MR Imaging Biomarkers to Monitor Early Response to Hypoxia-Activated Prodrug TH-302 in Pancreatic Cancer Xenografts.

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RESEARCH ARTICLE MR Imaging Biomarkers to Monitor Early Response to Hypoxia-Activated Prodrug TH302 in Pancreatic Cancer Xenografts Xiaomeng Zhang1*, Jonathan W. Wojtkowiak1, Gary V. Martinez1, Heather H. Cornnell1, Charles P. Hart2, Amanda F. Baker3, Robert Gillies1 a11111 1 H. Lee Moffitt Cancer Center, Tampa, FL, United States of America, 2 Threshold Pharmaceuticals, South San Francisco, CA, United States of America, 3 Arizona Cancer Center, Tucson, AZ, United States of America * Abstract OPEN ACCESS Citation: Zhang X, Wojtkowiak JW, Martinez GV, Cornnell HH, Hart CP, Baker AF, et al. (2016) MR Imaging Biomarkers to Monitor Early Response to Hypoxia-Activated Prodrug TH-302 in Pancreatic Cancer Xenografts. PLoS ONE 11(5): e0155289. doi:10.1371/journal.pone.0155289 Editor: Roger Chammas, Universidade de São Paulo, BRAZIL Received: June 11, 2014 Accepted: April 27, 2016 Published: May 26, 2016 Copyright: © 2016 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper, and its Supporting Information files are available through Figshare ( 6084/m9.figshare.3179140). Funding: This work was supported by: US National Institutes of Health: R01CA125627, R01CA077575 and U54CA143970;; the funding above was used in the study design, data collection and analysis; Moffitt Cancer Center Small Animal Imaging Laboratory, a CCSG funded core facility, CCSG Grant number: P30-CA76292;; the funding above was used for part of small animal imaging. TH-302 is a hypoxia-activated prodrug known to activate selectively under the hypoxic conditions commonly found in solid tumors. It is currently being evaluated in clinical trials, including two trials in Pancreatic Ductal Adenocarcinomas (PDAC). The current study was undertaken to evaluate imaging biomarkers for prediction and response monitoring of TH302 efficacy in xenograft models of PDAC. Dynamic contrast-enhanced (DCE) and diffusion weighted (DW) magnetic resonance imaging (MRI) were used to monitor acute effects on tumor vasculature and cellularity, respectively. Three human PDAC xenografts with known differential responses to TH-302 were imaged prior to, and at 24 h and 48 hours following a single dose of TH-302 or vehicle to determine if imaging changes presaged changes in tumor volumes. DW-MRI was performed at five b-values to generate apparent diffusion coefficient of water (ADC) maps. For DCE-MRI, a standard clinically available contrast reagent, Gd-DTPA, was used to determine blood flow into the tumor region of interest. TH302 induced a dramatic decrease in the DCE transfer constant (Ktrans) within 48 hours after treatment in the sensitive tumors, Hs766t and Mia PaCa-2, whereas TH-302 had no effect on the perfusion behavior of resistant SU.86.86 tumors. Tumor cellularity, estimated from ADC, was significantly increased 24 and 48 hours after treatment in Hs766t, but was not observed in the Mia PaCa-2 and SU.86.86 groups. Notably, growth inhibition of Hs766t was observed immediately (day 3) following initiation of treatment, but was not observed in MiaPaCa-2 tumors until 8 days after initiation of treatment. Based on these preclinical findings, DCE-MRI measures of vascular perfusion dynamics and ADC measures of cell density are suggested as potential TH-302 response biomarkers in clinical trials. Introduction The five-year survival rate for pancreatic adenocarcinoma (PDAC) is less than 6% and most survivors are those patients with a surgical option.[1–5] Thus, the majority of pancreatic PLOS ONE | DOI:10.1371/journal.pone.0155289 May 26, 2016 1 / 13 Imaging Biomarkers to Monitor Early Response to Prodrug TH-302 Threshold Pharmaceuticals provided support in the form of a salary for author CPH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Charles P. Hart is employed by and a stockholder of Threshold Pharmaceuticals. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. cancers are treated systemically with chemotherapy, generally gemcitabine (GEM), in combination with other agents. Notably, specific targeted therapies and biologicals such as cetuximab, trastuzamab or bevacizumab have shown little effect against PDAC. A regimen combining 5-Fluorouracil, leucovorin, irinotecan and oxaliplatin (FOLFIRINOX) increased median overall survival (OS) to 11.1 months, compared to 6.8 months for GEM alone.[6] Recently, nab-paclitaxel (ABI-007; nanoparticle-albumin-paclitaxel; Abraxane) has shown significant survival benefit in combination with GEM, and is being considered for front-line status.[7, 8] Even so, response still remains only fleeting and, hence, alternative therapeutic approaches are needed. One promising avenue is to target the cancer’s phenotype, such as hypoxia, which is often observed in pancreatic cancers.[9] This was investigated in Phase I/II (NCT01833546; NCT02047500) trials of TH-302 in combination with GEM, which are complete and a Phase III (NCT01746979) trial of the TH-302 + GEM doublet is underway. Further, a Phase I/II dose escalation trial of TH-302 + GEM + Abraxane triplet combination has recently been opened (NCT02047500). Tumor hypoxia, characterized by reduced oxygen concentrations, is a significant factor driving tumor physiology and resistance to cancer treatment.[10] The major difference from normal tissue is the irregular structured vasculature matrix found in solid tumors. The microvasculature is characterized by large openings in the endothelium and absence of smooth muscle layer, leading to Enhanced Permeability and Retention (EPR) of macromolecules. These factors result in an unbalanced supply of oxygen and nutrients. This exacerbates the sprouting and inefficient growth of new vessels, which leads to a reduced tumor oxygenation.[11, 12] Although several cancers are known to be hypoxic, pancreatic cancers are known to be profoundly so [13] and intratumoral hypoxia is related to a poor outcome.[14–16] This may be due to the increased levels of the survival factor, HIF-1α [15–18], or selection for defects in the apoptotic machinery.[17] Furthermore, the effect of hypoxia may not be mediated solely by the cancer cells, but may also involve the stroma. Pancreatic cancer is characterized by excessive desmoplastic fibroblasts (stellate cells), whose migration, type I collagen expression, and vascular endothelial growth factor (VEGF) production are all induced by hypoxia.[18] Fundamentally, hypoxia results from an imbalance between oxygen supply and demand. Hypoxic tumor regions are generally resistant to cytotoxic chemotherapy. In part this is due to the direct effects of hypoxia in upregulating cell survival pathways but may also be due to regional perfusion deficits, which result in both hypoxia and sub-therapeutic drug delivery. [14–16, 19, 20] Furthermore, hypoxic tumor cells tend to be quiescent, which also leads to resistance to therapies that are targeted to cell division.[21] Because of the relationship between hypoxia and poor response, one solution for hypoxia-related cancer is introducing hypoxia-activated prodrugs (HAPs), which are selectively activated under extreme hypoxia and deliver high-dose chemotherapy into the hypoxic regions of the tumor with minimal damage in healthy tissue. The preponderance of hypoxia in PDAC makes it an attractive target for HAPs [22], which have been developed as a family of compounds over the past two decades.[23] HAPs are converted into an active form in reduced oxygen conditions, as they are designed to undergo 1-eor 2-e- bio-reductions that are reversible in the presence of oxygen. These can be non-specifically catalyzed by a variety of oxoreductases, such as NADPH cytochrome p450 reductase, POR, or inducible nitric oxide synthase, iNOS.[24] The most common class of HAPs is based on 2-nitroimidazoles, exemplified by TH-302. TH-302 is a second-generation HAP that is currently being tested in 12 different clinical trials from Phase I-Phase III. TH-302 releases bromo-isophosphoramide mustard (Br-IPM) under hypoxic conditions, inducing DNA alkylation and cross-linking. Br-IPM can also diffuse into nearby oxygenated regions to prevent tumor progression via “bystander effects”. Both in vitro and in vivo experiments have demonstrated that TH-302 is selectively activated in the tumor hypoxic regions and is also present in PLOS ONE | DOI:10.1371/journal.pone.0155289 May 26, 2016 2 / 13 Imaging Biomarkers to Monitor Early Response to Prodrug TH-302 the surrounding tissue, but less so in healthy tissues[25]. Pre-clinical studies suggest that TH302 has significant anti-cancer effect when combined with anti-angiogenic therapies.[26] Hypoxic regions are often characterized by highly disorganized, dilated and tortuous vessels.[27] This chaotic pattern may result from imbalances of angiogenic mediators such as vascular endothelial growth factor (VEGF) and angiopoietins. Highly permeable tumor vessels may affect cytokines and angiogenic factors, which can dynamically alter the structure of the microvessel walls.[28] Accumulating evidence has already demonstrated that vascular permeability is most likely elevated in hypoxic regions.[27] Thus, evidential and empirical concordance between vessel permeability and hypoxia is established. DCE-MRI uses kinetic modeling of injected contrast reagents to characterize perfusion.[29] It can yield information on the flow, volume, and most importantly the permeability of vessels. Thus it is suitable to assume that DCE-MRI could be a useful surrogate biomarker of hypoxia.[30] Diffusion-weighted MRI (DW-MRI) detects relatively small changes in tissue structure at the cellular level based on the application of motion sensitive gradients that can trace the movement of water protons. Many preclinical studies have shown that DW-MRI has tremendous potential for monitoring early changes in tumor cellularity that may relate to treatment response.[31] Combination of DCE and DW-MRI may provide more accurate information for early diagnosis and detection of early therapeutic response. [32] Furthermore, these two imaging methods are readily deployed in daily clinic protocols. As such, it is very likely that the results of this study could be easily translated to additional clinic trials. Previous studies have demonstrated xenograft tumors derived from SU.86.86 human pancreatic cancer cell line are highly vascularized and well-perfused compared to tumors derived from Hs766t cell line.[33] Thus, hypoxic regions observed in Hs766t tumors are larger than those observed in SU.86.86, while tumors derived from Mia PaCa-2 cell line are hypoxic to a medium level. As hypoxia is considered to be a limiting factor for TH-302 activity, it has been experimentally shown that Hs766t-derived tumors are highly sensitive to TH-302 and that SU.86.86-derived tumors are highly resistant, with Mia PaCa-2 exhibit intermediate sensitivity. [34] The present study compared the efficacy of TH-302 in these pancreatic cancer xenograft models with measurements of tumor perfusion and cellularity by using two imaging methods: DW-MRI and DCE-MRI. Materials and Methods Cell culture Su.86.86, Hs766t and Mia-PaCa2 cells were acquired from ATCC (Manassas, VA). All cells lines were resuscitated from low passage with all experiments carried out with cells of passage number less than 15. Su.86.86 cells were cultured in RPMI-1640 (Life Technologies) supplemented with 10% FBS (HyClone Laboratories). Hs766t and Mia-PaCa2 cell lines were cultured in DMEM/F12 (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin solution (Sigma, St. Louis, MO). All cell lines were grown at 37°C and 5% CO2. Xenografts All procedures were in compliance with the Guide for the Care and Use of laboratory Animal Resources (1996), National Research Council, and approved by the Institutional Animal Care and Use Committee, University of South Florida under the approved protocol R4033. Immunocompromised mice were housed in a clean facility with special conditions that include HEPA filtered ventilated cage systems, autoclaved bedding, autoclaved housing, autoclaved PLOS ONE | DOI:10.1371/journal.pone.0155289 May 26, 2016 3 / 13 Imaging Biomarkers to Monitor Early Response to Prodrug TH-302 water, irradiated food and special cage changing procedures. Mice were handled under aseptic conditions including the wearing of gloves, gowns and shoe coverings. Mice were sacrificed by inhalation of CO2 from a pressurized tank in a mouse chamber. Female SCID mice of age 5–6 weeks were inoculated with SU.86.86, Hs766t or Mia-PaCa2 cells (5x106) subcutaneously on the left hind leg. Tumors were allowed to grow for an average of three weeks to an average size of ~150 mm3, as estimated using electronic calipers and tumor volumes calculated as π/6[(short axis in mm)2 x (long axis in mm)]. Mice were then randomized and placed into cohorts and treated with saline (control) or TH-302 (50 mg/kg) injected intraperitoneally. Mice were imaged as described in the magnetic resonance imaging methods section. A total of 34 mice underwent MR imaging studies. The SU.86.86 group consisted of 5 TH-302 treated and 5 control animals; Mia-PaCa2 consisted of 6 TH-302 treated and 5 control animals; Hs766t consisted of 7 TH-302 treated and 6 control animals. No significant animal weight loss was observed during this study. Animals were sacrificed when tumors reached 2000 mm3. Imaging Both DCE-MRI and DW-MRI were performed 24 hours prior to the treatment (TH-302 or vehicle), DW-MRI was scheduled 24 hours and 48 hours after treatment. In order to ensure adequate time for contrast agent washout and physiologic recovery, DCE-MRI was only performed 48 hours after treatment. All MRI experiments were performed with a Varian 7 T MRI scanner equipped with a maximum gradient amplitude of 400 mT/m. All animals were anesthetized by inhaled isoflurane (1.5% in O2) at 2.0 LPM and cannulated at the tail vein. A pressure-transducer balloon taped to the animal's chest was used to continuously monitor its respiration rate. The body temperatures were continuously monitored using a rectal fluoroptic thermometer (SAII1, SA Instruments, Stony Brook, NY). An external heater was used to maintain body temperature at 37.0 ± 0.2°C and respiratory rate was monitored at a range of 60–90 breaths pre minute during the course of the imaging experiments. The animal was gently secured in a plastic holder and loaded into a 35-mm-ID small-animal imaging Litz coil (Doty Scientific, Columbia, SC, USA). MRI session for both DCE- and DW-MRI scan was approximate 1.5 hours; 0.5 hours for DW-MRI only section. DCE-MRI protocol includes T2-weighted images for anatomical view of tumor, parametric maps of endogenous T1 relaxation time and a series of T1-weighted images which trace the dynamic change of contrast agent including AIF and wash-in and -out within tumor site. T2weighted images were acquired using a fast spin echo (FSE) pulse sequence with an echo factor of 4, giving an effective TE of 72 ms. Saturation recovery method for T1 mapping with five different recovery time (TR) values was acquired prior to injection of the contrast agent (TR = 5000, 2000, 800, 300, 150 ms, TE = 7.2 ms, FOV = 35 x 70 mm, matrix = 128 x 256, spatial resolution = 273 x 273 μm). Nine 1-mm-thick coronal slices were oriented over the tumor and artery to ensure a valid arterial input function (AIF). The animal was tightly secured with tape to avoid motion artifacts. Before, during and after bolus administration of a single dose of CA (0.15 mmol/kg Gd-DTPA), a dynamic series of spin echo T1-weighted images (TR = 150 ms, TE = 7.2 ms) were acquired for 35minutes. DCE-MRI data was processed using a general kinetic model as described by Tofts et al. A complete description of these methods has been described previously.[29, 35] DW-MRI was performed to measure the quantitative ADC values with same geometry as DCE-MRI. Sequence parameters include five b-values: 12, 50, 500, 800, 1500 s/mm2 with TR = 1500 ms and TE = 35; Six average were used to reduce the motion artifacts. ADC maps were generated by a home-made Matlab program that fits b-values to the Stejskal-Tanner PLOS ONE | DOI:10.1371/journal.pone.0155289 May 26, 2016 4 / 13 Imaging Biomarkers to Monitor Early Response to Prodrug TH-302 equation, S = S0 EXP(-b ADC), where S0 is the signal amplitude without diffusion weighting and S is the signal amplitude with diffusion weighting. Regions of interest (ROIs) were determined by T2w images with exactly same geometry of DCE- and DW-MRI acquisition. These ROIs were superimposed onto DCE- and DW-MRI data to minimize the variation of anthropic factor. Distribution histograms were obtained for each tumor ROIs of DCE- or DW-MRI. Data are presented as the mean and its standard error. Student’s t tests, ANOVA were used where appropriate. P < 0.05 was considered to be statistically significant. Immunohistochemistry Following completion of therapeutic and MRI imaging protocols, pimonidazole hydrochloride (60 mg/kg; Hypoxyprobe Inc.) was injected I.P. one hour prior to tumor removal, subsequently fixed in 10% formalin, paraffin embedded, and further processed for immunohistochemistry. Tumor cross sections were stained with a rabbit primary antibody against gamma-H2AX (Novus Biologicals, Littleton, CO) and the Ventana OmniMap anti-rabbit secondary antibody (Ventana Medical enfocar la importancia de la producción mediática de los niños en su descubrimiento del mundo, sobre todo utilizando el periódico escolar y la imprenta. Asimismo las asociaciones de profesores trabajaron en esta línea e incluso la enseñanza católica se comprometió desde los años sesenta realizando trabajos originales en el marco de la corriente del Lenguaje Total. Páginas 43-48 45 Comunicar, 28, 2007 En el ámbito de los medios, también desde el principio del siglo XX hay ciertas corrientes de conexión. Pero es a lo largo de los años sesenta cuando se constituyeron asociaciones de periodistas apasionados por sus funciones de mediadores, que fomentaron la importancia ciudadana de los medios como algo cercano a los jóvenes, a los profesores y a las familias. Así se crearon la APIJ (Asociación de Prensa Información para la Juventud), la ARPEJ (Asociación Regional de Prensa y Enseñanza para la Juventud), el CIPE (Comité Interprofesional para la Prensa en la Escuela) o la APE (Asociación de Prensa y Enseñanza), todas ellas para la prensa escrita Estas asociaciones fueron precedidas por movimientos surgidos en mayo de 1968, como el CREPAC que, utilizando películas realizadas por periodistas conocidos, aclaraba temas que habían sido manipulados por una televisión demasiado próxima al poder político y realizaba encuentros con grupos de telespectadores. cipio del siglo XX, y nos han legado textos fundadores muy preciados, importantes trabajos de campo y muchos logros educativos y pedagógicos. La educación en medios ha tenido carácter de oficialidad de múltiples maneras, aunque nunca como una enseñanza global. Así la campaña «Operación Joven Telespectador Activo» (JTA), lanzada al final de los años setenta y financiada de manera interministerial para hacer reflexionar sobre las prácticas televisuales de los jóvenes, la creación del CLEMI (Centro de Educación y Medios de Comunicación) en el seno del Ministerio de Educación Nacional en 1983, la creación de la optativa «Cine-audiovisual» en los bachilleratos de humanidades de los institutos en 1984 (primer bachillerato en 1989) y múltiples referencias a la educación de la imagen, de la prensa, de Internet. La forma más visible y rápida de evaluar el lugar de la educación en medios es valorar el lugar que se le ha reservado en los libros de texto del sistema educa- 2. Construir la educación en los medios sin nombrarla El lugar que ocupa la edu- La denominación «educación en medios», que debería cación en los medios es muy ambiguo, aunque las cosas están cambiando recientemente. entenderse como un concepto integrador que reagrupase todos los medios presentes y futuros, es a menudo percibida En principio, en Francia, co- por los «tradicionalistas de la cultura» como una tendencia mo en muchos otros países, la educación en los medios no es hacia la masificación y la pérdida de la calidad. una disciplina escolar a tiempo completo, sino que se ha ido conformado progresivamente a través de experiencias y reflexiones teóricas que han tivo en Francia. Una inmersión sistemática nos permi- permitido implantar interesantes actividades de carác- te constatar que los textos oficiales acogen numerosos ter puntual. Se ha ganado poco a poco el reconoci- ejemplos, citas, sin delimitarla con precisión. miento de la institución educativa y la comunidad es- colar. Podemos decir que ha conquistado un «lugar», 3. ¿Por qué la escuela ha necesitado casi un siglo en el ámbito de la enseñanza transversal entre las dis- para oficilializar lo que cotidianamente se hacía en ciplinas existentes. ella? Sin embargo, la escuela no está sola en esta aspi- Primero, porque las prácticas de educación en me- ración, porque el trabajo en medios es valorado igual- dios han existido antes de ser nombradas así. Recor- mente por el Ministerio de Cultura (campañas de foto- demos que no fue hasta 1973 cuando aparece este grafía, la llamada «Operación Escuelas», presencia de término y que su definición se debe a los expertos del colegios e institutos en el cine ), así como el Minis- Consejo Internacional del Cine y de la Televisión, que terio de la Juventud y Deportes que ha emprendido en el seno de la UNESCO, definen de esta forma: numerosas iniciativas. «Por educación en medios conviene entender el estu- Así, esta presencia de la educación en los medios dio, la enseñanza, el aprendizaje de los medios moder- no ha sido oficial. ¡La educación de los medios no apa- nos de comunicación y de expresión que forman parte rece oficialmente como tal en los textos de la escuela de un dominio específico y autónomo de conocimien- francesa hasta 2006! tos en la teoría y la práctica pedagógicas, a diferencia Este hecho no nos puede dejar de sorprender ya de su utilización como auxiliar para la enseñanza y el que las experiencias se han multiplicado desde el prin- aprendizaje en otros dominios de conocimientos tales Páginas 43-48 46 Comunicar, 28, 2007 como los de matemáticas, ciencias y geografía». A pe- mente en todas las asignaturas. Incluso los nuevos cu- sar de que esta definición ha servido para otorgarle un rrículos de materias científicas en 2006 para los alum- reconocimiento real, los debates sobre lo que abarca y nos de 11 a 18 años hacen referencia a la necesidad no, no están totalmente extinguidos. de trabajar sobre la información científica y técnica y En segundo lugar, porque si bien a la escuela fran- el uso de las imágenes que nacen de ella. cesa le gusta la innovación, después duda mucho en Desde junio de 2006, aparece oficialmente el tér- reflejar y sancionar estas prácticas innovadoras en sus mino «educación en medios» al publicar el Ministerio textos oficiales. Nos encontramos con una tradición de Educación los nuevos contenidos mínimos y las sólidamente fundada sobre una transmisión de conoci- competencias que deben adquirir los jóvenes al salir mientos muy estructurados, organizados en disciplinas del sistema educativo. escolares que se dedican la mayor parte a transmitir Este documento pretende averiguar cuáles son los conocimientos teóricos. La pedagogía es a menudo se- conocimientos y las competencias indispensables que cundaria, aunque los profesores disfrutan de una ver- deben dominar para terminar con éxito su escolaridad, dadera libertad pedagógica en sus clases. El trabajo seguir su formación y construir su futuro personal y crítico sobre los medios que estaba aún en elaboración profesional. Siete competencias diferentes han sido te- necesitaba este empuje para hacerse oficial. nidas en cuenta y en cada una de ellas, el trabajo con Aunque el trabajo de educación en los medios no los medios es reconocido frecuentemente. Para citar esté reconocido como disciplina, no está ausente de un ejemplo, la competencia sobre el dominio de la len- gua francesa definen las capa- cidades para expresarse oral- La metodología elaborada en el marco de la educación en mente que pueden adquirirse con la utilización de la radio e, medios parece incluso permitir la inclinación de la sociedad incluso, se propone fomentar de la información hacia una sociedad del conocimiento, como defiende la UNESCO. En Francia, se necesitaría unir el interés por la lectura a través de la lectura de la prensa. La educación en los medios las fuerzas dispersas en función de los soportes mediáticos y orientarse más hacia la educación en medios que al dominio adquiere pleno derecho y entidad en la sección sexta titulada «competencias sociales y cívi- técnico de los aparatos. cas» que indica que «los alum- nos deberán ser capaces de juz- gar y tendrán espíritu crítico, lo que supone ser educados en los las programaciones oficiales, ya que, a lo largo de un medios y tener conciencia de su lugar y de su influencia estudio de los textos, los documentalistas del CLEMI en la sociedad». han podido señalar más de una centena de referencias a la educación de los medios en el seno de disciplinas 4. Un entorno positivo como el francés, la historia, la geografía, las lenguas, Si nos atenemos a las cifras, el panorama de la las artes plásticas : trabajos sobre las portadas de educación en medios es muy positivo. Una gran ope- prensa, reflexiones sobre temas mediáticos, análisis de ración de visibilidad como la «Semana de la prensa y publicidad, análisis de imágenes desde todos los ángu- de los medios en la escuela», coordinada por el CLE- los, reflexión sobre las noticias en los países europeos, MI, confirma año tras año, después de 17 convocato- información y opinión rias, el atractivo que ejerce sobre los profesores y los Esta presencia se constata desde la escuela mater- alumnos. Concebida como una gran operación de nal (2 a 6 años) donde, por ejemplo, se le pregunta a complementariedad source of circulating FGF-21. The lack of association between circulating and muscle-expressed FGF-21 also suggests that muscle FGF-21 primarily works in a local manner regulating glucose metabolism in the muscle and/or signals to the adipose tissue in close contact to the muscle. Our study has some limitations. The number of subjects is small and some correlations could have been significant with greater statistical power. Another aspect is that protein levels of FGF-21 were not determined in the muscles extracts, consequently we cannot be sure the increase in FGF-21 mRNA is followed by increased protein expression. In conclusion, we show that FGF-21 mRNA is increased in skeletal muscle in HIV patients and that FGF-21 mRNA in muscle correlates to whole-body (primarily reflecting muscle) insulin resistance. These findings add to the evidence that FGF-21 is a myokine and that muscle FGF-21 might primarily work in an autocrine manner. Acknowledgments We thank the subjects for their participation in this study. Ruth Rousing, Hanne Willumsen, Carsten Nielsen and Flemming Jessen are thanked for excellent technical help. The Danish HIV-Cohort is thanked for providing us HIV-related data. 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