Orthotopic Animal Model of Pseudomyxoma Peritonei
2014; Elsevier BV; Volume: 184; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2014.03.004
ISSN1525-2191
AutoresAnthony Dohan, R. Lousquy, Clarisse Eveno, Diane Goèré, Dong Broquères-You, Rachid Kaci, Jacqueline Lehmann‐Che, Jean‐Marie Launay, Philippe Soyer, Philippe Bonnin, Marc Pocard,
Tópico(s)Hepatocellular Carcinoma Treatment and Prognosis
ResumoPseudomyxoma peritonei (PMP) is an uncommon peritoneal mucinous carcinomatosis confined to the peritoneal cavity. The rarity of PMP in humans makes evaluation of the disease biological features and new therapeutic strategies difficult. Accordingly, there is a need for animal models of PMP. Human PMP tissue was i.p. grafted and grown into nude mice, then constituted into reliable and reproducible orthotopic models. Histological and immunostaining analysis was performed. Bevacizumab was injected twice a week either during tumor growth or after cytoreductive surgery. In vivo imaging of tumor angiogenesis was performed using barium sulfate or isolectin microangiography and Doppler ultrasonography of the superior mesenteric artery. Tumor angiogenesis was confirmed by the presence of tortuous vascular networks with high levels of expression of CD31, vascular endothelial cadherin, and desmin. Doppler ultrasonography of the superior mesenteric artery revealed a twofold increase in blood flow velocity compared with tumor-free mice (P < 0.001). Bevacizumab administration was correlated with the normalization of tumor vascularity when injected during tumor growth and with the stabilization of the histological and hemodynamic findings when injected after cytoreductive surgery. Our PMP models mimic human PMP. Our results confirmed the presence of tumor angiogenesis related to PMP growth. Our murine model allows researchers to actually bench test and evaluate, in preclinical studies, the efficacy of new therapeutic strategies and anti-angiogenic therapies. Pseudomyxoma peritonei (PMP) is an uncommon peritoneal mucinous carcinomatosis confined to the peritoneal cavity. The rarity of PMP in humans makes evaluation of the disease biological features and new therapeutic strategies difficult. Accordingly, there is a need for animal models of PMP. Human PMP tissue was i.p. grafted and grown into nude mice, then constituted into reliable and reproducible orthotopic models. Histological and immunostaining analysis was performed. Bevacizumab was injected twice a week either during tumor growth or after cytoreductive surgery. In vivo imaging of tumor angiogenesis was performed using barium sulfate or isolectin microangiography and Doppler ultrasonography of the superior mesenteric artery. Tumor angiogenesis was confirmed by the presence of tortuous vascular networks with high levels of expression of CD31, vascular endothelial cadherin, and desmin. Doppler ultrasonography of the superior mesenteric artery revealed a twofold increase in blood flow velocity compared with tumor-free mice (P < 0.001). Bevacizumab administration was correlated with the normalization of tumor vascularity when injected during tumor growth and with the stabilization of the histological and hemodynamic findings when injected after cytoreductive surgery. Our PMP models mimic human PMP. Our results confirmed the presence of tumor angiogenesis related to PMP growth. Our murine model allows researchers to actually bench test and evaluate, in preclinical studies, the efficacy of new therapeutic strategies and anti-angiogenic therapies. CME Accreditation Statement: This activity ("ASIP 2014 AJP CME Program in Pathogenesis") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("ASIP 2014 AJP CME Program in Pathogenesis") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("ASIP 2014 AJP CME Program in Pathogenesis") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("ASIP 2014 AJP CME Program in Pathogenesis") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Pseudomyxoma peritonei (PMP) is a rare malignant disease characterized by the progressive accumulation of mucinous tumor tissue in the peritoneal cavity without extraperitoneal growth, leading to intra-abdominal compression and a fatal outcome. The consensus is that PMP has an intestinal origin and results from the perforation of a mucinous appendiceal neoplasm.1Cuatrecasas M. Matias-Guiu X. Prat J. Synchronous mucinous tumors of the appendix and the ovary associated with pseudomyxoma peritonei: a clinicopathologic study of six cases with comparative analysis of c-Ki-ras mutations.Am J Surg Pathol. 1996; 20: 739-746Crossref PubMed Scopus (98) Google Scholar, 2Guerrieri C. Franlund B. Fristedt S. Gillooley J.F. 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Sardi A. Liauw W. Yan T.D. Barrios P. Gomez Portilla A. de Hingh I.H. Ceelen W.P. Pelz J.O. Piso P. Gonzalez-Moreno S. Van Der Speeten K. Morris D.L. Early- and long-term outcome data of patients with pseudomyxoma peritonei from appendiceal origin treated by a strategy of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.J Clin Oncol. 2012; 30: 2449-2456Crossref PubMed Scopus (728) Google Scholar, 6Elias D. Gilly F. Quenet F. Bereder J.M. Sideris L. Mansvelt B. Lorimier G. Glehen O. Pseudomyxoma peritonei: a French multicentric study of 301 patients treated with cytoreductive surgery and intraperitoneal chemotherapy.Eur J Surg Oncol. 2010; 36: 456-462Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 7Miner T.J. Shia J. Jaques D.P. Klimstra D.S. Brennan M.F. Coit D.G. Long-term survival following treatment of pseudomyxoma peritonei: an analysis of surgical therapy.Ann Surg. 2005; 241: 300-308Crossref PubMed Scopus (280) Google Scholar, 8Moran B. Baratti D. Yan T.D. Kusamura S. Deraco M. Consensus statement on the loco-regional treatment of appendiceal mucinous neoplasms with peritoneal dissemination (pseudomyxoma peritonei).J Surg Oncol. 2008; 98: 277-282Crossref PubMed Scopus (168) Google Scholar, 9Smeenk R.M. Verwaal V.J. Antonini N. Zoetmulder F.A. Progression of pseudomyxoma peritonei after combined modality treatment: management and outcome.Ann Surg Oncol. 2007; 14: 493-499Crossref PubMed Scopus (46) Google Scholar, 10Sugarbaker P.H. New standard of care for appendiceal epithelial neoplasms and pseudomyxoma peritonei syndrome?.Lancet Oncol. 2006; 7: 69-76Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar Despite the improved survival rate achieved with the combined treatment, this therapeutic option is not applicable to all patients, especially for those with coexisting causes of morbidity. Moreover, treatment failure and recurrence are not rare.11Yan T.D. Chu F. Links M. Kam P.C. Glenn D. Morris D.L. Cytoreductive surgery and perioperative intraperitoneal chemotherapy for peritoneal carcinomatosis from colorectal carcinoma: non-mucinous tumour associated with an improved survival.Eur J Surg Oncol. 2006; 32: 1119-1124Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar Histopathologically, PMP is graded from disseminated peritoneal adenomucinosis (DPAM or low grade) to peritoneal mucinous carcinomatosis (PMCA or high grade), with an intermediate histological type.12Ronnett B.M. Yan H. Kurman R.J. Shmookler B.M. Wu L. Sugarbaker P.H. Patients with pseudomyxoma peritonei associated with disseminated peritoneal adenomucinosis have a significantly more favorable prognosis than patients with peritoneal mucinous carcinomatosis.Cancer. 2001; 92: 85-91Crossref PubMed Scopus (294) Google Scholar PMP presents as large patches of mucinous deposits that contain a few cells organized as a low-proliferative epithelium. The paucity of tumor cells may explain the relative lack of efficacy of the common cytotoxic chemotherapy regimens on PMP. To date, we are aware of only two case reports that have studied the potential impact of an anti-angiogenic agent (ie, bevacizumab) in PMP and reported a partial response.13Sun W.L. Hutarew G. Gradl J. Gratzl M. Denz H. Fiegl M. Successful antiangiogenic combination therapy for pseudomyxoma peritonei with bevacizumab and capecitabine.Cancer Biol Ther. 2009; 8: 1459-1462Crossref PubMed Scopus (22) Google Scholar, 14Winer I. Buckanovich R.J. Bevacizumab for the treatment of non-resectable Pseudomyxoma peritonei associated with mucinous ovarian tumor of low malignant potential: a comparison of two cases.Case Rep Oncol. 2009; 3: 1-8Crossref PubMed Scopus (4) Google Scholar To improve the overall survival and recurrence-free survival rate, the development of new therapeutic strategies that combine chemotherapy and/or an anti-angiogenic drug with cytoreductive surgery is of major importance. Animal models of PMP should mirror the peritoneal growth pattern of PMP in humans and should also mirror the histopathological classification used in humans. In addition, such animal models should be appropriate for testing new anti-angiogenic therapies and evaluating the tumor response. To our knowledge, there are only three published animal studies that describe the development of orthotopic xenografted PMP in animal models.15Chua T.C. Akther J. Yao P. Morris D.L. In vivo model of pseudomyxoma peritonei for novel candidate drug discovery.Anticancer Res. 2009; 29: 4051-4055PubMed Google Scholar, 16Flatmark K. Davidson B. Kristian A. Stavnes H.T. Forsund M. Reed W. Exploring the peritoneal surface malignancy phenotype: a pilot immunohistochemical study of human pseudomyxoma peritonei and derived animal models.Hum Pathol. 2010; 41: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar To date, the evaluation of patients with PMP is based on computed tomography and no functional imaging modality has a proven efficacy in the follow-up of these patients. Doppler ultrasonography (DUS) has been successfully used to monitor the tumor blood supply in primary and secondary liver cancers.17Berge M. Bonnin P. Sulpice E. Vilar J. Allanic D. Silvestre J.S. Levy B.I. Tucker G.C. Tobelem G. Merkulova-Rainon T. Small interfering RNAs induce target-independent inhibition of tumor growth and vasculature remodeling in a mouse model of hepatocellular carcinoma.Am J Pathol. 2010; 177: 3192-3201Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 18Delorme S. Haberkorn U. Kinscherf R. Zuna I. Bahner M.L. van Kaick G. Changes of tumor vascularity during gene therapy monitored with color Doppler US.Ultrasound Med Biol. 2001; 27: 1595-1603Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 19Eveno C. Le Henaff C. Audollent R. Soyer P. Rampanou A. Nemeth J. Brouland J.P. Dupuy E. Pocard M. Bonnin P. Tumor and non-tumor liver angiogenesis is traced and evaluated by hepatic arterial ultrasound in murine models.Ultrasound Med Biol. 2012; 38: 1195-1204Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 20Van de Veire S. Stalmans I. Heindryckx F. Oura H. Tijeras-Raballand A. Schmidt T. et al.Further pharmacological and genetic evidence for the efficacy of PlGF inhibition in cancer and eye disease.Cell. 2010; 141: 178-190Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 21Vincent F. Bonnin P. Clemessy M. Contreres J.O. Lamande N. Gasc J.M. Vilar J. Hainaud P. Tobelem G. Corvol P. Dupuy E. Angiotensinogen delays angiogenesis and tumor growth of hepatocarcinoma in transgenic mice.Cancer Res. 2009; 69: 2853-2860Crossref PubMed Scopus (49) Google Scholar The main blood supply of the PMP tumor arises from the superior mesenteric artery (SMA). We, thus, hypothesized that DUS of the SMA would provide an evaluation of the blood flow supplying the tumor vascular network. Indeed, it has been previously demonstrated that measurement of the blood flow velocity in the feeding artery upstream of the tumor vascular network allows for the semiquantitative analysis of the development or the involution of the tumor vasculature.17Berge M. Bonnin P. Sulpice E. Vilar J. Allanic D. Silvestre J.S. Levy B.I. Tucker G.C. Tobelem G. Merkulova-Rainon T. Small interfering RNAs induce target-independent inhibition of tumor growth and vasculature remodeling in a mouse model of hepatocellular carcinoma.Am J Pathol. 2010; 177: 3192-3201Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 21Vincent F. Bonnin P. Clemessy M. Contreres J.O. Lamande N. Gasc J.M. Vilar J. Hainaud P. Tobelem G. Corvol P. Dupuy E. Angiotensinogen delays angiogenesis and tumor growth of hepatocarcinoma in transgenic mice.Cancer Res. 2009; 69: 2853-2860Crossref PubMed Scopus (49) Google Scholar, 22Bonnin P. Villemain A. Vincent F. Debbabi H. Silvestre J.S. Contreres J.O. Levy B.I. Tobelem G. Dupuy E. Ultrasonic assessment of hepatic blood flow as a marker of mouse hepatocarcinoma.Ultrasound Med Biol. 2007; 33: 561-570Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar The goals of our study were threefold. First, we wanted to develop a murine model of PMP to investigate tumor angiogenesis in PMP. Second, we wanted to evaluate the capabilities of DUS in depicting and quantifying the development or the involution of the tumor vasculature in this experimental murine model. Third, we wanted to investigate the effects of anti-angiogenic therapies alone or as adjuvant treatments of PMP after incomplete surgical resection. All of the tissue samples were collected after Institutional Review Board approval and after informed consent was obtained from all of the patients. Tumor samples were obtained from 18 patients with histologically proven PMP that originated from an appendiceal mucinous neoplasm; patients were treated with surgery at two different institutions between November 2009 and May 2012. Five-week-old female athymic Swiss nude mice (Charles River Laboratories International Inc., Arbresle, France), weighing between 16 and 18 g, were acclimatized for 1 week before tumor transplantation. The animals were maintained under specific pathogen-free conditions, and irradiated food and water were supplied ad libitum. During surgical procedures, the animals were anesthetized with a mixture of 80 mg/kg ketamine and 16 mg/kg xylazine delivered i.p. (Virbac, Carros, France). All of the experimental protocols and the animal procedures for the care and use of laboratory animals were conducted according to the guidelines of our institution and the Federation of European Laboratory Animal Science Association. The tumor samples were collected and immediately brought to the animal facility. The fresh tumor tissue was cut into 3 × 3 × 3-mm pieces. The tumor fragments were i.p. implanted in two mice for each sample. A 1-cm midline laparotomy was performed, and six tumor pieces were placed in the peritoneal cavity in both sides of the subdiaphragmatic area, the flank, and the pelvis. After implantation, the peritoneum and muscle were closed with one layer of Monocryl 5/0 (Ethicon, Somerville, NJ), and the skin was closed with staples. The mice were carefully monitored and weighed twice a week. The mice were sacrificed when they weighed >40 g. After sacrificing the mice, the macroscopic assessment of PMP was performed, during which a search for primary and metastatic tumors was performed; these tumors were sampled for further analyses. The tumor samples were embedded in optimal cutting temperature compound or fixed in formalin. Cytoreductive surgery was performed at 33 g. Each mouse was anesthetized with 1.5% isoflurane in 100% O2. A large midline incision was made, and maximum resection was performed with a clip applier. Lesions adjacent to the digestive tract were not resected because of the high risk of a lethal wound. The peritoneum and muscle were closed with one layer of Monocryl 5/0, and the skin was closed with staples. Immediately after surgery, mice were given an analgesic drug (buprenorphine; Schering-Plough Europe, Brussels, Belgium) in a single s.c. dose of 0.1 mg/kg. Tissue from the mouse tumor was explanted after sacrifice, fixed with formalin, and stained with H&E-safran and Alcian Blue. Histological classification was performed according to the three groups, as described by Winer and Buckanovich14Winer I. Buckanovich R.J. Bevacizumab for the treatment of non-resectable Pseudomyxoma peritonei associated with mucinous ovarian tumor of low malignant potential: a comparison of two cases.Case Rep Oncol. 2009; 3: 1-8Crossref PubMed Scopus (4) Google Scholar: 1, DPAM; 2, PMCA; and 3, intermediate histological type PMCA. Archival paraffin-embedded tissue blocks were cut into sections (4 to 5 μm) and transferred to glass slides. After deparaffinization, antigen retrieval was performed using citrate solution at pH 6.0. The slides were stained on an automated Benchmark Ultra immunostainer (Ventana Medical Systems, Tucson, AZ). The monoclonal anti–intestinal mucin (MUC)-2 antibody (cloner Ccp58; Novocastra, Newcastle, UK) was used at 1:100 dilutions. The epitope specifically recognized by the anti–MUC-2 antibody is human MUC-2 glycoprotein (no cross-reaction with MUC-1– or MUC-3–derived peptides). Slides were incubated with the previously described primary antibody for 1 hour at room temperature; thereafter, goat anti-mouse secondary antibody was applied, and the reaction was developed using diaminobenzidine. In addition, tumor samples from each passage through passage 5 were embedded in optimal cutting temperature compound for the preparation of frozen tissue sections (5 μm thick), which were then fixed in acetone at 4°C for 10 minutes and immunostained as described. After washing, the non-specific sites were saturated for 20 minutes with bovine serum albumin (10 g/L in PBS–1% Tween). The sections were then incubated for 1 hour with the following primary antibodies: anti–pan-cytokeratin antibody (1:100; Abcam, Cambridge, MA) and anti-cytokeratin 20 (CK20) antibody (prediluted) (Abcam), which are specific for colon cancer; an antibody against Ki-67 (1:100; Neomarker Inc., Fremont, CA), which is a nuclear protein expressed in proliferating cells; antibodies against platelet endothelial cell adhesion molecule (CD31) (1:50; BD Pharmingen, Le Pont-de-Claix, France); an antibody against vascular endothelial (VE) cadherin (1:50; Santa Cruz Biotechnology Tebu SA, Le Perray-en-Yvelines, France), which is a protein localized at the intercellular junctions of endothelial cells; and an antibody against desmin (1:200; Neomarker Inc.), which is a protein specific for smooth and striated muscle. After washing, the sections were incubated for 30 minutes with the appropriate secondary antibody (1:200) [Alexa Fluor 555 goat anti-mouse, goat anti-rabbit, or donkey anti-goat, or Alexa Fluor 488 goat anti-mouse, donkey anti-rat, or donkey anti-goat (Interchim, Asnieres, France)] and then washed with PBS–1% Tween. The primary antibody was omitted on sections that were used as negative controls. To perform double immunostaining, the sections were initially incubated with the first primary antibody, then with the second primary antibody, and, finally, with the appropriate secondary antibody. The histological sections were analyzed with a fluorescence microscope equipped with the appropriate filters (Observer.Z1; Carl Zeiss MicroImaging GmbH, Göttingen, Germany). Mutational analysis of TP53 was performed using functional analysis of separated alleles in yeast, in accordance with the method described by Flaman et al.23Flaman J.-M. Frebourg T. Moreau V. Charbonnier F. Martin C. Chappuis P. Sappino A.-P. Limacher I.-M. Bron L. Benhattar J. Tada M. Van Meir E.G. Estreicher A. Iggo R.D. A simple p53 functional assay for screening cell lines, blood, and tumors.Proc Natl Acad Sci U S A. 1995; 92: 3963-3967Crossref PubMed Scopus (429) Google Scholar This method requires frozen tumor samples and evaluates the transactivation activity of p53 on a p53-responsive promoter stably integrated in the yeast genome. RNA was extracted by the phenol-chloroform method and reverse transcribed, and p53 transcripts were amplified by PCR and transfected into yeast. Yeast colonies transformed with wild-type or mutated TP53 sequences appear white and large or red and small, respectively. TP53 status was considered mutated when >10% of the yeast colonies were red and analysis using the split versions of the test could identify the defect in the 5′ or 3′ part of the gene. The seven most frequent KRAS mutations on codons 12 and 13 were analyzed by allelic discrimination after DNA extraction from selected formalin-fixed, paraffin-embedded sections (>20% tumor cells) of the tumor samples using the QIAamp DNA minikit (Qiagen, Hilden, Germany), according to manufacturer's instructions. Specific probes for each allele (mutated or not) were labeled with fluorescent reporter dyes at their 5′ end and analyzed by real-time PCR on an LC480 instrument (Roche Diagnostics, Meylan, France).24Lievre A. Bachet J.B. Boige V. Cayre A. Le Corre D. Buc E. Ychou M. Bouche O. Landi B. Louvet C. Andre T. Bibeau F. Diebold M.D. Rougier P. Ducreux M. Tomasic G. Emile J.F. Penault-Llorca F. Laurent-Puig P. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab.J Clin Oncol. 2008; 26: 374-379Crossref PubMed Scopus (1335) Google Scholar Conventional microangiography was performed in five grafted mice to characterize the tumor vascular network. Under anesthesia, a longitudinal laparotomy was performed, and the abdominal aorta was exposed by moving the tumor to the left side and then ligating upstream of the iliac bifurcation. A polyethylene catheter with monitored pressure and volume was introduced into the abdominal aorta to inject a contrast material (barium sulfate, 1 g/mL). Image acquisition was performed with a digital X-ray transducer (model 2100; Kodak Dental Systems, Atlanta, GA).22Bonnin P. Villemain A. Vincent F. Debbabi H. Silvestre J.S. Contreres J.O. Levy B.I. Tobelem G. Dupuy E. Ultrasonic assessment of hepatic blood flow as a marker of mouse hepatocarcinoma.Ultrasound Med Biol. 2007; 33: 561-570Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Alternatively, performing the same surgical procedure in another five mice, we perfused 0.5 mL of heparinized saline and 0.8 mL of bandeiraea simplicifolia isolectin B4 (IB4) conjugated to fluorescein isothiocyanate (Sigma, Saint Quentin Fallavier, France), diluted with PBS to a concentration of 0.25 mg/mL. This tracer binds selectively to terminal α-galactose (IB4)25Goldstein I.J. Winter H.G. The Griffonia simplicifolia I-B4 isolectin: a probe for alpha-D-galactosyl end groups.Subcell Biochem. 1999; 32: 127-141PubMed Google Scholar and is an endothelial cell-specific marker that was used to identify and characterize the neovessels in the tumor. Fifteen minutes later, the mice were sacrificed, and the tumors were harvested. After fixation, the tumor samples were analyzed with a computer-assisted Nikon fluorescence microscope with a digital camera (Eclipse TE200; Nikon Inc., Melville, NY). Because of the heterogeneous distribution of the vasculature and the presence of wide ranges of mucinous content, we did not quantify the density of the vessels. Ten Swiss, nude, PMCA tumor-grafted and 10 age-matched normal mice had ultrasonographic measurements using a DUS instrument (Vivid 7; GE Medical Systems Ultrasound, Horten, Norway) equipped with a 12-MHz linear transducer, as previously described.22Bonnin P. Villemain A. Vincent F. Debbabi H. Silvestre J.S. Contreres J.O. Levy B.I. Tobelem G. Dupuy E. Ultrasonic assessment of hepatic blood flow as a marker of mouse hepatocarcinoma.Ultrasound Med Biol. 2007; 33: 561-570Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Briefly, after sedation, the mice were placed on a heating blanket (38°C) in the left lateral prone position to avoid placing pressure on the abdomen with the transducer. The color Doppler mode was activated with an anterior longitudinal view of the abdomen crossing the longitudinal axis of the aorta. The SMA and celiac trunk were localized on the screen by their color-coded blood flow. A pulsed Doppler sample was then placed on the longitudinal axis of each vessel, and pulsed Doppler velocity waveforms were recorded. Peak-systolic, end-diastolic, and time-averaged mean blood flow velocities (BFVs) were measured according to the pulsed Doppler spectrum. The DUS recordings were repeated every 2 weeks from the initiation of the tumor graft up to 6 weeks. After sacrificing the mice, blood was collected into chilled tubes containing heparin (Sarstedt, Marnay, France). The plasma concentrations of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), and transforming growth factor β (TGF-β) were determined with ELISA tests (MMV00, DPG00, and MB100B, respectively), according to the manufacturer's instructions. In another set of experiments, we applied an anti-angiogenic treatment in two models of PMCA (PMCA-1 and PMCA-2). Four weeks after the PMCA surgical graft, the mice were randomly assigned to two different groups. The mice in the treated groups were i.p. injected twice a week with 5 mg/kg bevacizumab (n = 10 for PMCA-1 and n = 10 for PMCA-2), according to prior publications. The mice in the control groups (n = 10 for each experiment) received an equivalent volume of PBS. The mice were sacrificed when they weighed >40 g. The serum angiogenesis markers (VEGF, PlGF, and TGF-β) were quantified in these two groups 6 weeks after the tumor graft and compared with five age-matched, non-grafted mice. In another set of experiments, after cytoreductive surgery was performed at the weight of 33 g, the grafted mice were submitted to an anti-angiogenic treatment with bevacizumab (n = 11) versus PBS (n = 9), i.p. injected twice a week in an adjuvant setting. The mice were carefully monitored for 4 weeks with DUS and weighed weekly. The development of tumor growth was evaluated by following the increase in weight. Serum angiogenesis markers were also quantified. Results were expressed as the means ± SD. Heart rate, blood flow velocities, and concentrations of serum angiogenesis markers were compared using an analysis of variance. Post hoc paired and unpaired Student's t-tests were performed to identify which time point or which group differences accounted for the significant analysis of variance. The survival probabilities were estimated using the Kaplan-Meier product-limit method (MedCalc, version 10.4.3.0; MedCalc Software, Ostend, Belgium). P < 0.05 was considered to be significant. Among the 18 i.p. grafted, patient-retrieved PMP samples, nine succeeded in growing, but growth occurred only once for one of the samples. Among the eight remaining samples, seven were similar to a PMCA (PMCA-1 to PMCA-7), and one was representative of a DPAM. These eight models were considered to be reliable and reproducible orthotopic mouse models of PMP. The delay between successive tumor growths decreased from 136 days (range, 116 to 166 days) after the first transplant to 78 days (range, 42 to 115 days) after the fifth transplant. The take rates ranged from 80% to 100% for PMCA-1 to PMCA-7 and from 40% to 90% for DPAM. As observed in humans, the mice presented abdominal distension due to tumor growth. PMCA-1 to PMCA-7 were considered as more aggressive compared with DPAM because these tumors grew more quickly. The typical initial i.p. growth pattern was characterized by mucinous ascites, accompanied by a variable number of solid mucinous tumor lesions of varying sizes. The solid tumor components were attached to the peritoneum and serosa of all of the i.p. organs. No animal presented any metastatic lesions. The animal models of PMP reflect the histopathological classification after each transplant generation. In the mice, we confirmed with H&E-safran staining that DPAM developed from a low-grade appendiceal mucinous tumor. This subtype was characterized by widespread peritoneal deposits of abundant pools of extracellular mucin with strips of proliferative mucinous columnar epithelium w
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