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Inhibitorii caii de semnalizare Hedgehog in cancerul pancreatic

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Inhibition of Hedgehog Signaling Enhances Delivery of Chemotherapy in a Mouse Model of Pancreatic Cancer Kenneth P. Olive 1 , Michael A. Jacobetz 1,* , Christian J. Davidson 2,* , Aarthi Gopinathan 1,2,* , Dominick McIntyre 1 , Davina Honess 1 , Basetti Madhu 1 , Mae A. Goldgraben 1 , Meredith E. Caldwell 1 , David Allard 1 , Kristopher K. Frese 1 , Gina DeNicola 1,2 , Christine Feig 1 , Chelsea Combs 2 , Stephen P. Winter 1 , Heather Ireland 1 , Stefanie Reichelt 1 , William J. Howat 1 , Alex
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  Inhibition of Hedgehog Signaling Enhances Delivery of Chemotherapy in a Mouse Model of Pancreatic Cancer  Kenneth P. Olive 1, Michael A. Jacobetz 1,*, Christian J. Davidson 2,*,  AarthiGopinathan 1,2,*, Dominick McIntyre 1, Davina Honess 1, Basetti Madhu 1, Mae A.Goldgraben 1, Meredith E. Caldwell 1, David Allard 1, Kristopher K. Frese 1, GinaDeNicola 1,2, Christine Feig 1, Chelsea Combs 2, Stephen P. Winter  1, Heather Ireland 1, Stefanie Reichelt 1, William J. Howat 1,  Alex Chang 3, Mousumi Dhara 3, Lifu Wang 2,4, FelixRückert 5, Robert Grützmann 5, Christian Pilarsky 5, Kamel Izeradjene 6, Sunil R. Hingorani 6, Pearl Huang 7, Susan E. Davies 8, William Plunkett 9, Merrill Egorin 10, Ralph H. Hruban 3, Nigel Whitebread 11, Karen McGovern 11, Julian Adams 11, Christine Iacobuzio-Donahue 3, John Griffiths 1, and David A. Tuveson 1,† 1  Cancer Research UK, Cambridge Research Institute, The Li Ka Shing Centre, Robinson Way,Cambridge, CB2 ORE, UK 2  Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA19104, USA 3  Departments of Oncology and Pathology, The Sol Goldman Pancreatic Cancer ResearchCenter, Sidney Cancer Center and Johns Hopkins University, Baltimore MD 21287, USA 4  Department of Gastroenterology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China 5  Department of Surgery, University Hospital Dresden, Fetscherstr. 74, 01307 Dresden, Germany 6  Clinical Research and Public Health Sciences Division, Fred Hutchinson Cancer ResearchCenter, and University of Washington, Seattle, WA, 98109, USA 7  Oncology Franchise, Merck and Co, North Wales, PA 19454, USA 8  Department of Histopathology, Addenbrooke’s Hospital, Cambridge University Hospitals NHSFoundation Trust, Cambridge, CB2 2QQ, UK 9  Univ. of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 10  Division of Hematology and Oncology, University of Pittsburgh Medical Center, Pittsburgh, PA15213, USA 11  Infinity Pharmaceuticals Inc, Cambridge, MA 01239, USA  Abstract Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers, in part becauseit is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that isrefractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by co-administration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibiting the † To whom correspondence should be addressed. david.tuveson@cancer.org.uk.*These authors contributed equally to this work   NIH Public Access Author Manuscript Science . Author manuscript; available in PMC 2010 December 7. Published in final edited form as: Science  . 2009 June 12; 324(5933): 1457–1461. doi:10.1126/science.1171362. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    Hedgehog cellular signaling pathway. The combination therapy produced a transient increase inintratumoral vascular density and intratumoral concentration of gemcitabine, leading to transientstabilization of disease. Thus, inefficient drug delivery may be an important contributor tochemoresistance in pancreatic cancer.Pancreatic ductal adenocarcinoma (PDA) is among the most intractable of humanmalignancies. Decades of effort have witnessed the failure of many chemotherapeuticregimens and the current standard-of-care therapy, gemcitabine, extends patient survival byonly a few weeks (1–3). Oncology drug development relies heavily on mouse models bearing transplanted tumors for efficacy testing of novel agents. However, such models of PDA respond to numerous chemotherapeutic agents, including gemcitabine (4–9),suggesting that their predictive utility may be limited. Genetically engineered mouse (GEM)models of PDA offer an alternative to transplantation models for preclinical therapeuticevaluation. We have previously described KPC mice, which conditionally expressendogenous mutant Kras and p53 alleles in pancreatic cells (10), and which develop pancreatic tumors whose pathophysiological and molecular features resemble those of human PDA (11). Here we have used the KPC mice to investigate why PDA is insensitive tochemotherapy.We first compared the effect of gemcitabine on the growth of pancreatic tumors in four mouse models: the KPC mice and three distinct tumor transplantation models (12)(13).Gemcitabine inhibited the growth of all transplanted tumors, irrespective of their human or mouse srcin (Fig. 1A), but did not induce apoptosis (Fig. 1B). Rather, proliferation wassubstantially reduced in all transplanted tumors (fig. S1A). In contrast, most KPC tumors(15/17) in gemcitabine-treated mice showed the same growth rate as in saline-treated controls (Fig. 1C). This is consistent with clinical results wherein only 5–10% of patientstreated with gemcitabine demonstrate an objective radiographic response at the primarytumor site (3). Two KPC tumors demonstrated a transient response by high resolutionultrasound (13), which correlated with high levels of apoptosis (Fig. 1D)(fig S1).Additionally, proliferation was diminished in gemcitabine-treated KPC tumors shortly after treatment, but to a lesser extent than in transplantation models (fig. S1).Transplantation of low-passage cells derived from KPC tumors yielded subcutaneous tumorsthat were sensitive to gemcitabine treatment (see “Syngeneics”, Fig 1A), suggesting thatinnate cellular differences is unlikely to explain the chemoresistance of KPC tumors. Wetherefore assessed the metabolism of gemcitabine (2 ′ ,2 ′ -difluorodeoxycytidine, dFdC) to itsactive, intracellular metabolite, gemcitabine triphosphate (2 ′ ,2 ′ -difluorodeoxycytidinetriphosphate, dFdCTP), by high pressure liquid chromatography (HPLC). Consistent withthe results of clinical studies (14), circulating gemcitabine in wild-type mice was rapidlydeaminated to its inactive metabolite, 2 ′ 2 ′ -difluorodeoxyuridine (dFdU), resulting in a shorthalf-life for gemcitabine (fig. S2A-B). dFdCTP was present in transplanted tumor tissuesand control tissues, but was undetectable in KPC tumors (table S1). Thus, dFdCTPaccumulation in pancreatic tumor tissue distinguished transplantation and KPC models of PDA and correlated with the responsiveness to gemcitabine. Changes in expression of genesinvolved in gemcitabine transport are unlikely to explain the difference in gemcitabineaccumulation in transplanted and KPC pancreatic tumors (fig S2C-D).Impaired drug delivery is another possible mechanism of chemoresistance (15,16). Weinvestigated drug delivery by characterizing tumor perfusion in each model. First wedelineated functional vasculature through the intravenous infusion of a plant lectin (  L.esculentum)  in anesthetized mice, followed by the coimmunofluorescent detection of blood vessels in harvested tissues with a CD31 antibody (fig. S3). We found that transplanted tumors demonstrated a patent vasculature, whereas KPC tumors had a dysfunctional Olive et al.Page 2 Science . Author manuscript; available in PMC 2010 December 7. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    vasculature. Indeed, only 32% of CD31 +  blood vessels in KPC tumors were labeled withlectin, compared to 78% and 100% of vessels in transplanted tumors and normal pancreas,respectively. Second, to evaluate whether intravascular delivery and penetration of smallmolecule drugs is impeded in KPC tumors, we intravenously co-administered lectin with theautofluorescent drug doxorubicin (Figs. 2A-B)(fig. S4)(17). Confocal microscopy revealed amarked decrease in doxorubicin delivery to KPC pancreatic tumors compared to adjacentcontrol tissues and transplanted tumors, confirming inefficient drug delivery over a shorttime-course. Third, using high resolution contrast ultrasound, we found that the delivery of 5 μ m gas-filled liposomes (microbubbles) was more efficient in transplanted tumors thanKPC tumors (Figs. 2C-D)(fig. S5). Finally, we performed dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) on transplanted and KPC tumors (Fig. 2E-F)(fig.S6). Following administration of gadolinium-diethyltriaminepentaacetic acid (Gd-DTPA),we observed significant enhancement (of what???) in the periphery of transplanted tumors,while the tumor cores exhibited a variable, heterogeneous pattern of enhancement,consistent with the central necrosis observed by histology (fig. S6C). In contrast, weobserved efficient delivery of Gd-DTPA to the tissues surrounding KPC tumors, with littleor no enhancement (of what??) within the tumor body, despite little necrosis in these tumors(fig S6D). Collectively, these results suggest that drug delivery is impaired in KPC pancreatic tumors.We next investigated the vascular content and tissue architecture in transplanted and KPCtumors. The viable (peripheral) portions of transplanted tumors were densely vascularized (fig. S7A) and neoplastic cells made direct contact with blood vessels (Fig. 3A). In contrast,within the parenchyma of KPC and human pancreatic tumors, blood vessel density wasmarkedly decreased and vessels were embedded within the prominent stromal matrix that ischaracteristic of these tumors and of primary human ductal pancreatic cancer (Figs. 3B-D)(fig. S7)(fig. S8). Neoplastic cells in both KPC and human pancreatic tumors were widelyspaced from blood vessels compared to those in transplanted tumors, reflecting thesedifferences in stromal content (Fig. 3E). Using computer aided image analysis, weconfirmed the paucity of vasculature in an independent cohort of 18 human PDA specimens,compared to normal pancreatic tissues and chronic pancreatitis (CP) samples (Fig. 3F)(figS9). Our findings demonstrate that increased vascular content is not a prerequisite for ductal pancreatic cancer progression and suggest that the hypovascularity and vascular architectureof PDA tumors may impose an additional limitation to therapeutic delivery.We hypothesized that disrupting the stroma of pancreatic tumors might alter the vascular network and thereby facilitate delivery of chemotherapeutic agents. We studied an inhibitor of the hedgehog (Hh) pathway because paracrine Hh signaling from neoplastic cells tostromal cells promotes stromal desmoplasia (18,19). Binding of Hh ligands to the Patched1receptor relieves repression of the 12-transmembrane protein Smoothened (Smo), resultingin activation of the Gli family of transcription factors. While Sonic Hedgehog (Shh) isoverexpressed in the neoplastic cells of both human (20) and KPC (10) pancreatic tumors,Gli activity is restricted to the stromal compartment (21).IPI-926 is a semisynthetic derivative of cyclopamine (chemical structure shown in fig. S10)that potently inhibits Smo (EC 50 = 7nM) with a long half life (10.5 hours, CD1 mice) and ahigh volume of distribution (11L/kg, CD1 mice). The detailed characterization of IPI-926 incell-based and in vitro assays will be published separately. Daily oral administration of 40mg/kg IPI-926 to KPC mice resulted in a measurable accumulation of drug in PDAtissues (fig. S11A) and a significant decrease in the expression of Gli1, a transcriptionaltarget of the Hh pathway (fig. S11B). The effects of Smo inhibition on tumor histopathologyand perfusion were investigated in KPC mice after 8–12 days of treatment with IPI-926 or gemcitabine, alone or in combination (IPI-926/gem). In contrast to mice treated with vehicle Olive et al.Page 3 Science . Author manuscript; available in PMC 2010 December 7. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    or gemcitabine, which exhibited profuse desmoplastic tumor stroma, mice treated withIPI-926 or IPI-926/gem were depleted of desmoplastic stroma, resulting in densely packed ductal tumor cells (fig. S12A-D). The effect of Smo inhibition on the stroma was alsoevidenced by a decrease in Collagen I content (fig. S12E-H). Interestingly, these differenceswere not apparent in mice treated for only four days (fig. S13I-L). However,coimmunofluorescence performed on tumors treated for four days with vehicle or IPI-926found reduced proliferation in α -smooth muscle actin ( α SMA) positive stromalmyofibroblasts (fig. S11C)(fig S13A-B). This decrease in proliferation was balanced by anincrease in proliferation of α SMA negative cells (fig. S11D).Smo inhibition also had a profound effect on the tumor vasculature, with a significantlyhigher mean vessel density (MVD) noted in the tumors from IPI-926 treated mice (Fig. 4A)(figs. S14A-D). This effect was most significant in IPI-926/gem treated mice, where theMVD approximated that of normal pancreatic tissue. An increased CD31 content was also present after four days of treatment (fig. S14E-H). At this early timepoint, numerous isolated CD31 positive cells were noted, consistent with the active formation of new endothelial precursors after Smo inhibition. Indeed, coimmunofluorescence for proliferation and endothelial markers confirmed a significant increase in proliferating endothelial cellsfollowing IPI-926 treatment (fig. S11E)(fig. S13C,D). The increased MVD observed inIPI-926 treated mice also correlated with more effective delivery of doxorubicin to tumor tissues (Fig. 4B)(fig. S14I-L). Importantly, we found that the concentration of gemcitabinemetabolites in KPC tumors was elevated by 60% following 10 days pretreatment withIPI-926/gem (Fig. 4C)(P=.04, Mann-Whitney U). These data suggest that depletion of  pancreatic tumor stroma stimulates angiogenesis and consequently augments drug delivery.Interestingly, whole tissue mRNA microarray analysis revealed no significant differences in proangiogenic VEGF expression between what and what????(table S2).We then investigated the effects of Smo inhibition on cell proliferation and apoptosis.Although IPI-926 alone specifically decreased the proliferation of stromal myofibroblasts, ithad little effect on overall cellular proliferation, consistent with the finding that conditionalSmo deletion in pancreatic cells does not alter the progression of mutant Kras-induced  pancreatic tumors (23). Importantly, IPI-926/gem treated tumors harbored many dead and dying cells as evidenced by a significant increase in staining for the apoptotic marker Cleaved Caspase 3 (Fig. 4D).Finally, we performed an intervention survival study on KPC mice, monitoring tumor volume bi-weekly by 3D-ultrasonography. KPC mice treated with gemcitabine alone or IPI-926 alone showed no survival benefit in comparison with vehicle-treated controls. Incontrast, combination treatment with IPI-926/gem extended the median survival of KPCmice from 11 days to 25 days (P= .001, Log Rank Test), yielding a hazard ratio of 0.157±0.458 95%CI (Fig. 4E). Most IPI-926/gem-treated tumors (14/17) exhibited a transientdecrease in size within 1–2 weeks of treatment (fig. S15). In contrast, only a minority of gemcitabine (2/10) and IPI-926 (2/10) treated mice demonstrated objective ultrasonographicresponses to treatment. Interestingly, IPI-926/gem treatment resulted in a significantdecrease in metastases to the liver (Fig. 4F, P=.015, Fisher’s Exact). Investigating the biology of tumors at endpoint, we found no differences in the expression of genes associated with gemcitabine resistance in IPI-926/gem treated tumors (fig. S11G). However, thehypovascularity of IPI-926/gem treated tumors was restored at endpoint as the MVD inIPI-926 was similar to controls (fig. S11H).The general resistance of pancreatic cancer to systemic therapies is unusual among commoncarcinomas and was not predicted by preclinical models (24). Chemotherapeutic agentsshare two properties: a short half-life and small therapeutic index (the range of concentration Olive et al.Page 4 Science . Author manuscript; available in PMC 2010 December 7. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  
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