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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Malignant fibrous histiocytoma (MFH) of the bone is an aggressive tumor with high rates of local recurrence and metastasis. The development of novel therapeutic approaches is critical to improve the prognosis of patients with MFH. We reported previously that the side population (SP) cells of the MFH2003 bone MFH cell line have the characteristics of cancer stem-like cells (CSC)/cancer-initiating cells. In the present study, to establish immunotherapy targeting CSC, we analyzed cell surface immune molecules on SP cells of the MHF2003 cell line, as well as autologous CTL responses against these SP cells in the tumor microenvironment and peripheral circulating lymphocytes, using autologous tumor-infiltrating lymphocytes and autologous CTL clones derived from peripheral blood, respectively. We found that the SP cells expressed human leukocyte antigen (HLA) Class I molecules on the cell surface. The autologous tumor-infiltrating lymphocyte line TIL2003 recognized both the SP and main population cells of the MFH2003 cell line. Next, we induced the CTL clone Tc4C-6 by mixed lymphocyte tumor cell culture using autologous peripheral blood mononuclear cells and freshly isolated SP cells, followed by a limiting dilution procedure. The Tc4C-6 clone showed specific cytotoxicity against the SP cells. Moreover, the cytotoxicity against SP cells was blocked by the anti-HLA Class I antibody W6/32. In conclusion, the findings of the present study support the idea that CSC of bone MFH are recognized by autologous CTL in the tumor microenvironment and peripheral circulating lymphocytes. Thus, CTL-based immunotherapy could target CSC of bone sarcoma to help prevent tumor recurrence. (Cancer Sci 2011; 102: 1443–1447)

Malignant fibrous histiocytoma (MFH) of the bone is a rare primary neoplasm, accounting for <5% of primary bone malignancies.(1,2) Histologically, MFH of the bone is composed of fibroblasts and pleomorphic cells with a prominent storiform pattern. It is an aggressive tumor, with high rates of local recurrence and metastasis and a poor prognosis; the 5-year survival has been reported to be <60%.(3,4) Therefore, the development of novel therapeutic approaches is critical to improve the outcomes of patients with MFH.

It was thought that all neoplastic cells within a tumor were capable of tumorigenic growth. However, recent studies have demonstrated that malignant tumors can be generated by a distinct subpopulation of tumor cells, the so-called cancer stem cells (CSC)/cancer-initiating cells (CIC), which have self-renewal ability, differentiation potential, and tumorigenic capacity.(5,6) Thus, CSC could be a therapeutic target for the complete eradication of tumor cells. However, CSC have been reported to be resistant to standard therapeutic modalities, including radiation and drugs.(7,8)

Recently, many clinical trials of CTL-based immunotherapy using peptide vaccination have demonstrated the potency of this new therapeutic modality for various cancers that are resistant to standard chemotherapy.(9) However, it remains unknown whether CTL-based immunotherapy can kill CSC. Previously, we demonstrated that the side population (SP) cells from the bone MFH cell line MFH2003 have CSC characteristics.(10) The SP cells of the MFH2003 cell line exhibited cancer-initiating activity, with in vitro sphere formation and in vivo tumorigenesis in NOD/SCID mice. In the present study, to characterize the immunogenicity of CSC, we analyzed autologous CTL responses against SP cells of the MFH2003 cell line in the tumor microenvironment, as well as in peripheral circulating blood, using autologous tumor-infiltrating lymphocytes and a CTL clone.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

The present study was approved under the institutional guidelines for the use of human subjects in research. The patients and their families, as well as healthy donors, provided informed consent for the use of blood samples and tissue specimens in our research.

Cell lines and culture.  The cell lines used in the present study were a bone human MFH cell line (MFH2003), an erythroleukemia cell line (K562), and Epstein-Barr virus-transformed B cell lines (LG2-EBV, B2003-EBV). The OS2000, KIKU, MFH2003, and B2003-EBV cell lines were established in our laboratory.(11) The K562 cell line was purchased from American Type Culture Collection (Manassas, VA, USA). ThILG2-EBV cell line was donated by Dr. PG Coulie (Christian de Duve Institute of Cellular Pathology, University of Louvain, Brussels, Belgium). The MFH2003 cells were cultured in Iscove’s modified Dulbecco’s Eagle’s medium (IMDM; Gibco BRL, Grand Island, NY, USA) containing 10% FBS. The LG-2-EBV, B2003-EBV, and K562 cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, USA) containing 10% FBS. All other cell lines were maintained in DMEM (Sigma-Aldrich) containing 10% FBS in a 5% CO2 incubator at 37°C.

Purification of side population cells.  The CSC of the MFH2003 cell line were purified by side population analysis, as described previously.(10) Briefly, cell suspensions were labeled with Hoechst 33342 dye (Cambrex Bio Science, Walkersville, MD, USA) at a final concentration of 5.0 μg/mL in the presence or absence of verapamil (75 μM; Sigma-Aldrich) as an inhibitor of the ATP-binding cassette (ABC) transporter. Cells were incubated at 37°C for 90 min with continuous shaking. At the end of the incubation period, cells were washed with ice-cold PBS with 5% FBS, centrifuged at 440g for 5 min at 4°C, and resuspended in ice-cold PBS containing 5% FBS. Propidium iodide (final concentration 1 μg/mL; Life Technologies, Carlsbad, CA, USA) was used to gate viable cells. Flow cytometry and cell sorting were performed using a FACSAria II cell sorter (BD Biosciences, Bedford, MA, USA). The Hoechst 33342 dye was excited at 357 nm and its fluorescence was analyzed using dual wavelengths (blue, 402–446 nm; red, 650–670 nm).

When the proportion of SP cells was low (≤5%), the SP cells were sorted and subjected to in vitro culture in 10 mL IMDM containing 10% FBS for enrichment. Then, after at least 14 days culture, SP analysis and cell sorting were performed again.

Analysis of expression of cell surface molecules.  Expression of cell surface molecules was assayed as described previously(11) using an anti-human leukocyte antigen (HLA)-A24 mAb (C7709A2.6), anti-HLA-B&C mAb (B1.23.2), anti-HLA Class I mAb (W6/32), anti-HLA-Class II mAb (L243), and an anti-CD80 mAb (Hybridoma cells for C7709A2.6 were donated by Dr. PG Coulie [Christian de Duve Institute of Cellular Pathology, University of Louvain, Brussels, Belgium] and those for B1.23.2 and L243 were purchased from the American Type Culture Collection [Manassas, VA, USA]. An anti-CD80 mAb was purchased from Immunotech [Marseille, France].). SP, and main population (MP) cells of the MFH2003 cell line, as well as LG2-EBV, B2003-EBV, and K562 cells, were incubated with appropriate mAb for 40 min on ice. Then, the cells were incubated with FITC-labeled secondary antibodies and analyzed using a FACSCalibur flow cytometer (BD Biosciences).

ELISA.  Target cells (1–2 × 104) were plated in flat-bottomed 96-microwell plates (Corning, Corning, NY, USA) in DMEM containing 10% FBS. Then, TIL2003 cells (5 × 104) in AIM-V medium were added. After 24 h incubation at 37°C, the amount of granulocyte–macrophage colony stimulating factor (GM-CSF) in the supernatant (100 μL) was measured using an ELISA Development kit (TechneCorp, Minneapolis, MN, USA) according to the manufacturer’s instructions. All experiments were performed in duplicate.

Establishment of autologous CTL clones against SP cells of the MFH2003 cell line.  Autologous CTL clones against SP cells of the MFH2003 cell line were established as described previously. Briefly, peripheral blood mononuclear cells (PBMC) were obtained from an MFH2003 donor patient. The CD8+ T cells were collected from PBMC using magnetic anti-CD8 beads (Miltenyi Biotec, Gladbach, Germany). A total of 5 × 105 irradiated (100 Gy) SP cells of the MFH2003 cell line and 5 × 106 CD8 T cells were distributed into five wells of a 24-well flat-bottomed culture plate containing 2 mL/well AIM-V and cultured at 37°C. The following day, 20 U/mL recombinant human interleukin-2 (rhIL-2; a kind gift from Takeda Chemical Industries, Osaka, Japan) and 10% AB human serum (HS) were added. The stimulation of T cells was repeated at intervals of 7–10 days using SP cells. After the fourth stimulation, the CTL were plated from all five culture wells at various dilutions in round-bottomed 96-microwell plates (Corning) in AIM-V supplemented with rhIL-2 (200 U/mL) and phytohemagglutinin (PHA; 5 μg/mL; Wako Chemicals, Osaka, Japan). Irradiated LG-2 EBV cells (1 × 104 cells/well) and allogeneic PBMC (1 × 105 cells/well) were added as feeder cells. Cells were incubated at 37°C. After 42 days, three resultant CTL clones were used in the cytotoxicity assays. One CTL clone showing specific cytotoxicity against SP cells of the MFH2003 cell line was selected and designated Tc4C-6. The cytotoxicity assay was performed as described below. Cell surface phenotypes of Tc4C-6 were assayed using an FITC-conjugated anti-CD3 antibody (BD Biosciences), phycoerythrin (PE)-conjugated anti-CD4 antibody (BD Biosciences), PE-Cy5-conjugated anti-CD8 antibody (eBioscience, San Diego, CA, USA), FITC-conjugated anti-CD45RA (BD Biosciences) antibody, and PE-conjugated anti-CCR7 antibody (BD Biosciences). The Tc4C-6 clone and healthy donor PBMC were incubated with these antibodies for 30 min on ice in the dark. After washing with PBS, cells were fixed with 1% paraformaldehyde in PBS and analyzed by flow cytometry.

Cytotoxicity assay.  The specific cytotoxicity of CTL clones was measured using the non-radioactive aCella-TOX assay (Cell Technology, Mountain View, CA, USA) according to the manufacturer’s instructions. Target cells were plated in triplicate (5000 cells/well) in round-bottomed 96-well microwells in IMDM containing 50 U/well rhIL-2. Effector cells were added at various effector:target (E/T) ratios, as indicated. Spontaneous effector and target cell death was achieved by including control wells of effector and target cells at numbers corresponding to those of their various E/T ratios. To determine maximum release, calculated as total glyceralehyde-3-phosphate dehydrogenase (G3PDH) released, 10 μL lysis reagent (0.5% Nonidet P-40 per 100 μL sample) was added to the target cell positive control 10 min after the end of the assay incubation. After 12 h incubation at 37°C, the culture supernatant from each well was transferred into a corresponding well containing Enzyme Assay Reagent reacting against G3PDH on a white OptiPlate-96 (PerkinElmer, Waltham, MA, USA) and detection reagent was added to each well. The luminescence of each well was analyzed immediately using an ARVO MX/Light 1420 Multilabel Luminescence Counter (PerkinElmer). All experiments were performed in triplicate. Cytotoxicity (%) was calculated as [(experimental G3PDH release − spontaneous G3PDH release from effector cells − spontaneous G3PDH release from target cells)/(maximum G3PDH release from target cells − spontaneous G3PDH release from target cells) × 100].

In blocking experiments, the target cells was incubated with an anti-HLA Class I mAb (W6/32) or anti-HLA Class II mAb (L243) for 30 min at 37°C before the cytotoxicity assay was performed.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Enrichment of SP cells in the MFH2003 cell line.  To isolate SP cells as CSC/CIC, SP analysis and cell sorting were performed 95 times. As shown in Figure 1, in independent experiments the proportion of SP cells in the MFH2003 cell line ranged from 0.3% to 7.2% (mean 4.0 ± 1.7%). Figure 1(a) shows typical results from two independent SP analysis experiments. The number of SP cells isolated from bulk MFH2003 cells ranged from 0.1 to 6.7 × 105 (mean 2.4 ± 1.4 × 105). Because more than 5 × 105 SP cells were required for each experiment in the present study, the variance in the proportion of SP cells and the low number isolated sometimes made it difficult to complete the experiments. To overcome these problems, we enriched SP cells using in vitro SP cell culture. After 7–10 days, the proportion of SP cells increased to between 9.4% and 36.2% (mean 18.6 ± 7.4%). In addition, the resultant number of sorted SP cells increased more than sixfold, ranging from 3.3 to 38.2 × 105 (mean 15.3 ± 7.7 × 105; Fig. 1a,b). This improvement in the isolation efficiency of SP cells was useful for further experiments.

image

Figure 1.  Enrichment of the side population (SP) cells of the MFH2003 cell line. (a) The SP cells before and after enrichment, in the presence or absence of verapamil, in two independent experiments. The SP cells are encircled by black lines. The proportion of SP cells among total living cells is indicated in each case. (b) Summary of the enrichment of SP cells giving the mean proportion (%) of SP cells in the MFH2003 cell line, the mean number of bulk MFH2003 cells stained with Hoechst 33342 dye, and the mean number of sorted SP cells. Data show the mean ± SD with the range given in parentheses.

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Expression profiles of immune molecules on SP and MP cells of the MFH2003 cell line.  First, we analyzed the immune molecules on SP and MP cells of the MFH2003 cell line (Fig. 2). The SP cells expressed HLA Class I, HLA-A24, B, and C molecules on their cell surface. The expression of these molecules was greater than that for MP cells. Although MHC Class I and CD80, which provide costimulatory signals necessary for T cell activation and survival, were not expressed on SP cells, the higher expression of MHC Class I on SP cells suggests that CSC can be recognized by the host cellular immunity.

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Figure 2.  Expression profile of immune molecules on side population (SP) and main population (MP) cells showing the cell surface expression of human leukocyte antigen (HLA) Class I (HLA-A24, B&C), HLA Class II, and CD80 molecules on MFH2003 (bulk, SP, and MP cells), LG-2, EB-B (B2003-EBV) and K562 cells.

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Autologous TIL2003 recognized both SP and MP cells of MFH2003.  Next, to evaluate whether CTL can recognize SP cells in the tumor microenvironment, we assessed the response of the autologous tumor-infiltrating lymphocyte line TIL2003 against SP and MP cells. The TIL2003 cell line is a CTL line we established previously from the metastatic lymph nodes of the MFH2003 patient.(11) As shown in Figure 3, TIL2003 cells recognized both SP and MP cells. Although we could not completely rule out the possibility that MP cells triggered the immune response against both SP and MP cells in the context of some antigens expressed in both SP and MP cells, the results do suggest that the CTL response against SP cells was triggered by SP cells in the tumor microenvironment.

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Figure 3.  Autologous tumor-infiltrating lymphocyte TIL2003 cells recognized both side population (SP) and main population (MP) cells. Freshly isolated SP and MP cells from the MFH2003 cell line, autologous EB-B cells (B2003-EBV), and negative control K562 cells were cocultured with TIL2003. After 24 h, the culture supernatant was harvested and the granulocyte–macrophage colony stimulating factor (GM-CSF) released from the TIL2003 was determined using ELISA.

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Specific response of the CTL clone derived from peripheral blood against SP cells.  To detect the peripheral specific CTL response against SP cells of the MFH2003 cell line, we attempted to induce an autologous CTL clone that recognized the SP cells using SP cells as the stimulatory antigen. Cells were stimulated four times by mixed lymphocyte–tumor cell culture using purified SP cells of the MFH2003 cell line and autologous PBMC. Subsequently, conventional limiting dilution was performed. As a result, we obtained one CTL clone, namely Tc4C-6, which showed specific cytotoxicity against SP cells of the MFH2003 line. The Tc4C-6 clone expressed a single Vβ-chain (Vb5.2–3) mRNA, which was also expressed by TIL2003 cells (data not shown). The phenotype of the Tc4C-6 clone was CD3+CD4CD8+CCR7CD45RA+, a typical effector phenotype (Fig. 4a). Moreover, the Tc4C-6 clone exhibited higher cytotoxicity against SP cells than MP cells of the MFH2003 cell line, as purified by cell sorting (Fig. 4b,c). In addition, the anti-HLA Class I W6/32 antibody apparently blocked the cytotoxicity of the Tc4C-6 clone against MFH2003SP cells (Fig. 4d). These results suggest that SP cells can be killed by autologous CTL in an HLA Class I-restriction manner.

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Figure 4.  The autologous CTL clone Tc4C-6 recognized side population (SP) cells. (a) Cell surface expression of CD4, CD8, CCR7, and CD45RA on Tc4C-6 and allogeneic peripheral blood mononuclear cells (PBMC) from a healthy donor. (b) Reanalysis of sorted SP and main population (MP) cells. (c) Cytotoxicity of the CTL clone Tc4C-6. Freshly isolated SP and MP cells, autologous EB-B cells (B2003-EBV), and negative control K562 cells were used as target cells and cocultured with Tc4C-6 at the specified effector:target (E/T) ratios. After 12 h, CTL-mediated cytotoxicity was measured using the aCella-TOX assay (Cell Technology), as described in the Materials and Methods. (d) Blocking assay of Tc4C6-mediated recognition of SP cells from the MFH2003 cell line using anti-human leukocyte antigen (HLA) Class I (W6/32 and anti-HLA Class II (L243) mAbs. Cytotoxicity was also measured with the aCella-TOX assay.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

In the present study, we showed that: (i) SP cells, as CSC of the MFH200 cell line, expressed more HLA Class I on their cell surface than did MP cells (non-CSC); (ii) SP cells could be recognized by autologous tumor-infiltrating lymphocytes; and (iii) an autologous CTL clone could be induced by mixed lymphocyte–tumor cell culture using SP cells as antigens and that this induced clone killed SP cells rather than MP cells. These results indicate that CTL-recognizing CSC certainly exist in the tumor microenvironment and circulating peripheral blood and that SP cells can be killed by CTL. Thus, CTL-based immunotherapy against the CSC of bone sarcoma is a promising option.

Previous reports have suggested that CSC may be a candidate target for immunotherapy. For example, Pellegatta et al.(12) reported that dendritic cell based vaccine therapy resulted in an efficient anti-tumor immune response against glioma stem cells; Todaro et al. showed that γδ T cells killed human colon CSC; Pietra et al. demonstrated that natural killer (NK) cells killed human melanoma CSC;(13,14) and Weng et al.(15) induced CTL against ovarian CSC from HLA-A2+ healthy donors using CSC-DC fusion cells and demonstrated that the CTL killed ovarian CSC. However, until now, the autologous CTL response against CSC had not been investigated.

It is well documented that tumors can escape T cell-mediated elimination by downregulating molecules essential for immune recognition.(16) The downregulation of HLA Class I molecules in tumor tissues is a major prognostic factor and has an important role in tumor immune escape.(17) We have reported previously on the relationship between downregulation of HLA Class I and the poor prognoses of patients with osteosarcoma and Ewing’s sarcoma.(18,19) However, as shown in the present study, the expression profile of immune molecules, including HLA Class I molecules on CSC, is preserved. Therefore, CSC may not escape from cellular immune surveillance activated by CTL-based immunotherapy.

The identification of CSC-associated antigens recognized by autologous CTL is very important, especially for the establishment of CTL-based immunotherapy in the adjuvant setting for the prevention of recurrence and metastasis. To this end, establishment of anti-CSC-specific CTL lines is a prerequisite. Although Weng et al.(15) assessed the CTL response against allogeneic ovarian CSC, there are no reports regarding CTL lines induced by autologous CSC. Therefore, the CTL clone Tc4C-6 is the first CTL clone against CSC induced by autologous CSC and could serve as a good probe against autologous CTL clone-defined CSC-associated antigen. We are currently trying to isolate the cDNA of the T-cell receptor (TCR) α- and β-chains to develop a permanent probe for cDNA library expression cloning.

In the present study, we evaluated SP cells of the MFH2003 cell line in 72 independent experiments. The proportion of SP cells in the MFH2003 cell line varied among experiments and this was often the main obstacle to completing the experiments using SP cells; thus, we needed to enrich the SP cells. The isolation of SP cells requires high-level technical skills and intensive, hard laboratory work. Although we do not know why the proportion of SP cells in the MFH2003 cell line is so variable, differentiation of SP cells into MP cells in cell culture in vitro may contribute to the variance in the proportion of SP cells. Recently, the dynamic regulation theory of cancer stem cells was proposed.(20) A subpopulation of SP cells that were jumonji AT-rich interactive domain 1B (JARID1B) positive was shown to have high proliferative ability. However, not only could individual JARID1B-positive cells become JARID1B negative, but individual JARID1B-negative cells could become JARID1B positive and acquire tumorigenicity. Such a dynamic change in the characteristics of SP and MP cells is another possible reason for the variability of the proportion of SP cells among independent experiments.

In conclusion, we have demonstrated the immunogenicity of CSC of bone MFH using autologous tumor-infiltrating lymphocytes and a peripheral CTL clone. On the basis of our results, we propose that CTL-based immunotherapy could target the CSC of bone sarcoma to prevent tumor recurrence.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

The authors thank Dr Hisami Ikeda (Hokkaido Red Cross Blood Center, Sapporo, Japan) for the kind donation of human sera. This work was supported by Grants-in-Aid for Scientific Research (A) (grant no. 21249025 to N. Sato), for Scientific Research (B) (grant no. 20390403 to T. Wada), and for Young Scientists (A) (grant no. 22689041 to T. Tsukahara), as well as a grant from the Ministry of Health, Labour and Welfare (grant no. 200910002A to N. Sato; grant no. 200925049A to T. Wada) and Management Expenses Grants from the Government to the National Cancer Center (grant no. 14 to T. Wada), Takeda Science Foundation (grant no. 2010-Igakukei-Kenkyu-Syorei to T. Tsukahara), and Akiyama Memorial Foundation (grant no. H22-Shorei-Josei-7 to T. Tsukahara).

Disclosure Statement

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

All authors declare that they have no conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References