The Immunomodulatory Effects Of CXCR4 Antagonist AMD3100 In Treatment Of Tumors And Its Mechanism For Promoting Tumor Cell Growth

CXCR4 is a specific receptor of chemokine CXCL12.CXCL12/CXCR4 signal axis plays an important role in tumor cell proliferation,migration,angiogenesis and invasiveness.CXCL12/CXCR4 signal axis may represent a promising therapeutic target in blood disorders and solid tumors.As a highly specific and effective CXCR4antagonist,AMD3100 has been used to study the therapeutic potential for many diseases related to CXCL12/CXCR4 signal axis.Previous studies in our laboratory have demonstrated that AMD3100 shows unique advantages in combination with other drugs in the treatment of tumors.We have found that AMD3100 in combination with PD-1/PD-L1 checkpoint inhibitors can increase effector T cell infiltration in the tumor microenvironment of ovarian cancer,reduce intratumoral Treg and reprogram them into helper T cells.AMD3100 in combination with VIC008（a fusion protein with tumor antigen targeting and immune activation function）can reduce the PD1 expression on infiltrating CD8⁺T cells in mesothelioma mice and prolong the survival time of tumor-bearing mice.Through bioinformatics analysis using public data,we have found that the regulatory mechanism of AMD3100 on breast cancer is the most complex.Based on the previous studies in our laboratory,the current study attempts to explore the therapeutic effect of AMD3100 in combination with immune checkpoint inhibitors on breast cancer.After new phenomenons have been found from the combination therapy,we focused on theregulatory effects of AMD3100 alone on the immune microenvironment of breast cancer in vivo and the underlying mechanisms of AMD3100 on regulation of breast cancer and mesothelioma cells in vitro.Objective:In this study,breast cancer and mesothelioma cell lines were used to investigate the regulation of CXCR4 on the cellular phenotype of tumor cells,such as proliferation,migration,apoptosis,cell cycle,and the underlying molecular mechanisms.The mouse breast cancer model was used to study the effect of AMD3100 in combination with immune checkpoint inhibitor on tumor growth and survival time of tumor-bearing mice,and the regulatory effect of AMD3100 alone on tumor growth and tumor immune microenvironment in mouse breast cancer and mesothelioma models.The purpose of this study is to explore whether AMD3100 can interact with other molecules or pathways in addition to targeting the CXCL12/CXCR4 axis to regulate the biological behavior of tumors.This study can provide new ideas for the preclinical study of AMD3100 and a new theoretical basis for its clinical use of taboos or combination with other drugs.Methods:1.The effects of AMD3100 on tumor growth and survival in mice with breast cancer and mesothelioma,and the use of public data to analyze the clinical feature distribution,immune score and prediction of patients’response to immune checkpoint blockage in patients with high and low expression of CXCR4（1）The orthotopic tumor model of breast cancer was established in female BALB/c mice and treated with AMD3100in combination with anti-TIGIT antibody and anti-PD-1 antibody to observe the tumor growth and survival of tumor-bearing mice.（2）The mesothelioma model of C57BL/6J mice was established and treated with AMD3100 alone.The tumor growth and survival of tumor-bearing mice were observed by in vivo imaging technique.（3）Bioinformatics techniques were used to analyze the relationship between CXCR4expression levels and prognosis of breast cancer patients in TCGA database,the relationship between CXCR4 expression level and tumor clinical stages and survival proportion of patients（Sankey diagram:The width of the extended branches in the figure corresponds to the size of the data traffic）,the distribution of clinical characteristics and immune score of patients with high and low expression of CXCR4,the score coefficient of AMD3100 involved in regulating various diseases,and prediction of the disease phenotype that AMD3100 may be involved.2.Regulatory effect of AMD3100 on multiple tumoral phenotypes（1）Cy QUANT assay was used to verify the effect of AMD3100 on the proliferation of mesothelioma cell lines 40L and AE17 in vitro before and after CXCR4 m RNA was knocked down by small interference RNA.（2）Colony formation assay and CCK8 assay was used to verify the effect of AMD3100 on the proliferation of breast cancer cell lines（4T1,E0771,MDA-MB-231,MCF7）in vitro.（3）The effects of AMD3100 on the migration ability of breast cancer cell lines（4T1,E0771,MDA-MB-231,MCF7）were verified by scratch and Transwell migration assays.（4）The effects of AMD3100 on apoptosis（Annexin V-PI or Annexin V-7AAD）and cell cycle（PI）of breast cancer cell lines（4T1,E0771,MDA-MB-231,MCF7）were evaluated by flow cytometry.（5）4T1,E0771 and MDA-MB-231 cell lines with low expression of CXCR4 were transfected with CXCR4 lentivirus to construct breast cancer cell lines stably expressing CXCR4.The stable expression level of CXCR4 in the transfected cell lines was identified by flow cytometry.（6）CCK8 assay was used to verified the effect of AMD3100 on the proliferation of breast cancer cell lines stably expressing CXCR4 in high or low CXCL12 environment.In addition,Balixafortide（TFA）（another highly effective antagonist of CXCR4）was used as a positive control while Ed U assay was used to detect the effect of AMD3100on the proliferation of different breast cancer cells.3.Study on the mechanism of AMD3100 promoting the proliferation of mesothelioma and breast cancer cell linesWestern blot was used to detect the expression level of related pathway proteins.4.Regulatory effect of AMD3100 on tumor microenvironment in mouse breast cancer model（1）The subcutaneous tumor model of breast cancer was established in 6-8-week-old female C57BL/6J or female BALB/c mice.Four days after tumor injection,mice were treated with drugs via tail vein injection every other day,and the size of the tumor was measured and recorded accordingly.28 days after tumor establishment,the mice were CO₂-anesthetized.Peripheral blood,tumor tissue,spleen and tumor draining lymph nodes,liver and lung were taken and weighed,and the data were statistically analyzed by Graph Pad Prism 9.（2）Flow cytometry was used to measure the proportion of CD8⁺T cells and its surface PD-1,TIGIT and TIM3 in peripheral blood,spleen,tumor draining lymph nodes and tumor,and the proportion of M1 and M2 macrophages and myeloid inhibitory cells MDSC in tumor microenvironment.（3）Immunofluorescence was used to detect the deposition of infiltrating CD8⁺T cells,angiogenesis（CD31）,hypoxia（HIF-1α）,fibroblasts（αSMA）and typeⅠcollagen（collagenⅠ）in tumor tissue.Results:1.In the mouse model of breast cancer,AMD3100 in combination with immune checkpoint inhibitors（anti-TIGIT antibody and anti-PD-1 antibody）could not inhibit the growth of tumor.AMD3100 alone had a tendency to slightly promote the growth of tumor and slightly prolong the survival time of mice,but the statistical results were not significant.In the two mesothelioma mouse models,AMD3100 also slightly promoted tumor growth and slightly prolonged the survival time of mice,but the statistical results were not significant.Bioinformatics analysis showed that the expression of CXCR4 in breast cancer patients was higher than that in normal subjects,which however could not be used as an independent prognostic factor.Sankey diagram showed that there was no direct relationship among the four stages of primary cancer（T1-4）,the expression level of CXCR4 and the number of deaths.The patients were further divided into CXCR4low expression group and CXCR4 high expression group.Compared with the distribution of clinical features in the two groups（p T stage,p N stage,p TNM stage,tumor type,radiotherapy and treatment type）,it was found that all kinds of clinical features and CXCR4 expression were not significantly statistically correlated.The results of immune cell infiltration score showed that patients with high expression of CXCR4 had more immune cell infiltration in tumor,while patients with low expression of CXCR4 showed low level of immune cell infiltration in tumor.Breast cancer patients with high expression of CXCR4 were accompanied by high expression of immunosuppressive molecules on intratumoral immune cells.The prediction results of TIDE algorithm showed that the group with high expression of CXCR4 had a stronger response to the immune checkpoints blockade.The results of CTD and DGIdb database show that the regulation of AMD3100 on breast cancer was the most complex.The main phenotypes involved in the regulation of AMD3100 on breast cancer cells are cell proliferation,migration,apoptosis,epithelial-mesenchymal transformation（EMT）and so on.2.AMD3100 could promote the proliferation of mesothelioma cell lines 40L and AE17 in a concentration-dependent manner.AMD3100 still promoted the proliferation of 40L and AE17 cells after knockdown of CXCR4 using si RNA.CCK8 experiment showed that AMD3100 could promote the proliferation of breast cancer cells（4T1,E0771,MDA-MB-231,MCF7）.Transwell and scratch experiments showed that AMD3100 could inhibit the migration of breast cancer cells.Annexin V-PI apoptosis assay showed that AMD3100 had little effect on the apoptosis of the four breast cancer cell lines.Cell cycle experiments showed that AMD3100 did not affect the cycle of the four breast cancer cell lines.q RT-PCR and flow cytometry showed that compared with normal breast epithelial cells with low expression of CXCR4,4T1,E0771,and MDA-MB-231 had low expression of CXCR4 while MCF7 had high expression of CXCR4.The expression of CXCR4 of all those cell lines did not change after AMD3100treatment.The results of flow and fluorescence showed that the breast cancer cell line stably overexpressing CXCR4 was successfully constructed.We used TFA as a control.The results of CCK8 and Ed U showed that AMD3100 could promote the proliferation of 4T1-CXCR4 and E0771-CXCR4 regardless of the high or low expressiom level of CXCL12 or CXCR4,while TFA did not promote cell proliferation.3.Western Blot results showed that AMD3100 could promote the phosphorylation of AKT in 40L and AE17 cells in a concentration-dependent manner.In both low and high CXCL12 environments,AMD3100 could promote the oxidative inactivation of upstream inhibitory molecule PTEN in AKT regardless of the level of CXCR4 expression while the total PTEN level remained unchanged.It was also found that 40L did not express CXCR7 while AE17 overexpressed CXCR7.In addition,in the high CXCL12 environment,in 40L cells the level of ERK phosphorylation in AMD3100 group and si RAN-CXCR4 group was lower than that in wild type cell group.In AE17 cells the level of ERK phosphorylation did not change in all groups.In 4T1and E0771 mouse breast cancer cell lines,AMD3100 could promote the phosphorylation of AKT,but there was no significant change in ox-PTEN level.When the expression of CXCR4 is low,AMD3100 could promote the phosphorylation of PI3K and ERK in 4T1 cells.When the expression of CXCR4 was high,AMD3100could inhibit the phosphorylation of PI3K and ERK.4.The results of in vivo experiments showed that when the expression of CXCR4was low,AMD3100 at 3 mg/kg could significantly promote tumor growth,lung metastasis in 4T1 breast cancer model and tumor angiogenesis.When CXCR4 was highly expressed,AMD3100 did not significantly promote tumor growth and tumor metastasis into lung.The results of flow cytometry showed that AMD3100 had little effect on the proportion of CD4⁺T/CD8⁺T cells in peripheral blood,spleen,lymph nodes and tumors in each group.When CXCR4 was highly expressed,AMD3100down-regulated the proportion of tumor infiltrating MDSC cells and promoted the polarization from pro-tumor M2 macrophages to pro-inflammatory and anti-tumor M1macrophages.Immunofluorescence results also showed that when CXCR4 was highly expressed,AMD3100 could inhibit tumor fibrosis and type I collagen deposition,and increase CD8⁺T cell intratumoral infiltration.Conclusions:AMD3100 plays a dual role of promoting and inhibiting cancer in tumor regulation.When CXCR4 expression is low,AMD3100 can inactivate PTEN by oxidization and then activate AKT signal pathway,which promotes the proliferation of tumor cells.When CXCR4 is highly expressed,AMD3100 can partially reverse tumor growth by blocking CXCL12/CXCR4 signal axis in vitro.Moreover,in vivo,AMD3100 can regulate tumor immune microenvironment by promoting the polarization from M2macrophages to M1 macrophages,reducing intratumoral MDSC,reducing tumor fibrosis and promoting CD8⁺T cell infiltration,thus plays a role in tumor inhibition.This study provides a theoretical basis for the safe,precisional and combinatorial use of CXCL12/CXCR4-targeted drugs such as AMD3100 in preclinical and clinical settings.