Jonathan Cools-Lartigue

Metastasis accounts for most cancer-related mortality, and regional lymph nodes (LNs) are among the earliest sites of dissemination for many solid tumours. Yet tumour-draining lymph nodes (TDLNs) are also sites of adaptive immune activation, raising the question of how these immune organs become permissive to metastatic growth. We investigated whether neutrophil extracellular traps (NETs), which accumulate in TDLNs before detectable tumour-cell arrival, contribute to immune conditioning of histologically tumour-negative nodes and whether the consequences of this conditioning extend beyond the initiating tumour. Using multiplex immunofluorescence and spatial transcriptomic profiling of TDLNs from 19 patients with gastroesophageal adenocarcinoma, we found that NET positivity in non-metastatic TDLNs was associated with inferior overall survival and a distinct immunoregulatory state characterized by closer NET–Treg spatial relationships, coordinated increases in Treg-associated transcriptional programs, altered immune composition and redistribution of CD8⁺ T cells away from Tregs. NET-positive nodes also showed reduced CD8⁺ naïve/stem-like transcription, while greater peri-Treg CD8 redistribution was associated with lower cytotoxic-effector transcription. In vitro, direct NET exposure shifted human T cells toward regulatory and dysfunction-associated phenotypes and impaired tumour-cell control. NET-conditioned Tregs further suppressed CD8⁺ T-cell proliferation and granzyme B expression, while depletion of Tregs attenuated NET-associated CD8⁺ apoptosis. In tumour-bearing mice, neutrophil elastase inhibition altered activated Treg distribution across tumour-draining and contralateral LNs. Importantly, prior H59 tumour conditioning increased susceptibility to a subsequent heterologous MC38 metastatic challenge after removal of the initiating tumour; this phenotype was attenuated by TDLN resection and PAD4 deficiency. Adoptive transfer of contralateral-LN T cells from conditioned donors further identified a transferable component of this metastasis-permissive state that was sensitive to NET-directed intervention. These findings support a model in which NET-associated nodal immune conditioning alters Treg and CD8⁺ T-cell organization and function before overt nodal metastasis. The consequences of this conditioning extend beyond the draining node and can shape host susceptibility to a subsequent heterologous tumour challenge, broadening the premetastatic-niche concept toward conditioning of the host immune state.

Background: Successive advances in perioperative systemic therapy have improved outcomes in gastroesophageal adenocarcinoma, yet a substantial proportion of patients fail to complete treatment, and omission of postoperative therapy has been associated with poorer survival. We assessed whether perioperative changes in CBC-derived neutrophil-to-lymphocyte ratio (cNLR) and albumin added information about treatment initiation beyond tumour pathology and whether initiation was associated with survival. Methods: This retrospective cohort included 243 patients undergoing resection at the McGill University Health Centre from 2006 to 2025. Sequential logistic models evaluated pathological characteristics, pretreatment host variables and perioperative changes in cNLR and albumin. Postoperative measurements preceded adjuvant initiation. Nested comparisons used matched complete cases. Day-90 landmark and time-varying Cox models assessed associations with overall survival (OS) and disease-free survival (DFS). Results: Seventy-eight patients (32.1%) did not initiate adjuvant therapy. Adding host variables improved discrimination beyond pathology (AUC 0.672 to 0.763; P=.002). Greater perioperative cNLR increase was associated with lower odds of initiation (adjusted OR 0.67 per unit increase in Δlog₂ cNLR, 95% CI 0.47–0.95), whereas more favourable albumin change was associated with higher odds (OR 1.07 per g/L, 95% CI 1.01–1.13). Neither marker remained independently associated when modelled together. Initiation by day 90 was associated with longer OS (HR 0.53, 95% CI 0.36–0.78) and DFS (HR 0.57, 95% CI 0.39–0.84). Conclusions: Host inflammatory and albumin changes added information about adjuvant initiation beyond tumour pathology, and initiation was associated with longer survival. Prospective evaluation should determine whether host-directed perioperative strategies improve postoperative treatment delivery.

Background: Circulating tumor DNA (ctDNA) has emerged as a clinically valuable biomarker for cancer detection, treatment monitoring, and minimal residual disease assessment. Despite its growing clinical utility, the biological mechanisms governing ctDNA release remain incompletely understood. The objective of this study was to investigate how chemotherapy-induced cytotoxicity and chemoresistance influence the kinetics and fragmentation patterns of cfDNA released by cancer cells in vitro. Methods: Human lung adenocarcinoma (A549) and esophageal adenocarcinoma (FLO-1, OE19) cell lines were used to investigate cell-free DNA (cfDNA) release and fragmentation in vitro. Cancer cells were treated with chemotherapy (cisplatin and 5-fluorouracil), and cfDNA released into the culture medium was quantified as total cfDNA by Qubit fluorometry and mutation-specific cfDNA by droplet digital PCR (ddPCR). A cisplatin-resistant OE19 model was generated to directly compare cfDNA release kinetics between chemosensitive and chemoresistant cells. Fragment size distributions were determined and evaluated alongside cell death mechanisms assessed by flow cytometry. Results: cfDNA release correlated positively with viable tumor cell number across all cell lines. Chemotherapy increased per-cell cfDNA release in all cell lines. Chemosensitive OE19 cells exhibited significantly higher cfDNA release compared to chemoresistant OE19 cells following cisplatin exposure, with distinct temporal release kinetics. Additionally, chemotherapy treatment induced a shift toward the release of larger DNA fragments in both chemosensitive and resistant cells. This was accompanied by changes in PI-positive and Annexin V/PI double-positive cell populations, suggesting altered cell death processes following cisplatin treatment. Conclusions: Chemotherapy-induced cytotoxicity significantly influences cfDNA release kinetics and fragmentation patterns, with distinct effects in chemosensitive and chemoresistant cancer cells. These findings provide mechanistic insight into tumor-derived DNA release biology and may have important implications for the interpretation, timing, and standardization of liquid biopsy testing during treatment. Furthermore, they establish in vitro cancer models as a valuable platform for studying cfDNA dynamics and informing preclinical therapeutic development.

GEC risk-associated index SNVs and loci.

List of genes and their LRT scores surrounding each GWAS index SNV.

The rs147518036 SNV genotypes for the gastric cancer cell lines.

List of files used from ENCODE

Verification of the inducible UXS1 expression and knockdown by RT-qPCR.

Expression of UXS1 in normal and tumor tissues.