CCL5 Neutralization Restricts Cancer Growth and Potentiates the Targeting of PDGFRβ in Colorectal Carcinoma

Increased CCL5 levels are markers of an unfavourable outcome in patients with melanoma, breast, cervical, prostate, gastric or pancreatic cancer. Here, we have assessed the role played by CCL5/CCR5 interactions in the development of colon cancer. To do so, we have examined a number of human colorectal carcinoma clinical specimens and found CCL5 and its receptors over-expressed within primary as well as liver and pulmonary metastases of patients compared to healthy tissues. In vitro, CCL5 increased the growth and migratory responses of colon cancer cells from both human and mouse origins. In addition, systemic treatment of mice with CCL5-directed antibodies reduced the extent of development of subcutaneous colon tumors, of liver metastases and of peritoneal carcinosis. Consistently, we found increased numbers of CD45-immunoreactive cells within the stroma of the remaining lesions as well as at the interface with the healthy tissue. In contrast, selective targeting of CCR5 through administration of TAK-779, a CCR5 antagonist, only partially compromised colon cancer progression. Furthermore, CCL5 neutralization rendered the tumors more sensitive to a PDGFRβ-directed strategy in mice, this combination regimen offering the greatest protection against liver metastases and suppressing macroscopic peritoneal carcinosis. Collectively, our data demonstrate the involvement of CCL5 in the pathogenesis of colorectal carcinoma and point to its potential value as a therapeutic target

Amphiphilic block copolymers enhance the cellular uptake of DNA molecules through a facilitated plasma membrane transport

Amphiphilic block copolymers have been developed recently for their efficient, in vivo transfection activities in various tissues. Surprisingly, we observed that amphiphilic block copolymers such as Lutrol® do not allow the transfection of cultured cells in vitro, suggesting that the cell environment is strongly involved in their mechanism of action. In an in vitro model mimicking the in vivo situation we showed that pre-treatment of cells with Lutrol®, prior to their incubation with DNA molecules in the presence of cationic lipid, resulted in higher levels of reporter gene expression. We also showed that this improvement in transfection efficiency associated with the presence of Lutrol® was observed irrespective of the plasmid promoter. Considering the various steps that could be improved by Lutrol®, we concluded that the nucleic acids molecule internalization step is the most important barrier affected by Lutrol®. Microscopic examination of transfected cells pre-treated with Lutrol® confirmed that more plasmid DNA copies were internalized. Absence of cationic lipid did not impair Lutrol®-mediated DNA internalization, but critically impaired endosomal escape. Our results strongly suggest that in vivo, Lutrol® improves transfection by a physicochemical mechanism, leading to cellular uptake enhancement through a direct delivery into the cytoplasm, and not via endosomal pathways

Normalisation to Blood Activity Is Required for the Accurate Quantification of Na/I Symporter Ectopic Expression by SPECT/CT in Individual Subjects

The utilisation of the Na/I symporter (NIS) and associated radiotracers as a reporter system for imaging gene expression is now reaching the clinical setting in cancer gene therapy applications. However, a formal assessment of the methodology in terms of normalisation of the data still remains to be performed, particularly in the context of the assessment of activities in individual subjects in longitudinal studies. In this context, we administered to mice a recombinant, replication-incompetent adenovirus encoding rat NIS, or a human colorectal carcinoma cell line (HT29) encoding mouse NIS. We used 99mTc pertechnetate as a radiotracer for SPECT/CT imaging to determine the pattern of ectopic NIS expression in longitudinal kinetic studies. Some animals of the cohort were culled and NIS expression was measured by quantitative RT-PCR and immunohistochemistry. The radioactive content of some liver biopsies was also measured ex vivo. Our results show that in longitudinal studies involving datasets taken from individual mice, the presentation of non-normalised data (activity expressed as %ID/g or %ID/cc) leads to ‘noisy’, and sometimes incoherent, results. This variability is due to the fact that the blood pertechnetate concentration can vary up to three-fold from day to day. Normalisation of these data with blood activities corrects for these inconsistencies. We advocate that, blood pertechnetate activity should be determined and used to normalise the activity measured in the organ/region of interest that expresses NIS ectopically. Considering that NIS imaging has already reached the clinical setting in the context of cancer gene therapy, this normalisation may be essential in order to obtain accurate and predictive information in future longitudinal clinical studies in biotherapy

Organ-specific inhibition of metastatic colon carcinoma by CXCR3 antagonism

Liver and lung metastases are the predominant cause of colorectal cancer (CRC)-related mortality. Recent research has indicated that CXCR3/chemokines interactions that orchestrate haematopoetic cell movement are implicated in the metastatic process of malignant tumours, including that of CRC cells to lymph nodes. To date, however, the contribution of CXCR3 to liver and lung metastasis in CRC has not been addressed. To determine whether CXCR3 receptors regulate malignancy-related properties of CRC cells, we have used CXCR3-expressing CRC cell lines of human (HT29 cells) and murine (C26 cells) origins that enable the development of liver and lung metastases when injected into immunodeficient and immunocompetent mice, respectively, and assessed the effect of CXCR3 blockade using AMG487, a small molecular weight antagonist. In vitro, activation of CXCR3 on human and mouse CRC cells by its cognate ligands induced migratory and growth responses, both activities being abrogated by AMG487. In vivo, systemic CXCR3 antagonism by preventive or curative treatments with AMG487 markedly inhibited the implantation and the growth of human and mouse CRC cells within lung without affecting that in the liver. In addition, we measured increased levels of CXCR3 and ligands expression within lung nodules compared with liver tumours. Altogether, our findings indicate that activation of CXCR3 receptors by its cognate ligands facilitates the implantation and the progression of CRC cells within lung tissues and that inhibition of this axis decreases pulmonary metastasis of CRC in two murine tumour models

Les copolymères à blocs pour le transfert de gènes dans le muscle squelettique

Le transfert de gènes dans le muscle squelettique à l'aide de vecteurs synthétiques représente une stratégie prometteuse pour le traitement de maladies génétiques héréditaires ou acquises. Parmi les vecteurs synthétiques utilisés pour le transfert de gènes dans cet organe, les copolymères à blocs présentent de nombreux avantages en terme d'efficacité et de toxicité. Ces vecteurs se sont révélés être très efficaces pour permettre l'expression de la dystrophine et la restauration du complexe dystroglycoprotéique membranaire chez la souris myopathe. Le muscle squelettique peut également être utilisé comme organe sécréteur de protéines thérapeutiques systémiques. Le transfert du gène de l'érythropoïétine à l'aide de copolymères à blocs a permis la correction de l'anémie chez un modèle de souris en insuffisance rénale chronique. L'utilisation d'un promoteur inductible par la doxycycline permet de réguler l'expression du gène de l'érythropoïétine jusqu'à 4 mois après le transfert de gène avant la mise en place d'une réaction immunitaire contre le transactivateur chimérique. La répétition d'injections intramusculaires du gène codant l'érythropoïétine formulé avec des copolymères à blocs, permet d'adapter durablement le niveau de sécrétion d'érythropoïétineThere is conclusive evidence that muscle gene therapy using synthetic vectors can lead to real clinical benefit for treatment of genetic or acquired diseases. Block copolymers are new efficient and non toxic vectors used for gene transfer in this organ. These vectors have been used to restore efficiently the expression ofdystrophin and led to the restoration of the dystrophin proteins complex in dystrophic mice. This approach using new synthetic vectors is particularly appropriate to obtain long-term circulating therapeutic protein. Intramuscular injections of a plasmid DNA encoding this protein formulated with block copolymer prevent or correct anemia induced by acquired renal failure in adenine treated mice. The temporal regulation of erythropoietin gene expression was obtained by the use of a tetracyclin inducible promoter. However, an immune response against the chimeric transactivator was observed 4 months post-injection stopping the EPO synthesis. The development of a protocol consisting in repeated intramuscular injections of block copolymer/DNA formulations provides an efficient method for a durable and an adjustable secretion of erythropoietin.NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Adrenal gland infection by serotype 5 adenovirus requires coagulation factors.

Recombinant, replication-deficient serotype 5 adenovirus infects the liver upon in vivo, systemic injection in rodents. This infection requires the binding of factor X to the capsid of this adenovirus. Another organ, the adrenal gland is also infected upon systemic administration of Ad, however, whether this infection is dependent on the cocksackie adenovirus receptor (CAR) or depends on the binding of factor X to the viral capsid remained to be determined. In the present work, we have used a pharmacological agent (warfarin) as well as recombinant adenoviruses lacking the binding site of Factor X to elucidate this mechanism in mice. We demonstrate that, as observed in the liver, adenovirus infection of the adrenal glands in vivo requires Factor X. Considering that the level of transduction of the adrenal glands is well-below that of the liver and that capsid-modified adenoviruses are unlikely to selectively infect the adrenal glands, we have used single-photon emission computed tomography (SPECT) imaging of gene expression to determine whether local virus administration (direct injection in the kidney) could increase gene transfer to the adrenal glands. We demonstrate that direct injection of the virus in the kidney increases gene transfer in the adrenal gland but liver transduction remains important. These observations strongly suggest that serotype 5 adenovirus uses a similar mechanism to infect liver and adrenal gland and that selective transgene expression in the latter is more likely to be achieved through transcriptional targeting

Localization of the kidneys using ^99mTc-DMSA.

<p>Mice were injected intra-peritoneally with 95 MBq of ^99mTc-DMSA. Five hours later, the animals were anaesthetized and SPECT/CT scans performed. A) Transverse, coronal and sagittal sections centered on the kidneys. B) Volume rendering of the whole animal in which the kidneys appear in yellow. Legend: K: kidney, L: left, post: posterior side of the animal.</p