Mechanism Of Osteosarcoma Cells And Medicines Studied With Confocal Raman Microspectral Imaging

Osteosarcoma is a common and fatal malignant bone tumor in adolescents that is prone to recurrence and metastasis.The chemotherapeutic drugs gamma secretase inhibitors（N-[N-（3,5-Difluorophenacetyl-L-alany）]-S-phenylglycine t-butyl ester,DAPT）and cisplatin can inhibit the development of osteosarcoma,but their clinical application and the doses used are influenced by the inherent resistance and toxic effects of chemotherapeutic drugs,and their molecular mechanisms of action for the treatment of tumors have not been elucidated.Current research on cellular drug response imaging such as electron microscopy and immunofluorescence staining techniques can provide cellular image information,but they can cause damage to biological tissues or cells,and the use of exogenous fluorescent markers can disrupt cell structure or function,and their photobleaching phenomenon greatly limits the long-term,in vivo,dynamic observation of biological tissues or cells.Therefore,a non-destructive and label-free live cell detection imaging technique is needed to study the cellular and medicine action mechanisms at the molecular level.Confocal Raman Microspectral Imaging（CRMI）is a fast,nondestructive,label-free,and highly specific optical detection imaging technique that can interpret the material composition and molecular structural information of various biological samples at the molecular level and can quantitatively,qualitatively,and locally characterize the distribution of subcellular structural morphology and biochemical components.Therefore,in this paper,the dynamic response of osteosarcoma cells to DAPT and its combination with cisplatin was systematically investigated by combining spectral（molecular"fingerprint"spectroscopy）and graphical（Raman spectroscopy images）methods using CRMI technology and multivariate data analysis,and the synergistic anti-tumor mechanism of DAPT targeting Notch signaling pathway and its combination with cisplatin on osteosarcoma cells was elucidated.The main work and innovations of this paper are as follows:1.Raman spectroscopy and Raman images were used for the first time to clarify the relative contents of biochemical components of osteosarcoma cells,such as proteins,nucleic acids,lipids and carbohydrates,the molecular conformation,and the morphological information of subcellular structure nucleus,cytoplasm and cell membrane regions at the cellular and molecular levels.The differences in the content and structure of biochemical components between malignant osteoblasts and spindle-shaped osteosarcoma cells were compared,and the distribution characteristics of intracellular tryptophan,nucleic acid,lipid,and amide III were visualized.2.Detection and analysis of characteristic Raman spectra of osteosarcoma cells in the control and DAPT drug groups combined with Raman images elucidated the growth inhibitory effect of DAPT on osteosarcoma cells and monitored the dynamic response process of cells to different doses and different treatment times of DAPT.Analysis of the changes in the characteristic peaks at 787,1096,1315,and 1580 cm^-1 revealed that DAPT caused DNA changes in the nucleus,disrupting DNA base pairs,affecting DNA replication,and inhibiting cell growth.Spectral data models（PCA-LDA,PLS-DA and PCA-SVM）of cellular response to drugs were used to characterize the maximum response of osteosarcoma cells to 40μM DAPT action for 48 h and to distinguish different cell groups with 79.07%and 93.81%accuracy.Comparing the Raman images of different groups of cells,we found the changing patterns of nuclear and nucleic acid fragmentation distribution of 40μM DAPT-treated cells for 48 hours.3.A synergistic mechanism of apoptosis induction by DAPT in combination with cisplatin was developed.The induction of apoptosis by DAPT in combination with cisplatin involves two main pathways.First,DAPT combined with cisplatin acts on DNA in the nucleus,and cisplatin interacts with the N7 reaction center on purine residues to form a cross-linked complex,which changes the DNA double helix structure,causing it to break and become fragmented,inducing apoptosis.Second,it leads to the release of cytochrome c from mitochondria,which activates factors such as caspase-3 and promotes apoptosis.Raman spectra and Raman images were used to systematically elucidate the changes in intracellular protein,nucleic acid and lipid contents,molecular structures and intracellular distribution patterns,and found that the cellular effects of DAPT combined with cisplatin exhibited cell cycle arrest and induction of apoptosis.DAPT combined with cisplatin leads to a decrease in intracellular nucleic acid content and affects DNA replication,resulting in G1 phase block.