Tumor infiltrating NK cell (TINK) subsets and functional molecules in patients with breast cancer

NK cells have been introduced as the main innate arm of immunity against malignancies. Recent advances introduced new subsets of, and new effector molecules on NK cells suggesting new paradigms for NK cell functions in tumor immunity. Considering these new paradigms, in the current research we investigated the frequency of tumor infiltrating NK cell (TINK) subsets and their functional molecules in breast tumor tissues by flowcytometry method.

Breast tumor tissues were obtained from 32 untreated patients with breast cancer. The tissues were then minced mechanically to acquire a single cell suspension and surface-stained with monoclonal antibodies against CD3, CD56, CD11b, CD27, NKG2A, NKG2D and CXCR3. For intracellular staining (ICS), the surface-stained cells were then fixed, permeabilized and stained with anti-Perforin and anti-Granzyme B antibodies. The samples were run and the data were acquired on a four-color flowcytometer.

The cell suspension derived from tumor tissue encompassed 3.10 ± 0.52 % CD3^-CD56^{+(bright/dim)} total NK cells. Based on the conventional classification the percentages of cytotoxic (CD3^- CD56^dim) and regulatory (CD3^- CD56^bright) NK cells were respectively 1.74 ± 0.24 % and 1.36 ± 0.48 %. According to the new classification the percentages of cytotoxic (CD3- CD56⁺ CD11b⁺ CD27^-), regulatory (CD3^-CD56⁺ CD11b^+/- CD27⁺) and tolerant (CD3^-CD56⁺ CD27- CD11b^-) NK cells were respectively 0.48 ± 0.07, 1.55 ± 0.34 and 1.15 ± 0.51. A significant higher frequency of total NK cells (CD3^-CD56^{+ (bright/dim)}) in the breast tumor tissues of the patients whose tumor draining lymph nodes (TDLNs) has not been yet involved by tumor cells (LN^- patients) compared with the ones with lymph nodes involvement (LN⁺) (5.91 ± 1.79 % Vs. 2.20 ± 0.20 %, P < 0.004). Furthermore, NK cells with overexpressed activating receptor; NKGD2 (CD3- CD56^{+(bright/dim)} NKG2D⁺ NK cells) was observed to be elevated in LN^- patients compared with the LN⁺ ones (70.01 ± 7.96 Vs. 42.5 ± 4.81, P < 0.011). Correlation analysis revealed the percentages of conventional regulatory NK cells (CD3- CD56^bright) in breast tumor tissue to be in positive correlation with the tumor size (R = 0.380, P < 0.04). The mean percentage of this cell subset was also observed to be higher in patients with T3 tumor size compared with smaller T1 tumor size (1.61 ± 0.20 % vs. 0.75 ± 0.15 %, P < 0.023.

Our observations suggest that accumulation of NK cells as well as the expression of activating NKG2D receptor by TINKs may play roles in breast tumor regression especially in the LN^- patients. As the tumor growths and the size of tumor increases the accumulation of regulatory NK cells may facilitate the tumor improvement. These observations may have implications in cancer NK cell-based immunotherapy.