Timo L.M. ten Hagen

Anti-tumor efficacy of tumor vasculature-targeted liposomal doxorubicin

Angiogenesis is a key process in the growth and metastasis of a tumor. Disrupting this process is considered a promising treatment strategy. Therefore, a drug delivery system specifically aiming at angiogenic tumor endothelial cells was developed. Alpha v beta 3-integrins are overexpressed on actively proliferating endothelium and represent a possible target. For this, RGD-peptides with affinity for this integrin were coupled to the distal end of poly(ethylene glycol)-coated long-circulating liposomes (LCL) to obtain a stable long-circulating drug delivery system functioning as a platform for multivalent interaction with alpha v beta 3-integrins. The results show that cyclic RGD-peptide-modified LCL exhibited increased binding to endothelial cells in vitro. Moreover, intravital microscopy demonstrated a specific interaction of these liposomes with tumor vasculature, a characteristic not observed for LCL. RGD-LCL encapsulating doxorubicin inhibited tumor growth in a doxorubicin-insensitive murine C26 colon carcinoma model, whereas doxorubicin in LCL failed to decelerate tumor growth. In conclusion, coupling of RGD to LCL redirected these liposomes to angiogenic endothelial cells in vitro and in vivo. RGD-LCL containing doxorubicin showed superior efficacy over non-targeted LCL in inhibiting C26 doxorubicin-insensitive tumor outgrowth. Likely, these RGD-LCL-doxorubicin antitumor effects are brought about through direct effects on tumor endothelial cells.

Current uses of isolated limb perfusion in the clinic and a model system for new strategies

Isolated limb perfusion with melphalan is the treatment of choice for multiple (small) melanoma-in-transit metastases. The use of tumour necrosis factor alpha (TNFalpha) in isolated limb perfusion is successful for treatment of locally advanced limb soft-tissue sarcomas and other large tumours; this approach can avoid the need for amputation. TNFalpha was approved in Europe after a multicentre trial in patients with locally advanced soft-tissue sarcomas, deemed unresectable by an independent review committee; the response rate to isolated limb perfusion with TNFalpha plus melphalan was 76% and the limb was saved in 71% of patients. Moreover, the trial showed the efficacy of isolated limb perfusion of TNFalpha and melphalan against various other limb-threatening tumours such as skin cancers and drug-resistant bony sarcomas. Laboratory models of isolated limb perfusion have helped to elucidate mechanisms of action and to develop new treatment modalities. They have identified TNFalpha-mediated vasculotoxic effects on the tumour vasculature and have shown that addition of TNFalpha to the perfusate results in an increase of three to six times in uptake of melphalan or doxorubicin by tumours. New vasoactive drugs and new mechanisms of action are being discovered. Moreover, isolated limb perfusion is an effective modality for gene therapy mediated by an adenoviral vector. Various clinical phase I-II studies can be expected in the next few years.

Overcoming limitations in nanoparticle drug delivery: triggered, intravascular release to improve drug penetration into tumors.

Traditionally, the goal of nanoparticle-based chemotherapy has been to decrease normal tissue toxicity by improving drug specificity to tumors. The enhanced permeability and retention effect can permit passive accumulation into tumor interstitium. However, suboptimal delivery is achieved with most nanoparticles because of heterogeneities of vascular permeability, which limits nanoparticle penetration. Furthermore, slow drug release limits bioavailability. We developed a fast drug-releasing liposome triggered by local heat that has already shown substantial antitumor efficacy and is in human trials. Here, we show that thermally sensitive liposomes (Dox-TSL) release doxorubicin inside the tumor vasculature. Real-time confocal imaging of doxorubicin delivery to murine tumors in window chambers and histologic analysis of flank tumors illustrates that intravascular drug release increases free drug in the interstitial space. This increases both the time that tumor cells are exposed to maximum drug levels and the drug penetration distance, compared with free drug or traditional pegylated liposomes. These improvements in drug bioavailability establish a new paradigm in drug delivery: rapidly triggered drug release in the tumor bloodstream.

Distinct gene mutation profiles among luminal-type and basal-type breast cancer cell lines.

Breast cancer has for long been recognized as a highly diverse tumor group, but the underlying genetic basis has been elusive. Here, we report an extensive molecular characterization of a collection of 41 human breast cancer cell lines. Protein and gene expression analyses indicated that the collection of breast cancer cell lines has retained most, if not all, molecular characteristics that are typical for clinical breast cancers. Gene mutation analyses identified 146 oncogenic mutations among 27 well-known cancer genes, amounting to an average of 3.6 mutations per cell line. Mutations in genes from the p53, RB and PI3K tumor suppressor pathways were widespread among all breast cancer cell lines. Most important, we have identified two gene mutation profiles that are specifically associated with luminal-type and basal-type breast cancer cell lines. The luminal mutation profile involved E-cadherin and MAP2K4 gene mutations and amplifications of Cyclin D1, ERBB2 and HDM2, whereas the basal mutation profile involved BRCA1, RB1, RAS and BRAF gene mutations and deletions of p16 and p14ARF. These subtype-specific gene mutation profiles constitute a genetic basis for the heterogeneity observed among human breast cancers, providing clues for their underlying biology and providing guidance for targeted pharmacogenetic intervention in breast cancer patients.

Mild hyperthermia inhibits homologous recombination, induces BRCA2 degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition

Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41–42.5 °C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized on-demand induction of HR deficiency, an approach that we term induced synthetic lethality.

Hyperthermia and Thermosensitive Liposomes for Improved Delivery of Chemotherapeutic Drugs to Solid Tumors

ABSTRACTLipid-based nanocarriers or liposomes have been proven successful in the delivery of chemotherapeutic agents and are currently applied clinically in the treatment of various types of cancer. Liposomes offer the advantage of a high drug payload, decreased drug toxicity and enhanced drug accumulation at tumor sites. Increased accumulation is due to the relatively leaky tumor vasculature that allows liposome extravasation. Between different types of tumors and even within one tumor, vascular permeability and thus liposome extravasation may differ greatly. Furthermore, upon accumulation of liposomes in the tumor area, drug bioavailability is not guaranteed. At present, these are the major issues for clinically used liposomal drugs.Mild hyperthermia (HT), the heating of tumor tissue to temperatures of up to 43°C, has been developed in the past decades as an established and efficacious treatment modality in combination with chemo- and radiotherapy. HT can be used to further improve liposomal chemotherapy in two ways: HT is known to increase vascular permeability in solid tumors and may therefore increase levels of liposome accumulation, and thermosensitive liposomes have been developed that can be triggered to release their contents upon hyperthermia. By applying these two strategies, drug delivery to tumors can be strongly enhanced.

Triggered content release from optimized stealth thermosensitive liposomes using mild hyperthermia

Liposomes are potent nanocarriers to deliver chemotherapeutic drugs to tumors. However, the inefficient drug release hinders their application. Thermosensitive liposomes (TSL) can release drugs upon heat. This study aims to identify the optimum 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG(2000) (DSPE-PEG(2000)) concentration in stealth TSL to improve content release efficiency under mild hyperthermia (HT). TSL were prepared with DSPE-PEG(2000) from 1 to 10 mol%, around 80 nm in size. Quenched carboxyfluorescein (CF) in aqueous phase represented encapsulated drugs. In vitro temperature/time-dependent CF release and TSL stability in serum were quantified by fluorometry. In vivo CF release in dorsal skin flap window chamber models implanted with human BLM melanoma was captured by confocal microscopy. In vitro heat triggered CF release increased with increasing DSPE-PEG(2000) density. However, 6 mol% and higher DSPE-PEG(2000) caused CF leakage at physiological temperature. TSL with 5 mol% DSPE-PEG(2000) were stable at 37 degrees C, while released 60% CF in 1 min and almost 100% CF in 1h at 42 degrees C. In vivo optical intravital imaging showed immediate massive CF release above 41 degrees C. In conclusion, incorporation of 5 mol% DSPE-PEG(2000) optimized stealth TSL content release triggered by HT.

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.

Mild hyperthermia triggered doxorubicin release from optimized stealth thermosensitive liposomes improves intratumoral drug delivery and efficacy.

Liposome mediated anticancer drug delivery has the advantage of reducing cytotoxicity in healthy tissues. However, undesired slow drug release impedes the therapeutic efficacy of clinically applied PEG-liposomal doxorubicin (Dox). The aim of this study is to combine stealth thermosensitive liposomes (TSL) and local mild hyperthermia (HT) to increase bioavailable Dox levels in tumors. Dox was encapsulated in stealth TSL (~80nm) with optimized PEG concentration in the membrane, and compared with lysolipid-based Dox-LTSL for in vitro stability, release kinetics, and in vivo tumor growth control. In vitro cytotoxicity of Dox-TSL against murine BFS-1 sarcoma and, human BLM melanoma cell lines and Human Umbilical Vein Endothelial Cells (HUVEC) under normothermia (37°C) and HT (42°C) was compared with non-encapsulated Dox. In vitro Dox uptake in nuclei was imaged in BLM and HUVEC. In vivo intravascular Dox release from TSL in BFS-1 tumors under local mild HT in dorsal skin flap window chamber models was captured by intravital confocal microscopy. Intravascular Dox-TSL release kinetics, penetration depth and interstitial Dox density were subjected to quantitative image analysis. Systemic Dox-TSL administration in combination with local mild HT on subcutaneous tumor growth control was compared to Dox-LTSL plus local mild HT. Dox-TSL was stable at 37°C, while released over 95% Dox within 1min in 90% serum at 42°C. Dox-TSL demonstrated efficient in vivo intratumoral Dox release under local mild HT, followed by significant Dox uptake by tumor and tumor vascular endothelial cells. Dox-TSL plus mild HT showed improved tumor growth control over Dox-LTSL plus mild HT. Survival after a single treatment of Dox-TSL plus mild HT was 67%, while survival after Dox-LTSL plus mild HT was 22%. This combination of Dox-TSL and local mild HT offers promising clinical opportunities to improve liposomal Dox delivery to solid tumors.

Improved intratumoral nanoparticle extravasation and penetration by mild hyperthermia.

Accumulation of nanoparticles in solid tumors depends on their extravasation. However, vascular permeability is very heterogeneous within a tumor and among different tumor types, hampering efficient delivery. Local hyperthermia at a tumor can improve nanoparticle delivery by increasing tumor vasculature permeability, perfusion and interstitial fluid flow. The aim of this study is to investigate hyperthermia conditions required to improve tumor vasculature permeability, subsequent liposome extravasation and interstitial penetration in 4 tumor models. Tumors are implanted in dorsal skin flap window chambers and observed for liposome (~85 nm) accumulation by intravital confocal microscopy. Local hyperthermia at 41°C for 30 min initiates liposome extravasation through permeable tumor vasculature in all 4 tumor models. A further increase in nanoparticle extravasation occurs while continuing heating to 1h, which is a clinically relevant duration. After hyperthermia, the tumor vasculature remains permeable for 8h. We visualize gaps in the endothelial lining of up to 10 μm induced by HT. Liposomes extravasate through these gaps and penetrate into the interstitial space to at least 27.5 μm in radius from the vessel walls. Whole body optical imaging confirms HT induced extravasation while liposome extravasation was absent at normothermia. In conclusion, a thermal dose of 41°C for 1h is effective to induce long-lasting permeable tumor vasculature for liposome extravasation and interstitial penetration. These findings hold promise for improved intratumoral drug delivery upon application of local mild hyperthermia prior to administration of nanoparticle-based drug delivery systems.