Eradication of Staphylococcus aureus in Implant-Associated Osteomyelitis by an Injectable In Situ-Forming Depot Antibiotics Delivery System.

BACKGROUND: Bone infections with Staphylococcus aureus are notoriously difficult to treat and have high recurrence rates. Local antibiotic delivery systems hold the potential to achieve high in situ antibiotic concentrations, which are otherwise challenging to achieve via systemic administration. Existing solutions have been shown to confer suboptimal drug release and distribution. Here we present and evaluate an injectable in situ-forming depot system termed CarboCell. The CarboCell technology provides sustained and tuneable release of local high-dose antibiotics. METHODS: CarboCell formulations of levofloxacin or clindamycin with or without antimicrobial adjuvants cis-2-decenoic acid or cis-11-methyl-2-dodecenoic acid were tested in experimental rodent and porcine implant-associated osteomyelitis models. In the porcine models, debridement and treatment with CarboCell-formulated antibiotics was carried out without systemic antibiotic administration. The bacterial burden was determined by quantitative bacteriology. RESULTS: CarboCell formulations eliminated S. aureus in infected implant rat models. In the translational implant-associated pig model, surgical debridement and injection of clindamycin-releasing CarboCell formulations resulted in pathogen-free bone tissues and implants in 9 of 12 and full eradication in 5 of 12 pigs. CONCLUSIONS: Sustained release of antimicrobial agents mediated by the CarboCell technology demonstrated promising therapeutic efficacy in challenging translational models and may be beneficial in combination with the current standard of care.

Diffusion weighted magnetic resonance imaging (DW-MRI) as a non-invasive, tissue cellularity marker to monitor cancer treatment response.

BACKGROUND: Diffusion weighted magnetic resonance imaging (DW-MRI) holds great potential for monitoring treatment response in cancer patients shortly after initiation of radiotherapy. It is hypothesized that a decrease in cellular density of irradiated cancerous tissue will lead to an increase in quantitative apparent diffusion coefficient (ADC) values. DW-MRI can therefore serve as a non-invasive marker of cell death and apoptosis in response to treatment. In the present study, we aimed to investigate the applicability of DW-MRI in preclinical models to monitor radiation-induced treatment response. In addition, we compared DW-MRI with ex vivo measures of cell density, cell death and apoptosis. METHODS: DW-MRI was tested in two different syngeneic mouse models, a colorectal cancer (CT26) and a breast cancer (4 T1). ADC values were compared with quantitative determinations of apoptosis and cell death by flow cytometry. Furthermore, ADC-values were also compared to histological measurement of cell density on tumor sections. RESULTS: We found a significant correlation between ADC-values and apoptotic state in the CT26 model (P = 0.0031). A strong correlation between the two measurements of ADC-value and apoptotic state was found in both models, which were also present when comparing ADC-values to cell densities. CONCLUSIONS: Our findings demonstrate that DW-MRI can be used for non-invasive monitoring of radiation-induced changes in cell state during cancer therapy. ADC values reflect ex vivo cell density and correlates well with apoptotic state, and can hereby be described as a marker for the cell state after therapy and used as a non-invasive response marker.

A tumorsphere model of glioblastoma multiforme with intratumoral heterogeneity for quantitative analysis of cellular migration and drug response.

Glioblastoma multiforme (GBM) is the most common and malignant type of primary brain tumor and is characterized by its sudden onset and invasive growth into the brain parenchyma. The invasive tumor cells evade conventional treatments and are thought to be responsible for the ubiquitous tumor regrowth. Understanding the behavior of these invasive tumor cells and their response to therapeutic agents could help improve patient outcome. In this study, we present a GBM tumorsphere migration model with high biological complexity to study migrating GBM cells in a quantitative and qualitative manner. We demonstrated that the in vitro migration model could be used to investigate both inhibition and stimulation of cell migration with oxaliplatin and GBM-derived extracellular vesicles, respectively. The intercellular heterogeneity within the GBM tumorspheres was examined by immunofluorescent staining of nestin/vimentin and GFAP, which showed nestin and vimentin being highly expressed in the periphery of tumorspheres and GFAP mostly in cells in the tumorsphere core. We further showed that this phenotypic gradient was present in vivo after implanting dissociated GBM tumorspheres, with the cells migrating away from the tumor being nestin-positive and GFAP-negative. These results indicate that GBM tumorsphere migration models, such as the one presented here, could provide a more detailed insight into GBM cell biology and prove highly relevant as a pre-clinical platform for drug screening and assessing drug response in the treatment of GBM.

Liposome accumulation in irradiated tumors display important tumor and dose dependent differences.

Radiation therapy may affect several important parameters in the tumor microenvironment and thereby influence the accumulation of liposomes by the enhanced permeability and retention (EPR)-effect. Here we investigate the effect of single dose radiation therapy on liposome tumor accumulation by PET/CT imaging using radiolabeled liposomes. Head and neck cancer xenografts (FaDu) and syngenic colorectal (CT26) cancer models were investigated. Radiotherapy displayed opposite effects in the two models. FaDu tumors displayed increased mean accumulation of liposomes for radiation doses up to 10 Gy, whereas CT26 tumors displayed a tendency for decreased accumulation. Tumor hypoxia was found negatively correlated to microregional distribution of liposomes. However, liposome distribution in relation to hypoxia was improved at lower radiation doses. The study reveals that the heterogeneity in liposome tumor accumulation between tumors and different radiation protocols are important factors that need to be taken into consideration to achieve optimal effect of liposome based radio-sensitizer therapy.

HER2-targeted, enzyme-activated liposomes show superior in vivo efficacy in an ovarian cancer model.

Liposomes carrying chemotherapeutic drugs can accumulate passively in solid tumors at high levels. However, additional targeting of the liposomes towards e.g. receptors expressed on cancer cells may improve their interaction and therapeutic properties. In this study, we designed a liposomal delivery system, which utilizes the intrinsic characteristics of HER2-positive tumors to ensure efficient delivery of oxaliplatin to the cancer cells. On the liposome surface, trastuzumab, an antibody specific to the HER2 receptor, was shown to facilitate internalization by the cancer cells. A polyethylene glycol (PEG) layer on the liposome surface provides protection from mononuclear phagocyte system uptake. To optimize the interaction between liposomes and cancer cells, a protease-sensitive cleavable peptide linker was inserted at the base of each PEG. The PEG layer is then cleaved off by intra- and extracellular matrix metalloproteinases (MMPs) upon accumulation in the tumor. Our data demonstrate that the removal of PEG significantly destabilizes the liposomes and leads to substantial oxaliplatin release. The proposed beneficial effect of combining antibody-mediated internalization with MMP sensitivity was confirmed in a series of in vivo studies using ovarian cancer xenograft models. The results demonstrated that HER2-targeted MMP-sensitive liposomes have superior anticancer activity compared to non-targeted and non-cleavable liposomes.

Neoadjuvant Gold Nanoshell-Based Photothermal Therapy Combined with Liposomal Doxorubicin in a Mouse Model of Colorectal Cancer.

Introduction: Traditional cancer treatments, such as chemotherapy, are often incapable of achieving complete responses as standalone therapies. Hence, current treatment strategies typically rely on a combination of several approaches. Nanoparticle-based photothermal therapy (PTT) is a technique used to kill cancer cells through localized, severe hyperthermia that has shown promise as an add-on treatment to multiple cancer therapies. Here, we evaluated whether the combination of gold nanoshell (NS)-based PTT and liposomal doxorubicin could improve outcome in a mouse model of colorectal cancer. Methods: First, NS-based PTT was performed on tumor-bearing mice. Radiolabeled liposomes were then injected at different timepoints to follow their accumulation in the tumor and determine the ideal injection time after PTT. In addition, fluorescent liposomes were used to observe the liposomal distribution in the tumor after PTT. Finally, we combined PTT and doxorubicin-loaded liposomes and studied the effect of the treatment strategy on the mice by following tumor growth and survival. Results: PTT significantly improved liposomal accumulation in the tumor, but only when the liposomes were injected immediately after the therapy. The liposomes accumulated mostly in regions adjacent to the ablated areas. When PTT was combined with liposomal doxorubicin, the mice experienced a slowdown in tumor growth and an improvement in survival. Conclusion: According to our preclinical study, NS-based PTT seems promising as an add-on treatment for liposomal chemotherapy and potentially other systemic therapies, and could be relevant for future application in a clinical setting.

Development of a 64Cu-labeled CD4+ T cell targeting PET tracer: evaluation of CD4 specificity and its potential use in collagen-induced arthritis.

BACKGROUND: CD4+ T cells are central inflammatory mediators in the pathogenesis of autoimmune rheumatoid arthritis (RA), as they are one of the dominating cell types in synovial inflammation. Molecular imaging of CD4+ T cells has potential role for early detection and monitoring of RA. Here, we developed a new radiotracer for in vivo immunoPET imaging of murine CD4+ T cells and tested it in the collagen-induced arthritis (CIA) mouse model of human RA. RESULTS: The tracer, [64Cu]Cu-NOTA-CD4-F(ab)'2 ([64Cu]Cu-NOTA-CD4), was generated from F(ab)'2 fragments of R-anti-mouse CD4 antibodies conjugated to the 2-S-(isothiocyanatbenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) chelator and radiolabeled with copper-64. Accumulation of the tracer and isotype control was evaluated in the CIA model and mice receiving whole-body irradiation (WBI) (5 Gy). The potential of [64Cu]Cu-NOTA-CD4 for response assessment was evaluated in CIA induced mice treated with dexamethasone (DXM). Imaging data were compared with flow cytometry and immunohistochemistry (IHC) of inflammatory cells including CD4+ T cells. [64Cu]Cu-NOTA-CD4 showed increased accumulation in T cell-rich tissues compared with isotype control (p < 0.0001). In addition, reduced accumulation of [64Cu]Cu-NOTA-CD4 was observed in T cell-depleted tissue (p < 0.0001). Flow cytometry and IHC confirmed the increased infiltration of CD4+ T cells in CIA mice. CONCLUSIONS: We developed and evaluated a new radiotracer, [64Cu]Cu-NOTA-CD4, for immunoPET imaging of murine CD4+ T cells. [64Cu]Cu-NOTA-CD4 was successfully synthesized by F(ab)'2 fragments of R-anti-mouse CD4 antibodies conjugated to a chelator and radiolabeled with copper-64. We found that our novel CD4 PET tracer can be used for noninvasive visualization of murine CD4+ T cells.

Matrix effect in tumor lysates - Does it affect your cytokine ELISA and multiplex analyses?

Quantification of cytokines in cancerous tissue is important for understanding basic tumor biology and for deciphering anti-cancer mechanisms in drug development. Cytokine measurements on protein-level are often done by immunoassays such as enzyme-linked immunosorbent assay (ELISAs) and multiplex assays. However, immunoassays are prone to interference due to the presence of perturbing factors. The sum of these factors is known as the matrix effect, which results in a deviation of the measured cytokine concentration from the actual concentration. In this study, we demonstrated that matrix effects are present in tumor lysates from 11 different syngeneic murine tumors and that it can greatly affect cytokine measurements in ELISAs and multiplex assays. Dilution of tumor lysates and careful selection of lysis buffer components may decrease matrix effects. However, matrix effects are still present, and care should be taken when analyzing cytokine measurements of tumor lysates.

Corrigendum to 'quantitative determination of 64Cu-liposome accumulation at inflammatory and infectious sites: Potential for future theranostic system': [journal of controlled release 327 (2020) 737-746].

BACKGROUND: Therapeutic interventions for infectious and inflammatory diseases are becoming increasingly challenging in terms of therapeutic resistance and side-effects. Theranostic systems to ameliorate diagnosis and therapy are therefore highly warranted. The pathophysiological changes in inflammatory lesions provide an attractive basis for extravasation and accumulation of PEGylated liposomes. The objective of this study was to provide direct quantitative information on the theranostic potential of radiolabeled liposome for accumulation in inflammatory models using position emission tomography (PET). METHOD: Preclinical murine models of inflammation (turpentine and LPS), infection (Staphylococcus aureus) and collagen-induced arthritis (CIA) was established and monitored using bioluminescence imaging (BLI). Across all models PET imaging using radiolabeled PEGylated liposomes (64Cu-liposomes) were performed and evaluated in terms of accumulation properties in inflammatory and infectious lesions. RESULTS: BLI demonstrated that the inflammatory and infectious models were successfully established and provided information on lesion pathology. Activity of 64Cu-liposomes were increased in inflammatory and infectious lesions between early (10-min or 3-h) and late (24-h) PET scans, which validates that a continuous extravasation and accumulation of long circulation PEGylated liposomes occurs. CONCLUSION: The theranostic potential of long circulating PEGylated radiolabeled liposomes was shown in multiple preclinical models. Impressive accumulation was seen in both inflammatory and infectious lesions. These results are encouraging towards advancing PEGylated liposomes as imaging and drug delivery systems in inflammatory and infectious diseases.

Noninvasive Molecular Imaging of the Enhanced Permeability and Retention Effect by 64Cu-Liposomes: In vivo Correlations with 68Ga-RGD, Fluid Pressure, Diffusivity and 18F-FDG.

BACKGROUND: The accumulation of liposome encapsulated chemotherapy in solid cancers is dependent on the presence of the enhanced permeability and retention (EPR) effect. Positron emission tomography (PET) imaging with a liposome encapsulated radioisotope, such as liposome encapsulated Cu-64 (64Cu-liposome) may help to identify tumors with high liposome accumulation, and thereby stratify patients based on expected benefit from liposomal chemotherapy. However, intravenous administration of liposomes without a cytotoxic content is complicated by the accelerated blood clearance (ABC) phenomenon for succeeding therapeutic liposome dosing. Alternative markers for assessing the tumor's EPR level are therefore warranted. MATERIALS AND METHODS: To increase our understanding of EPR variations and to ultimately identify an alternative marker for the EPR effect, we investigated the correlation between 64Cu-liposome PET/CT (EPR effect) and 68Ga-RGD PET/CT (neoangiogenesis), 18F-FDG PET/CT (glycolysis), diffusion-weighted MRI (diffusivity) and interstitial fluid pressure in two experimental cancer models (CT26 and COLO 205). RESULTS: 64Cu-liposome and 68Ga-RGD SUVmax displayed a significant moderate correlation, however, none of the other parameters evaluated displayed significant correlations. These results indicate that differences in neoangiogenesis may explain some EPR variability, however, as correlations were only moderate and not observed for SUVmean, 68Ga-RGD is probably insufficient to serve as a stand-alone surrogate marker for quantifying the EPR effect and stratifying patients.

Injectable iodine-125 labeled tissue marker for radioactive localization of non-palpable breast lesions.

We have developed a 125I-radiolabeled injectable fiducial tissue marker with the potential to replace current methods used for surgical guidance of non-palpable breast tumors. Methods in routine clinical use today such as radioactive seed localization, radio-guided occult lesion localization and wire-guided localization suffers from limitations that this injectable fiducial tissue marker offers solutions to. The developed 125I-radiolabeled injectable fiducial tissue marker is based on highly viscous sucrose acetate isobutyrate. The marker was readily inserted in NMRI mice and proved to be spatially well-defined and stable over a seven day period with excellent CT contrast (>1500 HU), enabling fluoroscopic visualization of the marker during placement. The radioactivity remains strongly associated with the marker during the implantation period, which limits exposure to healthy tissue. Biodistribution studies show that there is negligible radioactivity in all non-tumor tissues sampled, with the exception of the thyroid gland, where limited accumulation was observed (0.06% of injected dose after 7 days). Based on the excellent performance of the marker and the fact that it can be delivered through thin hypodermic needles (≥27G), the marker holds great promise for clinical application, since patient discomfort is reduced significantly compared to current methods. STATEMENT OF SIGNIFICANCE: A new type of tissue marker for local administration to non-palpable breast tumors has been developed. The surgical guidance marker is based on derivatives of the biomaterial sucrose acetate isobutyrate and unlike currently used markers it is injectable in the tissue using thin needles, reducing the discomfort to the patients significantly. The marker confers CT contrast and has radioactive properties, meaning it also could find use in brachytherapy. The design of the iodine-125 labeled fiducial tissue marker enables control of dosimetry as well as a choice of iodine isotope used. The marker is anticipated to be clinical applicable due to its contrast performance in mice and its potential for enhanced flexibility in surgical procedures, compared to current methods.

Secretory phospholipase A2 responsive liposomes exhibit a potent anti-neoplastic effect in vitro, but induce unforeseen severe toxicity in vivo.

The clinical use of liposomal drug delivery vehicles is often hindered by insufficient drug release. Here we present the rational design of liposomes optimized for secretory phospholipase A2 (sPLA2) triggered drug release, and test their utility in vitro and in vivo. We hypothesized that by adjusting the level of cholesterol in anionic, unsaturated liposomes we could tune the enzyme specificity based on membrane fluidity, thus obtaining liposomes with an improved therapeutic outcome and reduced side effects. Cholesterol is generally important as a component in the membranes of liposome drug delivery systems due to its stabilizing effects in vivo. The incorporation of cholesterol in sPLA2 sensitive liposomes has not previously been possible due to reduced sPLA2 activity. However, in the present work we solved this challenge by optimizing membrane fluidity. In vitro release studies revealed enzyme specific drug release. Treatment of two different cancer cell lines with liposomal oxaliplatin revealed efficient growth inhibition compared to that of clinically used stealth liposomes. The in vivo therapeutic effect was evaluated in nude NMRI mice using the sPLA2 secreting mammary carcinoma cell line MT-3. Three days after first treatment all mice having received the novel sPLA2 sensitive liposome formulation were euthanized due to severe systemic toxicity. Thus the present study demonstrates that great caution should be implemented when utilizing sPLA2 sensitive liposomes and that the real utility can only be disclosed in vivo. The present studies have clinical implications, as sPLA2 sensitive formulations are currently undergoing clinical trials (LiPlaCis®).

[Canine cancer patients are included in translational research]. / Hunde med spontan cancer indgår i translationel forskning

Cancer bearing dogs represent a unique clinical cancer model with a direct potential for accelerating translation into human patients. A research collaboration between the veterinary and human medical facilities at Copenhagen University and Rigshospitalet has taken offset in this. Canine cancer patients are implemented for development of new strategies in molecular imaging and radiotherapy. The obtained results will be used to guide human clinical trials.

Dose painting based on tumor uptake of Cu-ATSM and FDG: a comparative study.

BACKGROUND: Hypoxia and increased glycolytic activity of tumors are associated with poor prognosis. The purpose of this study was to investigate differences in radiotherapy (RT) dose painting based on the uptake of 2-deoxy-2-[(18) F]-fluorodeoxyglucose (FDG) and the proposed hypoxia tracer, copper(II)diacetyl-bis(N(4))-methylsemithiocarbazone (Cu-ATSM) using spontaneous clinical canine tumor models. METHODS: Positron emission tomography/computed tomography scans of five spontaneous canine sarcomas and carcinomas were obtained; FDG on day 1 and (64)Cu-ATSM on day 2 and 3 (approx. 3 and 24 hours pi.). Sub-volumes for dose escalation were defined by a threshold-based method for both tracers and five dose escalation levels were formed in each sub-volume. Volumetric modulated arc therapy plans were optimized based on the dose escalation regions for each scan for a total of three dose plans for each dog. The prescription dose for the GTV was 45 Gy (100%) and it was linearly escalated to a maximum of 150%. The correlations between dose painting plans were analyzed with construction of dose distribution density maps and quality volume histograms (QVH). Correlation between high-dose regions was investigated with Dice correlation coefficients. RESULTS: Comparison of dose plans revealed varying degree of correlation between cases. Some cases displayed a separation of high-dose regions in the comparison of FDG vs. (64)Cu-ATSM dose plans at both time points. Among the Dice correlation coefficients, the high dose regions showed the lowest degree of agreement, indicating potential benefit of using multiple tracers for dose painting. QVH analysis revealed that FDG-based dose painting plans adequately covered approximately 50% of the hypoxic regions. CONCLUSION: Radiotherapy plans optimized with the current approach for cut-off values and dose region definitions based on FDG, (64)Cu-ATSM 3 h and 24 h uptake in canine tumors had different localization of the regional dose escalation levels. This indicates that (64)Cu-ATSM at two different time-points and FDG provide different biological information that has to be taken into account when using the dose painting strategy in radiotherapy treatment planning.