Isolation of Pancreatic Cancer Cells from a Patient-Derived Xenograft Model Allows for Practical Expansion and Preserved Heterogeneity in Culture
2016; Elsevier BV; Volume: 186; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2016.02.009
ISSN1525-2191
AutoresKien Pham, Daniel Delitto, Andrea E. Knowlton, Emily R. Hartlage, Ricky Madhavan, David Hernández Gonzalo, Ryan M. Thomas, K.E. Behrns, Thomas J. George, Steven J. Hughes, Shannon M. Wallet, Chen Liu, José G. Treviño,
Tópico(s)Cancer Research and Treatments
ResumoCommercially available, highly passaged pancreatic cancer (PC) cell lines are of limited translational value. Attempts to overcome this limitation have primarily consisted of cancer cell isolation and culture directly from human PC specimens. However, these techniques are associated with exceedingly low success rates. Here, we demonstrate a highly reproducible culture of primary PC cell lines (PPCLs) from patient-derived xenografts, which preserve, in part, the intratumoral heterogeneity known to exist in PC. PPCL expansion from patient-derived xenografts was successful in 100% of attempts (5 of 5). Phenotypic analysis was evaluated with flow cytometry, immunofluorescence microscopy, and short tandem repeat profiling. Importantly, tumorigenicity of PPCLs expanded from patient-derived xenografts was assessed by subcutaneous injection into nonobese diabeteic.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Morphologically, subcutaneous injection of all PPCLs into mice yielded tumors with similar characteristics to the parent xenograft. PPCLs uniformly expressed class I human leukocyte antigen, epithelial cell adhesion molecule, and cytokeratin-19. Heterogeneity within each PPCL persisted in culture for the frequency of cells expressing the cancer stem cell markers CD44, CD133, and c-Met and the immunologic markers human leukocyte antigen class II and programmed death ligand 1. This work therefore presents a reliable method for the rapid expansion of primary human PC cells and, thereby, provides a platform for translational investigation and, importantly, potential personalized therapeutic approaches. Commercially available, highly passaged pancreatic cancer (PC) cell lines are of limited translational value. Attempts to overcome this limitation have primarily consisted of cancer cell isolation and culture directly from human PC specimens. However, these techniques are associated with exceedingly low success rates. Here, we demonstrate a highly reproducible culture of primary PC cell lines (PPCLs) from patient-derived xenografts, which preserve, in part, the intratumoral heterogeneity known to exist in PC. PPCL expansion from patient-derived xenografts was successful in 100% of attempts (5 of 5). Phenotypic analysis was evaluated with flow cytometry, immunofluorescence microscopy, and short tandem repeat profiling. Importantly, tumorigenicity of PPCLs expanded from patient-derived xenografts was assessed by subcutaneous injection into nonobese diabeteic.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice. Morphologically, subcutaneous injection of all PPCLs into mice yielded tumors with similar characteristics to the parent xenograft. PPCLs uniformly expressed class I human leukocyte antigen, epithelial cell adhesion molecule, and cytokeratin-19. Heterogeneity within each PPCL persisted in culture for the frequency of cells expressing the cancer stem cell markers CD44, CD133, and c-Met and the immunologic markers human leukocyte antigen class II and programmed death ligand 1. This work therefore presents a reliable method for the rapid expansion of primary human PC cells and, thereby, provides a platform for translational investigation and, importantly, potential personalized therapeutic approaches. Pancreatic cancer (PC) is projected to be the second leading cause of cancer deaths by 2030.1Rahib 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 (4267) Google Scholar Systemic cytotoxic and kinase-targeted regimens represent the standard of care for most patients presenting with PC. Most tumors, however, will develop rapid resistance to these regimens and will continue to progress by unknown mechanisms.2Koay E.J. Truty M.J. Cristini V. Thomas R.M. Chen R. Chatterjee D. Kang Y. Bhosale P.R. Tamm E.P. Crane C.H. Javle M. Katz M.H. Gottumukkala V.N. Rozner M.A. Shen H. Lee J.E. Wang H. Chen Y. Plunkett W. Abbruzzese J.L. Wolff R.A. Varadhachary G.R. Ferrari M. Fleming J.B. Transport properties of pancreatic cancer describe gemcitabine delivery and response.J Clin Invest. 2014; 124: 1525-1536Crossref PubMed Scopus (141) Google Scholar, 3Moore 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 (3142) Google Scholar As a result, both the median survival and annual death rate for patients with PC have remained unchanged over the past 20 years.4Baxter N.N. Whitson B.A. Tuttle T.M. Trends in the treatment and outcome of pancreatic cancer in the United States.Ann Surg Oncol. 2007; 14: 1320-1326Crossref PubMed Scopus (122) Google Scholar To this end, an analysis of phase 1 cancer trials, which used agents with demonstrated efficacy in models derived from established commercially available cancer cell lines, revealed an overall objective response rate in only 3.8% of patients. For the past 40 years, commercially available PC cells (PCCs) such as MIA-PaCa2 (established in 1975),5Yunis A.A. Arimura G.K. Russin D.J. Human pancreatic carcinoma (MIA PaCa-2) in continuous culture: sensitivity to asparaginase.Int J Cancer. 1977; 19: 128-135Crossref PubMed Scopus (280) Google Scholar CFPAC-1 (liver metastases established from a cystic fibrosis patient in 1990),6McIntosh J.C. Schoumacher R.A. Tiller R.E. Pancreatic adenocarcinoma in a patient with cystic fibrosis.Am J Med. 1988; 85: 592Abstract Full Text PDF PubMed Scopus (48) Google Scholar PANC-1 (established in 1975),7Lieber M. Mazzetta J. Nelson-Rees W. Kaplan M. Todaro G. Establishment of a continuous tumor-cell line (panc-1) from a human carcinoma of the exocrine pancreas.Int J Cancer. 1975; 15: 741-747Crossref PubMed Scopus (423) Google Scholar and BxPC-3 (K-ras wild-type established in 1986)8Tan M.H. Nowak N.J. Loor R. Ochi H. Sandberg A.A. Lopez C. Pickren J.W. Berjian R. Douglass Jr., H.O. Chu T.M. Characterization of a new primary human pancreatic tumor line.Cancer Invest. 1986; 4: 15-23Crossref PubMed Scopus (171) Google Scholar have been widely used in PC models and derived from an extremely small set of PC patients from >30 years ago. Thus, the poor predictive value of studies with the use of established cancer cell lines is a major barrier to the development of new interventions.9Johnson 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 (692) Google Scholar, 10Daniel 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 (357) Google Scholar Our understanding of PC responses to therapy is also complicated by the marked molecular heterogeneity that exists among primary tumors from different patients and intrapatient tumor cell heterogeneity, neither of which can be captured with currently available commercial cell lines.11Cassidy J.W. Caldas C. Bruna A. Maintaining tumor heterogeneity in patient-derived tumor xenografts.Cancer Res. 2015; 75: 2963-2968Crossref PubMed Scopus (225) Google Scholar To begin to address some of these barriers, groups have attempted direct isolation and culture of patient-derived primary human PCCs from viable surgical specimens. The success rate of this method was reported to be approximately 10% in experienced hands,12Ruckert F. Aust D. Bohme I. Werner K. Brandt A. Diamandis E.P. Krautz C. Hering S. Saeger H.D. Grutzmann R. Pilarsky C. Five primary human pancreatic adenocarcinoma cell lines established by the outgrowth method.J Surg Res. 2012; 172: 29-39Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar although most groups do not publish failed attempts.13Holloway S. Davis M. Jaber R. Fleming J. A clinically relevant model of human pancreatic adenocarcinoma identifies patterns of metastasis associated with alterations of the TGF-beta/Smad4 signaling pathway.Int J Gastrointest Cancer. 2003; 33: 61-69Crossref Google Scholar, 14Chifenti B. Morelli M. Zavaglia M. Coviello D.A. Guerneri S. Santucci A. Paffetti A. Masetti M. Locci M.T. Bertacca G. Capodanno A. Collecchi P. Campani D. Mosca F. Bevilacqua G. Cavazzana A.O. Establishment and characterization of 4 new human pancreatic cancer cell lines: evidences of different tumor phenotypes.Pancreas. 2009; 38: 184-196Crossref Scopus (14) Google Scholar, 15Kyriazis A.P. McCombs 3rd, W.B. Sandberg A.A. 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Transcriptional dissection of pancreatic tumors engrafted in mice.Genome Med. 2014; 6: 27Crossref PubMed Scopus (35) Google Scholar Thus, to address these limitations, we leveraged our recently developed PC–patient-derived xenograft (PDX) model, whereby we have demonstrated that the early PDX is morphologically similar to the original cancer and retains both interpatient and intrapatient heterogeneity of the human disease.17Tentler 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 (927) Google Scholar, 21Delitto D. Pham K. Vlada A.C. Sarosi G.A. Thomas R.M. Behrns K.E. Liu C. Hughes S.J. Wallet S.M. Trevino J.G. Patient-derived xenograft models for pancreatic adenocarcinoma demonstrate retention of tumor morphology through incorporation of murine stromal elements.Am J Pathol. 2015; 185: 1297-1303Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Specifically, here, we demonstrate the expansion of human PCCs through a PDX model that preserves the tumor heterogeneity with a 100% success rate. These established PDX-derived primary cell lines display uniform markers associated with a human PC origin with retained tumorigenicity. In addition, after several passages, these PCCs continue to exhibit significant heterogeneity in the expression of markers associated with their initial molecular phenotype and PC-immune cell interactions. In summary, this method of primary PCC isolation may provide a critical model that enhances clinical relevance by augmenting preclinical investigations and affording a personalized therapeutic examination. Informed written consent was obtained from all patients, and the collection of all patient material was approved by the University of Florida Institutional Review Board. All animal studies were performed in accordance with the guidelines of the University of Florida Institutional Animal Care and Use Committee. Human PC specimens and all xenografts were evaluated by expert gastrointestinal pathologists, in accordance with the World Health Organization Classification of Tumors of the Digestive System. Tumors were classified according to site of origin and tumor stage. The implantation of surgical tumor tissue into immunocompromised mice was described previously.21Delitto D. Pham K. Vlada A.C. Sarosi G.A. Thomas R.M. Behrns K.E. Liu C. Hughes S.J. Wallet S.M. Trevino J.G. Patient-derived xenograft models for pancreatic adenocarcinoma demonstrate retention of tumor morphology through incorporation of murine stromal elements.Am J Pathol. 2015; 185: 1297-1303Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Briefly, a viable portion of resected tissue 2 × 2 mm in size was isolated immediately from surgically resected primary PC specimens with care to minimize critical ischemia time. PC tissue was then implanted subcutaneously into an 8-week-old female nonobese diabetic.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mouse (The Jackson Laboratory, Bar Harbor, ME). Xenografts were allowed to grow to a maximum diameter of 1.5 cm before passage and/or in vitro culture. Tissue collected from xenograft tumors was minced into small pieces and enzymatically dissociated into single cells with 2 mg/mL STEMxyme 1 Collagenase/Neutral Protease solution (Worthington Biochemical Corporation, Lakewood, NJ) for 30 minutes at 37°C. After washing with Hank's balanced salt solution that contained 2% of fetal bovine serum, cells were filtered through a 100-μm filter to remove nondissociated cell clumps. The single cell suspension was then cultured in advanced Dulbecco's Modified Eagle Medium with nutrient mixture F12, 10% fetal bovine serum, 6 mmol/L glutamine, 1% of penicillin/streptomycin, and 40 ng/mL dexamethasone, at a density of 105 cells/mL on culture plates coated with rat tail collagen I. Media were changed every second or third day, and cells were passaged with Accutase (Innovative Cell Technologies, San Diego, CA) at 80% confluence. To avoid the outgrowth of contaminating fibroblasts, differential trypsinization was performed until a homogenous population of cytokeratin (CK)19/class I human leukocyte antigen (HLA)-expressing human PCCs was achieved by flow cytometry analysis. DNA fingerprinting assays were performed to establish a unique genetic identification for each patient's PCC population. Total genomic DNA was extracted from cell pellets with Qiagen DNeasy Blood and Tissue kit (Qiagen, Valencia, CA) according to the manufacturer's protocol, and sent to the Sanger Sequencing Core at the University of Florida for cell line authentication. The identity of the DNA profiles was determined by short tandem repeat profiling with the use of GenePrint 10 System (Promega, Madison, WI). This kit amplifies nine short tandem repeat human loci (TPOX, vWA, D21S11, TH01, CSF1PO, D16S539, D7S820, D13S317, and D5S818) and AMEL for sex identification, which was cross-referenced against patient sex. The samples were processed on an ABI 3130XL Genome Analyzer (Applied Biosystems, Foster City, CA), and data were analyzed with GeneMapper software version 4.0 (Applied Biosystems). Fingerprints were then compared with an established database of DNA profiles in the ATCC (Manassas, VA) to verify uniqueness. Isolated DNA was also profiled for 38 somatic mutations within the following genes associated with PC: APC, BRAF, CDKN2A, CTNNB1, KRAS, NRAS, PIK3CA, SMAD4, and TP53 with the use of the Pancreatic Cancer qBiomarker Somatic Mutation PCR Array (Qiagen). Data were collected on the Applied Biosystems 7900HT instrument, and analyzed with the use of the Sequence Detection Systems software version 2.4.1 (Applied Biosystems). Samples were evaluated for the presence or absence of a specific somatic mutation and were normalized to gene copy reference assays for each gene present in the array. To establish tumorigenicity of cultured primary PCCs, 3 × 106 cells from each patient's cell population was suspended in 200 μL of a 1:1 mixture of Dulbecco's Modified Eagle Medium with nutrient mixture F12 and Matrigel Matrix (Corning, Corning, NY) and inoculated subcutaneously into the right flank of NSG mice. Mice were inspected twice a week, and tumor size was measured with a digital caliper. Mice were euthanized 40 days after the implantation. Tumors were fixed in 10% formalin and subjected to histologic analysis with hematoxylin and eosin staining. The growth rate (%/day) was used to evaluate the growth kinetics of primary PCC. Short-term proliferation assays were performed to determine growth rates in culture. Cells were seeded in 24-well plates at 1 × 105 cells/well on day 0. Cell number was determined with the TC20 Automated Cell Counter (Bio-Rad, Hercules, CA) on days 1 through 6. The growth curve was fitted to Gompertz growth model with GraphPad Prism software version 6 (GraphPad Software, La Jolla, CA). Doubling time was determined from the exponential phase with the use of the equation ln(2)/k, in which k is the rate constant. Assays were performed in triplicate and repeated at least three times. Tissue staining was performed by the University of Florida Molecular Pathology Core. Briefly, formalin fixed, paraffin-embedded patient tumors and PDX specimens were deparaffinized and cut into 5-μm sections. Sections were stained with hematoxylin and eosin. Additional 5-μm sections of PDX specimens were probed with anti–programmed death ligand 1 (PDL1; Abcam, Cambridge, MA) according to the manufacturer's recommended protocol. Primary PCCs were cultured on collagen-coated 24-well plates and fixed in 4% paraformaldehyde for 15 minutes at room temperature. After fixation, cells were permeabilized and blocked in 3% bovine serum albumin and 0.1% Triton X-100 for 1 hour at room temperature. The cells were then incubated in 1% bovine serum albumin and 0.1% Triton X-100 overnight at 4°C with the following antibodies: Alexa Fluor 647 (AF647) anti-human CK18, AF647 anti-human epithelial cell adhesion molecule (EpCam), AF647 anti-human CK19 (BioLegend, San Diego, CA), and rabbit anti-human CD45 (Cell Signaling Technologies, Danvers, MA) followed by Alexa Fluor 568 goat anti-rabbit secondary for 2 hours at room temperature. The DNA dye DAPI was used to counterstain nuclei. Images were collected on an EVOS FL digital inverted microscope (Life Technologies, Carlsbad, CA) and processed with ImageJ software version 1.48V (NIH, Bethesda, MD; https://imagej.nih.gov/ij/download.html). Cells were dissociated from 24-well culture dishes with the use of Accutase, washed in Dulbecco's phosphate-buffered saline that contained 5% fetal bovine serum, 5 mmol/L EDTA, and 0.1% sodium azide (Sigma-Aldrich, St. Louis, MO), and probed for CD45, EpCam, CD44, CD133, HLA-ABC, HLA-DR/DP/DQ, CD80, CD86, and PDL1 with the use of the following antibodies: Pacific Blue-conjugated CD45, Alexa Fluor 488 (AF488)-conjugated CD44, phycoerythrin (PE)-conjugated CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany), PE-cyanin 7–conjugated EpCam, allophycocyanin-cyanin 7–conjugated HLA-ABC, PE-conjugated CD86, AF647-conjugated HLA-DR/DP/DQ, PE-cyanin 7–conjugated PDL1, and allophycocyanin-H7–conjugated CD80 (BD Biosciences, Franklin Lakes, NJ). All antibodies were purchased from BioLegend unless otherwise indicated and used at a 1:100 dilution. Manufacturer's recommended isotype controls were used as negative controls for all antibodies used. A total of 10,000 events per sample were acquired with a BD LSR II (BD Biosciences), and data were analyzed with FlowJo data analysis software version 9 (FlowJo LLC, Ashland, OR). Cell were seeded in 96-well plate at a density of 5 × 103 cells/well in a total of 100 μL of culture medium and treated with gemcitabine (Selleck Chemicals, Houston TX) at multiple concentrations of 3, 10, 30, 100, and 300 nmol/L and 1 μmol/L. After 6ix days, viability was analyzed with the Cell Counting Kit-8 (Dojindo Molecular Technologies Inc., Rockville, MD), according to the manufacturer's protocol. The absorbance of yellow formazan dye, a derivative of water-soluble [2-(2-methox y-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] monosodium salt from dehydrogenase activity in cells was detected with colorimetric microplate reader at 450 nm. This assay was performed in triplicate and was repeated at least three times. Statistical analyses were performed with GraphPad Prism software version 6 (GraphPad Software). Data are presented as means ± SEM. P values were calculated with the Student's t-test with two-tailed distribution. P < 0.05 was considered significant. Primary pancreatic cancer cell lines (PPCLs) were isolated from PDX tumors which are enriched in patient-derived PCCs, contrary to previous attempts at direct isolation from resected PC specimens which are limited by a preponderance of myofibroblastic elements and relatively few cancer cells by volume.22Erkan M. Michalski C.W. Rieder S. Reiser-Erkan C. Abiatari I. Kolb A. Giese N.A. Esposito I. Friess H. Kleeff J. The activated stroma index is a novel and independent prognostic marker in pancreatic ductal adenocarcinoma.Clin Gastroenterol Hepatol. 2008; 6: 1155-1161Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar In addition, hepatic metastases from PC primaries were expanded with the PDX model and similarly isolated in culture. The isolation and expansion technique had a 100% success rate (5 of 5 lines) (Figure 1). Early depletion of murine tumor-associated stroma through differential trypsinization was critical to allow for the isolation of low-passage PCCs without the need for clonal expansion methods. All PPCLs were passaged through at least 10 generations without any sign of growth decline or morphologic change. The patient demographic and clinicopathologic data associated with each patient from whom the PDXs and PPCLs were derived are displayed in Table 1. PPCLs presented in this study were derived from PDX established from both primary tumors (PPCL-46, PPCL-59, and PPCL-68) and hepatic metastases (PPCL-LM1, PPCL-LM2). All PPCLs demonstrated mutational profiles consistent with PC, and all identified K-Ras and TP53 mutations were conserved between primary tumor, PDX, and PPCL (Table 1), short tandem repeat profiling with 10 loci and cross-referenced against a known ATCC database, demonstrated that all PPCLs generated were unique and sex matched to the original patient. DNA fingerprinting results are displayed in Table 2.Table 1Clinicopathologic Characteristics and Mutation Profiles of Five PPCLs Established from Patient-Derived XenograftsIDAge, yearsSexTumor sitePositive lymph nodes/totalOverall survival, moK-RasTP53PPCL-4675FemalePrimary7/3410G12VWTPPCL-5973FemalePrimary4/15Alive at 4 moG12VWTPPCL-6864FemalePrimary2/18Alive at 3 moG12DR248WPPCL-LM165MaleMetastasisNA8G12VWTPPCL-LM263MaleMetastasisNA2G12VWTmo, month; NA, not applicable; PPCL, primary pancreatic cancer cell line; WT, wild type. Open table in a new tab Table 2Short Tandem Repeat Profiling of PPCLsLocusPPCL-46PPCL-59PPCL-68PPCL-LM1PPCL-LM2D5S81812119, 129, 1111, 12D13S3179, 149, 128, 1011, 1311D7S8209, 11911108D16S53911, 139, 131310, 1311, 12vWA2014, 1815, 1715, 1615, 17TH018, 99.366, 9.36TPOX10, 119, 108, 10119CSF1PO12, 13121010, 1110, 11D21S112929282928, 32.2AMELXXXX, YX, YPPCL, primary pancreatic cancer cell line. Open table in a new tab mo, month; NA, not applicable; PPCL, primary pancreatic cancer cell line; WT, wild type. PPCL, primary pancreatic cancer cell line. Because of the nature of the technique, contamination by immune cells and fibroblasts from both human and murine sources was evaluated. Flow cytometric analysis was used to examine the expression of HLA class I, the immune cell marker CD45, and the myofibroblast marker α-smooth muscle actin (α-SMA). All PPCLs expressed HLA class I, whereby all cultures were negative for both the leukocyte antigen CD45 and the mesenchymal antigen α-SMA, indicating the presence of human cells in the absence of immune and fibroblastic cells, respectively (Figure 2). PPCLs were further assessed for the expression of the epithelial cell markers EpCam and CK19, whereby flow cytometric analysis and immunofluorescence microscopy demonstrated that all cultures were universally positive (Figure 2). Together these data demonstrate a culture that consisted of human epithelial cells free from immune and fibroblast contamination. To confirm tumorigenicity of PPCL, the ability of these cultures to form tumors in NSG mice was determined. Indeed, all PPCs were able to grow in vivo, albeit at different rates (Figure 3A). Histologic analysis displayed similar architectural structure to the parental PDX and original tumors (Figure 1). All PPCLs isolated from PDX tumors formed adherent monolayers in culture and also demonstrated variable exponential growth in vitro (Figure 3B). Here, PPCLs were broadly categorized into three subgroups as a function of growth kinetics: slow (doubling time > 38 hours), intermediate (doubling time 28 to < 38 hours), and fast (doubling time < 28 hours). Interestingly, the in vitro growth kinetics were comparable with the in vivo growth dynamics of PPCLs as measured by the previously validated specific growth rate (Figure 3D).23Mehrara E. Forssell-Aronsson E. Ahlman H. Bernhardt P. Specific growth rate versus doubling time for quantitative characterization of tumor growth rate.Cancer Res. 2007; 67: 3970-3975Crossref PubMed Scopus (167) Google Scholar Collectively, these data demonstrate that all PPCL retain their tumorigenicity, regardless of the heterogeneity in the growth rates observed. These results support the tumor variability commonly observed in the clinical setting. Because chemosensitivity of PC is hypothesized to be directly related to the proliferative capacity of cancer cells, the chemosensitivity to the clinically relevant antimetabolite, gemcitabine, was evaluated for each PPCL established. Interestingly, although all PPCLs were sensitive to gemcitabine treatment (Figure 3C), an inverse correlation between proliferation rate and sensitivity was observed (Figure 3D). These findings suggest that more diverse mechanisms than rate of proliferation are responsible for antimetabolite sensitivity.24de Sousa Cavalcante L. Monteiro G. Gemcitabine: metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer.Eur J Pharmacol. 2014; 741: 8-16Crossref PubMed Scopus (344) Google Scholar Dissociated PDX tumors have enabled careful experimental probing of intratumoral heterogeneity, critical to the discovery of PC stem cell markers.25Li C. Heidt D.G. Dalerba P. Burant C.F. Zhang L. Adsay V. Wicha M. Clarke M.F. Simeone D.M. Identification of pancreatic cancer stem cells.Cancer Res. 2007; 67: 1030-1037Crossref PubMed Scopus (2758) Google Scholar To assess whether the heterogeneity observed above in the PPCL cultures extends to PC stem cell markers, the established PC stem cell markers CD44, CD133, and c-Met were examined by flow cytometry.26Li C. Wu J.J. Hynes M. Dosch J. Sarkar B. Welling T.H. Pasca di Magliano M. Simeone D.M. c-Met is a marker of pancreatic cancer stem cells and therapeutic target.Gastroenterology. 2011; 141: 2218-2227.e5Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar Interestingly, each PPCL demonstrated a unique profile of CD44 and CD133 expression (Figure 4A), exhibiting from 1% to 96% CD44highCD133+ cells. Similarly, differential c-Met expression was observed among PPCLs, with each line demonstrating between 6% and 62% c-Met− cells (Figure 4B). Taken together, these experiments demonstrate the continued presence of intratumoral cancer stem cell heterogeneity in primary culture. The properties of PC epithelial cells may allow for evasion of immune clearance through the down-regulation of HLA and/or T-cell–activating costimulatory molecules and/or up-regulation of T-cell inhibitory molecules.27Delitto D. Perez C. Han S. Gonzalo D.H. Pham K. Knowlton A.E. Graves C.L. Behrns K.E. Moldawer L.L. Thomas R.M. Liu C. George Jr., T.J. Trevino J.G. Wallet S.M. Hughes S.J. Downstream mediators of the intratumoral interferon response suppress antitumor immunity, induce gemcitabine resistance and associate with poor survival in human pancreatic cancer.Cancer Immunol Immunother. 2015; 64: 1553-1563Crossref Scopus (23) Google Scholar Thus, to evaluate the extent to which the patient-derived PPCLs express this antigen presentation machinery and costimulatory molecules, flow cytometry was performed to examine HLA class II (HLA-DR/DP/DQ), the activating costimulatory molecules CD80, CD86, and the inhibitory costimulatory molecule PDL1. Interestingly, all PPCLs expressed the inhibitory costimulatory molecule PDL1 variably, with the highest expression observed in PPCLs of metastatic
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