Induction of Solid Mammary, Subcutaneous and Lung Cancers in Rats and Mice. To investigate rodent models of cancer

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1 THE UNIVERSITY OF QUEENSLAND Page 1 of 6 SOP No: AHP 30 DATE ISSUED: 29/09/2017 SUBJECT: REASON FOR USE: Induction of Solid Mammary, Subcutaneous and Lung Cancers in Rats and Mice To investigate rodent models of cancer This procedure may only be performed by operators skilled in the technique. Isolation of cells during cell cture and injection of cells into animals must be performed under aseptic conditions Monitoring of animals for tumour growth and wellbeing must be appropriate for the specific cell line and animal model used and where possible, animals shod be monitored for cancer metastases (cancers in secondary sites other than those intended to develop primary tumours). Injection of mammary cells into the fourth mammary fat pad represents an othotopic breast cancer mode, where breast cancer cells (from the same species/strain or humans) are injected into the same tissue from where they originate. Injection of cells via a lateral tail vein leads to selective lodgement of cells in the lungs as a rest of the narrow capillaries located within this organ. This represents a good model of secondary lung cancer metastases (where tumours located in one area of the body can metastasise via the blood to the lungs and form secondary tumours). Monitoring of lung tumour growth can only be done by using bioluminescent or fluorescent cancer cells and by imaging the animal over time (to track increases in bioluminescence/fluorescence signal). In general, 25G needles are ideal for minimising damage of the cells during syringe filling and injection. However, for small sized cancer cells, needle gauges down to 29G may be used if care is taken to ensure that cells do not clump together and the cells are not undy agitated while filling syringes and injecting the solutions. PRECAUTIONS: Gloves sprayed with a 10% chlorhexidine in 70% ethanol solution, hair net, long-sleeved gown,

2 THE UNIVERSITY OF QUEENSLAND Page 2 of 6 closed in shoes. All instruments and materials must be sterilised before use in 70% ethanol Cells must be isolated under sterile cell cture conditions EQUIPMENT: Biosafety cabinet Small animal clippers (optional) 1 ml syringe 25 to 29G needles Sterile gauze aseptic solution such as 10% chlorhexidine in 70% ethanol, 70% ethanol, 70% isopropanol Forceps (optional) Hot pad (37 deg C) Isoflurane Sterile container to hold final cell suspension, such as an Eppendorf tube or cryovial. 0.5 ml syringe permanently fitted with a 29G needle containing a ring of silicon or polyvinyl canna 0.5 cm from the tip of the needle. Because attachment of the silicon or polyvinyl compromises sterility of the needle, the needle must be soaked in 70% ethanol for 1 h and dried in a biosafety cabinet before being UV irradiated for 20 mins and resheathed. PROCEDURE: Isolate Cells 1) Isolate cells under sterile cell cture conditions (in a biosafety cabinet) according to recommended subcture procedures for the specific cell line to be used. 2) Prepare cell suspensions to the required density of cells in solution according to the number of cells required to be injected and according to table 1 in sterile isotonic buffer (Hank s balanced salt solution (), phosphate buffered saline () or other) or isotonic buffer mixed with Matrigel. 3) Keep the cell suspensions on ice at all times until injected into the animal. This will maintain good cell viability for approx. 1h. Ideally, Ca+2/Mg+2 free buffer shod be used to minimise cell clumping that can prevent the reproducible

3 THE UNIVERSITY OF QUEENSLAND Page 3 of 6 injection of a defined number of cells in all rodents. 4) Invert the cell suspension periodically (approx. every few mins) to redisperse the cells that have pelleted to the bottom of the sterile container/vial. Table 1: Recommended details about cell injection numbers, injection vehicles, volumes and needle gauges for the induction of tumours from isolated cells in rodents. Species Rat Mouse Injection site Flank (SC) Fourth mammary fat pad Flank (SC) Fourth mammary fat pad Left lung lobe # cells to be injected Up to 6 Up to 3 Up to 5 Up to 1 Up to 3 Recommended injection vehicle 1:1 matrigel/buffer Injection volume Needle gauge G Up to 50 Up to 50 Up to 30 Up to G 27G 29G 29G Injection of Cells into Flank 1) Anaesthetise animals under isoflurane (5% induction, 2-3% maintenance). 2) If necessary, shave the area surrounding the injection site with small animal clippers (this is not necessary for nude animals). 3) Place animals on a 37 deg C heated pad (lying on the left side) and clean the injection site with a suitable aseptic solution and sterile gauze 4) Draw the cell solution into the syringe slowly to avoid damaging cells. Gently tap out any air bubbles, taking care not to damage the cells. 5) Lift a flap of skin just over the right flank with sterile forceps or between the forefinger and thumb and insert the needle under the skin horizontal to the animal.

4 THE UNIVERSITY OF QUEENSLAND Page 4 of 6 6) Release grip on the skin and slowly inject the cell solution. Leave the needle in place for a few seconds and then gently remove it, taking care to prevent cells from flushing out of the small hole left by the needle (this can be prevented by the use of matrigel which forms a solid lump at 37 deg C). 7) Remove the animal from isoflurane and place it on its left side in the cage. Monitor the animal until fl consciousness is regained. Injection of cells into the mammary fat pad 1) Anaesthetise animals under isoflurane (5% induction, 2-3% maintenance). 2) If necessary, shave the area surrounding the injection site with small animal clippers (this is not necessary for nude animals). 3) Place animals on a 37 deg C heated pad (lying on their backs) and clean area around the 4 th mammary fat pad with a suitable aseptic solution and sterile gauze 4) Draw the cell solution into the syringe slowly to avoid damaging cells. Gently tap out any air bubbles, taking care not to damage the cells. 5) Gently lift the nipple of the 4 th left mammary fat pad with sterile forceps and insert the needle under the skin horizontal to the animal so that the bevel lies directly under the nipple. 6) Release grip on the skin and slowly inject the cell solution. Leave the needle in place for a few seconds and then gently remove it, taking care to prevent cells from flushing out of the small hole left by the needle (this can be prevented by the use of matrigel which forms a solid lump at 37 deg C, but is not required when injecting cells directly into the mammary fat pad since this site intrinsically supports the growth of mammary cancer cells). 7) Remove the animal from isoflurane and place it on its side in the cage. Monitor the animal until fl consciousness is regained. Injection of cells into the left lung lobe of mice 1) Anaesthetise mouse under isoflurane (5% induction, 2-3% maintenance).

5 THE UNIVERSITY OF QUEENSLAND Page 5 of 6 2) Place mouse on a 37 deg C heated pad (lying on the right side with the head facing the left side of the investigator) and clean the injection site with a suitable aseptic solution and sterile gauze. 3) Draw the cell solution into the syringe slowly to avoid damaging cells. Gently tap out any air bubbles, taking care not to damage the cells. 4) Identify the injection site which is located at the lateral dorsal axillary line, approx cm above the lower rib line and just below the inferior border of the scapa. 5) After the mouse exhales, insert the needle into the lung at a right angle to the rib cage and up to the silicon/polyvinyl mark. This prevents the needle from being pushed too far in. 6) Wait for the mouse to take another breath and at the conclusion of the breath, push in the entire contents of the syringe. Wait for another breath and then remove the needle. 7) While the mouse is still under isoflurane, monitor its breathing and nose for the next 1 min. If breathing does not appear normal or the nose starts to bleed, immediately euthanise the animal according to SOP AHT39. 8) Remove the animal from isoflurane and place it on its right in the cage. Monitor the animal until fl consciousness is regained. 9) The growth of lung metastases can only be monitored via in vivo bioluminescence/fluorescence measurement while the animal is under isoflurane anaesthesia. Monitoring of in vivo bioluminescence involves the tail vein injection of 150 mg/kg D-luciferin (not toxic and no adverse effects at this dose) via a lateral tail vein immediately prior to imaging. SOPs AHT28c (Foot transportation of laboratory rats and mice) and AHP56 (Fluorescence/bioluminescence imaging of live rodents) shod be followed to track lung tumour growth associated with this protocol. RECOMMENDATIONS:

6 THE UNIVERSITY OF QUEENSLAND STANDARD OPERA TING PROCEDURES Page 6 of6 CHAIR OF AEC REFERENCES 1. NHMRC. Australian code of practice or the care and use of animals for scientific purposes. 2013, Canberra. Section A Onn et al (2003) Development of an orthotopic model to study the biology and therapy of primary human lung cancer in nude mice. Clin Cancer Res 9: 5532.