Over the last decade, a variety of imaging technologies have been investigated as tools for cancer diagnosis and monitoring response to cancer therapies . Molecular imaging offers the potential for noninvasive assessment therapeutic responses and real-time monitoring of tumor procession simultaneously [22, 41]. In addition to this, molecular imaging provides the possibility to visualize and monitor cellular and molecular processes, such as metabolism, biosynthesis, angiogenesis, cell proliferation, and apoptosis [42–44]. In this study, we evaluated the effect of ES-CM on cancer procession by dual imaging. 4T1 cancer cells were transduced with a vector carrying a ubiquitin promoter driving Fluc followed by a seven-repeat Stat3-binding sequence (enhancer) and minimal TA (promoter) driving Rluc reporter genes (4T1-Fluc/GFP-pStat3/Rluc). Fluc and Rluc have different substrates, d-luciferin and coelenterazine respectively. Using this bioluminescence imaging system, we can simultaneously track the ES-CM on tumor growth (Fluc) and Stat3 signal pathway activation (Rluc) in vivo and in vitro. This model enabled us to obtain more tangible options for the ES microenvironment on tumor growth and Stat3 signaling pathway activation. Based on this model, we demonstrated that the ES cell microenvironment played a critical antitumor role in vitro and in vivo. This may be mediated by the Stat3 pathway.
As already mentioned, certain phenotypic characteristics are shared by ES cells and some aggressive cancer cells, such as unlimited self-renewal and expression of some pluripotent genes (NANOG, OCT4, SOX2) . However, cancer cells lack the appropriate regulatory mechanisms to maintain normal differentiation. This difference might be attributed to the different microenvironments surrounding them. Since the embryonic microenvironment possesses key regulatory cues and signaling molecules that function to maintain and regulate the growth of the stem cell population, we hypothesized that an ES cell microenvironment might be able to influence cancer cells by normalizing their plastic phenotype. Some researchers have revealed that the microenvironment of human ES cells is able to change and reprogram aggressive cancer cells to a less aggressive state. Some mechanisms involved in the phenotypic changes have been proved to associate with the Nodal signaling pathway, which plays a key role in tumor cell plasticity [20, 46]. However, as for mES cells, several other molecular mechanisms might be related directly and/or indirectly to these changes, including the Stat3 signaling pathway. Therefore in this article we investigated in detail of the effect of the ES cell microenvironment on breast tumor progression and metastasis. The results of our study demonstrate that exposure of breast cancer cells to the ES cell microenvironment downregulates Stat3 signaling, associated with a reduction in clonogenicity and tumorigenicity.
Stat3 has been identified with critical importance for maintaining cancer stemness . Stat3 is an oncogene expressed in many cancers including breast cancer, prostate cancer, lung cancer, head cancer, liver cancer, pancreatic cancer, and multiple myeloma [47–49]. Stat3 has also been found to be involved in cancer cell growth, survival, invasion, and migration through regulation of the expression of E-cadherin, VEGF, and MMPs [19, 50]. In addition to these kinds of roles, Jak–Stat3 signaling has recently been demonstrated to have central roles in premetastatic niche formation [51–54]. Evidence is also accumulating for the important roles of Stat3 in breast cancer stem cells . Stat3 ablation leads to decreased tumor cell proliferation and growth . In this study, we identify important functions of Stat3 and their implications in the antitumor effect of ES-CM. We show that the environment created by ES cells has a suppressive effect on 4T1 cells by downregulation of Stat3 in these tumor cells. The Stat3 signaling pathway stimulates cell proliferation and migration/invasion. Using a bioluminescent imaging system, we have shown that inhibiting Stat3 mediated by ES-CM in tumor-bearing mice dramatically decreased both the growth rates and volumes of the tumor. We provided evidence that downregulation of Stat3 was critical for the inhibition of cancer cells in vivo.
Activation of Stat3 is associated with metastasis in many tumors. This association may be attributed to the overexpression of several growth factors, MMP2 and VEGF, which are induced by Stat3 activation and subsequently promote tumor invasion and angiogenesis . Consistent with previous findings, the inhibition of Stat3 in our breast cancer mouse model resulted in a lower metastasis rate. Mmp2 and Vegf expression was downregulated in the ES-CM-treated group. The weight and volume of the tumor formed by 4T1 cells treated with ES-CM were also significantly decreased. Our in-vitro assays confirmed that ES-CM weakened the migration, metastasis, and angiogenesis of 4T1 cells, which may act by inhibiting Stat3. We thus propose that the microenvironment created by ES cells could inhibit the tumor growth possibly through downregulating the Stat3 signal pathway.
It has been commonly agreed that cancer cell behavior largely depends on the tumor microenvironment, which is very complex and consists of cells, growth factors, extracellular matrix, and extracellular vesicles (EVs) [38, 58, 59]. A growing number of studies suggest that ES-CM efficiently suppresses the invasive potential of cancer cells [2, 5]. The human ES cell microenvironment suppresses melanoma tumor cells by secretion of Lefty into the matrix . Our data suggested that some factors secreted by ES cells could efficiently suppress the Stat3 pathway in breast cancer, resulting in a loss of tumorigenicity. Besides these, some other mechanisms might be also involved in the antitumor effect of the ES cell microenvironment. Understanding the plastic phenotype expressed by the aggressive tumor cells in response to their environment is helpful to develop therapeutic strategies in patients with cancer [1, 2]. More detailed future studies are therefore required to illustrate the detailed mechanisms involved in the suppression effect of the ES cell microenvironment on cancer cells.