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. 2010 Mar 16;468(5):1373–1383. doi: 10.1007/s11999-010-1302-z

Cryoimmunologic Antitumor Effects Enhanced by Dendritic Cells in Osteosarcoma

Masanori Kawano 1,2, Hideji Nishida 1, Yasunari Nakamoto 3, Hiroshi Tsumura 2, Hiroyuki Tsuchiya 1,✉
PMCID: PMC2853649  PMID: 20232181

Abstract

Background

We previously reported a limb-salvage technique by treating tumor-bearing bone with liquid nitrogen. We also reported systemic antitumor immunity was enhanced by cryotreatment in a murine osteosarcoma (LM8) model. We therefore combined the cryotreatment of tumor with dendritic cells to promote tumor-specific immune responses.

Questions/purposes

We determined whether our technique could enhance systemic immune response and inhibit metastatic tumor growth in a murine osteosarcoma model.

Materials and Methods

To evaluate activation of the immune response, we prepared six groups of C3H mice (80 mice total): (1) excision only, (2) dendritic cells without reimplantation of the cryotreated primary tumor, (3) reimplantation of the cryotreated primary tumor alone, (4) dendritic cells combined with reimplantation of the cryotreated primary tumor, (5) dendritic cells exposed to cryotreated tumor lysates without reimplantation of the cryotreated primary tumor, and (6) dendritic cells exposed to cryotreated tumor lysates with reimplantation of the cryotreated primary tumor. We then compared and verified the activation state of each group’s antitumor immunity.

Results

Mice that received dendritic cells exposed to cryotreated tumor lysates with reimplantation of the cryotreated primary tumor group had high serum interferon γ, reduced pulmonary metastases, and increased numbers of CD8(+) T lymphocytes in the metastatic areas.

Conclusions

Combining tumor cryotreatment with dendritic cells enhanced systemic immune responses and inhibited metastatic tumor growth.

Clinical Relevance

We suggest immunotherapy could be developed further to improve the treatment of osteosarcoma.

Introduction

The standard treatment of osteosarcoma consists of preoperative chemotherapy, surgical tumor excision, and postoperative chemotherapy. Limb-saving surgery is feasible in most cases. Advances in osteosarcoma treatment have now achieved a 5-year survival rate of 60% to 90% for patients, and limb function after reconstruction continues to improve with time [3, 16, 30, 46, 47, 49].

Tsuchiya et al. developed a new approach using frozen autografts [48] to improve reconstruction after osteosarcoma resection. The tumor is resected with an adequate margin, and the resected specimen is immersed in liquid nitrogen for 20 minutes to kill all tumor cells. After thawing, the specimen is returned to the original place with appropriate internal fixation to reconstruct the defect. Compared with heat-treated bones [8, 14], bone genetic proteins and native biomechanical structures are preserved after cryotreatment [53]. In one report limb function using the technique of Tsuchiya et al. was rated as excellent in 71.4% of patients, and good in 10.7%, as assessed by the functional evaluation system of Enneking [11]. Two studies suggest the approach enhanced bone formation when compared histologically with pasteurized bone and irradiated bone [43, 48]. Another advantage in reimplanting cryotreated tumor tissue is its effect on the immune system [50]: tumor tissue after cryoablation in situ provokes an immune reaction in patients with breast and prostate cancer [6, 8, 39]. Brewer et al. reported metastatic tumors sometimes disappear or shrink after in situ cryoablation of the primary tumor with liquid nitrogen [4]. The structure of tumor antigens is retained in frozen tumor, and leukocytes probably can recognize these antigens. Similar antitumor effects can be expected from our reconstructive procedure of reimplanting tumor-bearing bone after cryotreatment with liquid nitrogen.

Nishida et al. observed an inadequate antitumor effect after reimplantation of frozen tumor tissue alone [35]. However, the antitumor effect was enhanced by promoting nonspecific immune activation by intraperitoneal injection of OK-432, a substance extracted from alpha-Streptococcus pyogenes. This approach promotes inflammation and activation of dendritic cells (DCs) that initiate the specific antitumor effect [19]. This type of immunotherapy reportedly is effective for breast and prostate cancers [6, 8, 39]. Many groups have reported successful immunotherapy for osteosarcoma [5, 15, 18, 20, 22, 24, 25, 33, 34, 36, 42, 51, 52]. However, the ability to control metastatic lesions and local recurrence does not appear to be superior to other adjuvant treatments [2, 7, 13, 23, 29].

We therefore wondered whether combining cryotreatment and immunotherapy might enhance tumor response. We specifically determined whether: (1) antitumor immunity could be enhanced through activation and transfer of DCs combined with reimplantation of the cryotreated primary tumor, and (2) metastatic lesions could be prevented owing to the involvement of T lymphocytes in a murine osteosarcoma model (LM8).

Materials and Methods

Using a reported method to induce osteosarcoma [1, 35], we hypodermically implanted 1 × 106 LM8 cells (a murine osteosarcoma cell line) into the subcutaneous gluteal region of 80 female C3H mice, 6 to 8 weeks old. Tumors developed in all animals. Two weeks after inoculation, we surgically excised the tumors and cryotreated them with liquid nitrogen. We established the following six groups (Fig. 1): (1) the tumor was excised with wide margins 14 days after inoculation (n = 15); (2) the tumor was excised with wide margins 14 days after inoculation and bone marrow-derived DCs then were injected into the contralateral subcutaneous gluteal region without reimplantation of the cryotreated primary tumor twice a week (n = 15); (3) the tumor was excised with wide margins 14 days after inoculation and reimplanted after cryotreatment with liquid nitrogen into the contralateral gluteal region to evaluate for local recurrence from frozen tumor tissue (n = 15); (4) the tumor was excised 14 days after inoculation and reimplanted after cryotreatment into the contralateral gluteal region to evaluate for local recurrence, and DCs then were injected twice a week into this secondary site (n = 15); (5) the tumor was excised with wide margins 14 days after inoculation and DCs exposed to cryotreated tumor lysates were injected twice a week into the contralateral gluteal region without reimplantation of the cryotreated primary tumor (n = 15); and (6) the tumor was excised with wide margins 14 days after inoculation and reimplanted after the treatment with liquid nitrogen into the contralateral gluteal region to evaluate for local recurrence (same as Group 3) with the addition of DCs exposed to cryotreated tumor lysates injected twice a week (n = 15). We harvested tumor from 30 mice, and then the tumor was treated with liquid nitrogen to create the lysates. We presumed a systemic immune response would be induced by injecting DCs around the frozen tumor tissue. We microscopically determined the presence of metastases in the lungs 2 weeks after the tumor inoculation. We had previously confirmed the presence of pulmonary metastases in an additional 20 mice in a preliminary experiment in advance. We also confirmed that there were no viable cells after cryotreatment using liquid nitrogen, in agreement with a previous study [35]. We observed no recurrence of the tumor at the primary site of inoculation after excision. All experiments were performed under the guidelines for animal experiments as stipulated by the Kanazawa University Graduate School of Medical Science [37].

Fig. 1.

Fig. 1

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A diagram of the experimental protocol and treatment schedule is shown. Two weeks after tumor inoculation, tumors were treated by one of the following methods: (1) excision only (n = 15); (2) DCs without reimplantation of the cryotreated primary tumor (n = 15); (3) reimplantation of the cryotreated primary tumor (n = 15); (4) DCs pulsed with cryotreated tumor lysates and reimplantation of the cryotreated primary tumor (n = 15); (5) DCs pulsed with cryotreated tumor lysates without reimplantation of the cryotreated primary tumor (n = 15); or (6) DCs pulsed with cryotreated tumor and reimplantation of the cryotreated primary tumor (LN) (n = 15). The mice were euthanized and evaluated 6 weeks after tumor inoculation. sc = subcutaneous.

LM8 cells, derived from Dunn osteosarcoma, were provided by the Riken BioResource Center (Saitama, Japan). The cells were maintained in complete medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 μg streptomycin per mL, and 100 units penicillin per mL and were cultured at 37°C in 5% CO2. To establish local implantation of the tumor and subsequent lung metastasis, the LM8 cells (1 × 106) were suspended in 0.2 mL phosphate-buffered saline (PBS) and subcutaneously inoculated into the right gluteal region of the mice. All animals had macroscopically and microscopically confirmed lung metastases within 4 weeks [1].

C3H mice were purchased from Sankyo Labo Inc (Toyama, Japan) and housed in a specific pathogen-free animal facility in our laboratory. We were not able to accurately determine the survival time of each group because the guidelines for animal experiments concerning pain required euthanasia in distressed animals.

Liquid nitrogen (−196°C) was used for cryotreatment. Tumor tissue was collected on gauze and soaked in liquid nitrogen for 20 minutes for en bloc tumor tissue freezing. The tumor was prethawed at room temperature (20°C) for 15 minutes and then thawed in distilled water (20°C) for 15 minutes. The liquid nitrogen-treated tumor tissue was transplanted subcutaneously in the left gluteal region of the same mouse.

Because the mice were genetically identical, the structure of the major histocompatibility complex (MHC) Class I molecules was such that the T cells would be able to recognize the MHC Class I with antigens on the antigen-presenting cells (APCs) [17, 27]. Bone marrow-derived DCs were generated as described by Lutz and Rössner [28] with minor modifications. Briefly, erythrocyte-depleted mouse bone marrow cells obtained from flushed marrow cavities (1 × 106 cells/mL) were cultured in complete medium with 20 ng/mL recombinant mouse GMCSF (PeproTech EC Ltd, London, UK) in 10-cm tissue culture dishes at 37°C in an atmosphere containing 50 mL CO2 per L. On Days 3 and 6, half of the medium was added to the same volume of fresh complete medium and used to replenish the original plates. The freeze-thawed tumor lysate was added to the DC cultures on Day 6 at a ratio of five DC equivalents to one tumor cell (ie, 5:1) and incubated at 37°C in an atmosphere containing 50 mL CO2 per L. After 24 hours of incubation, nonadherent cells including DCs were harvested by gentle pipetting.

For fluorescence activated cell sorting (FACS) analysis, DCs were counted with a FACSCalibur™ Flow Cytometer (Becton-Dickinson, San Jose, CA) and stained with fluorochrome-conjugated antibodies (BD Pharmingen, Tokyo, Japan) for the following markers: cluster of differentiation (CD)11c, CD80, CD86, I-Ad, and CD40. CD11c was used as a marker for all DCs regardless of the degree of maturation, whereas CD80, CD86, I-Ad, and CD40 are markers for DCs. Data analysis was performed with CELLQuest™ software (Becton-Dickinson). The corresponding labeled isotype antibodies served as controls. DCs used for vaccination were washed twice, enumerated, and resuspended in PBS at 1 × 106/mL.

We inoculated LM8 cells (5 × 106) in a mouse to make the tumor lysate. After 4 weeks, we resected the tumor mass and soaked the entire tumor in liquid nitrogen to kill the tumor cells. We mixed cryonecrotic tissue with DCs at Culture Day 6, after the tumor was defrosted, and the homogenate was prepared using PBS. The homogenate was passed through a 0.2-μm filter to remove bacteria and tissues and mixed with the DCs for 24 hours.

After intraperitoneal injection of 5 mL sodium pentobarbital (Somnopentyl®; Kyontsu Seiyaku, Tokyo, Japan), mice were euthanized by cervical dislocation and their blood was collected. Murine interferon (IFN)-γ and interleukin (IL)-4 release were measured by ELISA using Quantikine® (R & D Systems, Minneapolis, MN) according to the manufacturer’s protocol using an Easy Reader EAR340 microtest plate reader (SLT-Labinstruments, Salzburg, Austria).

We estimated the area of the pulmonary metastatic lesion on 50 serial histologic sections of each lung by manually drawing orthogonal lines delimiting the edges of the pulmonary metastatic lesion and selected the widest part of the specimen. The area was determined by multiplying the maximum orthogonal dimensions using ImageJ software (NIH, Bethesda, MD; http://rsb.info.nih.gov/ij/). We compared the mean areas between the six groups.

For immunohistochemistry, lung specimens were fixed in 20% formalin and embedded in paraffin. For each case, we examined all the blocks of lung tissues of formalin-fixed, paraffin-embedded tumor tissue. All specimens were decalcified, although we found the decalcification step did not influence the immunohistochemistry for any of the stains. Five sections for each mouse were cut 4-μm thick. Each section was cut at the maximum diameter. CD8(+) T lymphocytes and natural killer (NK) cells in the pulmonary metastatic lesion were quantified by measuring the immunohistochemistry-positive cells per unit area in each group. Rehydrated tissue sections were incubated with rat monoclonal antibody raised against CD8(+) T lymphocytes of mouse origin (Santa Cruz Biotechnology, Santa Cruz, CA) and rat monoclonal antibody raised against NK cells of mouse origin (Abcam Plc, Cambridge, UK). The two antibodies were diluted 1:50 with PBS. Color reactions were performed at room temperature for 15 minutes and cover slips were mounted with glycerol and gelatin.

We determined differences in serum IFN-γ, serum IL-4, pulmonary metastatic area, and number of CD8(+) lymphocytes and NK cells in the metastatic area among the six groups using a nonrepeated-measures ANOVA and the Scheffe test. All analyses were conducted with SPSS® 11.0 software (SPSS Japan Inc, Tokyo, Japan).

Results

We activated antitumor immunity by combining DCs exposed to lysates of cryotreated tumor and reimplantation of the cryotreated primary tumor. On Culture Day 7, the ratio of mature DCs to immature DCs was increased compared with the ratio at Culture Day 6 (Fig. 2; immature DCs, upper left; mature DCs, upper right). Moreover, this increase was more apparent in groups incubated with tumor lysate. Serum IFN-γ levels were greater (p < 0.0001) in the mice that received DCs combined with reimplantation of the cryotreated primary tumor (119.0 ± 7.61 pg/mL) than in the cryotreated primary tumor alone group (37.33 ± 2.58 pg/mL). Moreover, the group that received tumor lysate-exposed DCs combined with reimplantation of the cryotreated primary tumor (157.33 ± 14 pg/mL) had a greater (p < 0.0001) IFN-γ level than the group that received only tumor lysate-exposed DCs without reimplantation of the cryotreated primary tumor (120.27 ± 11.29 pg/mL) (Fig. 3). Serum IL-4 was lower (p < 0.0001) in the mice that received DCs exposed to the lysates of cryotreated tumor and reimplantation of the cryotreated primary tumor group (13.33 ± 9.75 pg/mL) than in the excision-only group (45.06 ± 5.71 pg/mL) (Fig. 4).

Fig. 2A–C.

Fig. 2A–C

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DC activation status was examined using flow cytometry. DCs at Culture Day 7 (Group B) were more mature than DCs at Culture Day 6 (Group A). On Culture Day 7, DC maturity was greatest in the groups receiving lysate-primed DCs (Group C) than in those not receiving lysate-primed DCs (Group B).

Fig. 3.

Fig. 3

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A graph of the serum IFN-γ levels in the six treatment groups is shown. The samples were collected 28 days after the reimplantation surgery and/or DC adoptive transfer. Mice that received DCs exposed to the lysates of cryotreated tumor and reimplantation of the cryotreated primary tumor group showed a highest IFN-γ level. Error bars represent SD.

Fig. 4.

Fig. 4

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A graph of the serum IL-4 in the six treatment groups is shown. Sera were collected 28 days after the reimplantation surgery and/or DC adoptive transfer. DCs exposed to the lysates of cryotreated tumor and reimplantation of the cryotreated primary tumor group showed a lower level than any of the other groups. Error bars represent SD.

The enhanced immune response by T lymphocytes reduced metastatic lesions. Reduction of the metastatic area was greater (p < 0.0001) in the group that received DCs without reimplantation of the cryotreated primary tumor (15.99 ± 3.93 mm2) than in the excision-only group (24.12 ± 3.60 mm2). The reduction of the metastatic area was greater (p < 0.0001) in the DCs combined with reimplantation of the cryotreated primary tumor group (5.39 ± 1.49 mm2) than in the reimplantation of the cryotreated primary tumor alone group (13.22 ± 2.59 mm2) (Fig. 5). CD8(+) T lymphocytes gathered in the pulmonary metastatic area in DC-treated groups, however, NK cells were not recruited to the metastatic area in the DC-treated groups compared with the nonDC-treated groups (Fig. 6). The number of CD8(+) T lymphocytes per unit area was greater (p < 0.0001) in the DCs combined with reimplantation of the cryotreated primary tumor group (8.33 ± 2.57 cells/mm2) than in the reimplantation of the cryotreated primary tumor alone group (2.44 ± 0.53 cells/mm2). Mice that received DCs exposed to the lysates of cryotreated tumor and reimplantation of the cryotreated primary tumor (12.79 ± 2.14 cells/mm2) showed higher (p < 0.0001) levels than the group that received DCs exposed to the lysates of cryotreated tumor without reimplantation of the cryotreated primary tumor (8.71 ± 2.39 cells/mm2) (Fig. 7). The number of NK cells per unit area was greater (p < 0.0001) in the group that received DCs exposed to the lysates of cryotreated tumor without reimplantation of the cryotreated primary tumor (3.90 ± 2.17 cells/mm2) than in the excision-only group (1.20 ± 0.30 cells/mm2) (Fig. 8). The CD8(+)T lymphocyte, CD4(+) T lymphocyte, and DC infiltrations in reimplanted tumors were similar to those seen with pulmonary metastases (data not shown).

Fig. 5.

Fig. 5

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Reduction of the metastatic area in the six treatment groups is shown. The samples were gathered 28 days after the reimplantation surgery and/or DC adoptive transfer. Error bars represent SD.

Fig. 6A–L.

Fig. 6A–L

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To evaluate CD8(+) T lymphocytes and NK cells in pulmonary metastasis, immunostaining was performed: (A) CD8(+) T lymphocytes in Group 1, (B) CD8(+) T lymphocytes in Group 2, (C) CD8(+) T lymphocytes in Group 3, (D) CD8(+) T lymphocytes in Group 4, (E) CD8(+) T lymphocytes in Group 5, (F) CD8(+) T lymphocytes in Group 6, (G) NK cells in Group 1, (H) NK cells in Group 2, (I) NK cells in Group 3, (J) NK cells in Group 4, (K) NK cells in Group 5, and (L) NK cells in Group 6. CD8(+) T lymphocytes gathered in Groups D,E, and F. However, they did not gather in Groups A, B, and C. However, NK cells were recruited only in Groups A, B, and C. (Original magnification, ×200).

Fig. 7.

Fig. 7

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The numbers of CD8(+) T lymphocytes per unit area in the six treatment groups are shown. The samples were gathered 28 days after the reimplantation surgery and/or DC adoptive transfer. DCs exposed to the lysates of cryotreated tumor and reimplantation of the cryotreated primary tumor group showed a higher level than any other groups. Error bars represent SD.

Fig. 8.

Fig. 8

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The numbers of NK cells per unit area in the six treatment groups are shown. The samples were gathered 28 days after the reimplantation surgery and/or DC adoptive transfer. Error bars represent SD.

Discussion

Various immunotherapies for osteosarcoma have been tried. As standard treatments for osteosarcoma are ineffectual for many patients, new treatments need to be developed. In the 1970s, immunotherapy for osteosarcoma was reported by Southam et al. [42], Neff and Enneking [34], and Campbell et al. [5]. In the 1980s, new methods such as the use of interferons and Bacille de Calmette et Guérin were reported [22, 24, 36]. Another approach used antiidiotypic antibodies using T cells and liposome encapsulation [18, 51, 52]. Current methods of immunotherapy for osteosarcoma include peptide therapy or gene transfer therapy combined with hyperthermia therapy [10, 15, 21, 25, 33]. We asked whether (1) antitumor immunity could be achieved through activation of DCs combined with reimplantation of the cryotreated primary tumor and (2) if metastatic lesions would be prevented owing to enhanced T lymphocyte involvement.

We acknowledge limitations in this study. First, we used mice with an identical genetic makeup. The structure of the MHC Class I molecules was similar and the T cells could recognize the MHC Class I. However, we needed to use DCs from a different (albeit genetically identical) mouse to accomplish our adoptive transfer experiments. We minimized the potential for an immune response to nonself antigens by using genetically identical tumor tissue and mice. It would be necessary to use DCs derived from the same individual in clinical application, but this could not be achieved in our mouse model. In humans, however, monocytes are separated from the patient’s own peripheral blood and DCs can be induced from these monocytes. Second, we could not completely replicate the clinical approach used in humans in our mouse model. In clinical cases frozen bone always is returned to the same site. However, it was impossible to replicate this in our experimental mouse model in which transplanted tumor cells were removed from the tibia and then returned to the same place after cryotreatment. In a preliminary experiment we attempted to do just that and these 20 mice could not move and died of starvation. We therefore used the contralateral gluteal region to check for local recurrence after tumor excision or recurrence from frozen tissue.

Antitumor immunity appeared to be activated through DCs combined with reimplantation of the cryotreated primary tumor or by exposing the transferred DC to lysates of cryotreated tumor. The use of lymphokine-activated killer (LAK) therapy has been used with other types of tumors [26]. However, T lymphocytes, which are the effectors, do not accumulate inside osteosarcoma tumors as expected. Autoclaving supplemented by DCs is thought to enhance the antitumor effect, but hyperthermia causes proteins to denature, and activation of the antitumor effect is often insufficient [37]. Several studies [12, 31, 41] report peptide vaccine therapy, but many patients apparently develop immunotolerance [45]. Thus, immunotherapy for malignant tumor achieved by these various methods has not been established definitively although investigations continue to try to overcome the major hurdles associated with immunotherapy (Table 1). We emphasize the immune response is activated by cryotreatment but not by heat-treated tissue. Our method differs from those described by others [7, 9, 10, 14]. In some regards DCs are believed to be the principal APCs for initiating immune responses in vivo [32]. In comparison with other traditional adjunct therapeutic options for cancer, such as radiation therapy and chemotherapy, immunotherapy provides a more targeted treatment to the cancer, with potentially fewer detrimental effects on noncancerous cells [30, 40]. DCs without sufficient cancer antigens may not have the ability to kill tumor cells and present the antigen to T lymphocytes by themselves. Our data suggest the antitumor effect in the group that received DCs without reimplantation of cryotreated primary tumor was almost the same as that in the reimplantation of cryotreated primary tumor alone group. The data further suggest the effects increased only when exposing the DCs to tumor lysates in the absence of cryonecrotic primary tumors. However, combining reimplantation of cryotreated primary tumor and DCs exposed to cryotreated tumor lysates produced synergistic effects. Using reimplantation of cryotreated primary tumor is more appropriate for clinical applications. We therefore believe an efficient immune response will be activated when DCs recognize tumor antigens appropriately. CD8(+) T cells act as an effector by the Th1 route, and this is promoted mainly by IFN-γ and IL-12 [38]. However, IL-4 [21], IL-6, and IL-10 strengthen humoral immunity. Levels of IFN-γ, IL-2, and IL-12 generally increase when cell-mediated immunity is activated, and IL-4, IL-6, and IL-10 increase when humoral immunity is activated. These cytokines act in opposition to maintain an immune balance.

Table 1.

Immunotherapeutic trials of malignant tumors

Tumor Immune intervention Route Immunologic response Comments References
Osteosarcoma BCG SC NC No consistent clinical effect [22, 24]
Osteosarcoma Interferon α SC, IV PR–NC Osteosarcoma-associated antigens have potential for targeted immunotherapy [36]
Unknown LAK IV NC T lymphocytes were unable to penetrate the tumor [26]
Osteosarcoma Antiidiotypic antibodies IV NC It may be possible to circumvent this heterogeneity by activation of tissue macrophages to the tumoricidal state [18, 51, 52]
Breast cancer, osteosarcoma Peptide therapy combined with hyperthermia therapy SC, IV NC It may be a potential agent for use in immunotherapy [15, 20]
Osteosarcoma Gene transfer therapy combined with hyperthermia therapy IV NC IL-23 seems to be a less effective immunotherapeutic for adjuvant treatment of osteosarcomas [25, 33]
Unknown Peptide vaccine therapy SC NC–PD Many patients have peptide-induced tolerance develop [45]
Osteosarcoma Cryoimmunology and DCs SC PR Combining cryotreatment with DCs resulted in enhanced antitumor effects Our data
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BCG = Bacille de Calmette et Guérin; SC = subcutaneous; NC = no change; IV = intravenous; PR = partial response; LAK = lymphokine-activated killer; IL = interleukin; PD = progressive disease; DCs = dendritic cells.

Our data suggest enhanced T lymphocyte recruitment and function reduce metastatic lesions in a murine osteosarcoma model. Immunoreactivity increased slightly in mice that received DCs exposed to lysates of cryotreated tumor combined with reimplantation of the cryotreated primary tumor. NK cells attack the tumor independently of APCs. NK cells attack cells that downregulate MHC Class I expression or have a stressed appearance [44]. We observed a reduced tumor burden in the groups that received transplanted DCs, which correlated with recruitment of CD8 lymphocytes to the tumor site as observed with immunohistochemistry.

Returning the frozen bone after liquid nitrogen treatment to its original place can be readily used in the clinic. After the first cryotreatment, it is possible to perform the treatment again using cultured DCs if a patient’s tumor cells have been preserved. This approach therefore still can be used even after other methods (such as chemotherapy, radiation therapy, or surgery) no longer are reasonable. Combining DCs pulsed with lysates of cryotreated tumor and reimplantation of the cryotreated primary tumor enhanced antitumor effects. We believe the approach may be a useful alternative for patients with osteosarcoma when other treatment options including chemotherapy, radiotherapy, and surgical treatment have been ineffective.

Acknowledgments

We thank Katsuro Tomita, Akihiko Takeuchi, Shuichi Kaneko, and Yohei Marukawa for supervision in this study.

Footnotes

Each author certifies that he or she has no commercial associations that might pose a conflict of interest in connection with the submitted article.

Each author certifies that his or her institution has approved the animal protocol for this investigation, and that all investigations were conducted in conformity with ethical principles of research.

This work was performed at the Department of Orthopaedic Surgery, Graduate School of Medical Science, Kanazawa University, and the Department of Orthopaedic Surgery, Faculty of Medicine, Oita University.

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