Cancer Immunology, Immunotherapy

, Volume 59, Issue 5, pp 769–777 | Cite as

Immunogenic chemotherapy with cyclophosphamide and doxorubicin against established murine carcinoma

  • Miki Tongu
  • Nanae Harashima
  • Takaya Yamada
  • Takayuki Harada
  • Mamoru Harada
Original Article


Mitigation of regulatory T cell-mediated immunosuppression and elicitation of immunogenic tumor cell death are crucial events for optimal anti-tumor immune activity in vivo. This study was designed to investigate the potential synergistic activity of the combined use of cyclophosphamide (CP) and doxorubicin (DR), both of which are known to resolve these two issues. BALB/c mice were inoculated subcutaneously with CT-26 carcinoma cells in the bilateral flank and treated with an intraperitoneal injection of a low dose of CP followed by an intratumoral injection of DR into one side of the tumor. We found that, in addition to a significant suppression of growth on the DR-treated side of the tumor, combination therapy suppressed the growth of DR-untreated remote tumors in both tumor-specific and T cell-dependent manners. Mitomycin C showed no such synergistic anti-tumor activity with CP treatment. Combination therapy increased the frequency of interferon (IFN)-γ-producing T lymphocytes specific to a CT-26-associated class I-binding tumor peptide in the tumor-draining lymph nodes. Real-time PCR analysis revealed that combination therapy led to an increase in IFN-γ and tumor necrosis factor-α mRNA expression; however, levels of Foxp3 and transforming growth factor-β within the remote tumor tissues were decreased. In addition, knock down of calreticulin expression in CT-26 cells using small interfering RNA attenuated anti-tumor vaccine effects induced by DR-treated CT-26 cells. These results provide an immunological rationale for the combined use of chemotherapeutic drugs, i.e., CP and DR, and further recommend their use with current cancer vaccines.


Cyclophosphamide Doxorubicin Regulatory T cells Calreticulin 



We thank Ms. Yoko Ono and Ms. Yoshimi Fujii for their technical assistance. The English in this document has been checked by at least two professional editors, both native speakers of English. This study was supported in part by grants from the Ministry of Education, Science, Sport, Culture, and Technology of Japan (no. 18591449 to M. H. and no. 20790930 to N. H) and from the Shimane University Medical Education and Research Foundation.


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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Miki Tongu
    • 1
    • 2
  • Nanae Harashima
    • 1
  • Takaya Yamada
    • 2
  • Takayuki Harada
    • 3
  • Mamoru Harada
    • 1
  1. 1.Department of ImmunologyShimane University Faculty of MedicineIzumoJapan
  2. 2.Experimental Animal CenterShimane UniversityIzumoJapan
  3. 3.Department of PathologyShimane University Faculty of MedicineIzumoJapan

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