Lymph nodes (LNs) are frequently the first sites of metastasis. Currently, the only prognostic LN assessment is determining metastatic status. However, there is evidence suggesting that LN metastasis is facilitated by the formation of a pre‐metastatic niche induced by tumour derived extracellular vehicles (EVs). Therefore, it is important to detect and modify the LN environmental changes. Earlier work has demonstrated that neutrophil extracellular traps (NETs) can sequester and promote distant metastasis. Here, we first confirmed that LN NETs are associated with reduced patient survival. Next, we demonstrated that NETs deposition precedes LN metastasis and NETs inhibition diminishes LN metastases in animal models. Furthermore, we discovered that EVs are essential to the formation of LN NETs. Finally, we showed that lymphatic endothelial cells secrete CXCL8/2 in response to EVs inducing NETs formation and the promotion of LN metastasis. Our findings reveal the role of EV‐induced NETs in LN metastasis and provide potential immunotherapeutic vulnerabilities that may occur early in the metastatic cascade.
Metastasis is responsible for 90% of all cancer-related deaths, making it the most significant challenge in cancer treatment for clinicians worldwide. Metastasis is thought to occur in a stepwise process, in which local lymph nodes (LNs) are first colonized by tumour cells before proceeding to distal organs. However, the mechanism by which LN metastasis facilitates distal metastasis is poorly understood. LNs undergo environmental changes to accommodate tumour cell growth within, notably by shifting the environment towards immunosuppression to shut down anti-tumour immune cells. This immunosuppressive environment is critical for the establishment of LN metastases, as cytotoxic CD8 T cells will otherwise neutralize incoming circulating tumour cells. Neutrophils are among the first cells recruited to tumour-draining LNs to mediate the environmental shift, and yet their method of action has not been fully elucidated. They have recently been found to secrete NETs within LNs during cancer, which are pro-inflammatory web-like formations of DNA decorated with antimicrobial peptides. While NETs have been shown to exert pro-tumour effects in the tumour microenvironment and to facilitate metastasis as a whole, their role in LNs has never been explored. Using a NETs deficient mouse model (PAD4-/-), we report that NETs deposition within the tumour-draining LNs of mice upregulates the Treg population while simultaneously downregulating anti-tumour CD8 T cell proliferation and activation. Following these findings, we performed an in vitro suppression assay and found that NETs-educated Tregs hinder the expansion of CD8 T cells more than non-NETs-educated Tregs. Finally, through an induced metastasis mouse model, we have observed that tumour-draining LN resection as well as the absence of NETs protects against the development of distal metastasis, and decreases body-wide inflammation as seen by a lowered Neutrophil-to-Lymphocyte ratio in the circulation. Taken together, these findings highlight the role of NETs deposition in LNs as a key player in disease progression and bring forward a potential target for anti-metastatic drug development. Citation Format: Ariane Brassard, Xin Su, Iqraa Dhoparee-Doomah, Sabrina Leo, Lixuan Feng, France Bourdeau, Betty Giannias, Corissa Larson, Qian Qiu, Jonathan Spicer, Lorenzo Ferri, Jonathan Cools-Lartigue. NETs act as immunosuppressive agents within lymph nodes during cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1315.
Background: While the incidence of esophageal adenocarcinoma (EAC) is rapidly increasing, the 5-year survival rate of this disease remains poor at less than 25%, with a substantial number of patients exhibiting inherent or acquired resistance to chemotherapy. Circulating tumor DNA (ctDNA) isolated from liquid biopsies of EAC patients holds clinical biomarker potential in this disease, but our understanding of how chemotherapy affects ctDNA release remains limited. Methods: In order to study the impact of cancer treatment on ctDNA emission in vitro, we first sought to establish a chemo-resistant model of EAC. As such, the EAC cell line OE19 was exposed to discontinuous cisplatin treatments for three months in order to establish drug resistance. Following this, established chemo-resistant OE19 cells and parental (chemo-sensitive) OE19 cells were exposed to five daily cisplatin treatments, and cell culture supernatant was collected every day throughout treatment. ctDNA was subsequently isolated from cell culture supernatant and quantified by Qubit fluorometer dsDNA HS assay. ctDNA quantities were additionally assessed by droplet digital PCR (ddPCR) assay using primers and probes targeting the TP53 N310K mutation in the OE19 cell line. Finally, ctDNA fragment lengths were analyzed using the Agilent Bioanalyzer 2100. Results: A chemo-resistant model of the OE19 cell line was established, as exemplified by marked morphological changes in the cells and significantly increased cell viability determined by CCK8 assay after 48-hour treatments of various doses of cisplatin (p<0.05). ctDNA levels, as measured by both Qubit fluorometer and mutant copies detected by ddPCR assay, increased after chemotherapy treatment for both chemo-sensitive and resistant cells. Notably, ctDNA emission was higher with larger amounts cell death, with chemo-sensitive cells emitting significantly higher levels of ctDNA compared to chemo-resistant cells throughout treatment (p<0.05). Fragmentomics analysis revealed ctDNA peaks corresponding to apoptosis (~167 bp) and necrosis (~10,000 bp). Moreover, chemotherapy treatment caused a shift in average fragment size, with larger fragments being observed during chemotherapy treatment in both chemo-sensitive and resistant cells. Conclusion: This study reveals that in vitro cancer models can be used to successfully study ctDNA emission during pre-clinical drug analyses. ctDNA release was found to be correlated to cytotoxicity and cell death, providing us with valuable insight into how clinical liquid biopsy data should be interpreted. Finally, fragment size analysis of ctDNA reveals a potential novel biomarker for treatment response in cancer patients. Citation Format: Alexandra Bartolomucci, Sarah Tadhg Ferrier, Thupten Tsering, Xin Su, Jonathan Cools-Lartigue, Julia V. Burnier. Characterizing circulating tumor DNA release kinetics and fragment length in a chemotherapy resistant model of esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6700.
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