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3D bioprinting complex models of cancer

Cite this: Biomater. Sci., 2023, 11, Ruchi Sharma, *a,c,d,e Milena Restan Perez, b Victor Allisson da Silva, a,c,d

3414 Jess Thomsen,d Lavanya Bhardwaj,f Thiago A. M. Andrade, a,c,d

Abdulaziz Alhussan h and Stephanie M. Willerth a,b,c,d,e,g

Cancer is characterized by the uncontrolled division of cells, resulting in the formation of tumors. The
Published on 14 March 2023. Downloaded on 10/27/2024 [Link] AM.

tumor microenvironment (TME) consists of a variety of cell types present within a heterogeneous extra-
cellular matrix (ECM). Current 2D culture methods for mimicking this microenvironment remain limited
due to spatial constraints. Many different types of 3D cancer models have been developed in recent years
using spheroids/organoids, biomaterial scaffolds, and cancer-on-chip systems. However, these models
cannot precisely control the organization of multiple cell types inside of complex architectures.
Bioprinted cancer models can incorporate both stromal and cancer cells inside of 3D constructs to gene-
rate custom models of this complex disease. 3D bioprinting can generate complex, multicellular, and
reproducible constructs where the matrix composition and rigidity are tailored locally to the tumor. These
capabilities make 3D bioprinting an attractive method for reproducing the tumor TME found in vivo.
Received 15th December 2022, Recent advancements in biomaterial-based bioinks enable the generation of 3D bioprinted cancer
Accepted 9th March 2023
models that accurately mimic the TM. Here we discuss recent examples of such 3D-bioprinted cancer
DOI: 10.1039/d2bm02060b models, including those of the lungs, prostate, skin, brain, and colon. We then highlight the advantages of
[Link]/biomaterials-science using 3D bioprinting compared to other in vitro modeling techniques and detail its limitations.

Introduction colorectal (10.0%), prostate (7.3%), and stomach (5.6%)

cancers are the next most frequently diagnosed types of
Cancer is the second-leading cause of death globally.1 Tumors cancer.5,6 An estimated 46% of all cancer diagnoses in 2021
occur when cells develop the ability to penetrate and destroy were for lung, breast, colorectal, and prostate. Even though
healthy human tissue while uncontrollably proliferating – thyroid cancer is less prevalent than many other types of
which is one of the hallmarks of cancer.2 Accordingly, cancer cancers, the National Cancer Institute estimates that 44 000
has the propensity to spread throughout the body.3 The overall new cases are reported each year in the United States.
five-year net cancer survival rate is currently 64%.4 It was esti- However, it has a very high 5-year survival rate regardless of
mated that 19.3 million new cases of cancer worldwide the stage of ∼98% overall. Additionally, the incidence of mela-
(18.1 million excluding nonmelanoma skin cancer) and nearly noma (skin) cancer continues to rise despite being a largely
10 million cancer deaths (9.9 million excluding nonmelanoma preventable disease. Lung cancer (25%), colorectal cancer
skin cancer) occurred in 2020.5,6 With an estimated 2.3 million (11%), and pancreatic cancer (7%) are estimated to be the top
new cases (11.7%), female breast cancer has surpassed lung three causes of mortality due to cancer in 2021.7
cancer as the most frequently diagnosed cancer. Lung (11.4%), Most lung cancers develop from cells lining the bronchi
and other areas of the lung, such as the bronchioles or alveoli.
The most prevalent types of lung cancer are mesothelioma,
a
Department of Mechanical Engineering, University of Victoria, Victoria, BC, non-small cell lung cancer, small cell lung cancer, and lung
Canada. E-mail: ruchis0983@[Link]
b
nodules.8 Surgical procedures, radiotherapy, chemotherapy,
Axolotl Biosciences, 3800 Finnerty Road, Victoria, BC, V8W 2Y2, Canada
c
Division of Medical Sciences, University of Victoria, Victoria, BC, Canada
and immunotherapy are the most frequently used forms of
d
Department of Biomedical Engineering, University of Victoria, Victoria, BC, Canada treatment for lung cancer. Prostate cancer (PCa) can impair
e
Centre for Advanced Materials and Technology, University of Victoria, Victoria, BC, urinary system function because the prostate functions as a
Canada muscle-driven mechanical switch for urination. PCa is the
f
Cognitive Science Program, UC Berkeley, 101 Stephens Hall, Berkeley,
second most common type of cancer in men and the fifth
CA 94720-2306, USA
g
School of Biomedical Engineering, University of British Columbia, Vancouver, BC,
leading cause of death worldwide.9–12 A lot of androgen-depen-
Canada dent PCa initially responds to hormone therapy.13,14 Hormone
h
Department of Physics and Astronomy, University of, Victoria, BC, V8P5C2, Canada therapy, chemotherapy, and radiotherapy are currently the

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treatments for prostate cancer. Unfortunately, these treatments 2.5 months. Although this is a significant increase in survival,
ultimately make it difficult to prevent tumor metastasis and there is room for new therapeutic approaches to increase
androgen resistance. Additionally, these forms of therapy patient survival rates.16 There are three main types of skin
are extremely toxic to normal tissues and promote drug cancer: melanoma, squamous cell carcinoma (SCC), and basal
resistance in addition to being unable to stop the growth and cell carcinoma (BCC). SCC and BCC belong to the non-mela-
metastasis of tumors. Consequently, the search for reliable, safe, noma skin cancer (NMSC) group and represent most skin
and especially androgen-targeting treatments continues to be cancers. Melanoma is caused by UV-induced damage to the
important. Colorectal cancer (CRC) is the third most prevalent melanocytes’ DNA.19,20 SCC starts in the squamous cells of the
cancer and the second leading cause of death epidermis, arising from the accumulation of dysplastic kerati-
among carcinomas. Poor prognosis persists, especially for nocytes that display frequent mutations in the tumor suppres-
advanced diseases or specific molecular subtypes of CRC, empha- sor gene p53, primarily caused by UV radiation, that grow to
sizing the immediate need for improved therapeutic approaches. form a tumor faster than BCC.19 Finally, BCC arises from
Glioblastoma (GBM), the most malignant type of primary abnormal epidermal basal cells and tends to grow slowly.
brain tumor, occurs primarily in the cerebral hemisphere.15,16 However, prompt treatment of BCC is vital because as the
Published on 14 March 2023. Downloaded on 10/27/2024 [Link] AM.

GBM tumors arise from astrocytes, and these tumors can grow tumor grows, it becomes more dangerous and may grow
quickly and spread throughout the brain to the cerebellum, into nearby tissue leading to deformity and significant
brainstem, or spinal cord.16,17 Complete removal of GBM morbidity.19,21
tumors can be difficult and even after partial removal, its Malignant melanoma arises from melanocytes, which are
recurrent nature can be fatal. Additionally, chemotherapeutic epidermis cells that produce skin pigments. Thus, it typically
drugs sometimes cannot cross the blood–brain barrier (BBB), affects the skin, the uvea and retinal pigmented epithelium,
making it difficult to treat. Current treatments for GBM consist mucosae, and very rarely visceral organs.22,23 Therefore, early
of surgery to remove the tumor, radiation to treat any remain- diagnosis and early therapies are crucial for a positive
ing tumor, and chemotherapy using temozolomide (TMZ). The outcome. Sun exposure, mainly during childhood and adoles-
prognosis for patients varies according to age, size of tumor, cence, can induce melanocytic naevi (benign melanocytic
response to treatment and other patient-specific health tumors), and increased numbers of naevi are associated with a
factors.15,16 A recent study showed that patients who under- higher risk of melanoma.24 Currently, there are several types of
went a combination of TMZ chemotherapy and radiation therapeutic treatments available for patients diagnosed with
therapy had a median survival rate of 14.6 months, and melanoma. The standard treatments used for the removal of
patients with no chemotherapy had a median survival rate of tumors include surgery, immunotherapy, targeted therapy,
12.1 months.18 Thus, a combination of chemotherapy and and less frequently chemotherapy. Other forms of therapy,
radiation therapy only increased the survival rate by such as oncolytic virus therapy, are also being researched for

Ruchi Sharma earned her Ph.D. Milena has a Bachelor of

degree in June 2022 from the Engineering degree in
Department of Mechanical Biomedical Engineering from the
Engineering at the University of University of Victoria and she
Victoria, Canada. She holds an currently works as an engineer at
[Link]. degree in Biotechnology Axolotl Biosciences. Her research
from Taipei, Taiwan, a [Link]. work has been focused on engin-
degree in Life Science, and also eering tissues using 3D bioprint-
an MBA degree in Marketing and ing and stem cells. She has also
Finance from Delhi, India. She is worked with patient-derived glio-
a Vice President of the Canadian blastoma cells and is currently
Biomaterials Society (CBS), BC testing a potential drug delivery
Ruchi Sharma Chapter. She has attended Milena Restan Perez system for glioblastoma multi-
several conferences in Canada forme.
and outside. She won several awards for her research during her
Ph.D. and masters. Her research work is published in the esteemed
journal Frontiers in Bioengineering and Biotechnology. Her
doctoral work explores the significance of 3D bioprinted neural
tissue models for drug screening and cell therapies for Parkinson’s
disease. Her research in biomedical engineering focuses on neural
tissue engineering and regenerative medicine using biomaterials,
stem cells, and drug delivery systems.

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and progression is based on histological investigations of

tumors developed in vitro in 2D.13,20,31 Animal models of
cancer have limitations including species differences, difficulty
in mimicking human disease progression, ethical concerns,
and limited predictiveness for human response to treatment.
These limitations hinder the accuracy and applicability of
findings obtained from animal studies to human cancer.34
On the other hand, the ability of 2D culture techniques to
imitate the cancer cell environment is severely constrained as
cancer exists in an inherently 3D environment.36 Cancer
drugs that reach the clinical trial phase have a high rate of
failure.37–40 Several studies report that roughly 95% of anti-
cancer drugs evaluated in phase I clinical trials never make it
to the market.20,41–45 This high rate of failure can partially be
Fig. 1 Schematic displaying the complexity of the tumor microenvi-
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attributed to inadequate pre-clinical methods for developing

ronment (TME).
new pharmaceutical candidates.3,26,38 2D cell culture models
are commonly used to screen potential drug targets for
cancer.36 However, 2D cultures cannot accurately replicate
the treatment of melanoma.25 Among the different skin the original TME, complex cellular composition, and
cancers that affect the human body, melanoma is one of the dynamic interactions.26–30,46,47 Typically, cells are cultured in
most aggressive and has a poor prognosis when it invades the a 2D system on a plate or dish mainly made of polystyrene.
dermis. The primary environmental driving factor of The physiological characteristics of cells in 2D, such as inter-
cutaneous melanoma arising over hair-bearing skin is actions between the cellular and extracellular environments
exposure to UVR (direct sun exposure or through devices, such and changes in the cell morphology and polarity, and the cell
as tanning beds).25 division method, can differ significantly from the 3D environ-
The tumor microenvironment (TME) plays an important ment. In addition to the limitations of tissue complexity and
role in how cancers form and progress.26–31 The TME consists cell behaviour in 2D, 3D models also offer a high control for
of tumor cells, tumor stromal cells, including stromal fibro- drug screening, tumor heterogeneity and the possibility to
blasts, endothelial cells, immune cells like microglia, macro- generate patient specific models. Other techniques such as
phages, and lymphocytes, as well as extracellular matrix (ECM) the use of spheroids, organoids, cancer-on-a-chip technology,
substances like collagen, fibronectin, hyaluronan, and tissue engineered scaffolds, and bioprinting have been intro-
laminin, among others as shown in Fig. 1. duced to cancer models to create 3D microenvironments.30–33
Biomaterials offer a way to create microenvironments both The use of spheroids in cancer research allows for a more
in vitro and in vivo.27,32–35 Important elements of the TME can detailed analysis of molecular diffusion, while organoids can
be reconstructed utilizing 3D matrices in an in vitro cancer accurately replicate the organization of various cell types
model.32,35 Most of our understanding of cancer development found in the body.33–38 Nevertheless, these models have the

As a Biomedical Engineer with a Jess has a bachelor’s degree in

Master’s degree in Applied Biomedical Engineering from the
Science in Mechanical University of Victoria. She
Engineering, Victor has a worked in the Willerth Lab in
passion for developing innova- 2022, including completing a co-
tive solutions to complex op term in collaboration with
medical problems, particularly Axolotl Biosciences. Her main
in the field of cancer screening research revolved around 3D bio-
and 3D bioprinting applications. printing of stem cells, as well as
He brings a unique blend of tech- investigating the mechanical pro-
nical expertise, real-world perties of different types of 3D
experience, and a well-rounded, bio-ink.
Victor Alisson da Silva interdisciplinary perspective to Jess Thomsen
the table. His background in
data science and experience in working in R&D have equipped him
with the skills to drive progress in these fields through the use of
machine learning and advanced technologies.

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drawback of not being able to precisely control the arrange- heterogeneity of real tumors to provide cell–cell and cell–
ment of various cell types in complex architecture and are matrix interactions that are physiologically relevant. Cell func-
difficult to produce, especially spheroids. However, organoids tion can be improved by using biomaterials that contain
are pluripotent stem cell-derived 3D cell culture systems that elements of the ECM. As a result of the interaction with bioma-
include tissue-specific cell types and mimic the character- terials (both natural and synthetic biopolymers), cells will be
istics of developing tissue. better able to proliferate, differentiate, interact with one
Organoids may also serve as a platform for testing thera- another, synthesize the ECM, and perform biological func-
peutics in human cells and be a source for cell replacement tions, resulting in the realization of cancer cell–environment
therapies for conditions such as injury or disease. Despite the interactions. A promising method for screening personalized
promising characteristics of organoids, their wide range of cancer therapies is to use bioprinted cancer models that
applications is constrained by several challenges that still need include stromal and cancer cells from the patient along with
to be overcome. These obstacles include a lack of high-fidelity genetic material, extracellular matrix proteins, and growth
cell types, limited maturation, uncommon physiology, and a factors. 3D cancer models hold great potential for advancing
lack of realization, all of which may reduce their reliability for the study of cancer biology, which has been historically
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particular applications. To overcome the challenge of assessing investigated in 2D cultures of tumor cells. Advanced 3D bio-
a drug’s cytotoxicity in an in vivo environment, cancer-on-chip printed cancer models have the potential to revolutionize
technology employs microfluidics strategies to mimic the flow the way to discover therapeutic targets, develop new drugs
and migration patterns of cancer cells and bioprinting offers a and personalize anticancer therapies in an accurate, repro-
highly reproducible approach to creating intricate architectural ducible, clinically translatable and robust manner. These
structures with promising implications for personalized ex vivo cancer models are already replacing existing in vitro
medicine.15–20 Complex, multicellular, and reproducible con- systems and could, in the future, diminish or even replace
structs can be created using 3D bioprinting with the matrix’s the use of animal models.48 Although animals are physio-
rigidity and composition being uniformly adjusted to match logically competent, they often fail to reproduce human
the tumor model. 3D bioprinting can potentially serve as an behaviours and responses, and 2D cell models are extremely
improved method for mimicking the TME because we can simplified and not representative of what may happen
control the factors much more. Like all tissues, cancer behaves in vivo. Therefore, 3D cell models are believed to better
very differently outside of the human body than it does inside. emulate the complex native tumor microenvironments.49 In
In terms of drug discovery, 3D bioprinted constructs accurately this review, we will discuss 3D modelling of cancer using
mimic the in vivo cancer environment. The 3D bioprinting conventional techniques, 3D bioprinting techniques and
process entails the precise deposition of biomaterials and cells bioinks used to develop 3D-bioprinted models of colorectal,
in specific patterns and layers which is based on a computer- lung, prostate, skin, and brain cancers as well as their advan-
aid design (CAD) model. These models can simulate the 3D tages and challenges.

Lavanya Bhardwaj is currently in Thiago received his undergradu-

her second year of undergradu- ate degree in Biology, Master’s
ate studies at UC Berkeley. She is degree, and Ph.D. in Medical
majoring in Cognitive Science Sciences from the University of
and has worked with mamma- Sao Paulo (Brazil). He was
lian cells at the Willerth Lab. Professor of Pathology and
Immunology and Professor/
Researcher at the Graduate
Program in Biomedical Sciences
at the University Center of
Herminio Ometto Foundation –
FHO|UNIARARAS (Brazil). His
Lavanya Bhardwaj Thiago Andrade main research field has been
about skin and wound healing
therapeutics involving in vivo (animal experimentation and
human clinical trials) and in vitro (skin cell and tissue culture)
investigations. During his career, he supervised 21 graduate stu-
dents and published more than 60 papers in the skin/wound
healing field. He joined the Willerth Lab in September/2021 as a
Postdoc working with 3D bioprinting of human skin co-culture
models.

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3D models of cancer experimentation and the production of spheroids of 1–2 mm

diameter, which are not easily reproduced using the hanging
Many ways exist to model cancer in 3D settings in addition to drop method due to its inconsistency.51 Cells seeded within
the use of bioprinting. The variety of available models makes hydrogels such as collagen and Matrigel can also form tumors
it hard to compare results stemming from different models. that are similar to traditional tissue engineering. Naturally
The choice of which model to use often stems from the sourced hydrogels, which are 95% water by volume, provide a
intended application of the cancer model.50 Many different 3D cell–liquid interface and have natural adhesive properties,
in vitro models have been described in the literature with allowing for high cell viability and controlled proliferation.29,55
different uses and benefits.51 Each method has its own advan- Cancer cells can grow within the hydrogels, and combine to
tages and disadvantages. The methods that will be examined form 3D cancer systems, However, Matrigel is derived from
in detail are as follows: the use of spheroids/organoids and mouse tumors and contains many components that are not
microfluidic devices, focusing on organ-on-a-chip devices. well defined. Accordingly, these matrices can have different
compositions, leading to the expression of different cancer
Spheroids/organoids phenotypes.56–58 As with most 3D models, a lack of vasculature
remains a challenge to be addressed.37 The structure of spher-
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Scaffold-free methods of 3D modelling tumors rely on self-

aggregation of the cells in specialized plates, such as ultra- oids causes an oxygen gradient to form,59 where there is less
lower attachment microplates. These aggregates are known as oxygen in the centre of the spheroid in comparison with the
spheroids/organoids, and have some similarities to in vivo outermost environment. This can result in cell death in the
tumors, including volume growth kinetics, cellular heterogen- core of the spheroid where hypoxia occurs. In contrast, the
eity, and cell secretions.52 Spheroids derived from patient- outside borders of a spheroid are in close contact with cell
specific cancer cells retain their genomic structure, allowing media, which means that this environment has good nutrient
for the facilitation of patient-specific testing and drug develop- and oxygen exchange.37,59 One benefit is that the 3D geometry
ment.53 The hanging drop method uses a specialized plate of spheroids does mimic the structure of tumors found in vivo.
that allows for the formation of drops of media, with cells However, the only ECM present is secreted by the tumor cell,
encapsulated, forcing self-aggregation due to the lack of an which does not accurately mimic the native environment of a
adhesion surface.54 Over the span of several days, spheroids tumor. Additionally, the distance of invasion cannot be deter-
form tumor-like structures. An ultra-low attachment plate mined using a spheroid model since there is limited compart-
relies on the same principle, allowing for a spheroid to form mentalization between the tumor and the stromal cells. This
due to the lack of an adhesive surface. However, ultra-low issue remains even when a co-culture is performed. Finally,
attachment microplates have the advantage of allowing a there is limited collagen density within spheroids, other than
larger diversity of cell growth capabilities, such as prolonged what is produced by the cells themselves.59 Thus, these spher-

Abdulaziz Alhussan is currently Dr Willerth, a Full Professor in

a PhD student in Medical Biomedical Engineering, holds a
Physics at the University of Canada Research Chair in
Victoria. He holds an M.S. Biomedical Engineering at the
degree in Medical Physics, an M. University of Victoria where she
S. degree in Radiation Health has dual appointments in the
Physics, and a Nuclear Department of Mechanical
Engineering B.S. degree from the Engineering and the Division of
United States. His current Medical Sciences. She also holds
project involves using a com- an appointment with the School
bined modality of radiotherapy, of Biomedical Engineering at the
nanotechnology, and chemo- University of British Columbia.
Abdulaziz Alhussan therapy in an in vitro co-culture Stephanie M. Willerth She recently founded the start-up
environment that mimics the company Axolotl Biosciences,
in vivo tumor microenvironment. The application will involve which sells high quality bioinks for bioprinting human tissue
modeling the complexity of cancer in a 3D structure and analyzing models. She served as the Acting Director of the Centre for
the effectiveness of such targeted therapy. The project will acceler- Biomedical Research and the Biomedical Engineering undergradu-
ate transitioning from cell culture preclinical studies to animal ate program at the University of Victoria from 2018 to 2021 and
models and eventually to clinical trials. as the President of the Canadian Biomaterials Society from 2018
to 2019. She was elected to the Royal Society of Canada’s College
of New Scholars and the Engineers and Geoscientists of B.C.
awarded her their Teaching Award of Excellence in 2021.

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oid models have certain advantages and limitations as a tool the model.34 There are many factors that influence the success
for modelling cancer. of these models, and it is imperative that they all work
together to achieve the desired functionality.37,62
Microfluidic devices – organ-on-a-chip
3D in vitro systems can address the shortcomings of conven-
tional 2D in vitro systems while providing novel biological per- 3D bioprinting
spectives. However, cell–cell interactions and spatial structures
are only partially supported by conventional 3D devices. Advances in tumor biology require a 3D microenvironment to
Additionally, a system’s effectiveness as a high throughput replicate the complex interactions of tumor cells with their
screening platform is constrained by the substantial sample microenvironment.36 3D bioprinting holds great promise for
volume needed. As a result, there has been an increase in the generation of cancer models as it can replicate the 3D
interest in innovative 3D culture systems that can offer better microenvironment and enable diverse cultures under carefully
biological models and functionality while lowering necessary regulated chemical and mechanical conditions. It creates 3D
quantities and cost. Microscale 3D in vitro models provide a biological structures by depositing biomaterials combined
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way to increase the functionality and throughput of conven- with cells in predefined patterns, layer by layer, using a
tional 3D systems. The advancement of microfluidic 3D cancer bottom-up assembly approach. Several bioprinting strategies
models, as well as their benefits and multiple applications to have already been used for these applications, however, the
cancer, might be advantageous.38,60,61 Microfluidics uses min- ones that stand out most are extrusion, droplet, and laser-
iature devices that process or manipulate small volumes of based.30,63,64 Fig. 2 shows a summary of these techniques.
fluids through channels that have sizes between a few tens and Extrusion-based bioprinting, a widely used technique, utilizes
many hundreds of micrometres. To create microfluidic chips, air pressure, mechanical pistons, or screws to extrude bioinks
thin grooves or small wells are typically made on the surface of into desired patterns. This method allows for the use of a wide
one layer, which is then enclosed by a second layer to create a range of materials and is relatively simple and cost-effective.
chamber containing microchannels. It is essential for chan- However, the pressure applied during the process can be detri-
nels to be leak-proof, so the layers must be tightly bonded.61 mental to the cells, resulting in lower survival rates and
However, their miniature nature means that organ-on-a-chip reduced function of the printed tissue. This type of approach
models do not allow for the recreation of the tumor at an accu- provides versatility in cancer research as they can create
rate scale.34 They also lack ECM components unless they are models that mimic in vivo tissue architecture and represent
added to the channels on the devices.9 Microfabrication limit- different stages of cancer, allowing for a comprehensive under-
ations lead to a limited possible range of dimensions and standing of the disease and potential treatment options.63 By
structures.37 Furthermore, long term studies pose challenges, utilizing extrusion bioprinters to deposit cells in the creation
due to the inclusion of multiple tissue and cell types. When of compartmental tumoroids, Mazzaglia and Sheng closely
using media and consistent fluid flow, it is difficult to main- mimicked the in vivo tumor environment.65 When combined
tain cell viability, functionality, and the structural integrity of with a collagen matrix rich in immune cells, this allows for the

Fig. 2 (A) Extrusion-based bioprinter: filaments of the bioink are deposited in response to pressure. (B) Droplet-based bioprinter: external energy
causes fine droplets of the bioink to be ejected from the nozzle. (C) Laser-based bioprinter: a pulsed laser beam scans the surface of the reservoir
and generates a specific cure of the bioink. Reproduced under Creative Commons Attribution License (CC BY) ([Link]
licenses/by/4.0/).70

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study of the interactions between the immune cells and the taneously possess bioactivity.59 Ideally, the polymers should be
tumoroids, providing valuable insights into the mechanistic optimized so that they exhibit appropriate functional pro-
understanding of stromal cancer interactions as well as future perties, mechanical strength, and cytotoxicity while still main-
drug testing approaches. In addition, these models can mimic taining printability with 3D bioprinters. However, it is impor-
different tumor stages and study the role of specific bio- tant to create 3D tissue models in vitro for the drug develop-
molecules and cell lines under controlled conditions.9,64 Inkjet ment process. Accordingly, it is essential to use biomaterials to
or droplet bioprinting offers an alternative to extrusion-based enhance cell function and activity. For high-throughput drug
bioprinters. This is a technique that uses energy sources such screening, recent developments in 3D bioprinting technology
as thermal and piezoelectric to deposit precise droplets of cell- have been extensively used to produce representative bioengi-
encompassing bioinks onto a supporting material. This allows neered tumor in vitro models that accurately reproduce the
for the creation of high-resolution structures at a relatively low tumor tissues and microenvironment. Furthermore, bio-
cost. However, the technique’s success is dependent on the printed patient-specific cancer models can be used as a pre-
physical properties of the bioink, such as viscosity and surface clinical immunotherapy screening tool. The tumor microenvi-
tension, which may limit the range of biomaterials that can be ronment, which is made up of cancer cells, stromal tissue, and
used.65 This approach has been effectively used to print
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various immune cells like T cells and macrophages, can vary

uniform drops containing human breast cancer cellular spher- from patient to patient. The development, progression, and
oids into concave wells, providing a way to generate models for treatment of cancer are thought to be significantly influenced
bio-imaging during cancer studies.37 Additionally, this tech- by the interactions between immune and tumor cells in this
nique can be used to print highly complex combinations of microenvironment. Furthermore, 3D bioprinted models are
biomaterials such as hydroxyapatite,66 polyethyleneimine expensive to prepare at a large scale, and the collection of cells
(PEI), and riboflavin sodium phosphate (HE) to create more may present a challenge.37 3D cancer models for a preclinical
realistic and informative cancer models.67 Likewise, laser- screening tool would require creating a dynamic environment
based bioprinting has also shown promising results when gen- within the hydrogel environment.28,66
erating cancer models. Laser-based bioprinting is a cutting-
edge technique that offers exceptional precision and accuracy
in the placement of cells. It allows for the creation of highly
detailed structures with unparalleled resolution. The laser Case studies of bioprinting cancer
beam is used to generate small, precise droplets of cell-laden
materials, which are deposited according to the intended Bioprinted lung cancer models
design. However, this method is complex and can only be used 3D bioprinting lung cancer models can enable the simulation
with a limited range of materials. Additionally, the wavelength of their invasion and metastasis characteristics. Wang et al.
of the laser used can impact the viability of the cells, leading used a suspension of the human lung cancer cell A549/95-D to
to lower cell survival rates. create a cell-laden hydrogel grid scaffold structure using the
The ability of this technique to produce complex images low-temperature molding principle of biological fabrication
using light, highly complex designs, such as honeycomb and 3D bioprinting.8 A549/95-D cells were collected in suspen-
branched structures with microchannels ranging from 25 to sion at a density of roughly 3 × 106 ml−1, and mixed with
120 μm, were generated to compare the behaviour of cancer gelatin–alginate solution at a volumetric ratio of 1 : 2 to create
cells and non-cancerous cell lines.68 Laser-induced forward bio-inks for printing, resulting in a final cell density in the
transfer (LIFT) is a widely used laser-based bioprinting tech- hydrogel of 106 ml−1. The printing parameters such as the
nique that has been shown to enhance drug-screening studies. speed and extruding speed were set to 3.5 mm s−1 and 0.15 ml
By utilizing this technique, Kanaki and collaborators have been s−1, respectively. In this experiment, standard dispensing
able to bioprint gemcitabine, a chemotherapy drug used to treat needles with an inner diameter of 260 μm were used as print-
cancer, into microneedles. This allows for the investigation of ing needles. The eight-layer structure was 12 mm wide and
ways to minimize side effects and optimize drug applications.69 12 mm long. After printing, this structure was immersed for
3D bioprinting begins with selecting the appropriate 3 minutes in a 3% (w/v) sterile calcium chloride solution to
bioink, whether it be natural or synthetic. However, there is no strengthen the cross-linking between the calcium ions and
universal bioink, which makes it difficult to compare results sodium alginate. After being washed 2 times in phosphate-
between studies since it adds another variable. Artificially buffered saline (PBS), the printed structures were cultured in
manufactured bioinks can cause low cell viability, but have DMEM containing 10% fetal bovine serum (FBS). These cells
good mechanical properties and printability, and the opposite were evenly dispersed throughout the hydrogel that was
is true for naturally occurring bioinks.71 Another example of printed and were still viable after printing, indicating that the
inconsistencies in 3D bioprinted models includes the usage of cells were not significantly affected by the pressure and temp-
different seeding densities. While there is currently a big push erature changes that occurred during printing. Furthermore,
to develop new functional polymers to be used in bioinks, they matrix metalloproteinases 2 (MMP2) and MMP9, which are
may not have ideal mechanical properties such as rheological related to invasion migration, were chosen as target genes for
and viscoelastic properties for bioprintability and simul- quantitative PCR (qPCR) detection. This study performed a

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preliminary evaluation of the biological characteristics of the of about 6 mm; it could be used as a homing site for PCa cell
printed cells, focusing on cell invasion and migration abilities, metastasis. The recombinant human bone morphogenetic
using a scratch test and the assessment of MMP2 and protein-7 (rhBMP-7) growth factor was used to stimulate the
MMP9 genes. The results showed that both types of cells metabolism and production of ECM components after human
showed greater expression of both genes in the 3D than in the mesenchymal progenitor cells were seeded on the bone struc-
2D. After printing and culturing, the relative gene expression ture. PCa cells showed a propensity for the engineered bone
of 95-D cells in the 3D group increased several times, to more structures, proliferated, and developed macro-metastases fol-
than ten times, while it was even higher for A549 cells. This lowing further culture. The mechanism of PCa cell behavior
result shows that A549 and 95-D cells both had higher inva- after metastasizing to the bone microenvironment might be
sion and migration potential in the bioprinted in vitro 3D investigated using this model.76 3D bioprinted prostate
tumor model than in 2D. Thus, 3D printed cells outperformed models are also feasible, effectively representing its TME
2D cultured cells in terms of both in vitro and gene properties. which could help with pre-clinical drug testing. The use of 3D
These findings demonstrate that it is possible to create a 3D printed prostate cancer models is becoming more common for
bioprinted lung cancer model that resembles a tumor. patient education and urological surgical planning.10 Multi-
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Although the ECM and environment could be somewhat colour extrusion fused deposition modeling (FDM) can be
mimicked by the gelatin–sodium alginate system, it was used to produce 3D prostate cancer models. These studies on
difficult to keep the constructed model in vitro for longer than 3D printed models frequently focus on creating expensive,
three weeks because the structure would disintegrate, confirm- high-quality models. The use of new generation FDM printers
ing the crucial roles of biomaterial selection in 3D with multiple extrusion is a promising path to 3D printed
bioprinting.8,72,73 Furthermore, more specific 3D bioprinted patient-specific models in the field of medicine.11
alveolar lung models could be used as disease models.
Colorectal cancer (CRC)
Bioprinted prostate cancer models Using tumor-associated stromal cells, Chen et al. created an
Studying intercellular interactions and performing drug devel- in vitro 3D model of tumor tissue that replicated in vivo cell
opment studies will be a lot easier with the help of a reliable, physiological function.77 The collagen-PCL scaffolds, which
reproducible 3D bioprinted model of a prostate tumor.9,11,13,74 were prepared, were used to assemble the 3D bioprinted
The use of a 3D model allows for high throughput testing in a tumor tissue. A uniquely engineered electro-hydrodynamic jet
far more accurate way than presently capable in monolayer (E-jet) 3D printing system, which includes a 3D collection plat-
models widely used in the lab. Spheroids, for example, allow form, a liquid feed system, a high voltage power supply, and
for the inclusion of a concentration gradient as well as the an observation system, was used to create the 3D scaffolds
ECM, which are all present in the in vivo tumor environment. used in this study. An electric field was used to induce fluid
The inclusion of this more complex method of testing will flow from micronozzles to print fibres at the micro- and nano-
accelerate the appropriate modelling of the TME and theoreti- scale levels. These scaffolds were put in a Petri dish with a dia-
cally reduce the cost and time for translation into the clinic. meter of 10 cm and sterilised for one hour with UV light. The
3D spheroid models have the advantage of creating smaller tumor cells (HCT116) and activated stromal cells (CAFs and
tumor sizes, that might not be easily attainable using 3D-bio- TECs) were combined in a 5 : 1 : 1 (HCT116 : CAF : TEC) ratio
printers, to suit the application it is intended to be used for. before being seeded (1 × 106 cells per mL) onto the 3D
For example, for testing the IC-50 of chemotherapeutic drugs scaffolds. In order to prevent uneven cell seeding brought on
such as docetaxel (DTX), a size of ∼300–400 μm is the by scaffold floating, the scaffolds were incubated for 2 mL of
optimum choice, because larger spheroid sizes introduce a DMEM in an incubator for 2–4 hours. 8 mL of medium was
necrotic core and a rise in the percentage of quiescent cells added following the cell adhesion to the scaffolds. The
could thus impact the experimental findings.75 scaffolds were seeded with colorectal cancer cells, cancer-
3D bioprinted prostate models are also feasible, effectively associated fibroblasts, and tumor-associated endothelial cells
representing its TME which could help with pre-clinical drug to create an ECM that facilitated cell processes like adhesion,
testing.74,76 Using the advantages of bioprinting, in vitro engin- cell survival, proliferation, and vascularization. Cancer stem
eered bone structure can be prepared and used as a platform cells (CSCs)-enriched spheroid models for drug screening are
for PCa metastasis research. Collagen accounts for more than created using 3D bioprinting with cell-laden gelatin methacry-
12% of the 120 types of ECM proteins in the prostate, and late (GelMA)-nanoclay hydrogels. GelMA-nanoclay hydrogels
deregulation of collagen metabolism was also associated with were highly porous with appropriate mechanical properties
the PCa Gleason score and may be a determinant of patient and good cytocompatibility with CRC cells. Additionally, the
survival. Collagen is a major ECM component in urological CRC CSCs were stimulated and enriched by the hydrogels,
organs and has been linked to the progression of urological which endowed them with better in vitro self-renewal capacity
disease.9,10,76 To mimic the morphological and functional and in vivo tumorigenic potential. In addition, the mechanism
humanized organ bone, Holzapfel et al. used a 3D printing governing stemness regulation was investigated, and GelMA-
extrusion system along with a rotating structure to prepare a nanoclay hydrogel-activated Wnt/-catenin signaling was found
hollow tube using medical-grade PCL (mPCL) with a diameter to contribute to the induction and enrichment of CSCs.78 To

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construct a TME, normal stromal cells were activated and tumor cell behaviour and cause different molecular subtypes
reprogrammed into tumor-related stromal cells. Their results of GBM to differentiate, with stiffer models causing higher
showed that tumor-related markers were overexpressed by the drug resistance to TMZ.85 Another study found that GSCs that
activated stromal cells, and they modified the ECM. were incorporated into a bioprinted construct that had a dia-
Furthermore, tumor progression results in 3D in vitro models meter of 3 mm, showed more resistance to chemotherapeutic
were similar to those found in vivo tumor models. These drugs in 3D when compared to 2D monolayer cultures.79 Most
tumor progression results were assessed by metabolic activities multicellular models also include endothelial cells such as
such as up regulation of glycolysis, glutaminolysis, lipid human umbilical vein endothelial cells (HUVECs) for vascular-
metabolism, the pentose phosphate pathway, and mitochon- ization since without this, the model would lack angiogenesis
drial biogenesis. In conclusion, this model is appropriate for which is a critical characteristic of cancer.18,79,82,85
CRC tumor biology research studies and the creation of highly Vascularization was assessed by staining the printed tissues
personalized cancer treatments.2,77,78 with anti-CD31 which is a protein that is expressed in intercel-
lular junctions when cells form capillary tubes. Finally, some
Glioblastoma multiforme (GBM) studies incorporate stromal cells, microglia, and/or healthy
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Current GBM 3D bioprinted models aim to better under- astrocytes into their multicellular 3D models. A study by Smits
stand disease progression and as a potential tool for drug et al. bioprinted a half-spherical construct containing U87-MG
testing. This is being done by engineering improved cells at a concentration of one million cells per millilitre of
methods to control the spatial organization of the tumor and bioink, with human astrocytes to determine what effect an
by printing multiple cell types to create a co-culture model N-cadherin antagonist had on the cells. They found that the
that better mimics the tumor. The most common GBM cell antagonist prevented spheroid formation, but it also caused the
line used when creating 3D models are U87-MG’s which was astrocytes to detach from the substrate and become rounded;
obtained from a male patient who had GBM.17,18,79,80 This however, at a lower concentration of the antagonist, the astro-
cell line is readily available and easy to handle, hence why it cytes did not become detached.80 This study shows how 3D bio-
is the most common cell type when constructing GBM printed co-culture models with healthy and diseased cells can
models. However, a 2016 study showed that this cell line had a be used to study new drugs and determine an appropriate con-
different DNA profile when compared to the original tumor. The centration that will not adversely affect healthy cells.
next most common cell lines are (i) U118-MG, derived from a Several biomaterials used to properly mimic the ECM of a
50-year-old male patient with grade IV GBM;13,14,81 and U251-MG, GBM tumor include alginate, collagen, and fibrin. The most
derived from a malignant GBM tumor.82 Although less common, commonly used hydrogel is alginate, a biocompatible, non-
mouse glioblastoma cells (GL261) have been used to study the toxic material whose mechanical properties can be manipu-
crosstalk between GBM cells and GBM-associated macrophages lated to resemble native tissue. However, cells placed in algi-
which were also derived from mouse.16,31,79,83–85 In this case, nate do not show any receptor-mediated engagement with the
since the macrophages were mouse-derived, it was most ideal to hydrogel which causes a spherical cell morphology. To prevent
use a mouse-derived GBM cell line rather than a human cell line. this from happening, a study modified alginate with peptide
Patient-derived GBM cells are the most ideal for the study of sequences which allowed U87-MG GBM cells to spread and
patient-specific disease progression and drug testing. For adhere to the alginate matrix.79 Other studies have incorpor-
example, Maloney et al. used patient-derived GBM cells at a con- ated biomaterials such as fibrin into an alginate-based bioink
centration of ten million cells per construct, an extrusion- to promote cell growth and stem cell-like expression in tumor
based bioprinter, and a collage-hyaluronic acid bioink to bioprint models.17,18 An issue with fibrin is that it does not provide
ununiformed droplets. This showed how bioprinted tumor structural integrity after extrusion. For this reason, Heinrich
models could be used for patient-specific drug screening, which et al. added gelatin to their bioink so that the construct is
would allow oncologists to choose the best drug for the specific stable immediately after extrusion.83 Another study included
tumor.3 gelatin in their GAF (gelatine/alginate/fibrin) bioink because it
In addition to GBM cells, various studies use glioblastoma could be easily crosslinked at low temperatures.18 Additionally,
stem cells (GSCs) which self-renew and have multi-lineage gelatin has been included in several bioinks for extrusion-
differentiation capacity. They are also associated with tumor based bioprinting because it has shear-thinning properties,
initiation, contribute to disease progression, and cause thera- which protects the cells from the inherent shear stress in extru-
peutic resistance.31,79,85–88 For example, Tang et al. bioprinted sion-based printing.87 Another popular material in GBM
cylindrical constructs containing human patient-derived GSCs models is GelMA because its mechanical properties can easily
(TS576) at a concentration of ten billion cells per millilitre of be adjusted while having negligible effects on biochemical
bioink in the center of the cylinder, and endothelial cells in cues is highly biocompatible and has shear-thinning
co-culture in the perimeter of the construct, using a digital properties.31,85 In bioinks where GelMA is used, hyaluronic
light processing (DLP) bioprinter and a bioink consisting of acid (HA) is typically integrated into the bioink at a constant
glycidyl methacrylate hyaluronic acid (GMHA) and GelMA to concentration.85 HA can influence various cellular behaviours
create a biochemically relevant microenvironment.85 This such as survival, proliferation, adhesion, and migration. Thus,
study showed that biophysical cues contribute to varying the concentration of GelMA is adjusted to mimic the stiffness

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of the tumor and HA is kept constant to prevent any poten- glands, sweat glands, and hair follicles rise to the surface of
tial impacts on the GBM cells.85 Finally, collagen is the least the skin from the dermis and subcutaneous layer where they
common biomaterial used when modelling GBM. However, originate. The dermis has the following sublayers: the papillary
type I collagen has been used in a collagen-HA bioink layer, the upper sublayer of the dermis, loosely connected
because it allows cells to reorganize by bundling collagen tissue and includes a large number of nerve fibers, capillaries,
fibrils, which generates collagen architectures that play a water and fibroblasts. Collagen fibers form a finer network
critical role in diseased tissue where the ECM changes over than those of the reticular layer; the reticular layer is the lower
time. Most studies with bioinks that are alginate-, fibrin-, or part of the dermis that undergoes continuous transition to the
gelatin-based utilize an extrusion-based bioprinter, such as a hypodermis. Hypodermis (or subcutis) is an elastic layer and
coaxial and/or a microfluidic bioprinter. On the other hand, includes a large number of fat cells that work as a shock absor-
studies using gelatin-methacrylate (GelMA) bioinks utilize a ber for blood vessels and nerve endings. However, the actual
digital light processing-based (DLP-based) bioprinter. This thickness differs from person to person, along the age, and it
bioprinter projects a still image using light that causes the also depends on each body region.93Then, real and standar-
bioink to gel in the projected pattern.31,85 In summary, the dized construction of the skin layers and of the skin cancer is
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ability to accurately replicate the in vivo characteristics of possible using 3D bioprinting and human cells, and it is
GBM 3D models in vitro is dependent on the appropriate crucial to have accuracy and more practical model systems.95,96
integration of bioinks, cellular components, and mechanical However, engineering these complex multi-layered, multicellu-
properties.17,31,83 lar anatomic structures of the skin as realistic as possible is
technically challenging. In order to promote the physiological
Skin cancer conditions of epidermal differentiation (keratinocyte differen-
Accurate and practical model systems are crucial aspects to be tiation into corneocytes for stratum corneum formation), the
considered when developing potential skin cancer treatments. 3D bioprinted skin tissue97 (and in 3D non-printed models as
Animal models are limited by their ethical concerns and accu- well) is raised to the air–liquid interface (ALI). In contrast to
racy. Every different skin region of an animal’s body presents ordinary immersed cultures, ALI culture cells in some layers of
some differences among them in terms of quantity and thick- the 3D bioprinted construct are grown on a basement mem-
ness of the dermis and epidermis layers, which differ when brane, that is raised to the air–medium interface after eligible
compared to similar regions of the human body.89 The ex vivo culture periods, resulting in exposure to air. For the modelling
alternative is restricted by the availability of living tissue. 2D of skin equivalents, this technique is the gold standard. These
cultures are anchorage-dependent systems where the cells equivalents offer high-end complexity and provide physiologi-
grow attached to a surface, and despite their simplicity, the cal behaviour regarding the interplay of fibroblasts and kerati-
cells are restricted by their incapacity to mimic the 3D in vivo nocytes, resulting in an abundant case of use for modelling
structure and behavior. Moreover, cell stratification and differ- cancer.98 Carcinoma cell lines, for instance, can be grown as
entiation are poor and they may show hyperproliferative pure cultures at an air–liquid interface to simulate a carcinoma
growth, making 2D cultures a poor choice for studying skin in situ, or they can be grown as mixtures with normal keratino-
biology. Several important signaling pathways function only cytes to mimic an earlier stage of epithelial dysplasia. The
when the cells are placed in a 3D structure. In vitro 3D models, reason is that the ALI accelerates the invasive growth of
on the other hand, provide an in vivo like structure containing cutaneous squamous cell carcinomas relative to invasive
human skin cells and components of the ECM like growth under submerged conditions. The ALI is thus con-
collagen.19–21,90–92 sidered to be a critical factor for both normal and neoplastic
Then, recent advances in tissue engineering have provided growth of skin cells.99
novel models to study skin cancer treatments in a more 3D culture models that reflect the architecture and cellular
reliable fashion. The human skin in general is divided histo- composition of a tumor are essential in immuno-oncology
logically into the epidermis, the dermis, and the hypodermis. studies. Nowadays, there are several 3D culture methods of
The epidermis consists of the basal cell layer (stratum basale), skin cancer such as spheroids and organoids, which are appli-
the deepest sublayer of the epidermis composed of basal kera- cable to immuno-oncology.72 To further drug discovery and
tinocytes (stem cells), which renews the strata above and starts improve clinical translation, a 3D human melanoma model
SCC when there is an accumulation of dysplastic keratinocytes; was developed as an alternative to animal testing, based on
the stratum spinosum, the prickle cell layer above basal cells primary human skin cells and melanoma cell lines (A375,
that make their shape somewhat flatter; the stratum granulo- Malme 3M, RPMI 7951 and SK-MEL 28 from ATCC) while
sum, granular cells where cornification or keratinization of including a key feature for tumor progression: blood and lym-
keratinocytes begins; the stratum lucidum: it can only be phatic capillaries. Chronic treatment with vemurafenib was
found in soles and palms, where cells become flatter and more applied to the model and elicited a dose-dependent response
densely packed during turn-over; stratum corneum, corneo- on proliferation and apoptosis of tumor cells, making it a
cytes (mortal differentiation of keratinocytes) responsible for promising tool to test new compounds in a human-like
the barrier function of the skin that are constantly renewed.93 environment.91 For an accurate evaluation of potential treat-
The dermis has a rich supply of blood vessels.94 Sebaceous ment efficacy, it is important to study 3D models as close as

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possible to the in vivo conditions. Then a 3D model of B16F10 Advantages of 3D bioprinted cancer
spheroids was used. The behavior of a well-known cytostatic,
doxorubicin (DOX), was evaluated in spheroids as compared to models
classical 2D culture conditions. It was confirmed that a much
higher DOX concentration is necessary to produce similar Cancer research has shown significant improvements in recent
effects compared with the monolayer. The 3D model developed years. The ability to detect the disease early and the methods
in this study was suitable to investigate drug penetration in used to treat cancer have been revamped. Nevertheless, the
time. Those findings may explain the decrease of the doxo- demand for customized therapies has grown over time given
rubicin therapeutic effect, suggesting the need for maintaining that each patient has a distinct set of inter-patient variations
the drug concentration at the tumoral place for at least 2 h in terms of prognosis, clinical outcomes, and reactions.32 Due
upon administration.90 to the recent advancements, the need for preclinical models
3D bioprinting skin models are very much needed for the that can diagnose each patient with a personalized regime has
testing of drugs and cosmetics and to investigate cancer skin, grown rapidly. In recent years, 3D bioprinting has been uti-
in view of bans being imposed on product testing on animals. lized for the progression of improved cancer research cellular
models as detailed in this review.103 With the use of 3D bio-
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This has been mainly motivated by the unprecedented ability

of these technologies in depositing cells, biomaterials, and printing, researchers may simulate the disease in a lab setting
bioactive molecules in predefined 3D locations with high pre- using models created from cancer cells taken from actual
cision and reproducibility. Given this greater need, 3D bio- patients. The ability to automate the fabrication of these 3D
printing is a promising technology that can achieve rapid and bioprinted models while maintaining high repeatability and
reliable production of biomimetic cellular skin substitutes, reliability matches up well with scalable production. The long-
satisfying both clinical and industrial needs.95,96 The analysis term objective of 3D bioprinting is the layer-by-layer fabrica-
of the skin cancer complexity is an important parameter to tion of highly functional 3D tissue-engineered constructs. As a
consider, not only in the search for new therapies, but also for result, crucial cell–cell and cell–ECM interactions would be
the patients’ identification and stratification likely to respond stimulated, and the complex ECM structures found in 3D
to immunotherapy. 3D bioprinting cancer models exhibit tissue-engineered constructs would be mimicked. Despite
varying levels of biological function and complexity at struc- being in its infancy, 3D bioprinting has a lot of potential for
tural, material, and cellular levels, and have been fabricated automating the fabrication of extremely complex 3D tissue
using different bioprinting technologies including vat poly- constructs in a scalable and repeatable manner. Some of the
merisation, inkjet, laser-assisted, and extrusion. There are bioprinting methods used to create 3D cancer models include
different types of cell lines, such as fibroblasts, endothelial extrusion-based, laser-based, and droplet-based. Each form of
cells, adipocytes, pericytes, stem cells, induced pluripotent technology has its own set of benefits, and its utilization is
stem cells (iPSCs), melanocytes, and keratinocytes.25 A major determined by how well it matches the demands of the tissue
advantage of bioprinting skin models relies on the ability to being produced.84 Although each type of technology has its
closely mimic the architecture of the native skin through own feature, a commonality they all tend to share is the plat-
the deposition of bioinks comprised of skin cells and bioin- form they have provided which has been crucial for the study
structive materials into a 3D construct with a tissue-specific of cancer pathology and biology. Moreover, these technologies
organisation. It has been demonstrated that increasing the have permitted the screening of anticancer drugs, have the
complexity and biomimicry of bioprinted skin translates into potential to address a variety of medical research problems
improved biological function, predictive value, and healing and have been used in various areas, including functional
ability.100 organ replacement, regenerative medicine, and drug deliv-
The feasibility of skin bioprinting has been demonstrated ery.103 Additionally, 3D bioprinted cancer constructs have
not only in vitro but also in situ by pre-clinical studies showing proved to present the physiological cancer tissues better when
that the deposition of cells (stem cells, fibroblasts and/or kera- it comes to the microenvironment of the tumor and cellular
tinocytes) directly onto the wound bed of mouse or porcine behavior with the characteristic spatial distribution of
models stimulates healing.101,102 However, the reported cells.32,60
achievements in bioprinting diseased skin models are more 3D bioprinting enables the creation of artificial culture
modest so far. To engineer a realistic TME that can recapitu- environments with a specialized spatial arrangement of
late not only cancer progression but also angiogenesis and specific cell types. The biophysical properties of the tissue can
metastasis, many complex interacting factors must be con- also be fine-tuned by adjusting the porosity, stiffness, and bio-
sidered. Strategies based on 3D bioprinting are now being chemical properties of the ECM.84 Chen et al. showed that a
investigated, which could simulate the TME by bioprinting 3D porous model promoted the formation of tumor cell spher-
living human cells. These approaches allow the accurate place- oids compared to 2D models, and it also immensely increased
ment of normal cancer cells and bioactive macromolecules the invasiveness and chemotherapeutic resistance of tumor
to monitor cancer progression, facilitate drug screening, cells.77 Compared to 2D models, 3D cancer models can better
and provide the design of new generations of anticancer mimic the TME which includes metastasis, anti-cancer drug
therapies.62 resistance, and angiogenesis.84 This is possible since the 3D

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constructs can accomplish biophysical characteristics which associated with creating heterogeneous cancer constructs,
are like the microenvironment of the native tissue, which including the isolation of a significant number of cells, main-
allows for the study of tumorigenesis and cancer progression. taining the heterogeneity of primary cells, bioprinting, and
Also, the 3D construct itself degrades over time as cells secrete rapidly conducting drug testing that will enable the selection
the native ECM, which supports the original bioprinted struc- of the most suitable chemotherapy. In most cases, the develop-
ture. Moreover, in relation to the study of cell–cell and cell– ment of 3D cancer models is commonly accomplished using
matrix interactions, 3D bioprinting provides a stronger patient derived cells. However, these cells are accustomed to
solution by yielding biomimetic microenvironments.35,84 the native environment from which they are obtained. This
Furthermore, 3D bioprinted models may include multiple cell tumor environment is specific to each patient, and therefore
types which are capable of secreting the ECM, such as cancer using patient derived cells is often better for creating personal-
and normal cells related to the microenvironment of the ized therapeutics rather than the development of a generalized
tumor. It allows for the formation of vessel-like structures 3D cancer model using cell lines. Additionally, the use of
which are vital to study the metastatic process and evaluate immortalized cell lines poses an issue since these cells are
anti-cancer drug delivery and responses. It also helps modu- manipulated over a long period of time, which results in very
high passage numbers.28
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late the composition of the exogenous ECM which consists of

numerous growth factors or signalling molecules and the in-
organic matrix.104,105 Furthermore, 3D cancer modelling
allows for the use of human immune cells, rather than using Conclusions
traditional animal models. For example, cancer models can be
created with patient derived xenografts using mice with The creation and application of 3D bioprinting to produce
deficient immunity, however, this work is time consuming, heterogeneous and intricate tumor models for drug screening,
laborious, and the difference in microenvironments between therapeutic interventions, and cancer diagnosis were reviewed
mice and humans must be taken into consideration.106 in this article. 3D bioprinting has catalysed the in vitro devel-
Creating a 3D cancer model with human immune cells is a opment of a high throughput TME. The integration of pre-
great advantage over traditional animal models using immu- cisely tailored biomaterials, 3D biofabrication technologies,
nocompromised mice.28 and computational tools might lead to the development of
improved integrated 3D in vitro platforms. Biomaterials may be
mixed to create composite bioinks with more realistic, adapt-
able, and customizable microphysical properties, which better
Challenges of 3D bioprinted models imitate the distinctive aspects of in vivo tumors, such as
First and foremost, an important factor of 3D bio-modelling is mechanical signals, which in turn affect cell responsiveness to
that all materials used need to be biocompatible and sterile. therapy. 3D bioprinting technology can provide a level of cus-
These factors may seem trivial to mention, however, this limits tomization, architectural control, and biomimicry that cannot
the number of materials that one can choose from when bio- be achieved with other 3D cell culture methods. Overall, 3D
printing. For example, polymers are frequently used in 3D bio- bioprinting may open the door to a revolutionary, yet effective,
printing. However, many polymers have limited ways to be ster- morphodynamical hallmark of tumor severity and a novel bio-
ilized.39 This necessity leads to restricted options when it physical approach to predicting the tumor spreading potential.
comes to what materials can be used to fabricate 3D bio-
printed cancer models. Furthermore, there are different tech-
niques for creating 3D cancer models, and this can be con- Author contributions
sidered another limitation. Since there is no standardized
Conceptualization: R. S. and S. M. W.; project administration:
method, there are challenges when it comes to the comparison
R. S. and M. R. P.; writing – original draft: R. S., M. R. P.,
and reproducibility of results. There are an increasingly large
V. A. S., J. T., L. B., T. A., and A. A.; writing – review and
number of methods to choose from, and the choice is often
editing: R. S. and S. M. W.
made depending on the intended application of the 3D
model.50 Finally, the ability to observe and oversee cells in real
time is currently unavailable and acquiring this capability
would enable researchers to monitor 3D cancer models more
Conflicts of interest
closely. The variability of 3D cancer models presents a chal- S. M. W. is the co-founder and C. E. O. of Axolotl Biosciences.
lenge as it limits the standardization, reproducibility, and
ability to use the models as tools for drug development.26
Furthermore, the ability to vascularize 3D cancer models Acknowledgements
remains one of the foremost limitations in this field of study.
This is a limitation for almost all 3D cancer models.37 S. M. W. acknowledges the funding from the Canada Research
Vascularization is critical for cell proliferation and metastasis Chairs Program, the UVIC Health Research Grant program, the
in tumor tissues.66 Moreover, there are several other challenges Natural Sciences and Engineering Research Council, and the

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Canadian Institutes of Health Research. R. S. acknowledges 14 H. Suzuki, et al., Hormonal therapy for prostate cancer:
support from MITACS, and T. A. acknowledges funding from current topics and future perspectives, Int. J. Urol., 2010,
FAPESP. 17(4), 302–313, DOI: 10.1111/j.1442-2042.2010.02460.x.
15 F. G. Davis, T. R. Smith, H. R. Gittleman, Q. T. Ostrom,
C. Kruchko and J. S. Barnholtz-Sloan, Glioblastoma inci-
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