Laura S. Chubb

Anti-angiogenic effects of interleukin-12 delivered by a novel hyperthermia induced gene construct

Purpose: Interleukin-12 (IL-12) is a pro-inflammatory cytokine possessing anti-cancer and anti-angiogenic properties. This study quantitatively assessed the anti-angiogenic effect of IL-12 delivered using an adenoviral vector with murine IL-12 placed under control of a heat shock promoter. This approach limits systemic toxicity by restricting IL-12 delivery locally to the tumour. The kinetics of the downstream cytokines interferon-γ (IFN-γ) and interferon inducible protein-10 (IP-10) and other molecules affecting angiogenesis, vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) were also studied. Materials and methods: 4T1 tumours were grown in Balb/C mice and the AdhspmIL-12 construct was injected intra-tumourally. The tumours were heated after 24 h using a water bath. At various time points post-heating the tumours were collected and quantitatively assessed for cytokine production and vascularity. Results: A significant reduction was seen in the tumour vasculature of the treated group vs. the control group mice. Systemic effects of IL-12 were limited to generalized immunostimulation. No hepatoxicity was noted. Conclusions: This study suggests that IL-12 can be effectively delivered using a gene-based approach with a heat shock promoter. This results in quantitatively measurable anti-angiogenesis and general immunostimulation. The complex inter-play of other pro- and anti-angiogenic factors (IFN-γ, IP-10, VEGF and PAI-1) was also studied.

Regional Induction of CYP1A1 in Rat Liver Following Treatment with Mixtures of PCB 126 and PCB 153

Liver enzyme induction has been shown previously to be regional with clear borders between induced and uninduced regions in vivo, and cells either fully induced or not induced in vitro. The current study examined this phenomenon in vivo by evaluating enzyme induction after exposure to PCB 126 and PCB 153 in female Fisher 344 (F344) and male Sprague—Dawley (SD) rats. IHC revealed a regional induction of CYP1A1 after exposure to PCB 126, apparent in the centrilobular region at lower doses and progressing to panlobular with higher doses. PCB 153 exposure induced CYP2B1/2 in the centrilobular region, which spread to the midzonal region as the dose increased, but never became panlobular even at the highest dosage tested. In rats treated with PCB 126 in combination with high doses of PCB 153, induction of CYP1A1 occurred preferentially in the periportal region, a reversal from the pattern seen with PCB 126 alone. This CYP1A1 induction pattern reversal is a unique example of complex biological interactions between coplanar (PCB 126) and noncoplanar (PCB 153) halogenated aromatic hydrocarbons.

Transcription Factor Ets1 Cooperates with Estrogen Receptor α to Stimulate Estradiol-Dependent Growth in Breast Cancer Cells and Tumors

The purpose of this study was to explore the role of transcription factor Ets1 in estrogen receptor α (ERα)-positive breast cancer progression. We expressed human Ets1 or empty vector in four human ERα-positive breast cancer cell lines and observed increased colony formation. Further examination of cellular responses in stable Ets1-expressing MCF7 clones displayed increased proliferation, migration, and invasion. Ets1-expressing MCF7 tumors grown in the mammary fat pads of nude mice exhibited increased rates of tumor growth (7.36±2.47 mm3/day) compared to control MCF7 tumors (2.52±1.70 mm3/day), but maintained their dependence on estradiol for tumor growth. Proliferation marker Ki-67 staining was not different between control and Ets1-expressing tumors, but Ets1-expressing tumors exhibited large necrotic centers and elevated apoptotic staining. Ets1 was shown to cooperate with ERα and the p160 nuclear receptor coactivator (NCOA/SRC) family to increase activation of a consensus estrogen response element luciferase reporter construct. Ets1-expressing MCF7 cells also exhibited elevated expression of the ERα target genes, progesterone receptor and trefoil factor 1. Using GST-pulldown assays, Ets1 formed stable complexes containing both ERα and p160 nuclear receptor coactivators. Taken together, these data suggest that the Ets1-dependent estradiol sensitization of breast cancer cells and tumors may be partially due to the ability of Ets1 to cooperate with ERα and nuclear receptor coactivators to stimulate transcriptional activity of estrogen-dependent genes.

Use of a medium-term liver focus bioassay to assess the hepatocarcinogenicity of 1,2,4,5-tetrachlorobenzene and 1,4-dichlorobenzene.

1,2,4,5-Tetrachlorobenzene (TeCB) and 1,4-dichlorobenzene (DCB) are important environmental contaminants that have been used extensively for a variety of industrial applications. Limited data are available in the literature regarding the carcinogenicity of TeCB. DCB has been shown to cause renal adenocarcinomas in rats and hepatic adenomas and carcinomas in mice at high doses in a 2-year study. In the studies presented here, we report that TeCB can promote the formation of preneoplastic foci and DCB cannot in a medium-term initiation/promotion assay. These results suggest that TeCB is a liver tumor promoter and that DCB is not at fairly low doses (0.1 and 0.4 mmol/kg per day).

Do Mesenchymal Stromal Cells Influence Microscopic Residual or Metastatic Osteosarcoma in a Murine Model

BackgroundMesenchymal stromal cells (MSCs) have been shown in rodent models to promote primary and pulmonary metastatic sarcoma growth when injected in the presence of gross tumor. In theory, this would limit their use in a clinical setting after limb salvage treatment for osteosarcoma. Although concerning, these models do not translate to the clinical setting wherein MSCs could be used after primary tumor resection to aid in bone healing and incorporation of tumor endoprostheses. If we can determine whether the use of MSCs in this setting is safe, it might improve our ability to augment bone healing in patients undergoing limb salvage.Questions/purposesThe purpose of this study was to determine (1) whether MSCs promote pulmonary metastatic disease progression in a murine osteosarcoma model; and/or (2) whether they affect local disease recurrence in the presence of microscopic residual osteosarcoma.MethodsAn orthotopic model of luciferase-expressing osteosarcoma was developed. At 10 days, resection of the primary tumor was performed. One hundred fourteen female C3H mice were inoculated with DLM8-luc osteosarcoma in the proximal tibia. Ninety-four mice developed orthotopic osteosarcoma with luciferase expression. Mice with bioluminescent evidence of a primary tumor received either a microscopically “clean” amputation at a time when residual microscopic metastatic disease was present in the lungs (pulmonary metastasis group; n = 65) or a “dirty” amputation (local recurrence group; n = 29). Mice were randomized to receive intravenous MSCs, MSCs at the surgical site, or no MSCs. Mice were monitored for development and progression of pulmonary metastasis and local recurrence by bioluminescence imaging and daily measurements at the surgical site. The number of pulmonary nodules, time to first evidence of metastasis, and size of recurrent tumor were compared using Kruskal-Wallis, analysis of variance, Welch’s, t-tests, or Mann-Whitney tests as appropriate for the specific data sets with p < 0.05 considered significant.ResultsMice receiving intravenous MSCs had a faster time to first detection of pulmonary metastasis (2.93 ± 1.90 days) compared with mice with local injection of MSCs (6.94 ± 6.78 days) or no MSCs (5.93 ± 4.55 days) (p = 0.022). MSC treatment did not influence whether mice developed local recurrence (p = 0.749) or size of recurrent tumors (p = 0.221).ConclusionsMSCs delivered to the surgical site did not promote local recurrence or size of recurrent tumors, but intravenous injection of MSCs did hasten onset of detection of pulmonary metastatic disease. Although local administration of MSCs into a surgical site does not appear to promote either pulmonary metastatic disease or local recurrence, large variation within groups and small numbers diminished statistical power such that a Type II error cannot be ruled out.Clinical RelevanceIf MSCs are to be used to augment bone healing in the postlimb salvage setting in patients with osteosarcoma, it will be important to understand their influence, if any, on pulmonary micrometastsis or residual microscopic local disease. Although murine models do not completely recapitulate the clinical scenario, these results suggest that intravenous delivery of MSCs may promote micrometastatic pulmonary disease. Local administration into a surgical wound, even in the presence of residual microscopic disease, may be safe, at least in this murine model, but further investigation is warranted before considering the use of MSCs for clinical use in patients with osteosarcoma.

Combined delivery of FGF-2, TGF-β1, and adipose-derived stem cells from an engineered periosteum to a critical-sized mouse femur defect

Critical-sized long bone defects suffer from complications including impaired healing and non-union due to substandard healing and integration of devitalized bone allograft. Removal of the periosteum contributes to the limited healing of bone allografts. Restoring a periosteum on bone allografts may provide improved allograft healing and integration. This article reports a polysaccharide-based tissue engineered periosteum that delivers basic fibroblast growth factor (FGF-2), transforming growth factor-β1 (TGF-β1), and adipose-derived mesenchymal stem cells (ASCs) to a critical-sized mouse femur defect. The tissue engineered periosteum was evaluated for improving bone allograft healing and incorporation by locally delivering FGF-2, TGF-β1, and supporting ASCs transplantation. ASCs were successfully delivered and longitudinally tracked at the defect site for at least 7 days post operation with delivered FGF-2 and TGF-β1 showing a mitogenic effect on the ASCs. At 6 weeks post implantation, data showed a non-significant increase in normalized bone callus volume. However, union ratio analysis showed a significant inhibition in allograft incorporation, confirmed by histological analysis, due to loosening of the nanofiber coating from the allograft surface. Ultimately, this investigation shows our tissue engineered periosteum can deliver FGF-2, TGF-β1, and ASCs to a mouse critical-sized femur defect and further optimization may yield improved bone allograft healing.

Orthotopic model of canine osteosarcoma in athymic rats for evaluation of stereotactic radiotherapy

OBJECTIVE To develop an orthotopic model of canine osteosarcoma in athymic rats as a model for evaluating the effects of stereotactic radiotherapy (SRT) on osteosarcoma cells. ANIMALS 26 athymic nude rats. PROCEDURES 3 experiments were performed. In the first 2 experiments, rats were injected with 1 × 10(6) Abrams canine osteosarcoma cells into the proximal aspect of the tibia (n = 12) or distal aspect of the femur (6). Tumor engraftment and progression were monitored weekly via radiography, luciferase imaging, and measurement of urine pyridinoline concentration for 5 weeks and histologic evaluation after euthanasia. In the third experiment, 8 rats underwent canine osteosarcoma cell injection into the distal aspect of the femur and SRT was administered to the affected area in three 12-Gy fractions delivered on consecutive days (total radiation dose, 36 Gy). Percentage tumor necrosis and urinary pyridinoline concentrations were used to assess local tumor control. The short-term effect of SRT on skin was also evaluated. RESULTS Tumors developed in 10 of 12 tibial sites and all 14 femoral sites. Administration of SRT to rats with femoral osteosarcoma was feasible and successful. Mean tumor necrosis of 95% was achieved histologically, and minimal adverse skin effects were observed. CONCLUSIONS AND CLINICAL RELEVANCE The orthotopic model of canine osteosarcoma in rats developed in this study was suitable for evaluating the effects of local tumor control and can be used in future studies to evaluate optimization of SRT duration, dose, and fractionation schemes. The model could also allow evaluation of other treatments in combination with SRT, such as chemotherapy or bisphosphonate, radioprotectant, or parathyroid hormone treatment.

Abstract A22: The impact of dose reduction upon local tumor control for stereotactic radiation therapy in an orthotopic rat model of osteosarcoma

Purpose: Primary bone tumors such as osteosarcoma (OSA) are rare but devastating tumors that most commonly affect young people. For adolescents and dogs with OSA, local tumor control can be difficult to achieve with finely fractionated radiation therapy. Stereotactic radiation therapy (SRT) is a promising treatment modality that results in the delivery of ablative radiation dose to the tumor, while preferentially minimizing dose to the surrounding normal tissues. In our clinical experience with SRT in dogs, a total dose of 36Gy delivered in three fractions has been associated with excellent local tumor control based upon clinical response and histopathology of the irradiated tumor. The risk of post radiation fracture is related to pre-existing tumor associated osteolysis and the effect of irradiation upon normal bone. A previously established orthotopic rat model was used to perform a dose reduction study in an effort to optimize the SRT protocol. Methods & Materials: Twenty-four nude rats were injected with 1 x 10^6 canine osteosarcoma cells into the distal femoral metaphyses. Two weeks after tumor inoculation, rats were treated with SRT delivered once daily in three equal fractions. The tumor affected bone of six rats each were treated to a total dose of 36Gy (12Gy/fx), 33Gy (11Gy/fx), 30Gy (10Gy/fx), or 27Gy (9Gy/fx). Tumor viability and response to SRT were evaluated once weekly for six weeks via bioluminescence imaging, radiographs of the femur, and urine pyridinoline levels (PYD). Acute radiation effects of the skin were evaluated by Veterinary Radiation Therapy Oncology Group (VROTG) normal tissue toxicity scoring rubric. Post mortem histology provided an objective measure of tumor necrosis. Results: In all treatment groups, PYD levels were significantly increased two weeks after tumor inoculation (p Conclusions: All four protocols were well tolerated and associated with mild, transient skin toxicity. In canine and human osteosarcoma data, improved duration of local tumor control is correlated with tumor necrosis of 80% or higher. While there was no statistical significance between treatment groups, only the 36Gy group achieved that benchmark of tumor control. Consideration of SRT as a therapeutic option for adolescents with OSA requires further evaluation via orthotopic models. Future dose reduction studies evaluating the clinically relevant concurrent administration of carboplatin and SRT are warranted. Citation Format: James T. Custis, Anthony L. Schwartz, Joseph F. Harmon, Barbara E. Powers, Laura S. Chubb, Susan M. LaRue, Nicole P. Ehrhart, Stewart D. Ryan. The impact of dose reduction upon local tumor control for stereotactic radiation therapy in an orthotopic rat model of osteosarcoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A22.

Abstract B48: Subcutaneous delivery of docetaxel and carboplatin accumulate preferentially in lymphatic circulation as compared to intravenous delivery in rats with surgically created lymph and venous fistulae

Purpose: Intravenous delivery of chemotherapy is an indisputable pillar of cancer therapy, but associated with unfavorable systemic toxicity. Locally delivered chemotherapy – via subcutaneous, surgical placement, or intracavitary routes – is effective in animal models of breast cancer and in client-owned dogs receiving treatment for bone tumors. There are unexplained beneficial effects of chemotherapy delivered via nontraditional routes resulting in decreased systemic toxicity and positive tumor control at dosages less than traditionally given. One role of the lymphatic system is to return large particulate matter and proteins in the interstitial space to the vascular system which cannot be absorbed by adjacent blood capillaries. This unidirectional circulatory pathway for particles deposited in areas outside of blood capillaries has not been characterized to date for nontraditionally delivered chemotherapy agents. Experimental Design: In this study the pharmacokinetics of docetaxel and carboplatin in the blood and lymph following subcutaneous (SQ) versus intravenous (IV) delivery was determined in a surgical lymphatic and hemovascular cannulated rat model. In anesthetized adult male Sprague Dawley rats, fixed length polyurethane catheters were surgically placed simultaneously into a jugular vein and the lymphatic thoracic duct in each animal. Either docetaxel (5 mg/kg) or carboplatin (14 or 28 mg/kg) were delivered IV via tail vein catheter or SQ into the mammary fat pad. Rats were permitted free movement and access to water and food ad libitum. Paired blood and lymph samples were serially collected in animals at 0, ½, 1, 2, 3, 4, 12, and 24 hrs. Drug levels were determined via LC/MS/MS or ICP/MS for docetaxel and carboplatin, respectively. Pharmacokinetic parameters were determined via noncompartmental analysis. Results: The maximum concentration measured (Cmax) and the area under the time-concentration curve (AUC0-24hr) in the plasma and lymph for docetaxel delivered IV were 2.86 µg/ml and 1160 hr*ng/ml, and 172 ng/ml and 1020 hr*ng/m, respectively. When administered SQ, the Cmax and AUC0-24hr were 0.644 µg/ml and 815 hr*ng/ml, and 107 ng/ml and 1650 hr*ng/ml, for plasma and lymph respectively, which demonstrates preferential lymphatic accumulation when delivered SQ. The terminal half-life (t1/2) in lymph for docetaxel is greater with either IV or SQ delivery (18.3 hr and 22.6 hr, respectively) as compared to t1/2 in blood for docetaxel with either IV or SQ delivery (9.6 hr and 6.0 hr, respectively), possibly suggesting lipophilic docetaxel resides in the lymph for longer periods. The Cmax and AUC0-24hr in the plasma and lymph for total platinum following delivery of carboplatin (14 mg/kg) IV were 23.0 µg/ml and 28.8 hr*µg/ml, and 16.4 µg/ml and 36.1 hr*µg/m, respectively. When delivered SQ, the Cmax and AUC0-24hr for the plasma and lymph were 6.99 µg/ml and 16.6 hr*µg/ml, and 9.39 µg/ml and 24.3 hr*µg/ml, respectively. While a dose response was observed in the plasma regardless of route of administration with the Cmax in the plasma following carboplatin (28 mg/kg) of 40.0 µg total platinum/ml when given IV and 12.9 µg total platinum/ml when given SQ, the AUC0-24hr did not when administered IV. Additionally, the AUC0-24hr in the lymph was 31.0 hr*µg/ml following IV dosing at 28 mg/kg which is similar to that observed in the plasma. However, when dosed SQ at 28 mg/kg, the Cmax and AUC0-24hr for total platinum in the lymph responded in a dose dependent manner with 17.3 µg/ml and 76.7 hr*µg/ml, respectively. This demonstrates preferential lymphatic accumulation of carboplatin delivered SQ and that there is a dose response with regards to the level of total platinum in the lymph when delivered SQ. Conclusions: Nontraditional SQ delivery of docetaxel and carboplatin achieves targeted lymphatic accumulation greater than with traditional IV delivery. This study has broad implication for dosing strategies of chemotherapeutics for cancers metastasizing predominantly via lymphatic pathways and for cancer patients susceptible to toxicity resulting from peak maximum hemovascular concentrations that may be lessened via SQ delivery. Citation Format: Deanna R. Worley, Ryan J. Hansen, Laura S. Chubb, Daniel L. Gustafson. Subcutaneous delivery of docetaxel and carboplatin accumulate preferentially in lymphatic circulation as compared to intravenous delivery in rats with surgically created lymph and venous fistulae. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B48.