Establishment And Preliminary Application Of Breast Cancer And Paracancerous Organoids

Objective:To improve the success rate of constructing tumor organoids(TO)and paired adjacent normal tissue-derived organoids(NO)derived from tissue samples,and verify the consistency between organoids and their source tissues through experiments to ensure that the experimental results in vitro can maximize the simulation of situation in vivo.A visual analysis process compatible with high-throughput atmospheric mass spectrometry imaging data was proposed.In terms of timeliness and safety,paired organoids may provide a well-characterized model for drug sensitivity test in vitro.Through the design of experimental procedures,the efficacy of drugs and adverse reactions to patients can be more comprehensively evaluated.Methods:We established TO and NO organoids from 88 Chinese people with different subtypes of breast cancer(BC),studied the effect of enzymatic digestion and mechanical filtration of tissue samples on the construction of organoids.For optimization of the organoids’passage method,after releasing and recovering the organoids in the Matrigel,we used repeated aspiration with a pipetting gun or transient digestion with TrypLE Express for the cystic or compact organoids.The survival and proliferation of organoids after cryopreservation and resuscitation were detected under the condition of programmed cryopreservation technology.At the validation level,organoids were identified at multiple levels by histology,flow cytometry,and whole-exome DNA sequencing.We explored the application of mass spectrometry imaging to organoid metabolomics.Organoids were transferred to a slide,sprayed with 2,5-dihydroxybenzoic acid matrix,and then used for mass spectrometry analysis.The atmospheric pressure mass spectrometry imaging data were converted to format and imported into MSiReader software.After data preprocessing,two-dimensional and three-dimensional mass spectrometry imaging data visualization and co-localization analysis,the visual analysis of mass spectrometry imaging data were realized.In addition,we combined the paired organoid model with Incucyte living cell analysis system,added Annexin V green dye for fluorescence staining of apoptotic areas,The responses of TO and NO to chemotherapeutic drugs were monitored in real time by microscope photography and fluorescence intensity detection to obtain the drug sensitivity curve of paired organoids.Results:Tumor and matched paracancerous tissues of BC patients were processed with mechanical filtration method and enzymatic digestion method,which were beneficial to the acquisition of paired organoids.In addition,before organoids were planted with matrix glue,adding the overnight suspension step was more conducive to the effective acquisition of nutrients and self-assembly to form organoids.The success rate of TO was 71.3%(62/87),and that of NO was 65.8%(52/79).The success rate of organoid resuscitation could reach 91.3%by decreasing 1℃ per minute using the programmed cryopreservation device.We also verified that paired organoids could replicate the characteristics of the corresponding tissues and maintained a high degree of similarity with the corresponding tissues at different levels.MSiReader software could convert.Raw into.imzML format data for facilitating reading.The data preprocessing showed that the background removal effect was the most significant when Mean was selected for data normalization.In MSiReader software,the heterogeneous spatial distribution of different molecules on the organoid surfaces could be visually observed in twodimensional mass spectrometry.Mass spectrometry imaging data could not only be displayed the signal intensity of molecules in the form of two-dimensional pseudo-color images,but also be displayed more clearly and intuitively in three-dimensional form.The RGB co-location analysis tool in MSiReader software could not only view the distribution of a single molecule on the organoid surface,but also help to find the co-location of molecules distributed in a specific space area.Through fluorescence intensity detection in the Incucyte system,it was found that half-maximal effect concentrations(concentration for 50%of maximal effect,EC50)of the same chemotherapy drug varied among different patients.Meanwhile,EC50 values of NO and TO derived from the same patient were also different.Conclusions:In this study,we initially established a culture and identification platform for patient-derived BC and paracancerous organoids.A mass spectrometry data analysis system of atmospheric mass spectrometry was proposed on organoids using MSiReader software,which could visualize the metabolic changes of organoids at the molecular level in situ.A rapid workflow for real-time monitoring of drug toxicity based on primary paired organoids was also developed,and drug sensitivity testing of paired organoids could be achieved within a week.It was expected to provide a technically feasible strategy for optimizing the relative dose of chemotherapy drugs in the future,and open up a new way to guide the personalized treatment strategy for each clinical patient.