Patient-Derived Xenograft Models for Pancreatic Adenocarcinoma Demonstrate Retention of Tumor Morphology through Incorporation of Murine Stromal Elements

Direct implantation of viable surgical specimens provides a representative preclinical platform in pancreatic adenocarcinoma. Patient-derived xenografts consistently demonstrate retained tumor morphology and genetic stability. However, the evolution of the tumor microenvironment over time remains poorly characterized in these models. This work specifically addresses the recruitment and incorporation of murine stromal elements into expanding patient-derived pancreatic adenocarcinoma xenografts, establishing the integration of murine cells into networks of invading cancer cells. In addition, we provide methods and observations in the establishment and maintenance of a patient-derived pancreatic adenocarcinoma xenograft model. A total of 25 histologically confirmed pancreatic adenocarcinoma specimens were implanted subcutaneously into nonobese diabetic severe combined immunodeficiency mice. Patient demographics, staging, pathological analysis, and outcomes were analyzed. After successful engraftment of tumors, histological and immunofluorescence analyses were performed on explanted tumors. Pancreatic adenocarcinoma specimens were successfully engrafted in 15 (60%) of 25 attempts. Successful engraftment does not appear to correlate with clinicopathologic factors or patient survival. Tumor morphology is conserved through multiple passages, and tumors retain metastatic potential. Interestingly, despite morphological similarity between passages, human stromal elements do not appear to expand with invading cancer cells. Rather, desmoplastic murine stroma dominates the xenograft microenvironment after the initial implantation. Recruitment of stromal elements in this manner to support and maintain tumor growth represents a novel avenue for investigation into tumor-stromal interactions. Direct implantation of viable surgical specimens provides a representative preclinical platform in pancreatic adenocarcinoma. Patient-derived xenografts consistently demonstrate retained tumor morphology and genetic stability. However, the evolution of the tumor microenvironment over time remains poorly characterized in these models. This work specifically addresses the recruitment and incorporation of murine stromal elements into expanding patient-derived pancreatic adenocarcinoma xenografts, establishing the integration of murine cells into networks of invading cancer cells. In addition, we provide methods and observations in the establishment and maintenance of a patient-derived pancreatic adenocarcinoma xenograft model. A total of 25 histologically confirmed pancreatic adenocarcinoma specimens were implanted subcutaneously into nonobese diabetic severe combined immunodeficiency mice. Patient demographics, staging, pathological analysis, and outcomes were analyzed. After successful engraftment of tumors, histological and immunofluorescence analyses were performed on explanted tumors. Pancreatic adenocarcinoma specimens were successfully engrafted in 15 (60%) of 25 attempts. Successful engraftment does not appear to correlate with clinicopathologic factors or patient survival. Tumor morphology is conserved through multiple passages, and tumors retain metastatic potential. Interestingly, despite morphological similarity between passages, human stromal elements do not appear to expand with invading cancer cells. Rather, desmoplastic murine stroma dominates the xenograft microenvironment after the initial implantation. Recruitment of stromal elements in this manner to support and maintain tumor growth represents a novel avenue for investigation into tumor-stromal interactions. A recent analysis of phase 1 cancer trials conducted from 2001 to 2012 revealed an overall objective response rate in only 3.8% of patients.1Roberts Jr., T.G. Goulart B.H. Squitieri L. Stallings S.C. Halpern E.F. Chabner B.A. Gazelle G.S. Finkelstein S.N. Clark J.W. Trends in the risks and benefits to patients with cancer participating in phase 1 clinical trials.JAMA. 2004; 292: 2130-2140Crossref PubMed Scopus (264) Google Scholar At some point, most agents included in this analysis demonstrated efficacy in xenograft models derived from cancer cell lines. The poor predictive value of cancer cell lines in vivo has been directly investigated and largely attributed to genetic instability resulting from primary culture.2Johnson J.I. Decker S. Zaharevitz D. Rubinstein L.V. Venditti J.M. Schepartz S. Kalyandrug S. Christian M. Arbuck S. Hollingshead M. Sausville E.A. Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials.Br J Cancer. 2001; 84: 1424-1431Crossref PubMed Scopus (719) Google Scholar, 3Daniel V.C. Marchionni L. Hierman J.S. Rhodes J.T. Devereux W.L. Rudin C.M. Yung R. Parmigiani G. Dorsch M. Peacock C.D. Watkins D.N. A primary xenograft model of small-cell lung cancer reveals irreversible changes in gene expression imposed by culture in vitro.Cancer Res. 2009; 69: 3364-3373Crossref PubMed Scopus (369) Google Scholar In contrast, patient-derived xenografts implant viable sections of cancer tissue directly into an immunocompromised host, thus avoiding the cell culture process altogether. In addition, patient-derived xenotransplantation demonstrates up to 10 times the success rate of cell line derivation from cancer specimens.4Tentler J.J. Tan A.C. Weekes C.D. Jimeno A. Leong S. Pitts T.M. Arcaroli J.J. Messersmith W.A. Eckhardt S.G. Patient-derived tumour xenografts as models for oncology drug development.Nat Rev Clin Oncol. 2012; 9: 338-350Crossref PubMed Scopus (980) Google Scholar, 5Dangles-Marie V. Pocard M. Richon S. Weiswald L.B. Assayag F. Saulnier P. Judde J.G. Janneau J.L. Auger N. Validire P. Dutrillaux B. Praz F. Bellet D. Poupon M.F. Establishment of human colon cancer cell lines from fresh tumors versus xenografts: comparison of success rate and cell line features.Cancer Res. 2007; 67: 398-407Crossref PubMed Scopus (151) Google Scholar Therefore, patient-derived xenografts represent a greater proportion of human malignancies, demonstrate reliable genetic stability, and are more predictive of clinical outcomes.6DeRose Y.S. Wang G. Lin Y.C. Bernard P.S. Buys S.S. Ebbert M.T. Factor R. Matsen C. Milash B.A. Nelson E. Neumayer L. Randall R.L. Stijleman I.J. Welm B.E. Welm A.L. Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.Nat Med. 2011; 17: 1514-1520Crossref PubMed Scopus (754) Google Scholar, 7Martinez-Garcia R. Juan D. Rausell A. Munoz M. Banos N. Menendez C. Lopez-Casas P.P. Rico D. Valencia A. Hidalgo M. Transcriptional dissection of pancreatic tumors engrafted in mice.Genome Med. 2014; 6: 27Crossref PubMed Scopus (36) Google Scholar In particular, reliable preclinical models are desperately needed in pancreatic cancer (PC). PC is the fourth leading cause of cancer death in the United States, projected to be second only to lung cancer by 2030.8Rahib L. Smith B.D. Aizenberg R. Rosenzweig A.B. Fleshman J.M. Matrisian L.M. Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States.Cancer Res. 2014; 74: 2913-2921Crossref PubMed Scopus (4812) Google Scholar Cytotoxic chemotherapy represents the major treatment modality in 80% of patients presenting with PC, extending survival to only 5 to 7 months,9Conroy T. Desseigne F. Ychou M. Bouche O. Guimbaud R. Becouarn Y. Adenis A. Raoul J.L. Gourgou-Bourgade S. de la Fouchardiere C. Bennouna J. Bachet J.B. Khemissa-Akouz F. Pere-Verge D. Delbaldo C. Assenat E. Chauffert B. Michel P. Montoto-Grillot C. Ducreux M. Groupe Tumeurs Digestives of Unicancer, PRODIGE Intergroup: FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer.N Engl J Med. 2011; 364: 1817-1825Crossref PubMed Scopus (5662) Google Scholar, 10Burris 3rd, H.A. Moore M.J. Andersen J. Green M.R. Rothenberg M.L. Modiano M.R. Cripps M.C. Portenoy R.K. Storniolo A.M. Tarassoff P. Nelson R. Dorr F.A. Stephens C.D. Von Hoff D.D. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial.J Clin Oncol. 1997; 15: 2403-2413Crossref PubMed Scopus (5312) Google Scholar and the addition of recently approved targeted therapies, such as erlotinib or nab-paclitaxel, only prolongs survival by an estimated 1 to 2 months.11Von Hoff D.D. Ervin T. Arena F.P. Chiorean E.G. Infante J. Moore M. Seay T. Tjulandin S.A. Ma W.W. Saleh M.N. Harris M. Reni M. Dowden S. Laheru D. Bahary N. Ramanathan R.K. Tabernero J. Hidalgo M. Goldstein D. Van Cutsem E. Wei X. Iglesias J. Renschler M.F. Increased survival in pancreatic cancer with nab-paclitaxel plus gemcitabine.N Engl J Med. 2013; 369: 1691-1703Crossref PubMed Scopus (4643) Google Scholar, 12Moore M.J. Goldstein D. Hamm J. Figer A. Hecht J.R. Gallinger S. Au H.J. Murawa P. Walde D. Wolff R.A. Campos D. Lim R. Ding K. Clark G. Voskoglou-Nomikos T. Ptasynski M. Parulekar W. Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group.J Clin Oncol. 2007; 25: 1960-1966Crossref PubMed Scopus (3255) Google Scholar Thus, annual death rates from PC continue to increase, underlining a global need for more representative preclinical models in the development of novel therapies.13Siegel R. Ma J. Zou Z. Jemal A. Cancer statistics, 2014.CA Cancer J Clin. 2014; 64: 9-29Crossref PubMed Scopus (11239) Google Scholar Notably, a small series investigating patient-derived xenografts in PC indicated a high degree of genetic stability when compared to the original PC specimen.14Rubio-Viqueira B. Jimeno A. Cusatis G. Zhang X. Iacobuzio-Donahue C. Karikari C. Shi C. Danenberg K. Danenberg P.V. Kuramochi H. Tanaka K. Singh S. Salimi-Moosavi H. Bouraoud N. Amador M.L. Altiok S. Kulesza P. Yeo C. Messersmith W. Eshleman J. Hruban R.H. Maitra A. Hidalgo M. An in vivo platform for translational drug development in pancreatic cancer.Clin Cancer Res. 2006; 12: 4652-4661Crossref PubMed Scopus (374) Google Scholar We sought to expand on this method in a cohort of 23 patients with PC, including two patients with metastatic lesions. Indeed, our results indicate that xenotransplantation of patient-derived PC specimens into immunocompromised mice successfully generated tumor grafts in most cases. Patient-derived xenografts retain morphological characteristics of the original PC specimen as well as metastatic potential from the implantation site. Furthermore, our results indicate that murine stroma is integrated into networks of expanding PC cells. Implications from this model are globally applicable to investigations into new pharmacological agents against PC, specifically agents that target tumor-stromal interactions. A viable 2 × 2-mm portion of tissue was immediately isolated from a surgically resected primary PC specimen with minimal ischemia time. PC tissue was then implanted subcutaneously into an 8-week-old female nonobese diabetic severe combined immunodeficiency mouse (Jackson Laboratory, Bar Harbor, ME). Xenografts were allowed to grow to a maximum diameter of 1.5 cm before passage. Herein, we define a passage as explantation of a PC xenograft and implantation into the flank of a new host. Tumor dimensions were measured three times per week using calipers. Tumor volumes were calculated using the following equation: v = (xy)2/2, where v is volume, x is tumor length, and y is tumor width. Final growth rate was determined using the amount of time taken to reach 1.5 cm in maximum diameter. Histological analysis of specimens was performed using hematoxylin and eosin staining. Cells were isolated from the blood of tumor-bearing mice by cardiac puncture and subjected to purification using a Ficoll Paque gradient (Sigma-Aldrich, St. Louis, MO). Live cells were then cultured in RPMI 1640 medium, 10% fetal calf serum, 20 ng/mL epithelial growth factor, and antibiotic antimycotic solution (Sigma-Aldrich) for 2 months before fixation and staining. A review of an institutional review board–approved, prospectively maintained PC database at the University of Florida (Gainesville) was performed. Clinicopathologic data were analyzed for patients who underwent pancreatic resection for PC. All statistical analysis was performed using SPSS version 22.0 (IBM SPSS Statistics for Windows; IBM Corp., Armonk, NY). All clinical data were tested for normality using the Shapiro-Wilk test and displayed nonparametric distributions (P < 0.05). U-tests and χ2 coefficients were, therefore, used to determine significance between groups for continuous and categorical variables, respectively. Kaplan-Meier survival curves and a Cox proportional hazards model examined the respective effects of successful engraftment and xenograft growth rate on overall survival. The log-rank test was used to determine survival differences between groups in Kaplan-Meier analysis. P < 0.05 was considered statistically significant. Immunohistochemical staining of tissue specimens was performed by the University of Florida's Molecular Pathology Core Facility. Briefly, patient tumors, xenografts, and murine organ specimens were analyzed in formalin-fixed, paraffin-embedded sections (5 μm thick). Hematoxylin and eosin stains were performed on all specimens, and subsequent stains were performed in serial sections (5 μm thick). Primary antibodies against human-specific vimentin and human leukocyte antigen (HLA)-A (Abcam, Cambridge, UK) were used after antigen retrieval with citrate buffer (pH 6.0) in tissue immunohistochemical analysis. Immunocytochemistry was similarly performed after methanol fixation of cells in culture. In total, 15 (60%) of 25 patient-derived PC specimens were successfully engrafted subcutaneously into nonobese diabetic severe combined immunodeficiency mice. Wounds healed by postoperative day 7. Tumor invasion into muscular tissue was typically observed by week 2, palpable growth was observed by week 3, and growth end point (1.5 cm) was reached within 8 to 16 weeks (Figure 1A). Because each xenograft represents a specific patient, we hypothesized that clinically significant markers would correlate with successful engraftment and subsequent growth kinetics. Interestingly, all examined clinicopathologic parameters displayed no correlation with successful engraftment or PC xenograft growth rate (Tables 1 and 2 and Figure 1B). Accordingly, neither successful engraftment nor growth rate correlated with overall patient survival (Figure 1C). Although the precise conclusions from these data are limited, we speculate that successful PC engraftment is likely dependent on technical, rather than patient, factors. Specifically, the reduction of tumor ischemia time to an absolute minimum is critical to successful engraftment.Table 1Clinicopathologic Parameters Do Not Correlate with Successful Tumor EngraftmentParameterSuccessful engraftmentP valueNo (n = 10)Yes (n = 15)Age, years∗Data are given as means ± SEM.67.2 ± 3.867.2 ± 2.60.765Tumor location†Data are given as number (percentage). Pancreas10 (100)13 (87)0.500 Hepatic metastasis02 (13)N1 stage†Data are given as number (percentage).8 (80)11 (85)1.00Positive lymph node ratio∗Data are given as means ± SEM.0.22 ± 0.070.14 ± 0.030.522Poor tumor differentiation†Data are given as number (percentage).4 (40)6 (46)1.00Tumor size (cm)∗Data are given as means ± SEM.3.05 ± 0.443.59 ± 0.270.563CA 19-9 (U/mL)∗Data are given as means ± SEM.1514 ± 9941437 ± 7750.546Neoadjuvant therapy†Data are given as number (percentage).2 (20)4 (31)0.660R1 resection†Data are given as number (percentage).4 (40)5 (39)1.00Clinicopathologic variables were assessed for effects on successful pancreatic cancer engraftment using χ2 coefficients for categorical variables or the Wilcoxon rank sum test for continuous variables because of nonparametric distributions.∗ Data are given as means ± SEM.† Data are given as number (percentage). Open table in a new tab Table 2Among Clinicopathologic Parameters Do Not Correlate with lymph node size 19-9 lymph node tumor and 19-9 were compared with xenograft growth rate growth in maximum successfully engrafted xenografts (n = using rank Open table in a new tab Clinicopathologic variables were assessed for effects on successful pancreatic cancer engraftment using χ2 coefficients for categorical variables or the Wilcoxon rank sum test for continuous variables because of nonparametric lymph node tumor and 19-9 were compared with xenograft growth rate growth in maximum successfully engrafted xenografts (n = using rank Patient-derived PC xenografts maintained characteristics of the original PC specimen after continuous results demonstrated of tumor specifically with to tumor and (Figure stromal elements through multiple of PC xenografts into new mice. We human tumor cells in mice patient-derived PC cells were isolated from the blood of tumor-bearing mice and immunofluorescence analysis revealed human that these cells were of human (Figure In addition, the metastatic potential of PC xenografts is demonstrated in this is similarly maintained through multiple We observed and in mice patient-derived xenografts, confirmed by hematoxylin and eosin (Figure This therefore, desmoplastic generated from stromal elements in the Furthermore, human cells in these tumors and metastasis to in murine this was not observed in all patient-derived PC in murine tissue are displayed for xenografts derived from patients with PC human PC tumors a to to tissue in mice. However, our observations demonstrate no specific correlation between clinicopathologic parameters and metastasis in murine of PC Xenografts from survival in to at 15 to at 7 liver, and from patients were examined for metastatic were from mice tumors at size end and analyzed hematoxylin and eosin Clinicopathologic characteristics of each patient are displayed as well as metastatic of pancreatic Open table in a new tab Xenografts from patients were examined for metastatic were from mice tumors at size end and analyzed hematoxylin and eosin Clinicopathologic characteristics of each patient are displayed as well as metastatic of PC, pancreatic in the microenvironment represent a in PC. therefore, stromal incorporation into expanding PC xenografts was of human this we immunohistochemical and immunofluorescence staining using human-specific method reveals that human stromal elements vimentin in PC specimens (Figure 3, A and However, on incorporation into a PC a is consistently observed a 2-mm of tissue the implanted PC staining for human-specific vimentin in this (Figure 3, of a PC xenograft the of human PC cells and the of human-specific stroma (Figure these results indicate that PC xenografts murine stromal Therefore, despite of tumor-stromal stromal elements in patient-derived PC xenografts are of murine We sought to the of patient-derived PC xenotransplantation and potential for in preclinical We initial results with to patient-derived PC xenograft metastatic and incorporation of murine data indicate that these xenografts are an method of investigating a representative of PC We demonstrate that xenografts maintain the of the original with to desmoplastic elements that are in PC. murine stromal elements are into expanding xenografts, an for preclinical work with this model. is to that patient-derived xenografts been characterized in models. In success rates of up to to been in multiple J.J. Tan A.C. Weekes C.D. Jimeno A. Leong S. Pitts T.M. Arcaroli J.J. Messersmith W.A. Eckhardt S.G. Patient-derived tumour xenografts as models for oncology drug development.Nat Rev Clin Oncol. 2012; 9: 338-350Crossref PubMed Scopus (980) Google Scholar, 5Dangles-Marie V. Pocard M. Richon S. Weiswald L.B. Assayag F. Saulnier P. Judde J.G. Janneau J.L. Auger N. Validire P. Dutrillaux B. Praz F. Bellet D. Poupon M.F. Establishment of human colon cancer cell lines from fresh tumors versus xenografts: comparison of success rate and cell line features.Cancer Res. 2007; 67: 398-407Crossref PubMed Scopus (151) Google Scholar, B. Jimeno A. Cusatis G. Zhang X. Iacobuzio-Donahue C. Karikari C. Shi C. Danenberg K. Danenberg P.V. Kuramochi H. Tanaka K. Singh S. Salimi-Moosavi H. Bouraoud N. Amador M.L. Altiok S. Kulesza P. Yeo C. Messersmith W. Eshleman J. Hruban R.H. Maitra A. Hidalgo M. An in vivo platform for translational drug development in pancreatic cancer.Clin Cancer Res. 2006; 12: 4652-4661Crossref PubMed Scopus (374) Google Scholar This to that successful engraftment in PC is likely more dependent on technical, rather than the reduction of ischemia time to an absolute PC, of human stroma has been by R. Juan D. Rausell A. Munoz M. Banos N. Menendez C. Lopez-Casas P.P. Rico D. Valencia A. Hidalgo M. Transcriptional dissection of pancreatic tumors engrafted in mice.Genome Med. 2014; 6: 27Crossref PubMed Scopus (36) Google Scholar, M. A. W. S. Y. L. A. An Z. Zhang Y. S. K. D. P. Molecular of patient-derived human pancreatic tumor xenograft models for preclinical and translational development of cancer 2013; 15: PubMed Scopus Google Scholar This work demonstrates the of human stroma by murine because these elements are the original implanted Interestingly, desmoplastic elements are retained in xenografts, that an of tumor-stromal not be work with the work of groups in that we demonstrate metastatic potential from patient-derived J.J. Tan A.C. Weekes C.D. Jimeno A. Leong S. Pitts T.M. Arcaroli J.J. Messersmith W.A. Eckhardt S.G. Patient-derived tumour xenografts as models for oncology drug development.Nat Rev Clin Oncol. 2012; 9: 338-350Crossref PubMed Scopus (980) Google Scholar, Wang H. J.L. J.B. of and human pancreatic cancer xenografts in 2009; PubMed Scopus Google Scholar is to that these typically displayed invasion into However, we observed human cells and metastatic to multiple in the of of the primary Notably, xenografts been to a of observed metastatic potential. However, in to implantation into the murine xenografts provide major with to the and of a and the of xenografts as a preclinical model. In we demonstrate that the of a patient-derived PC xenograft model is with a high rate of successful We two for preclinical murine stromal elements are into expanding xenografts early after implantation. PC xenografts demonstrate metastatic potential from implantation support the investigations of establishing patient-derived tumor xenografts as an preclinical model in PC.

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