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[Cancer Research 65, 2560-2564, April 1, 2005]
© 2005 American Association for Cancer Research

Priority Reports

CY15, a Malignant Histiocytic Tumor That Is Phenotypically Similar to Immature Dendritic Cells

Thomas Kammertoens1, Ralf Willebrand1, Bettina Erdmann2, Liangping Li2, Yongping Li4, Boris Engels3, Wolfgang Uckert2,3 and Thomas Blankenstein1,2

1 Institute of Immunology, Charité Campus Benjamin Franklin; 2 Max-Delbrück Center for Molecular Medicine; 3 Institute of Biology, Humbold University Berlin, Berlin, Germany; and 4 Zhongshan Ophthalmic Center Sun Yat-sen University, Guangzhou, China

Requests for reprints: Thomas Kammertoens, Institute of Immunology, Hindenburgdamm 30, 12200 Berlin, Germany. Phone: 49-30-8445-3639; Fax: 49-30-8445-4613; E-mail: tkamm{at}mdc-berlin.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The origin and pathogenesis of histiocytic malignancies and the biology of the tumor cells are poorly understood. We have isolated a murine histiocytic tumor cell line (CY15) from a BALB/c IFN{gamma}–/– mouse and characterized it in terms of phenotype and function. The morphology, as judged by electron microscopy, and the surface marker phenotype suggests that CY15 cells are similar to immature dendritic cells (CD11c low, MHC II low, CD11b+, B7.1+, B7.2+, and CD40+). The cells form tumors in BALB/c mice and metastasize to spleen, liver, lung, kidney, and to a lesser extend to lymph nodes and bone marrow, as judged by the growth of green fluorescent protein transfected tumor cells in mice. CY15 cells are capable of actively taking up antigen (FITC-ovalbumin) and can stimulate T lymphocytes in an allogenic mixed lymphocyte reaction but less effectively than their normal counterparts (immature dendritic cells). They respond to interleukin 4 (IL-4) with up-regulation of CD11c. If stimulated with IFN{gamma} the cells up-regulate MHC II, CD40 B7.1, and B7.2. Lipopolysaccharide induces the cells to up-regulate B7.1 and B7.2 and to secrete tumor necrosis factor {alpha} and IL-12. Based on these data, CY15 is a dendritic cell–like tumor cell line and may serve as a transplantable tumor model for histiocytosis in humans.

Key Words: dendritic cell neoplasm • histiocytic malignancy


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
By definition, histiocytes are tissue resident cells (macrophages, dendritic cell, and Langerhans cells) that ingest foreign antigens. Malignant growth of any of these cells results in histiocytic malignancy, which is a rare disease in humans. Of the malignant histiocytoses in humans, Langerhans cell histiocytosis is best studied (1). In general, the etiology and pathogenesis of histiocytic malignancy and the biology of the histiocytoma cells are poorly understood. It is not known how histiocytomas compare with their normal counterparts and whether mouse and human diseases are related. There are few reports on histiocytic malignancy in mice. These tumors have been reported to occur in IFN{gamma}–/– mice (2), this occurrence may result from chronic infection (3). Furthermore, the malignant histiocytosis sarcoma retrovirus can induce histiocytic malignancy in mice (4). Currently, there is no histiocytoma model with a dendritic cell–like phenotype available. Notably, there are only few murine dendritic cell–like cell lines available, all of which were immortalized in vitro (5, 6). We isolated a tumorigenic cell line with an immature dendritic cell phenotype. To our knowledge, there are currently no in vivo generated dendritic cell–like cell lines available. Therefore, we decided to analyze this tumor in more detail. CY15 cells can actively take up antigen, secrete cytokines, and change surface markers after stimulation. Furthermore, CY15 cells can stimulate T cells in an allogenic mixed lymphocyte reaction. When transplanted, the cells showed a similar metastasis pattern as histiocytoma in humans. Taken together, we have identified and characterized the first histiocytic dendritic cell–like tumor cell line that may serve as a transplantable mouse model for this type of histiocytic malignancy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Mice. BALB/c, C57Bl/6, and RAG1–/– BALB/c mice were obtained from Charles River (Sulzfeld, Germany). BALB/c IFN{gamma}–/– (Jackson Laboratory strain 002286) mice were bred at the animal facility of the Max-Delbrück Center, Berlin, Germany.

Cells and stimulation of cells. CY15 cells were isolated from a BALB/c IFN{gamma}–/– mouse treated with 200 µg of 7,12-dimethylbenz(a)anthracene (DMBA) and subsequently for 13 weeks with 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly (10 µg per animal). The cells were isolated from the spleen and cultured in RPMI 1640, 10% FCS. After establishing a B220– CD11b+ phenotype (around passage 4) the cells were stable for at least 20 passages and have a doubling time of 16 hours. J558L is a plasmacytoma derived from BALB/c. For stimulation, CY15 cells were seeded at 106 cells in 5 mL medium and 1 ng/mL of recombinant interleukin 4 (IL-4) or IFN{gamma}, or 5 µg/mL lipopolysaccharide (LPS, Sigma, Deisenhofen, Germany) was added. Forty-eight hours later, cells were analyzed by fluorescence-activated cell sorting (FACS), or alternatively, supernatant was analyzed by ELISA for IL-12 p40 or tumor necrosis factor {alpha} (TNF{alpha}). Recombinant cytokines and the ELISA reagents were purchased from BD and ELISA was done according to the supplier's instructions (BD, Hamburg, Germany).

Fluorescence-activated cell sorting analysis. Cells were first treated with Fc-block ({alpha}-CD16/CD32) for 15 minutes followed by 30 minutes of staining with the specific fluorochrome-labeled antibody or isotype control monoclonal antibody (mAb). The following mAbs (BD) were used: CD1d (1B1), CD2 (RM2-5), CD4 (RMA4-5), CD8{alpha} (53-6.7), CD11c (HL3), I-Ad/IEd (2G9), H-2Kd (SF1-1.1), CD11b (M1/70), CD14 (rmC5-3), CD40 (3/23), CD80 (16-10A1), and CD86 (G11). {alpha}-F4/80 (MCA497F) was purchased from Serotec (Duesseldorf, Germany). Cells were analyzed on a FACSCalibur using CellQuestPro software (BD).

Generation of immature dendritic cells from bone marrow. Dendritic cells were generated as described before (7). Briefly, cells were prepared from the femurs of BALB/c, BALB/c IFN{gamma}–/–, or C57Bl/6 mice and were incubated with granulocyte macrophage colony-stimulating factor (10-20 ng/mL) for 10 days.

Phagocytosis assay. CY15, dendritic cells or J558L cells were seeded in 96-well plates (1 x 105 cells per well). FITC-OVA (Molecular Probes, Karlsruhe, Germany) was added at 0.1 mg/mL in duplicates. Incubations were done for 40 minutes at 4°C or 37°C. After incubation, cells were washed and fixed with 2% formaldehyde. FITC-OVA uptake was evaluated by FACS.

T-cell proliferation in mixed lymphocyte reactions. Single cell suspensions of the responder cells (C57Bl/6 splenocytes) were prepared. 0.33 x 104 or 1 x 104 stimulator cells (bone marrow–derived dendritic cells from BALB/c or C57Bl/6 mice or CY15 tumor cells that had been irradiated with 90 Gy) were cocultured with 2 x 105 responder cells in 200 µL RPMI 1640/10% FCS, 10 U IL-2/mL, in 96-well plates for 5 days. Separate wells with stimulators or responder cells only served as negative controls. Responder cells stimulated with {alpha}-CD3 (2 µg/mL) and {alpha}-CD28 (1 µg/mL) mAbs served as positive controls. On day 5, 1 µCi per well 3H-thymidine was added and incubated over night. On day 6, plates were harvested and 3H-thymidine uptake analyzed.

Electron microscopy. Cells were fixed in 2% glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.4) for 20 hours, post-fixed with 1% osmium tetroxide for 2 hours, dehydrated, and embedded in Poly/Bed 812 (Polysciences, Inc., Eppelheim, Germany). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips EM 400T at an acceleration voltage of 80 kV.

Metastasis of CY15-GFP cells. CY15 cells were transduced to express green fluorescent protein (GFP) as described elsewhere (8). The resultant bulk culture was subcloned by limiting dilution. The clone with the highest GFP expression (designated CY15-GFP) was isolated and used for the experiments. Because the GFP expressing subclone did not grow in BALB/c mice, CY15-GFP cells were injected into RAG1–/– BALB/c mice. To analyze the percentage of GFP-positive cells in different organs, 106 tumor cells were injected s.c. and 30 days later mice were euthanized and perfused with PBS. Liver, lung and kidney were digested with 1 mg/mL collagenase D for 4 hours at 37°C. Splenocytes and bone marrow cells were prepared using standard protocols. Erythrocytes were lysed in all samples, and the percentage of CY15-GFP cells in each organ was determined by FACS.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Identification of CY15 tumor cells. CY15 cells were derived from a BALB/c IFN{gamma}–/– mouse during a two-stage skin carcinogenesis experiment (DMBA, followed by a biweekly treatment with TPA). After 13 weeks of TPA treatment, the animal had to be euthanized and it displayed spleenomegaly. The initial FACS analysis showed 99% B220+, CD4–, CD8–, and CD3– splenocytes. The cells were taken into culture and 3 weeks later the percentage of B220+ cells was reduced to 30%. After 4 weeks in culture, 100% of the cells were B220– and CD11bhigh/CD11clow. Whether the histiocytic cells grew out or whether the culture had converted from B220+ to CD11b+ is not clear. It has been observed that human histiocytic sarcomas can develop from acute B-cell lymphoblastic leukemia (9, 10). The morphology of CY15 cells observed in culture resembled that of the in vitro immortalized dendritic cell–like cell line CB1 (5).

Cytofluorimetric analysis of CY15 cells. We compared the expression of CD11c, CD11b, and MHC class I and II of immature bone marrow–derived dendritic cells from either BALB/c or BALB/c IFN{gamma}–/– mice with the expression observed in CY15 cells. The expression of CD11b and MHC I is similar, but CY15 cells show slightly lower expression of MHC II and CD11c (Fig. 1A). The expression of all surface molecules mentioned above was stable when analyzed, even in five subclones of CY15 cells (data not shown). There was one exception, MHC II was always low but displayed a little variability. We extended the cytofluorimetric analysis to other surface molecules and found that the cells express CD1d and CD14 and they show low expression of CD2, CD40, B7.1, B7.2, and F4/80 (Fig. 1A and B). These analyses confirmed the similarity between immature dendritic cell lines (5, 6) and our tumor cell line. Because expression of CD40, CD11c, and MHC II was low, we next analyzed whether these molecules could be up-regulated in response to the recombinant cytokines IL-4, IFN {gamma}, or in response to LPS. We found that IL-4 (1 ng/mL) induced CD11c up-regulation on CY15 cells (Fig. 1C, top). IFN{gamma} (1 ng/mL) led to an increased surface expression of CD40, MHC II and B7.2. Whereas stimulation with LPS (5 µg/mL) induced up-regulation of B7.1, CD40, and to a lesser extend B7.2 (Fig. 1C, bottom). Taken together, these results confirm the similarity of CY15 cells to immature dendritic cells.



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  		Figure 1. Flowcytometric and electron microscopic analysis of CY15 reveals dendritic cell (DC) phenotype of CY15 cells. A-C, CY15 cells or immature bone marrow–derived dendritic cell (A) were stained with specific mAbs (red lines) as indicated or isotype controls (black lines) and analyzed by flow cytometry. C, CY15 cells were stimulated with either 1 ng/mL IL-4 or IFN{gamma} or with 5 µg/mL LPS and the expression of B7.1, B7.2, MHC II, CD40, and CD11c was analyzed by FACS. Without stimulation (w/o). D, ultrastructure of CY15 cells. Note typical dendritic cell features like numerous dendritic prolongations, the indented nucleus and multilamellar bodies (inset). Bar, 1 µm. Magnification, x10,000; x30,000 (inset).

 
Ultrastructural analysis of CY15 cells. Because surface marker expression on tumor cells can be aberrant and by itself does not prove origin of the cells, we went on to determine the ultrastructure of CY15 cells by electron microscopy. As shown in Fig. 1D, the cells have dendritic prolongations, they have indented nuclei and some of the cells showed inclusions (like multilamellar bodies, Fig. 1D, inset; or lysosomes, data not shown). Therefore, the ultrastructural analysis corroborates that the cells resemble dendritic cells.

In vivo growth and metastasis of CY15 cells. Having characterized the cells in vitro, we wanted to know whether these cells grow as tumors in mice. To this end, CY15 cells were injected into BALB/c mice. Within 4 weeks after injecting 106 cells, or within 6 to 8 weeks after injecting 104 cells, all animals developed large numbers of macroscopically visible metastases in spleen, liver, and kidney, regardless if injected i.v., i.p., or s.c. From this, we conclude that we have identified a metastatic tumor cell line. To evaluate the structure of metastatic tumors, histologic analysis of liver metastasis was done. CY15 cells were s.c. injected into BALB/c mice and H&E staining of paraffin-embedded sections of liver metastasis (Fig. 2A and B) was done. Histology confirms that the cells are malignant histiocytes. To analyze whether the cells metastasize to similar organs as histiocytic malignancies observed in humans, CY15 cells were transduced to express the GFP using a GFP encoding retrovirus MP71-GFP-PRE (8). CY15-GFP cells were injected into RAG1-deficient BALB/c mice, because the transduced cells did not grow in lymphocyte-competent mice, probably due to the immunogenicity of GFP. As shown in Fig. 2C, the cells metastasize to lymph nodes and bone marrow and furthermore as seen macroscopically before to spleen, liver, kidney, and lung.



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  		Figure 2. CY15 cells are malignant histiocytes. A and B, 106 CY15 cells were injected s.c. into BALB/c mice. Thirty days later, the liver was isolated, fixed, and tissue sections of paraffin embedded tissue from liver metastasis of CY15 were stained with H&E. 40-fold and 200-fold magnification of a liver section. Arrows, metastasis. C, CY15 cells metastasize to lung, liver, spleen, kidney, bone marrow, and lymph nodes. CY15 cells were transduced to express GFP and 106 cells were s.c. injected into RAG1–/– BALB/c. Thirty days later, mice were euthanized and the percentage of CY15-GFP cells in the respective organs was determined by flow cytometry. Four mice were analyzed. One representative animal is shown.

 
In vitro functions of CY15 cells. After we had established that CY15 cells are a dendritic cell–like neoplasm metastasing similar to spontaneous histiocytic malignancy in humans, we wanted to analyze the functional properties of the cells in vitro. We found that like immature bone marrow derived dendritic cells, CY15 cells actively take up FITC coupled ovalbumin (Fig. 3A) and also fluorescently labeled latex beads (data not shown). Furthermore, we found that CY15 cells can stimulate T cells. This was shown in a 5-day allogenic mixed lymphocyte culture, coculturing 105 responder cells (from C57Bl/6 mice) with either 104 or 0.33 x 104 CY15 cells. However, this stimulation was less effective than that of immature dendritic cells (Fig. 3B). To analyze whether CY15 cells like their normal counterparts can secrete cytokines, 106 cells were stimulated for 48 hours with 5 µg/mL LPS. The tissue culture supernatants were analyzed for TNF{alpha} and IL-12 p40 by ELISA. As shown in Fig. 3C, CY15 cells produced 1026 pg/mL IL-12 p40 and 620 pg/mL TNF{alpha}, whereas from nonstimulated cells no IL-12 and only very little production (78 ng/mL) of TNF{alpha} was detectable. We conclude from the functional analysis, that CY15 cells still retained some of the functions of immature dendritic cells such as phagocytosis, cytokine secretion, and stimulation of T cells.



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  		Figure 3. CY15 cells take up antigen, stimulate T cells, and secrete cytokines. A, FITC-OVA uptake. CY15, dendritic cell (DC), or J558L cells were seeded at 1 x 105 cells per well in 96-well plates, FITC-OVA was added at 0.1 mg/mL, and incubated for 40 minutes at 4°C or 37°C. Cells were washed and fixed. FITC-OVA uptake was evaluated by FACS. B, mixed lymphocyte reaction with CY15 cells as stimulators. Responder cells (C57Bl/6 splenocytes) were cocultured with three different stimulator cells (bone marrow–derived dendritic cells from BALB/c (black columns), from C57Bl/6 mice (open columns) or CY15 tumor cells that had been irradiated with 90 Gy (grey columns). Stimulator cells (0.3 or 1 x 104) were cocultured with 2 x 105 responder cells for 5 days. Separate wells with stimulator cells, responder cells or responder cells stimulated with mAb {alpha}-CD3 and {alpha}-CD28 served as controls (hatched columns). On day 5, 1 µCi per well 3H-thymidine was added overnight. 3H-thymidine uptake as cpm. C, cytokine secretion by LPS stimulated CY15 cells. CY15 cells were stimulated with 5 µg/mL LPS. Forty-eight hours later, supernatants from the cells were harvested and analyzed by ELISA for IL-12 p40 or TNF{alpha}. Grey columns, IL-12 p40. Black columns, TNF{alpha}. 3 A, B, and C, one representative experiment of two.

 
Finally, we wanted to analyze whether either the genotype of the mouse CY15 cells originated from (IFN{gamma}–/–) or the DMBA/TPA treatment we used had played a role in the development the histiocytic malignancy. There is a possibility that IFN{gamma} deficiency or the carcinogenesis protocol used may affect histiocytic malignancy development. It has recently been suggested, that IFN{gamma}–/– mice on the BALB/c background are prone to develop either B-cell lymphomas or alternatively histiocytic malignancies (2). Furthermore, histiocytic sarcomas have been reported to be induced by coal tar (11). To analyze whether DMBA/TPA treatment induces histiocytic malignancies particularly in IFN{gamma}–/– animals, we repeated the experiment comparing tumor incidence in 14 IFN{gamma}–/– and 14 IFN{gamma}+/– animals that were treated as before. Because none of the animals analyzed developed another histiocytic tumor (data not shown) even 40 weeks after the beginning of the treatment, we conclude that the appearance of the CY15 histiocytoma was an isolated incidence, rather than a regular feature of this carcinogenesis protocol in these specific mice.

Altogether we have identified and characterized a new tumor cell line with features of immature dendritic cells. How these cells compare to human histiocytic malignancy and what their mechanisms of immune evasion are and whether they can serve as a mouse model for histiocytic malignancy remains to be shown.


   Acknowledgments
 
Grant support: Deutsche Krebshilfe, Mildred-Scheel-Stiftung e.V. grant 10-1535-BL2, and Deutsche Forschungsgemeinschaft grant BL 288.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Thomas Schueler for discussion and suggestions.

Received 11/29/04. Accepted 1/12/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Laman JD, Leenen PJ, Annels NE, Hogendoorn PC, Egeler RM. Langerhans-cell histiocytosis "insight into DC biology". Trends Immunol 2003 Apr;24:190–6.[CrossRef][Medline]
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  3. Enzler T, Gillessen S, Manis JP, et al. Deficiencies of GM-CSF and interferon {gamma} link inflammation and cancer. J Exp Med 2003;197:1213–9.[Abstract/Free Full Text]
  4. Franz T, Lohler J, Fusco A, et al. Transformation of mononuclear phagocytes in vivo and malignant histiocytosis caused by a novel murine spleen focus-forming virus. Nature 1985;315:149–51.[Medline]
  5. Paglia P, Girolomoni G, Robbiati F, Granucci F, Ricciardi-Castagnoli P. Immortalized dendritic cell line fully competent in antigen presentation initiates primary T cell responses in vivo. J Exp Med 1993;178:1893–901.[Abstract/Free Full Text]
  6. Paglia P, Chiodoni C, Rodolfo M, Colombo MP. Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vivo. J Exp Med 1996;183:317–22.[Abstract/Free Full Text]
  7. Schuler T, Blankenstein T. Naive CD8(+) but not CD4(+) T cells induce maturation of dendritic cells. J Mol Med 2002;80:533–41.[Medline]
  8. Engels B, Cam H, Schuler T, et al. Retroviral vectors for high-level transgene expression in T lymphocytes. Hum Gene Ther 2003;14:1155–68.[CrossRef][Medline]
  9. Feldman AL, Minniti C, Santi M, Downing JR, Raffeld M, Jaffe ES. Histiocytic sarcoma after acute lymphoblastic leukaemia: a common clonal origin. Lancet Oncol 2004;5:248–50.[CrossRef][Medline]
  10. Bouabdallah R, Abena P, Chetaille B, et al. True histiocytic lymphoma following B-acute lymphoblastic leukaemia: case report with evidence for a common clonal origin in both neoplasms. Br J Haematol 2001;113:1047–50.[CrossRef][Medline]
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