Establishment of a chemoradio-resistance colorectal cancer cell model and its changes in biological behavior

Introduction

Colorectal cancer (CRC) is the third most cancer worldwide, with more than 2 million people diagnosed each year and close to 1 million deaths from CRC (1). Although early screening has reduced the incidence and mortality of CRC in recent years, there are still CRC patients with advanced disease at the time of diagnosis, and about half of the patients with early CRC have cancer metastasis (2,3). CRC remains the third leading cause of cancer-related death (4). Although simultaneous chemoradiotherapy before surgical intervention is currently an important means of comprehensive treatment for CRC, especially rectal cancer (5,6), drug resistance of chemotherapy is a major problem (7-9). Previous studies have mainly focused on elucidating the mechanism by which tumor cells develop resistance to radiotherapy or chemotherapy (10-12), but the development of resistance to concurrent chemoradiotherapy involves unique factors. Currently, there is little research on developing a clinical model that can accurately simulate the concurrent administration of chemotherapy and radiotherapies in CRC, thereby facilitating an understanding of the key mechanisms through which tumor cells acquire resistance to this combined therapeutic approach.

In this study, an in vitro model of concurrent chemoradiotherapy resistance in CRC cells was established, and the changes in their biological behavior were explored. Through the thorough study of these changes, it is helpful to further elucidate the mechanism underlying chemoradiotherapy resistance in CRC, which has the potential to become a new viewpoint in the molecular diagnosis and gene therapy strategies of CRC and lay the foundation for further exploration of rational interventions to reverse CRC chemoradiotherapy resistance in the future. Furthermore, based on the in vitro model, an in vivo nude mouse xenograft model was established to validate the biological characteristics of chemoradiotherapy resistance and its relevance to the clinical setting.

Objectives

This study systematically addresses the core challenge of concurrent chemoradiotherapy resistance in CRC, with the following key components clearly defined.

Clear research question

This research aims to answer a critical question: how can a resistance model that accurately simulates clinical concurrent chemoradiotherapy stress be constructed, and how can this model be utilized to unveil the key biological mechanisms and phenotypic characteristics driving the acquisition of resistance?

Defined research objectives

To address the above question, we established two core objectives:

• Primary objective: to successfully establish in vitro cell models and in vivo animal models of concurrent chemoradiotherapy resistance in CRC by simulating clinical high-dose shock therapy.
• Core objective: to comprehensively characterize and compare the differences between resistant cells and parental cells in terms of proliferation, colony formation, invasion, migration, as well as in vivo tumorigenicity and metastatic capacity.

Testable research hypothesis

This study is founded on a central scientific hypothesis: compared to parental cells, CRC cells that survive concurrent chemoradiotherapy will acquire a more aggressive malignant phenotype, driven by distinct molecular mechanisms. All experimental designs in this study are intended to systematically verify this hypothesis. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-812/rc).

Methods

Cell lines and culture

The human CRC cell lines HT29 and HCT116 were purchased from the Cell Bank of Kunming Institute of Zoology, Chinese Academy of Sciences, in February 2020. These cell lines were preserved and authenticated by the Yunnan Cancer Institute. Cell identity was confirmed by Short Tandem Repeat (STR) profiling at the same cell bank in June 2020, prior to the induction of chemoradiotherapy resistance. HT29 and HCT116 cells, along with their derived resistant lines, were routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. All cells were cultured in a humidified incubator at 37 °C with 5% CO₂. The medium was refreshed every 2–3 days, and cells were subcultured upon reaching 80–90% confluence using 0.25% trypsin-EDTA. All experiments were performed with cells in the logarithmic growth phase.

Establishment of chemoradiotherapy-resistant (CRR) cell models

To simulate clinical high-dose concurrent chemoradiotherapy, we established resistant variants of HT29 and HCT116 cells. Wells containing actively growing cells in the logarithmic growth phase were selected for the induction protocol. The cells were cultured in a medium containing 10 µmol/L of 5-fluorouracil (5-FU). The induction protocol was as follows: two hours after the 5-FU addition, the cells were irradiated with 6 Gy of 6 MV X-rays using a medical linear accelerator [dose rate: 400 Gy/min, source-to-skin distance (SSD) =100 cm]. The cells were subsequently maintained in the 5-FU-containing medium for an additional 22 hours (making a total 5-FU exposure time of 24 hours). The culture medium was then replaced with fresh, drug-free complete medium, allowing for the continuous cultivation of the residual cancer cells. The medium was monitored and changed based on cell apoptosis status. Once the residual cells exhibited robust growth and reached approximately 70% confluence, the entire treatment cycle was repeated. After a total of 20 cycles, the stable CRR cell lines, designated HT29CRR and HCT116CRR, were successfully established.

Colony formation assay

Chemoradiotherapy-resistant (HCT116CRR/HT29CRR) and parental cell lines in the logarithmic growth phase were selected for this study. After trypsinization and counting, cells were seeded into 6-well plates at densities of 300, 400, 600, 1,000, 2,000, and 20,000 cells per well, with three replicates per density. To control for the effects of continuous passaging, parental cells were also cultured for 14 days as a parallel control. After a culture period of 24 hours, the medium was replaced with one containing 10 µmol/L 5-FU. The cells were then irradiated with increasing doses of X-ray (0, 2, 4, 6, 8, and 10 Gy). Following irradiation treatment and an additional culture period of 24 hours, the medium containing 5-FU was replaced with a drug-free fresh medium, and cells were cultured for a duration of 14 days. After 14 days, the colonies formed by surviving cells were fixed with methanol, stained with crystal violet for visualization, and counted under microscopy. The plating efficiency (PE) was calculated as follows: PE = (number of colonies per well in the control group/number of cells planted per well in the control group) ×100%. The surviving fraction (SF) was calculated using the PE from the control group (0 Gy) as a correction factor: SF = number of colonies per well in the experimental group/(number of cells planted per well × PE). The survival curve was fitted using SigmaPlot software with a multi-target single-hit model, represented by SF =1−(1−e^−D/D0)^N, where D is the radiation dose, D₀ is the mean lethal dose, and N is the extrapolation number representing the target number or repair capacity. This model was used to calculate radiosensitivity parameters, including the quasi-threshold dose (D_q), mean lethal dose (D₀), and survival fraction at 2 Gy (SF₂). Furthermore, the dose-response curve was analyzed by nonlinear curve fitting using GraphPad Prism (version 10.1.2) to calculate the half maximal inhibitory concentration (IC₅₀) value, providing an additional assessment of the radiation resistance in the resistant cell lines.

Cell migration assay

Cell migration was assessed using Transwell chambers. The residual HT29/HCT116 cell lines following radiotherapy and chemotherapy treatments, along with their parental controls, all in the long-term logarithmic growth phase, were selected for analysis. Cells from both the radiochemotherapy-resistant and the control groups were trypsinized, centrifuged, and resuspended to achieve a density of 2×10⁵ cells/mL. A 200 µL aliquot of this cell suspension was added to the upper chamber, while the lower chamber was filled with 500 µL of medium containing 10% FBS as a chemoattractant. The plates were then cultured for 24 hours. After incubation, the culture medium in the upper chamber was discarded, and the cells were washed with phosphate-buffered saline (PBS), fixed in methanol for 30 minutes, and the chamber was air-dried. The non-migrated cells on the upper surface of the membrane were carefully removed by wiping with a cotton swab. The chamber was then soaked in 0.1% crystal violet solution for 20 minutes, followed by three washes with PBS. The migrated cells on the lower surface were observed and imaged under a microscope at 400× magnification

Cell invasion assay

Cell invasion ability was assessed using Matrigel-coated Transwell chambers. The radiochemotherapy-resistant and parental cells were routinely cultured to the logarithmic growth phase. The cell density was adjusted to 3×10⁵ cells/mL. Subsequently, 100 µL of 1 mg/mL Matrigel was added to the upper chamber of a Transwell insert and incubated at 37 °C for 30 minutes to allow polymerization. Then, 200 µL of cell suspension was seeded onto the polymerized Matrigel in the upper chamber, and 500 µL of cell culture medium containing 10% FBS was added to the lower chamber as a chemoattractant. After 24 hours of incubation, the inserts were processed as follows: they were fixed in methanol for 30 minutes and then soaked in 0.1% crystal violet solution for 20 minutes. The non-invading cells and the residual matrix gel on the upper surface of the membrane were carefully removed by wiping with a cotton swab. The inserts were then washed three times with PBS. The invaded cells on the lower surface of the membrane were visualized and imaged under a microscope at 400× magnification.

MTT assay to detect cell proliferation resistance to radiotherapy and chemotherapy

Cell proliferation was assessed using the MTT assay. Cells from the radiochemotherapy-resistant group and the parental control group, both in the logarithmic growth phase, were digested, centrifuged, and resuspended. The cell density was adjusted to 2.5×10⁴ cells/mL. A volume of 200 µL of cell suspension (containing 5×10³ cells) from each group was inoculated into the central wells of a 96-well plate. The surrounding perimeter wells were filled with an equal volume of PBS to minimize evaporation edge effects. The plate was then cultured for 24 hours. Every 24 hours for a total of 6 days, the assay was performed on three replicate wells per group. Specifically, 20 µL of 5 mg/mL MTT solution was added to each well and incubated for 4 hours. Subsequently, the medium was carefully removed, and 150 µL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. The plates were shaken for 10 minutes, and the absorbance was measured at 490 nm using a microplate reader. On the 3rd day, if the absorbance values were outside the measurable range, the medium in those wells was replaced with fresh culture medium, and the measurement process was repeated until the 6th day.

Establishment of a subcutaneous Xenograft Model

Healthy 6-week-old female BALB/c nude mice (weighing 18–20 g) were purchased from ChuShang Technology Co., Ltd. () [Animal Quality Certificate No.: SYXK (Dian) K2020-0006]. All mice were maintained under specific pathogen-free (SPF) conditions in a controlled environment (temperature: 22±2 °C; humidity: 50%±10%; 12 h light/dark cycle) with access to autoclaved food and water ad libitum. All animal experiments were performed under a project license (No. kmmu20230737) granted by the Ethics Committee of Kunming Medical University, in compliance with institutional guidelines for the care and use of animals.

HCT116CRR cells and their parental HCT116 cells, both in the logarithmic growth phase, were trypsinized, centrifuged, and resuspended in PBS to a final concentration of 2.5×10⁶ cells/mL. A volume of 200 µL cell suspension (containing 0.5×10⁶ cells) was subcutaneously injected into the dorsal skin of the right forelimb of each mouse after disinfection with 70% alcohol. Each group (HCT116CRR and HCT116) consisted of 10 mice. When tumor volume reached approximately 50 mm³ (estimated using the formula V = a × b²/2, where ‘a’ is the long diameter and ‘b’ is the short diameter), five mice from each group were randomly selected for irradiation. Irradiation was performed using a 4 MV X-ray source at a dose of 2 Gy per session, delivered every other day for a total of four sessions. Tumor growth was monitored every three days by measuring the two perpendicular diameters with vernier calipers.

Construction of luciferase-labeled HCT116-Luc and HCT116CRR-Luc cells

HCT116 and HCT116CRR cells in the logarithmic growth phase were digested, centrifuged, and resuspended in culture medium at densities ranging from 1×10⁵ to 5×10⁵ cells/mL. Then, 2×10⁵ cells in 2 mL medium were seeded per well in 6-well plates. After overnight incubation, the medium was replaced with 1 mL fresh medium containing lentivirus carrying the Luc-GFP-Puro cassette. The virus volume was calculated based on a multiplicity of infection (MOI) of 20, using the formula: virus volume (µL) = (MOI × cell count)/virus titer. After 12 hours of infection, the virus-containing medium was replaced with standard culture medium. Transduction efficiency was observed 72 hours post-infection using fluorescence microscopy. Luciferase expression was confirmed by adding D-luciferin potassium salt and detecting bioluminescence with a small animal in vivo imaging system. To establish stably transduced polyclonal populations, cells were cultured in medium supplemented with 1.5 µg/mL puromycin for 10 days, with medium changes every 2–3 days and regular passaging.

Establishment of liver metastasis model via splenic injection

Nude mice were divided into two groups (n=5 per group): HCT116-Luc and HCT116CRR-Luc. Cells in logarithmic growth phase were trypsinized and resuspended in PBS at a concentration of 1×10⁷ cells/mL. Mice were anesthetized by intraperitoneal injection of 100 µL of 1% pentobarbital sodium and placed in the right lateral position. A 1 cm incision was made to expose the spleen, and 100 µL of cell suspension (1×10⁶ cells) was slowly injected into the splenic parenchyma. The injection site was pressed with a medical cotton swab dipped in povidone-iodine for 3 minutes to prevent cell leakage and potential peritoneal dissemination. The wound was then closed, and all animals were allowed to recover postoperatively.

Establishment of lung metastasis model via tail vein injection

For the lung metastasis model, ten nude mice were similarly allocated into two groups (n=5 per group): HCT116-Luc and HCT116CRR-Luc. Cells in logarithmic growth phase were digested, centrifuged, and resuspended in PBS at a final concentration of 5×10⁷ cells/mL. A volume of 100 µL cell suspension was injected into the tail vein of each mouse under restraint in a dedicated holder.

In vivo monitoring and imaging

After tumor cell inoculation, mice were monitored daily for general condition, activity, diet, and clinical signs such as posture, fur condition, and spontaneous movement. Body weight was measured every three days, and tumor volume growth curves were plotted. To minimize discomfort, all invasive procedures and imaging sessions were performed under transient isoflurane anesthesia. Soft diet and hydration gel were provided on the cage floor if animals exhibited weight loss or lethargy.

In vivo bioluminescence imaging was performed on day 28 for the liver metastasis model and day 42 for the lung metastasis model, based on body weight and activity changes. Before imaging, mice were intraperitoneally injected with 200 µL of D-luciferin potassium salt (30 mg/mL) and anesthetized with isoflurane. After 10 minutes of substrate distribution, mice were placed in an in vivo imaging system, and luminescence signals were acquired.

Adverse events: no unexpected adverse events occurred during the study. Expected adverse events were associated with tumor burden and metastatic progression, including gradual body weight loss (not exceeding 20% of peak weight) and reduced activity in later stages. These manifestations were predefined as part of the humane endpoint criteria.

Humane endpoints: the following humane endpoints were strictly enforced to prevent severe suffering—

• Body weight loss >20% of peak weight;
• Inability to access food or water voluntarily;
• Ulcerated or necrotic tumors exceeding 1.5 cm in any diameter;
• Severe lethargy or dyspnea.

Animals were monitored daily for these signs. No animal reached these endpoints before the scheduled imaging time points, and thus no early euthanasia was required in this study phase.

The liver metastasis model (via splenic injection) and lung metastasis model (via tail vein injection) are schematically summarized in Figure 1.

Figure 1 The models construction. (A) The models of liver metastasis by splenic injection. (B) The models of lung metastasis by caudal vein injection.

Statistical analysis

All statistical analyses in this study were performed using SPSS 25.0 software. Differences between groups were analyzed using independent samples t-test, with statistical significance denoted as *, P<0.05; **, P<0.01; ***, P<0.001.

Results

Establishment of HCT116/HT29 cell lines with acquired resistance to concurrent chemoradiotherapy

Following 20 cycles of induction via concurrent chemoradiotherapy, stable CRR cell lines (HT29CRR and HCT116CRR) were successfully established. As shown in Figure 2, the parental HCT116 and HT29 cells without any chemoradiotherapy treatment exhibited a plump morphology, tight arrangement, and no suspended cells in the culture medium (Figure 2A,2F,2G,2H). After treatment with 10 µmol/L 5-FU and 6 Gy irradiation using 6 MV X-rays for 24 hours, a large number of apoptotic cells were observed in suspension (Figure 2B,2K). Upon replacing the medium with fresh complete culture medium, a small number of residual cancer cells were visible (Figure 2C,2L). After continued culture for 24 hours, these residual cells proliferated vigorously, displaying regular morphology and orderly arrangement (Figure 2D,2E,2J,2H). After repeating this cycle 20 times, stable resistant cell lines (HT29CRR and HCT116CRR) were obtained.

Figure 2 Establishment of chemoradiotherapy-resistant cell lines through iterative selection. (A,G) Representative phase-contrast images of untreated parental HCT116 (A) and HT29 (G) cells, exhibiting plump morphology and tight arrangement (magnification ×). (F,H) Higher-magnification views of the boxed areas in (A) and (G), respectively (magnification ×). (B,K) Massive apoptotic cell death (floating cells) observed 24 hours after concurrent treatment with 10 µmol/L 5-FU and 6 Gy X-ray irradiation in HCT116 (B) and HT29 (K) cultures (magnification ×). (C,L) A small number of surviving adherent cells remained after replacing the treatment medium with fresh complete medium [HCT116 in (C); HT29 in (L)] (magnification ×). (D,J) The surviving cells resumed vigorous proliferation with a regular, orderly morphology after a 24-hour recovery period [HCT116 in (D); HT29 in (J)] (magnification ×). (E,I) Higher-magnification views of the boxed areas in (D) and (J), respectively (magnification ×). This cycle of treatment and recovery was repeated 20 times to establish the stable resistant cell lines HCT116CRR and HT29CRR. 5-FU, 5-fluorouracil; CRR, chemoradiotherapy-resistant.

Enhanced clonogenic survival of HCT116/HT29 cells after acquiring chemoradiotherapy resistance

The clonogenic capacity of cells following concurrent chemoradiotherapy was assessed. As shown in Figure 3A, the resistant HCT116CRR and HT29CRR cell lines formed a significantly greater number of colonies compared to their parental counterparts post-treatment. Cell survival curves were generated by fitting the data with the multi-target single-hit model [SF =1−(1−e^−D/D₀)^N, where N is the extrapolation number] (Figure 3B).

Figure 3 Validation of acquired chemoradioresistance of HCT116CRR/HT29CRR cells by clonogenic survival assay. (A) Representative images of colonies formed by HCT116/HT29 parental cells and HCT116CRR/HT29CRR cells after concurrent chemoradiotherapy (magnification × stained). (B) Clonogenic survival curves of HCT116/HT29 parental and resistant cells following irradiation combined with 5-FU treatment. Data were fitted using the multi-target single-hit model. *. 5-FU, 5-fluorouracil; CRR, chemoradiotherapy-resistant.

Radiobiological parameters derived from the model are summarized in Tables 1,2. The resistant cell lines exhibited marked increases in both the mean lethal dose (D₀) and the quasi-threshold dose (D_q). Specifically, for HT29 cells, D₀ increased from 1.73 to 2.37 Gy and D_q from 1.58 to 1.74 Gy in the resistant variant. Similarly, in HCT116 cells, D₀ rose from 2.30 to 2.79 Gy and D_q from 1.13 to 1.80 Gy upon acquisition of resistance.

Complementing these findings, the IC₅₀ for 5-FU was significantly elevated in both resistant lines (Table 3). The IC₅₀ for HCT116CRR cells was 5.85±0.21 µM, which was significantly higher than that for parental HCT116 cells (4.31±0.05 µM) (P<0.001), corresponding to a resistance fold of approximately 1.36. HT29CRR cells exhibited an IC₅₀ of 4.60±0.08 µM, a 1.21-fold increase over parental HT29 cells (3.80±0.05 µM), with the difference being statistically significant (P<0.001).

Enhanced migratory capacity of CRR CRC cells

The migratory ability of HCT116/HT29 parental and CRR cells was evaluated using a Transwell assay. As shown in Figure 4A, microscopic examination revealed a substantially greater number of migrated cells in the HCT116CRR and HT29CRR groups compared to their parental counterparts. The number of migrated cells was quantified by counting three random fields per replicate, and the results are presented as the mean ± standard deviation (n=3). For the HCT116 models, the number of migrated cells was significantly higher in the HCT116CRR group (67.67±18) than in the parental HCT116 group (21.33±4) (P<0.001; Figure 4B). A more pronounced increase was observed in the HT29 models, with the HT29CRR group (91±13) exhibiting a nearly 7-fold higher migration compared to the parental HT29 group (13±4) (P<0.001; Figure 4B). These results demonstrate that acquired resistance to concurrent chemoradiotherapy is associated with a significant enhancement of migratory capacity in CRC cells.

Figure 4 Enhanced migration of chemoradioresistant HCT116CRR and HT29CRR cells. (A) Representative images of migrated HCT116/HT29 parental and HCT116CRR/HT29CRR cells (magnification × stained). (B) Quantitative analysis of cell migration. Data are presented as the mean ± SD (n=3). ***, P<0.0001. CRR, chemoradiotherapy-resistant; SD, standard deviation.

Enhanced invasive capacity of CRR CRC cells

Cell invasion was assessed using a Matrigel-coated Transwell assay. As illustrated in Figure 5A, microscopic analysis revealed a substantially greater number of invaded cells in the HCT116CRR and HT29CRR groups compared to their parental counterparts. The number of invaded cells was quantified by counting three random fields per replicate. Quantitative analysis demonstrated that the invasive capacity was significantly enhanced in both resistant cell lines (Figure 5B). Specifically, the HCT116CRR group exhibited a marked increase in invasion (103.67±11) compared to the parental HCT116 cells (30±8) (P<0.001). A more profound enhancement was observed in the HT29 models, where the number of invaded HT29CRR cells (153.67±17) was approximately 7-fold higher than that of the parental HT29 cells (22±8) (P<0.001). Collectively, these results indicate that acquired resistance to concurrent chemoradiotherapy confers a more aggressive invasive phenotype on CRC cells.

Figure 5 Enhanced invasion of chemoradioresistant HCT116CRR and HT29CRR cells. (A) Representative images of invaded HCT116/HT29 parental and HCT116CRR/HT29CRR cells (magnification × stained). (B) Quantitative analysis of cell invasion. Data are presented as the mean ± SD (n=3). ***, P<0.0001. CRR, chemoradiotherapy-resistant; SD, standard deviation.

Chemoradiotherapy-resistant CRC cells exhibit enhanced proliferative capacity

Cell proliferation was assessed over a 6-day period using the MTT assay. The proliferation rate was calculated as (OD value on day n − mean OD value on day 0)/mean OD value on day 0. As shown in Figure 6, the CRR cell lines demonstrated a significantly higher proliferation rate compared to their parental counterparts throughout the course of the experiment. Specifically, the HCT116CRR cells exhibited a markedly accelerated growth rate from day 1 to day 6 relative to the parental HCT116 cells. A similar, and in later stages even more pronounced, enhancement in proliferation was observed in the HT29CRR cells compared to the parental HT29 cells. The differences in proliferation rates between the resistant cells and their respective parental controls were statistically significant (P<0.01) across all measured time points. These results collectively indicate that acquired resistance to concurrent chemoradiotherapy is associated with a sustained proliferative advantage in CRC cells.

Figure 6 Enhanced proliferation of chemoradioresistant HCT116CRR and HT29CRR cells. Growth curves of HCT116/HT29 parental and HCT116CRR/HT29CRR cells as determined by the MTT assay. Data are presented as the mean proliferation rate. The resistant cell lines of both models demonstrated significantly higher proliferation rates from day 1 to day 6 compared to their parental controls (P<0.01). CRR, chemoradiotherapy-resistant.

HCT116CRR cells exhibit enhanced tumorigenicity and radioresistance in vivo

To assess the tumorigenic potential and radiation response in vivo, nude mice bearing subcutaneous xenografts of HCT116 parental or HCT116CRR cells were established. The animals in each group were then randomized to receive either radiotherapy (RT) or no treatment (control), resulting in four experimental groups: HCT116-Control, HCT116-RT, HCT116CRR-Control, and HCT116CRR-RT. As shown in Figure 7, tumor volume in both control groups increased progressively over time. Notably, the HCT116CRR-Control group demonstrated a significantly faster tumor growth rate compared to the HCT116-Control group, indicating enhanced tumorigenicity of the resistant cells. Radiotherapy significantly inhibited tumor growth in both models. However, the degree of inhibition was markedly different. Tumors in the HCT116-RT group showed a strong response to radiation, whereas tumors in the HCT116CRR-RT group exhibited a substantially attenuated response, confirming their acquired radioresistance. On day 21, the difference in tumor volume between the HCT116CRR-RT and HCT116-RT groups was statistically significant (P<0.05), demonstrating that the inhibition of radiotherapy on resistant cells was significantly less than that on parental cells.

Figure 7 HCT116CRR cells demonstrate enhanced tumor growth and radioresistance in vivo. Growth curves of subcutaneous xenografts from HCT116 parental and HCT116CRR cells, with or without RT. Data are presented as mean ± SD. The HCT116CRR-control group showed accelerated growth compared to the HCT116-control group. While RT inhibited growth in both models, the HCT116CRR-RT group exhibited attenuated response. CRR, chemoradiotherapy-resistant; CRT, chemoradiotherapy; N; RT, radiotherapy; SD, standard deviation.

CRC cells with acquired chemoradioresistance exhibit enhanced liver metastatic potential in vivo

A liver metastasis model was established via splenic injection of luciferase-labeled HCT116-Luc or HCT116CRR-Luc cells to evaluate the metastatic capability of CRR cells in vivo. Bioluminescence imaging of liver metastases revealed a significantly higher tumor burden in the HCT116CRR-Luc group compared to the HCT116-Luc control group (Figure 8A). Beginning in the third week post-inoculation, mice in both groups exhibited decreased food intake and body weight loss. By the fourth week, the weight loss in the HCT116CRR-Luc group was significantly more pronounced than in the HCT116-Luc group (Figure 8B). Quantitative analysis confirmed that the mean luminous intensity in the HCT116CRR-Luc group was approximately 2.1-fold higher than that in the HCT116-Luc group [(4.17±1.11)×10⁸ vs. (1.97±0.68)×10⁸ p/s/cm²/sr, P=0.005] (Figure 8C, Table 4).

Figure 8 Liver metastasis model was established by splenic injection to observe and compare the liver metastasis ability of HCT116CRR and HCT116 cells. (A) Representative in vivo bioluminescence images of liver metastases. (B) Body weight changes of nude mice over time. Data are presented as mean ± SD. (C) Quantitative statistics of bioluminescence intensity from liver metastases. **, P<0.01; ***, P<0.001. CRR, chemoradiotherapy-resistant; SD, standard deviation.

CRC CRR cells exhibit enhanced lung metastatic ability in vivo

To compare the lung metastatic potential of CRR cells and their parental cells, we established an experimental lung metastasis model by intravenously injecting luciferase-labeled HCT116-Luc and HCT116CRR-Luc cells into nude mice via the tail vein (Figure 9A). Starting from the 4th week, mice in both groups exhibited decreased body weight loss, which was most pronounced at the 6th week. However, there was no statistically significant difference in body weight between the two groups at any time point (P>0.05) (Figure 9B). Bioluminescence imaging revealed that the luminous intensity of lung metastases in the HCT116CRR-Luc group was significantly higher than that in the HCT116-Luc group [(2.63±0.33)×10⁸ vs. (1.85±0.29)×10⁸ p/s/sr, P=0.004] (Figure 9C; Table 5). To provide in vivo validation, the lungs were dissected and examined. The HCT116CRR-Luc group exhibited a significantly greater number of lung metastatic foci (5.80±2.59) compared to the HCT116-Luc group (2.20±0.80) (P=0.02; Figure 9D, Table 5). Collectively, these results demonstrate that HCT116CRR cells possess a stronger lung metastasis ability and higher malignancy than the parental HCT116 cells.

Figure 9 HCT116CRR cells exhibit enhanced lung colonization in an experimental metastasis model. (A) Representative in vivo bioluminescence images of lung metastases. (B) Body weight changes of nude mice over time. No significant difference was observed between the groups (P>0.05). (C) Quantitative analysis of bioluminescence intensity from lung metastatic foci. (D) Quantitative analysis of the number of macroscopic lung metastatic foci. ns, not significant; *, P<0.05; **, P<0.01. CRR, chemoradiotherapy-resistant.

Discussion

CRC is recognized as the third most prevalent cancer globally, posing a significant threat to millions of lives worldwide. The primary challenges in CRC treatment, which lead to poor prognosis, are metastasis, recurrence, and drug resistance (13). Neoadjuvant radiotherapy and chemotherapy aim to reduce the risk of local recurrence, shrink the tumor to achieve R0 resection, and improve the rate of anal preservation, thereby prolonging patients’ disease-free survival (14). However, the response rate to chemotherapeutic agents like 5-FU and L-OHP in advanced CRC is 40% or less, with efficacy decreasing and side effects increasing with patient age (15,16). Currently, there are few reports on the mechanism of CRC resistance to concurrent radiotherapy and chemotherapy. Therefore, this study aimed to establish a novel in vitro model of radiochemotherapy resistance in CRC to investigate the reasons for decreased treatment sensitivity and characterize the associated changes in biological behavior.

To investigate the potential reasons for the decrease in CRC sensitivity to radiotherapy and chemotherapy, we established a model of concurrent radiochemotherapy resistance in vitro. Human CRC HT29 and HCT116 cells were subjected to iterative stimulation using 10 µmol/L 5-Fu combined with 4 Gy of 6 Mv X-ray irradiation for 20 passages, simulating clinical concurrent radiotherapy and chemotherapy using a high-dose impact method. The resulting radiochemotherapy-resistant cell lines, designated HT29CRR and HCT116CRR, were subsequently characterized. A plate clone formation assay demonstrated that HT29CRR and HCT116CRR cells formed a significantly greater number of clones than their wild-type counterparts after treatment, indicating enhanced insensitivity and an improved ability to repair sublethal damage, confirming that the cells had developed insensitivity and enhanced repair capacity following the high-dose rest treatment. Furthermore, cell migration, Transwell invasion, and MTT proliferation assays consistently revealed that the HT29CRR and HCT116CRR cells possessed significantly stronger migration, invasion, and proliferation capabilities compared to the parental cell lines.

We next assessed the in vivo metastatic potential of the resistant cells. Distant metastasis of CRC, especially to the liver and lung, is a major cause of poor prognosis, with 20% to 70% and 10% to 20% of patients developing liver and lung metastases, respectively (17,18). CRC liver metastasis models were constructed by performing intrasplenic injection of luciferase-labeled HCT116 Luc and HCT116CRR Luc cells into nude mice. Similarly, CRC lung metastasis models were established via tail vein injection of the same cells. The metastatic ability was evaluated using in vivo bioluminescence imaging.

In the liver metastasis model, nude mice in both groups exhibited mild weight loss by week 4, with the loss being more pronounced in the HCT116CRR Luc group. Consistent with this, small animal live imaging at week 4 revealed a significantly higher luminescence intensity in the livers of mice injected with HCT116CRR Luc cells, indicating more prominent liver metastases and suggesting that CRR cells are more capable of liver metastasis and more malignant. In the lung metastasis model, obvious weight loss was observed in both groups at week 6, though the difference between groups was not significant. Nevertheless, live imaging at week 6 showed that the luminescence intensity and the number of lung metastases were significantly higher in the HCT116CRR Luc group. We found that the cells retained their resistant and aggressive phenotype even in vivo, demonstrating that CRC cells with acquired radiochemotherapy resistance possess superior liver and lung metastatic abilities.

In summary, we successfully established stable radiochemotherapy-resistant human CRC cell lines that exhibit enhanced malignant potential. After 20 cycles of simulated treatment, we obtained stable resistant cell lines that demonstrated significantly enhanced resistance to radiotherapy and chemotherapy, along with stronger migration, invasion, and proliferation capabilities. These cells not only demonstrated stronger proliferative, migratory, and invasive capabilities in vitro but also showed more aggressive metastatic behavior in animal models. These findings corroborate the clinical observation that chemoradiotherapy resistance plays a critical role in local recurrence and metastasis in CRC patients (19) and are consistent with our experimental results showing that resistant cells possess superior malignant potential. Our established model provides a valuable tool for future research aimed at elucidating the molecular mechanisms of resistance, which is essential for developing strategies to reverse this process and improve patient outcomes.

While this study successfully established models of CRR CRC cells and validated their enhanced invasive and metastatic potential both in vitro and in vivo, we fully acknowledge its current limitations, which primarily lie in the following two aspects, as highlighted by the reviewers. These points also importantly guide future research directions.

First, regarding the mechanisms underlying the acquired invasive and metastatic traits in resistant cells. The current work focuses predominantly on phenotypic confirmation and lacks in-depth mechanistic insights. This is a significant shortcoming of our study. During experiments, we did observe a more mesenchymal-like morphological shift in resistant cells, suggesting a potential involvement of epithelial-mesenchymal transition. To explore the mechanisms, we have initiated preliminary RNA-sequencing analysis. Initial data indicate alterations in several pathways related to cell migration, extracellular matrix remodeling, and DNA damage repair. However, these data are still under validation and have not yet formed a complete mechanistic narrative for inclusion in this manuscript. We completely agree that uncovering the root causes—whether genetic mutations, epigenetic reprogramming, alternative splicing variants, or other adaptive changes—is crucial for understanding clinical resistance and metastasis. Future work will systematically employ integrated multi-omics analyses, functional gain- and loss-of-function experiments targeting key signaling pathways, and validation in clinical samples to elucidate the core mechanisms driving this aggressive phenotype.

Second, concerning the absence of the tumor immune microenvironment in our models. We fully recognize that the immunodeficient mouse models used in this study cannot recapitulate the complex interplay between the tumor and the immune system. Substantial evidence underscores the central role of this interplay in CRC development, therapy resistance, and metastasis, particularly in the context of radiotherapy. This limitation restricts our model’s ability to fully mimic the complete picture of resistance and metastasis in humans. Our initial rationale for choosing this model was to first isolate and define the intrinsic malignant progression properties acquired by tumor cells themselves under the selective pressure of chronic chemoradiotherapy in a controlled setting. This establishes the cellular foundation for studying tumor-immune interactions. We openly acknowledge that neglecting the immune microenvironment is a major drawback of our model, limiting the generalizability of our conclusions. Therefore, the findings presented here should be interpreted as describing the inherent “seed” properties of the resistant cells. The logical and essential next step is to study these resistant cells within the “soil”—that is, in immunocompetent hosts or co-culture systems incorporating immune cells. We plan to investigate whether the tumorigenicity, metastatic potential, and response to immunotherapy of these cells alter in immune-competent mice, thereby providing a more comprehensive assessment of their biological behavior.

Conclusions

In conclusion, this study provides a preliminary preclinical model and phenotypic evidence for understanding the invasive progression of CRR CRC cells. However, elucidating the underlying mechanisms and investigating their interaction with the tumor microenvironment require further depth and breadth. These acknowledged limitations clearly define the focus of our subsequent work: first, to delve deeper into the intrinsic molecular mechanisms of the cells, and second, to expand outward by validating and extending the current findings in more complex models incorporating the immune context.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-812/rc

Data Sharing Statement: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-812/dss

Peer Review File: Available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-812/prf

Funding: This work was supported by National Natural Science Foundation of China (No. 81960557), Clinical Collaboration Pilot Project of Traditional Chinese and Western Medicine for Major and Difficult Diseases in Yunnan Province, Applied Basic Research Program of Yunnan Province (No. 202201AY070001-160), Research Foundation of Yunnan Educational Committee (No. 2022J0235), Applied Basic Research Program of Yunnan Province [No. 2018FE001(-256)], Research Program of Yunnan medical and health institutions (No. 2018NS0057), Applied Basic Research Program of Yunnan Province [No. 2017FE468(-219)], Research Program of Yunnan medical and health institutions (No. 2017NS182), and Yunnan Province’s “Ten Thousand Talents Program” Special Project for Renowned Doctors.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jgo.amegroups.com/article/view/10.21037/jgo-2025-812/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license (No. kmmu20230737) granted by the Ethics Committee of Kunming Medical University, in compliance with institutional guidelines for the care and use of animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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