Korean J Urol.  2008 Nov;49(11):965-973. 10.4111/kju.2008.49.11.965.

Antitumor Effect of in Situ Cryoablation with Systemic Immunotherapy on Murine Renal Cell Tumor

• Shin DS
• Ko YH
• Choi H
• Kang SH
• Bae JH
• Park HS
• Moon DG
• Cheon J
• Yoon DK

• KMID: 1328166
• DOI: http://doi.org/10.4111/kju.2008.49.11.965

Abstract

PURPOSE: To investigate synergistic effect of local cryoablation with systemic immunotherapy, the tumor control ability and immunologic responses of combining these two modalities was compared with that of cryoablation, surgical excision, and immunotherapy only group in a tumor re-challenge model.
MATERIALS AND METHODS
Preliminary experiments were performed in two stages. The first stage consisted of 36 Balb/c mice with Renca bearing tumors imbedded in the right thigh, and was treated with interleukin-2 (IL-2) and interferon-alpha(IFN-alpha) to evaluate the efficacy of immunotherapy and to determine the adequate dosage. The second stage was performed on 10 mice, to evaluate histological changes and efficacy after cryoablation. The main experiment was performed on 48 mice, divided into 6 groups of control with tumor implantation, excision of tumor, excision combined with immunotherapy, cryoablation of tumor, cryoablation with immunotherapy and control without tumor. After treatment, tumor re-challenge was performed with Renca cell, then the growth pattern was evaluated with physical measurements, and immune response was investigated with fluorescent activated cell sorter and cytotoxicity assay.
RESULTS
Preliminary studies on immunologic efficacy revealed that IL-2 and IFN-alpha have a dose dependent inhibition of tumor growth. The main experiment evaluating the efficacy of combination treatment revealed that cryoablation with immunotherapy proved to be most effective in terms of tumor recurrence and tumor growth inhibition, yet the difference was not statistically significant from monotherapy with cryoablation. However, cytotoxicity was significantly increased cryoablation with immunotherapy compared with other groups.
CONCLUSIONS
Cryoablation on tumor re-challenge mice model showed advantages with immunotherapy most prominently in cytotoxicity.

Keyword

Cryoablation; Immunotherapy; Renal cell carcinoma

MeSH Terms

Mice
Animals

Figure

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  Fig. 1. Changes of the tumor volume in the preliminary studies to evaluate an adequate dose of the immunologic agents. (A) Changes of tumor volume in the IL-2 treated groups. (B) Changes of tumor volume in the IFN-α treated groups. ∗: significant difference of the tumor volume in the high dose administration group for each agent, as compared to the low dose administered group (p＜0.05 by the Mann-Whitney U test). †: significant difference of the tumor volume of the intermediate dose administered group for each agent, as compared to the low dose administered group (p＜0.05 by the Mann-Whitney U test). IL-2: interleukin-2, IFN-α: interferon-alpha.

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  Fig. 2. Results of the histologic examination, and the samples were taken at 2 days and 7 days after cryosurgery. Histologic change at (A-C) 2 days after cryoablation and (D-F) 7 days after cryoablation. (A) Central coagulative necrosis caused by cryoablation (H&E, x100). (B) At high magnification, shrunken and condensed cells with compact nuclei and pyknotic chromatin was found in the periphery of this zone, which are typical features of apoptosis (H&E, x200). (C) The areas of necrosis and apoptosis were surrounded by intact tumor cells, which were larger and they had slack chromatin (H&E, x200). (D) 7 days after the cryoablation was conducted, there was a wide area of necrosis without viable tumor cells (H&E, x40). (E) At high magnification, the central necrotic area was characterized by ghost cells and no apparent nuclei (H&E, x200). (F) This infiltration of neutrophils was a prominent finding at the peripheral muscular tissue, which was located below the previous injection site (H&E, x200).

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  Fig. 3. The tumor growth pattern for each treatment after tumor re-challenge. At day 15, the tumor volume was significantly lower in the cryoablated group (G4, G5) than that in the surgical excised group of G2 and G3, respectively. ∗: significant decrease of the tumor volume in the cryoablation only group (G4), as compared to the excision only group (G2) (p=0.02, by the Mann-Whitney U test), †: significant decrease of tumor volume in the cryoablation with immunotherapy group (G5), as compared to the excision group plus immunotherapy group (G3) (p=0.04 by the Mann-Whitney U test), ‡: the difference of tumor volume in the excision with immunotherapy group (G3), as compared to the excision only group (G2), did not reach statistic significance (p=0.62 by the Mann-Whitney U test). §: the difference of tumor volume in the cryoablation with immunotherapy group (G5), as compared to the cryoablation group (G4) did not reach statistic significance (p=0.9 by the Mann-Whitney U test).

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  Fig. 4. Fluorescent activated cell sorter (FACS) results after tumor re-challenge. On comparison with the excision group, the percentages of gated CD4 and CD8 T cells of group 3, which was treated with surgical excision with immunotherapy, were significantly increased (∗: p=0.021 and 0.047 by the Mann-Whitney U test for the CD4 and CD8 cells, respectively). However, the difference between the cryoablation group and the combination group of cryoablation with immunotherapy did not reach a significant value (†: p=0.19 for CD4, below 0.05 for the other cytokines).

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  Fig. 5. Result of the cytotoxicity assay, after tumor re-challenge. (A) The cryoablation group showed increased cytotoxicity at effector: the target cell ratios were 33:1, 11:1, 3.7:1 and 1.2:1, respectively. ∗: significantly increased compared to the excision group (p＜0.05, by the Mann-Whitney U test). (B) The excision with immunotherapy group showed increased cytotoxicity at effector: the target cell ratios were 33:1, 11:1 and 3.7:1, respectively ∗: significant increased compared to the excision only group (p＜0.05, by the Mann-Whitney U test). (C) The cryoablation group with immunotherapy group showed increased cytotoxicity at effector: the target cell ratios were 11:1, 3.7:1 and 1.2:1, respectively. ∗: significant increased compared with the cryoablation only group (p＜0.05, by the Mann-Whitney U test).

Reference

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