Study On The Tumor-targeting Property Of An Aptamer Specifically Binding To Myeloid-derived Suppressor Cells

[Background]Breast cancer is the most common cancer type in women,raising serious health issues worldwide.Massive studies focus on the pathological mechanism and prevention of breast cancer.Although so many anti-tumor drugs come to the market every year,drug therapeutics for solid tumors are not as efficacious as expected,because tumor-blood barriers impede drug penetration.Here,we introduce cell-mediated drug delivery as a new strategy for enhanced drug penetration.Polymorphonuclear Myeloid-derived suppressor cells(PMN-MDSCs)play a critical role in tumor growth and metastasis.Since they constantly migrate to the tumor tissue,MDSCs can serve as an ideal vehicle for targeting and penetrating tumors.We recently identified an MDSC-specific thioaptamer(T1)with tumor accumulating activity,demonstrating its potential for tumor-targeted drug delivery.Based on such,in the current study,we have carried out a structure-activity relationship analysis to further optimize the aptamer for tumor-targeted drug delivery.[Methods]1.Distribution of T1 in circulation was tested with Synergy?H4plate reader,distribution of cell-bound T1 was detected with flow cytometry,and distribution of T1 in vivo was observed through IVIS Imaging.2.Cy5 labeled T1 was given to mice i.v.by tail and bound to blood cells,blood and tumor tissue were harvested after in vivo binding,cells were sorted into two subsets:one with higher binding affinity to T1,and the other had a lower binding affinity to T1.To compare their abilities to migrate to tumors,the mobility of the sorted cells was measured with a transwell migration assay.To compare their activity in cytokine secretion,the sorted cells were treated with several ELISA assays.To study the transcriptome difference between the two sorted cells,the RNA-seq analysis was performed on cells of each group.On the other hand,the target cells infiltrating the tumor tissues were also analyzed with Luminex multi-cytokine/chemokine assay.3.The secondary structure of T1 was predicted with Mfold software,and studied by structure-activity relationship analysis to find the exact binding site of T1,DNA gel electrophoresis presented a piece of evidence for a tetramer formation.with a tumor spheroid 3D model,aptamers’penetration into the tumor by hitchhiking was observed.4.Statistical analysis:Data is presented as mean±s.d,Unpaired t test,two tailed was applied to comparison between two groups.Multiple t test was applied to multiple comparisons.One-way ANOVA with Turkey’s correction was applied to comparison between multiple groups at the same timepoint,The Two-way ANOVA was applied to comparison between multiple groups at different timepoint.For correlation analysis,data were fitted with linear regression,and Pearson correlation coefficients were calculated.[Results]1.within 4 hours,in vivo retention of Cy5-T1 aptamer was prolonged and their cell binding rate increased.T1 mainly bound to PMN-MDSC and M-MDSC in blood circulation(P<0.0001).In vivo distribution of T1 was dispersed and relatively stable.Ex vivo organs of the liver,spleen,bone,and tumor showed significant T1 accumulation.2.PBMCs were sorted according to their in vivo binding affinity to T1,those T1-binding cells preferentially migrated to tumors(P=0.0380),and secreted more immunosuppressive cytokines of CCL5(P<0.0001),IL-6(P<0.001)and IL-10(P<0.001).Based on the results of RNA-seq and transcriptome analysis,the differential gene expression of these two populations was mainly enriched in the GO pathways of taxis,chemotaxis,cytokine-cytokine receptor reaction,and cell motility.3.Tumor-infiltrated myeloid cells were sorted according to their in vivo binding affinity to T1.From the result of RNA seq and transcriptome analysis,the differential gene expression of these two populations mainly enriched in the pathways of taxis,chemotaxis,cytokine-cytokine receptor reaction,and cell motility.From the outcome of the Luminex assay,chemokines IL-1β(P<0.001),MIP1α(P<0.0001),MIP1β(P=0.019)and MIP2(P<0.0001)were found to be excessively secreted by T1-binding target cells.4.Through a structure-activity relationship analysis,we found that the binding activity of an aptamer depends on its special G-rich motif,and a G-rich aptamer always showed a secondary structure-related tetramolecular band on the electrophoresis gel.And the propensity to form a tetramolecular structure of an aptamer was positively relevant to its binding activity(R~2=0.8258,P=0.0046).5.A mutated aptamer M1 was obtained from SAR of T1.M1 had a much higher binding affinity than T1 while sharing the same responsive binding site,the proteoglycans syndecan-1 on the target cell,M1 also presented a typical binding pattern among those blood cells similarly to T1.In the tumor spheroid penetration assay,binding with PBMCs improved the penetration efficacy of aptamers compared to free diffusion,and M1 can infiltrate into tumors by hitchhiking more efficiently than T1.[Conclusions]1.aptamers binding to cells prolong their circulation time and delay clearance from the body.2.T1 preferentially binds to the myeloid cells closely associated with tumors.3.The binding activity of T1 depends on a tetramolecular G-quadruplex structure.Optimized aptamer M1 penetrates tumors more efficiently,and thus has an enhanced potential to deliver drugs into tumors.