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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5346_Библиотеки_им_академика_М_И_Перельмана
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156 Xuan Mu and Yu Shrike Zhang
immune cells, tumor antigens, and immune cell suppression, which underlie complex
interactions within the immune system and therapeutic optimization. Another challenge
is the lack of organ/tissue-specific microenvironment that is a driving force in the growth
and metastasis of tumors [4]. Many tumors exhibit tissue-specific mechanisms for metastasis and escaping immune surveillance, which is associated with prognostic outcomes.
The cancer immunotherapy, targeting the organ/tissue-specific microenvironment, is
expected to show high efficacy [5]. The third challenge is the production and expansion
of therapeutic cells for cancer immunotherapy. The conventional methods are largely
based on bulky and population-based analysis of cells, which are time-consuming, expensive, and low in efficiency [6], leading to the cost of half a million dollars for one infusion
of engineered T cells. On the contrary, single-cell analysis is promising to enhance the
production efficiency of therapeutic cells.
These challenges represent bottlenecks in the development and the widespread use of
canner immunotherapy, thus not trivial. Yet, addressing these challenges also presents
vast opportunities for the development and adoption of novel engineered materials
and devices. In particular, tumor-on-chips, broadly based on the technology of microfluidics, represent a promising tool for tackling several challenges of cancer immunotherapy and has shown a vast potential for benefiting cancer immunotherapy in multiple
aspects, such as cell screening, modulating the microenvironment, and producing therapeutic cells (Fig. 1) [7–9]. A tumor-on-a-chip is a bioengineered microdevice, as an in
Fig. 1 Overview of Tumor-on-a-Chip technology for cancer immunotherapy. The broad utility of
tumor-on-a-chip is illustrated in several specific examples, including extravasation, single-cell screening, and intracellular delivery, which are discussed in this chapter.
vitro model, created by advanced microfabrication techniques and the use of human/
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patient cells. Tumor-on-a-chip enables the detailed and dynamic studies of cellular
responses, often inaccessible to the conventional method of tissue culturing; tumoron-a-chip also allows the recapitulation of pathophysiological features of one or multiple
tissues instead of a whole animal. Furthermore, by using patient-derived cells, tumor-onchip is promising to emulate patient-specific conditions for personalized medicine.
Tumor-on-a-chip thus is roughly positioned between ex vivo tissue culture and in vivo
animal studies. Besides mimicking the physiological essentials, tumor-on-a-chip leverages the microfabricated structures and unique flow phenomena at the microscale to
manipulate cells with great flexibility [10].
In this chapter, we introduce essential concepts and state-of-art techniques of microfluidics and then discuss the crucial roles of microfluidics in cancer immunotherapy,
including the recapitulation of the tumor microenvironment, the production of therapeutic cells/materials, and the screening of immune cells. A focus is placed on translating technical advances into meeting the biomedical and clinical needs in cancer
immunotherapy. At the end of this chapter, we briefly summarize the current challenges
and envision the future development of tumor-on-a-chip techniques for cancer
immunotherapy.
157Tumor-on-a-chip devices for cancer immunotherapy
2. Microfluidics
Microfluidics refers to the control and manipulation of flow at the microscale
[11,12]. A key feature of microfluidics is a network of microchannels integrated into a
palm-sized or smaller chip. At the early stage, microfluidic chips are fabricated in glass
and silicone by photolithography and thermal bonding; the glass microfluidic chips
are widely used for chemical analysis, for example, electrophoresis [13,14]. Later, microfluidic chips are fabricated in polydimethylsiloxane (PDMS), a silicone rubber [15,16],by
soft lithography (Fig. 2) [17,18]. The PDMS microfluidic chips enable high-density and
miniaturized valves on the chip [19,20], and the recapitulation of physiological-relevant
tissue deformation, such as the distention of lung alveoli during gas-exchanging [21] and
the dilation of blood vessel accompanied with the beating heart [22]. New fabrication
techniques, such as three-dimensional (3D) printing [23–26], and new materials, such
as hydrogels [27,28], and extracellular matrix (ECM) [29], have been introduced into
the field of microfluidics [30], further extending the overall utility of microfluidic chips.
Besides the microfabricated structures, the fluid flow within the microchannel has been
exploited as potent engineering tools for manipulating cells in an unprecedented manner
[10]. By harnessing the bioengineered microstructures and flow phenomena, micro-
fluidics has been shown to mimic a range of physiological structures and functions, i.e.,
organ-on-chips [31–36] and tumor-on-chips [8,9,37,38].
158 Xuan Mu and Yu Shrike Zhang
Fig. 2 (A) Illustration of fabrication procedure of PDMS microfluidic chips via soft lithography.
(B) Laminar flow is a spontaneous phenomenon in the microchannels due to the small length scale,
apposite to turbulent flow. (C) Multiple (5 illustrated) streams merge in the main channel flowing in
parallel with each other. (D) Highly uniform aqueous droplets are continuously generated by flowfocusing within intersected microchannels. The aqueous droplets are wrapped by the oil phase.
2.1 Microfabrication
A wide range of fabrication methods and materials are available for rendering microfluidic
chips with various properties and functions [39]. Soft lithography with PDMS is likely the
most widely used approach for creating microfluidic chips due to the convenient fabrication process and the intriguing properties of PDMS, including elasticity, gas permeability, and visible transparency. Soft lithography gives rise to a mold with fine and embossed
structures by spin coating of photoresist on a substrate, light exposure, and photoresist
development (Fig. 2A). Other fabrication methods, including machining, laser cutting,
and 3D printing, may also apply to microfluidic chips, depending on the desired resolution and the used materials. After obtaining the mold, the PDMS solution is poured onto
the mold and cured by heating to replicate the structure inversly; the embossed structures
of the mold become a concaving one. The PDMS solution contains two mixed components: base resin and curing agent [40]. The mass ratio between the base resin and the
curing agent is usually 10:1 and is associated with the mechanical property of PDMS.
A higher ratio of the curing agent leads to a higher stiffness [41]. The elasticity of PDMS
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facilitates the demolding, and thus, the fine structure at the submicron resolution can be
faithfully replicated. One PDMS slab with concave structures is treated with air/oxygen
plasma, followed by bonding with another PDMS slab. The plasma treatment leads to
temporary, reactive silanol groups on the surface of the PDMS slab that forms covalent
bonding (SidOdSi). Despite the widespread use, several drawbacks have limited the
applications of PDMS. The production of PDMS-based structures or chips is challenging
to scaleup, in comparison to other thermoplastics, such as polystyrene (PS) and polypropylene. PDMS also suffers from the bulk absorption of small hydrophobic molecules [42]
and the swelling in certain organic solvents [16], limiting the scope of utility. An increasing number of materials have been adopted for making microfluidic chips, including
hydrogels [28,43], Teflon [44], and a range of thermoplastics [30,45,46]. These materials
usually exhibit advantages in specific aspects of fabrication and utility; however, none of
them can fully replace PDMS in the field of microfluidics.
Another emerging fabrication approach for microfluidics is 3D printing and bioprinting [47–51]. Compared with soft lithography, 3D printing is flexible in 3D complex
structures and rapid turnaround time due to the absence of molds and photomasks. One
light-assisted 3D printing approach has been developed to fabricate PDMS microfluidic
chips by using methacryloxypropyl-substituted PDMS macromers and a PDMSdissolvable photoinitiator, ethyl (2,4,6-trimethylbenzoyl) phenylphosphinate [52] . The
3D-printed PDMS microfluidic chips are comparable to that made by conventional
moldable PDMS (Sylgard 184) and soft lithography in terms of optical transparency, elasticity, and cytocompatibility. Note that 3D printing is advantageous in fabrication resolution and automation processes. Besides PDMS, other polymers, such as poly(ethylene
glycol)-diacrylate (PEG-DA), can be 3D printed for making microfluidic chips [53,54].
PEG-DA with different molecular weights is commercially available, which provides a
convenient way to tune the material properties of the PEG-DA microfluidic chips. For
example, PEG-DA with a molecular weight of 700 Da is more permeable than that with
280-Da molecular weight and provides suitable material for the investigation of mass
transfer between channels [54].
On the other hand, hydrogels, especially ECM-derived ones, have become the predominant ink components for 3D-bioprinted microfluidic systems due to the high content of water and the capability to encapsulate cells [55]. For extrusion-based 3D printing,
sacrificial materials, such as Pluronic F127 (PF127), are frequently used to generate hollow hydrogel microchannels [56–58]. PF127 is printed in the shape of the microfilament,
followed by printing or casting of another hydrogel, such as collagen/gelatin, to cover the
PF127 print. After elevating temperature from room temperature to 37°C, collagen/gelatin forms gels from solution; at a low temperature of 4°C, PF127 liquefies and is conveniently removed by aspiration. The hollow microchannel, embedded in a hydrogel,
can also be made using a liquid mold [28] or a metal needle [59]. Although sacrificial
159Tumor-on-a-chip devices for cancer immunotherapy
160 Xuan Mu and Yu Shrike Zhang
materials are often necessary, the structure of hollow channels can be printed by optimizing the gelation dynamics [60], which streamlines the printing process and eliminates the
need to remove the sacrificial materials. For light-based printing, it is possible to print
hollow channels due to the rapid dynamics of photo-crosslinking. Of note, it is necessary
to flush out the uncured ink from the channels to prevent clogging [50].
2.2 Flow at the microscale
Another essential feature of microfluidics is the intriguing fluid flow, including laminar
flow and droplets, within channels at the small length scale [61–63]. These fluid flow phenomena are associated with the small length scale and the property of liquids, thus occurring in the absence of expensive instruments and providing a convenient
engineering tool.
Laminar flow is characterized by the orderly and parallel streamline, opposite to the
eddy-like and random ones in the turbulent flow (Fig. 2B). One dimensionless number,
Reynolds number (Re), is used to roughly differentiate laminar and turbulent flow [61],as
described:
ρvd
Re ¼
μ
3
where ρ is the density (kg m
microchannel or object in the fluid (m), and μ is the viscosity of the liquid (kg m
Re has no unit, thus dimensionless, and refers to the balance between inertial and viscous
forces [61]. When Re is small (usually <2300), the inertial force is dominant, leading to
the dominance of laminar flow; when Re is large (>4000), the viscous force is dominant,
leading to the dominance of turbulent flow. Of note, even under a small Re number and
thus a laminar flow-dominated condition, the turbulent flow may still occur in tapered
and curving channels, such as Dean vertex. The synergy of the dominated laminar flow
and the controlled turbulent flow is useful for screening cells with different sizes and densities [64]. One typical example to demonstrate laminar flow is to flow multiple streams
into one main channel (Fig. 2B). Due to the parallel streamlines, all streams flow in a
parallel and orderly manner. In particular, the orderly pattern of the multistream laminar
flow has been exploited for a wide range of applications, including cell and material
patterning [65–68], solvent extraction [69,70], and fuel cell [71]. In addition, the mass
transfer under the condition of laminar flow is dominated by diffusion rather than convection. Using diffusion distance and time provides a convenient way to manipulate
gradients of a range of soluble cues relevant to immune surveillance and the tumor
microenvironment [72].
Unlike the multistream laminar flow that is composed of miscible liquids, microfluidic droplets, at the volume range of nanoliter to femtoliter, formed in two immiscible
liquids (Fig. 2D) [73,74]. One liquid phase is discrete and wrapped in the other
), v is the flow velocity (m s1), d is the length scale of
1s1
).
continuous liquid phase. The formation of droplets within microchannels has been found
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to relate to a dimensionless number, capillary number (Ca) [75], as shown,
vμ
Ca ¼
γ
where v is the flow velocity (m s
the surface tension between the two immiscible liquids (kg s
1
), μ is the viscosity of the liquid (kg m1s1), and γ is
2
). Ca number also refers to
the balance between the viscous force and the interfacial force. In most cases, a smaller Ca
number indicates a higher interfacial force between two immiscible liquids, thus leading
to droplets with minimized contacting surface between two immiscible liquids. In contrast, a higher Ca number implies a higher viscous force, leading to flow instability and
even two parallel immiscible flows [65,75]. The droplets can be generated in a highly
uniform and high-throughput manner, for example, up to 10,000 droplets per second
[74]. Each droplet is an individual bioreactor compatible with isolating and labeling single
cells. Thus, microfluidic droplets show promise for the efficient screening and sorting of
immune cells, as discussed in Section 5.
3. Recapitulating the tumor microenvironment
161Tumor-on-a-chip devices for cancer immunotherapy
The tumor microenvironment consists of the extracellular matrix, soluble biochemical cues, supporting stromal cells, and lymphatic and blood vessels, playing crucial
roles in the survival and escaping mechanisms of cancer cells. For example, the cancerassociated stromal cells can impede the accumulation and proliferation of therapeutic
T cells within the tumor [76] through physical and chemical approaches, such as the
secretion of a dense matrix and immune-suppressive signaling molecules of transforming
growth factor-β (TGF-β) and interleukin-10 (IL-10) [77]. The accumulation of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death
ligand-1 (PD-L1) within the microenvironment has been found to inhibit T cell
costimulation and activation, thus suppressing the immune response and the attack
against cancer cells [77]. Besides, the irregular and leaky blood vessels that are associated
with the tumor microenvironment prevent T cells from infiltrating into the tumor and
the intratumoural immobilization, subverting T cell-mediated immunosurveillance [78] .
Thus, it is necessary to consider the sophisticated organization and components of the
tumor microenvironment in developing and improving immunotherapies. Indeed, the
recapitulation of the tumor microenvironment is often missed in conventional methods
to evaluate the efficacy of immunotherapy.
Conventional tumor models mainly rely on two-dimensional (2D)/3D cell cultures
and animals. 2D cell culture-based models are characteristic of the monolayers of cancer
cells grown on flat, plastic surfaces (sometimes with a coating of a thin layer of ECM proteins). The 2D cell culture is convenient to operate and image and is advantageous in
162 Xuan Mu and Yu Shrike Zhang
producing in large quantities. However, the 2D configuration is often challenging to
mimic the 3D ECM and blood vessels, found in tumors. As a result, immunes cells that
effectively target cancer cells in the 2D models often become ineffective and even fail in
3D tumors. This immunotherapy failure is ascribed to the distinct cell architectures [79],
immune-cancer cell interaction, and specific antigen expressions between 2D models and
3D tumors [80,81].
In contrast, the 3D cell culture models can potentially mimic the 3D structural configuration of tumors by enabling and culturing the aggregation of cells (spheroids and
organoids). The spheroids can be achieved by methods, such as hanging-drop [82],
low-adhesion microwells [83], encapsulation [84], and rotating-wall vessel bioreactors
[85]. However, these 3D models are often limited in the recapitulation of other essential
features of the tumor microenvironment, such as vasculature and the gradient of soluble
environmental cues [72]. Animal-based models can mimic the complexity of the in vivo
environment in comparison to 2D/3D cell cultures. However, small animals, such as
mice and rabbits, often show inherently distinct mechanisms of immune responses
[86]. Thus, animal-based models lead to less accurate prediction of the efficacy of the
immunotherapy. Moreover, animal-based models are often costly, time-consuming,
and low in throughput, restricting their broad utility.
Microfluidics represents a new and potent engineering approach, compared to 2D/
3D cell culture and animal models, to mimic the complex, dynamic in vivo tumor microenvironment. By harnessing advanced microfabrication strategies and human-derived
cells, microfluidics is able to, in an unprecedented manner, recapitulate essential structural features of the tumor microenvironment, including angiogenesis, extravasation,
and the spatial-temporal manipulation of chemical gradients. The recapitulation of these
tissues and sometimes organ-level physiological features makes microfluidic-based tools
competitive to construct a native-like immunogenic tumor microenvironment for testing and optimizing immunotherapy.
3.1 Angiogenesis
Angiogenesis is the formation of new blood vessels by endothelial cells, which provides
the necessary delivery of nutrients and removes metabolic wastes, thus essential to the
growth and survival of malignant tumor cells [87]. Moreover, the blood vessel is a critical
pathway in the metastasis of cancer cells, as it allows the migration of the cancer cells
through the circulation system to other tissues [88]. Thus, tumor angiogenesis is an effective target for immunotherapy. However, it is often challenging for conventional tumor
models to induce a perfusable, functional 3D network of blood vessels within tumors and
evaluate angiogenesis in a quantitative and high(er)-throughput manner.
A quantitative microfluidic angiogenesis screen (QMAS) chip has been developed to
monitor and quantify the formation of angiogenic sprouts of endothelial cells (Fig. 3A–C)
[89]. The QMAS is made with PDMS via soft lithography, consisting of three main
163Tumor-on-a-chip devices for cancer immunotherapy
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Fig. 3 (A) Schematics of in vivo antiangiogenesis treatment of tumors with bortezomib. (B) Schematics
of the QMAS chip consisting of three primary chambers: blood vessel chamber, ECM chamber, and
tumor chamber. Blood vessel-like lumen structures are formed in the ECM chamber, which is a result
of the gradient of cancer-relevant signaling cues, such as VEGF. (C) Fluorescence image of the blood
vessel-like lumen structures formed by red fluorescence protein-expressing HUVECs. White boundaries
outline the position of the microposts. (D) and (E) Schematics of the tumor-induced angiogenesis (top
view and side view). The tumor spheroid mixed with fibrin gel is located in the middle; HUVECs are
seeded on the surface of the fibroin gel. (F) Fluorescence projection images of the tumor spheroid
of green fluorescent protein (GFP)-expressing U87MG and red fluorescent protein (RFP)-expressing
HUVECs. (A–C: Reproduced with permission from Kim C, Kasuya J, Jeon J, Chung S, Kamm RD.
A quantitative microfluidic angiogenesis screen for studying anti-angiogenic therapeutic drugs. Lab Chip
2015;15(1):301–10. Copyright 2016 Royal Society of Chemistry. D–F: Reproduced with permission from Ko
J, Ahn J, Kim S, Lee Y, Lee J, Park D, Jeon NL. Tumor spheroid-on-a-chip: a standardized microfluidic culture
platform for investigating tumor angiogenesis. Lab Chip 2019;19(17):2822–2833. Copyright 2019 Royal
Society of Chemistry.)
164 Xuan Mu and Yu Shrike Zhang
chambers separated by small pillars. The first chamber mimics the blood vessel, where the
monolayer of human endothelial cells is cultured. The second chamber is in the middle,
containing a 3D collagen gel for mimicking the interstitial ECM. The third chamber, at the
other side of the middle channel, is used to culture cancer cells or provide cancer-relevant
soluble cues, such as proangiogenic vascular endothelial growth factor (VEGF), for mimicking the cytokine gradient found in the tumor microenvironment. VEGF diffuses across
the middle ECM-mimetic chamber to reach the blood vessel chamber. The gradient of
VEGF induces the migration of endothelial cells toward the tumor-mimetic chamber, thus
capable of forming lumen-like structures in the ECM-mimetic chamber. Furthermore, an
antiangiogenetic drug and 26S proteasome-inhibitor, bortezomib [90], is used to impede
the angiogenesis. The number and projected area of migrated cells are obtained by image
analysis, which shows a linear relationship with the concentration of bortezomib largely
between 1 and 10 nM. Bortezomib, higher than 10 nM, not only inhibits the migration
of endothelial cells but also leads to the death of endothelial cells. This result provides new
insights to the antiangiogenesis mechanism of bortezomib.
The angiogenesis of tumor spheroids has also been investigated in a thermoplastic
microfluidic chip (Fig. 3D–F) [91]. This plastic microfluidic chip is made in PS via injection molding, which demonstrates several advantages in comparison to previous ones
made in PDMS via soft lithography. First, PS is a biocompatible thermoplastic widely
used for culturing of tissue and cells in the past decades; it shows minimal absorption
of small hydrophobic molecules and bare leaching of uncured monomers, which is critical to eliminate the contentious issues of PDMS to bias cellular behaviors [92,93]. Second, injection molding is an industrialized fabrication technique that allows the quick
fabrication of chips in large quantities, demonstrating a considerable capability to scale
up the production of microfluidic chips, in contrast to soft lithography that is usually
restricted in research settings.
Besides, the PS-based device has other designing features that are desirable for tissue
cultures. PS microstructures are designed to guide fluid flow through hydrophilic capillary force. The pipetted liquid droplet spontaneously fills the microfluidic chamber
in an easy to perform, rapid, and reproducible manner, in comparison to the PDMSbased burst valve. The PS microfluidic chips are made in the standard biomolecule
screening (SBS) format of 96-well plates, which facilitates automated and high throughput operations and imaging. Besides the advances in the material and fabrication of the PS
microfluidic chips, the adoption of tumor spheroids, instead of suspended cells, is highly
desirable to recapitulate the 3D microenvironment of tumors. In comparison to the 2D
cultured cancer cells, tumor spheroids are made of multiple cells in 3D, which enable
essential conditions of solid tumors [94,95], including proliferative gradients, limited
drug diffusion, and enhanced cell-cell and cell-ECM interactions. All these properties
of the in vitro models are thus critical to the accuracy and efficiency of drug screening
and therapeutics evaluation.
By using the PS microfluidic chip, a tumor spheroid of brain glioblastoma (U87MG)
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is cultured in the center of a 3D fibrin hydrogel and cocultured with human umbilical
vein endothelial cells (HUVECs) that are on the circumference of the fibroin hydrogel
[91]. Induced by the tumor spheroid, HUVECs form sprouts and a 3D vascular network
within the fibroin hydrogel and toward the tumor spheroid. Two antiangiogenetic drugs,
bevacizumab, and sunitinib, demonstrate the reduction of the vascular network area,
number of sprouts, and sprouting length, which are statistically different from the control
and the use of another drug, cetuximab, that is an epidermal growth factor receptor
inhibitor. This work highlights the promise of PS microfluidic chips for constructing
tumor spheroid-mediated angiogenesis and the efficient drug screening.
Microfluidic droplet array is useful to realize automated and high-throughput investigation of tumor angiogenesis and the screening of antiangiogenetic drugs [96]. For example, a microfluidic droplet array consists of multiple cell-laden Matrigel droplets (500 nL)
embedded in a chemically stable fluorocarbon-based fluid (FC-40, Fluorinert, 3 M) by
robotic liquid operation, which is an alternative to microfabrication and may streamline
the liquid operations [97]. The cancer cells (rat glioma cell line, C6) and HUVEC are cultured in distinct droplets. The suspended C6 cells spontaneously form spheroid-like aggregates after 48-h incubation. The aggregates have a diameter of approximately 1 mm and a
hypoxic inner center, both of which are characteristics of native solid tumors [98].
HUVECs aggregate to form vessel-like structures. Two droplets laden with cancer cells
and HUVECs can be paired and fused by using the relative movement of the dispensing
capillary and the deposited droplets, which shows no interference to the internal aggregates
and vessel-like structures. Consequently, the paired and fused droplets serve as a coculture
model. The angiogenetic capability of HUVECs, in terms of vessel area, is influenced by
cocultured cancer cells and is associated with the concentration of human VEGF factor
(hVEGFf ) secreted by the cancer cells. In particular, three types of cells (colon cancer cells,
LoVo and HT29, and embryonic kidney cells, HEK293) with the increased secretion of
hVEGFf (approximately 8, 3.5, and 0.2 ng mL
(approximately 0.2, 0.05, and 0.01 mm
1
) lead to the increased area of vessels
2
). Moreover, an antiangiogenetic drug,
fingolimod, is used in the microfluidic droplet-based coculture model. Fingolimod inhibits
angiogenesis by blocking VEGF-mediated sphingosine-1-phosphate receptor [99].The
antiangiogenetic efficacy of fingolimod is verified in all three types of cancer cells
(LoVo, HT29, and HEK293). The efficacy of fingolimod is concentration-dependent,
but activity is lost over the concentration of 0.2 μM, as ascribed to the colloidal aggregates
of fingolimod at higher concentrations [100].
165Tumor-on-a-chip devices for cancer immunotherapy
3.2 Extravasation
Extravasation is one of the critical steps in cancer metastasis, which is responsible for the
dissemination of cancer cells within the body and is the leading cause of cancer-related
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