Tumor-associated neutrophils and macrophages interaction contributes to intrahepatic cholangiocarcinoma progression by activating STAT3

Background Tumor-associated neutrophils (TANs) and macrophages (TAMs) can each influence cancer growth and metastasis, but their combined effects in intrahepatic cholangiocarcinoma (ICC) remain unclear. Methods We explored the distributions of TANs and TAMs in patient-derived ICC samples by multiplex immunofluorescent staining and tested their separate and combined effects on ICC in vitro and in vivo. We then investigated the mechanistic basis of the effects using PCR array, western blot analysis and ELISA experiments. Finally, we validated our results in a tissue microarray composed of primary tumor tissues from 359 patients with ICC. Results The spatial distributions of TANs and TAMs were correlated with each other in patient-derived ICC samples. Interaction between TANs and TAMs enhanced the proliferation and invasion abilities of ICC cells in vitro and tumor progression in a mouse xenograft model of ICC. TANs and TAMs produced higher levels of oncostatin M and interleukin-11, respectively, in co-culture than in monoculture. Both of those cytokines activated STAT3 signaling in ICC cells. Knockdown of STAT3 abolished the protumor effect of TANs and TAMs on ICC. In tumor samples from patients with ICC, increased TAN and TAM levels were correlated with elevated p-STAT3 expression. All three of those factors were independent predictors of patient outcomes. Conclusions TANs and TAMs interact to promote ICC progression by activating STAT3.

Quantitative RT-PCR was done using a SYBR PrimeScript RT-PCR Kit (Takara Bio, Shiga, Japan) in accordance with the manufacturer's instructions. We used GAPDH as an internal control. We calculated relative mRNA levels based on the Ct values and   expression was analyzed using the dedicated Web-based software package (http://www.superarray.com/pcr/arrayanalysis.php), which automatically performs all 2 -ΔCt based fold-change calculations from the specific uploaded raw threshold cycle data.

Western blot
Western blotting was performed as previously described 1 . Briefly, we generated total cell lysates, and proteins were separated on 10% SDS-PAGE, and then transferred the proteins to polyvinylidene difluoride (PVDF) membranes. The membranes were washed and blocked. Primary antibodies were applied, followed by horseradish-peroxidase-conjugated secondary antibodies. Antibody binding was detected by enhanced chemiluminescence assays.

Enzyme-linked immunosorbent assay (ELISA)
We determined the level of cytokines/chemokines in cell culture supernatants by using the corresponding quantikine human ELISA kit (R&D Systems) in accordance with the manufacturer's instructions. Briefly, we added 100 μL of sample to each well and incubated the plates for 2.5 h at RT. The plates were washed and incubated with the conjugate for 2 h. After washing, we determined immunoreactivity by adding substrate solution, and the absorbance was determined using a Microplate Spectrophotometer (Bio-Rad). A curve of the absorbance versus the concentrations of cytokines/chemokines in the standard wells was plotted.

TMA and immunohistochemistry
TMAs were constructed as previously described 2 . We took two 2-mm diameter core biopsies from the donor blocks and transferred these to the recipient paraffin block at Immunohistochemical staining was performed by the avidin-biotin-peroxidase complex method. Briefly, after rehydration and microwave antigen retrieval, primary antibodies were applied to slides, incubated at 4°C overnight, and followed with secondary antibody incubation (GK500705, Gene Tech, China) at 37°C for 30 min.
Staining was carried out with DAB and counter-staining was performed with Mayer's hematoxylin. In all assays, we included negative control slides with the primary antibodies omitted.

Evaluation of immunohistochemical variables
Immunohistochemical staining was assessed by three independent investigators who were blinded to patient characteristics, and discrepancies were resolved by consensus.
Under 200× magnification, photographs of three representative fields were captured by the Leica QWin Plus v3 software; identical settings were used for each photograph. For the CD66b and CD68 staining in TMAs, the number of positive cells was calculated in each 2-mm-diameter cylinder and expressed as the mean value of the triplicates (cells/spot) as described previously 3 . Median values were used as a cut-off in subsequent analyses unless specified. STAT3 density in TMA was determined using Image-Pro Plus v6.2 software (Media Cybernetics, Inc., Bethesda, MD). Integrated optical density of all positive STAT3 staining in each photograph was measured and its ratio to the total area of each photograph was calculated as the STAT3 density. The median STAT3 density was determined using immunohistochemistry and used as the cut-off in subsequent analyses. To assess the colony formation abilities of these cells, 500-1000 cells were seeded into each well of 6-well plates and incubated at 37°C for 12-16 d. Cells were then fixed with 100% methanol before staining with 0.1% crystal violet. Image-Pro Plus v6.2 (Media Cybernetics) was used to count the megascopic cell colonies.

In vivo assays for tumor growth and metastasis
For mouse ICC models, 1×10 7 HuCCT1, RBE and SG231 cells transfected with or without shRNA-STAT3 alone, or these cells were co-injected with TANs and/or TAMs  biweekly for 3 consecutive weeks from day 14 after inoculation. In some experiment groups, mice were given STAT3 inhibitor, S3I-201, intraperitoneally at 10 mg/kg every other day for 3 consecutive weeks from day 14 after inoculation. All mice were monitored once every 5 days and killed 5 weeks later. The volume of tumors was calculated in cm 3 as follows: V = ab 2 ⁄ 2 (with a and b representing the largest and smallest tumor diameters) 4 . Upon sacrifice, the tumours were recovered and the volume of each tumour was further determined. Lungs were removed and embedded in paraffin and the total number of lung metastases was counted under the microscope as described previously 5 . The metastases were classified into four grades on the basis of the number of tumor cells present at the maximal section for each metastatic lesion: grade I, ≤20 tumor cells; grade II, 20-50 tumor cells; grade III, 50-100 tumor cells; and grade IV, >100 tumor cells.