Identification and potential role of telocytes in human uterine leiomyoma
ULMs, despite being the most common gynecologic tumors, and associated with significant reproductive morbidity, their exact etiology is not understood.
In the present study, we have characterized, for the first time to the best of our knowledge, a specialized interstitial cell type; telocytes; in ULM and Myo-F. Our results show that as compared to Myo-F or Myo-N, ULMs contain higher number of telocytes with ultrastructural features suggestive of increased cellular activity including loss of the heterochromation clumps, extremely long telopods, large numbers of mitochondria, dilated rough endoplasmic reticulum cisternae, and clusters of shed vesicle.
Every effort has been made to correctly identify telocytes in our study. Positive immunostaining to c-Kit (CD 117) was employed as a marker to recognize telocytes [10, 30]. Mast cells, which are also c-KIT positive, were easily distinguished as large round or oval cells with abundant granular cytoplasm and large round nuclei [10, 31]. Moreover, strict ultrastructural criteria were applied to define telocytes under electron microscope. These included: 1) small cell body (9–15 ?m), with scanty cytoplasm, 2) spindle or triangular shape, 3) nucleus with moderately dense chromatin, and, 4) most characteristically, the presence of telopodes, identified as very long cytoplasmic processes (2–5 per cell) with a branching pattern and a “bead on string” appearance [9, 10].
In agreement with our results are the findings of previous studies in which telocytes have been identified in the human myometrium [10, 30, 32–34]. The function of telocytes in the myometrium is still controversial. Being morphologically similar to Interstitial Cells of Cajal (ICC) of the GIT that regulate gut motility, uterine telocytes are assumed to be the pace maker controlling myometrial contractions. Supporting this concept is electrophysiological data showing that myometrial telocytes of the non pregnant uterus exhibit hyperpolarizing chloride inward current with calcium dependence indicating a role in myometrial excitability [10, 26, 35]. Telocytes are also involved in intercellular signaling throughout the myometrium and involved in the process of myometrial regeneration [10, 36]. As telocytes communicate with immune cells within the myometrium, these specialized cells may play a role in the immune tolerance to pregnancy [37].
Although our study lacked functional experiments, but our observation of increased telocyte cell number and activity in ULM point out an active role of these cells in ULM development and/or growth.
The cellular origin of ULM is not exactly known. Early genetic studies have indicated that ULMs are monoclonal in origin [38]. It is reported that mouse and human myometrium contain multipotent stem cells that are responsible for tissue regeneration [39, 40]. Similarly, uterine leiomyomas were shown to contain a side population of cells that show phenotypic and functional characteristics of fibroid stem cells [41], which are believed to give origin to fibroid tumors under influence of steroid hormones [42].
To be accepted as cells of origin of such highly steroid dependent tumors, fibroid stem cells should have a mechanism to respond to estrogen and progesterone. However, the side population of fibroid stem cells contains very little amounts of steroid hormone receptors [41, 42]. It is postulated that estrogen and progesterone work on the nearby steroid hormone receptor positive mature myometrial and fibroid cells releasing paracrine factors that stimulate the fibroid stem cells to mediate tumor growth [1]. Telocytes can fit nicely into that model. They have been shown to express estrogen and progesterone receptors in their cell bodies and in cellular processes [43, 44]. Moreover, they keep an extensive network of intercellular communication within the myometrium, and as our findings indicate, within the ULM as well. Based on our findings and those of others, we can postulate that telocytes may work as hormonal sensors for uterine leiomyoma stem cells [44]. Telocytes can respond to estrogen and progesterone hormones through their expressed steroid hormone receptors, and relay their signals to the leiomyoma stem cell through their extensively branching processes.
Telocytes work as part of stem cell niche to form stem cell/telocyte tandem. In leiomyoma, their interstitial network of telopodes can build a dynamic scaffold surrounding the stem cells, which helps to guide the newly formed leiomyoma cells to form a coherent 3D architecture. Such a role of telocytes in nursing stem cells has been shown in cardiac [45, 46], and skeletal muscles [47, 48].
c-KIT (CD 117) expressed by telocytes is a member of type III trans-membrane receptor tyrosine kinase family. Binding of the c-KIT receptor to its ligand, stem cell factor (SCF), results in receptor dimerization and kinase activation. This leads to subsequent activation of two important molecular pathways: the mitogen-activated protein (MAP) kinase pathway (RAF, MEK and ERK), and phosphoinositide 3-kinase (PI3K) pathway (AKT, mammalian target of rapamycin (mTOR), S6 kinase). In addition, signal transducer and activator of transcription 3 (STAT3) is activated. The collective impact favors an increase in cell metabolism, cell cycle progression, and a decreased sensitivity to apoptosis [49].
The ERK signaling pathway, (a downstream of MAP kinase activation) has been shown to be a mediator of ULM growth [50]. In addition, mTOR signaling, (a downstream of PI3K activation), is activated in high frequency in ULM [51, 52]. The role of STAT3 in ULM was elucidated in a study in which tyrosine kinase inhibitor reduced leiomyoma cell proliferation in vitro mediated through phosphorylated STAT3 inhibition [53].
Taken together, it appears that c-KIT receptor activation can stimulate important intracellular pathways that have been implicated in ULM pathogenesis.
Of interest in our study is the finding that Myo-F contained fewer telocytes than the Myo-N, although the difference did not reach statistical significance. In the gastrointestinal tract, disorders characterized by excessive fibrosis, such as ulcerative colitis, Crohn’s disease, are associated with marked reduction in number and/or ultrastructural damage to the affected tissue telocytes [29, 54, 55]. This contributes to the loss of function and dysmotility encountered in the affected structures such as colon or stomach [54]. A similar concept can be extrapolated to the fibroid uterus, where excessive extracellular matrix deposition in ULM is associated with reduction in telocytes number in Myo-F. This can lead to disturbed tissue homeostasis and abnormal contraction waves in the uterus which can be associated with infertility or dysmenorrhea observed in ULM patients.
Alternatively, as telocytes show some degree of motility [13], the reduced number of telocytes in Myo-F compared to Myo-N, may be explained on the basis of migration and recruitment of myometrial telocytes to the region of the ULM.
Further supporting our data on ULMs are previous studies implicating telocytes in the pathogenesis of cancer [56]. Telocytes had potential functions in self-assembly of normal breast stromal, and breast cancer cells to self- assembly breast cancer tissue in vitro. Telocyte-like cells closely communicated with breast cancer cells as well as other stromal cells, and might serve as a bridge that directly linked the adjacent cells [57]. In the skin, and in comparison to telocytes from normal dermis, telocytes from basal and squamous cell carcinoma of the skin showed limitation in their heterocellular junctions. This suggests a possible involvement in induction of cell-cell communication into the tumor [58].
c-KIT expressed by telocytes is amenable for pharmacological manipulation. Imatinib, a 2-phenyl amino pyrimidine derivative, is a tyrosine kinase inhibitor with activity against c-KIT. It works by binding close to the ATP binding site on the tyrosie kinase, locking it in a closed conformation. This process ultimately results in “switching-off” the downstream signaling pathways [59], some of which are essentially important in uterine ULM growth. Imatinib is used as a molecularly targeted therapy for the treatment of cancers such as chronic myeloid leukemia or gastrointestinal stromal tumors (GIST). The drug is given orally and generally well tolerated, with serious side effects rarely happening [60].