Data acquisition

Measure Tumor Volume

1. Tumor volume（mm³）= 0.5 × Long diameter × short diameter ²
2. Tumor volume （mm³）= π/6 × Long diameter × short diameter × height

Animal imaging

Lumina LT Inst，Series III

Subcutaneous PDX tumor engraftment

Operation Steps

1. Select suitable tumor tissue for inoculation and trim it into 2*2*2 mm3-sized nodules.
2. Sterilization of the inoculation site.
3. Load the trimmed nodules into the inoculation needle.
4. Pierce the mouse skin with the inoculation needle and implant the nodules at the predetermined location.
5. Rotate the inoculation needle, with the tip facing downwards, and complete the inoculation procedure.

Number of Inoculations

1. For a drug efficacy experiment, one inoculation site is sufficient.
2. For model maintenance, four inoculation sites are suggested.

Intraductal inoculation of breast

Operation Steps

1. Preoperative local hair removal.
2. After anesthesia of the mouse, place it in a supine position and disinfect the surgical area of the skin.
3. Sterilization of the inoculation site.
4. Make an incision of about 0.5 cm beside the fourth mammary gland. Gently use sterile ophthalmic forceps to lift one side of the mammary gland and slowly inject the cells into the mammary gland. Pause briefly and withdraw the needle.
5. Suture and disinfect the wound, then place the postoperative mouse on a heating pad and return it to the cage after it has fully recovered from anesthesia.

Evaluation method
Palpation and tumor volume measurement.

Liver in situ inoculation

Operation Steps

1. Preoperative local hair removal.
2. After anesthesia of the mouse, place it in a supine position and disinfect the surgical area of the skin.
3. Sterilization of the surgical site.
4. Cut the skin and peritoneum of the liver area laterally, expose the liver, stab the syringe needle into the left lobe of the liver at a small angle, pay attention to avoid stabbing the lobe of the liver, slowly push the cell suspension, pause after the injection, slowly pull out the needle, press with a cotton swab to stop bleeding for a moment, and then put the lobe of the liver back into the mouse.

Evaluation method
Tumor imaging , CT , and Survival period of mouse.
Intrasplenic inoculation

Operation Steps

1. Preoperative local hair removal.
2. After anesthesia, the mice were placed in the right lateral decubitus position, and the skin of the surgical area was disinfected.
3. Sterilization of the surgical site.
4. Cut the skin and abdominal wall of the spleen area laterally, expose part of the spleen, insert the syringe needle into the tail of the spleen, slowly push in the cell suspension, pause after the injection, slowly pull out the needle, press with a cotton swab to stop bleeding for a while, and then put the spleen back into the mouse.
5. The wound was sutured and disinfected, and the postoperative mice were placed on a hot table and returned to the cage after awakening.

Evaluation method
Tumor imaging , CT , and Survival period of mouse.
Intra-colonic inoculation

Operation Steps

1. Preoperative local hair removal.
2. After anesthesia of the mouse, place it in a supine position and disinfect the surgical area of the skin.
3. Sterilization of the surgical site.
4. Cut the skin and peritoneum of the colon area laterally, expose the colon, stab the injection needle into the membrane of the intestinal wall, slowly push the cell suspension, pause after the injection, slowly pull out the needle, press the colon with a cotton swab for a moment, and then put the colon back into the mouse.
5. The wound was sutured and disinfected, and the postoperative mice were placed on a hot table and returned to the cage after awakening.

Evaluation method
Tumor imaging , CT , and Survival period of mouse.
In situ pancreatic transplantation

Operation Steps

1. Preoperative local hair removal.
2. After anesthesia, the mice were placed in the right lateral decubitus position, and the skin of the surgical area was disinfected.
3. Sterilization of the surgical site.
4. Cut the skin and abdominal wall of the spleen area laterally, separate the pancreas under the spleen, stab the syringe needle into the pancreatic tissue in parallel, be careful not to puncture, slowly push in the cell suspension, pause after the injection, slowly pull out the needle, press the pancreas and spleen with a cotton swab for a moment, and then put them back into the mouse.
5. The wound was sutured and disinfected, and the postoperative mice were placed on a hot table and returned to the cage after awakening.

Evaluation method
     Tumor imaging , CT , and Survival period of mouse.
Gastric gavage administration

Technical Description

Drugs are directly sent into the stomach through the mouth and esophagus, and enter the systemic circulation through the digestion process, and then the blood.
Operation Steps

1. Keep the mice well and try to keep upright.
2. The needle was inserted into the mouth of the mouse through the corner of the mouth, reached the posterior pharynx along the upper jaw, gently touched its upper jaw, and the gavage needle was slowly inserted into the esophagus along the upper jaw, with a slight sense of resistance when passing through the diaphragm of the esophagus.
3. If the mouse struggles, or has abnormal breathing, or encounters resistance, suspend the administration, pull out the gavage needle and reinsert it. If the animal has no abnormal breathing, it can slowly inject the drug solution.
4. At the end of the administration, the animals were put back into the cage after observing that there was no abnormality.

Advantages: Accurate dosing, commonly used, relatively safe, and convenient method of administration.
Disadvantages: Uneven absorption. Certain drugs may not be absorbed due to their physical properties.
Maximum dose: 0.2 mL / 10g
Subcutaneous Injection

Technical Description

The method of injecting drug solution into subcutaneous connective tissue and absorbing it into blood circulation through connective tissue, capillaries and lymphatic vessels. It is suitable for easily soluble and non irritating drugs as well as vaccines, vaccines, cells, etc.
Operation Steps
The left index finger and thumb grasped the skin of the back neck and back of the mouse.
Hold a 1mL syringe in the right hand, and the needle is parallel to the triangular area under the left thumb and index finger. Feel the needle passing through the left thumb and index finger, hold the needle, and inject. The thumb and index finger can feel full when injecting.
After the injection, pinch the needle position with your fingers and exit the needle.
Advantages: The absorption rate is uniform and slow, and the effect is long-lasting.
Disadvantages: It is only applicable to drugs that are not irritating to tissues, otherwise it may cause severe pain and tissue necrosis.
Maximum dose: 1.5 mL
Intraperitoneal injection

Technical Description

The method is suitable for water-soluble drugs with low irritation.
Operation Steps

1. Fix the mouse so that the abdomen of the mouse is upward;
2. Adjust the posture and slightly lower the animal head.
3. Sterilization of the inoculation site.
4. The needle was inserted in the right or left position of the mouse abdominal midline. The needle was inserted into the skin at an angle of 45 ° with the abdominal skin. Pay attention to the depth of the needle to avoid injury to organs.
5. Slowly inject the liquid medicine, and then exit the needle after the injection.

Advantages: Accurate dosage, large absorption area and high bioavailability.
Maximum dose: 1.0 mL
Tail vein injection

Technical Description

It is a common way of administration, suitable for water-soluble drugs with little irritation.
Operation Steps

1. The mice were fixed in the fixator.
2. Wipe the tail blood vessels with alcohol cotton ball to dilate the blood vessels and soften the surface horniness.
3. Hold the tail with the thumb and index finger of the left hand, and hold the syringe with the right hand, so that the needle position is parallel to the vein, and the needle tip section is upward.
4. From the plane position of the index finger, the needle tip gently presses the vein, and the needle is inserted horizontally. After insertion, slowly inject a small amount of liquid medicine. If there is no resistance, continue to inject.
5. After the injection, press the sterile dry cotton ball into the needle hole and take out the needle.

Advantages: The drug solution directly enters the blood without digestion process, and the efficacy is rapid and reliable.
Disadvantages: High concentrations of drugs rapidly reach the plasma and tissues, increasing the risk of adverse reactions. The method is not suitable for oil solutions or insoluble substances.
Maximum dose: 0.5 mL
Intramuscular injection

Operation Steps

1. Fixed mice, and then expose the hind limbs of mice.
2. Sterilization of the surgical site.
3. Clench the hind paw of the mouse and adjust the needle entry position.
4. With the right hand holding the syringe, the needle was injected from the distal end of the tibialis anterior muscle to start the injection.

Advantages: This method is suitable for oil solutions and irritants. 
Maximum dose: 0.05 mL
Intratumoral injection

Operation Steps

1. Restrain the mouse with the tumor to be treated using the left hand, and expose the side of the tumor that needs to be injected.
2. Wipe the injection site with an alcohol cotton ball to disinfect it. 
3. Hold the 1mL insulin syringe with the right hand and draw up the experimental drug. Insert the needle with the bevel up. 
4. Select the injection site according to the size of the tumor. 
5. After the administration is completed, rotate the syringe 180° degrees so that the section is facing down, and remove the needle. 

1. Single point injection: The syringe enters the tumor along its long diameter through a single injection port, passing through the subcutaneous tissue and reaches the middle of the tumor tissue for injection.
2. Two point injection: When the tumor volume ranges between 80 mm3 and 140 mm3, the syringe passes through a single injection port in the subcutaneous tissue, and the injection points are at 1/3 and 2/3 of the tumor’s long diameter direction respectively.
3. Three point injection: When the tumor volume exceeds 140 mm3, the syringe passes through a single injection port in the subcutaneous tissue. The injection points are at 1/3 and 2/3 of the tumor’s long diameter direction respectively. After inserting the needle, the needle tip is kept subcutaneous. When the second needle is used, it goes directly into the tumor tissue, with the injection point located in the middle and outer part of the tumor tissue.
4. Maximum dose : 0.05 mL

Nasal administration

Picture Source ：https://iacuc.ucsf.edu/sites

Operation Steps
After using anesthetics to deeply anesthetize mice, place them supine in the left heart.
Use a pipette with a gun head to suck the liquid medicine.
Perform nasal drip operation as the animal breathes, maintaining consistent droplet size and uniform velocity, alternating between bilateral nostrils.
Protect the mice and prevent the medication from entering the oral cavity.
After completion, the mice were placed back to observe their breathing status until they woke up.
Advantages: The nasal mucosa is rich in blood vessels, and the effect of intranasal administration is similar to intravenous injection.
Disadvantages: The method is easy to cause suffocation. Sneezing can cause inaccurate dosage.

Intrapulmonary nebulization administration

Picture Source：https://cn.bing.com/images

Operation Steps

1. After anesthesia, place the mouse in a supine position with the head up at nearly a 90-degree angle and hang it on the surgical board. Use a laryngoscope or other suitable tool to open the mouse's mouth, press the base of the tongue to locate the trachea.
2. Insert the needle into the trachea at a suitable depth.
3. Immediately and forcefully administer the injection.
4. Remove the needle and complete the administration.
5. After a single administration, wipe the needle with alcohol or a disinfectant, dry it, and then proceed with the next injection.

Advantages: Quantitative aerosol is administered to the lungs of mice, with good absorption effect and convenient operation.
Disadvantages: There are surgical risks, including bleeding, incision cracking, etc. which can easily be injected into surrounding tissues or esophagus.
Blood sample collection requirements

Note: The collected blood samples are directly handed over to the in vitro group for subsequent processing. If the samples are not tested within 2 hours, they are stored at -20 °C. The collected blood volume changes with the number of detection indicators.

Mouse circulating blood volume : The amount of blood entering the circulation through the cardiovascular system in unit time. And does not include the amount of blood in the liver, spleen and lymph or stagnated in capillaries. The circulating blood volume of mice was 5.5-7.0% of the animal weight (55-70 mL/kg).
Mouse circulating blood volume : 72 mL/kg

Blood collection from Retro orbital venous plexus

Operation Steps

1. Grasp the mouse from the back, and at the same time, gently press the mouse’s neck skin with the thumb and index finger (to prevent the animal from suffocating), so that the mouse’s face skin on the side of blood collection is tightened, making the eyeball protrude.
2. Hold a hard capillary glass tube (inner diameter 0.5-1.0 mm) in the right hand, stab it into the bottom of the orbit along the corner of the eye, gently twist the capillary tube to stab it into the orbital venous plexus, and blood outflow can be seen.
3. After collecting the required blood volume, remove the pressure on the neck, pull out the capillary tube, and press the eyeball with sterile dry cotton ball to stop bleeding.

Advantages：Simple, minimal wound and fast healing. High success rate and low mortality.
Disadvantages： Requires anesthesia and cannot avoid the mixing of tissue fluid.

Mandibular blood collection

Operation Steps

1. Hold the mouse and expose the head on the side where blood collection is required. 
2. At the position shown in the right figure, find the location of the submandibular venous plexus blood vessels, quickly puncture the blood sampling needle into the submandibular venous plexus blood vessels, and blood outflow can be seen.
3. After collecting the required amount of blood, use sterile dry cotton ball to compress and stop bleeding.

Advantages: Rapid and efficient blood collection with alternating sites, resulting in a higher volume of blood samples obtained. 
Cardiac blood collection

Operation Steps

1. Mice were anesthetized before blood collection.
2. The left hand gently pinched the abdominal skin of the mouse, as the focus, the right hand held a 1 mL syringe, the needle tip inclined upward, and the abdomen was about 10-20 ° angle, from the junction of xiphoid process and left costal arch into the syringe, at this time, blood can be seen to flow into the syringe.
3. After seeing the blood, keep the syringe still, pull the syringe backward at a constant speed, keep the distance between the needle core and the blood plane about 0.1 mL, that is, there is a certain negative pressure, until the suction is completed, pull out the syringe.

Advantages： Efficient blood collection, Minimizes risk of clotting, High quantity of blood obtained.
Saphenous vein blood collection

Operation Steps

1. Depilation of the medial hindlimb of mice before blood collection.
2. Grasp the hind limb of the mouse on the side of blood collection.
3. Puncture the saphenous vein vertically with a needle, wait for the blood to flow out in a ball, and collect the blood with a capillary tube.
4. After blood collection, gently press the wound to stop bleeding

Advantages: This method does not need to anesthetize mice, and blood sampling can be repeated.
Heart perfusion 

Operation Steps

1. The mice were fixed supine, spray with 75% medical alcohol on the chest and abdomen. And then open the chest, and completely exposed the heart of mice .
2. Connect normal saline and formalin to the infusion set through a three-way valve, and mark the corresponding connection to prevent wrong liquid infusion. Connect a 10 stainless steel needle at the outlet to confirm that the liquid flows out.
3. The needle was inserted into the left ventricle of the animal from the apex of the heart (as shown in Figure D). Open the valve and cut open the animal's right atrial appendage. Adjust the flow rate of normal saline to about two to three drops per second, first fast and then slow, and inject 50-100mL of normal saline.
4. Quality control point: the liver and limbs of animals turn white, and perfusion is completed.
5. Internal fixation: rotate the three-way valve so that formalin liquid can be injected into the mouse through the infusion device, and the mouse limbs and tail are stiff, that is, the fixation is successful.

Liver perfusion

Picture source：Aparicio-Vergara M,. Methods Mol Biol. 2017;1639:161-171. doi: 10.1007/978-1-4939-7163-3_16

Operation Steps

1. After the mice were deeply anesthetized, they were placed on the operation board.
2. The abdominal cavity was cut U-shaped, and the abdominal visceral organs were moved to the left side of the body, exposing the bile duct and hepatic portal vein.
3. The bile duct was separated, inserted and fixed.
4. The portal vein was isolated, the liver and spleen branches were ligated, the surgical silk thread was threaded between the proximal end of the portal vein and the pyloric vein branch, and the flexible sleeve was punched. The portal vein was cannulated, and perfusion was started after rapid fixation.
5. Cut the abdominal segment of the inferior vena cava, quickly open the chest, cut the thoracic segment of the inferior vena cava on the side close to the diaphragm, and flush the residual blood in the liver.
6. The liver was isolated intact and transferred to the organ tray of the perfusion instrument or placed in situ.
7. The residual blood in the liver was rinsed with PBS.