Abstract: Angiogenesis has been associated with disease progression in many solid tumours, however the statement that tumours need angiogenesis to grow, invade and metastasise seems no longer applicable to all tumours or to all tumour subtypes. Prognostic studies in pancreatic cancer are conflicting. In fact, pancreatic cancer has been suggested an example of a tumour in which angiogenesis is less essential for tumour progression. The aim of the present study was therefore to measure angiogenesis in two anatomically closely related however prognostically different types of pancreatic cancer, pancreatic head and periampullary cancer, and investigate its relation with outcome. Vessels were stained by CD31 on original paraffin embedded tissue from 206 patients with microscopic radical resection (R0) of pancreatic head (n=98) or periampullary cancer (n=108). Angiogenesis was quantified by microvessel density (MVD) and measured by computerised image analysis of three randomly selected fields and investigated for associations with recurrence free survival (RFS), cancer specific survival (CSS), overall survival (OS) and conventional prognostic factors. MVD was heterogeneous both between and within tumours. A higher MVD was observed in periampullary cancers compared with pancreatic head cancers (p<.01). Furthermore, MVD was associated with lymph node involvement in pancreatic head (p=.014), but not in periampullary cancer (p=.55). Interestingly, MVD was not associated with RFS, CSS or with OS. In conclusion, angiogenesis is higher in periampullary cancer and although associated with nodal involvement in pancreatic head cancer, pancreatic cancer prognosis seems indeed angiogenesis independent.
Abstract: Polyphenols in red wine are supposed to improve endothelial function. We investigated whether daily red wine consumption improves in-vivo vascular function by reducing endothelin-1 (ET-1). Additional pathways mediating this effect were studied using porcine coronary arteries (PCAs).
Abstract: The high mortality rate and minimal progress made in the treatment of pancreatic cancer over the last few decades, warrant an alternative approach. Treatment protocols should be individualised to the patient guided by prognostic markers. A particularly interesting target would be the architectural transcription factor high mobility group A1 (HMGA1), that is low or undetectable in normal tissue, induced during neoplastic transformation and consequently often exceptionally high in cancer. The aim of the current study was therefore to determine the differential expression of HMGA1 in pancreatic head and periampullary cancer and investigate its relation with outcome. HMGA1 expression was determined by immunohistochemistry on original paraffin embedded tissue from 99 pancreatic head- and 112 periampullary cancers (with R0). Expression was investigated for associations with recurrence free (RFS), cancer specific (CSS) and overall survival (OS) and conventional prognostic factors. HMGA1 was expressed in 47% and 26% of pancreatic head- and periampullary cancer, respectively and associated with poor RFS, CSS and OS in periampullary cancer. CSS 5years following surgery was 25% and 44% for patients with tumours which were positive or negative for HMGA1 protein, respectively. HMGA1 expression was not associated with survival in pancreatic head cancer. In conclusion HMGA1 was identified as an independent prognostic marker predicting poor outcome in periampullary cancer. Although expressed to a higher extent as compared to periampullary cancer, HMGA1 was not associated with survival in pancreatic head cancer.
Abstract: Improving vascularization of engineered adipose tissue constructs is a major challenge in the field of plastic surgery. Although human adipose-derived stromal cells (hASCs) are known to release factors that stimulate new blood vessel formation, detailed information about the effects of adipogenic differentiation on the angiogenic potential of hASCs remains largely unknown. In the present study, we studied the expression and secretion of a large panel of angiogenic factors during hASC differentiation and evaluated the effects of hASC-conditioned medium (hASC-CM) on endothelial cells.
Abstract: Solid tumor therapy with chemotherapeutics greatly depends on the efficiency with which drugs are delivered to tumor cells. The typical characteristics of the tumor physiology promote but also appose accumulation of blood-borne agents. The leaky tumor vasculature allows easy passage of drugs. However, the disorganized vasculature causes heterogeneous blood flow, and together with the often-elevated interstitial fluid pressure, this state results in poor intratumoral drug levels and failure of treatment. Manipulation of the tumor vasculature could overcome these barriers and promote drug delivery. Targeting the vasculature has several advantages. The endothelial lining is readily accessible and the first to be encountered after systemic injection. Second, endothelial cells tend to be more stable than tumor cells and thus less likely to develop resistance to therapy. Third, targeting the tumor vasculature can have dual effects: (i) manipulation of the vasculature can enhance concomitant chemotherapy, and (ii) subsequent destruction of the vasculature can help to kill the tumor. In particular, tumor necrosis factor alpha is studied. Its action on solid tumors, both directly through tumor cell killing and destruction of the tumor vasculature and indirectly through manipulation of the tumor physiology, is complex. Understanding the mechanism of TNF and agents with comparable action on solid tumors is an important focus to further develop combination immunotherapy strategies.
Abstract: Growth of solid tumors depends largely on the development of a functional vasculature, which has been the focus in anti-tumor therapy since Folkman in 1971 proposed that prohibiting the formation of new vessels could inhibit tumor growth. The recognition of the tumor vascular bed as an important target led to the development of 3 vascular-targeted strategies. I) The anti-angiogenesis strategy that prevents the formation of new blood vessels and normalizes the remaining vessels. II) Applying vasculo-destructive agents to induce apoptosis in the endothelium of the tumor-associated vasculature that results in vascular collapse and tumor necrosis. III) Promoting further abnormalization of the already abnormal features of the tumor-associated vasculature with vaso-active agents to enhance vessel permeability. Tumor necrosis factor alpha (TNF) is a very promising vaso-active agent because of its anti-tumor effects but its severe systemic toxicity is a major drawback. Therefore a new setting, in which the optimal therapeutic benefit of TNF could be exploited, needed to be found. Through an isolated perfusion high dose of TNF can be administered in the blood circulation of the tumor-bearing extremity or organ. Alternatively, systemically low doses can be safely administered for several times. Importantly, TNF has no anti-tumor effect by itself and the combination with a conventional chemotherapeutic drug that targets the tumor cell is a prerequisite for a good tumor response. In this dual approach, TNF enhances intratumoral accumulation of the chemotherapeutic drug resulting in an impressive tumor response.
Abstract: Airway remodeling and associated angiogenesis are documented features of asthma, of which the molecular mechanisms are not fully understood. Angiotensin (ANG)II and endothelin (ET)-1 are potent vasoconstricting circulatory hormones implicated in asthma. We investigated the effects of ANG II and ET-1 on human airway smooth muscle (ASM) cells proliferation and growth and examined the mRNA expression and release of the angiogenic peptide, vascular endothelial growth factor (VEGF). Serum deprived (48 h) human ASM cells were incubated with ANG II (100 nM) or ET-1 (10 nM) for 30 min, 1, 2, 4, 8, 16, and 24 h and the endogenous synthesis of VEGF was examined in relation to control cells receiving serum free culture medium. ET-1 induced time dependent DNA biosynthesis as determined by [3H]-thymidine incorporation assay. Using northern blot hybridization, we detected two mRNA species of 3.9 and 1.7 kb encoding VEGF in the cultured smooth muscle cells. Both ANG II and ET-1 induced the mRNA expression (two- to threefold) and secretion (1.8- to 2.8-fold) of VEGF reaching maximal levels between 4-8 h of incubation. Induced expression and release of VEGF declined after 8 h of ANG II incubation while levels remained elevated in the case of ET-1. The conditioned medium derived from ET-1-treated ASM cells induced [3H]-thymidine incorporation and cell number in porcine pulmonary artery endothelial as well as human umbilical vein endothelial cells. Moreover, the VEGF tyrosine kinase receptor inhibitor blocked the conditioned medium induced mitogenesis in endothelial cells. Our results suggest a potential role for ANG II and ET-1 in ASM cell growth and upregulation of VEGF that may participate in endothelial cell proliferation via paracrine mechanisms and thus causing pathological angiogenesis and vascular remodelling seen during asthma.
Abstract: 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.
Abstract: 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.
Abstract: Tumor response is strongly enhanced by addition of tumor necrosis factor (TNF)-alpha to chemotherapy in local-regional perfusion. TNF primarily targets the endothelial lining of the tumor-associated vasculature, thereby improving permeability of the vascular bed. This augments uptake of the coadministered chemotherapeutic drug in the tumor. In vitro, however the high dose of TNF did not directly affect endothelial cells, indicating that other factors, most likely TNF-induced, are involved in the antivascular activities observed in vivo. This is supported by in vivo studies in our laboratory in which depletion of leukocytes resulted in loss of the antivascular activity of TNF. The present study examined the role of peripheral blood mononuclear cells (PBMCs) on endothelial cells by exposing them to TNF, interferon (IFN)-gamma, and PBMCs. We observed morphological changes of the endothelial cells when exposed to TNF in combination with IFN. Endothelial cells became elongated. and gaps between the cells formed. Addition of PBMCs enhanced these alterations. The endothelial layer became disrupted with highly irregular-shaped cells displaying large gap formations. PBMCs also contributed to an increased permeability of the endothelial layer without augmenting apoptosis. Replacing PBMC by interleukin (IL)-1beta produced similar effect with regard to inhibition of cell growth, morphological changes, and induction of apoptosis. Blocking IL-1beta with a neutralizing antibody diminished the effects inflicted of PBMCs. These observations indicate that endogenously produced IL-1beta by primed PBMCs plays an important role in the antivascular effect of TNF.
Abstract: 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.
Abstract: 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.
Abstract: We have previously shown how tumor response of isolated limb perfusion (ILP) with melphalan was improved when tumor necrosis factor alpha (TNF-alpha) was added. Taking into account that other vasoactive drugs could also improve tumor response to ILP, we evaluated histamine (Hi) as an alternative to TNF-alpha.
Abstract: Previously we reported that encapsulation of tumor necrosis factor-alpha (TNF) in pegylated (STEALTH) liposomes (TNF-PEGL) dramatically improved circulation times of the protein and augmented accumulation in tumor tissue. We and others have demonstrated enhanced antitumor activity of doxorubicin or melphalan by free TNF when used in high doses in an isolated limb perfusion setting. In the present study the antitumor activity of TNF-PEGL was studied in combination with liposomal chemotherapy. BN rats with subcutaneous BN175 sarcomas (8-12 mm diameter) received no treatment or pegylated liposomal doxorubicin (Doxil) alone or in combination with various doses of TNF-PEGL (15-200 microg/kg). The evaluated endpoints were tumor response and toxicity of the treatment regimens. Here we demonstrate that TNF-PEGL at a dose of 15 microg/kg markedly augments the antitumor activity of liposomal doxorubicin, without resulting in the increased toxic side effects observed with free TNF at doses resulting in a similar enhancement of the antitumor effects. Even at a TNF dose of 200 microg/kg TNF, repeated administration of TNF-PEGL did not result in severe weight loss or cause diarrhea. Repeated dosing of free TNF at this dose resulted in severe, life-threatening weight loss and occurrence of diarrhea in all animals. These results indicate that pegylated liposomal encapsulation may be effective in systemic application of TNF for combined treatment with liposomal chemotherapy of advanced solid tumors.
Abstract: Previously we demonstrated that addition of Tumour Necrosis Factor-alpha to melphalan or doxorubicin in a so-called isolated limb perfusion results in synergistic antitumour responses of sarcomas in both animal models and patients. Yet, 20 to 30% of the treated tumours do not respond. Therefore agents that synergise with tumour necrosis factor alpha must be investigated. Actinomycin D is used in combination with melphalan in isolated limb perfusion in the treatment of patients with melanoma in-transit metastases and is well known to augment tumour cell sensitivity towards tumour necrosis factor alpha in vitro. Both agents are very toxic, which limits their systemic use. Their applicability may therefore be tested in the isolated limb perfusion setting, by which the tumours can be exposed to high concentrations in the absence of systemic exposure. To study the beneficial effect of the combination in vivo, BN-175 soft tissue sarcoma-bearing rats were perfused with various concentrations of actinomycin D and tumour necrosis factor alpha. When used alone the drugs had only little effect on the tumour. Only when actinomycin D and tumour necrosis factor alpha were combined a tumour response was achieved. However, these responses were accompanied by severe, dose limiting, local toxicity such as destruction of the muscle tissue and massive oedema. Our results show that isolated limb perfusion with actinomycin D in combination with tumour necrosis factor alpha leads to a synergistic anti-tumour response but also to idiosyncratic locoregional toxicity to the normal tissues. Actinomycin D, in combination with tumour necrosis factor alpha, should not be explored in the clinical setting because of this. The standard approach in the clinic remains isolated limb perfusion with tumour necrosis factor alpha in combination with melphalan.
Abstract: The toxicity associated with tumor necrosis factor-alpha (TNF-alpha) has limited its usefulness as an anticancer agent. However, encapsulation of TNF-alpha in Stealth (SL) liposomes can minimize risk for toxicity and thus increase its potential as an adjuvant treatment. Our recent studies have shown that SL-TNF-alpha plus radiation is more effective at inhibiting LS174T colon tumor growth than either radiation alone or free TNF-alpha plus radiation. This increase in efficacy was coincident with a modulation of immune parameters in blood and spleen. The aim of this study was to determine if infiltration of natural killer (NK) cells, macrophages, and neutrophils into LS174T tumors was altered by SL-TNF-alpha treatment and whether any observed changes could potentially contribute to the enhanced antitumor efficacy seen with SL-TNF-alpha plus radiation treatment. Sections of excised tumors were examined histologically and quantitative analysis was performed using laser scanning cytometry. The data showed that the group receiving multiple treatments with SL-TNF-alpha plus radiation had the smallest tumors, but yet the level of necrosis was similar to that found in groups with much larger tumors. Furthermore, the necrotic areas in the SL-TNF-alpha plus radiation group had signs of recent and/or continuing cell death and the highest levels of NK cell and macrophage infiltrates. In time course experiments, a single injection of SL-TNF-alpha (but not free TNF-alpha) induced fluctuations in leukocyte infiltration into tumors that correlated inversely with our previous findings in blood and spleen. Overall, the data indicate that the mechanisms underlying the increased efficacy of SL-TNF-alpha compared to free TNF-alpha include a rapid and relatively sustained recruitment of NK cells, macrophages, and neutrophils.
Abstract: Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) encapsulated in sterically-stabilized PEGylated STEALTH liposomes (SL) can better and more safely augment the efficacy of other treatment modalities than free TNF-alpha. The aim of this study was to examine the effects of SL-TNF-alpha in the LS174T human colon tumor xenograft model and to correlate its administration with alterations in innate immune system parameters.
Abstract: Addition of tumor necrosis factor-alpha (TNF-alpha) to chemotherapy enhances tumor response in several treatment modalities. However, it has been shown that TNF-alpha, and several other cytokines, exert inhibitory effects on cell-cycle progression and by doing so may attenuate sensitivity of these cells to cell-cycle dependent cytotoxic drugs (e.g., doxorubicin). Here, we determined the cytotoxic effect of TNF-alpha on several tumor cell lines in vitro in combination with doxorubicin (cell-cycle dependent) or melphalan (cell-cycle independent), and its effect on cell-cycle progression. The rat cell lines were prepared from tumors, which were used previously in animal studies, in which synergy was shown between TNF-alpha and the cytotoxic drugs. Results demonstrate that the addition of TNF-alpha to doxorubicin or melphalan in vitro had no attenuating effect on the cytotoxic drugs. Depending on the cell type used, addition of TNF-alpha induced no or only an additive cytotoxic effect. Only the tested rat osteosarcoma tumor cells demonstrated a cell arrest in the G2 phase, which did not result in attenuation of the cytotoxicity of doxorubicin towards these cells.
Abstract: Recent reports have shown that tumor necrosis factor-alpha (TNF-alpha) can augment the effects of radiation against certain tumor types. However, the high concentrations of intravenous infusion of TNF-alpha needed to cause tumor regression can induce many systemic side effects. The aims of this study were to determine if TNF-alpha encapsulated in sterically stabilized (Stealth, ALZA Corporation, Mountain View, CA), PEGylated liposomes (SL) augments the antitumor effects of radiation and to compare its efficacy and possible toxicity with free TNF-alpha in the LS174T human colon tumor xenograft model. Nude mice were injected subcutaneously (s.c.) with LS174T cells and treated intravenously (i.v.) with Stealth-liposomal TNF-alpha (SL-TNF-alpha) with and without radiation or TNF-alpha with or without radiation when tumor size was approximately 200 mm(3). In phase 1, a significant decrease (p = 0.047) in tumor growth was observed with radiation at day 21 but not with SL-TNF-alpha or free TNF-alpha alone. By the end of phase 1 (day 27) with continued treatments, the SL-TNF-alpha plus radiation group had significantly smaller tumors (p = 0.044) than those in the free TNF-alpha plus radiation group. In phase 2, where a similar tumor growth reduction pattern was observed, the addition of TNF-alpha to radiation, either as free protein or within SL, increased lymphocyte activation and natural killer (NK) cell numbers in both blood and spleen. The effect was generally more pronounced with SL-TNF-alpha. Systemic toxicity, based on hematologic analyses and body weight, was absent or minimal. Collectively, the data show that pretreatment with SL-TNF-alpha can enhance more effectively, and possibly more safely, the effects of radiation against human colon tumor xenografts than can free TNF-alpha and that the increased antitumor action may involve upregulation of lymphocytes.
Abstract: An isolated limb perfusion model (ILP) using soft tissue sarcoma bearing rats (BN175) was used to study antitumour activity of a tumour necrosis factor alpha mutant (TNF-SAM2) in combination with melphalan and doxorubicin. Progressive disease was demonstrated after ILP without agents (sham) or with 50 micrograms TNF-SAM2. ILP with 40 micrograms melphalan or 400 micrograms doxorubicin resulted in no change of tumour volume or progressive disease five days after perfusion. Partial and complete response rates were demonstrated in 76% of rats when the combination of TNF-SAM2 and melphalan was used. TNF-SAM2 in combination with doxorubicin was synergistic as well with a 70% response rate. Histopathologically these responses consisted of hemorrhagic necrosis of the coagulative type. 2 In conclusion, TNF-SAM2 has similar antitumour activity in combination with melphalan or doxorubicin as rHuTNF in sarcoma-bearing rats and is eligible to be tested in clinical ILP or organ perfusion settings because of its potential decreased toxicity.
Abstract: We have shown previously that isolated limb perfusion (ILP) in sarcoma-bearing rats results in high response rates when melphalan is used in combination with tumour necrosis factor alpha (TNF-alpha). This is in line with observations in patients. Here we show that ILP with doxorubicin in combination with TNF-alpha has comparable effects in two different rat sarcoma tumour models. The addition of TNF-alpha exhibits a synergistic anti-tumour effect, resulting in regression of the tumour in 54% and 100% of the cases for the BN175-fibrosarcoma and the ROS-1 osteosarcoma respectively. The combination is shown to be mandatory for optimal tumour response. The effect of high dose TNF-alpha on the activity of cytotoxic agents in ILP is still unclear. We investigated possible modes by which TNF-alpha could modulate the activity of doxorubicin. In both tumour models increased accumulation of doxorubicin in tumour tissue was found: 3.1-fold in the BN175 and 1.8-fold in the ROS-1 sarcoma after ILP with doxorubicin combined with TNF-alpha in comparison with an ILP with doxorubicin alone. This increase in local drug concentration may explain the synergistic anti-tumour responses after ILP with the combination. In vitro TNF-alpha fails to augment drug uptake in tumour cells or to increase cytotoxicity of the drug. These findings make it unlikely that TNF-alpha directly modulates the activity of doxorubicin in vivo. As TNF-alpha by itself has no or only minimal effect on tumour growth, an increase in local concentrations of chemotherapeutic drugs might well be the main mechanism for the synergistic anti-tumour effects.
Abstract: Nitric oxide (NO) is an important molecule in regulating tumour blood flow and stimulating tumour angiogenesis. Inhibition of NO synthase by L-NAME might induce an anti-tumour effect by limiting nutrients and oxygen to reach tumour tissue or affecting vascular growth. The anti-tumour effect of L-NAME after systemic administration was studied in a renal subcapsular CC531 adenocarcinoma model in rats. Moreover, regional administration of L-NAME, in combination with TNF and melphalan, was studied in an isolated limb perfusion (ILP) model using BN175 soft-tissue sarcomas. Systemic treatment with L-NAME inhibited growth of adenocarcinoma significantly but was accompanied by impaired renal function. In ILP, reduced tumour growth was observed when L-NAME was used alone. In combination with TNF or melphalan, L-NAME increased response rates significantly compared to perfusions without L-NAME (0-64% and 0-63% respectively). An additional anti-tumour effect was demonstrated when L-NAME was added to the synergistic combination of melphalan and TNF (responses increased from 70 to 100%). Inhibition of NO synthase reduces tumour growth both after systemic and regional (ILP) treatment. A synergistic anti-tumour effect of L-NAME is observed in combination with melphalan and/or TNF using ILP. These results indicate a possible role of L-NAME for the treatment of solid tumours in a systemic or regional setting.
Abstract: It has previously been demonstrated in the setting of an isolated limb perfusion that application of high-dose TNF-alpha in combination with chemotherapy (melphalan, doxorubicin) results in strong synergistic antitumor effects in both the clinical and preclinical settings. In this study, we demonstrate that systemic administration of low-dose TNF-alpha augments the antitumor activity of a liposomal formulation of doxorubicin (DOXIL(R)). Addition of TNF-alpha to a DOXIL(R) regimen, which by itself induced some tumor growth delay, resulted in massive necrosis and regression of tumors. Furthermore, we could demonstrate a significant increase of liposomal drug in the tumor tissue when TNF-alpha had been co-administered. Administration of TNF-alpha augmented DOXIL(R) accumulation only after repeated injections, whereas accumulation of free doxorubicin was not affected by TNF-alpha. Drug levels in the tumor interstitium appeared crucial as intracellular levels of free or liposome-associated doxorubicin were not increased by TNF-alpha. Therefore, we hypothesize that low-dose TNF-alpha augments leakage of liposomal drug into the tumor interstitium, explaining the observed improved antitumor effects. Regarding the effects of systemic administration of low doses of TNF-alpha, these findings may be important for enhanced tumor targeting of various liposomal drug formulations.
Abstract: Isolated perfusion of the extremities with high-dose tumour necrosis factor alpha (TNF-alpha) plus melphalan leads to dramatic tumour response in patients with irresectable soft tissue sarcoma or multiple melanoma in transit metastases. We developed in vivo isolated organ perfusion models to determine whether similar tumour responses in solid organ tumours can be obtained with this regimen. Here, we describe the technique of isolated kidney perfusion. We studied the feasibility of a perfusion with TNF-alpha and assessed its anti-tumour effects in tumour models differing in tumour vasculature. The maximal tolerated dose (MTD) proved to be only 1 microg TNF-alpha. Higher doses appeared to induce renal failure and a secondary cytokine release with fatal respiratory and septic shock-like symptoms. In vitro, the combination of TNF-alpha and melphalan did not result in a synergistic growth-inhibiting effect on CC 531 colon adenocarcinoma cells, whereas an additive effect was observed on osteosarcoma ROS-1 cells. In vivo isolated kidney perfusion, with TNF-alpha alone or in combination with melphalan, did not result in a significant anti-tumour response in either tumour model in a subrenal capsule assay. We conclude that, because of the susceptibility of the kidney to perfusion with TNF-alpha, the minimal threshold concentration of TNF-alpha to exert its anti-tumour effects was not reached. The applicability of TNF-alpha in isolated kidney perfusion for human tumours seems, therefore, questionable.
Abstract: The blood residence half-life and organ distribution of recombinant human tumor necrosis factor-alpha (TNF-alpha) encapsulated in sterically stabilized liposomes, were investigated in rats bearing a soft tissue sarcoma in the hind leg. We studied the decay in blood concentration of "empty" liposomes using the aqueous marker 67gallium-desferal, as well as the blood concentration of soluble TNF-alpha and liposome encapsulated TNF-alpha using l25I. Encapsulation efficacy of TNF-alpha was 24%. The pharmacokinetics of TNF-alpha were markedly altered after encapsulation in liposomes, with a 33-fold increase in mean residence time of TNF-alpha in the blood, and a concomitant 14-fold increase in the area under the plasma concentration vs. time curve for liposomal TNF-alpha. Although the liposomes exhibit Stealth characteristics, uptake by mononuclear phagocyte-rich organs (e.g., liver and spleen) was noticeable, especially at later time points. Encapsulation of TNF-alpha in sterically stabilized liposomes resulted in a marked increase in localization of the cytokine in tumor measured as total uptake over time. However, peak TNF-alpha concentration levels in tumor were not significantly enhanced compared with free TNF-alpha. Besides the augmented localization of TNF-alpha after encapsulation in sterically stabilized liposomes, a diminished toxicity was observed.
Abstract: Achieving an adequate drug concentration at the tumor site is a major challenge in systemic therapy. Besides massive dilution in the blood and uptake by other organs, the pathophysiology of the tumor hampers drug uptake. Vasoactive compounds, like tumor necrosis factor-alpha (TNF) or Cilengitide manipulates the tumor-associated vasculature improving the permeability of these vessels. In this process, we have called tumor vessel abnormalization, the co-administered chemotherapeutic drug, melphalan or liposomal doxorubicin (Doxil), can traverse more easily from the blood circulation into the surrounding tumor tissue. This increased intratumoral drug accumulation ultimately results in an improved tumor response.
