Proteomics Research Of Anti-tumor Immunity Induced By Cryo-thermal Therapy On 4T1 Metastatic Breast Cancer

Breast cancer has emerged as the most frequent malignancy for women;its metastasis accounts for the most death and is incurable.Currently,immunotherapy has been developed as a promising tool to improve patients' survival and is the focus of research.Alternatively,thermal therapy,exhibiting characteristics quite distinct from traditional targeted therapies,has presented as a systematic therapeutics to affect tumor environment via energy interference,showing its power in raising antitumor immunity.However,the mechanism of thermal induced anti-tumor immunity is still unclear.To investigate it on a global scale,not only a suitable tumor model,but also a high throughput technique is needed.Tumor subpopulation cell lines(67NR,168 FARN and 4T1),derived from a single,spontaneously arising mammary tumor from a Balb/c mouse,share identical tumorigenesis,while increasing metastatic capabilities,and well mimic the development of metastasis.What's more,4T1 is one of the few breast cancer models with the capacity to metastasize efficiently to sites similar to human breast cancer.Recently,the high throughput proteomics has emerged as a powerful tool to systematically study protein proprieties and profiles in a complex biological system.Thus,in this dissertation,based on various proteomics techniques,we first studied the metastatic characteristics of 4T1 subpopulations,especially 4T1,at the molecular level,and then we utilized the spontaneously metastatic 4T1 murine breast cancer model to investigate the anti-tumor immunity induced by thermal therapy to provide novel methodology for metastatic therapy.First,based on label free shotgun membrane proteomics,we studied the metastatic characteristics of 4T1 subpopulations.We established the biotin-streptavidin technology as an efficient approach to enrich and purify cell membrane proteins,in which 79% membrane proteins could be captured,higher than those previously reported.In total,1677 membrane proteins were identified and 472 of them were defined as significant proteins,in which metabolism was the most enriched GO function.Among significant metabolic proteins,12 of them were linearly up-or downregulated accompanied by the increasing metastatic capability.Finally,we focused on two metabolic proteins,FASN(fatty acid synthase)and NSDHL(NAD(P)dependent steroid dehydrogenase-like).These two proteins translocated from cytoplasm to cell membrane during the development of metastatic capability in 4T1 subpopulations.In particular,such translocation was remarkable on 4T1 cells.In this case,we suggested that FASN and NSDHL are essential in metastatic acquisiton for 4T1 subpopulations,especially 4T1 cells.They might serve as the malignant markers for 4T1 cells.The understanding of metastatic characteristics upon 4T1 subpopulations at the molecular level could benefit the developennt of a suitable metastatic breast cancer model for cryo-thermal therapy.Next,to better understand the mechanism that cryo-thermal boosts the anti-tumor immunity,we utilized a spontaneously metastatic 4T1 murine breast cancer model and iTRAQ labeled shotgun proteomics to compare the N-glycoproteome changes in serum samples with and without cryothermal treatment over time.Hydrazide chemistry was used to improve the efficiency of glycopeptide capture(> 90%).Finally,we quantified 231 highly confident N-glycosylated serum proteins.Among them,53 showed significantly discriminated regulatory patterns over the time course,in which the acute phase response emerged as the most enhanced pathway.Unsupervised hierarchical clustering distinguished the proteome to early and late responses and acute phase response was the first event.Next,to better understand how acute phase response acts in the antitumor immunity,PRM targeted proteomics coupled with ELISA,RT-PCR and flow cytometry were carried out in a total of 94 enriched serum samples over 23 significant proteins and 8 time points.Eventually,we found that cryo-thermal therapy reshaped the tumor chronic inflammatory microenvironment to an acute phenotypic state,with up-regulated expression of acute phase proteins.Such acute inflammatory microenvironment played a key role to stimulate dendritic cells maturation and enhance the proliferation of Th1 memory cells,and thus consequently shifted the tumor inflammatory microenvironment from Th2 immunosuppressive to Th1 immunostimulatory,which attenuated tumor growth and metastasis.Lastly,we investigated the uniqueness of anti-tumor immunity induced by acute phase response under cyro-thermal stimulation.We compared the pattern of acute phase response and corresponding anti-tumor immunity under different thermal treatments,that is,1)cryo-thermal therapy on tumor bearing or healthy mice and 2)hyperthermia,cryosurgery and cryothermal therapy on tumor bearing mice,respectively.We found that such “acute” magnitude induced by cryo-thermal on healthy mice was much weaker than that on tumor tissues,and what's more,T lymphocyte response was not effectively stimulated.This finding suggested that tumor releasing danger signals,such as antigens and cytokines do matter in cryothermal induced acute phase response and corresponding anti-tumor immunity.In addition,cryo-thermal treatment elicited the strongest acute response,accompanied by the most pronounced T lymphocyte response and the most inhibited immunosuppressive state(MDSC).As observed previously,cryo-thermal treatment could cause the most damage on tumors,thus these results indicated that cryo-thermal treatment caused the most significant releasing of tumor danger signals,which could augment the inflammatory state,and markedly alter the tumor microenvironment,and thus enhance acute phase response and facilitate anti-tumor immunity.In summary,investigation with high throughput proteomics allowed for a global view on cryo-thermal therapy induced anti-tumor immunity upon metastatic breast cancer,in which the acute phase respone and its magnitude could be critical.All of these findings help us to develop a novel thermal immunotherapy for metastatic breast cancer treatment.