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BACKGROUND: In high-grade chondrosarcoma, 5-year survival is lower than 50%. Therefore, it is important that preclinical models that mimic the disease with the greatest possible fidelity are used to potentially develop new treatments. Accumulating evidence suggests that two-dimensional (2-D) cell culture may not accurately represent the tumor's biology. It has been demonstrated in other cancers that three-dimensional (3-D) cancer cell spheroids may recapitulate tumor biology and response to treatment with greater fidelity than traditional 2-D techniques. To our knowledge, the formation of patient-derived chondrosarcoma spheroids has not been described. QUESTIONS/PURPOSES: (1) Can patient-derived chondrosarcoma spheroids be produced? (2) Do spheroids recapitulate human chondrosarcoma better than 2-D cultures, both morphologically and molecularly? (3) Can chondrosarcoma spheroids provide an accurate model to test novel treatments? METHODS: Experiments to test the feasibility of spheroid formation of chondrosarcoma cells were performed using HT-1080, an established chondrosarcoma cell line, and two patient-derived populations, TP19-S26 and TP19-S115. Cells were cultured in flasks, trypsinized, and seeded into 96-well ultra-low attachment plates with culture media. After spheroids formed, they were monitored daily by bright-field microscopy. Spheroids were fixed using paraformaldehyde and embedded in agarose. After dehydration with isopropanol, paraffin-embedded spheroids were sectioned, and slides were stained with hematoxylin and eosin. To compare differences and similarities in gene expression between 2-D and 3-D chondrosarcoma cultures and primary tumors, and to determine whether these spheroids recapitulated the biology of chondrosarcoma, RNA was extracted from 2-D cultures, spheroids, and tumors. Quantitative polymerase chain reaction was performed to detect chondrosarcoma markers of interest, including vascular endothelial growth factor alpha, hypoxia-inducible factor 1α, COL2A1, and COL10A1. To determine whether 2-D and 3-D cultures responded differently to novel chondrosarcoma treatments, we compared their sensitivities to disulfiram and copper chloride treatment. To test their sensitivity to disulfiram and copper chloride treatment, 10,000 cells were seeded into 96-well plates for 2-D culturing and 3000 cells in each well for 3-D culturing. After treating the cells with disulfiram and copper for 48 hours, we detected cell viability using quantitative presto-blue staining and measured via plate reader. RESULTS: Cell-line and patient-derived spheroids were cultured and monitored over 12 days. Qualitatively, we observed that HT-1080 demonstrated unlimited growth, while TP19-S26 and TP19-S115 contracted during culturing relative to their initial size. Hematoxylin and eosin staining of HT-1080 spheroids revealed that cell-cell attachments were more pronounced at the periphery of the spheroid structure than at the core, while the core was less dense. Spheroids derived from the intermediate-grade chondrosarcoma TP19-S26 were abundant in extracellular matrix, and spheroids derived from the dedifferentiated chondrosarcoma TP19-S115 had a higher cellularity and heterogeneity with spindle cells at the periphery. In the HT-1080 cells, differences in gene expression were appreciated with spheroids demonstrating greater expressions of VEGF-α (1.01 ± 0.16 versus 6.48 ± 0.55; p = 0.003), COL2A1 (1.00 ± 0.10 versus 7.46 ± 2.52; p < 0.001), and COL10A1 (1.01 ± 0.19 versus 22.53 ± 4.91; p < 0.001). Differences in gene expressions were also noted between primary tumors, spheroids, and 2-D cultures in the patient-derived samples TP19-S26 and TP19-S115. TP19-S26 is an intermediate-grade chondrosarcoma. With the numbers we had, we could not detect a difference in VEGF-α and HIF1α gene expression compared with the primary tumor. COL2A1 (1.00 ± 0.14 versus 1.76 ± 0.10 versus 335.66 ± 31.13) and COL10A1 (1.06 ± 0.378 versus 5.98 ± 0.45 versus 138.82 ± 23.4) expressions were both greater in the tumor (p (COL2A1) < 0.001; p (COL10A1) < 0.0001) and 3-D cultures (p (COL2A1) = 0.004; p (COL10A1) < 0.0001) compared with 2-D cultures. We could not demonstrate a difference in VEGF-α and HIF1α expressions in TP19-S115, a dedifferentiated chondrosarcoma, in the tumor compared with 2-D and 3-D cultures. COL2A1 (1.00 ± 0.02 versus 1.86 ± 0.18 versus 2.95 ± 0.56) and COL10A1 (1.00 ± 0.03 versus 5.52 ± 0.66 versus 3.79 ± 0.36) expressions were both greater in spheroids (p (COL2A1) = 0.003; p (COL10A1) < 0.0001) and tumors (p (COL2A1) < 0.001; p (COL10A1) < 0.0001) compared with 2-D cultures. Disulfiram-copper chloride treatment demonstrated high cytotoxicity in HT-1080 and SW-1353 chondrosarcoma cells grown in the 2-D monolayer, but 3-D spheroids were highly resistant to this treatment. CONCLUSION: We provide preliminary findings that it is possible to generate 3-D spheroids from chondrosarcoma cell lines and two human chondrosarcomas (one dedifferentiated chondrosarcoma and one intermediate-grade chondrosarcoma). Chondrosarcoma spheroids derived from human tumors demonstrated morphology more reminiscent of primary tumors than cells grown in 2-D culture. Spheroids displayed similar expressions of cartilage markers as the primary tumor, and we observed a higher expression of collagen markers in the spheroids compared with cells grown in monolayer. Spheroids also demonstrated greater chemotherapy resistance than monolayer cells, but more patient-derived spheroids are needed to further conclude that 3-D cultures may mimic the chemoresistance that chondrosarcomas demonstrate clinically. Additional studies on patient-derived chondrosarcoma spheroids are warranted. CLINICAL RELEVANCE: Chondrosarcomas demonstrate resistance to chemotherapy and radiation, and we believe that if they can be replicated, models such as 3-D spheroids may provide a method to test novel treatments for human chondrosarcoma. Additional comprehensive genomic studies are required to compare 2-D and 3-D models with the primary tumor to determine the most effective way to study this disease in vitro.
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Neoplasias Ósseas , Condrossarcoma , Modelos Biológicos , Esferoides Celulares , Células Tumorais Cultivadas , Estudos de Viabilidade , HumanosRESUMO
BACKGROUND: Surgical resection with negative margins is the foundation of extremity sarcoma management. Failure to achieve negative surgical margins can result in local recurrence (LR), a potentially devastating complication. Indocyanine green (ICG) is a US FDA-approved fluorophore previously used to guide carcinoma resections. We investigated the potential of ICG as an intraoperative guide during experimental sarcoma resection. METHODS: Fifty 6-week-old immunocompetent Balb/c female mice received left proximal tibia paraphyseal injections of 5 × 105 K7M2 murine osteosarcoma cells. Animals were separated into two groups (n = 25 each): (1) ICG-assisted surgical resection; and (2) no ICG-assisted resection. Resections were performed 4 weeks after primary tumor engraftment. All animals received 7.5 ug ICG via retro-orbital injection 12 h prior to surgery. ICG fluorescence measurements and clinical evaluations were performed 4 weeks after resection to detect LR. RESULTS: Eleven of 25 animals from each group developed gross tumors. Four weeks after resection, group 1 had 0/11 tumor recurrences, while group 2 had recurrences in 9/11 (81.8%) experimental mice (p < 0.0002) (Fig. 2). There was a 100% NPV in group 1, and no tumor recurrence with fluorescence-free margins after the primary surgery. Group 2 had a 100% positive predictive value for the development of an LR if any fluorescent signal was present at the surgical margin after resection. CONCLUSION: Intraoperative ICG guidance led to reliably negative surgical margins and a diminished LR rate. Given the benign safety profile of ICG and its prior clinical success, these results could be immediately translatable to the clinical realm.
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Neoplasias Ósseas/cirurgia , Angiofluoresceinografia/métodos , Fluorescência , Recidiva Local de Neoplasia/prevenção & controle , Imagem Óptica/métodos , Osteossarcoma/cirurgia , Cirurgia Assistida por Computador/métodos , Animais , Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/patologia , Feminino , Corantes Fluorescentes , Verde de Indocianina/metabolismo , Cuidados Intraoperatórios , Margens de Excisão , Camundongos , Camundongos Endogâmicos BALB C , Recidiva Local de Neoplasia/diagnóstico por imagem , Recidiva Local de Neoplasia/patologia , Osteossarcoma/diagnóstico por imagem , Osteossarcoma/patologiaRESUMO
BACKGROUND: Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined. METHODS: Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment. RESULTS: Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline phosphatase staining. CONCLUSIONS: Lung endothelial HULEC-5a cells are attractants for OS cell migration, proliferation, and survival. The SJSA-1 osteosarcoma cell line demonstrated greater metastatic potential than Saos-2 and U-2 cells. ALDH appears to be involved in the interaction between lung and OS cells, and ALP may be a valuable biomarker for monitoring functional OS changes during metastasis.
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Fosfatase Alcalina/genética , Biomarcadores Tumorais/genética , Isoenzimas/genética , Osteossarcoma/genética , Retinal Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Técnicas de Cocultura , Dissulfiram/administração & dosagem , Humanos , Pulmão/citologia , Pulmão/metabolismo , Camundongos , Metástase Neoplásica , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologiaRESUMO
Purpose: To test cefiderocol, a siderophore-cephalosporin antibiotic for topical monotherapy treatment of experimental extensively drug-resistant (XDR) Pseudomonas aeruginosa keratitis. Design: Preclinical study. Subjects and Controls: Deidentified P. aeruginosa keratitis isolates, XDR P. aeruginosa from eye drop outbreak, rabbits, saline, cefiderocol 50 mg/ml, ciprofloxacin 0.3%, and tobramycin 14 mg/ml. Methods Intervention or Testing: Cefiderocol antibacterial activity against P. aeruginosa keratitis isolates (n = 135) was evaluated by minimum inhibitory concentration (MIC) testing. Ocular toxicity/tolerability and antibacterial efficacy were tested in vivo with experimental rabbit models. Corneal concentrations and stability were assessed using a bioassay. Main Outcome Measures: Minimum inhibitory concentration analysis for susceptibility, graded tests for ocular toxicity/tolerability, colony-forming unit (CFU) analysis for bacterial burden, corneal cefiderocol concentrations. Results: One hundred percent of P. aeruginosa keratitis isolates were susceptible to cefiderocol (n = 135), the MIC90 was 0.125 µg/ml including the XDR isolate (MIC = 0.125 µg/ml). Topical cefiderocol 50 mg/ml was minimally toxic to the ocular surface and was well tolerated. For the XDR P. aeruginosa isolate, topical cefiderocol 50 mg/ml, significantly decreased corneal CFU compared with ciprofloxacin 0.3%, tobramycin 14 mg/ml, and saline. In addition, tobramycin 14 mg/ml was more effective than the saline control. Mean cefiderocol corneal concentrations were 191× greater than the MIC90 of the P. aeruginosa keratitis isolates. Refrigerated cefiderocol maintained antimicrobial activity over a 1-month period. Conclusions: These results demonstrate that cefiderocol is well tolerated on rabbit corneas and is effective against P. aeruginosa keratitis isolates in vitro and was effective in vivo against an XDR isolate in a rabbit keratitis model. Given the recent outbreak of keratitis caused by this XDR P. aeruginosa, cefiderocol is a promising additional antibiotic that should be further evaluated for topical treatment of keratitis caused by antibiotic resistant P. aeruginosa. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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Purpose: To test cefiderocol, a siderophore-cephalosporin antibiotic for topical monotherapy treatment of experimental extensively drug resistant (XDR) Pseudomonas aeruginosa keratitis. Design: Preclinical study. Subjects and Controls: Deidentified P. aeruginosa keratitis isolates, XDR P. aeruginosa from eye drop outbreak, rabbits, saline, cefiderocol 50 mg/ml, ciprofloxacin 0.3%, and tobramycin 14 mg/ml. Methods Intervention or Testing: Cefiderocol antibacterial activity against P. aeruginosa keratitis isolates (n=135) was evaluated by minimum inhibitory concentration (MIC) testing. Ocular toxicity/tolerability and antibacterial efficacy were tested in vivo with experimental rabbit models. Corneal concentrations and stability were assessed using a bioassay. Main Outcome Measures: MIC analysis for susceptibility, graded tests for ocular toxicity/tolerability, CFU analysis for bacterial burden, corneal cefiderocol concentrations. Results: 100% of P. aeruginosa keratitis isolates were susceptible to cefiderocol (n=135), the MIC90 was 0.125 µg/ml including the XDR isolate (MIC = 0.125 µg/ml). Topical cefiderocol 50 mg/ml was minimally toxic to the ocular surface and was well tolerated. For the XDR P. aeruginosa isolate, topical cefiderocol 50 mg/ml, significantly decreased corneal CFU compared to ciprofloxacin 0.3%, tobramycin 14 mg/ml, and saline. In addition, tobramycin 14 mg/ml was more effective than the saline control. Mean cefiderocol corneal concentrations were 191x greater than the MIC90 of the P. aeruginosa keratitis isolates. Refrigerated cefiderocol maintained antimicrobial activity over a one-month period. Conclusions: These results demonstrate that cefiderocol is well tolerated on rabbit corneas and is effective against P. aeruginosa keratitis isolates in vitro and was effective in vivo against an XDR isolate in a rabbit keratitis model. Given the recent outbreak of keratitis caused by this XDR P. aeruginosa, cefiderocol is a promising additional antibiotic that should be further evaluated for topical treatment of keratitis caused by antibiotic resistant P. aeruginosa.
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Antibiotic stewardship is viewed as having great public health benefit with limited direct benefit to the patient at the time of administration. The objective of our study was to determine if inappropriate administration of antibiotics could create conditions that would increase the rates of surgical infection. We hypothesized that sub-MIC levels of vancomycin would increase Staphylococcus aureus growth, biofilm formation, and rates of infection. S. aureus MRSA and MSSA strains were used for all experiments. Bacteria were grown planktonically and monitored using spectrophotometry. Quantitative agar culture was used to measure planktonic and biofilm bacterial burden. A mouse abscess model was used to confirm phenotypes in vivo. In the planktonic growth assay, increases in bacterial burden at » MIC vancomycin were observed in USA300 JE2 by 72 h. Similar findings were observed with ½ MIC in Newman and SH1000. For biofilm formation, USA300 JE2 at » and ½ MIC vancomycin increased biofilm formation by approximately 1.3- and 2.3-fold respectively at 72 h as compared to untreated controls. Similar findings were observed with Newman and SH1000 with a 2.4-fold increase in biofilm formation at ½ MIC vancomycin. In a mouse abscess model, there was a 1.2-fold increase with sub-MIC vancomycin at 3 days post infection. Our study showed that Sub-optimal vancomycin dosing promoted S. aureus planktonic growth and biofilm formation, phenotypic measures of bacterial virulence. This phenotype induced by sub-MIC levels of vancomycin was also observed to increase rates of infection and pathogenesis in our mouse model. Risks of exposure to sub-MIC concentrations with vancomycin in surgical procedures are greater as there is decreased bioavailability in tissue in comparison to other antibiotics. This highlights the importance of proper antibiotic selection, stewardship, and dosing for both surgical prophylaxis and treatment of infection.
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Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Camundongos , Vancomicina/farmacologia , Vancomicina/uso terapêutico , Staphylococcus aureus , Infecção da Ferida Cirúrgica , Abscesso , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/prevenção & controle , Infecções Estafilocócicas/microbiologia , Biofilmes , Testes de Sensibilidade MicrobianaRESUMO
Twenty-four dogs with OS underwent limb amputation. Serum, OS tumour, and normal bone were harvested at time of surgery. RNA was extracted and gene expression was performed using quantitative polymerase chain reaction (qPCR). Tissue and blood copper concentrations were also determined with spectrophotometry. Compared to bone, tumour samples had significantly higher expressions of antioxidant 1 copper chaperone (ATOX1, p = .0003). OS tumour copper levels were significantly higher than that of serum (p < .010) and bone (p = .038). Similar to our previous observations in mouse and human OS, dog OS demonstrates overexpression of genes that regulate copper metabolism (ATOX1), and subsequent copper levels. Dogs with OS may provide a robust comparative oncology platform for the further study of these factors, as well as potential pharmacologic interventions.
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Neoplasias Ósseas , Doenças do Cão , Osteossarcoma , Humanos , Cães , Animais , Camundongos , Cobre , Antioxidantes , Osteossarcoma/genética , Osteossarcoma/veterinária , Osteossarcoma/metabolismo , Doenças do Cão/genética , Doenças do Cão/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/veterinária , Expressão Gênica , Proteínas de Transporte de Cobre/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismoRESUMO
Background: Osteosarcoma (OS) and chondrosarcoma (CS) are primary bone malignancies whose prognoses have stagnated despite advancements in surgical management, chemotherapy, radiation therapy, and immunotherapy. The role of the immune system in generating anti-cancer physiologic responses is critical to prognosis. Prior studies have explored if immune system activation via infection enhances survival in bone sarcomas without a clear consensus. Methods: This study sought to (I) retrospectively examine the effect of postoperative infection on survival in OS and CS and (II) systematically review the effect of postoperative infection on survival in primary bone malignancies. We performed a retrospective case-control study of 192 patients treated between 1/2000-12/2015 at a single academic sarcoma referral center. Patients with OS or CS undergoing operative resection were included. Eligible patients were grouped by presence of metastasis, and survival was compared between patients with or without postoperative infection. Furthermore, we performed a systematic review following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines investigating the effect of infection on primary bone malignancy survival. Risk of bias assessment was performed utilizing the ROBINS-I (Risk of Bias in Non-randomized Studies-of Interventions) assessment tool. All presented studies included author information, study population, and overall or disease-free survival results. Results: One hundred and four patients were included, with 85 without infection (26 metastatic, 59 non-metastatic) and 19 with infection (10 metastatic, 9 non-metastatic). Five-year survival was greatest in patients without metastasis with a postoperative infection (100%), followed by patients without metastasis who were infection-free (80%). Five-year survival was comparatively lower in patients with metastasis who were infection-free (35%) and lowest in patients with metastasis with a postoperative infection (20%). No significant differences were present (P=0.17) on log-rank analysis. Our systematic review collected six studies exploring the impact of infection on primary bone malignancy survival, with two studies reporting significant findings of infection improving survival. Limitations of this review included risk of bias due to confounding, inconsistency comparing outcomes, and differences in patient populations. Conclusions: This retrospective study and systematic review suggests postoperative infection may play a role in modulating immune response to malignancy. Understanding the synergy between anti-pathogen and anti-cancer responses warrants further investigation as an alternative method of targeted cancer treatment.
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Aldehyde dehydrogenase 1A1 (ALDH) is a cancer stem cell marker highly expressed in metastatic cells. Disulfiram (Dis) is an FDA-approved antialcoholism drug that inhibits ALDH and has been studied as a candidate for drug repurposing in multiple neoplasia. Dis cytotoxicity in cancer cells has been shown to be copper-dependent, in part due to Dis's ability to function as a bivalent metal ion chelator of copper (Cu). The objectives of this research were to test ALDH expression levels and Cu concentrations in sarcoma patient tumors and human osteosarcoma (OS) cell lines with differing metastatic phenotypes. We also sought to evaluate Dis + Cu combination therapy in human OS cells. Intracellular Cu was inversely proportional to the metastatic phenotype in human OS cell lines (SaOS2 > LM2 > LM7). Nonmetastatic human sarcoma tumors demonstrated increased Cu concentrations compared with metastatic tumors. qPCR demonstrated that ALDH expression was significantly increased in highly metastatic LM2 and LM7 human OS cell lines compared with low metastatic SaOS2. Tumor cells from sarcoma patients with metastatic disease displayed significantly increased ALDH expression compared with tumor cells from patients without metastatic disease. Serum Cu concentration in canine OS versus normal canine patients demonstrated similar trends. Dis demonstrated selective cytotoxicity compared with human multipotential stromal cells (MSCs): Dis-treated OS cells demonstrated increased apoptosis, whereas MSCs did not. CuCl2 combined with Dis and low-dose doxorubicin resulted in a superior cytotoxic effect in both SaOS2 and LM7 cell lines. In summary, ALDH gene expression and Cu levels are altered between low and highly metastatic human OS cells, canine samples, and patient tumors. Our findings support the feasibility of a repurposed drug strategy for Dis and Cu in combination with low-dose anthracycline to specifically target metastatic OS cells.
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The absence of novel antibiotics for drug-resistant and biofilm-associated infections is a global public health crisis. Antimicrobial peptides explored to address this need have encountered significant development challenges associated with size, toxicity, safety profile, and pharmacokinetics. We designed PLG0206, an engineered antimicrobial peptide, to address these limitations. PLG0206 has broad-spectrum activity against >1,200 multidrug-resistant (MDR) ESKAPEE clinical isolates, is rapidly bactericidal, and displays potent anti-biofilm activity against diverse MDR pathogens. PLG0206 displays activity in diverse animal infection models following both systemic (urinary tract infection) and local (prosthetic joint infection) administration. These findings support continuing clinical development of PLG0206 and validate use of rational design for peptide therapeutics to overcome limitations associated with difficult-to-drug pharmaceutical targets.
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Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Biofilmes , Preparações FarmacêuticasRESUMO
The high antibiotic tolerance of Staphylococcus aureus biofilms is associated with challenges for treating periprosthetic joint infection. The toxin-antitoxin system, YefM-YoeB, is thought to be a regulator for antibiotic tolerance, but its physiological role is unknown. The objective of this study was to determine the biofilm and antibiotic susceptibility phenotypes associated with S. aureus yoeB homologs. We hypothesized the toxin-antitoxin yoeB homologs contribute to biofilm formation and antibiotic susceptibility. Disruption of yoeB1 and yoeB2 resulted in decreased biofilm formation in comparison to Newman and JE2 wild-type (WT) S. aureus strains. In comparison to yoeB mutants, both Newman and JE2 WT strains had higher polysaccharide intercellular adhesin (PIA) production. Treatment with sodium metaperiodate increased biofilm formation in Newman WT, indicating biofilm formation may be increased under conditions of oxidative stress. DNase I treatment decreased biofilm formation in Newman WT but not in the absence of yoeB1 or yoeB2. Additionally, WT strains had a higher extracellular DNA (eDNA) content in comparison to yoeB mutants but no differences in biofilm protein content. Moreover, loss of yoeB1 and yoeB2 decreased biofilm survival in both Newman and JE2 strains. Finally, in a neutropenic mouse abscess model, deletion of yoeB1 and yoeB2 resulted in reduced bacterial burden. In conclusion, our data suggest that yoeB1 and yoeB2 are associated with S. aureus planktonic growth, extracellular dependent biofilm formation, antibiotic tolerance, and virulence.
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PLG0206 is an engineered antimicrobial peptide that has completed phase 1 clinical studies. A prospective study was completed on explanted implants from chronic periprosthetic joint infections (n = 17). At a concentration of 1 mg/mL for 15 min, there was a mean 4-log10 reduction (range, 1 to 7) in the bacterial CFU identified from the implants. IMPORTANCE Chronically infected prosthetics of the knee were exposed to PLG0206, an engineered antimicrobial peptide, at a concentration of 1 mg/mL for 15 min. A mean 4-log10 reduction (range, 1 to 7) in the number of bacteria occurred, which may translate to improved clinical outcomes for persons with prosthetic joint infection of the knee.
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Peptídeos Antimicrobianos/administração & dosagem , Artroplastia do Joelho/efeitos adversos , Bactérias/efeitos dos fármacos , Complicações Pós-Operatórias/tratamento farmacológico , Infecções Relacionadas à Prótese/tratamento farmacológico , Bactérias/crescimento & desenvolvimento , Humanos , Técnicas In Vitro , Joelho/microbiologia , Joelho/cirurgia , Complicações Pós-Operatórias/microbiologia , Estudos Prospectivos , Infecções Relacionadas à Prótese/microbiologiaRESUMO
Periprosthetic joint infection of total knee arthroplasties represents a major challenge to the field of orthopedic surgery. These infections are commonly associated with antibiotic-tolerant Staphylococcus aureus biofilms. Engineered cationic amphipathic peptide WLBU2 has shown the ability to kill antibiotic-resistant pathogens and drug-tolerant bacterial biofilms. The novelty of using WLBU2 during the direct irrigation and debridement of periprosthetic joint infections led our group to investigate the optimal washout conditions for treatment of S. aureus biofilms. S. aureus mature biofilms were grown on metal implant material and treated with WLBU2 dissolved in differing irrigation solvents. Mature biofilms were treated both in vitro as well as in a periprosthetic joint infection murine model. WLBU2 activity against S. aureus biofilms was increased when dissolved in diphosphate-buffered saline (dPBS) with pH of 7.0 compared with normal saline with pH of 5.5. WLBU2 activity was decreased in acidic dPBS and increased in alkaline dPBS. WLBU2 activity could be decreased in hypertonic dPBS and increased in hypotonic dPBS. WLBU2 dissolved in less acidic dPBS displayed increased efficacy in treating periprosthetic joint infection (PJI) implants ex vivo. WLBU2 demonstrated the ability to eliminate PJI associated S. aureus biofilms on arthroplasty material. The efficacy of engineered cationic amphipathic peptide WLBU2 for intraoperative elimination of S. aureus biofilms can be further optimized when kept in a less acidic and more physiologic pH adjusted saline. Understanding optimal physical washout conditions are vital for the success of WLBU2 in treating S. aureus biofilms in PJI clinical trials going forward.
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Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Biofilmes/efeitos dos fármacos , Infecções Relacionadas à Prótese/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Concentração de Íons de Hidrogênio , Camundongos , Concentração Osmolar , Solução SalinaRESUMO
Both Staphylococcus aureus and Staphylococcus epidermidis are commonly associated with periprosthetic joint infections (PJIs). The treatment of PJI can be challenging because biofilms are assumed to have an increased intolerance to antibiotics. This makes the treatment of PJI challenging from a clinical perspective. Although S. aureus has been previously demonstrated to have increased biofilm antibiotic tolerance, this has not been well established with Staphylococcus epidermidis. A prospective registry of PJI S. epidermidis isolates was developed. The efficacy of clinically relevant antibiotics was quantified against these isolates. S. epidermidis planktonic minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were collected using clinical laboratory standard index (CLSI) assays for eight antibiotics (doxycycline, vancomycin, daptomycin, clindamycin, rifampin, nafcillin, and trimethoprim/sulfamethoxazole). Mature biofilms were grown in vitro, after which minimum biofilm inhibitory concentration (MBIC) and minimum biofilm bactericidal concentration (MBBC) were quantified. Only rifampin and doxycycline had a measurable MBIC across all tested isolates. Based on MBBC, 64% of S. epidermidis biofilms could be eliminated by rifampin, whereas only 18% by doxycycline. S. epidermidis biofilm was observed to have a high tolerance to antibiotics as compared to planktonic culture. Isolate biofilm antibiotic tolerance varied to a larger degree than was seen in planktonic cultures.
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Cutibacterium acnes (formerly Propionibacterium acnes) is a significant pathogen in periprosthetic joint infections (PJIs) in total shoulder arthroplasty. Poor outcomes seen in PJIs are due to the established C. acnes bacterial biofilms. The prolonged nature of C. acnes infections makes them difficult to treat with antibiotics. The goal of this study was to determine the relative efficacy of vancomycin compared with penicillin and doxycycline against planktonic and mature biofilms. Clinical isolates from PJI patients as well as a laboratory strain of C. acnes were tested. Planktonic minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were obtained using modified clinical laboratory standard index assays. Biofilm MICs and MBCs were also obtained. The MIC was determined for both using the PrestoBlue viability stain. The MBC was determined using differential reinforced clostridial medium agar plates for colony-forming unit analysis. Using the PrestoBlue viability reagent, the planktonic MIC values for vancomycin were significantly higher than doxycycline. Across 10 strains of C. acnes, all three antibiotics had decreased efficacy when comparing planktonic and biofilm cultures. Although effective antibiotic doses ranged from 1 to 1,000 µg/mL, only doxycycline achieved inhibitory and bactericidal concentrations in all tested strains. Penicillin failed to achieve the minimum biofilm inhibitory concentration (MBIC) in 60% of tested strains, whereas vancomycin failed in 80% of tested strains. Penicillin, doxycycline, and vancomycin have similar abilities in inhibiting C. acnes growth planktonically. The MBIC for doxycycline was within the clinical dosing range, suggesting C. acnes biofilm offers minimal tolerance to these antibiotics. The MBIC for penicillin was within clinical dosing ranges in only 60% of trials, suggesting the relative tolerance of C. acnes to penicillin. The minimum biofilm bactericidal concentration (MBBC) of doxycycline showed efficacy in 90% of trials, whereas penicillin and vancomycin achieved MBBC in 15% of samples.
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Although many cancer cells have significantly higher copper concentrations compared with normal cells and tissues, the role of copper in cancer biology and metastatic disease remains poorly understood. Here, we study the importance of copper in osteosarcoma, which frequently metastasizes to the lungs and is often chemoresistant. K12 and K7M2 are murine OS cells with differing metastatic phenotypes: K7M2 is highly metastatic, whereas K12 is much less so. Intracellular copper levels were determined using atomic absorption. Copper transporters were quantified by qPCR. Cytotoxicity of doxorubicin, disulfiram, and copper(II) chloride was assessed with a cell viability fluorescence stain. Additionally, K7M2 viable cell counts were determined by trypan blue exclusion staining after 72 hours of treatment. Copper levels were found to be significantly higher in K12 OS cells than in K7M2 cells. qPCR showed that K12 cells upregulate the copper influx pump CTR1 and downregulate the copper efflux pump ATP7A compared to K7M2 OS cells. Combination treatment of copper chloride (50 nM) with disulfiram (80 nM) was only cytotoxic to K12 cells. Triple treatment with doxorubicin, disulfiram, and copper displayed potent and durable cytotoxicity of highly metastatic K7M2 cells. We demonstrate here that murine OS cell lines differing in metastatic potential also vary in endogenous copper levels and regulation. Additionally, these differences in copper regulation may contribute to selective cytotoxicity of K12 cells by extremely low doses of copper-potentiated disulfiram. The combination of doxorubicin, disulfiram, and copper should be explored as a therapeutic strategy against OS metastases.
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Staphylococcus aureus biofilms have a high tolerance to antibiotics, making the treatment of periprosthetic joint infection (PJI) challenging. From a clinical perspective, bacteria from surgical specimens are cultured in a planktonic state to determine antibiotic sensitivity. However, S. aureus exists primarily as established biofilms in PJI. To address this dichotomy, we developed a prospective registry of total knee and hip arthroplasty PJI S. aureus isolates to quantify the activity of clinically important antibiotics against isolates grown as biofilms. S. aureus planktonic minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were assessed using clinical laboratory standard index assays for 10 antibiotics (cefazolin, clindamycin, vancomycin, rifampin, linezolid, nafcillin, gentamicin, trimethoprim/sulfamethoxazole, doxycycline, and daptomycin). Mature biofilms of each strain were grown in vitro, after which biofilm MIC (MBIC) and biofilm MBC (MBBC) were determined. Overall, isolates grown as biofilms displayed larger variations in antibiotic MICs as compared to planktonic MIC values. Only rifampin, doxycycline, and daptomycin had measurable biofilm MIC values across all S. aureus isolates tested. Biofilm MBC observations complemented biofilm MIC observations; rifampin, doxycycline, and daptomycin were the only antibiotics with measurable biofilm MBC values. 90% of S. aureus biofilms could be killed by rifampin, 50% by doxycycline, and only 15% by daptomycin. Biofilm formation increased bacterial antibiotic tolerance nonspecifically across all antibiotics, in both MSSA and MRSA samples. Rifampin and doxycycline were the most effective antibiotics at killing established S. aureus biofilms. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1604-1609, 2019.
Assuntos
Antibacterianos/farmacologia , Artrite Infecciosa/microbiologia , Biofilmes/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecções Relacionadas à Prótese/microbiologia , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Staphylococcus aureus is the major organism responsible for surgical implant infections. Antimicrobial treatment of these infections often fails, leading to expensive surgical intervention and increased risk of mortality to the patient. The challenge in treating these infections is associated with the high tolerance of S. aureus biofilm to antibiotics. MazEF, a toxin-antitoxin system, is thought to be an important regulator of this phenotype, but its physiological function in S. aureus is controversial. Here, we examined the role of MazEF in developing chronic infections by comparing growth and antibiotic tolerance phenotypes in three S. aureus strains to their corresponding strains with disruption of mazF expression. Strains lacking mazF production showed increased biofilm growth and decreased biofilm antibiotic tolerance. Deletion of icaADBC in the mazF::Tn background suppressed the growth phenotype observed with mazF-disrupted strains, suggesting the phenotype was ica dependent. We confirmed these phenotypes in our murine animal model. Loss of mazF resulted in increased bacterial burden and decreased survival rate of mice compared to its wild-type strain demonstrating that loss of the mazF gene caused an increase in S. aureus virulence. Although lack of mazF gene expression increased S. aureus virulence, it was more susceptible to antibiotics in vivo Combined, the ability of mazF to inhibit biofilm formation and promote biofilm antibiotic tolerance plays a critical role in transitioning from an acute to chronic infection that is difficult to eradicate with antibiotics alone.IMPORTANCE Surgical infections are one of the most common types of infections encountered in a hospital. Staphylococcus aureus is the most common pathogen associated with this infection. These infections are resilient and difficult to eradicate, as the bacteria form biofilm, a community of bacteria held together by an extracellular matrix. Compared to bacteria that are planktonic, bacteria in a biofilm are more resistant to antibiotics. The mechanism behind how bacteria develop this resistance and establish a chronic infection is unknown. We demonstrate that mazEF, a toxin-antitoxin gene, inhibits biofilm formation and promotes biofilm antibiotic tolerance which allows S. aureus to transition from an acute to chronic infection that cannot be eradicated with antibiotics but is less virulent. This gene not only makes the bacteria more tolerant to antibiotics but makes the bacteria more tolerant to the host.
Assuntos
Antitoxinas/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Biofilmes , Farmacorresistência Bacteriana , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/fisiologia , Animais , Antibacterianos/farmacologia , Antitoxinas/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Biofilmes/efeitos dos fármacos , Doença Crônica , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Sistemas Toxina-AntitoxinaRESUMO
Cancer-associated cachexia is a wasting syndrome that affects up to 50% of cancer patients. It is defined as unintentional weight loss ≥5% over 6 months and characterized by muscle atrophy, fatigue, and anorexia that are refractory to nutritional support. Sarcoma describes a diverse group of malignancies arising from the connective tissues. Sarcoma patients are uniquely susceptible to cancer-associated cachexia given its origins in the musculoskeletal system. Our previous research suggests that sarcoma cells may contribute to sarcoma-associated cachexia (SAC) via establishment of TNF-α-mediated inflammation and dysregulation of muscle homeostasis by abnormal Notch signaling. Here, we examine the role of the Notch pathway and pro-inflammatory cytokines in cells derived from cachectic and non-cachectic human sarcoma patients. We observed increased expression of Notch pathway genes in the cachexia group while no differences in pro-inflammatory cytokines were observed. Co-culture of muscle-derived stem cells (MDSCs) and sarcoma cells demonstrated the inhibition of MDSC maturation with both cachectic and non-cachectic patient cells, corresponding to elevated Pax7 and Notch pathway expression in MDSCs. Our findings suggest that there is no difference in inflammatory profile between cachexia and non-cachexia sarcoma samples. However, Cachectic sarcoma samples express increased Notch that mediates muscle wasting possibly through inhibition of myogenesis.
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INTRODUCTION: The overall survival rate of patients with osteosarcoma (OS) and pulmonary metastases has remained stagnant at 15-30% for several decades. Disulfiram (DSF) is an FDA-approved aldehyde dehydrogenase inhibitor that reduces the metastatic phenotype of OS cells in vitro. Here we evaluate its in vivo efficacy, as compared to doxorubicin chemotherapy, in a previously-validated orthotopic model of metastatic OS. RESULTS: All treatment groups displayed a significantly reduced quantitative OS metastatic burden compared with controls. The metastatic burden of Lo DSF-treated animals was equivalent to the DXR group. Ninety-five percent of control animals displayed evidence of metastatic disease, which was significantly greater than all treatment groups. DISCUSSION: Disulfiram treatment resulted in a reduced burden of OS metastatic disease compared with controls. This was statistically-equivalent to doxorubicin. No additive effect was observed between these two therapies. MATERIALS AND METHODS: One-hundred twenty immunocompetent Balb/c mice received proximal tibia paraphyseal injections of 5 × 105 K7M2 murine OS cells. Therapy began three weeks after injection: saline (control), low-dose disulfiram (Lo DSF), high-dose disulfiram (Hi DSF), doxorubicin (DXR), Lo DSF + DXR, and Hi DSF + DXR. Transfemoral amputations were performed at 4 weeks. Quantitative metastatic tumor burden was measured using near-infrared indocyanine green (ICG) angiography.