Intrahepatic Xenograft of Cutaneous T-Cell Lymphoma Cell Lines
2016; Elsevier BV; Volume: 186; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2016.03.012
ISSN1525-2191
AutoresLaëtitia Andrique, Sandrine Poglio, Martina Prochazkova‐Carlotti, Marshall E. Kadin, Alban Giese, Yamina Idrissi, M. Beylot‐Barry, Jean‐Philippe Merlio, Edith Chevret,
Tópico(s)Fungal Infections and Studies
ResumoCutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of diseases primarily involving the skin that could have an aggressive course with circulating blood cells, especially in Sézary syndrome and transformed mycosis fungoides. So far, few CTCL cell lines have been adapted for in vivo experiments and their tumorigenicity has not been adequately assessed, hampering the use of a reproducible model for CTCL biological evaluation. In fact, both patient-derived xenografts and cell line xenografts at subcutaneous sites failed to provide a robust tool, because engraftment was dependent on mice strain and cell line subtype. Herein, we describe an original method of intrahepatic injection into adult NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice liver of both aggressive (My-La, HUT78, HH, MAC2A, and MAC2B) and indolent (FE-PD and MAC1) CTCL cell lines. Six of the seven CTCL cell lines were grafted with a high rate of success (80%). Moreover, this model provided a quick (15 days) and robust assay for in vivo evaluation of CTCL cell lines tumorigenicity and therapeutic response in preclinical studies. Such a reproducible model can be therefore used for further functional studies and in vivo drug testing. Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of diseases primarily involving the skin that could have an aggressive course with circulating blood cells, especially in Sézary syndrome and transformed mycosis fungoides. So far, few CTCL cell lines have been adapted for in vivo experiments and their tumorigenicity has not been adequately assessed, hampering the use of a reproducible model for CTCL biological evaluation. In fact, both patient-derived xenografts and cell line xenografts at subcutaneous sites failed to provide a robust tool, because engraftment was dependent on mice strain and cell line subtype. Herein, we describe an original method of intrahepatic injection into adult NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice liver of both aggressive (My-La, HUT78, HH, MAC2A, and MAC2B) and indolent (FE-PD and MAC1) CTCL cell lines. Six of the seven CTCL cell lines were grafted with a high rate of success (80%). Moreover, this model provided a quick (15 days) and robust assay for in vivo evaluation of CTCL cell lines tumorigenicity and therapeutic response in preclinical studies. Such a reproducible model can be therefore used for further functional studies and in vivo drug testing. Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of diseases primarily involving the skin, which presumably derives from skin-homing T-cells exhibiting important differences according to their circulating capacities in the peripheral blood and lymph node involvement.1Rook A.H. Gottlieb S.L. Wolfe J.T. Vowels B.R. Sood S.S. Niu Z. Lessin S.R. Fox F.E. Pathogenesis of cutaneous T-cell lymphoma: implications for the use of recombinant cytokines and photopheresis.Clin Exp Immunol. 1997; 107: 16-20PubMed Google Scholar, 2Watanabe R. Gehad A. Yang C. Scott L.L. Teague J.E. Schlapbach C. Elco C.P. Huang V. Matos T.R. Kupper T.S. Clark R.A. Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells.Sci Transl Med. 2015; 7: 279ra239Crossref Scopus (356) Google Scholar The present research focuses on the three main CTCL subtypes: mycosis fungoides (MF), Sézary syndrome (SS), and primary CD30+ cutaneous anaplastic large-cell lymphoma. Patients with cutaneous anaplastic large-cell lymphoma present solitary or localized skin nodules or tumors of nonepidermotropic infiltrate of large CD30+ T-cells.3Willemze R. Jaffe E.S. Burg G. Cerroni L. Berti E. Swerdlow S.H. Ralfkiaer E. Chimenti S. Diaz-Perez J.L. Duncan L.M. Grange F. Harris N.L. Kempf W. Kerl H. Kurrer M. Knobler R. Pimpinelli N. Sander C. Santucci M. Sterry W. Vermeer M.H. Wechsler J. Whittaker S. Meijer C.J. WHO-EORTC classification for cutaneous lymphomas.Blood. 2005; 105: 3768-3785Crossref PubMed Scopus (3224) Google Scholar Despite a good prognosis,4Bekkenk M.W. Geelen F.A. van Voorst Vader P.C. Heule F. Geerts M.L. van Vloten W.A. Meijer C.J. Willemze R. Primary and secondary cutaneous CD30(+) lymphoproliferative disorders: a report from the Dutch Cutaneous Lymphoma Group on the long-term follow-up data of 219 patients and guidelines for diagnosis and treatment.Blood. 2000; 95: 3653-3661Crossref PubMed Google Scholar cutaneous anaplastic large-cell lymphoma may be harmful in approximately one-third of patients presenting large tumor skin lesions and/or multiple skin relapses. On the other hand, at early or patch/plaque stage, MF is a skin-limited variant of CTCL with fixed lesion(s) containing malignant cells with a skin resident effector memory T-cell phenotype. In contrast, SS is an aggressive leukemic CTCL subtype where malignant cells coexpress both T-central memory markers and skin-homing addressing with recirculating capacities between blood and skin.2Watanabe R. Gehad A. Yang C. Scott L.L. Teague J.E. Schlapbach C. Elco C.P. Huang V. Matos T.R. Kupper T.S. Clark R.A. Human skin is protected by four functionally and phenotypically discrete populations of resident and recirculating memory T cells.Sci Transl Med. 2015; 7: 279ra239Crossref Scopus (356) Google Scholar, 5Clark R.A. Watanabe R. Teague J.E. Schlapbach C. Tawa M.C. Adams N. Dorosario A.A. Chaney K.S. Cutler C.S. Leboeuf N.R. Carter J.B. Fisher D.C. Kupper T.S. Skin effector memory T cells do not recirculate and provide immune protection in alemtuzumab-treated CTCL patients.Sci Transl Med. 2012; 4: 117ra117Crossref Scopus (272) Google Scholar Together with advanced stage MF (tumor and/or large-cell transformed MF), SS represents the most aggressive form of CTCL. In these aggressive forms, malignant T-cells spread to several sites, including skin, blood, and lymph nodes. However, the mechanisms of tumor cell migration out of the skin are so far poorly understood, especially in patients with MF who may have a circulating dominant clone.6Delfau-Larue M.H. Laroche L. Wechsler J. Lepage E. Lahet C. Asso-Bonnet M. Bagot M. Farcet J.P. Diagnostic value of dominant T-cell clones in peripheral blood in 363 patients presenting consecutively with a clinical suspicion of cutaneous lymphoma.Blood. 2000; 96: 2987-2992Crossref PubMed Google Scholar To date, there is no model to assess cellular changes associated with migration and spreading into different tissue compartments. Few CTCL cell lines are available and most of them have been derived from blood samples of patients with CTCL (Table 1).7Kaltoft K. Bisballe S. Dyrberg T. Boel E. Rasmussen P.B. Thestrup-Pedersen K. Establishment of two continuous T-cell strains from a single plaque of a patient with mycosis fungoides.In Vitro Cell Dev Biol. 1992; 28A: 161-167Crossref PubMed Scopus (116) Google Scholar, 8Bunn Jr., P.A. Foss F.M. T-cell lymphoma cell lines (HUT102 and HUT78) established at the National Cancer Institute: history and importance to understanding the biology, clinical features, and therapy of cutaneous T-cell lymphomas (CTCL) and adult T-cell leukemia-lymphomas (ATLL).J Cell Biochem Suppl. 1996; 24: 12-23Crossref PubMed Scopus (55) Google Scholar, 9Starkebaum G. Loughran Jr., T.P. Waters C.A. Ruscetti F.W. Establishment of an IL-2 independent, human T-cell line possessing only the p70 IL-2 receptor.Int J Cancer. 1991; 49: 246-253Crossref PubMed Scopus (50) Google Scholar, 10Kadin M.E. Cavaille-Coll M.W. Gertz R. Massague J. Cheifetz S. George D. Loss of receptors for transforming growth factor beta in human T-cell malignancies.Proc Natl Acad Sci U S A. 1994; 91: 6002-6006Crossref PubMed Scopus (204) Google Scholar, 11del Mistro A. Leszl A. Bertorelle R. Calabro M.L. Panozzo M. Menin C. D'Andrea E. Chieco-Bianchi L. A CD30-positive T cell line established from an aggressive anaplastic large cell lymphoma, originally diagnosed as Hodgkin's disease.Leukemia. 1994; 8: 1214-1219PubMed Google Scholar, 12Dirks W.G. Zaborski M. Jager K. Challier C. Shiota M. Quentmeier H. Drexler H.G. The (2;5)(p23;q35) translocation in cell lines derived from malignant lymphomas: absence of t(2;5) in Hodgkin-analogous cell lines.Leukemia. 1996; 10: 142-149PubMed Google Scholar, 13Drexler H.G. MacLeod R.A. Malignant hematopoietic cell lines: in vitro models for the study of anaplastic large-cell lymphoma.Leukemia. 2004; 18: 1569-1571Crossref PubMed Scopus (14) Google Scholar In our hands, compared with other leukemia cell lines, CTCL cell lines present a low mitotic index. This phenomenon could be linked to intrinsic biological or genetic properties, such as the shortening of telomeres in aggressive CTCL cell lines,14Chevret E. Andrique L. Prochazkova-Carlotti M. Ferrer J. Cappellen D. Laharanne E. Idrissi Y. Boettiger A. Sahraoui W. Ruiz F. Pham-Ledard A. Vergier B. Belloc F. Dubus P. Beylot-Barry M. Merlio J.P. Telomerase functions beyond telomere maintenance in primary cutaneous T-cell lymphoma.Blood. 2014; 123: 1850-1859Crossref PubMed Scopus (18) Google Scholar and several of them are rather difficult to transduce. These constraints hamper biological investigation. Improvement in development of cell lines together with the generation of robust in vivo mouse xenograft models are needed to test phenotypic or biological changes during tumor formation and migration as well as to evaluate new therapies targeting biological pathways. Some rare patient-derived xenografts or cell line xenografts have been obtained by subcutaneous injection or intravenous tail injection of cells in grafted animals but did not generate a new cell line or a generalized model because their success was found to be mice strain and/or cell line dependent.15Tun-Kyi A. Qin J.Z. Oberholzer P.A. Navarini A.A. Hassel J.C. Dummer R. Dobbeling U. Arsenic trioxide down-regulates antiapoptotic genes and induces cell death in mycosis fungoides tumors in a mouse model.Ann Oncol. 2008; 19: 1488-1494Crossref PubMed Scopus (21) Google Scholar, 16Ito A. Ishida T. Utsunomiya A. Sato F. Mori F. Yano H. Inagaki A. Suzuki S. Takino H. Ri M. Kusumoto S. Komatsu H. Iida S. Inagaki H. Ueda R. Defucosylated anti-CCR4 monoclonal antibody exerts potent ADCC against primary ATLL cells mediated by autologous human immune cells in NOD/Shi-scid, IL-2R gamma(null) mice in vivo.J Immunol. 2009; 183: 4782-4791Crossref PubMed Scopus (63) Google Scholar, 17Yano H. Ishida T. Inagaki A. Ishii T. Ding J. Kusumoto S. Komatsu H. Iida S. Inagaki H. Ueda R. Defucosylated anti CC chemokine receptor 4 monoclonal antibody combined with immunomodulatory cytokines: a novel immunotherapy for aggressive/refractory Mycosis fungoides and Sezary syndrome.Clin Cancer Res. 2007; 13: 6494-6500Crossref PubMed Scopus (58) Google Scholar, 18Thaler S. Burger A.M. Schulz T. Brill B. Bittner A. Oberholzer P.A. Dummer R. Schnierle B.S. Establishment of a mouse xenograft model for mycosis fungoides.Exp Dermatol. 2004; 13: 406-412Crossref PubMed Scopus (26) Google Scholar, 19Krejsgaard T. Kopp K. Ralfkiaer E. Willumsgaard A.E. Eriksen K.W. Labuda T. Rasmussen S. Mathiesen A.M. Geisler C. Lauenborg B. Becker J.C. Zhang Q. Wasik M.A. Odum N. Woetmann A. A novel xenograft model of cutaneous T-cell lymphoma.Exp Dermatol. 2010; 19: 1096-1102Crossref PubMed Scopus (35) Google Scholar, 20Doebbeling U. A mouse model for the Sezary syndrome.J Exp Clin Cancer Res. 2010; 29: 11Crossref PubMed Scopus (14) Google Scholar, 21Han T. Abdel-Motal U.M. Chang D.K. Sui J. Muvaffak A. Campbell J. Zhu Q. Kupper T.S. Marasco W.A. Human anti-CCR4 minibody gene transfer for the treatment of cutaneous T-cell lymphoma.PLoS One. 2012; 7: e44455Crossref PubMed Scopus (32) Google Scholar In 2012, van der Fits et al22van der Fits L. Rebel H.G. Out-Luiting J.J. Pouw S.M. Smit F. Vermeer K.G. van Zijl L. Tensen C.P. Weijer K. Vermeer M.H. A novel mouse model for Sezary syndrome using xenotransplantation of Sezary cells into immunodeficient RAG2(-/-) gammac(-/-) mice.Exp Dermatol. 2012; 21: 706-709Crossref PubMed Scopus (14) Google Scholar described an original mouse model by xenotransplantation of two SS cell lines (HUT78 and SeAx) and fresh patient cells in newborn RAG2−/−γc−/− mice liver (2 to 7 days old). This model gave some interesting preliminary results, but the mice number was low and since their report, no further data were claimed using this model for CTCL engraftment.Table 1In Vitro Clonogenicity and in Vivo Intrahepatic Tumorigenicity of CTCL Cell LinesCTCL cell linePathologyIn vitroIn vivoCell originCell growth in culture (+ to +++)Soft agar clonogenicity (yes/no)HLA-ABC positive cells in liverMice liver/body ratio (%)Xenograft reproducibility (mice no.)CTCL cell lineAggressive CTCL My-La†My-La cell line was in our hand the only cell line giving a reproducible engraftment after subcutaneous injection.T-MFPlaque7Kaltoft K. Bisballe S. Dyrberg T. Boel E. Rasmussen P.B. Thestrup-Pedersen K. Establishment of two continuous T-cell strains from a single plaque of a patient with mycosis fungoides.In Vitro Cell Dev Biol. 1992; 28A: 161-167Crossref PubMed Scopus (116) Google Scholar+++Yes (5 × 104 cells/well)Yes13.77 ± 3.0∗∗∗60/60My-La†My-La cell line was in our hand the only cell line giving a reproducible engraftment after subcutaneous injection. HUT78SSPeripheral blood8Bunn Jr., P.A. Foss F.M. T-cell lymphoma cell lines (HUT102 and HUT78) established at the National Cancer Institute: history and importance to understanding the biology, clinical features, and therapy of cutaneous T-cell lymphomas (CTCL) and adult T-cell leukemia-lymphomas (ATLL).J Cell Biochem Suppl. 1996; 24: 12-23Crossref PubMed Scopus (55) Google Scholar+++Yes (5 × 104 cells/well)Yes16.91 ± 4.7∗∗∗10/10HUT78 HHcATLLPeripheral blood9Starkebaum G. Loughran Jr., T.P. Waters C.A. Ruscetti F.W. Establishment of an IL-2 independent, human T-cell line possessing only the p70 IL-2 receptor.Int J Cancer. 1991; 49: 246-253Crossref PubMed Scopus (50) Google Scholar++Yes (5 × 104 cells/well)Yes12.56 ± 3.6∗∗∗10/10HH MAC2ACD30+ ALCLSkin tumor, aggressive terminal phase10Kadin M.E. Cavaille-Coll M.W. Gertz R. Massague J. Cheifetz S. George D. Loss of receptors for transforming growth factor beta in human T-cell malignancies.Proc Natl Acad Sci U S A. 1994; 91: 6002-6006Crossref PubMed Scopus (204) Google Scholar+++NoYes4.60 ± 0.21/10MAC2A MAC2BCD30+ ALCLSkin tumor, aggressive terminal phase10Kadin M.E. Cavaille-Coll M.W. Gertz R. Massague J. Cheifetz S. George D. Loss of receptors for transforming growth factor beta in human T-cell malignancies.Proc Natl Acad Sci U S A. 1994; 91: 6002-6006Crossref PubMed Scopus (204) Google Scholar++Yes (5 × 105 cells/well)No4.70 ± 0.20/10MAC2BIndolent CTCL FE-PDCD30+ HL-ALCLPeripheral blood11del Mistro A. Leszl A. Bertorelle R. Calabro M.L. Panozzo M. Menin C. D'Andrea E. Chieco-Bianchi L. A CD30-positive T cell line established from an aggressive anaplastic large cell lymphoma, originally diagnosed as Hodgkin's disease.Leukemia. 1994; 8: 1214-1219PubMed Google Scholar, 12Dirks W.G. Zaborski M. Jager K. Challier C. Shiota M. Quentmeier H. Drexler H.G. The (2;5)(p23;q35) translocation in cell lines derived from malignant lymphomas: absence of t(2;5) in Hodgkin-analogous cell lines.Leukemia. 1996; 10: 142-149PubMed Google Scholar, 13Drexler H.G. MacLeod R.A. Malignant hematopoietic cell lines: in vitro models for the study of anaplastic large-cell lymphoma.Leukemia. 2004; 18: 1569-1571Crossref PubMed Scopus (14) Google Scholar+NoYes6.44 ± 1.8∗∗∗5/10FE-PD MAC1CD30+ ALCLPeripheral blood, early stage10Kadin M.E. Cavaille-Coll M.W. Gertz R. Massague J. Cheifetz S. George D. Loss of receptors for transforming growth factor beta in human T-cell malignancies.Proc Natl Acad Sci U S A. 1994; 91: 6002-6006Crossref PubMed Scopus (204) Google Scholar++NoYes6.01 ± 0.5∗∗10/10MAC1All CTCLYes96/120All CTCLLiver/body weight ratio is expressed as means ± SD.∗∗P < 0.01, ∗∗∗P < 0.001.cATLL, cutaneous adult T-cell leukemia/lymphoma; CTCL, cutaneous T-cell lymphoma; HL-ALCL, Hodgkin-like anaplastic large-cell lymphoma; SS, Sézary syndrome; T-MF, transformed mycosis fungoides; +, slow; ++, medium; +++, fast.† My-La cell line was in our hand the only cell line giving a reproducible engraftment after subcutaneous injection. Open table in a new tab Liver/body weight ratio is expressed as means ± SD. ∗∗P < 0.01, ∗∗∗P < 0.001. cATLL, cutaneous adult T-cell leukemia/lymphoma; CTCL, cutaneous T-cell lymphoma; HL-ALCL, Hodgkin-like anaplastic large-cell lymphoma; SS, Sézary syndrome; T-MF, transformed mycosis fungoides; +, slow; ++, medium; +++, fast. In the current study, we developed a highly reproducible intrahepatic model of CTCL in a large cohort of adult immunodeficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). For six of seven CTCL cell lines, mice rapidly developed liver tumors with a high successful rate of engraftment (80%) with cell spreading into organs. The present model opens the door to further preclinical models to assess single or multiagent therapies and in vivo evaluation of CTCL proliferation, tumor growth, and migration. Experiments were conducted on five aggressive CTCL cell lines, My-La (kindly gifted by Dr. Kaltof, Aarhus University, Denmark), HUT78 (ATCC, Molesheim, France), HH (DSMZ, Braunschweig, Germany), MAC2A, and MAC2B (DSMZ), and two indolent CTCL cell lines, FE-PD (Prof. Delsol, Inserm U563, Toulouse, France) and MAC1 (DSMZ). Cells were grown at 37°C in 5% CO2 in RPMI 1640 media containing 10% fetal bovine serum, except FE-PD, which required Iscove's modified Dulbecco's medium supplemented with 20% fetal bovine serum. Cell lines were regularly tested for the absence of mycoplasma contamination. A total of 5 × 104, 10 × 104, or 5 × 105 cells per well (6-well plates) were cultured in soft agar to determine CTCL cell line clonogenicity. Soft agar assays were performed as previously described.14Chevret E. Andrique L. Prochazkova-Carlotti M. Ferrer J. Cappellen D. Laharanne E. Idrissi Y. Boettiger A. Sahraoui W. Ruiz F. Pham-Ledard A. Vergier B. Belloc F. Dubus P. Beylot-Barry M. Merlio J.P. Telomerase functions beyond telomere maintenance in primary cutaneous T-cell lymphoma.Blood. 2014; 123: 1850-1859Crossref PubMed Scopus (18) Google Scholar Animal experiments were performed in level 2 animal facilities, Bordeaux University, in accordance with national institutional guidelines and with the agreement of the local Ethic Committee on Animal Experiments CEEA50 of Bordeaux (agreement number 50120151-A). A total of 5 × 106 cells were intrahepatically xenografted in adult (aged 8 to 12 weeks) NSG immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) under 2.5% isoflurane anesthesia (Belamont, Piramal Healthcare, Northumberland, UK). Body weight was daily measured with a global surveillance of animal health. At 4 to 5 weeks after engraftment, mice were sacrificed, body and liver weights were measured, and liver/body ratio was then determined. Liver, spleen, kidneys, heart, and lungs were fixed in 4% formaldehyde (MERCK Millipore, VWR International S.A.S, Fontenay-sous-Bois, France) for immunohistochemistry (IHC). A total of 60 mice were intrahepatically grafted for My-La cell line. Ten mice were grafted for each other cell line (HUT78, FE-PD, HH, MAC1, MAC2A, and MAC2B). Mayer's hematoxylin labeling and human leukocyte antigen (HLA-ABC) IHC were performed on mice organ sections (3 μm thick) (1:100 of Antibody 70328; Abcam, Paris, France) of formalin-fixed, paraffin-embedded organs. Slides were analyzed using Pannoramic Scan (3DHISTECH, Budapest, Hungary) and Pannoramic Viewer software version 1.15.4 (3DHISTECH) or using microscope LEICA DMR coupled with a camera NIKON DS-FI2 and NIS BR imaging software version 4.0 (Nikon, Champigny sur Marne, France). My-La cells were treated for 72 hours with methotrexate (MTX), 10 and 50 μmol/L (Mylan Laboratory, Saint-Priest, France), or left untreated, as control. Cell number was counted and analyzed at 48 and 72 hours. A total of 2.5 × 106 My-La cells were intrahepatically grafted in 20 NSG mice. Three days after engraftment, 10 mice were treated with 0.5 mg/kg of MTX, or left untreated as control. Treatment was daily conducted by intraperitoneal injection during 12 days. At sacrifice (day 15), organs were fixed in 4% formaldehyde for IHC or mechanically dissociated before fluorescence-activated cell sorter (FACS) analysis. HLA-ABC was detected in single-cell suspensions obtained from mice organs, using a PE-Cy7 antibody (561349; BD Biosciences, Le Pont de Claix, France). Detection was conducted using BD FACS Canto II, and results were analyzed by FlowJo software version 10 (Tree Star Inc., Ashland, OR). Apoptotic/necrotic cell proportion was measured using annexin V–PE antibody (BD Biosciences), according to the manufacturer's recommendations, and Hoechst 33342 (H3570; Molecular Probes, Eugene, OR) was added 5 minutes before sample acquisition. Apoptosis/necrosis was analyzed on FACS Canto II cytometer (BD Biosciences) using FlowJo software. Statistical analysis was performed with MedCalc software version 12.3 (MedCalc Software, Mariakerke, Belgium). For nonparametric statistical analysis, the Wilcoxon–Mann-Whitney test was used. P < 0.05 was considered statistically significant. To test the feasibility of a new intrahepatic xenograft model of CTCL in adult immunodeficient mice, we used My-La cells previously reported as the most aggressive CTCL cell line for both in vitro and in vivo subcutaneous xenograft models.18Thaler S. Burger A.M. Schulz T. Brill B. Bittner A. Oberholzer P.A. Dummer R. Schnierle B.S. Establishment of a mouse xenograft model for mycosis fungoides.Exp Dermatol. 2004; 13: 406-412Crossref PubMed Scopus (26) Google Scholar Herein, 5 × 106 My-La cells were xenografted intrahepatically in 60 adult mice (8 to 12 weeks old). Tumor formation was characterized by rapid macroscopically visible liver tumors (3 weeks) and disseminated intrahepatic nodules in all of the 60 grafted mice (Figure 1A). IHC of formalin-fixed, paraffin-embedded mice liver sections (Figure 1B) was used to detect ubiquitous HLA-ABC in all of the 60 mice livers. Liver/body weight ratio significantly increased between My-La xenografted mice and nongrafted control mice (13.77% ± 3.0% versus 4.60% ± 0.5%; P < 0.001) (Table 2). This ratio indicates a large liver mass increase because of tumor formation with a loss of animal weight, representing the rapid engraftment of My-La cells.Table 2Efficient My-La Intrahepatic Xenograft Mouse ModelVariablePathologyLiver macroscopic tumorMice liver/body weight ratio (%)Xenograft reproducibility (mice no.)Intrahepatic xenograft efficiencyControlNANo4.60 ± 0.5NANAMy-LaT-MFYes13.77 ± 3.0∗∗∗60/60100%Mice liver/body weight ratio was calculated and reproducibility of experiments was determined; 100% of the mice (60/60) developed liver tumors.∗∗∗P < 0.001.NA, not applicable; T-MF, transformed mycosis fungoides. Open table in a new tab Mice liver/body weight ratio was calculated and reproducibility of experiments was determined; 100% of the mice (60/60) developed liver tumors. ∗∗∗P < 0.001. NA, not applicable; T-MF, transformed mycosis fungoides. On the basis of the above results, we hypothesized that this intrahepatic route could be efficient for six other available CTCL cell lines. Thus, four aggressive (HUT78,8Bunn Jr., P.A. Foss F.M. T-cell lymphoma cell lines (HUT102 and HUT78) established at the National Cancer Institute: history and importance to understanding the biology, clinical features, and therapy of cutaneous T-cell lymphomas (CTCL) and adult T-cell leukemia-lymphomas (ATLL).J Cell Biochem Suppl. 1996; 24: 12-23Crossref PubMed Scopus (55) Google Scholar HH,9Starkebaum G. Loughran Jr., T.P. Waters C.A. Ruscetti F.W. Establishment of an IL-2 independent, human T-cell line possessing only the p70 IL-2 receptor.Int J Cancer. 1991; 49: 246-253Crossref PubMed Scopus (50) Google Scholar MAC2A,10Kadin M.E. Cavaille-Coll M.W. Gertz R. Massague J. Cheifetz S. George D. Loss of receptors for transforming growth factor beta in human T-cell malignancies.Proc Natl Acad Sci U S A. 1994; 91: 6002-6006Crossref PubMed Scopus (204) Google Scholar and MAC2B10Kadin M.E. Cavaille-Coll M.W. Gertz R. Massague J. Cheifetz S. George D. Loss of receptors for transforming growth factor beta in human T-cell malignancies.Proc Natl Acad Sci U S A. 1994; 91: 6002-6006Crossref PubMed Scopus (204) Google Scholar) and two indolent CTCL cell lines (FE-PD11del Mistro A. Leszl A. Bertorelle R. Calabro M.L. Panozzo M. Menin C. D'Andrea E. Chieco-Bianchi L. A CD30-positive T cell line established from an aggressive anaplastic large cell lymphoma, originally diagnosed as Hodgkin's disease.Leukemia. 1994; 8: 1214-1219PubMed Google Scholar, 12Dirks W.G. Zaborski M. Jager K. Challier C. Shiota M. Quentmeier H. Drexler H.G. The (2;5)(p23;q35) translocation in cell lines derived from malignant lymphomas: absence of t(2;5) in Hodgkin-analogous cell lines.Leukemia. 1996; 10: 142-149PubMed Google Scholar, 13Drexler H.G. MacLeod R.A. Malignant hematopoietic cell lines: in vitro models for the study of anaplastic large-cell lymphoma.Leukemia. 2004; 18: 1569-1571Crossref PubMed Scopus (14) Google Scholar and MAC110Kadin M.E. Cavaille-Coll M.W. Gertz R. Massague J. Cheifetz S. George D. Loss of receptors for transforming growth factor beta in human T-cell malignancies.Proc Natl Acad Sci U S A. 1994; 91: 6002-6006Crossref PubMed Scopus (204) Google Scholar) were grafted according to the same protocol. To compare in vitro clonogenicity of these cell lines with in vivo intrahepatic tumorigenicity, we first performed a soft agar assay for all cells using 5 × 104, 1 × 105, or 5 × 105 cells per well of 6-well plates. Aggressive (My-La,7Kaltoft K. Bisballe S. Dyrberg T. Boel E. Rasmussen P.B. Thestrup-Pedersen K. Establishment of two continuous T-cell strains from a single plaque of a patient with mycosis fungoides.In Vitro Cell Dev Biol. 1992; 28A: 161-167Crossref PubMed Scopus (116) Google Scholar HUT78,8Bunn Jr., P.A. Foss F.M. T-cell lymphoma cell lines (HUT102 and HUT78) established at the National Cancer Institute: history and importance to understanding the biology, clinical features, and therapy of cutaneous T-cell lymphomas (CTCL) and adult T-cell leukemia-lymphomas (ATLL).J Cell Biochem Suppl. 1996; 24: 12-23Crossref PubMed Scopus (55) Google Scholar HH,9Starkebaum G. Loughran Jr., T.P. Waters C.A. Ruscetti F.W. Establishment of an IL-2 independent, human T-cell line possessing only the p70 IL-2 receptor.Int J Cancer. 1991; 49: 246-253Crossref PubMed Scopus (50) Google Scholar and MAC2B10Kadin M.E. Cavaille-Coll M.W. Gertz R. Massague J. Cheifetz S. George D. Loss of receptors for transforming growth factor beta in human T-cell malignancies.Proc Natl Acad Sci U S A. 1994; 91: 6002-6006Crossref PubMed Scopus (204) Google Scholar) and one indolent (FE-PD)11del Mistro A. Leszl A. Bertorelle R. Calabro M.L. Panozzo M. Menin C. D'Andrea E. Chieco-Bianchi L. A CD30-positive T cell line established from an aggressive anaplastic large cell lymphoma, originally diagnosed as Hodgkin's disease.Leukemia. 1994; 8: 1214-1219PubMed Google Scholar, 12Dirks W.G. Zaborski M. Jager K. Challier C. Shiota M. Quentmeier H. Drexler H.G. The (2;5)(p23;q35) translocation in cell lines derived from malignant lymphomas: absence of t(2;5) in Hodgkin-analogous cell lines.Leukemia. 1996; 10: 142-149PubMed Google Scholar, 13Drexler H.G. MacLeod R.A. Malignant hematopoietic cell lines: in vitro models for the study of anaplastic large-cell lymphoma.Leukemia. 2004; 18: 1569-1571Crossref PubMed Scopus (14) Google Scholar CTCL cell lines presented a correlation between cell growth capacity in culture and cell clonogenicity ability (Table 1). Indeed, four aggressive cell lines were able to form colonies in soft agar, whereas FE-PD, which proliferated slower in vitro compared with other cell lines, was not able to form colonies (Table 1).11del Mistro A. Leszl A. Bertorelle R. Calabro M.L. Panozzo M. Menin C. D'Andrea E. Chieco-Bianchi L. A CD30-positive T cell line established from an aggressive anaplastic large cell lymphoma, originally diagnosed as Hodgkin's disease.Leukemia. 1994; 8: 1214-1219PubMed Google Scholar, 12Dirks W.G. Zaborski M. Jager K. Challier C. Shiota M. Quentmeier H. Drexler H.G. The (2;5)(p23;q35) translocation in cell lines derived from malignant lymphomas: absence of t(2;5) in Hodgkin-analogous cell lines.Leukemia. 1996; 10: 142-149PubMed Google Scholar, 13Drexler H.G. MacLeod R.A. Malignant hematopoietic cell lines: in vitro models for the study of anaplastic large-cell lymphoma.Leukemia. 2004; 18: 1569-1571Crossref PubMed Scopus (14) Google Scholar Regarding MAC cell lines, they exhibited different biological properties. Only MAC2B was found to be clonogenic in soft agar assay, using a high number of cells per well (5 × 105 cells), whereas MAC1 and MAC2A were not clonogenic in these conditions. Independently of their clonogenic status, all cell lines were intrahepatically xenografted in adult immunodeficient mice. Except for MAC2B, all CTCL cell lines significantly induced liver tumor formation with high reproducibility: 5 of 10 mice for FE-PD (50%), 10 of 10 mice for MAC1 (100%), and 10 of 10 mice for HUT78 and HH (100%). Interestingly, although FE-PD and MAC2A cells were not clonogenic in vitro, they were tumorigenic in vivo with high incidence for FE-PD and poor incidence for MAC2A (Table 1).10Kadin M.E. Cavaille-Coll M.W. Gertz R. Massague J. Cheifetz S. George D. Loss of receptors for transforming growth factor beta in human T-cell malignancies.Proc Natl Acad Sci U S A. 1994; 91: 6002-6006Crossref PubMed Scopus (204) Google Scholar, 11del Mistro A. Leszl A. Bertorelle R. Calabro M.L. Panozzo M. Menin C. D'Andrea E. Chieco
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