Construction Of Gastric Cancer Patient-derived Organoids And Their Utilization In Individualized Efficacy Prediction Of Nanoformulations

Aims:（1）construction of gastric cancer（GC）patient-derived organoids（PDOs）from surgically resected tumor tissues or endoscopic biopsies;（2）the constructed GC PDOs and their corresponding primary tumors were systematically verified by histopathological and genomic profiles;（3）GC PDOs were used as an in vitro model to investigate the efficacies of the clinically used paclitaxel（PTX）nanoformulations,albumin-bound PTX（Albu-PTX）and liposomal PTX（Lipo-PTX）;（4）to explore the relevant mechanisms of the difference in efficacy between Albu-PTX and Lipo-PTX on GC PDOs;（5）construction of patient-derived xenografts（PDXs）from GC samples to further verify the efficacy difference between the two PTX nanoformulations.Methods:Part I:the improved multiple-batch dissociation method was used to collect cells from the tumor samples of GC patients,mixed them with Matrigel,conducted by adding PDOs medium to construct GC PDOs,and subjected to long-term culture,passage,cryopreservation,and recovery.Part II:through the analysis of GC PDOs and their corresponding primary tumors by hematoxylin eosin（HE）staining,Alcian blue staining,immunohistochemical staining and whole exome sequencing（WES）to systematically identify the success of GC PDOs construction.Part III:first,the two PTX nanoformulations were characterized by dynamic light scanning and transmission electron microscopy.Secondly,the killing efficacies of Albu-PTX and Lipo-PTX in eight cases of GC PDOs were compared through the improved drug sensitivity analysis protocol for the nanoformulations,with the detection of cell viability as an index.Part IV:the different killing mechanisms of Albu-PTX and Lipo-PTX against GC PDOs were elucidated by transcriptome profiling analysis,Live/Dead staining analysis,and dynamic monitoring of the distributions of fluorescent-labeled PTX nanoformulations in GC PDOs.Part V:fresh tumor sample from GC patient was transplanted subcutaneously into severely immunodeficient mice to construct GC PDXs model.The tumor growth and survival period of tumor-bearing mice were compared by using intratumoral injection of PTX nanoformulations,and the tumor pathological sections were further analyzed by apoptotic cell staining,immunohistochemical staining and HE staining.Results:Part I:the improved multiple-batch dissociation method resulted in significantly improved viability of cells harvested from the tissue compared to the single-batch dissociation method.Using the improved approach,we constructed GC PDOs from tumor tissues derived from ten patients.Eight of these GC PDO lines were sourced from resected tissues,and two were obtained from endoscopic biopsies.Eight of the ten GC PDO lines could be stably passaged,and four of them were well recovered after cryopreservation.During long-term passaging,the morphology of the GC PDOs was well preserved.Part II:HE staining images showed that GC PDOs maintained the histopathological characteristics of the primary GC.Immunohistochemical staining images（CEA,Ki-67,LGR5,and E-cadherin）showed that PDOs could maintain the biological characteristics of the primary GC.Alcian blue staining images showed similar patterns of distribution of extracellular components in PDOs and primary GC.We subjected GC PDOs,the primary tumor sample,and the corresponding para-carcinoma tissue for WES,which revealed that GC PDOs largely recapitulated the primary GC in terms of copy number variations.We further found that the copy number variations of common driver genes exhibited a high degree of similarity in GC PDOs and primary GC.Moreover,we conducted mutational spectrum analysis and found that GC PDOs retained most of the mutations in genes that are observed in primary GC.GC PDOs and the primary GC also showed similarity with respect to the overall trend in the type of point mutations.Part III:transmission electron microscopy and dynamic light scanning analysis showed that Lipo-PTX and Albu-PTX were very similar in size and had similar zeta potentials.The drug sensitivity analysis of Albu-PTX and Lipo-PTX were performed on eight GC PDO lines.Based on dose-response kill curves,the IC₅₀of Albu-PTX in the eight GC PDO lines ranged from 0.019μM-0.67μM,and the IC₅₀of Lipo-PTX in the eight GC PDO lines ranged from 0.005μM-0.24μM,which emphasized the intrapatient heterogeneity recapitulated by these GC PDO lines.On comparing the performances of the two nanoformulations in each PDO line,we observed that the IC₅₀ values for Albu-PTX were always higher than those for Lipo-PTX.Quantitatively,the IC₅₀ratios of Albu-PTX/Lipo-PTX fluctuated from 2.3 to 10.8 in the eight GC PDO lines,indicating that Lipo-PTX outperformed Albu-PTX in killing the GC PDOs.Part IV:we conducted transcriptome profiling analysis of GC PDOs after treatment with Albu-PTX or Lipo-PTX.The heatmap showed that tubulin-related genes were highly expressed,and the up-regulation in Lipo-PTX group was greater than that in Albu-PTX group.DNA replication and repair-related genes were low in expression,and the down-regulation in Lipo-PTX group was greater than that in Albu-PTX group.Kyoto encyclopedia of genes and genomes（KEGG）pathway analysis also revealed that exposure to these formulations affects pathways involved in DNA replication,the cell cycle and gap junctions.Gene set enrichment analysis（GSEA）showed that compared to the Albu-PTX group,the Lipo-PTX group particularly showed upregulation of sets of genes related to apoptosis,cellular responses to chemical stimulus,and positive regulation of cellular metabolic process and downregulation of sets of genes related to DNA repair,DNA replication,and the cell cycle.We also performed Live/Dead staining analysis of GC PDOs.In the Albu-PTX group,the three-dimensional structure of the whole PDO was well preserved,and only a few dead cells were detected inside the PDO lumen.In sharp contrast,treatment with Lipo-PTX induced substantial destruction of the three-dimensional structure of the PDO,and dead cells dominated the cell populations.We dynamically monitored the distributions of fluorescent-labeled PTX nanoformulations in GC PDOs by confocal laser scanning microscopy,and found that Albu-PTX was mainly localized in the PDO lumen,while Lipo-PTX was mainly localized in the PDOs cells.This phenomenon was further confirmed by fluorescence colocalization analysis and three-dimensional reconstruction of GC PDOs.Part V:we utilized GC primary tumor tissue to construct a PDX model.Through intratumoral injection of PTX nanoformulations,it was found that the killing efficacies of Lipo-PTX on the GC PDXs model was stronger than that of Albu-PTX（P<0.01）.From the analysis of survival curve,the survival time of tumor-bearing mice treated with Lipo-PTX was significantly longer than that of tumor-bearing mice treated with Albu-PTX（P<0.001）.In accordance with HE staining images,apoptotic cell staining images showed that only about 20%of the cells in Albu-PTX-treated PDX tissue were apoptotic,whereas this value increased to 70%in the Lipo-PTX group.Reverse trends were found in immunohistochemical staining（Ki-67）images.Conclusions:we constructed GC PDOs from surgically resected tumor tissues and endoscopic biopsies,and the successful construction was systematically verified by histopathological and genomic profiles.We chose two representative PTX nanoformulations for comparative study and found that Lipo-PTX outperformed Albu-PTX in killing GC PDOs.Our results further showed that Albu-PTX was mainly localized in the PDO lumen,while Lipo-PTX was mainly localized in the PDOs cells.We also used the GC PDX model to reproduce the therapeutic superiority of Lipo-PTX over Albu-PTX,the results again supported the idea that our PDOs are a reliable model for predicting the efficacies of nanoformulations.