Spatial distribution analysis of tertiary lymphoid structures in esophageal squamous cell carcinoma predicts patient survival and response to  neoadjuvant chemo-immunotherapy

Background: The prognostic and predictive significance of tertiary lymphoid structures (TLSs) exhibits spatial specificity in various cancers. However, the spatial distribution, phenotypic characteristics of TLSs in esophageal squamous cell carcinoma (ESCC) and their impact on prognosis and prediction are not yet fully understood. Methods: We performed multiplex immunofluorescence staining on 87 untreated ESCC specimens to simultaneously analyse TLS expression and phenotypic characteristics, CD8+ T cells and PNAD+ high endothelial venules (HEVs) in different spatial regions of ESCC tissues using Panel-1 (CD20/CD21/CD23/PNAD/CD8/Pan CK/DAPI). Panel-2 (CD20/ CD3/Foxp3/DC-LAMP/Pan CK/DAPI) was used to evaluate the spatial distribution of TLS-related immune cells (CD20+ B cells, CD3+ T cells, Foxp3+ Treg cells, and LAMP+ mature DCs) within the tumor microenvironment of ESCC. Furthermore, the predictive value of TLSs and the clinical prognostic significance of TLSs at different spatial locations were assessed in an independent cohort of 15 ESCC patients who received neoadjuvant chemo- immunotherapy (NACI). Results: In untreated ESCC, a high number/density of mature follicular TLSs (F-TLSs) at the distal (＞500μm) was significantly associated with better overall survival (OS) in patients (number: p=0.0092; density: p=0.0268). Patients with distal high F-TLSs not only exhibited high densities of CD3+ T cells, CD8+ T cells, CD20+ B cells, LAMP+ mature DCs, and PNAD+HEVs in the stromal regions, but this was also associated with increased CD8+ T cell infiltration within the tumor nests (p<0.05). Additionally, it was correlated with a reduced proportion of Foxp3+ Treg cells in the distal stromal regions. In patients with ESCC receiving NACI, the partial response (PR) group exhibited higher numbers and densities of F-TLSs post-treatment than the non-PR group (p<0.05). High numbers/densities of total TLSs, early-TLSs, and F-TLSs in the proximal stromal region (≤500μm) of post-treatment specimens were significantly associated with better OS (p<0.05). Conclusions: In untreated ESCC, distal mature F-TLSs are critical prognostic indicator, driving "favorable" immune infiltration by modulating the spatially heterogeneous immune microenvironment. After NACI, TLS quantity and spatial distribution were reshaped; mature F-TLSs predicted treatment response. Treatment-induced expansion of TLSs localised at the proximal tumour-stroma interface is a key indicator for predicting favourable long-term survival.