Comparative proteomics analysis of biofilms and planktonic cells of Enterococcus faecalis and Staphylococcus lugdunensis with contrasting biofilm-forming ability.

Biofilms make it difficult to eradicate bacterial infections through antibiotic treatments and lead to numerous complications. Previously, two periprosthetic infection-related pathogens, Enterococcus faecalis and Staphylococcus lugdunensis were reported to have relatively contrasting biofilm-forming abilities. In this study, we examined the proteomics of the two microorganisms' biofilms using LC-MS/MS. The results showed that each microbe exhibited an overall different profile for differential gene expressions between biofilm and planktonic cells as well as between each other. Of a total of 929 proteins identified in the biofilms of E. faecalis, 870 proteins were shared in biofilm and planktonic cells, and 59 proteins were found only in the biofilm. In S. lugdunensis, a total of 1125 proteins were identified, of which 1072 proteins were found in common in the biofilm and planktonic cells, and 53 proteins were present only in the biofilms. The functional analysis for the proteins identified only in the biofilms using UniProt keywords demonstrated that they were mostly assigned to membrane, transmembrane, and transmembrane helix in both microorganisms, while hydrolase and transferase were found only in E. faecalis. Protein-protein interaction analysis using STRING-db indicated that the resulting networks did not have significantly more interactions than expected. GO term analysis exhibited that the highest number of proteins were assigned to cellular process, catalytic activity, and cellular anatomical entity. KEGG pathway analysis revealed that microbial metabolism in diverse environments was notable for both microorganisms. Taken together, proteomics data discovered in this study present a unique set of biofilm-embedded proteins of each microorganism, providing useful information for diagnostic purposes and the establishment of appropriately tailored treatment strategies. Furthermore, this study has significance in discovering the target candidate molecules to control the biofilm-associated infections of E. faecalis and S. lugdunensis.

Assessment of the biofilm-forming ability on solid surfaces of periprosthetic infection-associated pathogens.

Biofilm formation is one of the leading causes of complications after surgery in clinical settings. In this study, we profiled the biofilm-forming ability of various periprosthetic infection-associated pathogens on medically relevant surfaces, polystyrene (PS) and titanium (Ti). We also explored how a specific environmental stressor, epigallocatechin gallate (EGCG), affected biofilm formation. First, Congo red tests revealed that all microorganisms formed biofilms within 72 h. Then, the amounts of biofilm formation on PS at 24, 48 and 72 h and also on a Ti plate for 72 h were determined. Some microbes preferred one surface over the other, whereas other microbes formed consistent levels of biofilm regardless of the surface material. Staphylococcus lugdunenensis was the most potent, while Enterococcus faecalis and Staphylococcus aureus were the weakest. Bacterial adhesion to hydrocarbon (BATH) tests indicated that the biofilm-forming abilities were not directly correlated with cell surface hydrophobicity (CSH). Finally, an external signal, EGCG, was applied to challenge the biofilm formation of each microorganism. EGCG regulated each microorganism's ability differently, though the change was consistent across surfaces for most pathogens. This study can help a better understanding of a broad spectrum of periprosthetic infection-associated pathogens by relative comparison of their biofilm-forming abilities.

GPCR19 Regulates P2X7R-Mediated NLRP3 Inflammasomal Activation of Microglia by Amyloid ß in a Mouse Model of Alzheimer's Disease.

Amyloid ß (Aß) and/or ATP activate the NLRP3 inflammasome (N3I) via P2X7R in microglia, which is crucial in neuroinflammation in Alzheimer's disease (AD). Due to polymorphisms, subtypes, and ubiquitous expression of P2X7R, inhibition of P2X7R has not been effective for AD. We first report that taurodeoxycholate (TDCA), a GPCR19 ligand, inhibited the priming phase of N3I activation, suppressed P2X7R expression and P2X7R-mediated Ca++ mobilization and N3I oligomerization, which is essential for production of IL-1ß/IL-18 by microglia. Furthermore, TDCA enhanced phagocytosis of Aß and decreased the number of Aß plaques in the brains of 5x Familial Alzheimer's disease (5xFAD) mice. TDCA also reduced microgliosis, prevented neuronal loss, and improved memory function in 5xFAD mice. The pleiotropic roles of GPCR19 in P2X7R-mediated N3I activation suggest that targeting GPCR19 might resolve neuroinflammation in AD patients.

Skin Wound Healing Rate in Fish Depends on Species and Microbiota.

The skin is a barrier between the body and the environment that protects the integrity of the body and houses a vast microbiota. By interacting with the host immune system, the microbiota improves wound healing in mammals. However, in fish, the evidence of the role of microbiota and the type of species on wound healing is scarce. We aimed to examine the wound healing rate in various fish species and evaluate the effect of antibiotics on the wound healing process. The wound healing rate was much faster in two of the seven fish species selected based on habitat and skin types. We also demonstrated that the composition of the microbiome plays a role in the wound healing rate. After antibiotic treatment, the wound healing rate improved in one species. Through 16S rRNA sequencing, we identified microbiome correlates of varying responses on wound healing after antibiotic treatment. These findings indicate that not only the species difference but also the microbiota play a significant role in wound healing in fish.

Effect and Biocompatibility of a Cross-Linked Hyaluronic Acid and Polylactide-co-glycolide Microcapsule Vehicle in Intratympanic Drug Delivery for Treating Acute Acoustic Trauma.

The treatment of acute hearing loss is clinically challenging due to the low efficacy of drug delivery into the inner ear. Local intratympanic administration of dexamethasone (D) and insulin-like growth factor 1 (IGF1) has been proposed for treatment, but they do not persist in the middle ear because they are typically delivered in fluid form. We developed a dual-vehicle drug delivery system consisting of cross-linked hyaluronic acid and polylactide-co-glycolide microcapsules. The effect and biocompatibility of the dual vehicle in delivering D and IGF1 were evaluated using an animal model of acute acoustic trauma. The dual vehicle persisted 10.9 times longer (8.7 days) in the middle ear compared with the control (standard-of-care vehicle, 0.8 days). The dual vehicle was able to sustain drug release over up to 1 to 2 months when indocyanine green was loaded as the drug. One-third of the animals experienced an inflammatory adverse reaction. However, it was transient with no sequelae, which was validated by micro CT findings, endoscopic examination, and histological assessment. Hearing restoration after acoustic trauma was satisfactory in both groups, which was further supported by comparable numbers of viable hair cells. Overall, the use of a dual vehicle for intratympanic D and IGF1 delivery may maximize the effect of drug delivery to the target organ because the residence time of the vehicle is prolonged.

The characterization of exosomes from fibrosarcoma cell and the useful usage of Dynamic Light Scattering (DLS) for their evaluation.

Exosomes are a type of extracellular vesicles containing mRNA, miRNA, and proteins of origin cells, which can control the characteristics of other cells or surroundings. Despite increasing evidence on oncogenic properties of tumor-derived exosomes, fibrosarcoma-derived exosomes remain largely unrevealed. While the proper extraction and characterization of exosomes is critical in exosomes research, there are various limitations in techniques to measure the size and homogeneity of exosomes. Here, we analyzed exosomes from a fibrosarcoma cell line WEHI-164 compared with a breast cancer cell line MDA-MD-231 as a control. Results from dot blot and western blot analysis demonstrated that GM1 ganglioside, and TSG101, HSC70 and GAPDH proteins were contained in exosomes from the WEHI-164 fibrosarcoma cell line. The existence of tetraspanins such as CD81, CD63 and CD9 was confirmed in the exosomes by ExoView analysis. The results obtained from TEM showed their sphere-like shapes of around 50 to 70 nm in radius. Through DLS, we found out that the mean radius of the exosomes derived from WEHI-164 and MDA-MB-231 cell lines was 94.4 nm and 107.8 nm, respectively, with high homogeneity. When comparing the radius measured by TEM with the radius measured by DLS, it was revealed that the difference between the two methods was about 40 nm. This study has significance in characterizing the molecular properties of exosomes from a fibrosarcoma, which has not been researched much before, and in providing clear evidence that DLS can be used as an efficient, convenient and noninvasive technique to simply check the homogeneity and size of exosomes.

Publisher Correction: MAOA variants differ in oscillatory EEG & ECG activities in response to aggression-inducing stimuli.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

MAOA variants differ in oscillatory EEG & ECG activities in response to aggression-inducing stimuli.

Among the genetic variations in the monoamine oxidase A (MAOA) gene, upstream variable number tandem repeats (uVNTRs) of the promoter have been associated with individual differences in human physiology and aggressive behaviour. However, the evidence for a molecular or neural link between MAOA uVNTRs and aggression remains ambiguous. Additionally, the use of inconsistent promoter constructs in previous studies has added to the confusion. Therefore, it is necessary to demonstrate the genetic function of MAOA uVNTR and its effects on multiple aspects of aggression. Here, we identified three MAOA alleles in Koreans: the predominant 3.5R and 4.5R alleles, as well as the rare 2.5R allele. There was a minor difference in transcriptional efficiency between the 3.5R and 4.5R alleles, with the greatest value for the 2.5R allele, in contrast to existing research. Psychological indices of aggression did not differ among MAOA genotypes. However, our electroencephalogram and electrocardiogram results obtained under aggression-related stimulation revealed oscillatory changes as novel phenotypes that vary with the MAOA genotype. In particular, we observed prominent changes in frontal γ power and heart rate in 4.5R carriers of men. Our findings provide genetic insights into MAOA function and offer a neurobiological basis for various socio-emotional mechanisms in healthy individuals.

A novel supportive assessment for comprehensive aggression using EEG and ECG.

Aggression is a complex, ubiquitous phenomenon that impacts behavioral traits and psychological health. Assessing aggression is challenging because aggression constitutes multiple subtraits, such as anger, reactive aggression, and overt aggression. Conventional methods of assessing aggression are susceptible to bias because they mainly rely upon self-reports. Thus, more objective methods that provide a multifaceted understanding of aggression in individuals are required. Here, we propose a supportive method of assessing specific aggression subtraits in Koreans using electroencephalography (EEG) and electrocardiography (ECG). Our evaluations and statistical analyses revealed that EEG and ECG signals in subjects responding to video cues that induced aggression are associated with aggression subtraits. In particular, we identified spectral differences in EEG signals in response to stimuli with situation-dependent aggression. The α and ß signals of the Fp2 site (the right ventromedial prefrontal region) are highly associated with anger, reactive aggression, and overt aggression. Moreover, ECG signals are associated with anger and overt aggression. These results link neurobiological findings to psychological explanations of aggression and multiple aspects of human behavior. Our findings can potentially be applied to supportive assessment methods for psychological counseling or psychiatric diagnoses.

Taurodeoxycholate Increases the Number of Myeloid-Derived Suppressor Cells That Ameliorate Sepsis in Mice.

Bile acids (BAs) control metabolism and inflammation by interacting with several receptors. Here, we report that intravenous infusion of taurodeoxycholate (TDCA) decreases serum pro-inflammatory cytokines, normalizes hypotension, protects against renal injury, and prolongs mouse survival during sepsis. TDCA increases the number of granulocytic myeloid-derived suppressor cells (MDSCLT) distinctive from MDSCs obtained without TDCA treatment (MDSCL) in the spleen of septic mice. FACS-sorted MDSCLT cells suppress T-cell proliferation and confer protection against sepsis when adoptively transferred better than MDSCL. Proteogenomic analysis indicated that TDCA controls chromatin silencing, alternative splicing, and translation of the immune proteome of MDSCLT, which increases the expression of anti-inflammatory molecules such as oncostatin, lactoferrin and CD244. TDCA also decreases the expression of pro-inflammatory molecules such as neutrophil elastase. These findings suggest that TDCA globally edits the proteome to increase the number of MDSCLT cells and affect their immune-regulatory functions to resolve systemic inflammation during sepsis.

Cardiolipin activates antigen-presenting cells via TLR2-PI3K-PKN1-AKT/p38-NF-kB signaling to prime antigen-specific naïve T cells in mice.

Mitochondrial defects and antimitochondrial cardiolipin (CL) antibodies are frequently detected in autoimmune disease patients. CL from dysregulated mitochondria activates various pattern recognition receptors, such as NLRP3. However, the mechanism by which mitochondrial CL activates APCs as a damage-associated molecular pattern to prime antigen-specific naïve T cells, which is crucial for T-cell-dependent anticardiolipin IgG antibody production in autoimmune diseases is unelucidated. Here, we show that CL increases the expression of costimulatory molecules in CD11c+ APCs both in vitro and in vivo. CL activates CD11c+ APCs via TLR2-PI3K-PKN1-AKT/p38MAPK-NF-κB signaling. CD11c+ APCs that have been activated by CL are sufficient to prime H-Y peptide-specific naïve CD4+ T cells and OVA-specific naïve CD8+ T cells. TLR2 is necessary for anti-CL IgG antibody responses in vivo. Intraperitoneal injection of CL does not activate CD11c+ APCs in CD14 KO mice to the same extent as in wild-type mice. CL binds to CD14 (Kd = 7 × 10-7 M). CD14, but not MD2, plays a role in NF-kB activation by CL, suggesting that CD14+ macrophages contribute to recognizing CL. In summary, CL activates signaling pathways in CD11c+ APCs through a mechanism similar to gram (+) bacteria and plays a crucial role in priming antigen-specific naïve T cells.

Gender Differences in Aggression-related Responses on EEG and ECG.

Gender differences in aggression viewed from an evolutionary and sociocultural perspective have traditionally explained why men engage in more direct and physical aggression, and women engage in more indirect and relational aggression. However, psychological and behavioral studies offer inconsistent support for this theory due to personal or social factors, and little is known about the gender-based neurobiological mechanisms of aggression. This study investigates gender differences in aggression through an analysis of electroencephalography (EEG) and electrocardiography (ECG) based neurobiological responses to commonly encountered stimuli, as well as psychological approaches in healthy Korean youth. Our results from self-reports indicate that overall aggression indices, including physical and reactive/overt aggression, were stronger in men. This agrees with the results of previous studies. Furthermore, our study reveals prominent gender-related patterns in γ signals from the right ventrolateral frontal cortex and changes in heart rate through stimulation by aggressive videos. In particular, gender differences in EEG and ECG responses were observed in response to different scenes, as simple aversion and situation-dependent aggression, respectively. In addition, we discovered decisive gender-distinct EEG signals during stimulation of the situation-dependent aggression regions within the right ventromedial prefrontal and ventrolateral frontal regions. Our findings provide evidence of a psychological propensity for aggression and neurobiological mechanisms of oscillation underlying gender differences in aggression. Further studies of oscillatory responses to aggression and provocation will expand the objective understanding of the different emotional worlds between men and women.

miR-34a inhibits differentiation of human adipose tissue-derived stem cells by regulating cell cycle and senescence induction.

MicroRNAs (miRNAs) are critical in the maintenance, differentiation, and lineage commitment of stem cells. Stem cells have the unique property to differentiate into tissue-specific cell types (lineage commitment) during cell division (self-renewal). In this study, we investigated whether miR-34a, a cell cycle-regulating microRNA, could control the stem cell properties of adipose tissue-derived stem cells (ADSCs). First, we found that the expression level of miR-34a was increased as the cell passage number was increased. This finding, however, was inversely correlated with our finding that the overexpression of miR-34a induced the decrease of cell proliferation. In addition, miR-34a overexpression decreased the expression of various cell cycle regulators such as CDKs (-2, -4, -6) and cyclins (-E, -D), but not p21 and p53. The cell cycle analysis showed accumulation of dividing cells at S phase by miR-34a, which was reversible by co-treatment with anti-miR-34a. The potential of adipogenesis and osteogenesis of ADSCs was also decreased by miR-34a overexpression, which was recovered by co-treatment with anti-miR-34a. The surface expression of stem cell markers including CD44 was also down-regulated by miR-34a overexpression as similar to that elicited by cell cycle inhibitors. miR-34a also caused a significant decrease in mRNA expression of stem cell transcription factors as well as STAT-3 expression and phosphorylation. Cytokine profiling revealed that miR-34a significantly modulated IL-6 and -8 production, which was strongly related to cellular senescence. These data suggest the importance of miR-34a for the fate of ADSCs toward senescence rather than differentiation.

Exosomes from breast cancer cells can convert adipose tissue-derived mesenchymal stem cells into myofibroblast-like cells.

Exosomes are small membrane vesicles secreted into the extracellular environment by various types of cells, including tumor cells. Exosomes are enriched with a discrete set of cellular proteins, and therefore expected to exert diverse biological functions according to cell origin. Mesenchymal stem cells (MSCs) possess the potential for differentiation into multilineages and can also function as precursors for tumor stroma including myofibroblast that provides a favorable environment for tumor progression. Although a close relationship between tumor cells and MSCs in a neoplastic tumor microenvironment has already been revealed, how this communication works is poorly understood. In this study, we investigated the influence of tumor cell-derived exosomes on MSCs by treating adipose tissue-derived MSCs (ADSCs) with breast cancer-derived exosomes. The exosome-treated ADSCs exhibited the phenotypes of tumor-associated myofibroblasts with increased expression of α-SMA. Exosome treatment also induced increased expression of tumor-promoting factors SDF-1, VEGF, CCL5 and TGFß. This phenomenon was correlated with increased expression of TGFß receptor I and II. Analysis of SMAD2, a key player in the TGFß receptor-mediated SMAD pathway, revealed that its phosphorylation was increased by exosome treatment and was inhibited by treatment with SB431542, an inhibitor of the SMAD-mediated pathway, resulting in decreased expression of α-SMA. Taken together, our results show that tumor-derived exosomes induced the myofibroblastic phenotype and functionality in ADSCs via the SMAD-mediated signaling pathway. In conclusion, this study suggests that tumor-derived exosomes can contribute to progression and malignancy of tumor cells by converting MSCs within tumor stroma into tumor-associated myofibroblasts in the tumor microenvironment.

Exosomes from ovarian cancer cells induce adipose tissue-derived mesenchymal stem cells to acquire the physical and functional characteristics of tumor-supporting myofibroblasts.

OBJECTIVE: Most tumor tissue is composed of parenchymal tumor cells and tumor stroma. Mesenchymal stem cells (MSCs) can function as precursors for tumor stromal cells, including myofibroblasts, which provide a favorable environment for tumor progression. A close relationship between tumor cells and MSCs in a tumor microenvironment has been described. Exosomes are small membrane vesicles that are enriched with a discrete set of cellular proteins, and are therefore expected to exert diverse biological functions according to cell origin. METHODS: In the current study, we determined the biological effect of exosomes from two ovarian cancer cell lines (SK-OV-3 and OVCAR-3) on adipose tissue-derived MSCs (ADSCs). RESULTS: Exosome treatment induced ADSCs to exhibit the typical characteristics of tumor-associated myofibroblasts, with increased expression of α-SMA, and also increased expression of tumor-promoting factors (SDF-1 and TGF-ß). This phenomenon was correlated with an increased expression of TGF-ß receptors I and II. Analysis of TGF-ß receptor-mediated downstream signaling pathways revealed that each exosome activated different signaling pathways, showing that exosomes from SK-OV-3 cells increased the phosphorylated form of SMAD2, which is essential in the SMAD-dependent pathway, whereas exosomes from OVCAR-3 cells increased the phosphorylated form of AKT, a representative SMAD-independent pathway. Taken together, exosomes from ovarian cancer cells induced the myofibroblastic phenotype and functionality in ADSCs by activating an intracellular signaling pathway, although the activated pathway could differ from exosome-to-exosome. CONCLUSION: The current study suggested that ovarian cancer-derived exosomes contribute to the generation of tumor-associated myofibroblasts from MSCs in tumor stroma.

Cell cycle regulators are critical for maintaining the differentiation potential and immaturity in adipogenesis of adipose-derived stem cells.

A single cell division is governed by the catalytic reactions of cyclins and cyclin-dependent kinases (CDKs). Stem cells are unique in that they can differentiate into tissue-specific cell types (lineage commitment) during cell division (self-renewal). In this study, we analyzed changes in the differentiation potency of adipose tissue-derived stem cells (ADSCs) according to the number of spontaneous cell divisions. We used low passage number (p3) to late passage number (p60) adipose-derived stem cells (ADSCs) as our model system. A preliminary investigation of the typical stem cell phenotypes revealed that CD44 expression decreased remarkably as the passage number of the ADSCs increased. Further examinations revealed that the higher the cell passage number, the lower the cell proliferation capability, differentiation potency, and expression of stem cell transcriptional factors and cell cycle regulators such as cyclins E, A, B, CDK2, and CDK1/CDC2. To verify if the observed changes in differentiation potency according to the number of cell divisions were related to cell cycle regulators, p3 ADSCs were treated with the selective CDK2 and CDK1/CDC2 inhibitor Purvalanol A. Inhibitor treatment of p3 ADSCs induced changes in the morphology, differentiation potency, and pattern of stem cell transcriptional factor expression so that these low passage ADSCs more closely resembled high-passage ADSCs. Collectively, our results indicate that cell cycle regulators control the differentiation potency of ADSCs, and provide insights into the cell biology and differentiation potency of ADSCs according to the number of cell divisions.

MicroRNA expression profiling in neurogenesis of adipose tissue-derived stem cells.

Adipose tissue-derived stem cells (ADSCs) are one population of adult stem cells that can self renew and differentiate into multiple lineages. Because of advantages in method and quantity of acquisition, ADSCs are gaining attention as an alternative source of bone marrow mesenchymal stem cells. In this study, we performed microRNA profiling of undifferentiated and of neurally-differentiated ADSCs to identify the responsible microRNAs in neurogenesis using this type of stem cell. MicroRNAs from four different donors were analysed by microarray. Compared to the undifferentiation control, we identified 39-101 microRNAs with more than two-fold higher expression and 3-9 microRNAs with two-fold lower expression. The identified microRNAs were further analysed in terms of gene ontology (GO) in relation with neurogenesis, based on their target mRNAs predicted by computational analysis. This study revealed the specific microRNAs involved in neurogenesis via microRNA microarray, and may provide the basic information for genetic induction of adult stem cell differentiation using microRNAs.

Introduction of the CIITA gene into tumor cells produces exosomes with enhanced anti-tumor effects.

Exosomes are small membrane vesicles secreted from various types of cells. Tumor-derived exosomes contain MHC class I molecules and tumor-specific antigens, receiving attention as a potential cancer vaccine. For induction of efficient anti-tumor immunity, CD4+ helper T cells are required, which recognize appropriate MHC class II-peptide complexes. In this study, we have established an MHC class II molecule-expressing B16F1 murine melanoma cell line (B16F1- CIITA) by transduction of the CIITA (Class II transactivator) gene. Exosomes from B16-CII cells (CIITA- Exo) contained a high amount of MHC class II as well as a tumor antigen TRP2. When loaded on dendritic cells (DCs), CIITA-Exo induced the increased expression of MHC class II molecules and CD86 than the exosomes from the parental cells (Exo). In vitro assays using co-culture of immunized splenocytes and exosome-loaded DCs demonstrated that CIITA-Exo enhanced the splenocyte proliferation and IL-2 secretion. Consistently, compared to B16-Exo, CIITA-Exo induced the increased mRNA levels of inflammatory cytokines such as TNF-α, chemokine receptor CCR7 and the production of Th1-polarizing cytokine IL-12. A tumor preventive model showed that CIITA-Exo significantly inhibited tumor growth in a dose-dependent manner. Ex vivo assays using immunized mice demonstrated that CIITA-Exo induced a higher amount of Th1-polarized immune responses such as Th1-type IgG2a antibodies and IFN-γ cytokine as well as TRP2-specific CD8+ T cells. A tumor therapeutic model delayed effects of tumor growth by CIITA-Exo. These findings indicate that CIITA-Exo are more efficient as compared to parental Exo to induce anti-tumor immune responses, suggesting a potential role of MHC class II-containing tumor exosomes as an efficient cancer vaccine.

Hyperthermia-treated mesenchymal stem cells exert antitumor effects on human carcinoma cell line.

BACKGROUND: Mesenchymal stem cells (MSCs) possess the potential for differentiation into multilineages. MSCs have been reported to play a role as precursors for tumor stroma in providing a favorable environment for tumor progression. Hyperthermia destroys cancer cells by raising the temperature of tumor-loaded tissue to 40 degrees C to 43 degrees C and causes indirect sensitizing effects when combined with chemo- and/or radiotherapy. However, how hyperthermia affects the tumor-supportive stroma is unknown. Here, the authors investigated the effects of hyperthermia-treated MSCs, from different sources, on the human ovarian cancer cell line SK-OV-3. METHODS: MSCs from adipose tissue and amniotic fluid were untreated or heat-treated (HS-MSCs). The culture supernatant of each treatment group was collected and transferred to the SK-OV-3 cells. RESULTS: The morphological analysis and cell proliferation assay showed a reduced viability of the tumor cells in the conditioned medium with the HS-MSCs. Further investigations revealed that the conditioned medium of the HS-MSCs induced a higher nuclear condensation and a greater number of sub-G1 cells among the tumor cells. Analysis of the mRNA expression demonstrated that the conditioned medium of the HS-MSCs induced up-regulation or down-regulation of several tumor-associated molecules. Finally, the cytokine array of each conditioned medium showed that angiogenin, insulin-like growth factor binding protein 4, neurotrophin 3, and chemokine (C-C motif) ligand 18 are involved as main factors. CONCLUSIONS: This study showed that the conditioned medium of the HS-MSCs exerted a suppressive effect on tumor progression and malignancy, suggesting that hyperthermia enables tumor stromal cells to provide a sensitizing environment for tumor cells to undergo cell death.