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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 3 of 9

Addition of recombinant cytokines
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2 × 10 of each of MCF-7 and RAW 264.7 cells were seeded in T25 flasks. Next day, the medium was removed
and the different recombinant cytokines IL-4, IL-6, IL-8 (Peprotech, Rocky Hill, NJ, USA), all added at a
final concentration of 40 ng/mL, or undiluted PHA- stimulated Jurkat supernatant was added to the cell
culture flasks. One flask served as control with cells and medium alone. The cells were harvested after 3 days
of incubation by trypsinization, two washes with phosphate buffered saline (PBS, Invitrogen) and thereafter
stained with SA-MIPs according to below.

Preparation of polymers
The polymers (SA-MIPs) were prepared as described previously by Shinde et al . Before the SA-MIPs were
[13]
used for cell-based experiments, the dried SA-MIPs were resuspended in PBS and sonicated for 8 min with a
VWR sonicator. The stock solution of 1 mg/mL was further sonicated and diluted in PBS prior to use.

Flow cytometry analysis
SA-MIPs
1 × 10 cells/sample were stained with SA-MIPs or left unstained as a control. The cells were washed twice
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with 2 mL PBS and thereafter 100 µL of SA-MIPs with indicated concentration was added to the cells. 100 µL
of PBS was used as a negative control. The cells were incubated with SA-MIP for 30 min at 4 °C in the dark,
and were thereafter washed three times with 2 mL PBS and analyzed using flow cytometry (BD Biosciences,
Accuri C6 Flow Cytometry, NJ).

Lectins conjugated with biotin
1 × 10 cells/sample were stained with biotin-conjugated lectins (Vector Labs, Burlingame, CA, USA) or left
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unstained as a control. The cells were washed twice with 2 mL PBS. 100 µL of biotin-conjugated lectins, MAL
I (from Maackia amurensis) or SNA (from Sambucus nigra) at concentrations of 10 µg/mL was added to the
cells and 100 µL of PBS was used as a negative control. The cells were incubated with biotin- conjugated
lectins for 30 min on 4 °C and were thereafter washed three times with 2 mL PBS. Thereafter 100 µL of a
1:100 dilution of streptavidin-FITC (Agilent Technologies, Santa Clara, CA, USA) was added to the cells and
incubated for 20 min at 4 °C in the dark. After the incubation, the cells were washed three times with 2 mL
PBS and analyzed using flow cytometry (Accuri C6 Flow Cytometry).

ELISAs for IL-2, IL-6, IL-8 and TNF-a
96-well ELISA plates were coated overnight at room temperature with 100 μL/well of diluted capture
antibody against IL-2, IL-6, IL-8 or TNF-a, respectively, according to the manufacturer´s instructions (R&D
Systems, Minneapolis, MN, USA). The plates were washed three times with PBS-Tween (PBS-T), blocked by
adding 300 μL/well of blocking buffer (1% BSA in PBS) and thereafter incubated at room temperature (RT)
for 1 hr. After three washes with PBS-T, the plates were incubated with 100 μL/well IL-2, IL-6, IL-8 or TNF-
standards, respectively, diluted in reagent diluent, and 100 μL/well of samples collected from the Jurkat cell
supernatant. After an incubation of 2 h at RT, the plates were washed three times and thereafter100 μL/well
of 50 ng/mL biotin-linked detection antibody against IL-2, IL-6, IL-8 or TNF-a, respectively, were added
and incubated at RT for 2 h. Then washed the plates three times and incubated with streptavidin-HRP at
room temperature for 20 min. After three washes with PBS-T, the plates were incubated with 100 μL/well
with substrate solution (1:1 mixture of H2O2 and tetramethylbenzidine, TMB) at RT for 20 min. After that,
100 μL/well of stop solution was added and the plates were analyzed with a spectrophotometer at 450 nm.

Statistical methods
Mean and standard deviation were used for statistical analysis of all calculations.