Redirecting immunity via covalently incorporated immunogenic sialic acid on the tumor cell surface

Anti-tumor immunity was achieved via metabolically incorporated non-self antigen-labelled sialic acid on the tumor surface glycocalyx.

The reaction mixture was refluxed at 80 ℃ for 24 h. The solvent was then lyophilized and the residue solid was purified by silica gel chromatography using methylene chloride/methanol (10:1) as the eluent to give DNP-Tz Sia methyl ester as a yellow solid (1 g, 59%   sequentially stained with biotin-labeled anti-DNP Ab (10 μg ml -1 ) and PE-labeled streptavidin (10 μg ml -1 )

Incorporation of DNP Sia and DNP-Tz Sia into cells surface and immunostaining of cell surface DNP
for 15 min at 4 °C. The cells were washed with PBS and analyzed by a confocal fluorescence microscope. Bar: 10 μm.

Retention of cells surface DNP Sia
Catabolism of cell surface DNP Sia: B16F10 cells were first cultured for 24 h in DMEM containing DNP Sia methyl ester (100 μM

Western blot analysis of covalent incorporation of DNP Sia and DNP-Tz Sia into glycoconjugates
B16F10 cells were first cultured for 24 h in DMEM spiked with no sugar, DNP Sia (0.1, 1 mM) or DNP-Tz Sia methyl ester (0.1, 1 mM). The cells were collected, washed with PBS and then lysed with 1 ml of cell lysis buffer. The mixture were pelleted by centrifugation at 10,000 g for 10 min and the supernatant were diluted with lysis buffer to protein concentration of 1 mg ml -1 as determined by BCA protein assay.
The diluted samples were respectively added to 2 x SDS-PAGE loading buffer, resolved on 10% SDS-PAGE gels, transferred to nitrocellulose, and blocked with 5% bovine serum albumin in PBST (Dulbecco's Phosphate Buffered Saline with 0.05% Tween-20) for 1 h at 37 ℃. The blocked membrane was incubated with biotin-labeled anti-DNP Ab (1 μg ml -1 ) in blocking buffer for 2 h at 37 ℃, followed by HRP-conjugated streptavidin (1 μg ml -1 ) in blocking buffer for 2 h at 37 ℃, washed with PBST (3 x 10 min per wash), and developed using Super ECL Plus Chemiluminescent substrate.

Induction of anti-DNP antibodies in mice with DNP KLH
C57BL/6 mice were subcutaneously injected at the base of the tail with DNP KLH (50 μg) in 100 μl TiterMax Gold adjuvant (emulsions prepared according to the manufacturer's instructions). Two weeks later, Mice were further injected with 80% of the primary antigen dose injected on the back of the neck for 14 days after the first injection. The mice were maintained for following experiments.

ELISA analysis for anti-DNP Ab in mice
Blood samples were collected from the immunized mice and the levels of DNP-specific antibodies in the serum were analyzed by enzyme linked immunosorbent assay (ELISA) performed according to manufacturer's recommendation. Briefly, 96-well plates were coated with DNP-conjugated bovine serum albumin ( DNP BSA) at doses of 2 μg well -1 overnight and then blocked with 5% BSA in PBST for 1 h at 37 ℃. The serum from immunized or control mice were collected 2 weeks after second immunization and diluted using PBS (1:10 4 ). 100 μL of diluted sera or PBS was added to each well, and the plate was

Effects of DNP Sia on cell proliferation
Dose dependent cytotoxicity: B16F10 cells were cultured in DMEM containing DNP Sia methyl ester Absorbance at 590 nm was measured. In addition, Cell number was determined by Trypan Blue Assay.

Inhibition of tumor growth in mice mediated by cell surface-anchored DNP Sia
B16F10 cells were cultured in DMEM spiked without or with DNP Sia methyl ester (1 mM) for 24 h.
The resultant DNP Sia-displaying B16F10 cells (2.5x10 6 ) or control DNP Sia-free B16F10 cells inoculated into both flanks of mice that have been immunized with or without DNP KLH. The mice were anesthetized 7 days post inoculation, and the tumors were dissected from mice. In parallel experiments, immunized or control mice that have been implanted with DNP Sia-displaying B16F10 cells (2.5x10 6 ) or DNP Sia-free B16F10 cells (control) were maintained. The tumor volumes in living mice were measured over time (3-14 days post-treatment).
The resultant mice were treated with PBS (100 μl) or DNP Sia (30 mg kg -1 ) at 3 rd , 6 th and 9 th day after tumor transplantation. The tumor volumes in mice were measured over time (6-14 days after cell implantation).

In vivo distribution of DNP Sia in tumor-bearing mice
A cohort of C57BL/6 mice was subcutaneously injected with B16F10 cells and then maintained for 5-7 days to allow formation of tumor xenografts. The mice were injected intravenously with DNP Sia (60 mg kg -1 ) via tail vein. At 1 h following injection, the mice were anesthetized. The tumors and selected organs were excised, washed with PBS, and then sectioned. The slices were blocked with 5% bovine serum albumin in PBST (Dulbecco's Phosphate Buffered Saline with 0.05% Tween-20) for 30 min at 37 ℃, stained with biotin-labeled DNP-specific Ab (10 μg ml -1 ) for 30 min at 37 ℃, washed with PBST and then stained with PE-labeled streptavidin (10 μg ml -1 ) for 30 min and DAPI (1 μM) for 15 min at 37 ℃, and then probed for ex vivo PE fluorescence.

Retention of DNP Sia in tumors in mice
A cohort of C57BL/6 mice bearing subcutaneous B16F10 tumors were injected intravenously with DNP Sia (60 mg kg -1 ) via tail vein. At 0-24 h following injection, the mice were anesthetized and the tumor were excised, washed with PBS, and then sectioned. The slices were blocked with 5% bovine serum albumin in PBST for 30 min at 37 ℃, stained with biotin-labeled DNP-specific Ab (10 μg ml -1 ) for 30 min at 37 ℃, washed with PBST and then stained with PE-labeled streptavidin (10 μg ml -1 ) for 30 min and DAPI (1 μM) for 15 min at 37 ℃, and then probed for ex vivo PE fluorescence.

Cytotoxicity of DNP Sia in vivo
For cell toxicity: B16F10 cells were respectively cultured for 0-72 h in DMEM medium containing DNP Sia (0-1 mM) as described above. The cell number and cell viability were determined by MTT assay.
For systemic toxicity: Healthy mice were intravenously injected with DNP Sia (300 mg kg -1 ) via tail vein.
The mice were monitored regularly for adverse physiological effects. At 14 days post-injection, the mice were sacrificed and the tumor and selected organ was harvested, sectioned, and then stained with hematoxylin and eosin, and then recorded for visual images.