Transcriptomic profiles of tissues from rats treated with anticancer drug combinations

Bioinformatics

Animals and Treatments

The 96 male Sprague Dawley rats designated for use in each study were selected from 104 males received from Charles River Laboratories, Inc. (Raleigh, NC). Animals were given a unique identification number for this study by ear punch. The Sprague Dawley rat is an accepted species and strain that is commonly used in preclinical pharmacological and toxicological evaluations of drugs used or intended for use in humans. On Day 1 of the study, the rats were:

  1. approximately 7–8 weeks of age and weighed between 197.6–274.4 grams in the Temsirolimus and Sorafenib or Sunitinib study.

  2. approximately 8 weeks of age and weighed between 251.5–336.2 grams in the Oxaliplatin and Sorafenib or Sunitinib study.

  3. approximately 8–9 weeks of age and weighed between 256.6–307.3 grams in the Gemcitabine and Sorafenib or Erlotinib study.

Teklad Certified Rodent Diet 2016C (Harlan; Madison, WI) and tap water (Birmingham public water supply) were provided ad libitum to the rats during the prestudy and study periods. Analysis of the Certified Rodent Diet 2016C was conducted by the manufacturer. The results of the feed and water analyses are located in the facility records at Southern Research. The animals were individually housed in solid-bottom polycarbonate cages on stainless steel racks in a room maintained at a:

  1. temperature of 68–76 °F and a relative humidity of 41–82% in the Temsirolimus and Sorafenib or Sunitinib study.

  2. temperature of 64–79 °F and a relative humidity of 27–70% in the Oxaliplatin and Sorafenib or Sunitinib study.

  3. temperature of 70–76 °F and a relative humidity of 25–53% in the Gemcitabine and Sorafenib or Erlotinib study.

Cage size and animal care conformed to the guidelines of the Guide for the Care and Use of Laboratory Animals (National Research Council 2011), the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 99–198), and to the applicable Standard Operating Procedures (SOPs) of Southern Research.

The experimental designs and dose procedures used for the three studies are as follows:

Study 1: Temsirolimus and Sorafenib or Sunitinib

Test Articles

Temsirolimus (80 mg; NSC-683864; NSC/sample: 683864/6; Lot No.: BTM-105; SRI Lot No: M30/L-1; expiration date: September 22, 2015), sorafenib, tosylate salt (2.0 g; NSC-724772; NSC/sample: 724772-F/2; Lot No.: BSF-105; SRI Lot No.: M26/L-1; expiration date: April 24, 2019), and sunitinib, malate salt (160 mg; NSC-736511; NSC/sample: 736511-0/2; Lot No.: BST-104; SRI Lot No.: M31/L-1; expiration date: April 27, 2019) were supplied by the National Cancer Institute (Rockville, MD) and received at Southern Research on October 19, 2010. Each test article was shipped on dry ice and subsequently stored frozen (≤−20 °C) under inert gas.

Vehicle/Vehicle Control Articles

The following reagents were used in the preparation of dose formulations:

  • Ethyl Alcohol, 200 Proof Absolute Alcohol, ACS/USP Grade (Lot No. KID24C; Pharmco-AAPER; Brookfield, CT; recommended retest date April 2012)

  • Pluronic® F-68, solid cell culture tested, insect cell culture tested (Lot No. 020M0029; Sigma Aldrich; St. Louis, MO; retest date March 2014)

  • Polyethylene glycol, average M.W. 400 (PEG 400) (Lot No. A0284717; Acros Organics; Fair Lawn, NJ; assigned expiration date: September 23, 2011)

  • 1, 2 Propanediol, ACS reagent ≥99.5% (propylene glycol; Lot No. MKBD2071; Sigma Aldrich; St. Louis, MO; assigned expiration date: October 21, 2011)

  • Saline Solution 0.9% (Lot No. 090615A3; Nova-Tech, Inc.; Grand Island, NE; expiration date: June 2011)

  • Sterile Water for Injection; (Lot No. 090818A2; Vedco, Inc.; St. Joseph, MO; expiration date: August 2011)

  • Deionized Water (Southern Research Institute; Birmingham, AL; in-house deionized water system)

All reagents were received and stored at room temperature. In addition, polyethylene glycol was stored under nitrogen, protected from light.

Vehicle Control Formulation Preparation

Temsirolimus Vehicle Control (TEM vehicle)

The temsirolimus vehicle control formulation was prepared in three steps. First, a vehicle stock solution was prepared by mixing 1 volume of absolute ethanol with 1 volume of propylene glycol. For the preparation of an intermediate vehicle stock solution, the vehicle stock solution was diluted 1:4 with diluent (5% Tween 80/5% PEG 400 in sterile water) and then swirled/sonicated until a clear solution was obtained. The intermediate stock solution was diluted 1:82 with sterile saline to yield the temsirolimus vehicle control formulation.

10% Pluronic F68/propylene glycol/PEG 400 (15:42.5:42.5, w:w:w)

A solution containing 10% Pluronic F68 was prepared by dissolving Pluronic F68 in deionized water. The 10% Pluronic F68 solution was added to the appropriate amount of propylene glycol, followed by stirring/sonication, as required. Then the appropriate amount of PEG 400 was added to the 10% Pluronic F68/propylene glycol solution and the mixture was stirred/sonicated until the contents were visibly homogenous. The final solution contained 10% Pluronic F68/propylene glycol/PEG 400 (15:42.5:42.5, w:w:w; F68/PG/PEG400).

Test Article Formulation Preparation

Temsirolimus

Dose formulations of temsirolimus were prepared in three steps. First a stock solution of temsirolimus (20 mg/mL) was prepared by dissolving temsirolimus in absolute alcohol and then mixing with an equal volume of propylene glycol, with sonication until a clear solution was obtained. The stock solution of temsirolimus was prepared once and used for preparation of the dose formulation of temsirolimus prepared on each day of dosing; the stock solution of temsirolimus was stored refrigerated and protected from light between each day of use. An intermediate stock solution (5 mg/mL) of temsirolimus was prepared by mixing, with stirring and sonication, as required, one volume of the temsirolimus stock solution (20 mg/mL) with 3 volumes of diluent (5% Tween 80/5% PEG 400 in sterile water). Each temsirolimus dose formulation was prepared by mixing, with stirring and sonication, as required, 1 volume of the intermediate stock solution (5 mg/mL) with 82 volumes of sterile saline. The nominal concentration of temsirolimus in each dose formulation was 0.06 mg/mL.

Sorafenib

Dose formulations of sorafenib were prepared in 10% Pluronic F68:propylene glycol:PEG 400 (15:42.5;42.5; w:w:w) to contain a nominal concentration of sorafenib tosylate of 2.5 mg/mL. For preparation, the required amount of sorafenib tosylate was added to the vehicle and the mixture was stirred/sonicated until a visually uniform solution was obtained.

Sunitinib

Dose formulations of sunitinib were prepared in sterile water to contain a nominal concentration of sunitinib malate of 0.5 mg/mL. For preparation, the required amount of sunitinib malate was weighed and mixed with the appropriate amount of sterile water. The formulation was stirred until a clear solution was obtained.

Dose Formulation Concentration Analysis

Upon preparation, duplicate samples (0.5 mL) of each dose formulation were obtained and stored frozen (approximately −20 °C) for possible future analysis.

Randomization and Group Assignment

Rats were assigned to their respective treatment group using a computer-generated randomization procedure. Body weights required for randomization were obtained during Week -1. After randomization, each of the 96 rats was assigned to one of eight treatment groups as indicated in study 1 experimental design (Table 3).

Treatments

Dosing of the study animals was accomplished on two consecutive days, wherein two rats per dose group were dosed on the individual days of dosing. For each rat, the day of dosing was designated as Day 1. Each rat was administered a single bolus intravenous (iv) dose of temsirolimus or appropriate vehicle formulation, as described in the table. Thirty (30) minutes after iv administration of temsirolimus or vehicle formulation, sorafenib, sunitinib, or respective vehicle formulation was administered orally (per os; PO). IV doses were administered in a dose volume of 5 mL/kg. PO doses were administered in a dose volume of 10 mL/kg. Dose volumes were based on the most recent individual body weights taken.

Study 2: Oxaliplatin and Sorafenib or Sunitinib

Test Articles

Oxaliplatin (1.2 g; NSC-266046; NSC/sample: 266046-0/7; Lot No.: 100401; SRI Lot No.: M32/L-1; expiration date: unknown), sorafenib, tosylate salt (2.0 g; NSC-724772; NSC/sample: 724772-F/2; Lot No.: BSF-105; SRI Lot No.: M26/L-1; expiration date: April 24, 2019), and sunitinib, malate salt (160 mg; NSC-736511; NSC/sample: 736511-0/2; Lot No.: BST-104; SRI Lot No.: M31/L-1; expiration date: April 27, 2019) were supplied by the National Cancer Institute (Rockville, MD) and received at Southern Research on September 30, 2010 (sorafenib) or October 19, 2010 (oxaliplatin and sunitinib). Each test article was shipped on dry ice. Subsequently, oxaliplatin was stored refrigerated; sorafenib and sunitinib were stored frozen (≤−20 °C) under inert gas.

Vehicle/Vehicle Control Articles

The following reagents were used in the preparation of the dose formulations:

  • Deionized Water (DI-Water) (Southern Research Institute; Birmingham, AL; in house deionized water system)

  • 5% Dextrose Injection, USP (PSS World Medical, Inc.; Kennesaw, GA; Lot No. J0K942; expiration date: November 30, 2011)

  • Pluronic® F-68, solid cell culture tested, insect cell culture tested (Sigma-Aldrich; St. Louis, MO; Lot No. 020M0029; retest date: March 2014)

  • Polyethylene glycol, average M.W. 400 (PEG 400) (Acros Organics; Fair Lawn, NJ; Grouping No. A0284717; expiration date: September 23, 2011)

  • 1, 2 Propanediol, ACS reagent ≥99.5% (propylene glycol) (Aldrich; Milwaukee, WI; Lot No. MKBD1803; expiration date: October 21, 2011)

  • Sterile Water for Injection (Vedco Inc.; St. Joseph, MO; Lot No. 090818A2; expiration date: August 2011)

All reagents were received and stored at room temperature. In addition, polyethylene glycol was stored under nitrogen and propylene glycol was stored protected from light.

Vehicle Control Formulation Preparation

10% Pluronic F68/propylene glycol/PEG 400 (15:42.5:42.5, w:w:w)

A solution containing 10% Pluronic F68 was prepared by dissolving Pluronic F68 in deionized water. The 10% Pluronic F68 solution was added to the appropriate amount of propylene glycol, followed by stirring/sonication, as required. Then the appropriate amount of PEG 400 was added to the 10% Pluronic F68/propylene glycol solution and the mixture was stirred/sonicated until the contents were visibly homogenous. The final solution contained 10% Pluronic F68/propylene glycol/PEG 400 (15:42.5:42.5, w:w:w; F68/PG/PEG400).

Test Article Formulation Preparation

Oxaliplatin

Dose formulations of oxaliplatin were prepared in 5% dextrose in water (D5W) to contain a nominal concentration of oxaliplatin of 3.75 mg/mL. For preparation, the required volume of D5W was added to a weighed amount of oxaliplatin and the mixture was stirred until a visually uniform solution was obtained.

Sorafenib

Dose formulations of sorafenib were prepared in 10% Pluronic F68:propylene glycol:PEG 400 (15:42.5:42.5; w:w:w) to contain a nominal concentration of sorafenib tosylate of 2.5 mg/mL. For preparation, the required amount of sorafenib tosylate was added to the vehicle and the mixture was stirred/sonicated until a visually uniform solution was obtained.

Sunitinib

Dose formulations of sunitinib were prepared in sterile water to contain a nominal concentration of sunitinib malate of 0.5 mg/mL. For preparation, the required amount of sunitinib malate was weighed and mixed with the appropriate amount of sterile water. The formulation was stirred until a clear solution was obtained.

Dose Formulation Concentration Analysis

Upon preparation, duplicate samples (0.5 mL) of each dose formulation were obtained and stored frozen (approximately −20 °C) for possible future analysis.

Randomization and Group Assignment

Prior to dosing on Day 1, the catheter of each rat was checked for patency. All patent catheters were flushed with 0.2 to 0.3 mL of 0.9% saline, followed by 0.1 mL of heparin lock solution. Each of the 96 rats with patent catheters was arbitrarily assigned to one of eight treatment groups as indicated in study 2 experimental design (Table 4).

Treatments

Dosing of the study animals was accomplished on two separate days. Rats in Groups 1, 3, 5, and 7 were dosed on one day and rats in Groups 2, 4, 6, and 8 were dosed two days later. For each rat, the day of dosing was designated as Day 1.

Each rat was administered the drug by iv infusion over a 30-minute interval, of oxaliplatin or corresponding vehicle formulation (D5W), as described in Table 4. Thirty (30) minutes after iv administration of oxaliplatin or corresponding vehicle formulation, sorafenib, sunitinib, or the respective vehicle formulation was administered PO. IV infusion doses were administered at a flow rate of approximately 4 mL/kg/30 min infusion. Actual infusion rates were based on the mean body weight of animals dosed on each day of dosing. PO doses were administered in a dose volume of 10 mL/kg.

Study 3: Gemcitabine and Sorafenib or Erlotinib

Test Articles

Gemcitabine, hydrochloride salt (500 mg; NSC-613327; NSC/sample: 613327-S/2; Lot No.: ML-10-53; SRI Lot No.: M24/L-1; expiration date: unknown); sorafenib, tosylate salt (2 g; NSC-724772; NSC/sample: 724772-F/2; Lot No.: BSF-105; SRI Lot No.: M26/L-1; expiration date: April 24, 2019), and erlotinib, hydrochloride salt (1.6 g; NSC-718781; NSC/sample: 718781/4; Lot No.: BBE-104; SRI Lot No.: M25/L-1; expiration date: November 19, 2018) were supplied by the National Cancer Institute and received from Fisher BioServices (Rockville, MD; gemcitabine, sorafenib) or SAIC-Fredrick (Frederick, MD; erlotinib) on September 30, 2010.

All three test articles were received on dry ice and subsequently stored frozen at approximately −20 °C. Sorafenib and erlotinib were stored frozen (−20 °C) under inert gas.

Vehicle/Vehicle Control Articles

The following reagents were used in the preparation of dose formulations:

  • Carboxymethylcellulose sodium salt, medium viscosity (CMC; 400–800 cps; Sigma Aldrich, St. Louis, MO; Lot No.: 010M0089; assigned expiration date: June 22, 2011).

  • Deionized water (Southern Research Institute; Birmingham, AL; in-house deionized water system).

  • Pluronic® F-68 (solid, cell culture tested, insect cell culture tested; Sigma Aldrich; St. Louis, MO; Lot No.: 020M0029; retest date: March 2014)

  • Polyethylene glycol (PEG 400; average M.W. 400; Acros Organics; Fair Lawn, NJ; Lot No.: A0284717; assigned expiration date: September 23, 2011)

  • 1, 2-Propanediol, ACS reagent ≥99.5% (propylene glycol; Sigma Aldrich; St. Louis, MO; Lot No.: MKBD1803; assigned expiration date: September 23, 2011)

  • Sterile water for injection (Vedco, Inc.; St. Joseph, MO; Lot No.: 090818A2; expiration date: August 2011)

  • Saline solution 0.9% (Nova-Tech, Inc.; Grand Island, NE; Lot No.: 090615A3; expiration date: June 2011)

All reagents were received and stored at room temperature. In addition, polyethylene glycol was stored under nitrogen; propylene glycol was stored protected from light.

Vehicle Control Formulation Preparation

0.5% CMC

A weighed quantity of CMC was slowly added, with stirring, to approximately 90% of the target volume of sterile water. After addition of the total quantity of CMC, the formulation was brought to final volume with sterile water and stirred until a visually uniform solution was obtained.

10% Pluronic F68/propylene glycol/PEG 400 (15:42.5:42.5; w:w:w)

A solution containing 10% Pluronic F68 was prepared by dissolving Pluronic F68 in deionized water. The 10% Pluronic F68 solution was added to the appropriate amount of propylene glycol, followed by stirring/sonication, as required. Then the appropriate amount of PEG 400 was added to the 10% Pluronic F68/propylene glycol solution and the mixture was stirred/sonicated until the contents were visibly homogenous. The final solution contained 10% Pluronic F68/propylene glycol/PEG 400 (15:42.5:42.5; w:w:w).

Test Article Formulation Preparation

Gemcitabine

The dose formulation of gemcitabine was prepared in 0.9% saline to contain a nominal concentration of gemcitabine hydrochloride of 4 mg/mL. For preparation, the required amount of gemcitabine hydrochloride was mixed with saline until dissolution of the gemcitabine hydrochloride occurred.

Sorafenib

The dose formulation of sorafenib was prepared in 10% Pluronic F68:propylene glycol:PEG400 (15:42.5;42.5; w:w:w) to contain a nominal concentration of sorafenib tosylate of 2.5 mg/mL. For preparation, the required amount of sorafenib tosylate was added to the vehicle and the mixture was stirred/sonicated until a visually uniform solution was obtained.

Erlotinib

The dose formulation of erlotinib was prepared in 0.5% CMC to contain a nominal concentration of erlotinib hydrochloride of 15 mg/mL. For preparation, the required amount of erlotinib hydrochloride was mixed with the appropriate volume of 0.5% CMC. The formulation was stirred, sonicated (approximately 10 min), and homogenized (approximately 2 min) until a uniform suspension was obtained.

Dose Formulation Concentration Analysis

Upon preparation, duplicate samples (0.5 mL) of each dose formulation were obtained and stored frozen (approximately −20 °C) for possible future analysis.

Randomization and Group Assignment

Rats were assigned to their respective treatment group using a computer-generated randomization procedure. Body weights required for randomization were obtained during Week -1. After randomization, each of the 96 rats was assigned to one of eight treatment groups as indicated in study 3 experimental design (Table 5). On the day of randomization, the hair was clipped from the inguinal area of each animal to facilitate skin collection at necropsy. Re-clipping was accomplished prior to necropsy as required.

Treatments

Dosing of the study animals was accomplished on two consecutive days, wherein two rats per dose group were dosed on the individual days of dosing. For each rat, the day of dosing was designated as Day 1. Each rat was administered a single bolus iv dose of gemcitabine or appropriate vehicle formulation as described Table 5. Thirty (30) minutes after iv administration of gemcitabine or vehicle formulation, sorafenib, erlotinib, or vehicle formulation was administered PO. IV doses were administered in a dose volume of 5 mL/kg. PO doses were administered in a dose volume of 10 mL/kg. Dose volumes were based on the most recent individual body weights taken. Dose formulations containing erlotinib were suspensions and these were stirred for at least 5 min prior to use for dosing and continuing throughout their period of use for dosing.

Clinical Observations

Rats were observed twice daily during the quarantine and study periods for signs of mortality and moribundity. Detailed clinical examinations of each rat were collected prior to euthanasia.

Body Weights

Each animal was weighed on the day prior to dosing (for the calculation of infusion rate) and on Day 1 prior to dosing.

Tissue Collections and RNA Isolation

At 1, 6, or 24 hours (h) after the completion of dosing, four (4) rats per dose group were euthanized by CO2 asphyxiation. Immediately after euthanasia, selected tissues were collected for RNA isolation and for histopathology. Tissue samples for RNA isolation were collected first and collected as quickly as possible following animal euthanasia.

Tissues for RNA Isolation

Sections of each of the following tissues were collected from each rat for RNA isolation:

Study 1: Bone marrow (left femur), heart (apex, with left and right ventricles), liver (left lateral and median lobes).

Study 2: Kidney (left), heart (apex, with left and right ventricles), liver (left lateral and median lobes).

Study 3: Liver (left lateral and median lobes), heart (apex, with left and right ventricles), skin (inguinal area).

Samples of kidney, heart and liver collected for RNA isolation were cut into sections ≤2 mm in thickness and immediately placed into pre-filled tubes containing RNAlater® (Applied Biosystems; Foster City, CA). Bone marrow was flushed from the bone using RNAlater®.

After collection into RNAlater®, tissues were maintained refrigerated (approximately 4-5 °C) for at least 24 h and then stored at or below −20 °C.

Tissues for Histopathology

Samples of each of the following tissues were collected for histopathology and were fixed in 10% neutral buffered formalin:

Study 1: Bone marrow (right femur), heart, liver.

Study 2: Liver, heart, kidney.

Study 3: Liver, heart, skin (inguinal area).

RNA Isolation and Shipment of Samples

RNA isolation from individual tissues was accomplished using an RNeasy microarray kit (QIAGEN Inc.; Valencia, CA). After extraction, the RNA concentration of each sample was determined using a RiboGreen assay. The RNA Integrity Number (RIN)7 of each sample was also determined using an Agilent 2100 Bioanalyzer with 2100 Expert Software (Version B.02.06.S1418; Agilent; Santa Clara, CA). Only samples with an RIN of 7 or higher were deemed acceptable for gene expression analysis (Data Citation 1: Figshare http://dx.doi.org/10.6084/m9.figshare.7272536). Each RNA sample was diluted to the required concentration (250 ng/μL) and then stored at or below −70 °C prior to shipment to Expression Analysis (Durham, NC) for microarray analysis.

Microarray Analysis

RNA samples were converted into labeled target antisense RNA (cRNA) using the Single-Round RNA Amplification and Biotin Labeling System (Enzo Life Sciences, Farmingdale, NY). Microarray analysis was performed as previously described1. Briefly, 2.5 μg of total RNA was converted into double stranded cDNA via reverse transcription using an oligo-d(T) primer-adaptor. This cDNA was purified and used as a template for in vitro transcription using T7 RNA polymerase and biotinylated ribonucleotides. The resulting cRNA was purified using magnetic beads and quantitated using spectrophotometry. Next, 11 μg of purified cRNA was fragmented using a 5X fragmentation buffer (200 mM Tris-Acetate, pH 8.1, 500 mM KOAc, 150 mM MgOA), then a hybridization cocktail was prepared and added to the fragmentation product using the Hybridization, Wash and Stain kit (Affymetrix, Santa Clara, CA), applied to AffymetrixTM GeneChip® Rat Genome 230 2.0 Arrays, and incubated at 45 °C for 16 h. Following hybridization, arrays were washed and stained using standard Affymetrix procedures before scanning on the Affymetrix GeneChip Scanner 3000 using factory PMT settings to generate array images. Data extraction from the images was completed with the GeneChip Operating Software (GCOS) to generate CEL and CHP files. The microarray analysis method is an expanded version of descriptions in our related work1.

Affymetrix raw CEL files were preprocessed using the robust multichip average (RMA) algorithm5,6 which includes background correction, quantile normalization, summarization by the median polish approach and log base 2 transformation. The Bioconductor8 R packages “affy”, “affyPLM”, “affyQCReport”, “simpleaffy” and “arrayQualityMetrics” were used for array data quality assessment and visualization.

Code availability

No custom code was used for analysis. Microarray analysis and technical validation were performed using R function calls in the publicly available Bioconductor8 R packages.

Histopathology

Histology

All collected tissues from rats in each dose group were processed for histopathological examination. The fixed tissues were trimmed, processed, and microtomed (approximately 5-µm sections). The tissue sections were mounted on glass slides, stained with hematoxylin and eosin, and coverslipped. In Study 1 (Temsirolimus and Sorafenib or Sunitinib) special stains (trichrome and Congo red) were performed on the kidney of one animal (2M14) at the discretion of the pathologist in order to establish a diagnosis.

Microscopic Observations

All slides were examined microscopically by a pathologist. Records of gross findings for a specimen from postmortem observations were available to the pathologist when examining that specimen histopathologically. Indication of a histopathology observation and description of the finding are in Data Citation 1: Figshare http://dx.doi.org/10.6084/m9.figshare.7272536.

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