Head and neck cancer organoids established by modification of the CTOS method can be used to predict in vivo drug sensitivity

Abstract

Objectives: Currently there are no standard biomarkers of head and neck squamous cell carcinoma (HNSCC) response to therapy. This is, due to a lack of adequate predictive tumor models. To this end, we established cancer organoid lines from individual patient's tumors, and characterized their growth characteristics and response to different drug treatments with the objective of using these models for prediction of treatment response.

Materials and methods: Forty-three patients' samples were processed to establish organoids. To analyze the character of these organoids, immunohistochemistry, Western blotting, drug sensitivity assays, clonogenic survival assays, and animal experiments were performed. The HPV status and TP53 mutational status were also confirmed in these lines.

Results: HNSCC organoids were successfully established with success rate of 30.2%. Corresponding two-dimensional cell lines were established from HNSCC organoids at higher success rate (53.8%). These organoids showed similar histological features and stem cell, epithelial and mesenchymal marker expression to the original tumors, thus recapitulating many of the characteristics of the original tumor cells. The cisplatin and docetaxel IC50 were determined for HNSCC organoids and the corresponding 2D cell lines using drug sensitivity and clonogenic survival assays. Responses to drug treatment in vivo were found to be similar to the IC50 calculated from organoids by drug sensitivity assays in vitro.

Conclusion: We established novel in vitro HNSCC cancer organoid lines retaining many properties of the original tumors from they were derived. These organoids can predict in vivo drug sensitivity and may represent useful tools to develop precision treatments for HNSCC.

Keywords: 3D culture; Cancer organoid; Cancer tissue-originated spheroid (CTOS); Cisplatin; Docetaxel; Drug sensitivity assay; HPV-positive; Head and neck cancer.

Conflict of interest statement

Potential Conflicts of Interest: None declared

Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Figures

Figure 1.

Establishment of organoids from HNSCC cancer tissue, and HNSCC organoids recapitulate the characteristics of original tumor tissues. (A) Scheme of preparation of organoids by CTOS method. (1. Washing the specimens with HBSS well. 2. Necrotic tissue was removed with scalpel or scissors. 3. The specimens were minced into small pieces by scalpel or scissors. 4. The minced specimens were digested for 2 hours with shaking at 37°C. 5. The digested specimens were sequentially filtered through 100-µm mesh. 6. Then, filtered through 40-µm cell strainer. 7. The fragments on 40-µm cell strainer were collected and cultured with growth media. 8. After confirmation of CTOS formation, CTOSs were transferred into Matrigel and cultured with growth media.) (B) Representative phase contrast images of HNSCC organoid lines. The shapes of organoids are different among organoid lines. (C) Representative phase contrast image of 2D HNSCC cell lines established from HNSCC organoids. (D) Representative H&E sections of HNSCC organoids, original tumor tissues and xenograft tumors. (Magnification × 100) (E) Immunohistochemical staining of pankeratin, vimentin and CD68. (Magnification × 100) (F) Immunohistochemical staining of cancer stem cell markers, ALDH1A1 and CD44. (Magnification × 100) (G) Quantified graphs of (F).

Figure 2.

HPV and TP53 mutational status in HNSCC organoids and corresponding 2D cell lines. (A) PCR images detecting high-risk HPV positivity. MDA-HN-2C, −16C, −55C, and MDA–HN2016–2 showed bands indicative of high-risk HPV positive cell lines. Image of digested PCR products with Ava II showed different size of bands enable identification of specific HPV genotype. (B) Western blotting images of HNSCC organoids and corresponding 2D cell lines. HPV positive cell lines, MDA-HN-2C, −16C, −55C and MDA-HN2016–2 showed strong p16 and weak p53 bands respectively. MDA-HN-4C, −21C and MDA-HN2016–21 showed no p53 bands because of frame shift or splicing. (C) Detail information about HPV status and TP53 mutational status of all organoid lines and 2D cell lines.

Figure 3.

Drug sensitivity assays of cisplatin and docetaxel in MDA-HN-2C, −18C, −21C and corresponding 2D cell lines, and PCI-13 G245D cell line and corresponding organoid MDA-HN-1C. (A) Representative images of clonogenic survival assays. (B) Clonogenic survival curves of cisplatin and docetaxel responses in MDA-HN2016–2, −18 and −21. Cisplatin IC50 for MDA-HN2016–2, −18 and −21 are 0.80 µmol/L, 1.12 µmol/L and 0.42 µmol/L, and docetaxel IC50 are 0.59 nmol/L, 0.49 nmol/L and 0.30 nmol/L, respectively. (C) Representative images of drug sensitivity assay of HNSCC organoids. Arrows show same organoid at Day 1 and Day 8. (D) Drug sensitivity of cisplatin and docetaxel for MDA-HN-2C, −18C and −21C. Cisplatin IC50 for MDA-HN-2C, −18C and −21C are 1.02 µmol/L, 0.94 µmol/L and 0.92 µmol/L, and docetaxel IC50 are 3.75 nmol/L, 1.90 nmol/L and 1.46 nmol/L, respectively. (E) Clonogenic survival curves of docetaxel for PCI-13 G245D. Docetaxel IC50 for PCI-13 G245D is 0.21 nmol/L. (F) Drug sensitivity of cisplatin and docetaxel for MDA-HN-1C. Cisplatin IC50 is 0.76 µmol/L, and docetaxel IC50 is 1.57 nmol/L, respectively.

Figure 4.

Differential sensitivity to cisplatin and docetaxel in MDA-HN-2C and PCI-13 G245D in vivo. (A) The graph of body weight of MDA-HN-2C injected mouse. Some mice in docetaxel group showed rapid body weight loss 2 weeks after starting treatment. (B) Tumor growth curves of MDA-HN-2C showing resistance to cisplatin and docetaxel. (C) The graph of body weight of PCI-13 G245D injected mouse. (D) Tumor growth curves of PCI-13 G245D following treatment with cisplatin or docetaxel. Docetaxel showed significant tumor growth inhibition. (**; p<0.01) (E) Representative H&E section of MDA-HN-2C mice tumors. All groups showed similar histological features. (Magnification × 100) (F) Representative H&E section of PCI-13 G245D mice tumors. Only docetaxel group showed non-viable cells. (Magnification × 200)