A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing (Neo)adjuvant Therapies

Summary

In this protocol, we describe a mouse model of incomplete surgical resection of soft tissue sarcoma for testing (neo)adjuvant therapies.

Abstract

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant or neo-adjuvant therapies. This occurs when surgical margins are insufficiently tumor-free, resulting in residual cancer cells. From a biological and immunological perspective, surgery is not a null event; the wound healing environment is known to induce both pro- and anti-tumorigenic pathways. As a consequence, preclinical models for drug development aimed at preventing local relapse should incorporate surgical resection when testing new (neo)adjuvant therapies, to model the clinical settings in patients treated with surgery.

Here, we describe a mouse model of incomplete surgical resection of WEHI 164 soft tissue sarcoma that allows testing of (neo)adjuvant therapies in the setting of a wound healing response. In this model, 50% or 75% of the tumor is removed, leaving behind some cancer tissue in situ to model gross residual disease after surgery in the clinical setting. This model allows testing therapies in the context of surgery while also considering the wound healing response, which may affect the efficacy of (neo)adjuvant treatments. The incomplete surgical resection results in reproducible regrowth of the tumor in all mice in the absence of adjuvant therapy. Adjuvant treatment with checkpoint blockade results in reduced tumor regrowth. This model is thus appropriate for testing therapies in the context of debulking surgery and its associated wound healing response and can be extended to other types of solid cancer.

Introduction

Surgery remains the main treatment option for many solid tumors¹, including soft tissue sarcoma^2,3. Despite improvements in cancer surgery techniques, and combinations with (neo)adjuvant therapies, there is still a high risk of cancer relapse and metastasis following primary tumor resection^4,5. In soft tissue sarcoma, relapses occur particularly locoregionally, at the site of surgery, resulting in increased morbidity and mortality. In the clinical setting, it can be difficult to obtain wide enough margins (e.g., due to anatomica....

Protocol

Animals used in these experiments were obtained from the Animal Resource Centre (Perth, Western Australia). Animals were maintained under standard pathogen-free conditions at the Harry Perkins Institute of Medical Research Bioresources North Facility (Perth, Western Australia). All experiments were carried out following the protocol as approved by the Harry Perkins Institute of Medical Research Animal Ethics Committee. BALB/c mice of 8-12 weeks of age were used in these experiments. The WEHI 164 fibrosarcoma cell line was obtained from CellBank Australia (Westmead, NSW).

1. Inoculation of cells

1. Preparation of cells and animals ....

Results

Tumor growth to a size of 50 mm² is an ideal size for partial debulk. The incomplete surgical resection of 50 mm² tumors results in 100% (n=5) reproducible regrowth of the tumors in the absence of adjuvant immunotherapy (Figure 4A). We next used the model to test adjuvant immunotherapies using antibodies against checkpoint molecules Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4) and Programmed Death Receptor 1 (PD-.......

Discussion

We provide a protocol for a mouse model of incomplete surgical resection of soft tissue sarcoma to test peri-operative therapies. We also standardized the surgical incision to allow assessment of wound healing between mice following treatment.

Tumor placement is an important part of this protocol. We have opted for a subcutaneous tumor model to allow easy surgical access to the tumor site and administration of local therapies with minimal burden on the mice. It is also important to ensure that.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
26 gauge 0.5 mL insulin syringe	Becton Dickinson, Australia	326769	None
2-Mercaptoethanol	Life Technologies Australia Pty Ltd	21985023	None
Anaestetic gas machine	Darvall Vet, Australia	SKU: 2848	None
Anti-CTLA-4	BioXcell, USA	BE0164	None
Anti-PD-1	BioXcell, USA	BP0273	None
Buprenorphine Hydrochloride Injection, 0.3mg/mL	RB healthcare UK Limited, UK	55175	Prescription order
Chlorhexidine Surgical Scrub 4%	Perigo Australia, Australia	CHL01449F(scrub	None
Fetal Bovine serum	CellSera, Australia	AU-FBS-PG	None
Forceps Fine 10.5 cm	Surgical house, Western Australia	CC74110	None
Forceps Fine 12 cm Serrated	Surgical house, Western Australia	CC74212	None
Forceps Halsted 14 cm	Surgical house, Western Australia	CD01114	None
Heating chamber	Datesand Ltd, UK	Mini-Thermacage	None
HEPES (1M)	Life Technologies Australia Pty Ltd	15630080	None
Isoflurane	Henry Schein Animal Health, Australia	SKU: 29405	Prescription order
Lubricating Eye Ointment	Alcon	n/a	None
Penicillin/streptomycin 1000X	Life Technologies Australia Pty Ltd	15140122	None
Phosphate Buffered Solution 10x	Life Technologies Australia Pty Ltd	70013-032	None
Reflex 7mm Clips	Able scientific, Australia	AS59038	None
Reflex 7mm Wound Clip Applicator	Able scientific, Australia	AS59036	None
Reflex Wound Clip Remover	Able scientific, Australia	AS59037	None
Rodent Qube Anesthesia Breathing Circuit	Darvall Vet, Australia	#7885	None
Roswell Park Memorial Institute (RPMI) 1640 Medium + L-glutamine	Life Technologies Australia Pty Ltd	21870092	None
Scissors Iris STR 11 cm	Surgical house, Western Australia	KF3211	None
Scissors Iris STR 9 cm	Surgical house, Western Australia	JH4209	None
Small Induction Chamber	Darvall Vet, Australia	SKU: 9630	None
TrypLE express 1x	Life Technologies Australia Pty Ltd	12604-021	None
Germinator 500 Glass Bead Sterilizer	Cellpoint Scientific Inc., USA	5-1460-DK