An Oxygen-Concentration-Controllable Multiorgan Microfluidic
Platform for Studying Hypoxia-Induced Lung Cancer-Liver
Metastasis and Screening Drugs
Lulu Zheng,# Bo Wang,# Yunfan Sun, Bo Dai, Yongfeng Fu, Yule Zhang, Yuwen Wang, Zhijin Yang,
Zhen Sun, Songlin Zhuang, and Dawei Zhang*
Cite This: ACS Sens. 2021, 6, 823−832 Read Online
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ABSTRACT: Various cancer metastasis models based on organ-on-a-chip platforms
have been established to study molecular mechanisms and screen drugs. However,
current platforms can neither reveal hypoxia-induced cancer metastasis mechanisms nor
allow drug screening under a hypoxia environment on a multiorgan level. We have
developed a three-dimensional-culture multiorgan microfluidic (3D-CMOM) platform
in which the dissolved oxygen concentration can be precisely controlled. An organ-level
lung cancer and liver linkage model was established under normoxic/hypoxic
conditions. A transcriptomics analysis of the hypoxia-induced lung cancer cells (A549
cells) on the platform indicated that the hypoxia-inducible factor 1α (HIF-1α) pathway
could elevate epithelial-mesenchymal transition (EMT) transcription factors (Snail 1
and Snail 2), which could promote cancer metastasis. Then, protein detection
demonstrated that HIF-1α and EMT transcription factor expression levels were
positively correlated with the secretion of cancer metastasis damage factors alphafetoprotein (AFP), alkaline phosphatase (ALP), and gamma-glutamyl transpeptidase (γ-
GT) from liver cells. Furthermore, the cancer treatment effects of HIF-1α inhibitors (tirapazamine, SYP-5, and IDF-11774) were
evaluated using the platform. The treatment effect of SYP-5 was enhanced under the hypoxic conditions with fewer side effects,
similar to the findings of TPZ. We can envision its wide application in future investigations of cancer metastasis and screening of
drugs under hypoxic conditions with the potential to replace animal experiments.
KEYWORDS: hypoxia, microfluidic chip, 3D culture, cancer metastasis, drug screen
Cancer has become the leading cause of death worldwide,1
and cancer metastasis is one of the primary causes of
cancer-related death.2 An important aspect of cancer metastasis
is the widespread hypoxia phenomenon, which is due to the
rapid proliferation of cancer cells and the insufficient blood
oxygen supply in solid tumors.3 During lung cancer metastasis
induced by hypoxia, primary site cancer cells transition into
metastatic cancer cells through the blood stream via epithelialmesenchymal transitions (EMTs),4 subsequently producing a
secondary tumor in distant organs by means of the
mesenchymal-epithelial transition (MET).5 Studying the
signaling effects between the tumor and normal organs is
important to develop new treatment strategies for the
inhibition cancer metastasis.6 Moreover, hypoxia reduces the
effectiveness of chemotherapy and radiotherapy in tumors,7
and hypoxia target therapy has been employed to enhance
cancer treatment effects. Therefore, research on the hypoxic
cancer environment promoting cancer metastasis by signaling
pathways and optimizing hypoxia target cancer treatment
strategies has become an important research direction to
identify cancer metastasis mechanisms and evaluate the effects
of the hypoxia-related target drugs.8
Organ-on-a-chip platforms are miniaturized 3D human
microfluidic tissues used as organ-level models to recapitulate
the crucial biological parameters and functions of related in
vivo models. These platforms have many advantages over
traditional methods, including monitoring the microenvironmental parameters of miniaturized organs using biosensors,
providing conditions with excellent control for studying the
signaling effects between two different organs in cancer
metastasis, and more accurately detecting human responses.9
Therefore, there is a need for a seamlessly assembled
multiorgan platform to mimic cancer organs for investigations
of cancer metastasis mechanisms. Recently, studies have
reported single-organ microfluidic chips that can generate a
hypoxic environment in vitro to mimic the true dissolved
Received: September 3, 2020
Accepted: January 15, 2021
Published: March 4, 2021
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oxygen (DO) concentration of cancers in vivo. However, there
are some limitations in these reported models. For example,
cultured cells on a flat surface cannot mimic real 3D cell
growth patterns in the body;10,11 the oxygen concentration of
the cell culture chamber of the multiorgan chip cannot be
precisely controlled and detected;12,13 the system cannot offer
precisely controlled hypoxic conditions for studying the
signaling effects between two different organs during
hypoxia-induced cancer metastasis and for screening hypoxiarelated target drug.14,15 Thus, there is an urgent need for an
oxygen-concentration-controlled multiorgan microfluidic chip
platform to mimic a hypoxic microenvironment for the
investigation of the cancer metastasis mechanism and
evaluations of hypoxia-related target drugs.
In this study, we developed a 3D-CMOM platform to realize
lung cancer-liver linked organ culture that could precisely
control DO concentration in the different 3D cell culture
chambers. The performance of the regulation of the oxygen
concentration in the 3D cell culture chamber was analyzed.
Subsequently, we studied the influence of metastasis of lung
cancer on the liver using the 3D-CMOM platform with a
hypoxic microenvironment and investigated the effects of
cancer cell cocultures with fibroblasts in cancer metastasis by
means of transcriptomics (RNA-seq) and protein expression
detection. Furthermore, we evaluated the cancer treatment
effects of hypoxia-induced HIF-1α inhibitors (TPZ, SYP-5, and
IDF-11774) under normoxic and hypoxic environments on the
3D-CMOM platform. We believe that the oxygen-controlled
multiorgan microfluidic chip provides a platform for
researchers to study the mechanism of hypoxia-induced cancer
metastasis and the therapeutic effects of hypoxia-related target
anticancer drugs.
■ MATERIALS AND METHODS
Microfluidic Chip Fabrication. The microfluidic chip (Figure 1)
was made with polydimethylsiloxane (PDMS; Sylgard 184, Dow
Corning, USA) using a well-known soft lithography technique. First, a
negative photoresist (SU-82050, MicroChem, USA) was spin-coated
onto a silicon wafer. Then, the wafer was soft-baked on a heating plate
and cooled to room temperature. Subsequently, the wafer was
exposed using a photolithenic machine (MJB4, SUSS, Germany)
through a corresponding photomask, and then appropriate development was performed. Then, the patterned silicon wafer was fumigated
with trimethylchlorosilane (Sigma-Aldrich) for 10 min in a sealed box.
The PDMS prepolymer and curing agent were uniformly mixed at a
ratio of 10:1 and degassed.16 PDMS was poured onto the wafer of the
gas, isolation, and chamber layers. Simultaneously, PDMS was spincoated on the wafer of the fluid layer. The entire chip was cured in an
oven at 80 °C for 1 h. The gas and chamber layers were peeled off,
cut, and hole-punched. The bonding between the gas and fluid layers
was carried out by an oxygen plasma treatment (PDC-FMG-2,
Harrick Plasma), it was then peeled away from the wafer, and the
excess PDMS was removed and then bonded with the isolation layer
using oxygen plasma. The chamber layer was bonded with the glass
substrate using oxygen plasma. The three layers (the gas, fluid, and
isolation layers) were bonded as the lid of the chip, providing a
structure for gas−liquid diffusion. Bonding of the chamber layer and
the glass substrate as the bottom of the chip provided a structure for
the 3D cell culture (Figure 1B). The lid structure and the bottom
structure were clipped by PMMA and fixed with screws to make a
reassembleable chip (Figure 1D).
Cell Lines and Culture. To investigate the effects of lung cancer
metastasis to the liver, lung cancer and liver organ modules were
reconstructed using related cell lines in vitro. A549 and HFL-1
(fibroblasts) cell lines were obtained from the American Type Culture
Collection (ATCC, USA). The red fluorescent protein-transfected
A549 cell line (RFP-A549) was produced by our laboratory. Human
normal liver cells (L02) were kindly provided by the Chinese
Academy of Sciences (Shanghai, China). All cell lines were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum, 1% penicillin, and 1% streptomycin (all from
Gibco, Invitrogen, Inc., USA). Cells were cultured at 37 °C in an
incubator under an atmosphere of 5% CO2 and 95% humidity.
Establishment of 3D-CMOM. The chip surface and the
micropipes were cleaned three times with deionized water, with a
resistivity of 18 MΩ cm at 25 °C, and pasteurized before seeding the
cells. We used Gelatin Methacryloyl (GelMA, Engineering for Life,
China), which was cured using short UV-light irradiation, as the
scaffold to 3D culture cells. A549 and L02 were suspended in 5 wt %
GelMA at a density of 4 × 106 cells/mL, and then 35 μL of A549 and
L02 cell suspensions was added to the lung cancer chambers and liver
chambers, respectively, and immediately cross-linked using UV light
(405 nm, 800 mW for 10 s) to prevent cell deposition. Subsequently,
these chambers were covered with the gas−liquid diffusion lid and
locked with PMMA, and this model was named A549-L02. To better
mimic the real conditions in lung cancer, HFL-1 and A549 cells were
cocultured in the lung cancer chamber of the microfluidic chip to
form a two-layer cancer structure, and its downstream chamber was
seeded with L02 cells, resulting in a model named HFL-1/A549-L02.
The creation steps of this model are as follows: the lung cancer
chamber was coated with a 0.1 mg/mL poly-L-lysine solution at 4 °C
overnight and then washed three times with phosphate-buffered saline
(PBS; Gibco, Invitrogen). Subsequently, 35 μL of HFL-1 cell
suspension (2 × 106 cells/mL) was injected into the lung cancer
chamber and placed in the incubator overnight to allow HFL-1 to
adhere and spread to the bottom. The excess medium and
nonadherent HFL-1 cells in the lung chambers were aspirated, and
then A549 and L02 cells in the chambers were seeded as previously
described. A two-layer structure of lung cancer cells on the top and
fibroblasts on the bottom was formed. As the cells were cultured in
Figure 1. 3D-CMOM platform. (A) Schematic diagram displays the
functional description of each area of the platform. (B) A diagram of
the multilayer structure that comprises the platform. (C) The details
of the function of each hole in the platform. (D) Image of the
microfluidic platform.
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the chip, the medium outlets 1 and 2 needed to be blocked with
sealed needles to prevent the medium from spilling out from these
two holes. The chip was then placed in the cell incubator and injected
with DMEM using the syringe pump (PHD 22/2000, Harvard, USA)
to the chamber with a perfusion model.
During the culturing of cells in the chip, the lung cancer chamber
was maintained under hypoxic conditions by pumping in gas
containing 0% oxygen from gas inlet 1. The control lung cancer
chamber was under normoxic environmental conditions by pumping
in gas containing 20.9% oxygen from gas inlet 2. All the liver
chambers were under normoxic conditions by pumping in gas
containing 20.9% oxygen from gas inlet 3 (Figure 1A,C).
Generation of Different DO Concentrations and Characterization. PDMS has good gas permeability.17 The developed chip has
a thin layer of PDMS between the liquid and gas layers, and the DO
concentration of the medium flowing into the cell culture chamber
was adjusted by pumping gas with different oxygen concentrations
into the gas pipe. The left and right of the chip are symmetrical
structures that can simultaneously produce hypoxic and normoxic
environments in the cancer cell chamber (Figure 1).
An oxygen sensing system (NeoFox, Ocean Optics, USA) with an
oxygen probe (R-Series, HIOXY coating) was used to measure the
DO concentration in the chambers and was analyzed using NeoFox
Viewer. The oxygen sensing system used the two-point calibration
method to determine the DO concentration according to the
manufacturer’s instructions.18 Measuring the DO concentration in
the cell chambers requires that a hole be punched in the
corresponding chamber and after that the oxygen probe is inserted
into the chamber to measure the oxygen concentration of the
chamber in real time.
The medium was injected from the medium inlet with different
unilateral flow rates (1, 10, 20, 30, and 40 μL/min) and was regulated
by an injection pump (PHD 22/2000, Harvard, USA). Before the
measurement, air was passed into the medium overnight to saturate
the medium. Gas containing oxygen (0 and 20.9%) was supplied from
gas inlets 1 and 3, respectively, with a pressure of 5 psi and was
controlled by a pressure reducing valve (A-2H, Aerotech, USA), and
the DO probe was inserted into the corresponding chamber to
measure the DO concentration of the corresponding chamber.
Gene Expression Profiling. The hydrogel containing the A549
cells was extracted from the microfluidic platform after the cells were
cultured for 2 days and then dissolved using Collagenase A (SigmaAldrich) at 37 °C in an incubator. Then the cells were collected using
a centrifuge, and TRIzol (Thermo Scientific) was added to extract
RNA immediately. A bioanalyzer was used to quantify all samples and
assess their purity through a NanoDrop (Thermo Scientific). Nextgeneration sequencing was done using a hiSeq 3000 to measure the
gene expression level. The RNA-seq data were processed and further
analyzed by the R statistical programming language. The data analysis
was based on the Kyoto Encyclopedia of Genes and Genomes
(KEGG) database and the Gene Ontology (GO) database.
Immunofluorescence. After culturing the cells for 48 h in the
chip, we characterized three cancer metastasis-related markers (e.g.,
HIF-1α, transforming growth factor-beta1 (TGF-β1), and AFP) using
immunofluorescence (IF). Briefly, cells were fixed in the chamber and
permeabilized with a 0.1% PBS-Triton solution, blocked with 3%
bovine serum albumin (BSA), and separately incubated with the
following antibodies: monoclonal mouse antihuman HIF-1α (Abcam,
Cambridge, UK), polyclonal rabbit antihuman TGF-β1 (Abcam,
Cambridge, UK), and polyclonal rabbit antihuman AFP (Abnova,
Taiwan, China). Then, the cells were washed with PBS before being
incubated with an FITC-conjugated secondary antibody. After
incubation, the cells were washed with PBS and imaged with a
confocal microscope (LSM 900, ZEISS, Germany). We also
normalized the intensity ratio of protein of interest to Hoechst
(Figure S2), and the fluorescence intensity was analyzed using ImageJ.
Enzyme-Linked Immunosorbent Assay (ELISA). The expression levels of cancer metastasis-related markers (e.g., AFP, ALP, γ-GT,
and TGF-β1) were measured (cells cultured in the chambers and
culture supernatant, which mimics serum). The secretion samples of
the lung cancer chambers and the entire systems were collected from
medium outlets 1, 2, 3, and 4 (Figure 1C) after culturing the cells for
48 h in the chip. The samples were assayed by traditional ELISA kits
(Abnova, Taiwan, China) according to the manufacturer’s instructions. We use DMEM containing 10% FBS as a background control,
and the background values were subtracted from the values obtained
for the other groups.
Hypoxia-Related Anticancer Drug Treatment. TPZ, SYP-5,
and IDF-11774 were dissolved in dimethyl sulfoxide (DMSO; SigmaAldrich, USA) and stored at −80 °C until use. After cells were seeded
in the device, the chambers were injected with fresh DMEM
containing 0 and 100 μM TPZ, 50 μM SYP-5, and 50 μM IDF-11774.
The gas inlets of the lung cancer chambers were used to pump in gas
with different oxygen concentrations (0, 10, and 20.9%), and the gas
inlet of the liver chambers was used to pump in gas with 20.9%
oxygen. After treatments, the cell viabilities were calculated using a
Calcein-AM/PI Double Staining Kit (Shanghai Dojindo Laboratories,
Japan). Briefly, 2 μM Calcein-AM and 1.5 μM PI were mixed and
added into the cell chambers. Then, after incubating at 37 °C for 15
min and washing with PBS, fluorescence images were taken using a
confocal fluorescence microscope.
■ RESULTS AND DISCUSSION
Establishment of the Lung Cancer-Liver Linked
Organ Platform. We designed and manufactured a 3DCMOM platform to link the culture of the lung and liver
organs. The apparatus can steadily regulate the oxygen
concentration in the cell culture chamber and can be used to
investigate the mechanism of lung cancer metastasis to the liver
and the effects of hypoxia-related target anticancer drugs. The
cell culture medium is pumped via the microchannel to mimic
blood flow. The 3D-CMOM platform (Figure 1A) contains
four gas−liquid diffusion areas and four 3D cell culture
chambers. This platform (Figure 1B) is like a detachable
sandwich structure composed of a gas layer (3 mm), a fluid
layer (0.15 mm), an isolation layer (0.5 mm), a chamber layer
(2 mm), and a glass substrate (1 mm). The PMMA splint
bonds the system together and simultaneously prevents the
oxygen in the air from interfering with the DO concentration
regulation of the platform. The width of the liquid micropipes
in the gas−liquid diffusion areas is 0.2 mm, and the width of
the gas micropipes is 0.3 mm. The different widths of
micropipes ensure that the gas micropipes perfectly cover the
fluid micropipes to precisely control the DO concentration.
The overlap length of the gas−liquid diffusion micropipes in
each area is approximately 70 mm, and the height of each
micropipe is approximately 50 μm. The 3D cell culture
chambers are cylindrical with a diameter of 5 mm and a depth
of 2 mm.
The composition of tumors is very complicated, being
composed of cancer cells, immune cells, fibroblasts, the
extracellular matrix, lymphatic vessels, and blood vessels.3
3D-cultured lung cancer cells cocultured with fibroblasts on a
microfluidic chip is more representative of the true cancer
microenvironment than monocultured lung cancer cells.
Cancer cells and fibroblasts in tumors are not uniformly
mixed in vivo, and the fibroblasts surround the cancer cells.3
Therefore, we fabricated a two-layer structure of the HFL-1
cells at the bottom and the A549 cells in the above (Figure
S1). The 2D-cultured HFL-1 cells were attached to the bottom
of the lung cancer chambers, and the 3D-cultured A549 cells
were above them to form a two-layer lung cancer organ
structure. In addition, the two-layer structure can better
distinguish cancer cells and fibroblasts during the detection
period than direct coculture in hydrogel. L02 mixed with 5 wt
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% GelMA was added into the liver chambers to form a 3D liver
culture model in the chip. Thus, a 3D-CMOM platform was
fabricated.
Characterization of Oxygen Concentration. Due to the
permeability of PDMS, the oxygen concentration of the liquid
in the fluid layer will eventually be consistent with the oxygen
concentration of the gas being pumped in. The DO probe was
used to measure the actual DO concentration in the
corresponding chamber. To investigate the DO regulatory
effects of the device, a 0% oxygen gas mixture was pumped in,
and real-time DO concentration changes in the cell chamber
were recorded with a DO probe at different flow rates. The
data shown in Figure 2A indicate that as the flow speed of the
liquid increased, the DO concentration in the responding
chamber decreased faster. The medium with adjusted DO
concentration in the gas−liquid diffusion area could replace
the liquid in the cell culture chambers faster with a higher
liquid flow rate, resulting in the faster drop of the oxygen
concentration in the cell culture chamber. The DO
concentration of the medium in the chamber decreased to
0% after 3 h when the flow rate of the liquid was 1 μL/min. In
addition, the DO concentration of the chambers decreased
from 20.7 to 10% within less than 10 min and then decreased
to approximately 1% within 100 min as the flow rate was 20
μL/min. However, the final DO concentrations of the chamber
were stable at 1.4 and 1.6% when the flow rates of the liquid
were 30 and 40 μL/min, respectively. All the above results
indicated that the liquid flow rate was too fast to diffuse
sufficiently between the gas and liquid when the flow rate was
≥30 μL/min, resulting in the DO concentration of the cell
chamber being higher than that of the gas being pumped in.19
We inferred that the most appropriate liquid flow rate was 20
μL/min. Therefore, the system was operated with a flow rate of
20 μL/min during the first 2 h and then changed to 1 μL/min
for long-term cell culturing to avoid the shear stress damaging
the cells.20
In addition, gas containing 10% oxygen was pumped into the
gas micropipes to verify the ability of the device to regulate the
oxygen concentration. The results demonstrated that the DO
concentration stabilized after a maximum of 3 h at 9.8, 9.4,
10.1, 10.6, and 12.4% at liquid flow rates of 1, 10, 20, 30, and
40 μL/min, respectively. The stabilized DO concentration of
the chamber did not exactly match the oxygen concentration of
the gas pumped in, which may be due to the two-point
calibration detection method used. From these results, we
inferred that the most appropriate liquid flow rate was 20 μL/
min (Figure 2B).
The platform is a dual-organ culture system in which the
upstream lung cancer organ module culture chamber is
regulated to form a hypoxic environment by pumping in gas
containing 0% oxygen. To ensure that the downstream normal
liver organ module culture chamber had a normoxic environment, we pumped in gas containing 20.9% O2 with liquid flow
rates of 1 and 40 μL/min to regulate the DO concentration of
the liver chamber. The results indicated that the DO
concentrations in the liver chamber were maintained at 20.6
± 0.2 and 19 ± 0.1% at flow rates of 1 and 40 μL/min,
respectively (Figure 2C). From these results, we inferred that a
normoxic environment could be maintained in the liver
chamber when the flow rate was ≤40 μL/min. Eventually,
this system provides an environment for the linked culturing of
lung cancer and liver organ under hypoxic and normoxic
conditions, respectively.
Gene Expression Profiles of A549 Cells Induced by
Hypoxia. RNA-seq was employed to demonstrate the
functional assessments of A549 cells collected from the 3DCMOM platform on a molecular level. For control (Ctrl)
versus A549 cells induced by hypoxia for 48 h, the results
indicated 887 significantly differentially expressed genes (fold
change ≥1.5 and P-value <0.05, 464 genes were upregulated,
and 423 genes were downregulated). Hierarchical clustering
using the different expressed genes demonstrated two main
clusters, Ctrl and A549 cells induced by hypoxia (Figure 3A).
Forty-three unique differentially expressed genes were
identified, and these genes could play an important role in
the EMT process and tumor metastasis, including p53,21 IL-6,
IL-11, IL-12, IL-13,22 JNK,23 NF-κB, MAPK, Wnt, SMAD,
MMPs, Claudins, SOX, and so on (Figure 3B, Table S1).24
GO categories of differentially expressed genes were analyzed,
including p53 signaling, NF-κB signaling, Wnt pathway,
response to hypoxia,25 and so on (Figure 3C, Table S2).
KEGG pathway analysis indicated enrichment for the HIF-1
pathway, VEGF pathway,26 apoptosis,27 p53 pathway, and so
on (Figure 3D, Table S3). These pathways are closely related
to the EMT process and tumor metastasis.
The EMT plays a vital role in enabling epithelial origin cells
to migrate to distant organs. Increasing evidence suggests that
the EMT states can be detected in migrating clusters. The
EMT could be activated in hypoxic environments.24 The
literature reported that Snail 1 and Snail 2 are major EMTinducing transcription factors, which promote cancer metastasis.28 The EMT could be activated by pathways and then
regulate metastatic ability and tumor growth. Furthermore,
Snail 1 can activate the mesenchymal genes on the transcription level. TGF-β1, NF-κB, and Wnt, which are all
Figure 2. Transient change in the DO concentration in cell culture
chambers. (A) 0% oxygen was pumped into the lung cancer chamber,
and the medium was pumped at different flow rates (1, 10, 20, 30, and
40 μL/min). (B) 10% oxygen was pumped into the lung cancer
chamber with different medium flow rates (1, 10, 20, 30, and 40 μL/
min). (C) 0% oxygen was pumped into the lung cancer chamber,
while normoxic gas (20.9%) was pumped into the liver chamber after
which the DO concentration was measured downstream of the liver
chamber. The medium flow rates were 1 and 40 μL/min.
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upregulated in this study (Figures 3B, 5), can elevate the Snail
1 expression level. HIF expression levels could be elevated
under the hypoxia environments, and HIF-1α could upregulate
the lysyloxidase (LOX) expression and then elevate the Snail 1
protein.29 The HIF-1α and LOX expression levels were
upregulated by IF detection and RNA-seq, respectively, in
this study (Figures 3B, 4). Snail 1 and Snail 2, which play vital
roles in the activation of the EMT during cancer progression,
could be associated with other transcription factors to regulate
gene expression. Snail 1 and Snail 2 repress the E-cadherin
(CDH1) transcription by binding to its promoter and then
promote cancer invasion and metastatic phenotypes.20 The
expression level of CDH1 was downregulated by RNA-seq
analysis in this study (Figure 3B). Claudins are members
responsible for epithelial cell polarity maintenance. The
literature indicated that Snail l could bind to the Claudin
gene promoters, leading to expression repression. Claudin-7,
Claudin-16, Claudin-2, Claudin-22, and Claudin-20 were all
downregulated by RNA-seq detection in this study (Figure
3B). Correlative studies reported that MMPs could be
upregulated by Snail 1 and Snail 2.30 Stable transfection
introduces Snail into cancer cell lines, and then MMP-1,
MMP-2, and MMP-7 expression levels were enhanced. In this
study, the expression levels of MMP1, MMP3, MMP9,
MMP10, MMP12, MMP13, MMP19, and MMP25 were
increased by RNA-seq detection (Figure 3B). Cells with EMT
features could invade via expression matrix metalloproteinases
(MMPs). During these processes, the epithelial genes
including CDH1 were lost; meanwhile, the expression levels
of vimentin and fibronectin were increased by RNA-seq
detection (Figure 3B), and these genes could define the
mesenchymal phenotype.
In short, in this study, TGF-β, HIF-1α, NF-κB, and Wnt can
elevate the EMT transcription factor (Snail 1 and Snail 2)
expression levels, and after that, Snail 1 and Snail 2 can
regulate the downstream CDH1, Claudins, Vimentin, and
MMP expression levels, resulting in the promotion of cancer
invasion and metastasis.
Expression of Cancer Metastasis-Related Markers
under Hypoxic Conditions. A hypoxic environment can
promote the metastasis of cancer cells by releasing cancer
metastasis-related biomarker proteins.8 HIF-1α regulates the
expression of a series of downstream genes and proteins to
promote cancer progression, primarily affecting the processes
of angiogenesis, erythropoiesis, metabolism, cell survival, and
cell proliferation. Its expression and transcriptional activity are
strictly controlled by the oxygen concentration in cells.6 HIF-
1α protein expression levels in cells in the HFL-1/A549-L02
model were assessed by immunofluorescence (IF) to
determine whether hypoxia-related signaling pathways were
activated in the hypoxic environments (Figure 4A). After 48 h,
the A549 and HFL-1 cells cultured in the chip under hypoxic
conditions expressed approximately 3.8- and 3-fold greater
levels of HIF-1α protein, respectively, than those observed cells
Figure 3. Gene expression profiling of A549 cells after being cultured
in the device. (A) Heatmap of 887 DEGs (fold change ≥1.5 and Pvalue <0.05 in any pairwise comparison) under hypoxia versus
normoxic environments. (B) Heatmap of 43 DEGs related to the
EMT process and tumor metastasis. (C) Significant different genes of
GO categories in tumor metastasis. (D) KEGG enrichment pathway
related to cancer metastasis. (E) Hypoxia-induced EMT-related signal
pathway.
Figure 4. Characterization of HIF-1α protein expression of cells in
the HFL-1/A549-L02 model. (A) HIF-1α protein expression levels of
cells under normoxic and hypoxic conditions. (B) Mean fluorescence
intensity analysis of protein expression levels of HIF-1α by IF
detection. Normo means normoxia. Hypo means hypoxia. ★★★p <
0.001 versus A549 cells cultured in a normoxic environment. ###p <
0.001 versus HFL-1 cells cultured in a normoxic environment. The
data are expressed as mean ± SEM.
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cultured under normoxic conditions (Figure 4B, Figure S2A).
No notable differences in HIF-1α protein expression levels
were observed in the L02 cells cultured under hypoxic and
normoxic conditions in the HFL-1/A549-L02 model. These
results indicate that the HIF-1α signal pathway of the A549
and HFL-1 cells in the lung cancer chamber was activated
under hypoxic conditions. The RNA-seq analysis also
demonstrated that the HIF pathway was activated (Figure
4A). Thus, the HIF-1α signaling pathway in the L02 cells
cultured under normoxic conditions would not be activated.
These results demonstrated the ability of the device to regulate
the DO concentration in the chamber, allowing for the study of
cancer metastasis.
TGF-β1, as a multifunctional cell regulatory factor, has been
shown to have numerous promoting effects on cancer
progression. Studies have reported that it plays a number of
important roles in stimulating cell growth,31 metastasis,32 and
differentiation.33 In this study, cells were cultured for 48 h, and
the TGF-β1 protein levels of cells and the culture media of the
A549-L02 and HFL-1/A549-L02 models were assessed
(Figure 5, Figure S2B). As shown in Figure 5B, both the
hypoxic environmental conditions and the coculture model
(HFL-1/A549) could significantly improve the TGF-β1
expression levels in the lung cancer A549 cells. The RNAseq analysis also demonstrated that the TGF-β1 expression
level was increased (Figure 3). Figure 5B shows that the TGF-
β1 secretion levels of HFL-1 cells did not significantly change
under hypoxic conditions. The literature reported that TGF-β1
is mainly secreted by cancer cells in solid tumors34 and
secreted expression levels of proteins varied among different
cell lines.35 In this study, compared to the A549 cells, the TGF-
β1 expression levels of the HFL-1 cells were not significantly
increased under hypoxic conditions. The coculture model
under hypoxic conditions led to A549 cells having the highest
expression of TGF-β1 protein. Furthermore, we also tested the
concentration of secreted TGF-β1 protein in the medium of
the lung cancer chamber and whole systems of the two models
by ELISA (Figure 5C). The results indicated that the amounts
of secreted TGF-β1 protein collected from the lung cancer
chamber and whole system were consistent with the cell
expression results assessed by IF.
AFP is one of the earliest recognized cancer markers, and its
high expression is typically associated with liver cancer.36 In
addition, cancer metastasis to the liver can also stimulate liver
cells to overexpress AFP proteins.37 A549 and HFL-1 cells are
AFP-negative cell lines,38 and we tested the AFP protein
expression of L02 cells by IF to verify the cancerous metastasis
to liver cells in the two models (Figure 6A). By analyzing the
average fluorescence intensity (Figure 6B, Figure S2C), the
results indicated that in the linked culture of L02 cells with
A549 cells, the L02 cells were stimulated to overexpress AFP
proteins. The A549 cells under hypoxic conditions can
Figure 5. Characterization of TGF-β1 protein expression levels in the
A549-L02 and HFL-1/A549-L02 models. (A) The TGF-β1
expression levels of cells by IF detection. (B) Intensity analysis of
IF detection. (C) TGF-β1 protein concentrations of the media from
the models detected by ELISA. ★★★p < 0.001, ★★p < 0.01, ★p < 0.05
versus the normoxia A549 group. ##p < 0.01 versus the normoxia
A549-L02 group. The data are expressed as mean ± SEM.
Figure 6. Characterization of AFP protein expression levels in the
microfluidic chip. (A) The expression of AFP protein determined by
IF detection. (B) Intensity analysis of IF detection. (C) The AFP
protein concentrations of the media from the models were quantified
by ELISA. ★★★p < 0.001, ★★p < 0.01 versus the L02 group. ###p <
0.001, ##p < 0.01, #
p < 0.05. The data are expressed as mean ± SEM.
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828
stimulate the L02 cells to secrete AFP protein at higher levels
than that observed under a normoxic condition. In addition,
the L02 cells of the coculture model under hypoxic conditions
showed the highest AFP expression levels. Furthermore,
because the concentration of AFP is typically measured in
the clinic from serum, we detected the AFP protein
concentration in the media (which mimics serum) of the
lung cancer chambers and the whole systems of the two
models by ELISA. The results demonstrated that the AFP
protein levels of the medium collected from the lung cancer
chamber was very low, whereas those observed in the medium
collected from the whole system were consist with the IF
results (Figure 6C).
Serum ALP39 and γ-GT40 detection is the commonly used
test for predicting liver metastases. The ALP and γ-GT
expression levels of media (lung cancer chambers and the
whole systems) were verified to demonstrate the cancer liver
metastasis by ELISA (Figure 7). The results showed that the
ALP and γ-GT protein levels of the medium collected from the
lung cancer chamber were very low; however, the expression
levels collected from the whole system were significantly
higher. In a hypoxic environment, the secretion levels of these
two proteins in the whole system will increase notably.
Hypoxia-induced cancer metastasis was previously reported
in many studies. In this study, hypoxic conditions could induce
cancer metastasis and EMT-related marker expression,
including HIF-1α, TGF-β1, NF-κB, Wnt, Snail 1, Snail 2,
Vimentin, Claudins, and MMPs, and promote cancer metastasis-associated damage (AFP, ALP, and γ-GT) of the
downstream liver organ. In addition, carcinoma-associated
fibroblasts (CAFs) play many important roles in cancer cell
invasion, metastasis, and angiogenesis.41 Studies have shown
that the transformation of normal fibroblasts into CAFs
depends on cytokines secreted by cancer cells, including TGF-
42 In this study, lung cancer cells cocultured with fibroblasts
under a hypoxic condition could further promote cancer
metastasis, possibly due to fibroblasts activated by cancer cells
to transform into CAFs, especially under hypoxic conditions,
being dependent on secretion TGF-β and then the CAFs could
promote A549 cancer cell metastasis.
Cytotoxicity Tests of HIF Target Anticancer Drugs
under Different Oxygen Concentrations. The hypoxic
microenvironment in solid tumors leads to resistance to many
conventional anticancer drugs.8 Thus, hypoxia-related target
anticancer drugs have been gradually developed to improve the
anticancer effect of these drugs,42 and hypoxia can also be used
as a targeted cancer treatment strategy by means of
suppression of the hypoxia-induced HIF-1α.
43 The developed
microfluidic chip can also be used to investigate cellular
responses to hypoxia-related target anticancer drugs to explore
its potential applications in clinical oncology.
TPZ is an anticancer drug that does not kill cells under
normoxic conditions, but a hypoxic environment can activate
TPZ to realize cancer treatment via suppression of the HIF-1α
accumulation induced by hypoxia.44,45 On the other hand,
SYP-546 and IDF-1177447 are HIF-1α inhibitors; however, the
effects of cancer treatment of these inhibitors under a hypoxia
environment have not been investigated. In this study, the
cytotoxicities of these HIF-1α inhibitors under hypoxia and
normoxic environments were detected on the 3D-CMOM.
TPZ (100 μM) was injected to cells to treat the cells for 6 h,
after which the cell viability was analyzed (Figure 8A,B). When
the oxygen concentration in the input gas mixture was 0%, the
Figure 7. Concentrations of γ-GT and ALP proteins were determined
by ELISA. (A) The concentration of γ-GT protein. (B) The
concentration of ALP protein. ★★★p < 0.001, ★★p < 0.01 versus
the L02 group. ##p < 0.01. The data are expressed as mean ± SEM.
Figure 8. Drug screening tests of TPZ, IDF-11774, and SYP-5 were
developed on the platform. (A) Fluorescence diagram of cell activity
after cells were treated with different oxygen concentrations. The
control group represented cells without drug treatment for 6 h. (B)
Cell viability analysis of cells in the control and TPZ groups. (C) Cell
viability analysis of cells in the control, IDF-11774-treated, and SYP-
5-treated groups. ★★★p < 0.01, ★★p < 0.001 versus the 20.9% O2 drug
group of A549 cells. &&p < 0.01 versus the 20.9% O2 drug group of
HFL-1 cells. ###p < 0.001. The data are expressed as mean ± SEM.
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A549 cell viability decreased from 90.3 to 44.2%, while that of
the HFL-1 cells decreased from 96 to 45.4% in the TPZ
treatment group. When the oxygen concentration of the input
gas mixture was 10%, the A549 cell viability decreased from
90.3 to 70.2%, while that of the HFL-1 cells decreased from 96
to 69.1% in the TPZ treatment group. When the oxygen
concentration of the input gas mixture was 20.9%, the A549
and HFL-1 cell viabilities were maintained at 88.9 ± 2 and 93.7
± 3%, respectively (Figure 8B). The A549 and HFL-1 cell
viabilities in the TPZ treatment groups increased in response
to higher oxygen concentration, and this result showed an
oxygen-dependent manner. The L02 cell viability in the TPZ
treatment groups was maintained around 90 ± 1.1%. In
addition, because the L02 cells were continuously cultured in a
normoxic environment, the L02 cell viabilities did not notably
decrease. These results demonstrated that TPZ can kill cancer
cells under hypoxic conditions and does not have side effects
on the downstream liver organ in a normoxic environment.
Then, IDF-11774 was injected to cells to treat the cells for 24
h. When the oxygen concentrations in the input gas mixture
were 0, 10, and 20.9%, the A549 cell viabilities decreased to 92,
80, and 72% in the drug treatment group, respectively (Figure
8C). The A549 viabilities in the IDF-11774 treatment groups
increased in response to higher oxygen concentration, and the
results also indicated an oxygen-dependent manner. However,
the L02 cell viability in the IDF-11774 treatment groups was
decreased to around 67 ± 1.1%. These results demonstrated
that IDF-11774 can kill cancer cells under hypoxic conditions
in an oxygen-dependent manner. However, the cytotoxicity of
IDF-11774 on the downstream liver organ was significant.
After that, SYP-5 was injected to cells to treat the cells for 24 h.
The A549 cell viabilities decreased to 81, 86, and 94% in the
drug treatment group with the oxygen concentrations being 0,
10, and 20.9%, respectively (Figure 8C). The A549 viabilities
in the SYP-5 treatment groups increased in response to the
higher oxygen concentration in an oxygen-dependent manner.
The L02 cell viability in the drug treatment groups did not
change significantly. These results showed that SYP-5 can kill
cancer cells under hypoxic conditions with fewer side effects on
the downstream liver organ. Also, the cell viability in the
control groups, without drug treatment, did not change
significantly.
Based on the mentioned above, IDF-11774 was more toxic
to the liver cells than tumor cells, and the side effect of this
drug was significant. The effects of the cancer treatment of
SYP-5, which was less efficient than TPZ, were increased under
the hypoxic condition with negligible side effects. Thus, the
3D-CMOM platform showed the enormous application
potential for the screening of hypoxia-related target anticancer
drugs.
■ CONCLUSIONS
In this study, we demonstrated a 3D-CMOM platform for 3D
lung cancer-liver-linked organ cultures that could precisely
perform the control of the DO concentration in the cell
chambers. The capability of regulation of the DO concentration was monitored with an oxygen sensing system. This
platform was used to investigate the signaling effects of lung
cancer under hypoxia metastasis to the liver under normoxia by
RNA-seq and protein detection assay. These results indicated
887 differentially expressed genes after A549 cells were
induced by hypoxia (464 genes up; 423 genes down). HIF-
1α, TGF-β, NK-κB, and Wnt can elevate EMT transcription
factor (Snail 1 and Snail 2) expression levels, and then, Snail 1
and Snail 2 can regulate the gene expression levels of the
downstream CDH1, Claudins, Vimentin, and MMPs, resulting
in the promotion of cancer invasion and metastasis. The
secretion of HIF-1α, TGF-β1, NF-κB, Wnt, Snail 1, Snail 2,
Vimentin, Claudins, and MMPs, which were representative,
was positively correlated with the secretion of the cancer
metastasis damage factors AFP, ALP, and γ-GT. Furthermore,
lung cancer cells cocultured with fibroblasts further strengthened lung cancer metastasis to liver cells, especially in hypoxic
conditions. In addition, the cancer treatment effects of the
three HIF-1α inhibitors were investigated under normoxic and
hypoxic environments on the 3D-CMOM platform. The effects
of TPZ and SYP-5 were enhanced under the hypoxic
conditions and harmless for the downstream organ under
normoxic conditions, but the tumor treatment effect of SYP-5
was poor than that of TPZ. On the other hand, the cytotoxicity
of IDF-11774 was significant. The results indicated that the
platform could be used to screen hypoxia-related target
anticancer drugs by establishing a microenvironment with
regulated oxygen concentration. We believe that the 3DCMOM platform can replace some in vitro animal experiments
to reveal the mechanism of cancer metastasis under hypoxic
conditions and could be used for screening hypoxia-related
anticancer drugs.
■ ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acssensors.0c01846.
Figure S1. Characterization of cells in the platform.
Figure S2. The normalized fluorescence intensity ratio of
protein of interest/Hoechst. Table S1. List of differentially expressed genes of A549 between Ctrl and
hypoxia environments. Table S2. List of GO enrichment
pathways in Ctrl vs hypoxia environment. Table S3. List
of KEGG enrichment pathways in Ctrl vs hypoxia
environment (PDF)
■ AUTHOR INFORMATION
Corresponding Author
Dawei Zhang − University of Shanghai for Science and
Technology, Shanghai 200093, China; Shanghai Institute of
Intelligent Science and Technology, Tongji University,
Shanghai 200092, China; orcid.org/0000-0002-0841-
7826; Email: [email protected]
Authors
Lulu Zheng − University of Shanghai for Science and
Technology, Shanghai 200093, China
Bo Wang − University of Shanghai for Science and
Technology, Shanghai 200093, China
Yunfan Sun − Department of Liver Surgery and
Transplantation, Liver Cancer Institute, Zhongshan Hospital,
Fudan University, Shanghai 200032, China
Bo Dai − University of Shanghai for Science and Technology,
Shanghai 200093, China; orcid.org/0000-0002-0029-
792X
Yongfeng Fu − Department of Medical Microbiology and
Parasitology, School of Basic Medical Sciences, Fudan
University, Shanghai 200032, China
ACS Sensors pubs.acs.org/acssensors Article
https://dx.doi.org/10.1021/acssensors.0c0184
Yule Zhang − University of Shanghai for Science and
Technology, Shanghai 200093, China
Yuwen Wang − University of Shanghai for Science and
Technology, Shanghai 200093, China
Zhijin Yang − University of Shanghai for Science and
Technology, Shanghai 200093, China
Zhen Sun − East China Sea Fisheries Research Institute,
Chinese Academy of Fishery Sciences, Shanghai 200090,
China
Songlin Zhuang − University of Shanghai for Science and
Technology, Shanghai 200093, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acssensors.0c01846
Author Contributions
L.Z. and B.W. contributed equally to this work. D.Z., L.Z. and
B.W. initiated the project. S.Z. and D.Z. supervised the project.
L.Z. and B.W. designed and conducted the experiments. Y.Z.,
Y.W., and Z.Y. provided help during cell culture and ELISA
experiments. L.Z., B.W., Y.S., B.D., Y.F., and Z.S. discussed,
edited, and revised the manuscript.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We sincerely thank Ming Jing at Carl Zeiss (Shanghai) Co.,
Ltd. for supporting us with the Zeiss LSM 900 confocal
microscopy. This work was supported by the China National
Key R&D Program 2018YFF0109603, Defense Industrial
Technology Development Program (No.
TSXK20180917058-C), National Natural Science Foundation
of China (No. 61775140), and Science and Technology
Commission of Shanghai Municipality (Nos. 19441904100,
18142200800).
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