Dendritic Cell Dysfunction

APPLICATION FOR THE USE OF ANIMALS IN RESEARCHSubmit an electronic version to [email protected] for veterinary review prior to submission to the University of South Florida, Institutional Animal Care & Use Committee,

MDC 35, phone 974-7106, fax 974-7091, email [email protected] PHS #A4100-01; USDA #58-15; AAALAC #434

(type all responses) IACUC#: R________________

1. TITLE OF PROJECT

Mechanism of Lipid-Induced Dendritic Cell Dysfunction in Cancer

(Note: The title of this IACUC application must match the title of the grant that supports this research. If the described activity involving animals is supported by multiple grants with different titles, or a grant is awarded later, during the 3-year approval period of this protocol, that supports this research, inform the IACUC by completing and submitting a Request to Amend an Animal Use Protocol form.)

2. PERSONNEL

Principal Investigator (including degrees) Position (Academic Appointment) Department

Campus Address PI Email Address PI Phone

Secondary Study Contact Secondary Study Contact Email Address Secondary Study Contact Phone 3. QUALIFICATIONS

Name & Degree Role Years Experience/Species Cert#Debby D. Doe, Ph.D. PI 15/Mice 456Carline Someone, B.S., M.S. T 8/Mice 789Raymond Notsmart T *0/Mice 1005James J. Julienne, Ph.D. SI 10/Mice 1112*PI and Comparative Medicine Training Coordinator will train in all techniques and animal handling procedures.

(Note: Indicate role of involved personnel as either Principal (PI), or Secondary (SI) Investigators, or technicians/assistants (T). Indicate each individual's years of experience with species described herein (e.g., 6 yrs/mice, 4 yrs/rabbits). Certification of orientation, training, and experience regarding the regulatory, occupational health and safety, and care and use aspects of the species requested herein is required prior to IACUC review of this protocol, and is available from the IACUC c/o the Division of Comparative Medicine, 974-9796. Certification numbers for each involved individual must be listed.)

4. FUNDING SOURCE(Indicate funding agency, grant #, and account # if known.)

5. LOCATION The Principal Investigator requests that this protocol be conducted at facilities within - Please check location(s) where animals will be housed for study:

ACH ALZ COM CPH CRI IDRB KRC LSA NCF PSY SCA SRB VAHX

6. CERTIFICATIONA search for alternatives and alternative methods, including a search of the following relevant database(s) indicated below, covering the indicated years (at least the last 10 yrs.), and using the indicated search term(s), demonstrates that suitable alternatives to these procedures, and to aspects of these procedures which may cause pain or discomfort to animals, or to this animal use are not available or applicable. The methods described will be used and continuously refined so as to reduce animal discomfort and use. Conduct will be in accordance with the PHS policy, AWR, Guide, AAALAC guidelines, DEA regulations, and IACUC Policies. This project was designed with the consultation of a veterinarian. The described animal use does not duplicate previous or existing studies. This description is complete and accurate. Implementing changes to this description requires prior written IACUC approval. Current biomedical supplies will be used. Complete animal procedural/surgical/testing records will be maintained. Personnel are certified as adequately trained and experienced, and have complied with IACUC occupational health & safety policies. Comparative Medicine will be notified whenever data from any pre-clinical study involving animals is submitted to the Food & Drug Administration.

Database: Years: Search Terms:

__________________________________________________________________________________Signature of Principal Investigator DateNote: A single addendum page of response is permissible, if it is identified as a response to which item # and page #.

CMDC #001.11Effective 5/09

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Agency: American Cancer Society Grant #: 999222333

mouse, tumor injection, subcutaneous immunization, CTL, myeloid cells, tumor immunology, dendritic cells, tumor, pain, alternatives and early endpoint, distress, inflammation, intraperitoneal thioglycollate, peritoneal elicitation of macrophages, peritonitis, gavage, stress

PubMed, National Library of Medicine, Canadian council on animal care CAAC, Johns Hopkins CAAT

1990-2009

Debby D. Doe, Ph.D. Professor Interdisciplinary Oncology

MRC 4444 [email protected]

Carline Someone [email protected] 555-1234

mailto:[email protected]

mailto:[email protected]

PROTOCOL CLASSIFICATION 7. Please check one.a.

X New Project. 3rd Yr renewal replacing previous approved IACUC Protocol #

b. Please provide a brief summary (a few sentences) describing work accomplished during the last approval period and how the work proposed in this renewal extends the previous studies.

JUSTIFICATION FOR THE USE OF ANIMALS8. Briefly state in lay terms the purpose of this request (i.e., research hypothesis or teaching objectives).(Note: Your response must be in language that a high school senior can understand.)

9. Briefly outline or describe in lay terms the procedures in which animals will be used (i.e., the general sequence and schedule of what will be done to the animals). Section 9 or Section 11 of the application should contain an explanation/rationale for assigning the specific number of animals to the different Categories in Section 10. (Note: For complicated experimental designs, it may be helpful to the IACUC if this response includes a flow chart, diagram, timeline, or table which depicts the experiments or sequence of events. Your response must be in language that a high school senior can understand.)


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The immune system is very important for protection against cancer, and immune cells should recognize and kill tumor cells. The ability of the immune system to fight existing tumors is currently in clinical use via cancer vaccines. Unfortunately, the immune system in cancer patients is defective. The same type of defect is seen in tumor-bearing mice. Therefore, mice are actively used as a model of the situation in cancer patients. One of the main features of the defective immune system in cancer is the inability of dendritic cells to recognize tumor cells as antigenic, or foreign. The dendritic cells do not therefore properly stimulate t-cells (other members of the immune system) to kill the tumor cells. The mechanisms of these abnormalities are not fully known. Our recent study has identified a novel mechanism of dendritic cell dysfunction linked to the lipid (fat) content of these cells. The overall goal of this research is to investigate the mechanism of the defects in dendritic cells in cancer and to test several drugs able to overcome described above defects. This can dramatically improve the immune response to cancer.

Attached is a flow chart for better clarification of these studies.This proposal aims to understand the mechanisms of defective function of immune system in cancer and to find ways to improve the function of the immune system in cancer. In the experiments to address this goal, tumors will be established either intraperitoneally (i.p.) or subcutaneously (s.c. by injecting tumor cells in the right flank) in healthy mice. Special care will be given to avoid injection of tumor cells into the leg when performing s.c. injections into the flank. When tumors reach 2 cm in diameter (approximately 2-5 weeks after injection), mice will be euthanatized, and cells collected from spleens, lymph nodes and tumor tissue to analyze in different experiments in vitro. We will use nontransgenic (wild type) mice (C57BL/6 and BALB/C) as well as transgenic mice. Tumor cell lines will include EL4, CT26, Meth-A and MC38. Functional assays will also be needed, and therefore T-cells (another population of immune cells) will be harvested from the spleens of naïve mice. These experiments will aid in establishing a time line of lipid accumulation as well as determining the lipid profile of the cells. In order to further understand the role of lipid accumulation in dendritic cells in cancer, we will perform in vitro and in vivo experiments with a variety of drugs. For the in vitro work, we will harvest bone marrow from naïve mice and culture the cells to generate dendritic cells, in the presence of tumor conditioned medium and or the drugs described herein. For in vivo work, mice will be injected with tumor cells, and then treated either immediately or 1-3 weeks after tumor injection (1 cm diameter), with a variety of drugs, targeting various aspects of lipid metabolism

9. Procedures (Continued)

CHARACTERISTICS OF ANIMALS AND CATEGORY OF RESEARCH10. List and describe the animals to be studied. Indicate the anticipated number of animals to be used in each Category of Research and the total number of animals involved during the 3-year approval period of this protocol. Indicate strain or line designation if rodents are requested. Indicate the number of each species/strains that will be involved in planned procedures that are anticipated to produce momentary, slight, or no pain, discomfort or distress (Category A); more than momentary or slight pain, discomfort or distress which is alleviated by the use of appropriate anesthetics and/or analgesics (Category B); or pain, discomfort, or distress, which cannot, or is not alleviated by the administration of appropriate anesthetics and/or analgesics (Category C). (Note: Refer to IACUC Policy XII. The same number of animals requested here must be justified in response to item #11, below.)

Species, Strain, or LineMice:C57BL/6

Mice: BALB/c

Mice: C57BL/6Cd45.1

Mice;OT-1 +/+

Mice:HA+/+

Mice: B6JCD36-/- Mice:

B6JCD204-/-Mice: B6JMARCO-/-

Characteristics(age, sex, weight)

6-8 weeks, either M/F








#used in Category A procedures (no or slight discomfort).

90+6+10490+24+42+104+20+54

32018+8+20+54

18+6+8 72 72 72

#used in Category C procedures (discomfort not alleviated).

72+96+80+80 72+24+96 72 72 72

Total Number Requested 528 526 320 100 32 144 144 144


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Prostanoids are lipid based biochemicals produced by a cell in response to stress and also play a critical role in lipid biosynthesis. Prostanoids are in turn regulated by an enzyme called cyclooxygenase (COX). COX-2 is a rate-limiting enzyme in the synthesis of prostaglandins and is over-expressed in multiple cancers. We hypothesized that increased production of prostaglandins (primarily PGE2) mediated by high level of COX-2 in tumor cells may be responsible for accumulation of lipids in DCs. To test this hypothesis, mice with established tumor (1 cm diameter) will be treated with the selective COX-2 inhibitor CelebrexTM (celecoxib) (Pfizer, New York, NY) at a dose of 500 mg/kg/day, or the non-selective COX-1/2 inhibitor indomethacin by i.p. administration. Mice will be euthanatized 3, 7, 10, and 14 days after start of the treatment and the level of lipids in immune cells from spleens and lymph nodes will be evaluated as described above. Tumor size will be carefully monitored.Acetyl-CoA carboxylase (ACC) is a critical enzyme responsible for the biosynthesis of lipids. Inhibition of ACC results in inhibition of fatty acid synthesis and stimulation of fatty acid oxidation. Recently, two novel ACC inhibitors CP-610431 and CP-640186 were identified (Pfizer). For this experiment, mice will be injected with tumor cells and tumors established for 1-3 weeks, or a size of 1cm diameter. The tumor bearing mice will then be treated by gavage at a volume of 0.25 ml/25 g of body weight with an aqueous solution containing 0.5% methyl cellulose (vehicle control groups), and the other group will be treated with an aqueous solution containing 0.5% methyl cellulose plus CP-610431/ CP-640186 (treatment groups). Also, as a control, similar treatment will be performed in naïve tumor-free mice. After two-weeks of drug treatment, the mice will be euthanatized, spleens and lymph nodes collected, and the immune cell function determined.Also, the role of diet in the accumulation of lipids in dendritic cells will be evaluated. Mice will be fed one of three diets (standard, low fat, and high fat) for two weeks, and then tumors established for a maximum of 5 weeks tumor growth or when tumors reach a diameter of 2 cm, with the diets continuing throughout the tumor growth period. Mice will be euthanatized and immune cells will then be isolated and evaluated as previously described.To understand the details of the immune response in cancer we will use adoptive transfer experiments to study the effects of the tumor microenvironment on lipid accumulation in dendritic cells. In this type of experiment, cells isolated from one animal are injected into another. In the proposed experiments, congenic dendritic cells (naïve mice having the same HLA/DR expression on their immune cells, and therefore permitting transplantation without rejection) will be transferred i.p. into mice bearing 1-3 week induced tumor. The donor cells will then be harvested not more than 4 weeks later, not allowing tumors to grow for more than 5 weeks total. Ascites, spleen, and lymph nodes will be collected, and different in vitro tests will be performed to evaluate immune responses. Control animals will be treated with thioglycollate (a single injection i.p., then euthanatized 1-21 days later) in order to trigger the infiltration of monocytes. Immune cells will be obtained from tissues using magnetically labeled specific antibodies and magnetic columns. Additionally, scavenger receptors have been implicated in the uptake of lipids into cells, as has been established in cardiovascular disease and foam cell formation. We will study the role of scavenger receptors in lipid accumulation in dendritic cells in tumor bearing mice using available knock out models for scavenger receptors. Mice will be injected with tumor cells, and tumors established for 1-5 weeks, or a maximum tumor diameter of 2 cm. Mice will then be euthanatized and immune cells studied collected from spleen, tumor and lymph node.

11. Briefly describe the rationale using statistical analysis, whenever possible, used to determine the total number of each species of animals declared above in response to item #10 that will be needed for use during the 3-year approval period of this protocol. Section 9 or Section 11 of the application should contain an explanation/rationale for assigning the specific number of animals to the different Categories in Section 10. (Note: For complicated experimental designs, it may be helpful to the IACUC if this explanation indicates the # of animals needed for each experimental group, the # of groups required, and the analyses conducted using each group. Alternatively, it may be helpful to the IACUC if a flow chart depicting the sequence of events and the number of animals required for each step is shown.)

12. Briefly describe the characteristics of the animals requested that justify its use in this protocol.


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Attached is a flow chart for better clarification of these studies.1. In order to establish the phenomenon of lipid laden dendritic cells in vivo, analysis will consist of isolation of

immune cells from naïve and tumor bearing mice. We wish to study four tumor models, and each experiment will require at least 4 control and 4 tumor-bearing mice, at three separate time points, each point in triplicate. Additional naïve mice will be used as donors for T-cells for functional assays for each experiment. Therefore, we will need 360 mice for these experiments.

1. To address the main question of this project we need to perform a lipid profile analysis that requires purified dendritic cells. According to our previous experience to obtain a sufficient number of dendritic cells from spleen, we will need to pool together cells from at least 8 mice. Since we are comparing control and tumor-bearing mice we would need 8 control and 8 tumor-bearing mice. Each experiment needs to be performed at least 3 times to assure a reproducibility of the results. Therefore, we will need 48 mice for these experiments.

2. For initial in vitro experiments we will use mice as a source of cells (bone marrow from naïve mice). We will need to run control and drug treated cells (four different drugs), as well as cells treated with high or low amount of lipid in the medium. A minimum of three experiments are necessary achieve statistically appropriate data, with enough cells to run both phenotypic and functional analysis. Additional naïve mice will be used as donors for T-cells for functional assays for each experiment. Therefore, we will need 54 mice total.

4. Next, we will study the effects of diet and drug therapy in vivo, using naïve and tumor bearing mice, and two different tumor models. For the diet study we will require 12 mice per group, both naïve and tumor bearing, with three diets (control, low and high fat). For the drug studies, we will require 12 mice per group, both naïve and tumor bearing, with five drug groups (control, celecoxib, indomethacin, CP-610431, and CP-640186). Additional naïve mice will be used as donors for T-cells for functional assays for each experiment. Therefore, we will need 416 mice for these experiments.

5. Adoptive transfer studies will use dendritic cells derived from the bone marrow of naïve CD45.1 mice. Approximately two donor mice are needed to provide sufficient dendritic cells for each recipient mouse. We wish to study two tumor models, and each experiment will require at least 4 control (thioglycollate injected i.p.) and 4 tumor-bearing (i.p.) recipient mice, at 10 separate time points, each point in triplicate. Additional naïve mice will be used as donors for T-cells for functional assays for each experiment. Therefore, we will need 520 mice for these experiments.

6. To address question of molecular mechanisms of the observed phenomena we will use three strains of knockout mice (CD204, MARCO, CD36). We wish to study two tumor models, and each experiment will require at least 4 control and 4 tumor-bearing mice, at three separate time points, each point in triplicate. Additional naïve mice will be used as donors for T-cells for functional assays for each experiment. Therefore, we will need 540 mice for these experiments.

TOTAL = 1938 mice

C57BL/6, CD45.1, and BALB/c mice are well-characterized inbred strains of mice. They are syngeneic for the tumor cells and transgenic mice described above that would allow us to address all scientific questions of the study. HA and OT-1 mice are transgenic mice with T cells expressing receptors specific for one peptide (HA or OT-1). This would allow us to evaluate specific immune responses in our experiments. C57BL/6CD36 are defective in the expression of the scavenger receptor B. C57BL/6CD204 are defective in the expression of the scavenger receptor A. C57BL/6MARCO are defective in the expression of the MARCO (macrophage receptor with collagenous structure) scavenger receptor. These mice will allow us to evaluate the roles of scavenger receptors in lipid accumulation in dendritic cells.

It is important that we evaluate several different strains of mice and tumor cell lines in order to show the validity of our experiments. The better we define the phenomena in mice, the more likely the theories will translate to the human cancer condition and ultimately to therapeutics.

LABORATORY POLICY CONSIDERATIONS13. Will animals be involved in procedures that are anticipated to have the potential to produce more than momentary or slight pain, discomfort, or distress, which cannot, or will not be alleviated by the administration of appropriate anesthetics and/or analgesics?

No: Proceed to Item #14. Yes: X

Within the space below, define the clinical criteria that will be

used to ensure timely intervention and treatment, or removal of the animals from the study, either in advance of, or immediately after recognition of the discomfort. The earliest possible clinical end point, which will contribute to the resolution of the hypothesis, must be identified and utilized. If avoidance or alleviation of animal pain or discomfort adversely affects the protocol, provide a detailed justification of why treatments cannot be initiated.

14. Is animal death (excluding death from euthanasia) an intentional endpoint in this protocol (e.g., survival analysis, radiation, toxicity, or carcinogenesis testing)?

No: X

Proceed to Item #15. Yes: Within the space, explain why an earlier endpoint is not possible.

15. Will any animal research/procedural/testing areas outside of the animal facility be used for this protocol?

No: X

Proceed to Item #16. Yes: Within the space, provide a scientific or logistical justification for

why animals must be removed from the facility, indicate the location, the approximate number of hours animals will be held at this site, and whether the animals will need to be returned to housing after the procedure, or that the procedure will be terminal.

16. Will this study be performed in accordance with 21 CFR 58 as a GLP study?

No: X

Proceed to item #17. Yes: Attach a copy of the GLP Study Protocol.

17. Will scheduled substances controlled by the Drug Enforcement Administration be used in the protocol?

No: X

Proceed to item #18. Yes: Within the space, list the controlled substances to be used.

EXPERIMENTAL PROCEDURES

18. Are other than standard and routine husbandry and handling practices required for this protocol (e.g., food, fluid, or calorie restriction, unique diets or nutritional supplements, specialized caging or environments, or non-standard health monitoring)? No: Proceed to Item #19. Yes:

X Attach "Appendix A: Special Husbandry."

19. Are test substances administered to animals as part of this protocol (e.g., radioisotopes, toxic, immunogenic, pharmacologic, infectious, or carcinogenic agents, biomaterials, or cells)?


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If tumor size reaches 2 cm in diameter, or if tumor ulcerates, or if tumors interfere with posture, locomotion or feeding, mice will be humanely euthanatized. Additional signs of distress that would warrant euthanasia include shallow, rapid/or labored breathing, hypoactivity, hyperactivity, restlessness, self-trauma, aggressiveness, isolation from cage mates, ataxia, rapid/or labored breathing, pale mucous membranes, cyanosis, failure to groom, soiled anogenital area, inactivity, failure to respond to stimuli, ruffled hair coat, piloerection, matted hair coat, lack of inquisitiveness, vocalization, and/or hunched posture.


Attach "Appendix B: Test Substances."

20. Are specimens collected from animals prior to euthanasia as part of this protocol (e.g., tissues, blood, lymph, or other body fluids)?

No: X

Proceed to Item #21. Yes: Attach "Appendix C: Specimen Collection, Ante Mortem.”

21. Will surgery be performed on animals as part of this protocol?

No: X

Proceed to Item #22. Yes: Attach "Appendix D: Surgery."

22. Will animals be subject to experimental procedures other than those described above (e.g., behavioral manipulations, noxious stimuli, forced exercise, or physical restraint)?


Attach "Appendix E: Other Experimental Procedures.”

23. Will a human patient procedural area be required for this protocol?

No: X

Proceed to item #24. Yes: Attach "Appendix F: Patient Procedural Area."

24. Will Veterans Affairs resources (e.g., VA lab space, VA vivarium, VA equipment, VA funding, VA personnel, VA time) be used in any aspect of this research?

No: Proceed to item #25. Yes: Attach "Appendix G: VA Hospital Resources Information."

EUTHANASIA

25. Are animals euthanatized for postmortem tissue collection, or are euthanatized at the completion of this study?

No: Within the space below indicate the final disposition of the involved animals.

Yes: X

Does the method of euthanasia and means of assuring death following euthanasia comply with IACUC

Policy XX which describes acceptable methods of animal euthanasia within these laboratories?

No: Within the space below indicate why a deviation from policy is necessary. Yes: X

Within the

space below describe the method of euthanasia used for each species requested, indicating dose and route if a chemical agent, or provide a justification if a physical method.

STOP here. Complete and attach any required appendixes as indicated above. This application is complete.


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Mice will be euthanatized in cages by inhalation of increased concentrations of CO2 delivered from high pressure tank via special gas regulator. The chamber will not be prefilled. The chamber will be gradually filled with CO2. Between uses, the chamber will be cleaned and dried. Death will be confirmed by prolonged observation at room air, or by employing a secondary method of euthanasia such as cervical dislocation, decapitation, or thoracotomy.

X

APPENDIX A: SPECIAL HUSBANDRY(Complete & Submit for Review Only If Response to Item #18 was "Yes")

1. Does this protocol involve food, fluid, or caloric restriction? No: Yes:

2. Within the space provided, describe all non-standard husbandry and handling practices which are a part of this protocol, including the length of time they will be implemented (e.g., suspended wire mesh caging of rodents, dietary manipulations, food or water deprivation, modified light cycle, specialized housing, confinement, isolation, restricted observation, restricted environmental enrichment, unique requirements of immunocompromised strains).

3. Within the space provided, justify the implementation of this requested husbandry practice.

4. Will this special husbandry practice cause more than momentary, slight pain or discomfort to the animals (see policy statement regarding avoiding and alleviating animal pain and discomfort for guidance)?

No: X

Proceed to Item #5. Yes: Within the space below, describe the methods that will be used

to minimize pain and discomfort.

4. Within the space provided, describe the methods for monitoring the condition of the animal during the procedure and during the post-procedural period, and indicate whether a log of observations will be kept.(Note: Log entries describing procedures involving nonrodent mammals must be kept by the PI in the animal facility on forms provided by Comparative Medicine.)

Description of Appendix A is complete.


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One set of experiments will examine the effects of diet on the lipid content and function of dendritic cells, both in naïve and tumor bearing mice. Harlan Teklad provides diets that are enriched in fat or protein in order to achieve a higher or lower fat content than the standard diet. The current standard diet is Harlan 2018, which contains 18% protein, 5% fat, and 3.8% fiber. Our choice for a higher fat diet is Harlan 7004 with 17% protein, 11% fat, and 3.5% fiber. Our choice for a lower fat diet is Harlan 2016 with 16% protein, 3.5% fat, and 3.9% fiber. All diets are fortified with vitamins and minerals, and all are part of Harlan’s standard diet repertoire and are suitable for maintaining healthy mice. The diets will be administered for up to 5 weeks.

These standard diets are necessary to test the hypothesis that dietary conditions may contribute to the accumulation of fat within immune cells.

No log required. Cages will be marked SPECIAL DIET.

APPENDIX B: TEST SUBSTANCES (Complete & Submit for Review Only If Response to Item #19 was "Yes")

1. Complete the table below. Name the test substance(s) that will be administered to animals.

Indicate the class of each substance as either an: (A) infectious agent, (B) primary human explant, uncharacterized human blood, lymph or tissue specimen, (C) recombinant DNA, (D) radioisotope, (E) carcinogen, (F) hazardous or toxic chemical, (G) biological toxin, (H) Cell line, (I) Adjuvant, (J) Antigenic Substance, (K) Pharmacologic Agent, or (L) Other class of substance by using the appropriate capital letter. Indicate to which species each substance will be administered. Indicate the dose (e.g., μg/gm bwt), volume per administration, route (e.g., i.v., i.p., s.c.), interval (e.g. 1x, daily, eod, every 3rd day), and duration of administrations (e.g., 5 wks).

Substance(list all substances including vehicle/control)

Class(above)

Species(administered to)

Dose/Volume/Route(3.0mg/kg bw/ 0.1ml / i.p.)

Interval(1x, eod, ev 3rd day)

Duration(5 weeks)

1. EL-4, CT26, MC38, C3 tumor cell lines

H mice5x105 cells/

0.15ml/s.c., i.p.1 X N/A

2. Thioglycollate I mice 3% w/v/ 2.0 ml/i.p. 1 X N/A

3. Dendritic cells H mice10 x 106 cells/ 2.0

ml/i.p.1 X N/A

4. Indomethacin K mice5 mg/kg/day/ 0.1 ml/

i.p.1 X/day 14 da

5. Celecoxib K mice 500 mg/kg/0.1 ml/ i.p. 1 X/day 14 da

6. CP-610431 K mice 10 mg/kg/0.1 ml/ oral 1 X/day 14 da

7. CP-640186 K mice 10 mg/kg/ 0.1 ml/ oral 1 X/day 14 da

8. Methylcellulose vehicle K mice0.5% w/v / 0.1 ml /

oral1 X/da 14 da

2. In the same order in which substances to be administered are listed in the table above, very briefly indicate below the purpose of substance administration in the relation to the hypothesis, and the expected effect to the animal(s). If none, so state.

Purpose and Expected Effect of Substance Administration to the Animal

1. - to induce tumor growth in mice.

2. – to induce benign monocyte infiltration into the peritoneum. No side effect/ adverse reaction is expected as described in Appendix E based on our experience with this procedure for 2 years.

3. - to evaluate induction and suppression of immune responses. No side effect/ adverse reaction is expected.

4. - to inhibit COX activity and monitor effects on prostaglandins. No side effect/ adverse reaction is expected. 5. - to inhibit COX activity and monitor effects on prostaglandins. No side effect/ adverse reaction is expected.

6. – to inhibit acetyl co-A carboxylase activity (inhibits fatty acid synthesis). No side effect/ adverse reaction is expected.

7. – to inhibit acetyl co-A carboxylase activity (inhibits fatty acid synthesis). No side effect/ adverse reaction is expected.8. – this is the solution (vehicle) in which the acetyl co-A carboxylase inhibitors are diluted. No side effect/ adverse reaction is expected.


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APPENDIX B: TEST SUBSTANCES (cont.)3. Is there a possibility that any of the test substance(s) could cause more than momentary or slight pain, discomfort, or distress to the animals, either immediately following substance administration, or as a consequence long after administering the test substance(s)?

No: Proceed to item #4. Yes: X

List those substances in the space below. For each such

substance describe the consequences of administration that have a potential to cause animal discomfort, pain, or distress, and how discomfort, pain or distress will be anticipated, minimized or alleviated (e.g., nude mice administered tumor cells will be humanely euthanatized if induced tumors become >1.0 cm in diameter, or if tumors ulcerate, or if tumors interfere with posture, locomotion or feeding). If discomfort, pain, or distress will not be alleviated, then justify how treatments interfere with the procedures or the interpretation of results.

(Note: Log entries describing health concerns or complications that develop as a consequence of substance administration to nonrodent mammals, and their treatment and resolution, or when animals are euthanatized must be kept by the PI in the animal facility on forms provided by Comparative Medicine.)

4. Are any of the test substances listed above in response to item 1, page 7, included in any of the classes A through G, and considered a regulated or potentially hazardous material to research or animal care personnel?

No: X

STOP here. This appendix is complete. Yes: List those substances in the space below

and describe procedures to ensure these substances and/or animals to which these substances have been administered will be handled safely (e.g., BSL-2, USF Chemical Hygiene Plan, Universal Precautions, etc.):

Test substance(s) of any of the classes A-G listed above under item 1, page 7 are either regulated or potentially hazardous and require prior authorization of use by the appropriate Safety Committee. Signature of the Radiation Safety, Biohazard/Recombinant DNA Safety, or Chemical Safety Compliance Officer below indicates that the applicant has consulted with the appropriate Safety Committee, and that that committee has approved the use of the test substance(s). Signature of the applicant PI on page 1 ensures that research personnel will abide by all relevant, universal precautions regarding blood-borne pathogens, appropriate biosafety level precautions, radiation safety procedures, and the chemical hygiene plan.

(Note: Approval of an application involving hazardous materials is often contingent on a pre-performance meeting involving staff that represents the applicant’s laboratory, the Division of Comparative Medicine, the IACUC, and the appropriate Safety Committee(s). This meeting is required in order to ensure that all involved personnel are aware of the precautions, containment practices, facilities, protective devices, disposal and decontamination procedures, and other necessary safety procedures that must be followed to protect personnel, and prevent accidental animal exposure to the hazardous material.)

___________________________________________________________________________________________Signature of Safety Officer(s) Date

Description of Appendix B is complete.


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Tumor growth in mice may cause animal discomfort. If tumor size reaches 2 cm in diameter, or if tumor ulcerates, or if tumors interfere with posture, locomotion or feeding, mice will be humanely euthanatized. No discomfort, pain or distress after administration of drugs, T cells, or myeloid cells is anticipated. However, if clear signs indicative of a generalized disease process (metastasis) or signs of distress became visible after mentioned drugs administration mice would be immediately euthanatized. The signs which will be used as criteria for timely intervention include shallow, rapid and/or labored breathing, hypoactivity, hyperactivity, restlessness, self-trauma, aggressiveness, isolation from cages mates, ataxia, rapid/or labored breathing, pale mucous membranes, cyanosis, failure to groom, soiled anogenital area, inactivity, failure to respond to stimuli, ruffled hair coat, piloerection, matted hair coat, lack of inquisitiveness, vocalization, and/or hunched posture.

Thioglycollate injections i.p.: We have over two years of experience with thioglycollate elicited macrophages. The injected mice do not exhibit more than momentary discomfort or pain, primarily immediately after injection. The mice do not exhibit sickness behavior of any type during the post-injection period. However, in view of veterinarian concerns, we will monitor injected animals for any signs of sickness behaviors and if clear signs indicative of a generalized disease process (infection) or signs of distress become visible after thioglycollate administration, mice would be immediately euthanatized.

APPENDIX E: OTHER EXPERIMENTAL PROCEDURES(Complete & Submit for Review Only If Response to Item #22 was "Yes")

1. Within the space provided, describe the procedure(s) (e.g., behavioral manipulation, forced exercise, noxious stimuli, and physical restraint) and the expected outcome.

2. How long will each procedure last?

3. Will the procedure(s) cause more than momentary, slight pain or discomfort to the animals?

No: X

Proceed to Item #4. Yes: Within the space provided, describe the methods that will be

used to minimize pain and discomfort.

4. Within the space provided, describe the methods for monitoring the condition of the animal during the procedure and during the post-procedural period, and whether a log of observations will be kept.(Note: Log entries describing procedures involving nonrodent mammals must be kept by the PI in the animal facility on forms provided by Comparative Medicine.)

Description of Appendix E is complete.


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Gavage: Mice will be monitored daily for overall health during the entire drug administration protocol. A log will be kept by the investigator’s technician as to drug administration and animal well-being during this period.

Administration by Oral Gavage Animals will be administered two drug substances, CP-610431 or CP-640186, via oral gavage for 14 days.

Liquid compounds may be administered directly into the stomach of mice via a technique called oral gavage. In this procedure a bulb tipped gastric gavage needle is attached to a syringe and used to deliver the compound into the stomach. The bulb tip helps prevent trauma to the mouth and esophagus as the needle is introduced. The correct needle length is equal to the distance from the mouth to just beyond the last rib.The animal should be firmly restrained (grasp the animal by the loose skin of the neck and back) to immobilize the head. Maintain the animal in an upright (vertical) position and pass the gavage needle through the side of the mouth. Following the roof of the mouth, advance the needle into the esophagus and toward the stomach. If resistance is encountered you may be attempting to enter the trachea and you should alter your needle position. After the needle is passed to the correct length, the compound may be injected. If the animal coughs, chokes or begins to struggle vigorously after compound administration begins you may be injecting material into the lungs. If this occurs, stop and withdraw the needle immediately. If it appears that material has been injected into the lungs, the animal should be euthanatized. Struggling during administration or excessive force in advancing the needle, may lead to rupture of the esophagus or pharynx. If you suspect this has occurred, the animal should be euthanatized.

Volumes of up to 10 ml/kg in the mouse may be administered via oral gavage.

Gavage: Less than 5 minutes per day, for 14 days.

IACUC Application Supplemental Flow Chart for Section 9 & Section 11; “Mechanism of Lipid-Induced Dendritic Cell Dysfunction in Cancer”

1. Time course in four models

In order to establish the phenomenon of lipid laden dendritic cells in vivo, analysis will consist of isolation of immune cells from naïve and tumor bearing mice. We wish to study four tumor models, and each experiment will require at least 4 control and 4 tumor-bearing mice, at three separate time points, each point in triplicate. Additional naïve mice will be used as donors for T-cells for functional assays for each experiment.

1 week 2 weeks 3 weeks

Tumor model: mouse strain

# naïve mice

# tumor bearing

mice

# naïve mice

# tumor bearing

mice

# naïve mice

# tumor bearing

miceCT26: BALB/c 12 12 12 12 12 12Meth-A: BALB/c 12 12 12 12 12 12MC38: C57BL/6 12 12 12 12 12 12EL4: C57BL/6 12 12 12 12 12 12

Naïve mice for functional assays:Allogenic Antigen Specific9 C57BL/6 9 HA Tg9 C57BL/6 9 HA Tg9 BALB/c 9 OT1 Tg9 BALB/c 9 OT1 Tg

Totals: BALB/c: 90 naïve category A + 72 tumor bearing category C = 162 totalC57BL/6: 90 naïve category A + 72 tumor bearing category C = 162 totalHA Tg: 18 naïve category A

OT1 Tg: 18 naïve category A

360 total


11

0 1w 2w 3w

Inject tumor s.c. Euth. Euth. Euth.

2. Lipid Profile Analysis

To address the main question of this project we need to perform a lipid profile analysis that requires purified dendritic cells. According to our previous experience, in order to obtain a sufficient number of dendritic cells from the spleen, we will need to pool together cells from at least 8 mice. Since we are comparing control and tumor-bearing mice we would need 8 control and 8 tumor-bearing mice.

Each experiment needs to be performed at least 3 times to assure reproducibility of the results.

Totals: BALB/c: 24 naïve category A + 24 tumor bearing category C = 48 total

3. In vitro experiments with drugs and lipid levels

For initial in vitro experiments we will use mice as a source of cells (bone marrow from naïve mice). We will need to run control and drug treated cells (four different drugs), as well as cells treated with high or low amount of lipid in the medium. A minimum of three experiments are necessary achieve statistically appropriate data, with enough cells to run both phenotypic and functional analysis. Additional naïve mice will be used as donors for T-cells for functional assays for each experiment.

ConditionBALB/c

# naïve mice Additional # naïve mice for functional assays

Control 6 6- C57BL/6 (allogenic)Indomethacin 6 6- HA Tg (antigen specific)

Celecoxib 6CP-610431 6CP-640186 6

High lipid envir. 6Low lipid envir. 6

Totals: BALB/c: 42 naïve category A miceC57BL/6: 6 naïve category AHA Tg: 6 naïve category ATotal 54


12

0 1w 2w 3w

Inject tumors.c.

Euth.

4. In vivo diet and drug studies

Next, we will study the effects of diet and drug therapy in vivo, using naïve and tumor bearing mice, and two different tumor models. For the drug studies, we will require 12 mice per group, both naïve and tumor bearing, with five drug groups (control, celecoxib, indomethacin, CP-610431, and CP-640186). For the diet study we will require 12 mice per group, both naïve and tumor bearing, with three diets (control, low and high fat). Additional naïve mice will be used as donors for T-cells for functional assays for each experiment.

Model: CT26 Model: MC38Strain: BALB/c Strain: C57BL/6

Condition# naïve

mice# tumor bearing

mice

# naïve mice

# tumor bearing

miceControl 12 12 12 12

Indomethacin 12 12 12 12Celecoxib 12 12 12 12CP-610431 12 12 12 12CP-640186 12 12 12 12

Control diet 12 12 12 12

High fat diet 12 12 12 12Low fat diet 12 12 12 12

Naive mice for functional assays

8- C57BL/6 8- HA Tg

8- BALB/c 8- OT1 Tg

Totals: BALB/c: 104 naïve category A + 96 tumor bearing category C = 200 totalC57BL/6: 104 naïve category A + 96 tumor bearing category C = 200 totalHA Tg: 8 naïve category AOT1 Tg: 8 naïve category A

Total 416


13

0 1w 2w 3w

Inject tumors.c.

Start drug treatment Euth.Euth.Euth.

0 1w 2w 3w

Start diet

Euth.

Inject tumors.c.

4w 5w

Euth. Euth.

5. Adoptive transfer studies We will use dendritic cells derived from the bone marrow of CD45.1 mice. Approximately two donor mice are needed to provide sufficient dendritic cells for each recipient mouse. We wish to study two tumor models, and each experiment will require at least 4 control (thioglycollate injected i.p.) and 4 tumor-bearing (i.p.) recipient mice, at 10 separate time points, each point in triplicate. Additional naïve mice will be used as donors for T-cells for functional assays for each experiment.

Part A Part B

Tumor Model

# naïve CD45.1 donors

# thioglycollate C57BL/6recipients

# tumor C57BL/6 recipients

# naïve CD45.1 donors

# thioglycollate C57BL/6recipients

# tumor C57BL/6 recipients

MC38 80 20 20 80 20 20EL4 80 20 20 80 20 20

Naive mice for functional assays:

10- BALB/c10- OT1 Tg

10- BALB/c10- OT1 Tg

Totals: BALB/c: 20 naïve category A miceC57BL/6: 80 thioglycollate category C and 80 tumor category C = 160 totalOT1 Tg: 20 naïve category A miceCD45.1: 320 naïve category A miceTotal: 520 naïve category A mice


14

0 2w 3w

Inject tumor i.p.+DCs i.p. Euth.

0

Inject tumor i.p.

Euth.Inject DCs

9d 10d7d 8d 14d

Euth. Euth.Euth.

5d 7d3d

Euth.Euth. Euth.Euth.

0

Inject Thioglycollate i.p. Euth.Euth.Euth. Euth. Euth.

2w 3w1d 2d 5d3d

Euth.

TumorBearingMice

ControlMice

7d

A.

B.

6. Scavenger receptor knock out mice

To address the question of molecular mechanisms of the observed phenomena we will use three strains of knockout mice (CD204, MARCO, CD36). We wish to study two tumor models, and each

experiment will require at least 4 control and 4 tumor-bearing mice, at three separate time points, each point in triplicate. Additional naïve mice will be used as donors for T-cells for functional assays

for each experiment.

1 week 2 weeks 3 weeks Functional assays

Mouse Strain

Tumor Model

# naïve mice

# tumor bearing

mice# naïve

mice

Tumor Bearing

# tumor bearing

mice# naïve

miceAllogenic Antigen

specific

CD204-/-MC38 12 12 12 9 BALB/c 12 12 9 BALB/c 9 OT1 TgEL4 12 12 12 9 BALB/c 12 12 9 BALB/c 9 OT1 Tg

CD36-/-MC38 12 12 12 9 BALB/c 12 12 9 BALB/c 9 OT1 TgEL4 12 12 12 9 BALB/c 12 12 9 BALB/c 9 OT1 Tg

MARCO-/-MC38 12 12 12 9 BALB/c 12 12 9 BALB/c 9 OT1 TgEL4 12 12 12 9 BALB/c 12 12 9 BALB/c 9 OT1 Tg

Totals: CD204-/- 72 naïve category A + 72 tumor bearing category C = 144 totalCD36-/- 72 naïve category A + 72 tumor bearing category C = 144 total

MARCO-/- 72 naïve category A + 72 tumor bearing category C = 144 totalBALB/c: 54 naïve category AOT1 Tg: 54 naïve category ATotal: 540


15

0 1w 2w 3w

Inject tumor s.c. Euth. Euth. Euth.

Health & Medicine

Dendritic Cell Dysfunction