Immunohistochemistry
Sections of 4 μm thickness were cut from
formalin-fixed paraffin-embedded tissue blocks. The slides were
deparaffinized in xylene, rehydrated, followed by an antigen retrieval
step by heating at 95 oC for 45 min in citrate buffer
(pH6). Endogenous peroxidase was blocked with peroxidase blocking
reagent (DAKO) followed by non-specific binding protein block (DAKO,
X0909). Sections were then incubated with mouse anti-human ENOX2 (1:500,
abcam ab210751) overnight at 4 oC. Slides were then
washed, and secondary staining was performed with DAKO REAL EnVision
Detection System (K5007, DAKO) and visualized with diaminobenzidine
(DAB) according to the kit’s
instructions.
In vivo tumor experiments
All mouse work was approved by the Committee on the Use of Live Animals
in Teaching and Research (CULATR) of the University of Hong Kong
(protocol 4592-18). Experiments were performed in 8-weeks-old nude mice,
in which thymus-derived T cells were not developed, but functional
extrathymic T cells differentiation pathways, and low levels of T cells
were retained. Thirty nude mice per group were inoculated once
subcutaneously with 5 x 106 NPC cell lines + matrigel
(1:1) using a 29-gauge needle under anaesthesia. Around day 10, when we
expect the tumors to start growing, mice were injected with IDX 10
mg/kg, 20 mg/kg, Cis (5 mg/kg) or combinations of IDX and cis
intraperitoneally. Drugs were injected every day for up to three weeks.
Tumor growth was monitored every 2 days at the indicated time. Tumors
were harvested to prepare paraffin tissue sections for
immunohistochemical staining. Spleen cells were mechanically
disaggregated, whereas isolation of tumor-infiltrating leukocytes
required both mechanical and enzymatic digestion by using 1.5 μg/mL
collagenase IV (Roche), 0.8 mg/mL dispase (Invitrogen), and 0.1 mg/mL
DNase I (Sigma). Tissues were incubated in digestion medium at 37 °C for
30 min.