Wee1 Inhibition by MK-1775 Leads to Tumor Inhibition and Enhances Efficacy of Gemcitabine in Human Sarcomas

Sarcomas are rare and heterogeneous mesenchymal tumors affecting both pediatric and adult populations with more than 70 recognized histologies. Doxorubicin and ifosfamide have been the main course of therapy for treatment of sarcomas; however, the response rate to these therapies is about 10–20% in metastatic setting. Toxicity with the drug combination is high, response rates remain low, and improvement in overall survival, especially in the metastatic disease, remains negligible and new agents are needed. Wee1 is a critical component of the G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by MK1775 has been reported to enhance the cytotoxic effect of DNA damaging agents in different types of carcinomas. In this study we investigated the therapeutic efficacy of MK1775 in various sarcoma cell lines, patient-derived tumor explants ex vivo and in vivo both alone and in combination with gemcitabine, which is frequently used in the treatment of sarcomas. Our data demonstrate that MK1775 treatment as a single agent at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. Additionally, MK1775 significantly enhances the cytotoxic effect of gemcitabine in sarcoma cells lines with different p53 mutational status. In patient-derived bone and soft tissue sarcoma samples we showed that MK1775 alone and in combination with gemcitabine causes significant apoptotic cell death. Magnetic resonance imaging (MRI) and histopathologic studies showed that MK1775 induces significant cell death and terminal differentiation in a patient-derived xenograft mouse model of osteosarcoma in vivo. Our results together with the high safety profile of MK1775 strongly suggest that this drug can be used as a potential therapeutic agent in the treatment of both adult as well as pediatric sarcoma patients.     

p21 limits S phase DNA damage caused by the Wee1 inhibitor MK1775

The Wee1 inhibitor MK1775 (AZD1775) is currently being tested in clinical trials for cancer treatment. Here, we show that the p53 target and CDK inhibitor p21 protects against MK1775-induced DNA damage during S-phase. Cancer and normal cells deficient for p21 (HCT116 p21^-/-, RPE p21^-/-, and U2OS transfected with p21 siRNA) showed higher induction of the DNA damage marker γH2AX in S-phase in response to MK1775 compared to the respective parental cells. Furthermore, upon MK1775 treatment the levels of phospho-DNA PKcs S2056 and phospho-RPA S4/S8 were higher in the p21 deficient cells, consistent with increased DNA breakage. Cell cycle analysis revealed that these effects were due to an S-phase function of p21, but MK1775-induced S-phase CDK activity was not altered as measured by CDK-dependent phosphorylations. In the p21 deficient cancer cells MK1775-induced cell death was also increased. Moreover, p21 deficiency sensitized to combined treatment of MK1775 and the CHK1-inhibitor AZD6772, and to the combination of MK1775 with ionizing radiation. These results show that p21 protects cancer cells against Wee1 inhibition and suggest that S-phase functions of p21 contribute to mediate such protection. As p21 can be epigenetically downregulated in human cancer, we propose that p21 levels may be considered during future applications of Wee1 inhibitors.     

A Feasibility Study of Parametric Response Map Analysis of Diffusion-Weighted Magnetic Resonance Imaging Scans of Head and Neck Cancer Patients for Providing Early Detection of Therapeutic Efficacy

The parametric response map (PRM) was evaluated as an early surrogate biomarker for monitoring treatment-induced tissue alterations in patients with head and neck squamous cell carcinoma (HNSCC). Diffusion-weighted magnetic resonance imaging (DW-MRI) was performed on 15 patients with HNSCC at baseline and 3 weeks after treatment initiation of a nonsurgical organ preservation therapy (NSOPT) using concurrent radiation and chemotherapy. PRM was applied on serial apparent diffusion coefficient (ADC) maps that were spatially aligned using a deformable image registration algorithm to measure the tumor volume exhibiting significant changes in ADC (PRM_ADC). Pretherapy and midtherapy ADC maps, quantified from the DWIs, were analyzed by monitoring the percent change in whole-tumor mean ADC and the PRM metric. The prognostic values of percentage change in tumor volume and mean ADC and PRM_ADC as a treatment response biomarker were assessed by correlating with tumor control at 6 months. Pixel-wise differences as part of PRM_ADC analysis revealed regions where water mobility increased. Analysis of the tumor ADC histograms also showed increases in mean ADC as early as 3 weeks into therapy in patients with a favorable outcome. Nevertheless, the percentage change in mean ADC was found to not correlate with tumor control at 6 months. In contrast, significant differences in PRM_ADC and percentage change in tumor volume were observed between patients with pathologically different outcomes. Observations from this study have found that diffusion MRI, when assessed by PRM_ADC, has the potential to provide both prognostic and spatial information during NSOPT of head and neck cancer.     

Diffusion-Weighted MRI as a Biomarker of Tumor Radiation Treatment Response Heterogeneity: A Comparative Study of Whole-Volume Histogram Analysis versus Voxel-Based Functional Diffusion Map Analysis

RATIONALE: Treatment of glioblastoma (GBM) remains challenging due in part to its histologic intratumoral heterogeneity that contributes to its overall poor treatment response. Our goal was to evaluate a voxel-based biomarker, the functional diffusion map (fDM), as an imaging biomarker to detect heterogeneity of tumor response in a radiation dose escalation protocol using a genetically engineered murine GBM model. EXPERIMENTAL DESIGN: Twenty-four genetically engineered murine GBM models [Ink4a-Arf^-/-/Pten^loxp/loxp/Ntv-a RCAS/PDGF(+)/Cre(+)] were randomized in four treatment groups (n = 6 per group) consisting of daily doses of 0, 1, 2, and 4 Gy delivered for 5 days. Contrast-enhanced T1-weighted and diffusion-weighted magnetic resonance imaging (MRI) scans were acquired for tumor delineation and quantification of apparent diffusion coefficient (ADC) maps, respectively. MRI experiments were performed daily for a week and every 2 days thereafter. For each animal, the area under the curve (AUC) of the percentage change of the ADC (AUC_ADC) and that of the increase in fDM values (AUC_fDM+) were determined within the first 5 days following therapy initiation. RESULTS: Animal survival increased with increasing radiation dose. Treatment induced a dose-dependent increase in tumor ADC values. The strongest correlation between survival and ADC measurements was observed using the AUC_fDM+ metric (R² = 0.88). CONCLUSION: This study showed that the efficacy of a voxel-based imaging biomarker (fDM) was able to detect spatially varying changes in tumors, which were determined to be a more sensitive predictor of overall response versus whole-volume tumor measurements (AUC_ADC). Finally, fDM provided for visualization of treatment-associated spatial heterogeneity within the tumor.     

Characterizing tumor response to chemotherapy at various length scales using temporal diffusion spectroscopy

Measurements of apparent diffusion coefficient (ADC) using magnetic resonance imaging (MRI) have been suggested as potential imaging biomarkers for monitoring tumor response to treatment. However, conventional pulsed-gradient spin echo (PGSE) methods incorporate relatively long diffusion times, and are usually sensitive to changes in cell density and necrosis. Diffusion temporal spectroscopy using the oscillating gradient spin echo (OGSE) sequence is capable of probing short length scales, and may detect significant intracellular microstructural changes independent of gross cell density changes following anti-cancer treatment. To test this hypothesis, SW620 xenografts were treated by barasertib (AZD1152), a selective inhibitor of Aurora B kinase which causes SW620 cancer cells to develop polyploidy and increase in size following treatment, ultimately leading to cell death through apoptosis. Following treatment, the ADC values obtained by both the PGSE and low frequency OGSE methods increased. However, the ADC values at high gradient frequency (i.e. short diffusion times) were significantly lower in treated tumors, consistent with increased intracellular restrictions/hindrances. This suggests that ADC values at long diffusion times are dominated by tumor microstructure at long length scales, and may not convey unambiguous information of subcellular space. While the diffusion temporal spectroscopy provides more comprehensive means to probe tumor microstructure at various length scales. This work is the first study to probe intracellular microstructural variations due to polyploidy following treatment using diffusion MRI in vivo. It is also the first observation of post-treatment ADC changes occurring in opposite directions at short and long diffusion times. The current study suggests that temporal diffusion spectroscopy potentially provides pharmacodynamic biomarkers of tumor early response which distinguish microstructural variations following treatment at both the subcellular and supracellular length scales.     

Quantitative Multi-Parametric Magnetic Resonance Imaging of Tumor Response to Photodynamic Therapy

ObjectiveThe aim of this study was to characterize response to photodynamic therapy (PDT) in a mouse cancer model using a multi-parametric quantitative MRI protocol and to identify MR parameters as potential biomarkers for early assessment of treatment outcome.MethodsCT26.WT colon carcinoma tumors were grown subcutaneously in the hind limb of BALB/c mice. Therapy consisted of intravenous injection of the photosensitizer Bremachlorin, followed by 10 min laser illumination (200 mW/cm²) of the tumor 6 h post injection. MRI at 7 T was performed at baseline, directly after PDT, as well as at 24 h, and 72 h. Tumor relaxation time constants (T₁ and T₂) and apparent diffusion coefficient (ADC) were quantified at each time point. Additionally, Gd-DOTA dynamic contrast-enhanced (DCE) MRI was performed to estimate transfer constants (K^trans) and volume fractions of the extravascular extracellular space (v_e) using standard Tofts-Kermode tracer kinetic modeling. At the end of the experiment, tumor viability was characterized by histology using NADH-diaphorase staining.ResultsThe therapy induced extensive cell death in the tumor and resulted in significant reduction in tumor growth, as compared to untreated controls. Tumor T₁ and T₂ relaxation times remained unchanged up to 24 h, but decreased at 72 h after treatment. Tumor ADC values significantly increased at 24 h and 72 h. DCE-MRI derived tracer kinetic parameters displayed an early response to the treatment. Directly after PDT complete vascular shutdown was observed in large parts of the tumors and reduced uptake (decreased K^trans) in remaining tumor tissue. At 24 h, contrast uptake in most tumors was essentially absent. Out of 5 animals that were monitored for 2 weeks after treatment, 3 had tumor recurrence, in locations that showed strong contrast uptake at 72 h.ConclusionDCE-MRI is an effective tool for visualization of vascular effects directly after PDT. Endogenous contrast parameters T₁, T₂, and ADC, measured at 24 to 72 h after PDT, are also potential biomarkers for evaluation of therapy outcome.     

The contribution of DNA replication stress marked by high-intensity,pan-nuclear γH2AX staining to chemosensitization by CHK1 and WEE1 inhibitors

Small molecule inhibitors of the checkpoint proteins CHK1 and WEE1 are currently in clinical development in combination with the antimetabolite gemcitabine. It is unclear, however, if there is a therapeutic advantage to CHK1 vs. WEE1 inhibition for chemosensitization. The goals of this study were to directly compare the relative efficacies of the CHK1 inhibitor MK8776 and the WEE1 inhibitor AZD1775 to sensitize pancreatic cancer cell lines to gemcitabine and to identify pharmacodynamic biomarkers predictive of chemosensitization. Cells treated with gemcitabine and either MK8776 or AZD1775 were first assessed for clonogenic survival. With the exception of the homologous recombination-defective Capan1 cells, which were relatively insensitive to MK8776, we found that these cell lines were similarly sensitized to gemcitabine by CHK1 or WEE1 inhibition. The abilities of either the CDK1/2 inhibitor roscovitine or exogenous nucleosides to prevent MK8776 or AZD1775-mediated chemosensitization, however, were both inhibitor-dependent and variable among cell lines. Given the importance of DNA replication stress to gemcitabine chemosensitization, we next assessed high-intensity, pan-nuclear γH2AX staining as a pharmacodynamic marker for sensitization. In contrast to total γH2AX, aberrant mitotic entry or sub-G1 DNA content, high-intensity γH2AX staining correlated with chemosensitization by either MK8776 or AZD1775 (R² 0.83 – 0.53). In summary, we found that MK8776 and AZD1775 sensitize to gemcitabine with similar efficacy. Furthermore, our results suggest that the effects of CHK1 and WEE1 inhibition on gemcitabine-mediated replication stress best predict chemosensitization and support the use of high-intensity or pan-nuclear γH2AX staining as a marker for therapeutic response.     

Evaluation of poly-mechanistic antiangiogenic combinations to enhance cytotoxic therapy response in pancreatic cancer

Gemcitabine (Gem) has limited clinical benefits in pancreatic ductal adenocarcinoma (PDAC). The present study investigated combinations of gemcitabine with antiangiogenic agents of various mechanisms for PDAC, including bevacizumab (Bev), sunitinib (Su) and EMAP II. Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. In vivo experiments were performed via murine xenografts. Inhibition of in vitro proliferation of AsPC-1 PDAC cells by gemcitabine (10 μM), bevacizumab (1 mg/ml), sunitinib (10 μM) and EMAP (10 μM) was 35, 22, 81 and 6 percent; combination of gemcitabine with bevacizumab, sunitinib or EMAP had no additive effects. In endothelial HUVECs, gemcitabine, bevacizumab, sunitinib and EMAP caused 70, 41, 86 and 67 percent inhibition, while combination of gemcitabine with bevacizumab, sunitinib or EMAP had additive effects. In WI-38 fibroblasts, gemcitabine, bevacizumab, sunitinib and EMAP caused 79, 58, 80 and 29 percent inhibition, with additive effects in combination as well. Net in vivo tumor growth inhibition in gemcitabine, bevacizumab, sunitinib and EMAP monotherapy was 43, 38, 94 and 46 percent; dual combinations of Gem+Bev, Gem+Su and Gem+EMAP led to 69, 99 and 64 percent inhibition. Combinations of more than one antiangiogenic agent with gemcitabine were generally more effective but not superior to Gem+Su. Intratumoral proliferation, apoptosis and microvessel density findings correlated with tumor growth inhibition data. Median animal survival was increased by gemcitabine (26 days) but not by bevacizumab, sunitinib or EMAP monotherapy compared to controls (19 days). Gemcitabine combinations with bevacizumab, sunitinib or EMAP improved survival to similar extent (36 or 37 days). Combinations of gemcitabine with Bev+EMAP (43 days) or with Bev+Su+EMAP (46 days) led to the maximum survival benefit observed. Combination of antiangiogenic agents improves gemcitabine response, with sunitinib inducing the strongest effect. These findings demonstrate advantages of combining multi-targeting agents with standard gemcitabine therapy for PDAC.     

In vivo imaging of apoptosis in oncology: an update

In this review, data on noninvasive imaging of apoptosis in oncology are reviewed. Imaging data available are presented in order of occurrence in time of enzymatic and morphologic events occurring during apoptosis. Available studies suggest that various radiopharmaceutical probes bear great potential for apoptosis imaging by means of positron emission tomography and single-photon emission computed tomography (SPECT). However, for several of these probes, thorough toxicologic studies are required before they can be applied in clinical studies. Both preclinical and clinical studies support the notion that 99mTc-hydrazinonicotinamide-annexin A5 and SPECT allow for noninvasive, repetitive, quantitative apoptosis imaging and for assessing tumor response as early as 24 hours following treatment instigation. Bioluminescence imaging and near-infrared fluorescence imaging have shown great potential in small-animal imaging, but their usefulness for in vivo imaging in humans is limited to structures superficially located in the human body. Although preclinical tumor-based data using high-frequency-ultrasonography (US) are promising, whether or not US will become a routinely clinically useful tool in the assessment of therapy response in oncology remains to be proven. The potential of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) for imaging late apoptotic processes is currently unclear. Neither 31P MRS nor 1H MRS signals seems to be a unique identifier for apoptosis. Although MRI-measured apparent diffusion coefficients are altered in response to therapies that induce apoptosis, they are also altered by nonapoptotic cell death, including necrosis and mitotic catastrophe. In the future, rapid progress in the field of apoptosis imaging in oncology is expected.     

Radioimmunotherapy for breast cancer: treatment of a patient with I-131 L6 chimeric monoclonal antibody.

We report the first treatment of metastatic breast cancer by systemic radioimmunotherapy. The serial therapy doses were chosen based on quantitative imaging data in a treatment planning approach. A terminally ill patient with aggressive, locally advanced breast cancer who had failed radiation treatment and chemotherapy was injected intravenously with radiolabeled I-131 chimeric L6, a human-mouse chimeric lgG1 monoclonal antibody to adenocarcinoma. Initially, an imaging 10 mCi dose of I-131 chimeric L6 (dose 1) deposited 8.8% of the injected dose in her chest wall tumor at 48 hours. Ten days later the patient was given a 150 mCi I-131 chimeric L6 dose (dose 2) followed three weeks later by a 100 mCi dose (dose 3). Tumor uptake and retention were comparable for doses 1 and 2, and decreased for dose 3. Following dose 3 the patient developed a manageable thrombocytopenia and transient Grade IV granulocytopenia. The tumor was observed to decrease in size with peak tumor regression occurring two weeks after dose 3. This partial response (PR) was achieved by radioimmunotherapy at a time when conventional therapy had been unable to impact the growth of the patient's massive and aggressive tumor.     

Diffusion-Weighted MRI in the Assessment of Early Treatment Response in Patients with Squamous-Cell Carcinoma of the Head and Neck: Comparison with Morphological and PET/CT Findings

Objective

To evaluate changes in apparent diffusion coefficients (ADC) as measured by magnetic resonance imaging (MRI) before and after the treatment of primary tumors and cervical metastases in patients with squamous-cell carcinoma (SCC) of the head and neck, and to compare these values to the results of widely used morphological criteria and [18F]-FDG PET/CT findings.

Material and Method

This was a longitudinal, prospective, single-center nonrandomized trial involving patients with head and neck SCC treated with chemotherapy alone or in combination with radiotherapy. Imaging examinations ([18F]-FDG PET/CT and diffusion-weighted MRI) were performed on the same day, up to one day prior to the beginning of the first treatment cycle, and on the 14th day of the first chemotherapy cycle. Treatment response was evaluated based on the Response Evaluation Criteria in Solid Tumors (RECIST) and World Health Organization (WHO) morphological criteria, as well as PET Response Criteria in Solid Tumors (PERCIST) metabolic criteria.

Results

Seventy-five lesions were examined in 23 patients. Pre- and post-treatment comparisons of data pertaining to all target lesions revealed reductions in tumor size and SUV, as well as increases in ADC values, all of which were statistically significant. The increase in ADC following treatment was significantly higher in patients classified as complete responders by both morphological criteria than that observed in any of the other patient groups of response. Patients with a complete metabolic response also showed greater increases in ADC values as compared to the remaining groups.

Conclusion

The assessment of tumor response based on diffusion-weighted MRI showed an increase in the ADC of cervical lesions following treatment, which was corroborated by morphological and metabolic findings. Associations between changes in ADC values and treatment response categories using morphologic criteria and [18F]-FDG PET/CT were only identified in complete responders.     

Multiparametric Characterization of Grade 2 Glioma Subtypes Using Magnetic Resonance Spectroscopic,Perfusion, and Diffusion Imaging

BACKGROUND AND PURPOSE: The purpose of this study was to derive quantitative parameters from magnetic resonance (MR) spectroscopic, perfusion, and diffusion imaging of grade 2 gliomas according to the World Health Organization and to investigate how these multiple imaging modalities can contribute to evaluating their histologic subtypes and spatial characteristics. MATERIALS AND METHODS: MR spectroscopic, perfusion, and diffusion images from 56 patients with newly diagnosed grade 2 glioma (24 oligodendrogliomas, 18 astrocytomas, and 14 oligoastrocytomas) were retrospectively studied. Metabolite intensities, relative cerebral blood volume (rCBV), and apparent diffusion coefficient (ADC) were statistically evaluated. RESULTS: The 75th percentile rCBV and median ADC were significantly different between oligodendrogliomas and astrocytomas (P < .0001) and between oligodendrogliomas and oligoastrocytomas (P < .001). Logistic regression analysis identified both 75th percentile rCBV and median ADC as significant variables in the differentiation of oligodendrogliomas from astrocytomas and oligoastrocytomas. Group differences in metabolite intensities were not significant, but there was a much larger variation in the volumes and maximum values of metabolic abnormalities for patients with oligodendroglioma compared with the other tumor subtypes. CONCLUSIONS: Perfusion and diffusion imaging provide quantitative MR parameters that can help to differentiate grade 2 oligodendrogliomas from grade 2 astrocytomas and oligoastrocytomas. The large variations in the magnitude and spatial extent of the metabolic lesions between patients and the fact that their values are not correlated with the other imaging parameters indicate that MR spectroscopic imaging may provide complementary information that is helpful in targeting therapy, evaluating residual disease, and assessing response to therapy.     

Multimodality Imaging of Tumor and Bone Response in a Mouse Model of Bony Metastasis

Cancer drug development generally performs in vivo evaluation of treatment effects that have traditionally relied on detection of morphologic changes. The emergence of new targeted therapies, which may not result in gross morphologic changes, has spurred investigation into more specific imaging methods to quantify response, such as targeted fluorescent probes and bioluminescent cells. The present study investigated tissue response to docetaxel or zoledronic acid (ZA) in a mouse model of bony metastasis. Intratibial implantations of breast cancer cells (MDA-MB-231) were monitored throughout this study using several modalities: molecular resonance imaging (MRI) tumor volume and apparent diffusion coefficient (ADC), micro-computed tomography (µCT) bone volume, bioluminescence imaging (BLI) reporting cancer cell apoptosis, and fluorescence using Osteosense 800 and CatK 680-FAST. Docetaxel treatment resulted in tumor cell kill reflected by ADC and BLI increases and tumor volume reduction, with delayed bone recovery seen in µCT prefaced by increased osteoblastic activity (Osteosense 800). In contrast, the ZA treatment group produced similar values in MRI, BLI, and Osteosense 800 fluorescence imaging readouts when compared to controls. However, µCT bone volume increased significantly by the first week post-treatment and the CatK 680-FAST signal was slightly diminished by 4 weeks following ZA treatment. Multimodality imaging provides a more comprehensive tool for new drug evaluation and efficacy screening through identification of morphology as well as function and apoptotic signaling.     

Inhibition of de novo pyrimidine synthesis augments Gemcitabine induced growth inhibition in an immunocompetent model of pancreatic cancer

Leflunomide (Lef) is an agent used in autoimmune disorders that interferes with DNA synthesis. De Novo pyrimidine synthesis is a mechanism of Gemcitabine (Gem) resistance in pancreatic cancer. This study aims to assess the efficacy and changes in the tumor microenvironment of Lef monotherapy and in combination with Gem, in a syngeneic mouse model of pancreatic cancer.Methods: MTS proliferation assays were conducted to assess growth inhibition by Gem (0-20 nM), Lef (0-40 uM) and Gem+Lef in KPC (KrasLSL.G12D/+;p53R172H/+; PdxCretg/+) cells in vitro. An in vivo heterotopic KPC model was used and cohorts were treated with: PBS (control), Gem (75 mg/kg/q3d), Lef (40 mg/kg/d), or Gem+Lef. At d28 post-treatment, tumor burden, proliferation index (Ki67), and vascularity (CD31) were measured. Changes in the frequency of peripheral and intratumoral immune cell subsets were evaluated via FACS. Liquid chromatography-mass spectrometry was used for metabolomics profiling.Results: Lef inhibits KPC cell growth and synergizes with Gem in vitro (P<0.05; Combination Index 0.44 (<1 indicates synergy). In vivo, Lef alone and in combination with Gem delays KPC tumor progression (P<0.001). CTLA-4+T cells are also significantly decreased in tumors treated with Lef, Gem or in combination (Gem+Lef) compared to controls (P<0.05). Combination therapy also decreased the Ki67 and vascularity (P<0.01). Leflunomide inhibits de novo pyrimidine synthesis both in vitro (p<0.0001) and in vivo (p<0.05).Conclusions: In this study, we demonstrated that Gem+Lef inhibits pancreatic cancer growth, decrease T cell exhaustion, vascularity and as proof of principle inhibits de novo pyrimidine synthesis. Further characterization of changes in adaptive immunity are necessary to characterize the mechanism of tumor growth inhibition and facilitate translation to a clinical trial.     

Comparison of Optical and Power Doppler Ultrasound Imaging for Non-Invasive Evaluation of Arsenic Trioxide as a Vascular Disrupting Agent in Tumors

Small animal imaging provides diverse methods for evaluating tumor growth and acute response to therapy. This study compared the utility of non-invasive optical and ultrasound imaging to monitor growth of three diverse human tumor xenografts (brain U87-luc-mCherry, mammary MCF7-luc-mCherry, and prostate PC3-luc) growing in nude mice. Bioluminescence imaging (BLI), fluorescence imaging (FLI), and Power Doppler ultrasound (PD US) were then applied to examine acute vascular disruption following administration of arsenic trioxide (ATO).During initial tumor growth, strong correlations were found between manual caliper measured tumor volume and FLI intensity, BLI intensity following luciferin injection, and traditional B-mode US. Administration of ATO to established U87 tumors caused significant vascular shutdown within 2 hrs at all doses in the range 5 to 10 mg/kg in a dose dependant manner, as revealed by depressed bioluminescent light emission. At lower doses substantial recovery was seen within 4 hrs. At 8 mg/kg there was >85% reduction in tumor vascular perfusion, which remained depressed after 6 hrs, but showed some recovery after 24 hrs. Similar response was observed in MCF7 and PC3 tumors. Dynamic BLI and PD US each showed similar duration and percent reductions in tumor blood flow, but FLI showed no significant changes during the first 24 hrs.The results provide further evidence for comparable utility of optical and ultrasound imaging for monitoring tumor growth, More specifically, they confirm the utility of BLI and ultrasound imaging as facile assays of the vascular disruption in solid tumors based on ATO as a model agent.     

Prophylactic dendritic cell vaccination controls pancreatic cancer growth in a mouse model

PurposePancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths with high recurrence after surgery due to a paucity of effective post-surgical adjuvant treatments. DC vaccines can activate multiple anti-tumor immune responses but have not been explored for post-surgery PDAC recurrence. Intraperitoneal (IP) delivery may allow increased DC vaccine dosage and migration to lymph nodes. Here, we investigated the role of prophylactic DC vaccination controlling PDAC tumor growth with IP delivery as an administration route for DC vaccination.MethodsDC vaccines were generated using ex vivo differentiation and maturation of bone marrow–derived precursors. Twenty mice were divided into four groups (n = 5) and treated with DC vaccines, unpulsed mature DCs, Panc02 lysates or no treatment. After tumor induction, mice underwent three magnetic resonance imaging scans to track tumor growth. Apparent diffusion coefficient (ADC), a quantitative magnetic resonance imaging measurement of tumor microstructure, was calculated. Survival was tracked. Tumor tissue was collected after death and stained with hematoxylin and eosin, Masson's trichrome, terminal deoxynucleotidyl transferase dUTP nick end labeling and anti-CD8 stains for histology.ResultsDC-vaccinated mice demonstrated stronger anti-tumor cytotoxicity compared with control groups on lactate dehydrogenase assay. DC vaccine mice also demonstrated decreased tumor volume, prolonged survival and increased ΔADC compared with control groups. On histology, the DC vaccine group had increased apoptosis, increased CD8+ T cells and decreased collagen. ΔADC negatively correlated with % collagen in tumor tissues.DiscussionProphylactic DC vaccination may inhibit PDAC tumor growth during recurrence and prolong survival. ΔADC may be a potential imaging biomarker that correlates with tumor histological features.     

Experimental Study of Nasopharyngeal Carcinoma Radionuclide Imaging and Therapy Using Transferred Human Sodium/Iodide Symporter Gene

Purpose

The aim of this study was to design a method of radionuclide for imaging and therapy of nasopharyngeal carcinoma (NPC) using the transferred human sodium/iodide symporter (hNIS) gene.

Methods

A stable NPC cell line expressing hNIS was established (CNE-2-hNIS). After ¹³¹I treatment, we detected proliferation and apoptosis of NPC cells, both in vitro and vivo. In vivo, the radioactivity of different organs of nude mice was counted and ^99mTc imaging using SPECT was performed. The apparent diffusion coefficient (ADC) value changes of tumor xenografts were observed by diffusion-weighted magnetic resonance imaging (DW-MRI) within 6–24 days of ¹³¹I treatment. The correlation of ADC changes with apoptosis and proliferation was investigated. Post-treatment expression levels of P53, Bax, Bcl-2, Caspase-3, and Survivin proteins were detected by western blotting.

Results

¹³¹I uptake was higher in CNE-2-hNIS than in CNE-2 cells. The proliferation and apoptosis rate decreased and increased respectively both in vitro and vivo in the experimental group after ¹³¹I treatment. The experimental group tumors accumulated ^99mTc in vivo, leading to a good visualization by SPECT. DW-MRI showed that ADC values increased in the experimental group 6 days after treatment, while ADC values were positively and negatively correlated with the apoptotic and Ki-67 proliferation indices, respectively. After treatment, CNE-2-hNIS cells up-regulated the expression of P53 and Survivin proteins and activated Caspase-3, and down-regulated the expression of Bcl-2 proteins.

Conclusions

The radionuclide imaging and therapy technique for NPC hNIS-transfected cell lines can provide a new therapy strategy for monitoring and treatment of NPC.     

Comparative Proteomic Analysis Identifies Key Metabolic Regulators of Gemcitabine Resistance in Pancreatic Cancer

Pancreatic adenocarcinoma (PDAC) is highly refractory to treatment. Standard-of-care gemcitabine (Gem) provides only modest survival benefits, and development of Gem resistance (GemR) compromises its efficacy. Highly GemR clones of Gem-sensitive MIAPaCa-2 cells were developed to investigate the molecular mechanisms of GemR and implemented global quantitative differential proteomics analysis with a comprehensive, reproducible ion-current–based MS1 workflow to quantify ～6000 proteins in all samples. In GemR clone MIA-GR8, cellular metabolism, proliferation, migration, and ‘drug response’ mechanisms were the predominant biological processes altered, consistent with cell phenotypic alterations in cell cycle and motility. S100 calcium binding protein A4 was the most downregulated protein, as were proteins associated with glycolytic and oxidative energy production. Both responses would reduce tumor proliferation. Upregulation of mesenchymal markers was prominent, and cellular invasiveness increased. Key enzymes in Gem metabolism pathways were altered such that intracellular utilization of Gem would decrease. Ribonucleoside-diphosphate reductase large subunit was the most elevated Gem metabolizing protein, supporting its critical role in GemR. Lower Ribonucleoside-diphosphate reductase large subunit expression is associated with better clinical outcomes in PDAC, and its downregulation paralleled reduced MIAPaCa-2 proliferation and migration and increased Gem sensitivity. Temporal protein-level Gem responses of MIAPaCa-2 versus GemR cell lines (intrinsically GemR PANC-1 and acquired GemR MIA-GR8) implicate adaptive changes in cellular response systems for cell proliferation and drug transport and metabolism, which reduce cytotoxic Gem metabolites, in DNA repair, and additional responses, as key contributors to the complexity of GemR in PDAC. These findings additionally suggest targetable therapeutic vulnerabilities for GemR PDAC patients.     

Radiotherapy combined with gemcitabine and oxaliplatin in pancreatic cancer cells

Clinical evidence suggests that gemcitabine (Gem) plus oxaliplatin (Ox) is superior to gemcitabine alone in advanced pancreatic carcinoma. The addition of radiation to gemcitabine improves response and is a standard treatment for locally advanced disease. We investigated the effect of oxaliplatin on gemcitabine-based chemoradiation by determining whether gemcitabine and oxaliplatin produced synergistic cytotoxicity using median effect analysis and radiosensitization using clonogenic survival assays. We analyzed the effects of gemcitabine and oxaliplatin on cell cycle distribution by DNA content and on radiation-induced DNA damage repair by phosphorylated H2AX (gamma-H2AX). Gemcitabine and oxaliplatin produced schedule-dependent synergistic cytotoxicity in BxPC-3 and Panc-1 cells (combination indices: 0.76 +/- 0.05, 0.61 +/- 0.11). In BxPC-3 cells, oxaliplatin did not affect gemcitabine-mediated radiosensitization (Gem 1.99 +/- 0.27; Gem + Ox 2.38 +/- 0.30). In Panc-1 cells, oxaliplatin significantly enhanced gemcitabine-mediated radiosensitization (Gem 1.31 +/- 0.05; Gem + Ox 2.90 +/- 0.31). Radiosensitization by gemcitabine was accompanied by early S-phase arrest and induction/persistence of gamma-H2AX protein, which were unaltered by oxaliplatin. Addition of oxaliplatin to gemcitabine produces radiosensitization equal to or greater than gemcitabine alone, supporting our clinical investigation of oxaliplatin with gemcitabine-radiation in pancreatic cancer aimed at improving systemic disease control while maintaining local tumor radiosensitization.     

Use of photoacoustic imaging for monitoring vascular disrupting cancer treatments

Vascular disrupting agents disrupt tumor vessels, blocking the nutritional and oxygen supply tumors need to thrive. This is achieved by damaging the endothelium lining of blood vessels, resulting in red blood cells (RBCs) entering the tumor parenchyma. RBCs present in the extracellular matrix are exposed to external stressors resulting in biochemical and physiological changes. The detection of these changes can be used to monitor the efficacy of cancer treatments. Spectroscopic photoacoustic (PA) imaging is an ideal candidate for probing RBCs due to their high optical absorption relative to surrounding tissue. The goal of this work is to use PA imaging to monitor the efficacy of the vascular disrupting agent 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) through quantitative analysis. Then, 4T1 breast cancer cells were injected subcutaneously into the left hind leg of eight BALB/c mice. After 10 days, half of the mice were treated with 15 mg/kg of DMXAA and the other half were injected with saline. All mice were imaged using the VevoLAZR X PA system before treatment, 24 and 72 hours after treatment. The imaging was done at six wavelengths and linear spectral unmixing was applied to the PA images to quantify three forms of hemoglobin (oxy, deoxy and met-hemoglobin). After imaging, tumors were histologically processed and H&E and TUNEL staining were used to detect the tissue damage induced by the DMXAA treatment. The total hemoglobin concentration remained unchanged after treatment for the saline treated mice. For DMXAA treated mice, a 10% increase of deoxyhemoglobin concentration was detected 24 hours after treatment and a 22.6% decrease in total hemoglobin concentration was observed by 72 hours. A decrease in the PA spectral slope parameters was measured 24 hours after treatment. This suggests that DMXAA induces vascular damage, causing red blood cells to extravasate. Furthermore, H&E staining of the tumor showed areas of bleeding with erythrocyte deposition. These observations are further supported by the increase in TUNEL staining in DMXAA treated tumors, revealing increased cell death due to vascular disruption. This study demonstrates the capability of PA imaging to monitor tumor vessel disruption by the vascular disrupting agent DMXAA.