Ann Dermatol.  2014 Feb;26(1):53-60. 10.5021/ad.2014.26.1.53.

Combined Treatment of Murine Fibrosarcoma with Chemotherapy (Paclitaxel), Radiotherapy, and Intratumoral Injection of Dendritic Cells

• Byun JW
• Lee HS
• Song SU
• Lee SW
• Kim SK
• Kim WC
• Lee MH
• Choi GS

• KMID: 2265697
• DOI: http://doi.org/10.5021/ad.2014.26.1.53

Abstract

BACKGROUND
New antitumor therapeutic strategies aim to combine different approaches that are able to induce tumor-specific effector and memory T cell responses that might control tumor growth. Dendritic cells (DCs) have the capacity to induce antigen-specific cytotoxic T lymphocytes. We have previously shown that the combined treatment of paclitaxel chemotherapy (Chemo) and injection of DCs led to complete tumor regression.
OBJECTIVE
The goal of this study was to evaluate synergistic antitumor effect of a triple combination treatment comprising radiotherapy, paclitaxel Chemo and intratumoral injection of syngeneic bone marrow-derived DCs on murine fibrosarcoma, compared to other single or double combination treatments.
METHODS
For the murine fibrosarcoma model, naive C57BL/6 mice were inoculated intradermally with 2x10(3) MCA102 cells in the right upper flank. Mice were assigned to five groups (untreatedcontrol, RT alone, RT+Chemo, RT+DC, and RT+Chemo+DC), with eight mice in each group. In vitro cytotoxicity assays were performed to assess the immune activity. The persistence of tumor-specific immunity was determined by second tumor challenge in mice with complete tumor regression.
RESULTS
The triple combination treatment showed a significantly enhanced therapeutic efficacy by decreasing tumor size and inducing complete tumor regression, resulting in a cure of 50% of mice. The results of in vitro cytotoxicity assays and the second tumor challenge experiment strongly indicated the induction of a tumor-specific cytotoxic T lymphocyte response and acquisition of prolonged tumor immunity.
CONCLUSION
These findings suggest that the triple combination treatment can be a promising strategy for the treatment of murine fibrosarcoma.

Keyword

Combined modality therapy; Dendritic cells; Fibrosarcoma; Paclitaxel; Radiotherapy

MeSH Terms

Animals
Combined Modality Therapy
Dendritic Cells*
Drug Therapy*
Fibrosarcoma*
Lymphocytes
Memory
Mice
Paclitaxel
Radiotherapy*
T-Lymphocytes, Cytotoxic
Paclitaxel

Figure

• 

  Fig. 1 Study design. Mice were assigned to five groups (control, RT alone, RT+CT, RT+DC and RT+CT+DC) in the study. MCA102 cells were inoculated subcutaneously in the upper right flank of a C57BL/6 mouse. On day 10, when the average tumor size reached about 4~5 mm in diameter, paclitaxel was intraperitoneally administered and RT was performed 6 hours later. The same treatment was administered on two more days; day 12 and day 14. DCs were injected intratumorally four times on days 11, 13, 15 and 18. RT: radiotherapy, CT: chemotherapy, DC: dendritic cell.

• 

  Fig. 2 Phenotyping of cultured dendritic cells (DCs). The phenotypes of the primary DCs were subsequently verified by flow cytometry. (A) The circle indicates the DCs gated in the flow cytometric analysis. The results showed that the isolated DCs were positive for CD11c (D), CD44 (E), and I-Ad (F). The low expression of the maturation surface markers CD40 (B) and CD80 (C) indicates that the DCs were in an immature state. The purity of DCs was >65%.

• 

  Fig. 3 Photographs of animals at the end of treatment (day 52). In the control group, tumor bulk in most of the mice grew at a rapid rate with severe central necrosis (white arrows). Significant suppression of tumor growth was observed in the treated mice compared to the control group. Some mice showed complete tumor regression (yellow circles). Among the treated groups, the triple combination treatment group showed the greatest tumor suppressive effect. DEATH means tumor directly causes immediate death and LOSS means tumor does not cause death. RT: radiotherapy, CT: chemotherapy, DC: dendritic cell.

• 

  Fig. 4 Significant suppression of tumor growth in the treated groups. Tumor growth was suppressed significantly in the treated groups in contrast to the control group. However, there was no statistically significant difference in the degree of tumor growth suppression between the treated groups. The data are expressed as the mean±standerd error. RT: radiotherapy, CT: chemotherapy, DC: dendritic cell.

• 

  Fig. 5 Persistent antitumor memory after combined treatment of dendritic cell (DC) and radiotherapy (RT) with/without chemotherapy (CT). A second challenge with the same MCA 102 tumor cells was performed in the mice that showed complete tumor regression in the previous experiment. A very small sized tumor appeared around day ten after the second injection, but it disappeared quickly thereafter. Age-matched normal mice, as the control group, were also injected with the same type of tumor cells and these mice showed rapid tumor growth. The data are expressed as the mean±standerd error.

• 

  Fig. 6 In vitro cytotoxicity assay with splenocytes from mice with complete tumor regression. The graph and table show the percentage of specific cell death at different E to T ratios in each experimental group. The PKH-26 flow cytometry evaluates MCA102 cells (T) after incubation with splenocytes after combination treatment (E). The PKH-26 assay results showed that the level of cytotoxicity was highest in the triple combination treatment group. T: target cells, E: effector cells, RT: radiotherapy, CT: chemotherapy, DC: dendritic cell.

Reference

1. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998; 392:245–252.

2. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol. 1991; 9:271–296.

3. Celluzzi CM, Mayordomo JI, Storkus WJ, Lotze MT, Falo LD Jr. Peptide-pulsed dendritic cells induce antigen-specific CTL-mediated protective tumor immunity. J Exp Med. 1996; 183:283–287.

4. Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD. Cancer immunoediting: from immunosurveillance to tumor escape. Nat Immunol. 2002; 3:991–998.

5. Restifo NP, Antony PA, Finkelstein SE, Leitner WW, Surman DP, Theoret MR, et al. Assumptions of the tumor 'escape' hypothesis. Semin Cancer Biol. 2002; 12:81–86.

6. Choi GS, Lee MH, Kim SK, Kim CS, Lee HS, Im MW, et al. Combined treatment of an intratumoral injection of dendritic cells and systemic chemotherapy (Paclitaxel) for murine fibrosarcoma. Yonsei Med J. 2005; 46:835–842.

7. Nowak AK, Robinson BW, Lake RA. Gemcitabine exerts a selective effect on the humoral immune response: implications for combination chemo-immunotherapy. Cancer Res. 2002; 62:2353–2358.

8. Correale P, Cusi MG, Tsang KY, Del Vecchio MT, Marsili S, Placa ML, et al. Chemo-immunotherapy of metastatic colorectal carcinoma with gemcitabine plus FOLFOX 4 followed by subcutaneous granulocyte macrophage colony-stimulating factor and interleukin-2 induces strong immunologic and antitumor activity in metastatic colon cancer patients. J Clin Oncol. 2005; 23:8950–8958.

9. Ghiringhelli F, Menard C, Puig PE, Ladoire S, Roux S, Martin F, et al. Metronomic cyclophosphamide regimen selectively depletes CD4+CD25+ regulatory T cells and restores T and NK effector functions in end stage cancer patients. Cancer Immunol Immunother. 2007; 56:641–648.

10. Obeid M, Tesniere A, Ghiringhelli F, Fimia GM, Apetoh L, Perfettini JL, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med. 2007; 13:54–61.

11. Friedman EJ. Immune modulation by ionizing radiation and its implications for cancer immunotherapy. Curr Pharm Des. 2002; 8:1765–1780.

12. Overwijk WW, Theoret MR, Finkelstein SE, Surman DR, de Jong LA, Vyth-Dreese FA, et al. Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells. J Exp Med. 2003; 198:569–580.

13. Gattinoni L, Finkelstein SE, Klebanoff CA, Antony PA, Palmer DC, Spiess PJ, et al. Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells. J Exp Med. 2005; 202:907–912.

14. Quarmby S, Kumar P, Kumar S. Radiation-induced normal tissue injury: role of adhesion molecules in leukocyteendothelial cell interactions. Int J Cancer. 1999; 82:385–395.

15. Ganss R, Ryschich E, Klar E, Arnold B, Hämmerling GJ. Combination of T-cell therapy and trigger of inflammation induces remodeling of the vasculature and tumor eradication. Cancer Res. 2002; 62:1462–1470.

16. Nikitina EY, Gabrilovich DI. Combination of gammairradiation and dendritic cell administration induces a potent antitumor response in tumor-bearing mice: approach to treatment of advanced stage cancer. Int J Cancer. 2001; 94:825–833.

17. Spira AI, Ettinger DS. The use of chemotherapy in soft-tissue sarcomas. Oncologist. 2002; 7:348–359.

18. Berezhnaya NM, Vinnichuk UD, Belova OB, Baranovich VV. Antitumor action of lymphokin-activated cells of patients with soft tissue sarcomas and melanomas in dependence on expression of MHC classes I and II antigenes. Exp Oncol. 2006; 28:231–234.

19. Kucera A, Pýcha K, Pajer P, Spísek R, Skába R. Dendritic cell-based immunotherapy induces transient clinical response in advanced rat fibrosarcoma - comparison with preventive anti-tumour vaccination. Folia Biol (Praha). 2009; 55:119–125.

20. Shin JY, Lee SK, Kang CD, Chung JS, Lee EY, Seo SY, et al. Antitumor effect of intratumoral administration of dendritic cell combination with vincristine chemotherapy in a murine fibrosarcoma model. Histol Histopathol. 2003; 18:435–447.

21. Finkelstein SE, Iclozan C, Bui MM, Cotter MJ, Ramakrishnan R, Ahmed J, et al. Combination of external beam radiotherapy (EBRT) with intratumoral injection of dendritic cells as neo-adjuvant treatment of high-risk soft tissue sarcoma patients. Int J Radiat Oncol Biol Phys. 2012; 82:924–932.

22. Derby E, Reddy V, Kopp W, Nelson E, Baseler M, Sayers T, et al. Three-color flow cytometric assay for the study of the mechanisms of cell-mediated cytotoxicity. Immunol Lett. 2001; 78:35–39.

23. Shah DA, Madden LV. Nonparametric analysis of ordinal data in designed factorial experiments. Phytopathology. 2004; 94:33–43.

24. Paulos CM, Kaiser A, Wrzesinski C, Hinrichs CS, Cassard L, Boni A, et al. Toll-like receptors in tumor immunotherapy. Clin Cancer Res. 2007; 13:5280–5289.

25. Fulda S, Los M, Friesen C, Debatin KM. Chemosensitivity of solid tumor cells in vitro is related to activation of the CD95 system. Int J Cancer. 1998; 76:105–114.

26. Apetoh L, Ghiringhelli F, Tesniere A, Obeid M, Ortiz C, Criollo A, et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med. 2007; 13:1050–1059.

27. Sauter B, Albert ML, Francisco L, Larsson M, Somersan S, Bhardwaj N. Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. J Exp Med. 2000; 191:423–434.

28. Albert ML, Sauter B, Bhardwaj N. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature. 1998; 392:86–89.

29. Tsujitani S, Kakeji Y, Watanabe A, Kohnoe S, Maehara Y, Sugimaghi K. Infiltration of S-100 protein positive dendritic cells and peritoneal recurrence in advanced gastric cancer. Int Surg. 1992; 77:238–241.

30. Machlenkin A, Goldberger O, Tirosh B, Paz A, Volovitz I, Bar-Haim E, et al. Combined dendritic cell cryotherapy of tumor induces systemic antimetastatic immunity. Clin Cancer Res. 2005; 11:4955–4961.

31. Candido KA, Shimizu K, McLaughlin JC, Kunkel R, Fuller JA, Redman BG, et al. Local administration of dendritic cells inhibits established breast tumor growth: implications for apoptosis-inducing agents. Cancer Res. 2001; 61:228–236.