New frontiers in anti-cancer drug testing: The need for a relevant In vitro testing model

Highlights

• •Reviews methods for generating 3D structured spheroids for anticancer drug testing.
• •Explores role of spheroids in understanding cancer cell to cell and cell to matrix interaction.
• •Highlights advances in spheroid models for mimicking tumor microenvironment.
• •Discusses cytotoxicity assays and apoptosis analysis using flow cytometry technique for characterizing spheroids.
• •Explores advancements in bioink formulations for 3D bioprinting of spheroids.

Abstract

Drug development is a complex process involving multiple stages, including discovery, preclinical testing and clinical trials to ensure safety and efficacy. In vitro testing is crucial for early-stage anti-cancer drug toxicity and efficacy evaluation, often utilizing traditional monolayer cell cultures. Traditional cell cultures involve growing cells in a two-dimensional monolayer, providing a controlled environment for initial drug testing. It does not entirely replicate or mimic human in vivo microenvironment conditions, where the cell-to-cell interaction and cell-to-matrix (Extra Cellular Matrix) interaction which are vital in cellular function are limited in the traditional cell culture method and the preclinical animal testing models. Also, there are ethical considerations to be faced while using animal models, and the spheroid model can be a potential in vitro model that can reduce the use of animal testing. Hence, the spheroid models, which offer a three-dimensional structure more accurately mimic the natural tumor environment enhancing the study of drug interactions and cellular responses. This review focuses on the various anti-cancer drugs such as 5-Fluorouracil (5-FU), Tirapazamine (TPZ), Cisplatin, Resveratrol, Irinotecan and Doxorubicin that are tested in traditional monolayer cell culture and Spheroid models for comparing the efficacy and toxicity results in both using various analytical methods which helps us understand each method's advantages and limitations in drug development.

Graphical abstract

1. Introduction to drug development

The drug is effectively used in many areas of treatment. The journey of developing new drugs is complicated and challenging, involving extensive research, thorough testing, and careful analysis to ensure both safety and effectiveness. A drug, by definition, is a substance intended for use in curing, improving, treating, or preventing disease. Before a drug can be made available to the public, it must go through a detailed and rigorous process. This process typically includes preclinical research, clinical trials, and obtaining regulatory approval. The drug development process starts with the discovery phase, where scientists identify potential therapeutic agents by screening various compounds. Once the needed lead compound is found, it is taken to preclinical testing, where laboratory experiments (in vitro) and tests on animal models (in vivo) take place. These tests help scientists to evaluate the drug's efficacy and toxicity. If the lead shows positive results, then the Phase 1 trials are done which focus on the drug's safety, determining safe dosage ranges and identifying side effects with a small group of healthy volunteers or patients. Phase II trials assess the drug's efficacy and further evaluate its safety with a larger group of patients who have the condition the drug is intended to treat, helping to establish the optimal dose. Finally, Phase III trials compare the new drug to existing standard treatments in large-scale studies involving thousands of participants, confirming its efficacy, monitoring side effects, and collecting comprehensive data to ensure safe use. Success in these trials is crucial for obtaining regulatory approval and bringing the drug to market (Gong et al., 2015; Takayama et al., 2013).

Clinical trials are important for validating the clinical utility of new drugs. Clinical trials, which are often divided into several phases, play a crucial role in evaluating a drug's safety and efficacy in humans. Monolayer cell cultures (traditional cell culture) and animal models are mostly used in In vitro clinical trials. But they are time-consuming, expensive, and have ethical considerations to face. In recent years, in vitro clinical trial drugs have been tested on traditional cell culture methods or three-dimensional cell cultures. The three-dimensional cell cultures method provides early detection of a drug's s efficacy and toxicity, potentially reducing the time and need for animal testing and speeding up the drug development process. In vitro clinical trials are mostly used in evaluating a drug's efficacy and toxicity, which is important during the drug development process. Efficacy is about how well a drug works to produce the future therapeutic outcome, whereas toxicity is testing if the drug produces any harmful side effects. Cytotoxicity assays are done in both traditional cell cultures and spheroids to test the drug's efficacy and toxicity. Many assays are used to assess these parameters, providing detailed insights into the drug's pharmacodynamics (how it affects the body) and pharmacokinetics (how the body processes it) (Lee et al., 2019; Nayak et al., 2023).

2. Cell culture models in drug testing: evaluating traditional cell cultures vs. spheroid models

Cell culture models, where cells are grown in a controlled environment outside their natural environment, are essential for in vitro testing. Traditional Cell culture models allow researchers to observe how cells respond to drugs in a controlled environment. Cell cultures, which are obtained from different tissues, are widely used in cancer research to screen potential anti-cancer agents and understand their mechanisms of action. There are various types of cell cultures, such as primary cultures taken directly from tissues and secondary cell cultures or cell lines which are sub-cultured from primary cell culture. Cell culture models are widely used in high-throughput drug screening, and target validation providing a rapid and cost-effective means of identifying capable drugs and explaining their cellular and molecular effects (Nguyen et al., 2023; Pinto et al., 2020).

Spheroid models are three-dimensional cell cultures of cells that more accurately mimic human tumour cells' complex architecture and microenvironment. Unlike traditional two-dimensional cell cultures, spheroids have variations in nutrients and oxygen, making them more like the natural environment of cells. This makes them useful for studying drug penetration, drug resistance, drug efficacy and toxicity as same as actual human tumour cells. Spheroids can be formed using various techniques, such as hanging drop, spinner flask, microfluidic systems and microwell arrays, allowing for the generation of spheroids with constant size and shape, which is important for reproducibility and comparability in experiments. The advantages of spheroids include better mimicking the 3D structure and microenvironment of tumors, the presence of cell-cell and cell-matrix interactions that affect drug penetration and response, making them valuable for studying complex biological processes and testing the efficacy of anti-cancer drugs (Adcock, 2015a; N. H. Baek et al., 2016).

Traditional cell cultures are monolayered and widely used but lack three-dimensional cell culture's complexity. However, Spheroid models provide a more physiologically relevant environment mimicking the natural conditions of human tumor cells more closely. This complexity allows for better study of drug interactions and cellular responses. Traditional cell cultures typically present cells in a flat, monolayer arrangement, which does not accurately reflect the 3D structure of tissues or tumors. Spheroids, on the other hand, offer a three-dimensional arrangement that incorporates gradients of nutrients, oxygen, and waste products, providing a more realistic model for studying drug effects. Each model has its own set of advantages and disadvantages. Traditional cell cultures are easier to set up and analyze, but they may not fully capture the complexities of drug interactions in a natural setting. Spheroids offer a more detailed and realistic model but can be more complex and costly to create and maintain. Understanding these differences helps researchers choose the most appropriate model for their specific needs in drug development and testing (Jubelin et al., 2022; Nath and Devi, 2016). Spheroid models have emerged as a powerful tool for more accurately mimicking the tumor microenvironment. They help researchers study tumor growth, drug resistance, and the efficacy of new therapies in a more realistic context. Spheroids' ability to simulate the 3D structure and cell interactions of tumors offers a significant advantage in developing and testing novel cancer treatments (Fig. 1) (Azar et al., 2021; Rimann and Graf-Hausner, 2012).

Fig. 1.

Traditional monolayer cell culture and Spheroid model. Fig 1 compares the traditional monolayer cell culture (A), where cells grow in a flat, two-dimensional layer, to the spheroid model (B), which features a three-dimensional, multicellular structure. The spheroid model better mimics the in vivo tumor microenvironment, highlighting differences in cell-cell interactions and nutrient gradients.

Research by Yoshinori Imamura et al. demonstrated the advantages of 3D spheroids over 2D cultures for drug testing in breast cancer. Their study showed that 3D cultures, particularly dense multicellular spheroids (MCSs), exhibited greater resistance to chemotherapy drugs like paclitaxel and doxorubicin. The dense 3D MCSs displayed characteristics such as hypoxia, reduced proliferation (as indicated by Ki-67), and decreased apoptosis (as shown by caspase-3 activity), all of which contributed to drug resistance. These findings highlight that 3D spheroids offer a more accurate representation of tumor characteristics such as dormancy, anti-apoptotic features, and hypoxic environments, making them a superior platform for drug testing and understanding tumor biology (Imamura et al., 2015).

Moreover, Bell et al. conducted a multicentred study comparing 2D hepatic sandwich cultures and 3D spheroids for assessing long-term drug toxicity. Their findings revealed that 3D spheroids, generated using primary human hepatocytes, exhibited superior cellular functionality and longevity, maintaining liver-specific activities like cytochrome P450 enzyme expression. This allowed for more reliable prediction of drug-induced hepatotoxicity, whereas 2D cultures showed a decline in metabolic functions over time. This further underscore the advantages of spheroid models in mimicking the in vivo microenvironment for cancer research and broader drug toxicity testing (Bell et al., 2018a).

Table 1 compares Traditional Cell Cultures and Spheroid Models, highlighting differences in structure, cell interactions, and physiological relevance. It provides insights to help researchers choose the most suitable model for their studies.

Table 1.

Comparison of traditional cell cultures and spheroid models.

Traditional Cell Cultures	Spheroid Models	References
Cells grow forming a monolayer.	Cells form a three-dimensional structure, mimicking natural tissues.	(Białkowska et al., 2020)
Dominated by cell-surface contact with limited cell-cell interactions.	Enhanced cell-cell interactions and cell-extracellular matrix (ECM).	(Kapałczyńska et al., 2018)
Lacks nutrient, oxygen, and waste gradients.	Features gradients of nutrients, oxygen, and waste, similar to in vivo conditions.	(Jubelin et al., 2022)
Does not replicate the tumor microenvironment.	Can recreate aspects of the tumor microenvironment, including drug resistance.	(Pinto et al., 2020)
Easier to maintain and analyze with standard techniques.	Requires more advanced methods and careful maintenance.	(Nath and Devi, 2016)
Poor representation of in vivo cell behaviour.	Better mimics in vivo cell morphology and physiological responses.	(Imamura et al., 2015)
Provides limited insights for studying complex drug penetration and efficacy	Ideal for evaluating drug penetration, resistance, and toxicity.	(Lovitt et al., 2014; Van Zundert et al., 2020)
Typically used for basic cell biology studies.	Widely applied in cancer research and advanced drug testing.	(Duval et al., 2017)

3. Various methods for the spheroid model generation

Over the years, diverse techniques have been developed to generate multicellular tumour spheroids (MCTS), each bringing its advantages and applications. These methods fall into two main categories: scaffold-free and scaffold-based, both of which play crucial roles in advancing 3D cell culture for cancer research.

3.1. Scaffold-Free techniques for the development of spheroid models

Scaffold-free methods for generating three-dimensional (3D) cell cultures represent innovative approaches that enable cells to form tissue-like structures without the use of external scaffolds. These techniques are favoured for their ability to replicate the natural cellular environment more accurately, making them particularly useful in cancer research and drug testing (Fig 2) (Brancato et al., 2020; Lee and Kim, 2021).

Fig. 2.

Scaffold-Free Techniques for Spheroid Models. Fig 2 illustrates key scaffold-free techniques used to generate spheroid models. (a) The Hanging Drop Method forms spheroids by suspending cells in droplets. (b) Ultra-Low Attachment Plates prevent cell adhesion, promoting aggregation. (c) Magnetic Levitation and Magnetic 3D Printing use magnetic fields to assemble spheroids. (d) The Liquid Overlay Method employs non-adherent surfaces to encourage cell clustering.

3.1.1. Hanging drop method

The hanging drop technique holds significance in cancer research and drug testing. It was initially developed in the early 20th century, enabling researchers to create 3D cellular environments that closely mimic in vivo tumor conditions. This technique involves placing small droplets of a cell suspension on an inverted lid or plate cover, allowing surface tension to sustain each drop as it hangs. Gravity-driven cell aggregation at the bottom of each droplet facilitates spheroid formation, closely simulating tumor behaviour observed in vivo (Biju et al., 2023; Lee and Kim, 2021). A primary strength of the hanging drop method is its accessibility, as it requires minimal specialized equipment, which allows for reproducible spheroid production of defined sizes, essential in drug screening and toxicity studies. Additionally, it promotes natural cell-cell interactions in a scaffold-free environment, yielding spheroids with enhanced gene expression and cellular functionality compared to traditional monolayer cultures. This physiological relevance is crucial for evaluating anticancer drugs more accurately (Pinto et al., 2020). The hanging drop technique has been instrumental in studying various cancer types, including breast, ovarian, and prostate cancers. Spheroids formed with this technique exhibit characteristics like chemoresistance and apoptosis evasion, which are essential for developing predictive models that replicate in vivo tumor responses better than monolayer cultures. This approach also supports the engineering of spheroids from patient-derived tissues, preserving the histopathological features of original tumors and advancing personalized medicine by enabling drug efficacy testing across diverse cancer cell lines (Jubelin et al., 2022). To meet the demand for models that replicate the 3D structure and tumor microenvironment, the hanging drop method has proven cost-effective and widely utilized in spheroid formation for drug testing. Research led by Yoon Jeong et al. at the University of Illinois introduced a flipped well-plate technique, which allows the generation of large spheroids using standard 96-well plates. This setup is adaptable for both scaffold-free and scaffold-based spheroids, accommodating colorectal carcinoma cells (HCT116) to create sizable, uniform spheroids suitable for high-throughput applications (Foty, 2011; Jeong et al., 2022; Tung et al., 2011a). Further research by Yılmaz Ö. and Sakarya S. leveraged a 96-well hanging drop plate to form spheroids from breast cancer cell lines (JIMT, MCF-7, T-47D, and BT-474) within 72 h, achieving high cell densities necessary for studying the dense cellular architecture of breast tumors. This flexibility enables researchers to adjust cell densities and tailor spheroid sizes for drug screening, tumor proliferation studies, and invasion assays, enhancing model accuracy. Adaptations like the flipped well-plate and 96-well hanging drop techniques have expanded the utility of the hanging drop method in cancer research, making 3D tumor modelling more accessible and scalable. This technique's versatility, scalability, and reproducibility make it integral to next-generation cancer research, bridging the gap between traditional monolayer cultures and more complex in vivo models (O and S, 2018).

3.1.2. Ultra-low attachment plates

Ultra-low attachment (ULA) plates provide another scaffold-free option by using surfaces coated with inert materials like agar or poly (2-hydroxyethyl methacrylate). These coatings prevent cell attachment, encouraging cells to aggregate into spheroids. ULA plates come in various shapes, such as round-bottom or conical wells, which support the formation of consistent spheroids (Nath and Devi, 2016; Pinto et al., 2020). This method is well-suited for high-throughput applications due to its ability to generate multiple spheroids simultaneously. However, some cell lines may not form well-defined spheroids in ULA plates, which can limit their application for certain research needs. The procedure for spheroid formation in ULA plates is simple and efficient. Cells are seeded at a defined density in each well, and over time, they naturally aggregate into spheroids due to the non-adherent surface. Spheroids typically form within 24–72 h, depending on the cell line and experimental conditions. A key advantage of round-bottom ULA plates is their ability to produce spheroids of uniform size, reducing variability in experimental results. Media changes and spheroid monitoring can be performed directly on the plate, minimizing handling and reducing stress on the spheroids. ULA plates have gained prominence in drug discovery due to their ability to more accurately mimic the 3D architecture of tumors, providing a model that better reflects in vivo conditions compared to traditional monolayer cultures. Spheroids cultured in ULA plates are frequently used to evaluate the cytotoxicity and efficacy of anticancer drugs. For instance, Bresciani et al. (2021) demonstrated the use of ULA plates to generate pancreatic neuroendocrine neoplasm (pNEN) spheroids for assessing the efficacy of the receptor tyrosine kinase inhibitor Sunitinib. The study highlighted the reliability of ULA plates in terms of spheroid formation, maintenance, and reproducibility, making them an ideal tool for drug response studies in realistic tumor models. In high-throughput drug screening, ULA plates serve as an ideal platform. Madoux et al. (2017) developed a 1536-well ULA plate-based assay to evaluate the cytotoxic effects of drugs on spheroids, enabling parallel formation of spheroids and assessment of drug-induced effects using a luminescence-based cytotoxicity assay. The results from high-throughput screening in ULA plates were significantly different from those obtained in monolayer cultures, emphasizing the relevance of spheroids in drug discovery. ULA plates' compatibility with automation makes them particularly useful for screening large drug libraries while maintaining physiological relevance. Spheroids generated in ULA plates are used for various applications in drug discovery, including cytotoxicity screening, drug penetration studies, mechanism of action investigations, and chemoresistance research. These models allow for testing anticancer drugs in more physiologically relevant conditions, evaluating drug penetration through dense Spheroid structures, investigating mechanisms like apoptosis and cell cycle arrest, and studying chemotherapy resistance, which may not be observed in monolayer cultures. ULA plates provide a robust, high-throughput solution for spheroid generation in drug discovery, offering an improved representation of tumor biology and predictive screening outcomes for anticancer therapeutics (Bresciani et al., 2019; Madoux et al., 2017).

3.1.3. Magnetic levitation and magnetic 3D printing

Magnetic levitation and magnetic 3D printing are cutting-edge scaffold-free techniques that utilize magnetic particles or nanoparticles to guide cell aggregation. In magnetic levitation, cells are treated with superparamagnetic iron oxide nanoparticles (SPIONs) and then exposed to a magnetic field. This field causes the cells to cluster into spheroids. Magnetic 3D printing employs a similar principle but often with greater precision in cell placement and spheroid formation (Nath and Devi, 2016; Pinto et al., 2020). These techniques are advantageous for their speed and precision in spheroid generation. However, they involve the preparation and handling of magnetic nanoparticles and may produce a smaller number of spheroids compared to other methods. The procedure for magnetic levitation begins with labelling the cells with magnetic nanoparticles, typically SPIONs, and exposing them to a magnetic field. This exposure forces the cells to aggregate, rapidly forming spheroids, often within 24 to 48 h. Magnetic 3D printing, on the other hand, involves precise manipulation of magnetized cells using a 3D printer, allowing researchers to build more complex 3D structures by layering the spheroids or organizing them in a specific pattern. Both techniques offer the advantage of creating uniform spheroids, with minimal manual handling, which helps to reduce variability in experimental outcomes (Jubelin et al., 2022). These techniques have shown significant promise in drug discovery and cancer research. As highlighted in the systematic review by Marques et al. (2022), magnetic 3D cell culture systems, including levitation and bioprinting, have been applied to mimic human tissues in a physiologically relevant manner. The review emphasized the potential of these methods to generate homotypic and heterotypic spheroids, allowing researchers to study complex tissue interactions and tumor microenvironments. Such models are crucial for drug discovery, particularly in cancer research, where they can replicate the 3D architecture of tumors and facilitate more accurate drug testing (Marques et al., 2022). In the context of drug discovery, these techniques are especially useful for evaluating the efficacy and toxicity of anticancer drugs. Magnetic levitation has been used to create spheroids that simulate the tumor microenvironment, enabling researchers to study how drugs penetrate the spheroid structure and impact tumor cells in a more realistic setting. Leonard and Godin (2022) describe the use of magnetic levitation to create 3D co-cultures of tumor cells and cells from the tumor microenvironment. This method allowed the rapid formation of tumor spheres with defined cellular compositions and densities, providing a robust platform for drug testing (Leonard and Godin, 2016). By using spheroids generated through magnetic methods, researchers can perform high-throughput screenings, analyze drug penetration, and investigate mechanisms like apoptosis and chemoresistance. These models are particularly valuable in understanding how drugs interact with tumor tissues and the microenvironment, offering insights that traditional monolayer cultures cannot provide. Thus, magnetic levitation and magnetic 3D printing are transforming the landscape of in vitro cancer research, enabling the development of more relevant and physiologically accurate models for drug testing and therapeutic development (Leonard and Godin, 2016; Marques et al., 2022).

3.1.4. Liquid overlay method

The liquid overlay method is another well-established technique used for generating scaffold-free spheroids. This method prepares a non-adhesive surface by coating culture plates with substances like agarose or poly-HEMA, which prevent cells from attaching to the plate. When a cell suspension is added to these coated wells, the lack of attachment encourages the cells to aggregate and form spheroids. This method is appreciated for its ease of use and the ability to produce a large number of spheroids simultaneously (Lee and Kim, 2021; Rimann and Graf-Hausner, 2012). It also offers the advantage of consistency, as the non-adhesive coating ensures uniform spheroid formation. It produces a large number of spheroids simultaneously, making it ideal for high-throughput applications, especially in drug screening and preclinical evaluations (Costa et al., 2018). Spheroids generated via the liquid overlay method are highly reproducible and physiologically relevant, enabling a more accurate assessment of drug efficacy and toxicity compared to traditional monolayer cell cultures. Jubelin et al. (2022) demonstrated the scalability and homogeneity of liquid overlay method-generated spheroids across multiple cancer models, such as lung adenocarcinoma, osteosarcoma, prostate adenocarcinoma, and glioblastoma, with consistent morphology and size, which is critical for reducing experimental variability in drug testing. In the drug discovery process, liquid overlay method-generated spheroids are valuable for studying drug penetration, resistance, and efficacy in a spheroid. Since spheroids exhibit distinct layers with hypoxic cores, they allow for the investigation of how drugs diffuse through solid tumors and how cells in different regions respond to treatment. This is particularly relevant in drug resistance studies, where spheroids are more resistant to treatments like doxorubicin compared to monolayer cell cultures, underscoring their importance in modelling tumor behaviour more accurately (Jubelin et al., 2023). Furthermore, the liquid overlay method's scalability makes it ideal for high-throughput drug screening, enabling researchers to test multiple compounds across different spheroid models simultaneously. In various stages of the drug discovery process—target validation, lead compound testing, mechanistic studies, and toxicity evaluation—spheroids offer a more realistic platform for assessing drug candidates' therapeutic potential and safety. Their ability to replicate the complex tumor microenvironment, including gradients of oxygen and nutrients, makes liquid overlay method-generated spheroids an essential tool for understanding drug resistance mechanisms and improving the effectiveness of anticancer therapies. However, one challenge with the liquid overlay method is that the spheroids can vary in size depending on the initial cell density, and careful optimization of cell concentration is needed to produce spheroids of uniform size. Additionally, while the method is less labour-intensive than others, it may still require optimization to ensure the formation of spheroids with consistent shapes and sizes (Costa et al., 2018; Jubelin et al., 2023).

3.2. Scaffold-based techniques for the generation of spheroid models

Scaffold-based tumor models are pivotal in advancing our understanding of cancer biology and improving drug testing methodologies. These models use various scaffolds to provide a supportive structure for cell growth, enabling researchers to replicate the complex microenvironment of tumors more accurately. By mimicking the natural tissue architecture, scaffold-based models offer enhanced insights into tumor behaviour and therapeutic responses (Fig 3) (Brancato et al., 2020; Lee and Kim, 2021).

Fig. 3.

Scaffold-Based Techniques for the development of Spheroid model. Fig 3 highlights scaffold-based techniques for developing spheroid models. (a) Spinner Flasks create dynamic environments to promote uniform spheroid formation. (b) Hydrogel-based models provide a supportive 3D matrix that mimics extracellular environments. (c) Microfluidics enables precise control of spheroid culture conditions, simulating physiological fluid dynamics.

3.2.1. Spinner flasks

Spinner flasks represent a key technology in the large-scale generation of spheroids, utilizing continuous rotating agitation to inhibit cell adhesion and promote the formation of spheroidal aggregates. This technique relies on either a magnetic stirrer or the rotation of the entire flask to ensure a homogeneous distribution of oxygen and nutrients within the culture medium. In the case of spinner flasks, a magnetic stirrer positioned inside the flask facilitates consistent mixing, which enhances the supply of essential nutrients to the cells. However, the agitation also introduces direct shear forces, which pose a risk of cellular damage. This method is particularly advantageous for the production of large quantities of spheroids under controlled nutritional conditions. Despite these benefits, continuous rotation can make it challenging to visualize and monitor spheroid formation, and the mechanical forces involved may still compromise cell integrity. Douple et al. (1987) explored spheroid models of human small cell carcinoma of the lung (SCCL) using spinner flasks. Their study highlighted that spheroid volume reduction after chemotherapy treatment served as a reliable indicator of drug efficacy. Additionally, they utilized a labelling assay to quantify cell death in SCCL spheroids 24 h post-exposure to chemotherapeutic agents, such as doxorubicin and etoposide, showing a heterogeneous tumor response to chemotherapy. Spinner flask-generated spheroids have thus been critical in evaluating drug activity across a variety of cell lines and drug types, with large-scale production capabilities making them ideal for such assays (Douple et al., 1985). He et al. (2019) investigated the formation of cellular aggregates in spinner flasks using mesenchymal stem cells (MSCs) and chondrocytes, and their findings highlighted the influence of agitation speed and inoculation density on aggregate formation. Reduced agitation speed led to larger aggregates, while an increased initial cell density also promoted aggregate growth. The study revealed that cell adhesion molecules like integrin β1 and cadherin play significant roles in the aggregation process. This molecular understanding is crucial for optimizing spinner flask conditions for generating large-scale cell aggregates for therapeutic applications, such as cartilage regeneration (He et al., 2019). This understanding helps optimize spinner flask conditions for producing spheroids in large quantities for applications such as tissue regeneration and drug testing. In drug discovery, spheroids formed in spinner flasks serve as valuable models due to their 3D structure, which closely mimics in vivo tumors. Cells are added to the flask and, under continuous agitation, form spheroids that replicate tumor features like hypoxic cores and proliferative outer layers. This setup allows researchers to study drug efficacy, diffusion, and resistance in a more physiologically relevant model compared to traditional monolayer cell cultures. Spinner flask spheroids are particularly useful in high-throughput drug screening, where their large-scale production makes it feasible to test multiple drugs simultaneously. Additionally, these spheroids help in studying the effectiveness of combination therapies, as well as exploring tumor heterogeneity, which can lead to better insights into how different cancer types respond to treatments (Douple et al., 1985; He et al., 2019).

3.2.2. Hydrogels-based models using 3D bioprinter

Hydrogels are highly adaptable materials that can be customized for various Spheroid model applications. They can encapsulate cells, be used in high-throughput screening (HTS) platforms, and serve as bio-inks for 3D bio-printing and microfluidic devices. For example, prostate cancer models have been developed using thiol-modified hyaluronic acid/poly (ethylene glycol)-diacrylate hydrogels, which allow for the detailed imaging of tumor spheroids. Hydrogels can be tailored to provide different mechanical properties and biochemical signals, making them versatile tools for mimicking the tumor microenvironment. Hydrogels serve as a highly adaptable scaffold, allowing for the encapsulation of cancer cells and enabling the formation of spheroids that better mimic the in vivo tumor architecture. This is essential for creating more accurate and physiologically relevant models for anti-cancer drug testing. Hydrogels can be fine-tuned to provide specific biochemical and mechanical properties, mimicking the tumor microenvironment and supporting complex 3D cell growth. Thakor et al. (2024) explored the use of 3D bioprinting techniques for spheroid formation, where cancer cells are suspended in bio-inks composed of hydrogels. These bio-inks are printed layer by layer, offering precise control over the shape, size, and cell distribution within the spheroids. This method also allows the inclusion of multiple cell types, facilitating the development of complex tumor models that incorporate stromal and immune cells. By employing this approach, researchers can closely replicate the extracellular matrix (ECM) and other key features of tumor tissues, improving the predictive accuracy of drug testing. 3D bioprinting enhances the scalability and reproducibility of spheroid formation, making it suitable for high-throughput drug screening platforms. By integrating these systems with automated imaging and analysis tools, researchers can rapidly assess drug efficacy in a physiologically relevant 3D environment, which offers a more accurate representation of in vivo tumor behaviour compared to monolayer cell cultures. Fernando et al. (2024) discussed how hydrogels support the sustained release of chemotherapeutic agents within the spheroid models. This is especially useful for testing novel drug formulations where the controlled release of the drug can be monitored over time. The incorporation of hydrogels in spheroid models allows for a more detailed analysis of how drugs are released, distributed, and metabolized within the tumor microenvironment, which is critical for optimizing therapeutic strategies. In brain tumor research, hydrogels are particularly effective in replicating the complex ECM of the brain. Thakor et al. (2024) showed that hydrogels-based models using a 3D bioprinter creates a more realistic brain tumor model for testing drugs targeting glioblastoma and other aggressive brain cancers. These models allow for the evaluation of drug resistance mechanisms and the identification of therapeutic gaps where drugs are most effective. The ability to simulate the highly specialized environment of brain tumors using hydrogel-based spheroids represents a significant advancement in cancer research. The ability of hydrogels to simulate the tumor microenvironment is key to improving the accuracy of preclinical studies. By incorporating hydrogels into bioprinting, and high-throughput screening, researchers can develop more complex and physiologically relevant models. These models lead to better predictions of drug efficacy and resistance in clinical settings, offering a valuable tool for advancing cancer therapy (Fernando et al., 2021; Maji and Lee, 2022; Thakor et al., 2020).

3.2.3. Microfluidics

Microfluidic technology has revolutionized cancer research by allowing precise manipulation of fluids at the microscale. This innovation enables the creation of complex channel networks that control small fluid volumes, fostering the formation of uniform spheroids—three-dimensional cell aggregates that closely replicate the in vivo tumor microenvironment. These spheroids are essential for studying cancer biology, evaluating drug responses, and understanding disease mechanisms (Tevlek et al., 2023; Vadivelu et al., 2017a). A major application of microfluidics in oncology is the generation of multicellular spheroids (MCS). Advanced platforms not only facilitate spheroid formation but also allow continuous monitoring and high-throughput drug screening. For example, Vadivelu et al. (2017) highlighted new microfluidic techniques that preserve the structural integrity of spheroids, vital for accurate drug response assessments (Vadivelu et al., 2017a). Microfluidic systems are crucial for engineering spheroids, providing controlled environments that simulate physiological conditions. Tevlek et al. (2020) noted that these devices enhance experimental reproducibility by producing uniform spheroids, making them critical for exploring cell interactions and drug responses (Tevlek et al., 2023). The versatility of microfluidics extends to the design of specific platforms for spheroid culture, as demonstrated by Moshksayan et al. (2020). Their research emphasizes optimizing channel geometry and fluid dynamics to ensure consistent spheroid formation, which impacts downstream assays and drug testing (Moshksayan et al., 2018). Kwapiszewska et al. (2021) also introduced a microfluidic platform for real-time monitoring of tumor spheroids in response to therapeutic agents, providing insights into treatment efficacy and resistance mechanisms (Kwapiszewska et al., 2014). Additionally, integrating microfluidics with scaffold-based models has enhanced cell culture technologies. Microfluidic chips that include agarose scaffolds allow for precise control over the culture environment. According to Patra et al. (2017), these devices continuously perfuse culture media and drugs, mimicking physiological conditions within tumors. Over 24 to 48 h, cancer cells aggregate into spheroids, which can then be treated with various drugs (Patra et al., 2016a). The capability to generate large numbers of uniformly sized spheroids facilitates high-throughput screening of anti-cancer compounds. Research by Huang et al. demonstrated improved production of glycosaminoglycans under perfused conditions compared to static cultures, highlighting the potential of microfluidic platforms for studying dynamic cellular processes (X. Liu et al., 2021). Microfluidic technology significantly enhances cancer research by creating models that closely resemble tumor environments, thus improving our understanding of tumor biology and the development of targeted therapies. As this technology evolves, it promises to provide deeper insights into tumor complexities and facilitate the discovery of more effective treatments.

Table 2 outlines a comparison of various 3D cell culture methods, highlighting the unique advantages and limitations of each technique, from ease of use and scalability to specific technical requirements. This summary helps identify suitable methods based on the research goals and the type of cell culture desired. The challenges and recent innovations in scaffold-free and scaffold-based techniques for developing spheroid models are summarized in Table 3. This table provides insights into the practical considerations and advancements that improve these methods’ reliability and relevance for complex biological studies.

Table 2.

Comparison of different methods for spheroid development: advantages and disadvantages.

Method	Advantages	Disadvantages	References
Hanging Drop Method	- Simple and cost-effective - Allows for high cell density - Good control over microenvironment	- Limited scalability - Technical skills required - Risk of contamination due to exposure to air	(Pinto et al., 2020; Tung et al., 2011a)
Ultra-Low Attachment Plates	- Easy to use and standardize - High-throughput capabilities - Supports large-scale spheroid formation	- Spheroid morphology may vary - Limited to certain cell types - Requires optimization of coating	(Biju et al., 2023; Madoux et al., 2017)
Magnetic Levitation	- Enables uniform spheroid formation - Minimal shear stress on cells - Can be integrated with other methods	- Requires specialized equipment - May have limitations in cell types used - Potential for magnetic field interference	(Leonard and Godin, 2016; Rahman et al., 2024)
Magnetic 3D Printing	- Allows for precise spatial arrangement of cells - Scalable and reproducible - Suitable for complex structures	- High initial setup costs - Limited cell types and materials - Complex protocols for 3D structures	(Biju et al., 2023; Marques et al., 2022)
Liquid Overlay Method	- Simple to implement - Suitable for a wide range of cell types - Good for assessing drug responses	- Spheroids may be less uniform - Liquid media may limit nutrient diffusion - Risk of shear stress	(Costa et al., 2018; Jubelin et al., 2022)
Spinner Flasks	- Good for large-scale production - Homogeneous mixing of media - Suitable for suspension cultures	- Increased risk of shear stress - Requires careful monitoring of cell density - Technical complexity	(He et al., 2019; Pinto et al., 2020)
Hydrogel-Based Models	- Mimics extracellular matrix - Supports cell viability and function - Versatile for different applications	- May have variable mechanical properties - Complex formulation - Limited scalability	(Fernando et al., 2021; Maji and Lee, 2022)
Microfluidics	- Precise control over culture conditions - High-throughput capabilities - Real-time monitoring	- High initial investment in equipment - Requires technical expertise - Limited cell viability in some designs	(Kwapiszewska et al., 2014; Tevlek et al., 2023)

Table 3.

Challenges and Innovations of Scaffold-Free Techniques and Scaffold-Based Techniques for Spheroid Models.

Technique	Challenges	Innovations	References
Scaffold-Free Techniques for Spheroid Models	- Difficulty in maintaining consistent spheroid size and shape. - Challenges in nutrient and oxygen diffusion in larger spheroids, leading to necrotic cores.	- Development of advanced bioreactors to improve nutrient distribution. - Use of microfluidic systems for a more controlled environment. - Refinement of magnetic levitation and printing technologies for enhanced precision in spheroid formation.	(Pinto et al., 2020)
Scaffold-Based Techniques for Spheroid Models	- Variability between batches of natural scaffolds. - Thick or opaque scaffolds complicate observation using high-content imaging (HCI) techniques.	- Use of synthetic polymers to achieve more consistent results. - Decellularization methods to preserve the extracellular matrix (ECM) from healthy and cancerous tissues, providing bioactive scaffolds. - Novel crosslinking methods like methylene blue-mediated photo-oxidation and functionality of acellular tumor matrices.	(Jubelin et al., 2022)

4. 3D bioprinting

3D bioprinting is a cutting-edge technology that leverages automated additive manufacturing to create tissue-like structures by precisely positioning cells, tissues, and biodegradable materials. This method allows for the construction of complex, customized architectures that closely resemble native tissue, making it a powerful tool in the realm of personalized medicine. By controlling the spatial arrangement of cells and materials, bioprinting offers the flexibility to bridge the gap between artificially engineered tissue and natural tissue, potentially revolutionizing the field of regenerative medicine. The core component of bioprinting is the use of bio-inks—hydrogel-based materials with suitable viscoelastic properties that allow for the precise layering required to build detailed 3D structures. The bio-inks can be solidified through various mechanisms, including physical (e.g., temperature or light), enzymatic, or chemical crosslinking. These methods help ensure that the printed structures maintain their integrity and function as intended (Jubelin et al., 2022; Nath and Devi, 2016). Bioprinters are categorized based on their bioprinting modalities, which include droplet-based, laser-based, extrusion-based, and stereolithography bioprinting (Melissaridou et al., 2019; Theodoraki et al., 2015).

4.1. Types of 3D bioprinting techniques

3D bioprinting offers several advanced techniques for spheroid development, each with unique strengths that contribute to creating accurate and functional 3D models for research and therapeutic purposes. The main types of 3D bioprinting used for spheroid generation include inkjet-based, droplet-based bioprinting, microextrusion-based, and laser-assisted.

4.1.1. Inkjet-based bioprinting

Adapted from traditional inkjet printing, this method uses droplets to create 3D structures with high precision. It is known for its high-speed printing, cost-effectiveness, and ability to control cell and growth factor concentrations. However, it is limited by the viscosity of the bio-inks it can handle, which restricts its ability to create thicker, more complex tissues. This method, adapted from traditional inkjet printing, involves the deposition of bio-inks in tiny droplets to build spheroids layer by layer. Inkjet bioprinting is known for its high precision, speed, and cost-effectiveness, making it suitable for producing spheroids quickly. However, it is limited by the viscosity of the bio-inks, which can affect the size and complexity of the spheroids that can be generated (Fig 4) (Brancato et al., 2020; Jubelin et al., 2022; Tung et al., 2011a). Kumar et al. (2021) discusses the role of inkjet-based bioprinting in tissue engineering, emphasizing its capability to precisely position mammalian cells for the construction of intricate and functional tissue structures. This approach enables high-resolution cell placement, which is critical for mimicking the complex architecture of natural tissues. By employing tailored bio inks, inkjet printing can generate both two-dimensional and three-dimensional constructs that promote cell viability and functionality. The precision in cell placement not only advances tissue engineering projects but also paves the way for new possibilities in regenerative medicine, where these engineered tissues can be utilized for transplantation and drug screening. Ultimately, the incorporation of inkjet bioprinting into tissue engineering marks a significant development, allowing for the creation of more advanced and applicable in vitro models that facilitate the investigation of cellular interactions and tissue dynamics (Lu et al., 2024; Murphy and Atala, 2014).

Fig. 4.

Spheroid development using 3D Bioprinting techniques. Fig 4 depicts the process of spheroid development using 3D bioprinting techniques, including Inkjet-Based, Laser-Assisted, Microextrusion-Based, and Stereolithography-Based bioprinting. Cells are cultured, combined with bioink components such as growth factors, hydrogels, and ECM materials, and loaded into the bioprinter. The chosen technique fabricates layered constructs, which are then incubated to form spheroids. The workflow consists of pre-bioprinting (cell and bioink preparation), processing (fabrication), and post-bioprinting (stabilization and modifications).

4.1.2. Droplet-based bioprinting

Similar to inkjet printing, droplet printing relies on the precise deposition of bio-inks in tiny droplets to build 3D structures. This method excels in achieving detailed tissue models with a high degree of accuracy and control over cell placement. However, like inkjet printing, it is constrained by the viscosity of the bio-inks, limiting the complexity of the tissues it can generate. (Jubelin et al., 2022). Gudapati et al. (2016) discuss the use of droplet-based bioprinting (DBB) in the fields of tissue engineering and regenerative medicine, emphasizing its capability to precisely deposit biological materials, including cells, growth factors, and biomaterials. This innovative approach facilitates the fabrication of intricate tissue structures that can closely resemble natural tissues. The flexibility of DBB allows for the creation of tailored constructs designed to meet specific therapeutic requirements, thereby proving to be an essential tool in regenerative medicine. However, challenges persist, such as a limited selection of bio inks and the risk of cellular damage during the printing process. Despite these hurdles, advancements in DBB technology hold promise for improving its effectiveness and expanding its use in developing functional tissues for therapeutic and transplantation purposes (Gudapati et al., 2016; Rimann and Graf-Hausner, 2012).

4.1.3. Microextrusion-based bioprinting

This technique involves extruding bio-inks through nozzles to build structures layer by layer. It is valued for its versatility, allowing for the use of a wide range of material viscosities and the printing of high cell concentrations. While this method offers better structural integrity than inkjet bioprinting, it can be slower and may subject cells to shear stress, affecting their viability. Microextrusion-based bioprinting is a highly versatile technique accommodating a wide range of bio-ink viscosities and cell concentrations. Microextrusion bioprinting offers better structural integrity for the spheroids, although the process can be slower, and the mechanical forces exerted on the cells during extrusion may impact cell viability (Fig 4) (Jubelin et al., 2022). The integration of microextrusion with other technologies, such as microfluidics, has also shown promise in fabricating intricate structures. Hong et al. combined microextrusion with a microfluidic emulsification system to create liver lobule-like microtissues, demonstrating enhanced cell viability and structural integrity compared to non-structured spheroids (Lu et al., 2024) (Banerjee et al., 2022).

4.1.4. Laser-assisted bioprinting

This modality uses a laser to deposit bio-inks with high precision and resolution, making it ideal for applications requiring single-cell accuracy. However, the technique is limited by the need for low-viscosity materials and the potential cytotoxicity of metallic nanoparticles used in the process. A laser is used to precisely deposit bio-inks, allowing for high-resolution printing and single-cell accuracy. Laser-assisted bioprinting is particularly useful for creating complex spheroids that require a precise spatial arrangement of cells. However, it typically requires low-viscosity bio-inks and can involve metallic nanoparticles, which might pose cytotoxicity concerns (Fig 4) (Jubelin et al., 2022). Laser-assisted bioprinting has proven to be an effective method for constructing intricate three-dimensional tissue models, particularly in the realm of cancer research. In their study, Hakobyan et al. demonstrated its application by creating exocrine pancreas spheroid arrays that closely simulate the initial stages of pancreatic ductal adenocarcinoma (PDAC) development. This technique enabled the precise placement of both acinar and ductal cells within the spheroids, facilitating comprehensive phenotypic analysis over time. By overcoming the limitations associated with conventional 2D cell cultures, this advanced method offers a significant tool for exploring the cellular dynamics involved in PDAC initiation, which could ultimately lead to improved therapeutic approaches (Mandrycky et al., 2016) (Hakobyan et al., 2020).

4.1.5. Stereolithography-based bioprinting

This method uses light to cure photo-cross-linkable materials, creating highly detailed 3D structures with strong interlayer bonding. While stereolithography offers unparalleled resolution, it is limited to materials that can be photo-crosslinked, which restricts the variety of bio-inks available for use. Stereolithography involves using light to cure photo-cross-linkable materials, creating highly detailed spheroids with strong interlayer bonding. This method offers unmatched resolution, making it ideal for generating complex spheroid structures. However, the technique is limited to materials that can be photo-crosslinked, which restricts the range of bio-inks that can be used (Jubelin et al., 2022).

4.2. Bioink preparation

In preparing bio-inks for 3D bioprinting, particularly in cancer research and tumor modelling, a blend of cellular components, extracellular matrix (ECM) mimics, and biochemical factors come together to form a matrix supporting cell growth and organisation. The bio-ink preparation process is essential because it determines the printed model's bioactivity, printability, and structural stability, which are critical to faithfully mimicking the in vivo environment. To design an effective bioink for spheroid-based 3D bioprinting, researchers typically begin with a hydrogel base that closely resembles the ECM in terms of its mechanical properties and biochemical composition. Hydrogels, often derived from natural polymers like collagen, gelatin, alginate, or fibrin, provide a soft, hydrated network that can encapsulate cells, allowing them to proliferate and interact as they would within a tissue. These materials are also commonly chosen for their biocompatibility and biodegradability, ensuring that the bioink scaffold supports cell viability while gradually breaking down as new tissue forms. The bioink is also engineered to provide the correct stiffness and porosity for the specific tumor model, as these physical properties can influence cell behaviour. For example, a stiffer matrix might better replicate certain types of tumor environments, aiding the study of cell migration and invasion. Modulating the hydrogel composition to achieve the right viscoelastic properties is particularly important in bioprinting, as the bio-ink must be capable of flowing through a printer nozzle while still maintaining its shape after deposition. Biochemical cues are integrated into the bioink to further support cellular functions. Growth factors, cytokines, and adhesion molecules are added to mimic the biochemical signals in the tumor microenvironment, guiding cell differentiation, growth, and organization within the printed structure (Biju et al., 2023). For case, adding vascular endothelial growth factor (VEGF) to the bioink can stimulate angiogenic responses, while specific ECM proteins like laminin or fibronectin may promote adhesion and proliferation of particular cell types (Banerjee et al., 2022; Zhuang et al., 2021). In anti-cancer drug testing, this tailored bioink preparation enables the creation of highly controlled and reproducible tumor models. These bioinks allow researchers to study drug responses within a 3D structure that better represents the complexities of real tumors compared to traditional cell cultures. As bioink preparation advances, we can expect even more sophisticated models that mirror tumour architecture and provide insight into therapeutic resistance mechanisms, offering new frontiers for in vitro drug testing.

4.3. Spheroid development using 3D bioprinting techniques

Spheroid development through 3D bioprinting represents a cutting-edge technique in tissue engineering, with significant applications in cancer research and drug testing. The process begins by culturing cells and preparing a suitable bioink—a mixture of living cells, biomaterials, and growth factors designed to support cell viability and promote tissue formation. It is crucial that bio-ink maintains the integrity of the cells during the printing process, allowing them to proliferate and organize into structured forms after printing. Using a 3D bioprinter, the spheroids are built layer by layer according to a predefined model that mimics the natural tissue architecture. The printer's nozzle precisely deposits the bio-ink in specific locations, allowing the cells to aggregate and self-organize into three-dimensional structures. As time progresses, these printed cell clusters mature into spheroids with features similar to in vivo tissues, including gradients of nutrients, oxygen, and waste products, which simulate the tumor microenvironment. To support spheroid development, additional conditions such as mechanical forces or specialized culture setups (e.g., low adhesion surfaces or rotating systems) are introduced to encourage cell aggregation and spheroid formation. The outcome is a strong 3D spheroid model suitable for high-throughput drug screening, tumor modelling, and the study of cellular behaviours in a more biologically relevant environment compared to traditional monolayer cultures (Banerjee et al., 2022; Zhuang et al., 2021). Fig 4 illustrates the process of spheroid development using 3D bioprinting techniques, showcasing the sequential deposition of bioink and the self-organization of cells into spheroids that closely resemble in vivo conditions. This figure visually demonstrates the various stages of spheroid creation, from the initial bio-ink deposition by the printer to the formation of complex, tissue-like structures, emphasizing the potential of this technology in advancing cancer research and tissue engineering.

4.4. Application of spheroids in 3D bioprinting

Spheroids generated through 3D bioprinting techniques hold considerable potential in advancing cancer research and drug testing, providing more accurate in vitro models that better replicate the tumor microenvironment. Traditional 2D cell cultures fail to capture the intricate interactions between cells and the extracellular matrix (ECM), limiting their ability to accurately predict the effects of therapeutic compounds. In contrast, 3D bioprinted spheroids offer a more realistic platform to study cancer cell behaviour, drug responses, and tumor progression. One of the main uses of 3D bioprinted spheroids is in high-throughput drug screening. These models simulate the heterogeneous nature of tumors, enabling researchers to assess how drugs penetrate the tumor, how cells respond to therapy, and how drug resistance may develop. By better mimicking in vivo conditions compared to traditional 2D models, 3D spheroids provide more reliable predictions of drug efficacy and toxicity, leading to more consistent results in the early stages of drug development. Another key application is tumor modelling, where 3D bioprinted spheroids mimic important features of human tumors, such as cellular diversity, nutrient gradients, and oxygen limitations. This allows for a more accurate study of tumor microenvironment interactions, cancer cell invasion, and metastasis. By printing spheroids from various cancer cell lines, researchers can investigate specific tumor behaviours and gain insights into cancer biology that are difficult to observe in simpler 2D cultures. Furthermore, 3D bioprinted spheroids are being explored for personalized medicine. By creating spheroids from a patient's tumor cells, researchers can test different drugs on a patient-specific model, facilitating more tailored and effective treatment plans. This approach not only helps identify the most suitable therapies but also enables monitoring of drug resistance development, which is a major challenge in cancer treatment. In the realm of cancer immunotherapy, spheroids serve as an important model for studying the interactions between immune cells and cancer cells. 3D bioprinted spheroids can be used to evaluate the efficacy of immunotherapies, including checkpoint inhibitors and CAR-T cell therapies. These models provide a more accurate evaluation of immune cell infiltration, cytokine release, and tumor destruction, all of which are crucial factors in the success of immunotherapy. Additionally, 3D bioprinted spheroids are instrumental in studying cancer cell metabolism. The 3D structure of spheroids replicates the nutrient and oxygen gradients found within tumors, enabling researchers to observe metabolic changes in response to treatments. This is particularly important for understanding metabolic reprogramming, such as the Warburg effect, which is common in cancer cells (Lu et al., 2024). In conclusion, the use of 3D bioprinted spheroids has significantly advanced cancer research and drug testing by offering a more accurate and reliable in vitro model. These spheroids allow for more detailed studies of drug efficacy, tumor biology, immune responses, and metabolic changes, offering insights that traditional 2D cultures cannot provide. This innovative approach is paving the way for more effective anti-cancer drug development and personalized treatment strategies.

5. Characterisation techniques used for drug testing in spheroids models

5.1. Cytotoxicity assays

5.1.1. MTT assay

The MTT assay is a widely used technique for evaluating cell viability and proliferation. It operates on the principle that living cells can reduce the yellow dye MTT (3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide) to a purple formazan product through mitochondrial enzymes. After treatment, cells are incubated with MTT and the formazan crystals that form are dissolved in a solvent. The intensity of the purple colour is then measured using a spectrophotometer, and this intensity is proportional to the number of viable cells. This method is effective for assessing how various substances, including drugs, affect cell survival. However, the process requires organic solvents to dissolve the formazan crystals, which can potentially introduce toxicity and affect cell viability (Fig 5) (Mosmann, 1983). In drug discovery, the MTT assay is particularly valuable when integrated with 3D spheroid models, providing a more accurate reflection of how drugs affect tumour-like structures. According to Ho et al. (2024), multicellular tumor spheroids (MCTS) formed from breast cancer cells, particularly MCF-7, offer a model that better mimics the complexity of in vivo tumors compared to traditional monolayer cultures. In these models, the MTT assay involves incubating the spheroids with the MTT reagent, allowing the viable cells within the spheroid to reduce the dye into purple formazan crystals, which are then dissolved and measured. Spheroids for MTT assays are typically cultivated using low-adhesion or specialized spheroid plates, ensuring uniform size and consistency, which is critical for high-throughput screening (HTS). The modified MTT assay allows researchers to explore drug penetration and evaluate cytotoxic effects in 3D tumor models, offering a scalable approach for preclinical drug testing (Ho et al., 2012)

Fig. 5.

MTT assay. Fig 5 illustrates the protocol for the MTT assay, a colorimetric method to assess cell viability. Cells are seeded into a 96-well plate, followed by the addition of the MTT reagent. After incubation, viable cells convert MTT into a purple formazan product. The absorbance of the formazan is measured to quantify cell viability.

5.1.2. XTT assay

The XTT assay is a colorimetric method designed to measure cell viability and metabolic activity. This technique uses the yellow dye XTT, which is converted into an orange formazan dye by the enzymes present in living cells. One of the key benefits of the XTT assay is that it does not require toxic solvents for solubilizing the formazan dye, simplifying the procedure and reducing potential interference. The amount of formazan dye produced is measured by its absorbance, which reflects the number of viable cells. The XTT assay is suitable for high-throughput screening and can be used with various cell types, including both adherent and suspension cells (Fig 6) (Riss et al., 2016). In drug discovery, the XTT assay proves particularly useful when applied to 3D spheroid models that mimic the structural and functional characteristics of solid tumors. Spheroids, such as those generated via the aqueous two-phase system (ATPS), as demonstrated by Ham et al. (2024), replicate key features of tumors, including oxygen and nutrient gradients that create hypoxic cores. These 3D structures also exhibit extracellular matrix deposition and express cancer stem cell (CSC) markers, making them a robust model for evaluating potential anti-cancer therapies. The XTT assay, applied to these spheroid models, allows researchers to assess drug efficacy in a more physiologically relevant setting, especially when testing treatments targeting drug-resistant cancer cells or hypoxic regions within tumors. For instance, the assay has proven effective in evaluating combination therapies, such as the use of doxorubicin and the hypoxia-activated prodrug TH-302, to target triple-negative breast cancer (TNBC) spheroids (Ham et al., 2016).

Fig. 6.

XTT Assay. Fig 6 outlines the procedure for the XTT assay, a colorimetric assay used to evaluate cell viability. Cells are seeded into a 96-well plate, and the XTT reagent is added, allowing metabolically active cells to reduce XTT into an orange-colored formazan product. After incubation, the intensity of the color is measured spectrophotometrically. The absorbance correlates with cell viability.

Fig. 7.

Fig. 7.

WST-1 Assay. Fig 7 depicts the protocol for the WST-1 assay, a colorimetric method to assess cell proliferation and viability. Cells are seeded in a 96-well plate, followed by the addition of the WST-1 reagent, which is metabolized by viable cells into a soluble, yellow formazan product. After incubation, the absorbance of the formazan is measured to determine cell activity. The intensity of the color is proportional to the number of viable cells.

5.1.3. WST-1 assay

The WST-1 assay is a modern colorimetric method used for assessing cell viability and proliferation. In this assay, the tetrazolium salt WST-1 is converted into a soluble formazan dye by cellular enzymes. This conversion happens directly in the assay solution, eliminating the need for additional steps to dissolve the formazan crystals. The amount of formazan dye, which can be measured by its absorbance (450 nm), correlates with the number of viable cells. This assay is versatile and can be applied to both traditional cell cultures and complex spheroid models, making it a valuable tool for drug testing and cell viability studies (Niles et al., 2009; Riss et al., 2016). One of the key advantages of the WST-1 assay is its non-toxic nature, which allows for repeated measurements and makes it particularly suitable for long-term viability studies. It is especially valuable for investigating slow-growing cells or complex tissue models. In the context of 3D spheroid models, the WST-1 assay enables researchers to evaluate the effects of potential therapies in a more physiologically relevant setting. Spheroids, such as those derived from lung cancer cell lines (e.g., A549), closely mimic the biological characteristics of solid tumors, including gradients in oxygen and nutrient distribution and regions of hypoxia. These conditions promote the development of chemotherapy-resistant phenotypes, such as those expressing claudin-1 (CLDN1), a tight junction protein. As demonstrated by Akizuki et al. (2024), the overexpression of CLDN1 in spheroid models of lung adenocarcinoma significantly reduces the penetration and effectiveness of chemotherapeutic agents like doxorubicin (DXR) in the inner regions of the spheroids. By employing the WST-1 assay in these spheroids, researchers can assess drug efficacy, penetration, and resistance mechanisms in an environment that closely resembles actual tumors. The study also indicated that inhibiting CLDN1 expression could enhance drug accumulation and toxicity, thereby underscoring the utility of the WST-1 assay in evaluating the effects of tight junction proteins on drug resistance and the overall therapeutic response in spheroid models. This makes the WST-1 assay a valuable tool in the drug discovery process, particularly for developing effective treatments against resistant cancer phenotypes (Akizuki et al., 2018).

5.2. Flow cytometry analysis of drug-treated spheroids

Flow cytometry is a highly effective analytical technique for evaluating the impact of drugs on spheroids. This approach allows researchers to analyze large numbers of cells with high accuracy and speed, offering detailed insights into how drugs affect cellular behaviour within complex spheroid models. Unlike traditional monolayer cell cultures, spheroids better mimic the natural conditions of tumors, making them a more realistic model for drug testing. Tung et al. (2011) developed a high-throughput system using a 384-well hanging drop array for spheroid culture and drug testing. This system, when combined with flow cytometry, enables the simultaneous analysis of many spheroids, facilitating efficient drug screening. This method helps to assess drug interactions within the 3D environment of spheroids, providing a more accurate representation of in vivo drug effects. Patra et al. (2015) extended this approach by integrating flow cytometry with microfluidic devices to test drugs on a large number of uniformly sized spheroids. This combination allows for precise control over drug exposure and the ability to analyze thousands of spheroids in parallel. Flow cytometry in this setup offers valuable information on how drugs penetrate and affect cells in spheroids, aiding in the evaluation of drug efficacy and toxicity. By using this technique, researchers can gain a more comprehensive understanding of drug effects, improving the accuracy of drug testing and supporting more effective drug development (Patra et al., 2016b; Tung et al., 2011).

5.2.1. Apoptosis

Flow cytometry provides a detailed and efficient way to study how drugs induce apoptosis in spheroids. This technique allows researchers to analyze large numbers of cells simultaneously, offering insights into how drugs trigger cell death within the complex structure of spheroids. Unlike traditional cultures, spheroids better mimic the conditions found in actual tumors, making them a more accurate model for drug testing. For instance, advancements like the 384-well hanging drop array developed by Tung et al. (2011) and colleagues have significantly improved the ability to test and analyze many spheroids at once. When combined with flow cytometry, this system enables a thorough examination of drug effects on apoptosis, revealing how well drugs penetrate and affect cells in the 3D environment of spheroids (Patra et al., 2016).

5.2.2. Cell cycle

Flow cytometry is also effective for studying how drugs impact the cell cycle in spheroids. This method allows researchers to assess various phases of the cell cycle and detect any disruptions caused by drug treatment. Patra et al. (2015) and others have enhanced this approach by using microfluidic devices to handle and test a large number of uniformly sized spheroids. This setup provides precise control over drug exposure and enables the analysis of thousands of spheroids simultaneously. By employing flow cytometry in this context, researchers can gain valuable insights into how drugs influence cell cycle dynamics and identify potential issues such as cell cycle arrest or abnormal proliferation within the spheroid model (Sargenti et al., 2020).

5.3. Acid phosphatase assay (APH) for cell viability in spheroid models

The Acid Phosphatase Assay (APH) is an effective method for assessing cell viability in three-dimensional (3D) spheroid cultures. By quantifying the activity of acid phosphatase, an enzyme indicative of cell health, APH provides valuable insights into the effects of drugs on cell survival. Spheroids better mimic the complex environment of tumors than traditional monolayer cultures. Friedrich et al. (2007) demonstrated that APH is particularly suited for high-throughput drug screening in spheroids, as it measures cell viability without disrupting their structure. To enhance drug testing, Tung et al. (2011) introduced a high-throughput 384-well hanging drop array for efficient spheroid culture and drug testing. When paired with APH, this system allows extensive drug screening while maintaining spheroid integrity. Patra et al. (2015) further advanced this approach by integrating microfluidic devices with flow cytometry, enabling detailed analysis of drug effects on uniform-sized spheroids. This combination provides complementary data on drug penetration and cellular responses within 3D models (Barbosa et al., 2022; Friedrich et al., 2007a; Perche and Torchilin, 2012a). Collectively, these analytical techniques enhance our ability to evaluate drug efficacy and toxicity in spheroids, providing a comprehensive understanding of drug interactions. In drug discovery, spheroids derived from cancer cell lines mimic solid tumors, facilitating the assessment of therapeutic efficacy against drug-resistant phenotypes. By employing the APH assay, researchers can evaluate cell viability after exposure to various therapeutic agents, particularly in environments characterized by nutrient gradients and hypoxia, common in solid tumors. Additionally, the APH assay aids in elucidating mechanisms of drug resistance by analyzing enzyme activity changes in response to treatment. Integrating the APH assay with spheroid models significantly enhances the translational relevance of preclinical drug testing, offering insights into the performance of potential anti-cancer therapies in conditions that closely mimic human tumor (Friedrich et al., 2007b; Wen et al., 2013).

6. Comparing analytical techniques: sensitivity, specificity, and practicality

When comparing analytical methods for drug testing in spheroid cultures, it is crucial to consider their sensitivity, specificity, and practicality, as each technique has its advantages and limitations. The Acid Phosphatase Assay (APH) is a widely used method for assessing cell viability in spheroids. While it is efficient for large-scale experiments, it may only partially capture the responses of individual cells within the spheroid. On the other hand, the high-throughput 384-well hanging drop array, as described by Tung et al. (2011), allows for simultaneous testing of multiple spheroids. This method is beneficial for handling numerous samples and ensuring spheroid uniformity, which is essential for achieving consistent results. Although it provides good sensitivity for detecting drug effects, careful calibration is required to maintain reliability under varying conditions. Microfluidic devices combined with flow cytometry, as explored by Patra et al. (2015), offer a highly precise and detailed analysis of drug effects on large populations of uniform spheroids. This method excels in specificity and sensitivity, providing valuable insights into drug penetration and cellular responses at the single-cell level. However, its complexity and resource-intensive nature make it less practical for routine use when compared to simpler methods like APH or high-throughput arrays. Each of these methods serves a unique purpose, and selecting the right one depends on the specific goals of the drug testing process. By choosing the appropriate technique, researchers can gather accurate and meaningful data to advance drug development and improve therapeutic efficacy. The comparison of these characterization techniques for anti-cancer drug testing in spheroids is summarized in Table 2. The table presents a detailed comparison of three critical tools used in 3D cell culture for drug testing: a high-throughput culture platform (384-Well Hanging Drop Array), a viability assay (Acid Phosphatase Assay), and an advanced analytical tool (Microfluidic Flow Cytometry). Although each method fulfils different functions—culturing, viability assessment, and analysis—they all play essential roles in evaluating drug efficacy and toxicity in 3D models. APH assesses cell viability while preserving spheroid structure, making it ideal for drug screening. The 384-Well Hanging Drop Array supports high-throughput culture and ensures consistency, while Microfluidic Flow Cytometry provides single-cell precision, allowing for deeper analysis of drug effects. The comparison highlights sensitivity, specificity, and practicality, with APH and Microfluidic Flow Cytometry rated highly for sensitivity. Microfluidic Flow Cytometry has the highest specificity due to its single-cell precision, while APH stands out for its practicality due to simplicity. This comprehensive comparison illustrates the unique strengths and limitations of each method, guiding the selection of appropriate techniques for 3D drug testing models (Hait, 2010; Mosmann, 1983; Riss et al., 2016).

Table 4.

Table 4.

Comparison of analytical techniques used in drug testing.

Technique	Acid Phosphatase Assay (APH)	384-Well Hanging Drop Array	Microfluidic Flow Cytometry	References
Sensitivity	High	Moderate	High	(Patra et al., 2016b, 2016a; Tung et al., 2011a)
Specificity	Moderate	Moderate	High	(Patra et al., 2016b, 2016a; Tung et al., 2011a)
Practicality	High	High	Moderate	(Patra et al., 2016b, 2016a; Tung et al., 2011a)
Key Advantages	Measures cell viability without disrupting spheroid structure	Allows simultaneous testing of multiple spheroids; maintains uniformity	Provides detailed analysis of drug effects at the single-cell level; precise	(Mittler et al., 2017)
Limitations	May not capture detailed single-cell responses	Requires careful calibration for consistency	Complex and resource-intensive; less practical for routine use	(Sirenko et al., 2015)
Cost (range)	Rs 22,233.30 (Sigma)	$1675 (Akura plus – Insphero)	Rs 36,000–60,000 (Sigma)	(Adcock, 2015b)

7. Significance of spheroid cultures in cancer research

Clinical trials are essential for evaluating the effectiveness of new drugs. Historically, drug testing has relied on monolayer cell cultures and animal models, which can be time-consuming, costly, and raise ethical concerns. Recently, three-dimensional (3D) cell cultures have emerged as a valuable alternative, offering several advantages. Three-dimensional spheroid cultures better mimic the complex structure and environment of human tumors compared to traditional 2D cell cultures. These models provide a more accurate representation for studying drug behavior, such as how drugs penetrate tissues, their resistance mechanisms, efficacy, and toxicity. Techniques like hanging drop arrays, spinner flasks, and microfluidic systems help create spheroids with consistent size and shape, which is crucial for reliable results. Research has shown that drugs can behave differently in spheroid cultures than in 2D cultures. For instance, 5-Fluorouracil (5-FU), a well-known anti-cancer drug, is more effective in 2D cultures, while Tirapazamine (TPZ), a hypoxia-activated prodrug, shows better efficacy in spheroid cultures. This demonstrates the importance of Spheroid models in obtaining a more accurate understanding of drug responses. Various assays, such as the acid phosphatase assay (APH), are used to evaluate drug efficacy and toxicity in spheroids. APH is particularly useful because it measures cell viability in Spheroid models without needing to dissociate the spheroids, making it suitable for high-throughput testing. While traditional monolayer cell cultures are easier to manage, they do not capture the full complexity of Spheroid models. Monolayer cultures present cells in a flat layer, which does not reflect the true 3D structure of tissues or tumors. In contrast, spheroid models offer a more realistic environment with gradients of nutrients, oxygen, and waste products, allowing for a better understanding of drug interactions and cellular responses. Spheroids provide a more precise model for studying tumor growth and drug resistance, offering significant benefits for developing and testing new cancer treatments. They enable researchers to understand drug effects in a more realistic setting. However, spheroids can be more complex and expensive to create and maintain than traditional cell cultures. Recognizing these differences is crucial for selecting the most appropriate model for drug development. The use of spheroid cultures marks a significant advancement in cancer research, providing a more comprehensive understanding of drug efficacy and resistance. The different anti-cancer drugs’ Cancer Targeted, Mechanism of Action and Key Findings are listed below in Table 1. (Adcock, 2015a; Lovitt et al., 2014; Riss et al., 2016) (Fig 4). The various Anti-Cancer Drugs and Their Efficacy in Traditional Monolayer cell culture and Spheroid model are listed in Table 5. The various anti-cancer drugs tested in different models are listed in Table 6.

Table 5.

Anti-cancer drugs and their efficacy in traditional monolayer cell culture and spheroid model.

Drug Name	Target Cancer	Mechanism of Action	Key Findings	References
5-Fluorouracil (5-FU)	Colorectal, Breast, Stomach, Pancreatic, and Cervical Cancers	Anti-metabolite that inhibits thymidylate synthase, interfering with DNA synthesis and leading to cell death.	Higher anti-proliferative effect in monolayer cultures compared to spheroid models.	(Erden Tayhan, 2024)
Tirapazamine (TPZ)	Hypoxic Tumors, such as those in Lung Cancer	Hypoxia-activated prodrug that generates cytotoxic free radicals in low oxygen environments, leading to DNA damage.	More effective in spheroid models than in cultures.	(Tung et al., 2011a)
Cisplatin	Testicular, Ovarian, Bladder, Lung, and Head and Neck Cancers	Platinum-based drug that forms DNA adducts, leading to DNA crosslinking and apoptosis.	Tested on tumor spheroids, evaluating its efficacy in a high-throughput system.	(Patra et al., 2016b)
Resveratrol	Breast, Prostate, and Colon Cancers	Naturally occurring polyphenol that induces apoptosis and inhibits cancer cell proliferation by modulating various signaling pathways.	Tested in combination with other drugs on tumor spheroids.	(Patra et al., 2016b)
Irinotecan	Colon and Rectal Cancer	Topoisomerase I inhibitor that prevents DNA unwinding, leading to DNA damage and apoptosis.	Reduced efficacy in spheroid models compared to monolayers.	(Perche and Torchilin, 2012a)
Imidazoacridinone, C-1311	Breast, Ovarian, and Lung Cancers	Novel anti-cancer agent that inhibits topoisomerase II, leading to DNA damage and cell death.	Effectiveness evaluated in spheroid models.	(Perche and Torchilin, 2012a)
Doxorubicin	Breast Cancer, Leukemia, Lymphoma, and Sarcomas	Anthracycline antibiotic that intercalates into DNA, inhibiting topoisomerase II and generating free radicals, causing DNA damage and apoptosis.	Tested in spheroid models, showing differential responses depending on spheroid size and environment.	(Hait, 2010)

Table 6.

Efficacy of anticancer drugs on monolayer and spheroids models.

Drug Name	Monolayer cell culture Model Efficacy	Spheroid Model Efficacy	Key Insights	References
5-Fluorouracil (5-FU)	Higher anti-proliferative effects	Less effective	Anti-proliferative effects are significantly higher in monolayer cultures	(Tung et al., 2011b)
Tirapazamine (TPZ)	Less effective	More effective	Spheroid cultures better mimic hypoxic conditions, enhancing drug effectiveness	(Patra et al., 2016c)
Cisplatin	Evaluated in spheroids for drug efficacy	Insights into realistic performance	Provides valuable data on drug performance in realistic tumor models	(N. Baek et al., 2016)
Resveratrol	Tested with other drugs	Demonstrated anti-cancer properties	Effective when tested in combination with other drugs on spheroids	(Patra et al., 2016c)
Irinotecan	Higher efficacy	Reduced efficacy	Highlights differences in drug responses when tested in 3D cultures	(Perche and Torchilin, 2012b)
Imidazoacridinone C-1311	Assessed effectiveness	Valuable data in spheroids	Important for understanding the performance of the drug in 3D contexts	(Perche and Torchilin, 2012a)
Doxorubicin	Enhanced efficacy	Further Enhanced efficacy	Increased efficacy in spheroid cultures; provides insights into realistic model performance	(N. H. Baek et al., 2016b)

8. Applications of spheroid models in anti-cancer drug discovery and delivery

Three-dimensional (3D) cell culture models, particularly tumor spheroids, have emerged as powerful tools in cancer research due to their ability to more closely simulate the in vivo tumor environment. These models replicate the complexity of actual tumors, including cellular diversity, gene expression, differentiation, and the creation of hypoxic conditions. This makes them particularly useful for studying various aspects of tumor biology, including drug resistance, cell migration, invasion, and drug delivery strategies (Han et al., 2021; S. W. Lee et al., 2019; Takayama et al., 2013).

8.1. Chemoresistance

A significant challenge in cancer therapy is overcoming drug resistance, especially in aggressive and metastatic cancers. Tumor spheroids grown in 3D cultures provide a more accurate representation of the tumor environment, better capturing the factors that contribute to drug resistance. The 3D structure enhances cell-to-cell and cell-to-extracellular matrix (ECM) interactions, leading to changes in gene expression that can increase resistance to chemotherapy. For example, specific adhesion molecules may be upregulated in these 3D environments, resulting in greater resistance compared to monolayer cell cultures. The structural and environmental features of spheroids, such as hypoxia, altered metabolism, an acidic microenvironment, and the presence of cancer stem cells, all contribute to their heightened drug resistance. While many studies suggest that cells within spheroids exhibit more resistance to chemotherapy than those in 2D cultures, there are exceptions, highlighting the complexity of chemoresistance mechanisms (Perche and Torchilin, 2012a; Takayama et al., 2013).

8.2. Migration and invasion

Tumor cell migration and invasion are critical processes in the metastasis of cancer. Spheroid models are particularly well-suited for studying these processes because they more accurately mimic the 3D architecture and interactions found in tumors. These models allow for the examination of how cells move and invade surrounding tissues, and they closely replicate the behavior of tumors in vivo, including the expression of adhesion molecules and metastasis-associated proteins. By co-culturing spheroids with non-cancerous cells such as fibroblasts and immune cells, researchers can gain deeper insights into the interactions within the tumor microenvironment that drive migration and invasion. Various assays have been developed to measure these capabilities in spheroids, including methods where spheroids are placed on ECM-coated filters to observe cell movement or embedded within ECM gels to study tissue invasion. These assays provide valuable information about the mechanisms of invasion and potential therapeutic targets (Adcock, 2015b; Pinto et al., 2020).

8.3. Spheroids in nanomedicine

Nanomedicines offer a promising approach to improving drug delivery to tumors while reducing side effects. Nanoparticles (NPs) can be engineered for controlled drug release, targeted delivery, and enhanced accumulation in tumors. However, the translation of promising preclinical results into clinical success has been limited, in part because traditional 2D culture models do not accurately predict in vivo behavior. Spheroids, with their more accurate representation of tumor architecture and microenvironment, have become increasingly popular for evaluating nanomedicines. Research indicates that the penetration of NPs into spheroids depends on factors such as size, surface charge, and coating. Smaller NPs tend to penetrate deeper and distribute more evenly within spheroids, whereas larger NPs often remain confined to the outer layers. The surface charge of NPs also affects their penetration, with negatively charged particles generally penetrating more effectively than positively charged ones. Coating NPs with ECM-degrading enzymes has been shown to enhance their penetration into spheroids significantly. These findings highlight the importance of using 3D culture systems to assess the potential of nanomedicines, as they offer a more realistic prediction of how these therapies might perform in clinical settings (Bell et al., 2018b; Pinto et al., 2020). In summary, spheroid models provide a more physiologically relevant platform for studying cancer biology and evaluating anti-cancer therapies, including chemotherapeutic drugs and nanomedicines. Their ability to replicate the tumor microenvironment and provide insights into drug resistance and metastatic behavior makes them an invaluable tool in developing more effective cancer treatments.

8.4. Spheroids application in cancer vaccine testing

Spheroids have emerged as a critical tool in cancer vaccine research due to their ability to closely mimic the tumor microenvironment, far surpassing traditional two-dimensional models in accuracy. These three-dimensional (3D) cell culture systems allow for more realistic interactions between tumor cells and immune components, offering valuable insights into immune responses. This has proven essential for the advancement of cancer vaccines, as the spheroids' structure enables better diffusion of nutrients, oxygen, and waste, reflecting the in vivo tumor conditions more effectively. The unique advantages of spheroids have been instrumental in advancing personalized cancer immunotherapies. By growing tumor cells in a 3D architecture that mirrors the structure of tumors found in patients, spheroids facilitate more accurate antigen presentation and immune modulation. Immune cells, such as dendritic cells and T-cells, interact with these spheroids in a manner that better represents natural tumor-immune cell communication. This interaction is key for the development of cancer vaccines designed to stimulate the immune system to identify and attack tumor cells efficiently. Rahman et al. (2024) found that spheroids derived from cell culture systems generate stronger antitumor immune responses when compared to traditional monolayer models. Their study demonstrated that these spheroids promote the natural expression of tumor antigens, which significantly enhances the effectiveness of cancer vaccines in activating immune cells such as dendritic cells and cytotoxic T-cells. The findings suggest that using spheroids to develop dendritic cell vaccines led to improved immune activation and memory, making them a promising platform for personalized immunotherapy. The study highlights the advantages of spheroid models in preclinical vaccine evaluation and their potential in advancing cancer vaccine efficacy (Rahman et al., 2024). Additionally, Niavarani et al. (2024) explored the use of spheroids alongside immunomodulators in a heterologous prime-boost vaccination approach targeting triple-negative breast cancer (TNBC). This study utilized spheroids as part of an oncolytic virus-infected cell vaccine (ICV) strategy, which significantly improved the immunogenicity of tumor cells. By priming mice with a vaccine containing the influenza virus and boosting with Vesicular Stomatitis Virus (VSVd51), they observed enhanced recruitment and activation of dendritic cells. The use of spheroids in this approach resulted in higher frequencies of effector and memory T-cells, leading to robust antitumor immune responses. The strategy successfully prevented tumor recurrence in re-challenged mice, and combining it with early surgical tumor resection led to improved survival rates. This demonstrates the value of 3D spheroid models in enhancing cancer vaccine development, especially for hard-to-treat cancers like TNBC (Niavarani et al., 2023). In conclusion, the application of spheroids in cancer vaccine development provides a more accurate representation of the tumor microenvironment. By enhancing immune cell activation and antigen presentation, spheroids play a pivotal role in improving vaccine efficacy and driving innovation in personalized cancer immunotherapy (Rahman et al., 2024).

8.5. Spheroids application in cancer - precision medicine

Spheroids are revolutionizing precision medicine by offering advanced models that closely mimic the tumor microenvironment (TME). These three-dimensional (3D) cellular structures provide a more accurate representation of the complexity and heterogeneity of tumors compared to traditional monolayer cultures. By utilizing spheroids derived from patient-specific tumor samples, researchers can evaluate treatment responses and tailor therapeutic strategies to individual patients, ultimately enhancing treatment efficacy and minimizing adverse effects. The functioning of spheroids is grounded in their ability to replicate the architecture and cellular interactions found in actual tumors. Spheroids are formed by aggregating cells into spherical structures that allow for cell-cell and cell-matrix interactions, resulting in gradients of nutrients, oxygen, and waste products that are more representative of in vivo conditions. The application of spheroids and organoids enables a deeper exploration of tumor heterogeneity and the intricate interactions within the TME. This understanding is crucial for identifying specific biomarkers and therapeutic targets, which can facilitate personalized treatment plans. According to Gilazieva et al. (2020), the development of spheroid models represents a significant advancement in cancer research, providing insights that are closer to in vivo conditions. These models not only enhance our understanding of tumor biology but also play a critical role in personalizing treatment strategies. Spheroids can be integrated with high-throughput screening methods, allowing researchers to assess drug responses rapidly and effectively on a patient-by-patient basis. For instance, researchers can expose spheroids or organoids to various chemotherapeutic agents and assess cell viability or other response markers, enabling them to identify the most effective treatment regimens for individual patients. This capability is vital for advancing the concept of personalized medicine, where therapies are tailored based on the unique characteristics of an individual's cancer (Gilazieva et al., 2020). El Harane et al. (2024) highlight the importance of selecting appropriate techniques for generating spheroids based on tissue origin and disease state, ensuring that the models are relevant for drug screening and therapeutic evaluation (El Harane et al., 2023). A significant innovation in organoid technology is the ability to co-culture tumor cells with components of the TME, enabling researchers to study the interactions between cancer cells and immune cells. Homicsko et al. (2020) emphasizes that this co-culture approach provides insights into the complex dynamics that influence treatment responses. By simulating the TME, researchers can observe how immune cells respond to therapies, paving the way for novel immunotherapy strategies. However, challenges remain, as this co-culturing can currently only be maintained for limited durations, affecting the potential for extended studies Applications of spheroids and organoids in cancer research extend beyond drug testing and personalization of treatment regimens. They are being used to explore mechanisms of drug resistance, investigate tumor progression, and study metastatic behavior. For example, spheroids can provide insights into how cancer cells behave in a 3D context, allowing researchers to investigate the factors that contribute to treatment resistance or tumor recurrence. Additionally, organoids can be employed to model genetic alterations specific to a patient's tumor, enabling researchers to test targeted therapies that address those mutations (Yang et al., 2021). Despite the promising applications of spheroids and organoids in precision medicine, some challenges need to be addressed. Homicsko et al. (2020) points out that the prospective validation of organoid assays as reliable predictors of therapeutic benefits is still lacking. Extensive studies are required to establish the consistency and predictive capabilities of these models, particularly for immune therapies. Current immune checkpoint blockade therapies have shown efficacy in only select patient populations, highlighting the need for ex vivo assays that utilize organoid technology to evaluate patient-specific responses before treatment (Homicsko, 2020). In summary, spheroids and organoids are transforming the field of precision medicine by providing relevant platforms for studying cancer biology, testing therapeutic responses, and tailoring treatment strategies to meet individual patient needs. The combination of advanced 3D culture systems with high-throughput approaches holds significant potential for uncovering new insights into tumor biology and optimizing therapeutic strategies. As research continues to validate these innovative models, they may play a pivotal role in advancing personalized cancer therapies and improving patient outcomes in oncology.

8.6. High-throughput screening in spheroids

High-throughput screening (HTS) has emerged as a vital methodology in drug discovery, allowing researchers to efficiently assess the effects of a vast array of compounds on biological systems. In cancer research, HTS is especially critical for evaluating the effectiveness of anti-cancer agents utilizing three-dimensional (3D) cell models, such as spheroids. Unlike traditional monolayer cell cultures, spheroids create a more physiologically relevant environment that closely mimics the tumor microenvironment. This feature enhances the accuracy of drug response evaluations and helps elucidate underlying mechanisms of action. Integrating HTS with spheroid technology significantly improves the drug screening process. Automated systems and microplate formats enable researchers to rapidly test numerous compounds across various spheroid models derived from different tumor types or patient samples. This efficiency accelerates the drug discovery timeline and facilitates the identification of potential therapeutic candidates that might be overlooked when using monolayer cell cultures. Spheroids replicate key aspects of in vivo environments, such as cell-cell interactions, nutrient gradients, and the dynamics of drug penetration. Recent studies, including the work of Kondo et al. (2018), have underscored the benefits of employing cancer tissue-originated spheroids (CTOS) in HTS. This research introduced a system capable of evaluating over 2400 drugs derived from xenograft tumors. A significant hurdle when utilizing patient-derived organoids for drug screening is the limited availability of samples and the substantial time and costs associated with their culture. The CTOS methodology addresses these challenges by generating multiple spheroids from xenograft tumors, thus allowing for extensive drug screenings across diverse tumor models. Furthermore, the CTOS method maintains the morphological and genomic characteristics of the original tumors, ensuring that the drug responses observed in the spheroids accurately reflect the heterogeneity present in cancers, such as colorectal cancer (Kondo et al., 2019). The need for high-throughput 3D tumor spheroids has led to advancements in culturing techniques that facilitate efficient drug screening. For instance, Griner et al. (2019) demonstrated methods for generating 3D tumor spheroids in high-throughput formats, particularly utilizing 1536-well plates. This approach resolves the limitations associated with traditional 2D assays and complex matrix-based systems, providing a more efficient solution for screening various cancer cell lines with different genetic mutations. By assessing compound efficacy through a curated library that targets the Mitogen-Activated Protein Kinase (MAPK) pathway, researchers can compare the responses of spheroids to those of cells grown in 2D, revealing differential activities that offer insights into drug effectiveness (Griner et al., 2018). One notable advantage of employing HTS in spheroid models is the capacity to investigate combination therapies. Researchers can simultaneously test multiple drugs or drug combinations, identifying synergistic effects that may enhance treatment outcomes. This capability is particularly significant in oncology, where combination therapies are often essential to overcome drug resistance. Moreover, utilizing spheroids in HTS allows for a comprehensive analysis of how the tumor microenvironment influences treatment responses, offering valuable insights into the complex interactions that occur within tumors. In summary, high-throughput screening in spheroids, particularly through approaches like CTOS and high-throughput 3D culturing, signifies a major advancement in anti-cancer drug testing. This innovative strategy bridges the gap between traditional 2D cultures and complex in vivo models, enhancing the efficiency of drug discovery while providing a relevant platform for evaluating therapeutic responses that closely align with actual tumor behaviour. As research continues to refine these methodologies, the integration of HTS with spheroids is expected to play a crucial role in advancing cancer therapeutics and improving patient outcomes in the field of precision medicine (Abolhassani et al., 2023; Kunz-Schughart et al., 2004).

8.7. Successful spheroid models

Currently, a variety of effective spheroid models are being utilized in cancer research, each providing unique benefits that deepen our understanding of tumor behavior and drug effectiveness. A notable example is the cancer tissue-originated spheroid (CTOS), which is generated from patient tumors or xenograft tissues. CTOS models preserve the genetic and structural diversity of the original tissues, enabling researchers to assess drug responses in an environment that closely resembles in vivo conditions. This model is particularly advantageous for investigating colorectal cancer, as it captures the variations in treatment sensitivity that characterize the disease's inherent heterogeneity (Kondo et al., 2011). Another prevalent model is the cell line-derived tumor spheroid, which is formed from established cancer cell lines such as HeLa (cervical cancer), MCF-7 (breast cancer), and A549 (lung cancer). These spheroids allow for standardized evaluations of drug responses, enabling researchers to delve into specific mechanisms of action and compare various therapeutic strategies (Morimoto et al., 2021). Organoid cultures signify a major advancement in spheroid technology. These models are derived from stem or progenitor cells and can differentiate into diverse cell types present in tumors, thereby preserving the histological structure and genetic makeup of the original tissues. This characteristic makes organoids especially valuable in personalized medicine applications, as patient-derived organoids enable tailored drug testing that aligns with the unique features of an individual's tumor, potentially enhancing treatment success (Homicsko, 2020; Liu et al., n.d.). Microfluidic spheroid models are gaining traction due to their ability to provide precise regulation of the tumor microenvironment. This capability facilitates the examination of fluid dynamics, nutrient distribution, and cellular interactions within a controlled setting, making these models crucial for studying the effects of the microenvironment on drug delivery and therapeutic efficacy (Patra et al., 2016b; Vadivelu et al., 2017b). Additionally, heterotypic spheroids, which incorporate various cell types (including cancer cells, stromal cells, and immune cells), provide insights into the intricate interactions occurring within the tumor microenvironment. These models are vital for elucidating the complex signaling pathways that drive tumor progression and resistance to therapies (Vinci et al., 2012). Recent advancements have also led to the emergence of 3D bioprinted spheroids, which allow for the precise arrangement and composition of cells. This innovation enhances the predictive capabilities of drug testing by closely mimicking the natural tumor environment. Bioprinting techniques enable the integration of extracellular matrix components, further increasing the relevance of these models. Moreover, spheroids embedded in biomaterial scaffolds can be employed to explore how mechanical properties and cellular interactions affect drug responses. These models facilitate investigations into the impact of tissue stiffness and structural integrity on cancer cell behavior and treatment outcomes (Mandrycky et al., 2016; Murphy and Atala, 2014). In summary, the diverse range of spheroid models currently employed in cancer research continues to evolve, offering significant advancements in preclinical drug testing. These models enhance the predictive reliability of therapeutic responses and contribute to the progress of personalized medicine. As ongoing improvements are made to these methodologies, the potential for developing more precise and tailored cancer treatments increases, ultimately leading to improved patient outcomes.

9. Discussion and conclusion

The use of traditional cell cultures and spheroid models has significantly advanced our understanding of anti-cancer drug efficacy and mechanisms of action. Traditional cell cultures have provided foundational insights into drug-induced apoptosis, cell cycle alterations, and cytotoxicity through various assays such as MTT, XTT, and WST-1. These assays have been instrumental in assessing drug effects in a simplified environment. Spheroid models, more closely mimic the in vivo conditions, including gradients of nutrients, oxygen, and drug penetration. They have emerged as valuable tools for studying chemoresistance, cell migration and invasion, and drug delivery. Their ability to recreate aspects of tumor biology that are missing in Traditional cell cultures makes them particularly useful for more accurate drug testing and development. Despite their advantages, both models have limitations. Traditional cell cultures lack the three-dimensional complexity of tissues, while spheroid models may not fully replicate the details of the tumor microenvironment. The integration of these models with advanced technologies, such as high-throughput screening and microfluidics, holds promise for overcoming these limitations.

10. Perspectives and future directions

The future of cancer research will likely involve a more integrated approach, combining traditional cell cultures with advanced 3D models to leverage the strengths of both systems. The development of more sophisticated spheroid models that incorporate elements such as extracellular matrix components, vascularization, and immune cell interactions will enhance their predictive power. Additionally, the refinement of cytotoxicity assays and cell analysis techniques will improve the accuracy of drug testing. High-resolution imaging, coupled with automated data analysis, can provide deeper insights into drug effects at the cellular and molecular levels. Embracing multi-omics approaches, including genomics, proteomics, and metabolomics, will enable a more comprehensive understanding of drug interactions and resistance mechanisms. The integration of personalized medicine into preclinical drug testing is another promising direction. Customizing spheroid models based on patient-specific tumor profiles can provide more relevant data for predicting drug responses and tailoring treatments. Ultimately, continued innovation in cell culture technologies, coupled with a better understanding of tumor biology, will drive forward more effective and personalized cancer therapies. The ongoing collaboration between researchers, clinicians, and technologists will be crucial in translating these advancements from the laboratory to the clinic, improving outcomes for cancer patients worldwide.

CRediT authorship contribution statement

Anu Varshini A M: Writing – original draft. Usha Nandini S: Supervision. V Ramesh Kumar: Supervision. Thangam T: Writing – review & editing. Krupakar Parthasarathy: Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No data was used for the research described in the article.