Subcutaneous Tumors In SCID Mice Research Articles

Abstract 2700: BRCA1-IRIS overexpression promotes formation of aggressive breast cancer.

Abstract Introduction: Women with Her2+ or triple negative/basal-like (TN/BL) breast cancers succumb to their cancer rapidly and early in life, due in part to Herceptin resistance that develops in Her2+ patients and because of the lack of appropriate therapies for TN/BL patients. Great effort is being made now to uncover appropriate drug targets to overcome these shortcomings. In this report we examine whether BRCA1-IRIS can be a valuable treatment target for Her2+ and/or TN/BL tumors. Methods: Immunohistochemistry (IHC) staining of breast tumor samples using a newly generated mouse monoclonal anti-BRCA1-IRIS antibody was performed. BRCA1-IRIS expression was correlated to that of AKT1, AKT2, p-AKT, survivin, ERα, PR, and BRCA1/p220 expression detected using commercial antibodies. Where available, patients’ tumor grade, tumor stage, age at diagnosis, and node positivity were also compared. We also generated subcutaneous tumors in SCID mice using human mammary epithelial (HME) cells overexpressing BRCA1-IRIS and then compared AKT and survivin expression, and tumor phenotype and aggressiveness to RasV12-induced tumors. Results: The majority of human breast tumors, especially Her2+ and TN/BL tumors, were BRCA1-IRIS-positive. These tumors also expressed high levels of AKT1, AKT2, p-AKT, and survivin, but were ERα-, PR-, and BRCA1/p220-negative. BRCA1-IRIS overexpressing Her2+ and TN/BL tumors were high-grade tumors, and showed lymph-node positivity and/or distant metastasis compared to those not overexpressing BRCA1-IRIS. BRCA1-IRIS overexpression also significantly reduced the age at which patients with Her2+ or TN/BL tumors were diagnosed compared to patients with the same tumors but negative for BRCA1/IRIS (51.4±16.2 vs. 64.5±13.2, p≤0.05 for Her2+ and 48.9±10.1 vs. 70.8±6.8, p≤0.01 for TN/BL). Surprisingly, TN/BL/BRCA1-IRIS-positive tumors were larger than Her2+/BRCA1-IRIS-positive tumors (30.6±19.3 vs. 16.1±9.7, p≤0.05). HME cells overexpressing BRCA1-IRIS developed tumors when injected subcutaneously in SCID mice. These tumors were invasive and showed high expression levels of AKT1, AKT2, and p-AKT when compared to RasV12-induced tumors. These tumors also overexpressed the mesenchymal protein vimentin, but did not express the epithelial proteins CK19 and p63, and were BRCA1/p220-negative. In contrast, the less aggressive RasV12-induced tumors expressed CK19, p63, and BRCA1/p220, but not vimentin. Conclusion: These data demonstrate that BRCA1-IRIS overexpression triggers aggressive breast tumor formation, especially in patients with tumors of the Her2+ or TN/BL subtypes. We propose that BRCA1-IRIS inhibition may be used as a novel therapeutic option for the treatment of aggressive breast tumors of the Her2+ and/or TN/BL subtype. Citation Format: Wael M. El Shamy. BRCA1-IRIS overexpression promotes formation of aggressive breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2700. doi:10.1158/1538-7445.AM2013-2700

Intratumoral acetic acid injection eradicates human prostate cancer tumors in a murine model

To treat localized prostate cancer without substantial morbidity, an ideal treatment would be an effective local therapy with minimal morbidity. Direct injections have been used to treat benign prostatic hyperplasia without major complications, but in limited cases. We evaluated the local oncotoxic effects of acetic acid in a prostate cancer xenograft murine model. PC3 and LNCaP human prostate cancer cell lines were used to grow subcutaneous tumors in SCID mice. For each cell line, 14 mice underwent intratumor injection with 25% acetic acid (0.05 ml/100 cm3 of tumor) after the tumor was >300 mm3. Post-treatment one mouse/group was euthanized after 2 h, 24 h, 1 and 2 weeks; remaining mice (n = 10) were killed at 120 days. Control mice (8/group) were euthanized after they met the humane criteria for tumor burden and overall health. Tumor necrosis was noted immediately post-injection; by 24 h, ulceration and crusting of overlying skin were noted, which healed into scars by 23 ± 5 days. Histological examination showed tumor degeneration and necrosis with blood vessel obstruction. Ten treated mice in both groups survived for 120 days, which was much longer than the mean survival of PC3 (40 ± 9 days) and LNCaP (56 ± 10) control mice. Direct injection of acetic acid successfully eradicated both tumors. This treatment option could potentially be used in humans for treatment of early localized prostate cancer and nonoperative management of locally advanced cases. This is the first report of successful local chemical therapy for prostate cancer.

Abstract 5319: BRCA1-IRIS overexpression promotes formation of aggressive breast cancers

Abstract Introduction: Women with Her2+ or triple negative/basal-like (TN/BL) breast cancers succumb to their cancer rapidly and early in life, due in part to Herceptin resistance that develops in Her2+ patients and because of the lack of appropriate therapies for TN/BL patients. Great effort is being made now to uncover appropriate drug targets to overcome these shortcomings. In this report we examine whether BRCA1-IRIS can be a valuable treatment target for Her2+ and/or TN/BL tumors. Methods: Immunohistochemistry (IHC) staining of breast tumor samples using a newly generated mouse monoclonal anti-BRCA1-IRIS antibody was performed. BRCA1-IRIS expression was correlated to that of AKT1, AKT2, p-AKT, survivin, ER[[Unsupported Character - Symbol Font ]], PR, and BRCA1/p220 expression detected using commercial antibodies. Where available, patients’ tumor grade, tumor stage, age at diagnosis, and node positivity were also compared. We also generated subcutaneous tumors in SCID mice using human mammary epithelial (HME) cells overexpressing BRCA1-IRIS and then compared AKT and survivin expression, and tumor phenotype and aggressiveness to RasV12-induced tumors. Results: The majority of human breast tumors, especially Her2+ and TN/BL tumors, were BRCA1-IRIS-positive. These tumors also expressed high levels of AKT1, AKT2, p-AKT, and survivin, but were ER[[Unsupported Character - Symbol Font ]]-, PR-, and BRCA1/p220-negative. BRCA1-IRIS overexpressing Her2+ and TN/BL tumors were high-grade tumors, and showed lymph-node positivity and/or distant metastasis compared to those not overexpressing BRCA1-IRIS. BRCA1-IRIS overexpression also significantly reduced the age at which patients with Her2+ or TN/BL tumors were diagnosed compared to patients with the same tumors but negative for BRCA1/IRIS (51.4±16.2 vs. 64.5±13.2, p≤0.05 for Her2+ and 48.9±10.1 vs. 70.8±6.8, p≤0.01 for TN/BL). Surprisingly, TN/BL/BRCA1-IRIS-positive tumors were larger than Her2+/BRCA1-IRIS-positive tumors (30.6±19.3 vs. 16.1±9.7, p≤0.05). HME cells overexpressing BRCA1-IRIS developed tumors when injected subcutaneously in SCID mice. These tumors were invasive and showed high expression levels of AKT1, AKT2, and p-AKT when compared to RasV12-induced tumors. These tumors also overexpressed the mesenchymal protein vimentin, but did not express the epithelial proteins CK19 and p63, and were BRCA1/p220-negative. In contrast, the less aggressive RasV12-induced tumors expressed CK19, p63, and BRCA1/p220, but not vimentin. Conclusion: These data demonstrate that BRCA1-IRIS overexpression triggers aggressive breast tumor formation, especially in patients with tumors of the Her2+ or TN/BL subtypes. We propose that BRCA1-IRIS inhibition may be used as a novel therapeutic option for the treatment of aggressive breast tumors of the Her2+ and/or TN/BL subtype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5319. doi:1538-7445.AM2012-5319

BRCA1-IRIS Overexpression Promotes Formation of Aggressive Breast Cancers

IntroductionWomen with HER2+ or triple negative/basal-like (TN/BL) breast cancers succumb to their cancer rapidly due, in part to acquired Herceptin resistance and lack of TN/BL-targeted therapies. BRCA1-IRIS is a recently discovered, 1399 residue, BRCA1 locus alternative product, which while sharing 1365 residues with the full-length product of this tumor suppressor gene, BRCA1/p220, it has oncoprotein-like properties. Here, we examine whether BRCA1-IRIS is a valuable treatment target for HER2+ and/or TN/BL tumors.Methodology/Principal FindingsImmunohistochemical staining of large cohort of human breast tumor samples using new monoclonal anti-BRCA1-IRIS antibody, followed by correlation of BRCA1-IRIS expression with that of AKT1, AKT2, p-AKT, survivin and BRCA1/p220, tumor status and age at diagnosis. Generation of subcutaneous tumors in SCID mice using human mammary epithelial (HME) cells overexpressing TERT/LT/BRCA1-IRIS, followed by comparing AKT, survivin, and BRCA1/p220 expression, tumor status and aggressiveness in these tumors to that in tumors developed using TERT/LT/RasV12-overexpressing HME cells. Induction of primary and invasive rat mammary tumors using the carcinogen N-methyl-N-nitrosourea (NMU), followed by analysis of rat BRCA1-IRIS and ERα mRNA levels in these tumors.High BRCA1-IRIS expression was detected in the majority of human breast tumors analyzed, which was positively correlated with that of AKT1-, AKT2-, p-AKT-, survivin, but negatively with BRCA1/p220 expression. BRCA1-IRIS-positivity induced high-grade, early onset and metastatic HER2+ or TN/BL tumors. TERT/LT/BRCA1-IRIS overexpressing HME cells formed invasive subcutaneous tumors that express high AKT1, AKT2, p-AKT and vimentin, but no CK19, p63 or BRCA1/p220. NMU-induced primary and invasive rat breast cancers expressed high levels of rat BRCA1-IRIS mRNA but low levels of rat ERα mRNA.Conclusion/SignificanceBRCA1-IRIS overexpression triggers aggressive breast tumor formation, especially in patients with HER2+ or TN/BL subtypes. We propose that BRCA1-IRIS inhibition may be pursued as a novel therapeutic option to treat these aggressive breast tumor subtypes.

Abstract 1578: A critical role for RhoA-GTPase signaling in the tumor vascular disrupting action of combretastatin-A4-phosphate in vivo

Abstract Background: Tubulin binding microtubule depolymerising agents form a growing group of tumor vascular disrupting agents (VDAs) in clinical trial, with combretastatin A-4-phosphate (CA4P) the lead compound. Signalling through RhoGTPase/ROCK-dependent pathways is central to CA4P-induced effects on endothelial cells in vitro (1). Here, we tested the hypothesis that RhoGTPase/ROCK signalling is also important in tumor vascular damaging effects in vivo. Experimental procedures: SW1222 human colorectal carcinoma cells were grown as solid sub-cutaneous tumors in SCID mice. The Rho kinase (ROCK) inhibitor, Y-27632 (50 mg/kg) or saline vehicle, was administered intraperitoneally (i.p.), 5 minutes prior to 100 mg/kg CA4P or saline i.p. Laser Doppler flowmetry was used to assess tumor vascular response from 0 – 2h post-treatment, under isofluorane anaesthesia. Intravenous administration of fluorescent tomato lectin was used for assessing tumor perfusion at 1, 3, 6 and 24 hours post-CA4P/saline treatment, unanaesthetised. Necrosis (H&E) and leukocyte infiltration (immunohistochemistry) were assessed at 24h from excised tumors. Mean arterial blood pressure (MABP) was monitored in unanaesthetised mice without tumors, using a tail cuff system. Results: Y-27632 alone significantly decreased MABP by 40% but did not significantly affect tumor necrosis at 24 hours (17±4% of tumor sectional area for treated versus 10±3% for controls). Y-27632 alone reduced relative red cell flux in the tumor (laser Doppler flowmetry) by approximately 50%, which was similar to the reduction observed for CA4P alone. However, pre-treatment with Y-27632 did not affect CA4P-induced laser Doppler or perfused vascular volume measurements in the first few hours after CA4P and significantly reduced the effect of CA4P on perfused vascular volume and necrosis measured at 6 and 24 hours. Necrosis was 61±5% for CA4P alone and 35±7% for Y-27632+CA4P. These changes were accompanied by a decrease in staining for the myeloid markers, myeloperoxidase and GR-1 with Y-27632 pre-treatment. Conclusions: The Y-27632-induced decrease in MABP is consistent with the decrease in relative red cell flux in the tumor, via a decrease in tumor perfusion pressure. Despite these effects of Y-27632 alone, its administration prior to CA4P was protective of CA4P-induced vascular damage at the later time-points, suggesting that ROCK inhibition is acting downstream from initial vascular shut-down, potentially via modulation of myeloid cell recruitment. These data indicate that RhoGTPase/ROCK-dependent signalling is a critical factor in determining extent of vascular disruption by CA4P – these results have significance for similar VDAs in development. References (1) Kanthou C. & Tozer GM, 2002, Blood 99: 2060-2069. Funded by Cancer Research UK Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1578. doi:10.1158/1538-7445.AM2011-1578

Optimizing prostate cancer suicide gene therapy using herpes simplex virus thymidine kinase active site variants.

The herpes simplex virus (HSV) thymidine kinase gene (tk) forms the basis of a widely used strategy for suicide gene therapy. A library of HSV thymidine kinase enzyme (TK) active site mutants having different affinities for guanosine analog prodrugs was developed. We sought to determine the optimal combination of tk variant and prodrug specifically for prostate cancer gene therapy, using in vitro and in vivo studies of adenovirally infected CL1, DU-145, and LNCaP tumor lines carrying wild-type tk, tk30, tk75, and sr39tk mutants expressed by a strong, constitutive cytomegalovirus promoter and treated with ganciclovir and acyclovir. In vitro experiments involving prostate cancer (CaP) cell line infection were carried out with a broad range of prodrug concentrations, and cell killing was determined by limiting dilution (colony-forming), MTT, and propidium iodide assays. In vivo studies based on CL1-GFP xenograft experiments were carried out to examine the ability of each TK variant to prevent tumor formation and to inhibit tumor growth and development of metastases in established orthotopic and subcutaneous tumors in SCID mice. Both in vitro and in vivo studies suggest improved killing with the sr39tk variant. Thus, the results suggest that the use of sr39tk in future trials of prostate cancer tk suicide gene therapy may be beneficial.

Tumor cell-specific transgene expression prevents liver toxicity of the adeno-HSVtk/GCV approach.

Treatment of colorectal liver metastases with the HSVtk/GCV approach and adenoviral vectors is highly toxic. We present a nontoxic alternative using the cell type-specific CEA promoter instead of the widely used hCMV immediate-early promoter to drive tk gene expression in the context of a recombinant adenovirus. Analysis of CEA promoter-dependent tk gene expression showed significant activity of this promoter in several human and rat tumor-derived cell lines but not in rat primary hepatocytes and in mouse liver, whereas the CMV promoter was highly active in all cell types and tissues investigated. CEA promoter-dependent tk gene expression was sufficient to kill 100% of cancer cells in vitro, even if less than 10% were infected by the adenoviral vector, indicating a significant bystander effect. Moreover, treatment of subcutaneous tumors in SCID mice with Ad.CEA-tk led to a several-fold reduction of tumor growth, and tail vein injection of a high dose of Ad.CEA-tk caused no side-effects in the liver. The CMV promoter was more potent than the CEA promoter in mediating GCV sensitivity to cancer cells in vitro and in vivo, but even a 20-fold reduction of the dose of Ad.CMV-tk did not prevent its liver cell toxicity after systemic application to mice and still resulted in the death of all animals within 4 days after the start of GCV treatment. These results indicate that restriction of tk gene expression to tumor cells in the liver prevents systemic toxicity. Moreover, the CEA promoter is a safe and efficient tool for tumor cell-specific expression of suicide genes in the liver.

RB-reconstituted human retinoblastoma cells form RB-positive intraocular and intracerebral but not subcutaneous tumors in SCID mice.

WERI-Rb27 human retinoblastoma cells were reconstituted with an intact RB gene by retrovirus-mediated gene transfer, in order to study the phenotypic effects of the protein in vitro and in vivo. Extensive morphological changes were observed, dominated by the formation of multinucleated giant cells. Six weeks after retroviral infection, the giant cells began to die and small cells emerged, resembling the parental non-reconstituted line. They expressed RB and continued to grow, although they showed an increased sensitivity to serum starvation. The original RB-negative cells grew progressively after subcutaneous inoculation into SCID mice, whereas the reconstituted cells failed to grow. RB-positive cells grew progressively in the corpus vitreum of the eye and in the brain, however. The RB-reconstituted cells grew more slowly and were less invasive than the parental cells and cells infected with a firefly luciferase (LUX) gene carrying retrovirus, used as controls. RB-reconstituted cells re-explanted from the intraocular and intracranial tumors continued to express full-length RB protein. RBeye2, an RB-positive cell line established from an eye tumor, was still unable to grow subcutaneously. The reduced tumorigenicity of the RB-reconstituted cells in the subcutaneous space may be due to the influence of locally acting growth-controlling signals or the absence of microenvironment-specific trophic factors. Alternatively, it may reflect the action of residual immune effectors in the SCID mice. If this is the case, these would have to be more effective at the subcutaneous site than in the eye or brain.

OMA-AML-1: a leukemic myeloid cell line with CD34+ progenitor and CD15+ spontaneously differentiating cell compartments

OMA-AML-1 was established from a patient with acute myelomonocytic (M4) leukemia at fifth relapse when blasts were greater than 85% CD34+, CD15-. Leukemic cells were established in suspension culture and independently grown as subcutaneous tumors in SCID mice. Cells growing in suspension culture underwent differentiation by phenotypic and morphologic criteria. In contrast, cells grown as subcutaneous solid tumors in SCID mice maintained progenitor cell characteristics with high-density CD34 expression and lack of morphologic differentiation. A tendency toward differentiation to CD15+, CD34- cells in vitro and self-renewal of CD34+, CD15- cells in vivo was consistently demonstrated regardless of whether cells were initially grown in vitro or in vivo. The cell line maintains both a CD34+, CD15- progentitor cell pool and a non-overlapping, CD15+, CD34- differentiating cell compartment after more than 1 year in continuous culture. Cell cycle analysis and cloning experiments were consistent with terminal differentiation occurring in the CD15+, CD34- population. The cell line shows concentration-dependent proliferative responses to interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-6, but not to granulocyte CSF (G-CSF). OMA-AML-1 appears to mimic several features of normal myeloid hematopoiesis and should prove useful for the study of normal and malignant myeloid differentiation.

OMA-AML-1: A Leukemic Myeloid Cell Line With CD34+ Progenitor and CD15+ Spontaneously Differentiating Cell Compartments

Abstract OMA-AML-1 was established from a patient with acute myelomonocytic (M4) leukemia at fifth relapse when blasts were greater than 85% CD34+, CD15- . Leukemic cells were established in suspension culture and independently grown as subcutaneous tumors in SCID mice. Cells growing in suspension culture underwent differentiation by phenotypic and morphologic criteria. In contrast, cells grown as subcutaneous solid tumors in SCID mice maintained progenitor cell characteristics with high-density CD34 expression and lack of morphologic differentiation. A tendency toward differentiation to CD15+, CD34- cells in vitro and self- renewal of CD34+, CD15- cells in vivo was consistently demonstrated regardless of whether cells were initially grown in vitro or in vivo. The cell line maintains both a CD34+, CD15- progentitor cell pool and a non-overlapping, CD15+, CD34- differentiating cell compartment after more than 1 year in continuous culture. Cell cycle analysis and cloning experiments were consistent with terminal differentiation occurring in the CD15+, CD34- population. The cell line shows concentration- dependent proliferative responses to interleukin (IL)-3, granulocyte- macrophage colony-stimulating factor (GM-CSF), and IL-6, but not to granulocyte CSF (G-CSF). OMA-AML-1 appears to mimic several features of normal myeloid hematopoiesis and should prove useful for the study of normal and malignant myeloid differentiation.