Cryoimmunologic antitumor effects enhanced by dendritic cells in osteosarcoma

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 gamma, 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.

Fig. 1

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.

Fig. 2A–C

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

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

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.

Fig. 5

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

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

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

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.