On the other hand, granuloma formation and ulceration have been considered as side effects in systemic anti-cancer vaccine trials. These observations suggest thatP. acnesvaccination induces both systemic and local antitumor responses. In conclusion, this study shows thatP. acnesvaccination may be a potent therapeutic alternative in MM. == Introduction == Malignant melanoma (MM) is a life-threatening disease that is commonly resistant to treatment. Early diagnosis followed by surgical resection improves the prognosis of patients with MM. However, despite careful follow-up and treatment with combination chemotherapy or adjuvant therapy, patients with MM frequently develop both local and distant metastases. Patients with distant metastases almost always have a poor clinical outcome. Due to the high frequency of spontaneous recurrence of MM lesions, adjuvant therapy is generally recommended. Starting with Coley toxins more than one hundred ago, adjuvant therapy or immunotherapy has come to be regarded as one of the effective methods for boosting anti-tumor immunity[1], and various compounds and therapeutic modalities have been tested against melanoma using experimental TW-37 mouse models[2],[3],[4]. However, the precise mechanisms driving the response have not been elucidated. Propionibacterium acnes (P. acnes)is a component of the normal bacterial flora of the skin; it is aerotolerant, anaerobic gram-positive bacteria that plays an important role in the pathogenesis of acne[5]. When dendritic cells (DC) phagocyte and processP. acnes, a powerful Th1-type cytokine immune response is elicited, leading to an increased production of IL-12, IFN- and TNF-. Injection ofP. acneshas been reported to shift a dominant Th2 response to a Th1 type response, with an associated improvement in skin symptoms[6]. Clinically, acne vulgaris, aP. acnes-associated disease, often occurs in patients after a recovery from severe atopic dermatitis[7]. In the present study, we developed a mouse model of MM and investigated the clinical and immunological effects ofP. acnesvaccination. == Materials and Methods == == Ethics Statement == Animal care was performed according to standard ethical guidelines, and all of the experimental protocols were approved by the Institutional Board Committee for Animal Care and Use of Mie University (Permit Number 21-27-1). == Mice == Female C57BL/6J(B6) mice were purchased from Japan SLC Co. (Shizuoka, Japan) and were bred under specific-pathogen-free conditions. == P. acnes == P. acneswas purified from normal healthy volunteers and cultured in brain-heart infusion medium supplemented with L-cysteine and Tween-80, as reported previously[8]. Cultured bacteria were washed with sterile distilled water, killed by heating at 60C for 60 min, and then lyophilized. A strain ofP. acnesthat effectively promotes inflammatory cytokine production was selected (P. acnes-Mie1) and used in the subsequent experiments[9]. == Melanoma cell and injection == The B16 melanoma cell line was purchased from ATCC (Manassas, VA), and cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine serum (HyClone Laboratories, INC., South Logan, UT) at 37C in a humidified incubator containing 5% CO2 and 95% air. 1106melanoma cells were injected in the dorsal skin of 8 week-old mice. == Vaccination schedule == 3.3 mg of heat-killedP. acneswere injected into the dorsal skin at the site where MM cells had been injected in 810 week old mice. Mice TW-37 were divided into five groups: 1) MM-bearing mice treated with PBS (20 l (MM, n = 14), 2) a group of MM-bearing mice treated withP. acnesat 10 weeks old (PMM1, n = 15), 3) another group treated withP. acnes-treated at 8 and 10 weeks old (PMM2, n = 15), 4) a control group of mice treated TW-37 withP. acnes(P1, n = 11) at 10 weeks old, and 5) another control group treated withP. acnesat 8 and 10 weeks old (P2, n = 13). == Clinical manifestations and histopathological study == Tumor growth was measured by the longest (L) point and the perpendicular diameter (W) of the dorsal skin tumor mass at 12 weeks of age, and the tumor volume was calculated Lysipressin Acetate according to the following formula: 4W2L/3[10]. The tumor tissue, lung, liver, and spleen were excised from the animals of each group, and samples were embedded in.