In vitro grafting of hepatic spheroids and organoids on a microfluidic vascular bed.

With recent progress in modeling liver organogenesis and regeneration, the lack of vasculature is becoming the bottleneck in progressing our ability to model human hepatic tissues in vitro. Here, we introduce a platform for routine grafting of liver and other tissues on an in vitro grown microvascular bed. The platform consists of 64 microfluidic chips patterned underneath a 384-well microtiter plate. Each chip allows the formation of a microvascular bed between two main lateral vessels by inducing angiogenesis. Chips consist of an open-top microfluidic chamber, which enables addition of a target tissue by manual or robotic pipetting. Upon grafting a liver microtissue, the microvascular bed undergoes anastomosis, resulting in a stable, perfusable vascular network. Interactions with vasculature were found in spheroids and organoids upon 7 days of co-culture with space of Disse-like architecture in between hepatocytes and endothelium. Veno-occlusive disease was induced by azathioprine exposure, leading to impeded perfusion of the vascularized spheroid. The platform holds the potential to replace animals with an in vitro alternative for routine grafting of spheroids, organoids, or (patient-derived) explants.

Organ Distribution and Bone Tropism of Cellulose Nanocrystals in Living Mice.

Their physicochemical properties and relatively low cost make cellulose nanocrystals (CNCs) a potential candidate for future large-scale production in many fields including nanomedicine. Prior to a sustained and responsible development as theranostic agents, robust and reliable data concerning their safety, biocompatibility, and tissue distribution should be provided. In the present study, CNCs were extracted from Whatman filters functionalized with a fluorescent dye, and their interaction with living organisms has been thoroughly assessed. Our experimental evidence demonstrated that CNCs (1) are well tolerated by healthy mice after systemic injection; (2) are rapidly excreted, thus avoiding bioaccumulation in filter organs such as the kidneys and liver; (3) transiently migrate in bones; and (4) are able to penetrate in the cytoplasm of cancer cells without inducing material-related detrimental effects in terms of cell survival. Our results strongly suggest that the peculiar tropism to the bones is due to the chemical interaction between the Ca(2+) of the bone matrix and the active surface of negatively-charged CNCs. This feature, together with the ability to penetrate cancer cells, makes CNCs a potential nanodevice for theranostics in bone tumors.

Direct but not indirect co-culture with osteogenically differentiated human bone marrow stromal cells increases RANKL/OPG ratio in human breast cancer cells generating bone metastases.

BACKGROUND: Bone metastases arise in nearly 70% of patients with advanced breast cancer, but the complex metastatic process has not been completely clarified yet. RANKL/RANK/OPG pathway modifications and the crosstalk between metastatic cells and bone have been indicated as potential drivers of the process. Interactions between tumor and bone cells have been studied in vivo and in vitro, but specific effects of the direct contact between human metastatic cells and human bone cells on RANKL/RANK/OPG pathway have not been investigated. FINDINGS: We directly co-cultured bone metastatic human breast cancer cells (BOKL) with osteo-differentiated human mesenchymal cells (BMSCs) from 3 different donors. BMSCs and BOKL were then enzymatically separated and FACS sorted. We found a significant increase in the RANKL/OPG ratio as compared to control, which was not observed in BOKL cultured in medium conditioned by BMSCs, neither in BOKL directly cultured with fibroblasts or medium conditioned by fibroblasts. Direct co-culture with osteo-differentiated BMSCs caused BOKL aggregation while proliferation was not affected by co-culture. To more specifically associate RANKL expression to osteogenic differentiation degree of BMSCs, we determined their osteogenic markers expression and matrix calcification relative to osteoblasts and fibroblasts. CONCLUSIONS: In conclusion, our co-culture model allowed to demonstrate for the first time that direct contact but not paracrine interactions between human metastatic breast cancer cells and bone cells has a significant effect on RANKL/OPG expression in bone metastatic cells. Furthermore, only direct contact with the bone microenvironment induced BOKL clustering without however significantly influencing their proliferation and migration.

The Healthcare Relationship during the Second Wave of the COVID-19 Pandemic: A Qualitative Study in the Emergency Department of an Italian Hospital.

The COVID-19 pandemic had an impact not only on people's lives but also on the healthcare system. This study aimed to investigate the healthcare relationship in the Emergency Department (ED) of a hospital in northern Italy, during the second wave of the COVID-19 pandemic. The participants (N = 43) consisted of 16 nurses, 6 doctors from the hospital ED, and 21 patients who accessed this department. Semi-structured interviews were carried out and a thematic analysis was conducted. The findings suggest that the COVID-19 pandemic brought both positive and negative changes to the healthcare relationship that are linked to changes in the organization of the ED and to participants' various experiences of the pandemic. The changes in this relationship should be monitored because they could have long-term effects on healthcare professionals' wellbeing, treatment outcomes, and the healthcare system. The findings from this study could be used to understand these changes and inform intervention strategies to improve the healthcare relationship.

Inhibition of Sp1-dependent transcription and antitumor activity of the new aureolic acid analogues mithramycin SDK and SK in human ovarian cancer xenografts.

OBJECTIVE: Increased activity of Sp family of transcription factors is a frequent and critical event in cancer development and progression. Genes governing tumor growth, invasion and angiogenesis are regulated by Sp factors, like Sp1, Sp3 or Sp4, and are frequently over-expressed in tumors. Targeting Sp factors has been explored as a therapeutic approach. Mithramycin (MTM) is a natural antibiotic that binds DNA and inhibit Sp1-dependent transcription. New analogues, named MTM-SDK and MTM-SK, were recently obtained by genetic engineering of the MTM biosynthetic pathway and have demonstrated improved transcriptional and antiproliferative activity in ovarian cancer cell lines in vitro. In the present study we evaluated the activity of the new compounds in human ovarian cancer xenografts. METHODS: Expression of Sp1 and target proteins in ovarian cancer specimens and tumor xenografts was assessed by immunohistochemistry. Drug-induced silencing of Sp1-regulated genes in cells and tumor xenograft samples was assessed by quantitative RT-PCR. Toxicity and antitumor activity of the compounds were investigated in healthy and tumor-bearing immunocompromised mice, respectively. RESULTS: Expression of Sp1 was frequently increased in human epithelial ovarian cancers. MTM-SDK and MTM-SK acted as potent inhibitors of Sp1-dependent transcription both in vitro and in tumor xenografts. Both compounds were well tolerated even after prolonged administration and delayed growth of ovarian tumor xenografts. MTM-SDK was particularly effective against orthotopic tumors leading to a significant increase of survival and delay of tumor progression. CONCLUSIONS: MTM-SDK and MTM-SK show relevant activity in vivo and represent interesting candidates for treatment of ovarian cancers.

Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency.

BACKGROUND: Argininosuccinate synthase (ASS)1 is a urea cycle enzyme that catalyzes the conversion of citrulline and aspartate to argininosuccinate. Mutations in the ASS1 gene cause citrullinemia type I, a rare autosomal recessive disorder characterized by neonatal hyperammonemia, elevated citrulline levels, and early neonatal death. Treatment for this disease is currently restricted to liver transplantation; however, due to limited organ availability, substitute therapies are required. Recently, extracellular vesicles (EVs) have been reported to act as intercellular transporters carrying genetic information responsible for cell reprogramming. In previous studies, we isolated a population of stem cell-like cells known as human liver stem cells (HLSCs) from healthy liver tissue. Moreover, EVs derived from HLSCs were reported to exhibit regenerative effects on the liver parenchyma in models of acute liver injury. The aim of this study was to evaluate whether EVs derived from normal HLSCs restored ASS1 enzymatic activity and urea production in hepatocytes differentiated from HLSCs derived from a patient with type I citrullinemia. METHODS: HLSCs were isolated from the liver of a patient with type I citrullinemia (ASS1-HLSCs) and characterized by fluorescence-activated cell sorting (FACS), immunofluorescence, and DNA sequencing analysis. Furthermore, their differentiation capabilities in vitro were also assessed. Hepatocytes differentiated from ASS1-HLSCs were evaluated by the production of urea and ASS enzymatic activity. EVs derived from normal HLSCs were purified by differential ultracentrifugation followed by floating density gradient. The EV content was analyzed to identify the presence of ASS1 protein, mRNA, and ASS1 gene. In order to obtain ASS1-depleted EVs, a knockdown of the ASS1 gene in HLSCs was performed followed by EV isolation from these cells. RESULTS: Treating ASS1-HLSCs with EVs from HLSCs restored both ASS1 activity and urea production mainly through the transfer of ASS1 enzyme and mRNA. In fact, EVs from ASS1-knockdown HLSCs contained low amounts of ASS1 mRNA and protein, and were unable to restore urea production in hepatocytes differentiated from ASS1-HLSCs. CONCLUSIONS: Collectively, these results suggest that EVs derived from normal HLSCs may compensate the loss of ASS1 enzyme activity in hepatocytes differentiated from ASS1-HLSCs.

Questioning the oncogenic role of DeltaNp73alpha in different cell lines expressing p53 or not.

The recent finding that the 1p36.3 locus gene encodes an array of different p73 isoforms with apparently distinct and sometimes opposing cellular functions, might explain the difficulty in establishing the protein's role as tumor suppressor. Therefore we need to investigate the roles of each of these splicing variants in cellular functions when expressed alone or in combination with other family members, as well as the genetic background on which the proteins are expressed. We investigated, in two p53 null cell lines, the human SCLC line H1299 and a subline derived from the human colon carcinoma cell line HCT116 (HCT116/379.2), the effects of DeltaNp73alpha overexpression on cell growth and the response to anticancer treatment. We generated three different clones overexpressing DeltaNp73alpha under a tetracycline inducible promoter. Immunofluorescent staining and luciferase reporter assays confirmed that clones HCT116/DeltaNA and H1299/DeltaN7 and H1299/DeltaN11 did express a functional, nuclear localized DeltaNp73alpha protein. The stable overexpression of DeltaNp73alpha protein did not confer any cell growth advantage. Doubling time of clones overexpressing DeltaNp73alpha were comparable to counterparts not expressing it. Clonogenic assays showed that the cytotoxic activity of different DNA damaging agents, such as cDDP, UV light and doxorubicin, were comparable in clones expressing DeltaNp73 or not. The overall data argue against an oncogenic role for this isoform. These findings are independent of the p53 status since they overlap with those previously obtained by our group in HCT116 cell lines, wild type for p53.

Modeling Staphylococcus epidermidis-Induced Non-Unions: Subclinical and Clinical Evidence in Rats.

S. epidermidis is one of the leading causes of orthopaedic infections associated with biofilm formation on implant devices. Open fractures are at risk of S. epidermidis transcutaneous contamination leading to higher non-union development compared to closed fractures. Although the role of infection in delaying fracture healing is well recognized, no in vivo models investigated the impact of subclinical low-grade infections on bone repair and non-union. We hypothesized that the non-union rate is directly related to the load of this commonly retrieved pathogen and that a low-grade contamination delays the fracture healing without clinically detectable infection. Rat femurs were osteotomized and stabilized with plates. Fractures were infected with a characterized clinical-derived methicillin-resistant S. epidermidis (10(3), 10(5), 10(8) colony forming units) and compared to uninfected controls. After 56 days, bone healing and osteomyelitis were clinically assessed and further evaluated by micro-CT, microbiological and histological analyses. The biofilm formation was visualized by scanning electron microscopy. The control group showed no signs of infection and a complete bone healing. The 10(3) group displayed variable response to infection with a 67% of altered bone healing and positive bacterial cultures, despite no clinical signs of infection present. The 10(5) and 10(8) groups showed severe signs of osteomyelitis and a non-union rate of 83-100%, respectively. The cortical bone reaction related to the periosteal elevation in the control group and the metal scattering detected by micro-CT represented limitations of this study. Our model showed that an intra-operative low-grade S. epidermidis contamination might prevent the bone healing, even in the absence of infectious signs. Our findings also pointed out a dose-dependent effect between the S. epidermidis inoculum and non-union rate. This pilot study identifies a relevant preclinical model to assess the role of subclinical infections in orthopaedic and trauma surgery and to test specifically designed diagnostic, prevention and therapeutic strategies.

In vivo evaluation of bone deposition in macroporous titanium implants loaded with mesenchymal stem cells and strontium-enriched hydrogel.

Bone-implant integration represents a major requirement to grant implant stability and reduce the risk of implant loosening. This study investigates the effect of progenitor cells and strontium-enriched hydrogel on the osseointegration of titanium implants. To mimic implant-bone interaction, an ectopic model was developed grafting Trabecular Titanium(™) (TT) implants into decellularized bone seeded with human bone marrow mesenchymal stem cells (hBMSCs). TT was loaded or not with strontium-enriched amidated carboxymethylcellulose (CMCA) hydrogel and/or hBMSCs. Constructs were implanted subcutaneously in athymic mice and osteodeposition was investigated with microcomputed tomography (micro-CT), scanning electron microscopy (SEM), and pull-out test at 4, 8, and 12 weeks. Fluorescence imaging was performed at 8 and 12 weeks, histology at 4 and 8 weeks. Micro-CT demonstrated the homogeneity of the engineered bone in all groups, supporting the reproducibility of the ectopic model. Fluorescence imaging, histology, SEM and pull-out mechanical testing showed superior tissue ingrowth in TT implants loaded with both strontium-enriched CMCA and hBMSCs. In our model, the synergic action of the bioactive hydrogel and hBMSCs increased both the bone deposition and TT integration. Thus, we suggest that using orthopedic prosthetic implant preloaded with strontium-enriched CMCA and seeded with BMSCs could represent a valid single-step surgical strategy to improve implant osseointegration.

Longitudinal tracking of triple labeled umbilical cord derived mesenchymal stromal cells in a mouse model of Amyotrophic Lateral Sclerosis.

The translational potential of cell therapy to humans requires a deep knowledge of the interaction between transplanted cells and host tissues. In this study, we evaluate the behavior of umbilical cord mesenchymal stromal cells (UC-MSCs), labeled with fluorescent nanoparticles, transplanted in healthy or early symptomatic transgenic SOD1G93A mice (a murine model of Amyotrophic Lateral Sclerosis). The double labeling of cells with nanoparticles and Hoechst-33258 enabled their tracking for a long time in both cells and tissues. Whole-body distribution of UC-MSCs was performed by in-vivo and ex-vivo analyses 1, 7, 21 days after single intravenous or intracerebroventricular administration. By intravenous administration cells were sequestered by the lungs and rapidly cleared by the liver. No difference in biodistribution was found among the two groups. On the other hand, UC-MSCs transplanted in lateral ventricles remained on the choroid plexus for the whole duration of the study even if decreasing in number. Few cells were found in the spinal cord of SOD1G93A mice exclusively. No migration in brain parenchyma was observed. These results suggest that the direct implantation in brain ventricles allows a prolonged permanence of cells close to the damaged areas and makes this method of tracking reliable for future studies of efficacy.

Does PGE1 vasodilator prevent orthopaedic implant-related infection in diabetes? Preliminary results in a mouse model.

BACKGROUND: Implant-related infections are characterized by bacterial colonization and biofilm formation on the prosthesis. Diabetes represents one of the risk factors that increase the chances of prosthetic infections because of related severe peripheral vascular disease. Vasodilatation can be a therapeutic option to overcome diabetic vascular damages and increase the local blood supply. In this study, the effect of a PGE1 vasodilator on the incidence of surgical infections in diabetic mice was investigated. METHODOLOGY: A S. aureus implant-related infection was induced in femurs of diabetic mice, then differently treated with a third generation cephalosporin alone or associated with a PGE1 vasodilator. Variations in mouse body weight were evaluated as index of animal welfare. The femurs were harvested after 28 days and underwent both qualitative and quantitative analysis as micro-CT, histological and microbiological analyses. RESULTS: The analysis performed in this study demonstrated the increased host response to implant-related infection in diabetic mice treated with the combination of a PGE1 and antibiotic. In this group, restrained signs of infections were identified by micro-CT and histological analysis. On the other hand, the diabetic mice treated with the antibiotic alone showed a severe infection and inability to successfully respond to the standard antimicrobial treatment. CONCLUSIONS: The present study revealed interesting preliminary results in the use of a drug combination of antibiotic and vasodilator to prevent implant-related Staphylococcus aureus infections in a diabetic mouse model.

In vivo fate of avidin-nucleic acid nanoassemblies as multifunctional diagnostic tools.

This study describes the formulation optimization and body-cell distribution and clearance in mice of a dually fluorescent biodegradable poly avidin nanoassembly based on the novel Avidin-Nucleic-Acid-Nano-ASsembly (ANANAS) platform as a potential advancement of classic avidin/biotin-based targeted delivery. The nanoformulation circulates freely in the bloodstream; it is slowly captured by filter organs; it is efficiently cleared within 24-48 h, and it is poorly immunogenic. The system displays more favorable properties than its parent monomeric avidin and it is a promising tool for diagnostic purposes for future translational aims, for which free circulation in the bloodstream, safety, multifunctionality and high composition definition are all necessary requirements. In addition, the assembly shows a time-dependent cell penetration capability, suggesting it may also function as a NP-dependent drug delivery tool. The ease of preparation together with the possibility to fine-tune the surface composition makes it also an ideal candidate to understand if and how nanoparticle composition affects its localization.

Combination of the c-Met inhibitor tivantinib and zoledronic acid prevents tumor bone engraftment and inhibits progression of established bone metastases in a breast xenograft model.

Bone is the most common metastatic site for breast cancer. There is a significant need to understand the molecular mechanisms controlling the engraftment and growth of tumor cells in bone and to discover novel effective therapeutic strategies. The aim of this study was to assess the effects of tivantinib and Zoledronic Acid (ZA) in combination in a breast xenograft model of bone metastases. Cancer cells were intracardially implanted into immunodeficient mice and the effects of drugs alone or in combination on bone metastasis were evaluated by in vivo non-invasive optical and micro-CT imaging technologies. Drugs were administered either before (preventive regimen) or after (therapeutic regimen) bone metastases were detectable. In the preventive regimen, the combination of tivantinib plus ZA was much more effective than single agents in delaying bone metastatic tumor growth. When administered in the therapeutic schedule, the combination delayed metastatic progression and was effective in improving survival. These effects were not ascribed to a direct cytotoxic effect of the combined therapy on breast cancer cells in vitro. The results of this study provide the rationale for the design of new combinatorial strategies with tivantinib and ZA for the treatment of breast cancer bone metastases.

Diabetic mouse model of orthopaedic implant-related Staphylococcus aureus infection.

BACKGROUND: Periprosthetic bacterial infections represent one of the most challenging orthopaedic complications that often require implant removal and surgical debridement and carry high social and economical costs. Diabetes is one of the most relevant risk factors of implant-related infection and its clinical occurrence is growing worldwide. The aim of the present study was to test a model of implant-related infection in the diabetic mouse, with a view to allow further investigation on the relative efficacy of prevention and treatment options in diabetic and non-diabetic individuals. METHODOLOGY: A cohort of diabetic NOD/ShiLtJ mice was compared with non-diabetic CD1 mice as an in vivo model of S. aureus orthopaedic infection of bone and soft tissues after femur intramedullary pin implantation. We tested control and infected groups with 1×10(3) colony-forming units of S. aureus ATCC 25923 strain injected in the implant site. At 4 weeks post-inoculation, host response to infection, microbial biofilm formation, and bone damage were assessed by traditional diagnostic parameters (bacterial culture, C-reactive protein and white blood cell count), histological analysis and imaging techniques (micro computed tomography and scanning electron microscopy). RESULTS: Unlike the controls and the CD1 mice, all the diabetic mice challenged with a single inoculum of S. aureus displayed severe osteomyelitic changes around the implant. CONCLUSIONS: Our findings demonstrate for the first time that the diabetic mouse can be successfully used in a model of orthopaedic implant-related infection. Furthermore, the same bacteria inoculum induced periprosthetic infection in all the diabetic mice but not in the controls. This animal model of implant-related infection in diabetes may be a useful tool to test in vivo treatments in diabetic and non-diabetic individuals.

Breast cancer-derived bone metastasis can be effectively reduced through specific c-MET inhibitor tivantinib (ARQ 197) and shRNA c-MET knockdown.

Breast cancer exhibits a propensity to metastasize to bone, resulting in debilitating skeletal complications associated with significant morbidity and poor prognosis. The cross-talk between metastatic cancer cells and bone is critical to the development and progression of bone metastases. We have shown the involvement of the HGF/c-MET system in tumor-bone interaction contributing to human breast cancer metastasis. Therefore, disruption of HGF/c-MET signaling is a potential targeted approach to treating metastatic bone disease. In this study, we evaluated the effects of c-MET inhibition by both an oral, selective, small-molecule c-MET inhibitor, tivantinib, and a specific short hairpin RNA (shRNA) against c-MET in a mouse model of human breast cancer. Tivantinib exhibited dose-dependent antimetastatic activity in vivo, and the 120 mg/kg dose, proven to be suboptimal in reducing subcutaneous tumor growth, induced significant inhibition of metastatic growth of breast cancer cells in bone and a noteworthy reduction of tumor-induced osteolysis. shRNA-mediated c-MET silencing did not affect in vitro proliferation of bone metastatic cells, but significantly reduced their migration, and this effect was further enhanced by tivantinib. Both observations were confirmed in vivo. Indeed, more pronounced tumor growth suppression with concomitant marked decreases of lytic lesions and prolongation of survival were achieved by dual c-MET inhibition using both tivantinib and RNA interference strategies. Overall, our findings highlighted the effectiveness of c-MET inhibition in delaying the onset and progression of bone metastases and strongly suggest that targeting c-MET may have promising therapeutic value in the treatment of bone metastases from breast cancer.

Multiple drug delivery hydrogel system for spinal cord injury repair strategies.

The multifactorial pathological progress of spinal cord injury (SCI) is probably the main reason behind the absence of efficient therapeutic approaches. Hence, very recent highlights suggest the use of new multidrug delivery systems capable of local controlled release of therapeutic agents. In this work, a biocompatible hydrogel-based system was developed as multiple drug delivery tool, specifically designed for SCI repair strategies. Multiple release profiles were achieved by loading gel with a combination of low and high steric hindrance molecules. In vitro, in vivo and ex vivo release studies showed an independent combination of fast diffusion-controlled kinetics for smaller molecules together with slow diffusion-controlled kinetics for bigger ones. A preserved functionality of loaded substances was always achieved, confirming the absence of any chemical stable interactions between gel matrix and loaded molecules. Moreover, the relevant effect of the cerebrospinal fluid flux dynamics on the drug diffusion in the spinal cord tissue was here revealed for the first time: an oriented delivery of the released molecules in the spinal cord tract caudally to the gel site is demonstrated, thus suggesting a more efficient gel positioning rostrally to the lesion.

Interaction between human-breast cancer metastasis and bone microenvironment through activated hepatocyte growth factor/Met and beta-catenin/Wnt pathways.

To clarify the reciprocal interaction between human-breast cancer metastatic cells and bone microenvironment, we studied the influence of HGF/Met system on a proposed-prognostic marker of aggressiveness, the beta-catenin/Wnt pathway. For in vitro and in vivo experiments we used 1833-bone metastatic clone, derived from human-MDA-MB231 cells. In osteolytic bone metastases and in metastatic cells, Met was expressed in nuclei and at plasma membrane, and abnormally co-localised at nuclear level with beta-catenin and the tyrosine phosphorylated c-Src kinase. Thus, in 1833 cells nuclear-Met COOH-terminal fragment and beta-catenin-TCF were constitutively activated, possibly by receptor and non-receptor tyrosine kinases. The activity of the gene reporter TOPFLASH (containing multiple TCF/LEF-consensus sites) was measured, as index of beta-catenin functionality. In 1833 cells, human and mouse HGF increased Met and beta-catenin tyrosine phosphorylation and expression in nuclear and perinuclear compartments, beta-catenin nuclear translocation via Kank and TOPFLASH transactivation. Human HGF was autocrine/intracrine in bone metastasis, and mouse HGF originating from the adjacent host-bone marrow, was found inside the metastatic nuclei. Parental MDA-MB231 cell nuclei did not show functional beta-catenin, for TCF-transactivating activity, and the regulation by HGF. Our study highlighted the importance of the metastasis-stroma interaction in human-breast cancer metastatisation and first identified the HGF/nuclear Met/phospho-c-Src/beta-catenin-TCF/Wnt pathway as a potential-therapeutic target to delay establishment/progression of bone metastases by affecting the aggressive phenotype.

Phospholipase Cgamma1 is required for metastasis development and progression.

Cell motility and invasion play an essential role in the development of metastasis. Evidence suggests that the enzyme phospholipase Cgamma1 (PLCgamma1) may be involved in tumor progression and possibly development of metastasis. In this study, we show that down-regulation of PLCgamma1 expression severely impairs activation of the small GTP-binding protein Rac and cell invasion in breast cancer cell lines and U87 in vitro. Experimental metastasis assays in nude mice show that inducible knockdown of PLCgamma1 strongly inhibits development of MDA-MB-231-derived lung metastasis and reverts metastasis formation. In addition, analysis of 60 breast cancer patients' tissues revealed an increase of PLCgamma1 expression in metastasis compared with the primary tumor in 50% of tissues analyzed. These data show a critical role of PLCgamma1 in the metastatic potential of cancer cells, and they further indicate that PLCgamma1 inhibition has a therapeutic potential in the treatment of metastasis dissemination.

In vivo evaluation of the role of DNp73alpha protein in regulating the p53-dependent apoptotic pathway after treatment with cytotoxic drugs.

The amino terminus truncated p73 isoform, DeltaNp73alpha, shows dominant negative behavior toward TAp73 and wild-type p53, and has oncogenic potential. By contrast, we recently showed that in HCT116 clones forced expression of DeltaNp73alpha did not increase in vitro cellular resistance to anticancer agents. The purpose of this study was to characterize in vivo models and to investigate the functional interaction between the DeltaNp73alpha isoform and the p53 pathway. Human colon carcinoma HCT116 clones expressing inducible DeltaNp73alpha (HCT116/DN3, HCT116/DN14) and HCT116/8a (transfected with the mock empty vector), transplanted in immunodeficient nude mice, were used to study the antitumor activity of cis-diammine-dichloro-platinum (cDDP) (4 mg/kg, i.v., q7d x 3) and Doxorubicin (DX) (7.5 mg/kg, i.v., q7d x 3), with or without tetracycline-induced DeltaNp73alpha overexpression. DeltaNp73alpha expression was confirmed by RT-PCR, immunoblotting and immunohistochemical analysis. DeltaNp73alpha subcellular localization after DX treatment was checked by an immunofluorescence assay. Western blot was used to analyze p53, p21, Bax, Bcl-2 and p53AIP1 expression. DeltaNp73alpha overexpression did not modify the antitumor activity of either DX or cDDP in xenograft models. DX reduced DeltaNp73alpha protein expression, without affecting its nuclear localization. p53, p21, Bax and p53AIP1 protein expression increased and Bcl-2 decreased in HCT116 clone derived tumors 24 hr after DX exposure, independently of the presence of DeltaNp73alpha. Overexpression of DeltaNp73alpha does not affect tumor growth in vivo, does not increase the resistance of established tumors to anticancer agents and does not antagonize p53 apoptotic functions.