ESCC cell lines KYSE140 (K140), K180, K410, K510, and K520 were obtained from DSMZ, the German Resource Center for Biological Material. ESCC cell lines EC18, HKESC1, and immortalized esophageal epithelial cell line NE1 were provided by Professors G. Srivastava and G.S. Tsao (The University of Hong Kong). HUVECs were provided by Dr Stephanie Ma (The University of Hong Kong). All experiments were done using endothelial cells between passages 3 and 8.
Patients and samples
Four TMAs (two slides each for TMA-1 and TMA-2) composed of samples from a total of 125 ESCC patients from the surgical pathology archives of Linzhou Cancer Hospital (Henan, China) were used for histological staining. Of these, 89 paired ESCC and corresponding normal tissue samples from TMA-1 were used to evaluate B-cell spatial analysis by mIHC. One hundred and eighteen matched normal/tumor pairs from a consecutive slide of TMA-1 (n = 89) and TMA-2 (n = 29) were chosen to perform HMGB1 chromogenic immunohistochemistry. All tumor cases positive for B cells (n = 74 from TMA-1 and n = 51 from TMA-2) were chosen for cooperative biomarkers prognostic analysis. A summary of cases used for TMA is shown in Supplementary Fig. 2a.
Three paired ESCC tissues were used for RNA sequencing. An additional 38 paired ESCC tissues were used for qRT-PCR analysis. No patient in the study had received preoperative radiation or chemotherapy. Studies using human tissues were approved by the committees for ethical review of research involving human subjects of Zhengzhou University (Zhengzhou, China) and the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU/HA HKW IRB).
Seven ESCC patients who had undergone upfront esophagectomy at the Department of Surgery, Queen Mary Hospital, Hong Kong were also recruited for tissue dissociation and subsequent flow cytometric analysis.
PBMC were isolated from healthy donors (buffy coats, Hong Kong Red Cross Blood Transfusion Service) for subsequent culture.
Sample collection and processing
PBMC were isolated from healthy donors by Ficoll-Paque gradient (GE Healthcare) and centrifuged at 400×g for 25 min. The interface containing PBMC was carefully removed and cells were washed twice with PBS + 2 mM EDTA.
The tumor and normal tissue samples were processed into single-cell suspensions by mechanical disaggregation followed by enzymatic digestion 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; released cells were collected and filtered, and the remaining tissue was further processed.
Purification of B cells
PBMC were then re-suspended in chilled MACS buffer (PBS, 0.5% FCS and 2 mM EDTA), washed and incubated with CD20 microbeads (130-091-104; Miltenyi Biotec) for 15 min at 4 °C, and subsequently passed through magnetic separation columns (LS; Miltenyi Biotec). The bead-bound cells were collected as enriched, positively selected B cells. Purified CD20+ B cells were either re-suspended in FACS buffer for flow cytometry or in DMEM supplemented with 10% FCS, 50 IU/mL penicillin–streptomycin, and 10 mM HEPES buffer (Gibco/Invitrogen) for subsequent culture. Preparations were typically > 95% pure.
Directional migration of B cells was evaluated in Costar Transwell permeable polycarbonate supports (5 μm pores) in 24-well plates. HMGB1-overexpressing tumor cells/different concentration of recombinant HMGB1 (rHMGB1) were used to compare with parental cell lines/medium alone. B cells (0.25 × 106 cells/ml) pre-treated with 100 ng/mL recombinant IL-4 and 5 µg/mL IgM for 24 h were washed, placed in the top chamber, and allowed to migrate for 4 h at 37 °C. After that, cells in the bottom chambers were collected, stained with FITC-labeled anti-CD20 antibody, and the number of CD20+ B cells was calculated by flow cytometry.
Supernatants from co-cultured cells were collected. Total IgG/M were detected using human IgG/IgM ELISA quantitation set (Bethyl Laboratories) according to the manufacturer’s instructions.
PBMC/purified B cells (1 × 106/well) were labeled with 5 µM CFSE (Biolegend) in PBS/0.1% BSA for 8 min at 37 °C. Unbound dye was quenched by washing three times with ice-cold complete medium, followed by stimulation with 100 ng/mL recombinant IL-4 and 5 µg/mL IgM. 24 h later, pre-stimulated CFSE-labeled PBMC/B cells (1 × 104) were washed and mixed with ESCC cell lines in 96-round bottom plate at effector-to-target ratios of 10:1. Proliferation of PBMC/B cells was monitored by CFSE partitioning 6 days post-co-culture. The total B-cell population from PBMC, stained with anti-CD20-APC antibodies, was analyzed with a FACSCanto II. In some experiments, pre-stimulated B cells were treated in the presence or absence of rHMGB1 (10 ng/mL) or anti-VEGF (10 µg/mL; Sino Biological), cells were collected for flow cytometric analysis, and conditioned medium (CM) was collected for subsequent functional assays.
Collection of conditioned medium
CM was collected and centrifuged at 2000×g for 10 min. CM was either used undiluted for characterization by protein array or concentrated for an in vivo mouse model by centrifugation at 4000×g for 15 min at 13 °C, using ultrafiltration units (Amicon Ultra-PL 10, Millipore, Bedford, MA, USA). Filter units were used only once to avoid membrane saturation. Concentrated CM were then sterilized on 0.22 μm filters (Millipore), aliquoted, and stored at –80 °C until use.
Matrigel tube formation assay
HUVECs were maintained in endothelial cell growth medium M200 (Invitrogen) supplemented with 2% FBS and endothelial cell growth supplements (LSGS Medium). 2 × 104 cells were re-suspended in 100 µL co-culture CM (1:5; diluted with serum-free M200) and seeded in 96-well plates pre-coated with 50 µL of growth factor-reduced Matrigel (BD Bioscience). The cells were incubated at 37 °C for 6–8 h to allow for the formation of tube-like structures. Enclosed networks of tube structures from three fields were photographed randomly in each well and quantified using ImageJ analysis.
Spheroid sprouting assay and imaging
Spheroids containing 6000 HUVECs were generated by incubating suspended cells in M200 in 96-well Corning® Spheroid Ultra-Low Attachment Microplates overnight, after which they were embedded into 3 mg/mL Matrigel for a fixed 3D cell culture. In brief, 70 µL hydrogel solution containing 3 mg/mL Matrigel and cells were dispensed into each well followed by 20 µL of CM. The plate was then warmed to 37 °C to induce gelation. Images of the spheroid within the polymerized gels were captured using InCell6500 (Perkin Elmer). Endothelial sprouts were characterized by measuring average branch lengths using Fiji distribution of ImageJ. Briefly, images were converted to 8-bit grayscale then converted to binary images with appropriate threshold values. Parameters for the binary mask, such as area and perimeter, were analyzed using the ‘Analyze Particles’ function. For end-point calculation, the areas covered by the spheroids were traced followed by ‘Skeletonize’ function of Fiji for analysis.
Starved HUVECs (2 × 104) were seeded in the top chambers of Transwell plates (8 µm pore size) in 400 µL of M200 medium without serum. The bottom chambers were filled with 400 µL co-culture CM (1:5 dilutions). After 24 h, cells were fixed, stained with 1% crystal violet, and counted. For gene expression assays, HUVECs were plated in 24-well plates (5 × 104cell/well) incubated with CM (1:5 dilutions) for 8 h and 24 h. For the μ-slide chemotaxis assay (Ibidi), HUVECs were cultured on 2 µg/cm2 Matrigel-coated μ-slides and allowed to adhere for 3 h. One reservoir was filled with M200/2% FBS/LSGS and the second reservoir with the indicated 1:5 diluted CM collected from the co-cultures. Directional migration was assessed after 16 h and tracked with the aid of time-lapse microscopy.
Angiogenesis antibody arrays
The relative levels of human angiogenesis-related proteins in CM were measured using a Human Angiogenesis Array Kit (R&D Systems Inc.). Aliquots of CM (500 µL) were added to the array and the results were analyzed with the ImageJ software.
Immunohistochemistry (IHC) and immunocytochemistry (ICC)
Sections of 4 μm thickness were cut from FFPE tissue blocks. The slides were deparaffinized in xylene, rehydrated, and this was followed by an antigen retrieval step by heating at 95 °C for 45 min in citrate buffer (pH6). Endogenous peroxidase was blocked with peroxidase blocking reagent (Dako) followed by a non-specific binding protein block (Dako, X0909). For double staining, EnVision G|2 Doublestain System was used, according to the manufacturer’s instructions. Sections were then incubated with either mouse anti-human HMGB1 (1:400, ab18256, Abcam) and/or anti-human CD20 (1:50, Dako) and a species-matched isotype control overnight at 4 °C. 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.
After deparaffinization and blocking steps, sections were incubated with rabbit anti-human CD31 (1:100, ab28364, Abcam), mouse anti-human CD20 (1:50, Dako), rabbit anti-human CD20 (1:400 Thermo Fisher), mouse anti-human VEGF (1:80, MA-13182, Thermo Fisher), and a species-matched isotype control. Slides were then washed and secondary staining was performed with donkey anti-mouse-Alexa-555 or goat anti-rabbit-Alexa-488 in the case of the double stain for CD20/CD31 and EBI-3. For double staining of CD20/VEGF, goat anti-mouse-Alexa-555 or goat anti-rabbit-Alexa-488 was used. All slides were counterstained with DAPI and examined under Carl Zeiss LSM 700.
B cells were collected following recombinant HMGB1 incubation or co-culture with tumor cells and then washed twice with cold PBS cytospin. Cells were then fixed in methanol/acetone 1:1 followed by blocking and antibody incubation as described above.
For tyramide signal amplification (TSA) IHC staining, slides were first deparaffinized and rehydrated in serial passage through xylene and alcohol. Antigen retrieval was performed by microwaving the samples for 2 min 20 s with 100% power, followed by 20% power for 15 min and the slide was cooled for 20 min. Then, the sections were incubated with blocking solution, Biocare Medical Background Sniper supplemented with 2% BSA, for 15 min at room temperature. Slides were incubated with primary antibodies: Ki67 (1:50, Dako pH6), CD20 (1:10,000, EDTA pH9) and Cytokeratin-5 (1:10, Diva), VEGF (1:100, EDTA pH9) for 1 h at room temperature. Multiplexed TSA was visualized using performing a triplex (CD20 in Opal 650, CK-5 in Opal 570,Ki67 in Opal 690, and/or VEGF in Opal 570), followed by a 5-plex (addition of HMGB1 Opal 520). All multiplex TSA analyses were performed by repeating staining cycles in series, microwaving in between each cycle and at the end of the multiplex TSA. Slides were then counterstained with DAPI for 5 min and mounted with VECTASHIELD.
Digital image acquisition and analysis
TMA sections were digitally scanned at an absolute magnification of × 20 using the Vectra 3.0 Vectra Polaris imaging system (Akoya Biosciences) and analyzed with inForm Tissue Finder software (Akoya Biosciences). Multispectral images were unmixed using the spectral libraries built from images of single-stained slides. Firstly, 15 TMA cores were selected to train machine learning algorithms for tissue segmentation, cell segmentation, and cell phenotyping, which were later applied on the whole TMA cohort. The software was first trained to segment tissue by manually to segment tumor tissues into carcinoma, the intra- (epithelial), and stromal, the peri-(non-epithelial), areas based on tumor marker, cytokeratin-5 (CK-5). To detect immune cells, an algorithm was designed based on pattern recognition that quantified CD20, VEGF, and Ki67 cells. After which the distribution of immune cells was analyzed and cell segmentation was based on the nuclear DAPI stain but assisted using membrane CD20 staining. Training sessions for tissue segmentation and phenotype recognition were carried out repeatedly until the algorithm reached the level of confidence recommended by the program supplier (at least 90% accuracy) before performing final evaluation. Each scanned image was examined by one observer under the supervision of an experienced pathologist. The area of each tissue category, carcinoma, and stroma was evaluated to assess the density of lymphocytes, represented by (number of lymphocytes)/(pixel area mm2) in each tumor cores. Spatial relationships between cellular phenotypes CD20+ and CD31+ cells in the peritumoral area were determined using the phenoptrReports package (Akoya Biosciences). Then, the distribution between CD20+ and CD31+ cells was identified in consecutive 10 µm steps (distance classes) within 200 µm.
HMGB1 immunohistochemistry was quantified in tissues using ImageJ (Ver. 1.52b). As tumorous samples consisted of mixed cell types including tumor cells and immune cells, image segmentation was first performed to isolate tumor and non-tumor regions. This allowed us to determine the regions and boundaries between the tumor cells and surrounding stroma. A supervised training of the Trainable Weka Segmentation (TWS) from Fiji (ver. 3.2.3)  was performed. A training set of five randomly selected 1604 × 1604 pixel images consisted of features from three classes: tumor, non-tumor, and background. The feature set used for training included a total of 80 attributes. A multithreaded implementation of random forest classifier with 200 trees and two features per node was used to build the model. The model was then applied to all images for classification. Representative results of segmentation are presented in Fig. 1A. Quantification of staining intensity of HMGB1 in tumorous or normal tissue were performed by first obtaining average pixel intensity of the identified tumor region in the tumorous samples or average intensity of the healthy tissue samples, respectively, followed by background subtraction.
Establishment of stable HMGB1 overexpression cell lines
K510 and EC18 ESCC cell lines were maintained in RPMI and DMEM supplemented with 10% FBS and antibiotics, respectively. To generate stable HMGB1-overexpressing clones, cells were transfected by lipofectamine 2000 with pcDNA3.1 vector. Stable cell lines were selected by adding 500 µg/mL G418 for EC18 tumor cells and 100 µg/mL for K510 tumor cells.
Co-culture of tumor and B cells
ESCC overexpressing the HMGB1 gene (K510H or EC18H) or vector (K510pc or EC18pc) were seeded into a 24-well plate (5 × 104/mL/well) and used for co-culture experiments at 80% confluence. CD20+ B cells (1 × 106 cells) were pre-treated with 5 µg/mL IgM and 100 ng/mL IL-4 for 24 h, washed, and co-cultured with the relevant ESCC cell line (5 × 104 cells/well) through microporous cell inserts (1 µm pore size) in a 24-well plate for 6 days. Where indicated, the HMGB1 inhibitor GL (0.5 mM; Sigma), anti-HMGB1 monoclonal antibody (αHMGB1; 10 µg/mL; Arigo), or DMSO were added to ESCC cell lines before co-culture.
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated using the RNeasy Mini kit (Qiagen). RNA was reversed transcribed (Takara) into cDNA, which served as a template for the amplification by qRT-PCR using the SYBR Green gene expression assay (Applied Biosystems 7900). Relative quantification was measured using the comparative Ct (threshold cycle) and the Ct values were normalized to β-actin or GAPDH, where appropriate. Primers used are shown in Supplementary Table 3.
RNA-Seq and transcriptomic expression analysis
CD20+ B cells isolated from two healthy donor PBMC samples were subjected to a migration study for 24 h. B cells that migrated toward recombinant HMGB1 or medium only as a control were collected and RNA was isolated for sequencing using the SMART-Seq™ v4 Ultra™ Low Input RNA Kit. PCR products were amplified and sequenced on an Illumina HiSeq™ 2500 platform by Novogene (Beijing China). High-quality clean reads from all two samples were merged together and mapped to the reference sequence. To determine the biological significance of the differentially expressed genes, which were defined as genes with log2 expression fold change ≥ 0.5,or ≤ − 0.5, functional classification and gene enrichment analysis were performed using GO Term (Biological Process level 5) with DAVID Bioinformatics Resources. Top ten highly enriched functional categories were listed and arranged in descending order of p-value of enrichment.
B cells collected from the transwell of co-cultured systems were washed and re-suspended in FACS buffer (PBS, 0.5% BSA, and 2 mM EDTA). Cells were stained with antibodies to the surface markers CD19, CD27, and CD38 (BD Biosciences) for 30 min on ice, followed by intracellular staining for Ki67 and VEGFA (BD Biosciences) using a BD Cytofix/Cytoperm kit. Antibodies were diluted FACS buffer for surface staining and in BD Perm/Wash buffer for intracellular staining. Cells were analyzed on a NovoCyte Quanteon and the data were analyzed using FlowJo Software.
Anti-phospho-ERK (1:500, sc-7383, Santa Cruz Biotechnology), anti-ERK (1:500, sc-94, Santa Cruz Biotechnology), anti-phospho-p38 (1:1000, 9211, Cell Signaling Technology), anti-p38 (1:1000, 9212, Cell Signaling Technology), anti-HMGB1 (1:1000, ab18256, Abcam), anti-beta-actin (1:5000, ab6276, Abcam), and anti-VEGF (1:1000, ab46154, Abcam) were used as primary antibodies. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitor on ice for 45 min and collected by centrifugation at 16000×g for 15 min at 4 °C. Protein concentrations were measured by the bicinchoninic acid protein assay kit (Pierce). Cell lysates containing 30 µg of total protein were separated by 10% SDS-PAGE and subsequently transferred to nitrocellulose membranes. The membranes were subsequently probed with the indicated antibodies and proteins were detected using the ECL Plus Western Blotting Detection System (GE Healthcare).
In vivo tumor experiments
For co-implanting tumor cells with B cells, 6-week-old NOD/SCID mice were irradiated at 300 cGy, then 4 h later, purified CD20+ B cells pre-treated with IgM and IL-4 were mixed with tumor cells (1 × 106 HMGB1-overexpressing/empty vectors in 100 µl of PBS, n = 6 per group) in growth factor-reduced Matrigel (BD Biosciences) at a 5:1 ratio, and then implanted subcutaneously into mice under anesthesia. B cells or tumor cells alone served as controls. Tumor growth was monitored every 2 days for the indicated time. Excised tissues were divided into portions for mIHC and FACS. To increase the yield of B cells, two tissues from the same group were pooled together for enzymatic dissociation and B cells were sorted based on Epcam (epithelial cell marker), CD45 (immune cell marker), and CD20 (B-cell marker), followed by qRT-PCR analysis. A loss-of-function analysis was performed by subcutaneous injection with 5 × 106 tumor cells. Five days later, mice were treated with 50 μL CM collected from co-culture by intratumoral injection for consecutive 5 days. The control group received CM from co-culture in the absence of GL. Tumors were harvested to prepare paraffin tissue sections for immunofluorescent staining.
Differences in quantitative variables were analyzed by the Mann–Whitney U test when comparing two groups and by the Kruskal–Wallis with Dunn’s post hoc test when comparing more than two groups. All analyses were performed using GraphPad Prism software. Survival curves were generated according to the Kaplan–Meier method, and statistical analysis was performed using the log-rank test. The association between HMGB1 expression and clinicopathological characteristics was tested by Pearson’s Chi-Square test. p value < 0.05 was considered statistically significant.