Name: a mouse model of incompletely resected soft tissue sarcoma for testing (neo)adjuvant therapies
Uploaded: 2020-07-28T13:00:00
Description: Watch this Scientific Journal Video about A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing Neoadjuvant Therapies at JoVE.com

This work is supported by grants from the Sock it to Sarcoma! Foundation, the Australian and New Zealand Sarcoma Association, the Children&#39;s Leukemia &#38; Cancer Research Foundation and Perpetual Philanthropy. W.J.L is supported by a Simon Lee Fellowship and a research fellowship from the National Health and Medical Research Council, and the Cancer Council WA.

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 wa...

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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...

Surgery remains the main treatment option for many solid tumors1, including soft tissue sarcoma2,3. 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 resection4,5. 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 anatomical constraints), resulting in incomplete resection and subsequent tumor recurrence6. Surgical stress and the subsequent process of wound healing are known to create an immunosuppressive tumor microenvironment favorable for tumor recurrence7,8. Therefore, the discovery and development of new therapies for soft tissue sarcoma, particularly immunotherapies, should ideally take the surgical wound healing response into account.
Most preclinical studies for adjuvant therapies are initially carried out using subcutaneous syngeneic or xenotransplant mouse models, without incorporating the surgical stress and wound healing response9,10. Therefore, we developed a syngeneic subcutaneous mouse soft tissue sarcoma model incorporating incomplete surgical resection. WEHI 164 fibrosarcoma cells are inoculated subcutaneously, and once tumors are established, we remove 50-75% of the tumor bulk (Figure 1A-E). Tumors consistently re-grow from the remaining tumor. This model allows for testing adjuvant therapies while considering the effect of surgical stress and wound healing. Similar surgical models of incomplete resection have been used in a number of studies by several groups and found to be reproducible and effective11,12,13. Here, we provide a detailed description of this protocol.

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

a mouse model of incompletely resected soft tissue sarcoma for testing (neo)adjuvant therapies

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.

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-...

Watch this Scientific Journal Video about A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing Neoadjuvant Therapies at JoVE.com

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing Neoadjuvant Therapies

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

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

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant ...

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