Research On Mechanisms Of Tumor-associated Neutrophils-secreted AGR2 In Promoting Colorectal Cancer Metastasis

Objective: Reciprocal cellular crosstalk within the tumour microenvironment(TME)actively participates in tumor metastasis.The anterior gradient-2(AGR2)can be secreted to extracellular compartments and contribute to colorectal cancer(CRC)metastasis.Nevertheless,the cellular source for secreted AGR2 in the TME and how secreted AGR2 functionally facilitates CRC metastasis were largely unclear.In this study,we aim to investigate the cellular source of secreted AGR2 in the TME and the underlying mechanisms mediating secreted AGR2’s effects.Methods:1.Aim to investigate the cellular source of secreted AGR2 in the TME.(1)Tumor tissues were collected from CRC patients and the colocalization of AGR2 with cell-type markers were observed in paraffin-fixed CRC tissues using immunofluorescence costaining.Furthermore,the m RNA and protein expression levels of AGR2 were quantified in tumor-associated neutrophils(TANs)collected from tumor tissues of CRC patients.(2)The relationships of TANs infiltration(the number and percentage of infiltrating AGR2+TANs)with the clinicopathological features and outcomes of CRC patients were analyzed in the tissue microarray cohort.(3)The transgenic mice with conditional Agr2 knockout in neutrophils(Agr2f/f;Mrp-Cre)were generated and the experimental liver metastasis model(intrasplenic injection of mouse CRC cells)was established in Agr2f/f;Mrp-Cre mice to determine the role TANs-secreted AGR2 in CRC metastasis in vivo.(4)Transwell migration assay was used to detect the impact of secreted AGR2(Recombinant human AGR2,rAGR2)on CRC cells migration.The phalloidin(F-actin)staing was used to assess the impact of rAGR2 on CRC cells cytoskeletal rearrangement(Factin-rich stress fibres and filopodium-like protrusions).2.Aim to explore the underlying mechanisms mediating secreted AGR2’s effects.(1)The potential binding partners of TANs-secreted AGR2 were identified by liquid chromatography-mass spectrometry analysis.The interaction of secreted AGR2 with the receptor was further validated using co-immunoprecipitation and immunofluorescence costaining.(2)Transwell migration assay and F-actin staining were used to determine the role of potential receptor in secreted AGR2’s effect on CRC cells migration and cytoskeletal rearrangement.(3)The experimental lung metastasis model(intravenous injection of CRC cells and intraperitoneal injection of rAGR2)was established to detect the role of the receptor in secreted AGR2’s effect on CRC metastasis in vivo.(4)The intracellular mechanisms mediating secreted AGR2’s effects on CRC metastasis were further analyzed based on literature search,RNA-seq analysis and experiments of molecular biology.Results:(1)AGR2 is preferentially expressed in TANs within the TME of CRC.(2)Both the number and percentage of infiltrating AGR2+TANs within tumour tissues were positively and negatively correlated with American Joint Committee on Cancer tumour(AJCC)stages and overall survival of patients with CRC,respectively.(3)Neutrophils specific ablation of Agr2 in mice ameliorated CRC liver metastases in vivo.(4)rAGR2 promoted cell migration and increased the number of F-actin-rich stress fibres and filopodium-like protrusions in CRC cells.(5)Secreted AGR2 physically interacted with the heavy chain of CD98(CD98hc)at the cell membrane of CRC cells.(6)On rAGR2 stimulation,CD98 hc knockout significantly reduced CRC cells’ migration and decreased the number of F-actin-rich stress fibres and filopodium-like protrusions.(7)The number of metastatic foci increased in mice receiving intraperitoneal injection of rAGR2,and stable CD98 hc knockdown in CRC cells drastically reversed rAGR2-induced lung metastasis.(8)x CT(CD98 light chain)was involved in secreted AGR2’s migration-promoting effect.Moreover,secreted AGR2 increased x CT’s cystine-transport activity and thus activated GSH-Rho A-ROCK2 cascade to facilitate CRC metastasis.Conclusions: TANs were a predominant cell type for secreting AGR2 in the TME of CRC.TANs-secreted AGR2 promotes CRC metastasis through interacting with the functional receptor CD98hc-x CT,subsequently increasing x CT activity to activate GSH-Rho AROCK2 cascade.Our study defines a novel mechanism mediating TANs-secreted AGR2 ’s effect on CRC metastasis.Targeting this cascade might be a promising approach for inhibiting CRC metastasis.