Role of Oxidative Stress-Dependent C/EBPβ Expression on CAF Transformation Inducing HCT116 Colorectal Cancer Cell Progression; Migration and Invasion

Objective: To investigate oxidative stress-related CAF transformation through C/EBPβ, which affects CRC progression and may have a potential implication for CRC treatment. Methods: The conditioned media (CM) from HCT116, CRC cells, was used to activate CCD-18Co, colon fibroblasts, then the ability of activated FBs to induce HCT116 growth and progression was assessed using MTT assay, transwell migration, and matrix invasion assay. Alteration of the cytokine profile and oxidative stress of the activated FBs were studied with cytokine arrays and DCFH-DA assay, respectively. The protein expressions of the CAF markers (α-SMA and FAP) and C/EBPβ were investigated with immunofluorescence and western blotting. Result: It was found that CM from HCT116 cells induced oxidative stress, change of cytokine profile, CAF markers, and the C/EBPβ expression of activated FBs. Furthermore, when the oxidative stress of the activated FBs was suppressed, FAP and C/EBPβ expression were downregulated, correlating with the disabling of their capability to support the cancer progression. The C/EBPβ and prognosis for CRC patients were accessed using the GEPIA dataset, in which high C/EBPβ expression was associated with a poor prognosis. Conclusion: These findings suggest that C/EBPβ expression has a role in CAF transformation in an oxidative stress-related manner and might be used as a target to improve aggressive CRC treatment outcomes.


Introduction
Cancer is one of the leading causes of death worldwide, after cardiovascular disease, while colorectal cancer (CRC) is the second most common cause of cancer-related death (Keum and Giovannucci, 2019).The challenge of CRC treatment is distant metastasis, which involves a poor prognosis for cancer (Tauriello et al., 2017).Nowadays, the tumor microenvironment (TME), consisting of cells and extracellular matrix components, has been proven to influence the aggressiveness of cancer (Wu and Dai, 2017).Chronic inflammation, one of the hallmarks of the TME, causes oxidative stress to cancer cells which contribute to cancer development and progression (Lim and However, there has been little research on how oxidative stress can induce FBs to become CAFs.Therefore, identifying the oxidative stress-related regulators involved in how FBs are regulated to become CAFs would be beneficial for aggressive cancer treatment.Since, the TME has a niche that resembles the site of an unhealing wound, consisting of chronic inflammation and fibrosis, myofibroblasts are commonly present in the TME, which is considered to be activated FBs or CAFs (Rybinski et al., 2014).Thus, in the CAF transformation process, FB activation may resemble the activation of myofibroblasts in chronic inflammation and fibrosis sites.
CCAAT/enhancer binding protein β (C/EBPβ) is a transcriptional factor that regulates diverse cell processes and cell differentiation.Interestingly, the dysregulation of C/EBPβ is associated with oncogenic roles in many cancers, including gastrointestinal malignancy (Regalo et al., 2016).Moreover, some studies have mentioned that C/EBPβ is found to be upregulated in activated FBs in the fibrosis area of the lung and cardiac myofibroblasts of autoimmune myocarditis.These studies also suggested that C/EBPβ plays a role in the myofibroblast transformation (Hu et al., 2007;Li et al., 2018).However, the role of C/EBPβ in CAF transformation is still unknown and needs to be elucidated.
The aim of this study was to investigate the oxidative stress-related CAF transformation mechanism via C/EBPβ expression, which affects CRC progression.Therefore, HCT116, a high progressive capacity CRC cell line, was used to activate the FBs.Then, the CAF characteristics of the activated FBs in the aspect of morphology, CAF markers, and cytokine profile were examined.Furthermore, the ability to induce the HCT116 CRC progression of activated FBs was studied along with cellular oxidative stress and C/EBPβ expression.Additionally, the prognosis for CRC patients with differential C/EBPβ expression was accessed using the Gene Expressing Profiling Interactive Analysis (GEPIA) database.

Cell lines and cell culture
HCT116 (ATCC ® CCL-247TM), human colon cancer cells which have a high growth rate and are commonly used for migration and invasion experiments (Meng et al., 2019;Tao et al., 2019;Chen and Liu, 2021), were cultured with McCoy's 5A.CCD-18Co (ATCC ® CRL-1459™), human colorectal fibroblasts, was cultured with EMEM.Both mediums were supplemented with 10% FBS, 1% L glutamine, 1% penicillin-streptomycin, and non-essential amino acid.The cells were maintained at 37°C in a humidified atmosphere that was supplied with 5% CO 2 in an incubator.

Conditioned medium (CM)
The cells were cultured until they reached 70-80% confluency.The culture medium was replaced with a serum free medium, and the cells were cultured for 24 h.Then the culture medium was collected and centrifuged at 2,000 rpm for 10 min.After that, the supernatant was separated and filtered through a 0.22 µm filter, aliquoted, and kept at -20 °C for a few weeks.Finally, the CM was diluted with completed medium at a ratio of 1:1 when used for the experiments.

Fibroblast with morphological change counting
CCD-18Co cells were seeded in 6 well plates (10,000 cells/well) and cultured for 24 h.The cells were treated with CM from HCT116 for 24 h and were observed under an inverted light microscope at 40X magnification and randomly count for 100 cells from each independent experiment.The activated FBs with morphological change are considered as the criteria in Table 1.

Cytokine arrays
The cytokine array membranes (ab133996, Abcam, UK) were incubated with 1X blocking buffer at RT for 30 min.Then, the CM was incubated with membranes at 4 °C overnight and washed five times at RT for 5 min each time.The membranes were incubated with 1X Biotin-conjugated Anti-cytokines at 4 °C overnight and washed five times at RT for 5 min each time.After that, the membranes were incubated with 1X HRP-Conjugated Streptavidin at RT for 2 h and washed five times.Finally, the membranes were mixed with detection buffers, incubated at RT for 2 min, washed, detected with a Gel documentation analyzer, and had their densitometry analyzed using ImageJ software.
The densitometry was normalized with the control.According to the instruction of the cytokine array membrane, the normalized values were calculated as Eq.1.
X Ny = X y *P 1 /P y Eq.1 P 1 = mean signal density of positive control spots on reference array P y = mean signal density of positive control spots on array 'y' X y = mean signal density of spot 'X' on array of sample 'y' XN y = normalized signal intensity of spot 'X' on array 'y'

3-(4,5-dimethythiazol-2-yl)-2,5 diphenyltetrasodium bromide (MTT) assay
HCT116 cells were seeded in 96-well plates (20,000 cells/well) and cultured for 24 h.The cells were treated with CM from activated FBs for 24 h.Then, the medium was replaced by 100 μl per well of serum free medium, which contained 10 μl of 5 mg/ml MTT stock solution and incubated for 2 h.After that, the medium was replaced with 100 μl of dimethyl sulfoxide (DMSO) and the formazan soluble was detected by a microplate reader at an absorbance of 570 nm in wavelength.The results were shown as percentages of the viable cells (% cell viability) of the control.

Transwell migration and matrix invasion assay
CCD-18Co cells were cultured with CM from HCT116 in the lower part of the 24-well plates for 24 h.For the transwell migration assay, HCT116 cells (100,000 cells/ insert) were seeded into cell culture inserts (Corning, Eq.3 Eq.4

Gene expression profiling interactive analysis (GEPIA)
Differentiating the mRNA expression of C/EBPβ in colon adenocarcinoma (COAD) and in rectum adenocarcinoma (READ) was performed by using online software, Gene Expression Profiling Interactive Analysis (GEPIA), based on comparing tumors and normal samples from the TCGA (The Cancer Genome Atlas).The overall survival analysis of COAD and READ patients with high and low C/EBPβ expression was performed based on the Kaplan Meier curve.

Statistical analysis
R (version 4.1.0)and Rstudio (version 1.4.1717)were used for all statistical analyses.Comparisons between two groups were based on analysis using the student T-test.Comparisons among more than 3 groups were based on analysis using one-way ANOVA and the Tukey's post-hoc test to identify the significant differences between each group.Data are expressed as means±SD (n=3).Significant statistical difference was considered when P<0.05.

Conditioned media from HCT116 induced morphological change and CAF marker expression of fibroblasts
The morphology of activated FBs was observed, randomly counted as the criteria (Table 1) under inverted microscopy and presented as a percentage.When the FBs were treated with CM from HCT116 cells, the morphology of the FBs was changed.In the control, most FBs had an elongated-spindle shape with a smooth USA) with 8.0 μm pores and co-culture with activated FBs for 24 h, while for matrix invasion assay, the HCT116 cells were seeded into cell culture inserts coated with extracellular matrix (ECM) gel (Sigma-Aldrich, USA) and co-cultured with activated FBs for 72 h (Justus et al., 2014).The cell cultured inserts were collected, and the upper part was washed with PBS.Then, the lower part of the inserts was fixed with 4% paraformaldehyde and stained with crystal violet before being observed under a light microscope at 200X magnification and count with the ImageJ.

Wound healing assay
The HCT116 cells were seeded into 6-well plates and cultured until the cells reached 100% confluence with the monolayer.Then the wound gap was created using a pipette tip and the cells were further incubated with or without H 2 O 2 50 μM at 37 o C in a humidified atmosphere supplied with 5% CO 2 .After 12 h and 24 h the closure of the wound gap was recorded randomly in three fields.The closure of the wound was measured using ImageJ and calculated as a % of the control, as Eq.2.

Cell migration % of control = (D i -D t ) /D i * 100
Eq.2 D i = distance of wound gap at initiation D t = distance of wound gap at time t

DCFH-DA assay
Briefly, CCD-18Co cells were seeded in 96-well plates (4,000 cells/well) and incubated for 24 h.Then cells were treated with CM from HCT116, CM with H 2 O 2 , or CM with vitamin C for 24 h.The cultured medium was removed and replaced with 10 μM of DCFH-DA media and incubated for 1 h.In this process, DCF fluorescence intensity was assessed for cellular oxidants with a fluorescent microscope and a fluorescence microplate reader at excitation/emission wavelengths of 485/535 nm.

Immunofluorescent assay
CCD-18Co cells were cultured on a coverslip in 6-well plates for 24 h and treated with CM from HCT116 for 24 h.The cells were fixed in 100% methanol at 4 °C for 30 min, and then washed with PBS.After that, the cells were incubated with blocking buffer (1% BSA in PBS) for 1 h at RT, washed, and stained with primary antibodies: α-SMA (1:500, ab5694; Abcam), FAP (1:100, ab28244; Abcam), C/EBPβ (1:100, SAB2702381; Sigma-Aldrich) for 1 h at RT.The cells were washed and stained with secondary antibody conjugated with Alexa Fluor 488 (1:500, ab150077, ab150113; Abcam) for 1 h at RT.The samples were washed with PBS.Then, the cells were counter stained with Hoechst fluorescent stain.Finally, coverslips were mounted, and the slides were observed under a fluorescence microscope (Model BX53, Olympus, Tokyo, Japan).
The quantification of nuclear localization was calculated by measuring the mean fluorescence intensity and %Nuclear as Eq.3 and Eq.4,respectively (Kelley and Paschal, 2019).border and regular size, while in the CM-treated group, the activated FBs had expanded cytoplasm and irregular shapes with more dendritic borders than in the control group (Figure 1A).Moreover, when we observed the arrangement of α-SMA, the cytoskeleton of cells, we found that the activated FBs with morphological change had distinctly dense stress fibers with a crossing pattern when compared with the normal FBs (Figure 1B).Further investigation of the CAF markers, α-SMA, and FAP, by IFA showed that α-SMA was clearly upregulated in the   activated FBs with morphological changes while the FAP was slightly upregulated when the FBs were treated with CM (Figure 1C).

The cytokine profile of activated FBs was changed
The CM from the activated FBs was investigated with cytokine arrays in order to study the cytokine profile and compared it with the control (Figure 2).The activated FBs released cytokines including MCP1, IL-6, IL-8, GRO (α, β, γ), and GRO-α, while in the control, only MCP1 and IL-8 were found and there were significantly more MCP1, IL-6, IL-8, GRO (α, β, γ), and GRO-α from the activated FBs than in the control.

C/EBPβ was localized around nucleus of activated FBs
To examine the C/EBPβ localization of the activated FBs, the FBs were incubated with the CM from HCT116 for 24 h and stained with C/EBPβ antibody (Figure 3).The results show that when the FBs were activated with CM, C/EBPβ was localized in the nucleus of the FBs more than was found in the control group.

Activated fibroblast induced cancer cell proliferation and progression
To confirm that activated FBs can be representative of the CAFs which have the ability to induce cancer proliferation and progression, CM from the activated FBs was used to treat cancer cells and observe the  proliferation using MTT assay (Figure 4A).Furthermore, cancer progression was assessed by transwell migration (Figure 4B) and transwell invasion assay (Figure 4C).The results show that treated cancer cells could proliferate significantly more than when compared with the control group.Additionally, when the activated FBs were co-cultured with HCT116 cells, they were able to induce HCT116 cell migration and invasion significantly more than when compared with the control group.

Oxidative stress was increased in the activated fibroblasts and the decreased oxidative stress of activated FBs could suppress cancer progression
From studying the cellular oxidative stress of the activated FBs with DCFH-DA assay, it was found that oxidative stress increased significantly compared with the control.Additionally, to investigate the association between oxidative stress and CAF transformation, the activated FBs (CM) were manipulated under different oxidative stress conditions: oxidative stress was induced  by 50μM H 2 O 2 (CMH) and reduced oxidative stress with antioxidant, 5μM vitamin C (CMV) (Figure 5A).Furthermore, to investigate whether oxidative stress is related to the FB activation process, which affects cancer progression, activated FBs with different stress conditions were co-cultured with HCT116, and then the cancer invasion was observed.Interestingly, when the activated FBs were induced with oxidative stress, the HCT116 cells could invade the chamber significantly more than the HCT116 cells co-cultured with activated FBs could do.Also, when the oxidative stress of the activated FBs was reduced, HCT116 cell invasion was significantly suppressed compared with the HCT116 cells co-cultured with activated FBs (Figure 5B).

C/EBPβ and CAF marker expressions were upregulated in activated FBs and associated with oxidative stress condition
After the activated FBs were manipulated under different stress conditions, the α-SMA, FAP, and C/EBPβ expression levels were investigated with western blotting.The expressions of the CAF markers and C/EBPβ were significantly upregulated in the activated FBs, which had higher oxidative stress than the control.When the oxidative stress of the activated FBs was increased, none of the markers showed significant change, but the C/EBPβ and α-SMA expression tended to be upregulated.Additionally, when the oxidative stress of the activated FBs was decreased, the expression of FAP and C/EBPβ was significantly downregulated, while there was no significant change to α-SMA (Figure 6).

C/EBPβ was upregulated in migrated HCT116 colorectal cancer cells and associated with oxidative stress
Because oxidative stress has been known to be involved with cancer progression, HCT116 cell migration induced with H 2 O 2 50 μM was studied using a wound healing assay.Then, to study the correlation between oxidative stress and C/EBPβ expression on the aggressiveness of CRC, the C/EBPβ expression of HCT116 in different oxidative stress conditions harvested from wound healing assay, which migrated for different times, was investigated with western blotting.When HCT116 cells were induced with oxidative stress, they migrated faster than the control (Figure 7A, 7B).Furthermore, the C/EBPβ expression of HCT116 upregulate when HCT116 cells were induced with oxidative stress with the most significant upregulation observed at 24 h compared with the control at 0 h (Figure 7C, 7D).

C/EBPβ has higher expression in colorectal cancer and associated with low survival rate of colorectal cancer patients
The mRNA expression of the C/EBPβ in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) was compared with normal tissue near the tumor using a GEPIA database (Figure 8A).The data showed that COAD and READ had significantly higher C/EBPβ expression.Furthermore, the overall survival rate of COAD and READ patients with high C/EBPβ expression was lower than for their counterparts with low C/EBPβ expression.In addition, the hazard ratio (HR) of COAD and READ patients with high C/EBPβ expression was more than 1, which suggests that high C/EBPβ expression increased the risk of death in COAD and READ patients (Figure 8B).

Discussion
Previous studies have shown that besides the aggressiveness of cancer itself, CAFs are a crucial player supporting cancer growth and progression related to the prognosis of cancer patients (Bussard et al., 2016;Gieniec et al., 2019;Sahai et al., 2020).Therefore, more knowledge about the transformation of FBs to CAFs is necessary in order to further support the achievement of the objective of inhibiting CAF formation, which may improve the treatment outcome of patients with aggressive cancer.Because the TME niche is considered to be an unhealing wound with chronic inflammation (Greten and Grivennikov, 2019), the process of FB activation into CAFs may resemble FB activation in cases of chronic inflammation.Thus, we hypothesized that any C/EBPβ found upregulated in the FBs of chronic inflammatory disease (Hu et al., 2007;Li et al., 2018) would be related to the FB activation process in CAFs.This study supports the hypothesis because when the FBs were activated with CM from CRC, the activated FBs showed morphological change similar to the finding of previous research, which mentioned that some activated FBs underwent a morphological change that left them appearing flat with a leaf-like shape (Shimura et al., 2018).The cells which underwent a morphological change upregulated α-SMA and had cytoskeletal rearrangement, which represents an increase in the contractility of the cells (Ribatti and Tamma, 2019).When the cytokine profile of activated FBs was investigated, it was found that many pro-inflammatory cytokines, including GRO (α, β, γ), GRO-α, IL-6, IL-8, and MCP1, were upregulated compared with the normal FBs.These cytokines were observed in CAFs and found to be related to cancer proliferation, progression, and poor prognosis in cancer patients (Li et al., 2020;Chen et al., 2022;Cui et al., 2022;Gundlach et al., 2022).Moreover, the activated FBs showed CAF phenotypes, including upregulated CAF markers and enhanced cancer proliferation and progression, which correspond with the pro-inflammatory cytokine released by activated FBs.
Oxidative stress that is increased in chronic inflammation leads to cancer progression and is involved in the CAF transformation process (Lim and Moon, 2016;Tejada et al., 2019).Therefore, it has been suggested that oxidative stress might also be related to the regulation of signaling molecules in activated FBs.Previous research has shown that FBs with generated antioxidant defectives increased cellular oxidative stress and expressed more CAF phenotypes through HIF-1/CXCR4/CXCL12 pathway (Toullec et al., 2010).This is consistent with previous research which shows that prostate cancer induces the TGFβ-NOX4 pathway of FBs which then causes intracellular ROS production, resulting in CAF transformation (Sampson et al., 2018).Also, C/EBPβ was reported to be regulated by oxidative stress (Lei et al., 2020;Liu et al., 2021).Therefore, we assumed that in the process of CAF transformation, C/EBPβ might control the differentiation of the FBs in an oxidative stress-related manner.
Interestingly, the results partially support this assumption because activated FBs increased oxidative stress more than was observed with the control, while C/ EBPβ expression was upregulated and re-located into the nucleus, which suggests an association between C/EBPβ and CAF transformation.When the oxidative stress of the activated FBs was increased by H 2 O 2 , the expressions of the C/EBPβ and CAF markers were not significantly upregulated.This might be due to the duration of the oxidative stress induction not being long enough to replicate the chronic inflammatory environment.However, when the oxidative stress was reduced with an antioxidant, vitamin C, the expressions of FAP and C/EBPβ were significantly downregulated, correlating with the capability of the activated FBs to induce cancer progression being disabled.These results suggest that oxidative stress is required for C/EBPβ expression and the CAF transformation process.
According to the results, even though activated FBs induced with H 2 O 2 did not undergo significant increases in their CAF markers and C/EBPβ expression when compared with activated FBs, they could induce significantly more CRC cell invasion than the activated FBs could do.This may be explained by previous studies, which have reported that besides growth factors and cytokines, oxidative stress can induce cells to release extracellular vesicle (EV) secretions (Berumen Sánchez et al., 2021).The efficiency of the CAF-derived EV usually comes from the miRNAs contained in the EVs (Vokurka et al., 2022), which have various roles in relation to cancer cells, including influencing the cancer cells to increase their migration and invasion capability (He et al., 2021;Li et al., 2021).Therefore, the reduction of oxidative stress by an antioxidant might suppress CRC invasion through two mechanisms: suppressing both CAF transformation and EV release from the FBs.Another unexpected result is the α-SMA expression of activated FBs which was not suppressed when vitamin C was added.Because the CAFs still have no specific marker, using different combined markers is necessary to identify the CAFs (Shiga et al., 2015).The α-SMA is originally the marker of myofibroblasts, which was found in the wound healing process.One previous study found that vitamin C influences myofibroblast phenotypes including α-SMA expression and collagen secretion (Piersma et al., 2017).This effect of vitamin C other than as an antioxidant may explain why α-SMA expression was not regulated as had been expected.
The dysregulation of C/EBPβ has been found in various types of cancer, including gastrointestinal cancer (Regalo et al., 2016;Cao et al., 2021;Sterken et al., 2022).Moreover, increased cellular oxidative stress has been found in the cancer parenchymal and stromal parts (Prasad et al., 2017).For that reason, the effect of oxidative stressrelated C/EBPβ expression on CRC progression was directly investigated in CRC cells.Because metastases are the most problematic aspect of CRC, causing a low 5-year survival rate among CRC patients (Chibaudel et al., 2015;Tauriello et al., 2017), HCT116 cells were selected to represent highly aggressive CRC for studying the role of oxidative stress-related C/EBPβ in CRC progression.The results show that HCT116 induced with oxidative stress migrated faster than the control, which corresponds to the findings for C/EBPβ expression.Previous research has reported that C/EBPβ could activate the ubiquitin4/Wnt/β-catenin signaling pathway, which promotes CRC progression (Tang et al., 2021).In addition, C/EBPβ can mediated IL-6 transcription (Poli, 1998), the well-known pro-inflammatory cytokine that has a role in the EMT of cancer cells (Bharti et al., 2016;Li et al., 2020).These are the possible pathways for oxidative stress-related C/EBPβ expression inducing HCT116 progression.
From the GEPIA dataset, the mRNA of C/EBPβ was found to be upregulated in CRC and associated with a poor survival rate among CRC patients.These data correspond with the results from this study which showed that C/EBPβ plays roles in CRC progression and CAF transformation, contributing to the aggressiveness of the cancer.
Because C/EBPβ is a transcription factor induced by pro-inflammatory cytokines and responds by binding with the promoter of inflammatory cytokines (Vanoni et al., 2017), the interplay between inflammation, the pro-inflammatory cytokines, oxidative stress, and C/EBPβ expression is the key to understanding the role of C/EBPβ in CAF transformation and CRC progression.Therefore, besides the consistency of C/EBPβ expression, CAF transformation, and the aggressiveness of CRC, more knowledge about oxidative stress-related C/EBPβ pathways in CAF transformation will benefit TME-targeted therapy in patients with aggressive colorectal cancer.
In conclusion, this study proposed a novel aspect of C/EBPβ in CAF transformation which affects HCT116 CRC cell growth and progression (Figure 9) and showed that the expression of C/EBPβ depends on cellular oxidative stress in both CAFs and CRC cells.Furthermore, high C/EBPβ expression is associated with a poor prognosis for CRC patients.This knowledge can be applied to improve the treatment outcomes of aggressive CRC.

Figure 1 .
Figure 1.Morphology and CAF Markers of Activated FBs.(A) Morphology observed under inverted microscopy at 40X magnification after 24 h of CM treatment with the graph of percentage of morphological change at 12 h and 24 h.Scale bar: 400 μm (B) Cytoskeletal arrangement of normal FBs and activated FBs with morphological change.Scale bar: 100 μm (C) Immunofluorescence of α-SMA and FAP.Scale bar: 200 μm.Results were statistically significant at *P<0.001.

Figure 2 .
Figure 2. Cytokine Array of the CM from Activated FBs Comparing with the Control.Results were statistically significant at *P <0.05 and.ND = Not detected.

Figure 3 .
Figure 3. Immunofluorescence of C/EBPβ after being Incubated with CM from HCT116 for 24 h.The arrows sign indicated cells that C/EBPβ was localized in the nucleus.Scale bar: 200 μm.Results were statistically significant at *P <0.001.

Figure 4 .
Figure 4. Cancer Proliferation and Progression.(A) MTT assay of HCT116 cells after incubated with CM from activated FBs for 24 h.(B) Transwell migration assay of HCT116 cells co-cultured with activated FBs for 24 h.(C) Matrix invasion assay of HCT116 cells co-cultured with activated FBs for 72 h.Results were statistically significant at *P<0.05 and **P<0.01.

Figure 5 .
Figure 5. Oxidative Stress of Activated FBs and Effects of Activated FBs with Different Oxidative Stress on Cancer Progression.(A) DCFH-DA assay of activated FBs incubated with CM from HCT116 (CM), CM with H 2 O 2 50μM (CMH), and CM with vitamin C 5μM (CMV).(B) Matrix invasion assay of HCT116 cells co-cultured with activated FBs in different oxidative stress conditions.Results were statistically significant at *P <0.05, **P<0.01,and ***P<0.001.

Figure 6 .
Figure 6.C/EBPβ and CAF Marker Expressions in Activated FBs Related with Oxidative Stress.CM from HCT116 promotes CAF markers (α-SMA and FAP) and C/EBPβ expression in different oxidative stress conditions.The CAF markers and C/EBPβ expression levels were measured with western blotting and used β-actin as the loading control.Results were statistically significant at *P<0.05 and **P<0.001.

Figure 7 .
Figure 7. HCT116 Migration and C/EBPβ Expression of HCT116 with Oxidative Stress Induction.(A) Wound healing migration of HCT116 when induced with H2O2 50 μM at 12 h and 24 h.(B) Cell migration (% of control) of HCT116 when induced with H2O2 50 μM at 12 h and 24 h.(C) C/EBPβ expression of HCT116 at different time and oxidative stress conditions.(D) Relative C/EBPβ expression of HCT116 at different time and oxidative stress conditions.The C/EBPβ expression level was measured with western blotting and used β-actin as the loading control.Results were statistically significant at *P<0.05, **P<0.01,and ***P<0.001.

Figure 8 .
Figure 8.The mRNA Expression and Overall Survival Rate Analysis of Colon Adenocarcinoma (COAD) and Rectum Adenocarcinoma (READ) Patients Using the GEPIA Database.(A).The box plot shows the relative C/EBPβ expression in COAD and READ (red) compared with normal tissue near the tumor (gray).COAD; n (tumor)=275, n (normal)=41.READ; n (tumor)=92, n (normal)=10.(B) The C/EBPβ expression and overall survival analysis of COAD and READ patients.n=181/group.Results were statistically significant at *P<0.01.

Figure 9 .
Figure 9. Roles of Oxidative Stress-Related C/EBPβ Expression in Aggressive CRC.The migration of HCT116, colorectal cancer cells, is influenced by oxidative stress-related C/EBPβ expression.Additionally, HCT116 induces CAF transformation through oxidative stress-related C/EBPβ expression and the CAFs eventually support cancer growth and progression (migration and invasion) by secreting pro-inflammatory cytokines.

Table 1 .
Criteria for Counting Activated FBs under Inverted Light Microscopy at 100X Magnification.The activated FBs with morphological change are considered when score≥2.Scale bar: 150 μm.