell lines has features of the two SCs and invasive malignant tumors such as self-renovation, tumorigenic potentials, migratory abilities, and radioresistance.^[24]

The isolation of CSCs from oral cancers has mainly been performed with the CD44 marker. CD44 is an adhesion molecule that binds itself to hyaluronan and its expression is necessary for the maintenance of the CSC's properties. CSCs lose their “stemness” when CD44 is experimentally reduced. However, a problem with CD44, and also with all other CSC markers that have been identified so far, is that they are not entirely specific. No single marker is capable of specifically recognizing CSCs and additional markers have therefore been sought. ALDH1 is an intracellular enzyme involved in detoxification and drug resistance through the oxidation of aldehydes, and ALDH-positive cells in HNSCC are reported to have typical CSC behavior and increased tumorigenic ability. The combination of CD44 with other markers, such as ALDH1, may improve the specificity of CSCs' recognition and isolation.^[16]

Multiple researchers published information on the successful separation of oral CSC populations through different markers. In general, CSCs in OSCC are possibly separated through the cell-surface markers or the respective specific practical features.^[24]

A particular population of CSCs is constituted by MSCs that differentiate into cells of mesodermal characteristics. Cells capable to differentiate into mesodermal-derived tissues, such as adipocytes, chondrocytes, and osteoblasts, are called MSCs and they are suggested to reside in all human organs.^[4]

Oral Cancer Stem Cells and Their Signaling Pathways

CSCs have the shared features with normal SCs and several certain traits maintaining tumor growth and invasion. One of the primary features of CSCs is their self-renewal capacities so that it apparently is one of the motives to begin and maintain tumorigenicity. CSCs self-renewal may be retained through multiple endogenous signaling paths, including Wnt, Bmp, Pten, Notch, B cell-specific Moloney murine leukemia virus integration site 1 (Bmi1), transforming growth factor (TGF)-β, and Hedgehog that would be often actuated in human cancers.^[24]

Origin and History of Mesenchymal Stem Cells

Originated from the mesoderm and first found in BM, MSCs can be isolated from multifarious postnatal tissues, including adipose, UC, UC blood, amniotic fluid, and other tissues, as stated by Whiteside, 2018; Almeida-Porada et al., in 2020; and Tavakoli et al., in 2020.^{[25],[26],[27]} Apart from these tissues, MSCs have also been isolated from dental tissues, including dental pulp, dental follicle, apical papillae, periodontal ligament, and gingiva.^[5]

MSCs provide a great potential for its involvement in tissue regeneration (da Silva Meirelles et al., 2008). Many studies have been conducted to validate the use of MSCs in bone regeneration and nerve regeneration as proved by Liau et al., in 2020 and Fu et al., in 2021. MSCs exhibit chemotactic properties similar to immune cells in response to tissue injury and inflammation. MSCs can release various bioactive factors, such as immunosuppressive molecules, growth factors, chemokines, and complement components to regulate the inflammatory process and create a balanced inflammatory and regenerative microenvironment in damaged tissues, thereby treating various degenerative and inflammatory diseases.^[28] Due to their inherent ability to migrate and colonize into tumor tissues, MSCs were reported to closely interacted with tumor and tumor cell. Thus, MSCs are attracting increasing interest in the field of oncology.^[5]

Characterization of Mesenchymal Stem Cells

MSCs are positive for CD105, CD90, CD73, CD146, CD29, STRO-1, but are negative for CD14, CD34, CD45.^{[29],[30],[31]} Under a suitable inductive medium, MSCs are capable of differentiating into osteoblasts, adipocyte, chondrocytes, and many other cells (Whiteside, 2018). MSCs are localized throughout the adult body as a small population in the stroma of the tissue concerned, and the microenvironment protects their self-renewal potential and undifferentiated state (Urbanek et al., 2006; Wang et al., 2011). On tissue injury or inflammation insult, MSCs are activated and leave their ecological niche and migrate to the site of injury, inflammation, and tumors, where they are able to secrete various cytokines, chemokines, and growth factors that closely interact with the inflammatory environment and the tumor environment (TME), respectively (Sun et al. 2014). With the accumulation of data about the interaction between MSCs and tumor cells, MSCs have been demonstrated the natural anti-tumor functions, which is the basis for intensive research for new methods using MSCs as a tool to inhibit tumor growth and invasion, although the dualistic role of MSCs on tumors still exists.^[5]

Functional Properties of Mesenchymal Stem Cells

MSC's peculiar biological properties have been exploited for SC-based therapeutic approaches both in preclinical and clinical studies. The favorable properties such as extensive proliferation, multipotency, ability to migrate and home to the site of injury or inflammation, and immune-modulation boost MSC's use in regenerative medicine, and as vehicle for gene and drug delivery into diseased areas. As therapeutic agents, MSC can act directly by cell–cell interactions or indirectly, by secreting factors (growth factors, chemokines, cytokines, and exosomes), which modulate cell and tissue function. In vivo, MSCs display complex biological features and behavior highly dependent on their genetic profile and the surrounding microenvironment of the anatomic sites in which they reside that are formed by different cell types and extracellular matrix (ECM) composition. Therefore, both humoral and cell-cell signaling mechanisms control MSC growth, mobilization, and differentiation. MSC's ability to migrate and accumulate into inflamed, ischemic, or injured tissues, as well as in tumors, contributes to tissue repair or regeneration. MSC homing has been definite as the active or passive arrest within vasculature followed by transmigration across the endothelium.

A mutual influence exists between migratory and immunomodulatory properties since immune-related factors released by MSCs regulate immune response and simultaneously MSCs are affected by paracrine modulation of immune cells (T and B lymphocytes, NK cells). MSCs exert immunomodulatory activity on both the innate and adaptive immune systems.

Overall, the biological properties of MSCs evidence a perspective role in oncology, based on their innate tumor tropism and release of relevant factors to modulate tumor microenvironment (TME), also considering genetically modified and loaded MSCs able to transport and deliver therapeutics to cancer sites. In this context, the oncogenic risk and prometastatic effects represent crucial limitation factors for clinical applications.^[7]

Role of MSCs in Neoplastic Microenvironment

It is well known that interactions between cancer cells and stroma are fundamental importance in promoting both the development and invasiveness of tumors. For instance, cancer cells may lead to modifications of topography and molecular composition of stroma during early tumor development and this, in turn, can affect the properties of the cancer cells. Therefore, the bidirectional interplay between cancer cells and cells of stroma, including MSCs, endothelial, immune, and fibroblast-like stromal cells, plays a key role in tumor progression and metastasis and creates a complex microenvironment called tumor niche. In the normal stroma, predominant cells are fibroblasts that secrete an ECM providing a natural barrier against tumor progression.^[32],[33] On the other hand, the ECM is able to support and promote tumor progression by modifications of the same ECM. In this context, both fibroblasts and myofibroblasts, denominated cancer-associated fibroblasts (CAFs) produce proteins such as collagen, fibronectin, a-smooth muscle actin, and others, creating alterations of ECM architecture. As a result, the cancer cells start to change their morphology becoming invasive and metastatic. It has been reported that MSCs can originate from tumor resident stroma progenitors, or can be recruited from other tissues as BM by circulation. Interestingly, MSCs have the tendency to migrate into damaged tissues or organs, driven by chemotactic gradients of cytokines/chemokines released from the same damaged tissues. Once arrived at injured sites, MSCs provide structural support and secrete factors for tissue repair. Therefore, this physiological behavior happens also for the tumor that can be considered as a “wounds that never heal.” Circulating MSCs from BM, AT, or MSCs derived from tumor stroma cells that are able to differentiate in CAFs.^[4] Another key feature of cancer is that the metabolism is an anaerobic glycolysis even under aerobic conditions. Furthermore, cancer cells can increase their mitochondria mass and activity and consequently increase their energetic metabolism. MSCs can also regulate the metabolism of cancer cells through the secretion of exosomes. Different types of immune cells are also identifiable within the tumor microenvironment. These cells play both stimulatory and inhibitory roles on cancer growth. Both T-cells and B-cells infiltrations may represent an important favorable prognostic factor.^[34] During tumor progression, another class of immune cells to be considered is macrophages. In fact, monocytes and macrophages can be recruited into tumors site altering the tumor microenvironment and accelerating tumor progression. Macrophages can be subdivided into two categories: classic M1 and alternative M2 macrophages. The M1 macrophage is involved in the inflammatory response, pathogen clearance, and antitumor immunity. On the contrary, the M2 macrophage is involved in an anti-inflammatory response, wound healing, and has protumorigenic properties. The tumor-associated macrophages (TAMs) closely resemble the M2-polarized macrophages and are critical modulators of the tumor microenvironment. Several studies have suggested that TAM accumulation in tumors correlates with a poor clinical outcome and provide a favorable microenvironment to support tumor development and progression regulating tumor angiogenesis, invasion, metastasis, immune-suppression, and chemotherapeutic resistance. Together, MSCs and TAMs promote tumor growth. Thus, MSCs and TAMs can engage in a bidirectional interaction resulting in tumor promotion and progression.^[4]

Effects of Mesenchymal Stem Cells On Oral Cancer

In recent years, there have been gradually increasing studies on the relationship between the MSCs and oral cancer, and in these studies, it was found that, on the one hand, MSCs have an inhibitory effect on oral cancer, while on the other hand, MSCs can promote the progression of oral cancer.^[5]

Dental tissues derived MSCs (DMSCs): Ji et al. showed that gingival tissue-derived MSCs (GMSCs) inhibited the growth of tongue squamous cell carcinoma (TSCC) cell lines CAL27 and WSU-HN6 in vitro, and the soluble factors in CM of GMSCs played a key role in this process by inducing apoptosis of tumor cells.^[31] MSCs can influence tumor progression by regulating intra-tumor angiogenesis. Anti-angiogenic therapy may be a possible treatment strategy of tumors in the future. Liu et al. treated TSCC CAL27 cells with exosomes derived from human deciduous exfoliated teeth by direct multi-point intratumoral injection and found that the tumor volume was significantly smaller than that of the control group.^[35] Further experiments showed that exosomes from human deciduous exfoliated teeth downregulated VEGF-A expression through miR-100-5p and miR-1246, which significantly reduced the generation of microvasculature around TSCC. Dental pulp SCs (DPSCs)-derived CM-induced apoptosis in tumor cells, inhibited the proliferation of TSCC cells AW13516 by enhancing the expression of p16, enhanced the invasion, adhesion, and multidrug resistance of AW13516 by upregulating angiopoietin-2, epidermal growth factor, macrophage-stimulating factor, PDGF-AA, PDGF-BB, TNF-α, and IL-2, downregulating the anti-inflammatory cytokines TNFβ1, and pro-inflammatory cytokine IL-4.^[36] Based on Ki-67 assays, Raj et al. indicated the dual effect of MSCs on tumors. DPSCs-CM inhibited the proliferation of TSCC cells AW13516 at 50% and 100% concentrations and promoted the proliferation at a 20% concentration.^[37],[38] Several growth factors, including VEGF, hepatocyte growth factor, angiopoietin-2, TGF-α, SC factor, erythropoietin, colony-stimulating factor, fibroblast growth factor, PDGF-BB, pro-inflammatory cytokines, TNF-α, and IL-8, may play a dominant role in promoting the proliferation of tumor cells^[5]
Bone marrow mesenchymal stem cells (BMSCs): Promotional effects on oral cancer: Human BMSCs inhibited the proliferation of TSCC cells HSC-3 but promoted the invasion of tumor cells by upregulating the expression of the C-C Motif Chemokine 5. In addition, BMSCs-induced production of type I collagen after interaction with HSC3 is associated with poor prognosis in TSCC cells patients^[38]
BMSCs: Inhibitory effects on oral cancer: Using the hamsters OSCC model, Bruna et al. demonstrated that the systematic administration of allogeneic BMSCs did not aggravate the progression of precancerous lesions.^[39] On the contrary, the administration of BMSCs at the hypoplasia stage of precancerous lesions inhibited tumor growth, whereas only a small proportion of lesions transformed to OSCC by applying BMSCs at the papilloma stage. Bagheri et al. (2021) demonstrated that human BMSCs-CM exhibited a time-dependent inhibitory effect on TSCC cells CAL27, which showed the lowest cells viability after 72 h of coculture with BMSCs-CM. The decrease of proliferation marker proliferating cell nuclear antigen, anti-apoptotic marker BCL-2 and Ki67-positive cells at 24 and 72 h of coculture indicated that BMSCs-CM decreased the proliferation of CAL27 cells and increased apoptosis. Another study demonstrated that 3 weeks after oral tissue injection of human OSCC (moderately differentiated tumor of buccal mucosa) in nude mice, direct intra-tumor injection of mice-derived BMSCs in combination with cisplatin revealed an inhibition of tumor growth and an increase in the lifespan of the mice, these effects may be due to BMSCs' promotion of cisplatin distribution for better anti-cancer action and increased apoptosis of tumor cells^[40]
Other MSCs: Among the applications of MSCs for cancer treatment, AT-derived MSCs (AMSCs) have received increasing attention due to the advantages of relatively easy collection and production. Sinha et al.^[41] demonstrated that after co-culture with human TSCC cell HSC-3, AMSCs did not induce proliferation, migration, and invasion of tumor cells, which providing preliminary evidence that AMSCs may have more suitable properties for tumor therapy relative to other types of MSCs. Because there are relatively few studies on the interaction of AMSCs and tumors, more studies are needed before further applications. In a study of human amniotic membrane MSCs (HAMCSs)-derived CM, its promoting effect on TSCC cells CAL27 was demonstrated after a 24 h of co-culture with human HAMSCs-CM, manifested by an increase in CAL27 cell viability. After 72 h of co-culture, the increased expression of proliferating cell nuclear antigen indicated that HAMSCs-CM promoted the proliferation of CAL27, and the results of flow cytometry showed a decrease in the number of apoptotic CAL27 cells.^[42]

Increased expression of microRNA-8485 in exosomes derived from human oral leukoplakia with dysplasia and oral cancer (species not specified) derived-MSCs promotes the proliferation, migration, and invasion of SCC15 cells in vitro and reduces the expression of the oncogene p53 (Li et al., 2019a). Ji et al. (2021) showed that OSCC-derived MSCs promoted the migration and invasion of OSCC cell lines CAL27 and WSU-HN6. With a significant decrease in E-cadherin, alpha E catenin, and a significant increase in N-cadherin, OSCC-MSCs may promote OSCC metastasis through EMT. Further experiments showed that upregulation of CPNE7, a calcium-dependent phospholipid-binding protein in tumor-derived MSCs, promotes phosphorylation of p65 and IκBα as well as nuclear translocation of p65, which activates the NF-κB pathway, promotes the expression of IL-8, and thus promotes tumor metastasis. Chen et al. (2019) demonstrated that rat oral mucosa malignancy-derived MSCs can inhibit the proliferation of T cells and promote the apoptosis of T cell through soluble factors and intercellular contacts, whereas T cell migration was not affected. The immunosuppressive effect of MSCs on T cells is enhanced with increasing tumor malignancy. The higher the number of MSCs at the tumor sites, the higher the proliferative status of tumor cells, showing that tumor-derived MSCs play an important role in the malignant progression of oral mucosa.^[5]

Cancer Stem Cells and Treatment Failure

As they are the cells stimulating tumor growth, the elimination of CSCs is necessary for the elimination of tumors. However, many studies have now shown that CSCs are more resistant than other tumor cells to ChT and RT. In vitro assays show that when CD44-high CSCs are irradiated or exposed to ChT, they may be over 10 times more resistant to apoptosis than CD44-low cells. The sensitivity of surrounding normal tissues to high doses of chemo- and radio-therapies restricts the dose levels that can be administered and, despite the various methods of targeting, the dose provided may be sufficient to kill many tumor cells but not all of the CSCs. Clinically, therefore, the tumor may appear to shrink, and even perhaps disappear, only for a few remaining CSCs to begin to divide and subsequently regenerate it. Local tumor recurrence is a major problem for OSCC therapy and the elimination of CSCs is a target of therapy but one that is made more complex by the heterogeneity of CSCs.^[16]

Oral Cancer Stem Cell Therapy

Cancer therapy is a very important aspect of the public health field. Many researchers developed a variety of therapeutic approaches such as immune cell therapy, SC therapy, gene therapy, nanotechnology-based therapy, and utilization of natural compounds in the treatment of various cancers. In this regard, several fields emphasize the identification and specific targeting of the head and neck CSCs. However, the new therapeutic regimes carried considerable morbidities such as defacement and functional modifications from surgical operations to the systemic toxicity caused by chemotherapy as well as radiation-induced consequences due to radiotherapy. In addition, as a result of diverse innate systems, the CSCs frequently resisted to the conventional radiation and chemotherapy. Such cells have been capable of surviving through treatment and repopulating the tumors with the chemo-radio resistant cells, thus, specifically targeting head-and-neck CSCs provided a potent device of the ameliorated cancer outputs by demonstrating the organ conservation and declining the off-target toxicity.^[24]

As seen in the literature, CD44 is one of the well-known exploration targets for the targeted therapies against CSCs. In fact, researchers utilized hyaluronic acid (with its selective binding to CD44) as one of the agents to deliver the directed treatments as opposed to the CD44-positive cells such as the hyaluronic acid conjugated chemotherapeutics as well as the hyaluronic acid-guided NPS. Moreover, hyaluronic acid induced the interactions between CD44 and the SC transcription factors Nanog, Sox2, and Oct-4.^[43] Thus, additional investigations should be performed for showing advantages of the hyaluronic acid targeting with any induction of more activation of the CSCs. Consequently, experts in the field explored the anti-CD133 treatments as the targeted head-and-neck anti-CSC therapies. One of the studies on the bacterial toxin (cytolethal distending toxin) to an antihuman CD133 monoclonal antibody revealed inhibiting the cells proliferation while other investigation, which utilized a single-chain variable fragment targeting CD133 demonstrated remarkable diminishment in the rapid growth of the tumors in the cells and rat models As a result, CD271 inhibited in the cell models for decreasing the formation of the tumors.^[44]

Finally, one of the encouraging options to treat this condition would be targeting the CSC surface markers and the best performance in relation to the remaining treatments would be as a delivery mechanism. Notably, one of the today's main investigation fields is the addition of the novel agents or targeted treatment related to the standard cisplatin chemotherapy. Moreover, salinomycin with paclitaxel and cisplatin functioned for increasing apoptosis in the head-and-neck CSCs.^[45] In addition, GRP78 has been considered to be one of the multifunctional proteins that contributed to the cell survival as well as resistance to chemotherapy. Inhibiting the GRP78 would sensitize the head-and-neck CSCs for radiation and ChT. In this regard, Huang et al. revealed the greater sensitivity to cisplatin by small hairpin RNA knock-down of Nanog.^[46] Furthermore, researchers indicated that CSCs had lower levels of ROS, assisting in the maintenance of the stem-like features and chemoresistance. Finally, inhibiting the ROS scavenging proteins (SOD2 and Catalase) enhanced the ROS and the following enhancement in the sensitivity to cisplatin.

Experts in the field are growingly applying the epidermal growth factor receptor (EGFR) inhibition (with cetuximab) in the advanced and recurring HNSCC therapeutic guidelines. The former investigations also suggested the potent contribution to the EGFR-targeted treatment especially as opposed to the head and neck CSCs. However, in the nasopharyngeal carcinomas, EGFR acted using CTNNB1 and AKT pathways for driving the CSC phenotypes. Moreover, activating EGFR in the head-and-neck CSCs enhanced the expressing of the genes engaged in the CSC rapid growth or proliferation (OCT4, BMI1, CD44, NANOG), and CSCs treatment through inhibiting EGFR declined the tumor growth and augmented the sensitivity to cisplatin. Greater abilities for the efflux cytotoxic agents have been considered as one of the main devices of CSC resistance to chemotherapy. Therefore, researchers examined the cellular efflux proteins as the potent targets to sensitize the CSCs to the current chemotherapy agents. It is notable that suppressors to the ABC transporter family in case of application to the head and neck and CSC populations, enhanced sensitivity to the chemotherapy.^[47] Moreover, the authors largely explored the enhanced CSC sensitivity to the radiation. However, today's examinations target ATRA (a retinoid involved in cell terminal differentiation) and CHEK1/2 DNA damage repair genes in the head and neck CSCs. Such explorations demonstrate greater responses to the radiation in the CSCs following the CHEK1/2 suppression and ATRA utilization. Finally, inhibiting the SHH/MTOR/RPS6KB1 pathways augmented radio-sensitivity to CSCs, reflecting the contribution of the above pathways and potent targetable choices to enhance the CSC radiosensitivity.^[24]

Mesenchymal Stem Cells in the Treatment of Oral Cancer

The treatment of oral cancer depends mainly on the severity of the disease. OSCC is the most common type of oral cancers with a poor prognosis and a high recurrence rate (Satija et al., 2009) and a huge potential for regional metastasis even in the early stages (Vargas-Ferreira et al., 2012). A proportion of OSCC can be detected at an early stage, but current treatment modalities adversely affect patients physically and psychologically, severely affecting their quality of life. Most OSCC is not detected until advanced stage, by which time the survival rate of patients has been markedly reduced. Among the many treatment modalities, surgical treatment is the main modality for oral cancer. When the primary tumor is large and incomplete resection or signs of infiltration are suspected, radiation adjuvant therapy is administered after surgery. Molecularly targeted therapy with cetuximab added to postoperative RT, targeting the epidermal growth factor receptor, has been approved for the treatment of OSCC. Docetaxel, cisplatin and 5-fluorouracil, and other anticancer drug-induced ChT are usually used as induction therapy before surgery or alone or in combination with RT after surgery. Combined surgery-RT has become the standard procedure for the treatment of advanced oral cancer (Colevas et al., 2018). In recent years, antitumor immunotherapies such as the programmed death-1 inhibitor, which block tumor immunosuppressive signals and enhance antitumor immune responses by targeting the programmed death-1/programmed death-ligand 1 pathway, have played an important role in recurrence and metastasis of oral cancer (Cramer et al., 2019).

Although currently available treatment strategies include excision of malignant tissue in combination with RT and ChT, the 5-year survival rate is still about 50% (Sasahira et al., 2014). The oral cavity as a functional organ of mastication, speech, and articulation, surgery may lead to serious esthetic and functional problems, as well as psychological trauma for the patient. RT can cause altered taste, dysphagia, dry mouth, and hypothyroidism, causing temporary or permanent damage to healthy tissues. ChT can also lead to severe systemic reactions such as nausea, vomiting, hair loss, infection, and diarrhea, which can seriously affect patients' health and quality of life. Therefore, it is particularly important to find new methods for the treatment of oral cancer. As previously mentioned, MSCs can inhibit tumor progression in multiple ways, such as inhibition of angiogenesis, suppression of cell proliferation and metastasis, induction of apoptosis, cell cycle arrest, inflammatory infiltration, and regulation of oncogenes. In light of these studies on MSCs, there has been increasing interest in MSCs-based cancer therapy in recent years, and advanced approaches to modify MSCs to become powerful and precise targeting tools for killing cancer cells rather than normal healthy cells have been continuously explored.

As mentioned earlier, MSCs show both promoting and inhibiting effects on oral cancer; therefore, their mechanisms should be further explored before applying MSCs to the treatment of oral cancer. In addition, circumventing the promotive effects of MSCs on oral cancer and making full use of the effects of MSCs on oral cancer, such as homing and inhibition, and using them as therapeutic drug carriers or MSCs to directly inhibit tumors would be beneficial to discover the potential applications of MSCs for oral cancer treatment. Both wild-type MSCs and modified MSCs have been used for the treatment of oral cancer. MSCs can be used as carriers for the delivery of therapeutic proteins or anticancer drugs, and are genetically modified to over-express several anti-tumor factors, such as IL, IFN, pro-drugs, oncolytic viruses, pro-apoptotic proteins, anti-angiogenic agents, and growth factor antagonists (Shah, 2012). MSCs can selectively migrate and aggregate at the tumor sites, thus exerting a therapeutic effect, improving therapeutic efficacy and reducing systemic toxicity. Unlike cell therapies, MSCs-derived secretomes can be better evaluated for their safety, dosage, and potency. Secretomes, aside from avoiding the inconveniences of administering living proliferating cells, show other additional advantages, including cheaper, safer, and more practical for clinical use. For instance, exosomes derived from MSCs, one of the secretomes, have been evaluated for their potential to be used as drug-delivery vehicles (Eiro et al., 2021).

Direct application of MSCs: Isolated from gingival tissues, GMSCs possessed the properties of easy isolation, rapid expansion, profound immunomodulatory, and anti-inflammatory functions, making them a potential source for SC-based therapy (Ji et al., 2016). GMSCs can inhibit the growth of oral cancer cells in vitro and in vivo by altering the microenvironment of surrounding oral cancer cells, suggesting that GMSCs have potential applications in the treatment of oral dysplasia and oral cancer (Ji et al., 2016). MSCs can influence tumor progression by regulating intratumor angiogenesis. Anti-angiogenic therapy may be a possible treatment strategy of tumors in the future. Liu et al. (2022) treated TSCC CAL27 cells with exosomes derived from human deciduous exfoliated teeth by direct multi-point intratumor injection and found that the tumor volume was significantly smaller than that of the control group. Further experiments showed that exosomes from human deciduous exfoliated teeth downregulated VEGF-A expression through miR-100-5p and miR-1246, which significantly reduced the generation of microvasculature around TSCC. Up to date, the influence of BMSCs on cancer remains uncertain. Some studies have shown that BMSCs promote cancer progression, whereas others show that BMSCs suppress cancer progression, and also some studies found BMSCs have no significant impact on cancer progression (Barcellos-de-Souza et al., 2013; Gao et al., 2016; Mi and Gong, 2017; Wu et al., 2019; Zhang et al., 2019)
MSCs as drug delivery vehicles: GMSCs exhibit the capability to encapsulate and release anticancer drugs without any genetic changes. Cocce et al. (2017) demonstrated by in vitro experiments that GMSCs can efficiently bind three important antitumor drugs and then release them in active form: paclitaxel, doxorubicin, and gemcitabine, thereby significantly inhibiting the growth of TSCC cell SCC154, indicating that MSCs-mediated drug delivery systems have potential applications in the field of oral oncology. Sun et al.^[48] used MSCs membrane-encapsulated oxygen-carrying perfluorocarbon and sonosensitizer verteporfin to develop a novel biomimetic sonosensitizer, named M/LPV/O2, which could induce cancer cell death by increasing uptake cancer cells and stimulating intracellular reactive oxygen species production under hypoxic conditions. M/LPV/O2 can accumulate in oral tumor, relieved hypoxia, and effectively inhibited tumor. In addition, exhibiting minimal systemic side effects, M/LPV/O2 can successfully maintain oral function without causing esthetic problems.

Zhou et al.^[49] showed that OSCC recruits DPSCs through the CXCL8-CXCR2 axis. They used DPSCs membranes (DPSCM) modified with metal-organic framework nanoparticles (MOFs) to create a novel nanoparticle, [email protected], which can effectively deliver antitumor drugs to target OSCC. [email protected] carries doxorubicin (DOX) can induce the death of CAL27 cells and block the growth of CAL27 tumor. The data suggest that this novel [email protected] nanoparticle is a potential targeted drug delivery system that can be used for OSCC. Du et al.^[50] constructed IFN-β gene-modified GMSCs (GMSCs/IFNβ) and found that GMSCs/IFN-β inhibited the proliferation of CAL27 cells in vitro. Through in vivo experiments, they showed that GMSCs/IFN-β expressed high levels of IFN-β, which significantly inhibited tumor growth. Further experiments demonstrated that GMSCs/IFN-β inhibited the growth of TSCC xenografts by suppressing cell proliferation and inducing apoptosis. In addition, GMSCs are genetically modified to release other cytokines, such as IFN-γ, IL-2, IL-12, and IL-24 (Matsuzuka et al., 2010; Zhang et al., 2013; Yang et al., 2014; You et al., 2015), to achieve antitumor effects.

Exosomes from human BMSCs with upregulated miR-101-3p may serve as a promising new direction for the development of oral cancer therapeutics. Human BMSCs can transfer microRNA-101-3p to human TSCC cells TCA8113 through exosomes to negatively regulate the collagen X-type α1 chain of the target gene, thereby inhibiting the proliferation, invasion, and migration of TCA8113 cells (Xie et al., 2019).

Oral potentially malignant disorders are usually asymptomatic clinical lesions that appear before OSCC.^[51] Put mice genetically modified BMSCs-derived extracellular vesicles (EVs) highly expressing microRNA-185 (BMSCs-EVs-miR-185) pasted on oral potentially malignant disorders induced with dimethylbenzanthracene, and found that BMSCs-EVs-miR-185 could attenuate the inflammatory condition and reduce the dysplasia in the lesion tissue. In addition, BMSCs-EVs-miR185 may inhibit disease progression by suppressing proliferation, angiogenesis and promoting the activation of the Akt pathway to increase the apoptosis of tumor cells.^[5]

Future treatment perspectives

Surgical resection is still a major therapy for OSCC and is effective, especially in treating smaller lesions. Current anti-cancer therapies for more advanced lesions are typically based on radio-and chemo-therapeutic agents that target proliferative cancer cells. However, compared to the bulk of tumor cells, the resistance of EPI-CSC populations to such therapies is greatly enhanced due to their slow cell cycle and their mechanisms for rapid DNA repair and drug exclusion. Consequently, although most non-CSC tumor cells may be eradicated with standard therapies, the therapy-resistant CSCs may selectively survive the doses of radio-and chemotherapies that are achievable without major damage to the surrounding normal structures. With such partially effective therapies, CSCs can be expected to survive through a process similar to natural selection, and their self-renewal capacity can then enable them to regenerate themselves and stimulate the growth of a new tumor. To avoid such recurrence, therapy, therefore, needs to employ agents, or combinations of agents, which provide widely effective actions, along with better assays which may allow the effective screening of new and existing drugs for their differential effects on all sub-types of CSCs and non-CSCs. The molecular advances in tumor biology studies are guiding an individualized treatment approach. For example, a clinically validated chemopredictive assay (ChemoID®) is now being tested for HNSCC, in which both CSCs and bulk tumor cells are challenged by various FDA-approved drugs and their combinations to determine the most effective ChT scheme. This assay, although still not FDA approved, was recently published as a new complementary procedure to HNSCC drug treatment, aiming at both the elimination of unnecessary toxicity in patients as well as avoiding ineffective ChT regimens. A better understanding of CSC properties is crucial for the development of effective alternative strategies, for example, targeting SC maintenance, signaling pathways, or blocking EMT/MET to prevent the switching of CSCs between drug-resistant phenotypes.^[16]

Discussion

The overall 5-year survival of oral cavity squamous cell carcinoma has remained at 50%, largely unchanged for 40 years, despite intensive research. This high mortality has been largely attributed to high rates of locoregional recurrence.^[52] An emerging hierarchical concept of carcinogenesis proposes that CSCs sit atop a hierarchy of a heterogeneous population of cells within cancer and are defined functionally as a subset of cells that display stemness characteristics, including the ability to asymmetrically divide, resulting in self-renewal of CSCs and the production of heterogeneous populations of cancer cells that are further down the hierarchical ladder. CSCs are highly tumorigenic compared to the other cancer cells and are believed to be largely responsible for the biological characteristics of cancer, namely, rapid growth, invasion, and metastasis.^[53]

CSCs are predominantly in the inactive G0 phase and thus avoid destruction by RT and ChT that target active cells. CSCs in OSCCC are resistant to both RT and ChT agents such as cisplatin, carboplatin, docetaxel, paclitaxel, etoposide, gemcitabine, and 5-fluorouracil.^{[54],[55],[56]} Thus, treatment results in an enriching effect on CSCs within the posttreatment cancer cell population, providing a plausible rationale for locoregional recurrence and distant metastasis from RT- and ChT-resistant cells, despite aggressive treatment.^[53]

MSCs are essential components of tumor stromal cells and have a pivotal role in tumor microenvironment, by modulating tumor growth and development. These cells are able to become tumor-associated fibroblasts (TAFs), which contribute to fibrovascular network expansion and tumor progression.^[57],[58] MSCs are also capable of differentiating into pericytes and mural cells around tumor blood vessels, thus contributing to the formation of tumoral vasculature.^[59] MSCs are generally believed to reach the tumor from the BM,^[60] as much as a strong evidence demonstrates that MSCs home to injury sites in various pathological conditions, including inflammation, tissue repair, and neoplasms.^{[61],[62],[63]} During the progression and development of tumors, MSCs can be recruited in large numbers to the tumor site. On the other hand, it is well known that BM-MSCs exert immunosuppressive activity on various cells of the immune system.^[64]

If the diagnosis can be performed based on the origin of the cancer cells, a more specific therapeutic strategy may be implemented to improve prognosis. This would be a paradigm shift in diagnostic and therapeutic strategies for OSCC.^[65]

Conclusion

The identification of the mechanisms underlying oral cancer initiation and progression is of the utmost importance. Despite advances in therapeutic options for OSCC over the last decades, mortality and morbidity rates have not been markedly improved. Therefore, the search for new and better CSC markers that relate comprehensively to the known alterations of tumor progression seems to be necessary. CSC markers that could act as a therapeutic target could play an important role in the effective treatment strategies of OSCC.

Regenerative, immunomodulatory, and tumor-homing properties of MSCs have been exploited to repair tissue injuries, interfere with cancer, immune-based disorder, and neurodegenerative disease development. This large use was also sustained by the relatively easiness of harvest and processing from different sources, and the abundant availability after in vitro expansion. Beyond regenerative medicine, MSC tumor tropism and nonimmunogenicity laid the groundwork for their application in oncology research. In particular, MSCs release soluble factors or EVs that acting through autocrine and paracrine mechanisms are able to modulate cancer cell survival and proliferation, migratory pathways, and induce host immunomodulation. Despite initial enthusiasm, current literature highlights growing data inconsistencies and divergences on whether and how MSCs may promote or inhibit tumor growth in various cancers. A new frontier for MSC application is the achievement of genetic engineering-based methodology to convert MSCs into therapeutic vehicles to graft into the tumor and produce or release engineered EVs and nanoparticles, or cytotoxic agents. These strategies supported by some initial preclinical studies were confirmed in few human cancer studies.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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