Journal of Controlled Release 333 (2021) 91–106

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Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Review article

3D bioprinting of engineered breast cancer constructs for personalized and

targeted cancer therapy
Majid Sharifi a, b, c, 1, Qian Bai a, 1, Mohammad Mahdi Nejadi Babadaei d, Farhan Chowdhury e,
Mahbub Hassan f, Akbar Taghizadeh c, Hossein Derakhshankhah g, Suliman Khan a, *,
Anwarul Hasan h, i, **, Mojtaba Falahati j, *
a
Department of Anesthesiology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou, China
b
Department of Tissue Engineering, School of Medicine, Shahroud University of Medical Science, Shahroud, Iran
c
Department of Animal Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran
d
Department of Molecular Genetics, Faculty of Biological Science, North Tehran Branch, Islamic Azad University, Tehran, Iran
e
Department of Mechanical Engineering and Energy Processes, Southern Illinois University Carbondale, Carbondale, IL 62901, USA
f
The University of Sydney, School of Chemical and Biomolecular Engineering, NSW 2006, Australia
g
Pharmaceutical Sciences Research Center, Health Institute, Kermanshah University of Medical Sciences, Kermanshah 6714415153, Iran
h
Department of Mechanical and Industrial Engineering, College of Engineering, Qatar University, Doha 2713, Qatar
i
Biomedical Research Center, Qatar University, Doha 2713, Qatar
j
Department of Nanotechnology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: The bioprinting technique with specialized tissue production allows the study of biological, physiological, and
3D bioprinting behavioral changes of cancerous and non-cancerous tissues in response to pharmacological compounds in
Breast cancer personalized medicine. To this end, to evaluate the efficacy of anticancer drugs before entering the clinical
Stromal components
setting, tissue engineered 3D scaffolds containing breast cancer and derived from the especially patient, similar
Tumor models
to the original tissue architecture, can potentially be used. Despite recent advances in the manufacturing of 3D
Polymeric and composite scaffolds
Drug screening bioprinted breast cancer tissue (BCT), many studies still suffer from reproducibility primarily because of the
uncertainty of the materials used in the scaffolds and lack of printing methods. In this review, we present an
overview of the breast cancer environment to optimize personalized treatment by examining and identifying the
physiological and biological factors that mimic BCT. We also surveyed the materials and techniques related to 3D
bioprinting, i.e, 3D bioprinting systems, current strategies for fabrication of 3D bioprinting tissues, cell adhesion
and migration in 3D bioprinted BCT, and 3D bioprinted breast cancer metastasis models. Finally, we emphasized
on the prospective future applications of 3D bioprinted cancer models for rapid and accurate drug screening in
breast cancer.

1. Introduction
With the advent of 3D bioprinting, drug screening on biopsy sample-
derived tissues can be vastly benefitted [1]. Although 3D bioprinting has
been primarily used in tissue engineering and regenerative medicine
applications like engineered tissue generation [2], the use of this tech­
nique has tremendous potential for application in personalized treat­
ments with a focus on controlling drug release, screening drugs for
treating cancer cells as well as determining probable side effects, and
studying the metastasis and invasion of cancer cells [3,4]. In recent
years, due to several key achievements in the field of biocompatibility
studies for cellular adaptation [5], tissue scaffolds and their constituents
[6], and other biological components to support tissues [7], 3D bio­
printing applications in personalized or general therapies of cancer are
increasingly becoming suitable [8]. Nevertheless, there are several key
challenges to overcome before 3D bioprinted tissues can be used as in
vitro 3D cancer models. For example, precise positioning of cells, loading
of biological and chemical factors, and creating structures with

* Corresponding authors.
** Corresponding author at: Department of Mechanical and Industrial Engineering, College of Engineering, Qatar University, Doha 2713, Qatar.
E-mail addresses: suliman.khan18@mails.ucas.ac.cn (S. Khan), ahasan@qu.edu.qa (A. Hasan), mojtaba.falahati@alumni.ut.ac.ir (M. Falahati).
1
These authors are joint first authors and contributed equally to this work.

https://doi.org/10.1016/j.jconrel.2021.03.026
Received 17 November 2020; Received in revised form 21 March 2021; Accepted 22 March 2021
Available online 25 March 2021
0168-3659/© 2021 Elsevier B.V. All rights reserved.
M. Sharifi et al. Journal of Controlled Release 333 (2021) 91–106

mechanical strength and flexibility similar to real tissue architecture.
Consequently, mimicking native 3D microenvironment or the hetero­
geneous composition of cancer tissues such as BCT by 3D bioprinters still
remains in its infancy [9].
At present, cancer treatment approaches are mostly trial-and-error
basis, and patients are sometimes subjected to a time-consuming and
overwhelming treatment regimen. The treatment regimen will be more
effective and successful if it is personalized for each patient so that safety
can be predicted beforehand likely upon its favorable response. This is
where the use of 3D bioprinters can be leveraged to create an in vitro 3D
model that can reproduce many of the features of in vivo tumors. This
way, individual treatment response can be monitored in vitro before
administering any treatment regimen [10]. The current use of 3D bio­
printers in cancer research is presented in Fig. 1A along with a com­
parison of global cancer incidence worldwide in Fig. 1B. These two pie
charts show how the 3D bioprinters can be leveraged more effectively
for many cancers in the future.
Moreover, 3D bioprinted in vitro models can be utilized in addition to

Fig. 1. A: Application of 3D bioprinters in cancer research from a statistical point of view. The information is based on the PubMed Database. B: Global cancer
incidence in both sexes (Men & Women) worldwide from a statistical point of view, Lung, Breast and Colorectal cancers have the highest ranks (The outcomes were
analyzed based on the worldwide cancer data (https://www.wcrf.org/).

92
M. Sharifi et al.
producing or developing anti-cancer drugs to optimize them for
personalized therapies. While conventional methods to evaluate the
anti-cancer drug efficacy may take several weeks affecting treatment
outcome, the 3D bioprinter can place tumor tissues similar to a patient’s
real tumor in a chamber, connected to a network of fluid-passage
channels, within hours [11]. It is possible to use 3D bioprinting any­
where and anyone can have 3D bioprinted cancer tissues replicated as
needed [12]. Therefore, 3D bioprinting may have a profound impact on
cancer therapy studies to test efficacy and potency of thousands of newly
discovered drugs on specific patient. However, the main challenge is to
fabricate the heterogeneous microenvironment of extracellular matrix
(ECM) components and different cell types adequately to recapitulate in
vivo biological activity [13]. Here we present an overview on the utili­
zation of 3D bioprinting for BCT engineering. We first discuss the main
approaches for printing BCT constructs. Next, we describe the different
types of biomaterials and scaffolds and their impact on the printed tissue
constructs. Finally, we overview the 3D bioprinted breast cancer models
for drug discovery and therapeutic screening as well as the challenges of
current technologies and the future application of 3D bioprinting in
breast cancer studies.
2. Breast cancer environment
In general, the BCT environment includes metastatic sites, cellular
junctions between tumor components, and even the association of can­
cer cells with other surrounding cells as stromal components. The ac­
tivity of BCT cells can be checked by by-products derived from metabolic
activities, exosomes, signaling molecules, proteins or enzymes secreted
from tumor cells (Fig. 2) [14]. Along with the activity of BCT, the
stromal components also significantly influence cancer progression.
2.1. Stromal components
Generally stromal components include capillaries and lymph vessels,
immune cells, fibroblasts, adipose tissue, pericytes, endothelial cells,
mesenchymal stem cells (MSCs) and ECM [15]. The detailed investiga­
tion of biological, biochemical and biophysical aspects of the stromal
component of breast and BCT is beyond the scope of this literature. The
stromal components which can be observed in each patient at different
ratios due to the variable biological conditions are important to
construct a cohesive tissue from breast cancer through various 3D-print­
ing methods are described below.
2.1.1. Extracellular matrix
The ECM is an interconnected network of proteins that encompass all
Fig. 2. Scheme of breast cancer tumor designed by 3D-bioprinting and drug screening by bioprinting pathway v.s. conventional pathway.
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Journal of Controlled Release 333 (2021) 91–106
types of stromal cells and breast cancer cells. Generally, this network is
composed of three classes of structural proteins such as collagen, pro­
teoglycans, and glycoproteins (Fig. 2) [16]. ECM is found dynamic,
renewable, and has a major role in cellular processes such as growth,
proliferation, migration and stabilization of cells [16,17]. In BCT, this
network becomes disordered and leads to aberrant features in tissues
and cells which is attributed to the alteration of ECM metabolism caused
by the activity of breast cancerous cells and immune cells [18]. The most
important protein component in ECM is collagen, whose degradation by
the proteases increases the invasion of breast cancer cells [19]. On the
other hand, alteration of the chemical properties of ECM changes the
physical and textural properties of the BCT [19]. For example, lysyl
oxidase activity on collagen fibers makes BCT stiffer and changes the
feature of ECM [20]. Alteration of physical and chemical properties in
ECM was also reported to stimulate the metastatic activity of cancerous
cells and tumor angiogenesis [21].
2.1.2. Immune cells
Immune cells are involved in the development of cancerous cells
through alteration of BCT inflammation and angiogenesis based on
altered signaling activities. The most important immune cells in breast
cancer are macrophages, lymphocytes, dendritic cells, and neutrophils.
Macrophages associated with BCT form the main population of immune
cells that play a key role in tumor growth and angiogenesis, tissue
regeneration and immunosuppression [22]. Many tumor-stimulating
factors such as ECM remodeling factors (TGF-b), proangiogenic factors
(vascular endothelial growth factor (VEGF) and CXCL12), inflammatory
factors (TNFα and kinds of interleukin), and invasion and metastasis
intensifying enzymes which are secreted by these cells [23–26]. Lym­
phocytes are another major factor in the immune cells in the BCT
environment, the most important of which are T cells that are divided
into three groups of T-helper cells, Treg cells (CD4+, CD25+ phenotype),
and effector cells (natural killer cells) [27]. T-helper cells secrete in­
flammatory and proangiogenic agents that adsorb macrophages for their
modulating [28]. Thus, T cells that induce auto-immune responses to
disease, increase metastatic activities in breast cancer by blocking anti-
tumor responses in BCT. Adult dendritic cells play an important role in
inducing antitumor responses by invading activated neoplastic cells
[29]. The presence of the immature dendritic cells in BCT increases the
migration of endothelial cells, which are very effective in the growth of
tumors [30].
2.1.3. Adipocytes
Since adipocyte cells make up an average of 48% of breast tissue,
their contribution to the design of normal breast tissue and BCT is
M. Sharifi et al.
significant [31]. In addition to energy storage, adipocytes are important
in the secretion of leptin and estrogen due to the exacerbation of breast
cancer [32]. Also, adipose cells produce growth factors such as VEGF
and IGF-I, which are important for breast vascular development [33].
Therefore, breast fats can be effective in responding to ECM remodeling.
Adipocytes were associated to make breast cancerous cells more inva­
sive through inflammatory cytokines such as interleukin, TNFα and
matrix metalloproteinases (MMPs) [34–37]. Thus, adipocytes become
active in response to tumor cells and stimulate the secretion of tumori­
genic factors.
2.1.4. Fibroblasts
Fibroblasts form an important part of stromal cells that are critical
for cancer initiation, development and therapeutic resistance [38]. Fi­
broblasts are involved in the synthesis of ECM as a physical scaffold. In
principle, fibroblasts secrete fibronectin, kinds of collagen, and laminin
along with proteases [38]. Fibroblasts recruited by breast cancer cells
secrete pro-inflammatory and angiogenesis agents such as TGF-b, VEGF,
IL-6, and SDF-1 [39,40]. Also, fibroblasts degrade the matrix proteins by
MMP [41] and thereby reduce the uptake of drugs and change the es­
trogen signaling via secreting collagens type I, III, V, and VI.
2.1.5. Endothelial cells
Vascular and lymphatic endothelial cells recruited by breast cancer
play an important role in producing vessels for transferring nutrients
and materials into BCT, controlling blood pressure in BCT, and leuko­
cyte motility or adhesion [42]. Compared to normal vessels, vessels
developed in BCT possess intercellular gaps or abnormal buds for
tumorigenesis and metastasis activities. Endothelial cells also interact
with integrin and protein glycan to reinforce the ECM [43]. In addition,
endothelial cells in BCT regulate VEGF and EGF receptors [28] and
stimulate CXCL1/2 secretion by secreting TNFα [44].
2.2. Matrix stiffness of BCT
The breast has a soft tissue but in cancer progression based on the
specific situation of each patient it becomes evidently stiffer [45,46] that
in BCT it is correlated with altered matrix biochemical properties such as
change of desmoplastic response [47]. Since cancer cells degrade and
regenerate ECM for life cycle advancement, the tissue stiffening is
stemmed from increased collagen deposition, repositioning of parallel
collagen fibers, and increased crosslinking collagen fibers due to lysyl
oxidase activity [48,49]. In this regard the tumors with metastatic nodes
possessed notably higher stiffness up to 150–200 kPa [50,51]. Abnormal
binding of collagen increases the signaling of growth factor and tumor
extension into adjacent tissue. Reports show that AKT/PI3K and MAPK
pathways that cause matrix hardness are related to survival and prolif­
eration of breast cancer cells, respectively [52,53]. This increase in tis­
sue firmness as well as TAZ pathway increases the proliferation of cancer
stem cells [54].
Mammography is a standard clinical practice in diagnosis and
screening of breast cancer which shows that normal breast tissue is less
dense than BCT [55]. The soft BCT is found to respond more effectively
to chemotherapy than firm tissue [56]. Therefore, chemotherapeutic
outcomes depend on the development of BCT and their varying degree of
stiffness resulting from different stages of cancer development and
physiological condition of patients. Although the metastatic activity is
inversely correlated to tumor hardness [57], metastatic cells are trans­
ferred from a primary stiff tumor tissue to a soft tissue for development
[58]. The hydro- and cryo-gels containing glycosaminoglycan on poly­
ethylene glycol (PEG) scaffolds, Bray, et al. [59] demonstrated that
stiffness alone could turn normal breast cells into invasive cells. While it
has been proven that the BCT firmness may change based on stromal
components interactions [60]. Taken together, changes in matrix
hardness between the initial tumor and the developed and even meta­
static sites are less explored.
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2.3. Porosity of BCT
In addition to the examination of matrix density and stiffness in BCT
development [61], the porosity which is a reflection of matrix void
fractions of the ECM demands greater investigation to unveil its role in
the development and progression of breast cancer [62]. It is inferred that
ECM porosity will decrease if the breast tumor density and stiffness in­
crease upon cancer development, but this phenomenon is yet to be
established scientifically based on available porosity and pore size in
BCT. Changes in the pores can alter the ability of a matrix designed for
optimizing cell adhesion to transfer nutritional and signaling factors
[63].
3. Identification of physiological and biological factors in
mimicking BCT
Imitation of the patients’ BCT for medical practice requires the
incorporation of new technologies from multidisciplinary workgroups
such as tissue engineering, biology, materials engineering, chemistry,
physics, and nano-medicine. In this regard, experimental and research
activities in personalized treatments are generally based on identifica­
tion of two streams in BCT including physiological and biological
factors.
3.1. Mimicking BCT based on physiological factors
Optimizing tissue functions in tumors such as BCT are complex as the
instantaneous changes occur in physiological parameters including pH,
temperature, oxygen or CO2 concentration, ions control, and waste
elimination. Moreover, the frequent events of activation or inactivation
of some cellular functions directly influence signaling changes in the
tissue development of BCT which makes the BCT simulation more
challenging especially in personalized therapies. Because of the ambi­
guity in the full activities of the BCT physiology due to diversity in pa­
tients, 3D culture systems like a static culture still encounter some
behavioral challenges such as cellular displacements and spatial con­
figurations. For example, the extracellular pH of breast cancer cells is
observed more acidic than epithelial cells [64]. The acidic surrounding,
in addition to enhancing breast cancer cell transcription for further
development through increased expression of VEGF and IL-8, enhances
the expression of proteases such as MMPs to facilitate cell migration
[65]. Most static and some 3D patterns have tissue heterogeneity due to
inadequate nutrient distribution, oxygen, and waste elimination that
cause hypoxic conditions in the central part of the scaffolds and cell
growing areas [66]. In general, due to the importance of physiological
factors in BCT development, it is necessary to increase the access of cells
to nutrients and cellular messages, and then to cell migration.
3.2. Mimicking BCT based on biological factors
Breast cancer simulation activities like biological activities in the
body require a variety of biological factors such as growth factors, types
of hormone and chemokines for growth, differentiation and prolifera­
tion of cells, which is variable in patients. Almost all stromal compo­
nents release biological factors for activity and survival, which are
crucial for tissue homeostasis. The importance of each of the biological
factors secreted from stromal components is measurable based on
changes in cellular activity by indexes such as cell proliferation, intra­
cellular toxicity, protein induction, etc. Although the presence of each
biological factor is essential for the development and maintenance of
BCT, the amount of each biological factor depends on the time of cul­
ture, the type of cell being cultured, and the concentration of stimuli
[67]. Hence, the pattern of preservation of simulated BCT based on the
amount of secreted biological factors in each experiment is almost
unique, despite similarity in the pattern of secreted biological factors.
Therefore, co-culturing methods can be used to prevent monopoly in the
M. Sharifi et al. Journal of Controlled Release 333 (2021) 91–106

simulated BCT [68]. For instance, it is demonstrated that the pattern of providing a suitable substrate for the survival, proliferation, and dif­
secretion of biological factors such as IL-6, CXCL9 or 10, CCL2, IFN-γ, ferentiation of breast cancer cells, it is necessary to consider the merits
TNF-α and galectin-1 or − 3 in co-culturing media between blood and demerits of each designed scaffold.
mononuclear cells and adipose cells is very different from that of indi­
vidual cultures [69]. Taken together, although simulating BCT based on 4.1. Materials used in BCT 3D bioprinting
altering the pattern of biological factors is challenging, but the incor­
poration of various components such as ECM, growth factors, chemo­ One of the crucial drawbacks in the 3D bioprinting technology is that
kines and hormones can provide the three-dimensional BCT cultures materials used are not only biocompatible but also have required
with close resemblance to real tissue. biomechanical and bio-functional features for tissue constructs. The
bioprinting machineries usually use materials with well-developed
4. Materials and used techniques in 3D bioprinting characteristics for cell seeding. Therefore, material bioengineering can
be employed to control interaction of cells with materials. Nevertheless,
Despite the high diversity of biological materials available in print­ the leading activity of bio-printable materials is not only modulation of
ing activities, there are limitations and challenges based on tissue size, selective contact with a cell, but also to supply promising scaffolding for
biological and physiological complexity, and the resistance required for tissue engineering. In general, two groups of materials usually applied
processing. Among the materials used, polymer and ECM from real tis­ for 3D bioprinting include native ECM from each patient and polymers.
sues are more common due to their easy processing, high biodegrad­ Moreover, physicochemical properties of composites made of ECM and
ability, excellent biocompatibility and low cost. polymer are crucial when allocating a scaffold for 3D bioprinting.
On the other hand, based on the type of materials used, the tech­
nology available, and the cost of production, the most common methods 4.1.1. Native ECM
of tissue manufacturing are the extrusion, laser, acoustic, lithography The native tissues are primarily considered for scaffold fabrication
and inject methods, which generally fabricate six main groups of scaf­ with both decellularized or acellularized modes due to biological and
folds (Table 1). Since scaffolds provide the ability to simulate BCT by physiological similarities with the original tissue [71]. The commonly
used natural structures include collagen, fibronectin, chitosan, alginate,
gelatin and hyaluronic acid (HA) which have high biocompatibility, low
Table 1 toxicity and low cost. However, the presence of unknown agents in the
Advantages and disadvantages of scaffolds types for tissue engineering [70]. ECM produced as impurities in the structure can limit their application
Scaffold Advantage Disadvantage [71]. Among the native ECM derived from each patient, collagen is
widely used due to its biological adaptation in BCT simulation. Breast
Native ECM • Due to the production of these • The immunogenic response
scaffolds from real tissues, due to incomplete
and others cells are attached directly to collagen via receptors such as
contain desirable biological decellularization, glycoprotein VI and integrin or indirectly through ligands such as
and physiological properties • low stability in body fibronectin [72]. In order to enhance the physical and biological char­
such as high biocompatibility temperatures, acteristics of collagen-based scaffold, substances such as chitosan or
or high cell adhesion for the • high water solubility
elastin are mixed. Collagen also broadly provides the optimal stroma for
development of simulated • low mechanical strength
tissues. • uncontrolled degradation, breast cancer by stiffing the tissue and altering signaling [73]. The ad­
• The inflammatory response • Spatially random without vantages and disadvantages of major ECM components employed in
and immunogenicity are proper care and complex bioprinting are framed in Table 2.
minimal. molecular composition
• The high mechanical should be considered.
strength,
4.1.2. Polymers
• Remodeled and modulated by Polymeric materials made from repetitive monomeric structures are
cells and variable stiffness in one of the widely processed materials by 3D bio-printers because of their
these scaffolds. low cost, compatibility, degradability, and easy manipulation of me­
Hydrogel • High controlled • Low mechanical strength and
chanical, chemical and biological properties [80]. Polymers such as
biodegradation rate along reduced leakage rate.
with biocompatibility. • Imbalance between the rate of polycaprolactone (PCL), PEG, polylactide acid (PLA), poly­
• Moderate tensile strength. degradation and cell adhesion lactide–co–glycolide (PLGA) are fed as powder, hydrogel, filament and
time in the tissue. sheet to 3D printers (Table 3) [81]. Selection of polymers depends on the
Fibrous • High cell adhesion for • Surface modification is type of activity such as PLA, PCL, PLGA and even EAA-co-NIPAM
proliferation and crucial to enhance the surface
differentiation. performance of nanofibers in
copolymer for pharmaceutical purposes and alginate or gelatin for the
• The inflammatory responses this class. cell encapsulation [82,83]. PLA-based scaffold was found to enhance the
are very low. production of IL-6 compared to chitosan scaffolds, while chitosan scaf­
Porous • High porosity with specific • Poor cell adhesion folds exhibit increased expression of TNF-α [84].
pore sizes that can prevent • High porosity can reduce the
cell death in the center of the homogeneous distribution of
scaffold. simulated cancer cells. 4.1.3. Composites
• Tunable degradability Composite materials in the design of BCT consist of at least two
• Good ductility different materials to increase the mechanical strength of the scaffolds
• Easy processability and to create stiffness in the tissue. As mentioned earlier, BCT devel­
Composite • High mechanical strength • By-products produced by the
• High absorbability decomposition of scaffolds are
opment and induction of metastasis activity are directly related to
• High biodegradability along inappropriate due to the cancer tissue stiffness. On the other hand, the combination of two
with the presence of surface acidic nature. different materials, such as hydrogels (PCL, PEG, PLGA) with ECM
agents have made use of these • The interaction between the compounds (collagen, laminin, HA) increase the adhesion of cancerous
scaffolds. cancer cell and the scaffold is
cells on scaffolds as well as enhance drug delivery and tissue tensile
slightly lower.
• Expensive strength. In addition, toxicity in scaffolds can be controlled by the in­
• Time-consuming production clusion of varying contents of composite materials during the fabrication
Microsphere • Easy production • The high toxicity stage. Also, manufacturing nanocomposites from various polymers
• controlled physicochemical enhanced the biocompatibility of scaffolds for the signaling, delivery of
properties for drug delivery
catalysts, and cellular products [89].

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Table 2 4.1.4. The biodegradability and biocompatibility of scaffolds

Advantages of native ECM in tissue engineering. One of the most important challenges in the construction and use of
Items Advantage Disadvantages Ref. scaffolds is their biocompatibility and then biodegradability in biolog­
ical processes. The biocompatibility of scaffolds generally means the
Collagen • Similar to fibrous protein • Less stable [74]
scaffolds in breast cancer • Low mechanical and absence of any inflammation and immunogenicity due to the use of
• Biocompatible melting point foreign materials in tissues engineering [90,91]. Due to the degradation
• Biodegradable • Nanofiber fusion in of polymers to prevent immunogenicity and other medical abnormal­
• Easily cut into various aqueous. ities, a great deal of attention has been given to research in this field
shapes,
• No immunogenic
[92–94], but the study of the mechanism of scaffold degradation in drug
response. delivery and tissue repair activities has received less attention. While
Fibrinogen • Protein present in • Fibronectin clearance is [75] having sufficient information about the mechanism of scaffold degra­
plasma often disturbed dation can reduce the challenges of scaffold stability and by-products of
• No immunogenic • Requires chemical cross-
polymer degradation. The most important polymer degradation mech­
response linking in resistance of
• Degradable scaffold anisms reported in several literatures are summarized in Table 4
• Biocompatible • Less stable in aqueous [95,96]. Modeling the mechanisms of degradation in addition to pro­
• Average stability in vivo solution. ducing microtumors in a stable and efficient manner for drug delivery/
Gelatin • Derivative of collagen • High solubility at [76] drug side effects studies, enable the study of the actual structure of tu­
• Versatile physiological temperature
• Biocompatible • Requires chemical cross-
mors during the expansion and reduction of tumor volume by drugs and
• Cheap linking other compounds.
• Resorbable, • Less stable
• Antithrombogenic • Soluble in aqueous
• No immunogenicity solution. 4.2. Fabrication methods of 3D bioprinting
under physical
condition.
Unlike acellular printing methods, 3D bioprinters simultaneously
Hyaluronic • Controlled degradation • Highly soluble at 23 ◦ C [77]
acid rates • High surface tension deposit living cells and scaffold-forming matrix by preserving
• Naturally present in • High rate of elimination biochemical and biophysical integrity of fabricated cell- materials
ECM and turnover in the body construct. The modes of bioprinting are based on extrusion, laser,
• High biocompatibility • High viscosity acoustic, lithography and injection mechanism, each of which has its
• Biodegradable • Requires crosslinking and
modification for
advantages and disadvantages as listed in Table 5.
enhancing the scaffold.
Chitosan • Bioactive • Immunogenic [78] 5. Breast cancer cell interaction on 3D bioprinted scaffolds
• Biodegradable • Variable degrees of
• High availability impurities
• Good mechanical • Difficulty electrospinning. Cell adhesion in fabricated tissues is a multistep process that pri­
strength, marily occurs between the interaction of the scaffold and the breast
• Biocompatibility cancer cell wall. In general, integrins are a major factor in the adhesion
• Non-toxic of breast cancer cells, which by signaling induce a coordinated activity
High porosity
of cells to focalize the scaffold [1,102]. On the other hand, determining
•
Alginate • High water trapping • Possibility of an [79]
capacity immunogenic response the levels of adhesion proteins in BCT may provide another criterion for
• High flexibility determining the aggressive potential of breast cancer cells as metastases
• Biodegradable [47]. Studies have shown that to promote breast cancer, the tumor needs
Biocompatible
•
to interact more precisely with the scaffold in order to survive and then
• High availability
• Various cross-linking
move, in addition to altering the peripheral tissue [103,104]. In this
options regard, binding of breast cancer cells to scaffolds through activation of
protein kinase is applied. Integrin beta-1 (ITGB1) undergoes structural
alteration by binding to collagen I or III-containing ECM scaffolds and
Table 3
then activates adhesion kinases such as FAK (focal adhesion kinase) and
Advantages of used polymers in tissue engineering. ILK (integrin linked kinase) [1,47,102]. Therefore, the lack of binding
Items Advantage Disadvantages Ref.
Table 4
PLA Flexibility, No immune rejection or High melting point [85] Summary of mechanism and duration of polymer degradation in tissue engi­
cytotoxic effects, (200–230 ◦ C), Slow
neering scaffolds.
degradation.
PCL Low melting temperature Slow degradation [86] Scaffolds Material Mechanisms Degradation
(55–60 ◦ C), excellent viscoelastic time
and rheological properties, Long-
Ceramics Dicalcium Cell-mediated 5–60 months
term zero-order release, Low glass-
phosphate
transition temperatures (− 60 ◦ C).
Calcium carbonate Dissolution and cell- 3–50 months
PLGA Higher processability and High Tg [87]
mediated
mechanical strength, High
Bioglass partial dissolution 24–80 months
degradation, highly porous scaffold
Polymers Chitosan Lysozyme 1–3 months
PVA Bioinert, sintered at a low Low mechanical [88]
Collagen Collagenase 2–24 months
temperature (65 ◦ C)
Fibrin Plasmin 1–2 months
PEEK High elastic modulus High melting point [87]
Hyaluronan Hyaluronidase 1–3 months
(350 ◦ C)
Polylactides Hydrolysis 12–48 months
PEG Good mechanical properties, High No biological activities. [82]
polyglycolides Hydrolysis 3–12 months
surface area, highly porous scaffold
Polycaprolactone Hydrolysis 24–36 months
Polyanhydrides Hydrolysis 12–18 months
Metals Iron Corrosion 36–90 months
Titanium Practically no degradation 48–120 months

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Table 5
Advantages of used methods in 3D bioprinting.
Items Advantage Disadvantages Ref.

Extrusion • This method is based on two principles of pneumatic and mechanical pressure that • Shear stress with cell viability of 40–95% [97]
in both methods, high density cells are printed by abrasives. • Moderate cost
• Disposable syringes in mechanical method • Thermodynamic restrictions of droplet formation
• Disposable cartridges in pneumatic method • Deposition of one material could be incompatible with
• Simple operation without the need to operator expertise other Materials in printable
• High speed of print • Limited control on cell–cell and cell– matrix interactions
• Print multiple bio-inks • The moderate accuracy
• Layer-by-layer deposition • Variable resolution (15–400 μm)
• Mechanical way is more fragile and parts need cleaning
Lithography • High printing speed • Challenge of using various cells in one print [98]
• No shear stresses • Poor hollow structure
• High resolution • The design of the printer for non-biological activities
• Cost-effectiveness • Non-monotonous mechanical stiffness in the tissue due to
• Cell viability above 85% the change of polymerization length
Inkjet • The precise control of the amount of material injected per drop • Limited to non-complex architecture due to the low [99]
• High resolution (10–100 μm) viscosity
• Low cost • Nozzle clogging when using solutions with high cell
• Medium fabrication speed, density
• Widely available, • Need to fast polymerization procedure post-printing which
• Viability of more than 90% of cells has a Negative effect on ECM
• Cross-contamination in bioprinting of multiple bio-ink
• Fragility of micro-valves
Laser • High resolution (10–100 μm) • The high cost [100]
• Cell deposition individually • Low fabrication speed
• Cell viability above 95% • The lack of commercialization
• Side effects of laser exposure to cells
• The long time in fabrication
• The low biodiversity of the inks
Core-shell • Precise • Requires cell seeding post-print [101]
Microfluidic • High cell viability • Moderate resolution
• High Resolution
Acoustic • High precision, • Requires to low viscosity bioink
• High resolution (37–150 μm)
• Controlled directionality

proteins such as collagen, fibronectin and elastin in scaffolds does not
allow for proper adhesion and accumulation of breast cancer cells in
scaffolds. It was determined that there are more than 180 proteins in the
ECM that are used to bind cancer cells to the ECM scaffolds [105]. For
instance, PCL despite possessing high biocompatibility and biodegrad­
ability Mondal, et al. [106] showed poor adhesion behavior due to the
absence of surface compounds such as proteins and minerals in the
fabricated scaffolds. Likewise, Pal, et al. [107] the surface modification
of PLGA scaffolds by gelatin methacrylamide hydrogel and natural fil­
aments from native ECM, revealed the enhanced cell adhesion compared
to native ECM and hydrogel scaffolds which was examined by the
change of fluorescent intensity on the modified scaffold. This work also
showed that the modified scaffold increased breast cancer cell prolif­
eration by 2-fold along with migration of breast cancer cells as meta­
static activity compared to the native ECM and hydrogel scaffolds [107],
thereby reducing the use of topographic status and fiber nature of ECM
scaffolds can effectively adhere with cancer cells. However, most cancer
cell adhesion studies on the scaffold used the hydrogel pathway for cell
seeding and retention, which makes the tissue production process more
complex and sensitive to environmental reactions. Recently, Eslami
Amirabadi, et al. [108] E-cadherin protein on PCL scaffolds, not only
demonstrated improved adhesion of breast cancer cells, but also enabled
the study of metastasis by changing the morphology of breast cancer
cells from normal to invasive (Fig. 3A). However, different breast cancer
cells exposed to E-cadherin showed different behaviors on scaffolds. For
example, MCF-7 cells with a little delay showed the highest concentra­
tion and cell adhesion based on E-cadherin expression compared to
CAMA-1 cells. Whereas, MDA-MB-231 cells showed remarkable
response at the beginning to E-cadherin expression and decreased their
concentration over time [108].
Along with the surface modification and composition, the angle of
scaffold is also observed to influence the activities of breast cancer cells.
For instance, the adhesion, proliferation, and migration of MCF7 cells in
60◦ -angle PCL scaffolds were greater than that of 45◦ and even 90◦
angles [109]. The angular changes in scaffolds have been suggested to
affect adhesion and proliferation of cancer cells through changes in the
size of the cavities that alter the surface area available, and pore
deformation (from square at 90◦ to equilateral triangle at 60◦ and tri­
angles or irregular polygons at 45◦ ) (Fig. 3B) [109]. Despite the differ­
ences between two published results Domingos, et al. [110] Palomeras,
et al. [109], the optimal and high adhesion of breast cancer cells at 90◦
angles of PC scaffolds, Domingos, et al. [110] revealed the positive role
of the altering angles of the scaffold on adhesion, migration, and
development of breast cancer cells. Moreover, it was found that the
deformation of the cavities from square to Hexagonal reduced the
growth of breast cancer cells due to decreased adhesion and prolifera­
tion of cells on the PEG-diacrylate/hydroxyapatite scaffold [103].
Regardless of the cell type, the increase in the number of cavities
compared to their size due to the increase in surface area available en­
hances the efficiency of adhesion and migration of breast cancer cells
[103].
6. 3D bioprinting systems
Three key systems such as biomimicry, autonomous self-assembly
and mini-tissue building blocks have been used as designing ap­
proaches for bioprinting studies. In the following, the application of
each approach will be discussed regarding the bioprinting of breast
cancer cell.
6.1. Biomimetic models
Biomimetic models derived from each person, which are well known
in bioprinting for personalized treatments, are generally developed to

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Fig. 3. (A): a; Microfluidic chip. It consists of two microfluidic layers that are separated by thick electrospun matrices. b; Quantified fluorescence E-cadherin signal
from the cells on top of the electrospun matrices. c; Invasion depth of breast cancer cell lines MCF-7, CAMA-1 and MDA-MB-231 under the gradient after 3 days. d;
Invasion of breast cancer cell lines into the electrospun PCL matrices inside the microfluidic chip. The cross sections show the invading cells under the positive control
condition after 3 days [108]. (B): a; Scaffolds designs with different deposition angles: 90◦ ; 60◦ ; 45◦ . b; Counted cells (%) in 3D conditions compared to 2D control
conditions three days after seeding. Adherent and non-adherent wells were assayed. c; Inverted optical microscopy images of MCF7 cells seeded on scaffolds with
adherent and non-adherent wells. (a) 45◦ and (b) 60◦ scaffolds in adherent conditions. Cells were attached both at scaffolds and at the surface; (1 and 3) 45◦ and (2
and 4) 60◦ scaffolds [109].

simulate one or more tissue properties that can be controlled and eval­
uated. 3D bioprinting mainly aims to refabricate the ECM of a tissue and
this can be achieved by refabricating specified cellular functional parts
of tissues, for example, by imitating the spheroid morphology of the
breast cancer cells or engineering the biologically relevant bioactive
materials. Here it is reiterated that for the potential reproduction of
bioactive tissues, the exploration of biological and physiological activ­
ities of cells, stromal component and organization of the ECM, regula­
tion of the soluble or insoluble items, and the type of the stress in the
microenvironment, are essentially required to consider in each patient.
Moreover, the expansion of this information is critical for the develop­
ment of this system and should be exploited from fundamental bio­
research in the fields of bioactive materials, cellular and molecular
biology, biochemistry and biophysics, bioengineering, and biomedicine.
To assess BCT cells proliferation, the conventional methods involve
the easy control of mechanical stiffness of 2D substrate, which, however,
is not similar to the 3D mechanical microenvironment in native tissue
and hence could produce outcomes different from those of 3D models
[111]. The breast mechanical features were examined within 3D scaf­
fold models, however, the clinically approved stiffness range and the

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geometry of breast cancer cells were not taken into account and thus
exhibited outcomes with limited reliability in exploring BCT progression
[57,112]. In addition to the significance of an applicable 3D mechanical
microenvironment, the material applied to explore BCT progression and
metastasis has also been demonstrated to play a pivotal role in con­
trolling cancer proliferation [113,114]. Unlike the native ECM of BCT,
both natural or synthetic polymers scaffolds which are used to regulate
stiffness do not possess the biochemical and biophysical cues, therefore
development of an integrated biomimetic model consisting of ECM of
BCT as a tissue-specific agent and a 3D mechanical microenvironment
with tissue-scale geometry linked to diseased tissue is essential to
examine the biomechanical participation of the cirrhotic microenvi­
ronment in BCT growth and metastasis.
Native ECM of BCT is controlled by a wide range of biomolecules
especially collagens and growth factors which essentially stimulate a
multiplex microenvironment to support BCT growth and bioactivity
compared to less complex protein networks employed in 2D or 3D in
vitro models. Furthermore, it has been extensively documented that BCT
decellularized ECM (dECM) regulates the culture of breast cancer cells
and is considered as a potential natively-derived material for in vitro cell
culture [115,116]. To date, the application of breast cancer dECM in
vitro is mostly restricted to 2D coatings or 3D gels with simple structures
[116], which do not show a biomimetic development that imitates BCT
microenvironment with nested fibrous biomolecules. Moreover, the
shortage of approaches controlling the mechanical characteristics of
dECM materials limit their implementations to pathological

Fig. 4. (A) Schematic illustration of 3D bioprinting of dECM with different mechanical features and biomimetic structures [13]. (B) Schematic diagram of direct, 3D
bioprinted, cell-laden bone matrix as a biomimetic model for breast cancer (BrCa) metastasis study [104].

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environments where tissue geometry and mechanical features are both
crucial in influencing disorder progression. 3D bioprinting can be used
to model several bioactive items such as breast cancer cells, biological
macromolecules, and some other necessary materials exhibit con­
descending merit for the production of complex 3D structures with ac­
curate control over material characteristics. Therefore, 3D bioprinting
method can be used to produce decellularized breast cancer dECM-based
models with controllable mechanical features. These systems can act as a
premise for exploring the impacts of biologically applicable 3D matrix
mechanical strength on BCT progression and invasion. Indeed, a new
proof-of-concept BCT platform with a biomimetic dECM development
can be achieved to assess breast cancer cell invasive responses at the
cellular and molecular levels.
These ECM-based 3D biomimetic systems can be developed to study
the responses of different cancer cells under selective environments to
reveal cancer mechanisms and their utilization in preclinical drug
screening. Recently a dECM based scaffold was Ma, et al. [13] developed
via 3D bioprinting with regionally different mechanical characteristics
and biomimetic geometry (Fig. 4A).
6.1.1. Biomimetic models in metastasis
Biomimetic models imitate the features of bioactive materials having
reset similar natural bioactivity against the native compound which is
absent or impotent to execute satisfactorily. Therefore, biomimetic ap­
proaches can be used to represent an organ specific model for exploring
breast cancer metastasis by bioprinting. BCT metastasis occurs due to
the transfer of cancerous cells through the bloodstream to the lung,
bone, liver or brain, and can disrupt the function of these vital organs in
the body. Metastasis is known as one of the deadliest outcomes of breast
cancer cells, with bone being one of the primary sites of occurrence.
Inadequate 3D biomimetic systems at current state hinder to imitate this
system in vitro. A biomimetic bone matrix was fabricated utilizing bio-
printer devices to reveal the interaction between breast cancer cells
and bone stromal cells Zhou, et al. [104] (Fig. 4B). A stereo-lithography
bio-printer device was used to produce bone matrices supplemented
with osteoblasts or MSCs entrapped into hydrogel with hydroxyapatite.
This work exhibited that the spread of BCT was increased by the pres­
ence of osteoblasts or MSCs, however the growth of the osteoblasts or
MSCs was decreased by the presence of breast cancer cells. The inter­
action of breast cancer cells with MSCs or osteoblasts also increased the
level of VEGF secretion. These results suggested that the bioprinted
scaffold, with breast cancer and bone stromal cells, generated a unique
system characterizing the interactive impacts of cells in an artificial
bone environment and hence can be used as a standard model for the
studying of post-metastatic BCT.
The role of epithelial-adipose interaction can be considered Vinson,
et al. [117] as an important step in BCT metastasis. In this regard a laser
direct-write was employed to reveal the capability of producing
patterned breast cancer cell-laden hydrogel microbeads supplemented
with differentiated adipocytes. The resultant data supported the fabri­
cated matrix as a promising scaffold for breast cancer cell invasion into
adipose tissue. This model revealed adipocytes and served as an
outstanding approach to investigate cellular and tissue interactions to­
wards the early diagnosis of BCT metastasis.
6.2. Self-assembly
Exploring the basics of self-assembly in the biological systems, it is
pivotal to employ step by step procedure to construct living tissues and
organs. The self-organizing capability of tissues allows fabricating
functional living microstructures with patterned morphologies. For
example, multicellular spheroids can be placed into biocompatible and
biodegradable scaffolds by the application of a 3D bio-printer. This
strategy imitates early morphogenesis and is based on the concept of the
genetic regulation of self-assembly approaches. The process of self-
assembly can be carried out by fusion of spheroid cells, to produce 3D
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breast cancer cell tumors. The self-assembly of breast cancer cells lead to
the concurrence of overcoming solid tissue blocks and induction of early
vascularization [118].
In this approach, the cellular components of a growing tissue
generate their own ECM materials, relevant cell signaling and autono­
mous association and combination to achieve the expected biological
structures and corresponding bioactivity. Self-assembling cellular
spheroids can be fused and experience cellular reorganization to imitate
growing BCT.
Recently scaffold-free production has received a great deal of
attention due to the capability of recapitulating tissue engineering by
applying self-assembly, which imitates the embryonic growth system.
Despite efforts, bioprinted scale-up organs with the features of recapit­
ulated tissue engineering and bioactivity are still lacking. Therefore, Yu,
et al. [119] fabricated and developed a scaffold-free expandable tissue
strand as an outstanding bioink agent for robotic-aided bioprinting
methods (Fig. 5A). It has been well documented that tumors are het­
erogeneous and the tumor microenvironment stimulates tumor growth
and metastasis. Solid tumor in vivo grows in a 3D environment with
other cells like stromal cells. In this context, Jiang, et al. [120] self-
assembly driven multicellular tumor spheroids were formed after
extrusion bioprinting of MDA-MB-231 breast cancer cell and IMR-90
fibroblasts containing hydrogel matrix.
6.3. Mini-tissue building blocks
Biological tissues comprise bio-functional building blocks which can
be manufactured and developed into larger structures by logical as­
sembly, self-assembly, or their association. 3D bioprinting approaches
can be employed to bioprint these components and fabricate 3D bio-
functional microstructures; for example, spheroids samples have been
assembled using ECM-bio-mimicking scaffolds [121].
Indeed, cells can be used as biomaterial building blocks [122] for
fabrication of a wide range of tissues. The widely used direct approach
for examining biomaterial characteristics of microstructures is managed
compression of cell population [123]. Nevertheless, the precondition of
ideal spherical geometry is a restriction of this method. Therefore,
another advanced approach called tissue aspiration procedure was
introduced (Fig. 5B). This method potentially examined cushion micro
tissue explants that revealed an outstanding level of responsivity [124].
Fluorescent probes were embedded in micro tissue fabricated constructs
for exploring biomaterial features derived from microbead-stimulated
mobility [125]. The fabricated tissue spheroid will be covered by the
spheroid with low efficient biomaterial.
The feasibility of direct bioprinting of multicellular building blocks
was Swaminathan, et al. [126] carried out in alginate-based scaffold to
produce a BCT model singly or co-cultured with vascular endothelial
cells. This approach showed that 3D cellular microstructure bioprinting
held a great promise in creating tumor models. Cell membranes and
nuclei integrity of all spheroids was further tested by using cellular as­
says over 96 h post-printing. MCF10A and MCF10A-NeuN spheroids
continued the pre-printed form over the full examination period; how­
ever, spheroids that placed in close proximity started to contact each
other. MDA-MB-231 and MCF-7 spheroids continued their pre-printed
morphology for 72 h and subsequently proliferated and sprouted from
the spheroids [126].
7. 3D bioprinting for producing metastasis models for breast
cancer
Since the multi focalization of breast cancer cells create different
roles leading to the spreading of breast cancer cells to adjacent tissues,
induction of such behavior in simulated BCT is essential for metastasis
studies. Generally breast cancer metastatic activities increase with the
following conditions: 1- cell-matrix interactions (adhesion-degradation-
regeneration interaction), 2- mechanical stimulation (by matrix stiffness
M. Sharifi et al.
Fig. 5. (A) Schematic presentation of the tissue bioprinting employing tissue strands [119]. (B) Schematic representation of the creation a heterogeneous tumor
model by MDA-MB-231 breast cancer cells as well as IMR-90 fibroblasts [120].
and porosity, stress and compression), 3- cancer cell interaction with
fibroblasts and immune cells 4- oxygen gradient and nutrients, and 5-
scaffold structural features such as endothelial barriers or pore size and
shape [127]. Therefore, for the development of metastasis activity in
designed 3D tissue should provide the development of heterogeneous
multicellular tissue. The mechanism of multi focalization of breast
cancer cells is based on the destruction of the ECM tissue, the formation
of defective capillaries, and the cloning of new tissue as a cluster [128].
The secondary colony created in BCT is susceptible to metastatic activity
[127]. However, the uneven distribution of colonies formed during the
multi focalization of breast cancer cells changes the behavior of the
cancerous tissue relative to the primary tumor. On the other hand, the
lack of proper angiogenesis in 3D-designed BCT can cause cancerous
cells to die in the center of the tissue or scaffold. In addition, the oxygen
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gradient in the cancerous tissue causes the cellular migration and met­
astatic activity of breast cancer which is depicted in 3D tissues. To
overcome this limitation, hollow tube networks similar to vasculature
were designed Bertassoni, et al. [129] Hinton, et al. [130] by hydrogel-
supported extrusion technique, which were found to improve the multi
focalization of cancer tissue for metastasis through agarose exit from
gelatin. But it was observed that vessels with multiple ramifications
were not possible in this method. In order to increase vascularization
and its branches, in addition to the use of vascular tube-producing
scaffolds, vascular-producing cells on the scaffolds can be used based
on heterocellular live cell mixing techniques. The tendency to increase
vessels leads to the further development of cancer cells and their
migration to other areas. In this regard, Cui, et al. [131] with the
assistance of stereolithography technique, the endothelial cells with
M. Sharifi et al. Journal of Controlled Release 333 (2021) 91–106

breast cancer cells on hyaluronic acid, hydrogel gelatin, and PEG poly­
mer were bioprinted to produce vessels in the 3D scaffold that increased
metastatic activity along with adhesion and proliferation. This study
particularly determined that the cell type response varied the invasive
activity, therefore in the presence of endothelial cells, the MDA-MB-
231cells exhibited higher invasive activity over MCF-7 cells within
7–14 days [131].
8. Application of 3D bioprinted breast cancer models in
pharmaceutics
Traditionally, the efficacies of anticancer drugs are assessed first in
cell culture systems and after showing appropriate initial responses, are
evaluated on animals and eventually in clinical trials. However, most
drugs are noted effective in cell culture while failing in animals or
clinical trials. The main reason for this disparity in responses can be
attributed to the lack of 3D dynamic activities of real tissue in cell

Fig. 6. (A): a; Cell survival, morphology, and growth status on polymeric scaffolds. b; The number of live and dead cells in the 2D and 3D cultures were quantified. c;
Proliferation rate of MCF10A and MDA-MB-231 cells on porous PLGA scaffolds. d; Sensitivity of cancer cells grown on 3D PLGA scaffolds to anticancer drugs [12].
(B): a; Representative IF staining images for vimentin (red) and cleaved Caspase-3 (green), and nuclei (blue) of 21PT and ADMSC within bioprinted constructs with
different ADMSC layer thickness after 21-day culture and with addition of doxorubicin (DOX) for another 3-day culture. b; Positive cleaved Caspase-3 cells density in
the middle region in the bioprinted constructs with different ADMSC layer thickness. c; Stiffness of different regions in the bioprinted constructs without top and
bottom ADMSC layers, with and without the LOX inhibitor [136]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)

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cultures. Evidence suggests that 3D tissue culture as experimental
models not only determine the efficacy of drugs similar to clinical trials
along with the mechanism of drug entry into tissues and cells, but also
illustrate the potential for screening and side effects of drugs on vital
cancer systems [132,133]. In this regard, the chemotherapeutic effects
of cisplatin, paclitaxel, methotrexate and tamoxifen were tested King,
et al. [134] by employing ECM and stromal component including fi­
broblasts, endothelial cells and adipocytes that influence drug resistance
of breast cancer through growth factor secretion and other hormones,.
The system showed advantages over conventional screening methods
such as the ability to simultaneously measure the effects of drugs on
cancer cells and the responses of different cells. Using the adenosine
triphosphate luciferase assay, the chemotherapeutic effects of breast
cancer cells after 14 days revealed that cancer and stromal cells expe­
rienced enhanced toxicity of drugs in conventional cellular cultures over
the cells growing in 3D scaffolds [134]. Based on this experiment, it can
be concluded that co-activation of breast cancer cells with other stromal
cells and dense multicellular breast cancer in a more dynamic environ­
ment than previous methods can reduce the deleterious effects of drugs
on cancer cells. Another study showed that MDA-MB-231 cancer cells
cultured on collagen-containing PCL scaffolds bespoke a clearer
response to the drug resistance of breast cancer when exposed to
different concentrations of 0–100 μg/ml carboplatin [135]. 3D culture
versus other cultures allowed simultaneous evaluation of morphological
changes of MDA-MB-231 cancer cells, cell cycles, toxicity and cell sur­
vival in the presence of carboplatin. Western blot assay of carboplatin in
MDA-MB-231 described an increase in Bcl-2, Oct-4 and Sox-2, which
suggests the protection of breast cancer cells from apoptosis. On the
other hand, during the first 7 days, the study showed that using fiber
scaffolds could change the morphology of breast cancer cells from
normal to invasive in the presence of cyclin D1, which was not found in
normal cultures [135]. In the following, Rijal, et al. [12] the designed
BCT on PLGA and PCL scaffolds, were used to investigate the effect of
hydroxy-tamoxifen on estrogen receptor inhibition in MCF10A and
MDA-MB-231 cells that play a critical role in breast cancer development
and metastasis activity (Fig. 6A). In this study, in addition to increasing
the duration of the experiment compared to conventional cell culture,
was also conducted to examine the processes of breast cancer spread
[12]. The drug resistance observed in this experiment could be due to
the heterogeneous population of cancer cells and the complex properties
of ECM environments that may influence drug permeability and over­
expression of drug resistant protein in the cancer cell. On the other hand,
despite the effect of obesity on the activity of breast cancer cells, it was
not possible to evaluate it by previous cellular models. Therefore,
adipose-derived mesenchymal stromal cells were used to design a 3D
BCT Wang, et al. [136] which not only improved the long-term viability
of cancerous cells compared to other methods, but also provided the
ability to affect obesity on cancer cell development through increasing
the thickness of the designed tissue (Fig. 6B). In this study, the response
of cancerous cells to doxorubicin in obesity (thickness of average and
high) to thin condition revealed that breast cancer cells expressed less
Caspase-3 when exposed to doxorubicin [136]. As a result, breast cancer
cells will show less apoptosis. On the other hand, the study exposed that
doxorubicin did not have a significant effect on lysyl oxidase expression,
such as slimming and obesity states [136]. Therefore, the use of a lysyl
oxidase inhibitor can prevent tumor stiffness, which plays a critical role
in reducing cancer metastasis. This study further found that the use of a
lysyl oxidase inhibitor could increase the effect of doxorubicin on breast
cancer cells in all conditions [136]. However, the effect of doxorubicin
reports on BCT in previous cell culture models were unable to express
the effect of obesity as well as the limiting effect of lysyl oxidase on
breast cancer cells.
Taken together, the results of the literature suggest that the use of
BCT designed on 3D scaffolds due to their structural and behavioral
similarity to real conditions can more favorably investigate the un­
known drug resistance of breast cancer and the complete screening.
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9. Challenges
In this review, we discuss the 3D bioprinting techniques in person­
alized treatments that have attracted much attention to mimic the BCT
environment. Despite the significant benefits of 3D environments
compared to current cell cultures such as the ability to obtain more
accurate and reliable data, greater cell viability, high proliferation,
displaying processes related to protein expression simultaneously with
other biomolecules, showing how cells are resistant to drugs, the pos­
sibility of a realistic examination of the metastatic activities, etc., are
important challenges in 3D bioprinting scaffolds that will make future
studies more beneficial by removing any of them.
1- The first challenge is the expensive and complex production of
scaffold-based BCT that make public access more difficult. However,
paying attention to all the details of a simulated tissue can provide
more accurate information on breast cancer. But more detail requires
over-engineering of manufactured tools and textures that further
reduces public access.
2- The lack of standardization due to the diversity of imitation of the
patients’ BCT for medical practice among patients makes it difficult
to compare among different studies. The lack of standardized
printers and supplies in laboratories has limited the scalability and
architectural aspects resolution. In addition, the need to optimize 3D
bioprinting conditions with the lack of integrity of materials and
even cells during the printing is a challenge.
3- The use of natural polymers for the production of BCT in accordance
with actual conditions is very common. However, many of the bio­
polymers used in the manufacture of scaffold such as gelatin, algi­
nate, and dextran cannot provide part of the physical, chemical, and
functional properties of BCT. Also, for more robustness of the
designed BCT, it requires the presence of some compounds that are
not found in real tissue and can affect cell activity. Using composites
can reduce the incidence of these problems based on crosslinks. But
the impact of these crosslinks on cell viability, duration of composite
presence, and cell density in these scaffolds still pause unanswered
questions.
4- The use of native ECM instead of polymers in tissue production is in
the focus of attention because of their greater adaptation to BCT.
However, the presence of different cells and compounds, even toxins,
makes it impossible to fully screen and evaluate the effect of each
component on cancer cells. In this context, it is recommended that by
reducing the stromal components in natural scaffolds, the control of
the development and morphological change of the breast cancer cell
based on the scaffold components is explained. However, the
reduction of the components results in a mismatch of the experi­
mental tissue versus the actual tissue.
5- The vascular structure that is required in tissues to reduce cell death
at the center of the scaffolds and increase the metastasis likelihood is
another challenge, has not been fully resolved. Since scaffolds are
generally produced by layer-by-layer printing, it is difficult to
construct vascular structures due to the collapse of scaffolds in hol­
low sites and the incorrect structure of scaffolds. Although this
problem has been partially solved by mechanisms such as the use of
alginate-containing hydrogels. Nevertheless, the presence of addi­
tional materials or processes not only increases the complexity of
production, but can also alter the number of print nozzles and even
the incompatibility of materials used when removing from the scaf­
fold. The method of using cells simultaneously in scaffold printing
also encounters the challenge of cell viability during scaffold pro­
duction and the time constraint of scaffold production to maintain
cell viability without altering gene expression and even cell
morphology.
6- Although 3D models allow biological, physiological, and behavioral
manipulations to examine BCT, however, they do not supply access
to milk producing cells to investigate the effect of hormones,
M. Sharifi et al.
especially prolactin or estrogen, on development of breast cancer
cells. This challenge is very evident in all previous cell cultures.
10. Future applications
Based on breast cancer 3D bioprinting technology, the ability to
create a small and dynamic cancer tissue on a chip is provided for
extensive investigations of the pharmacological and toxicological effects
of drugs. The microfluidic flow in this chip can provide long-term,
continuous delivery of the drug with a more permanent presence of
BCT without stress generating agents such as shear stress of cells in the
simulation [137,138]. This technique can provide a promising ground
for drug tests even for personal medicine. In this way, by sampling the
breast cancer and stromal cells of the patient and culturing them on the
designed scaffolds, it is possible to examine more closely the pharma­
cological effects on the cancer tissue and its adverse effects. On the other
hand, the dose of anticancer drugs depend on the time and condition of
the BCT can be identified and administered with the least toxic effects in
the body by breast cancer 3D bioprinting on a chip.
Despite its complexity and exaggeration in fluidic properties, this
pattern is able to provide close and checkable connections between
cultured breast cancer cells with other cells in the vicinity of induced
drugs or compounds [139]. For instance, based on the microfluidic
spaces of Jeong, et al. [140] with the simultaneous culture of colon
cancer and fibroblast cells, they eventually revealed that cancerous cells
exhibit very high drug resistance in the presence of paclitaxel compared
to individual cultures. Therefore, recurrence of cancerous tumor after
treatment with anticancer drugs may be attributed to its weak effect on
cancerous cells in the vicinity of other cells in the real tissue which can
be investigated in 3D bioprinting of cancer cells and other cells on de­
vices such as microfluidics or chips.
11. Conclusion
In this literature, recent research on the advancement of BCT
designed on 3D scaffolds, bioprinting methods as well as the current
state of this technique are discussed. Consecutive research from the last
decade on 3-dimensionalization of BCT cultures is indicative of the
strategic application of this method in the treatment of breast cancer via
controlling the type of drug used and the concentration required.
However, the challenges facing this method, such as vascularization,
scaffold production, and its effects on BCT growth, cost and the limita­
tions of using materials in scaffolds indicate that further research on the
development of BCT on scaffolding is required. Further expansion of
native ECM, hydrogels development and surface modification of mate­
rials used in scaffolds, and the combination of printing techniques are
among the most important that can help advance 3D bioprinting ap­
plications in the personalized and general treatments of breast cancer.
Based on the rapid development of this technique, it is expected that
bioprinting products in the field of breast cancer will be commercialized
faster than anticipated so that patients with this disease can increase
their life expectancy by carefully processing the drug’s effects on the
cancer cell.
Acknowledgment
This article was made possible by Qatar National Research Fund (a
part of Qatar Foundation) through NPRP10-0120-170211 and
NPRP12S-0310-190276 grants. We acknowledge Key R&D and promo­
tion projects (Science and Technology Project) from Henan Science and
Technology Department (grant number 212102310127) to QB, and
Research grant from China Postdoctoral Science Foundation (grant
number 2020M672291) to SK. FC acknowledges the support from Elsa
U. Pardee Foundation. The statements made here are the sole re­
sponsibility of the authors.
104
Journal of Controlled Release 333 (2021) 91–106
References
[1] G. Rijal, W. Li, 3D scaffolds in breast cancer research, Biomaterials 81 (2016)
135–156.
[2] N. Wickramasinghe, 3D Printing in healthcare: opportunities, benefits, barriers,
and facilitators, in: Handbook of Research on Optimizing Healthcare
Management Techniques, IGI Global, 2020, pp. 220–227.
[3] C. Kengla, S.J. Lee, J.J. Yoo, A. Atala, 3-D bioprinting technologies for tissue
engineering applications, in: Rapid Prototyping of Biomaterials, Elsevier, 2020,
pp. 269–288.
[4] L. Xu, Z. Chu, H. Wang, L. Cai, Z. Tu, H. Liu, C. Zhu, H. Shi, D. Pan, J. Pan,
Electrostatically assembled multilayered films of biopolymer enhanced
nanocapsules for on-demand drug release, ACS Appl. Bio Mater. 2 (2019)
3429–3438.
[5] K. Fakhruddin, S.I.A. Razak, N.H.M. Nayan, M.R.A. Kadir, 3D bioprinting of a
tissue engineered human heart, in: Cardiovascular Engineering, Springer, 2020,
pp. 243–259.
[6] W.L. Ng, J.M. Lee, M. Zhou, W.Y. Yeong, Hydrogels for 3-D bioprinting-based
tissue engineering, in: Rapid Prototyping of Biomaterials, Elsevier, 2020,
pp. 183–204.
[7] M. Santschi, A. Vernengo, D. Eglin, M. D’Este, K. Wuertz-Kozak, Decellularized
matrix as a building block in bioprinting and electrospinning, Curr. Opin.
Biomed. Eng. 1 (2019) 1–10.
[8] P. Li, W. Zhang, L.J. Smith, D. Ayares, D.K. Cooper, B. Ekser, The potential role of
3D-bioprinting in xenotransplantation, Curr. Opin. Organ Transpl. 24 (2019) 547.
[9] S. Mehrotra, J.C. Moses, A. Bandyopadhyay, B.B. Mandal, 3D printing/
bioprinting based tailoring of in vitro tissue models: recent advances and
challenges, ACS Appl. Bio Mater. 2 (2019) 1385–1405.
[10] T. Liu, C. Delavaux, Y.S. Zhang, 3D bioprinting for oncology applications, J. 3D
Print. Med. 3 (2019) 55–58.
[11] F. Yu, D. Choudhury, Microfluidic bioprinting for organ-on-a-chip models, Drug
Discov. Today 1 (2019) 1–10.
[12] G. Rijal, C. Bathula, W. Li, Application of synthetic polymeric scaffolds in breast
Cancer 3D tissue cultures and animal tumor models, Int. J. Biomater. 2017
(2017).
[13] X. Ma, C. Yu, P. Wang, W. Xu, X. Wan, C.S.E. Lai, J. Liu, A. Koroleva-Maharajh,
S. Chen, Rapid 3D bioprinting of decellularized extracellular matrix with
regionally varied mechanical properties and biomimetic microarchitecture,
Biomaterials 185 (2018) 310–321.
[14] M.I. Diaz Bessone, M.J. Gattas, T. Laporte, M. Tanaka, M. Simian, The tumor
microenvironment as a regulator of endocrine resistance in breast cancer, Front.
Endocrinol. 10 (2019).
[15] S.S. Dykes, V.S. Hughes, J.M. Wiggins, H.O. Fasanya, M. Tanaka, D. Siemann,
Stromal cells in breast cancer as a potential therapeutic target, Oncotarget 9
(2018) 23761–23779.
[16] J. Insua-Rodríguez, T. Oskarsson, The extracellular matrix in breast cancer, Adv.
Drug Deliv. Rev. 97 (2016) 41–55.
[17] A.S. Dias, C.R. Almeida, L.A. Helguero, I.F. Duarte, Metabolic crosstalk in the
breast cancer microenvironment, Eur. J. Cancer 121 (2019) 154–171.
[18] M.K. Jena, J. Janjanam, Role of extracellular matrix in breast cancer
development: a brief update, F1000Research 7 (2018).
[19] M. Nawaz, N. Shah, B.R. Zanetti, M. Maugeri, R.N. Silvestre, F. Fatima, L. Neder,
H. Valadi, Extracellular vesicles and matrix remodeling enzymes: the emerging
roles in extracellular matrix remodeling, progression of diseases and tissue repair,
Cells 7 (2018) 167.
[20] F. Salvador, A. Martin, C. López-Menéndez, G. Moreno-Bueno, V. Santos,
A. Vázquez-Naharro, P.G. Santamaria, S. Morales, P.R. Dubus, L. Muinelo-Romay,
Lysyl oxidase–like protein LOXL2 promotes lung metastasis of breast cancer,
Cancer Res. 77 (2017) 5846–5859.
[21] K.A. Johnston, K.M. Lopez, Lysyl oxidase in cancer inhibition and metastasis,
Cancer Lett. 417 (2018) 174–181.
[22] J. Choi, J. Gyamfi, H. Jang, J.S. Koo, The role of tumor-associated macrophage in
breast cancer biology, Histol. Histopathol. 33 (2018) 133–145.
[23] S. Aras, M.R. Zaidi, TAMeless traitors: macrophages in cancer progression and
metastasis, Br. J. Cancer 117 (2017) 1583.
[24] Y. Ma, Y. Ren, Z.-J. Dai, C.-J. Wu, Y.-H. Ji, J. Xu, IL-6, IL-8 and TNF-α levels
correlate with disease stage in breast cancer patients, Adv. Clin. Exp. Med. 26
(2017) 421–426.
[25] P.K. Mandal, S. Biswas, G. Mandal, S. Purohit, A. Gupta, A. Majumdar, S.
R. Chowdhury, A. Bhattacharyya, CCL2 conditionally determines CCL22-
dependent Th2-accumulation during TGF-β-induced breast cancer progression,
Immunobiology 223 (2018) 151–161.
[26] H. Sun, C. Miao, W. Liu, X. Qiao, W. Yang, L. Li, C. Li, TGF-β1/TβRII/Smad3
signaling pathway promotes VEGF expression in oral squamous cell carcinoma
tumor-associated macrophages, Biochem. Biophys. Res. Commun. 497 (2018)
583–590.
[27] E.S. Stovgaard, D. Nielsen, E. Hogdall, E. Balslev, Triple negative breast
cancer–prognostic role of immune-related factors: a systematic review, Acta
Oncol. 57 (2018) 74–82.
[28] M. De Palma, D. Biziato, T.V. Petrova, Microenvironmental regulation of tumour
angiogenesis, Nat. Rev. Cancer 17 (2017) 457.
[29] Á. de Mingo Pulido, A. Gardner, S. Hiebler, H. Soliman, H.S. Rugo, M.F. Krummel,
L.M. Coussens, B. Ruffell, TIM-3 regulates CD103+ dendritic cell function and
response to chemotherapy in breast cancer, Cancer Cell 33 (2018) 60–74 (e66).
[30] M. Mego, H. Gao, E.N. Cohen, S. Anfossi, A. Giordano, S. Tin, T.M. Fouad, U. De
Giorgi, M. Giuliano, W.A. Woodward, Circulating tumor cells (CTCs) are
M. Sharifi et al.
associated with abnormalities in peripheral blood dendritic cells in patients with
inflammatory breast cancer, Oncotarget 8 (2017) 35656.
[31] Y.Y. Wang, C. Attané, D. Milhas, B. Dirat, S. Dauvillier, A. Guerard, J. Gilhodes,
I. Lazar, N. Alet, V. Laurent, Mammary adipocytes stimulate breast cancer
invasion through metabolic remodeling of tumor cells, JCI Insight 2 (2017).
[32] M. Hosney, S. Sabet, M. El-Shinawi, K.M. Gaafar, M.M. Mohamed, Leptin is
overexpressed in the tumor microenvironment of obese patients with estrogen
receptor positive breast cancer, Exp. Ther. Med. 13 (2017) 2235–2246.
[33] J. Incio, J.A. Ligibel, D.T. McManus, P. Suboj, K. Jung, K. Kawaguchi, M. Pinter,
S. Babykutty, S.M. Chin, T.D. Vardam, Obesity promotes resistance to anti-VEGF
therapy in breast cancer by up-regulating IL-6 and potentially FGF-2, Sci. Transl.
Med. 10 (2018) (eaag0945).
[34] D.-T. Chu, T. Nguyen Thi Phuong, N.L.B. Tien, D.-K. Tran, T.-T. Nguyen, V.
V. Thanh, T. Luu Quang, L.B. Minh, V.H. Pham, V.T.N. Ngoc, The effects of
adipocytes on the regulation of breast cancer in the tumor microenvironment: An
update, Cells 8 (2019) 857.
[35] J. Gyamfi, M. Eom, J.-S. Koo, J. Choi, Multifaceted roles of interleukin-6 in
adipocyte–breast cancer cell interaction, Transl. Oncol. 11 (2018) 275–285.
[36] M. Sharifi, A. Hasan, N.M.Q. Nanakali, A. Salihi, F.A. Qadir, H.A. Muhammad, M.
S. Shekha, F.M. Aziz, K.M. Amen, F. Najafi, Combined chemo-magnetic field-
photothermal breast cancer therapy based on porous magnetite nanospheres, Sci.
Rep. 10 (2020) 1–15.
[37] M. Sharifi, S. Jafari, A. Hasan, B.A. Paray, G. Gong, Y. Zheng, M. Falahati,
Antimetastatic activity of lactoferrin-coated mesoporous maghemite
nanoparticles in breast cancer enabled by combination therapy, ACS Biomater.
Sci. Eng. 6 (2020) 3574–3584.
[38] N. Eiro, L. González, A. Martínez-Ordoñez, B. Fernandez-Garcia, L.O. González,
S. Cid, F. Dominguez, R. Perez-Fernandez, F.J. Vizoso, Cancer-associated
fibroblasts affect breast cancer cell gene expression, invasion and angiogenesis,
Cell. Oncol. 41 (2018) 369–378.
[39] J. Suh, D.-H. Kim, Y.-J. Surh, Resveratrol suppresses migration, invasion and
stemness of human breast cancer cells by interfering with tumor-stromal cross-
talk, Arch. Biochem. Biophys. 643 (2018) 62–71.
[40] A. Bahrami, S.M. Hassanian, M. Khazaei, M. Hasanzadeh, S. Shahidsales,
M. Maftouh, G.A. Ferns, A. Avan, The therapeutic potential of targeting tumor
microenvironment in breast cancer: rational strategies and recent progress,
J. Cell. Biochem. 119 (2018) 111–122.
[41] T. Alkasalias, L. Moyano-Galceran, M. Arsenian-Henriksson, K. Lehti, Fibroblasts
in the tumor microenvironment: shield or spear? Int. J. Mol. Sci. 19 (2018) 1532.
[42] Q. Ma, L.C. Dieterich, M. Detmar, Multiple roles of lymphatic vessels in tumor
progression, Curr. Opin. Immunol. 53 (2018) 7–12.
[43] A.B. Hanker, M.V. Estrada, G. Bianchini, P.D. Moore, J. Zhao, F. Cheng, J.P. Koch,
L. Gianni, D.R. Tyson, V. Sánchez, Correction: extracellular matrix/integrin
signaling promotes resistance to combined inhibition of HER2 and PI3K in HER2
+ breast cancer, Cancer Res. 79 (2019) 873.
[44] H. Ha, B. Debnath, N. Neamati, Role of the CXCL8-CXCR1/2 axis in cancer and
inflammatory diseases, Theranostics 7 (2017) 1543.
[45] H. Lv, L. Li, M. Sun, Y. Zhang, L. Chen, Y. Rong, Y. Li, Mechanism of regulation of
stem cell differentiation by matrix stiffness, Stem Cell Res Ther 6 (2015)
(103–103).
[46] M.C. Lampi, C.A. Reinhart-King, Targeting extracellular matrix stiffness to
attenuate disease: From molecular mechanisms to clinical trials, Sci. Transl. Med.
10 (2018) (eaao0475).
[47] V. Gkretsi, T. Stylianopoulos, Cell adhesion and matrix stiffness: coordinating
cancer cell invasion and metastasis, Front. Oncol. 8 (2018).
[48] C.E. Barcus, K.A. O’Leary, J.L. Brockman, D.E. Rugowski, Y. Liu, N. Garcia, M. Yu,
P.J. Keely, K.W. Eliceiri, L.A. Schuler, Elevated collagen-I augments tumor
progressive signals, intravasation and metastasis of prolactin-induced estrogen
receptor alpha positive mammary tumor cells, Breast Cancer Res. 19 (2017) 9.
[49] S. Fiorino, S. Di Saverio, P. Leandri, A. Tura, C. Birtolo, M. Silingardi, D. De Biase,
E. Avisar, The role of matricellular proteins and tissue stiffness in breast cancer: a
systematic review, Future Oncol. 14 (2018) 1601–1627.
[50] A. Evans, P. Rauchhaus, P. Whelehan, K. Thomson, C.A. Purdie, L.B. Jordan, C.
O. Michie, A. Thompson, S. Vinnicombe, Does shear wave ultrasound
independently predict axillary lymph node metastasis in women with invasive
breast cancer? Breast Cancer Res. Treat. 143 (2014) 153–157.
[51] M. Cavo, M. Fato, L. Peñuela, F. Beltrame, R. Raiteri, S. Scaglione,
Microenvironment complexity and matrix stiffness regulate breast cancer cell
activity in a 3D in vitro model, Sci. Rep. 6 (2016) 35367.
[52] M.-F. Pang, M.J. Siedlik, S. Han, M. Stallings-Mann, D.C. Radisky, C.M. Nelson,
Tissue stiffness and hypoxia modulate the integrin-linked kinase ILK to control
breast cancer stem-like cells, Cancer Res. 76 (2016) 5277–5287.
[53] W. Linthicum, M.-T. Thanh, M. Vitolo, Q. Wen, Effects of PTEN loss and activated
KRAS overexpression on mechanical properties of breast epithelial cells, Int. J.
Mol. Sci. 19 (2018) 1613.
[54] H.J.J. van Rensburg, D. Lai, T. Azad, Y. Hao, X. Yang, TAZ enhances mammary
cell proliferation in 3D culture through transcriptional regulation of IRS1, Cell.
Signal. 52 (2018) 12–22.
[55] J. Geisel, M. Raghu, R. Hooley, The role of ultrasound in breast cancer screening:
the case for and against ultrasound, seminars in ultrasound, Semin. Ultrasound CT
MR 39 (2018) 25–34.
[56] S.H. Medina, B. Bush, M. Cam, E. Sevcik, F.W. DelRio, K. Nandy, J.P. Schneider,
Identification of a mechanogenetic link between substrate stiffness and
chemotherapeutic response in breast cancer, Biomaterials 202 (2019) 1–11.
105
Journal of Controlled Release 333 (2021) 91–106
[57] J. Fenner, A.C. Stacer, F. Winterroth, T.D. Johnson, K.E. Luker, G.D. Luker,
Macroscopic stiffness of breast tumors predicts metastasis, Sci. Rep. 4 (2014)
5512.
[58] H. Qiao, T. Tang, Engineering 3D approaches to model the dynamic
microenvironments of cancer bone metastasis, Bone Res. 6 (2018) 3.
[59] L. Bray, C. Secker, B. Murekatete, J. Sievers, M. Binner, P. Welzel, C. Werner,
Three-dimensional in vitro hydro-and cryogel-based cell-culture models for the
study of breast-cancer metastasis to bone, Cancers 10 (2018) 292.
[60] X. Yue, T.D. Nguyen, V. Zellmer, S. Zhang, P. Zorlutuna, Stromal cell-laden 3D
hydrogel microwell arrays as tumor microenvironment model for studying
stiffness dependent stromal cell-cancer interactions, Biomaterials 170 (2018)
37–48.
[61] B.A. Morris, B. Burkel, S.M. Ponik, J. Fan, J.S. Condeelis, J.A. Aguirre-Ghiso,
J. Castracane, J.M. Denu, P.J. Keely, Collagen matrix density drives the metabolic
shift in breast cancer cells, EBioMedicine 13 (2016) 146–156.
[62] D. Huang, Y. Nakamura, A. Ogata, S. Kidoaki, Characterization of 3D matrix
conditions for cancer cell migration with elasticity/porosity-independent tunable
microfiber gels, Polym. J. (2019) 1–12.
[63] T.H. Qazi, D.J. Mooney, G.N. Duda, S. Geissler, Biomaterials that promote cell-
cell interactions enhance the paracrine function of MSCs, Biomaterials 140 (2017)
103–114.
[64] J. Azevedo-Silva, O. Queirós, A. Ribeiro, F. Baltazar, K.H. Young, P.L. Pedersen,
A. Preto, M. Casal, The cytotoxicity of 3-bromopyruvate in breast cancer cells
depends on extracellular pH, Biochem. J. 467 (2015) 247–258.
[65] M. Bohloli, A. Atashi, M. Soleimani, S. Kaviani, A. Anbarlou, Investigating effects
of acidic pH on proliferation, invasion and drug-induced apoptosis in
lymphoblastic leukemia, Cancer Microenviron. 9 (2016) 119–126.
[66] J.J. Campbell, A. Husmann, R.D. Hume, C.J. Watson, R.E. Cameron, Development
of three-dimensional collagen scaffolds with controlled architecture for cell
migration studies using breast cancer cell lines, Biomaterials 114 (2017) 34–43.
[67] O. Yersal, S. Barutca, Biological subtypes of breast cancer: prognostic and
therapeutic implications, World J. Clin. Oncol. 5 (2014) 412–424.
[68] M. Van Moorst, C.R. Dass, Methods for co-culturing tumour and endothelial cells:
systems and their applications, J. Pharm. Pharmacol. 63 (2011) 1513–1521.
[69] M. Patrikoski, J. Sivula, H. Huhtala, M. Helminen, F. Salo, B. Mannerström,
S. Miettinen, Different culture conditions modulate the immunological properties
of adipose stem cells, Stem Cells Transl. Med. 3 (2014) 1220–1230.
[70] F.N. Alaribe, S.L. Manoto, S.C. Motaung, Scaffolds from biomaterials: advantages
and limitations in bone and tissue engineering, Biologia 71 (2016) 353–366.
[71] E. Garreta, R. Oria, C. Tarantino, M. Pla-Roca, P. Prado, F. Fernández-Avilés, J.
M. Campistol, J. Samitier, N. Montserrat, Tissue engineering by decellularization
and 3D bioprinting, Mater. Today 20 (2017) 166–178.
[72] S. Radhakrishnan, S. Nagarajan, M. Bechelany, S.N. Kalkura, Collagen based
biomaterials for tissue engineering applications: a review, in: Processes and
Phenomena on the Boundary Between Biogenic and Abiogenic Nature, Springer,
2020, pp. 3–22.
[73] Z. Raglow, S.M. Thomas, Tumor matrix protein collagen XIα1 in cancer, Cancer
Lett. 357 (2015) 448–453.
[74] D. Zhang, X. Wu, J. Chen, K. Lin, The development of collagen based composite
scaffolds for bone regeneration, Bioact. Mater. 3 (2018) 129–138.
[75] M.P. Linnes, B.D. Ratner, C.M. Giachelli, A fibrinogen-based precision
microporous scaffold for tissue engineering, Biomaterials 28 (2007) 5298–5306.
[76] G. Yang, Z. Xiao, H. Long, K. Ma, J. Zhang, X. Ren, J. Zhang, Assessment of the
characteristics and biocompatibility of gelatin sponge scaffolds prepared by
various crosslinking methods, Sci. Rep. 8 (2018) 1616.
[77] M.N. Collins, C. Birkinshaw, Hyaluronic acid based scaffolds for tissue
engineering—a review, Carbohydr. Polym. 92 (2013) 1262–1279.
[78] S. Ahmed, A. Ali, J. Sheikh, A review on chitosan centred scaffolds and their
applications in tissue engineering, Int. J. Biol. Macromol. 116 (2018) 849–862.
[79] M. Farokhi, F. Jonidi Shariatzadeh, A. Solouk, H. Mirzadeh, Alginate based
scaffolds for cartilage tissue engineering: a review, Int. J. Polym. Mater. Polym.
Biomater. (2019) 1–18.
[80] L. Xu, V. Selin, A. Zhuk, J.F. Ankner, S.A. Sukhishvili, Molecular weight
dependence of polymer chain mobility within multilayer films, ACS Macro Lett. 2
(2013) 865–868.
[81] N. Iqbal, A.S. Khan, A. Asif, M. Yar, J.W. Haycock, I.U. Rehman, Recent concepts
in biodegradable polymers for tissue engineering paradigms: a critical review, Int.
Mater. Rev. 64 (2019) 91–126.
[82] J.W. Stansbury, M.J. Idacavage, 3D printing with polymers: challenges among
expanding options and opportunities, Dent. Mater. 32 (2016) 54–64.
[83] Y. Lu, A. Zhuk, L. Xu, X. Liang, E. Kharlampieva, S.A. Sukhishvili, Tunable pH and
temperature response of weak polyelectrolyte brushes: role of hydrogen bonding
and monomer hydrophobicity, Soft Matter 9 (2013) 5464–5472.
[84] C.R. Almeida, T. Serra, M.I. Oliveira, J.A. Planell, M.A. Barbosa, M. Navarro,
Impact of 3-D printed PLA-and chitosan-based scaffolds on human monocyte/
macrophage responses: unraveling the effect of 3-D structures on inflammation,
Acta Biomater. 10 (2014) 613–622.
[85] E. Polonio-Alcalá, M. Rabionet, X. Gallardo, D. Angelats, J. Ciurana, S. Ruiz-
Martínez, T. Puig, PLA electrospun scaffolds for three-dimensional triple-negative
breast cancer cell culture, Polymers 11 (2019) 916.
[86] G. Brunello, S. Sivolella, R. Meneghello, L. Ferroni, C. Gardin, A. Piattelli,
B. Zavan, E. Bressan, Powder-based 3D printing for bone tissue engineering,
Biotechnol. Adv. 34 (2016) 740–753.
[87] M. Maniruzzaman, 3D and 4D Printing in Biomedical Applications: Process
Engineering and Additive Manufacturing, Wiley-VCH, 2019.
M. Sharifi et al.
[88] H.N. Chia, B.M. Wu, Recent advances in 3D printing of biomaterials, J. Biol. Eng.
9 (2015) 4.
[89] A. Bharadwaz, A.C. Jayasuriya, Recent trends in the application of widely used
natural and synthetic polymer nanocomposites in bone tissue regeneration,
Mater. Sci. Eng. C 110698 (2020).
[90] M. Rücker, M.W. Laschke, D. Junker, C. Carvalho, F. Tavassol, R. Mülhaupt, N.
C. Gellrich, M.D. Menger, Vascularization and biocompatibility of scaffolds
consisting of different calcium phosphate compounds, J. Biomed. Mater. Res. Part
A 86 (2008) 1002–1011.
[91] X. Zhang, Z. Li, P. Yang, G. Duan, X. Liu, Z. Gu, Y. Li, Polyphenol scaffolds in
tissue engineering, Mater. Horizons 1 (2021) 1–10.
[92] A.K. Badekila, S. Kini, A.K. Jaiswal, Fabrication techniques of biomimetic
scaffolds in three-dimensional cell culture: a review, J. Cell. Physiol. 236 (2021)
741–762.
[93] J. He, X. Hu, J. Cao, Y. Zhang, J. Xiao, D. Chen, C. Xiong, L. Zhang, Chitosan-
coated hydroxyapatite and drug-loaded polytrimethylene carbonate/polylactic
acid scaffold for enhancing bone regeneration, Carbohydr. Polym. 253 (2021)
117198.
[94] F. Ghorbani, D. Li, S. Ni, Y. Zhou, B. Yu, 3D printing of acellular scaffolds for bone
defect regeneration: a review, Mater. Today Commun. 22 (2020) 100979.
[95] F. Zhang, M.W. King, Biodegradable polymers as the pivotal player in the design
of tissue engineering scaffolds, Adv. Healthc. Mater. 9 (2020) 1901358.
[96] H. Jodati, B. Yılmaz, Z. Evis, A review of bioceramic porous scaffolds for hard
tissue applications: effects of structural features, Ceram. Int. 46 (2020)
15725–15739.
[97] C. Mandrycky, Z. Wang, K. Kim, D.-H. Kim, 3D bioprinting for engineering
complex tissues, Biotechnol. Adv. 34 (2016) 422–434.
[98] Z. Wang, M.A. Andrews, Z.-X. Wu, L. Wang, C.T. Bauch, Coupled
disease–behavior dynamics on complex networks: a review, Phys Life Rev 15
(2015) 1–29.
[99] S.V. Murphy, A. Atala, 3D bioprinting of tissues and organs, Nat. Biotechnol. 32
(2014) 773.
[100] V. Keriquel, H. Oliveira, M. Rémy, S. Ziane, S. Delmond, B. Rousseau, S. Rey,
S. Catros, J. Amédée, F. Guillemot, In situ printing of mesenchymal stromal cells,
by laser-assisted bioprinting, for in vivo bone regeneration applications, Sci. Rep.
7 (2017) 1778.
[101] P. Agarwal, H. Wang, M. Sun, J. Xu, S. Zhao, Z. Liu, K.J. Gooch, Y. Zhao, X. Lu,
X. He, Microfluidics enabled bottom-up engineering of 3D vascularized tumor for
drug discovery, ACS Nano 11 (2017) 6691–6702.
[102] G. Bendas, L. Borsig, Cancer cell adhesion and metastasis: selectins, integrins, and
the inhibitory potential of heparins, Int. J. Cell Biol. 2012 (2012).
[103] W. Zhu, B. Holmes, R.I. Glazer, L.G. Zhang, 3D printed nanocomposite matrix for
the study of breast cancer bone metastasis, Nanomedicine 12 (2016) 69–79.
[104] X. Zhou, W. Zhu, M. Nowicki, S. Miao, H. Cui, B. Holmes, R.I. Glazer, L.G. Zhang,
3D bioprinting a cell-laden bone matrix for breast cancer metastasis study, ACS
Appl. Mater. Interfaces 8 (2016) 30017–30026.
[105] E.R. Horton, J.D. Humphries, J. James, M.C. Jones, J.A. Askari, M.J. Humphries,
The integrin adhesome network at a glance, J. Cell Sci. 129 (2016) 4159–4163.
[106] D. Mondal, M. Griffith, S.S. Venkatraman, Polycaprolactone-based biomaterials
for tissue engineering and drug delivery: current scenario and challenges, Int. J.
Polym. Mater. Polym. Biomater. 65 (2016) 255–265.
[107] M. Pal, H. Chen, B.H. Lee, J.Y.H. Lee, Y.S. Yip, N.S. Tan, L.P. Tan, Epithelial-
mesenchymal transition of cancer cells using bioengineered hybrid scaffold
composed of hydrogel/3D-fibrous framework, Sci. Rep. 9 (2019) (8997–8997).
[108] H. Eslami Amirabadi, M. Tuerlings, A. Hollestelle, S. SahebAli, R. Luttge, C.C. van
Donkelaar, J.W.M. Martens, J.M.J. den Toonder, Characterizing the invasion of
different breast cancer cell lines with distinct E-cadherin status in 3D using a
microfluidic system, Biomed. Microdevices 21 (2019) 101.
[109] S. Palomeras, M. Rabionet, I. Ferrer, A. Sarrats, M.L. Garcia-Romeu, T. Puig,
J. Ciurana, Breast cancer stem cell culture and enrichment using poly(epsilon-
Caprolactone) scaffolds, Molecules 21 (2016) 537.
[110] M. Domingos, F. Intranuovo, T. Russo, R. De Santis, A. Gloria, L. Ambrosio,
J. Ciurana, P. Bartolo, The first systematic analysis of 3D rapid prototyped poly
(ε-caprolactone) scaffolds manufactured through BioCell printing: the effect of
pore size and geometry on compressive mechanical behaviour and in vitro hMSC
viability, Biofabrication 5 (2013), 045004.
[111] Y. Imamura, T. Mukohara, Y. Shimono, Y. Funakoshi, N. Chayahara, M. Toyoda,
N. Kiyota, S. Takao, S. Kono, T. Nakatsura, Comparison of 2D-and 3D-culture
models as drug-testing platforms in breast cancer, Oncol. Rep. 33 (2015)
1837–1843.
[112] P. Casbas-Hernandez, In Vitro Coculture Models to Study Heterotypic Interactions
in Breast Cancer Microenvironments, 2013.
[113] S.P. Pathi, D.D. Lin, J.R. Dorvee, L.A. Estroff, C. Fischbach, Hydroxyapatite
nanoparticle-containing scaffolds for the study of breast cancer bone metastasis,
Biomaterials 32 (2011) 5112–5122.
[114] G.M. Balachander, S.A. Balaji, A. Rangarajan, K. Chatterjee, Enhanced metastatic
potential in a 3D tissue scaffold toward a comprehensive in vitro model for breast
cancer metastasis, ACS Appl. Mater. Interfaces 7 (2015) 27810–27822.
106
Journal of Controlled Release 333 (2021) 91–106
[115] G. Rijal, W. Li, A versatile 3D tissue matrix scaffold system for tumor modeling
and drug screening, Sci. Adv. 3 (2017), e1700764.
[116] T. Hoshiba, Decellularized extracellular matrix for cancer research, Materials 12
(2019) 1311.
[117] B.T. Vinson, T.B. Phamduy, J. Shipman, B. Riggs, A.L. Strong, S.C. Sklare, W.
L. Murfee, M.E. Burow, B.A. Bunnell, Y. Huang, Laser direct-write based
fabrication of a spatially-defined, biomimetic construct as a potential model for
breast cancer cell invasion into adipose tissue, Biofabrication 9 (2017), 025013.
[118] K. Jakab, C. Norotte, B. Damon, F. Marga, A. Neagu, C.L. Besch-Williford,
A. Kachurin, K.H. Church, H. Park, V. Mironov, Tissue engineering by self-
assembly of cells printed into topologically defined structures, Tissue Eng. A 14
(2008) 413–421.
[119] Y. Yu, K.K. Moncal, J. Li, W. Peng, I. Rivero, J.A. Martin, I.T. Ozbolat, Three-
dimensional bioprinting using self-assembling scalable scaffold-free “tissue
strands” as a new bioink, Sci. Rep. 6 (2016) 28714.
[120] T. Jiang, J.G. Munguia-Lopez, S. Flores-Torres, J. Grant, S. Vijayakumar, A. De
Leon-Rodriguez, J.M. Kinsella, Directing the self-assembly of tumour spheroids by
bioprinting cellular heterogeneous models within alginate/gelatin hydrogels, Sci.
Rep. 7 (2017) 4575.
[121] N.E. Fedorovich, J. Alblas, J.R. de Wijn, W.E. Hennink, A.J. Verbout, W.J. Dhert,
Hydrogels as extracellular matrices for skeletal tissue engineering: state-of-the-art
and novel application in organ printing, Tissue Eng. 13 (2007) 1905–1925.
[122] K.E. Kasza, A.C. Rowat, J. Liu, T.E. Angelini, C.P. Brangwynne, G.H. Koenderink,
D.A. Weitz, The cell as a material, Curr. Opin. Cell Biol. 19 (2007) 101–107.
[123] G. Forgacs, R.A. Foty, Y. Shafrir, M.S. Steinberg, Viscoelastic properties of living
embryonic tissues: a quantitative study, Biophys. J. 74 (1998) 2227–2234.
[124] J.T. Butcher, T.C. McQuinn, D. Sedmera, D. Turner, R.R. Markwald, Transitions in
early embryonic atrioventricular valvular function correspond with changes in
cushion biomechanics that are predictable by tissue composition, Circ. Res. 100
(2007) 1503–1511.
[125] L.Y. Leung, D. Tian, C.P. Brangwynne, D.A. Weitz, D.J. Tschumperlin, A new
microrheometric approach reveals individual and cooperative roles for TGF-β1
and IL-1β in fibroblast-mediated stiffening of collagen gels, FASEB J. 21 (2007)
2064–2073.
[126] S. Swaminathan, Q. Hamid, W. Sun, A.M. Clyne, Bioprinting of 3D breast
epithelial spheroids for human cancer models, Biofabrication 1 (2019) 1–10.
[127] I. Januškevičienė, V. Petrikaitė, Heterogeneity of breast cancer: the importance of
interaction between different tumor cell populations, Life Sci. 239 (2019)
117009.
[128] G. Turashvili, E. Brogi, Tumor heterogeneity in breast cancer, Front. Med.
(Lausanne) 4 (2017) (227–227).
[129] L.E. Bertassoni, M. Cecconi, V. Manoharan, M. Nikkhah, J. Hjortnaes, A.
L. Cristino, G. Barabaschi, D. Demarchi, M.R. Dokmeci, Y. Yang, Hydrogel
bioprinted microchannel networks for vascularization of tissue engineering
constructs, Lab Chip 14 (2014) 2202–2211.
[130] T.J. Hinton, Q. Jallerat, R.N. Palchesko, J.H. Park, M.S. Grodzicki, H.-J. Shue, M.
H. Ramadan, A.R. Hudson, A.W. Feinberg, Three-dimensional printing of complex
biological structures by freeform reversible embedding of suspended hydrogels,
Sci. Adv. 1 (2015), e1500758.
[131] H. Cui, T. Esworthy, X. Zhou, S.Y. Hann, R.I. Glazer, R. Li, L.G. Zhang,
Engineering a novel 3D printed vascularized tissue model for investigating breast
cancer metastasis to bone, Adv. Healthc. Mater. 1 (2019) 1–10.
[132] B. Subia, T. Dey, S. Sharma, S.C. Kundu, Target specific delivery of anticancer
drug in silk fibroin based 3D distribution model of bone–breast cancer cells, ACS
Appl. Mater. Interfaces 7 (2015) 2269–2279.
[133] L. Xu, H. Wang, Z. Chu, L. Cai, H. Shi, C. Zhu, D. Pan, J. Pan, X. Fei, Y. Lei,
Temperature-responsive multilayer films of micelle-based composites for
controlled release of a third-generation EGFR inhibitor, ACS Appl. Polym. Mater.
2 (2020) 741–750.
[134] S.M. King, S.C. Presnell, D.G. Nguyen, Development of 3D Bioprinted Human
Breast Cancer for In Vitro Drug Screening, AACR, 2014.
[135] K. Guiro, S.A. Patel, S.J. Greco, P. Rameshwar, T.L. Arinzeh, Investigating breast
cancer cell behavior using tissue engineering scaffolds, PLoS One 10 (2015)
(e0118724-e0118724).
[136] Y. Wang, W. Shi, M. Kuss, S. Mirza, D. Qi, A. Krasnoslobodtsev, J. Zeng, H. Band,
V. Band, B. Duan, 3D bioprinting of breast cancer models for drug resistance
study, ACS Biomater. Sci. Eng. 4 (2018) 4401–4411.
[137] M.C. Regier, E.T. Alarid, D.J. Beebe, Progress towards understanding heterotypic
interactions in multi-culture models of breast cancer, Integr. Biol. (Camb.) 8
(2016) 684–692.
[138] M. Sharifi, A. Hasan, F. Attar, A. Taghizadeh, M. Falahati, Development of point-
of-care nanobiosensors for breast cancers diagnosis, Talanta 217 (2020) 121091.
[139] M. Shang, R.H. Soon, C.T. Lim, B.L. Khoo, J. Han, Microfluidic modelling of the
tumor microenvironment for anti-cancer drug development, Lab Chip 19 (2019)
369–386.
[140] S.Y. Jeong, J.H. Lee, Y. Shin, S. Chung, H.J. Kuh, Co-culture of tumor spheroids
and fibroblasts in a collagen matrix-incorporated microfluidic chip mimics
reciprocal activation in solid tumor microenvironment, PLoS One 11 (2016),
e0159013.