Combination therapy of irreversible electroporation and cytokine-induced killer cells for treating mice bearing panc02 pancreatic-cancer xenografts

Abstract

The current study aimed to investigate the antitumor effects and potent mechanism of cytokine-induced killer (CIK) cells combined with irreversible electroporation (IRE) via Panc02 cell-bearing mouse model in vivo. CIK cells were isolated from the spleens of Panc02 pancreatic-cancer (PC) subcutaneous-xenograft model and the proportion of different lymphocytes was also determined. The antitumor effect of the combination of IRE and CIK cells in a PC subcutaneous-xenograft model was also investigated. The proportion of cells that were positive for CD3⁺CD8⁺ and the proportion of CD3⁺CD56⁺ cells were both significantly increased after 21 days of in vitro culture. Combined treatment of IRE and CIK cell significantly inhibited tumor growth and increased the survival rate of Panc02 cell-bearing mice. Furthermore, infiltration of lymphocytes into tumor tissue was significantly increased by this combination therapy compared with the untreated group or monotherapy group. In addition, IRE significantly enhanced the expression of chemokine receptors elicited by CIK cells. In conclusion, IRE combined with CIK cells showed superior antitumor efficacy in a PC xenograft model, which we attributed to the promotion of lymphocytic infiltration, as well as to upregulation of chemokine receptor expression and the regulators of CIK cell proliferation.

Keywords: Chemokine receptors; Combination therapy; Cytokine-induced killer cells; Irreversible electroporation; Pancreatic cancer.

Fig. 1

In vitro expansion of cytokine-induced killer cells, and treatment schedule. (A) In vitro growth curves of CIK cells for 24 days. (B) Percentages of CD3, CD4, CD8, and CD56 expressed on the surfaces of CIK cells on different days of culture. *P < 0.05; **P < 0.01; ***P < 0.001 versus 0 days (mean ± SD, n = 6). (C) Mice in IRE-alone group just received IRE on 0 day. And one CIK cells treatment was designed on 3 day in CIK-alone group. Mice in IRE + CIK group were received IRE plus one course of CIK cells.

Fig. 2

Antitumor effects of irreversible electroporation combined with cytokine-induced killer cells on pancreatic cancer in tumor-bearing mice. (A) Tumor growth, (B) mouse survival, and (C) tumor image per treatment group were monitored. ^###P < 0.001 versus IRE + CIK group (mean ± SD, n = 10); *P < 0.05, **P < 0.01, ***P < 0.001 versus untreated group (mean ± SD, n = 10).

Fig. 3

Combination therapy with irreversible electroporation and cytokine-induced killer cells increased the infiltration of lymphocytes into tumor tissue. Numbers of (A) CD3⁻ cells, (B) CD3⁺ cells, (C) CD8⁺ cells, and (D) CD4⁺ cells with tumor infiltration. (E–F) FCM analysis. Numbers of intratumoral CD3⁻ cells and CD3⁺ cells were higher in the IRE-alone and CIK cells-alone treatment groups than in the untreated group. Numbers of CD8⁺ and CD4⁺ cells were also increased to varying degrees. What is more, numbers of CD3⁻, CD3⁺, CD4⁺, and CD8⁺ cells in the combined IRE/CIK cell treatment group were even higher than those in the CIK cells-alone group, and the differences were statistically significant. ^###P < 0.001 versus IRE + CIK group (mean ± SD, n = 5); *P < 0.05, **P < 0.01, ***P < 0.001 versus untreated group (mean ± SD, n = 5).

Fig. 4

Percentages of chemokine receptor positivity in CD3⁺ cells. (A) CXCR3, (B) CCR5, and (C) CXCR4. CD3⁺ cells in the CIK cell treatment group highly expressed all three of these receptors, with the positive rate of CXCR4 exceeding 80%. All three chemokine receptors were significantly elevated in the combined IRE/CIK cell treatment group compared with the CIK cells-alone group. *P < 0.05, **P < 0.01, ***P < 0.001 versus untreated group (mean ± SD, n = 5); ^###P < 0.001 versus IRE + CIK group (mean ± SD, n = 5).

Fig. 5

Effect of irreversible electroporation on in vivo proliferation of cytokine-induced killer cells in tumor tissues. Proportions of CIK cells expressing (A) Ki-67, (B) ICOS, (C) GrB, (D) perforin, (E) IFN-γ, and (F) TNF-α in tumors were detected via intracellular staining with the corresponding aBs. *P < 0.05, **P < 0.01, ***P < 0.001 versus untreated group (mean ± SD, n = 5); ^###P < 0.001 versus IRE + CIK group (mean ± SD, n = 5).