The αVß3/αVß5 integrin inhibitor cilengitide augments tumor response to melphalan isolated limb perfusion in a sarcoma model.

Isolated limb perfusion (ILP) with melphalan and tumor necrosis factor (TNF)-α is used to treat bulky, locally advanced melanoma and sarcoma. However, TNF toxicity suggests a need for better-tolerated drugs. Cilengitide (EMD 121974), a novel cyclic inhibitor of alpha-V integrins, has both anti-angiogenic and direct anti-tumor effects and is a possible alternative to TNF in ILP. In this study, rats bearing a hind limb soft tissue sarcoma underwent ILP using different combinations of melphalan, TNF and cilengitide in the perfusate. Further groups had intra-peritoneal (i.p.) injections of cilengitide or saline 2 hr before and 3 hr after ILP. A 77% response rate (RR) was seen in animals treated i.p. with cilengitide and perfused with melphalan plus cilengitide. The RR was 85% in animals treated i.p. with cilengitide and ILP using melphalan plus both TNF and cilengitide. Both RRs were significantly greater than those seen with melphalan or cilengitide alone. Histopathology showed that high RRs were accompanied by disruption of tumor vascular endothelium and tumor necrosis. Compared with ILP using melphalan alone, the addition of cilengitide resulted in a three to sevenfold increase in melphalan concentration in tumor but not in muscle in the perfused limb. Supportive in vitro studies indicate that cilengitide both inhibits tumor cell attachment and increases endothelial permeability. Since cilengitide has low toxicity, these data suggest the agent is a good alternative to TNF in the ILP setting.

Macrophage phenotypes and monocyte subsets after destabilization of the medial meniscus in mice.

Macrophages play an important role in the development and progression of osteoarthritis (OA). The aim of this study was to identify macrophage phenotypes in synovium and monocyte subsets in peripheral blood in C57BL/6 mice by destabilizing the medial meniscus (DMM), and the association of macrophage subsets with OA features. DMM, sham, and non-operated knees were histologically assessed between 1 and 56 days for macrophage polarization states by immunohistochemistry (IHC), cartilage damage, synovial thickening, and osteophytes (n = 9 per timepoint). Naive knees (n = 6) were used as controls. Monocyte and polarized synovial macrophage subsets were evaluated by flow cytometry. CD64 and CD206 levels on IHC were higher at early timepoints in DMM and sham knees compared to naive knees. iNOS labeling intensity was higher in DMM and sham knees than in naive knees from d3 onwards. CD163 expression was unaltered at all timepoints. Even though macrophage polarization profiles were similar in DMM and sham knees, only in DMM knees the presence of iNOS and CD206 associated with synovial thickness, and CD163 staining inversely correlated with osteophyte presence. At day 14, monocyte subset distribution was different in peripheral blood of DMM mice compared with sham mice. In conclusion, monocyte subsets in blood and synovial macrophage phenotypes vary after joint surgery. High levels of iNOS+ , CD163+ , and CD206+ cells are found in both destabilized and sham-operated knees, and coexistence with joint instability may be a requirement to initiate and exacerbate OA progression.

Labelling of mammalian cells for visualisation by MRI.

Through labelling of cells with magnetic contrast agents it is possible to follow the fate of transplanted cells in vivo with magnetic resonance imaging (MRI) as has been demonstrated in animal studies as well as in a clinical setting. A large variety of labelling strategies are available that allow for prolonged and sensitive detection of the labelled cells with MRI. The various protocols each harbour specific advantages and disadvantages. In choosing a particular labelling strategy it is also important to ascertain that the labelling procedure does not negatively influence cell functionality, for which a large variety of assays are available. In order to overcome the challenges still faced in fully exploiting the benefits of in vivo cell tracking by MRI a good understanding and standardisation of the procedures and assays used will be crucial.

Tumor necrosis factor alpha mediates homogeneous distribution of liposomes in murine melanoma that contributes to a better tumor response.

Successful treatment of solid tumors with chemotherapeutics requires that adequate levels reach the tumor cells. Tumor vascular normalization has been proposed to enhance drug delivery and improve tumor response to chemotherapy. Differently, augmenting leakage of the tumor-associated vasculature, and as such enhance vascular abnormality, may improve tumor response as well. In the present study, we show that addition of low-dose tumor necrosis factor alpha (TNF) to systemic injections with pegylated long circulating liposomes augmented the tumor accumulation of these liposomes 5- to 6-fold, which strongly correlated with enhanced tumor response. Using intravital microscopy, we could study the liposomal distribution inside the tumor in more detail. Especially 100 nm liposomes effectively extravasate in the surrounding tumor tissue in the presence of TNF and this occurred without any effect on tumor vascular density, branching, and diameter. Next to that, we observed in living animals that tumor cells take up the liposomes intact, followed by intracellular degradation. To our knowledge, this is an unprecedented observation. Taken together, TNF renders more tumor vessels permeable, leading to a more homogeneous distribution of the liposomes throughout the tumor, which is crucial for an optimal tumor response. We conclude that delivery of nanoparticulate drug formulations to solid tumor benefits from augmenting the vascular leakage through vascular manipulation with vasoactive drugs like TNF.

In Vivo Stabilized SB3, an Attractive GRPR Antagonist, for Pre- and Intra-Operative Imaging for Prostate Cancer.

PURPOSE: The gastrin-releasing peptide receptor (GRPR), overexpressed on various tumor types, is an attractive target for receptor-mediated imaging and therapy. Another interesting approach would be the use of GRPR radioligands for pre-operative imaging and subsequent radio-guided surgery, with the goal to improve surgical outcome. GRPR radioligands were successfully implemented in clinical studies, especially Sarabesin 3 (SB3) is an appealing GRPR antagonist with high receptor affinity. Gallium-68 labeled SB3 has good in vivo stability, after labeling with Indium-111; however, the molecule shows poor in vivo stability, which negatively impacts tumor-targeting capacity. A novel approach to increase in vivo stability of radiopeptides is by co-administration of the neutral endopeptidase (NEP) inhibitor, phosphoramidon (PA). We studied in vivo stability and biodistribution of [111In]SB3 without/with (-/+) PA in mice. Furthermore, SPECT/MRI on a novel, state-of-the-art platform was performed. PROCEDURES: GRPR affinity of SB3 was determined on PC295 xenograft sections using [125I]Tyr4-bombesin with tracer only or with increasing concentrations of SB3. For in vivo stability, mice were injected with 200/2000 pmol [111In]SB3 -/+ 300 µg PA. Blood was collected and analyzed. Biodistribution and SPECT/MRI studies were performed at 1, 4, and 24 h postinjection (p.i.) of 2.5 MBq/200 pmol or 25 MBq/200 pmol [111In]SB3 -/+ 300 µg PA in PC-3-xenografted mice. RESULTS: SB3 showed high affinity for GRPR (IC50 3.5 nM). Co-administration of PA resulted in twice higher intact peptide in vivo vs [111In]SB3 alone. Biodistribution studies at 1, 4, and 24 h p.i. show higher tumor uptake values with PA co-administration (19.7 ± 3.5 vs 10.2 ± 1.5, 17.6 ± 5.1 vs 8.3 ± 1.1, 6.5 ± 3.3 vs 3.1 ± 1.9 % ID/g tissue (P < 0.0001)). Tumor imaging with SPECT/MRI clearly improved after co-injection of PA. CONCLUSIONS: Co-administration of PA increased in vivo tumor targeting capacity of [111In]SB3, making this an attractive combination for GRPR-targeted tumor imaging.

SPIO labeling of endothelial cells using ultrasound and targeted microbubbles at diagnostic pressures.

In vivo cell tracking of therapeutic, tumor, and endothelial cells is an emerging field and a promising technique for imaging cardiovascular disease and cancer development. Site-specific labeling of endothelial cells with the MRI contrast agent superparamagnetic iron oxide (SPIO) in the absence of toxic agents is challenging. Therefore, the aim of this in vitro study was to find optimal parameters for efficient and safe SPIO-labeling of endothelial cells using ultrasound-activated CD31-targeted microbubbles for future MRI tracking. Ultrasound at a frequency of 1 MHz (10,000 cycles, repetition rate of 20 Hz) was used for varying applied peak negative pressures (10-160 kPa, i.e. low mechanical index (MI) of 0.01-0.16), treatment durations (0-30 s), time of SPIO addition (-5 min- 15 min with respect to the start of the ultrasound), and incubation time after SPIO addition (5 min- 3 h). Iron specific Prussian Blue staining in combination with calcein-AM based cell viability assays were applied to define the most efficient and safe conditions for SPIO-labeling. Optimal SPIO labeling was observed when the ultrasound parameters were 40 kPa peak negative pressure (MI 0.04), applied for 30 s just before SPIO addition (0 min). Compared to the control, this resulted in an approximate 12 times increase of SPIO uptake in endothelial cells in vitro with 85% cell viability. Therefore, ultrasound-activated targeted ultrasound contrast agents show great potential for effective and safe labeling of endothelial cells with SPIO.

Synergistic antitumor response of interleukin 2 with melphalan in isolated limb perfusion in soft tissue sarcoma-bearing rats.

The cytokine interleukin 2 (IL-2) is a mediator of immune cell activation with some antitumor activity, mainly in renal cell cancer and melanoma. We have previously shown that tumor necrosis factor (TNF)-alpha has strong synergistic antitumor activity in combination with chemotherapeutics in the isolated limb perfusion (ILP) setting based on a TNF-mediated enhanced tumor-selective uptake of the chemotherapeutic drug followed by a selective destruction of the tumor vasculature. IL-2 can cause vascular leakage and edema and for this reason we examined the antitumor activity of a combined treatment with IL-2 and melphalan in our well-established ILP in soft tissue sarcoma-bearing rats (BN175). ILP with either IL-2 or melphalan alone has no antitumor effect, but the combination of IL-2 and melphalan resulted in a strong synergistic tumor response, without any local or systemic toxicity. IL-2 enhanced significantly melphalan uptake in tumor tissue. No signs of significant vascular damage were detected to account for this observation, although the tumor sections of the IL-2- and IL-2 plus melphalan-treated animals revealed scattered extravasation of erythrocytes compared with the untreated animals. Clear differences were seen in the localization of ED-1 cells, with an even distribution in the sham, IL-2 and melphalan treatments, whereas in the IL-2 plus melphalan-treated tumors clustered ED-1 cells were found. Additionally, increased levels of TNF mRNA were found in tumors treated with IL-2 and IL-2 plus melphalan. These observations indicate a potentially important role for macrophages in the IL-2-based perfusion. The results in our study indicate that the novel combination of IL-2 and melphalan in ILP has synergistic antitumor activity and may be an alternative for ILP with TNF and melphalan.

Gene therapy in in vivo isolated perfusion models.

Locoregional administration of a genetic construct by means of in vivo, in situ isolated perfusion (IP) of a target organ or extremity is a method that may increase in vivo efficacy. Vascular isolation and perfusion minimizes systemic exposure and thereby reduces unwanted side effects. Isolated hepatic perfusion (IHP) is the most extensively studied IP model, especially in gene therapy protocols for inborn errors of metabolism. To achieve stable transduction most frequently retroviruses are used in IHP. IHP is combined with hepatectomy or vascular ligation of liver lobes to induce liver regeneration increasing transduction efficacy. When adenoviruses are used in IHP high transduction percentages of hepatocytes can be achieved without significant toxicity. In tumor models adenoviral IHP has been performed, but has not been very successful up till now. Isolated limb perfusion (ILP) is a promising treatment modality in pre-clinical cancer gene therapy studies. After ILP a homogeneous distribution of transduced cells was demonstrated especially at the viable rim of the tumor and around tumor associated vessels. Moreover complete tumor responses have been observed. Isolated pulmonary perfusion (IPP) results in selective expression in the perfused lung and the duration of expression is longer than after systemic administration. In rats a significant decrease of tumor nodules upon IPP can be achieved. Furthermore other less studied perfusion models are discussed: isolated kidney perfusion (IKP), isolated spleen perfusion (ISP) and isolated cardiac perfusion (ICP). IP is a methodology that delivers vectors highly selectively, with a long exposure time and high concentrations at the target side. This results in higher transduction rates and thereby may improve therapeutic effects.

Effect of low-dose tumor necrosis factor-alpha in combination with STEALTH liposomal cisplatin (SPI-077) on soft-tissue- and osteosarcoma-bearing rats.

Cisplatin is a widely used agent for treatment of solid tumors, but its clinical utility is limited by toxicity. Preclinical studies have shown less acute toxicity when STEALTH liposomal cisplatin (SPI-077) is used, with antitumor effects equivalent to those of intravenously administered free cisplatin. We previously reported that systemic treatment with low-dose tumor necrosis factor-alpha (TNF) augments the activity of STEALTH liposomal doxorubicin (Doxil). In this study, we examined the effect of repeated systemic applications of low-dose TNF on the antitumor activity of SPI-077 in rats with soft-tissue sarcoma or osteosarcoma. Addition of TNF to SPI-077 treatment showed an improved tumor growth delay of the soft-tissue sarcoma. The combined SPI-077/TNF treatment resulted in a more prolonged antitumor activity, whereas free cisplatin showed a better tumor response, however with a rapid outgrowth a few days after the end of therapy. In the osteosarcoma, free cisplatin did not have an antitumor effect, but addition of TNF caused a clear tumor growth delay. SPI-077 alone resulted in a tumor growth delay, but combination with TNF had no additive effect. SPI-077 yielded less systemic toxicity than cisplatin. Depending on the type of tumor, the addition of TNF to SPI-077 results in a better tumor growth delay with a prolonged antitumor effect and, in combination with the reduced toxicity of SPI-077, this combination may be preferable to cisplatin.

Histamine combined with melphalan in isolated limb perfusion for the treatment of locally advanced soft tissue sarcomas: preclinical studies in rats.

PURPOSE: To evaluate the potential benefit of histamine combined with melphalan in the isolated limb perfusion (ILP) as an alternative to TNF-alfa and melphalan combination, for the treatment of irresectable soft tissue sarcomas of the limbs in Brown Norway (BN) rats. METHODS: 20 BN rats had small fragments of syngeneic BN-175 fibrosarcoma inserted on the right hind limb. In 7-10 days the tumor reached a median diameter of 12-15 mm and they were randomly divided in four groups (sham, melphalan, histamine and escalating doses of histamine combined to melphalan) being submitted to experimental ILP for 30 minutes. Tumors were measured daily with a caliper and the volume was calculated. RESULTS: Response curves showed a significant effect of the combination of histamine 200 mg/mL with melphalan, with 66% overall response, including 33% complete responses (p< 0.01). There were no systemic collateral effects and locally only mild temporary edema was observed for some animals treated with histamine. CONCLUSION: Histamine combined with melphalan had a promising effect in the ILP warranting future studies to better explore the mechanism of action as well as its potential use in organ perfusion.

Nanoparticles and clinically applicable cell tracking.

In vivo cell tracking has emerged as a much sought after tool for design and monitoring of cell-based treatment strategies. Various techniques are available for pre-clinical animal studies, from which much has been learned and still can be learned. However, there is also a need for clinically translatable techniques. Central to in vivo cell imaging is labelling of cells with agents that can give rise to signals in vivo, that can be detected and measured non-invasively. The current imaging technology of choice for clinical translation is MRI in combination with labelling of cells with magnetic agents. The main challenge encountered during the cell labelling procedure is to efficiently incorporate the label into the cell, such that the labelled cells can be imaged at high sensitivity for prolonged periods of time, without the labelling process affecting the functionality of the cells. In this respect, nanoparticles offer attractive features since their structure and chemical properties can be modified to facilitate cellular incorporation and because they can carry a high payload of the relevant label into cells. While these technologies have already been applied in clinical trials and have increased the understanding of cell-based therapy mechanism, many challenges are still faced.

Magnetic Resonance Detection of CD34+ Cells from Umbilical Cord Blood Using a 19F Label.

Impaired homing and delayed recovery upon hematopoietic stem cell transplantation (HSCT) with hematopoietic stem cells (HSC) derived from umbilical cord blood (UCB) is a major problem. Tracking transplanted cells in vivo will be helpful to detect impaired homing at an early stage and allows early interventions to improve engraftment and outcome after transplantation. In this study, we show sufficient intracellular labeling of UCB-derived CD34+ cells, with 19F-containing PLGA nanoparticles which were detectable with both flow cytometry and magnetic resonance spectroscopy (MRS). In addition, labeled CD34+ cells maintain their capacity to proliferate and differentiate, which is pivotal for successful engraftment after transplantation in vivo. These results set the stage for in vivo tracking experiments, through which the homing efficiency of transplanted cells can be studied.

Isolated limb perfusion based anti-p21ras gene therapy in a rat rhabdomyosarcoma.

BACKGROUND: Inhibition of ras oncogene is a promising new strategy. Gene therapy against ras proved successful in human and murine tumour cell lines. Previously we demonstrated effective targeted transfection of tumour in a rat model by using an isolated limb perfusion (ILP) for the delivery of adenoviral vectors. MATERIALS AND METHODS: This study explores the anti-tumour activity of an adenoviral construct encoding an intracellular single-chain antibody (scFv) against p21ras (Y28). In order to determine the influence of the ras status on the efficacy of the scFv, we used a wild-type rat rhabdomyosarcoma and its ras-oncogene transfectant, for in vitro studies. In vivo we used the ILP delivery method to study anti-tumour activity on established limb tumours. RESULTS: In vitro studies demonstrated an inhibition of growth caused by the Y28 construct. No significant difference between transfected and wild-type cell lines could be demonstrated. Upon ILP, homogeneous transduction was observed in 5% of tumour cells. Perfusion with the Y28 construct, however, did not result in any additional anti-tumour activity compared to controls. CONCLUSION: Despite in vitro activity and in vivo transfection, no significant tumour response could be detected using anti-p21ras gene therapy in this ILP-tumour model.

Cell quantification: evolution of compartmentalization and distribution of iron-oxide particles and labeled cells.

The purpose of the study was to show the feasibility of quantification in the case of cell death, cell migration and cell division by parametric MRI. We identify limitations for quantitative cell tracking owing to mixed parallel processes. Various intravoxel SPIO-labeled cell, super paramagnetic iron oxide particles (SPIO) and micron-sized paramagnetic iron oxide (MPIO) particle distributions were prepared by methods mimicking biologically relevant processes (compartmentalization, migration, division and cell death). R(2)* and R(2) relaxometry measurements were performed at 3.0 T; iron concentration was measured by optical emission spectrometry. The effects of spatial distribution and compartmentalization of paramagnetic iron-oxide particles on relaxivity were analyzed. Assessment of R(2)' (R(2)*-R(2)) allowed differentiation between intracellular and extracellular SPIO only if no high-iron-content extracellular particles were present. Relaxivity was sensitive to variations in cell labeling. Samples of the same cell types embedded in the same suspension media at the same cell density produced different relaxivity values, depending on the preparation of the labeled cells. In the case of cell division, a unique relationship between relaxation rate and iron concentration was found, where the relaxivity proved to be independent of initial cell labeling. In case of cell mixing, the cell density could be derived from relaxation values, even if iron concentration was undetermined. We demonstrated that relaxometry does not allow labeled cell quantification when multiple physiological processes such as cell division and cell migration coexist. The measured transversal relaxation rates were sensitive to the labeling technique. However, under special circumstances, despite the numerous limiting factors, quantification of the number of labeled cells by relaxometry was feasible.

Facilitating tumor functional assessment by spatially relating 3D tumor histology and in vivo MRI: image registration approach.

BACKGROUND: Magnetic resonance imaging (MRI), together with histology, is widely used to diagnose and to monitor treatment in oncology. Spatial correspondence between these modalities provides information about the ability of MRI to characterize cancerous tissue. However, registration is complicated by deformations during pathological processing, and differences in scale and information content. METHODOLOGY/PRINCIPAL FINDINGS: This study proposes a methodology for establishing an accurate 3D relation between histological sections and high resolution in vivo MRI tumor data. The key features of the methodology are: 1) standardized acquisition and processing, 2) use of an intermediate ex vivo MRI, 3) use of a reference cutting plane, 4) dense histological sampling, 5) elastic registration, and 6) use of complete 3D data sets. Five rat pancreatic tumors imaged by T2*-w MRI were used to evaluate the proposed methodology. The registration accuracy was assessed by root mean squared (RMS) distances between manually annotated landmark points in both modalities. After elastic registration the average RMS distance decreased from 1.4 to 0.7 mm. The intermediate ex vivo MRI and the reference cutting plane shared by all three 3D images (in vivo MRI, ex vivo MRI, and 3D histology data) were found to be crucial for the accurate co-registration between the 3D histological data set and in vivo MRI. The MR intensity in necrotic regions, as manually annotated in 3D histology, was significantly different from other histologically confirmed regions (i.e., viable and hemorrhagic). However, the viable and the hemorrhagic regions showed a large overlap in T2(*)-w MRI signal intensity. CONCLUSIONS: The established 3D correspondence between tumor histology and in vivo MRI enables extraction of MRI characteristics for histologically confirmed regions. The proposed methodology allows the creation of a tumor database of spatially registered multi-spectral MR images and multi-stained 3D histology.

Variations in labeling protocol influence incorporation, distribution and retention of iron oxide nanoparticles into human umbilical vein endothelial cells.

Various studies have shown that various cell types can be labeled with iron oxide particles and visualized by magnetic resonance imaging (MRI). However, reported protocols for cell labeling show a large variation in terms of labeling dose and incubation time. It is therefore not clear how different labeling protocols may influence labeling efficiency. Systematic assessment of the effects of various labeling protocols on labeling efficiency of human umbilical vein endothelial cells (HUVEC) using two different types of iron oxide nanoparticles, i.e. super paramagnetic iron oxide particles (SPIOs) and microparticles of iron oxide (MPIOs), demonstrated that probe concentration, incubation time and particle characteristics all influence the efficiency of label incorporation, label distribution, label retention and cell behavior. For SPIO the optimal labeling protocol consisted of a dose of 12.5 µg iron/2 ml/9.5 cm(2) and an incubation time of 24 h, resulting in an average iron load of 12.0 pg iron/per cell (uptake efficiency of 9.6%). At 4 h many SPIOs are seen sticking to the outside of the cell instead of being taken up by the cell. For MPIO optimal labeling was obtained with a dose of 50 µg iron/2 ml/9.5 cm(2). Incubation time was of less importance since most of the particles were already incorporated within 4 h with a 100% labeling efficiency, resulting in an intracellular iron load of 626 pg/cell. MPIO were taken up more efficiently than SPIO and were also better tolerated. HUVEC could be exposed to and contain higher amounts of iron without causing significant cell death, even though MPIO had a much more pronounced effect on cell appearance. Using optimal labeling conditions as found for HUVEC on other cell lines, we observed that different cell types react differently to identical labeling conditions. Consequently, for each cell type separately an optimal protocol has to be established.

Synergistic antitumor effects of histamine plus melphalan in isolated hepatic perfusion for liver metastases.

BACKGROUND: Nonresectable primary and metastatic liver tumors remain an important clinical problem. Melphalan-based isolated hepatic perfusion (M-IHP) leads to more than 70% objective responses in selective groups of patients with nonresectable metastases confined to the liver. Complete responses are rare and progression-free survival is limited. Tumor necrosis factor (TNF), a very active agent in isolated limb perfusion, is linked to serious hepatotoxicity, restricting its use in IHP. Because of its vasoactive properties, histamine (Hi) is an alternative to TNF. In this article we evaluate its potential synergistic effect in M-IHP, improving response rates. METHODS: Our experimental rat IHP model is used for the treatment of soft tissue sarcoma liver metastases. Blood samples are collected for monitoring liver enzymes. Livers are excised 72 h and 7 days after treatment for histologic evaluation. RESULTS: After sham-IHP and Hi-IHP, tumor progression was observed in 100% of treated animals, while after M-IHP this number fell to 62% and after Hi + M-IHP it fell to only 22% (P = 0.006). Overall response rates were of 55% for Hi + M-IHP vs. 25% for M-IHP, and, more importantly, complete responses (CR) were observed only after Hi + M-IHP (22%) (P = 0.009). Hepatotoxicity peaked within 24 h after IHP, independent of the treatment administered, recovered in 48 h, and was related mainly to the elevation of transaminases (grade 3 ASAT and grade 1 ALAT for control group and grades 3 and 4, respectively, for all other treatments). No serious systemic toxicity was observed. Histology of the liver showed no serious damage. CONCLUSION: Hi + M-IHP has synergistic antitumor effects without any increase in regional or systemic toxicity.

Early destruction of tumor vasculature in tumor necrosis factor-alpha-based isolated limb perfusion is responsible for tumor response.

Addition of high-dose tumor necrosis factor-alpha to melphalan-based isolated limb perfusion enhances anti-tumor effects impressively. Unfortunately, the mechanism of action of tumor necrosis factor-alpha is still not fully understood. Here, we investigated the effects of tumor necrosis factor-alpha on the tumor microenvironment and on secondary immunological events during and shortly after isolated limb perfusion in soft-tissue sarcoma-bearing rats. Already during isolated limb perfusion, softening of the tumor was observed. Co-administration of tumor necrosis factor-alpha in the isolated limb perfusion with melphalan induced a six-fold enhanced drug accumulation of melphalan in the tumor compared with isolated limb perfusion with melphalan alone. In addition, directly after perfusion with tumor necrosis factor-alpha plus melphalan, over a time-frame of 30 min, vascular destruction, erythrocyte extravasation and hemorrhage was detected. Interstitial fluid pressure and pH in the tumor, however, were not altered by tumor necrosis factor-alpha and no clear immune effects, cellular infiltration or cytokine expression were observed. Taken together, these results indicate that tumor necrosis factor-alpha induces rapid damage to the tumor vascular endothelial lining resulting in augmented drug accumulation. As other important parameters were not changed (e.g. interstitial fluid pressure and pH), we speculate that the tumor vascular changes, and concurrent hemorrhage and drug accumulation are the key explanations for the observed synergistic anti-tumor response.

Addition of low-dose tumor necrosis factor-alpha to systemic treatment with STEALTH liposomal doxorubicin (Doxil) improved anti-tumor activity in osteosarcoma-bearing rats.

Improved efficacy of Doxil (STEALTH liposomal doxorubicin) compared to free doxorubicin has been demonstrated in the treatment of several tumor types. We have shown that addition of low-dose tumor necrosis factor (TNF) to systemic Doxil administration dramatically improved tumor response in the highly vascularized rat soft tissue sarcoma BN175. Whether a similar enhanced efficacy can be achieved in less vascularized tumors is uncertain. We therefore examined the effect of systemic administration of Doxil in combination with low-dose TNF in intermediate vascularized osteosarcoma-bearing rats (ROS-1). Small fragments of the osteosarcoma were implanted s.c. in the lower limb. Treatment was started when the tumors reached an average diameter of 1 cm. Rats were treated with five i.v. injections at 4-day intervals with Doxil or doxorubicin and TNF. Systemic treatment with Doxil resulted in a better tumor growth delay than free doxorubicin, but with progressive diseases in all animals. The 3.5-fold augmented accumulation of Doxil compared to free doxorubicin presumably explains the enhanced tumor regression. Addition of low-dose TNF augmented the anti-tumor activity of Doxil, although no increased drug uptake was found compared to Doxil alone. In vitro studies showed that ROS-1 is sensitive to TNF, but systemic treatment with TNF alone did not result in a tumor growth delay. Furthermore, we demonstrated that treatment with Doxil alone or with TNF resulted in massive coagulative necrosis of tumor tissue. In conclusion, combination therapy of Doxil and low-dose TNF seems attractive for the treatment of highly vascularized tumors, but also of intermediate vascularized tumors like the osteosarcoma.

Tumor necrosis factor-alpha augmented tumor response in B16BL6 melanoma-bearing mice treated with stealth liposomal doxorubicin (Doxil) correlates with altered Doxil pharmacokinetics.

The application of tumor necrosis factor-alpha (TNF) for the treatment of solid tumors is limited by its severe, life-threatening, toxicity. Therefore, only low dosages of this cytokine can be applied systemically, which results in poor tumor response. It has been demonstrated previously that administration of high-dose TNF in a so-called isolated perfusion system markedly improved tumor response when combined with chemotherapy. It appeared that TNF had a major impact specifically on the tumor-associated vasculature. At these high concentrations, endothelial cell death is induced by TNF, resulting in complete collapse of the tumor vascular bed. Strikingly, this effect alone is not enough to induce a tumor response, but addition of a chemotherapeutic drug is mandatory to obtain an anti-tumor effect. We showed that TNF has no anti-tumor effect by itself but augmented drug accumulation mainly in the tumor, most likely by enhancing vascular leakage. It seems that enhanced vascular leakage, but not endothelial cell death, explains the interaction between TNF and the co-administered drug. We hypothesized that in a low-dose setting TNF could induce tumor accumulation of chemotherapeutic drugs and consequently improve tumor response. We demonstrate that free TNF has a strong effect on the pharmacokinetics of co-administered Doxil in B16BL6 melanoma-bearing mice, resulting in strongly augmented drug accumulation in the tumor and improved tumor response. Co-injection of Stealth liposomal TNF with Doxil resulted in comparable or less pronounced tumor responses as compared to free TNF. These results imply that systemic application of clinically tolerable doses of TNF may improve drug distribution and tumor response and could be useful in a number of anti-cancer therapies.