J Korean Med Sci.  2007 Sep;22(Suppl):S38-S46. 10.3346/jkms.2007.22.S.S38.

Inhibition of Lewis Lung Carcinoma Growth by Toxoplasma gondii through Induction of Th1 Immune Responses and Inhibition of Angiogenesis

• Kim JO
• Jung SS
• Kim SY
• Kim TY
• Shin DW
• Lee JH
• Lee YH

• KMID: 1785787
• DOI: http://doi.org/10.3346/jkms.2007.22.S.S38

Abstract

Toxoplasma gondii is an obligate intracellular protozoan parasite that induces antitumor activity against certain types of cancers. However, little information is available regarding the immunologic mechanisms that regulate these effects. For this purpose, C57BL/6 mice were administered either the T. gondii Me49 strain orally or Lewis lung carcinoma (LLC) cells intramuscularly. Survival rates, tumor size, histopathology, and immune responses were determined for each group, and angiogenesis was evaluated by in vivo Matrigel plug assay. Toxoplasma-infected (TG-injected) mice survived the entire experimental period, whereas cancer cell-bearing (LLC-injected) mice died within six weeks. Mice injected with both T. gondii and cancer cells (TG/LLC-injected group) showed significantly increased survival rates, CD8+ T-cell percentages, IFN-gamma mRNA expression levels, serum IgG2a titers, and CTL responses as compared to the LLC-injected mice. In addition, angiogenesis in the TG/LLC-injected mice was notably inhibited. These effects in TG/LCC-injected mice were similar or were increased by the addition of an adjuvant, Quil-A. However, TG/LLC-injected mice showed decreased percentages of CD4+ and CD8+ T cells, IFN-gamma mRNA expression levels, and serum IgG1 and IgG2a titers as compared to TG-injected mice. Taken together, our results demonstrate that T. gondii infection inhibits tumor growth in the Lewis lung carcinoma mouse model through the induction of Th1 immune responses and antiangiogenic activity.

Keyword

Toxoplasma gondii, Immunotherapy; Lewis Lung Carcinoma; Antitumor; Antiangiogenesis

MeSH Terms

Animals
Base Sequence
CD4-Positive T-Lymphocytes/immunology
CD8-Positive T-Lymphocytes/immunology
Carcinoma, Lewis Lung/blood supply/genetics/immunology/*therapy
Cell Line, Tumor
Cytotoxicity, Immunologic
DNA Primers/genetics
Female
Immunoglobulin G/blood
Immunotherapy/*methods
Interferon-gamma/genetics
Mice
Mice, Inbred C57BL
Neovascularization, Pathologic
RNA, Messenger/genetics/metabolism
Th1 Cells/*immunology
Toxoplasma/*immunology

Figure

• 

  Fig. 1 Survival rates of C57BL/6 mice injected with Toxoplasma gondii, Lewis lung carcinoma (LLC) cells or Quil-A. Mice were checked daily for eight weeks following cancer cell injection, to measure survival rates. The groups (n=10 per group) were as follows: LLC, injected in the femoral muscle with 1×105 viable LLC cells; TG, infected with five cysts of the Me49 strain of T. gondii; TG/LLC, received both Toxoplasma parasites and LLC cells; QA, received 20 µg/mouse of Quil-A twice weekly for 3 weeks; LLC/QA, received both LLC cells and Quil-A; TG/QA, received both T. gondii and Quil-A; TG/LLC/QA, received T. gondii, LLC cells, and Quil-A.

• 

  Fig. 2 Histopathologic findings for the muscles and lungs of C57-BL/6 mice injected with T. gondii, LLC cells or Quil-A. Paraffin-embedded tissues were stained with hematoxylin and eosin, and visualized under a microscope at ×400 magnification. (A) Normal muscle; (B) cancerous changes in the muscle at cancer cell-injected sites; (C) normal lung; (D) cancerous changes in the lung (cancer metastasis to the lungs); (E) tumor masses of mice injected with LLC cells (left and middle) and injected with both T. gondii and LLC cells (right). Ulceration was observed in cancer-bearing mice (arrow).

• 

  Fig. 3 Sizes of tumors in C57BL/6 mice injected with T. gondii, LLC cells or Quil-A. Tumor growth was measured weekly using sterile metric calipers. Tumor volume (µL)=tumor width (mm)2×tumor length (mm)×0.5. Data are presented as the means±SE of five mice (*, p<0.05 compared with LLC- or LLC/QA-injected group).

• 

  Fig. 4 Serum IgG1 (A) and IgG2a (B) titers of C57BL/6 mice injected with T. gondii, LLC cells or Quil-A, as measured by ELISA. Serum samples were obtained from mice at the indicated time-points, and the IgG subclasses against Toxoplasma lysate antigen were quantified. Data are presented as the mean±SE of five mice (*, p<0.05 compared with LLC- or LLC/QA-injected group; †, p<0.05 compared with TG- or TG/QA-injected group).

• 

  Fig. 5 Relative IFN-γ (A) and IL-10 (B) mRNA expression in splenocytes from C57BL/6 mice injected with T. gondii, LLC cells or Quil-A. The transcript levels are relative to those in splenocytes obtained from control mice (designated value of 1). Splenocytes were harvested at the time-points indicated and the expression of IFN-γ and IL-10 mRNAs was assayed by RT-PCR. Data are presented as mean±SE of five mice.

• 

  Fig. 6 Cytotoxic T-cell responses of C57BL/6 mice injected with T. gondii, LLC cells or Quil-A. One week after T. gondii (or cancer cell) injection, splenic lymphocytes were isolated from each mouse, labeled with 51Cr, and cultured with irradiated LLC cells at different cell ratios. Data are presented as mean±SE of five mice.

• 

  Fig. 7 Angiogenesis in C57BL/6 mice injected with T. gondii, LLC cells or Quil-A. One week after T. gondii (or cancer cell) injection, a Matrigel that was supplemented with heparin and basic fibroblast growth factor was injected into the mice. The gels were recovered on day 6 postinjection for hemoglobin measurement using the Drabkin reagent kit. Data are presented as the mean±SE of five mice. (A) Hemoglobin content of each group; (B) Matrigel plugs were removed 6 days after injection and photographed (arrows): LLC-bearing mice (left), Toxoplasma-infected mice (middle), and Matrigel alone (right).

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