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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2023 Mar 11;149(10):7235–7246. doi: 10.1007/s00432-023-04670-w

TIMP-2 as a predictive biomarker in 5-Fu-resistant colorectal cancer

Yaoqing Li 1, Chuchu Xu 1, Renjun Zhu 2, Liyijing Shen 3, Gengyuan Hu 1, Kelong Tao 1, Feng Tao 1,4,✉, Zengxin Lu 3,4,✉, Guolin Zhang 1,✉
PMCID: PMC11797206  PMID: 36905423

Abstract

Purpose

This study aims to evaluate the value of tissue inhibitors of MMPs-2 (TIMP-2) to indicate 5-Fluorouracil (5-Fu) resistance status in colorectal cancer.

Methods

The 5-Fu resistance of colorectal cancer cell lines was detected using Cell-Counting Kit-8 (CCK-8) and calculated using IC50. Enzyme-linked immunosorbent assay (ELISA) and real time-quantitative polymerase chain reaction (RT-qPCR) were used to detect TIMP-2 expression level in the culture supernatant and serum. Twenty-two colorectal cancer patients' TIMP-2 levels and clinical characteristics were analyzed before and after chemotherapy. Additionally, the patient-derived xenograft (PDX) model of 5-Fu resistance was used to evaluate the feasibility of TIMP-2 as a predictive biomarker of 5-Fu resistance.

Results

Our experimental results display that TIMP-2 expression is elevated in colorectal cancer drug-resistant cell lines, and its expression level is closely related to 5-Fu resistance. Moreover, TIMP-2 in colorectal cancer patient serum undergoing 5-Fu-based chemotherapy could indicate their drug resistance status, and its efficacy is higher than CEA and CA19-9. Finally, PDX model animal experiments reveal that TIMP-2 can detect 5-Fu resistance in colorectal cancer earlier than tumor volume.

Conclusion

TIMP-2 is a good indicator of 5-Fu resistance in colorectal cancer. Monitoring the serum TIMP-2 level can help the clinician identify 5-Fu resistance in colorectal cancer patients earlier during chemotherapy.

Keywords: TIMP-2, Biomarker, 5-Fu, Drug resistance, Colorectal cancer

Introduction

According to the American Cancer Society assessment in 2021, colorectal cancer ranks third in incidence among all cancers in America. Colorectal cancer also ranks third in cancer mortality (Siegel et al. 2021). The 5-fluorouracil (5-Fu) has become the first-line chemotherapy drug for colorectal cancer treatment by blocking cell DNA synthesis and histone deacetylation (Du et al. 2017; Lawrence et al. 1975). Chemotherapy with 5-Fu as the basic drug initially displays a perfect therapeutic efficacy for colorectal cancer, but patients develop 5-Fu resistance with long-term administration.

The mechanism of 5-Fu resistance has been extensively researched. Currently recognized mechanisms of 5-Fu resistance include, but are not limited to, 5-Fu metabolic enzyme destruction, drug transporter damage, epigenetic changes, and tumor microenvironment disorders (Blondy et al. 2020; Liu et al. 2019; Marjaneh et al. 2019). Cytokines regulate cell growth, differentiation, and immune response by binding to corresponding receptors. Several studies demonstrate their importance in regulating drug resistance (Zhang et al. 2020; Wang et al. 2019; Yin et al. 2017). We believe that further research into cytokines sheds more light on the mechanism of 5-Fu resistance.

Matrix metalloproteinases (MMPs) are a family of proteases that can target many extracellular proteins, including other proteases, cell surface receptors, and adhesion molecules. MMP-2, MMP-3, MMP-7, and MMP-9 are important factors for normal tissue remodeling during embryonic development, cancer invasion, angiogenesis, carcinogenesis, and apoptosis (Apte and Parks 2015; Ahmed et al. 2018). Tissue inhibitors of MMPs (TIMPs) primarily function to inhibit MMPs. TIMPs can irreversibly inactivate MMPs by binding directly to the catalytic zinc cofactor (Chirco et al. 2006; Escalona et al. 2018). TIMPs family is divided into TIMP-1, TIMP-2, TIMP-3, and TIMP-4, and they have certain homology. TIMP-2 functions as an intercellular signal messenger on MMP-2 at the cell membrane surface to regulate cellular physiological activities and pathophysiological processes (Shen et al. 2010).

Research reveals melanoma, liver, and breast cancer resistance to chemotherapy drugs are linked to TIMP-2 (Tavakoli et al. 2018; Bjornland et al. 1998; Waleh et al. 2010). Researchers have discovered that siRNA targeting TIMP-2 inhibits the invasiveness of the colorectal cancer tumor cell line HCT116 (Nishimoto and Nishida 2006). Clinical experiments revealed that MMP-2 and TIMP-2 are more expressed in colorectal cancer patient’s tumor tissues than in normal people. MMP-2 and TIMP-2 expression levels are higher in the tumor tissue of colorectal cancer patients with metastasis than in non-transformed colorectal cancer patients (Groblewska et al. 2014; Park et al. 2011). Additionally, elevated TIMP-2 levels have been detected in colorectal cancer patients with chemotherapy intolerance (Gentner et al. 2009). However, TIMP-2 mechanism of 5-Fu resistance remains poorly understood in colorectal cancer.

Materials and methods

Cell culture

HCT116 and DLD-1 colorectal cancer cell lines were purchased from American Type Culture Collection (ATCC, Manassas) and cultured in Dulbecco's Modified Eagle Medium (DMEM) with higher glucose levels (Genom) and RPMI-1640 (Genom) containing 10% fetal bovine serum (FBS, GIBCO). It was continuously cultivated in an incubator at 37 °C in a 5% CO2 atmosphere. 5-Fu was purchased from MedChemExpress.

Screening and culture of 5-Fu-resistant cell lines

HCT116 and DLD-1 cells were grown in the corresponding medium containing 5-Fu for 2–3 days and then cultured in the corresponding medium without 5-Fu for 2–3 days. HCT116 and DLD-1 cells were cultured with a 5-Fu concentration of 0.1 μM. The 5-Fu concentration was increased two to three times per cycle during alternating culture with 5-Fu-containing medium and without 5-Fu. Since cell survival was poor in the medium containing a higher 5-Fu concentration, we would continue to culture HCT116 and DLD-1 cells in the medium containing the initial 5-Fu concentration for a time. The 5-Fu concentration in the culture solution was continuously increased to achieve screening. Finally, HCT116 cells can survive in the medium with a 5-Fu concentration of 10 μM, while DLD-1 cells can survive in a medium with a 5-Fu concentration of 20 μM. The cell viability assay and IC50 were tested regularly to determine whether drug-resistant cell lines were formed.

Cell viability assay

Cell-Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies) was used to detect cell viability. The operation process was strictly conducted following the instruction. Each sample was tested at least three times.

Enzyme-linked immunosorbent assay (ELISA)

TIMP-2, CEA, and CA19-9 levels in the cell culture supernatant or serum were detected using the sandwich ELISA kit (Elabscience) and followed the operation manual. The absorbance was measured at 450 nm using the microplate reader. Each sample was tested at least three times.

RNA isolation and real-time-quantitative polymerase chain reaction (RT-qPCR)

The Trizol reagent (Invitrogen) was used to extract total RNA. The cDNA was synthesized using the cDNA reverse transcriptase kit (Takara). The LightCycler 480 real-time PCR system (Roche, Mannheim) was used to perform SYBR Green-based (Takara) RT-qPCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as the internal control. The mRNA levels of the target genes were analyzed using the 2−ΔΔCq relative quantification method.

Ethical considerations

The ethical approval was obtained from the ethical approval agency at Shaoxing People's Hospital (study number: 2021-K-Y-158–01). The standard animal care guidelines were followed in this study.

Study patients

Colorectal cancer patient serum was collected between 2010 and 2018 at Shaoxing People's Hospital. More than two senior pathologists were responsible for patient tumor pathology. The 5-Fu-based chemotherapy regimen was agreed upon by two or more oncologists to treat the patient. Twenty-two patients were included in the analysis and equally divided into two groups: 5-Fu sen and 5-Fu res. This classification standard was based on the treatment effect of patients within six months after entering the chemotherapy cycle. Patients who did not have tumor progression six months after chemotherapy were designated 5-Fu sen. If the patient's tumor progressed within six months, it was called 5-Fu res. The clinical tumor tissue was obtained from a 66-year-old with pathologic stage III adenocarcinoma rectal cancer in the patient-derived xenograft (PDX) model. The patient had not received radiotherapy or chemotherapy before surgery. The postoperative chemotherapy process suggested that his tumor tissue was sensitive to 5-Fu.

Animal experiments

Four-week-old BALB/c-nude mice were provided by SiBeiFu Biotechnology Co., Ltd (Beijing). Tumor tissue from colorectal cancer patients was subcutaneously implanted in the groin of the nude mice. In the experiment, 40 mg/kg of sodium pentobarbital was used as an anesthetic. According to our experimental procedure, nude mice were divided into three groups: -5-Fu, + 5-Fu sen, and + 5-Fu res. When the tumors planted under the skin of nude mice grew to the third week, part of the mice was injected intraperitoneally with 30 mg/kg of 5-Fu twice a week (n = 12), referred to as the experimental group; the remaining were injected with the same dose of normal saline, referred to as the -5-Fu group (n = 5). The tumor volume of some nude mice was controlled under continuous 5-Fu treatment. They were called the + 5-Fu sen group (n = 9). However, the tumor volume of the other mice was controlled first and then increased rapidly. They were called the + 5-Fu res group (n = 3). Mouse tumor volume was measured weekly as follows: V = Length × Width2 × /2. Blood was obtained from the eyelids of nude mice every week to analyze the changes in some cytokines and tumor markers, such as CEA and CA19-9. The experimental mice were killed when they reached the humanitarian end-of-animal life standards.

Statistical analysis

Statistical analysis was performed using SPSS 22.0 software and GraphPad Prism 8.0 software. The data from the independent experiment in triplicates were presented as means ± standard deviation (SD). The one-sample Kolmogorov–Smirnov test was used to analyze whether the experimental data conformed to the normal distribution. Then an independent sample t test or non-parametric test was used to analyze the two groups’ experimental results. One-way ANOVA multiple comparison analysis with Tukey’s posttest was also used. Two-way ANOVA with Fisher’s LSD test was used for independent sample two-way variance analysis. Simple linear regression was employed when studying the correlation between the two; r represented the correlation coefficient. In all cases, p values were two sided. P ≤ 0.05 was considered statistically significant.

Results

TIMP-2 level significantly correlated with 5-Fu resistance of colorectal cancer in vitro

As described in the method, we continuously increase 5-Fu concentration on the culture medium (CM) of colorectal cancer cell lines HCT116 and DLD-1 to obtain drug-resistant cell lines. Consequently, we designated the original colorectal cancer cell lines HCT116 5-FuS and DLD-1 5-FuS that were sensitive to 5-Fu. These 5-Fu-resistant cell lines were identified as HCT116 5-FuR and DLD-1 5-FuR after screening. We monitored the 50% inhibitory concentration (IC50) changes during the screening for up to 12 months. IC50 of drug-resistant cell lines increased as 5-Fu pressure increased, and sensitive and drug-resistant cell lines differ significantly (Fig. 1A and B). IC50 of HCT116 5-FuR was 4.93-fold that of HCT116 5-FuS by the 12th month, and IC50 of DLD-1 5-FuR was 11.58-fold that of DLD-1 5-FuS, demonstrating that drug-resistant cell lines were successfully constructed (Fig. 1C and D).

Fig. 1.

Fig. 1

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Screening and culture of colorectal cancer cell lines resistant to 5-Fu. A–B IC50 changes of HCT116 and DLD-1 cells by gradually increasing 5-Fu concentration in CM. C–D Relative cell viability of 5-Fu-sensitive and -resistant cell lines in increasing concentration of 5-Fu for three days. *p < 0.05, **p < 0.01, ***p < 0.001

During screening and culturing drug-resistant cell lines, we monitored the TIMP-2 protein expression level on the CM at various time points. As expected, colorectal cancer cell lines that had undergone 5-Fu screening expressed higher TIMP-2 levels on CM than those that had not experienced 5-Fu, both in HCT116 and DLD-1 cell lines after the first month of 5-Fu stress (Fig. 2A and B). We performed ELISA and RT-qPCR to detect the changes in TIMP-2 expression levels in each cell line in the 12th month to strengthen our verification. ELISA showed that secreted TIMP-2 expression in drug-resistant cell lines detected on CM was significantly increased (Fig. 2C). From the perspective of transcription level, the semi-quantitative mRNA analysis indicated that TIMP-2 transcription of HCT116 5-FuR and DLD-1 5-FuR were significantly higher than HCT116 5-FuS and DLD-1 5-FuS (Fig. 2D).

Fig. 2.

Fig. 2

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TIMP-2 level is significantly correlated with 5-Fu resistance in colorectal cancer cell lines. A–B TIMP-2 protein expression level differences in 5-Fu-sensitive and -resistant cell lines at different time points. C TIMP-2 protein expression level in the paired DLD-1 5-FuS and DLD-1 5-FuR cells, HCT116 5-FuS and HCT116 5-FuR cells in the 12th month. D Semi-quantitative TIMP-2 mRNA expression level in the paired DLD-1 5-FuS and DLD-1 5-FuR cells, HCT116 5-FuS and HCT116 5-FuR cells in the 12th month. (E–F) Scatter plots showing a correlation between IC50 and TIMP-2 levels in HCT116 and DLD-1 cells. *p < 0.05, **p < 0.01, ***p < 0.001

Furthermore, we explored the relationship between TIMP-2 expression level and drug resistance of colorectal cancer cell lines. Strikingly, there was a significant correlation between TIMP-2 expression levels and IC50 of tested colorectal cancer cell samples (Fig. 2E and F).

Serum TIMP-2 levels indicate colorectal cancer patients are resistant to 5-Fu

We tested the serum TIMP-2 expression levels in patients who were sensitive or resistant to 5-Fu treatment to study whether TIMP-2 could indicate the condition of colorectal cancer patients resistant to 5-Fu. Before the start of chemotherapy, the serum TIMP-2 expression level of patients who show 5-Fu resistance was higher than those who show 5-Fu sensitivity (Fig. 3A). This suggests that TIMP-2 may have a role in identifying primary resistance. Furthermore, after chemotherapy, TIMP-2 expression was significantly higher in the serum of 5-Fu-resistant patients than in 5-Fu-sensitive patients (Fig. 3B), proving the higher value of TIMP-2 in identifying colorectal cancer patients with acquired 5-Fu resistance. We compared the changes in TIMP-2 expression in 5-Fu-sensitive patients before and after chemotherapy and found no significant changes (Fig. 3C). However, we discovered that TIMP-2 expression level was significantly higher after chemotherapy in 5-Fu-resistant patients than before chemotherapy (Fig. 3D). These experimental results indicated that TIMP-2 expression level could prompt 5-Fu resistant in colorectal cancer patients.

Table1.

Patient characteristics of 5-Fu-sensitive or -resistance set

Patient Age (years) Sex Stage Histology Chemotherapy

5-Fu

sen

 P1375 67 M IVA Adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin + Bevacizumab
P4362 72 F IVA Adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin + Cetuximab
P2143 57 M IVB Adenocarcinoma 5-Fu + Oxaliplatin + Bevacizumab
P1237 68 F IIIC Mucus adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin
P2465 61 F IVB Adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin + Bevacizumab
P2434 76 M IIIC Mucus adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin + Bevacizumab
P1274 56 M IVA Adenocarcinoma 5-Fu + Oxaliplatin
P2358 77 M IVB Adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin
P0136 48 F IVA Adenocarcinoma 5-Fu + Oxaliplatin
P0941 66 M IVB Adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin + Bevacizumab
P1452 63 F IVB Adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin

5-Fu

res

 P1683 35 F IVB Adenocarcinoma 5-Fu + Oxaliplatin
P2346 68 M IIIC Adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin
P2298 41 F IVA Adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin + Bevacizumab
P2461 71 M IVA Adenocarcinoma 5-Fu + Irinotecan + Bevacizumab
P3119 56 M IVB Adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin + Cetuximab
P2391 69 F IVA Mucus adenocarcinoma 5-Fu + Oxaliplatin
P0843 52 F IVB Adenocarcinoma 5-Fu + Irinotecan + Bevacizumab
P0716 78 M IVA Adenocarcinoma 5-Fu + Irinotecan + Bevacizumab
P2711 45 F IVA Mucus adenocarcinoma 5-Fu + Oxaliplatin
P1185 80 M IVB Adenocarcinoma 5-Fu + Irinotecan + Bevacizumab
P2263 63 F IIIC Adenocarcinoma 5-Fu + Irinotecan + Oxaliplatin + Cetuximab
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Table 2.

Correlation between patient resistant to 5-Fu and clinical characteristics

Characteristics Total 5-Fu sen 5-Fu res OR 95% CI P value
All cases 22 11 (50.0%) 11 (50.0%)
Age (years)
  ≥ 65 11 6 (54.6%) 5 (45.4%)
  < 65 11 5 (45.4%) 6 (54.6%) 0.694 0.130–3.720 0.670
Gender
 Male 11 6 (54.6%) 5 (45.4%)
 Female 11 5 (45.4%) 6 (54.6%) 0.694 0.130–3.720 0.670
Stage
 IIIC 4 2 (60.0%) 2 (40.4%)
 IVA 9 4 (44.4%) 5 (55.6%)
 IVB 9 5 (55.6%) 4 (44.4%) 0.895
Histological type
 Adenocarcinoma 18 9 (50.0%) 9 (50.0%)
Mucus adenocarcinoma 4 2 (50.0%) 2 (50.0%) 1.000 0.580
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P value calculated by Chi-square test

Fig. 3.

Fig. 3

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TIMP-2 activity correlates with 5-Fu resistance in colorectal cancer patients. A–B Comparison of the serum TIMP-2 protein expression level in 5-Fu-sensitive (n = 11) and -resistant (n = 11) colorectal cancer patients in the pre-chemotherapy or after-chemotherapy. C–D Difference of the serum TIMP-2 protein expression level before and after chemotherapy in 5-Fu-sensitive (n = 11) and -resistant (n = 11) colorectal cancer patients. Patient details are displayed in Tables 1 and 2. Sen, sensitive patients. Res, resistant patients. *p < 0.05, **p < 0.01, ***p < 0.001

As CEA and CA19-9 play an important role in colorectal cancer diagnosis and prognosis, we evaluated their value as an indicator of drug resistance in colorectal cancer patients. Before chemotherapy, there was no difference in CEA and CA19-9 expression between 5-Fu-sensitive and -resistant patients (Fig. 4A and E), but after chemotherapy, 5-Fu-resistant patients have higher CEA and CA19-9 expression than 5-Fu-sensitive patients (Fig. 4B and F). CEA and CA19-9 levels were significantly reduced in 5-Fu-sensitive patients after chemotherapy (Fig. 4C and G). However, CEA and CA19-9 levels did not change significantly in 5-Fu-resistant patients after chemotherapy (Fig. 4D and H). CEA and CA19-9 also have a certain value in detecting 5-Fu resistance in colorectal cancer. However, they are as ineffective as TIMP-2 based on our clinical results.

Fig. 4.

Fig. 4

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Evaluations of tumor marker candidates for colorectal cancer patients resistant to 5-Fu. A–B Comparison of the serum CEA expression level in 5-Fu-sensitive (n = 11) and -resistant (n = 11) colorectal cancer patients before and after chemotherapy. C–D Difference of the serum CEA expression level before and after chemotherapy in 5-Fu sensitive (n = 11) and resistant (n = 11) in colorectal cancer patients. E–F Comparison of the serum CA19-9 expression level in 5-Fu-sensitive (n = 11) and -resistant (n = 11) colorectal cancer patients before and after chemotherapy. G–H Difference of the serum CA19-9 expression level before and after chemotherapy in 5-Fu-sensitive (n = 11) and -resistant (n = 11) colorectal cancer patients. Patient details are displayed in Tables 1 and 2. Sen, sensitive patients. Res, resistant patients. *p < 0.05, **p < 0.01, ***p < 0.001

TIMP-2 activity but not CEA or CA19-9 level early warns colorectal cancer resistant to 5-Fu in vivo

We used PDX model in vivo to prove that TIMP-2 has an early warning effect on 5-Fu resistance in colorectal cancer patients with chemotherapy. The PDX model can retain most primary tumor characteristics, including histopathology, molecular biology, and genetic level predictability. Figure 5A displays the basic steps for constructing a PDX model of colorectal cancer resistance.

Fig. 5.

Fig. 5

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Successful construction of a PDX model of colorectal cancer resistant to 5-Fu. A brief schematic presentation of PDX-drug resistance model setup. B Changes curves of tumor volume in Veh, 5-Fu sen, and 5-Fu res group PDX mice models during the experiment. C Differences of tumor volume in Veh, 5-Fu sen, and 5-Fu res group PDX mice models in the seventh and ninth week. -5Fu (Veh), not treated with 5-Fu. 5-Fu sen, treated with 5-Fu and sensitive to 5-Fu. 5-Fu res, treated with 5-Fu and resistant to 5-Fu. *p < 0.05, **p < 0.01, ***p < 0.001

We measured the tumor volume of nude mice every week for up to nine weeks. The tumor volume of nude mice in 5-Fu group continued to increase from the third week of drug treatment, while the tumor volume of the nude mice was controlled in the experimental group. In + 5-Fu res group, nude mice began to develop tumors around the seventh week as 5-Fu started to fail. The mice in + 5-Fu sen group had tumor volume that was always controlled by 5-Fu (Fig. 5B). The tumor volume change in nude mice was the best criterion to confirm 5-Fu resistance in nude mice. In our PDX model, it is evident that + 5-Fu res group nude mice exhibited resistance in the seventh week. Figure 5C displays the tumor volume changes of the three groups of nude mice in the seventh and ninth weeks.

We detected TIMP-2, CEA, and CA19-9 concentrations in nude mice serum every week. The TIMP-2 level in nude mice serum was significantly in + 5-Fu res group than in + 5-Fu sen group as early as the fourth week, let alone during the longer 5-Fu processing time (Fig. 6A). Figure 6B depicts the TIMP-2 expression level in the serum of nude mice during the fourth week when the expression difference started, and the ninth week, when the experiment ended. However, CEA and CA19-9 expression levels in the serum of nude mice did not differ during the experiment (Fig. 6C and D). In the PDX model, the serum TIMP-2 level of nude mice could be resistant to 5-Fu treatment three weeks earlier than the tumor volume, while CEA and CA19-9 in the serum of nude mice did not indicate drug resistance (Fig. 6E).

Fig. 6.

Fig. 6

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TIMP-2 activity early warns colorectal cancer resistant to 5-Fu in vivo. A Change curves of TIMP-2 protein expression levels in Veh, 5-Fu sen, and 5-Fu res group PDX mice models during the experiment. B Differences of TIMP-2 protein expression levels in Veh, 5-Fu sen, and 5-Fu res group PDX mice models in the fourth and ninth week. C-D Changes in curves of CEA, CA19-9 expression level in Veh, 5-Fu sen, and 5-Fu res group PDX mice models during the experiment. -5Fu (Veh), not treated with 5-Fu. 5-Fu sen, treated with 5-Fu and sensitive to 5-Fu. 5-Fu res, treated with 5-Fu and resistant to 5-Fu. *p < 0.05, **p < 0.01, ***p < 0.001. E Schematic diagram of TIMP-2 predicting colorectal cancer resistance to 5-Fu

Discussion

This study highlights the potential role of TIMP-2 as a non-invasive biomarker for 5-Fu resistance in colorectal cancer through cell experiments, clinical data, and PDX models. It demonstrates that TIMP-2 could indicate 5-Fu resistance in colorectal cancer several weeks before clinically observed changes in tumor size. It proves that TIMP-2 is a better indicator of 5-Fu resistance in colorectal cancer than CEA and CA19-9.

Non-anatomical factors are important for evaluating 5-Fu resistance in colorectal cancer patients. It can alert clinicians before chemotherapy drug resistance and change the treatment plan to improve the patient's prognosis (Na et al. 2021). Currently, there is no recognized biomarker to determine 5-Fu resistance in colorectal cancer patients. Many researches focus on the drug resistance mechanism of colorectal cancer and predict the prognosis after colorectal chemotherapy (Wasserman et al. 2019; Zhang et al. 2019; Blondy et al. 2021). We can extend patient life and improve their life quality by discovering the resistance status of colorectal cancer to chemotherapy drugs in advance and clinicians changing treatment plans in time (Woolston et al. 2019).

Numerous studies have confirmed that cytokines regulate tumor-related innate immunity, adaptive immunity, angiogenesis, tissue damage repair, and other functions (Propper and Balkwill 2022). Researchers have discovered that CXC family, HIF-1α, TGF-β, and other cytokines are closely related to the drug resistance of colorectal cancer (Cabrero-de Las Heras and Martínez-Balibrea 2018; Tang et al. 2018). TIMP-2 is one of the family members that regulate MMPs’ activity. It binds to MMPs on the cell membrane surface by forming a non-covalent stoichiometric complex and then transmits the next step of the intercellular signaling pathway. TIMP-2 has been linked to tumor growth, lymph node invasion, and distant metastasis (Peeney et al. 2020; Wu et al. 1990). In the preliminary study, we discovered that TIMP-2 could promote 5-Fu resistance in colorectal cancer through MAPK/ERK signaling pathway, and TIMP-2 inhibition may effectively reverse 5-Fu resistance in colorectal cancer.

This study focused on whether TIMP-2 can accurately indicate the 5-Fu resistance in colorectal cancer. We discovered that TIMP-2 expression is positively correlated with IC50 of drug-resistant colorectal cancer cell lines during the culture process, representing drug resistance levels. This provides solid experimental support for our cellular-level conclusion. We also evaluated the changes in TIMP-2 before and after drug resistance in clinical patients and PDX models. We attempted to use it as a clinically specific indicator of 5-Fu resistance in colorectal cancer. The experimental results demonstrated that TIMP-2 could be used as an excellent indicator of 5-Fu resistance in colorectal cancer than the most commonly used tumor size indicator.

CEA and CA19-9 are typically used in clinical practice to assess and predict the occurrence, progression, and metastasis of colorectal cancer (Das et al. 2017; Ushigome et al. 2020). This study compared TIMP-2 with CEA and CA19-9 to determine their accuracy, pros, and cons for the indication effect and to indicate drug resistance in colorectal cancer. Consistent with our conjecture, TIMP-2 revealed a better indicator of colorectal cancer for 5-Fu resistance status than CEA and CA19-9. However, the tumor volume change is our current gold standard for indicating drug resistance in colorectal cancer, and it cannot be compared to these molecular markers. Therefore, we cannot directly compare TIMP-2 with CEA and CA19-9 quantitatively. Additionally, if more clinical features were included for analysis, the experimental conclusions derived from our experimental results would be more convincing.

Consequently, this novel study proves the clinical significance of TIMP-2 in the blood of colorectal cancer patients as a non-anatomical cancer biomarker that may indicate the 5-Fu resistance in colorectal cancer. Additionally, TIMP-2 is superior to CEA and CA19-9 in detecting 5-Fu resistance in colorectal cancer through earlier prediction than tumor volume changes. Regarding the exploratory design of our experiments, we need further randomized clinical trials to confirm these findings. We believe it may be good news for colorectal cancer patients who need advanced-stage chemotherapy.

Conclusions

TIMP-2 expression is closely related to 5-Fu resistance in vitro and in vivo. According to our results, TIMP-2 is a better indicator of 5-Fu resistance in colorectal cancer than CEA or CA19-9. Accordingly, monitoring the serum TIMP-2 level can help the clinician identify 5-Fu resistance in colorectal cancer patients receiving chemotherapy during earlier stages.

Author contributions

YL and LS: performed the experiments, writing—original draft, and wrote the manuscript. CX and RZ: performed the experiments. GH and KT: formal analysis, analyzed the data, and conducted the experiments. GZ, FT, and ZL: supervised the study.

Funding

This work was supported by Zhejiang Province Health Science and Technology Plan, China (Grant Numbers: 2023KY1234, 2021RC133, and 2021KY1150), Shaoxing Health Science and Technology Program, Zhejiang Province, China (Grant Number: 2022SY014), and Shaoxing Bureau of Science and Technology, Zhejiang Province, China (Grant Number: 2020A13022).

Data availability

Data supporting the findings of this study are available from the Department of Gastrointestinal Surgery, Shaoxing People's Hospital, but the availability of these data is limited; they are used under the license of the current study and are, therefore, unavailable publicly available. Data are available from the authors upon reasonable request and with permission of the Department of Gastrointestinal Surgery, Shaoxing People's Hospital.

Declarations

Conflict of interest

The authors have no conflicts of interest to declare.

Ethical approval

The human and animal ethics involved in this experiment were approved by the ethical approval agency at Shaoxing People's Hospital. Study number: 2021-K-Y-158-01.

Consent to participate

Informed consent was obtained from all participants included in the study.

Consent to publish

The authors affirm that human research participants provided informed consent for publication.

Footnotes

Publisher's Note

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Contributor Information

Feng Tao, Email: tf_zjsx@yeah.net.

Zengxin Lu, Email: luzx777@163.com.

Guolin Zhang, Email: zhangguolin@zju.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data supporting the findings of this study are available from the Department of Gastrointestinal Surgery, Shaoxing People's Hospital, but the availability of these data is limited; they are used under the license of the current study and are, therefore, unavailable publicly available. Data are available from the authors upon reasonable request and with permission of the Department of Gastrointestinal Surgery, Shaoxing People's Hospital.

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