Nr-CWS was supplied by Greatest Biopharma Limited Company (purity>98%). Fluorescein conjugated anti-mouse antibodies APC-NK1.1, FITC-NK1.1, APC/Cy7-CD3ε, PE/Cy7-CD3ε, APC-CD4, PE-CD8α, PerCP/Cy5.5-CD11b, PE-CD27, FITC-TNF-α, and PerCP/Cy5.5-IFN-γ from BioLegend, and PerCP/Cy5.5-CD69, FITC-FasL, PE-TRAIL, PE-Perforin, and FITC-Granzyme-B from eBioscience were used in this study. TUNEL assay kit and MTS Cell proliferation, cytotoxicity or chemosensitivity assays kit (cat # 3580) were obtained from Promega. The LEGENDplex™ Multiplex mouse cytokine kit was provided by BioLegend. EasySep™ mouse NK cell isolation kit was from STEMCELL Technologies Inc. Recombinant murine IL-2 was from PeproTech Inc.

Female C57BL/6 mice (6–8 weeks, 18–20 g) supplied by Beijing Vital River Laboratory Animal Technology, were maintained in specific pathogen-free animal rooms and treated following the guideline for Care and Use of Laboratory Animals of Jinzhou Medical University. Lewis lung carcinoma (LLC) and B16F10 melanoma cell lines were bought from Cell Bank of Chinese Academy of Sciences in Shanghai. The cells were grown in DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, in a humidified atmosphere of 5% CO₂ at 37°C. LLC cells (5×10⁶/ml in PBS) were subcutaneously injected in the medial axillas of 22 C57BL/6 mice (0.1 ml/mouse) and after establishment of tumor model for 24 h (hrs), mice were divided into two groups randomly (11 mice/group, Nr-CWS group and control group). For experimental metastasis, B16F10 melanoma cells (1×10⁶/ml in PBS) were injected into C57BL/6 mice through tail-vein (0.2 ml/mouse) and the injected mice were divided into two groups randomly 24 h later (4 mice/group, Nr-CWS group, control group). Nr-CWS group were given Nr-CWS by intra-peritoneal injection (10 mg/kg in PBS, once-daily) for 7 or 14 successive days, and the control group were treated with equivalent PBS.

For the mouse tumor-bearing model (11 mice/group), 5 mice from each group were euthanized with an overdose of isoflurane on day 7 after tumor cell inoculation. Then solid tumors were dissected for analyses of tumor weight and the percentage of tumor-infiltrating lymphocytes. After 14 days, the remained mice (6 mice/group) were euthanized for sampling: the solid tumors were dissected, each tumor sample was sectioned for tumor morphology and tumor cell apoptosis assay. For experimental metastasis model (one mice in control group died during experiment was excluded), pulmonary metastases were assessed by comparing the numbers of metastases on day 14 after tumor cell injection. Blood serum and spleen were obtained from the metastasis model mice. Index for spleen was calculated as previously reported [18].

Tumor morphology was analyzed as previously described [19]: in brief, tumor tissues from Nr-CWS treated and control groups (day 14) were fixed using paraformaldehyde, Fixed samples were embedded in wax after dehydration and clearing using a series of ethanol solution and xylene, thin slices (4 μm) were cut and mounted on microscope slides, samples were stained using hematoxylin and eosin stain (H&E) after deparaffinized and rehydration. Finally slides were mounted using mounting medium and analyzed [19].

Tumor cell apoptosis was detected using TUNEL Assay Kit, the ratio of positive cells per specimen was analyzed under a light microscope. For each sample, five most obvious positive staining areas were analyzed [20]. TUNEL positive cells were counted and apoptotic index (AI) was calculated in reference of a previous publication [20].

Each tumor sample dissected on day 7 after tumor cell inoculation was digested using trypsin (0.05%) and mashed through moisturized cell strainer (70 μm), then the single-cell suspension was collected and counted. 1×10⁶ cells were taken for staining. The cocktail of antibodies (APC-CD4, PE/Cy7-CD3ε, FITC-NK1.1, and PE-CD8α) was added into the cell suspension and then the stained samples were incubated for 20 min at 4°C. For dead cell exclusion, 5 µl of 7-Aminoactinomycin D (7-AAD) was used for each sample before analysis. Then samples were detected using flow cytometry (FCM) (BD FACS Canto II) and 30,000 events were acquired and analyzed using BD Diva software.

Spleens from wild type C57BL/6 mice were mashed through a moisturized cell strainer. NK cell was purified with EasySep™ mouse NK cell isolation kit and its purity was evaluated by FCM. For the cytokines analysis in culture supernatant and NK cell cytotoxicity assay, purified NK cells were subjected to fluorescence-activated cell sorting (BD FACSAriaIII) to exclude other cell contamination using CD3ε and NK1.1 staining. Freshly isolated NK cells or sorted NK cells were seeded in 96-well flat-bottom plate (1×10⁵/well) containing RPMI-1640 complete medium supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin, 50 μM β-mercaptoethanol, 500 U/ml IL-2, and treated with or without 15 μg/ml Nr-CWS (the optimal dose for splenocytes from our previous experiments) in a humidified atmosphere of 5% CO₂ at 37°C. After 24 or 48 h incubation respectively, NK cells were collected for staining. The culture supernatant was collected for multi-cytokines analysis by FCM (BD Canto II).

For NK cells analysis from in vivo experiment, spleen single-cell suspension were made by mashing whole spleen through a moisturized cell strainer and RBC lysing. For cultured NK cells analysis in vitro, NK cell was purified from spleen single-cell suspension with EasySep™ mouse NK cell isolation kit. NK cells from spleen of Nr-CWS treated and control B16F10 metastasis mice (after 14 days treatment), and the cultured wild type (WT) spleen NK cells incubated with or without Nr-CWS (15 μg/ml) at indicated time-points were collected for analysis of surface molecules expression by FCM. And cells from each sample were suspended in 100 μl staining buffer. A cocktail of antibodies (combination of the fluorescent conjugated antibodies CD3ε, NK1.1, TRAIL, FasL, CD69 or CD3ε, NK1.1, CD11b, and CD27) was used and the stained samples were incubated for 20 min (4°C). After washing with PBS, the stained cells were analyzed by FCM.

Purified WT NK cells treated with or without Nr-CWS (15 μg/ml) were collected at indicated time-points. Monensin (BioLegend) was present for the last 5 h before collection. Then, samples were stained with APC-NK1.1 and APC/Cy7-CD3ε antibodies for surface markers. For intracellular staining, samples were fixed and permeabilized after surface staining using Fixation Buffer (cat # 420801) and Intracellular Staining Permeabilization Wash Buffer (10×, cat # 421002). Then, samples were further stained with the cocktail of cytotoxic molecules (PE-Perforin, FITC-Granzyme B) or cytokines (FITC-TNF-α, PerCP/Cy5.5-IFN-γ) antibodies. After intracellular staining and extensive washing, samples were subjected to FCM analysis.

The cytokines in the mouse serum of metastasis model and WT NK cell culture supernatants were measured using the LEGENDplex™ Multiplex mouse cytokine kit from BioLegend by FCM as we have reported [5]. The serum from Nr-CWS treated (for 14 days) metastasis mice and control mice were collected and stored at −80°C. The supernatants of cultured NK cells treated with or without Nr-CWS (15 μg/ml) were collected at indicated time-points. Cytokines (IL-2, IL-4, IL-5, IL-6, IL-10, IFN-γ and TNF-α) were evaluated in all serum and supernatant samples. Results were analyzed using LEGENDplex software as previously reported [4, 5].

The assay for NK cell cytotoxicity was performed as previously described by MTS assay [19]. Briefly, NK cells sorted from wild type C57BL/6 mice were stimulated with or without Nr-CWS (15 μg/ml) for 24 h and collected as effectors after washing. LLC cells were serviced as targets. The effector NK cells were mixed with LLC cells (5×10³/well) at effector:target ratios of 10:1 and 5:1, and incubated in 96-well flat-bottom plate (Corning-Costar) in 100 µl volume for 6 h. At the last 1 hour, 20 µl/well of CellTiter 96^® AQueous One Solution Reagent (MTS) was added. After 1 h at 37°C in a humidifi ed, 5% CO₂ atmosphere, the optical density (OD) was measured using multiskan spectrum microplate spectrophotometer at 490 nm from triple wells. The cytotoxicity was calculated as previous report [19]: Cytolytic activity ( % ) = { 1 - OD of  ( target cells  +  NK cells ) - OD of NK cells OD of target cells } × 100

Data were shown using mean ± standard deviation (SD). The Independent-Samples t Test was used to analyze results in SPSS 16.0 (SPSS, Inc., Chicago, IL, United States). The p-value <0.05 was considered as statistically significant, as shown in the figure legends. Data shown in figures are representative of three independent experiments.

To confirm the anti-tumor activity of Nr-CWS, C57BL/6 wild type mice were implanted with LLC cells and then treated with either Nr-CWS or PBS control. The results showed that the average tumor weight of the Nr-CWS group was decreased after 7 days treatment when compared to the control group (p < 0.05). However, it was comparable after 14 days treatment (p > 0.05) (Figure 1A; Supplementary Figure S1A). The histopathological examination of tumors in the Nr-CWS group (after 14 days treatment) showed larger necrotic areas and more prominent infiltration of inflammatory cells than that in the control group. H&E staining showed the tumor cells were poorly differentiated and heterogeneous, with hyperchromatic nuclei and scant cytoplasm. There were also binucleated or multinucleated cells in the tumor samples. The representative histopathological changes are shown in Supplementary Figures S1B,C.

The TUNEL System was used for detecting apoptotic cells and apoptotic bodies in situ of solid tumors. The morphological changes of apoptotic cells were observed as follows: apoptotic DNA fragments were stained brown mainly in the condensed nuclei (Supplementary Figure S1D). The apoptotic index was calculated using the percentage of stained brown cells at ×400 magnification. Results showed that Nr-CWS treatment caused more tumor cell apoptosis and higher apoptotic index compared with the control group (p < 0.05, Figure 1B, Supplementary Figure S1D).

For each tumor sample from the tumor-bearing mice after 7 days treatment with or without Nr-CWS, the percentage of infiltrated lymphocytes including CD3⁺, CD4⁺ T, CD8⁺ T, and NK cells, were analyzed by FCM (Figure 1C, Supplementary Figure S1E). The percentage of CD8⁺ T and NK cells were significantly increased after Nr-CWS treatment (p < 0.05), while the percentage of CD3⁺ and CD4⁺ T cells were only slightly up-regulated (p > 0.05) (Figure 1C, Supplementary Figure S1E).

The spleen index of the Nr-CWS treated group was significantly increased after 14 days treatment (p < 0.05), while the spleen index of the Nr-CWS and control group did not show any significant difference (data not shown) after 7 days treatment, as shown in Figure 1D.

To further investigate the role of Nr-CWS in vivo, we adopted the B16F10 melanoma metastasis model. Results showed Nr-CWS treatment can significantly reduce the lung metastasis compared to the control group in Figure 1E, Supplementary Figure S1F (p < 0.01).

The NK cells were purified using EasySep™ mouse NK cell isolation kit based on Magnetic-activated cell sorting (MACS). All living cells (7-AAD negative) were analyzed. The percentage of NK cell in spleen before purification was 2.63 ± 0.31%. After purification, the percentage of NK cell was >80% (81.97 ± 3.40%) as determined by FCM gating on NK1.1⁺ CD3ε⁻ population (Supplementary Figure S1G). For the cytokines analysis in culture supernatant and NK cell cytotoxicity assay, purified NK cells were subjected to fluorescence-activated cell sorting to exclude other cell contamination using CD3ε and NK1.1 staining. The purity of sorted NK cell was more than 98% (data not shown).

To assess the effect of Nr-CWS on the functional molecules of NK cells, FCM analysis was performed to determine the expression of functional markers on NK cells gated the population of NK1.1⁺ CD3ε⁻ cells. The in vivo samples showed that the expression of TRAIL and FasL on splenic NK cell were much higher in the Nr-CWS group than the control group (p < 0.05, Figure 2A, Supplementary Figure S2A). However, CD69 expression and splenic NK cell percentage were comparable between the two groups (p > 0.05, Figures 2A,B). Results of the in vitro experiment (Supplementary Figures S2B,C) showed Nr-CWS can strikingly increase the expression of CD69, TRAIL, and FasL on NK cells after incubation for 24 h (p < 0.01, Figure 2C) and increase CD69 expression after incubation for 48 h (p < 0.01, Figure 2D).

To investigate the effect of Nr-CWS on NK cell cytotoxicity, the expression of intracellular perforin and granzyme-B of NK cells (gated on NK1.1⁺ CD3ε⁻) treated with or without Nr-CWS for 24 or 48 h, were analyzed by FCM. Results showed that Nr-CWS significantly increased the expression of granzyme-B and perforin after incubation for 24 and 48 h, as shown in Figures 3A,B, Supplementary Figures S3A,B.

Purified normal spleen NK cells treated with or without Nr-CWS were collected at indicated time-points for subgroups analysis by FCM gating on NK1.1⁺CD3ε⁻ population. Our data showed that Nr-CWS treatment can significantly up-regulate the percentage of CD27^lowCD11b^high and CD27^lowCD11b^low cells, and down-regulate the percentage of CD27^highCD11b^low and CD27^highCD11b^high cells after incubation for 24 and 48 h, as shown in Figures 3C,D and Supplementary Figures S3C,D.

Purified normal spleen NK cells treated with or without Nr-CWS were collected at indicated time-points for intracellular cytokine analysis by FCM gating on NK1.1⁺CD3ε⁻ population. Results showed that Nr-CWS can stimulate NK cells to produce significantly more IFN-γ and TNF-α after treatment for 24 and 48 h (p < 0.01, Figures 4A,B, Supplementary Figures S4A,B).

The levels of cytokines in the supernatant and serum from NK cell culture and animals treated with or without Nr-CWS were analyzed by FCM using LEGENDplex™. The serum levels of IL-2, IFN-γ, and TNF-α were significantly higher after Nr-CWS treatment in vivo (p < 0.01, Figure 4C). Results of cytokines in the cultured supernatants showed Nr-CWS significantly increased the production of IFN-γ and TNF-α after treatment for 24 and 48 h in vitro. In contrast, the level of IL-2 was comparable between the two groups (p > 0.05). Nr-CWS can drive the expression of IL-6, which were 37.51 ± 8.32 and 57.16 ± 4.80 pg/ml after incubation for 24 and 48 h in vitro, whereas it was not detectable (ND) in the control group, as shown in Figures 4D,E.

To measure the cytotoxic activity, sorted NK cells were treated with or without Nr-CWS for 24 h. Then the NK cells were washed and the number was accounted, and mixed with LLC cells at indicated ratios for 6 h. The cytotoxic activity was measured by MTS assay. Results showed that Nr-CWS can strikingly increase NK cell-mediated cytotoxicity at both effector:target ratios of 10:1 and 5:1 (p < 0.05, Figure 5).