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. Author manuscript; available in PMC: 2019 Dec 1.
Published in final edited form as: Oral Oncol. 2018 Oct 23;87:49–57. doi: 10.1016/j.oraloncology.2018.10.018

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

Noriaki Tanaka a,1, Abdullah A Osman a, Yoko Takahashi a, Antje Lindemann a, Ameeta A Patel a, Mei Zhao a, Hideaki Takahashi a, Jeffrey N Myers a
PMCID: PMC6294331  NIHMSID: NIHMS1510403  PMID: 30527243

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 histlogical features and stem cell, epithelial and mesenchemal 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 culculated 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: Head and Neck Cancer, Cancer Organoid, Cancer Tissue-Originated Spheroid (CTOS), HPV Positive, 3D Culture, Drug Sensitivity Assay, Cisplatin, Docetaxel

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide with approximately 550,000 new cases being diagnosed each year [1]. In recent years, there have been many advances across all of the treatment modalities available for HNSCC patients such as extended resection made possible by microvascular free-tissue transfer reconstructions [2], intensity-modulated radiation therapy (IMRT) [2–4], intensity-modulated proton therapy (IMPT) [2], molecularly targeted therapy [2, 5] and immunotherapy [5, 6]. However, the cure rate for advanced HNSCC patients remains poor [7] mainly as a result of treatment resistance of tumors cells to conventional chemotherapy and radiotherapy.

Precision medicine is a relatively new strategy to identify the best therapy for each patient’s disease, based on the genomic characterization of an individual’s tumor, and while this approach holds tremendous promise, the complex genetic and epigenetic profiles of individual tumor cells has made this approach challenging. Another approach to predicting treatment response has been to directly test tumor cells from patients in established two-dimensional (2D) models in drug screening studies. Recent studies by Yoshii and colleagues indicated that three-dimensional (3D) culture conditions may be more reflective of tumor in vivo growth conditions than 2D cell cultures [8, 9]. To explore this, we set out to develop 3D culture models for HNSCC and to compare this to 2D cultures and in vivo tumors models.

Patient-derived xenografts (PDXs) have been also widely used for drug screening studies to overcome the limitations of 2D screening studies [10]. However, PDX models also have some limitations; including, low engraftment rates of cancer in nude mice, striking differences in pharmacokinetics between two humans and mice, and the lengthy time required for drug screening (4 to 8 months) studies in PDXs [10–12].

Recently, 3D cell culture models have also used for drug screening studies and biomarker analysis [9, 13, 14]. Establishment of cell lines from 2D cultures of tumors derived from patient tissue has a low success rate, and only select tumor cells can survive in the 2D environment which may be reflected in an altered tumor heterogeneity from the original tumor. Furthermore, it is well known that long term culture can select for genetic alterations [15–17]. Since most 3D culture models derived from established 2D cell lines do not maintain the original tumor phenotype, development of 3D cancer organoid cultures directly from a patient’s cancer tissue which includes cancer stem cells, can better recapitulate the character of the patient’s tumor than those derived from cells previously passaged as 2D cultures [18–21]. Although, there are published methods to generate cancer organoid in various cancer types, establishment of HNSCC cancer organoids has not been attempted. Therefore, we were interested to develop a HNSCC cancer organoid model and characterizes it properties and compare them to features of the original.

The Cancer Tissue-Originated Spheroids (CTOS) method is a technique to establish cancer organoid lines reported by Kondo et al. [22] which showed highly established rate in colon cancer [22], lung cancer [23] and bladder cancer [24]. In this method, the cell lines obtained are cultured and maintained as organoids. Organoids have the capabilities to maintain the same histological features of the original tumors. In addition, the population of cancer stem cell is very similar between the original tumor and organoids lines, indicating that the CTOS method can recapitulate the character of the original tumor. In this paper, we demonstrate the feasibility of establishment and characterization of HNSCC organoids, and the utility of these cell lines as a model for drug screening and testing.

Materials and Methods

Cancer tissue-originated spheroid preparation and culture

This study of human tumor samples was approved by the Institutional Review Board at The University of Texas MD Anderson Cancer Center (MDACC). Resected surgical specimens were obtained from HNSCC patients at MDACC after obtaining informed consent. After obtaining surgical specimens, preparation of CTOS was performed based on the method reported by Kondo et al. [22] with slight modifications (Fig. 1A). Briefly, after washing the specimens with HBSS (Invitrogen, Carlsbad, CA), necrotic tissue was removed with scalpel (Figure 1A–2). Then, the specimens were minced with scalpel and scissors into smaller than 2 mm cubic (Figure 1A–3). The minced specimens were washed with HBSS and digested with DMEM (Invitrogen) containing 0.26 U/ml Liberase DH (Roche, Basel, Switzerland) for 2 hours with shaking at 37 °C (Figure 1A–4). The digested specimens were sequentially filtered through 100-µm metal mesh (Figure 1A–5, Sigma Aldrich) and then through 40-µm cell strainer (Figure 1A–6, LabScientific inc. Highlands, NJ). The fragments on 40-µm cell strainer were collected and cultured with StemPro hESC (Invitrogen) supplementary added 8 ng/ml bFGF (Invitrogen), in an Ultra-low culture dish (Corning, Corning, NY) for 24 to 72 hours (Figure 1A–7). After confirmation of CTOS formation, CTOSs were transferred into Matrigel (Corning) and cultured with StemPro hESC supplemented with bFGF (Figure 1A–8). All established CTOS cell lines could be cultured more than 5 passages were confirmed genetically unique cell lines by STR profiling. We also established CTOS cell line from PCI-13 mutp53 G245D (PCI-13 G245D) mice tumor.

Figure 1.

Figure 1.

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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.

Figure 2.

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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.

Figure 3.

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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.

Figure 4.

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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)

Two-dimensional cell line establishment

After establishment of organoids by the CTOS method, organoids were further used to establish 2D cell lines. Experimental details are shown in supplemental information. All established 2D cell lines were also confirmed as genetically unique cell lines by STR profiling.

DNA extraction

Genomic DNA was isolated from organoids cultured in Matrigel and 2D cell lines using ArchivePure DNA purification kit (5 prime, Hamburg, Germany). Experimental details are shown in supplemental information.

STR profiling

STR profiling was performed as previously described [25]. For data comparison, we referred to well-characterized and validated reference data provided by the ATCC and others [26, 27]. Details are shown in supplemental information.

Western blotting

Whole-cell lysates were harvested from organoids cultured in Matrigel and 2D cell lines. Matrigel was completely removed when the organoids were collected. Whole-cell lysates were then subjected to Western blot analysis with primary antibodies described in supplemental information.

Immunohistochemistry

Immunohistochemistry was performed as previously described [28]. Details are shown in supplemental information.

Human Papilloma Virus (HPV) genotyping

PCR was performed with 0.5 μg genomic DNA using Human Papillomavirus PCR Typing Set (Takara Bio, Kusatsu, Japan) following the manufacture’s protocol. Experimental details are shown in supplemental information.

TP53 gene sequencing

Total RNA was isolated from organoids cultured in Matrigel and 2D cell lines using TRIzol Reagent (ThermoFisher Scientific, Waltham, MA). cDNA was synthesized from total RNA using high capacity cDNA Reverse Transcription kit (Applied biosystems). Then, PCR was performed using Phusion Flash High-Fidelity PCR Master Mix (ThermoFisher Scientific) with specific primers shown in supplementary Table 1. After amplification, PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Purified PCR products were loaded on Agarose gel to confirm their corresponding sizes, and were evaluated via direct DNA sequencing.

Clonogenic survival assay

MDA-HN2016–2, MDA-HN2016–18 and MDA-HN2016–21 cells were seeded in 6-well plates at 500 cells, 1000 cells and 2000 cells per well, respectively. Next day, the cells were exposed to different fixed concentrations of cisplatin (0.125–2 µmol/L) or docetaxel (0.0625–1 nmol/L) for 24 hours, and the clonogenic cell survival was determined as previously described [28].

Animal experiment

All animal experimentation was approved by the Institutional Animal Care and Use Committee of MDACC. A total of 18 mice per cell line were subcutaneously injected with 2 × 102 organoids suspended in 100 µl Matrigel or 2 × 106 cells suspended in a volume of 200 µL of PBS directly into the both sides of flank with a transplantation needle (Natsume Seisakusyo, Tokyo, Japan) for organoids or a 1-mL tuberculin syringe (Hamilton Co., Reno, NV) and a 27-gauge hypodermic needle. Tumors were measured in two dimensions and tumor volume was calculated as V = AB2 (π/6), where A is the longest dimension of the tumor and B is the dimension of the tumor perpendicular to A [29, 30]. Treatment was initiated when tumors were less than 150 mm3 in size. Mice were then treated with single agent cisplatin at a dose of 4 mg/kg administrated intravenously (i.v.) on day 1 for 4 weeks [29, 30], or docetaxel at a dose of 6mg/kg administrated intraperitoneal (i.p.) on day 1 and 4 for 2 weeks [31] or on day 1 for 4 weeks. Mice were examined twice a week for 4 weeks during which time tumor size and body weight were assessed and recorded. At the end of study, mice were euthanized when they lost more than 20% of their preinjection body weight or 4 weeks following the initiation of treatment. After mice were sacrificed, tumors were collected and embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E).

Cell lines

The STR-verified HNSCC cell lines UM-SCC-47, HMS001, HN30, PCI-13, and STAR were described previously [25, 30, 32]. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS. Stable cell lines expressing mutant p53 G245D (PCI-13 G245D) was established as described previously [29, 32].

Results

Establishment of head and neck cancer organoids by CTOS method

A modified CTOS method was used for preparation of head and neck cancer organoids from specimens obtained from head and neck cancer patients. A total of 43 specimens were processed (Table 1) and CTOS formation was seen in 41 specimens (formation rate; 95.3%). A total of 16 of the 41 original CTOS cultures continued to grow following transfer to Matrigel (growth rate; 37.2%) and 13 of the CTOS cultures were successfully expanded in vitro (passage; 30.2%). Three of 13 organoid cultures could be split and propagated several passages with a result of 10 of the original organoid cultures being used for further experiments. Representative images are shown in Figure 1B. The HNSCC organoids showed lower rates of passage when compared to passage rates reported for colon cancer [22] (68.6%) and lung cancer [23] (80.0%), respectively. There was no significant difference of success rate associated gender, pathological stage, histological grade, histological invasion pattern or primary site (Table 1). HNSCC organoids can be cultured and maintained for more than one month, whereas colon cancer CTOSs can be cultured and maintained for only 2 weeks [22]. In addition, these organoids can be stored at frozen conditions and easily recovered as 2D cell lines. Furthermore, 2D cell lines can be established from organoids and seeded onto regular culture dishes. Eventually, seven 2D cell lines (53.8%) have been established from 13 organoids (Figure 1C).

Table 1.

Success rate of CTOS formation, growth and passage in vitro from HNSCC specimen

Formation (%) Growth (%) Passage (%)
Gender
Male 31/32 (96.9) 11/32 (34.4) 10/32 (31.3)
Female 10/11 (90.9) 5/11 (45.5) 3/11 (27.3)
Pathological Stage
T1 and T2 21/22 (95.5) 8/22 (36.4) 6/22 (27.3)
T3 and T4 20/21 (95.2) 8/21 (38.1) 7/21 (33.3)
N0 19/20 (95.0) 6/20 (30.0) 4/20 (20.0)
N1 or greater 22/23 (95.7) 10/23 (43.5) 9/23 (39.1)
Histological Grade
G1 3/3 (100.0) 1/3 (33.3) 0
G2 22/22 (100.0) 8/22 (36.4) 7/22 (31.8)
G3 9/9 (100.0) 5/9 (55.6) 4/9 (44.4)
Post therapy 7/9 (77.8) 2/9 (22.2) 2/9 (22.2)
Perineural Invasion
Positive 17/19 (89.5) 6/19 (31.6) 4/19 (21.1)
Negative 24/24 (100.0) 10/24 (41.7) 9/24 (37.5)
Lymph-vascular Invasion
Positive 17/18 (94.4) 8/18 (44.4) 7/18 (38.9)
Negative 24/25 (96.0) 8/25 (32.0) 6/25 (24.0)
Site
Buccal Mucosa 2/2 (100.0) 1/2 (50.0) 1/2 (50.0)
Floor of Mouth 1/2 (50.0) 0 0
Gingiva 7/7 (100.0) 2/7 (28.6) 1/7 (14.3)
Tongue 16/16 (100.0) 9/16 (56.3) 7/16 (43.8)
Tonsil 4/4 (100.0) 2/4 (50.0) 2/4 (50.0)
Pharynx 2/2 (100.0) 0 0
Larynx 9/9 (100.0) 2/9 (22.2) 2/9 (22.2)
Metastatic LN 0/1 (0.0) 0 0
HPV status
Positive 9/9 (100.0) 3/9 (33.3) 3/9 (33.3)
Negative 32/34 (94.1) 13/34 (38.2) 10/34 (29.4)
Totals 41/43 (95.3) 16/43 (37.2) 13/43 (30.2)
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Organoids show characteristics similar to the original tumors

Next, to determine if organoids maintain characteristics similar to their original tumors, we compared the histological features between organoids and original tumors by H&E sections. All organoids showed similar histological patterns to their original tumors (Figure 1C). In addition, some organoids showed tumorigenicity in nude mice when 1–2×103 organoids were injected into the flank subcutaneously. Tumors which formed in mice also showed similar histological patterns to original tumors (Figure 1D). Cancer tissues are heterogeneous, therefore, we evaluated the purity of the cells in organoids. Pankeratin was used as an epithelial marker, vimentin as a mesenchymal marker, and CD68 as a marker of macrophage and Langerhans cells. Positive staining of vimentin is sometimes seen in epithelial cancer cells, so the consistency of expression pattern between organoids and original tumors is important for evaluation. Positive staining of pankeratin was found only in cancer cells in the HNSCC organoids and original tumors whereas CD68 showed positive reaction only in original tumors (Figure 1E). Vimentin showed positive staining only in mesenchymal cells of original tumor in MDA-HN-2C. Substantial positive reaction in mesenchymal and epithelial cells of the original tumor and epithelial cells of organoids in MDA-HN-16C was also observed. Vimentin expression patterns in epithelial cells between organoids and original tumor were very similar in MDA-HN-16C, suggesting that organoids are composed of pure cancer cells of the original tumor. In addition, we evaluated the rate of cancer stem cell markers in organoids and their tumors of origin to determine if organoids are composed of a specific population from of original tumors. Both CD44 and ALDH1A1 are well-known stem cell markers for head and neck cancer [34, 35], thus, we used these two markers to detect cancer stem cells. Positive ratios of these markers were different among cell lines, however, it was very close between organoids and original tumors (Figure 1F). Furthermore, quantification of the positive ratio of these markers, showed no significant difference between organoids and original tumors (Figure 1G).

HPV status and TP53 mutational status in organoids

The HPV positivity and TP53 mutational status are important because they can impact the biological behavior of the tumor [29, 30]. Therefore, to investigate these characteristics of the HNSCC organoids, we performed HPV genotyping using established HPV positive cell lines as positive controls (HMS001 and UM-SCC-47; HPV-16 positive, STAR; HPV-33 positive) and HPV negative cell lines as negative control (PCI-13 and HN30). Three of ten organoids showed high-risk HPV positive (Figure 2A), and the corresponding 2D cell lines also showed positive bands for HPV in the PCR test. To identify the exact genotype, the PCR products were digested with Ava II restriction enzyme and analyzed on agarose gel as indicated. Two of three organoids, MDA-HN-16C and MDA-HN-55C, showed exact same patterns of HMS001 and UM-SCC-47, indicating that these organoids are HPV-16 positive. The MDA-HN-2C organoid and corresponding 2D cell line showed the same pattern of the bands maintaining an HPV-18 positive identity (Figure 2A). We also confirmed overexpression of p16 in these HPV positive cell lines by Western Blot analysis (Figure 2B). TP53 cDNA sequencing and Western blot analysis were performed to identify the exact TP53 mutational status and protein expression levels. Cells bearing splice mutation showed no p53 protein expression (Figure 2B, 2C). HPV positive cells displayed very low level of p53 protein expression, suggesting wild-type p53 (Figure 2B, 2C). Cells harboring mutant p53 showed various degrees of p53 protein expression. Results of HPV genotyping and TP53 mutational analysis are summarized in Figure 2C.

Organoids and their corresponding 2D cell lines display differential sensitivities to cisplatin and docetaxel treatment

To determine if organoids are suitable models for drug studies, we evaluated organoids and cell lines response to cisplatin and docetaxel. For 2D cell lines, clonogenic cell survival assays were performed and the IC50s were calculated (Figure 3A, 3B). Our previous publication showed that wild-type p53 cell lines were very sensitive to cisplatin compared to p53 null and mutant p53 bearing cells [29]. MDA-HN2016–2 cell line was established from MDA-HN-2C organoid. The organoid was established from a relapsed patient following treatment with radiotherapy, cisplatin, docetaxel, and cetuximab. As anticipated, MDA-HN2016–2 showed the highest docetaxel IC50 among the three cell lines (Figure 3B). For organoids, we compared the area of organoids between Day 1 and Day 8 as shown in Figure 3C. The relative area of organoids were calculated and blotted on the graph to calculate the IC50 of the organoids. MDA-HN-2C demonstrated significant resistance to docetaxel in MDA-HN-2C compare to the 2D cell line (Figure 3D). We also evaluated the IC50 of cisplatin and docetaxel for PCI-13 G245D cell line and organoid line MDA-HN-1C established from PCI-13 G245D tumor grown in mice. The cisplatin IC50 for PCI-13 G245D was previously reported as 1.04 µmol/L [29]. While similar cisplatin IC50 was observed in MDA-HN2016–18 cell line bearing high risk p53 mutation compared to PCI-13 G245D, the same cell line had the lowest docetaxel IC50 among all four cell lines tested (Figure 3E). The 3D organoid cell line, MDA-HN-1C, displayed similar cisplatin and docetaxel IC50 to those of MDA-HN-18C and −21C respectively (Figure 3F).

Organoids can predict in vivo drug sensitivity

In vivo sensitivity to cisplatin and docetaxel treatment was evaluated in organoids, MDA-HN-1C, 2C, 18C and 21C, injected subcutaneously into the flank of nude mice. All injected organoids formed tumors in the flank, however, the tumor growth speed was so slow in MDA-HN-1C, 18C and 21C, suggesting that these organoids are not suitable for in vivo experiment. We also injected 2D cell lines, MDA-HN2016–18 and MDA-HN2016–21, and these cell lines also formed slow growing tumor in vivo. MDA-HN-1C was established from PCI-13 G245D mice tumor, meaning PCI-13 G245D mice tumor is mostly same as the original tumor of MDA-HN-1C. In addition, our previous work demonstrated that the PCI-13 G245D cell line possess aggressive in vivo growth phenotype [29]. Therefore, we utilized MDA-HN-2C and PCI-13 G245D cell lines for further in vivo drug testing. At first, we injected the MDA -HN-2C organoid line and the treatment regimens with cisplatin and docetaxel were designed based on our own previous and other studies [29, 30, 31] respectively. Two weeks following treatment with docetaxel, some mice showed excessive body weight loss due to an adverse effect (Figure 4A). Therefore, docetaxel treatment was discontinued for the subsequent third and fourth week as initially planned. In contrast, no mice showed body weight loss or adverse effects in the cisplatin treated group (Figure 4A). During the course of treatment, the size of tumors was consistently measured twice a week and no significant differences among untreated, cisplatin and docetaxel groups were observed (Figure 4B). Next, in an attempt to reduce docetaxel toxicity, we injected PCI-13 G245D cells and docetaxel was administered once a week using the same dosage. Docetaxel significantly reduced in vivo tumor growth and all mice tolerated this modified dosage very well with no obvious severe weight loss (Figure 4C) No significant response was seen in the cisplatin group (Figure 4D). To validate the effect of these drugs, the HE sections of all samples were evaluated. In the first experiment with MDA-HN-2C, there was no difference between untreated group and treated group, because all cells remained viable in all groups (Figure 4E). On the other hand, in the second experiment with PCI-13 G245D, we found a lot of non-viable cells only in docetaxel group although we couldn’t find any difference between untreated group and cisplatin group (Figure 4F).

Discussion

While established cell lines have been considered very useful tools for biological analysis for decades [10], some limitations of 2D cell lines for such analysis have also been reported [10]. Such limitations are attributed to the concept that 2D cell lines can’t exactly recapitulate the characteristics of the original tumors. Several studies have demonstrated the importance of 3D culture or in vivo models for cancer biology, that more faithfully recapitulates the biological features of original tumors [8–14, 18–24]. Cancer organoid 3D culture models have been successfully generated for various cancer types [18, 20, 21, 38–39]. However, to our knowledge, HNSCC organoid models have been yet to be described in the literature. For the first time, we provide data describing novel head and neck cancer organoid lines established by the CTOS method. Interestingly, these organoid lines show very similar histological features to original tumor tissues, and the expression rate of cancer stem cell markers are also similar to their original tumors. These data suggest that the organoids recapitulate many of the properties of the human tumors from which they were derived, which makes them useful tools for biological analysis and therapeutic studies. Similar to other tumor organoids, the formation rate of the HNSCC organoids was very high, but the growth rate and passage seemed to be lower when compared to those seen in colon, lung and bladder cancers [22–24]. Taken together, the results indicate that the 3D in vitro culture conditions may not be optimal for head and neck organoids, limiting continuous renewal and maintenance processes. We have also attempted original culture conditions for optimization using type I collagen gel, however, it didn’t work well and we are still investigating the optimal culture conditions to increase the success rate for growth and indefinite number of passages using growth factors and ECM substrate.

It is well documented that the mutational status of the TP53 tumor suppressor gene and HPV positivity can impact the biology of HNSCC cancer cells [29, 40]. Therefore, the HPV and TP53 mutational statuses were determined to gain insight into the biological behavior of the established HNSCC organoids. Our data provide evidence that HNSCC organoids can be established regardless of TP53 mutational or HPV status. There are very limited numbers of HPV+HNSCC cell lines. In this study, we were able to generate successfully three well characterized HPV positive organoid lines that can be used for further investigation. Another additional useful aspect of this method is that once organoids are established, one can obtain 2D cell lines easily from these organoids. As a result, we established seven 2D cell lines from 13 organoid lines. Thus, the CTOS method is valuable for generating cell lines not only organoid lines but also 2D cell lines.

Furthermore, we present evidence that the HNSCC organoids are useful tools for testing antitumor agents. Exposure of these organoids to cisplatin or docetaxel recapitulated and defined very accurately the range of sensitivity and resistance to these drugs reported in the 2D cell lines. Interestingly, the MDA-HN-2C displayed resistance to cisplatin and docetaxel consistent with the establishment of this organoid from a patient whose tumor recurred after prior treatment with cisplatin and docetaxel. Thus, all cells were considered cisplatin resistant, but their response to docetaxel is unknown. Cisplatin IC50s ranged from 0.42–1.12 µmol/L in 2D cell lines, and 0.76–1.02 µmol/L in organoid lines (Figure 3). We considered 0.8 µmol/L as physiological concentration of cisplatin in HNSCC patients [29], thus, organoids reflected accurately the response to cisplatin with IC50 ≥0.8 µmol/L. Docetaxel IC50 ranged from 0.21–0.59 nmol/L in 2D cell lines, and 1.46–3.75 nmol/L in organoids (Figure 3). In organoids, the difference was more significant, and only MDA-HN-2C showed IC50 >3 nmol/L. Brunsving et al. has reported that 3 nmol/L of docetaxel is the physiological serum concentration in patients achieved 72 hours after 20 mg/m2 docetaxel injection [41]. The In vivo experiments with MDA-HN-2C demonstrated resistance to cisplatin and docetaxel but PCI-13 G245D only showed resistance to cisplatin (Figure 4). Taken together, these results provide strong evidence that HNSCC organoids can predict drug sensitivity in vivo and further suggest that organoids established by the CTOS method recapitulate well the characteristics of the original tumors of their derivation, making them useful tools for analysis of cancer biology and drug testing. Additionally, these organoids are derived from patient tumors and can be subjected to whole genome sequencing and phosphoproteomic analysis to identify and explore useful biomarkers for HNSCC patients’ stratification.

Supplementary Material

Supplemental Material and Methods
Supplementary Table 1

Highlights.

  • The CTOS method is good for establishment of HNSCC cancer organoids.

  • HNSCC cancer organoids established by the CTOS method are well recaptured the character of the original tumor.

  • The results of drug sensitivity assay are different between cancer organoids and 2D cell lines.

  • The results of drug sensitivity assay with cancer organoids are similar to the response of in vivo drug treatment.

Acknowledgements

We wish to thank Dr. Masahiro Inoue for his excellent advice.

Funding: This study was supported by the National Institute of Health/NIDCR R01DE024601 (A.A. Osman and J.N. Myers), the University of Texas MD Anderson Cancer Center Stiefel Oropharyngeal Cancer Program (A.A. Osman and J.N. Myers) and Provost’s Office.

Footnotes

Potential Conflicts of Interest: None declared

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Reference

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Associated Data

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Supplementary Materials

Supplemental Material and Methods
NIHMS1510403-supplement-Supplemental_Material_and_Methods.docx (18.9KB, docx)
Supplementary Table 1
NIHMS1510403-supplement-Supplementary_Table_1.pdf (163.9KB, pdf)

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