Organoids derived from patients provide a new opportunity for research and individualized treatment of malignant peritoneal mesothelioma

Abstract

Background: Malignant peritoneal mesothelioma (MPM) is an extremely rare and highly invasive tumor. Due to the lack of accurate models that reflect the biological characteristics of primary tumors, studying MPM remains challenging and is associated with an exceedingly unfavorable prognosis. This study was aimed to establish a new potential preclinical model for MPM using patient-derived MPM organoids (MPMOs) and to comprehensively evaluate the practicality of this model in medical research and its feasibility in guiding individualized patient treatment.

Methods: MPMOs were constructed using tumor tissue from MPM patients. Histopathological analysis and whole genome sequencing (WGS) were employed to determine the ability of MPMOs to replicate the original tumor's genetic and histological characteristics. The subcutaneous and orthotopic xenograft models were employed to assess the feasibility of establishing an in vivo model of MPM. MPMOs were also used to conduct drug screening and compare the results with retrospective analysis of patients after treatment, in order to evaluate the potential of MPMOs in predicting the effectiveness of drugs in MPM patients.

Results: We successfully established a culture method for human MPM organoids using tumor tissue from MPM patients and provided a comprehensive description of the necessary medium components for MPMOs. Pathological examination and WGS revealed that MPMOs accurately represented the histological characteristics and genomic heterogeneity of the original tumors. In terms of application, the success rate of creating subcutaneous and orthotopic xenograft models using MPMOs was 88% and 100% respectively. Drug sensitivity assays demonstrated that MPMOs have different medication responses, and these differences were compatible with the real situation of the patients.

Conclusion: This study presents a method for generating human MPM organoids, which can serve as a valuable research tool and contribute to the advancement of MPM research. Additionally, these organoids can be utilized as a means to evaluate the effectiveness of drug treatments for MPM patients, offering a model for personalized treatment approaches.

Keywords: Drug screen; Malignant peritoneal Mesothelioma; Organoids; Patient-derived organoids (PDO); Patient-derived organoids xenograft (PDOX); Peritoneal orthotopic xenograft; Precision medicine; Primary cell lines; Translational medicine.

Fig. 1

Flow diagram of establishment and characterization of MPMOs. Including the generation of MPMOs and primary cell from patients, as well as the histological characterization, genomic analysis, drug screen, and xenografts models of MPMOs.

Fig. 2

Establishment of patient-derived Malignant peritoneal mesothelioma (MPM) organoid cultures. A Representative images of MPMOs culture process, including the generation of MPM primary cells. B The PET/CT images of MPM patients showed significantly enhanced dense shadows in multiple areas of the abdominal cavity, and there were radioactive concentrations of imaging agents, indicating that the patient’s abdominal cavity has been invaded by multiple tumors. C Heat map showing the fraction of the derived organoids in the D1 and D7. D Expansion potential of MPM- and CPM-derived organoid. Arrow, continuous expansion ; Dot, passage ; Cross, Stop growth/death; MPM, malignant peritoneal mesothelioma ; CPM, cystic peritoneal mesothelioma ; O, organoid. E Representative bright field images of MPM and CPM-derived organoids from 4 patients. Scale, 50 μm

Fig. 3

Culture characteristics of MPMOS and composition of culture medium. (A) MPMOs formed in different tumor-organoid culture medium. The image shown is from MPM-C1-O as a representative sample after 10 days of planting. Left, bright field images, scale, 200 μm;Right, Quantification of the MPMOs number per well in the different tumor-organoid culture medium. (B) The formation of MPM organoids culture in factor deletion medium after 10 days. The images shown are from MPM-C2-O as a representative sample. Left, bright field images, scale, 200 μm;Right, Quantification of the MPMOs number per well. (C) Time-lapse photography of images with different magnification of MPMOs. Left, bright field images, Scale bar, 400 μm, 200 μm, 100 μm, 50 μm;Right, Quantification of the MPMOs number per well for individual organoid. Data were showed as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 and each experiments were performed in triplicate

Fig. 4

Histological characterization and biomarkers expression analysis of MPMOs. A Bright-field (BF) microscopy images of MPMOs together with hematoxylin-eosin (HE) stain histological analysis of MPM tissues and MPMOs. All of the MPMOs were the second generation. HE Scale bar, 20 μm. BF Scale bar, 100 μm. B Multiple immuoflurescence staining of cytokeratin 5/6 (CK5/6), Wilms tumor (WT-1), calretinin on MPM-derived organoids and the paimary tumors. CK5/6, red;WT-1, green;Calretinin, pink. 40X Scale bar (20 μm), 100X Scale bar (10 μm), 200X Scale bar (5 μm)

Fig. 5

Analysis of MPM organoids xenotransplantation. A Representative image of subcutaneous xenograft tumor. B The growth curve of subcutaneous xenografts from three different MPM organoids. C The weight comparison of subcutaneous xenografts from three different MPM organoids. D Histologic assessment of parental tumors, MPM organoids, and xenograft tumors in Hematoxylin-eosin (HE) stain. Scale bar (20 μm). E Multiple immuoflurescence staining of cytokeratin 5/6 (CK5/6), Wilms Tumor (WT-1) and calretinin were performed on the parental tumors, MPM organoids, and xenograft tumors. CK5/6, red;WT-1, green;Calretinin, pink. 40X Scale bar (20 μm), 100X Scale bar (10 μm), 200X Scale bar (5 μm), 400X Scale bar (2.5 μm). Data were showed as mean ± SD,Each organoid line was transplanted into three mice

Fig. 6

Human derived MPM organoids orthotopic xenotransplantation model (MPM-PDOX). A Body weight changes of nude mice after intraperitoneal injection three different MPM organoids. B The subarea and scoring of the Experimental Peritoneal Cancer index (ePCI) scoring system [16]. C ePCi score of three different PDOX. D - O The gross pathology of PDOX. (D, F, liver; E, diaphragm; G, pancreas, stomach; H, the images show that the tumor invaded the peritoneal peritoneum and multiple organs of nude mice; I, spleen; J, mesentery; K, retroperitoneal vascular tissue; L, kidney; M,O, peritoneum; N, pelvic cavity.) The images shown are from MPM-C6-O as a representative sample. Data were showed as mean ± SD,Each organoid line was transplanted into three mice

Fig. 7

Genetic characterization of malignant peritoneal mesothelioma (MPM) organoids. A Somatic genomic landscape of 5 MPMOs (-O) and the corresponding parental tumors (-T). The types of genetic alterations are indicated in the legend. B Somatic variation types from MPM tumors and corresponding organoids. C The top 30 genes with the highest number of variations in MPM. D Proportion of genome mutation types in MPM and corresponding organoids. E Histogram illustrating the various contributions of point mutation types in MPMOs(-O) and their respective tumors(-T), the six types of point mutation types are represented. F CNVs landscape (red, gain; blue, loss) in MPMOs (-O) and tumor tissues (-T)

Fig. 8

Cytological characteristics of malignant peritoneal mesothelioma (MPM) primary cell lines. A Morphology was showed under the inverted phase contrast microscope of MPM-C6 primary cell. Scale bar, 100 μm. B The MPM-C6 primary cell morphology was revealed by phalloidin staining. Scale bar, 10 μm. C Multiple immuoflurescence staining of cytokeratin 5/6 (CK5/6), Wilms Tumor (WT-1), calretinin on MPM-C6 primary cells. CK5/6, red;WT-1, green;Calretinin, pink. Scale bar, 10 μm. D The genomic authentication of the MPM-C6 primary cell was confirmed by the short tandem repeats (STRs). E Heat map of logIC50 values for 14 anticancer drugs used to treat MPM-C6 primary cell and related MPM organoids. Putative targets of the tested anticancer drugs are listed on the left. F Representative dose response curves of the MPM-C6 primary cell and related MPM were treated with first-line chemotherapy drugs alone. Data were showed as mean ± SD and each experiment were performed in triplicate

Fig. 9

Drug response in malignant peritoneal mesothelioma (MPM) organoid lines. A Heat map of logIC₅₀ values of 14 anticancer drugs used to treat seven MPMOs and a CPMO from 8 patients. All organoids are of the first generation. B Dose response curves of first-line chemotherapy drugs for MPMOs and CPMO. C MPM organoids morphology after first-line chemotherapy drugs treated were showed under the inverted phase contrast microscope. The image shown are from MPM-C7-O (drug resistance) and MPM-C6-O (drug sensitivity) as a representative sample. D CT scan images show the tumor status of MPM patients during treatment. Primary tumor (circled in cyan), metastatic lesion (circled in red), ascites (circled in yellow). E Fitted dose–response curves illustrating the distinct responses of MPMOs to pemetrexed and cisplatin. F The serum CA125 levels in different MPM patients pre and post treatment. G The overall survival of the MPM and CPM patients in this study. Data were showed as mean ± SD and each experiment were performed in triplicate