Yunhua Ji, Linmeng Wang, Qi Xue, Zhirong Luo and Bo Zhang^{* *}Correspondence: Bo Zhang, Department of Urology, Air Force Military Medical University, Tangdu Hospital, China, Email:

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Abbreviations

NMIBC: Non-Muscle Invasive Bladder Cancer; TME: Tumor Micro-Environment; ECM: Cell-Extracellular Matrix; Alg-Gel: Alginate-Gelatin; GelMA: Methacrylate-Anhydride Gelatin; PDX: Patient-Derived Tumor Xenograft; HUVEC: Human Umbilical Vein Endothelial Cells

Introduction

Bladder cancer is one of the most common tumors in humans. It is estimated that there are about 5 million new cases and 10 million deaths each year. In the last 15 years, it has shown a year-onyear increase, with an average growth rate of 68.29% (Richters A, et al ., 2020). Among them, Non-Muscle Invasive Bladder Cancer (NMIBC), the most common type of bladder tumor, accounts for 70%.

Statistically, many patients do not die from the disease, but experience multiple recurrences. Therefore, patients have a higher survival rate. Among middle-aged and older men, bladder cancer is the second most common malignancy, after prostate cancer (Feldman AR, et al ., 1986; Vercelli M, et al ., 1999). Therefore, its high recurrence rate is not only a problem for urologists, but also a great psychological burden for patients (DeGeorge KC, et al ., 2017). In recent years, the situation has improved with increased awareness and treatment of recurrent tumors (Han J, et al ., 2020).

However, this is not for everyone. Even with optimal treatment, some patients will relapse (Kirkali Z, et al ., 2005), and it remains at a high risk of progression to Muscle-Invasive Bladder Cancer (MIBC) or advanced lesions. It is because of the heterogeneity of relapsed patients that it is difficult to determine the best treatment modality; therefore, the treatment is extremely important. In recent years, the aggressiveness, drug resistance, and metastasis of recurrent bladder cancer have increased, with data showing that for non-muscle invasive bladder cancer approximately 40% of patients are ineffective, and even 15% of patients exist (Ślusarczyk A, et al ., 2019). Despite the continuous development of advanced therapeutic measures, such as molecular therapy, immunotherapy, and gene therapy, however, this therapy are still in the early stages of research and their effectiveness in patients after recurrence remains to be tested, mainly related to the heterogeneity of the disease with the lack of targeted therapies (Medle B, et al ., 2022).

Currently, the success rate of clinical trial development of new drugs is only 3.4% (Wong CH, et al ., 2019). Most of the drug trial results are attributed to the widespread use of monolayers. 2D models have cell-cell and cell-matrix interactions that are very different from those in vivo . This phenomenon causes difficulties in preserving the original phenotype of the cells. Plate cultivation do not mimic the human in vivo microenvironment and do not reflect the true human response to drugs, thus there is an urgent need for models that can accurately screen the efficacy and safety of drugs (Sia D, et al ., 2015).

At present, most cancer cell experiments are based entirely on the immunology, molecular biology and genetics of individual tumor cells, ignoring the exploration of the interactions between mesenchymal cells and cancer cells (Mueller MM and Fusenig NE, 2004). The stromal cells include a complex system that contains not only the fibroblasts, endothelial cells, and immune cell (Kise K, et al ., 2006). The stromal cells can make modifications to cell fate, and cancer cells may also influence changes in growth factors within the stroma through some pathway to promote their own growth (Sahai E, et al ., 2020).The destruction of normal epithelial tissue may lead to the secretion of soluble factors, such as growth factors and cytokines, through tumor cells and stromal cells, which in turn may lead to the suppression of other cells by immune cells, and may also lead the proliferation, migration, and differentiation of other cells through some signaling pathway. The newly created environment formed by the destruction of epithelial cells, the Tumor Micro-Environment (TME), plays a vital role in tumor progression and metastasis (Mhaidly R and Mechta-Grigoriou F, 2020). It is a complex environment where dynamic cell-cell and cell-Extracellular Matrix (ECM) interactions help lead to more aggressive tumor cells. Metastasis occurs by destroying connective tissue while expressing the corresponding proteins to evade surveillance by the body’s immune cells (Hoekstra ME, et al ., 2021). Crosstalk between stromal cells and immune cells leads to a series of tumor-friendly events (Junttila MR and De Sauvage FJ, 2023). Under adverse conditions such as hypoxia tumor cells are able to alter their metabolism to adapt to the corresponding environmental changes, leading to the activation of abnormal signaling by oncogenes or oncogenes, and these environmental changes require information translation between cancer cells and the extracellular matrix, which is how the extracellular matrix affects the fate of cancer cells through changes in composition (Strickaert A, et al ., 2017). For bladder tumors, Immune Checkpoint Blockade (ICB) has become the mainstay of treatment at this stage. Bladder tumor cells can upregulate Programmed Cell Death Ligand 1 (PD-L1) expression and stimulate PD-L1 expression in cells within the TME (Alsaab HO, et al ., 2017), but lower neoantigens and tumor mutations in the TME inhibit T cells from destroying tumor cells, resulting in some patients not responding to ICB (Bellmunt J, et al ., 2017). Recent studies have pointed out that wild-type Tumor protein (p53) epithelial cell mutations, by releasing miR-1246 exosomes, can promote the reprogramming of macrophages within the tumor microenvironment to form distinct cell populations (Cooks T, et al ., 2018). In addition, it has also been shown that the tumor microenvironment plays an important role in the plasticity of tumor-associated macrophage expression and function through p19 (Abdollah F, et al ., 2013). As more and more research has focused on the interaction between the tumor Microenvironment and carcinoma cells, the process of establishing a high fidelity in vivo environment has become an ineradicable aspect.

3D bioprinting allows for highly accurate deposition of multi-elementalization in spatial locations (Ostrovidov S, et al ., 2019). Hydrogels, cells, growth factors and other substances can be integrated together to form functional constructs by extrusion, inkjet, laser, and so on (Yi HG, 2021). Cellular communication and efficient intra and extracellular transport of bioactive substances can be improved by the bioink (Rastogi P and Kandasubramanian B, 2019; Levy AF, et al ., 2014). Compared to traditional 2D culture, bioprinting can precisely control the in vivo microenvironment to approximate the human environment and reproduce the biological characteristics of urinary bladder tumors in vitro (Gungor-Ozkerim PS, et al ., 2018). A three-dimensional and biomimetic tumor microstructure model formed by a bioprinter requires biomaterials to mimic the stiffness and ultrastructure of the extracellular matrix in order to simulate the physiologically relevant tumor microenvironment (Salg AG, et al ., 2022). Multiple bioinks with different cell types are able to form functional tumor structures, and the three-dimensional microenvironment constructed by the model is able to induce the expression of genes and proteins similar to those in vivo . The fast and inexpensive manufacturing process enables it to be used as a tool for rapid diagnosis and analysis of patients’ tumor cells, and for more rapid examination of the pharmacological characteristics of drugs to be tested in clinical trials. It provides fully automated high-throughput production, high stability and highly reproducible batch models for disease modeling, drug screening, and personalized medicine, gradually becoming a bridge from basic research to clinical research (Matai I, et al ., 2020; Dey M and Ozbolat IT, 2020).

Currently, bioprinting has attracted significant attention in regenerative medicine, engineering, cell biology, and oncology. With multidisciplinary crossing and integration, it makes difficult clinical problems simple and solutions practical. For the intractable left renal vein compression syndrome, Zhang’s (He D, et al ., 2020) team applied printing technology to the clinic, allowing for an optimal solution to the surgical approach. In the future, bioprinting will become clinicians’ secret weapon to fight disease. Since most of the articles are about bioprinter principles and technology, bioink manufacturing and improvement, there is no detailed description of bioprinting in bladder tumors. Therefore, the aim of this systematic review is to determine the role and clinical potential of bioprinting in bladder tumor modeling for the subsequent construction of more complex bladder tumor models.

Literature Review

Literature screening

The systematic description is mainly searched in three databases according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, namely PubMed (www.pubmed.org), Scopus (www.scopus.com), Web of Science (www.webofscience.com) and take the (“bioprint” OR “3D print” OR “three-dimensional in vitro model”) AND (“bladder cancer” OR “Urinary Bladder Neoplasms”) as the keywords.

Inclusion and exclusion criteria

Titles and abstracts are selected independently by two authors (Ji and Wang). Conducting relevant review according to the previously specified inclusion and exclusion criteria. The different views were solved by the third author (Luo). The inclusion criteria were as follows-

• Application of bioprinter or biological printing methods

• Bladder cancer cells as the object

• Original article written in the English

• Publications from 2012 to 2022

• In vitro and in vivo research

The exclusion criteria were as follows-

• Systematic and narrative reviews, interpretations, case series, guidelines and technical reports

• Basic information about the model building process is missing

Quality assessment

The quality assessment of the included studies was conducted independently by two academics (Ji and Wang), using the Joanna Briggs Institute (JBI) (Tufanaru C, 2017) scale for the quality assessment of study methodology and analysis of possible biases in design, implementation, and analysis. Each item of the risk of bias tool was scored as A (low risk of bias), B (high risk of bias), C (unclear bias), or D (not applicable). The work is completely compliant with the PRISMA 2020 statement (Page MJ, et al ., 2021). Substantial compliance with AMSTAR 2 (Shea BJ, et al ., 2017). Unique identifying number is: Reviewregistry1680 (https://www.researchregistry.com/browse-the-registry#registryofsystematicreviewsmeta-analyses/ registryofsystematicreviewsmeta-analysesdetails/64d487ea066f7000284a ab96/).

Discussion

Based on the search formula, a total of 394 documents were retrieved, of which PubMed: 45, 257 using Scopus, and 92 using Web of Science. The search results were submitted to the Public Reference Manager (Mendeley 1.14, www.mendeley.com) to remove duplicates (n=27) and the initial search (n=367) was evaluated for literature by two researchers by reviewing titles and abstracts. Subsequently, screening was performed based on pre-determined inclusion and exclusion criteria, and the final number of articles was determined to be five literature articles.

Quality evaluation

The risk of bias in included studies was assessed using JBI’s checklist. Overall, almost all studies had a low risk of bias. However, one study did not specify whether a control group was available. The results of the risk assessment are summarized in Table 1 .

Table 1: Risk of bias assessment of the included studies

Characteristics of the included studies

Of the five included studies, two of the five papers present the process of bladder tumor modeling in detail, two articles correlate bioprinting with microfluidic systems using extruded bioprinters and explore their role in drug screening, and one paper uses an inkjet bioprinter for the first time and combines it with organoid culture to study bladder tumor heterogeneity. The main bladder tumor cell lines currently reported in the literature to be used are RT4 and patient derived xenografts bladder cancer cells and 5637, T24, Medical Research Council cell strain 5 (MRC-5), and Human Umbilical Vein Endothelial Cells (HUVEC). 5637 bladder tumor cell line is the most used. Table 2 summarizes the results.

Table 2: Characteristics of the included studies (n=5)

3D bioprinting and bioink methods

Reviewing the previous results, we found that four papers were identified as using extrusion-based biometric 3D printers. Although the current number of papers on bladder tumor modeling is few, it is clear from the results that the constructed models basically achieve the following-

• Precise control of the spatial distribution and layer-by-layer assembly of ECM, cells and other biomaterials

• Formation of 3D cellular arrangements

• Better conformity to the human internal environment compared to flat plate culture

The other used an inkjet bioprinter, which reproduced the heterogeneity of bladder tumors in vitro . Although different bioprinting technologies were utilized, the use of each technology played to its own strengths when constructing the models. The two main technologies currently used, inkjet and extrusion, are common techniques in the construction of cancer models (Yi HG, 2021) and are simple and fast 3D printing techniques. We observed that the piezoelectric printing technique is used when inkjet bioprinting is chosen. Because it has several characteristics-

• In order to eject picoliter droplets, it uses piezoelectric crystals to convert electrical signals into physical forces (Wijshoff H, 2010).

• The number of cells contained in each droplet can be controlled (Kim YK, et al ., 2016).

• Cell arrays are created by controlling the jet torque, allowing the separation of individual cells (The R, et al ., 2013).

• At high speeds of 10-1000 Hz, and resolution up to 100 mm, it can be able to produce accurate hydrogel patterns of multiple cell types (Park TM, et al ., 2017; Yoon S, et al ., 2018; Park JA, et al ., 2017; Park JA, et al ., 2020).

It is also more suitable for studying tumor heterogeneity because it has almost no effect on cell viability and growth (Ji Y, et al ., 2019) and is able to achieve single-cell separation within conventional cell culture medium (Xu T, et al ., 2005). In addition, the majority of the results used were extruded manufacturing bladder cancer models due to their greater proximity to a variety of inks including natural, synthetic and hybrid bioinks (Ersumo N, et al ., 2016). In particular, we found that the combination of microfluidic chips and bioprinting models in both papers can precisely control the flow rate, pH, CO2 and O2 to provide dynamic culture conditions that more realistically reflect the fluidity of human microstructures. Gelatin accounted for the majority of bioinks in five papers. The sodium Algin ate-Gelatin (Alg-Gel) hydrogel was used in one study, Methacrylate-Anhydride Gelatin (GelMA) was used in three studies, and Matrige was used in one study. Table 3 describes the inks in detail in the five literatures.

Table 3: Bioink properties and the experimental outcomes of the studies included (n=5)

Biocompatibility

2D culture affects the phenotype and function of cells (Hickman GJ, et al ., 2016), due to the lack of extracellular matrix, so it is necessary to immobilize them in an extracellular matrix where they can interact (Thomas D, et al ., 2018). Bioink, on the other hand, can fulfill this condition by encasing cells in a functional matrix that promotes cell survival, proliferation, and some functions. The included studies recorded cell survival, although pore size as well as porosity was not reported. The majority of bioprinted cells survived between 85%-90%, and in addition we found that 5637 cell line had a relatively high survival rate and Patient-Derived tumor Xenograft (PDX) cells survived the drug response at a much greater rate than conventional bladder cancer cell lines. Our study also found that cell survival is further improved by introducing microfluidic systems into the culture mode. In a way, the efficiency of screening for bladder tumors is also increased.

Bioprinting and microfluidic

Among the results, two papers combined bioprinting with microfluidic systems, where bioprinting provides a tumor model that mimics the human body and the microfluidic system provides a dynamic culture fluid that ensures cell growth and oxygen uptake. One of the papers stated that when the flow rate was at 2 μl/min, it was more suitable for bladder tumor survival and that co-culturing MRC-5 (fibroblasts), HUVEC increased the survival rate by 5%-10% compared to culture alone. To make screen bladder cancer efficiently, it made four BCOC-embedded microfluidic systems, using a single syringe pump to control the flow rate. It divides the flow control system into top case, top layer middle layer bottom layer bottom case nut case. Middle layer mold stores bio-printing cell models, and this layer has four chambers to screen four models. Compared with the previous study, the number of samples is increased, which enables more efficient screening of bladder tumors. In fact, it takes advantage of the deformability of Polydimethylsiloxane (PDMS), which makes the BCOC system easier to connect and greatly prevents fluid leakage. To stabilize the model position in the fluid, a polycarbonate trail etching film (GVS Filter Technology, Sanford, ME, USA) was used for separation. It places the diffusible drug in the middle layer, allowing full contact of the drug with the model.

Overview of the included studies

A systematic review of these five studies shows that bioprinting plays a major role in the construction of bladder tumor models. It allows precise control of cell density, good encapsulation of tumor cells, and enables the construction of multicellular models with co-culture of endothelial cells and fibroblasts. This not only improves the survival rate of tumor cells, but also effectively captures the complex bladder tumor microenvironment. And the combination of a bladder tumor model and a microfluidic system can be used to further facilitate efficient drug screening. The above findings suggest that the rapid and reproducible cancer tissue microarrays constructed by bioprinting hold potential for large-scale, more reliable and accurate preclinical drug screening in drug testing. According to the study, bladder tumor cells have shown good viability and cell proliferation activity after 3D printing. It is more resistant compared to the conventional 2D culture. This may be due to the co-printing of multiple cells and a more appropriate cell density, which integrates multiple previous single factors and gradually approaches the complex TME, resulting in a more appropriate fit with clinical drug (Wang X, et al ., 2019).

In conclusion, 3D bioprinting, as an emerging technology, has further improved the effectiveness and convenience in personalized oncology y drug screening. In the case of organoid, the complexity of the organoid culture process h and the inability to control the number of cells within the culture plate are improved. Currently, much of the literature suggests the advantages of bioprinting in establishing tumor models as well as the potential clinical applications. However, for clinical studies, there are fewer reports in the literature, especially in the establishment of bladder tumor models, cell lines are still used, humanized cells are less used, and there is a lack of clinical reports for individualization. In addition, the study of the interaction between cancer cells and vascular lymph is still in the preliminary stage. In advance, extrusion-based bioprinting can be used to construct embedded vascular systems, similar to the texture of a leaf, integration of endothelial, stromal and parenchymal cells to form vascularized tissues.

The importance of bioink

Living cells, a critical link in the biomanufacturing pathway. Current cell densities in human tissues and organs reach 200 million cell densities and it has been documented that typically 10-20 million cells per ml quantities (Wang X, et al ., 2019; Sun W, et al ., 2020). For tumor models, the vast majority of plate cultures do not accommodate such high cell densities. The inability to mimic human cell density in the study of antitumor drug development may be the reason why new drugs cannot be adapted to the human body. This low-density culture model is considered to be defective (Peng W, et al ., 2017). The more advanced organoid cultures with external scaffold cultures are also not able to precisely control the number of cells in the culture dish. However, bioprinting can improve the cell density problem by regulating the ink, and it can help regulate cell growth and facilitate cell-cell interactions for information exchange (Pradhan S, et al ., 2017). Most of the inks used for tumor models are sodium alginate, GelMA, which is commonly referred to as hydrogel. Hydrogels have good biological properties due to their similarity to the extracellular matrix composition and are widely used to construct models (Annabi N, et al ., 2010; Charbe N, et al ., 2017).

At present, bioinks are mainly classified into two categories, one is natural compounds, which have good mechanical properties and are more easily degradable, and the other is synthetic compounds with high mechanical properties and stability. Recently, some scholars (Ravanbakhsh H, et al ., 2020) reported the application of composite inks, which have shown satisfactory printability whether in tissue engineering, organ models, microfluidic artificial tumor models; especially in the application of tumor model construction, composite hydrogels can both simulate the tumor microenvironment and regulate cell migration in in vitro models (Cui H, et al ., 2020; Zhou X, et al ., 2016). It can approximate the pathophysiological microenvironment of tissues (Albritton JL and Miller JS, 2017). For example, in the study of breast cancer about bone metastases, scholar (Zhu W, et al ., 2016) used 3D printing technology to develop a skeletal-like environment to study the potential characteristics of bone metastases, and a composite hydrogel was utilized to create a biomimetic bone-specific environment. This bionic bone-specific environment provides a platform for further treatment of breast cancer bone metastases. Another scholar (Zhu W, et al ., 2016) prepared an in vitro bone matrix consisting of polyethylene glycol hydrogels and different concentrations of nano-hydroxyapatite, which successfully mimicked the microenvironment of natural bone. This is indication that the use of functional materials in combination with living cells is more beneficial for current tumor research.

The key of methods for bioprinting

Extruded bioprinting, due to its bioink viscosity range of 30 to 60 KPas, is widely used in various ink manufacturing processes. Gliomas (Tang M, et al ., 2021), cervical cancer (Gospodinova A, et al ., 2021), pituitary adenomas (Diao J, et al ., 2019), cholangiocarcinoma (Mao S, et al ., 2020), liver cancer (Xie F, et al ., 2021), and oral cancer (Almela T, et al ., 2021) have all taken advantage of their ability to be deposited as fine filaments. It can create complex geometric features relatively quickly.

It works by placing the bio-ink in a medical grade syringe and then extruding the ink from the container into a sterile petri dish by means of a pneumatic, piston, rotating rod. Pneumatic is more effective for highly viscous materials, but causes a delay in extrusion due to compressed gas; piston driven, allows more direct control of the hydrogel flow from the nozzle. The use of in situ sexual crosslinking methods (Ouyang L, et al ., 2017) can be widely applied to different photo-crosslinked hydrogel formulations. Screw-rod actuation further accommodates higher viscosity inks. There are also inkjet bioprinters, a technology more suitable for high-throughput single-cell printing, often used in drug screening and modelling of disease, but which can only be used with dilute forms of hydrogel-type inks and therefore cell densities are limited to a few million cells per ml. The majority of bladder cancer model is used by extrusion printing. Although its resolution is limited to 100 μm or less, it is adequate for modeling drug screening.

The limitation of bioprinting

Currently, bioprinting models have made great progress, but there is still a long way to go, before simulating the real human. For one thing, the model at this stage is single tumor cell lines, and the more complex ones are mixture of fibroblasts and endothelial cells. The immune system also exists in the body and its influence on oncogenesis development is considerable, particularly in the progression of bladder neoplasia. There are still no better models to study the mechanisms of action between immune cells and cancer cells.

Another challenge is the post-print survival rate of the cells. A survival rate of 90% or more is currently considered to be a successful model to construct. However, different tumor cells require different proportion of bio-ink, which makes it impossible to print easily with the ink. Each time, the optimum ratio of ink must be tested to adjust the shear forces caused to the cells. On top of this, stem cells are more sensitive to such shear forces and survival rates after printing are a concern. The integration of vascularisation and the vascular system is another obstacle to the further use of the model for personal drug screening and predictive disease modelling. Bladder tumor tissue also contains specific cells such as mesenchymal bladder cells and bladder smooth muscle cells, which are associated with bladder regeneration and involved in the complex cellular signaling processes that control bladder filling and emptying, and which play different roles in tumorigenesis (McCloskey KD, 2013). There is no suitable way to reproduce these multilevel structures in vitro . Thus, bio-3D printing is a promising field and although it faces significant challenges, and it is a key step from basic to applied research.

Conclusion

In this systematic review, bioinks are in a key position in the construction of the model. Cell viability and growth were promoted while mimicking the bladder cancer microenvironment in vivo . The bioprinting models are more resistant to drug use than conventional 2D cultures, possibly due to the co-printing of multiple cells and the loading of bioinks with appropriate cell densities, integrating multiple previously single factors to form a complex tumor microenvironment, resulting in a more relevant outcome to clinical drug use.

There are some limitations to this systematic review. Studies published in languages other than English were excluded. In addition, 3D bioprinting for cancer modelling, although extensively studied, has only been applied to a few studies, which has led to a clinical understanding of bioprinting in the clinic. In addition, the selection of cells, observation and duration of ongoing testing varies, making heterogeneity present in each study and not suitable for quantitative studies.

In conclusion, bioprinting as a novel approach provides a completely new platform to further unravel the mechanisms of tumor development and the drug resistance of tumors. The combination of different bioink phases in these studies to construct in vitro tumor microenvironments, as well as the introduction of microfluidics, has been more helpful in constructing precise tumor microenvironments with heterogeneity. Future studies should introduce vascularised components and enhance the deeper work of bioink parameters of patient-derived primary cells and tumor stem cells to further enhance the simulation of the model. Although bioprinting is still at the experimental research stage, its hidden power is unlimited and it’s potential to help patient custom personal urethral mucosal scaffolds to solve several clinical problems such as urethral strictures, even including bladder reconstruction. And there is great potential for personal screening of oncology drugs, with the ability to reconstitute patient-derived cells in vitro to further mimic the in vivo environment. In the future, as more and more researchers invest, immune cells are introduced, vascularisation emerges, and more humanized features will gradually be reflected in bioprinting models and individualized medical treatment of tumors will become a reality.

Author Contributions

All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by National Natural Science Foundation of China (NSFC), research on role and detailed mechanisms of transcription factor E2F3 in promoting metastasis and immune escape of invasive bladder cancer (Project No. 81872077).

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