Adipose-Derived Stem Cells from Fat Tissue of Breast Cancer Microenvironment Present Altered Adipogenic Differentiation Capabilities.
ABSTRACT: Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate into multiple cell types, including adipocytes, osteoblasts, and chondrocytes. The role of adipose-derived stem cells (ADSCs) in cancers is significantly relevant. They seem to be involved in the promotion of tumour development and progression and relapse processes. For this reason, investigating the effects of breast cancer microenvironment on ADSCs is of high importance in order to understand the relationship between tumour cells and the surrounding stromal cells. With the current study, we aimed to investigate the specific characteristics of human ADSCs isolated from the adipose tissue of breast tumour patients. We compared ADSCs obtained from periumbilical fat (PF) of controls with ADSCs obtained from adipose tissue of breast cancer- (BC-) bearing patients. We analysed the surface antigens and the adipogenic differentiation ability of both ADSC populations. C/EBP? expression was increased in PF and BC ADSCs induced to differentiate compared to the control while PPAR? and FABP4 expressions were enhanced only in PF ADSCs. Conversely, adiponectin expression was reduced in PF-differentiated ADSCs while it was slightly increased in differentiated BC ADSCs. By means of Oil Red O staining, we further observed an impaired differentiation capability of BC ADSCs. To investigate this aspect more in depth, we evaluated the effect of selective PPAR? activation and nutritional supplementation on the differentiation efficiency of BC ADSCs, noting that it was only with a strong differentiation stimuli that the process took place. Furthermore, we observed no response in BC ADSCs to the PPAR? inhibitor T0070907, showing an impaired activation of this receptor in adipose cells surrounding the breast cancer microenvironment. In conclusion, our study shows an impaired adipogenic differentiation capability in BC ADSCs. This suggests that the tumour microenvironment plays a key role in the modulation of the adipose microenvironment located in the surrounding tissue.
Project description:Although DNA modification is adaptive to extrinsic demands, little is known about epigenetic alterations associated with adipose differentiation and reprogramming. We systematically characterized the global trends of our methylome and transcriptome data with reported PPAR(γ) cistrome data. Our analysis revealed that DNA methylation was altered between induced pluripotent stem cells (iPSCs) and adipose derived stem cells (ADSCs). Surprisingly, DNA methylation was not obviously changed in differentiation from ADSCs to mature fat cells (FatCs). This indicates that epigenetic predetermination of the adipogenic fate is almost established prior to substantial expression of the lineage. Furthermore, the majority of the PPAR(γ) cistrome corresponded to the pre-set methylation profile between ADSCs and FatCs. In contrast to the pre-set model, we found that a subset of PPAR(γ)-binding sites for late-expressing genes such as Adiponectin and Adiponectin receptor2 were differentially methylated independently of the early program. Thus, these analyses identify two types of epigenetic mechanisms that distinguish the pre-set cell fate and later stages of adipose differentiation.
Project description:Adipose-derived stem cells (ADSCs) have been implicated in tumor growth and metastasis in breast cancer. ADSCs exhibit tumor tropism, and are of increasing clinical relevance due to the autologous fat grafting for breast reconstruction. Although we have previously shown that a high level of the adipocytokine visfatin in human breast cancer tissues correlated with tumor progression mediated by cAbl and STAT3, the effects of visfatin in the tumor microenvironment are unclear. To understand how visfatin modulates breast cancer within the tumor-stromal environment, we examined determinants of breast cancer progression using a visfatin-primed ADSCs-tumor co-culture model. ADSCs were isolated from tumor-free adipose tissue adjacent to breast tumors. ADSCs were treated with or without visfatin for 48 h and then collected for co-culture with breast cancer cell line MDA-MB-231 for 72 h in a transwell system. We found that the MDA-MB-231 cells co-cultured with visfatin-treated ADSCs (vADSCs) had higher levels of cell viability, anchorage independent growth, migration, invasion, and tumorsphere formation than that co-cultured with untreated ADSCs (uADSCs). Growth differentiation factor 15 (GDF15) upregulation was found in the co-culture conditioned medium, with GDF15 neutralizing antibody blocking the promoting effect on MDA-MB-231 in co-culture. In addition, a GDF15-induced AKT pathway was found in MDA-MB-231 and treatment with PI3K/AKT inhibitor also reversed the promoting effect. In an orthotopic xenograft mouse model, MDA-MB-231 co-injected with vADSCs formed a larger tumor mass than with uADSCs. Positive correlations were noted between visfatin, GDF15, and phosphor-AKT expressions in human breast cancer specimens. In conclusion, visfatin activated GDF15-AKT pathway mediated via ADSCs to facilitate breast cancer progression.
Project description:BACKGROUND:Spinal metastasis is a major challenge in patients with advanced lung cancer, but the mechanisms in the organotropism of metastasis are still unclear. Adipose-derived mesenchymal stem cells (ADSCs) exhibit cancer-promoting properties that influence the tumour microenvironment; however, there is no research on ADSCs from epidural fat thus far. METHODS:In this study, we isolated and identified ADSCs from epidural adipose tissue for the first time. We examined the activation of epidural ADSCs treated with lung cancer cell-conditioned medium by immunohistochemistry, western blot and qRT-PCR assays. The expression of interleukin (IL)-6 family cytokines in the supernatants of ADSCs were evaluated by enzyme-linked immunosorbent assay. The effects of epidural ADSCs on the growth and invasion of lung cancer cells were evaluated with the CCK-8 and Transwell assays. The expression of signal transducer and activator of transcription 3 (STAT3), matrix metalloprotease and epithelial-mesenchymal transition markers were measured by western blot assays. RESULTS:Our results showed that ADSCs treated with lung cancer cell-conditioned medium expressed higher levels of the myofibroblast marker α-smooth muscle actin and fibroblast activation protein than ADSCs cultured alone. Then, we found that lung cancer cells induced ADSCs to secrete high levels of IL-6 family cytokines and activate the STAT3 signalling pathway. Moreover, activated epidural ADSCs exhibited the ability to promote lung cancer cell proliferation and invasion by elevating matrix metalloprotease expression and epithelial-mesenchymal transition in cancer cells. Furthermore, blocking IL-6 can counteract the differentiation and tumour-promoting effects of ADSCs. CONCLUSION:Our results suggest that ADSCs respond to lung cancer cells and are involved in the crosstalk between primary tumours and pre-metastatic niches in epidural fat.
Project description:MicroRNAs (miRNAs) influence stem cell functions, including mobilization, proliferation, and differentiation. miR-150 is abundantly expressed in monocytes. Knockdown of miR-150 promotes bone marrow stem cell migration. The role of miR-150 in adipose-derived stem cells (ADSCs) is unclear. In this study, the effects of miR-150 on adipogenic differentiation and proliferation of ADSCs were investigated. ADSCs were isolated from the inguinal adipose tissue of wild-type (WT) and miR-150 knockout (KO) mice and were induced for adipogenic differentiation. The miR-150 level was detected by real-time PCR. ADSCs were transfected by miR-150 or small-interfering RNA (siRNA) of Notch3. MTT assay and colony formation assay were performed in miR-150 knockdown and control ADSCs. Real-time PCR showed that miR-150 was expressed in ADSCs. miR-150 knockdown significantly decreased the capacity of adipogenic differentiation of ADSCs, as compared with their counterparts from WT mice. It is intriguing that the overexpression of miR-150 significantly increased C/EBP? and PPAR-? expression and lipid formation in ADSCs with adipogenic induction. Overexpression of miR-150 significantly decreased Notch3 expression in ADSCs compared with the control groups. Furthermore, Notch3 inhibition promoted the adipogenic differentiation in ADSCs. miR-150 also suppressed proliferation potential and the expression of Nanog in ADSCs. In summary, this study demonstrates, for the first time, that miR-150 promotes adipogenic differentiation and inhibits proliferation of ADSCs. miR-150 regulates adipogenic differentiation of ADSCs, likely mediated by the downregulation of Notch3.
Project description:Klotho was originally discovered as an aging-suppressor gene. The purpose of this study was to investigate whether secreted Klotho (SKL) affects the proliferation and differentiation of adipose-derived stem cells (ADSCs). RT-PCR and Western blot analysis showed that short-form Klotho was expressed in mouse ADSCs. The Klotho gene mutation KL(-/-) significantly decreased proliferation of ADSCs and expression of pluripotent transcription factors (Nanog, Sox-2, and Oct-4) in mice. The adipogenic differentiation of ADSCs was also decreased in KL(-/-) mice. Incubation with Klotho-deficient medium decreased ADSC proliferation, pluripotent transcription factor levels, and adipogenic differentiation, which is similar to what was found in KL(-/-) mice. These results indicate that Klotho deficiency suppresses ADSC proliferation and differentiation. Interestingly, treatment with recombinant SKL protein rescued the Klotho deficiency-induced impairment in ADSC proliferation and adipogenic differentiation. SKL also regulated ADSCs' differentiation to other cell lineages (osteoblasts, myofibroblasts), indicating that SKL maintains stemness of ADSCs. It is intriguing that overexpression of SKL significantly increased PPAR-? expression and lipid formation in ADSCs following adipogenic induction, indicating enhanced adipogenic differentiation. Overexpression of SKL inhibited expression of TGF?1 and its downstream signaling mediator Smad2/3. This study demonstrates, for the first time, that SKL is essential to the maintenance of normal proliferation and differentiation in ADSCs. Klotho regulates adipogenic differentiation in ADSCs, likely via inhibition of TGF?1 and activation of PPAR-?. Stem Cells 2016;34:1615-1625.
Project description:Partial breast irradiation of early breast cancer patients after lumpectomy and the use of endogenous adipose tissue (AT) for breast reconstruction are promising applications to reduce the side effects of breast cancer therapy. This study tries to investigate the possible risks associated with these therapeutic approaches. It also examines the influence of adipose derived stem cells (ADSCs) as part of the breast cancer microenvironment, and endogenous AT on breast cancer cells following radiation therapy. ADSCs, isolated from human reduction mammoplasties of healthy female donors, exhibited multilineage capacity and specific surface markers. The promoting effects of ADSCs on the growth and survival fraction of breast cancer cells were reversed by treatment with high (8 Gy) or medium (2 Gy) radiation doses. In addition, a suppressing influence on breast cancer growth could be detected by co-culturing with irradiated ADSCs (8 Gy). Furthermore the clonogenic survival of unirradiated tumor cells was reduced by medium of irradiated ADSCs. In conclusion, radiation therapy changed the interactions of ADSCs and breast cancer cells. On the basis of our work, the importance of further studies to exclude potential risks of ADSCs in regenerative applications and radiotherapy has been emphasized.
Project description:BACKGROUND:Emerging evidence supports the pivotal roles of adipocytes in breast cancer progression. Tumour induced beige/brown adipose tissue differentiation contributes to the hypermetabolic state of the breast cancer. However, the mediators and mechanisms remain unclear. METHODS:Survival probabilities were estimated using the Kaplan-Meier method based on immunohistochemistry results. Biochemical studies were performed to characterize the novel interrelation between breast cancer cells and adipocytes. RESULTS:We show that tumour-surrounding adipocytes exhibit an altered phenotype in terms of upregulated beige/brown characteristics and increased catabolism associated with an activated state characterized by the release of metabolites, including free fatty acids, pyruvate, lactate and ketone bodies. Likewise, tumour cells cocultivated with mature adipocytes exhibit metabolic adaptation and an aggressive phenotype in vitro and in vivo. Mechanistically, we show that tumour cells induce beige/brown differentiation and remodel metabolism in resident adipocytes by exosomes from the co-culture system that carry high levels of miRNA-144 and miRNA-126. miRNA-144 promotes beige/brown adipocyte characteristics by downregulating the MAP3K8/ERK1/2/PPAR? axis, and exosomal miRNA-126 remodels metabolism by disrupting IRS/Glut-4 signalling, activating the AMPK/autophagy pathway and stabilizing HIF1? expression in imminent adipocytes. In vivo inhibition of miRNA-144 or miRNA-126 decreases adipocyte-induced tumour growth. CONCLUSIONS:These results demonstrate that by inducing beige/brown differentiation and enhancing catabolism in recipient adipocytes, exosomal miRNA-144 and miRNA-126 from the tumour-adipocyte interaction reprogram systemic energy metabolism to facilitate tumour progression.
Project description:Breast cancer (BC) is an important disease with high incidence as well as mortality among women, and critical socio-economic impacts. In the past 50 years, it has become a major health problem for women worldwide with over 2 million new cases diagnosed in 2018. This represents about 12% of all new cancer cases, 25% of all cancers in women and more than 600.000 cases of deaths worldwide in 2018 (Bray et al, 2018). If the cancer is located only in the breast, the 5-year relative survival rate of people with BC is 99% but this rate decreases if it is spread to lymph nodes (85%) and more dramatically if diagnosed with distant metastasis (26%) (Howlader et al, 2019; Siegel et al, 2017). Metastatic process, or the spread of tumor cells throughout the body, is responsible for about 90% of cancer patient deaths (Chaffer et al, 2011) and represents the central clinical challenge of solid tumor oncology. The development and progression of BC are complex processes that involve hormonal factors as well as numerous genetic and epigenetic alterations. During the past 10 years, many studies have focused on the role of the tumor microenvironment and the peritumoral stromal fraction, composed of adipose tissue, cancer-associated fibroblasts, endothelial cells and immune cells as macrophages and leukocytes. During tumor progression, cancer cells will deeply modify their microenvironment which in return will promote the growth and dissemination of the tumor (Allen et al, 2011; Polanska et al, 2013). Adipose tissue, consisting of mainly mature adipocytes and progenitors (preadipocytes and adipose-derived stem cells (ADSCs), is the most abundant component surrounding BC cells. Adipose tissue exerts a major endocrine and secretory role, and represents then an essential actor in the inflammatory, angiogenic or remodeling responses of the extracellular matrix, which influences tumor behavior.
Project description:Fat transplantation is increasingly used in breast augmentation; and recently, the issue of safety concerns from a cellular and molecular point of view has been raised. In this study, attentions were paid to the interaction between adipose-derived stem cells (ADSC) and mammary epithelial cells: human breast cancer cell line - 100 (HBL - 100) cells were used to simulate the normal microenvironment in breast tissue, ADSCs were harvest from human and co-cultured with HBL-100 cells. It was found that ADSCs formed tube-like structures in the co-culture with HBL-100 cells in contrast to the normal morphology of ADSCs in the control group. In addition, the immunofluorescence imaging showed that cytokeratin 18 and 19 (CK18 and 19) were significantly expressed in ADSCs after the co-culture with HBL-100 cells. The ultrastructure of those ADSCs also showed epithelial changes. In conclusion, ADSCs are not biological stable when co-cultured with HBL-100 cells. They differentiate into epithelial-like cells with the expression of epithelial surface marks (CK 18, 19) and form tube-like structures. This may offer an important evidence for the further study of clinical application of transplanting ADSCs rich adipose tissue into the breast in the future.
Project description:The stemness maintenance of adipose-derived stem cells (ADSCs) is important for adipose homeostasis and energy balance. Programmed cell death 4 (Pdcd4) has been demonstrated to be involved in the development of obesity, but its possible roles in ADSC function and adipogenic capacity remain unclear. In this study, we demonstrate that Pdcd4 is a key controller that limits the self-renewal and white-to-beige transdifferentiation of ADSCs. Pdcd4 deficiency in mice caused stemness enhancement of ADSCs as evidenced by increased expression of CD105, CD90, Nanog and Oct4 on ADSCs, together with enhanced in situ proliferation in adipose tissues. Pdcd4 deficiency promoted proliferation, colony formation of ADSCs and drove more ADSCs entering the S phase accompanied by AKT activation and cyclinD1 upregulation. Blockade of AKT signaling in Pdcd4-deficient ADSCs led to a marked decline in cyclinD1, S-phase entry and cell proliferation, revealing AKT as a target for repressing ADSC self-renewal by Pdcd4. Intriguingly, depletion of Pdcd4 promoted the transdifferentiation of ADSCs into beige adipocytes. A reduction in lipid contents and expression levels of white adipocyte markers including C/EBPα, PPAR-γ, adiponectin and αP2 was detected in Pdcd4-deficient ADSCs during white adipogenic differentiation, substituted by typical beige adipocyte characteristics including small, multilocular lipid droplets and UCP1 expression. More lactate produced by Pdcd4-deficient ADSCs might be an important contributor to the expression of UCP1 and white-to-beige transdifferentiation. In addition, an elevation of UCP1 expression was confirmed in white adipose tissues from Pdcd4-deficient mice upon high-fat diet, which displayed increased energy expenditure and resistance to obesity as compared with wild-type obese mice. These findings provide evidences that Pdcd4 produces unfavorable influences on ADSC stemness, which contribute to adipose dysfunction, obesity and metabolic syndromes, thereby proposing Pdcd4 as a potential intervening target for regulating ADSC function.