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International Journal of Bioprinting Increased ECM stiffness enhances chemoresistance

Since then, the 6%-3DP model maintained a high growth 3DP model (127.6 vs. 62.85 µM, 4.997 vs. 3.305 µM, 39.59
rate, while because there was not enough room to grow, vs. 27.17 µM, respectively) (Figure 7A–C).
the number of cells in the 2D model gradually decreases. To investigate the mechanism underlying the differences
Moreover, the amount of cells in the 6%-3DP model in the pharmacodynamics of 3D ovarian cancer models
increased approximately four times by day 10, while the with different stiffness, we collected RNA from 6%-3DP
number of cells on day 10 in the 2D model was similar and 8%-3DP cells after 10 days of culture and evaluated
to that on day 1 (Figure 3B). Notably, in both 3D models, the expression of multidrug resistance genes. Our results
the cells maintained a high growth rate. Nevertheless, revealed that the stiffness of ECM affects the expression of
differences in the rate of proliferation between the two drug resistance genes. In 8%-3DP model, the expression
models began to appear starting day 3, and by day 10, the of MDR1 and MRP1 genes were obviously increased,
number of cells in the 6%-3DP and 8%-3DP models had and their mRNA levels were approximately four and two
approximately quadrupled and tripled, respectively, as times higher than those in 6%-3DP model, respectively.
compared to the cell number on day 1 (Figure 3C).
However, there was no significant difference in ABCA1
3.3. Tumor-related protein and mRNA expression expression between the two groups (Figure 7D–F).
To assess the biological activity of tumor cells in the
3D-bioprinted models, we used immunofluorescence to 3.6. Transcriptional profiling of 3D-bioprinted
detect the expression of ovarian cancer-related proteins in models with different stiffness
6%-3DP and 8%-3DP models. Our findings revealed that Next, we found significant differences in transcriptional
Ki-67 and p53 proteins were expressed in both models, profile between the 6%-3DP and 8%-3DP models. A total
indicating high activity of tumor cells in these models of 217 DEGs were identified, including 145 upregulated
(Figure 4). genes and 72 downregulated genes (Figure 8A and 8B).
To explore the functional characteristics of DEGs, we
To investigate whether ECM stiffness affects tumor- performed GO pathway enrichment analysis. GO analysis
related genes, we extracted mRNA from cells in 6%-3DP results demonstrated that the upregulated genes were
and 8%-3DP models 10 days after printing. We evaluated obviously enriched in “metabolic process,” “response to
the expression of p53, IL-6, ALDH1, CD44 and CD133 chemical,” “phosphorus metabolic process,” “response to
genes. Our findings indicate that the levels of p53 and IL-6 organic substance,” and “response to endogenous stimulus.”
mRNA were approximately eight- and three-fold higher, For the downregulated DEGs, the most enriched GO
respectively, in 8%-3DP group than in 6%-3DP group. terms included “immune system process,” “interspecies
Additionally, as the stiffness of 3D tumor model increased, interaction between organisms,” “response to external
the expression of tumor stemness markers ALDH1 and stimulus,” “regulation of response to stress,” and “response
CD44 was upregulated, whereas the expression of CD133 to biotic stimulus” (Figure 9).
did not change significantly (Figure 5).
Additionally, bioinformatics analysis also revealed
3.4. Effects of antitumor drugs on 2D-Ov and that elevating the stiffness of the 3D-bioprinted ovarian
3D-bioprinted model cancer model led to increased expression of key genes
Initially, we evaluated the response of 2D-Ov and 6%-3DP associated with chemotherapy resistance, namely EGR1,
cells to anti-tumor drugs by treating both models with FOS, CRYAB, G6PD, DUSP1, PIM1, PPDPF, HDAC5,
carboplatin, paclitaxel, and olaparib for 72 h. Our results and FGFR4 (Figure 8C). These findings align with
revealed that the IC values of carboplatin, paclitaxel, and previous research that has confirmed the correlation
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olaparib in the 6%-3DP models were significantly higher between overexpression of these genes and enhanced
compared to those in 2D-Ov models (62.85 vs. 9.921 chemoresistance, which has implication on shortening the
µM, 3.305 vs. 0.004 µM, 27.17 vs. 8.119 µM, respectively) overall survival durations. 39-47
(Figure 6).
4. Discussion
3.5. Effects of antitumor drugs on 3D-bioprinted
models with different stiffness In this study, we used 3D bioprinting technology to
To further investigate the impact of the structural stiffness construct in vitro ovarian cancer tumor models and
of 3D tumor models on pharmacodynamics, we treated generated ovarian cancer models with varying degrees of
6%-3DP and 8%-3DP models with carboplatin, paclitaxel, stiffness by adjusting GelMA hydrogel content. Thus, we
and olaparib for 72 h. Our findings indicated that the IC were able to simulate ovarian cancer tissues with different
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values of the three anti-tumor drugs measured in the 8%- levels of stiffness that closely resembled those observed in
3DP model were higher than those measured in the 6%- clinical settings. Compared with traditional 2D models,

Volume 10 Issue 3 (2024) 235 doi: 10.36922/ijb.1673