S100A7 as a potential diagnostic and prognostic biomarker of esophageal squamous cell carcinoma promotes M2 macrophage infiltration and angiogenesis

Abstract Dysregulated expression of S100A7 is found in several cancers and plays an important role in tumor progression; however, its carcinogenic role in esophageal squamous carcinoma (ESCC) is still poorly understood. Here, we identified that the levels of S100A7 were remarkably upregulated in 341 tumor tissues (P < .001) and 274 serum samples (P < .001) of ESCC patients compared with normal control. It was an independent prognostic factor (P = .026). Furthermore, a new diagnostic model for ESCC based on serum S100A7, SCC, and crfra21‐1 was established with area under curve (AUC) up to 0.863 (95% CI: 0.802‐0.925). Mechanically, we found upregulated S100A7 could promote cell migration and proliferation through intracellular binding to JAB1 and paracrine interaction with RAGE receptors and then activates the downstream signaling pathways. In addition, exocrine S100A7 could promote M2 macrophage infiltration and polarization by up‐regulating M2 macrophage associated proteins, and tumor angiogenesis by enhancing the activation of p‐ErK and p‐FAK pathways. Further animal experiments confirmed the role of S100A7 in promoting M2 macrophage infiltration and angiogenesis in ESCC. In conclusion, these findings highlighted the potential diagnostic and prognostic value of S100A7 in patients with ESCC. Meanwhile, our results reveal that S100A7 promotes tumor progression by activating oncogenic pathways and remodeling tumor microenvironment, which paving the way for the progress of S100A7 as a therapeutic target for cancer treatment.

concentrated virus in the presence of 5μg/ml polybrene (Sigma-Aldrich) and selected with puromycin (1.5 ng/ml). The expression levels of S100A7 in the infected cells were confirmed by qRT-PCR and western blot 96 hrs after puromycin selection.

Transient transfection
SiRNAs targeted S100A7 and negative control siRNAs (Thermo Fisher) were introduced into cells with RNAiMAX (Life Technologies). For transfection in 6-well plates, 9μl transfection reagent per well was diluted in 150μl Opti-MEM(Sigma) and mixed with 25 pmol siRNA in 150μl Opti-MEM. Incubated for 5 minutes then add to the cell culture dish. The expression level of S100A7 was confirmed 72 hrs later via western blot. Transient transfection of plasmids was performed with Lipofectamine 3000 kit (Invitrogen) according to the manufacturer's instructions. The transient overexpression plasmids (5 μg/6-well plate) of S100A7, JAB1 and RAGE as well as their respective negative controls were introduced into cells. The cells were harvested at 72 hr after transfection.
RNA extraction, RT-qPCR, western blot, cell proliferation and transwell assays RNA extraction, RT-qPCR, western blot, cell proliferation and transwell assays were performed as previous study [19]. Total RNA was obtained with TRIzol RNA-Extraction reagent according to the instructions. First-Strand cDNA Synthesis kit (Life Technologies, #K1612) was used for reverse transcription. RT-qPCR was performed with SYBR™ Select Master Mix (Life Technologies, #4472908) on ABI 7900HT Real-Time PCR thermocycler (Life Technologies).
The Transwell assay was performed using the Transwell chamber coated with or without Matrigel (BD Biosciences, San Jose, CA, USA).

Cell cycle and apoptosis
The cell cycle analysis was performed with Cell Cycle Detection Kit (KeyGEN BioTECH, #KGA511) according to the manufacturer's introductions. To evaluate cell apoptosis, flow cytometry was performed using the Annexin V-FITC Apoptosis Detection Kit (KeyGEN BioTECH, #KGA107). Treatment with 5μM cisplatin for 24 hours was used to induce apoptosis.

Enzyme linked immunosorbent assay (ELISA)
The concentration of free S100A7 protein in serum of ESCC patients and control people was measured by CircuLex S100A7/Psoriasin ELISA Kit (#CY-8073) according with the instructions. For each well 100µl serum without dilution was used for the ELISA analysis of S100A7. Two biological duplicates were designed for each sample. The optical densities (OD) were measured immediately after adding stop solution at 450 nm using a spectrophotometer (SpectraMax® 190, Molecular Devices, Sunnyvale, CA, USA).

Cell chemotaxis assay
The chemotaxis experiment was implemented by Transwell chamber. PMA activated THP-1 M0-macrophages were seeded on the upper chamber with FBS free media. S100A7 overexpressing cells, control cells, 100ng/ml S100A7 protein and control medium were added to the lower chamber. The culture media added in the lower chamber was 1640 with zinc ions and 1% FBS. Twenty-four hours later, cells on the lower side of the chamber were fixed, stained and counted in five different areas at 100-fold magnification.

Tube formation assay
The µ-Slide Angiogenesis (ibidi #81506) was used to investigate angiogenesis in tube formation assays. Fill the inner well with 10μl growth factor reduced Matrigel (BD Biosciences) and given 30min to solidify. Apply 50 µl of HUVEC or EAhy926 cell suspension into the upper well. Cover the µ-Slide Angiogenesis with the supplied lid. Incubate at 37°C and 5% CO2 as usual. Cells were treated with 100 ng/ml S100A7 protein (R&D), indicated cell culture supernatant or PBS control. Images were captured under phase contrast microscopy. Five microscopic fields were randomly selected for each well, and the number of branch points of the tubes per field was counted. Images were automatically analyzed in ImageJ with the angiogenesis analyzer plug-in.
In vivo Matrigel plug assay.
Growth Factor Reduced Matrigel Matrix phenol Red-free (BD Biosciences) with 100ng/ml recombinant murine S100a7a protein and Matrigel blank control were injected subcutaneously into 6-week-old C57/BL6 mice. On day 7, the mice were sacrificed and Matrigels were completely excised to assess the angiogenic response. The excised Matrigel nodules were soaked with picric acid and embedded in paraffin for hematoxylin and eosin (H&E) staining and immunohistochemistry. The levels of angiogenesis determined via IHC analysis of endothelial marker CD31 (anti-CD31, CST #3528), and count the number of new blood vessels under the microscope.

Co-immunoprecipitation (Co-IP) Assay
For the JAB1-S100A7 interaction, JAB1-Flag and S100A7-Flag overexpression plasmids were transfected into KYSE30 cells. After 48 hours, IP Lysis Buffer (Thermo Scientific, #87787) was used to harvest cell lysates. For free S100A7 protein binding to cell membrane RAGE receptor, the recombinant human S100A7-GST protein and S100A7-Flag protein were added to RAGE-Flag or RAGE overexpressed HUVEC respectively, and harvested 4 hours later.  Heatmap showing correlation coefficients of differentially infiltrated immune cells between tumors with high S100A7 expression and tumors with low S100A7 expression. (D) The Transwell system was used to evaluate the chemotactic effect of S100a7a, the mouse homolog of S100A7, on the mouse macrophage cell line RAW264.7. The left side shows representative images, and the right side shows the number of migrating cells. (E) Representative images of migrated PMA-activated macrophages under the indicated chemotactic conditions. (F) Tumor nodes of S100A7-overexpressing and vector-containing control KYSE150 clones mixed with or without activated macrophages (Mφ). (G) Subcutaneous xenograft growth curves of the indicated treatment groups. ** p<0.01, *** p<0.001. Figure S4. S100A7 promotes tube formation and migration of endothelial cells.

(A)
Representative pictures of tubules formed by HUVECs and EAhy926 vascular endothelial cells treated with or without S100A7 protein or the indicated culture supernatants. (B) Representative pictures of HUVECs and EAhy926 cells passing through chambers treated with or without S100A7 protein or the indicated culture supernatants. Figure S5. The correlation between S100A7 and HMGB1 in ESCC. (A, B and C) The correlation between S100A7 and HMGB1 by the transcriptome data from TCGA database(A), GSE53622(B) and GSE23400(C). (D) Western blot analysis of the correlation between S100A7 and HMGB1 from vector-control and S100A7-overexpressing ESCC cells.