Whole Slide Image Analysis Quantification using Aperio Digital Imaging in a Mouse Lung Metastasis Model

poster abstractDigital whole slide imaging is the technique of digitizing a microscope slide at the highest resolution to produce a “digital virtual microscope slide”. This digital image can be viewed in three or four fields, from low to high power, which can be commonly used to evaluate the tissue. Many of these systems have whole slide software image analysis capability. The goal of this study was to determine if the Aperio positive pixel algorithm (image analysis) could effectively quantitate metastatic mouse lung tumors in a lung section using a H&E stain. Lung sections from a mouse lung metastasis model of 8 mice per group were evaluated: control, 50mg/kg, and 75mg/kg carboplatin. H&E and Ki67 immunostain slides were scanned using the Aperio whole slide scanning system (Scanscope CS). A single field of view from each slide representing a whole lung lobe with multiple lung metastases was selected for image analysis. The standard positive pixel algorithm was altered to read the H&E slides. Various histology slides were used to validate the altered algorithm. The immunostain (Ki67) was generated using the standard positive pixel algorithm analysis. The Aperio automated positive pixel count for a Ki67 immunostain was consistent with the H&E image analysis. The values decreased with a dose dependent treatment (control vs. 50mg/kg and 75mg/kg carboplatin) and were (H&E) 37%, 28%, and 22%, and (Ki67) 9%, 5%, and 3%. The analysis had decreasing values for both the H&E and Ki67 analysis on a dose dependent drug treatment. The metastases decreased in both treatment groups compared to controls with both the H&E and Ki67 analyses. The Aperio Image Analysis positive pixel algorithm allows large areas of the lung tissue section to be examined and not just a single 25x or 40x field like many common image analyses systems

Potentiation of Carboplatin-Mediated DNA Damage by the Mdm2 Modulator Nutlin-3a in a Humanized Orthotopic Breast-to-Lung Metastatic Model

Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC

Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions

Modulation of the Mdm2 signaling axis sensitizes triple-negative breast cancer cells to carboplatin

Triple-negative breast cancers (TNBCs) are highly refractive to current treatment strategies, and new multi-targeted treatments need to be elucidated. Combination therapy that includes targeting the murine double minute 2 (Mdm2) signaling axis offers a promising approach. Protein-protein interaction inhibitors such as Nutlin-3a block the binding of key signaling molecules such as p53, p73&agr;, and E2F1 to the hydrophobic hydrophobic pocket of Mdm2 and can lead to activation of cell-death signaling pathways. Since clinical trials for TNBC are evaluating the DNA damaging agent carboplatin, the objective of this thesis was to evaluate the therapeutic potential and mechanism of action of combination carboplatin and Nutlin-3a to treat TNBC. In TNBC cell lines with a mutant p53 background, we determined if modulation of Mdm2 function in the context of carboplatin-mediated DNA damage resulted in a synergistic inhibition of cell growth. Several ratios of carboplatin:Nutlin-3a were strongly synergistic in increasing cell death, with combination indices of 0.5 and lower. Mechanistic studies indicated that drug sensitivity and Mdm2 expression were dependent on p73. Mdm2 localized to a larger degree in the chromatin fraction isolated from cells treated with the combination treatment consistent with observations by others that Mdm2 binds to the Mre11/Rad50/Nbs1 complex, inhibits the DNA damage response, and increases drug sensitivity. In vivo efficacy experiments were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-PrkdcscidIl2rg tm1Wjl/SzJ (NSG) mice. For assessment of baseline tumor burden and randomization, fluorescent imaging of E2-Crimson expressing TMD231 cells was performed. Following Nutlin-3a and carboplatin combination treatment, there was a statistically significant reduction in primary tumor volume as well as lung metastases with significantly increased probability of survival compared to Vehicle and single drug treatments (p<0.001). While there was a decrease in bone-marrow cellularity, this did not lead to bone-marrow aplasia, and body weights recovered to normal levels within 7 days post-treatment. The present studies demonstrate the promise of Mdm2 as a therapeutic target in combination with conventional therapy, increase our understanding of how to potentiate DNA damage in cancers, and may lead to new clinical therapies for triple-negative primary and metastatic breast cancer