Modeling lung adenocarcinoma metastases using patient-derived organoids

Abstract

Approximately 50% of patients with surgically resected early-stage lung cancer develop distant metastasis. At present, there is no in vivo metastasis model to investigate the biology of human lung cancer metastases. Using well-characterized lung adenocarcinoma (LUAD) patient-derived organoids (PDOs), we establish an in vivo metastasis model that preserves the biologic features of human metastases. Results of whole-genome and RNA sequencing establish that our in vivo PDO metastasis model can be used to study clonality and tumor evolution and to identify biomarkers related to organotropism. Investigation of the response of KRAS^G12C PDOs to sotorasib demonstrates that the model can examine the efficacy of treatments to suppress metastasis and identify mechanisms of drug resistance. Finally, our PDO model cocultured with autologous peripheral blood mononuclear cells can potentially be used to determine the optimal immune-priming strategy for individual patients with LUAD.

Keywords: coculture; drug resistance; immune priming; in vivo LUAD metastasis; lung adenocarcinoma; metastasis marker; patient-derived organoids; tumor evolution.

Graphical abstract

Figure 1

Establishment and characterization of PDOs (A) Bars showing the success rate of generation of PDOs from different LUAD biospecimens. “Yes,” preservation of the major mutations of the parental tumor; “No,” major mutations in parental tumor are not preserved in the PDOs. (B) Bars indicating the success of generation of PDOs from the primary LUAD tumor with indicated clinicopathologic features. SUVmax, maximum standardized uptake value; “Yes” and “No,” as in (A). (C) H&E (rows 1 and 3), phase contrast (row 2), and Ki-67 (row 4) immunostaining of the primary tumor, noncancerous tissue, and matched PDOs. (D) Immunostaining of Sox2 in PDOs and their parental tumors. (E) Correlation heatmap of variant allele fractions across PDOs and their parental primary tumors. (F) Proportion of substitution context across PDOs and their parental primary tumors. (G) Correlation heatmap of the transcriptomic profiles across PDOs and their parental primary tumors.

Figure 2

Establishment of our in vivo PDO metastasis model (A) Schematic representation of the generation of our in vivo PDO metastasis model: (1) collection and dissociation of surgical LUAD specimens, (2) generation and characterization of PDOs, (3) labeling with luciferase-green fluorescence protein (GFP) (PDO_GL), (4) introduction into mice by intracardiac injection, (5) formation of metastatic site(s), (6) isolation of metastatic tumor and regeneration of organoids (MDOs), and (7) characterization of MDOs. (B) Bioluminescent images of mice injected with indicated PDOs. (C) Table showing organs in which metastases formed in mice after intracardiac injection of PDOs at 5 months. GB, gallbladder; LN, lymph node. (D) Representative H&E staining from healthy mice or mice with intracardiac injection of PDOs. (E) H&E and immunohistochemical staining of PDO_GL and MDOs.

Figure 3

Characterization of our in vivo PDO metastasis model (A) Phylogenetic trees of the sample sets of PDO_6 and PDO_23. Pie charts show the mutational signature of aggregate samples (along a branch) or an individual sample (at the end of a branch). SV, structure variation; TMB, tumor mutation burden. (B) Schematic of brain metastasis markers. Venn diagram overlapping the differentially expressed genes in (1) PDOs derived from human brain metastases (n = 3), compared with PDOs from human primary tumors (n = 8), and (2) MDOs from mouse brain metastases (n = 2), compared with their parental PDOs (n = 3). (C) Expressional heatmap of 10 overlapping genes of the putative “Brain Met. Markers” (shown in B). Green brackets, altered in all PDOs and MDOs from brain metastases, compared with PDOs from the primary tumor. (D) Table summarizing the prediction of outcomes of 10 overlapping genes. The table details the p values obtained (OS and PFS, log rank test). OS, overall survival; PFS, progression-free survival. (E) Bars indicating BTG2 mRNA in LUAD that metastasized to the brain (n = 6), compared with LUAD that metastasized to other organs (n = 4). Data are represented as mean ± SEM. (F–I) LUAD H460 cells were transfected with short hairpin RNA (shRNA) BTG2 or scramble (Sc.). (F) Immunoblots showing BTG2 and actin in H460 cells. (G) Bioluminescent images at 11 days after injection with H460 cells. (H) Curves indicating the average total bioluminescence (log region of interest [ROI]) after injection. Data are represented as mean ± SEM. (I) Bioluminescent brain images at 11 days (left) after injection. Curves indicate average bioluminescence (log ROI) in the brain (right). Data are represented as mean ± SEM.

Figure 4

PDOs as a platform for assessment of therapeutic efficacy in vitro and in vivo (A) Drug-response curves (top) showing the percentage of cell death and IC₅₀ of PDOs with KRAS^G12C treated with sotorasib in two independent runs (n = 3). Immunoblots (bottom) indicating p-ERK and total ERK in each PDO. (B) Representative pictures (top) showing metastases in mice with PDO_12 treated with or without sotorasib. The table (bottom) summarizes the locations of metastases formed. (C) Drug-response curves showing the percentage of cell death and IC₅₀ of the regenerated organoids from the metastatic sites of mice (MDOs) with PDO_23 treated with sotorasib in two independent runs (n = 3). (D) Heatmap showing genes differentially expressed in RNA sequencing. (E) Drug-response curves (left) showing the percentage of cell death and the IC₅₀ of PDO_23 and MDO_liver and MDO_brain treated with sotorasib in two independent runs (n = 3). Immunoblots (right) indicating FGFR1 in MDOs. (F) Schematic illustration of the immune-priming strategy. PDOs were pretreated with cisplatin plus vinorelbine (chemotherapy), X-ray, or DMSO 24 h before coculture with autologous PBMCs. After 2 weeks of coculture, T cell effector functions were evaluated using flow cytometry. Activated CD8⁺ T cell-mediated killing was evaluated using a PDO-killing assay. (G) Representative plots gated on CD8⁺ T cells (top) tested for reactivity against autologous PDOs. Bars (bottom) showing quantification of CD107a+CD8⁺ and CD137+CD8⁺ T cells obtained after 2-week coculture. (H) Microphotographs of untreated PDOs (left) after culture with activated CD8⁺ T cells. PDOs were labeled with CellTrace Yellow (magenta) before coculture; apoptotic cells appeared green. Bars show quantification of PDO killing (right). Data are represented as mean ± SEM (n = 5 fields/sample).