Retinoic acid-loaded PLGA nanocarriers targeting cell cholesterol potentialize the antitumour effect of PD-L1 antibody by preventing epithelial-mesenchymal transition mediated by M2-TAM in colorectal cancer.

Tumour-associated macrophages (TAMs) often promote cancer progression through immunosuppression in the tumour microenvironment (TME). However, the signalling pathways crosstalk responsible for this mechanism remain unclear. The aim of our study was to investigate whether the interaction between TAMs and colorectal cancer cells could be down-regulated by nanoparticles (NPs) loaded with retinoic acid (RA) and coated with cholesterol (CHO), in combination with an anti-PD-L1 immune checkpoint inhibitor. Tumours were evaluated by qRT-PCR and immunohistochemistry from allographic tumour growth model. In addition, human tumours were evaluated by Tissue Microarray (TMA) and immunohistochemistry. Complementary analysis of epithelial-mesenchymal transition, cell migration, and macrophage polarisation were evaluated in vitro. We showed that the IL-10R/IL-10 axis is involved in overstimulation of the STAT3 pathway as well as downregulation of the NF-κB signalling pathway, which supports a loop of immunosuppressive cytokines that induces the M2-TAM phenotype. Furthermore, our combined findings suggest that the upregulation of STAT3/NF-κB pathways crosstalk mediated by immunosuppressive cytokines, such as IL-10/PD-L1/TGF-ß, via M2-TAMs in the TME, leads to immunosuppression and epithelial-mesenchymal-transition of the colorectal cancer for stimulating Vimentin, CXCL12 and CD163 in the primary tumours. Importantly, NPs holding RA and coated with CHO in combination with anti-PD-L1 were more efficient in blocking this signalling pathway. These results contribute to our understanding of the immunological mechanisms, especially the re-educating of TAMs, and provide a novel management strategy for aggressive colorectal cancers using anti-PD-L1-conjugated nanocarriers.

Extracellular Vesicles from M1-Polarized Macrophages Combined with Hyaluronic Acid and a ß-Blocker Potentiate Doxorubicin's Antitumor Activity by Downregulating Tumor-Associated Macrophages in Breast Cancer.

One of the main reasons for cancer's low clinical response to chemotherapeutics is the highly immunosuppressive tumor microenvironment (TME). Tumor-ass ociated M2 macrophages (M2-TAMs) orchestrate the immunosuppression, which favors tumor progression. Extracellular vesicles (EVs) have shown great potential for targeted therapies as, depending on their biological origin, they can present different therapeutic properties, such as enhanced accumulation in the target tissue or modulation of the immune system. In the current study, EVs were isolated from M1-macrophages (M1-EVs) pre-treated with hyaluronic acid (HA) and the ß-blocker carvedilol (CV). The resulting modulated-M1 EVs (MM1-EVs) were further loaded with doxorubicin (MM1-DOX) to assess their effect in a mouse model of metastatic tumor growth. The cell death and cell migration profile were evaluated in vitro in 4T1 cells. The polarization of the RAW 264.7 murine macrophage cell line was also analyzed to evaluate the effects on the TME. Tumors were investigated by qRT-PCR and immunohistochemistry. MM1-DOX reduced the primary tumor size and metastases. NF-κB was the major gene downregulated by MM1-DOX. Furthermore, MM1-DOX reduced the expression of M2-TAM (CD-163) in tumors, which resulted in increased apoptosis (FADD) as well as decreased expression of MMP-2 and TGF-ß. These results suggest a direct effect in tumors and an upregulation in the TME immunomodulation, which corroborate with our in vitro data that showed increased apoptosis, modulation of macrophage polarization, and reduced cell migration after treatment with M1-EVs combined with HA and CV. Our results indicate that the M1-EVs enhanced the antitumor effects of DOX, especially if combined with HA and CV in an animal model of metastatic cancer.

Zinc-Phthalocyanine-Loaded Extracellular Vesicles Increase Efficacy and Selectivity of Photodynamic Therapy in Co-Culture and Preclinical Models of Colon Cancer.

Photodynamic therapy (PDT) is a promising and clinically approved method for the treatment of cancer. However, the efficacy of PDT is often limited by the poor selectivity and distribution of the photosensitizers (PS) toward the malignant tumors, resulting in prolonged periods of skin photosensitivity. In this work, we present a simple and straightforward strategy to increase the tumor distribution, selectivity, and efficacy of lipophilic PS zinc phthalocyanine (ZnPc) in colon cancer by their stabilization in purified, naturally secreted extracellular vesicles (EVs). The PS ZnPc was incorporated in EVs (EV-ZnPc) by a direct incubation strategy that did not affect size distribution or surface charge. By using co-culture models simulating a tumor microenvironment, we determined the preferential uptake of EV-ZnPc toward colon cancer cells when compared with macrophages and dendritic cells. We observed that PDT promoted total tumor cell death in normal and immune cells, but showed selectivity against cancer cells in co-culture models. In vivo assays showed that after a single intravenous or intratumoral injection, EV-ZnPc were able to target the tumor cells and strongly reduce tumor growth over 15 days. These data expose opportunities to enhance the potential and efficacy of PDT using simple non-synthetic strategies that might facilitate translation into clinical practice.

AT1 and AT2 Receptor Knockout Changed Osteonectin and Bone Density in Mice in Periodontal Inflammation Experimental Model.

BACKGROUND: The aim of this study was to evaluate the role of AT1 and AT2 receptors in a periodontal inflammation experimental model. METHODS: Periodontal inflammation was induced by LPS/Porphyromonas gingivalis. Maxillae, femur, and vertebra were scanned using Micro-CT. Maxillae were analyzed histopathologically, immunohistochemically, and by RT-PCR. RESULTS: The vertebra showed decreased BMD in AT1 H compared with WT H (p < 0.05). The femur showed increased Tb.Sp for AT1 H and AT2 H, p < 0.01 and p < 0.05, respectively. The Tb.N was decreased in the vertebra (WT H-AT1 H: p < 0.05; WT H-AT2 H: p < 0.05) and in the femur (WT H-AT1 H: p < 0.01; WT H-AT2 H: p < 0.05). AT1 PD increased linear bone loss (p < 0.05) and decreased osteoblast cells (p < 0.05). RANKL immunostaining was intense for AT1 PD and WT PD (p < 0.001). OPG was intense in the WT H, WT PD, and AT2 PD when compared to AT1 PD (p < 0.001). AT1 PD showed weak immunostaining for osteocalcin compared with WT H, WT PD, and AT2 PD (p < 0.001). AT1 H showed significantly stronger immunostaining for osteonectin in fibroblasts compared to AT2 H (p < 0.01). CONCLUSION: AT1 receptor knockout changed bone density, the quality and number of bone trabeculae, decreased the number of osteoblast cells, and increased osteonectin in fibroblasts.

Maximizing the potency of oxaliplatin coated nanoparticles with folic acid for modulating tumor progression in colorectal cancer.

One of the challenges of nanotechnology is to improve the efficacy of treatments for diseases, in order to reduce morbidity and mortality rates. Following this line of study, we made a nanoparticle formulation with a small size, uniform surfaces, and a satisfactory encapsulation coefficient as a target for colorectal cancer cells. The results of binding and uptake prove that using the target system with folic acid works: Using this system, cytotoxicity and cell death are increased when compared to using free oxaliplatin. The data show that the system maximized the efficiency of oxaliplatin in modulating tumor progression, increasing apoptosis and decreasing resistance to the drug. Thus, for the first time, our findings suggest that PLGA-PEG-FA increases the antitumor effectiveness of oxaliplatin by functioning as a facilitator of drug delivery in colorectal cancer.

STAT3/NF-κB signalling disruption in M2 tumour-associated macrophages is a major target of PLGA nanocarriers/PD-L1 antibody immunomodulatory therapy in breast cancer.

BACKGROUND AND PURPOSE: Inflammation associated with the tumour microenvironment (TME) is critical for cancer development, and immunotherapeutic strategies modulating the immune response in cancer have been crucial. In this study, a methotrexate-loaded (MTX) poly(lactic-co-glycolic acid)-based (PLGA) drug nanocarrier covered with polyethyleneimine (Pei) and hyaluronic acid (HA) was developed and combined with an PD-L1 antibody to investigate anti-cancer and immunomodulatory effects in breast cancer TME. EXPERIMENTAL APPROACH: Naked or HA-coated PeiPLGA-MTX nanoparticles (NPs) were assessed on 4T1 breast cancer cells grown in culture and in a mouse model of orthotopic tumour growth. Tumours were evaluated by qRT-PCR and immunohistochemistry. The cell death profile and cell migration were analysed in vitro in 4T1 cells. Polarization of murine macrophages (RAW cells) was also carried out. KEY RESULTS: Naked or HA-coated PeiPLGA-MTX NPs used alone or combined with PD-L1 antibody modified the tumourigenic course by TME immunomodulation, leading to reduction of primary tumour size and metastases. STAT3 and NF-κB were the major genes downregulated by NPs. In tumor-associated macrophages (TAM) such regulation switched M2 phenotype (CD163) towards M1 (CD68) and reduced levels of IL-10, TGF-ß and CCL22. Moreover, malignant cells showed overexpression of FADD, APAF-1, caspase-3 and E-cadherin, and decreased expression of Bcl-2, MDR-1, survivin, vimentin, CXCR4 and PD-L1 after treatment with NPs. CONCLUSION AND IMPLICATIONS: NPs-mediated STAT3/NF-κB signalling axis suppression disrupted crosstalk between immune and malignant cells, reducing immunosuppression and critical pro-tumour events. These findings provide a promising therapeutic approach capable of guiding the immune TME to suppress the development of breast cancer.

Exploiting the Natural Properties of Extracellular Vesicles in Targeted Delivery towards Specific Cells and Tissues.

Extracellular vesicles (EVs) are important mediators of intercellular communication that participate in many physiological/pathological processes. As such, EVs have unique properties related to their origin, which can be exploited for drug delivery applications in cell regeneration, immunosuppression, inflammation, cancer treatment or cardioprotection. Moreover, their cell-like membrane organization facilitates uptake and accumulation in specific tissues and organs, which can be exploited to improve selectivity of cargo delivery. The combination of these properties with the inclusion of drugs or imaging agents can significantly improve therapeutic efficacy and selectivity, reduce the undesirable side effects of drugs or permit earlier diagnosis of diseases. In this review, we will describe the natural properties of EVs isolated from different cell sources and discuss strategies that can be applied to increase the efficacy of targeting drugs or other contents to specific locations. The potential risks associated with the use of EVs will also be addressed.

Zirconia/hydroxyapatite (80/20) scaffold repair in critical size calvarial defect increased FGF-2, osteocalcin and OPG immunostaining and IL-10 levels.

The aim of this study was to characterize and evaluate zirconia/hydroxyapatite in a critical size calvarial defect model in rats. Zirconia/hydroxyapatite (80/20) scaffold was characterized by X-ray diffraction (XRD) and Scanning Electron Microscopy (SEM). Critical size (8 mm) calvarial defects were created in wistar rats (n=48) and divided into four groups (90 days): G0 Group: positive control; G1 Group: hydroxyapatite; G2 Group: Zirconia; G3 Group: Zirconia/hydroxyapatite (80/20). Calvaria were subjected to Micro CT, histological and immunohistochemical analyses (RANK, RANKL, OPG, osteocalcin and FGF-2). IL-1 beta, IL-10 and TNF-alpha levels were analyzed by Elisa Immunoassay. The XRD analysis confirmed the formation of a crystalline structure and SEM showed the presence of regions corresponding to Zirconia and Hydroxyapatite. The Micro CT showed increased bone volume (BV/TV) and bone mineral density (BMD) in the G3 group (P<0.05). In addition, discrete periosteal bone formation was found at the interface of the defect edge and the external surface of the scaffold in the G3 group, showing osteocytes inside and osteoblasts (P<0.05) with scarce mononuclear inflammatory cells (P<0.01) in the central region of the defect. The immunostaining was moderate for RANKL, Osteocalcin and FGF-2 in the G3 group (P<0.5), while it was intense for OPG (P<0.001). IL-1 beta levels were decreased and IL-10 levels increased (P<0.05). Zirconia/hydroxyapatite (80/20) scaffold repair in critical size calvarial defects increased bone density, osteoblast and osteoclast cell numbers, FGF-2, osteocalcin and OPG immunostaining and IL-10 levels.

Correction to: Ceramide and palmitic acid inhibit macrophage-mediated epithelial-mesenchymal transition in colorectal cancer.

The third and fifth author's affiliation was published incorrectly in the original article. Also, the Figure 5 and the Acknowledgement section was published incorrectly. The corrected affiliation, Figure 5 and the Acknowledgement section are provided in this correction.

Ceramide and palmitic acid inhibit macrophage-mediated epithelial-mesenchymal transition in colorectal cancer.

Accumulating evidence indicates that ceramide (Cer) and palmitic acid (PA) possess the ability to modulate switching of macrophage phenotypes and possess anti-tumorigenic effects; however, the underlying molecular mechanisms are largely unknown. The aim of the present study was to investigate whether Cer and PA could induce switching of macrophage polarization from the tumorigenic M2- towards the pro-inflammatory M1-phenotype, and whether this consequently altered the potential of colorectal cancer cells to undergo epithelial-mesenchymal transition (EMT), a hallmark of tumor progression. Our study showed that Cer- and PA-treated macrophages increased expression of the macrophage 1 (M1)-marker CD68 and secretion of IL-12 and attenuated expression of the macrophage 2 (M2)-marker CD163 and IL-10 secretion. Moreover, Cer and PA abolished M2 macrophage-induced EMT and migration of colorectal cancer cells. At the molecular level, this coincided with inhibition of SNAI1 and vimentin expression and upregulation of E-cadherin. Furthermore, Cer and PA attenuated expression levels of IL-10 in colorectal cancer cells co-cultured with M2 macrophages and downregulated STAT3 and NF-κB expression. For the first time, our findings suggest the presence of an IL-10-STAT3-NF-κB signaling axis in colorectal cancer cells co-cultured with M2 macrophages, mimicking the tumor microenvironment. Importantly, PA and Cer were powerful inhibitors of this signaling axis and, consequently, EMT of colorectal cancer cells. These results contribute to our understanding of the immunological mechanisms that underlie the anti-tumorigenic effects of lipids for future combination with drugs in the therapy of colorectal carcinoma.

Environmentally compatible bioconjugated gold nanoparticles as efficient contrast agents for inflammation-induced cancer imaging.

For many cancers, early detection is the key to improve survival and reduce the morbidity, which is associated with radical resections due to late diagnosis. Here, we describe the efficiency of primary antibody-conjugated gold nanoparticles (AuNPs) to specifically target chronic inflammatory processes, specially M2 macrophages, in tissue sections of ulcerative colitis (UC) and steatohepatitis in rats which may lead to colorectal cancer and liver carcinoma, respectively. In this study, we demonstrate that AuNPs synthesized by a simple, inexpensive, and environmentally compatible method can be easily conjugated with the antibodies anti-COX-2, anti-MIF, and Alexa Fluor® 488 (ALEXA) to perform immunofluorescence staining in inflamed tissues. Moreover, we showed that primary antibody-conjugated gold nanoparticles (AuNPs) can be used to target M2 macrophages by flow cytometry. We designed three immunofluorescence staining protocols of tissue section with AuNPs for 30 min and overnight incubation, as well as one flow cytometry protocol of M2 macrophage labeling with AuNPs for 30 min. Immunofluorescence and flow cytometry results suggest that conjugation was achieved by direct adsorption of antibodies on the AuNPs surface. When compared to the standard ALEXA protocol in immunofluorescence (IF) and flow cytometry (FC), our 30-min incubation protocol using AuNPs instead of ALEXA decreased from approximately 23 h to 5 h for IF and from 4 h to 1 h for FC, proving to be less laborious, which makes the method eligible for inflammation-induced cancer diagnostic.

Spherical neutral gold nanoparticles improve anti-inflammatory response, oxidative stress and fibrosis in alcohol-methamphetamine-induced liver injury in rats.

This study aimed to elucidate the anti-inflammatory, anti-oxidant and antifibrotic effects of gold nanoparticles (GNPs) in rats subjected to liver injury with ethanol and Methamphetamine (METH). The liver injury was induced by gavage administrations of 30% alcoholic solution (7 g/kg) once a day during 28 days, followed by METH (10 mg/kg) on the 20th and 28th days of treatment. GNPs treatment (724.96 µg/kg) during the ethanol and METH exposure was associated with reduced steatosis, hepatic cord degeneration, fibrosis and necrosis. Furthermore, there was a reduction in biochemical markers of liver damage and oxidative stress, and pro-inflammatory cytokines IL-1ß and TNF-α, compared to ethanol + METH group alone. A decrease of FGF, SOD-1 and GPx-1 expression was also observed. GNPs down-regulated the activity of Kupffer cells and hepatic stellate cells affecting the profile of their pro-inflammatory cytokines, oxidative stress and fibrosis through modulation of signaling pathways AKT/PI3K and MAPK in ethanol + METH-induced liver injury in a rat model.

Fluorescence- and multispectral optoacoustic imaging for an optimized detection of deeply located tumors in an orthotopic mouse model of pancreatic carcinoma.

A crucial point for the management of pancreatic ductal adenocarcinoma (PDAC) is the decrease of R1 resections. Our aim was to evaluate the combination of multispectral optoacoustic tomography (MSOT) with fluorescence guided surgery (FGS) for diagnosis and perioperative detection of tumor nodules and resection margins in a xenotransplant mouse model of human pancreatic cancer. The peptide cRGD, conjugated with the near infrared fluorescent (NIRF) dye IRDye800CW and with a trans-cyclooctene (TCO) tag for future click chemistry (cRGD-800CW-TCO), was applied to PDAC bearing immunodeficient nude mice; 27 days after orthotopic transplantation of human AsPC-1 cells into the head of the pancreas, mice were injected with cRGD-800CW-TCO and imaged with fluorescence- and optoacoustic devices before and 2, 6 and 24 hr after injection, before they were sacrificed and dissected with a guidance of FGS imaging system. Fluorescence imaging of cRGD-800CW-TCO allowed detection of the tumor area but without information about the depth, whereas MSOT allowed high resolution 3 D identification of the tumor area, in particular of small tumor nodules. Highly sensitive delineation of tumor burden was achieved during FGS in all mice. Imaging of whole-mouse cryosections, histopathological analysis and NIRF microscopy confirmed the localization of cRGD-800CW-TCO within the tumor tissue. In principle, all imaging modalities applied here were able to detect PDAC in vivo. However, the combination of MSOT and FGS provided detailed spatial information of the signal and achieved a complete overview of the distribution and localization of cRGD-800CW-TCO within the tumor before and during surgical intervention.

Apoptosis in human liver carcinoma caused by gold nanoparticles in combination with carvedilol is mediated via modulation of MAPK/Akt/mTOR pathway and EGFR/FAAD proteins.

In cancers, apoptosis signaling pathways and cell survival and growth pathways responsible for resistance to conventional treatments, such as Pi3K/Akt/mTOR and mitogen-activated protein kinase (MAPK) become dysregulated. Recently, alternative treatments to promote tumor cell death have become important. The present study reports on the antitumor and cytoprotective action of gold nanoparticles (GNPs) and carvedilol in combination and in isolated application. Apoptosis was analyzed by FITC/propidium iodide staining flow cytometry; caspase-3, caspase-8, Bcl-2 and MAPK/ERK activity by immunofluorescence microscopy; gene expression of proteins related to cell death as Akt, mTOR, EGFR, MDR1, survivin, FADD and Apaf, by the real-time PCR; and western blot analysis for MAPK/ERK, Akt and mTOR. Oxidative stress evaluation was performed by reduced glutathione (GSH) and malondialdehyde (MDA) levels. Intracellular GNPs targets were identified by transmission electron microscopy. After exposure to a combination of GNPs (6.25 µg/ml) and carvedilol (3 µM), death as promoted by apoptosis was detected using flow cytometry, for expression of pro-apoptotic proteins FADD, caspase-3, caspase-8 and sub-regulation of anti-apoptotic MAPK/ERK, Akt, mTOR, EGFR and MDR1 resistance. Non-tumor cell cytoprotection with GSH elevation and MDA reduction levels was detected. GNPs were identified within the cell near to the nucleus when combined with carvedilol. The combination of GNP and carvedilol promoted downregulation of anti-apoptotic and drug resistance genes, over-regulation of pro-apoptotic proteins in tumor cells, as well as cytoprotection of non-tumor cells with reduction of apoptosis and oxidative stress.

Evaluation of EphA2 and EphB4 as Targets for Image-Guided Colorectal Cancer Surgery.

Targeted image-guided oncologic surgery (IGOS) relies on the recognition of cell surface-associated proteins, which should be abundantly present on tumor cells but preferably absent on cells in surrounding healthy tissue. The transmembrane receptor tyrosine kinase EphA2, a member of the A class of the Eph receptor family, has been reported to be highly overexpressed in several tumor types including breast, lung, brain, prostate, and colon cancer and is considered amongst the most promising cell membrane-associated tumor antigens by the NIH. Another member of the Eph receptor family belonging to the B class, EphB4, has also been found to be upregulated in multiple cancer types. In this study, EphA2 and EphB4 are evaluated as targets for IGOS of colorectal cancer by immunohistochemistry (IHC) using a tissue microarray (TMA) consisting of 168 pairs of tumor and normal tissue. The IHC sections were scored for staining intensity and percentage of cells stained. The results show a significantly enhanced staining intensity and more widespread distribution in tumor tissue compared with adjacent normal tissue for EphA2 as well as EphB4. Based on its more consistently higher score in colorectal tumor tissue compared to normal tissue, EphB4 appears to be a promising candidate for IGOS of colorectal cancer. In vitro experiments using antibodies on human colon cancer cells confirmed the possibility of EphB4 as target for imaging.

Traumatic Brain Injury: Preclinical Imaging Diagnostic(s) and Therapeutic Approaches.

BACKGROUND: Traumatic brain injury (TBI) is the result of an external physical force to the head that harms the brain. TBI is a major public health problem worldwide and mainly results from falls, vehicle accidents and violence. Clinical problem: The management of TBI, causing a wide spectrum of possible health outcomes, has barely changed over the years as encouraging outcomes from many pre-clinical therapeutic and pharmacological studies have only rarely been translated to the clinical situation. New management options: In the last decades management of TBI is rapidly advancing and new innovative imaging modalities with sophisticated treatment options by using nanomedicine based drug delivery systems are under investigation. Nano formulations such as PLGA, exosomes and liposomes have the advantage of a targeted and controlled delivery of their cargo, such as diagnostic probes and/or therapeutic drugs. SUMMARY: Here we provide an overview of new promising pre-clinical developments in TBI management that may find their way to the clinic in the near future. Nanotechnology and nanomedicine in TBI intervention may establish new platforms for targeted drug delivery to the traumatized brain to improve the quality of life and survival of TBI patients.

The Necrosis-Avid Small Molecule HQ4-DTPA as a Multimodal Imaging Agent for Monitoring Radiation Therapy-Induced Tumor Cell Death.

PURPOSE: Most effective antitumor therapies induce tumor cell death. Non-invasive, rapid and accurate quantitative imaging of cell death is essential for monitoring early response to antitumor therapies. To facilitate this, we previously developed a biocompatible necrosis-avid near-infrared fluorescence (NIRF) imaging probe, HQ4, which was radiolabeled with 111Indium-chloride (111In-Cl3) via the chelate diethylene triamine pentaacetic acid (DTPA), to enable clinical translation. The aim of the present study was to evaluate the application of HQ4-DTPA for monitoring tumor cell death induced by radiation therapy. Apart from its NIRF and radioactive properties, HQ4-DTPA was also tested as a photoacoustic imaging probe to evaluate its performance as a multimodal contrast agent for superficial and deep tissue imaging. MATERIALS AND METHODS: Radiation-induced tumor cell death was examined in a xenograft mouse model of human breast cancer (MCF-7). Tumors were irradiated with three fractions of 9 Gy each. HQ4-DTPA was injected intravenously after the last irradiation, NIRF and photoacoustic imaging of the tumors were performed at 12, 20, and 40 h after injection. Changes in probe accumulation in the tumors were measured in vivo, and ex vivo histological analysis of excised tumors was performed at experimental endpoints. In addition, biodistribution of radiolabeled [111In]DTPA-HQ4 was assessed using hybrid single-photon emission computed tomography-computed tomography (SPECT-CT) at the same time points. RESULTS: In vivo NIRF imaging demonstrated a significant difference in probe accumulation between control and irradiated tumors at all time points after injection. A similar trend was observed using in vivo photoacoustic imaging, which was validated by ex vivo tissue fluorescence and photoacoustic imaging. Serial quantitative radioactivity measurements of probe biodistribution further demonstrated increased probe accumulation in irradiated tumors. CONCLUSION: HQ4-DTPA has high specificity for dead cells in vivo, potentiating its use as a contrast agent for determining the relative level of tumor cell death following radiation therapy using NIRF, photoacoustic imaging and SPECT in vivo. Initial preclinical results are promising and indicate the need for further evaluation in larger cohorts. If successful, such studies may help develop a new multimodal method for non-invasive and dynamic deep tissue imaging of treatment-induced cell death to quantitatively assess therapeutic response in patients.

Identification of receptor-type protein tyrosine phosphatase µ as a new marker for osteocytes.

Osteocytes are the predominant cells in bone, where they form a cellular network and display important functions in bone homeostasis, phosphate metabolism and mechanical transduction. Several proteins strongly expressed by osteocytes are involved in these processes, e.g., sclerostin, DMP-1, PHEX, FGF23 and MEPE, while others are upregulated during differentiation of osteoblasts into osteocytes, e.g., osteocalcin and E11. The receptor-type protein tyrosine phosphatase µ (RPTPµ) has been described to be expressed in cells which display a cellular network, e.g., endothelial and neuronal cells, and is implied in mechanotransduction. In a capillary outgrowth assay using metatarsals derived from RPTPµ-knock-out/LacZ knock-in mice, we observed that the capillary structures grown out of the metatarsals were stained blue, as expected. Surprisingly, cells within the metatarsal bone tissue were positive for LacZ activity as well, indicating that RPTPµ is also expressed by osteocytes. Subsequent histochemical analysis showed that within bone, RPTPµ is expressed exclusively in early-stage osteocytes. Analysis of bone marrow cell cultures revealed that osteocytes are present in the nodules and an enzymatic assay enabled the quantification of the amount of osteocytes. No apparent bone phenotype was observed when tibiae of RPTPµ-knock-out/LacZ knock-in mice were analyzed by µCT at several time points during aging, although a significant reduction in cortical bone was observed in RPTPµ-knock-out/LacZ knock-in mice at 20 weeks. Changes in trabecular bone were more subtle. Our data show that RPTPµ is a new marker for osteocytes.

Quantitative assessment of a novel flow-through porous microarray for the rapid analysis of gene expression profiles.

A novel microarray system that utilizes a porous aluminum-oxide substrate and flow-through incubation has been developed for rapid molecular biological testing. To assess its utility in gene expression analysis, we determined hybridization kinetics, variability, sensitivity and dynamic range of the system using amplified RNA. To show the feasibility with complex biological RNA, we subjected Jurkat cells to heat-shock treatment and analyzed the transcriptional regulation of 23 genes. We found that trends (regulation or no change) acquired on this platform are in good agreement with data obtained from real-time quantitative PCR and Affymetrix GeneChips. Additionally, the system demonstrates a linear dynamic range of 3 orders of magnitude and at least 10-fold decreased hybridization time compared to conventional microarrays. The minimum amount of transcript that could be detected in 20 microl volume is 2-5 amol, which enables the detection of 1 in 300,000 copies of a transcript in 1 microg of amplified RNA. Hybridization and subsequent analysis are completed within 2 h. Replicate hybridizations on 24 identical arrays with two complex biological samples revealed a mean coefficient of variation of 11.6%. This study shows the potential of flow-through porous microarrays for the rapid analysis of gene expression profiles in clinical applications.