br Every fraction was weighted and
Every fraction was weighted and completely dissolved with di-methyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) at the solubility of 200 mg/mL, and then diluted to the final concentrations of 2, 0.5, and 0.1 μg/mL with adenosine triphosphate tumor chemosensitivity assays (ATP-TCA) kit cell culture medium (Jinzijing Biotech Co. Ltd., Beijing, China) and Cellix 601 serum-free medium (SIMA, Beijing, China), re-spectively. To avoid aﬀecting cell activity, we kept DMSO concentra-tion < 0.1% (v/v). All solutions were filtered through 0.22 μm mem-brane filter (Pall, USA), stored at 4 °C, and brought to room temperature before use.
The human colorectal adenocarcinoma cell line HCT116 was ob-tained from the Tumor Epigenetics and Early Detection Laboratory, Chinese People's Liberation Army (PLA) General Hospital (Beijing, China) and cultured in RPMI-1640 medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco Inc.), 50 U/mL penicillin, and 50 mg/mL streptomycin at 37 °C with 5% CO2. The human gastric adenocarcinoma cell line BGC823 and human pancreatic International Immunopharmacology 70 (2019) 241–251
carcinoma cell line SW1990 were obtained from the Institute of General Surgery, Chinese PLA General Hospital (Beijing, China) and cultured in Dulbecco's Modified Eagle Medium (DMEM; Sigma) with 10% heat-inactivated FBS and 1 U/mL gentamicin at 37 °C with 5% CO2. Cells in the logarithmic phase of growth were collected for the experiment.
CIK UNC1999 were isolated and cultured as described previously [20,21]. Briefly, 50 mL of peripheral blood from healthy adult donors was drawn. Participants in our study all provided written informed consents. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient Ficoll-Paque centrifugation (TBD, Tianjin, China), washed with saline, and then cultured in Cellix 601 serum-free medium (SIMA) for 3 h. To prepare CIK cells, the non-adherent cells were har-vested, stimulated with 1000 U/mL recombinant interferon (IFN)-γ (SIMA) in Cellix 601 serum-free medium (SIMA, Beijing, China) at 37 °C with 5% CO2 for 24 h, and then activated with 20 μg/mL anti-CD3, 1 μg/mL anti-CD28 (Mabworks Biotech, Beijing, China), and 1000 U/ mL recombinant IL-2 for 12–14 days.
2.3. Co-culture and microscopy
To evaluate the eﬀects of 22 fractions on diﬀerent tumor cells and CIK cells, 2 × 103 tumor cells and 2 × 104 CIK cells were seeded into each well of a 96-well polypropylene microplate (Costar, USA), re-spectively. The standard solutions of 22 fractions were added in tri-plicate at three diﬀerent final concentrations of 2, 0.5, and 0.1 μg/mL. The plates were incubated for 72 h at 37 °C with 5% CO2. The cells were observed microscopically to check for overgrowth, and photographed by TE2000-U inverted microscope (Nikon, Japan) after 24, 48, and 72 h of co-culture.
2.4. ATP-TCA chemosensitivity assay
At the end of the 72-h incubation period, chemosensitivity was as-sessed using the ATP-TCA kit (Jinzijing Biotech Co. Ltd., Beijing, China). Briefly, remaining cells were lysed by the addition of 50 μL of tumor cell extraction reagent (trichloroacetic acid; Jinzijing Biotech). 50 μL of the lysate from each well was added to corresponding wells in a white 96-well microplate (Jinzijing Biotech) followed by the addition of 50 μL of luciferin-luciferase reagent (Jinzijing Biotech). The level of ATP present was measured on the basis of the absorbance (A) values at 562 nm using a BHP9504 Luminometer (Hamamatsu Photon Techniques Inc., Beijing, China). Luminescence measurements are di-rectly related to ATP levels and enable to determine the percentage of living cells. The cell growth inhibition rate (IR) was calculated using the following equation: IR = (1 −experimental group A value / control group A value) × 100%. Three categories of in vitro sensitivity were defined as: (a) strong sensitivity (SS), IR ≥ 70%; (b) partial sensitivity (PS), 50 ≤IR < 70%; (c) resistance (R), IR < 50%. Thus, IR ≥70% to tumor cells was defined as high eﬃciency and IR < 50% to CIK cells was defined as low toxicity.
2.5. Preparation of emulsion
Emulsion has lymphatic aﬃnity and also characteristic of coating drugs in the internal phase, for which drugs can be protected from hydrolysis and be improved of clinical eﬃcacy . Furthermore, previous studies have shown that lymphatic targeting of water in oil (W/O) emulsions after local injection is better than that of oil in water
(O/W) emulsions . Thus, the W/O emulsions with various con-centrations of HELT fractions were prepared following the previous methods . Briefly, to obtain the oil phase, 0.3 g Oleum Camelliae (Jinhaitang Medicinal Oil Co. Ltd., Jiangxi, China) and 0.1 g soybean lecithin (Sigma) were added into appropriate amount of ethanol and warmed up to 60 °C in the water bath. To obtain the water phase, F18 (25 mg for high-dose emulsion, 2.5 mg for middle-dose emulsion, and 0.5 mg for low-dose emulsion) dissolved in DMSO, 0.2 g poloxamer