Photophysical Characterization and In Vitro Evaluation of α-Mangostin-Loaded HDL Mimetic Nano-Complex in LN-229 Glioblastoma Spheroid Model.

Cytotoxic activity has been reported for the xanthone α-mangostin (AMN) against Glioblastoma multiforme (GBM), an aggressive malignant brain cancer with a poor prognosis. Recognizing that AMN's high degree of hydrophobicity is likely to limit its systemic administration, we formulated AMN using reconstituted high-density lipoprotein (rHDL) nanoparticles. The photophysical characteristics of the formulation, including fluorescence lifetime and steady-state anisotropy, indicated that AMN was successfully incorporated into the rHDL nanoparticles. To our knowledge, this is the first report on the fluorescent characteristics of AMN with an HDL-based drug carrier. Cytotoxicity studies in a 2D culture and 3D spheroid model of LN-229 GBM cells and normal human astrocytes showed an enhanced therapeutic index with the rHDL-AMN formulation compared to the unincorporated AMN and Temozolomide, a standard GBM chemotherapy agent. Furthermore, treatment with the rHDL-AMN facilitated a dose-dependent upregulation of autophagy and reactive oxygen species generation to a greater extent in LN-229 cells compared to astrocytes, indicating the reduced off-target toxicity of this novel formulation. These studies indicate the potential therapeutic benefits to GBM patients via selective targeting using the rHDL-AMN formulation.

Self-assembling peptides induced by eyes absent enzyme to boost the efficacy of doxorubicin therapy in drug-resistant breast cancer cells.

Enzyme-induced self-assembly (EISA) is a recently developed nanotechnology technique in which small molecules are induced by cellular enzymes self-assembling into nanostructures inside cancer cells. This technique can boost the efficacy of chemotherapy drugs by avoiding drug efflux, inhibiting the cells' DNA repair mechanisms, and targeting the mitochondria. In this work, we study the self-assembly of a short peptide and its fluorescence analogue induced by Eyes absent (EYA) tyrosine phosphatases to boost the efficacy of doxorubicin (DOX) therapy in drug-resistant types of breast cancer cells, MDA-MB-231 and MCF-7. The peptides Fmoc-FF-YP and NBD-FF-YP were synthesized with the solid-phase peptide synthesis (SPPS) method and analyzed with HPLC and MALDI-TOF. Dynamic light scattering was used to determine the size distribution of peptides exposed to the EYA enzyme in vitro. The presence of EYA enzymes in breast cancer cells was confirmed using the western blotting assay. The intracellular location of the peptide self-assembly was studied by imaging fluorescence NBD-tagged peptides. The efficacy of the peptide alone and with DOX was determined against MCF-7 and MDA-MB-231 using MTT and LIVE-DEAD assays. Nucleus and cytoplasm F-actin (Phalloidin) staining was used to determine cell morphology changes in response to the combination therapy of peptides/DOX. At an optimal concentration, the peptides are not toxic to the cells; however, they boost the efficacy of DOX against drug-resistant breast cancer cells. We used state-of-the-art computer-aided techniques to predict the molecular structure of peptides and their interactions with EYA. This study demonstrates an approach for incorporating non-cytotoxic components into DOX combination therapy, thereby avoiding increased systemic burden or adverse effects.

Discovery and Characterization of a Chemical Probe for Cyclin-Dependent Kinase-Like 2.

Acylaminoindazole-based inhibitors of CDKL2 were identified via analyses of cell-free binding and selectivity data. Compound 9 was selected as a CDKL2 chemical probe based on its potent inhibition of CDKL2 enzymatic activity, engagement of CDKL2 in cells, and excellent kinome-wide selectivity, especially when used in cells. Compound 16 was designed as a negative control to be used alongside compound 9 in experiments to interrogate CDKL2-mediated biology. A solved co-crystal structure of compound 9 bound to CDKL2 highlighted key interactions it makes within its ATP-binding site. Inhibition of downstream phosphorylation of EB2, a CDKL2 substrate, in rat primary neurons provided evidence that engagement of CDKL2 by compound 9 in cells resulted in inhibition of its activity. When used at relevant concentrations, compound 9 does not impact the viability of rat primary neurons or certain breast cancer cells nor elicit consistent changes in the expression of proteins involved in epithelial-mesenchymal transition.

The Expression of Adipogenic Marker Is Significantly Increased in Estrogen-Treated Lipedema Adipocytes Differentiated from Adipose Stem Cells In Vitro.

Lipedema is a chronic, idiopathic, and painful disease characterized by an excess of adipose tissue in the extremities. The goal of this study is to characterize the gene expression of estrogen receptors (ERα and ERß), G protein-coupled estrogen receptor (GPER), and ER-metabolizing enzymes: hydroxysteroid 17-beta dehydrogenase (HSD17B1, 7, B12), cytochrome P450 (CYP19A1), hormone-sensitive lipase (LIPE), enzyme steroid sulfatase (STS), and estrogen sulfotransferase (SULT1E1), which are markers in Body Mass Index (BMI) and age-matched non-lipedema (healthy) and lipedema ASCs and spheroids. Flow cytometry and cellular proliferation assays, RT-PCR, and Western Blot techniques were used to determine the expression of ERs and estrogen-metabolizing enzymes. In 2D monolayer culture, estrogen increased the proliferation and the expression of the mesenchymal marker, CD73, in hormone-depleted (HD) healthy ASCs compared to lipedema ASCs. The expression of ERß was significantly increased in HD lipedema ASCs and spheroids compared to corresponding healthy cells. In contrast, ERα and GPER gene expression was significantly decreased in estrogen-treated lipedema spheroids. CYP19A1 and LIPE gene expressions were significantly increased in estrogen-treated healthy ASCs and spheroids, respectively, while estrogen upregulated the expression of PPAR-ϒ2 and ERα in estrogen-treated lipedema-differentiated adipocytes and spheroids. These results indicate that estrogen may play a role in adipose tissue dysregulation in lipedema.

Bioreactor Culture to Create Adipose Tissue from Human Mesenchymal Stromal Cells.

Adipose tissue is a complex and multifaceted endocrine organ located throughout the body. The dysfunction of adipose tissue is known to induce a wide variety of comorbidities that can negatively impact one's health and quality of life. In addition to behavioral changes, drugs that target dysfunctional adipose tissue to treat associated diseases are clinically needed. Regarding drug-testing platforms, animal models are the most popular models, limited by known differences from humans in genetics and physiology. Two-dimensional and static three-dimensional (3D) cell cultures are also used. Still, these in vitro models with static culture fail to recapitulate the phenotype and function of adipocytes seen in vivo. To combat this, our lab has developed an adipose tissue microphysiological system. A perfusion bioreactor with dual-flow chambers is 3D printed, which enables individualized top and bottom medium flows after adipose tissues are inserted as a barrier. Human progenitor cells, such as human mesenchymal stem cells, are embedded within a gelatin scaffold and in situ adipogenic differentiation within the bioreactor. Medium flow is established via a syringe pump system, allowing in vivo-like conditions to be maintained. The novel bioreactor-cultured adipose tissues represent a versatile disease modeling and drug-testing system. Here, we present the step-by-step methods to generate the bioreactors and adipose tissues. We also show the process of collecting and analyzing samples. In addition, we highlight the critical steps that require particular attention in notes.

Isolation of Human Adipose-Derived Stem Cells.

Human adipose-derived stromal/stem cells (hASCs) are a promising source of adult stem cells used in numerous applications in regenerative medicine. We present the protocols from our laboratory for isolating and expanding hASCs. The isolation of hASCs involves the enzymatic digestion of adipose tissue and subsequent culturing of the isolated cells.

A Method for Screening a Kinase Inhibitor Drug Library on ASCs.

Breast cancer is an ongoing issue due to its high mortality rates. Obesity enhances the problems associated with breast cancer, meaning there must be a biological connection between them. This crosstalk may be the adipose-derived stem cell. If we can interrupt the communication between adipose-derived stromal/stem cells (ASCs) and breast cancer, we may be able to prevent cancer propagation. Specific kinase inhibition may allow us to downregulate signals, preventing ASC-mediated cancer growth. This chapter provides a critical method for screening a kinase inhibitor drug library for hits on ASCs.

A Spontaneous Assembling Lipopeptide Nanoconjugate Transporting the Anthracycline Drug N-Benzyladriamycin-14-valerate for Personalized Therapy of Ewing Sarcoma.

To meet the current need for a tumor-selective, targeted therapy regimen associated with reduced toxicity, our laboratory has developed a spontaneously assembled nanostructure that resembles high-density lipoproteins (HDLs). These myristoyl-5A (MYR-5A) nanotransporters are designed to safely transport lipophilic pharmaceuticals, including a novel anthracycline drug (N-benzyladriamycin-14-valerate (AD198)). This formulation has been found to enhance the therapeutic efficacy and reduced toxicity of drugs in preclinical studies of 2D and 3D models of Ewing sarcoma (EWS) and cardiomyocytes. Our findings indicate that the MYR-5A/AD198 nanocomplex delivers its payload selectively to cancer cells via the scavenger receptor type B1 (SR-B1), thus providing a solid proof of concept for the development of an improved and highly effective, potentially personalized therapy for EWS while protecting against treatment-associated cardiotoxicity.

Obesity-associated epigenetic alterations and the obesity-breast cancer axis.

Both breast cancer and obesity can regulate epigenetic changes or be regulated by epigenetic changes. Due to the well-established link between obesity and an increased risk of developing breast cancer, understanding how obesity-mediated epigenetic changes affect breast cancer pathogenesis is critical. Researchers have described how obesity and breast cancer modulate the epigenome individually and synergistically. In this review, the epigenetic alterations that occur in obesity, including DNA methylation, histone, and chromatin modification, accelerated epigenetic age, carcinogenesis, metastasis, and tumor microenvironment modulation, are discussed. Delineating the relationship between obesity and epigenetic regulation is vital to furthering our understanding of breast cancer pathogenesis.

Enhanced Angiogenesis in HUVECs Preconditioned with Media from Adipocytes Differentiated from Lipedema Adipose Stem Cells In Vitro.

Lipedema is a connective tissue disorder characterized by increased dilated blood vessels (angiogenesis), inflammation, and fibrosis of the subcutaneous adipose tissue. This project aims to gain insights into the angiogenic processes in lipedema using human umbilical vein endothelial cells (HUVECs) as an in vitro model. HUVECs were cultured in conditioned media (CM) collected from healthy (non-lipedema, AQH) and lipedema adipocytes (AQL). The impacts on the expression levels of multiple endothelial and angiogenic markers [CD31, von Willebrand Factor (vWF), angiopoietin 2 (ANG2), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMPs), NOTCH and its ligands] in HUVECs were investigated. The data demonstrate an increased expression of CD31 and ANG2 at both the gene and protein levels in HUVECs treated with AQL CM in 2D monolayer and 3D cultures compared to untreated cells. Furthermore, the expression of the vWF, NOTCH 4, and DELTA-4 genes decreased. In contrast, increased VEGF, MMP9, and HGF gene expression was detected in HUVECs treated with AQL CM cultured in a 2D monolayer. In addition, the results of a tube formation assay indicate that the number of formed tubes increased in lipedema-treated HUVECs cultured in a 2D monolayer. Together, the data indicate that lipedema adipocyte-CM promotes angiogenesis through paracrine-driven mechanisms.

The effect of obesity on adipose-derived stromal cells and adipose tissue and their impact on cancer.

The significant increase in the incidence of obesity represents the next global health crisis. As a result, scientific research has focused on gaining deeper insights into obesity and adipose tissue biology. As a result of the excessive accumulation of adipose tissue, obesity results from hyperplasia and hypertrophy within the adipose tissue. The functional alterations in the adipose tissue are a confounding contributing factor to many diseases, including cancer. The increased incidence and aggressiveness of several cancers, including colorectal, postmenopausal breast, endometrial, prostate, esophageal, hematological, malignant melanoma, and renal carcinomas, result from obesity as a contributing factor. The increased morbidity and mortality of obesity-associated cancers are attributable to increased hormones, adipokines, and cytokines produced by the adipose tissue. The increased adipose tissue levels observed in obese patients result in more adipose stromal/stem cells (ASCs) distributed throughout the body. ASCs have been shown to impact cancer progression in vitro and in preclinical animal models. ASCs influence tumor biology via multiple mechanisms, including the increased recruitment of ASCs to the tumor site and increased production of cytokines and growth factors by ASCs and other cells within the tumor stroma. Emerging evidence indicates that obesity induces alterations in the biological properties of ASCs, subsequently leading to enhanced tumorigenesis and metastasis of cancer cells. As the focus of this review is the interaction and impact of ASCs on cancer, the presentation is limited to preclinical data generated on cancers in which there is a demonstrated role for ASCs, such as postmenopausal breast, colorectal, prostate, ovarian, multiple myeloma, osteosarcoma, cervical, bladder, and gastrointestinal cancers. Our group has investigated the interactions between obesity and breast cancer and the mechanisms that regulate ASCs and adipocytes in these different contexts through interactions between cancer cells, immune cells, and other cell types present in the tumor microenvironment (TME) are discussed. The reciprocal and circular feedback loop between obesity and ASCs and the mechanisms by which ASCs from obese patients alter the biology of cancer cells and enhance tumorigenesis will be discussed. At present, the evidence for ASCs directly influencing human tumor growth is somewhat limited, though recent clinical studies suggest there may be some link.

Aberrant Expression of COX-2 and FOXG1 in Infrapatellar Fat Pad-Derived ASCs from Pre-Diabetic Donors.

Osteoarthritis (OA) is a degenerative joint disease resulting in limited mobility and severe disability. Type II diabetes mellitus (T2D) is a weight-independent risk factor for OA, but a link between the two diseases has not been elucidated. Adipose stem cells (ASCs) isolated from the infrapatellar fat pad (IPFP) may be a viable regenerative cell for OA treatment. This study analyzed the expression profiles of inflammatory and adipokine-related genes in IPFP-ASCs of non-diabetic (Non-T2D), pre-diabetic (Pre-T2D), and T2D donors. Pre-T2D ASCs exhibited a substantial decrease in levels of mesenchymal markers CD90 and CD105 with no change in adipogenic differentiation compared to Non-T2D and T2D IPFP-ASCs. In addition, Cyclooxygenase-2 (COX-2), Forkhead box G1 (FOXG1) expression and prostaglandin E2 (PGE2) secretion were significantly increased in Pre-T2D IPFP-ASCs upon stimulation by interleukin-1 beta (IL-1ß). Interestingly, M1 macrophages exhibited a significant reduction in expression of pro-inflammatory markers TNFα and IL-6 when co-cultured with Pre-T2D IPFP-ASCs. These data suggest that the heightened systemic inflammation associated with untreated T2D may prime the IPFP-ASCs to exhibit enhanced anti-inflammatory characteristics via suppressing the IL-6/COX-2 signaling pathway. In addition, the elevated production of PGE2 by the Pre-T2D IPFP-ASCs may also suggest the contribution of pre-diabetic conditions to the onset and progression of OA.

Liver Kinase B1 Regulates Remodeling of the Tumor Microenvironment in Triple-Negative Breast Cancer.

Liver kinase B1 (LKB1) is a potent tumor suppressor that regulates cellular energy balance and metabolism as an upstream kinase of the AMP-activated protein kinase (AMPK) pathway. LKB1 regulates cancer cell invasion and metastasis in multiple cancer types, including breast cancer. In this study, we evaluated LKB1's role as a regulator of the tumor microenvironment (TME). This was achieved by seeding the MDA-MB-231-LKB1 overexpressing cell line onto adipose and tumor scaffolds, followed by the evaluation of tumor matrix-induced tumorigenesis and metastasis. Results demonstrated that the presence of tumor matrix enhanced tumorigenesis in both MDA-MB-231 and MDA-MB-231-LKB1 cell lines. Metastasis was increased in both MDA-MB-231 and -LKB1 cells seeded on the tumor scaffold. Endpoint analysis of tumor and adipose scaffolds revealed LKB1-mediated tumor microenvironment remodeling as evident through altered matrix protein production. The proteomic analysis determined that LKB1 overexpression preferentially decreased all major and minor fibril collagens (collagens I, III, V, and XI). In addition, proteins observed to be absent in tumor scaffolds in the LKB1 overexpressing cell line included those associated with the adipose matrix (COL6A2) and regulators of adipogenesis (IL17RB and IGFBP4), suggesting a role for LKB1 in tumor-mediated adipogenesis. Histological analysis of MDA-MB-231-LKB1-seeded tumors demonstrated decreased total fibril collagen and indicated decreased stromal cell presence. In accordance with this, in vitro condition medium studies demonstrated that the MDA-MB-231-LKB1 secretome inhibited adipogenesis of adipose-derived stem cells. Taken together, these data demonstrate a role for LKB1 in regulating the tumor microenvironment through fibril matrix remodeling and suppression of adipogenesis.

Short-Term Autophagy Preconditioning Upregulates the Expression of COX2 and PGE2 and Alters the Immune Phenotype of Human Adipose-Derived Stem Cells In Vitro.

Human adipose-derived stem cells (hASCs) are potent modulators of inflammation and promising candidates for the treatment of inflammatory and autoimmune diseases. Strategies to improve hASC survival and immunoregulation are active areas of investigation. Autophagy, a homeostatic and stress-induced degradative pathway, plays a crucial role in hASC paracrine signaling-a primary mechanism of therapeutic action. Therefore, induction of autophagy with rapamycin (Rapa), or inhibition with 3-methyladenine (3-MA), was examined as a preconditioning strategy to enhance therapeutic efficacy. Following preconditioning, both Rapa and 3-MA-treated hASCs demonstrated preservation of stemness, as well as upregulated transcription of cyclooxygenase-2 (COX2) and interleukin-6 (IL-6). Rapa-ASCs further upregulated TNFα-stimulated gene-6 (TSG-6) and interleukin-1 beta (IL-1ß), indicating additional enhancement of immunomodulatory potential. Preconditioned cells were then stimulated with the inflammatory cytokine interferon-gamma (IFNγ) and assessed for immunomodulatory factor production. Rapa-pretreated cells, but not 3-MA-pretreated cells, further amplified COX2 and IL-6 transcripts following IFNγ exposure, and both groups upregulated secretion of prostaglandin-E2 (PGE2), the enzymatic product of COX2. These findings suggest that a 4-h Rapa preconditioning strategy may bestow the greatest improvement to hASC expression of cytokines known to promote tissue repair and regeneration and may hold promise for augmenting the therapeutic potential of hASCs for inflammation-driven pathological conditions.

Breast Cancer-Stromal Interactions: Adipose-Derived Stromal/Stem Cell Age and Cancer Subtype Mediated Remodeling.

Adipose tissue is characterized as an endocrine organ that acts as a source of hormones and paracrine factors. In diseases such as cancer, endocrine and paracrine signals from adipose tissue contribute to cancer progression. Young individuals with estrogen receptor-alpha positive (ER-α+) breast cancer (BC) have an increased resistance to endocrine therapies, suggesting that alternative estrogen signaling is activated within these cells. Despite this, the effects of stromal age on the endocrine response in BC are not well defined. To identify differences between young and aged ER-α+ breast tumors, RNA sequencing data were obtained from The Cancer Genome Atlas. Analysis revealed enrichment of matrix and paracrine factors in young (≤40 years old) patients compared to aged (≥65 years old) tumor samples. Adipose-derived stromal/stem cells (ASCs) from noncancerous lipoaspirate of young and aged donors were evaluated for alterations in matrix production and paracrine secreted factors to determine if the tumor stroma could alter estrogen signaling. Young and aged ASCs demonstrated comparable proliferation, differentiation, and matrix production, but exhibited differences in the expression levels of inflammatory cytokines (Interferon gamma, interleukin [IL]-8, IL-10, Tumor necrosis factor alpha, IL-2, and IL-6). Conditioned media (CM)-based experiments showed that young ASC donor age elevated endocrine response in ER-α+ BC cell lines. MCF-7 ER-α+ BC cell line treated with secreted factors from young ASCs had enhanced ER-α regulated genes (PGR and SDF-1) compared to MCF-7 cells treated with aged ASC CM. Western blot analysis demonstrated increased activation levels of p-ER ser-167 in the MCF-7 cell line treated with young ASC secreted factors. To determine if ER-α+ BC cells heightened the cytokine release in ASCs, ASCs were stimulated with MCF-7-derived CM. Results demonstrated no change in growth factors or cytokines when treated with the ER-α+ secretome. In contrast to ER-α+ CM, the ER-α negative MDA-MB-231 derived CM demonstrated increased stimulation of pro-inflammatory cytokines in ASCs. While there was no observed change in the release of selected paracrine factors, MCF-7 cells did induce matrix production and a pro-adipogenic lineage commitment. The adipogenesis was evident by increased collagen content through Sirius Red/Fast Green Collagen stain, lipid accumulation evident by Oil Red O stain, and significantly increased expression in PPARγ mRNA expression. The data from this study provide evidence suggesting more of a subtype-dependent than an age-dependent difference in stromal response to BC, suggesting that this signaling is not heightened by reciprocal signals from ER-α+ BC cell lines. These results are important in understanding the mechanisms of estrogen signaling and the dynamic and reciprocal nature of cancer cell-stromal cell crosstalk that can lead to tumor heterogeneity and variance in response to therapy.

Human Mesenchymal Stem Cell-Derived Miniature Joint System for Disease Modeling and Drug Testing.

Diseases of the knee joint such as osteoarthritis (OA) affect all joint elements. An in vitro human cell-derived microphysiological system capable of simulating intraarticular tissue crosstalk is desirable for studying etiologies/pathogenesis of joint diseases and testing potential therapeutics. Herein, a human mesenchymal stem cell-derived miniature joint system (miniJoint) is generated, in which engineered osteochondral complex, synovial-like fibrous tissue, and adipose tissue are integrated into a microfluidics-enabled bioreactor. This novel design facilitates different tissues communicating while still maintaining their respective phenotypes. The miniJoint exhibits physiologically relevant changes when exposed to interleukin-1ß mediated inflammation, which are similar to observations in joint diseases in humans. The potential of the miniJoint in predicting in vivo efficacy of drug treatment is confirmed by testing the "therapeutic effect" of the nonsteroidal anti-inflammatory drug, naproxen, as well as four other potential disease-modifying OA drugs. The data demonstrate that the miniJoint recapitulates complex tissue interactions, thus providing a robust organ chip model for the study of joint pathology and the development of novel therapeutic interventions.

Adipose-Derived Stromal/Stem Cell Response to Tumors and Wounds: Evaluation of Patient Age.

Tumors were characterized as nonhealing wounds by Virchow in 1858 and Dvorak in 1986. Since then, researchers have analyzed tumors from a new perspective. The parallels between tumorigenesis and physiological wound healing can provide a new framework for developing antitumor therapeutics. One commonality between tumors and wounds is the involvement of the stromal environment, particularly adipose stromal/stem cells (ASCs). ASCs exhibit dual functions, in which they stimulate tumor progression and assist in tissue repair and regeneration. Numerous studies have focused on the role of ASCs in cancer and wound healing, but none to date has linked age, cancer, and wound healing. Furthermore, very few studies have focused on the role of donor-specific characteristics of ASCs, such as age and their role in facilitating ASC behavior in cancer and wound healing. This review article is designed to provide important insights into the impact of donor age on ASC tumor and wound response and their role in facilitating ASC behavior in cancer and wound healing.

Adipose Tissue-Derived Mesenchymal Stem Cells.

The long-held belief about adipose tissue was that it was relatively inert in terms of biological activity. It was believed that its primary role was energy storage; however, that was shattered with the discovery of adipokines. Scientists interested in regenerative medicine then reported that adipose tissue is rich in adult stromal/stem cells. Following these initial reports, adipose stem cells (ASCs) rapidly garnered interest for use as potential cellular therapies. The primary advantages of ASCs compared to other mesenchymal stem cells (MSCs) include the abundance of the tissue source for isolation, the ease of methodologies for tissue collection and cell isolation, and their therapeutic potential. Studies conducted both in vitro and in vivo have demonstrated that ASCs are multipotent, possessing the ability to differentiate into cells of mesodermal origins, including adipocytes, chondrocytes, osteoblast and others. Moreover, ASCs produce a broad array of cytokines, growth factors, nucleic acids (miRNAs), and other macromolecules into the surrounding milieu by secretion or in the context of microvesicles. The secretome of ASCs has been shown to alter tissue biology, stimulate tissue-resident stem cells, change immune cell activity, and mediate therapeutic outcomes. The quality of ASCs is subject to donor-to-donor variation driven by age, body mass index, disease status and possibly gender and ethnicity. This review discusses adipose stromal/stem cell action mechanisms and their potential utility as cellular therapeutics.

A Role for Adipocytes and Adipose Stem Cells in the Breast Tumor Microenvironment and Regenerative Medicine.

Obesity rates are climbing, representing a confounding and contributing factor to many disease states, including cancer. With respect to breast cancer, obesity plays a prominent role in the etiology of this disease, with certain subtypes such as triple-negative breast cancer having a strong correlation between obesity and poor outcomes. Therefore, it is critical to examine the obesity-related alterations to the normal stroma and the tumor microenvironment (TME). Adipocytes and adipose stem cells (ASCs) are major components of breast tissue stroma that have essential functions in both physiological and pathological states, including energy storage and metabolic homeostasis, physical support of breast epithelial cells, and directing inflammatory and wound healing responses through secreted factors. However, these processes can become dysregulated in both metabolic disorders, such as obesity and also in the context of breast cancer. Given the well-established obesity-neoplasia axis, it is critical to understand how interactions between different cell types in the tumor microenvironment, including adipocytes and ASCs, govern carcinogenesis, tumorigenesis, and ultimately metastasis. ASCs and adipocytes have multifactorial roles in cancer progression; however, due to the plastic nature of these cells, they also have a role in regenerative medicine, making them promising tools for tissue engineering. At the physiological level, the interactions between obesity and breast cancer have been examined; here, we will delineate the mechanisms that regulate ASCs and adipocytes in these different contexts through interactions between cancer cells, immune cells, and other cell types present in the tumor microenvironment. We will define the current state of understanding of how adipocytes and ASCs contribute to tumor progression through their role in the tumor microenvironment and how this is altered in the context of obesity. We will also introduce recent developments in utilizing adipocytes and ASCs in novel approaches to breast reconstruction and regenerative medicine.

Macrophages Modulate the Function of MSC- and iPSC-Derived Fibroblasts in the Presence of Polyethylene Particles.

Fibroblasts in the synovial membrane secrete molecules essential to forming the extracellular matrix (ECM) and supporting joint homeostasis. While evidence suggests that fibroblasts contribute to the response to joint injury, the outcomes appear to be patient-specific and dependent on interactions between resident immune cells, particularly macrophages (Mφs). On the other hand, the response of Mφs to injury depends on their functional phenotype. The goal of these studies was to further explore these issues in an in vitro 3D microtissue model that simulates a pathophysiological disease-specific microenvironment. Two sources of fibroblasts were used to assess patient-specific influences: mesenchymal stem cell (MSC)- and induced pluripotent stem cell (iPSC)-derived fibroblasts. These were co-cultured with either M1 or M2 Mφs, and the cultures were challenged with polyethylene particles coated with lipopolysaccharide (cPE) to model wear debris generated from total joint arthroplasties. Our results indicated that the fibroblast response to cPE was dependent on the source of the fibroblasts and the presence of M1 or M2 Mφs: the fibroblast response as measured by gene expression changes was amplified by the presence of M2 Mφs. These results demonstrate that the immune system modulates the function of fibroblasts; furthermore, different sources of differentiated fibroblasts may lead to divergent results. Overall, our research suggests that M2 Mφs may be a critical target for the clinical treatment of cPE induced fibrosis.