Cancer-associated fibroblasts (CAFs) secrete various pro-tumorigenic cytokines, yet the role of these cytokines in the progression of endometrial cancer remains unclear. We found that CAFs isolated from human endometrial cancer (EC) tissues secreted high levels of interleukin-6 (IL-6), which promotes EC cell proliferation in vitro. Neutralizing IL-6 in CAF-conditioned media reduced (47% inhibition) while IL-6 recombinant protein increased cell proliferation (~2.4 fold) of both EC cell lines and primary cultures. IL-6 receptors (IL-6R and gp130) were expressed only in EC epithelial cells but not in CAF, indicating a one-way paracrine signaling. In the presence of CAF-conditioned media, Janus kinase/signal transducers and activators of transcription (JAK/STAT3) pathway was activated in EC cells. Treatment with JAK and STAT3 specific inhibitors, AD412 and STATTIC, respectively, significantly abrogated CAF-mediated cell proliferation, indicating the role of IL-6 activation in EC cell proliferation. We further showed that one of STAT-3 target genes, c-Myc, was highly induced in EC cells after exposure to CAF-conditioned medium at both mRNA (>105-fold vs. control) and protein level (>2-fold vs. control). EC cell proliferation was dependent on c-Myc expression, as RNAi-mediated c-Myc down-regulation led to a significant 46% reduction in cell viability when compared with scrambled control. Interestingly, CAF-conditioned media failed to promote proliferation in EC cells with reduced c-Myc expression, suggesting that CAF-mediated cell proliferation was also dependent on c-Myc expression. Subcutaneous tumor xenograft model showed that EC cells grew at least 1.4 times larger when co-injected with CAF, when compared to those injected with EC cells alone. Mice injected with EC cells with down-regulated c-Myc expression, however, showed at least 2.5 times smaller tumor compared to those in control group. Notably, there was no increase of tumor size when co-injected with CAFs. Further immunohistochemical staining on human tissues showed positive expression of IL-6 receptors, phosphorylated-STAT3 and c-Myc in human EC tissues with less signals in benign endometrium. Taken together, our data suggests that IL-6 secreted by CAF induces c-Myc expression to promote EC proliferation in vitro and in vivo. IL-6 pathway can be a potential target to disrupt tumor-stroma interaction in endometrial cancer progression.
Cell motility is a critical step in the metastasis cascade. However, the role of cancer-associated fibroblasts (CAFs) in facilitating endometrial cancer (EC) cell motility remains unclear. The present study aimed to investigate the role of CAFs in EC motility in a 3D environment. A co-culture model was established using an EC cell line (ECC-1) and CAFs on a Matrigel® matrix and compared to the respective individual monocultures. It was demonstrated that endometrial CAFs increased the motility of the EC cell line, compared with the monoculture. Using live cell imaging, CAFs were observed to form cell projections that served as contact guidance for ECC-1 cell locomotion in the spheroid formation process. These effects were specific to CAFs, as fibroblasts isolated from benign endometrial tissue samples did not form cell projections. Molecular analysis revealed that RhoA/Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) signaling activation partly contributed to CAF-mediated ECC-1 cell migration. The presence of Matrigel® increased the mRNA expression of RhoA, and the mRNA and protein expression levels of its downstream effectors, ROCK1 and p-MLC, respectively, in the ECC-1 and CAF co-culture, as well as the ECC-1 and CAF monocultures. Interestingly, high phosphorylation levels of myosin light chain mediated the activation of RhoA/ROCK1 signaling in the ECC-1 and CAF co-culture. The ROCK1 inhibitor Y-27632 attenuated the motility of tumor cells in ECC-1 and CAF co-cultures. However, similar treatment led to a significant inhibition in the motility of the CAF monoculture, but not the ECC-1 monoculture. Moreover, tumor spheroid formation was inhibited due to a reduction in stress fiber formation in ECC-1 and CAF co-cultures. Altogether, these findings suggest that the regulation of the RhoA/ROCK1 signaling pathway is required for CAFs to serve as cellular vehicles in order for EC cells to migrate and form spheroids in a 3D environment.
Obesity is strongly linked with increased risk and poorer prognosis of endometrial cancer (EC). Cancer-associated fibroblasts (CAFs) are activated fibroblasts that form a large component of the tumor microenvironment and undergo metabolic reprogramming to provide critical metabolites for tumor growth. However, it is still unknown how obesity, characterized by a surplus of free fatty acids drives the modifications of CAFs lipid metabolism which may provide the mechanistic link between obesity and EC progression. The present study aims to evaluate the utility of Raman spectroscopy, an emerging nondestructive analytical tool to detect signature changes in lipid metabolites of CAFs from EC patients with varying body mass index. We established primary cultures of fibroblasts from human EC tissues, and CAFs of overweight/obese and nonobese women using antibody-conjugated magnetic beads isolation. These homogeneous fibroblast cultures expressed fibroblast markers, including α-smooth muscle actin and vimentin. Analysis was made in the Raman spectra region best associated with cancer progression biochemical changes in lipids (600-1800 cm-1 and 2800-3200 cm-1). Direct band analysis and ratiometric analysis were conducted to extract information from the Raman spectrum. Present results demonstrated minor shifts in the CH2 symmetric stretch of lipids at 2879 cm-1 and CH3 asymmetric stretching from protein at 2932 cm-1 in the overweight/obese CAFS compared to nonobese CAFs, indicating increased lipid content and a higher degree of lipid saturation. Principal component analysis showed that CAFs from overweight/obese and nonobese EC patients can be clearly distinguished indicating the capability of Raman spectroscopy to detect changes in biochemical components. Our results suggest Raman spectroscopy supported by chemometric analysis is a reliable technique for characterizing metabolic changes in clinical samples, providing an insight into obesity-driven alteration in CAFs, a critical stromal component during EC tumorigenesis.
Since the first nationwide movement control order was implemented on 18 March 2020 in Malaysia to contain the coronavirus disease 2019 (COVID-19) outbreak, to what extent the uncertainty and continuous containment measures have imposed psychological burdens on the population is unknown. This study aimed to measure the level of mental health of the Malaysian public approximately 2 months after the pandemic's onset. Between 12 May and 5 September 2020, an anonymous online survey was conducted. The target group included all members of the Malaysian population aged 18 years and above. The Depression Anxiety Stress Scale (DASS-21) was used to assess mental health. There were increased depressive, anxiety and stress symptoms throughout the study period, with the depression rates showing the greatest increase. During the end of the data collection period (4 August-5 September 2020), there were high percentages of reported depressive (59.2%) and anxiety (55.1%) symptoms compared with stress (30.6%) symptoms. Perceived health status was the strongest significant predictor for depressive and anxiety symptoms. Individuals with a poorer health perception had higher odds of developing depression (odds ratio [OR] = 5.68; 95% confidence interval [CI] 3.81-8.47) and anxiety (OR = 3.50; 95%CI 2.37-5.17) compared with those with a higher health perception. By demographics, young people-particularly students, females and people with poor financial conditions-were more vulnerable to mental health symptoms. These findings provide an urgent call for increased attention to detect and provide intervention strategies to combat the increasing rate of mental health problems in the ongoing COVID-19 pandemic.