Subcutaneous Injection of Human Colorectal Cancer Cells in Athymic Nude Mice to Evaluate Antitumor Efficacy

Summary

This study focuses on the in vivo subcutaneous induction of luminescent colorectal cancer cells in a murine model and the subsequent luminescence measurement using an imaging system. The procedure aims to establish a reproducible tumor growth model for real-time monitoring and therapeutic evaluation.

Abstract

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide, highlighting the urgent need for improved preclinical models to evaluate potential therapies. Animal models are essential for elucidating tumor biology and assessing novel therapeutic interventions. Mouse models offer key advantages over existing models, including genetic manipulability, rapid reproduction, cost-effectiveness, and the availability of immunocompetent and immunodeficient strains, enabling precise modeling of human cancers.

This protocol focuses on the subcutaneous induction of the bioluminescent colorectal cancer cell line HCT-116 in an immunodeficient murine model, followed by non-invasive bioluminescence imaging using the IVIS system and caliper to monitor tumor progression and growth. Using luminescent cells provides a real-time, quantifiable approach for tracking tumor development and response to therapeutic interventions, reducing the need for invasive procedures. A cohort of mice (n = 12) is required for tumor cell implantation, and luminescence intensity is measured to establish a standardized model for therapeutic testing. Tumor volume is typically calculated using only length and width measurements or width.

The current methodology ensures consistent tumor establishment while minimizing° variability, ultimately enhancing the reliability of preclinical assessment. The findings of this study will contribute to refining tumor models for drug testing and improving the translational potential of preclinical research in clinical applications. By employing advanced imaging techniques and a reproducible experimental design, this study aims to improve the accuracy and efficiency of in vivo drug evaluation for colorectal cancer treatment.

Introduction

Colorectal cancer (CRC) is a major global health burden, ranking among the most prevalent and deadly malignancies¹. Developing reliable preclinical models is essential for evaluating new therapeutic approaches, as traditional in vitro assays fail to fully capture the complexity of tumor growth and drug response in a living system. In vivo models, particularly murine models, have been used to provide valuable insights into tumor biology, allowing researchers to assess the efficacy of potential treatments in a controlled environment.

The use of luminescent CRC cells in murine models represents a significant advancement in preclinical research, enabling real-time, noninvasive monitoring of tumor progression². This study aims to establish a standardized protocol for subcutaneous tumor induction and IVIS imaging to track tumor growth dynamically. A subcutaneous injection model was selected because of its technical simplicity, reproducibility, and ease of longitudinal tumor monitoring. By leveraging bioluminescence imaging, researchers can obtain precise, quantifiable data on tumor development and therapeutic responses without the need to euthanize animals at multiple time points³.

The IVIS imaging system is a powerful tool for assessing tumor burden with high sensitivity and specificity, enhancing the reproducibility and accuracy of in vivo studies. This study will serve as a foundation for future therapeutic evaluations, providing a standardized and efficient approach to preclinical drug testing for colorectal cancer treatment. By refining tumor induction methods and implementing advanced imaging techniques, this research contributes to bridging the gap between preclinical and clinical applications in oncology⁴.

Protocol

Animals were maintained under specific pathogen-free conditions according to the guidelines established by the Instituto Murciano de Investigación Biosanitaria (IMIB, Murcia) ethical committee (No A13240502). Inspections were conducted weekly by qualified personnel in compliance with R. D. 1201/2005.

1. Animals

NOTE: All animals must be securely and safely restrained before injection. If working with immunodeficient mice (i.e., nude athymic mice such as Crl: NU(NCr)-Foxn1nu), it is essential to maintain sterility. The current study utilized 12 athymic nude mice, 5 weeks old (20-22 g for females and 22-26 g for males) at the beginning of the experiment (50% males). All anesthesia and experimental procedures were conducted in the Guía de Ética y Buena Práctica en Investigación of the center, with approval from the institutional ethics committee (Protocol No. A13240502) and in compliance with Spanish legislation (R.D. 1201/2005).

1. Before initiating the assay, acclimate the mice, aged 4 weeks, to the animal center for approximately 1 week. Subsequently, carry out the following procedures.

2. D-luciferin stock preparation

1. Prepare a 15 mg/mL working solution of D-Luciferin Potassium Salt Bioluminescent Substrate in prewarmed Phosphate Buffered Saline (PBS) in a conical tube inside a biosafety cabinet. Invert the tube several times to homogenize the solution well.
2. Transfer the solution through a 0.2 µm syringe filter for sterilization.
3. Aliquot the sterilized solution in 1 mL opaque microtubes and store at -20 °C.
   NOTE: Luciferin is light-sensitive.

3. Extracellular matrix preparation

1. Thaw phenol-red-free commercial extracellular matrix (ECM) hydrogel overnight on ice inside a refrigerator between 2 °C and 8 °C⁵.
2. On the day of injection, keep the ECM on ice and, using a prechilled P1000 micropipette, aliquot 200 µL of ECM into sterile tubes.
   NOTE: It is recommended to use wide-bore microtubes. Minimize temperature changes and avoid repeated freeze-thaw cycles.
3. Prechill the syringes and needles on ice before preparing injections to prevent sudden temperature changes of the ECM.

4. Cell detachment and counting

1. Plate HCT-116 bioluminescent cells (ATCC cell line CCL-247-LUC2) in T75 flasks a few days before tumor induction to ensure sufficient cell number. Maintain cell culture at 37 °C, 5% CO₂, and replace medium if necessary⁶.
2. On the day of the experiment, aspirate the culture medium carefully to avoid disturbing adherent cells.
3. Wash the cells with 8 mL of sterile PBS (prewarmed at 30 °C in a water bath) to remove residual serum.
4. Add 2 mL per flask of warm Trypsin-EDTA (0.05%) and incubate at 37 °C for 4-5 min. Monitor cell detachment under an inverted microscope. If cells remain attached, gently tap the side of the flask to aid detachment.
5. Neutralize trypsin by adding 10 mL of complete growth medium containing fetal bovine serum (FBS).
6. Transfer cells into a 15 mL conical tube using a 10 mL serological pipette. Rinse flasks with an additional 2 mL of fresh medium to collect any remaining cells and add the washes into the 15 mL tube.
7. Centrifuge the cell suspension at 200 × g for 5 min at room temperature. Carefully aspirate the supernatant without disturbing the cell pellet.
8. Gently resuspend the cell pellet in 3 mL of seeding medium to achieve a homogeneous suspension.
9. Count the cells in a Neubauer chamber or an automatic cell counter. Determine cell viability by mixing 10 µL of the cell suspension with 10 µL of Trypan blue and count viable cells under an inverted microscope.
   NOTE: Prepare a sufficient number of cells for the study: at least 3 × 10⁶ viable cells per mouse.
10. Resuspend the cells in sterile PBS (prewarmed at 30 °C in a water bath) and modify the concentration of viable cells to a final concentration of 2 × 10⁷ cells/mL with PBS. Prepare aliquots of 200 µL.

5. Set up equipment items and anesthetize animals.

1. Turn on the imaging system at least 4 h before imaging.
   NOTE: Follow the system's instructions to allow the initialization step, including adjusting settings for light intensity and camera exposure time depending on signal-to-noise ratio. This setting must be set manually based on the experimental conditions.
2. Weigh the animals and calculate the volume of D-luciferin that must be administered (200 µL of a 15 mg/mL solution per 20 g of the animal).
3. Clean and disinfect the imaging chambers and cabinet.
4. Identify and safely restrain each mouse for anesthesia.
   NOTE: Wear two pairs of gloves to protect hands from accidental needle sticks during the subcutaneous injection.
5. Anesthetize mice with 2% isoflurane in oxygen at 1.8-2.0 L/min flow. Turn on the suction pump simultaneously with the anesthesia pump.

6. Preparation of the cells for injection

1. Mix 200 µL of 2 × 10⁷ bioluminescent CRC cells/mL with 200 µL of ECM in a cryovial inside a biosafety cabinet. Gently pipette with a P1000 pipette to avoid air bubble formation and dissociation.
2. Transfer the 400 µL mixture to a 1 mL syringe, remove air bubbles, and proceed immediately to injection.
3. Safely uncap the needle, always maintaining the sterility of the needle.
   NOTE: It is recommended to use individual needles for each animal to minimize contamination. A new needle is used for each mouse to prevent cross-mouse infection.
4. Attach a 25 G needle and adjust the syringe content to 200 µL, removing any air bubbles.
   NOTE: Injections are usually done with 200 µL, leading to 2 × 10⁶ cells per injection.
5. Slide the needle back into the cap loosely without handling either the cap or the needle while the animal is restrained.

7. Subcutaneous tumor induction

1. Remove the mouse from the chamber once it is anesthetized and place the animal's snout in an outlet supplying the same anesthetic.
   NOTE: In our case, the nozzle was made using a glove.
2. Place the animal on its right side on a flat surface. Using the tips of the fingers of the non-dominant hand, gently pinch and lift the animal's skin in the lower left quadrant.
3. Before injecting, aspirate first to ensure the appropriate placement of the needle. Insert the needle bevel-up with the dominant hand, two-thirds into the lifted skin, parallel to the body, being very careful not to prick fingers or puncture through the double gloves.
   NOTE: Proper placement should yield negative pressure and no aspiration in the hub of the needle.
4. Inject the contents of the syringe (200 µL) into the lifted skin, right between the two fingers. Hold briefly before withdrawing the needle slowly. Remove the needle after the subcutaneous injection and discard it safely into a Sharps container.
   NOTE: Prior to injection, sanitize the injection site using 70% alcohol or an equivalent skin disinfectant.

8. Intraperitoneal injection of D-luciferin

1. Before returning the animal to the anesthesia chamber, administer D-luciferin intraperitoneally (IP) at a concentration of 150 mg/kg of body weight with the 15 mg/mL stock solution in a 30 G insulin syringe (200 µL per 20 g of the animal).
   NOTE: Up to 0.5 mL injection is well tolerated.
2. Manually restrain, dorsal recumbence (abdomen side up), with the cranial (head) end of the animal pointed down. Allow the intestinal contents to move downward.
3. Keeping the needle bevel-side up and slightly angled at 15-20°, push the needle into the abdominal cavity so that the tip of the needle just penetrates the abdominal wall of the animal's left lower abdominal quadrant.
   NOTE: Prior to injection, sanitize the injection site using 70% alcohol or an equivalent skin disinfectant.
4. Slowly inject the D-luciferin into the intraperitoneal cavity, remove the needle, and discard it safely.
5. Place the mouse in the anesthesia chamber and allow it to stabilize for approximately 5 min. After 15 min, proceed with in vivo imaging.
   NOTE: Construct a D-luciferin kinetic curve for each new animal model to determine peak signal time. In our case, the light emission peaked at approximately 15 min in in vivo imaging.

9. In vivo imaging analysis

1. Place the fully anesthetized animal in the chamber of the imaging system, on the right flank. Ensure the animal is properly aligned with the camera and the light source to capture the tumor area.
2. Open the linked software. Choose the visual or photographic imaging mode to confirm the position of the animal. Save the data and change the folder´s name.
3. Choose the bioluminescence imaging mode and set it to automatic. Capture bioluminescence images and monitor the tumor light on the computer screen.
4. Once the imaging is complete, return the animal to its cage and observe any complications.
5. Perform postprocessing, first scale the bioluminescent light so that all the mice tumors show the same scale, usually between 1e⁶ and 1e⁸.
6. Set up a Region of Interest (ROI) around the tumor for analysis and copy it for further analyses. Measure the light of the ROI and export these results for further analysis. Paste previously created and copied ROI of the same size for subsequent tumors.
7. Shut down the system according to the manufacturer's instructions and clean the imaging chamber. Turn off the extraction and anesthesia pumps, together with the oxygen flow.

10. Tumor volume measurement

1. Anesthetize animals with isoflurane for tumor measurement. Place the animal in a stable position and cover its head. Quickly immobilize the animal.
2. Measure tumor length, width, and depth using a calibrated digital caliper.
   NOTE: Perform the measurements quickly, as the animal will begin to awaken approximately 1 min after removal from the anesthesia chamber.
3. Repeat measurements and calculate an average.
4. Calculate the volume according to the formula for spherical or elliptical tumors:
   For spherical tumors ⁷    (1)
   For ellipsoid tumors ⁸     (2)
   NOTE: To minimize measurement bias, all tumor measurements within the study should be performed by the same operator. Additionally, it should be noted that on the day of tumor induction, accurate measurement is often challenging because the tumor mass is not yet fully solidified.

Results

This protocol allows for the introduction of human cells into an immunodeficient mouse model, enabling the growth of these tumoral cells in vivo, which is always more physiologically relevant than in vitro. It is crucial to ensure that the experimental conditions are as reproducible as possible, that tumors contain a similar number of viable cells, and that they are implanted in a comparable and consistently accessible location.

To test the sensitivity of the bioluminescent H...

Discussion

The protocol for the subcutaneous induction of luminescent colorectal cancer cells in an athymic nude mouse model presents a standardized and reproducible approach for tumor formation and real-time monitoring. First, it is important to note that such studies are typically conducted over 28 days, with luminescence measurements performed on the day of tumor induction and subsequently weekly. Conversely, caliper measurements of tumor volume are not performed on the first day, as the cell suspension has not yet encapsulated;...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL opaque microtubes tubes	Eppendorf	30125215	D-luciferin storage
15 mL conical tube	Falcon, Thermo Fisher Scientific	11507411	Cell culture material
Advanced Dulbecco’s Modified Eagle Medium (DMEM)	Thermo Fisher Scientific	1152046	Cell culture reagent
Athymic Nude Mouse	Charles River	Crl:NU(NCr)-Foxn1nu	Immunodeficient nude mouse
Bioluminescent Human Colon cancer cells HCT116	ATCC	CCL-247-LUC2	HCT 116-Luc2 is ideal for in vivo bioluminescence
D-luciferin Pierce	Thermo Fisher Scientific	15215733	For bioluminescence measurement
Extracellular Matrix (ECM)	Corning	356231	Matrigel Matrix for cell invasion
Fetal Bovine Serum	Thermo Fisher Scientific	10500064	Cell culture reagent
Filter Pipette Tips	Finntip, Thermo Fisher Scientific	94056710	To avoid air bubble formation
Insulin Syringe 25 G 1 mL	Iberomed	JERINRAYS29	For HCT-116 implantation of cell line
Insulin Syringe 30 G 0.5 mL	DH Material medico	9.41652E+15	For D-luciferin administration
IVIS image system with Living Image software	PerkinElmer/Revvity	128201	Bioluminescence and fluorescence imaging scanner
Needle Sterican Heparin	VWR	612-0153 10	Administration of D-luciferin and HCT-116 implantation
Phosphate Buffered Saline (PBS)	VWR	392-0042P	Cell suspension solution for the detachment and implantation of HCT-116
Trypsin-EDTA	Thermo Fisher Scientific	11590626	Cell culture reagent
T-75 Flask, TC-Treated	STARLAB	CC7682-41	Cell culture material