Intrahepatic Xenograft of Cutaneous T-Cell Lymphoma Cell Lines

Intrahepatic Xenograft of Cutaneous T-Cell Lymphoma Cell Lines

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The American Journal of Pathology, Vol. -, No. -, - 2016

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Intrahepatic Xenograft of Cutaneous T-Cell Lymphoma Cell Lines A Useful Model for Rapid Biological and Therapeutic Evaluation Q30

Laetitia Andrique,* Sandrine Poglio,* Martina Prochazkova-Carlotti,* Marshall Edward Kadin,y Alban Giese,z Yamina Idrissi,* Marie Beylot-Barry,*x Jean-Philippe Merlio,*{ and Edith Chevret*

Q2 Q3 From the Cutaneous Lymphoma Oncogenesis Team,* INSERM U1053 BordeAux Research in Translational Oncology, and the Histology Platform UMS TMB

Core,z Bordeaux University, Bordeaux, France; the Department of Dermatology,y Boston University and Roger Williams Medical Center, Providence, Rhode Island; the Department of Dermatology,x CHU Bordeaux, Bordeaux, France; and the Tumor Bank and Tumor Biology Laboratory,{ CHU Bordeaux, Pessac, France Accepted for publication March 11, 2016.

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Address correspondence to Jean-Philippe Merlio, Cutaneous Lymphoma Oncogenesis team, INSERM U1053 BordeAux Research in Translational Oncology, Bordeaux University, Bâtiment 3B, 2ème étage, Zone Nord 146, rue Leo Saignat, F-33076 Bordeaux, France. E-mail: [email protected].

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. (Am J Pathol 2016, -: 1e10; http://dx.doi.org/10.1016/j.ajpath.2016.03.012)

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.1,2 The present research focuses on the three main CTCL subtypes: mycosis fungoides (MF), Sézary syndrome (SS), and primary CD30þ 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 CD30þ T-cells.3 Despite a good prognosis,4 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.2,5 Together with advanced stage MF (tumor and/or Supported by the CHU de Bordeaux PHRC CACLYT, the PHRC REVLEG (CHUBX 2011/28-REV-LEG), the Société Française de Dermatologie, Comité Dordogne, Ligue contre le Cancer, grant, and SIRIC-BRIO INCA DGOS-Inserm 6046 grant (L.A.). Disclosures: None declared.

Copyright ª 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2016.03.012

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Andrique et al 125 large-cell transformed MF), SS represents the most and SeAx) and fresh patient cells in newborn RAG2/gc/ 126 aggressive form of CTCL. In these aggressive forms, mamice liver (2 to 7 days old). This model gave some interesting 127 lignant T-cells spread to several sites, including skin, blood, preliminary results, but the mice number was low and since 128 and lymph nodes. However, the mechanisms of tumor cell their report, no further data were claimed using this model for 129 migration out of the skin are so far poorly understood, CTCL engraftment. 130 especially in patients with MF who may have a circulating In the current study, we developed a highly reproducible 131 dominant clone.6 To date, there is no model to assess intrahepatic model of CTCL in a large cohort of adult 132 immunodeficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/ cellular changes associated with migration and spreading 133 into different tissue compartments. SzJ). For six of seven CTCL cell lines, mice rapidly 134 135 Few CTCL cell lines are available and most of them have developed liver tumors with a high successful rate of 136 been derived from blood samples of patients with CTCL engraftment (80%) with cell spreading into organs. The 137 ½T1 (Table 1).7e13 In our hands, compared with other leukemia present model opens the door to further preclinical models 138 to assess single or multiagent therapies and in vivo evaluacell lines, CTCL cell lines present a low mitotic index. This 139 tion of CTCL proliferation, tumor growth, and migration. phenomenon could be linked to intrinsic biological or 140 genetic properties, such as the shortening of telomeres in 141 aggressive CTCL cell lines,14 and several of them are rather 142 Materials and Methods difficult to transduce. These constraints hamper biological 143 investigation. Improvement in development of cell lines Cell Lines 144 together with the generation of robust in vivo mouse xeno145 graft models are needed to test phenotypic or biological Experiments were conducted on five aggressive CTCL cell 146 147 changes during tumor formation and migration as well as to lines, My-La (Dr. Kaltoft), HUT78 (ATCC, Manassas, VA), 148 evaluate new therapies targeting biological pathways. Some HH (DSMZ), MAC2A, and MAC2B (DSMZ), and two 149 rare patient-derived xenografts or cell line xenografts have indolent CTCL cell lines, FE-PD (Prof Delsol) and MAC1 150 been obtained by subcutaneous injection or intravenous tail (DSMZ). Cells were grown at 37 C in 5% CO2 in RPMI 151 injection of cells in grafted animals but did not generate a 1640 media containing 10% fetal bovine serum, except 152 new cell line or a generalized model because their success FE-PD, which required Iscove’s modified Dulbecco’s me153 15e21 was found to be mice strain and/or cell line dependent. dium supplemented with 20% fetal bovine serum. Cell lines 154 22 In 2012, van der Fits et al described an original mouse were regularly tested for the absence of Mycoplasma 155 model by xenotransplantation of two SS cell lines (HUT78 contamination. 156 157 Table 1 In Vitro Clonogenicity and in Vivo Intrahepatic Tumorigenicity of CTCL Cell Lines 158 159 In vitro In vivo 160 HLA-ABC 161 positive Xenograft CTCL CTCL Cell growth Soft agar 162 Mice liver/body reproducibility cell cell in culture clonogenicity cells in 163 liver ratio (%) (mice no.) line line Pathology Cell origin (þ to þþþ) (yes/no) 164 165 Aggressive CTCL 166 My-Lay T-MF Plaque7 þþþ Yes (5  104 Yes 13.77  3.0*** 60/60 My-Lay 167 cells/well) 168 HUT78 SS Peripheral blood8 þþþ Yes (5  104 Yes 16.91  4.7*** 10/10 HUT78 169 cells/well) 170 þþ Yes (5  104 Yes 12.56  3.6*** 10/10 HH HH cATLL Peripheral blood9 171 cells/well) 172 MAC2A CD30þ ALCL Skin tumor, aggressive þþþ No Yes 4.60  0.2 1/10 MAC2A 173 terminal phase10 174 Skin tumor, aggressive þþ Yes (5  105 No 4.70  0.2 0/10 MAC2B MAC2B CD30þ ALCL 175 terminal phase10 cells/well) 176 Indolent CTCL 177 No Yes 6.44  1.8*** 5/10 FE-PD FE-PD CD30þ HL-ALCL Peripheral blood11e13 þ 178 MAC1 CD30þ ALCL Peripheral blood, early þþ No Yes 6.01  0.5** 10/10 MAC1 179 stage10 180 All CTCL Yes 96/120 All CTCL 181 Liver/body weight ratio is expressed as means  SD. 182 **P < 0.01, ***P < 0.001. 183 y My-La cell line was in our hand the only cell line giving a reproducible engraftment after subcutaneous injection. 184 cATLL, cutaneous adult T-cell leukemia/lymphoma; CTCL, cutaneous T-cell lymphoma; HL-ALCL, Hodgkin-like anaplastic large-cell lymphoma; SS, Sézary 185 syndrome; T-MF, transformed mycosis fungoides; þ, ---; þþ, ---; þþþ, ---. 186

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Intrahepatic Cutaneous Lymphoma Xenograft Q1

Soft Agar Assays 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.14

Ethics Statement 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).

Intrahepatic Mouse Model 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).

IHC

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Mayer’s hematoxylin labeling and human leukocyte antigen (HLA-ABC) IHC were performed on mice organ sections (3 mm thick) (1:100 of Antibody 70328; Abcam, Paris, France) of formalin-fixed, paraffin-embedded organs. Slides were analyzed using Pannoramic Scan (3DHISTECH, CA) and Pannoramic Viewer software version 1.15.4 or using microscope LEICA DMR coupled with a camera NIKON DS-FI2 and NIS BR imaging software.

MTX in Vitro Treatment My-La cells were treated for 72 hours with methotrexate (MTX), 10 and 50 mmol/L (Mylan Laboratory, Saint-Priest, France), or left untreated, as control. Cell number was counted and analyzed at 48 and 72 hours.

MTX in Vivo Mice Treatment 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

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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.

311 312 313 314 315 FACS Analysis 316 317 Human Cell Detection 318 HLA-ABC was detected in single-cell suspensions obtained 319 from mice organs, using a PE-Cy7 antibody (561349; BD 320 321 Biosciences, Le Pont de Claix, France). Detection was 322 conducted using BD FACS Canto II, and results were 323 Q18 analyzed by FlowJo software (Tree Star Inc., Ashland, OR). 324 325 Apoptosis/Necrotic Cell Detection 326 Apoptotic/necrotic cell proportion was measured using 327 annexin VePE antibody (BD Biosciences), according to 328 the manufacturer’s recommendations, and Hoechst 33342 329 (H3570; Molecular Probes, Eugene, OR) was added 5 minutes 330 before sample acquisition. Apoptosis/necrosis was analyzed 331 on FACS Canto II cytometer (BD Biosciences) using FlowJo 332 software. 333 334 335 Statistical Analysis 336 337 Q19 Statistical analysis was performed with MedCalc software. 338 For nonparametric statistical analysis, the WilcoxoneMann339 Whitney test was used. P < 0.05 was considered statistically 340 significant. 341 342 343 Results 344 345 Intrahepatic Xenograft Model for My-La Cells 346 347 To test the feasibility of a new intrahepatic xenograft model 348 of CTCL in adult immunodeficient mice, we used My-La 349 cells previously reported as the most aggressive CTCL cell 350 line for both in vitro and in vivo subcutaneous xenograft 351 18 6 models. Herein, 5  10 My-La cells were xenografted 352 intrahepatically in 60 adult mice (8 to 12 weeks old). Tumor 353 formation was characterized by rapid macroscopically 354 visible liver tumors (3 weeks) and disseminated intrahepatic 355 nodules in all of the 60 grafted mice (Figure 1A). IHC of ½F1 356 formalin-fixed, paraffin-embedded mice liver sections 357 358 (Figure 1B) was used to detect ubiquitous HLA-ABC in all 359 of the 60 mice livers. Liver/body weight ratio significantly 360 increased between My-La xenografted mice and nongrafted 361 control mice (13.77%  3.0% versus 4.60%  0.5%; P < 0.001) (Table 2). This ratio indicates a large liver mass ½T2 362 363 increase because of tumor formation with a loss of animal 364 weight, representing the rapid engraftment of My-La cells. 365 366 In Vitro Clonogenicity and in Vivo Intrahepatic 367 368 Tumorigenicity of Seven CTCL Cell Lines 369 370 On the basis of the above results, we hypothesized that this 371 intrahepatic route could be efficient for six other available 372

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Figure 1 Efficient My-La intrahepatic xenograft mouse model. A total of 5  106 My-La cells were intrahepatically xenografted in adult (8 to 12 weeks) NSG immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). A: At 4 to 5 weeks after engraftment, mice were sacrificed and large liver tumor growth was observed, with several disseminated intrahepatic nodules. B: Mayer’s hematoxylin labeling and human leukocyte antigen (HLA-ABC) immunohistochemistry on liver sections shows the presence of My-La lymphoid tumor cells. Scale bars Z 50 mm (B). HES, Mayer’s hematoxylin.

CTCL cell lines. Thus, four aggressive (HUT78,8 HH,9 MAC2A,10 and MAC2B10) and two indolent CTCL cell lines (FE-PD11e13 and MAC110) 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,7 HUT78,8 HH,9 and MAC2B10) and one indolent (FE-PD)11e13 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).11e13 Regarding MAC cell lines, they exhibited different biological properties. Only MAC2B was found to be Table 2

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).10e13 As well as for My-La cells, the liver/body weight ratio was higher for HUT78 (16.91%  4.7%), HH (12.56%  3.6%), and FE-PD (6.44%  1.8%), than in nongrafted mice control group (4.6%  0.5%) (Table 1).7e13 Liver engraftment of these three cell lines led to the development of several parenchymal tumor nodules formed by HLA-positive cells displaying malignant cell criteria, such as multilobular nucleus, prominent nucleoli, and, for HUT78, numerous mitotic figures (Figures 2 and 3). Several large macroscopic nodules ½F2 ½F2½F3 ½F3 were observed with My-La and HUT78, whereas HH cell line exhibited a periportal mode of nodular formation with microscopic nodules (Figures 2 and 3). Conversely, a single and large tumor nodule was observed in only one MAC2A xenografted animal with abundant central necrosis and peritumoral infiltration by many polymorphonuclear leukocytes. In other animals engrafted with MAC2A cells, there was no liver tumor or infiltration (Figure 3). Only MAC1 provided a reproducible liver infiltration without tumor formation. Such infiltration corresponded to a massive sinusoidal infiltration by many tumor cells and small microscopic embolus in centrolobular veins (Figure 3). No liver tumor or infiltration was observed after engraftment with MAC2B. In comparison and despite tumorigenic capacities for MAC1 and MAC2A, a significant enhancement of the liver/body weight ratio was only observed for MAC1 (6.01%  0.5%). A similar liver/ body weight ratio and sinusoidal pattern of infiltration was shown for the FE-PD cell line (Figure 3). Altogether, the model was successful for six of the seven CTCL cell lines tested. The rate of CTCL engraftment was 96 of 120 mice (80%).

CTCL Intrahepatic Xenografts Induce Metastases Intrahepatic xenograft is a common mouse model for hepatocellular carcinoma research,23 generally performed in

Efficient My-La Intrahepatic Xenograft Mouse Model

Variable

Pathology

Liver macroscopic tumor

Mice liver/body weight ratio (%)

Xenograft reproducibility (mice no.)

Intrahepatic xenograft efficiency

Control My-La

NA T-MF

No Yes

4.60  0.5 13.77  3.0***

NA 60/60

NA 100%

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.

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Intrahepatic Cutaneous Lymphoma Xenograft

Figure 2 Human leukocyte antigen (HLA-ABC) immunohistochemistry (IHC) in mice tissues after CTCL intrahepatic xenografts. Formalin-fixed, paraffinembedded tissue sections were deparaffinized, and IHC was performed with a HLA-ABC antibody (Ab70328). Sections were counterstained with Mayer’s hematoxylin (HES). Images outlined in green present a negative HLA-ABC immunostaining. Scale bars Z 50 mm.

athymic or immunodeficient mice. Because this model is informative for metastatic spreading properties of tumor cells, we chose to systematically analyze kidneys, spleen, lungs, and heart after CTCL intrahepatic xenografts both macroscopically and after HLA-ABC immunostaining of organ sections (Figure 2). Histological analysis of organs is ½T3 presented as tumor/infiltrate/embolus in Table 3. For My-La, HUT78, HH, and FE-PD, liver enlargement or tumor formation was associated with bilateral kidney colonization macroscopically detected at sacrifice (Figure 1A). Indeed, both kidneys of engrafted animals appeared less vascularized than in control mice, and tumor cell infiltration of the cortex was visible at the microscopic level (Figures 2 and 3). With the My-La cell line, a subcapsular infiltration by microscopic nodules corresponded to a generalized cortical infiltration of kidneys. For HUT78, HH, and FE-PD cell lines, HLA-ABC positive cells were detected on sections with a predominant infiltration of the cortical zone with

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clusters between glomeruli and tubules. The MAC1 cell line provided a predominant mode of vascular infiltration with numerous intravascular emboli and a specific mesangial infiltration of the glomeruli (Figure 2 and Supplemental Figure S1). There was no kidney involvement with MAC2A or MAC2B cell lines except the presence of rare human cells in the vessels for MAC2A cell line. Although no infiltration was seen for MAC2A and MAC2B cell lines in spleen, CTCL cells were present in the sinusoids of the red pulp after engraftment with My-La, HUT78, HH, FE-PD, and MAC1 cell lines (Figure 2 and Supplemental Figure S1). Spleen involvement was also associated with involvement of Billroth’s cords, as seen with HUT78 cell line (Supplemental Figure S1). A massive and diffuse infiltration of the red pulp was observed for MAC1. Regarding the lungs, a massive and generalized infiltration was observed in all grafted mice with My-La, HUT78, HH, FE-PD, and MAC1 cell lines, and in one mouse grafted with MAC2A. This infiltration mainly

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Figure 3 Human leukocyte antigen (HLA-ABC) immunostaining by immunohistochemistry (IHC) in livers. Mice were intrahepatically xenografted with CTCL cell lines: My-La, HUT78, HH, FE-PD, MAC1, and MAC2A. After animal sacrifice, formalin-fixed, paraffin-embedded tissue sections were deparaffinized, and IHC was performed with a HLA-ABC antibody (Ab70328). Images were captured with a LEICA DMR microscope coupled with a NIKON DS-FI2 camera and analyzed with NIS BR imaging software. Scale bars Z 100 mm. Original magnification: 10 (left column); 20 (middle column); 40 (right column).

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involved the alveoli septa and was sometimes associated with capillaries’ dilatation and fibrosis. The MAC1 cell line exhibited again a specific tropism with both septa infiltration and replacement of alveolar cells in a lepidic mode. Finally, no heart infiltration was observed, only sporadic cells were seen in the capillaries between cardiomyocytes for MAC1 (data not shown).

A Model for Rapid Therapeutic Evaluation in CTCL Methotrexate is a cytotoxic chemotherapy described as anticancer treatment in solid tumors (breast, head and neck, lung, stomach, and esophagus) also used to treat acute lymphoblastic leukemia and non-Hodgkin lymphoma as

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CTCL patients.24 We first studied in vitro the impact of MTX treatment on My-La cells using 10 and 50 mmol/L of MTX. We observed that MTX treatment decreased significantly My-La cell number in culture, as soon as 48 hours, irrespective of the MTX dose tested (Figure 4A). Cell ½F4 number diminution was associated with a higher proportion of late apoptotic cells after MTX treatment. Indeed, proportions of annexin V/Hoechst-positive cells were increased in MTX-treated cells compared with untreated cells (Figure 4, B and C, and Supplemental Figure S2), confirming a previous in vitro report.25 Then, we used MTX to validate our in vivo intrahepatic model of CTCL in a preclinical study. My-La cells were xenografted intrahepatically at 2.5  106 in adult mice at day 1 (Figure 5A). ½F5

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Intrahepatic Cutaneous Lymphoma Xenograft Table 3 CTCL cell line

Histological Analysis of Tumor Cell Spreading in Mice Tissues after CTCL Intrahepatic Xenografts HLA-ABCepositive cells in tissues of 10 mice Pathology

Aggressive CTCL My-La T-MF HUT78 SS HH cATLL MAC2A CD30þ MAC2B CD30þ Indolent CTCL FE-PD CD30þ MAC1 CD30þ

Cell spreading in organs (embolus/infiltrate/tumor)

Liver

Spleen

Kidneys

Lungs

Heart

Liver

Spleen

Kidneys

Lungs

Heart

10/10 8/10 10/10 0/10 0/10

5/10 8/10 10/10 0/10 0/10

8/10 5/10 9/10 1/10 0/10

5/10 4/10 9/10 0/10 0/10

T/I/E T/E T/I T

E I I

T/E T/E I/E

I/E T/E I E

/ E E

ALCL ALCL

60/60 10/10 10/10 1/10 0/10

ALCL ALCL

5/10 10/10

5/10 8/10

4/10 7/10

5/10 8/10

5/10 8/10

I/E I/E

E Iþþ

T/E I/E

I I/E

E E

ALCL, anaplastic large cell lymphoma; cATLL, cutaneous adult T-cell leukemia/lymphoma; CTCL, cutaneous T-cell lymphoma; E, embolus; I, infiltrate; SS, Sézary syndrome; T, tumor; T-MF, transformed mycosis fungoides.

Starting day 3, mice were daily treated intraperitoneally with 0.5 mg/kg of MTX or left untreated (in control mice) during 12 days. At day 15, mice were sacrificed (Figure 5A), body ½T4 and liver weights were measured (Table 4), organs were mechanically dissociated in single-cell suspension, and HLA-ABCepositive cells were detected by FACS analysis.

MTX treatment efficiently inhibited liver tumor formation of My-La cells, as shown by the decrease of liver/body weight ratio in MTX-treated mice (Table 4). This was confirmed by HLA-ABCepositive cells and FACS analysis (Figure 5, B and C). Moreover, MTX treatment significantly reduced My-La cells spreading in spleen and liver

Figure 4 Methotrexate induces apoptosis/necrosis in My-La cells. A: My-La cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and treated with methotrexate (MTX; 10 or 50 mmol/L) or left untreated. Cells were counted after 48 and 72 hours of incubation with or without MTX. B: Apoptosis/necrosis analysis of My-La cells after 48 and 72 hours of culture with or without 10 or 50 mmol/L MTX. Proportions of alive, early/late apoptotic, and necrotic cells were evaluated by annexin V and Hoechst 33342 staining and analyzed by fluorescence-activated cell sorting. C: Dot plots are representative of cell viability after 72 hours in culture for each condition. Apoptotic/necrotic cell proportion was measured using annexin VePE and Hoechst, respectively. *P < 0.05.

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Figure 5

CTCL intrahepatic xenograft, a useful model for in vivo preclinical study. A: Intrahepatic xenograft and methotrexate (MTX) in vivo treatment procedure. B: Methotrexate treatment strongly diminished liver tumor formation, as observed by a decrease of human leukocyte antigen (HLA-ABC) staining in liver sections by immunohistochemistry (IHC). C: HLA-ABC was detected by fluorescence-activated cell sorter analysis in mice tissues. A decrease of HLA-ABC positive cells in lungs, spleen, and liver in MTX treated mice is observed. Dot plots represent cell granularity [Side SCater (SSC) along y axis] versus HLA-ABC expression (x axis). Data are given as means  SEM. *P < 0.05.

(Figure 5C), whereas no effect was observed for organs with low infiltration (Figure 5C). Taken together, these results indicate that the intrahepatic mouse model for CTCL is a robust model for rapid in vivo evaluation of therapeutic efficiency on tumor formation, migration or spreading capacities, and organ infiltration. Table 4 Methotrexate Intrahepatically

Variable

Treatment

Mice average weight (g)

in

Mice

Engrafted

Liver/body Liver weight average weight (g) ratio (%)

My-La control 26.12  1.0 3.38  0.3 12.95  1.3 My-La þ methotrexate 29.82  1.4 2.18  0.1 7.32  0.5***

Q29

Ten adult mice were engrafted intrahepatically with 2.5  106 My-La cells. Starting day 3, mice were treated with 0.5 mg/kg of methotrexate (or dimethyl sulfoxide as control) during 12 days. At the end of experiment (day 15), mice were sacrificed, and liver/body weight ratio was determined. Data are expressed as means  SD. ***P < 0.001.

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Discussion For the past 10 years, few articles reported mouse xenograft models of fresh CTCL patient cells or cell lines.15e20 These models were based on a small number of animals and a highly variable rate of engraftment appeared to be mouse strain and cell line dependent. Indeed, most engraftments were obtained using My-La cells, an aggressive transformed MF cell line.14e20 For other CTCL cell lines, in 2010, Doebbeling20 obtained a low rate of engraftment in CB-17 mice (CB-17/lcr.Cg-Prkdcscid Lystbg/Crl), in two of seven mice for HUT78 and zero of seven for SeAx (another SS cell line) underlining the absence of reproducible model for CTCL cell line engraftment. Because CTCL are primary skin diseases, the subcutaneous site was primarily chosen for injection of aggressive CTCL cells My-La, HUT78, SeAx, or HH, although most of these cells derived from blood circulating tumor T-cells.7e11 The My-La cell line was widely used for local tumorigenesis evaluation in subcutaneous models, and there was no

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Intrahepatic Cutaneous Lymphoma Xenograft

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evidence of extracutaneous spreading or metastases.14e20 So far, only one study reported liver infiltration after subcutaneous injection of HH cells into NOG mice.16 Although a subcutaneous site facilitates tumor growth monitoring, this does neither represent a physiological site nor an ideal niche for the expansion of CTCL cell lines. Indeed, most experiments consisting in subcutaneous injection of fresh cells from patients or CTCL cell lines in immunodeficient mice failed. Others used intravenous injection in severe combined immunodeficiency mice of HH cell for therapeutic evaluation, but did not report visceral infiltration.17 We hypothesized that CTCL cells may need an appropriate homing site delivering the necessary growth factors and thus considered intrahepatic mouse route as interesting for CTCL in which malignant T-cells often circulate.26 It is known that with advanced stages of CTCL disease, liver is an organ where CTCL cells accumulated.27 Moreover, liver is a wellvascularized organ that can provide essential cytokines for CTCL cell proliferation and tumor growth. Intrahepatic mouse models are widely used to study hepatocellular carcinoma local development and metastatic potential.23 Indeed, van der Fits et al22 were the first to use such liver injection of CTCL in newborn mice. This model was originally developed for human immune system reconstitution in sublethally irradiated newborn mice engrafted with human CD34þ progenitor cells with a high rate (>80% after 8 weeks) of engraftment.28 Despite its interest for CD34þ progenitor cell engraftment, this model exhibited some clear technical limits for the expansion of differentiated or memory neoplastic T-cells. First, handling and grafting mice at this age (2 to 7 days old) present an evident high risk of animal death after injection. Second, the injected volume had to be reduced according to the small liver size, leading to a reduction of injected cell number, which may be critical for the engraftment of cells with low proliferative or selfrenewal capacities. Finally, it only worked for two SS cell lines (HUT78 and SeAx) with a small number of animals and has so far not been successfully reproduced for engraftment of other CTCL cell lines or fresh primary CTCL cells. In this study, we generated a mouse model for aggressive (My-La, HUT78, HH, MAC2A, and MAC2B) and indolent (FE-PD and MAC1) CTCL cell line engraftment. To the best of our knowledge, this is the first report of a working in vivo mouse model for FE-PD and MAC2A cells. With this model, cell spreading in mice organs like kidneys, lungs, spleen, and heart was variable for each cell line. These differences in migration/spreading capacities could be explained by CTCL subtype (SS, CD30þ ALCL, or transformed MF), cell origin (peripheral blood, tumor, plaque), and specific cell surface molecule expression.29 Indeed, MAC cell lines were generated by Kadin et al10 from the same patient at different time of the disease and, more important, at different body locations. Thus, MAC1 was obtained at early stage during the indolent phase from peripheral blood of a CD30þ ALCL, whereas MAC2A and

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MAC2B were obtained from two individual skin tumors at aggressive terminal stage.30 We hypothesized that MAC1 cell line originating from the blood retains a capacity to infiltrate hepatic, renal, and splenic sinusoids and centrolobular hepatic veins via endothelial cell addressins. Taken together, these results demonstrated that intrahepatic xenografts in adult immunodeficient mice are an efficient model to evaluate differences in CTCL tumorigenicity, migration, and spreading capacities. This may permit the evaluation of imaging or monitoring methods of CTCL cells. The use of our model for patient-derived CTCL xenografts may also facilitate the generation of new cutaneous lymphoma cell lines. However, a humanized bed could be necessary for the successful engraftment of fresh patient CTCL cells, as reported for human primary squamous carcinoma cells.31 For now, because of a lack of a relevant model to test drugs targeting CTCL, preclinical in vivo studies are realized with nonappropriate surrogate models. For example, a recent evaluation of the in vivo activity of a humanized KIR3DL2 antibody against CTCL required the ectopic expression of this marker in a B-cell lymphoma cell line before mice engraftment.32 In conclusion, this intrahepatic mouse model is an efficient model that can be used for extended in vivo preclinical studies. Therefore, this new model may be used for in vivo screening of therapeutic molecules in different compartments, as observed herein with methotrexate. This model also allows further functional investigations that may lead to a better understanding of the genomic and biological hallmarks of the different CTCL subtypes.33

Acknowledgments We thank Bordeaux University SFR Transbiomed for the Q21 use of the cytometry platform and A2 animal housing facility, Dr. Vincent Servant for providing methotrexate, and Dr. Anne Pham-Ledard and Prof. Pierre Dubus for their constant help to the cutaneous lymphoma project and general helpful advice.

Supplemental Data Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2016.03.012.

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Supplemental Figure S1 Human leukocyte antigen (HLA-ABC) immunostaining by immunohistochemistry (IHC) in organs after CTCL intrahepatic xenografts. Mice were intrahepatically xenografted with CTCL cell lines: My-La, HUT78, HH, FE-PD, MAC1, and MAC2A. After animal sacrifice, formalin-fixed, paraffin-embedded tissue sections were deparaffinized, and IHC was performed with a HLA-ABC antibody (Ab70328). Images were captured using a LEICA DMR microscope coupled with a NIKON DS-FI2 camera and analyzed with NIS BR imaging software. Scale bars Z 100 mm. Supplemental Figure S2 Evaluation of methotrexate (MTX) effects on My-La cell viability after 48 hours of incubation with or without MTX. Apoptosis/ necrosis analysis of My-La cells before and after cultured with or without MTX (10 or 50 mmol/L). Proportions of alive, early/late apoptotic, and necrotic cells were evaluated by annexin V and Hoechst 33342 staining and analyzed by fluorescence-activated cell sorting. Dot plots are representative of cell viability 48 hours in culture for each condition.

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