Accuracy and Precision of Iodine Quantification in Subtracted Micro-Computed Tomography: Effect of Reconstruction and Noise Removal Algorithms.

PURPOSE: To evaluate the effect of reconstruction and noise removal algorithms on the accuracy and precision of iodine concentration (CI) quantified with subtracted micro-computed tomography (micro-CT). PROCEDURES: Two reconstruction algorithms were evaluated: a filtered backprojection (FBP) algorithm and a simultaneous iterative reconstruction technique (SIRT) algorithm. A 3D bilateral filter (BF) was used for noise removal. A phantom study evaluated and compared the image quality, and the accuracy and precision of CI in four scenarios: filtered FBP, filtered SIRT, non-filtered FBP, and non-filtered SIRT. In vivo experiments were performed in an animal model of chemically-induced mammary cancer. RESULTS: Linear relationships between the measured and nominal CI values were found for all the scenarios in the phantom study (R2 > 0.95). SIRT significantly improved the accuracy and precision of CI compared to FBP, as given by their lower bias (adj. p-value = 0.0308) and repeatability coefficient (adj. p-value < 0.0001). Noise removal enabled a significant decrease in bias in filtered SIRT images only; non-significant differences were found for the repeatability coefficient. The phantom and in vivo studies showed that CI is a reproducible imaging parameter for all the scenarios (Pearson r > 0.99, p-value < 0.001). The contrast-to-noise ratio showed non-significant differences among the evaluated scenarios in the phantom study, while a significant improvement was found in the in vivo study when SIRT and BF algorithms were used. CONCLUSIONS: SIRT and BF algorithms improved the accuracy and precision of CI compared to FBP and non-filtered images, which encourages their use in subtracted micro-CT imaging.

Early detection of colorectal cancer somatic mutations using cfDNA liquid biopsies in a murine carcinogenesis model.

Colorectal cancer (CRC) is one of the top five cancers in incidence and mortality worldwide. The early detection of this neoplasm through analysis of circulating free DNA (cfDNA), which carries tumor genetic alterations, as a liquid biopsy, could have a major impact in enhancing early detection and reducing the mortality rate. The aim of this work was to demonstrate the feasibility of using cfDNA as a liquid biopsy for the early detection of CRC. For this purpose, we implemented an azoxymethane and dextran sodium sulfate-induced murine carcinogenesis model to detect oncogenic somatic mutations in Ctnnb1 and Kras during CRC development. To enhance the sensitivity in the detection, E-ice-COLD-PCR was utilized to selectively enrich for mutant alleles, followed by massively parallel sequencing. Driving somatic mutations were detected in Ctnnb1 and Kras in the liquid biopsies of early stages of tumor development, corresponding to the formation of aberrant crypt foci, the first histological alterations that can be identified throughout the formation of CRC. The concentration of cfDNA was increased along the carcinogenic process. Polyclonality in Ctnnb1 was found in tumor samples and cfDNA in this model. On the other hand, the use of cfDNA as a non-invasive test resulted in superior early detection compared to microPET/CT imaging. As a proof-of-principle, this study shows the great potential use of allelic-specific PCR for the detection and enrichment of pathogenic alleles present in cfDNA samples, as a test for early non-invasive detection of CRC. This work provides scientific evidence to set methodological bases that allow early detection of mutations in cfDNA obtained from plasma of CRC in humans.

Lipomics: A Potential Carrier for the Intravenous Delivery of Lipophilic and Hydrophilic Drugs.

In the present work, we propose the development of a novel carrier that does not need organic solvents for its preparation and with the potential for the intravenous delivery of lipophilic and hydrophilic drugs. Named lipomics, this is a mixed colloid of micelles incorporated within a liposome. This system was designed through ternary diagrams and characterized by physicochemical techniques to determine the particle size, zeta potential, shape, morphology, and stability properties. The lipomics were subjected to electron microscopy (SEM, TEM, and STEM) to evaluate their physical size and morphology. Finally, pharmacokinetic studies were performed by radiolabeling the lipomics with Technetium-99m chelated with BMEDA to evaluate the in vivo biodistribution through techniques of molecular imaging (microSPECT/CT) in rats. Radiolabeling efficiency was used to compare the encapsulation efficiency of the hydrophilic and lipophilic molecules in lipomics and liposomes. According to the results, lipomics are potentially carriers of lipophilic and hydrophilic drugs.

Evaluation of Traumatic Spinal Cord Injury in a Rat Model Using 99mTc-GA-5 as a Potential In Vivo Tracer.

Spinal cord injury (SCI) refers to the damage suffered in the spinal cord by any trauma or pathology. The purpose of this work was to determine whether 99mTc-GA-5, a radiotracer targeting Glial Fibrillary Acidic Protein (GFAP), can reveal in vivo the reactivation of astrocytes in a murine model with SCI. A method for the 99mTc radiolabeling of the mouse anti-GFAP monoclonal antibody GA-5 was implemented. Radiochemical characterization was performed, and radioimmunohistochemistry assays were used to evaluate the integrity of 99mTc-GA-5. MicroSPECT/CT was used for in vivo imaging to trace SCI in the rats. No alterations in the GA-5's recognition/specificity ability were observed after the radiolabeling. The GA-5's radiolabeling procedure implemented in this work offers a practical method to allow the in vivo following of this monoclonal antibody to evaluate its biodistribution and specificity for GFAP receptors using SPECT/CT molecular imaging.

Quantitative Imaging Parameters of Contrast-Enhanced Micro-Computed Tomography Correlate with Angiogenesis and Necrosis in a Subcutaneous C6 Glioma Model.

The aim of this work was to systematically obtain quantitative imaging parameters with static and dynamic contrast-enhanced (CE) X-ray imaging techniques and to evaluate their correlation with histological biomarkers of angiogenesis in a subcutaneous C6 glioma model. Enhancement (E), iodine concentration (CI), and relative blood volume (rBV) were quantified from single- and dual-energy (SE and DE, respectively) micro-computed tomography (micro-CT) images, while rBV and volume transfer constant (Ktrans) were quantified from dynamic contrast-enhanced (DCE) planar images. CI and rBV allowed a better discernment of tumor regions from muscle than E in SE and DE images, while no significant differences were found for rBV and Ktrans in DCE images. An agreement was found in rBV for muscle quantified with the different imaging protocols, and in CI and E quantified with SE and DE protocols. Significant strong correlations (Pearson r > 0.7, p < 0.05) were found between a set of imaging parameters in SE images and histological biomarkers: E and CI in tumor periphery were associated with microvessel density (MVD) and necrosis, E and CI in the complete tumor with MVD, and rBV in the tumor periphery with MVD. In conclusion, quantitative imaging parameters obtained in SE micro-CT images could be used to characterize angiogenesis and necrosis in the subcutaneous C6 glioma model.

Liposomes Co-Encapsulating Cisplatin/Mifepristone Improve the Effect on Cervical Cancer: In Vitro and In Vivo Assessment.

Cervical cancer is usually diagnosed in the later stages despite many campaigns for early detection and continues to be a major public health problem. The standard treatment is cisplatin-based chemotherapy plus radiotherapy, but patient response is far from ideal. In the research for new drugs that enhance the activity of cisplatin, different therapeutic agents have been tested, among them the antiprogestin mifepristone. Nevertheless, the efficacy of cisplatin is limited by its low specificity for tumor tissue, which causes severe side effects. Additionally, cervical tumors often become drug resistant. These problems could possibly be addressed by the use of liposome nanoparticles to encapsulate drugs and deliver them to the target. The aim of this study was to prepare liposome nanoparticles that co-encapsulate cisplatin and mifepristone, evaluate their cytotoxicity against HeLa cells and in vivo with subcutaneous inoculations of xenografts in nu/nu mice, and examine some plausible mechanisms of action. The liposomes were elaborated by the reverse-phase method and characterized by physicochemical tests. The nanoparticles had a mean particle size of 109 ± 5.4 nm and a Zeta potential of -38.7 ± 1.2 mV, the latter parameter indicating a stable formulation. These drug-loaded liposomes significantly decreased cell viability in vitro and tumor size in vivo, without generating systemic toxicity in the animals. There was evidence of cell cycle arrest and increased apoptosis. The promising results with the co-encapsulation of cisplatin/mifepristone warrant further research.

In vivo Regeneration of Mineralized Bone Tissue in Anisotropic Biomimetic Sponges.

In the last two decades, alginate scaffolds have been variously studied as extracellular matrix analogs for tissue engineering. However, relevant evidence is still lacking concerning their ability to mimic the microenvironment of hierarchical tissues such as bone. Hence, an increasing amount of attention has recently been devoted to the fabrication of macro/microporous sponges with pore anisotropy able to more accurately replicate the cell niche structure as a trigger for bioactive functionalities. This paper presents an in vivo study of alginate sponges with anisotropic microporous domains (MAS) formed by ionic crosslinking in the presence of different fractions (30 or 50% v) of hydroxyapatite (HA). In comparison with unloaded sponges (MAS0), we demonstrated that HA confers peculiar physical and biological properties to the sponge, depending upon the inorganic fraction used, enabling the sponge to bio-mimetically support the regeneration of newly formed bone. Scanning electron microscopy analysis showed a preferential orientation of pores, ascribable to the physical constraints exerted by HA particles during the pore network formation. Energy dispersive spectroscopy (EDS) and X-Ray diffraction (XRD) confirmed a chemical affinity of HA with the native mineral phase of the bone. In vitro studies via WST-1 assay showed good adhesion and proliferation of human Dental Pulp-Mesenchymal Stem Cells (hDP-MSC) that increased in the presence of the bioactive HA signals. Moreover, in vivo studies via micro-CT and histological analyses of a bone model (e.g., a rat calvaria defect) confirmed that the maximum osteogenic response after 90 days was achieved with MAS30, which supported good regeneration of the calvaria defect without any evidence of inflammatory reaction. Hence, all of the results suggested that MAS is a promising scaffold for supporting the regeneration of hard tissues in different body compartments.

Evaluation of bone regeneration in a critical size cortical bone defect in rat mandible using microCT and histological analysis.

GOAL: Evaluate bone regeneration in a critical size bone defect model in the jaw of healthy rats as a function of gender and defect location. DESIGN: A series of microCT and histological studies were performed to evaluate the process of bone regeneration in rats with a mandibular critical size defect. Rats were placed in two groups according to gender and sorted in terms of bone defect location. Bone regeneration rate and hydroxyapatite concentration were assessed with microCT imaging at specific times after surgery. Histological analysis was also performed to evaluate bone regeneration. RESULTS: No more that 85% of bone regeneration was observed after 60 days, with a low rate constant (K) indicating a slow restoration of the defect. Assessment of microCT images showed partial closure of the defect in all cases, which was confirmed by histological analysis. Hydroxyapatite concentration values revealed that regenerated bone was not fully calcified. No statistically significant differences in terms of gender or defect location were found. CONCLUSION: The defect model studied here, located in the jaw of healthy rats, shows potential as a preclinical critical size bone defect model to evaluate bone regeneration therapies in the fields of dentistry and maxillofacial surgery.

KRas4B-PDE6δ complex stabilization by small molecules obtained by virtual screening affects Ras signaling in pancreatic cancer.

BACKGROUND: The GTPase KRas4B has been utilized as a principal target in the development of anticancer drugs. PDE6δ transports KRas4B to the plasma membrane, where it is released to activate various signaling pathways required for the initiation and maintenance of cancer. Therefore, identifying new small molecules that prevent activation of this GTPase by stabilizing the KRas4B-PDE6δ molecular complex is a practical strategy to fight against cancer. METHODS: The crystal structure of the KRas4B-PDE6δ heterodimer was employed to locate possible specific binding sites at the protein-protein interface region. Virtual screening of Enamine-database compounds was performed on the located potential binding sites to identify ligands able to simultaneously bind to the KRas4B-PDE6δ heterodimer. A molecular dynamics approach was used to estimate the binding free-energy of the complex. Cell viability and apoptosis were measured by flow cytometry. G-LISA was used to measure Ras inactivation. Western blot was used to measure AKT and ERK activation. MIA PaCa-2 cells implanted subcutaneously into nude mice were treated with D14 or C22 and tumor volumes were recorded. RESULTS: According to the binding affinity estimation, D14 and C22 stabilized the protein-protein interaction in the KRas4B-PDE6δ complex based on in vitro evaluation of the 38 compounds showing antineoplastic activity against pancreatic MIA PaCa-2 cancer cells. In this work, we further investigated the antineoplastic cellular properties of two of them, termed D14 and C22, which reduced the viability in the human pancreatic cancer cells lines MIA PaCa-2, PanC-1 and BxPC-3, but not in the normal pancreatic cell line hTERT-HPNE. Compounds D14 and C22 induced cellular death via apoptosis. D14 and C22 significantly decreased Ras-GTP activity by 33% in MIA PaCa-2 cells. Moreover, D14 decreased AKT phosphorylation by 70% and ERK phosphorylation by 51%, while compound C22 reduced AKT phosphorylation by 60% and ERK phosphorylation by 36%. In addition, compounds C22 and D14 significantly reduced tumor growth by 88.6 and 65.9%, respectively, in a mouse xenograft model. CONCLUSIONS: We identified two promising compounds, D14 and C22, that might be useful as therapeutic drugs for pancreatic ductal adenocarcinoma treatment.

Biodistribution and Tumor Uptake of 67Ga-Nimotuzumab in a Malignant Pleural Mesothelioma Xenograft.

Malignant pleural mesothelioma (MPM) is the most common tumor of the pulmonary pleura. It is a rare and aggressive malignancy, generally associated with continuous occupational exposure to asbestos. Only a multimodal-approach to treatment, based on surgical resection, chemotherapy and/or radiation, has shown some benefits. However, the survival rate remains low. Nimotuzumab (h-R3), an anti-EGFR (epidermal growth factor receptor) humanized antibody, is proposed as a promising agent for the treatment of MPM. The aim of this research was to implement a procedure for nimotuzumab radiolabeling to evaluate its biodistribution and affinity for EGF (epidermal growth factor) receptors present in a mesothelioma xenograft. Nimotuzumab was radiolabeled with 67Ga; radiolabel efficiency, radiochemical purity, serum stability, and biodistribution were evaluated. Biodistribution and tumor uptake imaging studies by microSPECT/CT in mesothelioma xenografts revealed constant nimotuzumab uptake at the tumor site during the first 48 h after drug administration. In vivo studies using MPM xenografts showed a significant uptake of this radioimmunoconjugate, which illustrates its potential as a biomarker that could promote its theranostic use in patients with MPM.

Biodegradable poly(D,L-lactide-co-glycolide)/poly(L-γ-glutamic acid) nanoparticles conjugated to folic acid for targeted delivery of doxorubicin.

A novel targeted drug delivery nanoparticle system based on poly(D,L-lactide-co-glycolide) acid (PLGA) for delivery of doxorubicin (DOX) was developed. DOX-PLGA NPs were obtained by the emulsification-solvent evaporation technique. Then, their surface was modified with poly(L-γ-glutamic acid) (γ-PGA) and finally conjugated to modified folic acid (FA) as a targeting ligand. The surface modification and FA conjugation were followed by UV-Vis and FT-IR spectroscopies. Morphology was observed by TEM/SEM. Particle size, PDI and zeta potential were measured using DLS studies. Encapsulation and loading efficiencies, and DOX release kinetics were determined. Specific uptake and cell viability of DOX-PLGA/γ-PGA-FA NPs were tested in HeLa cells. Quasi-spherical nanoparticles with a particle size lower than 600nm (DLS) were obtained. Spectroscopic techniques demonstrated the successful surface modification with γ-PGA and FA conjugation. Release profile of DOX-PLGA/γ-PGA-FA NPs showed a release of 55.4±0.6% after seven days, in an acidic environment. HeLa cells exhibited a decrease in viability when treated with DOX-PLGA/γ-PGA-AF NPs, and cellular uptake was attributed to FA receptor-mediated endocytosis. These results suggest that DOX-PLGA/γ-PGA-FA NPs are a potential targeted drug carrier for further applications in cancer therapy.

Characterization and in vitro evaluation of nimotuzumab conjugated with cisplatin-loaded liposomes.

In this paper, we report the conjugation of the humanized monoclonal antibody nimotuzumab with cisplatin-loaded liposomes and the in vitro evaluation of its affinity for tumor cells. The conjugation procedure was performed through derivatization of nimotuzumab with N-succinimidyl S-acetylthioacetate (SATA) followed by a covalent attachment with maleimide groups at the end of PEG-DSPE chains located at the membrane of pre-formed liposomes. Confocal microscopy was performed to evaluate the immunoliposome affinity for EGFR antigens from human epidermoid carcinoma (A-431) and normal lung (MRC-5) cell lines. Results showed that the procedures implemented in this work do not affect the capability of the nimotuzumab-immunoliposomes to recognize the tumor cells, which overexpress the EGFR antigens.

Biodistribution and Lymphatic Tracking of the Main Neurotoxin of Micrurus fulvius Venom by Molecular Imaging.

The venom of the Eastern coral snake Micrurus fulvius can cause respiratory paralysis in the bitten patient, which is attributable to ß-neurotoxins (ß-NTx). The aim of this work was to study the biodistribution and lymphatic tracking by molecular imaging of the main ß-NTx of M. fulvius venom. ß-NTx was bioconjugated with the chelator diethylenetriaminepenta-acetic acid (DTPA) and radiolabeled with the radionuclide Gallium-67. Radiolabeling efficiency was 60%-78%; radiochemical purity ≥92%; and stability at 48 h ≥ 85%. The median lethal dose (LD50) and PLA2 activity of bioconjugated ß-NTx decreased 3 and 2.5 times, respectively, in comparison with native ß-NTx. The immune recognition by polyclonal antibodies decreased 10 times. Biodistribution of ß-NTx-DTPA-(67)Ga in rats showed increased uptake in popliteal, lumbar nodes and kidneys that was not observed with (67)Ga-free. Accumulation in organs at 24 h was less than 1%, except for kidneys, where the average was 3.7%. The inoculation site works as a depot, since 10% of the initial dose of ß-NTx-DTPA-(67)Ga remains there for up to 48 h. This work clearly demonstrates the lymphatic system participation in the biodistribution of ß-NTx-DTPA-(67)Ga. Our approach could be applied to analyze the role of the lymphatic system in snakebite for a better understanding of envenoming.

Newcastle Disease Virus: Potential Therapeutic Application for Human and Canine Lymphoma.

Research on oncolytic viruses has mostly been directed towards the treatment of solid tumors, which has yielded limited information regarding their activity in hematological cancer. It has also been directed towards the treatment of humans, yet veterinary medicine may also benefit. Several strains of the Newcastle disease virus (NDV) have been used as oncolytics in vitro and in a number of in vivo experiments. We studied the cytolytic effect of NDV-MLS, a low virulence attenuated lentogenic strain, on a human large B-cell lymphoma cell line (SU-DHL-4), as well as on primary canine-derived B-cell lymphoma cells, and compared them to healthy peripheral blood mononuclear cells (PBMC) from both humans and dogs. NDV-MLS reduced cell survival in both human (42% ± 5%) and dog (34% ± 12%) lymphoma cells as compared to untreated controls. No significant effect on PBMC was seen. Cell death involved apoptosis as documented by flow-cytometry. NDV-MLS infections of malignant lymphoma tumors in vivo in dogs were confirmed by electron microscopy. Early (24 h) biodistribution of intravenous injection of 1 × 10(12) TCID50 (tissue culture infective dose) in a dog with T-cell lymphoma showed viral localization only in the kidney, the salivary gland, the lung and the stomach by immunohistochemistry and/or endpoint PCR. We conclude that NDV-MLS may be a promising agent for the treatment of lymphomas. Future research is needed to elucidate the optimal therapeutic regimen and establish appropriate biosafety measures.

High liposomal doxorubicin tumour tissue distribution, as determined by radiopharmaceutical labelling with (99m)Tc-LD, is associated with the response and survival of patients with unresectable pleural mesothelioma treated with a combination of liposomal doxorubicin and cisplatin.

BACKGROUND: There are currently no available biomarkers for advanced pleural mesothelioma that determine which patients could benefit from a specific chemotherapy regimen. METHODS: Based on the results of a previously published phase II study, we associated the (99m)Technetium-labelled liposomal doxorubicin ((99m)Tc-LD) uptake value (75 % cut-off) with the response rate, progression-free survival and overall survival of patients treated with a combination of liposomal doxorubicin and cisplatin. RESULTS: Patients with tumours exhibiting increased (99m)Tc-LD uptake showed better response rates, progression-free survival and overall survival than those exhibiting lower uptake 73.3 versus 15 % (p < 0.001); 6.9 versus 3.2 months (p = 0.033) and 23 versus 6.6 months (p = 0.001), respectively. CONCLUSION: (99m)Tc-DL uptake in tumour tissue could define a set of patients who would benefit from this chemotherapy regimen.

Synthesis of glycerol-derived diallyl spiroorthocarbonates and the study of their antishrinking properties in acrylic dental resins.

In this work was evaluated the efficiency of an antishrinkage additive in a dental resin. This additive was a mixture 1:1 of five and six-membered ring spiroorthocarbonates functionalized with allylic groups (SOC DA). The aim of this study was to reduce the shrinkage of a typical dental resin composed of a blend of the dimethacrylates, Glycerolate bisphenol A dimethacrylate (Bis-GMA)/2-[(3,5,5-trimethyl-6-[2-(2-methyl prop-2-enoyloxy) ethoxycarbonylamino] hexyl) carbamoyloxy] ethyl, 2-methyl prop-2-enoate, (UDMA)/triethyleneglycol dimethacrylate (TEGDMA) in a 50:30.20 molar ratio, and silicon dioxide as filler. SOC DA was added at 5, 10 and 20 mol% to the already mentioned formulation. It was found that the addition of 20 mol% of SOC DA decreased 53 % the shrinkage of the cured composite material, in comparison with a formulation where it was not added the antishrinkage additive. Besides, the kinetics of photopolymerization determined by Real-Time infrared spectroscopy, demonstrated that the addition of increasing concentration of SOC DA improved the conversion of double bonds of dimethacrylates. Additionally, the presence of SOC DA at 10 % mol, helped to increase the flexural strength and the compressive strength of the composite, as a consequence of the augment of the crosslink density, induced by the ring opening polymerization of SOC DA.

Mifepristone improves chemo-radiation response in glioblastoma xenografts.

BACKGROUND: We have investigated the ability of Mifepristone, an anti-progestin and anti-glucocorticoid drug, to modulate the antitumor effect of current standard clinical treatment in glioblastoma xenografts. METHODS: The effect of radiation alone or combined with Mifepristone and Temozolamide was evaluated on tumor growth in glioblastoma xenografts, both in terms of preferentially triggering tumor cell death and inhibiting angiogenesis. Tumor size was measured once a week using a caliper and tumor metabolic-activity was carried out by molecular imaging using a microPET/CT scanner. The effect of Mifepristone on the expression of angiogenic factors after concomitant radio-chemotherapy was determined using a quantitative real-time PCR analysis of VEGF gene expression. RESULTS: The analysis of the data shows a significant antitumoral effect by the simultaneous administration of radiation-Mifepristone-Temozolamide in comparison with radiation alone or radiation-Temozolamide. CONCLUSION: Our results suggest that Mifepristone could improve the efficacy of chemo-radiotherapy in Glioblastoma. The addition of Mifepristone to standard radiation-Temozolamide therapy represents a potential approach as a chemo-radio-sensitizer in treating GBMs, which have very limited treatment options.

Volume-of-interest assessment of oncologic response using 18F-FDG PET/CT: a phantom study.

UNLABELLED: The aim of this study was to evaluate the volume-of-interest (VOI) technique in the measurement of volume radioactivity and in the differentiation of necrotic sites from residual tumor activity in a phantom. METHODS: PET/CT was performed on a phantom filled with (18)F-FDG solution at different concentrations. The VOI was quantified in 2 sessions to evaluate the VOI measurements as a function of activity concentration in the phantom. Software was used to build the VOI, determine the volume radioactivity of the cylindric inserts (cm(3)), and compare them with their real volumes. The VOI technique was also used to discern the mixed distribution of regions of (18)F-FDG activity from cold regions that represent areas of necrosis without tumor activity. RESULTS: Volumes measured with the VOI technique were similar to the actual volumes of cylinders in the phantom (no statistical differences; P > 0.05 after t test analysis). The diameter of cold inserts correlated positively with the percentage of visualization (P < 0.01); in both sessions, it was possible to visualize 100% of the 12.7-, 11.1-, and 9.5-mm cold rods. CONCLUSION: VOI technique has shown great potential for evaluating volume radioactivity and differentiating hot and cold regions in a phantom; clinical studies should be performed with this technique to evaluate its utility.

Use of an orthovoltage X-ray treatment unit as a radiation research system in a small-animal cancer model.

BACKGROUND: We explore the use of a clinical orthovoltage X-ray treatment unit as a small-animal radiation therapy system in a tumoral model of cervical cancer. METHODS: Nude mice were subcutaneously inoculated with 5 x 106 HeLa cells in both lower limbs. When tumor volume approximated 200 mm3 treatment was initiated. Animals received four 2 mg/kg intraperitoneal cycles (1/week) of cisplatin and/or 6.25 mg/kg of gemcitabine, concomitant with radiotherapy. Tumors were exposed to 2.5 Gy/day nominal surface doses (20 days) of 150 kV X-rays. Lead collimators with circular apertures (0.5 to 1.5 cm diameter) were manufactured and mounted on the applicator cone to restrict the X-ray beam onto tumors. X-ray penetration and conformality were evaluated by measuring dose at the surface and behind the tumor lobe by using HS GafChromic film. Relative changes in tumor volume (RTV) and a clonogenic assay were used to evaluate the therapeutic response of the tumor, and relative weight loss was used to assess toxicity of the treatments. RESULTS: No measurable dose was delivered outside of the collimator apertures. The analysis suggests that dose inhomogeneities in the tumor reach up to +/- 11.5% around the mean tumor dose value, which was estimated as 2.2 Gy/day. Evaluation of the RTV showed a significant reduction of the tumor volume as consequence of the chemoradiotherapy treatment; results also show that toxicity was well tolerated by the animals. CONCLUSION: Results and procedures described in the present work have shown the usefulness and convenience of the orthovoltage X-ray system for animal model radiotherapy protocols.