Translationally-Relevant Tumor Resection Model for Murine Preclinical Models of Oral Squamous Cell Carcinoma

Summary

A unique method to surgically resect oral squamous cell carcinoma tumors established in the oral cavity is shown here.

Abstract

Currently, the first-line treatment for the most common form of head and neck squamous cell carcinoma (HNSCC), oral cavity squamous cell carcinoma (OSCC), is surgical resection, followed by risk-adapted treatments, such as chemoradiotherapy. However, with the current standard of care and even the emergence of new treatments, such as checkpoint blockade, the overall survival for advanced, recurrent disease remains high, and the prevalence of head and neck cancer is projected to continue to increase in the coming years. Thus, deeper investigation into current standard-of-care modalities, in addition to novel therapeutics, is necessary to translate potential and promising treatment options to patients with HNSCC. Preclinical models are utilized to evaluate the safety and efficacy of potential treatment interventions. Unfortunately, current preclinical models fail to fully embody the surgical procedure many patients receive, as they primarily involve non-surgical treatment modalities, like radiation, chemotherapy, or immunotherapy. Furthermore, existing surgical models are limited by additional reagents needed to support the establishment of tumors in orthotopic sites and the requirement of microsurgery for tumor removal. This warrants the generation of improved and simplified preclinical models to investigate therapeutics and gain insight into locoregional tumor recurrence and treatment resistance. This article describes a novel syngeneic clinically relevant orthotopic murine subtotal resection model that recapitulates the standard of care in OSCC. This model simplifies existing models and results in measurable recurrence following surgical resection of buccal mucosal tumors. Use of this model may lead to valuable insights into tumor recurrence and treatment resistance in patients with OSCC. The establishment of this preclinical model paves the way for further work to investigate how OSCC responds to post-resection adjuvant therapies, capturing the tumor state at the clinically relevant window at which patients generally begin such subsequent treatments.

Introduction

Malignancies originating in the larynx, pharynx, hypopharynx, nasal cavity, salivary glands, and oral cavity are categorized as head and neck cancers. Head and neck squamous cell carcinoma (HNSCC), which accounts for over 90% of these cases, develops from the squamous epithelial cells that line these anatomical sites¹. More specifically, oral cavity squamous cell carcinoma (OSCC) is a malignancy confined to the oral cavity region. In 2022, head and neck cancer was the sixth most prevalent cancer globally, with an estimated 947,000 new cases recorded worldwide and 482,000 deaths annually².

Surgical resection is generally the first-line treatment modality for head and neck cancers, including OSCC, often followed by chemoradiotherapy^1,3,4. While these standard-of-care therapies are potentially curative in locally restricted or early-stage cases, there remains a high rate of locoregional and distant recurrence for patients with OSCC. In fact, fewer than two-thirds of patients with OSCC survive beyond five years and endure significant side effects^5,6,7. There is increasing evidence that the traumatic stress incurred during surgery triggers the physiological processes involved in tissue damage and wound healing^8,9. Moreover, studies have also reported that surgical resection contributes to immunological dysfunction that can, in turn, contribute to the potential for recurrence¹⁰.

In spite of a majority of patients undergoing surgical resection as first-line therapy for OSCC, there currently remains a need for relevant preclinical orthotopic resection models for OSCC that standardize the establishment of tumors and subtotal resection and preserve the associated immunological mechanisms for tumor recurrence in order to provide a critical context for research in this area^11,12,13. Prior studies reporting animal resection models include a 4T1 breast tumor resection model that has been used to study the immunosuppressive effects of surgery¹⁴. For studies in OSCC, the use of a heterotopic MOC1-OVA tumor subtotal resection model in the murine flank has been reported¹⁵. In orthotopic sites, the use of the HPV oropharyngeal SCC cell line, mEERL95, has been used to establish submental tumors for resection and recurrence modeling, while surgically transferring draining lymph nodes to naïve mice for further study¹⁶. Relevant orthotopic studies investigating recurrence using immunodeficient mice have also been reported¹³.

Herein is a newly established protocol for an immunocompetent, syngeneic, orthotopic OSCC preclinical model of standard-of-care tumor resection survival surgery and recurrence, observed within one week post-resection. This procedure further simplifies existing models by performing tumor removal without microsurgery and using the murine oral cancer tumor cell line (MOC2), which does not require the addition of cell or basement membrane matrix to establish tumors.

Protocol

The following procedure, as well as animal housing and maintenance, was conducted in the Center for Laboratory Animal Medicine and Care at The University of Texas Health Science Center at Houston (UTHealth), in coordination with the institutional Animal Welfare Committee. The associated protocol was approved by the Institutional Animal Care and Use Committee (IACUC) and is in accordance with the principles and ethical guidelines outlined in the US Public Health Service's policy on the humane care and use of laboratory animals. Please ensure observance of all policies set by local IACUC and ensure an IACUC-approved animal use protocol (or equivalent) is in place before proceeding with the following sections. The reagents and the equipment used are listed in the Table of Materials.

1. Establishing orthotopic buccal mucosal oral tumors

NOTE: For this specific protocol, MOC2 cells were used to establish tumors¹⁷. However, other cell lines or types may also be used. The quantity of tumor cells necessary to establish tumors in vivo is cell line-dependent and should be optimized prior to inoculation for surgery.

1. When the tumor cell culture reaches approximately 80% confluence, harvest cells by aspirating and discarding culture media.
2. To remove remaining serum, wash cells with phosphate-buffered saline (PBS) without magnesium and calcium, sufficient in volume to fully cover the vessel (e.g., flask) surface with the attached cells.
3. Add 0.05% trypsin to the culture flask to sufficiently cover the bottom of the flask. Incubate cells at 37 °C with 5% CO₂ for approximately 5 min for cell detachment.
   NOTE: Incubation period required (2-15 min) to detach cells may vary depending on the cells (i.e., cell line) used. However, prolonged exposure to trypsin may be damaging to cells. If additional time is needed for adequate cell detachment, monitor cells periodically and closely.
4. After cell detachment is observed, quench with at least 2 times the volume of trypsin used with complete media (e.g., for 5 mL trypsin, quench with at least 10 mL of complete media).
5. Centrifuge the cell suspension at 350 x g for 5 min at room temperature, and aspirate the supernatant.
6. Resuspend in cold PBS and take an aliquot for counting. Repeat steps 1.5-1.6 for an additional wash to remove any remaining media.
7. Using additional PBS, adjust to the optimized concentration of cells for inoculation (i.e., 3 x 10⁴ MOC2 cells per 30 µL volume). Keep cell suspension on ice until ready for use.
8. Anesthetize the mouse using 1%-3% isoflurane, supplemented with 1.5 L/min oxygen in a small animal chamber.
   NOTE: Hair removal is not necessary but recommended at this stage for help in visualizing the inoculation site and improvement of inoculation consistency. If removing at this stage, follow the instructions at steps 3.3-3.5.
9. Mix the cell suspension thoroughly to ensure even distribution of cells and collect in a 0.3 mL insulin syringe.
10. Restrain the sedated mouse by scruffing its posterior neck with one hand so the murine anterior side faces the handler and the head faces upward for the mouth to be open and accessible.
11. Carefully insert the syringe into the open mouth, at a downward angle toward the left lower mandibular vestibule. Continue to insert the needle through the lower buccal mucosa.
12. Deliver 30 µL of cell suspension into this region and carefully remove the syringe, hold this position for 5 s, then slowly withdraw the syringe to avoid tracking additional cell suspension upon removal.
13. Ensure the mouse is in normal physical and behavioral condition following inoculation.
14. Monitor the mouse's weight and tumor growth with a balance and digital calipers, respectively. Monitor for tumor development regularly, 2 times per week or per specified protocol.

2. Material setup for surgery

NOTE: It is highly recommended that at least one other surgeon or surgical assistant be present to assist in setup and organization throughout the procedure to help ensure maintenance of sterility.

1. Select a clean procedure room with a closable door and limited traffic.
2. Pre-warm a new cage, containing clean, dry bedding and a nest of paper towels, by setting it partially atop an electric warmer.
3. Warm a bead sterilizer to operating temperature (250-265 °C).
4. Set up the isoflurane induction chamber and portable anesthesia device, with the appropriate nose cone, and connect to isoflurane, oxygen, and the relevant scavenging system.
   NOTE: When using the induction chamber, anesthetize mice with the setting at approximately 3% isoflurane. When using the nose cone, anesthetize mice with 1%-2% isoflurane.
5. Turn on the water-heated surgical pad to 38.9 °C and secure the isoflurane source at the head of the pad.
6. Sanitize the intended work surface and surgical pad with a disinfectant and cover the workspace with a clean bench pad.

Figure 1: Selected surgical instruments and materials for tumor resection. (1) Surgical gloves, (2) drape, (3) gauze pad, (4) 0.3 mL insulin syringe, (5) towel drape, (6) isopropyl alcohol swab pad, (7) scalpel blade, (8) scalpel without blade, (9) Hartman hemostat, (10) Mosquito hemostatic forceps, (11) micro dissecting scissors, (12) micro dissecting forceps (13) Brown-Adson forceps (14) micro dissecting forceps, (15) cotton tip applicators, (16) trimmer, (17) 4-0 coated vicryl suture, (18) 0.25% Bupivacaine, and (19) eye lubricant ointment. Please click here to view a larger version of this figure.

3. Preparation of the animal for surgery

1. Weigh the mouse 24 h prior to the procedure to determine appropriate anesthesia and pain relief drug dosage, per specified protocol.
2. Anesthetize the mouse in the induction chamber for approximately 2 min, and then administer analgesics at least 30 min before surgery. This should be subcutaneously injected at the nape of the murine neck.
3. If not done previously at the time of inoculation or if previously cleared hair has begun to regrow, add a small amount of hair removal cream using a cotton-tip applicator directly to the hair at the tumor resection site, with sufficient margins to keep hair out of the incision space through the procedure.
4. Allow cream to sit for approximately 10 s and wipe hair away with dry, clean gauze.
5. Remove any remaining cream from the site with gauze dampened with saline or water, then dry the area with dry, clean gauze.
   NOTE: Hair removal by an electronic trimmer is an acceptable but less preferred alternative method, as the skin around the head and neck can be thin and delicate. If using a trimmer, be sure to avoid sensitive areas, such as the eyes, and keep any loose skin taut to help avoid unwanted injury to the mouse.
6. Administer systemic analgesic subcutaneously and local analgesic to the incision site.
7. Place the anesthetized mouse on the heating pad, lying on its back with its anterior side facing upward. Its nose should be just inside the nose cone cover.
8. Place a small amount (i.e., sufficient to cover the eye) of sterile lubricant eye ointment on each eye of the mouse to prevent drying of the eyes during the procedure.
9. Using a clean cotton-tip applicator, swab the site of resection with 70% isopropyl alcohol followed by 10% iodine 3 times, making sure to remove the iodine after the last pass with 70% isopropyl alcohol.
10. Wearing sterile gloves and appropriate personal protective equipment (robe, hair cover), prepare the sterile field of at least 18"W X 26"L, and lay out all appropriate tools and materials (sterile cotton gauze, sterile Q-tips, sterile saline, isopropyl alcohol applicators, surgical tools) (Figure 1).
11. Cover the mouse with a sterile drape and cut an access hole immediately above the resection site using sterile scissors.

4. Surgical resection of established oral tumors

NOTE: Throughout the procedure, the surgeon or assistant should check for breathing and reflex of the mouse to ensure an appropriate oxygenation while the mouse is under anesthesia. Adjust isoflurane in accordance with veterinary guidance and institutional policy as needed.

1. Using a new surgical scalpel, create a submandibular incision, just below the tumor and along the left jawline, taking up the length of the tumor (Figure 2A,B).
2. Carefully use the Hartman hemostats to widen the opening toward the tumor. As needed, dab any blood with sterile gauze.
3. Locate the tumor and use forceps to hold it in place (Figure 2B).
   NOTE: An assistant may assist at this point with using forceps to maintain access to the tumor site (i.e., pull skin away).
4. Using surgical scissors, snip around the base of the tumor to resect all visible portions (Figure 2C). Reposition the skin over the resected tumor site and close the incision with four to six interrupted sutures, using 4-0 long-lasting vicryl sutures (Figure 2D).
   NOTE: Be careful not to secure sutures too tightly to preserve skin without further hurting the mice, as well as to prevent potential injuries upon later suture removal.
5. To continue with additional surgeries, clean the surfaces of surgical instruments with 70% isopropyl alcohol and place them in the heated bead sterilizer for 10-15 s prior to each additional use. The same set of tools may be used to perform surgery on up to 5 mice or, as specified by institutional guidelines and practices.
   NOTE: Resected tumors may be discarded or taken for processing and further analysis (i.e., fixation for histology and imaging; tissue dissociation and immune staining for flow cytometry phenotyping analysis, per the specific research study experimental plans^18,19 (Figure 2E).

Figure 2: Oral tumors, established transorally through the lower buccal mucosa, can be resected via a submandibular approach without healing complications. (A) Established tumor (red arrow) in the oral cavity. (B) Submandibular incision exposing the tumor. (C) The resected tumor. (D) Wound closure with multiple interrupted vicryl sutures. (E) Excised tumor. (F) Summary schematic for transoral inoculation. Image created in BioRender. Please click here to view a larger version of this figure.

5. Post-resection care

1. After closing the incision, turn off the anesthesia flow to 0% and continue only O₂ at 1.5 L/min until the mouse begins to regain consciousness (less than 5 min). If the mouse remains motionless and unresponsive after several minutes, refer to institutional policies on veterinary intervention.
2. Gently transfer the mouse to the pre-warmed cage. Continue to monitor the mouse for post-operative consciousness and mobility. It should regain full consciousness and mobility by 10 min.
3. Perform daily post-operative monitoring in accordance with approved protocol and institutional policies.
4. Immediately following the surgery, provide "soft" food for the mouse by setting pellets soaked in water in an accessible container (i.e., plastic weigh boat) directly into the cage. If no complications arise, the mouse can resume eating normal solid food at the next routine feeding.
5. Monitor mice twice daily for the first 48 h (2 days) and once per day for a minimum of 96 h (4 days) post-operation. Subsequently, resume a normal monitoring schedule (i.e., 2 times weekly).
6. At day 7 post-surgery, remove sutures under anesthesia, being careful not to cut or injure the mice.
7. Continue to monitor mice and once they reach their humane endpoint, humanely euthanize animals in accordance with the approved protocol euthanasia criteria and the American Veterinary Medical Association.
   NOTE: Statistical analysis of tumor growth kinetics and animal survival are relevant metrics for this methodology. For Kaplan-Meier survival curves, statistical analyses using the log-rank/Mantel-Cox test were performed via statistical analysis software. For tumor growth curves, Šídák's multiple comparisons test by way of a two-way ANOVA analysis was used to compare tumor sizes between unresected and resected groups. P-values less than 0.05 were considered significant.

Results

Observations from MOC2 murine buccal mucosa
Using the MOC2 cell line, tumors were established in the murine buccal mucosa of the oral cavity (Figure 2A-F). Established primary tumors were resected at a tumor size of 25-36 mm². Upon resection, mice typically regained full mobility within 5-10 min. Mice were subsequently monitored for post-operative care to confirm healing and recovery and to observe tumor recurrence. During thi...

Discussion

Herein, this article describes a simple procedure for the establishment and subtotal resection of tumors in the buccal mucosa of the oral cavity in mice. Previous studies have similarly demonstrated the feasibility of establishing tumors through the murine tongue, buccal mucosa, or floor-of-mouth for downstream analysis of resected tumors, as well as to investigate subsequent local recurrence and regional and distant metastasis, each with its own limitations^12,13...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin	Corning	25-052-Cl	0.53 mM EDTA, 1 X [-] sodium bicarbonate
0.25% Bupivacaine HCl Injection	McKesson	1077280	2.5mg/kg-1mg/kg, ID/SC 20-30uL
0.9% Sodium Chloride, USP	McKesson	161736	5 mL/kg, SC, IV 1.5 mL
23 G Needle	Becton, Dickinson and Company [McKesson]	402
4-0 Coated Vicryl	McKesson	100700
50 mL conical tubes	Thermo Scientific	33965
Absorbent Bench Pads	VWR	115-0684
Alcohol Swab Isopropyl Alcohol 70%	Becton, Dickinson and Company [VWR]	326895
Autoclave Pouch Self-Sealing 5 ¼” x 10”	McKesson	960944
Balance	Ohaus [VWR]	30253019
Bioclave Mini	Benchmark Scientific	B4000-M
Brown-Adson Forceps	ROBOZ [VWR]	RS-5231
Cotton-Tipped Applicator Sterile 6 in	McKesson	999736
Dulbecco's Phophate Buffered Saline / Modified	Cytiva	SH30028.02	[-] calcium, [-] magnesium
Ethiqa XRÒ*	Fidelis Animal Health	NDC 86084-100-30	3.25 mg/kg SC
General Purpose Drape	McKesson	68107
Glass Bead Sterilizer	VWR	75999-324
Glass Beads	VWR	75999-332
Hartman Hemostats	Fine Science Tools [VWR]	13003-10
High-Quality, Portable Anesthesia Device	Patterson Veterinary	07-8915662
Isoflurane, USP	McKesson	803250	4-5% Induction, 1-3% Maintenance
MedLEDÒ Chrome (Headlight)	KLS Martin	ML-MC7-HT-HK-X21
Micro Dissecting Forceps	ROBOZ [VWR]	RS-5174
Micro Dissecting Scissors	ROBOZ [VWR]	RS-5914
Monoject Insulin Syringes	Cardinal Health [McKesson]	8881600145
Mosquito Hemostatic Forceps	ROBOZ [VWR]	RS-7100
Nair Body Cream Hair Remover	Church & Dwight Co., Inc.
Needle Holder	ROBOZ [VWR]	RS-7154
PVP Prep Solution 10% Povidone Iodine	McKesson	911740
Scalpel	ROBOZ [VWR]	RS-9843
Scalpel Blades Carbon Steel No. 10	McKesson	862685
Small Animal Heated Pad	K&H Pet Products [Amazon]		9 X 12in 20W
Small Mouse Chamber	Patterson Veterinary	07-893338
Small Nose Cone	Patterson Veterinary	78909681
Steri-Drape	McKesson	5713
Sterile Gauze Pads 2”x2” – 12 PLY	Dukal Corporation [Amazon]	REF: 1212
Sterile Lubricant Eye Ointment	McKesson	730979
Surgical Gloves	McKesson	20-2070N
Syringe 1cc	McKesson	1065984
Traceable Digital Carbon Fiber Calipers	Fischer Brand	15-077-957
Trimmer Kit	WAHL [Amazon]		BravMini