Lymph

CD45.2 C57BL/6 (B6) and CD45.1 B6 mice were from the National Cancer Institute or a colony maintained at the University of California, San Francisco. Mice lacking Sphk2 and carrying LoxP-flanked Sphk1 were on a B6/129 mixed background (Pappu et al., 2007 (link)). Lyve-1 Cre knockin mice on a B6/129 mixed background were generated as described in Fig. S1. Lyve-1 Cre⁺ Sphk1^{f/− or f/f} Sphk2^−/− mice were generated by intercrossing. Control mice were usually littermates and were always from the same intercross and carried at least one wild-type Sphk allele. Rosa26-YFP reporter mice (Srinivas et al., 2001 (link)) were provided by N. Killeen (University of California, San Francisco, San Francisco, CA). To generate BM chimeras, recipient CD45.2⁺ mice were lethally irradiated with 1,300 rads in two doses separated by 3 h and injected with 5 × 10⁶ wild-type BM cells prepared from a CD45.1⁺ donor. In some experiments, ∼2 × 10⁷ cells/ml were labeled with 3.3 µM CFSE (Invitrogen) or 10 µM 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR; Invitrogen) in RPMI 1640 containing 2% FCS for 20 min at 37°C, and were then washed by spinning through a layer of FCS. Labeled cells were resuspended at ∼2 × 10⁷ cells/ml, and were treated with 10 ng/ml OB or PTX at 37°C for 10 min, washed twice in warm RPMI 1640 with 2% FCS and 10 mM Hepes, and transferred to recipient mice. Lymph collection was performed as previously described (Matloubian et al., 2004 (link)). In brief, under a stereomicroscope, lymph was drawn from the cysterna chyli using a fine borosilicate glass microcapillary pipette (Sutter Instrument Co.). Cell numbers determined by flow cytometry were divided by the volume of collected lymph to determine the concentration. Protocols were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.

All eligible ESCC patients underwent esophagectomy via Ivor-Lewis or McKeown procedures with standard 2-field lymphadenectomy. All Siewert AEG procedures were performed via the Ivor-Lewis procedure or combined thoracoabdominal approach. Proximal gastrectomy was routinely performed in AEG patients. Patients with a combined thoracoabdominal approach did not have esophageal involvement of more than 3.0 cm, and upper mediastinal lymph node metastasis was evaluated by preoperative CT scan. Due to the surgical approach, these AEG patients did not undergo complete upper mediastinal lymphadenectomy. In this study, the mediastinal lymph nodes were divided into upper and lower areas. The upper mediastinal lymph area included the left and right recurrent laryngeal nerve, upper thoracic paraesophageal, paratracheal, and subcarinal lymph nodes. The depth of the primary tumor, grade of the tumor, degree of lymph node, and TNM staging were defined according to the Union Internationale Against Cancer (UICC)/American Joint Committee on Cancer (AJCC) TNM classification (8th edition) (3 ).

Pieces of tumors were collected and digested overnight in digestion buffer (2.1 mg/mL collagenase type I (Worthington), 75 µg/mL DNAase I (Worthington), 5 mM CaCl₂ and 1% P/S in RPMI). The following day, samples were incubated for 15 min at 37°C and filtered through a 70 µm nylon cell strainer and erythrocytes lysed using the red blood cells (RBC) lysing buffer (Sigma) for 1–2 min. Splenocytes (isolated from spleens of female mice) were instead generated by forcing freshly isolated spleens through 70 µm cell strainers and erythrocytes lysed using RBC lysing buffer for 1 min. Following centrifugation, cells (tumor/splenocytes) were washed and eventually resuspended in PBS. Single-cell suspensions from tumors/spleens were stained for 30 min (4°C in the dark) for relevant cell-surface markers in Fluorescence-activated cell sorter (FACS) staining buffer (PBS with 7% FBS). Next, cells were washed, resuspended in PBS and stored at 4°C until flow cytometric analyses were performed on the same day. All samples were acquired on Cytek Aurora equipped with four lasers (Cytek Biosciences) or by FACSCanto II cell Analyzer BD and analyzed with FlowJo V.10.6.1 or V.10.8 software. Viable cells were determined as a fraction of single cells negative for staining with Zombie Aqua Fixable Viability Dye. The immune cell populations were analyzed in three different panels: Panel 1 (general panel), Panel 2 (myeloid panel) and Panel 3 (lymphoid panel), which are described in online supplemental materials and methods. All cells were gated on singlets, living cells and the corresponding gating strategy for each cell population.