Physiological characterization of 3D MTSs
To monitor the growth dynamics of MTSs, the diameter, roundness and cell
growth of MTSs were recorded and assessed every day. In the initial
stage of MTSs formation, cells grew slowly and were easy to distinguish
individual cells (Figure. 2A ). Driven by the interaction and
contact between the cells, cells gradually gathered into clusters. After
2 days of culture, the cells entered the logarithmic growth phase,
proliferated rapidly and gradually formed smooth surfaces. On the 6th
day, MTSs reached the plateau stage, with the largest diameter of 532.21
± 21.42 μm and 592.54 ± 13.64 μm, respectively, when the seeding
densities were 1000 and 2000 cells/well (Figure. 2B ). The
roundness of obtained MTSs exceeded 0.9 (Figure. 2C ), with the
margin of error no more than 10%. The number of living cells in a
single MTS reached the maximum on day 6, approx. 17 times the initial
amount (Figure. 2D ). After that, the boundaries of MTSs began
to wrinkled and blurred, and the spheroids gradually disintegrated.
The morphology and microstructure of MTSs cultured for 6 days were
observed by the SEM, and the MTSs showed a good 3D structure and regular
spherical shape (Figure. 2E, Figure. S2 ). Interestingly, MTSs
of different cell types had different surface structures. The surface of
the single-component Hela cell spheroid was relatively smooth
(Figure. 2E ), while the multi-component MTSs retained the more
obvious tissue structures (Figure. S2 ). When co-cultured with
fibroblasts such as UCF, the fibroblasts extended outwards MTSs. Obvious
vesicles were observed in the MTSs co-cultured with immune cells such as
PBMC. This indicates that the more complex 3D model would preserve more
completely biological characteristics of tumor tissues in vivo .
Compared with 2D monolayer cells,
the cell proliferation rate in 3D MTSs was reduced. The specific growth
rate (µ) of cells cultured for 2 days under 2D conditions was 0.0455
h-1, while the µ of cells in MTSs cultured for 2, 4,
and 6 days were 0.0263 h-1, 0.0246
h-1, 0.00522 h-1, respectively, with
the margin of error no more than 13.6 % (Figure. S3 ). The
cellular viability of 3D MTSs that were cultured for 2 days was similar
to 2D monolayer cells, but the percentage of living cells and apoptotic/
necrotic cells in MTSs are gradually decreased and increased,
respectively, with the culture time (Figure. 2F ). Compared with
2D monolayer culture, the cell cycle in 3D MTSs was blocked throughout
the cultivation. In addition, cells in the 3D MTS are accumulated and
decreased over the culture age in the G1 and S phase, respectively
(Figure. 2F ). However, there was no significant change in the
proportion of cells in G2 phase between 2D monolayer culture and 3D
MTSs.
Chemosensitivity of Hela
tumor spheroids following 5-FU treatment
The cytotoxicity of 5-FU to Hela cells cultured under both 2D monolayer
and 3D MTSs was evaluated (Figure. 3A ). 3D MTSs showed stronger
resistance than 2D monolayer cells, and after the 5-FU treatment for 48
h the IC50 of MTSs (93.88 μM, 95 % confidence interval:
64.88 ~ 148.5 μM) was approximately 5.72 times that of
2D monolayer culture (16.42 μM, 95 % confidence interval: 12.73
~ 21.14 μM). Therefore, we treated the HeLa carcinoma
cells from both 2D and 3D models with 16 μM 5-FU for 48 h in the
following study. Next, the changes of cell apoptosis and cell cycle were
compared before and after the 5-FU treatment (Figure. 3B ). The
effect of 5-FU on cells in 3D MTSs was significantly reduced compared
with 2D monolayer culture. After the 5-FU treatment, the percentage of
living cells and apoptotic cells was decreased and increased,
respectively, in 2D monolayer, while there were no significant changes
in 3D MTSs. As is known, 5-FU mainly targeted S-phase cells (Ijichi,
Adachi, Ogawa, Hasegawa, & Murakami, 2014). After the 5-FU treatment,
the cell cycle was blocked in G1 phase in both 2D monolayer and 3D MTSs.
Meanwhile, we observed a decrease in the proportion of S phase cells and
a constant proportion of G2 phase cells in the 2D monolayer after the
5-FU treatment, while no significant changes were observed in the 3D
MTS.
To adapt to rapid metabolic requirements of tumor cells, the metabolic
pathways have significantly changed to metabolize nutrients in a manner
conducive to proliferation rather than efficient ATP production (Vander
Heiden et al., 2009). Mitochondrion is the energy factory and the main
position of oxygen consumption, and the abnormal mitochondrial
metabolism in tumor cells are often related to
drug
resistance (Yan & Li, 2018). According to the mitochondrial respiration
profile (Figure. 3C ), the oxygen consumption in 3D MTSs was
much higher than that of 2D monolayer cells, which was mainly due to the
significant increase of non-mitochondrial respiration. The results
showed that the non-mitochondrial respiration of MTSs was about 4.60 and
3.19 times that of 2D cultures under the control and 5-FU treatment
conditions, respectively. The possible reason for the increased
non-mitochondrial respiration was
that the main energy source
shifted from mitochondrial oxidative phosphorylation to glycolysis.
Contrary to this, the spare respiratory capacity of MTSs was reduced
about 74.98 % and 63.31 relative to the 2D culture, which was
associated with mitochondrial dysfunction. Furthermore, the
mitochondrial basal respiration and maximum respiration capacity were
reduced by 68.56 % and 71.81 % in 3D MTSs relative to the 2D culture
under the control conditions, while the ATP synthesis capacity was
almost constant. After the 5-FU treatment, the mitochondrial basal
respiration and maximum respiration capacity was almost constant, while
the ATP synthesis capacity were about 62.40 % reduced in 3D MTSs as
compared with the 2D cultures. The further inhibition of 5-FU on the
mitochondrial ATP production capacity of 3D MTS aggravated the
dependence of MTS on the glycolytic pathway. As the major hub of
cellular energy generation, mitochondrion, is also the main source of
reactive oxygen species (ROS) and it has been reported that the ROS
level can be reduced if glycolysis as the main energy source (Herst,
Tan, Scarlett, & Berridge, 2004). However, we found that the ROS
production capacity in 3D MTSs was significantly higher than 2D
monolayer cells (Figure. 3D ).
As can be seen from Figure.
3E, 3F , in the presence of glucose and glutamine, tumor cells preferred
to use glutamine. Under control conditions, cells cultured in 2D
monolayer cultures and 3D MTSs mainly used glutamine, and began to
consume a small amount of glucose after 60 h of culture. Under the 5-FU
treatment, glutamine was still the main energy source in 2D monolayer
culture, while cells consumed glutamine and glucose at the same time, in
3D MTSs. In addition, with 5-FU treatment reabsorption phenomenon was
found in the 3D MTSs, while HeLa cells continued to secrete ammonia
under 2D monolayer culture conditions (Figure. 3G ). It has been
reported that the reabsorption and reuse of ammonia was beneficial to
the growth of tumor cells (Spinelli et al., 2017). The secretion rate of
lactate in 3D MTSs was slightly higher than that in 2D culture under
both control and 5-FU treatment conditions (Figure. 3H ,Figure. S4) .
Identification of
transcriptional alterations for 5-FU resistance
The biological characteristics of 3D MTSs were more representative than
that of 2D monolayer culture, such as hypoxic regions and pH gradients
caused by mass transfer limitations, enhanced extracellular matrix (ECM)
secretion, drug permeation barriers caused by closely cells contact,
increased ECM deposition and the improvement of tumor cell stemness
(Dittmer & Leyh, 2015). Therefore, we measured the transcriptional
levels of drug resistance-related genes in Hela cells cultured under
both 2D cell cultures and 3D MTSs
(Figure. 4 ).
In the 3D MTS model, there is often an oxygen diffusion limit of
150~200 μm
(Oldham, Clish, Yang, & Loscalzo, 2015). Hence, exceeding this radius
would likely form an area of hypoxia in the MTSs, which would lead to
genetic and metabolic reprogramming regulated by hypoxic induction
factor (HIF1A ) (Denko & Nicholas, 2008). Compared with 2D
monolayer cells, after the 5-FU treatment, the transcript level ofHIF1A in the 3D MTSs were significantly increased by 1.60 times
(Figure. 4 ). It has been reported that tumor hypoxia
microenvironment would cause
abnormal activation of the oncogene MET , and consistent with
this, the transcription level of MET was 2.54 and 1.8 times
higher up-regulated in the 3D MTSs than in the 2D cultures under the
control and 5-FU treatment conditions, respectively, which would promote
angiogenesis and maintain tumor aggressiveness (Stella, Benvenuti, &
Comoglio, 2010). HIF1A could also induce the expression of
vascular endothelial growth factor (VEGFA ), which is central to
the growth and metastasis of tumors, thereby promoting the malignant
progression of tumors (Mohamed, Khalil, & Toni, 2020). As evidenced, we
observed that the transcription level of VEGFA was about 5.04
times and 14.87 times higher up-regulated in more 5-FU resistant 3D MTSs
than 2D monolayer cells under the control condition and the 5-FU
treatment conditions, respectively.
Compared with normal cells, tumor cells display upregulated glycolysis
for the provision of intermediates for rapid proliferation, which is
mainly manifested by enhanced glucose uptake and lactate
excretion (Yong, Stewart, &
Frezza, 2019). Similarly, we observed an increase in glucose utilization
and lactate excretion in 3D MTSs, especially following the 5-FU
treatment, compared with 2D monolayer culture (Figure. 3E, 3H ).
Therefore, the transcript level of the genes encoding glucose
transporter (GLUT ), lactate dehydrogenase (LDH ) and
phosphofructokinase (PFK1 ) were measured. As compared with the 2D
monolayer cultures, the transcript levels of GLUT1 , LDHA ,PFK1 were 4.20, 1.60 and 1.21 times higher upregulated in the 3D
MTSs under the control conditions (Figure. 4 ). As expected, the
transcript levels of these genes were more pronouncedly increased after
the 5-FU treatment, showing that 9.72, 2.45 and 1.88 times higher
upregulated in the 3D MTSs than in the 2D cultures. This result
indicated that 3D MTSs featured enhanced aerobic glycolysis, i.e., the
well-known Warburg effect.
The rapid proliferation of tumor cells required a large number of
nucleic acids. The only source of
thymine in cells is the de novo synthesis pathway, and the high
expression of thymidylate synthetase (TYMS ) is often associated
with poor prognosis of tumors (Donner et al., 2019). 5-FU blocks DNA
synthesis to induce cell death by inhibiting TYMS, while
dihydropyrimidine dehydrogenase (DPYD) could decompose and deactivate
5-FU before it was converted into active metabolites
(Negarandeh et al., 2020).
Compared with the 2D cultures, the transcription level of TYMSwere 2.15 and 3.48 times higher up-regulated, meanwhile the transcript
level of DPYD were 3.66 and 1.26 times higher up-regulated in 3D
MTSs before and after the 5-FU treatment conditions, respectively
(Figure. 4 ). Therefore, the up-regulation of the expression ofTYMS and DPYD were also the reasons for the enhanced
resistance of MTSs to 5-FU.
Increasing evidence has ever shown that conventional cancer chemotherapy
is seriously limited by the multidrug resistance (MDR) commonly
exhibited by tumor cells (Perez-Tomas, 2006). In drug-resistant tumor
cells, the main mechanism was the drug accumulation and efflux in which
the ATP binding cassette (ABC) transporters played an important role
(Orlando & Liao, 2020; Ye et al., 2016). However, we did not observe
the up-regulation of ABCB1 and ABCG2 transcription levels
in 3D MTSs as expected, but there may be differences in protein or
metabolite levels. Lysosome-associated transmembrane protein 4B
(LAPTM4B ), a multidrug resistance gene, could stimulate drug
resistance and promote cell growth and proliferation by regulating drug
efflux mechanism and activating PI3K/Akt signal transduction (Gu et al.,
2020). Consistent with this,
compared with the 2D monolayer cultures, the expression level ofLAPTM4B was 1.57 and 1.32 times higher up-regulated in 3D MTSs
before and after the 5-FU treatment (Figure. 4 ).
Apoptosis defects were also one of the reasons for drug resistance,
which were usually regulated by the BCL-2 protein family (Warren,
Wong-Brown, & Bowden, 2019). Compared with 2D monolayer cells, the
transcription level of the BCL2 encoding anti-apoptotic protein
in 3D MTSs was 1.55 times and 3.09 times higher up-regulated before and
after the 5-FU treatment, respectively, which contributed to the
progression of tumor drug resistance (Figure. 4 ). However, the
transcription level of the BAX encoding apoptotic protein was
also up-regulated in 3D MTSs, which might be related to the decrease of
cell proliferation activity.
Then, the expression of cytokines related to tumor cell proliferation
and progression was tested. Transforming growth factor
(TGFB1 )
was generally up-regulated in tumor cells, and could induce
epithelial-mesenchymal transition and promote tumor cell growth,
proliferation and invasion (Fuxe
& Karlsson, 2012). The mTOR pathway was a classical signal transduction
pathway regulating cell growth and metabolism, and was dysregulated in
many cancers (Gao et al., 2009).
It has been reported that the glycolytic pathway was affected by the
mTOR pathway through two key transcription factors, HIF1A and MYC
(Renner et al., 2017). As compared
with the 2D monolayer cultures, the transcript levels of TGFB1 ,MTOR , MYC were 1.92, 2.08 and 1.59 times higher
upregulated in the 3D MTSs, respectively (Figure. 4 ). As
expected, the transcript levels of these genes were more pronouncedly
increased after the 5-FU treatment, showing that 1.82, 3.21 and 3.51
times higher upregulated in 3D MTSs than in the 2D monolayer cultures,
respectively (Figure. 4 ).
ECM, such as laminin, fibronectin, vimentin, mediated interactions
between cells and participated in signal transduction in processes such
as cell adhesion, migration, invasion, proliferation and EMT to promote
the development of drug resistance, referred to as cell adhesion
mediated drug resistance (CAM-DR) (Valkenburg, de Groot, & Pienta,
2018; Wantoch von Rekowski et al., 2019). Although the transcript level
of laminin β1 (LAMB1 ) did
not change significantly, compared with the 2D cultures, we found 3.68
times higher up-regulation in the expression of
fibronectin (FN1 ) in 3D
MTSs under the control condition. Meanwhile, the transcript levels of
vimentin (VIM ) were 1.70 and 3.18 times higher
upregulated in 3D MTSs under the
control and 5-FU treatment conditions, respectively (Figure.
4 ).
It was found that ECM participated
in CAM-DR by stimulating integrin mediated PI3K activation to protect
tumor cells from damages caused by radiotherapy and
chemotherapy (Hodkinson,
Mackinnon, & Sethi, 2007). Compared with the 2D monolayer cultures, the
expression level of integrin β1 (ITGB1 ) was 1.76 and 3.59 times
higher up-regulated in 3D MTSs before and after the 5-FU treatment,
respectively (Figure. 4 ). ITGB1 (also known asCD29 ) and CD44 are reported as tumor stem cell markers,
and the presence of tumor stem cells could affect the drug treatment and
subsequent tumor recurrence (Tomasetti, Li, & Vogelstein, 2017). It has
been shown that CD44 can mediate the stemness of tumor cells and
participate in metastasis by binding to hyaluronic acid (Gomez et al.,
2020). Compared with 2D monolayer cultures, the expression level of andCD44 was 1.79 and 2.21 times higher upregulated in 3D MTSs than
in 2D monolayer culture under the control and 5-FU treatment conditions,
respectively (Figure. 4 ).