Ann L.B. Seynhaeve

Tumor Necrosis Factor α 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.

Cationic thermosensitive liposomes: A novel dual targeted heat-triggered drug delivery approach for endothelial and tumor cells

Developing selectively targeted and heat-responsive nanocarriers holds paramount promises in chemotherapy. We show that this can be achieved by designing liposomes combining cationic charged and thermosensitive lipids in the bilayer. We demonstrated, using flow cytometry, live cell imaging, and intravital optical imaging, that cationic thermosensitive liposomes specifically target angiogenic endothelial and tumor cells. Application of mild hyperthermia led to a rapid content release extra- and intracellularly in two crucial cell types in a solid tumor.

Low-dose tumor necrosis factor-α augments antitumor activity of stealth liposomal doxorubicin (DOXIL®) in soft tissue sarcoma-bearing rats

It has previously been demonstrated in the setting of an isolated limb perfusion that application of high‐dose TNF‐α 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‐α augments the antitumor activity of a liposomal formulation of doxorubicin (DOXIL®). Addition of TNF‐α to a DOXIL® 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‐α had been co‐administered. Administration of TNF‐α augmented DOXIL® accumulation only after repeated injections, whereas accumulation of free doxorubicin was not affected by TNF‐α. Drug levels in the tumor interstitium appeared crucial as intracellular levels of free or liposome‐associated doxorubicin were not increased by TNF‐α. Therefore, we hypothesize that low‐dose TNF‐α 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‐α, these findings may be important for enhanced tumor targeting of various liposomal drug formulations. Int. J. Cancer 87:829–837, 2000.

Pegylated liposomal tumor necrosis factor-alpha results in reduced toxicity and synergistic antitumor activity after systemic administration in combination with liposomal doxorubicin (Doxil) in soft tissue sarcoma-bearing rats.

Previously we reported that encapsulation of tumor necrosis factor‐α (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 μg/kg). The evaluated endpoints were tumor response and toxicity of the treatment regimens. Here we demonstrate that TNF‐PEGL at a dose of 15 μg/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 μg/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.

Biodistribution and tumor localization of stealth liposomal tumor necrosis factor-α in soft tissue sarcoma bearing rats

The blood residence half‐life and organ distribution of recombinant human tumor necrosis factor‐α (TNF‐α) 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‐α and liposome encapsulated TNF‐α using 125I. Encapsulation efficacy of TNF‐α was 24%. The pharmacokinetics of TNF‐α were markedly altered after encapsulation in liposomes, with a 33‐fold increase in mean residence time of TNF‐α in the blood, and a concomitant 14‐fold increase in the area under the plasma concentration vs. time curve for liposomal TNF‐α. 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‐α 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‐α concentration levels in tumor were not significantly enhanced compared with free TNF‐α. Besides the augmented localization of TNF‐α after encapsulation in sterically stabilized liposomes, a diminished toxicity was observed. Int. J. Cancer 77:901–906, 1998.© 1998 Wiley‐Liss, Inc.

A novel two-step mild hyperthermia for advanced liposomal chemotherapy ☆☆

Liposomal chemotherapy brings the advantage of minimizing systemic toxicity towards healthy organs and tissues, while has the drawbacks of limited nanoparticle accumulation and low drug bioavailability at targeted tumors. The aim of our study is to apply a clinically available mild hyperthermia (HT) treatment with thermosensitive liposomes (TSL) to tackle both issues. A two-step HT approach was combined with systemic administration of doxorubicin (Dox) TSL, in a first step to maximize nanoparticle accumulation in tumors and second step to actively trigger Dox release. The therapeutic activity of the two-step approach was compared to a one-step HT triggering intravascular Dox release from circulating TSL. Whereas the intravascular drug release approach requires fast releasing Dox-TSL (Dox-fTSL), the TSL formulation used in the two-step approach is fine-tuned to prolong Dox retention at physiological temperature in circulation, while releasing their drug content at mild HT at a slower rate (Dox-sTSL). Cytotoxicity assays show that a first-step HT at 41°C for 1h causes no drug resistance on murine BFS-1 sarcoma, human BLM melanoma cell lines and Human Umbilical Vein Endothelial Cells (HUVEC) towards subsequent exposure to Dox. However, HT sensitizes HUVEC towards Dox at higher concentrations (10-100μM). After 2h of intratumoral Dox-TSL accumulation, HT at 42°C for 1h was applied to trigger Dox release from Dox-sTSL. Quantification of intratumoral Dox accumulation revealed that the two-step HT approach increased TSL accumulation and Dox bioavailability reaching levels comparable to the intravascular release approach. The two-step HT in combination with Dox-sTSL delayed tumor growth for 12days compared to PBS group, however, was less effective compared to intravascular Dox release from Dox-fTSL using one-step HT. The two-step approach focuses on interstitial drug release upon mild HT, instead of intravascular drug release. This novel two-step approach represents an attractive alternative for the treatment of large and deep seated tumors, which are difficult to heat precisely and require loco-regional HT of the tumor area and accumulated Dox-sTSL therein to obtain a precise intratumoral drug delivery.

Intact Doxil is taken up intracellularly and released doxorubicin sequesters in the lysosome: evaluated by in vitro/in vivo live cell imaging.

Doxil, also known as Caelyx, is an established liposomal formulation of doxorubicin used for the treatment of ovarian cancer, sarcoma and multiple myeloma. While showing reduced doxorubicin related toxicity, Doxil does not greatly improve clinical outcome. To become biologically active, doxorubicin needs to be released from its carrier. Uptake and breakdown of the liposomal carrier and subsequent doxorubicin release is not fully understood and in this study we explored the hypothesis that Doxil is taken up by tumor cells and slowly degraded intracellularly. We investigated the kinetics of liposomal doxorubicin (Doxil) in vitro as well as in vivo by measuring cytotoxic effect, intracellular bioavailability and fate of the carrier and its content. To prevent fixation artifacts we applied live cell imaging in vitro and intravital microscopy in vivo. Within 8h after administration of free doxorubicin, 26% of the drug translocated to the nucleus and when reaching a specific concentration killed the cell. Unlike free doxorubicin, only 0.4% of the doxorubicin added as liposomal formulation entered the nucleus. Looking at the kinetics, we observed a build-up of nuclear doxorubicin within minutes of adding free doxorubicin. This was in contrast to Doxil showing slow translocation of doxorubicin to the nucleus and apparent accumulation in the cytoplasm. Observations made with time-lapse live cell imaging as well as in vivo intravital microscopy revealed the liposomal carrier colocalizing with doxorubicin in the cytoplasm. We also demonstrated the sequestering of liposomal doxorubicin in the lysosomal compartment resulting in limited delivery to the nucleus. This entrapment makes the bioavailable concentration of Doxil-delivered doxorubicin significantly lower and therefore ineffective as compared to free doxorubicin in killing tumor cells.

Tumor necrosis factor‐α 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‐α (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.

Angiogenic capacity of human adipose-derived stromal cells during adipogenic differentiation: an in vitro study.

BACKGROUND 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. METHODS hASCs were cultured on adipogenic medium or basal medium. Conditioned medium was collected, and cells were harvested following 0, 3, 7, 14, and 22 days of culture. The stage of adipogenic differentiation of hASC was assessed using Oil Red O staining, fatty acid binding protein-4 gene expression, and glycerol-3-phosphate dehydrogenase activity. RESULTS Gene expression of vascular endothelial growth factor (VEGF), placental growth factor, angiopoietin-1 (ANGPT1), angiopoietin-2 (ANGPT2), and protein secretion of VEGF significantly increased during short-term adipogenic differentiation of hASCs. Moreover, conditioned medium from differentiated hASCs strongly enhanced endothelial cell numbers compared to conditioned medium from undifferentiated hASCs. CONCLUSION In vitro adipogenic differentiation of hASCs improves their ability to support endothelial viable cell numbers and suggests that hASCs differentiated for a short period potentially improve angiogenic responses for in vivo implantation.

Tumor necrosis factor‐mediated interactions between inflammatory response and tumor vascular bed

Summary: 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 α 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.