Computational Simulation of Tumor Hypoxia Based on In Vivo Microvasculature Assessed in a Dorsal Skin Window Chamber.

Malignant growth usually leads to the depletion of oxygen (O2) supply in most solid tumors. Hypoxia can cause resistance to standard radiotherapy, some chemotherapy and immunotherapy. Furthermore, it can also trigger malignant progression by modulating gene expression and inducing genetic instability. The relationship between microvasculature, perfusion and tumor hypoxia has been intensively studied and many computational simulations have been developed to model tissue O2 transport. Usually simplified 2D phantoms are used to investigate tumor hypoxia and it is assumed that vessels are perpendicular to the region of interest and randomly distributed across the domain. Such idealistic topology overlooks vascular heterogeneity and is not accurate enough to approximate real scenarios. In addition, experimental verification of the spatial gradient of computational simulations is not directly feasible. Realistic vasculature obtained from fluorescence imaging imported as geometry for partial differential equations solving did not receive necessary attention so far. Therefore, we established a computational simulation of in vivo conditions using experimental data obtained from dorsal skin window chamber tumor preparations in nude rats for the verification of computational results. Tumor microvasculature was assessed by fluorescence microscopy. Since the conventional finite difference method can hardly satisfy the real measurements, we established a finite element method (FEM) for the experimental data in this study. Realistic 2D tumor microvasculature was reconstructed by segmenting fluorescence images and then translated into FEM topology. O2 distributions and the O2 gradients were obtained by solving reaction-diffusion equations. The simulation results show that the development of tumor hypoxia is greatly influenced by the irregular architecture and function of microvascular networks.

A novel immunological model for the study of prostate cancer.

The Dunning R-3327 rat prostatic adenocarcinoma is a widely accepted model for in vivo experimental studies of prostate cancer. We have previously derived phenotypically distinct cell lines from a s.c. tumor resulting from the inoculation of the R-3327-5 subclone into Copenhagen rats. In this study, we report studies using a gelatin sponge model for the delivery of tumor cells and the retrieval of tumor-specific leukocytes responsive to different prostatic cell lines. S.c. preimplanted sponges were inoculated with tumor cells previously selected for differential properties of tumor formation and metastasis and examined for leukocyte content at time points of 1, 3, and 5 weeks after tumor cell inoculation. Cytospin and flow cytometric analyses revealed fewer tumor-associated leukocytes present in sponges inoculated with tumorigenic R-3327-5' and R-3327-5'B lines, with lesser sponge degradation, than in experiments with the nontumorigenic R-3327-5'A line, suggestive of a tumor cell-induced immunomodulatory mechanism. Morphological studies indicate an intermittent tumor growth pattern that gradually disappears in sponges inoculated with the nontumorigenic R-3327-5'A cells but a robust growth pattern in sponges inoculated with the tumorigenic cell lines. Cytokine analyses show the secretion of higher levels of active transforming growth factor-beta by the more invasive and metastatic lines. Total transforming growth factor-beta levels are higher in the epithelial, tumorigenic R-3327-5'B line. Additionally, the more tumorigenic lines secrete interleukin 10, a potent immunosuppressive molecule. In this report, we demonstrate the ability to retrieve viable leukocyte populations from a prostate tumor line bearing sponges, which offers an important model for further in vitro and in vivo manipulations and holds promise for testing adoptive immunotherapeutic strategies.

Percutaneous ultrasound guided implantation of VX2 for creation of a rabbit hepatic tumor model.

Creation of a VX2 tumor model has traditionally required a laparotomy and surgical implantation of tumor fragments. Open surgical procedures are invasive and require long procedure times and recovery that can result in post-operative morbidity and mortality. The purpose of this study is to report the results of a percutaneous ultrasound guided method for creation of a VX2 model in rabbit livers. A total of 27 New Zealand white rabbits underwent a percutaneous ultrasound guided approach, where a VX2 tumor fragment was implanted in the liver. Magnetic resonance imaging was used to assess for tumor growth and necropsy was performed to determine rates of tract seeding and metastatic disease. Ultrasound guided tumor implantation was successful in all 27 rabbits. One rabbit died 2 days following the implantation procedure. Two rabbits had no tumors seen on follow-up imaging. Therefore, tumor development was seen in 24/26 (92%) rabbits. During the follow-up period, tract seeding was seen in 8% of rabbits and 38% had extra-hepatic metastatic disease. Therefore, percutaneous ultrasound guided tumor implantation safely provides reliable tumor growth for establishing hepatic VX2 tumors in a rabbit model with decreased rates of tract seeding, compared to previously reported methods.

An implantable guide-screw system for brain tumor studies in small animals.

OBJECT: To overcome the problems associated with using stereotactic techniques to establish intracranial xenografts in nude mice and to treat engrafted tumors with intratumoral therapies (such as gene or viral therapies), the authors developed an implantable guide-screw system. In this study, they describe the guide-screw system, its method of implantation, and their experience with establishing xenografts and delivering intratumoral therapy. METHODS: The system consists of a 2.6-mm guide screw with a central 0.5-mm diameter hole that accepts the 26-gauge needle of a Hamilton syringe. The screw is implanted into a small drill hole made 2.5 mm lateral and 1 mm anterior to the bregma. A stylet is used to cap the screw between treatments. Tumor cells or therapeutic agents are injected in a freehand fashion by using a Hamilton syringe and a 26-gauge needle fitted with a cuff to determine the depth of injection. To test this system, guide screws were successfully implanted in 44 (98%) of 45 nude mice. After 1 to 2 weeks of recovery, 38 mice were inoculated with U87MG cells and killed 5 days later. On histological studies in 37 (97%) of these animals, xenografts were evident within the caudate nucleus (mean diameter 2.5 mm). To determine whether injections into the center of an established xenograft could be reproducibly achieved with the guide-screw system, an adenovirus vector containing the beta-galactosidase gene was injected 3 days after cell implantation in 15 of the mice. All of these animals demonstrated transduced cells within the tumor. To demonstrate that engrafted animals have a uniform survival time that is indicative of reproducible tumor growth, the survival of six mice was assessed after engraftment with U87MG cells. All six animals died within 28 to 35 days. CONCLUSIONS: The guide-screw system allows a large number of animals to be rapidly and reproducibly engrafted and for intratumoral treatments to be accurately delivered into established xenografts.

[A method and equipment for the intraosseous implantation of sarcoma 45 for x-ray research]. / Metodika i tekhnika vnutrikostnoi implantatsii sarkomy-45 dlia rentgenologicheskikh issledovanii.

A procedure for sarcoma-45 suspension implantation into bone to study osteogenic tumors by X-ray and morphologic means is suggested. Isolation of growing tumors without dissemination to adjacent soft tissues is assured in 94.3% of cases if trepanation hole area is reliably sealed. Also, a special device for homogenizing tumor tissue and a trepanation needle are suggested.

Intracranial stereotaxic cannulation for development of orthotopic glioblastoma allograft in Sprague-Dawley rats and histoimmunopathological characterization of the brain tumor.

Glioblastoma is the most common brain tumor that causes significant mortality annually. Limitations of the current therapeutic regimens warrant development of new techniques and treatment strategies in orthotopic animal model for better management of this devastating brain cancer. There are only a few experimental orthotopic models of glioblastoma for pre-clinical testing. In the present investigation, we successfully implanted rat C6 cells via intracranial stereotaxic cannulation in adult Sprague-Dawley rats for development and histoimmunopathological characterization of an advanced orthotopic glioblastoma allograft model, which could be useful for investigating the course of glioblastoma development as well as for testing efficacy of new therapeutic agents. The orthotopic glioblastoma allograft was generated by intracerebral injection of rat C6 cells through a guide-cannula system and after 21 post-inoculation days the brain tumor was characterized by histoimmunopathological experiments. Histological staining and immunofluorescent labelings for TERT, VEGF, Bcl-2, survivin, XIAP, and GFAP revealed the distinct characteristics of glioblastoma in C6 allograft, which could be useful as a target for treatment with emerging new therapeutic agents. Our investigation indicated the successful development of intracranial cannulated orthotopic glioblastoma allograft in adult Sprague-Dawley rats, making it as a useful animal model of glioblastoma for pre-clinical evaluation of various therapeutic strategies for the management of glioblastoma.

Optical imaging of tumor cells in hollow fibers: evaluation of the antitumor activities of anticancer drugs and target validation.

The in vivo hollow fiber assay, in which semipermeable hollow fibers filled with tumor cells, are implanted into animals, was originally developed to screen for anticancer compounds before assessment in more complex tumor models. To enhance screening and evaluation of anticancer drugs, we have applied optical imaging technology to this assay. To demonstrate that tumor cells inside hollow fibers can communicate with the host mice, we have used fluorescence imaging in vivo and CD31 immunostaining ex vivo to show that angiogenesis occurs around cell-filled hollow fibers by 2 weeks after subcutaneous implantation. Bioluminescence imaging has been used to follow the number of luciferase-expressing tumor cells within implanted hollow fibers; proliferation of those cells was found to be significantly inhibited by docetaxel or irinotecan. We also used bioluminescence imaging of hollow fibers to monitor the nuclear factor kappaB (NFkappaB) pathway in vivo; NFkappaB activation by lipopolysaccharide and tumor necrosis factor-alpha was evaluated in tumor cell lines genetically engineered to express luciferase controlled by an NFkappaB-responsive element. These results demonstrate that optical imaging of hollow fibers containing reporter tumor cells can be used for the rapid and accurate evaluation of antitumor activities of anticancer drugs and for measurement of molecular pathways.

Xenografting tumour beneath the renal capsule using modern surgical equipment.

INTRODUCTION: The growth of human tumours under the renal capsule in animal models has been performed in the past. However, the use of modern surgical equipment has not always been translated into the laboratory. We report on a novel method for human renal tumour transplants using an automated biopsy gun to obtain tumour tissue and an epidural needle with introducer to easily deploy the grafts under the renal capsule. METHODS: Nude mice had human xenografted tumours grown subcutaneously after implantation of cells from culture. Tumours were then biopsied using a 16-gauge automated biopsy gun. Digital calipers were used to measure a 2-mm segment of the biopsy core that was cut and placed inside a hollow needle (epidural needle). The needle was placed under the renal capsule and the trocar introduced to deploy the graft beneath the capsule with minimal trauma. Further groups had tumour harvested similarly by automated biopsy gun but had the implants placed subcutaneously for comparison. RESULTS: Tumour grafts were established in 90% of grafted kidneys in this renal subcapsular model (229.68 +/- 118.32 mm(3); mean +/- 95% CI) which compared favourably to the subcutaneous model (163.81 +/- 43.3 mm(3)). Grafts were confirmed by direct observation and histology. CONCLUSION: Modern surgical equipment may be utilised to allow tumour transplantation to be precise, with an identifiable and reproducible tumour volume deployed. Surgical researchers and laboratory-based scientists need to embrace new techniques and utilise them to improve models. This model may be adapted to many situations in oncologic research involving xenografting.

A reproducible rat liver cancer model for experimental therapy: introducing a technique of intrahepatic tumor implantation.

To investigate therapeutic strategies for hepatoma, it is necessary to have a reproducible animal model with a tumor growth pattern allowing accurate assessment of results. Many techniques of intrahepatic tumor implantation (IHTI) have been devised for intrahepatic tumor models. Most of them, however, have the disadvantage of high rates of artificial tumor dissemination during tumor implantation, which interferes with the evaluation of therapy. To overcome this problem, we have developed a technique of IHTI in which a piece of Gelfoam is placed into a small incision in the liver for the purpose of both hemostasis and formation of a tension-free pocket to accept the tumor implant. In 583 ACI rats receiving IHTI with Morris hepatoma 3924A, the tumor take rate was 100%. Resembling the natural course of human hepatoma, the implanted tumor grows locally early in the course of disease and eventually invades the surrounding organs causing ascites and also metastasizes to the lung. Liver microangiography demonstrated that the tumor received blood supply mainly from the hepatic artery. This IHTI technique was also compared to two other methods of IHTI: insertion of fragments without using Gelfoam and implantation with a tumor cell suspension. A significantly lower rate of early lung metastases was achieved with our technique (0%) in comparison with other two techniques (41 and 80%). We conclude that this rat liver cancer model is reproducible and allows efficient evaluation of treatment modalities for liver cancer without interference from tumor at undesirable sites.

The tumor-cell-releasing machine.

We describe the construction of a machine for the injection of 10- to 100-microliter volumes of viable cells in suspension. The cells are maintained in a tissue culture environment and can be injected according to a preprogrammed schedule. Preliminary results show that the tumor incidence and metastasis pattern of TA3Ha cells injected intravenously into mice are the same if the cells are injected in a single dose, or administered in 20 fractions during 3 h.

Progression of a weakly tumorigenic mouse fibrosarcoma at the site of early phase of inflammation caused by plastic plates.

To elucidate tumor progression-enhancing factor(s), we examined the effects of host inflammation and host immunological status on in vivo tumor progression. One x 10(4) cells of QR clones (QR-32, -20 and -18), regressor tumor clones of 3-methylcholanthrene-induced fibrosarcoma, were unable to grow when injected s.c. into C57BL/6 mice in cell suspension form. However, QR clones grew and were lethal when s.c. implanted, attached to plastic plates. Furthermore, the tumor lines (QRpP) obtained from the tumors which had arisen from the plate-attached QR-32 clone cells no longer required plastic plates for their growth in normal mice, and had acquired stable malignant phenotypes. Although QR-32 cells became lethal when injected at the site of plastic plate implantation 1, 5 and 10 days before tumor injection, few tumors developed when plastic plates had been implanted 20 or 30 days before tumor injection. We established culture clones from the tumors arising in normal mice and mice immunosuppressed by irradiation. Clones derived from the tumors which had arisen in normal mice after implantation with plastic plates were lethal when re-implanted in normal mice (71%). On the other hand, clones derived from the tumors that arose in irradiated mice with or without plastic plates were lethal in only a few normal mice, when re-implanted (20 and 8%, respectively). These results indicate that QR clone cell progression is enhanced by the early phase of inflammation at the site of plastic plate implantation and that the progression-enhancing activity of co-implantation with a plastic plate is inhibited by previous whole-body irradiation of hosts.

A model for intratumoural chemotherapy in the rat brain.

To achieve the best reproducibility in rat brain tumour models several injection techniques have been used. Although stereotactic cell injections have proved to be effective and reliable, they are expensive and time consuming. A new permanently implanted device is presented here. It allows precise cell delivery for best tumour reproducibility, and it can be left in place for future injections at the exact same location, such as intratumoural chemotherapy. A Teflon tube was mounted on a disc, inserted into the rat brain and sealed to the skull. The device was tested in two rat strains (Wistar and New Zealand Nude rats) with two different glioma cell lines (9L and C6). Rats were treated with placebo to determine if repeated treatments had an effect on the device placement, or if device-related morbidity was induced. Analysis of brain sections showed that the device path was always within the tumour. The device never moved or came off the scalp. Both Wistar rats and NZ nude rats tolerated the device well. No morbidity or mortality was observed, regardless of the presence of the device; no infections were seen. Biocompatible, non-irritating and well tolerated, such a device can be used for reproducible tumour cell injections and repeated intralesional delivery of drugs.