Cryoimmunotherapy with local co-administration of ex vivo generated dendritic cells and CpG-ODN immune adjuvant, elicits a specific antitumor immunity

Abstract

Cryoablation is a low-invasive surgical procedure for management of malignant tumors. Tissue destruction is obtained by repeated deep freezing and thawing and results in coagulative necrosis and in apoptosis. This procedure induces the release of tumor-associated antigens and proinflammatory factors into the microenvironment. Local administration of immature dendritic cells (DCs) potentiates the immune response induced by cryoablation. To further augment the induction of long-lasting antitumor immunity, we investigated the clinical value of combining cryoimmunotherapy consisting of cryoablation and inoculation of immature DCs with administration of the immune adjuvant, CpG oligodeoxynucleotides. Injection of the murine Lewis lung carcinoma, D122-luc-5.5 that expresses the luciferase gene, results in spontaneous metastases, which can be easily monitored in vivo. The local tumor was treated by the combined treatment. The clinical outcome was assessed by monitoring tumor growth, metastasis in distant organs, overall survival, and protection from tumor recurrence. The nature of the induced T cell responses was analyzed. Combined cryoimmunotherapy results in reduced tumor growth, low metastasis and significantly prolonged survival. Moreover, this treatment induces antitumor memory that protected mice from rechallenge. The underlying suggested mechanisms are the generation of tumor-specific type 1 T cell responses, subsequent induction of cytotoxic T lymphocytes, and generation of systemic memory. Our data highlight the combined cryoimmunotherapy as a novel antitumor vaccine with promising preclinical results. Adjustment of this technique into practice will provide the therapeutic benefits of both, ablation of the primary tumor and induction of robust antitumor and antimetastatic immunity.

Fig. 1

Cryoimmunotherapy models. a Characterization of bone marrow-derived DCs generated ex vivo: expression of cell surface markers on naïve and LPS-treated cells. b Illustration of treatments and vaccination protocols

Fig. 2

Increased survival of mice treated with combined cryoimmunotherapy. Mice were inoculated i.f.p. with D122-luc-5.5 and then treated as described in materials and methods. a Tumor growth kinetics measured with calipers (tumor volume, mm³). b Post-surgical survival of mice treated with different protocols. Combined results of five independent experiments (n = 5–8 mice per group per experiment). *P < 0.05; **P < 0.001; ***P < 0.0001

Fig. 3

Combined cryoimmunotherapy induces anti-metastatic immunity. a Representative images of in vivo D122-luc-5.5 tumor metastasis monitored 57 and 72 days following tumor inoculation (captured with an IVIS 200 imaging system). Luciferase activity 57 (b) and 72 (c) days following tumor inoculation. d Lungs weight, as an indication for metastatic load, was detected 100–120 days post-amputation or when mice were moribund. The data are presented as mean lungs weight (mg). Lungs weight of naïve 25-week-old mouse is 200 mg. Combined results of five independent experiments (n = 5–8 mice per group per experiment). *P < 0.05; **P < 0.001; ***P < 0.0001

Fig. 4

Immunocyte populations in the dLNs of tumor-bearing mice following treatments. Mice were inoculated i.f.p. with D122-luc-5.5 and further treated as described in materials and methods. Eight days post-second treatment, the dLNs were excised and analyzed by flow cytometry. a Percentages of CD4⁺ effector T cells (CD62L^low CD44^high). b–e Percentages of CD4⁺ and CD8⁺ T cells secreting IFNγ (b), TNFα (c), IFNγ and TNFα (d), IL4 (e). f Percentages of Treg (CD4⁺CD25⁺Foxp3⁺) cells. Data presented as mean ± SEM, combined results of three independent experiments (n = 5–8 mice per group per experiment). *P < 0.05; **P < 0.001

Fig. 5

Protection against parental tumor rechallenge in mice that rejected the primary tumor. Mice were inoculated with D122-luc-5.5 cells and treated as previously described. Surviving mice (80–95 days following amputation of the primary tumor-bearing foot) and a control group of six age-matched naïve mice were rechallenged with 2 × 10⁵ D122-luc-5.5 cells in the other foot. a Tumor growth kinetics measured with calipers (tumor volume, mm³). Forty-five days after tumor rechallenge, cells were isolated from spleens and analyzed for: b percentages of CD4⁺ and CD8⁺ effector T cells (CD62L^low CD44^high), c percentages of IFNγ and TNFα secreting CD4⁺ and CD8⁺ T cells. Combined data of two independent experiments (n = 5–8 mice per group per experiment). ***P < 0.0001

Fig. 6

Induction of systemic antitumor immunity by combined cryoimmunotherapy. Mice were inoculated with D122-luc-5.5 cells and treated as previously described. Surviving mice (80–95 days following amputation of the primary tumor-bearing foot) and a control group of six age-matched naïve mice were rechallenged with 2 × 10⁵ D122-luc-5.5 cells in the other foot. Spleens were removed 45 days later, and cells were stimulated with irradiated D122-luc-5.5 cells for 5 days. Further, they were incubated with the following ³⁵S-methionine-labeled target cells: D122-luc-5.5 (a), K^b39.5 (b), and B16-F10.9 (c). CTL assays were carried out at four effector:target ratios ranging from 100:1 to 12.5:1, in triplicates. d Specific killing of D122-luc-5.5 at effector:target ratio of 100:1 is shown for the different CpG-ODN concentrations. n = 5–8 mice per group; *P < 0.05; **P < 0.001; ***P < 0.0001