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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		<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 ...

This protocol aims to mimic the clinical setting of residual disease after tumor debulking surgery, and therefore allows us to test new cancer therapies in the context of postoperative wound healing. The advantage of this technique is that it allows standardization of the amount of wound inflicted and the amount of tumor to remain. This technique is particularly important for preclinical studies in that identifying new treatments for cancers, that it relapses locally an example of which is soft tissue sarcoma.This visual demonstration is critical to demonstrate the inversion technique, which allows us to expose the tumor attached to the skin side. Begin this procedure with the subcutaneous inoculation of prepared cancer cells in mice, as described in the text protocol. On day 12, post inoculation, when tumors have reached a size of approximately 50 square millimeters, dose mice with 100 microliters of buprenorphine subcutaneously in the scruff of the neck, 30 minutes prior to surgery.Set up the surgical area with the heat pad covered with bench coat and set up a nose cone for anesthesia. Arrange clean surgical equipment so that they are within easy reach, including chlorhexadine, swab, gauze, eye gel, two curved forceps, scissors, clip applicator, clip remover, and clip refills. Warm the heating chamber to 37 degrees Celsius and set up another heat pad for recovery.Note that this procedure requires two people. An assistant must monitor the breathing of the mouse closely during the procedure. After anesthetizing the mouse as described in the text protocol, perform a pinch test and corneal reflex test to ensure that the mouse is fully anesthetized before commencing surgery.Cover the mouse's eyes with a small amount of ophthalmic gel to avoid eye dryness. Begin this surgery by swabbing the surgical area with chlorhexadine. Using forceps and a pair of scissors make a one centimeter straight incision along the dorsal side, three millimeters away from the tumor.Do not cut the skin directly covering the tumor as it will not heal well. Avoid using a scalpel to cut the tumor and use tweezers to scoop the pieces. Using tweezers, pull away the fascia and subcutaneous fatty tissue between the tumor and peritoneum.The subcutaneous tumor is normally attached to the skin side. Open the wound by gently holding the skin on the tumor bearing side using tweezers and invert the tumor so that it is visible outside. Using a pair of scissors, cut away the tumor capsule from the half to remove, starting from the base of the tumor closest to the opening.For 50%debulk surgery, cut across the middle of the tumor. Using curved forceps scoop up the section of the tumor to be removed. Scoop up any remnants from the debulk area.For 75%debulk, start by performing a 50%tumor debulk then cut in half the remaining 50%of tumor. Scoop up 25%of the tumor using curved forceps. To close the surgical site, place the remaining tumor back underneath the skin and using forceps, pull the skin flaps together and line up the skin along the wound.Hold the skin together five millimeters from the edge of the wound and use surgical clips to close the wound starting on the side, closest to the forceps. Apply as many clips as needed to ensure no underlying tissue is exposed. Generally, three to four clips are applied with two millimeter gaps between clips.Allow the mice to recover by putting them into the warm heating chamber. Place the mouse's cage on the heat pad. Monitor the mice in the heating chamber until they have recovered from the anesthetic.Then, put the mice back into the cage, leave the cage on the heat pad for a further 10 minutes until the mice have become more active. Give the mice wet and soft food. Monitor the mice one hour after surgery for recovery.Monitor the mice again at the end of the day and the following morning and ensure clips remain in place. Treat mice perioperatively with advent or neoadjuvant therapy at any given time, depending on the treatment of interest. For example, treat mice with one dose of 100 micrograms of anti-CTLA-4 intraperitoneally on day 15 after inoculation.Alternatively, treat the mice with three doses of 200 micrograms of anti-PD-1 intraperitoneally on days, 15, 17, and 19 after inoculation. The incomplete surgical resection of 50 square millimeter tumors, indicated here by the dotted line results in 100%reproducible regrowth of the tumors in the absence of adjuvant immunotherapy. Therefore, tumor growth to a size of 50 square millimeters is an ideal size for partial debulk.The model was then used to test adjuvant immunotherapies using antibodies against checkpoint modules, cytotoxic T lymphocyte associated protein four and programmed death receptor one. Treatment of mice with anti-CTLA-4, resulted in a cure rate of 80%Whereas treatment of mice with anti-PD-1 resulted in a cure rate of 25%The response with anti-PD-1 provides an opportunity to test it novel combinations, to further improve the response rate. It is important that the remaining tumor stays attached to the skin, otherwise the blood supply will be cut off and the tumor will become the necrotic.Following this procedure, tumors can be analyzed by a variety of methods, such as flow cytometry in order to assess how surgery affects tumor infiltrating cells. This mouse model has allowed us to explore how wound healing impacts cancer immunotherapies.

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Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...