Artificial tumor matrices and bioengineered tools for tumoroid generation

The tumor microenvironment (TME) is critical for tumor growth and metastasis. The TME contains cancer-associated cells, tumor matrix, and tumor secretory factors. The fabrication of artificial tumors, so-called tumoroids, is of great significance for the understanding of tumorigenesis and clinical cancer therapy. The assembly of multiple tumor cells and matrix components through interdisciplinary techniques is necessary for the preparation of various tumoroids. This article discusses current methods for constructing tumoroids (tumor tissue slices and tumor cell co-culture) for pre-clinical use. This article focuses on the artificial matrix materials (natural and synthetic materials) and biofabrication techniques (cell assembly, bioengineered tools, bioprinting, and microfluidic devices) used in tumoroids. This article also points out the shortcomings of current tumoroids and potential solutions. This article aims to promotes the next-generation tumoroids and the potential of them in basic research and clinical application.


Biotechnology in cancer research
Tumors containing malignant cells, so-called cancers.Cancer cells interact with CACs within the TME [1].Cancer cells reside in the specific sites of host tissues and cause physical and chemical changes to form TME and support cancer growth and metastasis [2].Like normal tissues, cancer tissues contain a heterogeneous cell population that secrete soluble tumor factors and non-soluble tumor ECM, which interact to each other synergistically within the TME [2].
The TME is an abnormal cellular environment.Cancer cells are not only divide rapidly themselves but also recruit surrounding normal cells such as stromal cells, immune cells, endothelial cells, pericytes, and CAFs for cancer development [3][4][5][6].Besides, tumor ECM is believed to be not just a silent bystander but an active promoter of cancer progression [7].Cancer cells and ECM together induce intratumorally angiogenesis, peripheral immune tolerance, and drug resistance [8,9].
Traditional 2D cell culture has been widely used for cancer research and drug discovery; however, this model cannot reproduce the tumor TME and fails to preserve their biological characteristics adequately.PDX is an allogeneic model established by transplanting surgically resected tumor tissue masses from cancer patients into immunodeficient mice [10].The PDX models have highly similar histopathological and genetic characteristics to the maternal tumors.Studies have shown that, the drug sensitivity of the PDX model to anticancer drugs is highly correlated with the clinical treatment outcomes of cancer patients [11].PDX models are still the most used models in the pharmaceutical industry and personalized medicine in the past few decades although they are time-consuming and cost-ineffective [12][13][14].
Tumoroids have been developed in the laboratories for drug screening and discovery.Tumoroids or tumor spheroids, indicating 3D tumor culture, are not a new concept.Thanks to the organoid technology, the terms of tumor organoid, PDTOs, or patient-derived cancer organoids (PDCOs) have frequently appeared in the last few years [15].However, in our opinion, tumors or cancers are not organs but 3D new abnormal tissues; thus, 'tumoroid' is a proper terminology in the cancer research.
Tumoroids are tumor avatars of cancer patients that have become a potential preclinical models circumventing the issues of 2D cell culture and PDX models [10].Nowadays, a variety of tumoroids have been reported, such as colorectal, pancreas, prostate, kidney, lung, and sarcoma [16].An ideal tumoroids should consider TME as similar to the original tumors to achieve high correlation of drug responses [17].Tumoroid technology is an multidisciplinary approaches that aims to preserve the characteristics of primitive cancer tissues.This approach now enter the era of reconstruction of TME with vasculature and immune cell components [18].The highthroughput sequencing and spatial omics technology play important roles nowadays and has been heavily applied to illustrate tumorigenesis and explore the heterogeneity of cancers [19].Due to the high heterogeneity of tumors, the therapeutic effects vary significantly from patient to patient.These findings indicate that ideal tumoroids cannot be just tumor spheroids using one type of cancer cells.Multiple cell types and optimal ATM are essential for the future tumoroids [20].Therefore, tumoroids stand as a 'firewall' in pre-clinical drug misuses [21].
Personalized drug screening is urgently needed for those with advanced cancers together with exhausted conventional treatments [22][23][24].Until now, the treatment of metastatic cancers is still challenging and the leading cause of death among cancer patients [25,26].The clinical application of tumoroid models and personalized drug screening is expected to provide tailored therapies for patients with metastatic tumors, thereby improving the therapeutic effects and prolonging the survival of patients.
This review discusses two types of tumoroids: (1) tumor slices from the surgery samples, and (2) tumoroids using cancer cell suspensions.Although tumor slices have their own TME, they still need a hydrogel substrate, such as Matrigel or collagen, to maintain the cell growth.To form tumoroids from cancer cell suspensions, cell types, matrix materials, and the biofabrication approaches are vital in determining the size, shape, and quality of the tumoriods.In terms of cells, both cancer cell lines and primary cancer tissues have been applied in tumoroids.Cancer cells and CACs can be co-cultured or assembled to form tumoroids, so called 'tumor assembloids' [27].In terms of matrix materials, a variety of ATMs, including nature and synthetic materials, have shown the potential in maintaining long-term cancer cell activity in vitro.In terms of biofabrication approaches, microscale models, nanoscale patterns, 2D/3D bioprinting, and microfluidic devices have been applied for various applications [28][29][30][31][32]. Figure 1 shows the overview of tumoroid fabrication and clinical application.

Tumoroid technology
As abovementioned, tumoroid is not a new term.However, thanks to the recent organoid technology, tumor organoids have become promising preclinical model for drug tests.The major advance of tumoroid is that TME is emphasized rather than just cell co-culture.Although the similarity between tumoroids and primary tumors is still questionable, tumoroids should recapitulate the TME to a certain extent.Together with low-cost and time-saving protocols compared to the PDX models, tumoroids have a broad prospect of clinical applications.In this chapter, tumoroids derived from cancer tissues and cancer cell suspensions are discussed.This review will also explore cancer cell suspensions with or without CACs.
In terms of ATMs, Matrigel is widely used in tumoroids [33].Matrigel is a gelatinous protein mixture extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma [34].Matrigel is a common ATM in cell culture, supporting the growth, differentiation, and organization of various cell types [35], including stem cells and malignant tumor cells [36,37].Matrigel primarily contains laminin (∼60%), collagen IV (∼30%), entactin (∼8%), heparin sulfate proteoglycan perlecan (∼2%) and small amount of insoluble growth factors [34,38,39].Due to its exceptional bioactivity and sol-gel properties, Matrigel has been in use for over 40 years since 1980.It has also served as a common positive control for evaluating new protocols or models, such as assessing cancer cell invasion [40].

Tumor fragments
Tumor fragments are obtained directly from chopped cancer tissues, which contains endothelial cells, stromal cells, immune cells, and TME of the original tumor [41].In this method, the tumor fragments are approximately 1 mm 3 , which ensures that sufficient nutrients and reagents can enter the core of the fragments.In addition, to preserve the stereo spatial structure of the original tumor and prevent immune cell efflux, this method requires tissue fragments embedded into Matrigel for culture.Therefore, this tumoroid model refer to the tumor fragments placing on the Matrigel.
This tumoroid model contains various tumorrelated cells and can provide a suitable TME for the growth of tumor cells; therefore, there is no need to add growth factors and pathway inhibitors required for the tumoroids culture.For instance, tumor tissue was obtained by injection of human hepatocyte carcinoma (Hep3B) cells into female BALB/c nude mice.The liver tumors were cut into small pieces, washed, placed on Matrigel, and incubated in DMEM for further histological analysis [42].Afterward, liver tumoroids were used for sorafenib drug testing to assess its effect on oncogenic kinases [42].Another study uses this model to perturb the TME and found that the responses of different tumors to anti-PD-1 therapy can be detected within 24-48 h.The immunological reactions to tumor fragments correlate closely with clinical outcomes [41].Small tumor fragments were manually cut into 1-2 mm 3 sizes and cultured on ice.Based on the culture data of these tumor fragments, it was shown that the presence of tertiary lymphoid structures and intra-tumor T cells can reactivate anti-tumor immunity through PD-1 blockade.This suggests that the structured presence of these immune cell aggregates may influence sustained anti-tumor immune responses [41].
A novel antitumor strategy involving tumor fragment interference and nanotechnology has been developed for particular tumor therapy [43].Tumor fragment-based nanoformulations and nanoparticleinduced immunogenic dying tumor cells can serve as a vaccine to stimulate immune responses in cancer patients [43].Recently, a wide range of nanoparticle-based methods, encompassing chemotherapeutic, photodynamic, photothermal, magnetic, and radiotherapeutic effects, have been utilized to induce immunogenic cell death within tumor cells in living organisms.These effects trigger DNA damage, generate cytotoxic reactive oxygen species, and induce localized hyperthermia damage in cancer cells.This sets off a series of responses, including heat shock protein expression due to altered intracellular stress, release of tumor-associated antigens, and production of pro-inflammatory factors.Ultimately, these events lead to immunogenic cell death in tumor cells, triggering the maturation of dendritic cells from an immature to a mature state.This maturation process then activates the production of cytotoxic T lymphocytes, contributing to the effective eradication of tumors [43].
The ALI is an alternative method for culturing tumor fragments, which resuspends sliced tissues with type I collagen or Matrigel and places them on top of a transwell insert [39,44,45].The growth medium is added to the outer cell culture well-plate and provides nutrients for the cells in the insert through the permeable membrane.A notable aspect of this method is that tumor fragments come into direct contact with air, enhancing oxygen diffusion and promoting tumor growth.In the ALI system, cancer cell growth can be achieved by supplementing with fetal bovine serum (FBS) alone, while the supply of other factors, such as WNT3A, EGF, NOGGIN, and RSPO1, can further promote the growth of tumor epithelial cells [45].Crucially, the immune and fibroblast stroma remained detectable within tumor fragments after 1-2 months of culture, when the addition of IL-2 or anti-CD3/anti-CD28 allowed TILs to be preserved for up to 2 months [45].These features render ALI a method to maintain TME of primary tumors, which help actualize the promise of precision cancer therapies [46].

Tumor slices
TSC is a method in which tumor tissues are sliced into thin sections (200-300 µm thick) and cultured on an ATM such as type I collagen [47][48][49][50][51][52].Similar to the ALI approach, TSC involves placing tumor samples on a type I collagen matrix using a standard culture medium.TSC is a physiologically relevant culture system that maintains tumor morphology, cellular composition, and the TME for several weeks.Previous studies have demonstrated that immune cells, including T cells and macrophages, as well as stromal myofibroblasts, can be preserved in this system during the brief culture period [51].The primary advantage of TSC is its real-time platform for testing cytotoxic or immunological drugs.Fresh tumor tissue sections can be used directly for drug testing without the requirement to expand tumor cells, and the evaluation process typically takes approximately 4 d.Rodriguez et al developed a microfluidic platform that enables the culture and multiplex drug testing of whole tumor slices grown on porous PDMS membranes [53].So far, TSC-based drug testing has been performed on multiple tumor types, including breast, pancreatic, liver, colon, and urological [52,54].
In 2021, Xing et al and Lei et al integrated labelfree metabolic imaging approaches and TSC technology to achieve a quantitative evaluation of the efficacy of anticancer drugs with visual pharmacodynamic methods to predict feasible personalized medicine for cancer patients [54,55] (figure 2).In 2022, Chakrabarty et al combined TSC and microfluidic technology with the concept of organ-on-achip; therefore, breast tumor slices can be perfused culture in vitro and predict the chemotherapy sensitivity of tumors [56].The data showed no significant changes in gene expression and immune components of tissue sections during culture.Importantly, in addition to cytotoxic drugs, tumors' responses to immune checkpoint PD1/PD-L1 inhibitors can also be tested using this system.Furthermore, the anti-tumor role of CAR-T has been validated in vivo through coculture with GBM slices [57].These findings indicate that TSC is a promising versatile platform to study the TME and assess the therapeutic efficacy of anti-cancer drugs for cancer patients which can reduce the risk of drug misuses [54].

Tumoroids without cancer-associated cells
Tumoroids can be prepared using a single type of cancer cells without additional CACs.Research-focused institutions often utilize cancer cell lines, whereas clinical-oriented institutes have access to primary tumor samples.While cancer cell lines may only partially resemble primary cancers, they offer convenience and benefits in pre-clinical studies.
Tumoroids without the ATM are defined as tumor spheroids in this review, such as pellet culture and hanging drop methods.Ganguli et al used modular micro-hanging drop culture to form LN229 GBM spheroids on a microchip.By designing the shape of the droplets, they were able to manipulate the geometry of the cultured spheroids for drug screening [58].Tung et al designed a 384-well format hanging drop culture plate using polystyrene and Pluronic F108, which promotes the formation of tumoroids from human epithelial carcinoma.They demonstrated that the anticancer drug 5-fluorouracil had a stronger anti-proliferative effect on 2D cultures, while the hypoxia-activated drug tirapazamine (TPZ) was more effective on 3D cultures [59].Although these methods were relatively simple, they lack mechanical supports for the cancer cells.Cancer cells are forced to attach to each other to grow.They produce a new ECM during the culture, which takes time.When the size of tumoroids increase, cell death or apoptosis becomes noticeable.Due to different inherent cellular properties and culture conditions, the tumoroids have various morphologies such as mass-like, grapelike, and stellate-like [60].
ATM provides mechanical supports for the growth of cancer cells, preserving tissue morphology and facilitating unique cell-matrix interactions.The advantages of using ATM have been summarized and discussed by others [60,61].In brief, tumoroids recapitulate key cellular and spatial characteristics of original tumors, such as cell-cell communication, the low concentration of nutrients, hypoxia and hypermetabolic environment.These characteristics make tumoroids an ideal model for studying drug efficacy [62][63][64][65][66].For example, Séraudie et al developed a scaffold-free tumoroid model for Clear cell Renal Cell Carcinoma (ccRCC).These models created using a scaffold-based approach with collagen I or Matrigel.Tumoroids were successfully generated from both mouse and human ccRCC samples with a high success rate (⩾90%).The researchers observed that these tumoroids exhibited self-organizing abilities and preserved various tumor-resident cell types, including endothelial progenitor cells.Transcriptomic analysis indicated the reproducibility of the method, with the majority of gene expression patterns conserved in tumoroids compared to their matching tumor tissue.Therefore, this tumoroid allows for the evaluation of drug effects and invasiveness of renal cancer cells in a 3D context, offering a robust preclinical tool for drug screening and biomarker discovery [67].Zheng et al designed an abiotic/biotic nano-thylakoid system that can effectively produce oxygen within tumoroid formed by CT26 cells (a murine colon carcinoma cell line).Under 660 nm laser excitation, the nanothylakoid system provides intracellular oxygen production while also inhibiting anaerobic respiration within tumoroids [68].

Tumoroids with cancer-associated cells
Tumoroids can be prepared using cancer cells and CACs [69].Tumoroids formed by co-culture with CACs, such as stromal fibroblasts and endothelial cells, exhibit a TME that more closely resembles in vivo tumors [70].Previous studies have demonstrated that in breast tissue, myoepithelial cells and fibroblasts can influence the behavior of tumor cells.Myoepithelial cells often exhibit robust tumorsuppressive activity, while fibroblasts frequently promote tumor growth, invasion, and the release of ECM-degrading enzymes [71].Holliday et al's research describes the development of a physiologically relevant 3D co-culture system involving mixed breast tumoroids derived from either normal or tumor sources.The study illustrates how such models can be used to dissect the interactions that influence cell behavior, particularly the disruption of normal structures mediated by tumor-associated fibroblasts [70].
The tumor tissues can come from surgical resection or patient biopsy.Tumor tissues are either mechanically dissociated or enzymatically digested into single cells or clusters.These cells are then embedded in ECM and cultured in a medium supplemented with various growth factors and/or pathway inhibitors to generate organoids.Tumoroids effectively preserve the characteristics of primary tumors, enabling the study of the dynamics of cancer development and progression in individualized patients.
In comparison to conventional 2D cell lines and PDX models, tumoroids offer several advantages for clinical applications: (i) a high success rate in establishing tumoroid models, with variations in success rates depending on the tumor type, but an overall success rate of approximately 80%.(ii) A relatively short time frame for establishing tumoroid models, typically ranging from 1 week to 3 months, which can meet clinical application needs.(iii) Tumoroids can effectively preserve the genomic, transcriptomic, and histological features of the primary tumors.(iv) It can be continuously cultured for a lone time in vitro and establish large living cancer biobanks.(v) Tumoroids can be used for high-throughput drug screening to provide treatment guidance for cancer patients.(vi) Tumoroids can simulate tumor progression and metastasis, enabling the validation of the therapeutic effects of target molecules.
So far, a variety of tumoroid models have been successfully established, such as breast cancer [72], bladder cancer [73], colon cancer [74,75], gastric cancer [76], liver cancer [77], pancreatic cancer [78], ovarian cancer [79], lung cancer [80] and nasopharyngeal cancer [81].There have been some important adjustments in the recent organoid-related research bills.Two important adjustments were made in the Food and Drug Amendments of 2022H.R.7667, (1) a uniform revision of the original act from 'animal experiments' to 'non-clinical experiments.'This change in the act reduces the traditional misunderstanding that 'animal experimentation' is 'preclinical experimentation,' and a clear definition of "nonclinical experiments.Nonclinical experiments refer to in vitro experiments or non-human in vivo experiments conducted before or during the clinical trial phase of a drug.(2) Organ Chips and microphysiological systems were included in the bill as an independent non-clinical drug experimental evaluation system for the first time.Organ Chips and cell models, computer and animal models were considered essential research methods.
The introduction of this amended bill reflects the FDA's favorable stance towards new technologies in drug development and underscores the significance of organ chips and microphysiological systems as models for non-clinical experimental research; it is anticipated that this human resource system, with its high level of bionics and high-throughput innovation, will catalyze a revolutionary breakthrough in the early development and clinical transformation of drugs.As a result, the potential utilization of tumoroids to guide personalized therapy for cancer patients, particularly those with advanced disease, has garnered significant attention from scientists and doctors.
According to the in vitro drug screening results of tumoroids, we can anticipate how tumors will respond to various cancer drugs, enabling the development of personalized therapy for individual patients.Several research groups have, in fact, employed this system to assess cancer responses to targeted therapy, chemotherapy, and radiotherapy [74,75,82,83].Vlachogiannis et al demonstrated that the responses of colorectal and gastroesophageal tumoroids to anticancer agents closely mimic patients' clinical responses [82].Yao et al discovered that RCOs predicate chemoradiation responses are highly matched to those in patients, with an accuracy of 84.43%, sensitivity of 78.01%, and specificity of 91.97% [75].
However, the majority of published studies lack of treatment guidance.For example, in a study done by Chen et al tumoroids derived from breast cancer patients were used as a real-time platform to inform the treatment of breast cancer patients.Clinical case studies have shown that tumoroid models from patients have been developed as an effective platform for in vitro assessment of patient-specific drug sensitivity, allowing for personalized treatment decisions for late-stage breast cancer patients.All patients achieved significant improvement [84].While this data indicates the potential feasibility of utilizing tumoroids as a platform to guide personalized therapy for cancer patients, prospective clinical trial is also needed in the future to assess whether all patients can be benefited from this approach.
The main limitation of the current tumoroids is the lack of immune cells and stromal cells, thus, they cannot completely preserve the TME of the primary tumors, which may lead to inconsistencies between some tumoroids drug testing results and clinical treatment outcomes of the patients [85].Some studies aim to reconstitute the TME in tumoroids by adding CACs for co-culture [86].This approach demonstrated that Wnt from CAFs is required for the growth of the Wnt-non-producing subtype of human ductal adenocarcinoma [87].The reconstitution of immune TME in tumoroids also has been performed.This reconstitution model provides a means to assess the response of tumor cells to immune cell-mediated attack at the individual level [88].Another drawback of tumoroids is that long-term culture is challenging to achieve in this model.
According to the results of the study, the researchers found the hydrophilicity and hydrophobicity of the material directly impact the growth of tumoroids.In 2020, Yin et al developed a tumoroids model using Teflon-coated plates.This model significantly influenced the aggregated growth of tumoroids.The selection of Teflon as the base material significantly increased the likelihood of tumoroid formation, especially for applications in precise oncology drug concentration testing.Based on the analysis of 59 patients, the overall predictive accuracy for clinical outcomes in gastric cancer, colorectal cancer, and breast cancer was 93% [89].This modified tumoroids model not only enables the expansion of tumor cells but also preserves primary fibroblast and immune cells, which play important roles in tumor formation and physiological functions.Importantly, the tumoroids have an overall accuracy of 93% in predicting clinical outcomes in different tumors.In general, immune cells and stromal cells can only survive for a short duration under conventional 3D culture conditions.Consequently, only early-passage tumoroids can partially preserve the TME of the original tumors.For this reason, some research groups, including ours, use early passage organoids (passages 1-2) for drug tests [79,81,90].Despite some limitations, these studies increasingly advocate the use of tumoroid models for studying the TME.
Cell co-culturing could be complicated but necessary for tumoroids.Ideally, all tumor-related cells (i.e.CSCs, CTCs, stromal cells, immune cells, and metastasis-supporting cells) should be isolated from the patient's tumor tissues or blood to address the heterogeneity of cancers in each patient.Wu et al collected blood samples from patients with highly invasive pancreatic ductal adenocarcinoma and cultured the patients' CTC tumoroids were cultured on materials with a specific topographic surface.The proliferation rate of the tumoroids reached 87.8%.Staining results for EpCAM and CD45 indicated a substantial presence of CTCs within the tumoroids [91].Similar extraction and cultivation techniques have also been applied by Lin et al in head and neck cancer patients [92], and by Lee et al in small cell lung cancer patients [93].These studies have further utilized the subsequent tumoroids for drug testing, using the tumoroids as 'avatars' for the tumors inside the patients to predict the efficacy of various drug treatments for the patients' tumors.According to the research, these CTC tumoroids are heterogeneous, containing epithelial cells, stromal cells, and immune cells [91][92][93].
The heterogeneous composition of tumoroids can reveal cellular composition using histopathological image analysis or spatial transcriptomics that researchers can use this information to reconstruct a patient's unique TME and tumor tissues [94].There are two major approaches to building tumoroids using various cell suspensions: (1) cell assembly in suspension or on a surface, and (2) cell culture within a scaffold.The first method requires cells to produce ECM from scratch within the tumoroids; so the process will be time-consuming.It may take a few weeks to months because various cells may be seeded sequentially but not simultaneously.Generally, stromal cells are seeded first, organized, and deposited in ECM before seeding tumor cells.The second method is cell culture within a scaffold, which involves preparing artificial tumor niches such as ATMs for tumoroids in advance.Both are valid ways to build tumoroids.

Artificial tumor matrices (ATMs)
As mentioned earlier, ATMs are essential for tumoroid formation, providing mechanical support, maintaining morphology, and facilitating cell-matrix interactions.In this chapter, various types of ATMs are discussed, including hydrogels, porous scaffolds, and decellularized tissues.This paper defines ATMs as substances that can assist tumor formation, including artificial auxiliary materials, artificial structures, artificially treated tissues, and artificially stimulated secretion of factors.For example, ATMs constructed with gelatin and electrospinning or porous materials are added with tumor nutrients and growth factors, then the ATMs are like a magnet, which could attract specific cells [95,96].Different ATMs can be created to represent different tumor types and subtypes.For example, a typical metastatic breast TME involves high infiltrations of TAMs, TANs, and CAFs (in the stroma, at the periphery) [97].Adipocytes may also be included to represent obesity-related breast cancers [98].
The next-generation tumoroids will involve preseeding these cells in the tumor compartment and inoculating circulating immune cells in the blood compartment.The pre-made ATMs could significantly reduce the time required for tumoroid formation.For instance, when a late-stage cancer patient requires personalized drug screening, tumor cells can be isolated from the blood (e.g.CTCs and other tumor-associated cells) and cultured in these pre-made ATMs [99].This approach offers several advantages, such as time-saving without surgery of tissue biopsies, convenience for patients with cancer metastasis and drug resistance, easy preparation for multiple cancer models, and supporting dynamic modeling during different cancer courses.Nevertheless, some disadvantages of pre-made ATM could be existed, such as different immune system and cancer niche between individuals.In the future, by integrating techniques such as genome sequencing, spatial transcriptomics, and drug screening, tumoroids technology has the potential to overcome inconsistencies between in vitro and in vivo models in clinical responses.
The ECM is a non-cellular 3D macromolecular network composed of collagens, proteoglycans (PGs)/glycosaminoglycans (GAGs), elastin, fibronectin, laminins, and several other glycoproteins [100,101].In tumors, the ECM interacts with cell adhesion receptors to affect cell behavior.In the past, the ECM was considered an inert cellular scaffold that only provided structure to cells.But in recent years, it has been found that more cell behavior, biochemical and biophysical functions are affected by ECM, and ECM can be used as a storage area and binding site for bioactive molecules.In tumor-related research, tumor-associated ECM can promote tumor cell growth, invasion, metastasis, and angiogenesis, reducing cell death and the diffusion of therapeutic drugs [101].Therefore, by interacting these ECMs with cell receptors and regulating the biomimetic microenvironment, these ATMs attract tumor cells like magnets to form tumoroids.Additionally, the use of topologically structured surfaces such as micropatterns and nanostructures can also promote the formation of tumoroids.Tumoroids can also be formed by adding stromal cells and immune cells to microenvironments containing topologically structured surfaces.
A variety of materials are available and facilitating for the generating of tumoroids, including natural and synthetic ATM in a form of hydrogel and scaffold, decellularized ECM (dECM), microscale molds, nanostructured surfaces, and bioprinting, through the cell-ECM interaction.In addition, TuChip is an integrated system where the dynamic flow is incorporated.In the TuChip system, tumoroids are cultured in a small chamber, where a more biomimetic TME, such as hypoxia and medium flow, is produced.Tumoroids under both static or dynamic culture conditions show their potential to be tumor avatars for cancer research and drug screening (figure 3).

Hydrogels
The success of organoid culture relies on various physical and chemical characteristics of the culture microenvironment.Hydrogels, due to its high biocompatibility, biodegradability, and function to simulate in vivo extracellular matrix, it is been used to construct a myriad of organoids.Due to the crosslinking network, hydrogels can swell and hold water, and the amount of water absorption is closely related to the degree of cross-linking.The higher the degree of crosslinking, the lower the water absorption.This property is much like a kind of soft tissue.The water content in hydrogels can range from a few percent to 99%.The aggregated state of the gel is intermediate between the solid and liquid [102].Ruiter et al revealed that the physical stiffness of hydrogel would influence the formation of kidney organoids, changing EMT.In detail, stiff hydrogel encapsulation caused certain renal cell types to lose EMT.At the same time, the soft one prompted major renal segments and primary cilia formation, increased apical proximal tubule polarization, and reduced fibrosis or EMT, based on which a better-engineered hydrogel with proper mechanics and dynamics characteristics is essential for organoid culture, making us closer to a more rational preclinical disease modeling design [103].Hydrogels can encapsulate one or more types of cells (e.g.endothelial cells, fibroblasts, and macrophages) as well as various proteins and growth factors, which allow hydrogels to reproduce intratumorally complexity and heterogeneity to a certain extent.According to its origins, hydrogels can be divided into decellularized tissue-derived, natural, and synthetic polymer-based hydrogels.Natural hydrogels are mainly composed of polysaccharides and protein synthesis.Synthetic hydrogels, on the other hand, are hydrogels formed through physical or chemical crosslinking of synthetically hydrophilic polymers.
Various natural hydrogels, such as proteins (gelatin and collagens) and polysaccharides (alginate, chitosan, and hyaluronic acid) have been applied in tumoroids [104].Collagen, a crucial ECM component, stands out due to its biodegradability and strong biocompatibility [105].On the other hand, collagen is the most abundant protein in the human body and a crucial component of connective tissues [106,107].To date, approximately 30 types of collagens have been identified, each with specific functions, for instance, type I collagen providing strength in the skin and type II collagen providing elasticity in articular cartilage.Collagens also constitute a major component of the ECM in tumors and cancers [36,45]; hence, collagens have been used as ATMs for tumoroid preparation.Caruso et al developed a new protocol for growing mammary organoids using a collagen I-based gel.The culture involved the periodic addition of FGF2 and EGF, resulting in highly branched 3D mammary organoids [108].
Hyaluronic acid (HA) is an essential component of the natural ECM and a polymer widely used to construct 3D tumor models.HA is a starting material for hydrogels with ideal morphology, stiffness, and bioactivity [109].One study constructed a hydrogel-derived PCa model using chemically modified HA carrying acrylate groups (HA-AC) or HA reactive thiols (HA-SH).The model was used for the in vitro evaluation of doxorubicin (Dox) loaded polymer nanoparticles (Dox-NPs).The model's design involved evaluating Dox-loaded nanoparticles for simulating the TME is versatile and not limited to a single usage.For instance, dECM can not only be directly used to simulate TME but also employed in the construction of hydrogels or scaffolds before being used for TME simulation.Microfluidic devices provide an integrated system where the dynamic culture is the main character of so-called tumoroids-on-a-chip.Tumoroids under static and dynamic cultures have shown their potential to be tumor avatars for cancer modeling and drug screening.Herein, this article proposes the concept of ATMs as a magnet.When ATMs (such as scaffolds, electrospun structures) are added with CAFs, TAMs, and tumor associate peptides, so that ATMs can attract specific cells (such as CSC, CTC) like a magnet.
(Dox-NPs) and utilized for engineering tumor models.After 7 d, LNCaP PCa cells cultured in the hydrogel formed tumor-like multicellular aggregates with an average diameter of 50 µm.The results showed that LNCaP PCa cells cultured in HA hydrogel were more resistant to the treatment of Dox and Dox-NPs (figure 4(A)) [110].In a different research effort focused on creating in vitro tumor models that closely mirror physiological conditions, Hao et al developed a biomimetic hydrogel.They achieved this by synthesizing and characterizing a hydrogel using thiolated HA (HA-SH) and an acrylated copolymer that contained numerous copies of a cell adhesive peptide (PolyRGD-AC).When HA-PolyRGD gels encapsulating LNCaP PCa cells were introduced as dispersed single cells, they amalgamated to form MCTs  C for 2 h.In 2D, free DOX (50 µg ml −1 ) was mainly located in the nucleus, while DOX-NPs (67 mg ml −1 , the corresponding DOX concentration was 50 µg ml −1 ) were mainly located in the cytoplasm.In 3D, both DOX and DOX NPs were in the cytoplasm.Reprinted from [110], Copyright (2013), with permission from Elsevier.(B) Typical confocal images of LNCaP cells at low (10×) and high (40×) magnification with immunofluorescence after 28 d of three-dimensional culture.The nuclei, ITGβ1, F-actin, and E-CAD were stained in blue, green, red, and magenta, respectively.Reprinted with permission from [111].Copyright (2016) American Chemical Society.(C) E-CAD and ITG were expressed in LNCaP cells cultured with 3D hydrogel.(a)-(i) Confocal sections of representative cells embedded in the sodium alginate/matrix gel complex at different time points (1, 4, and 7 d).The cells were stained with F-actin, and the nuclei were re-stained with propidium iodide.The cells were round in 100%A gel and did not express their spreading ability.It was elongated in 75%:25%A:M gel.They ended up showing starlike morphology and invading feet in the 50%:50%A:M gel (red arrow).(j)-(l) The shape of the nuclei isolated from the c-to-k plates indicated that the cells expressed nuclear fragmentation in the presence of stromal gel, which is characteristic of malignancy.Reproduced from [114].CC BY 4.0.(D) Fluorescence images show nuclei stained with DAPI.Reprinted from [116], Copyright (2016), with permission from Elsevier.(E) The phenotypes and biological characteristics of cells in vivo can be replicated by 3D culture in PEG fibrin hydrogels.On day 30 of culture, A549 hydrogel was embedded in paraffin.Sections were stained with IHC for cytokeratin 7 and cytokeratin 20.The same procedure was applied to a sample of lung adenocarcinoma.Display representative picture (40× magnification).Reprinted from [117], Copyright (2016), with permission from Elsevier.
by the fourth day.The RGD signal present in the HA matrix elevated cellular metabolism, facilitated the growth of larger tumoroids, and heightened the expression of E-cadherin and integrins [111].MCTS were formed after 4 d of LNCaP culture, and the average diameter of spheroids reached 95 µm on day 28.Unlike the PolyRDG control gel, LNCaP PCa cells encapsulated with HA-polyRGD gel formed a multicellular tumor (figure 4(B)).The multivalence of RGD peptides in the HA matrix increased cellular metabolism, promoted the development of larger carcinomas, and enhanced the expression of E-CAD and integrin [111].HA can also form ECM-derived hydrogels.Forsythe et al used a thiolated HA, thiolated gelatin, and polyethylene glycol (PEG) diacrylate-based hydrogel system (ESI-BIO, Alameda, CA) to create a three-dimensional micro tumor platform, which can support cell growth for a long time and can also be used for high-throughput drug screening [112].
Alginate is another polysaccharide hydrogel with the same physicochemical properties as HA.Alginate is a natural polymer extracted from brown algae.Alginate has been used for organoid formation due to its advantages of rapid gelation, high biocompatibility, bioinert and low cost [113].In a study, human invasive breast cancer cells (MDA-MB-231 cells) were cultured in a specific gel (50% alginate and 50% Matrigel) to obtain a structurally stable and biologically active substrate.In this way, the more similar microenvironment to the internal environment can be regulated.The results showed that the gel allows MDA-MB-231 cells to exhibit typical invasive behavior in vivo, and it may provide a new method for the study and drug detection of invasive breast cancer (figure 4(C)) [114].Fang et al used microfluidic technology to encapsulate tumoroids by passing non-adhesive alginate droplets into the lumen of fine tubes with mouse mammary tumor cells [113].High-throughput generation of tumoroids in alginate microbeads was successfully achieved in vitro and showed high similarity to the original fresh tumors in cell phenotype and lineages [113].De et al used alginate-based 3D microcapsules to provide spatial demarcation with microscaffolds that can be used to mimic the TME [115].Carbon-dot-based nanosensors were designed in microscaffolds to monitor pH changes in the TME in real time, so it can be used to form and monitor tumoroids in various cells [115].
Gelatin is a product of collagen denaturation and belongs to the category of protein macromolecules.It has similar properties to protein macromolecules, but its physicochemical properties are unique due to the particularity of its molecular structure.Gelatin is widely used to construct three-dimensional tumor models because it has no immunogenicity but has the same composition and biological properties as collagen.Nitish et al used the GelMA hydrogel and the two-step lithography technology for the microengineering design of the 3D breast tumor model.Breast cancer cell lines (MDA-MB-231 and MCF7 cells) and non-tumorigenic mammary epithelial cells (MCF10A) were embedded in a hydrogel, respectively.The results showed that MDA-MB-231 cells exhibited extensive migratory behavior and invaded the surrounding matrix, whereas MCF7 or MCF10a cells formed clusters and were confined within the round tumor area (figure 4(D)).The hydrogel shows good structure and adjustable stiffness and has a good application prospect in developing three-dimensional tumor models [116].
PEG-fibrinogen is another hydrogel mainly made of proteins.In a study, Francesca et al cultured A549, A549-HUVECs, A549-MRC5, and A549-HUVECs-MRC5 on 3D PEG-fibrin hydrogel, respectively.After 5 weeks of co-culture, the size of A549-HUVECs clusters can reach 0.3 mm-diameter, while the size of A549-HUVECs-MRC5 clusters can reach 1.0 mmdiameter.The results show that PEG fibrin hydrogel can mimic the growth and behavior of human lung adenocarcinoma and its interaction with interstitial elements, which has some potential advantages over other 3D systems (figure 4(E)) [117].
Furthermore, intestinal crypts or tumor tissues containing stem cells are isolated from mice and cultured in Matrigel, which can be used to mimic the microenvironment of stem cell growth in the gut and constructed as organoids/tumoroids that can be further used for drug analysis [118].Matrigel can be mixed with polysaccharides such as alginate to adjust its mechanical properties.The results show that brain organoids can be grown in Matrigel and alginate with the characteristic formation of neuroepithelial buds [119].An engineered composite hydrogel consisting of gelatin and alginate components was shown to be a bioink for cell culture, and this composite hydrogel can be used to construct 3D bioprinted in vitro breast tumor models [120].Alginate and gelatin concentrations can modulate the mechanical and cell adhesion properties of hydrogels, thereby affecting the TME [120].Gelatin and alginate composite hydrogels were applied to MDA-MB-231 breast cancer cells, and the results showed the dependence of the gel formulation on the rate and frequency of self-assembly into MCTS [120].Besides, commercially available hydrogel substrates (e.g.Matrigel) are primarily derived from animal tissue.Notably, these animal-derived hydrogel matrices are not amenable to controlled modification and present a risk of transfer of immunogens and pathogens, impairing their clinical application.However, these limitations can be overcome by using synthetic hydrogel substrates based on polymers such as PEG, nano-cellulose, alginate, hyaluronan, and polylactic acid-glycolic acid copolymers.In summary, hydrogels can further promote the formation of tumoroids by cells through mechanisms such as 3D culture, special functional groups, high biocompatibility, biological inertness, mechanical properties, and nutrient supply in the colloid.

Scaffolds
Porous scaffolds have spiral structures with controllable pore size, allowing cell migration, oxygen, and nutrients inlet to support cell growth and timely remove the waste materials.Accumulating publications have demonstrated that porous scaffolds can provide a favorable environment for tumor cell growth, division, and migration [121].3D tumor models using porous scaffolds are considered efficient for studying complex cancer mechanisms and therapy responses.They support in vitro cell culture with stromal-like ECM, including vascular elements, mimicking the in vivo biophysical microenvironment, unlike traditional 2D culture.Materials commonly utilized to construct the scaffolds include natural polymers (such as gelatin, collagen, and chitosan-alginate), synthetic polymers (such as poly (ε-caprolactone) (PCL) and polylactic acids (PLAs), colloidal crystal poly), and bioactive ceramics (hydroxyapatite and silica) [122,123].These materials were further used to fabricate porous scaffolds via various procedures, such as emulsion [124], electrospinning [125][126][127], solvent-casting particleleaching [127], 3D printing [128], stereolithography [129] and gas foaming [130].
Wang et al established a 3D endothelialized hepatic tumor microtissue model of multicellular aggregates of HUVECs and human hepatocellular carcinoma cells in poly(lactic-co-glycolic acid) (PLGA)based porous microspheres (figure 5(A)) [124].The PLGA microspheres with a uniform particle size of 395 µm were produced via microfluidics-assisted fabrication.Specifically, the microspheres were made through the W/O ratio of 1:2.4,gelatin concentration of 7.5% (w/v), PLGA concentration of 2% (w/v), and flow rates of the continuous phase and dispersion phase set at 0.05 and 2 ml min −1 , respectively.After removing aqueous gelatin droplets, porous structures would be created.Those microspheres possessed porous structures with interconnecting windows in the pore size distribution range of 10-60 µm, which would be substantially helpful for the entry of cells into microspheres' interiors.Compared with the traditional 2D culture, cells within the microspheres show higher half-maximal inhibitory concentration values for DOX, cisplatin, and other anticancer drugs.The IC 50 values for 2D-and 3D-cultured models were 15.94 ± 0.98 and 27.80 ± 7.53 µg ml −1 after 24 h of incubation and 3.07 ± 0.16 and 7.87 ± 2.16 µg ml −1 after 48 h incubation, respectively.The high density of the cells packed in the microspheres-based tumor models may contribute to the differences in the diffusion, resulting in different eventual availability of the drugs.Also, their tumor model is feasible to coculture with other types of cells with great potential for drug screening applications (figure 5(A)) [124].
Through electrospinning, Prieto et al built a 3D culture system that can mimic metastatic ECM to investigate the behavior of breast CSCs in vitro [125].In their study, polycaprolactone (PCL)-based nanofibers were fabricated in different forms, such as aligned, porous, and collagen-coated.To reproduce the tumor ECM assemble, scaffolds with aligned fibers were created by raising the speed of the rotating mandrel from 225 to 850 rpm.The average fiber radius was fixed in the nanometer range and was similar to the radius of collagen fibers in the TME.The MDA-MB-231 breast cancer cells cultured in the model perform, exhibit a cytoskeletal phenotype similar to invasive cancer cells.At the same time, the upregulation of mesenchymal markers, including Nestin, Oct-3/4, Sox2, CD49f, and ALDH1, suggests that cancer cells have experienced epithelialmesenchymal transition.Their study provides a culture platform for maintaining CSC propagation by mimicking tumor ECM [125].
To investigate mechanotransduction in tumor biology, Mikos et al utilized coaxial electrospinning to establish in vitro tumor models with tunable mechanical properties (figure 5(B)) [126].Different ratios (20%, 50%, and 80%) of gelatin and poly (ε-caprolactone) based coaxial electrospun meshes were fabricated and cultured with osteosarcoma cells.Their study utilized PCL as the core polymer and gelatin as the shell polymer.The PCL scaffolds had the lowest theoretical pore radii of 8.7 ± 2.1 µm, while coaxial fibers consisting of 50% gelatin had pores with mean radii of 28.0 ± 7.5 µm.The cells exhibit increased nuclear localization of Hippo pathway effectors, YAP and TAZ, while matrix stiffness was reduced.Moreover, these 3D models better reproduce the clinically observed results compared to 2D models, with downregulation of the IGF-1R/mTOR axis, which leads to increased resistance to combination chemotherapy and IGF-1R/mTOR targeted agents.Their results reveal that osteosarcoma cells sense an environment influenced by the substrate's stiffness and architecture, which could afford a framework to design the specialized tumor niches for relative applications (figure 5(B)) [126].
Fontoura et al investigate the influence of 2D and different 3D tumor cell culture models on cell behavior and drug responses.They fabricated the polyhydroxybutyrate (PHB) based scaffolds via solventcasting particle-leaching (SCPL) or electrospinning technique to generate two types of membranes [127].The SCPL membranes were formed in random shapes with pore sizes ranging from 0.56 to 48.36 µm, while electrospun membrane pores varied between 0.66-12.07µm.The electrospun fibers had diameters with a mean of 1.1 ± 0.47 µm.They also used conventional 2D tissue culture plates and commercial 3D culture gel derived from EHS tumor to compare with PHB-based scaffolds.Compared with 2D models, B16 F10 murine melanoma and 4T1 murine breast cancer cells cultured on all 3D models exhibited cell morphology and gene expressions, such as lymphoid enhancer-binding factor 1 (LEF1) and VEGF, more similar to in vivo models.In addition, increased resistance to dacarbazine and cisplatin was observed in these 3D models.The resemblance between the two synthetic systems and the commercial EHS gel reveals the potential application of PHB-based membranes as cost-effective alternatives in drug screening for 3D tumor culture [127].In summary, as porous scaffolds utilize various preparation methods, they provide an excellent substrate for cell attachment and the construction of an intracellular microenvironment.Therefore, they serve as an excellent structural basis for the construction of tumoroids.

Decellularized ECM
dECM made from tissue can preserve the primary tumor ECM's structure, composition, and biomechanical properties [131].Collagen, PG, laminin, elastin, and growth factors originally from the dECM can be retained [131].Decellularization procedures have been developed to degrade the cellular substance and the ECM [132,133].These techniques can be classified into physical (e.g.sonication, freezethawing), enzymatic (e.g.trypsin, peptidases, or nucleases), and chemical manner (e.g.acid/bases, hypertonic solutions, and detergents).These methods are seldom used alone and commonly conjugated with several treatments to confirm that no genetic substance remains.Decellularized tissues can be further digested into solutions containing macromolecules such as saccharides and proteins.In tumor modeling, decellularization techniques provide the in vitro scaffold-based models, which are construction analyzable and biologically relevant.Matrix stiffness is key role in regulating tumor activity, impacting extracellular mechanical property.dECM has following merits: (1) by removing cellular components, such as antigens and other proteins, immune rejection can be avoided; (2) retaining original binding points and extracellular skeleton, promoting cell adhesion and spatial distribution, activating signaling pathway; (3) convenient for in vitro fabricating organoid by providing a mimic environment [131,134].
In 2020, an increasing number of impressive works have been published, such as key methods of physicochemical modification for isolation of dECM, and the processing of dECM from murine breast, lung, muscle, and tumorous murine liver into bioinstructive recellularized scaffolds, soft hydrogels and for 3D bioprinting [132].The dECM was used to reconstruct tumor models such as lung, breast, colorectal, and Hepatocarcinoma [132].This research showcases the capacity of decellularized-based in vitro biomaterials to efficiently represent the essential communications of tumor cells and surrounding ECM in drug resistance [135][136][137], gene expression [138], and metastasis [137,139,140].
Ferreira et al integrated the microfibrillar dECM fragments from porcine mammary-derived adipose and connective tissues immediately adjacent to the mammary gland, MDA-MB-231 human breast cancer cell line, and human primary dermal fibroblasts cells in spheroids assembly, which generates the breast cancer model with tunable size and reproducible morphology (figure 6(A)) [135].The decellularization was conducted using Triton X-100 (1% v/v) combined with ammonium hydroxide (0.1% w/v).To generate 3D dECM microtumors with welldefined morphological, they applied the liquid overlay technique with some novel modifications, including sequential seeding/centrifugation of dECM and varied cell types in ultra-low adhesion substrates.Specifically, before cell seeding, freeze-dried dECM microfibrillar fragments were resuspended in a cell culture medium, seeded on ultra-low adhesion plates, and centrifuged.Then, cancer-stromal cocultured cells were seeded on a pelleted dECM microfibrillar and centrifuged.The resulting cell-stromal models were cultured for 14 d.Compared to conventional spheroids, this dECM tumor-stroma spheroid displays necrotic core formation, secretion of crucial biomarkers, and more resistance to various chemotherapeutics.Importantly, including both fibroblasts and dECM led to apparent changes in their exometabolomic profiles compared to 2D breast cancer.Cells actively consumed pyruvate, glucose, fructose, and several amino acids, while excreting format, shortchain fatty acids, alanine, and lactate.This profile indicated that this dECM system exhibits intense glycolytic activity, a hallmark of the native breast cancer microenvironment [135].
Di Blasio et al co-cultured the decellularized dermis with keratinocytes, fibroblasts, melanoma cells, and immune cells, reconstructing the TME [138].A de-epidermized human dermis was accomplished by incubating the dermis derived from donors who underwent surgery for one month in PBS containing gentamicin and antibiotic at 37 • C.Then, to remove all living cells in the dermis, the deepidermized dermis is exposed to additional freezing/thawing cycles and frozen for further use.Their culturing system enables the real-time study of hostmalignant cell interactions inside a multicellular tissue construction.They observed that the presence of a tumor in the system induces the transformation of conventional dendritic cells (cDC)2 s into CD14 + dendritic cells (DCs).The normal immunostimulatory DCs conversed into DCs with an impaired ability to stimulate T-cells.Their works can further identify the candidate genes and molecules relating to immune escape processes, resulting in the (iii) Different genotoxic treatments were investigated to determine the most effective method in suppressing the GBMs-on-chips (left and middle).After sorting the treatment candidates, the combination, CIS + KU + O 6 BG with radiation, was effective on the GBM-28-on-a-chip compared to GBM-37-on-a-chip (right).The GBM ID 28 or 37 indicated that the patient before or after received anti-cancer treatment, respectively.Abbreviations: O 6 -benzylguanine (O 6 BG), methoxyamine (MX), KU60019 (KU), temozolomide (TMZ), concurrent chemoradiation (CCRT), cisplatin (CIS).* * * P < 0.001, * * * * P < 0.0001 and n.s., not significant; one-way ANOVA and Bonferroni post-hoc tests.Reproduced from [141], with permission from Springer Nature.design of innovative therapeutic targeted strategies for overcoming the melanoma microenvironment [138].
Tian et al investigated organ-specific metastases by culturing colorectal cancer (CRC) cells on lung, and liver decellularized scaffolds [139].Those scaffolds were prepared by perfusion of decellularization reagents into the portal vein (liver-based scaffold) or inferior vena cava (lung-based scaffold).Specifically, the vasculature was perfused with basal medium, sodium deoxycholate containing phospholipase, and sodium chloride until the perfusate was negative for proteins as assessed by optical density.Then, organs were washed with basal medium and snap-frozen.Those frozen decellularized organs were pulverized into a fine powder and stored at −80 • C for further application.The retaining ECM components and bound signaling molecules on the decellularized scaffolds allow the spontaneous organization of 3D cell colonies that histologically, molecularly, and phenotypically mimic in vivo metastases.Their data demonstrate that the therapeutic response of metastatic CRC cell lines to standard treatment regimens (i.e.chemotherapy and radiotherapy) are affected by their in vitro acellular microenvironment; The cells cultured on lung dECMs are commonly more sensitive to treatment than that on liver dECMs, which are compatible with the clinical observation that liver metastasis is the primary reason for morbidity and mortality in metastatic CRC patients.Their system can provide the platform for high-throughput screening assays to identify the treatment to address organ-specific cancer metastases [139].
dECM techniques can further combine with bioprinting to reconstitute the ex vivo models simulating pathological properties and the complex environment of the native TME [141,142].Yi et al bioprinted the GBM tumors consisting of patientderived tumor cells, vascular endothelial cells, and brain tissue-based dECM (figure 6(B)) [141].They developed a bioink solution constituted of brain-dECM.Specifically, the porcine brain was decellularized via the sequential processing of chemical and enzymatic agents.Through adjusting the decellularization parameters, they established the suitable conditions for the effective removal of the doublestranded DNA content (nearly 99% reduction) while reducing the damage to the ECM components, such as GAG and HA.The dECM can solubilize as a viscous solution, which performs the thermosensitive sol-gel transition properties.The compartmentalized cancer-stroma concentric-ring structure fabricating in the model recapitulates the structural, biochemical, and biophysical characteristics of the native tumors.This GBM-on-chip analog reproduces clinically observed patient-specific resistances to treatment after concurrent chemoradiation using temozolomide.Also, the GBMs-on-chips displayed patient-specific sensitivity against potential drug combinations, which can be used to determine the effective therapies associated with superior tumor killing [141].In this section, the decellularized technology regulates the structure, composition, mechanical properties, and immune rejection of ECM.Therefore, constructing organoids by decellularized tissue can more easily simulate the cellular microenvironment in vitro.

Bioengineered tools 4.1. Microscale molds
Microscale molds (i.e.10-900 µm) is one of the most used methods to form cell aggregations.Using microscale molds to guide multicellular self-assembly is an simply but powerful way that can generate cell spheres with well-controlled size [143].By changing the size and property of the molds, cell shape and proliferation can be regulated within it.Using force aggregation (i.e.centrifugation and magnetic field) or sedimentation (i.e.gravity), tumoroids can be formed rapidly.These methods can lead to the formation of grape-like and mass-packed spheroids dependent on the degree of aggregation.
The aggregation method has been applied to generate homogenous cell spheroids such as AggreWell TM (STEMCELL Technology, USA) (figure 7(A)) and honeycomb microwells [144][145][146].This method uses force to concentrate the cell suspension into a high density of cells, which can facilitate cell aggregation.This method provides a simple procedure to obtain large and uniform cell spheroids.Also, the main advantage of this method is that the size of the cell spheroids can be adjusted by controlling the cell seeding density [144].Razian et al have established a system that can generate a large number of uniform spheres with multiple cell lines from different sources, including HT29 colon cancer cells, LNCaP PCa cells, and TE6 esophageal cancer cells (figure 7(A)) [145].The AggreWell TM , equipped with densely packed microwells shaped like pyramids, facilitates the exact creation of tumoroids from a suspension of cells through sedimentation.This tool ensures consistent development of tumoroids in different sizes and compositions [145,147].
Lee et al describe a method for forming 3D MCTs using concave microwell arrays with a cellloss-free platform.They designed the microwell array to improve the unbalanced cell-cell interaction in the initial aggregation process of the cells and the low-profile structure to minimize the space between microwells to prevent false trapping of cells (figure 7(B)).They further proceeded with the formation of MCTS from MRC-5 cells to evaluate the response to anticancer drugs, paclitaxel, and gemcitabine [148].To enable multiplexed imaging and analysis of spheroids, Monjaret et al used a variety of cancer cell lines to grow in 3D on a 96well plate with a micropattern.Each microwell could form spheroids automatically.The spheroid made by micropattern could be used in hydrogels (agarose and Matrigel) to enhance epithelial or stem cell characteristics or perform 3D invasion assays after the tumoroids are formed (figure 7(C)) [149].Mori et al developed a micropattern chip with homogeneous spheroids and cylindroids for forming liver tumoroids.The expression of liver functions (protein secretion and ammonia removal) was more significant in the spheroids and cylindroids than in the monolayer culture, and this expression was maintained for at least 2 weeks of culture (figure 7(D)) [150].They used collagen to create a cell adhesion area for liver cancer cell culture.Using the micropatterning techniques, ECM and artificial materials can be further utilized to delimit cell adhesion/nonadhesion areas, such as collagen, to design cell adhesion areas and PEG to create cell non-adhesion areas [150].Wu et al present a physiologically inspired design allowing microfluidic self-assembly of spheroids, formation of uniform spheroid arrays, and characterizations of spheroid dynamics all in one platform.This microfluidic device is based on the hydrodynamic trapping of cancer cells in controlled geometries, and the formation of spheroids is enhanced by maintaining compact groups of the trapped cells due to continuous perfusion [151].
Low cell adhesion or low-fouling surfaces can also facilitate tumoroid formation [152].Using lowadherent or hydrophilic treated cell culture plates has also been implemented.In this method, plates are treated further with agents such as covalently bound neutral hydrophilic hydrogels that inhibit cell attachment, protein absorption, and enzyme activation.HepG2 spheroids were cultivated by the hanging drop method on a nonadherent surface with initial cell densities of 50, 100, and 500 cells/well (figure 7(E)) [153].This treatment causes the cells to aggregate and form spheroids [154].Conventional bacterial culture dishes and certain ELISA 96-well plates are made of non-stick plastic and suitable for producing multicellular spheroids.Alternatively, culture ware can be made non-adhesive for cells by coating with agarose thin films, hydrophobic polymers, including poly(2-hydroxyethl methacrylate) (PolyHEMA), or poly-N-p-vinylbenzyl-Dlactonamide (PVLA).Culture parameters such as cell type, seeding density, medium composition, and static or stirred growth determine the efficiency of cell aggregation and the uniformity of the size and shape of the sphere.The cell spheroids generated by this method are usually difficult to control the size distribution of the spheroids [143].
In this summary, micropatterns and micropores, through the regulation of the surface structure, can facilitate the formation of cell spheroids, providing a fast and high-throughput method for constructing organoid tissues, especially for the formation of tumoroids.

Nanostructured surfaces
Manipulating cell expression and maintaining the pro-tumor phenotype in vitro culture is also a challenge to overcome.This section will introduce methods of forming tumoroids using nanostructures (i.e.10-900 nm).These nanostructures create a shape and space for cancer cell growth in vitro and induce cancer cells to form tumoroids through the influence of material properties.The ECM features various nanostructures such as fibers, filaments, nanopores, and ridges that can be simulated by topographic and 3D substrates for cell and tissue cultures in an environment closer to in vivo conditions.Several methods are employed to promote the formation of tumoroids on nanostructures, including the use of nanoparticles, nanoimprints, nanofiber, nano-scaffold, and nanostructure culture plates.Nanoimprinting techniques have advanced to allow topographical and even 3D substrates to be built that are critical for ex vivo cell and tissue culturing [156].Many researchers used nanoimprinting techniques to study 3D cell formation.More reviews focused on the effects of surface nanotopography and biointerfacial interaction, which is the interface between the cell and other fabricated material, on cell behavior regarding motility, alignment, adhesion, migration, differentiation, proliferation, and nerve regeneration [156].Yoshii et al developed a 3D culture system with inorganic nanoscale scaffolding using nanoimprinting technology (nano-culture plates).They used colon adenocarcinoma Colon-26 (mouse) and HT-29 (human) cells to form tumor cell spheroid and reproduced the characteristics of tumor cell growth in vivo (figure 8(A)) [157].According to the research results, reducing physical contact between cells and matrix will promote spontaneous tumor cell migration, cell adhesion, and multicellular 3D sphere formation while maintaining cell proliferation and viability.The multicellular spheroid structure formed by nanoimprinting is very similar to the hypoxic core area of a tumor growing in vivo [157].NCP is the product of using nanoimprinting technology.NCP is a cell/tissue culture plate with patterned nano-scale grids on the plate base [156].According to Arai et al research, this large field of NCP will limit the spread of cells on the substrate and allows tumor cells to migrate more than a monolayer cell culture system (figure 8(B)) [158].
Nanofibers are another common type of highly biocompatible nanostructure.Nanofibers have many advantages in cancer treatment, such as the ability to obtain fibers with diameters ranging from nanometers to sub-micrometers, surface modification, alignment variation, and drug encapsulation.Electrospun nanofibers provide a unique opportunity to build cell environments that mimic the TME in vivo (figure 8(C)) [159][160][161].Sims-Mourtada et al sought to characterize the chemoresistance and stem-like properties of breast cancer cell lines grown on nanofiber scaffolds.Cells cultured on three-dimensional templates of electrospun poly(εcaprolactone)-chitosan nanofibers showed increases in mammary stem cell markers and sphere-forming ability compared with cells cultured on polystyrene culture dishes [162].3D nanostructures are also another research hotspot.The 3D structure can better simulate the growth of cells in the body.Katti et al used PCL/nanoclay scaffolds seeded with a sequential culture of hMSCs followed by human PCa cells to form 3D tumoroids successfully [163].PCL/HAPclay scaffolds have over 86% porosity with pore sizes ranging from 100 mm to 300 mm, which is sufficient for cell infiltration.This nanocomposite system can be used as a test platform for studying cancer metastasis and the efficacy of anticancer drugs by using this kind of polymersome delivery method.The new continuous cell culture system in the three-dimensional in vitro bone model can provide a unique bone simulation environment.The PCL/HAPclay nano-scaffold system is used for the continuous culture of hMSCs and HPCCs, which can offer a new model system for studying the interaction between PCa cells and the bone microenvironment [163].
A new family of nanostructured surfaces composed of various types of particles known as colloidal self-assembled patterns (cSAPs) was developed by Wang et al and has been continuously used for manipulating the behavior of different mammalian cells and bacteria [164][165][166][167][168][169][170][171][172].Particles, including silica (SiO 2 ), polymers, and hydrogels, are easily fabricated and modified for various applications.For the  [160].CC BY-NC 3.0.Reproduced from [161].CC BY 4.0.(D) Fabrication of monolayer binary colloidal crystals (BCC) structure.Reprinted with permission from [170].Copyright (2019) American Chemical Society.BCCs drive capturing and enriching of CTCs from cancer patients into tumoroids.Reproduced from [93].CC BY 4.0.Reprinted from [157], Copyright (2011), with permission from Elsevier.first time, Wang et al tactfully assembled particles to form a cell culture substrate.cSAPs have large surface physicochemical signals that can control cell morphology from spherical to spread out.Using different particle combinations, cSAPs surfaces can be manufactured in different surface topographies, roughness, chemistry, and rigidity.They may direct cell behavior by varying surface properties, such as cell motility, cell differentiation, and even cell reprogramming [166].One recent study showed that cSAPs could modulate cell adhesion of A549 cells and then alter drug response indicating that tumor ECM is a potent modulator for chemotherapy [173].Interestingly, the cSAPs also showed their potential to capture patients' CTCs to self-assemble and form tumoroids (figure 8(D)) [91][92][93]170].It is hypothesized that cSAPs could display complex tumor ECM properties; therefore, CTCs can undergo the epithelialmesenchymal-transition (EMT) process and then adhere to the cSAPs.According to these results, cSAPs have shown their potential to generate CTC-derived tumoroids for personalized drug screening.In addition, 'NanoVelcro' developed by Tseng et al can also be used to capture CTCs.NanoVelcro uses the concept of cell-affinity substrates to efficiently fix and acquire CTCs using nanostructured substrates, similar to Velcro [173,174].
Nanostructured surfaces affect the interaction between the surface nanotopography and the biological interface.The transition between nanostructure penetration and cell attachment can further regulate and stimulate the cytoskeleton and nucleus, affecting cell structure, genetic expression, cell reprogramming, and cell fate changes.Due to the adjustability of the nanostructured surface's height, it is a powerful tool for constructing tumoroids.

2D/3D bioprinting
Bioprinting methods allow the creation of 3D structures with precise spatial arrangements of ECM materials, cells, and various bioactive factors in vitro [174].Therefore, 3D bioprinting is one of the effective ways to purchase and construct tumoroids in vitro [142].Meng et al demonstrate the precise positioning of tumor cells, stromal cells, and infused vascular cells based on their physiological functions using 3D bioprinting.3D bioprinting empowered them to fabricate stimuli-responsive capsules incorporating growth factors, facilitating controlled spatial and temporal dispersion of chemical signals.These models were utilized in preclinical screening to target immunotoxins and assess their anticancer effectiveness [175].Reynolds et al presented an innovative microporous structured matrix designed for embedded bioprinting.This matrix was tailored by incorporating sacrificial microparticles made of gelatin-chitosan complexes into a prepolymer collagen solution, affecting its rheological properties, printing behavior, and porosity.To showcase its applicability, they utilized this approach to generate a 3D tumoroids by embedding mouse melanoma cell ink within a microporous structured collagen matrix at 4 • C [176].
3D-printed CAFs tumoroids were seeded in a collagen matrix containing immune cells, which could allow the interaction between immune cells and tumoroids to be tracked in the 3D environment [177].Highly drug-resistant MCTs-ECM tumor grafts were analyzed in a 3D in vitro model by combining MCTs and collagen matrix.Compared with general MCTs, MCTs-ECM tumor grafts promoted the high activity of MMP2 and MMP9 and induced cancer cell motility [178].Through the embedding of collagen gel, PANC-1 cells (human pancreatic epithelioid carcinoma cell line) cultured into tumor spheres are co-cultured with pancreatic stellate cells, which can be used for ECM fiber network, cancer cell invasive migration, and EMT-related proteins expression effects [179].To further simulate the ECM in vitro, a serum-free 3D matrix composed of laminin, entactin, and type IV collagen was used to cultivate mature human endothelial cells, and the matrix will 'reset' these endothelial cell cultures to adaptability strong angiogenic cells.An adaptive vascular niche can be established and further cultured and adjusted tissue-specific to conform to organoid and tumoroid structures [180].
Magnetic 3D bioprinting represents an alternative approach for tumoroid formation, wherein cells grown in 2D are co-incubated with magnetic nanoparticles.Following cell re-suspension, cells, and ATM are uniformly distributed into the wells of a 96well plate.The plate is then placed on a 96-well magnetic drive, facilitating the 3D printing process [181].Spheres formed via magnetic 3D bioprinting undergo immediate contraction during cell rearrangement and compaction, a phenomenon intricately linked to cell viability and cytoskeletal organization.Tseng et al successfully utilized magnetic 3D bioprinting technology to perform CTCs enrichment from blood samples of prostate and kidney cancer patients.Cells were magnetized using nanoparticles and subsequently bioprinted into spherical structures, facilitating the growth of CTCs tumoroids [182].Based on the narrative in this chapter, table 1 listed few examples for the fabrication of tumoroids, using various ATMs, cell types, and methods.

Microfluidic system
The extravasation rate was significantly higher in the osteocyte-conditioned microenvironment than in collagen gel matrices.The breast cancer cell receptor CXCR2 and the bone-secreted chemokine CXCL5 play a major role in the extravasation rate.
Biomaterials 2014 [189] (Continued.)Androgen receptor-negative PC-3 cells will form clusters.Prostate cancer cells grown in 3D culture showed expression of markers that confirm their epithelial status and cell phenotypes.
Biomaterials 2019 [192] Human macrophages from whole blood Porous Scaffold (type I collagen) Generation of 3D scaffolds using collagen I from rat tail for human macrophage M1/M2 dichotomy influenced by physical environment.
Results indicate that integrin β2 regulates STAT1 phosphorylation in response to IFNγ/LPS.
Integrinβ2 is also responsible for inhibiting the expression of ALOX15 in response to IL-4/IL-13 in 3D.

Tumoroids-on-a-chip (TuChip)
To understand the fundamental mechanisms of malignant tumors and the way of tumor termination, scientists have developed various in vitro tumoroid culture methods [197].To date, most tumoroid cultures have been conducted using 3D cell culture and animal models, which could cause high costs and variations between the animal used and cause statistical data to become difficult.In addition, most in vitro cell culture systems can only focus on regulating various nutrient and matrix structures but cannot simulate complex vascular networks and blood fluidic mechanics [198,199].Hence, modeling tumoroid microenvironment is a vital crux for in vitro tumoroid culture systems.Conventional cell culture models lack their native TME, thus limiting the ability to offer an efficient in vitro culture.In contrast, microfluidic technologies provide a stable TME by mimicking cellular interactions, fluid flows, and mechanical stimulation.They also offer a sustainable, high-throughput 3D tumoroid formation platform, which can be applied to in vitro anti-cancer drug screening, co-culture analysis, model cellular study, and disease progression [57,200,201].
TuChip is a kind of culture system that can culture tumoroids with high throughput in vitro, which has become a powerful method for 3D tumoroid research and clinical drug development by adjusting the chamber size and low-attachment materials.The TuChip device is mainly made of PDMS, glass, 3D printing photosensitive resins, and other customized materials [201,202].Tuchip can be used for micro, high-throughput tumoroids culture, and easy to observe and count tumoroids.ATMs can be used to culture tumoroids in TuChip, which provides a laminar flow and mechanical shear stress in the cell culture niche.In addition, when culturing organoids on a chip, TuChip can facilitate the addition of growth factors, drugs, etc.In previous studies, Chih et al used an array of 1024 non-adherent microwells coated polyHEMA.PolyHEMA is a wide-used, biocompatible, and hydrophilic hydrogel used as a non-adherent coating material for cell aggregation.In this microfluidic platform, they used a high flow rate (300 µl min −1 ) with a high concentration (5 × 10 6 cells per ml) of T47D cells which were settled down and aggregated as tumoroids in each microwell when loaded into the channel (figure 9(A)) [203].To mimic tumoroid microenvironment, they used secretion of tumor-fibroblast as conditioned media to induce tumor drug resistance, combined with photodynamic therapy (PDT) and chemical drug treatment (i.e.cisplatin) for drug screening (figure 9(B)).They used 10 µM methylene blue as a photosensitizer solution and ambient oxygen level (20%) to treat T47D cancer cells in PDT treatment.After incubation with the drug for 1 h, tumoroids were illuminated with (0.1 J cm −2 ) exposure doses for 14 s to 1 h (total 43.8 J cm −2 ).In this work, the results showed no significant increase in PDT resistance when comparing conditioned media to standard culture media [203].However, due to cancerassociated fibroblast cells influencing tumoroids by increasing these drug resistance mechanisms, the efficacy of cisplatin treatment with conditioned media was lower than the standard culture method.To adjust tumoroid size, Patra et al designed different sizes of chambers in a microfluidic device that has high-throughput functions for anti-cancer drug testing.In this experiment, the microfluidic device was made from PDMS, designed with 300 × 300 µm 2 (width × length) and 200 × 200 µm 2 (width × length) tumoroid culture chamber (figure 9(C)) [204].PDMS is a universally used elastomeric material for microfluidic devices.Due to its excellent optical transparency, high biocompatibility, and gas permeability, it is suitable for cell culture.To make PDMS surface resistant to cell adhesion, 1% w/v Synperonic ® F-108 was coated before cell seeding.For tumoroids formation, human hepatocellular carcinoma cells (HepG2) suspension with a density of 2 × 10 7 cells ml −1 , and 6 × 10 7 cells ml −1 were loaded into 300 × 300 µm 2 and 200 × 200 µm 2 culture chambers.To confirm the capabilities of the microfluidic device for drug test, Cisplatin, Resveratrol and TPZ were prepared in culture medium with gradient concentrations of 20-200 µM were treated on the cultured tumoroids for 48 h.In this method, the bright field images and quantitative characterization show that tumoroid diameter has been reduced after anti-cancer drug treatment.For cell viability, the result was displayed by flow cytometry analysis that ani-cancer drugs cause more cell death in standard culture systems compared with tumoroid culture (figure 9(D)) [204].In addition, a trap shape array in microfluidic is a way to collect cancer cells together.Liz et al collected several MCF-7 breast cancer cells in U-shaped trapping sites for aggregate as tumoroids (figure 9(E)) [151].Recently, Schuster et al developed automated high-throughput microfluidics on tumoroids culture and anti-cancer drug screening from patients.Their system could provide hundreds of combinations for automated drug screening and enables real-time analysis of organoids (figure 9(F)) [205].To understand the drug screening capabilities of the platform, they treated tumoroids with different clinically relevant doses of gemcitabine and paclitaxel.Cellular death fluorescent dyes were used for tumoroids growth and image analysis of the reactions (figure 9(G)) [205].
Many other factors still influence the quality of TuChip, such as gradients of oxygen with hypoxia in the tumoroid core, cell co-culture, vascularization, pH value, and other environmental gradient factors.Hypoxia switches the metabolism of tumor cells and induces drug resistance.Palacio-Castaneda et al developed a new tumor-on-a-chip microfluidic platform, which could detect oxygen levels by oxygen-sensitive probes, and the effect on the 3D tumor cell culture was investigated by a pH-sensitive dual-labeled fluorescent dextran and a fluorescently labeled glucose analog [206].After two days of culture, the device detected decreased oxygen and pH levels and increased glucose consumption, indicating a rapid metabolic switch of the tumor cells under hypoxic conditions towards increased glycolysis [206].Ayuso et al developed a microfluidic tumor slice model to study cell behavior under metabolic starvation gradients [207].The result shows that environmental gradients could affect tumoroid growth, such as nutrient starvation and pH gradients, while hypoxia can play a secondary role [207].Moreover, Dadgar et al also used tumoroids formed by Matrigel as a standard when developing a microfluidic platform for in vitro culture of ovarian tumoroids [208].
Internal vascularization of tumor tissue is another crucial factor that affects tumoroid formation and growth [209].The fibroblasts in the tumoroid induced angiogenic.This behavior builds a perfusable vascular network in the tumoroids.Nashimoto et al presents a tumor-on-a-chip platform that enables the evaluation of tumor activities with the intraluminal flow in an engineered tumor vascular network [210].This finding demonstrates that perfusable vascular networks are important for evaluating tumoroids in a drug screening platform.The results are pretty different from the static conditions [210].The more prospective application in TuChip is the capture of specific cells [211].For example, ATM is added with CAF, TAM, and tumor-associated peptides, which can be used to test whether CTCs can be captured in vitro, and as a test model before implantation of biomaterials.
However, it is a big challenge to duplicate the complex integration and interaction of multiple cell types on a chip [212].Besides, many 3D organoidon-chip models is unable to decide the size and shape of organoids, and restricted operating space limits harvesting and expanding cells, making downstream morphological and molecular analysis difficult; for example, the limited cell number is a considerable obstacle for drug testing, while high-throughput drug screening chip is expensive.What's worse, the mostly existing chip models are double-layer elastic membrane structure coating ECM, which, strictly speaking, cannot support fabricating organoids with 3D system, but a purely 2D cell co-culture model.

Future perspectives
Despite the excellent characteristics of tumoroids, there are still some challenges.Firstly, ensuring the consistency and reproducibility of producing tumoroids with consistent shapes and sizes is a concern.Secondly, establishing effective methods for tumoroids growth and drug efficacy assessment is a priority.Thirdly, employing high-throughput approaches is essential.The development of highthroughput tumoroids cultivation and drug screening methods is a fundamental requirement for commercial applications [213,214].
Although protocols have been established to generate various tumoroids or tumor assembloids, the differences between laboratories and batches are still significant, and the proposed methods and materials need to be further improved.For instance, in lung cancer organoid culture, using the airway organoid medium can effectively enhance its success rate and effect [215].Static culture is easy and is a commonly used method in the laboratory, while spinning flasks or microfluidic devices provide dynamic flow, facilitating nutrition transportation and enabling the growth of tumoroids.However, dynamic culture is challenging to be standardized across laboratories.
Cell co-culturing remains challenging when the number of cell types increases.For studies on cell coculturing, a maximum of four types of cells were used.The medium will need to be re-designed for the coculture system.A study generates tumor assembloids by culturing human tumoroid in Matrigel containing patient-derived CAFs and endothelial cells [27].The tumor assembloids were cultured again into another Matrigel containing human smooth muscle cells to mimic the bladder and its tumors.The normal and tumor bladder assembloids media was not complex using advanced DMEM/F12 supplemented with nine supplements.This study illustrates how to generate tumoroids; however, the protocol for various cancers could differ.The tumor assembloid is still in a fetal stage that requires a lot of try-and-error experiments to optimize the culture protocol and media.
Tumor angiogenesis has been shown to play a crucial role in tumorigenesis and cancer metastasis.The abnormal growth of blood vessels can affect the metabolism in tumors and facilitate the formation of the anaerobic microenvironment, which has been used as one of the targets for anti-tumor therapy.Existing tumor-like models, such as tumor microspheres and tumoroids, cannot preserve the microvascular morphology and structure of the original tumor.To overcome this defect, the researchers established bioengineered blood vessels with HUVEC and combined them with tumoroids embedded in ECM to construct a microfluidic model of the solid tumor-blood vessel interface.This platform is helpful in screening antivascular drugs and studying the mechanism of tumor and vascular interaction [216].
The tumor immune microenvironment has become important in clinical therapy that regulates tumor initiation, growth, and metastasis and affects the therapeutic efficacy of various therapeutic modalities, including immunotherapy.Therefore, establishing tumoroid models that preserve the tumor immune microenvironment is important for new drug discovery and clinical therapy.Fresh tumoroids derived from patients' tumors keep immune cells to some extent and maintain a similar immune microenvironment of the tumors in vivo [54,89].Co-culture of tumor cells with peripheral blood lymphocytes could artificially establish the tumor immune microenvironment [88].Due to the rapid progress of immunotherapy (e.g.checkpoint inhibitors, PD1/PDL1), immune cell embedding within tumoroids becomes important.It needs to be noted that the composition and activity of immune cells in tumors could be significantly varied between individuals.Also, co-culturing immune cells will be more complex than tumorassociated cells in terms of medium composition and protocol.Building immune cells-containing tumoroids from cell suspensions is still a challenge.From a bioengineering perspective, ATMs and cell assembly technology could generate high-quality tumoroids.

Conclusion
Tumoroid technology has shown the potential to produce cancer avatars in vitro.Nevertheless, tumoroid is a multidisciplinary technology that needs the continuous cooperation of oncologists and bioengineers.Recent studies have proved that tumoroid technology is cost-and time-effective for tumor modeling and drug screening compared to the PDX models although challenges remain, such as vascularization and immune microenvironment.In addition, whether a perfect tumoroids is needed for clinical drug screening is still an open question.Even if tumoroids are non-perfect, they still have potential for in vitro drug screening and benefits cancer patients.Tumoroid expansion and biobanking is vital in the next-generation tumoroids and drug discovery, which needs advances of matrix materials and biofabrication.

Figure 1 .
Figure 1.Tumoroids fabrication and clinical application discussed in this review.(A) Scheme of tumoroids fabrication, including (1) tumoroids from the surgical samples, and (2) tumoroids using cancer cell suspensions.The method of producing tumoroids using cancer cell suspensions includes (3) artificial tumor matrices (ATMs), and (4) bioengineered tools.(B) Scheme of the clinical application.ATMs and bioengineered tools facilitate the fabrication of high-quality tumoroids.They are powerful tumor avatars to study tumorigenesis and personalized drug administration.

Figure 2 .
Figure 2. Establishment of a tumor slice culture (TSC) platform.(A) The procedure flowchart for tumor slices sectioning and 3D-TSCs.Tumor tissue cut into 300-µm-thick slices in D-PBS solution using a vibratome.The slices are transferred to culture medium and cultured on an ALI.Reproduced from [54].CC BY 4.0.(B) (a).Diagram depicting the workflow of preparation, culturing, and analysis of precision-cut tissue slices.(b).Measurement of cell proliferation of tissue at the processing time of 1, 2, 3, 4, 5, 6 and 7 d using MTT assay.Reproduced from [55].CC BY 4.0.© 2023 The Authors.Advanced Science published by Wiley-VCH GmbH.

Figure 3 .
Figure 3.The TME mimetic platforms use various ATMs (i.e.polymers, decellularized tissues, and structured substrates) and co-culturing technology (i.e.cancer stem cells and tumor-associated cells) to form tumoroids.The material depicted in the figurefor simulating the TME is versatile and not limited to a single usage.For instance, dECM can not only be directly used to simulate TME but also employed in the construction of hydrogels or scaffolds before being used for TME simulation.Microfluidic devices provide an integrated system where the dynamic culture is the main character of so-called tumoroids-on-a-chip.Tumoroids under static and dynamic cultures have shown their potential to be tumor avatars for cancer modeling and drug screening.Herein, this article proposes the concept of ATMs as a magnet.When ATMs (such as scaffolds, electrospun structures) are added with CAFs, TAMs, and tumor associate peptides, so that ATMs can attract specific cells (such as CSC, CTC) like a magnet.

Figure 4 .
Figure 4. Tumoroid generation using hydrogels.(A) LNCaP PCa cells were exposed to adriamycin at 37• C for 2 h.In 2D, free DOX (50 µg ml −1 ) was mainly located in the nucleus, while DOX-NPs (67 mg ml −1 , the corresponding DOX concentration was 50 µg ml −1 ) were mainly located in the cytoplasm.In 3D, both DOX and DOX NPs were in the cytoplasm.Reprinted from[110], Copyright (2013), with permission from Elsevier.(B) Typical confocal images of LNCaP cells at low (10×) and high (40×) magnification with immunofluorescence after 28 d of three-dimensional culture.The nuclei, ITGβ1, F-actin, and E-CAD were stained in blue, green, red, and magenta, respectively.Reprinted with permission from[111].Copyright (2016) American Chemical Society.(C) E-CAD and ITG were expressed in LNCaP cells cultured with 3D hydrogel.(a)-(i) Confocal sections of representative cells embedded in the sodium alginate/matrix gel complex at different time points (1, 4, and 7 d).The cells were stained with F-actin, and the nuclei were re-stained with propidium iodide.The cells were round in 100%A gel and did not express their spreading ability.It was elongated in 75%:25%A:M gel.They ended up showing starlike morphology and invading feet in the 50%:50%A:M gel (red arrow).(j)-(l) The shape of the nuclei isolated from the c-to-k plates indicated that the cells expressed nuclear fragmentation in the presence of stromal gel, which is characteristic of malignancy.Reproduced from[114].CC BY 4.0.(D) Fluorescence images show nuclei stained with DAPI.Reprinted from[116], Copyright (2016), with permission from Elsevier.(E) The phenotypes and biological characteristics of cells in vivo can be replicated by 3D culture in PEG fibrin hydrogels.On day 30 of culture, A549 hydrogel was embedded in paraffin.Sections were stained with IHC for cytokeratin 7 and cytokeratin 20.The same procedure was applied to a sample of lung adenocarcinoma.Display representative picture (40× magnification).Reprinted from[117], Copyright (2016), with permission from Elsevier.

Figure 6 .
Figure 6.Tumoroid generation using decellularized tissues.(A) (i)-(iv) The breast cancer model integrates through microfibrillar dECM fragments, MDA-MB-231 cells, and human primary dermal fibroblasts cells.(i) Schematic overview of the decellularization process and method for establishing tumor model.(ii) The scanning electron micrographs of porous microspheres scaffolds.(iii) The confocal microscopy images of monotypic (A1)-(A4) and heterotypic (A5)-(A8) dECM-spheroids.Reprinted from [135], Copyright (2021), with permission from Elsevier.(B) (i)-(iii) The bioprinting of the GBM tumors consisting of patient-derived tumor cells, vascular endothelial cells, and brain tissue-based dECM for simulation of native tumors microenvironment.(i) Schematic depicting methodology for bioprinting the GBM-on-a-chip with multiple bioinks and other materials to build up a compartmentalized structure.(ii) The fluorescent images of different regions staining by pimonidazole for the hypoxic cells, Ki67 for the proliferating cells, and DAPI for the cell nuclei.(iii)Different genotoxic treatments were investigated to determine the most effective method in suppressing the GBMs-on-chips (left and middle).After sorting the treatment candidates, the combination, CIS + KU + O 6 BG with radiation, was effective on the GBM-28-on-a-chip compared to GBM-37-on-a-chip (right).The GBM ID 28 or 37 indicated that the patient before or after received anti-cancer treatment, respectively.Abbreviations: O 6 -benzylguanine (O 6 BG), methoxyamine (MX), KU60019 (KU), temozolomide (TMZ), concurrent chemoradiation (CCRT), cisplatin (CIS).* * * P < 0.001, * * * * P < 0.0001 and n.s., not significant; one-way ANOVA and Bonferroni post-hoc tests.Reproduced from[141], with permission from Springer Nature.

Figure 7 .
Figure 7. Tumoroid generation using micropatterns.(A) Nanostructure made by UV nanoimprint lithography.Reprinted from [155], Copyright (2011), with permission from Elsevier.And schematic of spheroid production using the microwell system.Spheroids were generated in microwell plates with HT29 colon cancer cells, LNCaP prostate cancer cells, and TE6 esophageal cancer cells.Reproduced with permission from [145].© 2013 CC BY-NC-ND 3.0.(B) Fabrication of cell-loss-free (CLF) concave microwell array for multi-cellular tumoroids (MCTS) formation with A549 cells.Reproduced from [148].CC BY 4.0.(C) A variety of cancer cell lines to grow in 3D on a micropattern 96-well plate with HCT-116, T-47D, MCF-7, MDA-MB-231, A549, and HeLa cells.Reproduced with permission from [149].© 2016 Society for Laboratory Automation and Screening.Published by Elsevier Inc. (D) Phase-contrast micrographs of the morphology of HepG2 cells on the tissue culture dish, spheroid chip, and cylindroid chip.Reprinted from [150], Copyright (2008), with permission from The Society for Biotechnology, Japan.Published by Elsevier B.V. All rights reserved.(E) Formation of a HepG2 tumoroids with hanging drop technique by inoculation with 500 cells per drop.[153].John Wiley & Sons.Copyright © 2003 Wiley Periodicals, Inc.

Figure 9 .
Figure 9. Tumoroid-on-a-chip using microfluidic devices.(A) T47D breast tumoroids were cultured in microfluidic with non-adherent microwell-coated PolyHEMA.(B) The cell viability of T47D breast tumoroids after chemo-drug and PDT.(A) and (B) Reproduced from [203], with permission from Springer Nature.(C) HepG2 tumoroid were treated by the anti-cancer drug in high-throughput microfluidic.(D) Image of Hepg2 tumoroids was treated by the anti-cancer drug.(C) and (D) Reproduced from [204].CC BY 4.0.(E) MCF-7 tumor cells were captured and aggregated as a tumoroid in a U-shaped array in microfluidics.Reproduced from[151], with permission from Springer Nature.(F) Automated high-throughput microfluidics culture system for pancreatic tumoroids from patients.(G) Cellular death fluorescent dye analysis with different combination doses of anti-cancer drugs.(F) and (G) Reproduced from[205].CC BY 4.0.