Natural Killer Cells Combined With Trastuzumab Enhance The Cytotoxicity Of Trastuzumab To Resistant Breast Cancer Cells

Objective:Trastuzumab(Herceptin®) is an efficient antibody to HER2 positive breast cancer, but the resistance often formed within one year. Natural killer(NK) cells represent a critical component of innate immunity against virus and cancer. They are also efficient cells in antibody-dependent cell-mediated cytotoxicity(ADCC) of trastuzumab, which is the most significant way to kill tumor cells. To enhance the cytotoxicity of trastuzumab to resistant cells, we investigated the combinatorial effects of trastuzumab and NK cells on trastuzumab resistant human breast cancer cells.Methods:1. MTT assay was used to comfirm the resistance of SKBR3-pool2,BT474-HR20 and SKBR3-TR.2. Calcein-AM release assay was used to detect the synergistic cytotoxicity of trastuzumab combined with NK cells to compare the cytotoxicity before and after Fas blocking.3. Flow cytometry was the principle method to explore the mechanism of NK cells combined with trastuzumab. It’s used to detect the expression of activating receptors NKp44, NKp46 and NKG2D; activating ligands ULBP1, ULBP2, and MICA; inhibitory receptors KIR2DL1, KIR2DL2, KIR2DL3, and KIR3DL1;apoptosis receptor Fas and its ligand Fas L. In addition, the secretion of chemokine CCL5 and production of cytokines IL13 were spotted by CBA kit. Moreover, the expression of perforin and granzyme B were identified.4. ELISA kits were used to detect the secretion of cytokines IFN-γ and TNF-α.Results:The resistance to trastuzumab of SKBR3-pool2, BT474-HR20 and SKBR-TR were confirmed. In these resistant cells, the HER2 expression was lower than sensitive cells. Either NK cells or trastuzumab showed low cytotoxicity to the resistant cells, while NK cells combined with trastuzumab, the cytotoxicity to the resistant cells was significantly increased. Hence, the NK cells- trastuzumab combination treatment appearently enhances the cytotoxic effects.Furtherly, the mechanism was studied. Trastuzumab combined with NK cells increased the expression of activating receptors NKp44 and ligands ULBP1, ULBP2,and MICA in trastuzumab resistant cells; in contrast decreased the expression of inhibitory receptors KIR2DL1, KIR2DL2, KIR2DL3, and KIR3DL1 on NK cells.Trastuzumab increased IFN-γ, TNF-α, CCL5 and IL13 secretion by NK cells. The combination treatment also up-regulated the expression of the Fas receptors on cancer cells and Fas ligand on NK cells, and the cytotoxicity to the resistant cells were partrally reduced after blocking. The expression of perforin and granzyme B appearenced no differences between different groups due to the high basic level.Conclusion:1. The expression of HER2 was lower in the resistant cells than in the sensitive cells, and that may be the reason of resistance.2. HER2 expression was reduced without affecting the trastuzumab-induced ADCC, and the synergistic cytotoxicity increased with the large amount of NK cells.3. The mechanism between trastuzumab and NK cells are as following: 1 Up regulating activating receptors, activating ligands and down regulating inhibitory receptors; 2 Up regulating cytokines production of NK cells; 3 Up regulating apoptosis receptors Fas and ligand Fas L.