Thiomyristoyl – Selective JAK3 inhibitor

Assessment of SIRT2 Inhibitors in Mouse Models of Cancer

Yashira L. Negrón Abril, Irma Fernández, and Robert S. Weiss

Abstract
New therapeutics directed against established and novel molecular targets are urgently needed to intervene against cancer. Recently, it was reported that several members of the sirtuin family (SIRT1-7), the mam- malian orthologs of the silent information regulator 2 (Sir2) protein in Saccharomyces cerevisiae, play important roles in carcinogenesis. Although SIRT2 has been attributed both tumor-promoting and tumor- suppressing activities in different contexts, selective SIRT2 inhibition with a small molecule mechanism- based inhibitor known as Thiomyristoyl lysine (TM) repressed the growth of breast cancer cell lines. In light of the anticancer effect of SIRT2 inhibition in cell culture, it was critical to assess the efficacy of TM as a potential anticancer therapy in vivo. This was accomplished by testing the SIRT2 inhibitor in genetically engineered and xenotransplantation mouse models of breast cancer, using the procedures detailed in this chapter.

1 Introduction
Over 14 million people worldwide are diagnosed with cancer each year and cancer is the second leading cause of death [1]. Currently available chemotherapies often have damaging side effects and recurrence is common. Therefore, identifying new drug targets in cancers is motivated by the need for therapeutics with improved efficacy and greater selectivity. Previous research has implicated posttranslational modifications (PTMs), such as protein acetyla- tion, in cancer initiation and progression [2, 3]. Among the pro- teins known to regulate cancer-relevant PTMs is the Sirtuin family of NAD+-dependent lysine deacylases [4]. There are seven sirtuins, SIRT1-7, and they are known to regulate a myriad of cellular pro- cesses such as longevity, DNA repair, metabolism, and proliferation [5, 6]. Thus, sirtuins have emerged as important therapeutic tar- gets [7]. Some sirtuins have tumor suppressor activity whereas others act as tumor promoters [4]. In some cases, both activities have been assigned to the same sirtuins in different contexts.
Thus, potent and selective sirtuin inhibitors are necessary for therapeutic applications.
SIRT2 is primarily cytosolic and has been reported to have robust deacetylase activity [8, 9]. Among its cytosolic targets are tubulin and several metabolic enzymes LDH-A (lactate dehydro- genase A), PEPCK1 (phosphoenolpyruvate carboxykinase 1), ACLY (ATP-citrate lyase), and G6PD (glucose-6-phosphate dehy- drogenase) [10]. It was later found that SIRT2 can translocate to the nucleus and has important epigenetic roles. Its nuclear targets include histones, as well as transcription factors and coactivators such as p300, FOXO1, FOXO3, HIF-1?, NF-?B, and PGC-1 [11, 12]. Interestingly, the effect of SIRT2 on cancer progression can be either positive or negative, depending on the cancer context and possibly on the mode of SIRT2 inhibition (genetic vs. pharma- cological) [13-18]. Previous studies have shown that SIRT2 inhib- itors can reduce proliferation in cancer cell lines [19-28]. Unfortunately, most SIRT2 inhibitors are either not very potent (having high IC50 values) or not very selective (acting against multiple sirtuins), limiting their utility as anticancer agents. To examine the potential of SIRT2 as a therapeutic target, Lin and colleagues at Cornell University set out to develop SIRT2 inhibi- tors with improved potency and selectivity. Previous studies sug- gested that sirtuins have different acyl group specificity [29-32]. The Lin lab utilized this knowledge to synthesize several mechanism-based candidate compounds with differing acyl groups. These compounds are thioacyl lysine peptides that can react with NAD in the sirtuin active site, forming a stable intermediate that can inhibit sirtuin activity [33, 34]. This strategy yielded a SIRT2- selective mechanism-based inhibitor, Thiomyristoyl lysine (TM), that was also very potent with an IC50 value of 0.028 ?M [17].
The creation of TM provided an opportunity to investigate the anticancer therapeutic potential of selective SIRT2 inhibition in genetically engineered and xenograft mouse models of breast can- cer [17]. First, we used a well-established transgenic mouse mam- mary tumor model (MMTV-PyMT), which is characterized by mammary gland-specific expression of the polyoma middle T anti- gen (PyMT) oncogene under the mouse mammary tumor virus promoter (MMTV) [35]. MMTV-PyMT mice rapidly develop mammary adenocarcinoma that spontaneously metastasizes to the lung [35]. This and other genetically engineered mouse models for cancer have a few key advantages. First, tumorigenesis is initiated by genetic events, recreating the stepwise transformation of cells of origin as in human cancers. Second, these cancers arise in the equivalent tissue as in the human disease, replicating the natural tumor microenvironment. Lastly, creation of MMTV-PyMT transgenic mice can be accomplished in an immunocompetent background, allowing for the study of tumor-immune system interactions [36].
A complementary approach, the direct implantation of human cancer cell lines in immunocompromised mice, also can be uti- lized. This approach has the advantage that therapeutics are being tested on human cancer cells, as opposed to murine cells in the transgenic model. Xenograft models are easy to use and usually feature rapid tumor progression, but do so in the absence of a host immune response. The resulting neoplasms are often less hetero- geneous than malignancies in patients due to their derivation from established cell lines, and, in the case of ectopic xenografts, lack the natural tumor microenvironment [36].
We chose to rigorously test the anticancer activity of the SIRT2 inhibitor TM using both genetically engineered and xenograft mouse models and determined that SIRT2 inhibition with TM reduces tumor burden in both models [17]. The following narra- tive addresses experimental design considerations, treatment strat- egies and technical execution of treatment, tissue collection and endpoints, and potential follow-up studies.

2 Materials
2.1 Animals
MMTV-PyMT transgenic male mice on a pure FVB/N back- ground (FVB/N-Tg(MMTV-PyVT)634Mul/J) were obtained from the Jackson Laboratory [35]. NCr nude mice (CrTac:NCr- Foxn1nu) were purchased from Taconic Biosciences. All mice were maintained in compliance with the Guide for the Care and Use of Laboratory Animals and all procedures were in compliance with protocols approved by the Cornell University Institutional Animal Care and Use Committee [37].

2.2 Genotyping of MMTV-PyMT mice
1. Polymerase Chain Reaction (PCR) machine.
2. 10× Tris/Borate/EDTA (TBE) buffer: Dissolve 108 g Tris and 55 g Boric acid in 900 mL distilled water (ddH2O). Add 40 mL 0.5 M Na2EDTA (pH 8.0). Adjust volume to 1 L and store at room temperature.
3. 1× TBE buffer: Mix 100 mL of the 10× TBE buffer with 900 mL of ddH2O.
4. 0.8% Agarose solution: Add 100 mL of 1× TBE buffer and 0.8 g of agarose into a 250 mL flask. Swirl the agarose in the liquid and microwave for 1 min. Swirl the flask for several seconds and microwave the solution for an additional 1-2 min until the agarose is completely dissolved. Add ethidium bromide to a final concentration of 0.5 ?g/mL and swirl the flask.

2.3 Subcutaneous Injection of Human Breast Cancer Cells in Xenograft Mice
1. MDA-MB-231 cells can be purchased from American Type Culture Collection or other vendors.
2. Cell culture media: Mix 500 mL Dulbecco’s modified Eagle’s medium (DMEM) with 10% of bovine calf serum (57.5 mL), 1% nonessential amino acids (5.7 mL), 1% L-glutamate (5.7 mL), and 1% penicillin and streptomycin (5.7 mL).
3. 1× Cell Culture Phosphate Buffered Saline (PBS) buffer: Mix 100 mL of 10× sterile cell culture-grade PBS with 900 mL of ddH2O. Autoclave the solution for 25-30 min.
4. Trypsin-EDTA (0.05% Trypsin, 0.53 mM EDTA, 1×).
5. 150 mm tissue culture dishes.
6. 50 mL sterile conical tubes.
7. Centrifuge.
8. Hemocytometer.
9. Isoflurane, USP (Piramal Critical Care).
10. Matrigel Matrix: To avoid multiple freeze-thaws, prepare ali- quots of 200-400 ?L into prechilled 1.5 mL autoclaved Eppendorf tubes. Store at ?20 °C.
11. Single channel manual pipette (P-200).
12. Barrier filter pipette tips (200 ?L).
13. Disposable syringes (1 mL).
14. 18-gauge needles.
15. Ethanol.
16. Hand warmers.
17. Caliper.
18. Digital scale.

2.4 Preparation of TM Solution and Treatment of Mice
1. Thiomyristoyl lysine (TM) compound was provided by Dr. Hening Lin (Department of Chemistry and Chemical Biology, Cornell University). TM in its solid state was stored in ?20 °C freezer.
2. Tissue-culture-grade Dimethyl sulfoxide (DMSO) (see Note 1).
3. Whatman sterile syringe filter (0.2 ?m).
4. Disposable syringes (1 mL).
5. 0.052 M TM solution for intraperitoneal (IP) injection: Transfer 15 mg of TM (581.85 g/mol) powder to a 1.5 mL Eppendorf tube and dissolve it in 500 ?L DMSO. Draw the solution into a disposable 1 mL syringe. Carefully attach the syringe to a Whatman sterile syringe filter (0.2 ?m). Filter the solution into a sterile 1.5 mL Eppendorf tube. Store the TM solution at ?20 °C if not using right away (see Note 2).
6. 0.026 M TM solution for intratumoral (IT) injection: Transfer
7.5 mg of TM powder to a 1.5 mL Eppendorf tube and dissolve it in 500 ?L DMSO. Filter the solution and store it at 20 °C as indicated above.
7. Needles for IP injections (25 gauge, 5/8?) or IT injections (28.5 gauge, 1/2?).
8. Digital scale.

2.5 Necropsy and Tissue Collection
1. 10× PBS buffer, pH 7.4: Dissolve 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4 and 2.4 g KH2PO4 in 800 mL of ddH2O. Adjust the pH to 7.4 and adjust volume to 1 L with additional ddH2O.
2. Sterile 1× PBS buffer: Mix 100 mL of 10× PBS with 900 mL of ddH2O. Sterilize by autoclaving.
3. 4% Paraformaldehyde solution (PFA): Add 90 mL ddH2O to a 250 mL flask. Add 4 g paraformaldehyde and stir while heating using a magnetic stirrer. Add 6 ?L 10 N NaOH (paraformal- dehyde will not dissolve unless the pH is basic). When PFA is completely dissolved, remove the flask from heat and add 10 mL of 10× PBS. Cool to 4 °C for use.
4. Camera (optional).
5. Bucket containing liquid nitrogen.
6. Necropsy stage.
7. 3 scissors (Autoclaved).
8. 3 forceps (Autoclaved).
9. 6-8 Pins (Autoclaved).
10. 1.5 mL Eppendorf tubes (see Note 3).
11. 2.0 mL RNAse-free Eppendorf tubes (see Note 4).
12. Sterile 1× PBS buffer.
13. 60 mm tissue culture dishes.
14. Disposable syringes (1 mL) with needle (25 gauge × 1 in.)-(1 per mouse).
15. Disposable syringes (10 mL) with needle (25 gauge × 1 in.)- (1 per mouse).
16. Centrifuge.
17. Scintillation vials (1 per mouse).
18. Weigh boat.
19. 70% ethanol.
20. Caliper.
21. Digital scale.
22. Mouse necropsy data sheet (Fig. 1).

2.6 Measurement of TM by LC-MS
1. Sterile 1.5 mL Eppendorf safe-lock tubes (round bottom).
2. Pure methanol.
3. Stainless steel beads for tissue homogenization (5 mm).
4. TissueLyser bead homogenizer machine.
5. Microcentrifuge.
6. Sonicator.
7. High Performance Liquid Chromatography (HPLC) with Kinetex 5u EVO C18 100A 30 × 2.1 mm column and Mass Spectrometry (MS) using Thermo LCQ Fleet MS.
8. HPLC-grade water with 0.1% acetic acid: Mix 1 mL of Acetic acid (HPLC-grade) with 999.0 mL ddH2O (HPLC-grade).
9. HPLC-grade acetonitrile with 0.1% acetic acid: Mix 1 mL of Acetic acid (HPLC-grade) with 999.0 mL acetonitrile (HPLC-grade).

3 Methods
3.1 Generation of Experimental Mice
3.1.1 Generation, Maintenance, and Genotyping of Transgenic Mice
1. Breed MMTV-PyMT transgenic males on a pure FVB/N background with wild-type FVB/N female mice to obtain MMTV-PyMT hemizygote experimental females.
2. Before weaning age, apply ear tags in order to identify mice and collect tail biopsies for genotyping.
3. Perform polymerase chain reaction (PCR) amplification using allele-specific primers and genomic DNA extracted from mouse tail snips to genotype the mice.
4. To identify the MMTV-PyMT allele use the following primers and PCR reaction conditions:
(F) 5?-GGAAGCAAGTACTTCACAAGGG-3?.
(R) 5?-GGAAAGTCACTAGGAGAGGG-3?.
PCR reaction conditions are 94 °C (3 min), followed by 30 cycles of 94 °C (30 s), 64 °C (1 min), 72 °C (1 min), and 72 °C (2 min).
5. As a negative control, use DNA from any wild-type (transgene- negative) FVB mouse. As positive control, use DNA from a MMTV-PyMT hemizygote transgenic parent.
6. Conduct gel electrophoresis of PCR products using a 0.8% agarose gel run at 130 V and constant amperage for approxi- mately 30 min.
7. A single band at 556 bp is expected for MMTV-PyMT trans- gene positive pups.
8. Save MMTV-PyMT transgenic females for experimental use.
9. The minimum number of mice needed for an experiment should be estimated statistically using the desired confidence and power and an estimate of the relevant difference between experimental groups or effect size.
3.1.2 Subcutaneous Injection of Human Breast Cancer Cells in Xenograft Mice
1. Grow human cancer cells (MDA-MB-231; American Type Culture Collection) to approximately 80% confluence in a 150 mm dish (see Note 5).
2. Aspirate off cell culture media and rinse once with 1× sterile PBS.
3. Add 2.5 mL of 2× Trypsin-EDTA and let cells to detach for approximately 5 min at 37 °C (see Note 6).
4. Add approximately 10 mL of cell culture media to stop the trypsinization reaction and collect the cells.
5. Transfer the cell suspension into a 50 mL conical tube and pipette up and down to obtain a single cell suspension.
6. Spin down cells at 1500 rpm (378 × g) for 5 min.
7. Aspirate off the media and wash the cell pellet with cold 1× sterile PBS.
8. Spin down and resuspend cells in 10 mL of cold 1× cell culture PBS.
9. Transfer a small aliquot of the cell suspension to an Eppendorf tube and count the cells with a hemocytometer.
10. Spin down the remaining cells in the conical tube at 1500 rpm (378 × g) for 5 min.
11. Dilute cells in cold 1× cell culture PBS to a concentration of 2 × 107 cells/mL (see Note 7).
12. In the mouse room, weigh the mice to be injected and anes- thetize with isoflurane following standard rodent inhalant anesthesia protocols (see Note 8).
13. Draw up 100 ?L of cell suspension with a pipette and wedge the pipette tip firmly into the hub of a disposable 1 mL syringe. Draw the cell suspension into the syringe with the plunger instead of expelling with the pipette. The syringe will draw out all of the cell suspension from the pipette.
14. Repeat step 13 with 100 ?L of matrigel (see Note 9).
15. Add 18-gauge needle to syringe. Invert the assembly and gen- tly tap to bring the cells-matrigel suspension to the end of the syringe (see Note 10).
16. Slowly push the plunger until a drop appears at the end of the needle. Draw the drop just back into the syringe.
17. Clean and sterilize the injection site on the mouse with ethanol.
18. Grab a loose tent of skin at the lower flank of the anesthetized NCr nude mouse and insert the needle, bevel up, into the tent of skin created, just between the tips of the fingers.
19. Insert about 1 cm of the needle and slowly inject the cell sus- pension (2 million MDA MB-231 cells suspended in 100 ?L 1× sterile PBS and 100 ?L matrigel).
20. Continue to pinch the skin around the injection site while drawing out the needle slowly with a small twist to prevent leakage at the injection side.
21. Release the injection site a few moments after withdrawing the needle. Check for any sign of leakage.
22. Repeat steps 13-21 to perform the injection on the other flank of the mouse.
23. Let the mouse recover from anesthesia on hand warmers, and then return the mouse to its cage (see Note 11).
24. Repeat steps 12-23 for each experimental mouse.

3.2 IP Treatment of MMTV PyMT Mice
3.2.1 Establish the Appropriate Starting Point for IP Treatment
To test the efficacy of anticancer therapies in MMTV-PyMT mice, it is important to consider the starting point for treatment. MMTV-PyMT transgenic mice develop palpable mammary tumors as early as 5 weeks of age [35]. However, mammary tumors will arise in MMTV-PyMT females over a range of ages. Depending on the goal of the study, treat- ment can be initiated either before or after the onset of disease pathol- ogy. In the studies described here, the treatment was initiated based on age, with MMTV-PyMT females being treated beginning at 6 weeks of age. Alternatively, treatment of MMTV-PyMT mice can be initiated when the first tumor is palpated or at an established tumor size. The choice of when to initiate treatment will influence the logistics of the experiment and the results obtained (see Note 12).
3.2.2 Monitoring Mice Before Initiating IP Treatment
1. After weaning, weigh MMTV-PyMT females three times per week and randomly distribute mice into each treatment group (see Note 13).
2. At this stage, also palpate and monitor the mammary glands three times per week for the development of mammary tumors (see Note 14).
3. Record the date when the first mammary tumor is palpated and subsequently use a caliper to monitor the size of the tumors.
4. To measure the dimensions of the tumor, gently scruff and restrain the mouse smoothly without hesitation. Use a caliper to measure perpendicular axes of the tumor.
5. Use the following formula to determine the tumor volume [38]:
Tumor volume (mm3 ) = Length (mm) × Width2 (mm2 ) /2
3.2.3 IP Treatment of MMTV-PyMT Mice
1. When the experimental start point is reached, initiate treat- ment. In our experiments, the starting point was when mice reached 6 weeks of age.
2. Before removing the mouse from the cage, bring the solution containing 0.052 M TM to room temperature.
3. Weigh the mouse and calculate the amount of solution needed to inject 50 mg/kg (dose) using the following equation (see Note 15) [39]:
Injection volume (mL) = Animal weight (kg) × Dose (mg / kg) / Concentration mg / mL
where the dose = 50 mg/kg and the concentration = 1.5 mg/0.05 mL.
4. Draw up the amount of solution to be administered into the disposable syringe (1 mL) and needle (25 gauge, 5/8?).
5. Gently remove the mouse from the cage and restrain the mouse smoothly without hesitation.
6. Identify the appropriate area in the abdomen to perform the injection. IP injection is made in the left or right lower quadrant of the abdomen to avoid the liver and the bladder (see Note 16).
7. The needle should be angled (30-40° angle) to penetrate the intraperitoneal cavity.
8. Aspirate the syringe to ensure placement within the intraperi- toneal cavity and not within any organ or vascular structure (see Note 17).
9. Proceed with the injection by depressing the plunger until the solution has been fully administered (see Note 18).
10. Pull out the needle and place the animal back into its cage.
11. Observe the animal for a few minutes for any complications.
12. Repeat the same procedure (steps 3-11) for each experimen- tal mouse. Use a new needle and syringe for each animal.
13. Treat mice daily for 1 month and monitor tumor size and health status (see Note 19).

3.3 Treatment of Mice Bearing Human Breast Cancer Xenografts
3.3.1 Monitoring Xenograft Mice Before Initiating Treatment
1. Monitor tumor size and mouse weight twice a week.
2. To measure the dimensions of the tumors, follow steps 4 and 5 from Subheading 3.2.2.
3.3.2 IP Treatment of Xenograft Mice
1. Once the tumors reach a threshold size of 200 mm3, randomly distribute experimental xenograft mice into each treatment group.
2. Treat the mice with daily IP injections of vehicle alone (DMSO) or 0.052 M TM for 1 month as described in Subheading 3.2.3 (steps 2-13).
3. Monitor tumor size and mouse weight three times per week. Calculate tumor volume using the formula shown above in Subheading 3.2.2.
3.3.3 IT Treatment of Xenograft Mice
1. For IT injections, warm up the solution containing 0.026 M TM to room temperature.
2. Draw up the amount of solution to be administered (0.05 mL) into a disposable syringe (1 mL) and needle (28.5 gauge, 0.5?) (see Note 20).
3. Remove the mouse to be injected from the cage and anesthe- tize it with isoflurane.
4. Slowly insert the needle into the tumor and inject 0.05 mL of TM or vehicle control. Gently remove the needle from the tumor to ensure no leakage.
5. Repeat steps 2-5 in the other tumor.
6. Perform IT injections three times per week for a month (see
Note 21).
7. Monitor tumor size and mouse weight three times per week (see Note 22).
8. Use the formula provided above to calculate the tumor volume (Subheading 3.2.2).

3.4 Endpoint and Tissue Collection
In our studies, we established the following endpoint criteria. Mice were euthanized:
3.4.1 Define Endpoints for MMTV-PyMT and Xenograft Mice
1. After 1 month of treatment, or
2. If the tumor reached 1.5 cm in diameter or was ulcerated, or
3. If mouse met one of the following humane endpoint criteria:
(a) Excessive weight loss (20% reduction in body weight when compared to its weight before starting treatment).
(b) Tumor size or location interferes with normal physiologi- cal requirements (e.g., ability to walk, eat, drink, breathe, or perform other vital functions).
(c) Signs of pain and distress (significantly decreased body condition, ruffled coat, hunched posture).
(d) Animal found unexpectedly to be moribund, cachectic, or unable to obtain food or water.
3.4.2 Preparation Before Performing Necropsy of Treated Mice
Before euthanizing the mouse, assemble the instruments and pre- pare the solutions necessary to perform necropsy and tissue collection.
1. Wrap three sets of scissors and forceps in aluminum foil. Include 6-8 pins with one set.
2. Autoclave the three sets of necropsy utensils for 30 min (see
Note 23).
3. Transfer 2-4 mL of 1× PBS into a 60 mm tissue culture plate (1 per mouse). Load a 10 mL syringe with 1× PBS.
4. If planning to fix tissues, prepare and chill 4% PFA solution. Label a scintillation vial (1 per mouse) with the following information: tag number, treatment. Transfer 3-5 mL of 4% PFA solution into each scintillation vial (see Note 24).
5. If planning to freeze tissue for future experiments (e.g., pro- tein, DNA, or RNA extraction) label RNAse-free Eppendorf tubes with the following information: tag number, treatment, and tissue. Prepare at least one tube per tissue per mouse (liver, kidney, spleen, mammary tumors, fat, and serum).
6. Add liquid nitrogen (N2) into an appropriate container.
7. Assemble all instruments and solutions next to the necropsy stage.
3.4.3 Necropsy of MMTV-PyMT Mice
1. After 1 month of treatment or earlier if the mouse meets humane endpoint criteria, euthanize the mouse by CO2 asphyxiation. Euthanize treated mice 24 h after performing the last injection (see Note 25).
2. Avoid cervical dislocation if possible since it may damage major blood vessels in the neck and result in accumulation of blood in the lungs and mammary glands, rendering the dissection more difficult.
3. Spray the mouse generously with 70% ethanol.
4. Quickly perform cardiac puncture and collect blood using a dis- posable 1 mL syringe with a 25 gauge, 1? needle (see Note 26).
5. Place the needle, bevel up, into the chest and parallel to the spine.
6. Puncture the heart and apply slight back pressure with the syringe (see Note 27).
7. Aspirate the blood and place it into a 1.5 mL Eppendorf tube.
8. Let the blood clot for 10 min. Spin down at 15,000 rpm (21,130 × g) for 5 min at RT.
9. Collect the serum (supernatant) and flash-freeze in liquid nitrogen (see Note 28).
10. While waiting for the blood to clot, secure the body of the mouse with the pins.
11. Using the first set of scissors and forceps, pull up the abdominal skin at the midline and make a small incision.
12. Cut the skin up to the neck of the animal while avoiding puncturing the abdominal or thoracic cavities.
13. Cut the skin on the front legs to the midline incision, resulting in a “Y shape.” Cut the skin on the rear limbs the same way.
14. Gently separate the skin away from the body wall and pin back the skin flaps to expose the mammary glands and tumors (see Note 29).
15. Using the second set of utensils (scissor and forceps), carefully open the abdominal cavity by making a small incision in the midline of the peritoneum with the scissors. Extend the inci- sion to expose the organs.
16. Grasp the xiphoid process with forceps and cut bilaterally just above the diaphragm to expose the thoracic organs.
17. Use the 10 mL syringe containing 1× PBS buffer and slowly insert the needle into the trachea. Inflate the lungs with 1× PBS buffer before removal from the thorax (see Note 30).
18. Remove the lungs together with the heart and thymus with the third set of scissors and forceps.
19. Place them in a 60 mm petri dish containing sterile 1× PBS and separate each lung lobe from the heart and the thymus.
20. Examine the lungs for any visible metastasis (see Note 31).
21. Place the left lobe of the lungs in a RNAse-free tube and snap-freeze (see Note 32).
22. Place the other four lobes in a scintillation vial containing 4% PFA solution (see Note 33).
23. After removal of the lungs, quantify the number of mammary tumors.
24. Using the first set of utensils carefully collect mammary tumors and remove any fat surrounding the tumors.
25. Place mammary tumors in a weigh boat (see Note 34).
26. Weigh each tumor and measure the dimensions using a caliper. Calculate tumor volume using the formula described in Subheading 3.2.2.
27. After recording the weight, quickly place some tumor tissue in the scintillation vial containing 4% PFA solution and freeze other samples in liquid nitrogen for future experiments. Do not leave the tissues at room temperature for a long period of time (see Note 35).
28. Examine the abdominal cavity for any sign of irritation or tox- icity. Remove abdominal organs such as liver, kidney, spleen, intestines, and/or stomach for further analyses. Fat can also be collected (see Note 36).
29. Store frozen tissues in ?80 °C freezer.
30. Dispose of the mouse carcass and any hazardous materials according to Institutional guidelines.
31. Twenty-four hours after fixing tissues in 4% PFA, transfer them to a scintillation vial containing 70% ethanol (see Note 37).
3.4.4 Necropsy of Mice Bearing Human Breast Cancer Xenografts
1. After 1 month of treatment or when the mouse meets humane endpoint criteria, euthanize the mouse by CO2 asphyxiation.
2. Follow steps 3-9 from Subheading 3.4.3.
3. While waiting for the blood to clot, place the mouse on the necropsy stage facing down and secure the body of the mouse with the pins.
4. Using scissor and forceps pull up the skin near to the lower flanks and carefully remove the tumors from the mouse. Place tumors in a weigh boat (see Note 38).
5. Weigh each tumor and measure the dimensions using a caliper. Calculate tumor volume using the formula described in Subheading 3.2.2.
6. Slice the tumors into small pieces.
7. Fix small pieces of each tumor in 4% PFA solution.
8. Place additional pieces of tumor tissue into multiple labeled Eppendorf tubes and snap-freeze the tissues (see Note 39).
9. Follow steps 28-31 from Subheading 3.4.3.

3.5 Measurement of TM Using LC-MS
3.5.1 Extraction of TM from Serum and Tissue Samples
1. (a) To extract TM from serum, pipette 100 ?L of serum into a sterile 1.5 mL Eppendorf safe-lock tube and add 100 ?L of pure methanol.
(b) To extract TM from tissue samples, such as tumor tissue, fat, or any organ, transfer a small piece (<0.5 cm) of tissue into a sterile 1.5 mL Eppendorf safe-lock tube and add 100 ?L of pure methanol. 2. Homogenize for 30 s at an oscillation frequency of 30 s?1 using a TissueLyser machine (see Note 40). 3. Centrifuge the mixture at 14,000 rpm (18,407 × g) for 10 min at 4 °C. 4. Collect the supernatant and sonicate the solution for 5 min. 5. Repeat step 3. 6. Collect the supernatant and inject into LC-MS for analysis. 3.5.2 LC-MS Run and Analysis 1. Use a gradient of HPLC-grade water with 0.1% acetic acid and HPLC-grade acetonitrile with 0.1% acetic acid to run the samples. 2. Load 100 ?L of the supernatant and elute it using the follow- ing gradient of solvents: (a) 100% HPLC-grade water with 0.1% acetic acid for the first 2 min. (b) 20% HPLC-grade acetonitrile with 0.1% acetic acid for the next 2 min. (c) 40% HPLC-grade acetonitrile with 0.1% acetic acid for 15 min. (d) 100% HPLC-grade acetonitrile with 0.1% acetic acid for 10 min. 3. Monitor the elution at two wavelengths (215 and 254 nm). 4. After the run, use Thermo Xcalibur Qual program for further analysis. 5. Use purified TM compound dissolved in methanol as a positive control. Dilute it in a 1:1 mixture of water and acetonitrile to inject into the LC-MS. 4 Notes 1. Prepare an aliquot of the control vehicle by transferring 500 ?L of DMSO to a sterile 1.5 mL Eppendorf tube. Store the DMSO aliquots at ?20 °C. 2. Dosage might vary depending on the efficiency and toxicity of the compound tested [40]. After dissolving TM in DMSO, filter the solution inside a tissue culture hood and transfer the solution to a sterile Eppendorf tube. 3. If interested in freezing down tissues, label tubes with the fol- lowing information: mouse number, tissue, and treatment. Tumor tissues can be collected in multiple vials to avoid mul- tiple freeze-thaw cycles. Store the samples at ?80 °C. 4. If interested in extracting RNA from tissues, label RNAse-free tubes with the following information: mouse number, tissue, and treatment. Transfer a small piece of tissue into the RNAse- free tube and snap-freeze using liquid nitrogen. Store the sam- ples at ?80 °C. 5. Test cells for mycoplasma before injecting them into mice. 6. Trypsinization of cells should involve the most minimal exposure necessary to generate a single cell suspension. 7. The number of cells required depends on the particular tumor cell line and can vary by an order of magnitude. 8. Mice should be 4-6 weeks old. Inhalant anesthesia is recom- mend for ease of titration, rapid recovery, and lack of necessity for use of controlled drugs. If injectable anesthesia is desired, consult institutional veterinary staff. 9. It is recommended to place the matrigel in the refrigerator overnight at 4 °C and keep it on ice while using it to prevent polymerization. Matrigel solidifies rapidly at room tempera- ture. It is also recommended to store disposable 1 mL syringes and a box of pipette tips (200 ?L) in the freezer and keep them on ice the day of the injection to prevent the solidifica- tion of the matrigel when transferring from the pipette to the syringe. 10. Do not draw back. There should be a bubble of air against the plunger. This ensures that the 200 ?L volume is fully injected. 11. Monitor the mouse to avoid thermal damage to the skin. 12. Selection of when to initiate treatment is an important factor in carrying out appropriate in vivo efficacy testing. As discussed above, there are several options for initiating treatment of MMTV-PyMT mice. The timing of treatment initiation will depend on the goals of the study. (a) If a specific age is chosen for the initiation of treatment, the researcher must consider that tumor development in MMTV-PyMT mice varies from animal to animal. At the starting point, some animals might have palpable tumors, while others may not. There is also a possibility that some experimental animals will have bigger tumors than others. It is therefore necessary to randomly sort the mice into experimental groups. (b) If a specific tumor size is chosen for the initiation of treat- ment, the researcher may not be able to start the treatment of the control group at the same time as the experimental group. (c) If treatment is initiated immediately upon the first detec- tion of a palpable tumor mass, the researcher might encounter the same issue as in (b). If treatment initiation is based on tumor detection, it is recommended that one researcher be responsible for palpating the mice through- out the whole experiment for consistency. 13. Mammary tumors in MMTV-PyMT females arise with variable timing in different animals. It is recommended to distribute mice randomly into the two experimental groups to reduce experimental bias. 14. Use the tip of the index finger to palpate the mammary glands. Mammary tumors in MMTV-PyMT mice are first detected as a small solid mass. 15. Example of the calculation: If a mouse weighs 0.020 kg, the dose is 50 mg/kg and the concentration is 1.5 mg/0.05 mL, the injection volume will be: Injection volume (mL) = 0.020 kg × 50 mg / kg = 0.033 mL / 1.5 mg / 0.05 mL 16. Make sure that the bevel of the needle is facing "up" and the numbers on the syringe barrel can be read. It is acceptable to alternate the side of injection between right and left quadrant of the abdomen when performing daily injections for multiple days. Alternating the side of the injection might reduce dis- comfort in treated mice [41]. 17. If any material (e.g., fecal) or fluid (e.g., urine) is aspirated, do not inject the drug. Remove the needle and discard the needle and syringe. Draw up fresh drug with a new needle and syringe, then repeat steps 6-10 [41]. 18. When administering the solution, do not move the needle inside the abdominal cavity. 19. The treatment time will depend on the time required for the tested compound to affect the tumor cells, and whether its effect is reversible or irreversible. This will be highly dependent on the stability and bioavailability of the compound [40]. 20. Administer the same volume when performing IT injections of drug or vehicle solution. The volume injected is not adjusted for the weight of the mouse being treated. 21. Alternate the injection sites to avoid ulceration. 22. Record data in an appropriate notebook. 23. Keep the autoclaved utensils wrapped in the aluminum foil until use. 24. For most histopathological work, a good general fixative that is compatible with many downstream assays is 4% PFA. 10% neutral buffered formalin can be used as an alternative. 25. The length of time from performing the last injection to eutha- nasia can vary, depending in part on the half-life of the com- pound that is tested. In this work, we euthanized the mice 24 h after performing the last injection. 26. Needles smaller than 25 gauge may restrict the flow of blood into the syringe. Needles shorter than 2.54 cm may not reach the level of the heart when approaching from the diaphragm. 27. Excessive back pressure on the syringe may prevent blood flow into the syringe. 28. Detection of the compound in serum using LC-MS can be used to assess its pharmacokinetic properties. 29. It is recommended to photograph the tumors before removing them from the body. It is also recommended to measure the dimensions of the tumors. 30. Use forceps to pinch off the trachea rostral to the insertion so that 1× PBS is forced into the lungs and not out the nostrils. If the lungs fail to inflate, reinsert the needle into the trachea and slowly flush the lungs with 1× PBS buffer. 31. It is recommended to photograph the lungs. 32. Perform this step if interested in testing PyMT expression in lung tissues. The PyMT oncogene is exclusively expressed in the mam- mary epithelium of MMTV-PyMT transgenic mice. Detection of PyMT expression in lung tissues is a common assay used for sensitive detection of metastatic mammary epithelial cells into the lungs. It is important to change the utensils while performing steps 12-20 to avoid contamination of lung tissues. 33. Histological analysis can be performed on fixed lung tissues to assess the effect of the test compound on metastasis. 34. It is recommended to take photographs. It is also recom- mended to annotate the size and mass of each tumor, and the total weight of all tumors. 35. It is recommended to fix and flash-freeze multiple mammary tumor pieces for future analysis. To maintain consistency between experimental mice, it is recommended to designate which tumors will be frozen and which tumors will be fixed (e.g., snap-freeze tumors from mammary glands 1-3 (Left side), snap-freeze tumors from mammary glands 4-5 (Left side) for RNA extraction, and fix tumors from mammary glands 1-5 (Right side)). 36. It is recommended to collect and fix abdominal organs to per- form histopathological analysis in order to assess drug toxicity. Samples must be sliced to allow adequate fixation. It is also recommended to flash-freeze pieces of these organs to measure the concentration of the tested compound by LC-MS. 37. Following fixation, tissues can be stored in 70% ethanol until being cassetted and paraffin embedded for further histological analyses. 38. Only one set of utensils is required for the necropsy of these mice. It is recommended to take photographs and record the dimensions of each tumor as well as the weight. 39. If interested in performing RNA sequencing on tumor tissue from xenograft mice, snap-freeze a piece of tumor in a RNAse- free Eppendorf tube. 40. If the tissue is not completely broken apart after lysing for 30 s, place the Eppendorf tube on ice for 2 min and repeat this step one more time. References 1. National Cancer Institute (2018) Cancer statis- tics. https://www.cancer.gov/about-cancer/ understanding/statistics. Accessed 27 Mar 2018 2. Arif M, Senapati P, Shandilya J, Kundu TK (2010) Protein lysine acetylation in cellular function and its role in cancer manifestation. Biochim Biophys Acta 1799(10-12):702-716. h t t p s : / / d o i . o r g / 1 0 . 1 0 1 6 / j . bbagrm.2010.10.002 3. Krueger KE, Srivastava S (2006) Posttranslational protein modifications: current implications for cancer detection, prevention, and therapeutics. Mol Cell Proteomics 5(10):1799-1810. https://doi.org/10.1074/ mcp.R600009-MCP200 4. Chalkiadaki A, Guarente L (2015) The multi- faceted functions of sirtuins in cancer. Nat Rev Cancer 15(10):608-624. https://doi. org/10.1038/nrc3985 5. Chang HC, Guarente L (2014) SIRT1 and other sirtuins in metabolism. Trends Endocrinol Metab 25(3):138-145. https://doi. org/10.1016/j.tem.2013.12.001 6. Haigis MC, Sinclair DA (2010) Mammalian sirtuins: biological insights and disease rele- vance. Annu Rev Pathol 5:253-295. https:// d o i . o r g / 1 0 . 1 1 4 6 / a n n u r e v . pathol.4.110807.092250 7. Lavu S, Boss O, Elliott PJ, Lambert PD (2008) Sirtuins--novel therapeutic targets to treat age- associated diseases. Nat Rev Drug Discov 7(10):841-853. https://doi.org/10.1038/ nrd2665 8. Blander G, Guarente L (2004) The Sir2 family of protein deacetylases. Annu Rev Biochem 73:417-435. https://doi.org/10.1146/ annurev.biochem.73.011303.073651 9. Imai S, Guarente L (2010) Ten years of NAD- dependent SIR2 family deacetylases: implica- tions for metabolic diseases. Trends Pharmacol Sci 31(5):212-220. https://doi. org/10.1016/j.tips.2010.02.003 10. Houtkooper RH, Pirinen E, Auwerx J (2012) Sirtuins as regulators of metabolism and health- span. Nat Rev Mol Cell Biol 13(4):225-238. https://doi.org/10.1038/nrm3293 11. Imai S, Armstrong CM, Kaeberlein M, Guarente L (2000) Transcriptional silencing and longevity protein Sir2 is an NAD- dependent histone deacetylase. Nature 403(6771):795-800. https://doi. org/10.1038/35001622 12. de Oliveira RM, Sarkander J, Kazantsev AG, Outeiro TF (2012) SIRT2 as a therapeutic tar-get for age-related disorders. Front Pharmacol 3:82. https://doi.org/10.3389/ fphar.2012.00082 13. Park S-H, Zhu Y, Ozden O, Kim H-S, Jiang H, Deng C-X, Gius D, Vassilopoulos A (2012) SIRT2 is a tumor suppressor that connects aging, acetylome, cell cycle signaling, and carci- nogenesis. Transl Cancer Res 1(1):15-21 14. Zhou W, Ni TK, Wronski A, Glass B, Skibinski A, Beck A, Kuperwasser C (2016) The SIRT2 deacetylase stabilizes slug to control malig- nancy of basal-like breast cancer. Cell Rep 17(5):1302-1317. https://doi. org/10.1016/j.celrep.2016.10.006 15. Serrano L, Martinez-Redondo P, Marazuela- Duque A, Vazquez BN, Dooley SJ, Voigt P, Beck DB, Kane-Goldsmith N, Tong Q, Rabanal RM, Fondevila D, Munoz P, Kruger M, Tischfield JA, Vaquero A (2013) The tumor suppressor SirT2 regulates cell cycle progres- sion and genome stability by modulating the mitotic deposition of H4K20 methylation. Genes Dev 27(6):639-653. https://doi. org/10.1101/gad.211342.112 16. McGlynn LM, Zino S, MacDonald AI, Curle J, Reilly JE, Mohammed ZM, McMillan DC, Mallon E, Payne AP, Edwards J, Shiels PG (2014) SIRT2: tumour suppressor or tumour promoter in operable breast cancer? Eur J Cancer 50(2):290-301. https://doi. org/10.1016/j.ejca.2013.10.005 17. Jing H, Hu J, He B, Negron Abril YL, Stupinski J, Weiser K, Carbonaro M, Chiang YL, Southard T, Giannakakou P, Weiss RS, Lin H (2016) A SIRT2-selective inhibitor promotes c-Myc oncoprotein degradation and exhibits broad anticancer activity. Cancer Cell 29(3):297-310. https://doi.org/10.1016/j. ccell.2016.02.007 18. Chen J, Chan AW, To KF, Chen W, Zhang Z, Ren J, Song C, Cheung YS, Lai PB, Cheng SH, Ng MH, Huang A, Ko BC (2013) SIRT2 overexpression in hepatocellular carcinoma mediates epithelial to mesenchymal transition by protein kinase B/glycogen synthase kinase- 3beta/beta-catenin signaling. Hepatology 57(6):2287-2298. https://doi.org/10.1002/ hep.26278 19. Cheon MG, Kim W, Choi M, Kim JE (2015) AK-1, a specific SIRT2 inhibitor, induces cell cycle arrest by downregulating Snail in HCT116 human colon carcinoma cells. Cancer Lett 356(2 Pt B):637-645. https://doi. org/10.1016/j.canlet.2014.10.012 20. He B, Hu J, Zhang X, Lin H (2014) Thiomyristoyl peptides as cell-permeable Sirt6 inhibitors. Org Biomol Chem 12(38):7498-7502. https://doi.org/ 10.1039/c4ob00860j 21. Heltweg B, Gatbonton T, Schuler AD, Posakony J, Li H, Goehle S, Kollipara R, Depinho RA, Gu Y, Simon JA, Bedalov A (2006) Antitumor activity of a small-molecule inhibitor of human silent information regulator 2 enzymes. Cancer Res 66(8):4368-4377. https://doi.org/10.1158/0008-5472. Can-05-3617 22. Hoffmann G, Breitenbucher F, Schuler M, Ehrenhofer-Murray AE (2014) A novel sirtuin 2 (SIRT2) inhibitor with p53-dependent pro- apoptotic activity in non-small cell lung cancer. J Biol Chem 289(8):5208-5216. https://doi. org/10.1074/jbc.M113.487736 23. Kim WJ, Lee JW, Quan C, Youn HJ, Kim HM, Bae SC (2011) Nicotinamide inhibits growth of carcinogen induced mouse bladder tumor and human bladder tumor xenograft through up-regulation of RUNX3 and p300. J Urol 185(6):2366-2375. https://doi. org/10.1016/j.juro.2011.02.017 24. Mahajan SS, Scian M, Sripathy S, Posakony J, Lao U, Loe TK, Leko V, Thalhofer A, Schuler AD, Bedalov A, Simon JA (2014) Development of pyrazolone and isoxazol-5-one cambinol analogues as sirtuin inhibitors. J Med Chem 57(8):3283-3294. https://doi.org/10.1021/ jm4018064 25. McCarthy AR, Sachweh MC, Higgins M, Campbell J, Drummond CJ, van Leeuwen IM, Pirrie L, Ladds MJ, Westwood NJ, Lain S (2013) Tenovin-D3, a novel small-molecule inhibitor of sirtuin SirT2, increases p21 (CDKN1A) expression in a p53-independent manner. Mol Cancer Ther 12(4):352-360. https://doi.org/10.1158/1535-7163. Mct-12-0900 26. Neugebauer RC, Uchiechowska U, Meier R, Hruby H, Valkov V, Verdin E, Sippl W, Jung M (2008) Structure-activity studies on splitomi- cin derivatives as sirtuin inhibitors and compu- tational prediction of binding mode. J Med Chem 51(5):1203-1213. https://doi. org/10.1021/jm700972e 27. Rotili D, Tarantino D, Nebbioso A, Paolini C, Huidobro C, Lara E, Mellini P, Lenoci A, Pezzi R, Botta G, Lahtela-Kakkonen M, Poso A, Steinkuhler C, Gallinari P, De Maria R, Fraga M, Esteller M, Altucci L, Mai A (2012) Discovery of salermide-related sirtuin inhibi- tors: binding mode studies and antiproliferative effects in cancer cells including cancer stem cells. J Med Chem 55(24):10937-10947. https://doi.org/10.1021/jm3011614 28. Zhang Y, Au Q, Zhang M, Barber JR, Ng SC, Zhang B (2009) Identification of a small mol- ecule SIRT2 inhibitor with selective tumor cytotoxicity. Biochem Biophys Res Commun 386(4):729-733. https://doi.org/10.1016/j. bbrc.2009.06.113 29. Du J, Zhou Y, Su X, Yu JJ, Khan S, Jiang H, Kim J, Woo J, Kim JH, Choi BH, He B, Chen W, Zhang S, Cerione RA, Auwerx J, Hao Q, Lin H (2011) Sirt5 is a NAD-dependent pro- tein lysine demalonylase and desuccinylase. Science 334(6057):806-809. https://doi. org/10.1126/science.1207861 30. Feldman JL, Baeza J, Denu JM (2013) Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacyla- tion by mammalian sirtuins. J Biol Chem 288(43):31350-31356. https://doi. org/10.1074/jbc.C113.511261 31. Zhu AY, Zhou Y, Khan S, Deitsch KW, Hao Q, Lin H (2012) Plasmodium falciparum Sir2A preferentially hydrolyzes medium and long chain fatty acyl lysine. ACS Chem Biol 7(1):155-159. https://doi.org/10.1021/ cb200230x 32. Bheda P, Jing H, Wolberger C, Lin H (2016) The substrate specificity of sirtuins. Annu Rev Biochem 85:405-429. https://doi. org/10.1146/annurev-biochem-060815- 014537 33. Hawse WF, Hoff KG, Fatkins D, Daines A, Zubkova OV, Schramm VL, Zheng W, Wolberger C (2008) Structural insights into intermediate steps in the Sir2 deacetylation reaction. Structure 16(9):1368-1377. https:// doi.org/10.1016/j.str.2008.05.015 34. Cen Y, Falco JN, Xu P, Youn DY, Sauve AA (2011) Mechanism-based affinity capture of sirtuins. Org Biomol Chem 9(4):987-993. https://doi.org/10.1039/c0ob00774a 35. Guy CT, Cardiff RD, Muller WJ (1992) Induction of mammary tumors by expression of polyomavirus middle T oncogene: a trans- genic mouse model for metastatic disease. Mol Cell Biol 12(3):954-961 36. Kim WY, Sharpless NE (2012) Drug efficacy testing in mice. Curr Top Microbiol Immunol 355:19-38. https://doi.org/10.1007/82_ 2011_160 37. Institute for Laboratory Animal Research (2011) Guide for the care and use of laboratory animals, 8th edn. National Academies Press, Washington, DC 38. Faustino-Rocha A, Oliveira PA, Pinho- Oliveira J, Teixeira-Guedes C, Soares-Maia R, da Costa RG, Colaco B, Pires MJ, Colaco J, Ferreira R, Ginja M (2013) Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. Lab Anim 42(6):217-224. https://doi.org/10.1038/ laban.254 39. Earnest E, Ajaghaku D (2014) Guidelines on dosage calculation and stock solution prepara-tion in experimental animals' studies. J Nat Sci Res 4(18):100-106 40. He B, Hu J, Zhang X, Lin H (2014) Thiomyristoyl peptides as cell-permeable Sirt6 inhibitors. Org Biomol Chem 12(38):7498-7502. https://doi.org/ 10.1039/c4ob00860j
41. Machholz E, Mulder G, Ruiz C, Corning BF, Pritchett-Corning KR (2012) Manual restraint and common compound administration routes in mice and rats. J Vis Exp (67):e2771. https://doi.org/10.3791/2771