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Three-Dimensional In Vitro Cell Models of HNSCC: History
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Subjects: Oncology
Contributor: Irina Arutyunyan , Enar Jumaniyazova ,
Andrey Makarov
, Timur Fatkhudinov

Researchers have been trying to answer the demand of clinical oncologists to create an ideal preclinical model of head and neck squamous cell carcinoma (HNSCC) that is accessible, reproducible, and relevant. Over the past years, the development of cellular technologies has naturally allowed people to move from primitive short-lived primary 2D cell cultures to complex patient-derived 3D models that reproduce the cellular composition, architecture, mutational, or viral load of native tumor tissue. Depending on the tasks and capabilities, a scientific laboratory can choose from several types of models: primary cell cultures, immortalized cell lines, spheroids or heterospheroids, tissue engineering models, bioprinted models, organoids, tumor explants, and histocultures. HNSCC in vitro models make it possible to screen agents with potential antitumor activity, study the contribution of the tumor microenvironment to its progression and metastasis, determine the prognostic significance of individual biomarkers (including using genetic engineering methods), study the effect of viral infection on the pathogenesis of the disease, and adjust treatment tactics for a specific patient or groups of patients. 

  • head and neck squamous cell carcinoma
  • in vitro models
  • 3D models

1. Spheroids

Multicellular spheroids collected from tumor cells are considered the most simple and reproducible in vitro tumor model capable of reflecting the response of tumor tissue to therapy [1][2][3][4]. Changing cultivation conditions during the transition from 2D to 3D leads to the formation of volumetric multicellular conglomerates, in which cells interact with each other and the external environment without the participation of a culture substrate; as a result, pathophysiological gradients characteristic of tumor tissue are formed in the spheroids [5][6][7][8]. Spheroids are characterized by cellular zoning, which begins to clearly manifest itself as the size of the spheroid increases; upon reaching a diameter of 500 μm, a hypoxic zone and foci of necrosis are formed in the spheroid. Histological examination allows the identification of several layers in large spheroids: an outer layer represented by rapidly proliferating cells, a middle layer with senescent or quiescent cells, and an inner layer containing necrotic cells [1][8][9][10]. These heterogeneous layers are the result of limited diffusion of oxygen and nutrients into the multicellular structure. Cells in the outer layer multiply rapidly due to easier access to oxygen, nutrients, and growth factors; closer to the center of the spheroid, the supply of oxygen and nutrients decreases, and the amount of carbon dioxide and decay products increases [5][8][11].
Large spheroids with a diameter of 500 μm or more reproduce the oxygen gradient characteristic of tumor tissue, forming a focus of hypoxia in its center, which affects the processes of tumorigenesis, activating protumor transcription [12][13] and triggering mechanisms for the development of chemo- and radio-resistance, which lead to increased tumor growth and worsening patient outcomes [14][15][16]. Under conditions of insufficient tissue perfusion, protons and lactic acid accumulate in the extracellular space, leading to the formation of an acidic extracellular microenvironment and creating a pH gradient. This phenomenon is similar to the Warburg effect associated with lactate accumulation in solid tumors [17].
The formation of spheroids occurs spontaneously under conditions where cell–cell interactions predominate over interactions between cells and the culture substrate. In fact, all the main methods for forming spheroids are aimed at creating the following conditions: the hanging drop method, culturing cells in dishes or vials with an ultra-low adhesion surface, and culturing using rotational systems or magnetic fields [1][18][19][20]. In each of these methods, the main task is to minimize the contact of cells and the culture substrate; however, the methods for solving this problem differ:
(1)
completely remove the substrate, culturing cells in a drop supported by surface tension (hanging drop method) [1][21][22];
(2)
modify the substrate, eliminating the possibility of cells to attach to it (culturing in dishes or plates with an ultra-low adhesion surface) [23][24][25];
(3)
ensure continuous mixing of the cell suspension, preventing cells from settling and coming into contact with the substrate (agitation-based method, magnetic levitation) [8].
The first two methods are most commonly used to create spheroids from immortalized cell lines or primary cell cultures isolated from tumor tissue of patients with HNSCC [22][25]. Primary cultures are inherently heterogeneous and are obviously more relevant models for use in personalized medicine. However, the process of obtaining primary cultures from HNSCC biopsies does not always lead to the desired result:
(1)
part of the material may be initially contaminated with bacteria or fungi;
(2)
during the cultivation process, epithelial cells can be replaced by more rapidly proliferating stromal cells;
(3)
the rate of cell proliferation and the efficiency of spheroid formation decreases with an increase in the number of passages performed;
(4)
some methods for obtaining spheroids from such cultures are ineffective [26][27].
To reproduce the heterogeneous composition of tumor tissue, it was proposed to use heterospheroids (or heterotypic spheroids, co-culture spheroids) [28], consisting of a well-characterized immortalized line and an additional cellular component usually associated with tumor cells in vivo, for example, immune cells, fibroblasts, or endothelial cells. Currently, there is active research work using heterospheroids; however, in most cases they are used to model liver and pancreatic tumors, and only a few studies have been performed with heterospheroids based on HNSCC tumor lines.
Thus, reproducible and accessible protocols for obtaining spheroids from immortalized lines or primary cultures of HNSCC allow the creation of in vitro models, in which diffusion restrictions are reproduced not only for oxygen and nutrients, but also for the transport of biologically active substances, imitating the physiological barriers existing in vivo. In many respects, spheroids are closer to tumor tissue than 2D cell cultures, which allows them to be used as a relevant preclinical model for testing the efficacy and toxicity of many anticancer drugs [1][5][21][23][29][30].

2. Tissue-Engineered Models

The 2D cultures, spheroids, and heterospheroids described above have a significant drawback as an in vitro model, namely the lack of interaction between tumor cells and the extracellular matrix (ECM), which plays an important role in the occurrence and progression mechanisms of HNSCC [31]. In order to increase the relevance of in vitro tumor models, tissue engineering methods were used, namely cell culture on 3D scaffolds. The first attempts to cultivate HNSCC cells in collagen gel were made at the end of the 20th century [32][33], and the first model of HNSCC using a synthetic scaffold was created in 2007, based on oral squamous cell carcinoma (OSCC-3) cells cultured within a highly porous poly(lactide-co-glycolide) scaffold [34]. Currently, type I collagen in the form of a gel [35] or a porous matrix [36] is most often used as a scaffold material for modeling HNSCC. Such scaffolds are populated with both immortalized tumor cells (SCC090, UM-SCC6 [36], FaDu [37]), and primary cultures obtained from tumor tissue of patients with HNSCC [35][36].
A more complex technology for creating a 3D model of tumor tissue using TRACER (the Tissue Roll for Analysis of Cellular Environment and Response) is also described. It is a novel model in which cells (HNSCC cell lines CAL33 or FaDu co-cultured with cancer-associated fibroblasts) are embedded in a collagen hydrogel infiltrate into a porous cellulose scaffold that is then rolled around an aluminum core to generate a multi-layered 3D tissue [38][39].
Such models are very convenient for studying the interaction of tumor cells and individual proteins of the extracellular matrix or stromal cells [39], and the variability of the scaffolds used makes it possible to control the conditions of this interaction, for example, providing normoxia [38] or hypoxia [36]. By analogy with already developed tissue engineering models of other types of solid tumors, it is, in principle, possible to model HNSCC using a wide variety of scaffolds based on biocompatible natural or synthetic polymeric materials and their composites [40][41][42][43].
Another option for this type of in vitro model is the cultivation of tumor cells on decellularized ECM, when cellular components are removed, and the biological composition is mostly preserved. A study by He et al. compared the proteome of normal and tumor decellularized ECM from oral cavity tissues. It was found that 26 proteins only showed in tumor ECM, 14 proteins only showed in late-stage tumor ECM, and most variant proteins were linked to metabolic regulation and tumor immunity. Tumor ECM influenced the proliferation, apoptosis, and migration of tumor cells, as well as polarized macrophages towards an anti-inflammatory phenotype, which once again confirms the hypothesis of a significant influence of the ECM on the progression of HNSCC [31].
Thus, tissue-engineered in vitro tumor models provide a native-like tissue context for studies of HNSCC biology, but their applications are still at an early stage.

3. Bioprinted Models

In recent years, the method of 3D printing of tissues (including tumor tissues) has been actively developing. This method makes it possible to create complex, volumetrically defined structures, which are also anatomically accurate and relevant [44]. The method consists of constructing 3D structures by precise spatial superimposing layers of bioink that contain cells or cell conglomerates, cytokines, and extracellular matrix components [45][46]. Bioink is usually biocompatible hydrogel loaded with single cells or cell spheroids [47].
Three-dimensional printing was developed several decades ago, but only recently has this versatile technology been adapted to the biomedical field for the fabrication of complex structures using biocompatible materials [45][48]. Inkjet printing [49], extrusion printing [50], laser printing [51], and stereolithography [52] are used for 3D tissue bioprinting. These methods differ from each other in many respects: the cost of equipment and consumables, printing speed, resolution, restrictions on the maximum linear dimensions of the created object, the set of biogels suitable for work, etc. All these bioprinting methods enable a highly improved control of cell distribution within the 3D space compared to conventional approaches [45][53]. The created 3D models are structurally stable over a wide temperature range of 4 °C to 37 °C, which is a necessary condition for working with biological objects [44].
Currently, there are not many works describing the successful modeling of HNSCC using the bioprinting method. In a study by Kort-Mascort et al., emphasis was placed on the development of a new printable biogel containing alginate and gelatin as rheological modifiers, which impart mechanical integrity to the biologically active decellularized ECM (dECM), derived from porcine tongue after its decellularization and solubilization. The topographical characterization of the bioink showed a fibrous network with nanometer-sized pores. Immortalized cell lines UM-SCC-12 (larynx carcinoma) or UM-SCC-38 (oropharynx carcinoma) were selected as the cellular component. The model in the shape of discs with a 5 mm diameter and 500 µm height was printed using extrusion printing. The model was highly reproducible and allows proliferation and reorganization of HNSCC cells while maintaining cell viability above 90% for periods of nearly 3 weeks; cells produced spheroids having a cross-sectional area of at least 3000 μm2 by day 15 of culture and were positive for cytokeratin. The resulting 3D model was used to assess the response of tumor cells to cisplatin and 5-fluorouracil [54]. The authors continued their work in this direction and recently published data on an improved 3D model of HNSCC, in which the cellular component was represented by two types of cells—UM-SCC-38 and A8-HVFFs (immortalized human vocal fold fibroblasts) in a ratio of 1:2. In the process of culturing the model, spheroid development and growth over time with cancer cells in the core and fibroblasts in the periphery were observed [55].
It can be concluded that the 3D bioprinted model of HNSCC is essentially an improved tissue-engineered model, where a hydrogel is used as a scaffold, and the use of a printing method instead of the usual layering of the gel on a substrate allows for more accurate control of the compliance of the tissue architecture with the specified parameters. So far, bioprinted HNSCC models have been obtained only for immortalized lines, which is due to the need to use a large number of cells to obtain the desired result (in [55] cells were encapsulated in gel at a final concentration of 10 million cells per ml). Obviously, an important task for researchers is to obtain such 3D models with HNSCC patient-derived cells to closely match more biological features and potentially use this technique for personalized medicine.

4. Organoids

The technology of obtaining organoids, which represent an improved in vitro model that combines the advantages of spheroids (the ability of cells to 3D self-organization) and tissue engineering models (the presence of ECM) helped researchers to advance even further on the path to studying the interaction of tumor cells and the ECM [56]. Based on grammar, “-oid” is a suffix meaning “resembling”; organoid thus means “resembling an organ”, so organoids have to exhibit at least some organ functionalities of the modeled tissue [57]. Over the years of improving protocols for the production and use of organoids as an in vitro model of tumor tissue (in this case, the term “tumoroid” is also used), their main mandatory properties have been determined:
(1)
organoids contain tumor cells at different levels of differentiation, including cancer stem cells;
(2)
organoids consist of several cell types that self-organize in space, reproducing the architectonics of the original tumor tissue;
(3)
self-organization of the organoid occurs in the presence of the ECM;
(4)
the interaction of cells and the ECM allows reproduction of the functional properties of the simulated tumor tissue [57][58][59].
Thus, organoids have high genetic and phenotypic similarity to native tissue, maintaining the original intratumoral heterogeneity [60][61].
Most often, organoids for modeling HNSCC are obtained from resected tumor tissue of the patient. In the first stage, the biopsy sample is cleared of tissues of the peritumoral area (connective, muscle, fat), then washed very thoroughly, since the area of material collection (oral cavity, pharynx, nasal septum) is highly likely to be contaminated with bacteria or fungi. Next, the tissue is subjected to mechanical and enzymatic disaggregation using collagenases, dispase, hyaluronidase, trypsin, and EDTA (separately or as part of a cocktail of enzymes), after which sieved single cells or cell clusters are plated in a 3D extracellular matrix (ECM) hydrogel such as basement membrane extract (BME), where they form complex conglomerates [62]. This matrix is obtained from the basement membrane of sarcoma of Engelbreth-Holm-Swarm (EHS) mice, and is rich in such ECM proteins as laminin (a major component), collagen IV, heparan sulfate proteoglycans, entactin/nidogen, and a number of growth factors. It is easy to use and commercially available in standard, protein-enriched, or growth-factor reduced versions [63]. On the other hand, the EHS matrix is extremely complex; proteomic analysis shows that it contains more than 1800 unique proteins, the ratio of which varies from lot to lot, which can affect the reproducibility of the properties of organoids grown in it. In addition, the matrix obtained from mouse tumor tissue has an obvious drawback—it does not meet animal-derived-free standards, which will undoubtedly become mandatory for biomedical products in the near future, so researchers are actively looking for a replacement among synthetic or natural hydrogels [64][65].
Over the past few years, organoids have been successfully obtained from normal and tumor tissues of the brain, lungs, esophagus, stomach, intestines, liver, pancreas, kidneys, salivary glands, ovaries, mammary glands, prostate, and other organs [66]. For each source, a lengthy selection of optimal conditions for the formation of organoids was necessary. In protocols for obtaining organoids from head and neck tumor tissue, the steps for isolating individual cells or clusters do not differ fundamentally between different research groups, but the composition of the culture medium varies significantly.
The number of studies that resulted in the successful production of HNSCC organoids is small due to difficult logistics (the time from resection of tumor tissue to delivery to the laboratory must be minimized), an often insufficient volume of biomaterial, as well as high requirements for the qualitative and quantitative composition of the culture medium, which demands the mandatory presence of growth factors, cytokines, and inhibitors of signaling pathways leading to the triggering of apoptosis or epithelial-mesenchymal transition. At the same time, the efficiency of obtaining organoids from tumor tissue of the head and neck does not exceed 60–70%, even in laboratories that have been actively working with this material for many years. It is believed that the main factor influencing the efficiency of obtaining this 3D model of HNSCC is the quality of the initial biomaterial, since necrotic tumor tissue has poor organoid-forming efficacy. To minimize cell death and increase organoid outgrowth, Driehuis et al. recommend to transport tumor pieces in ice-cold basic medium containing 10 μM Rho kinase (ROCK) inhibitor compound Y-2763 or (less desired) ice-cold PBS [62]. It has also been shown that organoids can be established from cryopreserved tissues, although the efficacy of derivation is probably lower than that obtained when starting with fresh material [62]. According to Wang et al., the efficiency of obtaining HNSCC organoids is also affected by:
(1)
mass of biomaterial (for tumor samples weighing less than 50 mg, the efficiency of obtaining organoids from it is reduced to 3%);
(2)
time of transportation of tumor tissue to the laboratory (time exceeding 24 h leads to a decrease in efficiency from 60–70% to 22%);
(3)
composition of the culture medium (for example, concentration of Wnt3a, R-spondin-1, EGF, Y27632, Noggin, and FGF2);
(4)
ECM used (Matrigel is preferable to collagen I);
(5)
primary/recurrent tumor status (for nasopharyngeal carcinoma, the efficiency of obtaining organoids is 82% for recurrent tumor and only 47% for primary tumor) [67].
In the same study, Wang et al. showed that the effectiveness of obtaining organoids from nasopharyngeal carcinoma tissue does not depend on the gender and age of the patient, or on the clinical stage or lesion location (primary focus or metastasis) of the tumor [67].
Thus, obtaining organoids requires significant material and time, as well as highly qualified personnel and close coordination of clinicians and cell biologists. The result is a tumor model that can be maintained in vitro for a long time, while maintaining genetic stability [68][69] and fairly fully reproducing the morphological characteristics of the original tissue. For example, organoids from nasopharyngeal carcinoma do not express CK7, unlike organoids from normal mucosa obtained from the same patient, which corresponds to differential expression of this marker in the original tissues [67]. It has been shown that organoids, when cultivated, retain the expression of many other markers of head and neck tumors, including those used to identify cancer stem cells: CK13, CK18, ALDH1A1, BMI-1, CD44, and CD133 [67][69][70][71][72]. Interestingly, lactate, which is promoted by Wnt activity, is required to maintain the population of CD133+ cells in HNSCC organoids. Moreover, silencing monocarboxylate transporter 1 (MCT1), the prominent pathway for lactate uptake in human tumors, with siRNA significantly impaired organoid-forming capacity of oral squamous cell carcinoma cells, which allows MCT1 to be considered as a potential therapeutic target for the treatment of HNSCC [72].
Organoids are considered highly relevant in vitro tumor models because they are able to preserve a unique set of biomarkers from donor tissue [73][74]. Increasing evidence indicates that organoids can predict response to treatment of the tumor from which they are derived [75]. In a pilot study, organoids obtained from 21 patients with gastrointestinal tumors predicted response to various types of chemotherapy with 100% sensitivity and 93% specificity [76]. A number of studies have highlighted the prognostic value of organoids in predicting the response of solid tumors to radiotherapy and its combination with other treatments [77][78]. In addition, organoids can also be expanded in vitro and cryopreserved, facilitating the establishment of an organoid biobank of different cancer subtypes from a large number of patients, representing an extremely useful tool for preclinical research [9][79][80].

5. Tumor Explants and Histocultures

Another type of 3D cell model is patient-derived tumor explant (PDE) cultures, in which a tissue fragment is kept alive under ex vivo culture conditions. Explants require minimal manipulation to obtain an in vitro tumor model: the tissue, after mechanical grinding (usually to fragments of 1–3 mm3 in size), is placed in a culture medium. This approach allows preserving the heterogeneous composition of the original tumors, including the extracellular matrix and tumor-associated cells [81]. Explants are often used to obtain adherent 2D primary cell cultures, but in the case of the PDE model this is not done, so researchers cultivate explants under special conditions that exclude the migration of plastic-adherent cells from the tissue fragment. To do this, HNSCC explants are placed on an additional supporting matrix, for example, a collagen sponge [82] or a dermal equivalent consisting of human fibroblasts cultured on a viscose fiber fabric [83].
Despite the ease of production and the possibility of preserving the architecture of tumor tissue, the scope of application of PDE as an in vitro model is significantly limited by its fragility; most often, PDE is used for rapid testing of the effectiveness and/or toxicity of drugs within 5–7 days after being obtained [81]. It has been shown that the viability of HNSCC explants decreases from 90% to 30% within a week after the start of cultivation [84]. The survival rate of HNSCC explants can be increased several times by modifying the culture protocol, for example, adding hydrocortisone, aprotinin, ascorbic acid, EGF, or folic acid to the medium [83][84], or placing the explant in more physiological conditions that imitate contact with normal tissue, for example, onto the surface of the dermal equivalent [83] or into cell sheet composing of epithelium and subepithelial stroma [85]. Under such conditions, explants can be maintained in culture for 21 [83] or even more than 30 days [85], while cancer cell heterogeneity and the microenvironment, including vital immune cells, as well as tissue foci of hypoxia, are maintained.
Another option for this type of 3D model is histoculture. When preparing it, tumor tissue, as when obtaining explants, is crushed mechanically, not with surgical instruments, but with the help of a vibratome, which makes it possible to obtain thick sections of unfixed tissue. In these sections, as in explants, tumor cells are retained in their original microenvironment, including the extracellular matrix and immune and stromal cells [86]. The main problem with culturing histocultures is rapid deterioration of tissue condition and loss of cell viability (from 2 [87] to 6 days [88]). However, this time is sufficient to assess the response of tumor tissue to the action of, for example, cisplatin, docetaxel, and cetuximab, while apoptotic fragmentation, activation of caspase 3, and cell loss were observed in treated tumor slices, which demonstrated a heterogeneous individual response [87][88].
Thus, explants and histocultures of HNSCC are easy to obtain and effective for quickly assessing the response of tumor tissue to drug therapy, which suggests their promise for use in personalized medicine.

6. Microfluidic Devices (Tumor-on-Chip)

At the very beginning, researchers will make an important note: the cultivation of tumor cells in microfluidic devices is not a separate type of model; rather, it is a special technology for culturing the models described above, providing continuous perfusion and removal of waste from cells and mimicking the function of the circulatory system. Microfluidic devices exploit the physical and chemical properties of liquids and gases at a microscale; in such systems, liquids circulate through channels with dimensions of tens to hundreds of micrometers [89]. Thanks to their small size, microfluidics allow the analysis and use of lower volumes of samples, chemicals, and reagents, reducing the global fees of applications. In addition, this technology makes it possible to very accurately control the impact on the object under study; in the future, it will also make it possible to automate routine research work. To assess the tumor response to exposure, it is possible to analyze both the system effluent (sampling can be carried out at any selected interval, for example, every 2 h) and the object itself after extraction [89][90].
When modeling HNSCC, microfluidic devices are most often used for culturing tumor tissue fragments (explants and histocultures).
Thus, tumor-on-chip technology is a valuable tool for personalized assessment of the effectiveness of various antitumor agents (alone or in combination), allowing dynamic monitoring of the response of tissue samples from patients with HNSCC to treatment. The development of microfluidic platforms can improve patient outcomes through the selection of an optimal personalized treatment strategy.

This entry is adapted from the peer-reviewed paper 10.3390/jpm13111575

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