Staphylococcus aureus planktonic but not biofilm environment induces an IFN-ß macrophage immune response via the STING/IRF3 pathway.

Chronic implant-related bone infections are a severe complication in orthopaedic surgery. Biofilm formation on the implant impairs the immune response, leading to bacterial persistence. In a previous study, we found that Staphylococcus aureus (SA) induced interferon regulatory factor 3 (IRF3) activation and Ifnb expression only in its planktonic form but not in the biofilm. The aim of this study was to clarify the role of the stimulator of interferon genes (STING) in this process. We treated RAW 264.7 macrophages with conditioned media (CM) generated from planktonic or biofilm cultured SA in combination with agonists or inhibitors of the cyclic GMP-AMP synthase (cGAS)/STING pathway. We further evaluated bacterial gene expression of planktonic and biofilm SA to identify potential mediators. STING inhibition resulted in the loss of IRF3 activation and Ifnb induction in SA planktonic CM, whereas STING activation induced an IRF3 dependent IFN-ß response in SA biofilm CM. The expression levels of virulence-associated genes decreased during biofilm formation, but genes associated with cyclic dinucleotide (CDN) synthesis did not correlate with Ifnb induction. We further observed that cGAS contributed to Ifnb induction by SA planktonic CM, although cGAS activation was not sufficient to induce Ifnb expression in SA biofilm CM. Our data indicate that the different degrees of virulence associated with SA planktonic and biofilm environments result in an altered induction of the IRF3 mediated IFN-ß response via the STING pathway. This finding suggests that the STING/IRF3/IFN-ß axis is a potential candidate as an immunotherapeutic target for implant-related bone infections.

Staphylococci planktonic and biofilm environments differentially affect osteoclast formation.

INTRODUCTION: The pathophysiology of chronic implant-related bone infections is characterized by an increase in osteoclast numbers and enhanced bone resorption. Biofilms are a major reason for chronicity of such infections as the biofilm matrix protects bacteria against antibiotics and impairs the function of immune cells. Macrophages are osteoclast precursor cells and therefore linked to inflammation and bone destruction. OBJECTIVE AND METHOD: Investigations on the impact of biofilms on the ability of macrophages to form osteoclasts are yet missing and we, therefore, analyzed the effect of Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) planktonic and biofilm environments on osteoclastogenesis using RAW 264.7 cells and conditioned media (CM). RESULTS: Priming with the osteoclastogenic cytokine RANKL before CM addition enabled the cells to differentiate into osteoclasts. This effect was highest in SE planktonic or SA biofilm CM. Simultaneous stimulation with CM and RANKL, however, suppressed osteoclast formation and resulted in formation of inflammation-associated multinucleated giant cells (MGCs) which was most pronounced in SE planktonic CM. CONCLUSION: Our data indicate that the biofilm environment and its high lactate levels are not actively promoting osteoclastogenesis. Hence, the inflammatory immune response against planktonic bacterial factors through Toll-like receptors seems to be the central cause for the pathological osteoclast formation. Therefore, immune stimulation or approaches that aim at biofilm disruption need to consider that this might result in enhanced inflammation-mediated bone destruction.

Bacterial and Metabolic Factors of Staphylococcal Planktonic and Biofilm Environments Differentially Regulate Macrophage Immune Activation.

Biofilm formation is a leading cause for chronic implant-related bone infections as biofilms shield bacteria against the immune system and antibiotics. Additionally, biofilms generate a metabolic microenvironment that shifts the immune response towards tolerance. Here, we compared the impact of the metabolite profile of bacterial environments on macrophage immune activation using Staphylococcus aureus (SA) and epidermidis (SE) conditioned media (CM) of planktonic and biofilm cultures. The biofilm environment had reduced glucose and increased lactate concentrations. Moreover, the expression of typical immune activation markers on macrophages was reduced in the biofilm environment compared to the respective planktonic CM. However, all CM caused a predominantly pro-inflammatory macrophage cytokine response with a comparable induction of Tnfa expression. In biofilm CM, this was accompanied by higher levels of anti-inflammatory Il10. Planktonic CM, on the other hand, induced an IRF7 mediated Ifnb gene expression which was absent in the biofilm environments. For SA but not for SE planktonic CM, this was accompanied by IRF3 activation. Stimulation of macrophages with TLR-2/-9 ligands under varying metabolic conditions revealed that, like in the biofilm setting, low glucose concentration reduced the Tnfa to Il10 mRNA ratio. However, the addition of extracellular L-lactate but not D-lactate increased the Tnfa to Il10 mRNA ratio upon TLR-2/-9 stimulation. In summary, our data indicate that the mechanisms behind the activation of macrophages differ between planktonic and biofilm environments. These differences are independent of the metabolite profiles, suggesting that the production of different bacterial factors is ultimately more important than the concentrations of glucose and lactate in the environment.

Chronic Implant-Related Bone Infections-Can Immune Modulation be a Therapeutic Strategy?

Chronic implant-related bone infections are a major problem in orthopedic and trauma-related surgery with severe consequences for the affected patients. As antibiotic resistance increases in general and because most antibiotics have poor effectiveness against biofilm-embedded bacteria in particular, there is a need for alternative and innovative treatment approaches. Recently, the immune system has moved into focus as the key player in infection defense and bone homeostasis, and the targeted modulation of the host response is becoming an emerging field of interest. The aim of this review was to summarize the current knowledge of impaired endogenous defense mechanisms that are unable to prevent chronicity of bone infections associated with a prosthetic or osteosynthetic device. The presence of foreign material adversely affects the immune system by generating a local immune-compromised environment where spontaneous clearance of planktonic bacteria does not take place. Furthermore, the surface structure of the implant facilitates the transition of bacteria from the planktonic to the biofilm stage. Biofilm formation on the implant surface is closely linked to the development of a chronic infection, and a misled adaption of the immune system makes it impossible to effectively eliminate biofilm infections. The interaction between the immune system and bone cells, especially osteoclasts, is extensively studied in the field of osteoimmunology and this crosstalk further aggravates the course of bone infection by shifting bone homeostasis in favor of bone resorption. T cells play a major role in various chronic diseases and in this review a special focus was therefore set on what is known about an ineffective T cell response. Myeloid-derived suppressor cells (MDSCs), anti-inflammatory macrophages, regulatory T cells (Tregs) as well as osteoclasts all suppress immune defense mechanisms and negatively regulate T cell-mediated immunity. Thus, these cells are considered to be potential targets for immune therapy. The success of immune checkpoint inhibition in cancer treatment encourages the transfer of such immunological approaches into treatment strategies of other chronic diseases. Here, we discuss whether immune modulation can be a therapeutic tool for the treatment of chronic implant-related bone infections.

Stimulation of calvarial bone healing with human bone marrow stromal cells versus inhibition with adipose-tissue stromal cells on nanostructured ß-TCP-collagen.

Bioactive functional scaffolds are essential for support of cell-based strategies to improve bone regeneration. Adipose-tissue-derived-stromal cells (ASC) are more accessible multipotent cells with faster proliferation than bone-marrow-derived-stromal-cells (BMSC) having potential to replace BMSC for therapeutic stimulation of bone-defect healing. Their osteogenic potential is, however, lower compared to BMSC, a deficit that may be overcome in growth factor-rich orthotopic bone defects with enhanced bone-conductive scaffolds. Objective of this study was to compare the therapeutic potency of human ASC and BMSC for bone regeneration on a novel nanoparticulate ß-TCP/collagen-carrier (ß-TNC). Cytotoxicity of ß-TCP nanoparticles and multilineage differentiation of cells were characterized in vitro. Cell-seeded ß-TNC versus cell-free controls were implanted into 4 mm calvarial bone-defects in immunodeficient mice and bone healing was quantified by µCT at 4 and 8 weeks. Tissue-quality and cell-origin were assessed by histology. ß-TNC was non-toxic, radiolucent and biocompatible, lent excellent support for human cell persistence and allowed formation of human bone tissue by BMSC but not ASC. Opposite to BMSC, ASC-grafting significantly inhibited calvarial bone healing compared to controls. Bone formation progressed significantly from 4 to 8 weeks only in BMSC and controls yielding 5.6-fold more mineralized tissue in BMSC versus ASC-treated defects. Conclusively, ß-TNC was simple to generate, biocompatible, osteoconductive, and stimulated osteogenicity of BMSC to enhance calvarial defect healing while ASC had negative effects. Thus, an orthotopic environment and ß-TNC could not compensate for cell-autonomous deficits of ASC which should systematically be considered when choosing the right cell source for tissue engineering-based stimulation of bone regeneration. STATEMENT OF SIGNIFICANCE: Bone-marrow-derived-stromal cells (BMSC) implanted on bone replacement materials can support bone defect healing and adipose-tissue-derived-stromal cells (ASC) being more accessible and better proliferating are considered as alternate source. This first standardized comparison of the bone regeneration potency of human ASC and BMSC was performed on a novel nanoparticular ß-TCP-enriched collagen-carrier (ß-TNC) designed to overcome the known inferior osteogenicity of ASC. ß-TNC was non-toxic, biocompatible and osteoconductive supporting human bone formation and defect-closure by BMSC but not ASC. Long-term cell-persistence and the distinct secretome of ASC appear as main reasons why ASC inhibited bone healing opposite to BMSC. Overall, ASC-grafting is at considerable risk of producing negative effects on bone-healing while no such risks are known for BMSC.

Stimulation of a calcified cartilage connecting zone by GDF-5-augmented fibrin hydrogel in a novel layered ectopic in vivo model.

Tissue engineering approaches for reconstructing full-depth cartilage defects need to comprise a zone of calcified cartilage to tightly anchor cartilage constructs into the subchondral bone. Here, we investigated whether growth and differentiation factor-5-(GDF-5)-augmented fibrin hydrogel can induce a calcified cartilage-layer in vitro that seamlessly connects cartilage-relevant biomaterials with bone tissue. Human bone marrow stromal cells (BMSCs) were embedded in fibrin hydrogel and subjected to chondrogenesis with TGF-ß with or without GDF-5 before constructs were implanted subcutaneously into SCID mice. A novel layered ectopic in vivo model was developed and GDF-5-augmented fibrin with BMSCs was used to glue hydrogel and collagen constructs onto bone disks to investigate formation of a calcified cartilage connecting zone. GDF-5 significantly enhanced ALP activity during in vitro chondrogenesis while ACAN and COL2A1 mRNA, proteoglycan-, collagen-type-II- and collagen-type-X-deposition remained similar to controls. Pellets pretreated with GDF-5 mineralized faster in vivo and formed more ectopic bone. In the novel layered ectopic model, GDF-5 strongly supported calcified cartilage formation that seamlessly connected with the bone. Pro-chondrogenic and pro-hypertrophic activity makes GDF-5-augmented fibrin an attractive bioactive hydrogel with high potential to stimulate a calcified cartilage connecting zone in situ that might promote integration of cartilage scaffolds with bone. Thus, GDF-5-augmented fibrin hydrogel promises to overcome poor fixation of biomaterials in cartilage defects facilitating their long-term regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2214-2224, 2018.

Fibrinogen scaffolds with immunomodulatory properties promote in vivo bone regeneration.

The hypothesis behind this work is that fibrinogen (Fg), classically considered a pro-inflammatory protein, can promote bone repair/regeneration. Injury and biomaterial implantation naturally lead to an inflammatory response, which should be under control, but not necessarily minimized. Herein, porous scaffolds entirely constituted of Fg (Fg-3D) were implanted in a femoral rat bone defect and investigated at two important time points, addressing the bone regenerative process and the local and systemic immune responses, both crucial to elucidate the mechanisms of tissue remodelling. Fg-3D led to early infiltration of granulation tissue (6 days post-implantation), followed by bone defect closure, including periosteum repair (8 weeks post-injury). In the acute inflammatory phase (6 days) local gene expression analysis revealed significant increases of pro-inflammatory cytokines IL-6 and IL-8, when compared with non-operated animals. This correlated with modified proportions of systemic immune cell populations, namely increased T cells and decreased B, NK and NKT lymphocytes and myeloid cell, including the Mac-1+ (CD18+/CD11b+) subpopulation. At 8 weeks, Fg-3D led to decreased plasma levels of IL-1ß and increased TGF-ß1. Thus, our data supports the hypothesis, establishing a link between bone repair induced by Fg-3D and the immune response. In this sense, Fg-3D scaffolds may be considered immunomodulatory biomaterials.

Elevated ratio of MMP2/MMP9 activity is associated with poor response to chemotherapy in osteosarcoma.

BACKGROUND: Matrix metalloproteinases (MMPs) are crucially involved in the regulation of multiple stages of cancer progression. Elevated MMP levels have been associated with the development of metastases and poor prognosis in several types of cancer. However, the role of MMPs in osteosarcoma and their prognostic value is still unclear. Available data are conflicting, most likely due to different technical approaches. We hypothesized that in contrast to total mRNA or protein levels frequently analyzed in previous studies the enzymatic activities of MMPs and their inhibitors the tissue inhibitors of matrix metalloproteinases (TIMPs) are closer related to their biological functions. We therefore aimed to evaluate the reliability of different zymography techniques for the quantification of MMP and TIMP activities in osteosarcoma biopsies in order to investigate their distribution, possible regulation and prognostic value. METHODS: All analyses were done using cryo-conserved osteosarcoma pretreatment biopsies (n = 18). Gene and protein expression of MMPs and TIMPs were analyzed by RT-qPCR and western blot analysis, respectively. Overall MMP activity was analyzed by in situ zymography, individual MMP activities were analyzed by gelatin zymography. Reverse zymography was used to detect and quantify TIMP activities. RESULTS: Strong overall MMP activities could be detected in osteosarcoma pretreatment biopsies with MMP2 and MMP9 as predominant active MMPs. In contrast to total RNA or protein expression MMP2 and MMP9 activities showed significant quantitative differences between good and poor responders. While MMP9 activity was high in the good responder group and significantly decreased in the poor responder group, MMP2 activity showed a reverse distribution. Likewise, significant differences were detected concerning the activity of TIMPs resulting in a negative correlation of TIMP1 activity with MMP2 activity (p = 0.044) and negative correlations of TIMP2 and TIMP3 with MMP9 activity (p = 0.007 and p = 0.006). CONCLUSION: In contrast to mRNA or protein levels MMP and TIMP activities showed significant differences between the analyzed good and poor responder groups. A shift from MMP9 to predominant MMP2 activity is associated with poor response to chemotherapy suggesting that the ratio of MMP2/MMP9 activity might be a valuable and easily accessible marker to predict the response to chemotherapy in osteosarcoma.

Mesenchymal stromal cell implantation for stimulation of long bone healing aggravates Staphylococcus aureus induced osteomyelitis.

Large bone defects requiring long-term osteosynthetic stabilization or repeated surgeries show a considerable rate of infection. Mesenchymal stromal cells (MSCs) have been successfully used to enhance bone regeneration, but their powerful immunomodulatory effects may impose an enhanced risk for osteomyelitis development. In order to unravel whether implantation of MSCs aggravates a simultaneous bone infection, a hydrogel-supported osteomyelitis ostectomy model was developed in which rats received a femoral bone defect with rigid plate-fixation. After fibrin-assisted transfer of Staphylococcus aureus (SA), effects of MSC implantation on osteomyelitis development were quantified over 3-4 weeks. All SA-infected animals developed an acute local osteomyelitis with significantly increased blood neutrophil count, abscess formation and bone destruction. MSC-treatment of infected defects aggravated osteomyelitis according to a significantly elevated osteomyelitis score and enhanced distal bone loss with spongy alteration of cortical bone architecture. Increased attraction of macrophages, osteoclasts and regulation of pro- and anti-inflammatory mediators were potential MSC actions. Overall trophic actions of MSCs implanted into non-sterile bone defects may enhance an infection and/or exacerbate osteomyelitis. Studies on antibiotic carrier augmentation or antibiotic treatment are warranted to decide whether MSC implantation is a safe and promising therapy for orthopedic implant-stabilized bone defects at high risk for development of infection.

Bone quality assessment for total hip arthroplasty with intraoperative trabecular torque measurements.

BACKGROUND: In cases of poor bone quality, intraoperative torque measurement might be an alternative to preoperative dual-energy X-ray absorptiometry (DXA) to assess bone quality in total hip arthroplasty (THA). METHODS: Trabecular peak torque measurement was applied in 14 paired fresh frozen human femurs. Here, a 6.5 × 23 mm wingblade was inserted into the proximal femur without harming the lateral cortical bone. Further tests of the proximal femur also evaluated bone strength (DXA, micro-computed tomography (µCT), monoaxial compression test), and the results were compared to the trabecular torque measurement. Student's t-test was used to compare the values of the groups. Pearson product-moment was applied to correlate the values of the peak torque measurement with the bone strength measured by DXA, µCT, and monoaxial compression test. RESULTS: In the femoral head, the mean trabecular peak torque was 4.38 ± 1.86 Nm. These values showed a strong correlation with the values of the DXA, the µCT, and the biomechanical load test (Pearson's product-moment: DXA: 0.86, µCT-BMD: 0.80, load test: 0.85). Furthermore, the torque measurement showed a more pronounced correlation with the biomechanical load test compared to the DXA. CONCLUSIONS: The use of this method provides highly diagnostic information about bone quality. Since the approach was adjusted for THA, no harm of the lateral bone stock will result from this measurement during surgery. The results of this initial study employing small sample sizes indicate that this new method is as sensitive as DXA in predicting bone quality and may function as an intraoperative alternative to DXA in THA. Nevertheless, before this method will turn into clinical use, more research and clinical trials are necessary.

Melanocortin 1 receptor-signaling deficiency results in an articular cartilage phenotype and accelerates pathogenesis of surgically induced murine osteoarthritis.

Proopiomelanocortin-derived peptides exert pleiotropic effects via binding to melanocortin receptors (MCR). MCR-subtypes have been detected in cartilage and bone and mediate an increasing number of effects in diathrodial joints. This study aims to determine the role of MC1-receptors (MC1) in joint physiology and pathogenesis of osteoarthritis (OA) using MC1-signaling deficient mice (Mc1re/e). OA was surgically induced in Mc1re/e and wild-type (WT) mice by transection of the medial meniscotibial ligament. Histomorphometry of Safranin O stained articular cartilage was performed with non-operated controls (11 weeks and 6 months) and 4/8 weeks past surgery. µCT-analysis for assessing epiphyseal bone architecture was performed as a longitudinal study at 4/8 weeks after OA-induction. Collagen II, ICAM-1 and MC1 expression was analysed by immunohistochemistry. Mc1re/e mice display less Safranin O and collagen II stained articular cartilage area compared to WT prior to OA-induction without signs of spontaneous cartilage surface erosion. This MC1-signaling deficiency related cartilage phenotype persisted in 6 month animals. At 4/8 weeks after OA-induction cartilage erosions were increased in Mc1re/e knees paralleled by weaker collagen II staining. Prior to OA-induction, Mc1re/e mice do not differ from WT with respect to bone parameters. During OA, Mc1re/e mice developed more osteophytes and had higher epiphyseal bone density and mass. Trabecular thickness was increased while concomitantly trabecular separation was decreased in Mc1re/e mice. Numbers of ICAM-positive chondrocytes were equal in non-operated 11 weeks Mc1re/e and WT whereas number of positive chondrocytes decreased during OA-progression. Unchallenged Mc1re/e mice display smaller articular cartilage covered area without OA-related surface erosions indicating that MC1-signaling is critical for proper cartilage matrix integrity and formation. When challenged with OA, Mc1re/e mice develop a more severe OA-pathology. Our data suggest that MC1-signaling protects against cartilage degradation and subchondral bone sclerosis in OA indicating a beneficial role of the POMC system in joint pathophysiology.

Mesenchymal stroma cells trigger early attraction of M1 macrophages and endothelial cells into fibrin hydrogels, stimulating long bone healing without long-term engraftment.

Implantation of mesenchymal stroma cells (MSCs) is an attractive approach to stimulate closure of large bone defects but an optimal carrier has yet to be defined. MSCs may display trophic and/or immunomodulatory features or stimulate bone healing by their osteogenic activity. The aim of this study was to unravel whether fibrin hydrogel supports early actions of implanted MSCs, such as host cell recruitment, immunomodulation and tissue regeneration, in long bone defects. Female rats received cell-free fibrin or male MSCs embedded in a fibrin carrier into plate-stabilized femoral bone defects. Removed callus was analyzed for host cell invasion (day 6), local cytokine expression (days 3 and 6) and persistence of male MSCs (days 3, 6, 14 and 28). Fibrin-MSC composites triggered fast attraction of host cells into the hydrogel while cell-free fibrin implants were not invaded. A migration front dominated by M1 macrophages and endothelial progenitor cells formed while M2 macrophages remained sparse. Only MSC-seeded fibrin hydrogel stimulated early tissue maturation and primitive vessel formation at day 6 in line with significantly higher VEGF mRNA levels recorded at day 3. Local TNF-α, IL-1ß and IL-10 expression indicated a balanced immune cell activity independent of MSC implantation. Implanted MSCs persisted until day 14 but not day 28. Our results demonstrate that fibrin hydrogel is an attractive carrier for MSC implantation into long bone defects, supporting host cell attraction and pro-angiogenic activity. By this angiogenesis, implant integration and tissue maturation was stimulated in long bone healing independent of long-term engraftment of implanted MSCs.

Engineering hydrophobin DewA to generate surfaces that enhance adhesion of human but not bacterial cells.

Hydrophobins are fungal proteins with the ability to form immunologically inert membranes of high stability, properties that makes them attractive candidates for orthopaedic implant coatings. Cell adhesion on the surface of such implants is necessary for better integration with the neighbouring tissue; however, hydrophobin surfaces do not mediate cell adhesion. The aim of this project was therefore to investigate whether the class I hydrophobin DewA from Aspergillus nidulans can be functionalized for use on orthopaedic implant surfaces. DewA variants bearing either one RGD sequence or the laminin globular domain LG3 binding motif were engineered. The surfaces of both variants showed significantly increased adhesion of mesenchymal stem cells (MSCs), osteoblasts, fibroblasts and chondrocytes; in contrast, the insertion of binding motifs RGD and LG3 in DewA did not increase Staphylococcus aureus adhesion to the hydrophobin surfaces. Proliferation of MSCs and their osteogenic, chondrogenic and adipogenic differentiation potential were not affected on these surfaces. The engineered surfaces therefore enhanced MSC adhesion without interfering with their functionality or leading to increased risk of bacterial infection.

Multi-parametric approach to identify coffee components that regulate mechanisms of gastric acid secretion.

SCOPE: Chlorogenic acid (CA), caffeine (CAFF), pyrogallol (PYR), catechol (CAT), (ß)N-alkanoyl-hydroxytryptamides (C5HT) and N-methylpyridinium (N-MP) were evaluated for their influence on mechanisms of gastric acid secretion as single compounds and in biomimetic mixtures. METHODS AND RESULTS: Compounds were tested in coffee representative concentrations. Human gastric cancer cells (HGT-1) were used to study the proton secretory activity by Ussing chamber experiments and FACS analysis. For activation of EGFr, Akt1, ERK1/2, ATF-2 and cAMP levels, we performed pathway screening assays. Time-dependent expression of related genes were determined by real-time PCR. Part of the data was used for neural network modeling to identify the most relevant compounds. N-MP increased the expression of the anti-secretory somatostatin receptor by 114%, whereas C5HT decreased its expression by 52%. N-MP down-regulated the pro-secretory CHRM3 receptor by 36% and the H⁺,K⁺-ATPase by 36%. CAFF stimulated the secretory activity in the functional assays, whereas N-MP and CA decreased proton secretion. After applying a pathway analysis, we were able to discriminate between CAFF, CA, CAT, C5HT, PYR and histamine-activating EGFr signaling and N-MP-associated ERK1/2 signaling. CONCLUSION: By applying a multi-parametric approach, N-MP was shown to effectively down-regulate mechanisms of gastric acid secretion in human parietal gastric cells.

Measurement of the intracellular ph in human stomach cells: a novel approach to evaluate the gastric acid secretory potential of coffee beverages.

As the consumption of coffee beverages sometimes is reported to cause gastric irritation, for which an increased stomach acid secretion is one of the promoting factors, different processing technologies such as steam-treatment have been developed to reduce putative stomach irritating compounds. There is evidence-based data neither on the effect of detailed processing variations nor on individual coffee components affecting the proton secretory activity (PSA). This work aimed at developing a screening model suitable for investigating the effects of commercial coffee beverages and components thereof on human parietal cells. Human gastric cancer cells (HGT-1) were treated with reconstituted freeze-dried coffee beverages prepared from customary coffee products such as regular coffee (RC, n = 4), mild bean coffee (MBC, n = 5), stomach friendly coffee (SFC, n = 4), and SFC decaffeinated (SFCD, n = 3). PSA was analyzed by flow cytometry using the pH-sensitive dye SNARF-AM. Treatment of the cells with MBC did not result in a PSA different from RC treatment (p