Regulatory T lymphocytes are involved in the immunosupression generated by a malignant myoepithelial cell-containing breast tumor.

KRASNAPOLSKI MARTÍN; MAGLIOCO ANDREA F; PELUFFO G; DRAN, GRACIELA; BAL DE KIER JOFFE E; EIJAN A M

Miami, FL, USA

Congreso; Tumor immunology: New perspectives; 2008

American Association for Cancer Research (AACR)

Regulatory T lymphocytes are involved in the immunosuppression generated by a malignant myoepithelial cell-containing breast tumor KRASNAPOLSKI M A1, MAGLIOCO A F2, PELUFFO G1, DRAN G2, BAL DE KIER JOFFE E1 and EIJAN A M1 1 Área Investigación. Instituto de Oncología "Ángel H. Roffo”. Buenos Aires, Argentina. 2 ILEX-CONICET. División Medicina Experimental, IIHEMA, Academia Nacional de Medicina. Buenos Aires, Argentina. We obtained the LM38-LP (bicellular), LM38-HP (epithelioid) and LM38-D2 (myoepithelial clone) cell lines from the spontaneous murine mammary adenocarcinoma M38 comprising both, malignant luminal and malignant myoepithelial cells. Subcutaneous (s.c.) tumor take of LM38-LP and LM38-D2 cells is 100%, while LM38-HP cells are less tumorigenic (30%) and the few tumors that arise have a low growing rate and metastatic capacity. The aim of this work was to assess whether the difference in the in vivo behavior or the M38-derived cell lines can be explained by specific interactions with the host’s immune system. First, in vitro specific cytotoxicity assays were performed. Both, splenocytes from LM38-LP or LM38-HP tumor-bearing BALB/c mice (termed SLP and SHP) were challenged with the tumor cell lines (50:1). After 24h tumor cell viability was estimated with MTS. SLP were less cytotoxic than SHP (LM38-LP: 52.8% vs 63.6%, p=0.033; LM38-HP: 60.5% vs 77.5%, p=0.014). Being a well-known immunosuppressive factor, TGFβ activity was estimated using an in vitro bioassay in conditioned media from LM38-LP, LM38-HP and LM38-D2. Myoepithelial cell-containing cultures secreted more total and active TGFβ than LM38-HP cells. Since TGFβ can induce regulatory T lymphocytes differentiation, the number of Tregs in the draining lymph node was evaluated by flow cytometry. LM38-LP and LM38-D2 presented a higher number of CD4+CD25+FoxP3- (2.67 and 3.40 times over control) and CD4+CD25+FoxP3+ (2.03 and 2.24 times over control). Later, cytokines present in the draining lymph node were analyzed by a Cytokine Array. We showed that LM38-LP cells diminished IL-6, RANTES and SCF, and increased GM-SCF and IL-3. On the other side, LM38-HP increased TIMP1, IL-3 and IL-8 and LM38-D2 only increased TIMP1. The immunosuppressive activity of the myoepithelial cells was assessed by a concomitant challenge study in BALB/c mice. LM38-LP, LM38-D2 cells or vehicle were s.c. inoculated and 14 days later LM38-HP cells were inoculated s.c. in the other flank. The complementary assay was also conducted by inoculating LM38-HP cells in the first flank and LM38-HP or LM38-LP cells in the second. Tumor take was evaluated 40 days after the second inoculum. The presence of LM38-LP or LM38-D2 tumors enhanced LM38-HP tumorigenesis from 30% to 80%, while the presence of LM38-HP tumors did not affect LM38-LP or LM38-D2. Finally, we evaluated the s.c. growth of 2x106 LM38-HP cells in athymic or immunocompetent mice. LM38-HP cells had a higher tumor take [6/6 (100%) vs 2/7 (28%) p=0.038 chi square] and growth rate [74 (42 – 254) mm3/day vs 0.07 [0.01 – 0.13] mm3/day p=0.007 K-W] in nude than in BALB/c mice. Together, these results suggest that the low malignancy shown by LM38-HP cells is due, at least in part, to immune rejection, that may be prevented in the myoepithelial cell-containing LM38-LP and LM38-D2 cell lines by the production of active TGFβ that might lead to the recruitment and/or induction of Tregs.