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Zhang et al. J Cancer Metastasis Treat 2019;5:56 I http://dx.doi.org/10.20517/2394-4722.2018.112 Page 5 of 9

2,0E+05

1,6E+05

1,2E+05
MFI
8,0E+04

4,0E+04

0,0E+00
MCF-7+SNA MCF-7+MALI RAW 264.7+SNA RAW 264.7+MALI
Control Cocktail addition

Figure 2. Lectin binding to RAW 264.7 cells and MCF-7 cells. Results of cells RAW 264.7 cells and MCF-7 cells stained with the MAL
I and SNA lectins. Flow cytometry results present the mean fluorescence intensity for the lectins and a small increase in binding of the
lectins after treatment with the cytokine cocktail. The experiment shown represents the average of least three performed

3.0

2.5
Fold change SA-MIP binding 1.5
2.0

1.0

0.5

0.0
Control lL-4 lL-6 lL-8 cocktail
RAW 264.7 MCF-7

Figure 3. Flow cytometry of cytokine-treated RAW264.7 cells showing increase in SA-MIP binding after IL-4, IL-6, IL-8 or cocktail
treatment. Results of cells RAW 264.7 cells and MCF-7 cells stained with SA-MIP. Flow cytometry results were re-calculated to fold
change of response compared to control. The experiment shown represents the average of least three performed

an increase in SA-MIP binding in both cell lines, whereas IL-6 and IL-8 showed an even larger decrease
[Figure 3]. The cytokine cocktail with unknown content influenced the SA-MIP binding in a similar manner
as IL-6 and IL-8. All additions of cytokines to RAW 264.7 cells were found to be significantly different from
the control, as determined by statistical calculations.

For the MCF-7 cell line, addition of the recombinant cytokines IL-4, IL-6 or IL-8 did not influence any
increase of SA-MIP binding to the cells [Figure 3]. No statistical significant difference could be determined
for cytokine treatment of MCF-7 cells. However, addition of the cytokine cocktail induced a 2-fold significant
increase of SA-MIP binding in MCF-7 cells. To elucidate the cytokine content in the cocktail, ELISA was
performed.