A surgical orthotopic xenograft approach with immune response for colorectal cancer research

Abstract

The high morbidity and mortality of colorectal cancer (CRC) is a major challenge in clinical practice. Although a series of alternative research models of CRC have been developed, appropriate orthotopic animal models that reproduce the specific clinical response as well as pathophysiological immune features of CRC are still lacking. In the current study, we constructed a CRC orthotopic xenograft model by implanting the tumor tubes at the colorectum of mice and monitored the model development using bioluminescence imaging. This model successfully recapitulates the clinical chemotherapy efficacy, including reduced total flux, tumor weight, and the expression of Ki67 after treatment of the first‐line chemotherapy regime of CRC (FOLFOX: oxaliplatin and 5‐fluorouracil/calcium folinate). The model also reproduced the immunosuppressive effect of FOLFOX, indicated by decreased infiltration of macrophages and increased Treg cells in tumor. Additionally, the orthotopic xenograft approach may be applied in immunodeficient NCG/NSG mice for constructing patient‐derived xenografts, and being used in clinical precision medicine and drug evaluation. We believe the current model is a successful surgical orthotopic xenograft approach for cancer research and deserves to be popularized, which will provide a convenient and efficient platform for in‐depth mechanism exploration of CRC and preclinical drug evaluation.

INTRODUCTION

Colorectal cancer (CRC) is the third-most diagnosed cancer and is the second leading cause of cancer-related mortality worldwide. According to the latest data from Freddie Bray, there were more than 1.9 million new cases and 930 000 deaths in 2020, and it is projected to have 3.2 million new cases and 1.6 million deaths by 2040.¹ Despite increasing efforts to develop efficient strategies for CRC, the success rate of a novel anti-CRC drug in clinical trials is less than 10% () due to the huge obstacle between clinical reality and the preclinical in vivo models. There is an urgent need to develop effective and reproducible evaluation models to increase the efficiency of clinical therapies.

Preclinical models are indispensable for the evaluation of novel therapeutics before they are applied to human subjects in clinical trials. Common CRC animal models, including carcinogen-induced mouse models, genetically engineered mouse models, transplantation and metastases models, zebrafish-based models, and patient-derived models, are widely used for investigating mechanisms of action of drugs and evaluating drugs (Table 1). Considering the importance of immune regulation in cancer development, orthotopic xenograft models that mimic immune response are essential for preclinical studies. However, the existing evaluation models still cannot meet these demands. The most commonly used subcutaneous xenograft models are convenient to be constructed and monitored, but they cannot simulate the intestinal immune environment of CRC. Carcinogen-induced mouse model and genetically engineered mouse model simulate both the immune microenvironment and the development of tumor, transgenic mice can be applied to investigate specific targets, whereas mouse modeling processes are time-consuming and difficult for intravital monitor. Recently, the development of patient-derived models has significantly overcome the divide between animal models and clinical heterogeneity, but the high cost and the low success rate are barriers to their broader application. Moreover, patient-derived models are constructed based on immunodeficient mice, which cannot be used in immune-related studies. Therefore, it is urgent to develop orthotopic xenograft models that accurately mimic immune responses while remaining cost-effective and technically feasible for preclinical research studies.

TABLE 1 Comparison of different colorectal cancer models.

Model	Advantages	Disadvantages
Carcinogen-induced mouse models	AOM + DSS/TNBS2	Technically easy; economical; relatively high tumor heterogeneity; satisfied success rate; suitable for immune-related evaluation	Time consuming; mice tumor environment; heterogeneous gene between mice and human; low metastasis rate; unable to predict clinical response; unsuitable for high-throughput testing
DHM + DSS/TNBS3
Heterocyclic amines (PhIP, etc.)4
Aromatic amines (DMAB, etc.)5
Alkylating substances (MNNG, MNU, etc.)6
Genetically engineered mouse models	APCmin/+ model7	Does not require extensive surgical procedure; satisfied success rate; specific mutation sites can be edited; known genetic event; suitable for target exploration and high-throughput testing; orthotopic tumor development; reproduces early stages of tumor initiation; suitable for immune-related evaluation	High cost; time consuming; mice tumor and immune environment; heterogeneous gene between mice and human; limited strategy for tumor monitor; limited metastasis rate
KrasG12D+8
P53−/−9
Msh2−/−10
Smad4TKO11
Transplantation and metastases models	Subcutaneous injections12	Technically easy; economical; high success rate; easy to monitor	Unable to predict clinical response; low heterogeneity; no metastases form; ectopic and mice environment
Cecal-based injections13	Economical; high success rate; progress to proximal and distant metastases form; avoids intestinal occlusion during tumor development	Surgical skills required; different microenvironment to the colon; mice environment
Tail intravenous injection14	Technically easy; economical; high success rate; progress to distant metastases form	Mice environment; low heterogeneity; only simulate the extravasation and the organ colonization of tumor metastasis
Spleen injection15	Formation of liver metastasis; high success rate; economical; suitable for immune-related evaluation; avoids intestinal occlusion during tumor development	Surgical skills required; different microenvironment to the colon; mice environment; risk of bleeding
Renal capsule injections16	High success rate; high rate of vascularization; formed macrometastatic colonies; avoids intestinal occlusion during tumor development	Surgical skills required; mice environment; different microenvironment to the colon; no metastases form
Rectal injection via creation of prolapse17	Progress to distant metastases form; suitable for intravital monitor; colon microenvironment	Surgical skills required; mice environment; tumor development leads to intestinal occlusion
Mucosal damage model + rectal enema18	Progress to distant metastases form; uncomplex surgical procedure; colon microenvironment	Induce intestinal mucosa; mice environment
Colonoscopy-guided submucosal injections19	Without mucosal damage; colon microenvironment; high success rate	Colonoscopy device is required; surgical and colonoscopy skills required; limited metastasis rate
Animal-derived orthotopic organoids16	Preserve the parental tumor microenvironment; in vivo evaluation; editable genes; suitable for immune-related evaluation	High cost; difficult of operation; low heterogeneity; unable to predict clinical response
Zebrafish-based models	Zebrafish xenograft20	High-throughput testing; 70% similar to human proteins; easy drug exposure; economical; high fecundity; quick and easy gene editing	More than one orthologue for many human genes; rarely produces highly conserved proteins; limited published comparisons; different preference temperatures between tumor and zebrafish
Patient-derived models	Orthotopic patient-derived organoids16	High heterogeneity; in vivo evaluation; preserve the parental tumor microenvironment; maintains the genetic consistency; editable genes	High cost; time consuming; difficulty in performing operation; unsuitable for immune-related evaluation; limited success rate
PDX21–23	High heterogeneity; highly similar response to clinical; preserve the parental tumor microenvironment; maintains the genetic consistency; easy to monitor and operate	High cost; time consuming; limited success rate; only suitable for certain tumor types; unsuitable for immune-related evaluation
Humanized PDX24	High heterogeneity; highly similar response to clinical; preserve the parental tumor microenvironment; maintains the genetic consistency; suitable for immune-related evaluation; relatively easy to monitor and operate	High cost; time consuming; limited success rate; only suitable for certain tumor types

In the current study, the clinically preferred regime for CRC FOLFOX was selected to verify the clinical reproducibility of the model. We established a surgical orthotopic xenograft mouse model that reproduced the anti-CRC and the immunosuppressive effect of FOLFOX. We monitored the development of CRC and the efficacy of FOLFOX through in vivo bioluminescence imaging (BLI), tumor weight, and histopathology. Furthermore, flow cytometry integrated with multi-factor detection was utilized to investigate the immune infiltration levels of tumor-bearing mice. Overall, the current model can be a valuable platform for better understanding mechanisms underlying CRC progression as well as evaluation of novel therapeutic agents.

MATERIALS AND METHODS

Chemicals and reagents

Oxaliplatin, 5-fluorouridine, and calcium folinate were purchased from J&K Scientific (Beijing, China). VivoGlo luciferin was purchased from Promega (the United States). Type I and type IV collagenase were purchased from Solarbio (Beijing, China). Anti-Ki67 was purchased from Servicebio (Wuhan, China), anti-PCNA was purchased from ABclonal (Wuhan, China), and anti-CD45, anti-F4/80, and anti-FoxP3 were obtained from BioLegend (the United States).

Cell culture

Mouse colon cancer cell line CT-26 was purchased from the Cell Bank of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Luci-CT-26 was generated through the transduction of lentiviral vectors encoding firefly luciferase (LV16-NC), which was purchased from GenePharma Co., Ltd. (Shanghai, China), supplied with 5 μg/mL of polybrene (Sigma), and screened by puromycin (5 μg/mL, Sigma-Aldrich). The cells were cultured in DMEM (Gibco, Grand Island, the United States) supplied with 10% fetal bovine serum (FBS; Gibco) in a humidified atmosphere with 5% CO₂ at 37°C.

Animal study

Seven- to eight-week-old male and female BALB/c mice (20–24 g) were provided by the Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) with the permission number SCXK (Jing) 2021–0006. The study was conducted in accordance with the standards established by the Medical Ethics Committee of Jianghan University. All the mice were housed in a temperature-controlled environment (24 ± 2°C) under a 12/12 h-dark/light cycle.

For colorectal orthotopic xenograft model establishment, the male and female mice were separately divided into three groups: the sham group, the model group, and the FOLFOX group. The model and FOLFOX groups were orthotopically inoculated with Luci-CT-26 tumor tissues (day 1). Five days after tumor inoculation (day 6), in vivo spectral real-time imaging system (IVIS, USA) was applied to confirm tumor formation, and then the mice were randomly divided into groups (n = 5). FOLFOX (oxaliplatin 6 mg/kg, 2 h after 5-FU 50 mg/kg, and calcium folinate 90 mg/kg treatment) was intraperitoneally administrated once a week in the FOLFOX group.²⁵ Model individuals were treated with corresponding vehicles. Tumor volume was monitored using bioluminescence imaging (days 10 and 16). All the mice were killed at the end of the experiment (Day 17); tumor, colorectum, liver, spleen, and kidney tissues were collected and weighed for histopathology, flow cytometry, western blot, multi-factor detection, and pharmacodynamic evaluation. Tumor inhibition rate was calculated by total flux, relative total flux, and tumor weight according to our previous reports.²⁶ Organ coefficient (liver and spleen) was calculated by the following formulas: organ coefficient% = organ weight/body weight × 100%.

Histopathology

Tumor, colorectum, liver, spleen, and kidney tissues were fixed in formalin and embedded in paraffin, then subjected to hematoxylin and eosin (HE) staining. And tumor tissues were subjected to immunohistochemistry (Ki67) as we previously reported.²⁷

Flow cytometry

Tumor and colorectum tissues were carefully removed, cut into pieces, and incubated in the digestive solution with types I and IV collagenase (1 mg/mL) at 37°C (tumor: 30 min; colorectum: 60 min). After digested into the single-cell suspension, the solution was filtered with a 40-μm cell strainer (Biosharp, China). The cells were centrifuged at 5000 rpm for 5 min at 4°C, and washed with PBS twice. Then cell pellets were stained with CD45, F4/80, FoxP3 for 30 min, and detected using flow cytometry with C6 Plus system (BD Biosciences, the United States).²⁶

Multi-factor detection

Mouse serum multi-factors (IL-1β, IL-12p70, IL-17A) were detected by an ABplex Mouse Cytokine 8-Plex Assay Kit (RK04863, ABclonal) according to the manufacturer's instructions on a multi-index flow analyzer (ABplex-100, ABclonal).

Statistical analysis

Data analysis and graphing were performed by GraphPad Prism 8 software (GraphPad Software Inc., La Jolla, CA, USA). The results are presented as mean ± SD, and the independent unpaired two-tailed Student's t test was performed to evaluate the differences between two groups, unless otherwise specified.

RESULTS

Mice orthotopically inoculated with luciferase-labeled Luci-CT-26 tumor tissues to establish colorectal orthotopic xenograft model

About 1 × 10⁶ of Luci-CT-26 cells were injected subcutaneously into the flank of the mouse to form a colorectal cancer in vivo. When the tumor size reached approximately 500 mm³, the tumor tissue was isolated and cut into cubes of about 10 mm³ in sterile saline solution. Necrotic or suspected necrotic tissues in tumor were removed. Subsequently, both male and female BALB/c mice were anesthetized by isoflurane inhalation, disinfected with iodophor disinfectant, and then their colorectum was exposed. The above-mentioned tumor cube was implanted at about 2 cm below the cecum with 5–0 surgical sutures for both the model and FOLFOX groups. After the colorectum was returned to the abdominal cavity, the wounds of the abdomen and skin were sutured with 3–0 surgical sutures. The same procedure was carried out for sham mice except for tumor cube implantation (Figure 1A). During the surgical procedure, animals were kept at 37°C and sterile environment.

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Five days after orthotopic implantation (day 6), tumor formation was verified by BLI. Mice were randomly divided into the model group (male, n = 5; female, n = 5) and the FOLFOX group (male, n = 5; female, n = 5) based on body weights and flux. FOLFOX individuals were intraperitoneally administrated once a week in the FOLFOX group, whereas model individuals were treated with corresponding vehicles. Tumor volume was monitored using BLI (days 11 and 16). All the mice were killed at the end of the experiment (day 17), and tumor and organ tissues were collected and weighed for further examination (Figure 1B).

FOLFOX administration significantly inhibited colorectal cancer development

To examine the effect of gender in our study, the evaluation of model development and FOLFOX efficacy was carried out on male (Figures 2 and 3) and female (Figures S1 and S2) mice independently. As shown in Figures 2A,B and S1A,B, the body weight and total flux of male mice were consistent after randomization (day 6). Colorectal cancer development did not affect the body weight of mice, whereas the weekly administration of FOLFOX induced body weight loss (Figures 2A and S1A). Based on the bioluminescence imaging, FOLFOX treatment inhibited the development of tumor, with the inhibition rate of 73.63% at day 11 (Figure 2C) and 56.96% at day 16 (Figure 2D) in the male cohort. Comparing to the male model group, the CRC onset time was later in female model mice. And FOLFOX inhibition rate was 4.44% at day 11 (Figure S1C) and 33.91% at day 16 (Figure S1D), indicating the anti-cancer effects of FOLFOX were less prominent in female mice.

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Tumor tissues were photographed and weighed at the end of experiment (day 17). As shown in Figures 1,3 and S2A–C, FOLFOX significantly inhibited the development of tumor in both male (inhibition rate: 83.94%) and female (inhibition rate: 82.65%) mice. Model development and FOLFOX treatment did not change the liver index of male and female mice. Consistent with the pharmacodynamic evaluation index of BLI and tumor weight, the Ki67 level in tumor tissues also decreased after FOLFOX treatment (Figures 3D and S2D). Moreover, histopathology examination revealed no obvious injury in organs such as the liver, spleen, kidney, and colorectum in the model and FOLFOX groups (Figures 3E and S2E,F). Interestingly, the development of orthotopic xenograft significantly induced splenomegaly, and FOLFOX administration reversed this pathological phenomenon (Figure 3D,F), suggesting the involvement of immune reprogramming during model development and FOLFOX treatment. Overall, these results suggest that the current surgical orthotopic xenograft approach developed into rapidly growing malignant tumors within 5 days, and this model served as a good simulation of chemotherapy efficacy of FOLFOX. Furthermore, the splenomegaly was induced by the model development and reversed by FOLFOX treatment, indicating that the approach can be utilized for immune-related studies of CRC.

FOLFOX-induced immunosuppression of tumor-bearing mice

Immunosuppression is one of the most typical characteristics of chemotherapy in the clinic. Data show that more than 40% CRC patients suffered from myelosuppression (neutropenia, leukopenia, etc.) after FOLFOX chemotherapy.²⁸ To verify whether the current model could simulate the immunosuppression status caused by FOLFOX, we collected tumor tissues for flow cytometry at the end of the experiment. Results showed that the infiltration of macrophages (CD45⁺F4/80⁺) decreased, whereas the level of immunosuppressive Treg cells (CD45⁺FoxP3⁺) increased (Figures 4A,B and S3A,B) in both male and female tumor tissues after FOLFOX administration, which is consistent with the myelosuppressive effect of FOLFOX observed in patients. Serum samples were also collected for multi-factor detection at the end of the experiment. Results suggested that cancer development promoted secretion of IL-1β and IL-17A but not IL-12p70. And FOLFOX treatment reversed the tumor-induced release of IL-17A, decreased the level of IL-12p70, and did not affect serum concentration of IL-1β (Figures 4C and S3C). Overall, the current orthotopic xenograft model of colorectal cancer achieved efficient proliferation in vivo and simulated the therapeutic and immunosuppressive effect of FOLFOX, indicating that it can be applied as a reliable research model for immune-related preclinical studies.

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DISCUSSION

In the current study, we established a surgical orthotopic xenograft model based on colorectal cancer Luci-CT-26 cells in both male and female mice (Figure 1). Based on the bioluminescence imaging, all the model mice progressed to CRC 5 days after the surgery. Tumor development did not lead to significant changes in body weight and critical organs of mice. The subsequent treatment of the first-line CRC chemotherapy regime FOLFOX significantly inhibited tumor development in both male and female mice (Figures 2, 3, S1 and S2), indicating the efficient simulation of this model to patients.

Immune microenvironment reprogramming is one of the typical characteristics of cancer, which contributes to cancer development by avoiding the recognition and attack from the body's immune system.²⁸ Many studies have confirmed that immune regulation plays important roles in chemotherapy efficacy as well as in adverse effects. 5-Fluorouracil (5-FU), calcium folinate, and oxaliplatin combination strategy, known as FOLFOX, is the preferred chemotherapeutic regime for CRC. However, more than 40% CRC patients suffered from myelosuppression after FOLFOX therapy.²⁹ Meanwhile, studies showed that FOLFOX treatment leads to CD8⁺ T lymphocyte exhaustion in CRC patients, and the remodeling of tumor immune microenvironment significantly affects its sensitivity.^30,31 We tried to explore whether the current model could mimic the key features of clinical immune microenvironment and immune response of FOLFOX. As important components of tumor microenvironment, macrophages and Treg cells are involved in tumor immune escape and anti-tumor immune response.^32,33 Therefore, this study focused on the infiltration of macrophages and Treg cells in tumor tissues and related inflammatory factors in serum. Consistent with real data from patients,²⁸ we found that FOLFOX treatment decreased infiltrating macrophages in tumor tissues but increased the level of Treg cells (Figures 4 and S3). Meanwhile, when compared with sham mice, model mice had elevated levels of inflammatory factors IL-1β and IL-17A,^34,35 suggesting that the tumor development induces a systemic inflammatory response. On the contrary, FOLFOX treatment triggered inflammatory suppressive effect such as inhibition of IL-17A as well as IL-12p70 (the active form of IL-12), which may contribute to the altered infiltration of macrophages and Treg cells in microenvironment.^36,37

As a surgical orthotopic xenograft approach, our model may also be applied in clinical precision medicine. Recently, the development of PDXs has made great progress in drug evaluation and clinical individualized therapy of CRC. However, subcutaneous and renal capsule xenograft models are most commonly used in studies, in which the tumor cannot develop in colon microenvironment. Our orthotopic model could greatly offset the limitations of current PDXs when applied in severely immunodeficient mice (NCG/NSG) or immune system humanized mice (injected with Hu-PBL/PBMC) and contribute to clinical CRC therapy.

The current approach provides novel options for preclinical CRC mice model, but still has some limitations. First, basic surgical skills of small animals are necessary to construct this model. Second, about 2%–5% of model mice developed nonfatal intestinal obstruction during the experiment. Third, this model cannot be used to investigate CRC initiation as the tumor stitched onto colon is already a tissue. In addition, only about 20% of the model mice can progress to liver metastasis, and thus this model may not be suitable for metastasis evaluation.

CONCLUSIONS

In the current study, we constructed a novel CRC orthotopic xenograft model with high success rate, low toxicity, convenience, low complication, which can simulates clinical response to FOLFOX chemotherapy and allows for efficient in vivo imaging. Our approach provides a novel platform for in-depth CRC investigation and anti-CRC drug screening that may contribute to discovery and development of therapeutic targets.

AUTHOR CONTRIBUTIONS

Xiaoying Hou: Conceptualization; data curation; formal analysis; investigation; supervision; writing – original draft; writing – review and editing. Xiaoxuan Li: Formal analysis; investigation; writing – review and editing. Qian Fang: Data curation; investigation; writing – review and editing. Yufei Deng: Data curation; investigation; methodology; writing – review and editing. Haiping Wang: Supervision; writing – review and editing. Binlian Sun: Supervision; writing – review and editing. Chengliang Zhang: Resources; supervision. Hongzhi Du: Funding acquisition; resources; supervision; writing – review and editing. Yuchen Liu: Funding acquisition; supervision; writing – original draft; writing – review and editing.

ACKNOWLEDGMENTS

Not applicable.

FUNDING INFORMATION

This work was supported by the National Natural Science Foundation of China (82204416), the Natural Science Foundation of Hubei Province (2024AFD241, 2024AFB941), the Wuhan Science and Technology Project (2023010201020448), the Research Fund of Jianghan University (grant no.: 2021jczx-002).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

Available upon request through correspondence.

ETHICS STATEMENT

The study was conducted in accordance with the standards established by the Medical Ethics Committee of Jianghan University(Approval number: JHDXLL2023-100).

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