Antitumor activity of mixed heat shock protein/peptide vaccine and cyclophosphamide plus interleukin-12 in mice sarcoma

Abstract

Background: The immune factors heat shock protein (HSP)/peptides (HSP/Ps) can induce both adaptive and innate immune responses. Treatment with HSP/Ps in cancer cell-bearing mice and cancer patients revealed antitumor immune activity. We aimed to develop immunotherapy strategies by vaccination with a mixture of HSP/Ps (mHSP/Ps, HSP60, HSP70, Gp96 and HSP110) enhanced with cyclophosphamide (CY) and interleukin-12 (IL-12).

Methods: We extracted mHSP/Ps from the mouse sarcoma cell line S180 using chromatography. The identity of proteins in this mHSP/Ps was assayed using SDS-PAGE and Western blot analysis with antibodies specific to various HSPs. BALB/C mice bearing S180 cells were vaccinated with mHSP/Ps ×3, then were injected intraperitoneally with low-dose CY and subcutaneously with IL-12, 100 μg/day, ×5. After vaccination, T lymphocytes in the peripheral blood were analyzed using FACScan and Cytotoxicity (CTL) was analyzed using lactate dehydrogenase assay. ELISPOT assay was used to evaluate interferon γ (IFN-γ), and immune cell infiltration in tumors was examined in the sections of tumor specimen.

Results: In mice vaccinated with enhanced vaccine (mHSP/Ps and CY plus IL-12), 80% showed tumor regression and long-term survival, and tumor growth inhibition rate was 82.3% (30 days), all controls died within 40 days. After vaccination, lymphocytes and polymorphonuclear leukocytes infiltrated into the tumors of treated animals, but no leukocytes infiltrated into the tumors of control mice. The proportions of natural killer cells, CD8+, and interferon-γ-secreting cells were all increased in the immune group, and tumor-specific cytotoxic T lymphocyte activity was increased.

Conclusions: In this mice tumor model, vaccination with mHSP/Ps combined with low-dose CY plus IL-12 induced an immunologic response and a marked antitumor response to autologous tumors. The regimen may be a promising therapeutic agent against tumors.

Figure 1

SDS-PAGE and western blot analysis of mixed HSP/Ps from S180 sarcoma. A. SDS-PAGE of mHSP/P from S180; Lane1, molecular standard, Line2,3 collection of F3-F6 from Sephacryl S-200HR. There were many protein bands other than MW60, 70, 96 and110. B. Western blot: Lane 1, SDS-PAGE, molecular standard. 2, SDS-PAGE, collection of F3-F6, Line3 analysis with antibodies against HSP60, Line4 analysis with antibodies against HSP70, Line5 analysis with antibodies against Gp96, and Line6 analysis with antibodies against HSP110. Identified The mixture included HSP60, HSP70, Gp96 and HSP110.

Figure 2

Effect of various mHSP/P vaccinations on the survival of S180 tumor-bearing mice. * The number of mouse in each group is 10.

Figure 3

Tumor growth curve of S180 tumor in BALB/C mice after various treatments.

Figure 4

mHSP/P+Cy+IL12 vaccination elicits IFN-γ by ELISPOT assay ConA: stimulate lymphocyte proliferation in vitro with ConA. ConA+mHSP/P: stimulate lymphocyte proliferation in vitro with ConA and mHSP/P. IFN-γ elicited by mHSP/P+Cy+IL12 vaccination is significantly higher than tumor bearing mice and naïve mice, *P < 0.05.

Figure 5

mHSP/P+Cy+IL12 vaccination elicits a tumor-specific CTL response. The cytolysis percent of mHSP/P+Cy+IL12 vaccine is significantly higher than mHSP/P vaccine and naïve mice *P < 0.05, and tumor bearing mice, #P < 0.01.

Figure 6

Lymphocytes infiltration in tumor of mHSP/P immunized mice. A leukocytes infiltration into tumor lesion after mHSP/P immunization, X40. B lymphocytes in blood vessels after mHSP/P immunization, X40. C No lymphocytes infiltration in tumor lesion after NS treatment, X40. Which revolved preimmunization after mHSP/P immunization.