Construction of tumor organoids and their application to cancer research and therapy

Abstract

Cancer remains a severe public health burden worldwide. One of the challenges hampering effective cancer therapy is that the existing cancer models hardly recapitulate the tumor microenvironment of human patients. Over the past decade, tumor organoids have emerged as an in vitro 3D tumor model to mimic the pathophysiological characteristics of parental tumors. Various techniques have been developed to construct tumor organoids, such as matrix-based methods, hanging drop, spinner or rotating flask, nonadhesive surface, organ-on-a-chip, 3D bioprinting, and genetic engineering. This review elaborated on cell components and fabrication methods for establishing tumor organoid models. Furthermore, we discussed the application of tumor organoids to cancer modeling, basic cancer research, and anticancer therapy. Finally, we discussed current limitations and future directions in employing tumor organoids for more extensive applications.

Keywords: antitumor therapy; basic cancer research; cellular components; culture methods; tumor organoids.

Figure 1

Schematic illustration of cellular materials, various tumor organoid construction methods, and their applications to cancer research and therapy. CT: Chemotherapy; RT: radiotherapy; IT: immunotherapy.

Figure 2

Co-cultures of normal fibroblast (NF)-early-stage cancer-associated fibroblast (T1CAF) pairs and early-stage colorectal cancer (T1CRCs) organoids. (A) Procedures for the co-culture of organoids and fibroblasts. (B) Representative images of suspension co-cultures of T1CRC organoids with unmatched NF-T1CAFs. Epithelial cells and fibroblasts were stained with pan cytokeratin (Pan CK) and vimentin, respectively. (C) In situ zymography showing proteolytic activity with dye-quenched gelatin as a substrate on cryosections of Matrigel-embedded co-cultures of T1 CRC organoids with unmatched NF-T1CAFs after 12 days of culture. (D) Immunofluorescence staining of suspension co-cultures for CD44 (green), Pan CK (red), and DAPI (blue). Adapted with permission from , copyright 2023, Elsevier Ltd.

Figure 3

Gastrointestinal (GI) tissue-derived extracellular matrix (ECM) hydrogels via decellularization for organoid culture. (A) Schematic illustration of the preparation of GI organoids using ECM hydrogels (stomach extracellular matrix (SEM) and intestinal extracellular matrix (IEM)) derived from the decellularized GI tract. (B) (a) A principal component analysis (PCA) of matrisome proteins existing in Matrigel, SEM, and IEM. All the protein composition and the most abundant top 10 matrisome proteins in (b) Matrigel, (c) SEM, and (d) IEM. (C) Representative brightfield images showing (left) gastric and (right) intestinal organoids cultured in SEM/IEM hydrogel and Matrigel (MAT) at various passages. Scale bar: 100 μm. (D) Representative immunofluorescence images of GI tumor organoids derived from GI cancer cell lines. Adapted with permission from , copyright 2022, Nature Publishing Group.

Figure 4

(A) Spinner or (B) rotating flasks for organoid construction.

Figure 5

Construction and characterization of the integrated superhydrophobic microwell array chip (InSMAR-chip). (A) Schematics (left) and cross-section view (right) of the InSMAR-chip. (B) Photograph of an InSMAR-chip with a droplet array in the microwells. The contact angle of the superhydrophobic surface is > 160°. (C) Photographs of the droplet array in the microwells. (Top) When the excess medium was removed from the chip, the droplet array of culture medium formed spontaneously. (Middle) The droplet assay of the Matrigel loaded in the microwells. (Bottom) The Matrigel droplets are overlaid on the culture medium via the spot-cover method. (D) Representative H&E staining images of the parental tumor tissue and the corresponding LCOs cultured on the InSMAR-chip (on-chip) and the traditional multiwell plate (off-chip). Scale bar: 20 µm. (E) LCOs maintain continuous growth on the InSMAR chip for 21 days. Scale bar: 200 µm. (F) Correlation between patient response and on-chip drug sensitivity. Adapted with permission from , copyright 2021, Nature Publishing Group.

Figure 6

Patient-derived organoids from colorectal cancer (CRC) and paired liver metastasis (LM) predicted chemotherapeutic response. (A) Organoid culture success rate from CRC and LM tissues of patients with colorectal cancer liver metastasis (CRLM). (B) CRC and LM organoids from different CRLM patients showed three typical characteristics in the bright field. (C) H&E staining of CRC/LM organoids and corresponding parental tumors. T: parental tumors; O, CRC, or LM organoids. (D) Immunohistochemical staining of CRC/LM organoids and corresponding parental tumors with Ki-67, CDX2, β-catenin, CK-pan, and CK20. (O, CRC or LM organoids; T, parental tumors). (E) a, IC50 values of organoids for FOLFOX chemotherapy from SD/PR (n = 8) and PD patients (n = 5). b, An ROC curve showed the predictive efficacy of organoids for FOLFOX chemotherapy. (SD: stable disease; PR: partial response; PD: progressive disease). (F) a, Correlation between the IC50 values of organoids and progression‐free survival (PFS) for patients (n = 13). b, An ROC curve showed the predictive efficacy of organoids for the clinical prognosis of patients receiving FOLFOX treatment. (G) a, IC50 values of organoids for FOLFIRI chemotherapy from SD/PR (n = 5) and PD patients (n = 5). b, An ROC curve showed the predictive efficacy of organoids for FOLFIRI treatment response. (H) a, Correlation between IC50 values of organoids and PFS of patients (n = 10). b, An ROC curve showed the predictive efficacy of organoids for the clinical prognosis of patients receiving FOLFIRI treatment. Black scale bar, 200 µm; red scale bar, 100 µm. Adapted with permission from , copyright 2022, Wiley.

Figure 7

Tumor organoid-guided personalized treatment in pancreatic ductal adenocarcinoma (PDAC). (A) Procedures for the establishment of PDAC organoid-based platform. (B) Growth curves for 4 PDAC patient-derived tumor organoids. (C) Summary of PDO response to GA or FFX, as measured by the ex vivo ODSA at elevated drug doses. (D) Consistency of the ex vivo tumor organoid responses with carbohydrate antigen 19-9 (CA19-9) status in the corresponding tumor patients (“matched” indicates consistent findings). GA: gemcitabine plus nab-paclitaxel; FFX: 5-fluorouracil, irinotecan and oxaliplatin. Adapted with permission from , copyright 2022, Amer Soc Clinical Investigation Inc.