The Tumor-Educated-Macrophage Increase of Malignancy of Human Pancreatic Cancer Is Prevented by Zoledronic Acid

We previously defined macrophages harvested from the peritoneal cavity of nude mice with subcutaneous human pancreatic tumors as “tumor-educated-macrophages” (Edu) and macrophages harvested from mice without tumors as “naïve-macrophages” (Naïve), and demonstrated that Edu-macrophages promoted tumor growth and metastasis. In this study, Edu- and Naïve-macrophages were compared for their ability to enhance pancreatic cancer malignancy at the cellular level in vitro and in vivo. The inhibitory efficacy of Zoledronic acid (ZA) on Edu-macrophage-enhanced metastasis was also determined. XPA1 human pancreatic cancer cells in Gelfoam co-cultured with Edu-macrophages proliferated to a greater extent compared to XPA1 cells cultured with Naïve-macrophages (P = 0.014). XPA1 cells exposed to conditioned medium harvested from Edu culture significantly increased proliferation (P = 0.016) and had more migration stimulation capability (P<0.001) compared to cultured cancer cells treated with the conditioned medium from Naïve. The mitotic index of the XPA1 cells, expressing GFP in the nucleus and RFP in the cytoplasm, significantly increased in vivo in the presence of Edu- compared to Naïve-macrophages (P = 0.001). Zoledronic acid (ZA) killed both Edu and Naïve in vitro. Edu promoted tumor growth and metastasis in an orthotopic mouse model of the XPA1 human pancreatic cancer cell line. ZA reduced primary tumor growth (P = 0.006) and prevented metastasis (P = 0.025) promoted by Edu-macrophages. These results indicate that ZA inhibits enhanced primary tumor growth and metastasis of human pancreatic cancer induced by Edu-macrophages.     

Functional evaluation of therapeutic response for a mouse model of medulloblastoma

Medulloblastoma is an aggressive childhood cerebellar tumor. We recently reported a mouse model with conditional deletion of Patched1 gene that recapitulates many characteristics of the human medulloblastoma. Qualitative symptoms observed in the mouse model include irregular stride length, impaired cranial nerve function and decreased motor coordination and performance. In our current study, several quantitative behavioral assays including a mouse rotarod, a forced air challenge, a screen inversion test, a horizontal wire test, and stride length analysis were evaluated to determine the most sensitive and cost-effective functional assay for impaired neuromotor behavior associated with disease progression. Magnetic resonance imaging (MRI) was used to confirm and monitor tumor growth and as an anatomical biomarker for therapeutic response. Wild type mice or medulloblastoma-prone, conditional Patched1 knockout mice were observed by behavioral assays and MRI from postnatal weeks 3–6. Bortezomib treatment was administered during this period and therapeutic response was assessed using cerebellar volumes at the end of treatment. Of the behavioral tests assessed in this study, stride length analysis was best able to detect differences between tumor-prone mice and wild type mice as early as postnatal day 37 (P = 0.003). Significant differences between stride lengths of bortezomib treated and control tumor-bearing mice could be detected as early as postnatal day 42 (P = 0.020). Cerebellar volumes measured by MRI at the end of treatment validated the therapeutic effects seen by behavioral tests (P = 0.03). These findings suggest that stride length analysis may serve as one of the more sensitive and cost-effective method for assessing new therapeutic compounds in this and other preclinical model of brain tumors.     

Development of a novel preclinical pancreatic cancer research model: bioluminescence image-guided focal irradiation and tumor monitoring of orthotopic xenografts

PURPOSE: We report on a novel preclinical pancreatic cancer research model that uses bioluminescence imaging (BLI)-guided irradiation of orthotopic xenograft tumors, sparing of surrounding normal tissues, and quantitative, noninvasive longitudinal assessment of treatment response. MATERIALS AND METHODS: Luciferase-expressing MiaPaCa-2 pancreatic carcinoma cells were orthotopically injected in nude mice. BLI was compared to pathologic tumor volume, and photon emission was assessed over time. BLI was correlated to positron emission tomography (PET)/computed tomography (CT) to estimate tumor dimensions. BLI and cone-beam CT (CBCT) were used to compare tumor centroid location and estimate setup error. BLI and CBCT fusion was performed to guide irradiation of tumors using the small animal radiation research platform (SARRP). DNA damage was assessed by γ-H2Ax staining. BLI was used to longitudinally monitor treatment response. RESULTS: Bioluminescence predicted tumor volume (R = 0.8984) and increased linearly as a function of time up to a 10-fold increase in tumor burden. BLI correlated with PET/CT and necropsy specimen in size (P < .05). Two-dimensional BLI centroid accuracy was 3.5 mm relative to CBCT. BLI-guided irradiated pancreatic tumors stained positively for γ-H2Ax, whereas surrounding normal tissues were spared. Longitudinal assessment of irradiated tumors with BLI revealed significant tumor growth delay of 20 days relative to controls. CONCLUSIONS: We have successfully applied the SARRP to a bioluminescent, orthotopic preclinical pancreas cancer model to noninvasively: 1) allow the identification of tumor burden before therapy, 2) facilitate image-guided focal radiation therapy, and 3) allow normalization of tumor burden and longitudinal assessment of treatment response.     

Mesenchymal stromal cells alone or expressing interferon-β suppress pancreatic tumors in vivo,an effect countered by anti-inflammatory treatment

Background aimsBecause of the inflammatory nature and extensive stromal compartment in pancreatic tumors, we investigated the role of mesenchymal stromal cells (MSC) to engraft selectively in pancreatic carcinomas and serve as anti-tumor drug delivery vehicles to control pancreatic cancer progression.MethodsHuman pancreatic carcinoma cells, PANC-1, expressing renilla luciferase were orthotopically implanted into SCID mice and allowed to develop for 10 days. Firefly luciferase-transduced MSC or MSC expressing interferon (IFN)-β were then injected intraperitoneally weekly for 3 weeks. Mice were monitored by bioluminescent imaging for expression of renilla (PANC-1) and firefly (MSC) luciferase.ResultsMSC selectively homed to sites of primary and metastatic pancreatic tumors and inhibited tumor growth (P = 0.032). The production of IFN-β within the tumor site by MSC–IFN-β further suppressed tumor growth (P = 0.0000083). Prior studies indicated that MSC home to sites of inflammation; therefore, we sought to alter the tumor microenvironment through treatment with a potent anti-inflammatory agent. After treatment, inflammation-associated mediators were effectively down-regulated, including NFκB, vascular endothelial growth factor (VEGF) and interleukin (IL)-6 as well as chemokines involved in MSC migration (CCL3 and CCL25). Treatment with the anti-inflammatory agent CDDO-Me before and after MSC–IFN-β injections resulted in reduction of MSC in the tumors and reversed the positive effect of tumor inhibition by MSC–IFN-β alone (P = 0.041).ConclusionsThese results suggest that MSC exhibit innate anti-tumor effects against PANC-1 cells and can serve as delivery vehicles for IFN-β for the treatment of pancreatic cancer. However, these beneficial effects may be lost in therapies combining MSC with anti-inflammatory agents.     

SW1990和Capan-2红色荧光标记胰腺癌移植肿瘤模型的建立和比较

目的建立稳定表达红色荧光蛋白基因的人胰腺癌细胞系,为体内监测肿瘤的早期生长及抗肿瘤药物的药效评价建立一种新的肿瘤动物模型。方法以Lipofectamine 2000介导chickenβ-actin-RFP-NEO转染人胰腺癌细胞SW1990和Capan-2,经梯度浓度G418筛选获得稳定表达红色荧光蛋白的细胞克隆并扩大培养。BALB/cA-nu裸鼠皮下接种1×106个发光细胞使其成瘤,活体荧光成像系统观察肿瘤的生长情况。结果获得了稳定表达RFP的两种不同的人胰腺癌细胞株,将其接种到裸鼠体内可成瘤,利用活体成像系统观察了肿瘤的生长动态过程,并且SW1990肿瘤细胞的生长速度较Capan-2细胞快。结论用红色荧光蛋白标记的人胰腺癌细胞建立的裸鼠肿瘤模型为胰腺癌的研究和相关药物筛选提供了可进行荧光影像活体、动态分析的动物模型。     

Predicting Therapeutic Efficacy of Vascular Disrupting Agent CA4P in Rats with Liver Tumors by Hepatobiliary Contrast Agent Mn-DPDP-Enhanced MRI

To evaluate hepatobiliary-specific contrast agent (CA) mangafodipir trisodium (Mn-DPDP)–enhanced magnetic resonance imaging (MRI) for predicting the therapeutic efficacy of the vascular disrupting agent combretastatin A4 phosphate (CA4P) in rats with primary and secondary liver tumors, 36 primary hepatocellular carcinomas (HCCs) were raised by diethylnitrosamine gavage in 16 male rats, in 6 of which one rhabdomyosarcomas (R1) was intrahepatically implanted as secondary liver tumors. On a 3.0T MR scanner with a wrist coil, tumors were monitored weekly by T2-/T1-weighted images (T2WI/T1WI) and characterized by Mn-DPDP-enhanced MRI. CA4P-induced intratumoral necrosis was depicted by nonspecific gadoterate meglumine (Gd-DOTA)–enhanced MRI before and 12 h after therapy. Changes of tumor-to-liver contrast (ΔT/L) on Mn-DPDP-enhanced images were analyzed. In vivo MRI findings were verified by postmortem microangiography and histopathology. Rat models of primary HCCs in a full spectrum of differentiation and secondary R1 liver tumors were successfully generated. Mn-DPDP-enhanced ΔT/L was negatively correlated with HCC differentiation grade (P < 0.01). After treatment with CA4P, more extensive tumoral necrosis was found in highly differentiated HCCs than that in moderately and poorly differentiated ones (P < 0.01); nearly complete necrosis was induced in secondary liver tumors. Mn-DPDP-enhanced MRI may help in imaging diagnosis of primary and secondary liver malignancies of different cellular differentiations and further in predicting CA4P therapeutic efficacy in primary HCCs and intrahepatic metastases.     

MUC-1 aptamer targeted superparamagnetic iron oxide nanoparticles for magnetic resonance imaging of pancreatic cancer in vivo and in vitro experiment

This study aims to explore the ability of magnetic resonance imaging (MRI) in mucin 1 (MUC1) modified superparamagnetic iron oxide nanoparticle (SPION) targeting human pancreatic cancer (PC). The MUC1 target-directed probe was prepared through MUC1 conjugated to SPION using the chemical method to assess its physiochemical characteristics, including hydration diameter, surface charge, and magnetic resonance signal. The cytotoxicity of MUC1-USPION was verified by MTS assay. BxPC-3 was cultured with MUC1-USPION and SPION in different concentrations. The combined condition of the targeted probes and cells were observed through Prussian blue staining. The nude mice model of pancreatic cancer was established to investigate the application of the probe. MRI was performed to determine the intensity of the signal of the transplanted tumor, while immunohistochemistry and Western blot analysis were performed to detect the expression of MUC1 after taking the transplanted tumor specimen. The particle size of the prepared molecular probe was 63.5 ± 3.2 nm, and the surface charge was 10.2 mV. Furthermore, the probe solution could significantly reduce the MRI at T₂, and the magnetic resonance transverse relaxation rate (ΔR²) has a linear relationship with the concentration of iron in the solution. The cell viability of MUC1-USPION in different concentrations revealed no statistical difference, according to the MTS assay. In vitro, the MRI demonstrated decreased T2WI signal intensity in both groups, especially the targeting group. In vivo, MUC1 could selectively accumulate in the nude mice model, and significantly reduce the T₂ signal strength. In subsequent experiments, the expression of MUC1 was high in pancreatic cancer tissues, but low in normal pancreatic tissues, as determined by immunohistochemistry and Western blot analysis. The prepared samples can be combined with pancreatic cancer tissue specificity by in vivo imaging, providing reliable early in vivo imaging data for disease diagnosis.     

Cdh11 Acts as a Tumor Suppressor in a Murine Retinoblastoma Model by Facilitating Tumor Cell Death

CDH11 gene copy number and expression are frequently lost in human retinoblastomas and in retinoblastomas arising in TAg-RB mice. To determine the effect of Cdh11 loss in tumorigenesis, we crossed Cdh11 null mice with TAg-RB mice. Loss of Cdh11 had no gross morphological effect on the developing retina of Cdh11 knockout mice, but led to larger retinal volumes in mice crossed with TAg-RB mice (p = 0.01). Mice null for Cdh11 presented with fewer TAg-positive cells at postnatal day 8 (PND8) (p = 0.01) and had fewer multifocal tumors at PND28 (p = 0.016), compared to mice with normal Cdh11 alleles. However, tumor growth was faster in Cdh11-null mice between PND8 and PND84 (p = 0.003). In tumors of Cdh11-null mice, cell death was decreased 5- to 10-fold (p<0.03 for all markers), while proliferation in vivo remained unaffected (p = 0.121). Activated caspase-3 was significantly decreased and β-catenin expression increased in Cdh11 knockdown experiments in vitro. These data suggest that Cdh11 displays tumor suppressor properties in vivo and in vitro in murine retinoblastoma through promotion of cell death.     

Understanding Heterogeneity and Permeability of Brain Metastases in Murine Models of HER2-Positive Breast Cancer Through Magnetic Resonance Imaging: Implications for Detection and Therapy

OBJECTIVES: Brain metastases due to breast cancer are increasing, and the prognosis is poor. Lack of effective therapy is attributed to heterogeneity of breast cancers and their resulting metastases, as well as impermeability of the blood–brain barrier (BBB), which hinders delivery of therapeutics to the brain. This work investigates three experimental models of HER2 + breast cancer brain metastasis to better understand the inherent heterogeneity of the disease. We use magnetic resonance imaging (MRI) to quantify brain metastatic growth and explore its relationship with BBB permeability. DESIGN: Brain metastases due to breast cancer cells (SUM190-BR3, JIMT-1-BR3, or MDA-MB-231-BR-HER2) were imaged at 3 T using balanced steady-state free precession and contrast-enhanced T1-weighted spin echo sequences. The histology and immunohistochemistry corresponding to MRI were also analyzed. RESULTS: There were differences in metastatic tumor appearance by MRI, histology, and immunohistochemistry (Ki67, CD31, CD105) across the three models. The mean volume of an MDA-MB-231-BR-HER2 tumor was significantly larger compared to other models (F_2,12 = 5.845, P < .05); interestingly, this model also had a significantly higher proportion of Gd-impermeable tumors (F_2,12 = 22.18, P < .0001). Ki67 staining indicated that Gd-impermeable tumors had significantly more proliferative nuclei compared to Gd-permeable tumors (t[24] = 2.389, P < .05) in the MDA-MB-231-BR-HER2 model. CD31 and CD105 staining suggested no difference in new vasculature patterns between permeable and impermeable tumors in any model. CONCLUSION: Significant heterogeneity is present in these models of brain metastases from HER2 + breast cancer. Understanding this heterogeneity, especially as it relates to BBB permeability, is important for improvement in brain metastasis detection and treatment delivery.     

Lentivirus-mediated overexpression of TGF-β inducible early gene 1 inhibits SW1990 pancreatic cancer cell growth

TIEG1 (TGF-β inducible early gene 1) plays a significant role in regulating cell proliferation and apoptosis in various cell types. Previous studies have shown a close relationship between the expression level of TIEG1 and various cancers, including breast, prostate, colorectal and pancreatic cancer. In this study, we up-regulated the gene expression of TIEG1 in SW1990 pancreatic cancer cell line by a lentivirus transfection system and investigated its potential as a therapeutic target for pancreatic cancer. The results showed that lentivirus-mediated overexpression of TIEG1 gene inhibited human pancreatic cancer SW1990 cell proliferation and caused the cell cycle arrest at the G1-phase in vitro. SW1990 cells transduced with lenti-TIEG1 showed significant inhibition of colony formation and cancer cell growth in 3-D culture model. Moreover, overexpression of TIEG1 gene significantly slowed the growth of SW1990 xenografts in nude mice. Taken together, these data provided evidence that overexpression of TIEG1 gene by a lentivirus transfection system led to suppressed human pancreatic cancer cell growth and might therefore be a feasible approach in the clinical management of pancreatic cancer.     

Noninvasive Assessment of Losartan-Induced Increase in Functional Microvasculature and Drug Delivery in Pancreatic Ductal Adenocarcinoma

PURPOSE: Losartan, an angiotensin II receptor blocker, can reduce desmoplasia and enhance drug delivery and efficacy through improving interstitial transport and vascular perfusion in pancreatic ductal adenocarcinoma (PDAC) models in mice. The purpose of this study was to determine whether magnetic resonance imaging (MRI) of magnetic iron oxide nanoparticles (MNPs) and micro–positron emission tomography (PET) measurements could respectively detect improvements in tumor vascular parameters and drug uptake in orthotopic PDAC in mice treated with losartan. METHOD AND MATERIALS: All experiments were approved by the local Institutional Animal Care and Use Committee. FVB mice with orthotopic PDAC were treated daily with an i.p. injection of losartan (70 mg/kg) or saline (control vehicle) for 5 days. In order to calculate the fractional blood volume, vessel size index, and vessel density index, MRI was performed at 4.7 T following the injection of 3 mg/kg iron ferumoxytol (i.v.). Dynamic PET images were also acquired for 60 minutes using an ¹⁸F-5FU tracer dose of 200 μCi and analyzed for time activity curves normalized to muscle. Statistical analyses compared both cohorts using an unpaired two-tailed t test. RESULTS: In comparison to the control treatment, the losartan administration significantly increased the fractional blood volume (mean ± SEM) [12.1 ± 1.7 (n = 19) vs 6.7 ± 1.1 (n = 20); P < .02] and vessel size index (128.2 ± 35.6 vs 57.5 ± 18; P < .05). Losartan also induced a significant increase in the intratumoral uptake of ¹⁸F-5FU by 53% (P < .0001). CONCLUSION: MRI using FDA-approved MNPs provides a noninvasive, translatable means of assaying microvascular parameters induced by losartan in pancreatic cancer. PET measurements demonstrated that losartan significantly increased the uptake of ¹⁸F-5FU.     

Radiofrequency Ablation of Liver VX2 Tumor: Experimental Results with MR Diffusion-Weighted Imaging at 3.0T

Purpose

To evaluate the value of DWI in detecting the lesions of pre- and post-radiofrequency ablation (RFA) of the rabbit liver VX2 tumors.

Materials and Methods

Twenty-two New Zealand White rabbits were tested. The protocol was approved by the Committee on the Ethics of Animal Experiments. Twenty separate tumor fragments were implanted into the livers of 20 rabbits, the liver was exposed by performing midline laparotomy. 3.0T MR DWI (b = 0, 200, 400, 600, 800,1000 s/mm²) were performed 14–21 days after tumor implantation (mean, 17 days) in the 18 tumor-bearing animals. Then RFA was performed in the 18 tumor-bearing animals and in the two healthy animals. 3.0T MR DWI was performed 7–10 days after RFA (mean, 8 days). Pathology exam was performed immediately after the completion of post- RFA MR imaging. Analyzing the features of MRI and ADC values in the pre- and post- RFA lesions of the VX2 tumors, and histopathologic results were compared with imaging findings.

Results

The difference of ADC value between viable tumor and normal liver parenchyma was significant (P<.001). After RFA, when b = 200, 400, 600, 800, 1000 s/mm², the differences of ADC values of viable tumor, granulation tissue, necrosis, normal liver parenchyma were significant (P<.001). At the time the animals were sacrificed after RFA and MR imaging, histopathologic results of local viable tumors were found in 9 (50%) of the 18 treated tumors. Macroscopic viable tumors were found at the RFA sites in 3 (17%), all 3 macroscopic viable tumors were visualized at the periphery of the RFA areas.

Conclusions

3.0T MR DWI can be used to follow up the progress of the RFA lesion, it is useful in detecting different tissues after RFA, and it is valuable in the further clinical research.     

Tumor‐Targeting Salmonella typhimurium A1‐R Promotes Tumoricidal CD8+ T Cell Tumor Infiltration and Arrests Growth and Metastasis in a Syngeneic Pancreatic‐Cancer Orthotopic Mouse Model

The present study determined the effect of the tumor‐targeting strain Salmonella typhimurium A1‐R (S. typhimurium A1‐R) on CD8⁺ tumor‐infiltrating lymphocytes (TILs) in a syngeneic pancreatic‐cancer orthotopic mouse model. The effect of tumor‐targeting S. typhimurium A1‐R on CD8⁺ TILs was determined on the Pan02 murine pancreatic‐adenocarcinoma implanted orthotopically in the pancreatic tail of C57BL/6 immunocompromised mice. Three weeks after orthotopic implantation, mice were randomized as follows G1: untreated control group (n = 8); and G2: S. typhimurium A1‐R‐treatment group (n = 8, 1 × 10⁷ colony forming units [CFU]/body, iv, weekly, 3 weeks). On the 22nd day from initial treatment, all mice were sacrificed and tumors were harvested. The tumor‐volume ratio was defined as ratio of tumor volume on the 22nd day relative to the 1st day. The tumor volume ratio was significantly lower in the S. typhimurium A1‐R‐treated group (G2) (3.0 ± 2.8) than the untreated control (G1) (39.9 ± 30.7, P < 0.01). Hematoxylin and easin (H&E) staining on tumor sections was performed to evaluate tumor destruction which was classified according to the Evans grading system and found to be much greater in the S. typhimurium A1‐R‐treated mice (G2). Six mice in G1 had peritoneal dissemination, whereas no mice showed peritoneal dissemination in G2 (P < 0.01). Immunohistochemical staining with anti‐mouse CD8⁺ antibody was performed in order to detect TILs determined by calculating the average number of CD8⁺ cells in three high power fields (200×) in the treated and untreated tumors. The TIL score was significantly higher in G2 (133.5 ± 32.2) than G1 (45.1 ± 19.4, P < 0.001). The present study demonstrates that S. typhimurium A1‐R promotes CD8⁺ T cell infiltration and inhibition of tumor growth and metastasis. J. Cell. Biochem. 119: 634–639, 2018. © 2017 Wiley Periodicals, Inc.     

Meningiomas of the Cerebellopontine Angle: Radiological Differences in Tumors with Internal Auditory Canal Involvement and Their Influence on Surgical Outcome

This study explored the clinical, radiological, and pathological characteristics of cerebellopontine angle (CPA) meningiomas with internal auditory canal (IAC) involvement. The pre- and postoperative MR images of 193 consecutive patients with pathologically diagnosed meningioma centered around the IAC were analyzed, focusing on changes in the IAC, maximal axial tumor volume, peritumoral brain edema, and postoperative residual tumor. Patient age, sex, tumor volume, postoperative residual tumor, and pathological subtype were compared in patients with and without IAC involvement by the tumor and among the different types of IAC involvement. The results showed that the 71 patients (36.8%) with IAC involvement had a higher ratio of peritumoral edema (χ²=5.922, P=0.015), postoperative residual tumor (χ²=22.183, P< 0.001), and a predominance of the meningothelial subtype (χ²=5.89, P=0 .015). Peritumoral edema was a risk factor for IAC involvement (P=0.016, OR=2.186). Radiologically, IAC involvement could be distinguished as intruding (31%, 22/71), filled (29.6%, 21/71), and dilated (39.4%, 28/71). Patients with intruding IAC were significantly older (54.5±9.54 years, P=0.021) and had the lowest postoperative residual tumor values (42%, χ²=7.865, P= 0.005), while those with filled IAC were more likely to be female (95%, χ²=9.404, P=0.009).Our observations provide the basis for a morphological classification of IAC involvement by CPA meningiomas and further insight into the clinical features of these tumors.     

Mice Lacking NCF1 Exhibit Reduced Growth of Implanted Melanoma and Carcinoma Tumors

The NADPH oxidase 2 (NOX2) complex is a professional producer of reactive oxygen species (ROS) and is mainly expressed in phagocytes. While the activity of the NOX2 complex is essential for immunity against pathogens and protection against autoimmunity, its role in the development of malignant tumors remains unclear. We compared wild type and Ncf1 ^m1J mutated mice, which lack functional NOX2 complex, in four different tumor models. Ncf1 ^m1J mutated mice developed significantly smaller tumors in two melanoma models in which B16 melanoma cells expressing a hematopoietic growth factor FLT3L or luciferase reporter were used. Ncf1 ^m1J mutated mice developed significantly fewer Lewis Lung Carcinoma (LLC) tumors, but the tumors that did develop, grew at a pace that was similar to the wild type mice. In the spontaneously arising prostate carcinoma model (TRAMP), tumor growth was not affected. The lack of ROS-mediated protection against tumor growth was associated with increased production of immunity-associated cytokines. A significant increase in Th2 associated cytokines was observed in the LLC model. Our present data show that ROS regulate rejection of the antigenic B16-luc and LLC tumors, whereas the data do not support a role for ROS in growth of intrinsically generated tumors.     

RGD-Targeted Ultrasound Contrast Agent for Longitudinal Assessment of Hep-2 Tumor Angiogenesis In Vivo

Objective

To prepare arginine-glycine-aspartate (RGD)-targeted ultrasound contrast microbubbles (MBs) and explore the feasibility of their use in assessing dynamic changes in αvβ3 integrin expression in a murine model of tumor angiogenesis.

Methods

RGD peptides were conjugated to the surfaces of microbubbles via biotin-avidin linkage. Microbubbles bearing RADfK peptides were prepared as controls. The RGD-MBs were characterized using an Accusizer 780 and optical microscopy. The binding specificity of the RGD-MBs for ανβ3-expressing endothelial cells (bEnd.3) was demonstrated in vitro by a competitive inhibition experiment. In an in vivo study, mice bearing tumors of three different stages were intravenously injected with RGD-MBs and subjected to targeted, contrast-enhanced, high-frequency ultrasound. Subsequently, tumors were harvested and sectioned for immunofluorescence analysis of ανβ3 expression.

Results

The mean size of the RGD-MBs was 2.36 ± 1.7 μm. The RGD-MBs showed significantly higher adhesion levels to bEnd.3 cells compared to control MBs (P < 0.01). There was rarely binding of RGD-MBs to αvβ3-negative MCF-7 cells. Adhesion of the RGD-MBs to the bEnd.3 cells was significantly inhibited following treatment with anti-alpha(v) antibodies. The quantitative acoustic video intensity for high-frequency, contrast-enhanced ultrasound imaging of subcutaneous human laryngeal carcinoma (Hep-2) tumor xenografts was significantly higher in small tumors (19.89 ± 2.49) than in medium tumors (11.25 ± 2.23) and large tumors (3.38 ± 0.67) (P < 0.01).

Conclusions

RGD-MBs enable noninvasive in vivo visualization of changes in tumor angiogenesis during tumor growth in subcutaneous cancer xenografts.     

Noninvasive Identification of Viable Cell Populations in Docetaxel-Treated Breast Tumors Using Ferritin-Based Magnetic Resonance Imaging

Background

Cancer stem cells (CSCs) are highly tumorigenic and are responsible for tumor progression and chemoresistance. Noninvasive imaging methods for the visualization of CSC populations within tumors in vivo will have a considerable impact on the development of new CSC-targeting therapeutics.

Methodology/Principal Findings

In this study, human breast cancer stem cells (BCSCs) transduced with dual reporter genes (human ferritin heavy chain [FTH] and enhanced green fluorescence protein [EGFP]) were transplanted into NOD/SCID mice to allow noninvasive tracking of BCSC-derived populations. No changes in the properties of the BCSCs were observed due to ferritin overexpression. Magnetic resonance imaging (MRI) revealed significantly different signal intensities (R₂* values) between BCSCs and FTH-BCSCs in vitro and in vivo. In addition, distinct populations of pixels with high R₂* values were detected in docetaxel-treated FTH-BCSC tumors compared with control tumors, even before the tumor sizes changed. Histological analysis revealed that areas showing high R₂* values in docetaxel-treated FTH-BCSC tumors by MRI contained EGFP+/FTH+ viable cell populations with high percentages of CD44+/CD24− cells.

Conclusions/Significance

These findings suggest that ferritin-based MRI, which provides high spatial resolution and tissue contrast, can be used as a reliable method to identify viable cell populations derived from BCSCs after chemotherapy and may serve as a new tool to monitor the efficacy of CSC-targeting therapies in vivo.     

[18F]FLT PET for Non-Invasive Assessment of Tumor Sensitivity to Chemotherapy: Studies with Experimental Chemotherapy TP202377 in Human Cancer Xenografts in Mice

Aim

3′-deoxy-3′-[¹⁸F]fluorothymidine ([18F]FLT) is a tracer used to assess cell proliferation in vivo. The aim of the study was to use [18F]FLT positron emission tomography (PET) to study non-invasively early anti-proliferative effects of the experimental chemotherapeutic agent TP202377 in both sensitive and resistant tumors.

Methods

Xenografts in mice from 3 human cancer cell lines were used: the TP202377 sensitive A2780 ovary cancer cell line (n = 8–16 tumors/group), the induced resistant A2780/Top216 cell line (n = 8–12 tumors/group) and the natural resistant SW620 colon cancer cell line (n = 10 tumors/group). In vivo uptake of [18F]FLT was studied at baseline and repeated 6 hours, Day 1, and Day 6 after TP202377 treatment (40 mg/kg i.v.) was initiated. Tracer uptake was quantified using small animal PET/CT.

Results

TP202377 (40 mg/kg at 0 hours) caused growth inhibition at Day 6 in the sensitive A2780 tumor model compared to the control group (P<0.001). In the A2780 tumor model TP202377 treatment caused significant decrease in uptake of [18F]FLT at 6 hours (-46%; P<0.001) and Day 1 (-44%; P<0.001) after treatment start compared to baseline uptake. At Day 6 uptake was comparable to baseline. Treatment with TP202377 did not influence tumor growth or [18F]FLT uptake in the resistant A2780/Top216 and SW620 tumor models. In all control groups uptake of [18F]FLT did not change. Ki67 gene expression paralleled [18F]FLT uptake.

Conclusion

Treatment of A2780 xenografts in mice with TP202377 (single dose i.v.) caused a significant decrease in cell proliferation assessed by [18F]FLT PET after 6 hours. Inhibition persisted at Day 1; however, cell proliferation had returned to baseline at Day 6. In the resistant A2780/Top216 and SW620 tumor models uptake of [18F]FLT did not change after treatment. With [18F]FLT PET it was possible to distinguish non-invasively between sensitive and resistant tumors already 6 hours after treatment initiation.