Neoadjuvant photodynamic therapy augments immediate and prolonged oxaliplatin efficacy in metastatic pancreatic cancer organoids.

Effective treatment of advanced metastatic disease remains the primary challenge in the management of inoperable pancreatic cancer. Current therapies such as oxaliplatin (OxPt)-based chemotherapy regimens (FOLFIRINOX) provide modest short-term survival improvements, yet with significant toxicity. Photodynamic therapy (PDT), a light-activated cancer therapy, demonstrated clinical promise for pancreatic cancer treatment and enhances conventional chemotherapies with non-overlapping toxicities. This study investigates the capacity of neoadjuvant PDT using a clinically-approved photosensitizer, benzoporphyrin derivative (BPD, verteporfin), to enhance OxPt efficacy in metastatic pancreatic cancer. Treatment effects were evaluated in organotypic three-dimensional (3D) cultures, clinically representative models that bridge the gap between conventional cell cultures and in vivo models. The temporally-spaced, multiparametric analyses demonstrated a superior efficacy for combined PDT+OxPt compared to each monotherapy alone, which was recapitulated on different organotypic pancreatic cancer cultures. The therapeutic benefit of neoadjuvant PDT to OxPt chemotherapy materialized in a time-dependent manner, reducing residual viable tissue and tumor viability in a manner not achievable with OxPt or PDT alone. These findings emphasize the need for intelligent combination therapies and relevant models to evaluate the temporal kinetics of interactions between mechanistically-distinct treatments and highlight the promise of PDT as a neoadjuvant treatment for disseminated pancreatic cancer.

An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models.

While it is increasingly recognized that three-dimensional (3D) cell culture models recapitulate drug responses of human cancers with more fidelity than monolayer cultures, a lack of quantitative analysis methods limit their implementation for reliable and routine assessment of emerging therapies. Here, we introduce an approach based on computational analysis of fluorescence image data to provide high-content readouts of dose-dependent cytotoxicity, growth inhibition, treatment-induced architectural changes and size-dependent response in 3D tumour models. We demonstrate this approach in adherent 3D ovarian and pancreatic multiwell extracellular matrix tumour overlays subjected to a panel of clinically relevant cytotoxic modalities and appropriately designed controls for reliable quantification of fluorescence signal. This streamlined methodology reads out the high density of information embedded in 3D culture systems, while maintaining a level of speed and efficiency traditionally achieved with global colorimetric reporters in order to facilitate broader implementation of 3D tumour models in therapeutic screening.

Image-Based Quantification of Benzoporphyrin Derivative Uptake, Localization, and Photobleaching in 3D Tumor Models, for Optimization of PDT Parameters.

Photodynamic therapy (PDT) is a light-based treatment modality in which wavelength specific activation of a photosensitizer (PS) generates cytotoxic response in the irradiated region. PDT response is critically dependent on several parameters including light dose, PS dose, uptake time, fluence rate, and the mode of light delivery. While the systematic optimization of these treatment parameters can be complex, it also provides multiple avenues for enhancement of PDT efficacy under diverse treatment conditions, provided that a rational framework is established to quantify the impact of parameter selection upon treatment response. Here we present a theranostic technique, combining the inherent ability of the PS to serve simultaneously as a therapeutic and imaging agent, with the use of image-based treatment assessment in three dimensional (3D) in vitro tumor models, to comprise a platform to evaluate the impact of PDT parameters on treatment outcomes. We use this approach to visualize and quantify the uptake, localization, and photobleaching of the PS benzoporphyrin derivative monoacid ring-A (BPD) in a range of treatment conditions with varying uptake times as well as continuous and fractionated light delivery regimens in 3D cultures of AsPC-1 and PANC-1 cells. Informed by photobleaching patterns and correlation with cytotoxic response, asymmetric fractionated light delivery at 4 hours BPD uptake was found to be the most effective regimen assessed. Quantification of the spatial profile of cell killing within multicellular nodules revealed that these conditions also achieve the highest depth of cytotoxicity along the radial axis of 3D nodules. The framework introduced here provides a means for systematic assessment of PDT treatment parameters in biologically relevant 3D tumor models with potential for broader application to other systems.

Inhibition of ovarian cancer cell proliferation by a cell cycle inhibitory peptide fused to a thermally responsive polypeptide carrier.

Current treatment of solid tumors is limited by normal tissue tolerance, resulting in a narrow therapeutic index. To increase drug specificity and efficacy and to reduce toxicity in normal tissues, we have developed a polypeptide carrier for a cell cycle inhibitory peptide, which has the potential to be thermally targeted to the tumor site. The design of this polypeptide is based on elastin-like polypeptide (ELP). The coding sequence of ELP was modified by the addition of the cell penetrating peptide Bac-7 at the N-terminus and a 23 amino acid peptide derived from p21 at the C-terminus (Bac-ELP1-p21). Bac-ELP1-p21 is soluble in aqueous solutions below physiological temperature (37 degrees C) but aggregates when the temperature is raised above 39 degrees C, making it a promising thermally responsive therapeutic carrier that may be actively targeted to solid tumors by application of focused hyperthermia. While Bac-ELP1-p21 at 37 degrees C did not have any effect on SKOV-3 cell proliferation, the use of hyperthermia increased the antiproliferative effect of Bac-ELP1-p21 compared with a thermally unresponsive control polypeptide. Bac-ELP1-p21 displayed both a cytoplasmic and nuclear distribution in the SKOV-3 cells, with nuclear-localized polypeptide enriched in the heated cells, as revealed by confocal microscopy. Using Western blotting, we show that Bac-ELP1-p21 caused a decrease in Rb phosphorylation levels in cells treated at 42 degrees C. The polypeptide also induced caspase activation, PARP cleavage, and cell cycle arrest in S-phase and G2/M-phase. These studies indicate that ELP is a promising macromolecular carrier for the delivery of cell cycle inhibitory peptides to solid tumors.

Therapeutic peptides for cancer therapy. Part II - cell cycle inhibitory peptides and apoptosis-inducing peptides.

BACKGROUND: Therapeutic peptides have great potential as anticancer agents owing to their ease of rational design and target specificity. However, their utility in vivo is limited by low stability and poor tumor penetration. OBJECTIVE: The authors review the development of peptide inhibitors with potential for cancer therapy. Peptides that arrest the cell cycle by mimicking CDK inhibitors or induce apoptosis directly are discussed. METHODS: The authors searched Medline for articles concerning the development of therapeutic peptides and their delivery. RESULTS/CONCLUSION: Inhibition of cancer cell proliferation directly using peptides that arrest the cell cycle or induce apoptosis is a promising strategy. Peptides can be designed that interact very specifically with cyclins and/or cyclin-dependent kinases and with members of apoptotic cascades. Use of these peptides is not limited by their design, as a rational approach to peptide design is much less challenging than the design of small molecule inhibitors of specific protein-protein interactions. However, the limitations of peptide therapy lie in the poor pharmacokinetic properties of these large, often charged molecules. Therefore, overcoming the drug delivery hurdles could open the door for effective peptide therapy, thus making an entirely new class of molecules useful as anticancer drugs.

Application of thermally responsive elastin-like polypeptide fused to a lactoferrin-derived peptide for treatment of pancreatic cancer.

A well characterized, peptide derivative of bovine lactoferrin, L12, has been shown to possess anticancer properties in multiple cell lines. However, adverse side effects in normal tissues and poor plasma kinetics that hinder the clinical effectiveness of current chemotherapeutics also deter the potential for effective delivery of this L12 peptide. To overcome these limitations, we have developed an Elastin-like polypeptide (ELP) carrier that has the potential to thermally target therapeutic peptides and chemotherapeutics to a tumor site. The coding sequence of ELP was modified with the L12 peptide at the C-terminus and a membrane transduction domain derived from the HIV-1 Tat protein at the N-terminus (Tat-ELP-L12). The thermally responsive Tat-ELP1-L12 is soluble in aqueous solutions at 37 degrees C but aggregates near 41 degrees C, which makes Tat-ELP1-L12 ideal for targeting to solid tumors on application of focused hyperthermia. We observed that under hyperthermia conditions at 42 degrees C, Tat-ELP1-L12 mediated cytotoxicity in MIA PaCa-2 pancreatic adenocarcinoma cells was enhanced by nearly thirty-fold. We investigated the mechanisms of cell death and found evidence of mitochondrial membrane depolarization and caspase activation, which are characteristic of apoptosis, as well as, increased membrane permeability, as shown by LDH release. These results suggest that Tat-ELP1-L12 possesses cytotoxic properties to cancer cells in vitro and may have the potential to provide an effective vehicle to thermally target solid tumors.

Inhibition of ovarian cancer cell metastasis by a fusion polypeptide Tat-ELP.

Tumor cell metastasis is a complex, multi-step process that is a major cause of death and morbidity amongst cancer patients. Cell adhesion plays a critical role in the development of metastatic cancer, and it is mediated by interactions between receptors on the cell surface and ligands of the extracellular matrix or other surfaces. Therefore, inhibition of the cell adhesion process appears to be an effective method of preventing metastasis. This work describes a genetically engineered polypeptide with the potential to prevent cell adhesion and inhibit metastasis. We have found that the cell penetrating peptide Tat, fused with elastin-like polypeptide (ELP) inhibited adhesion, spreading, invasion and migration of SKOV-3 ovarian cancer cells in cell culture. Furthermore, we have also confirmed that Tat-ELP has anti-metastatic potential in an experimental ovarian cancer metastasis model in vivo, causing approximately 80% reduction in the tumor burden. Since cell attachment is an important step in tumor cell invasion and metastasis, these results suggest a novel role of Tat-ELP as a therapeutic intervention in cancer metastasis.

Thermally targeted delivery of chemotherapeutics and anti-cancer peptides by elastin-like polypeptide.

Current chemotherapy treatment of solid tumors is limited due to a lack of specific delivery of the drugs to the tumor, leading to systemic toxicity. Therefore, it is necessary to develop targeted cancer therapies and tumor-targeted drug carriers. The authors review the development of elastin-like polypeptide (ELP) as a potential carrier for thermally targeted delivery of therapeutics. The authors searched Medline for articles concerning the application of ELP as a drug delivery vector for small molecule drugs and therapeutic peptides. ELP has been demonstrated to be a promising thermally targeted carrier. Further examination of the in vivo biodistribution and efficacy will provide the necessary data to advance ELP technology toward the ultimate goal of human therapeutics.

A thermally responsive Tat-elastin-like polypeptide fusion protein induces membrane leakage, apoptosis, and cell death in human breast cancer cells.

The thermally responsive elastin-like polypeptide (ELP) has great potential as a macromolecular drug delivery vehicle due to its ability to be actively targeted to solid tumors by application of focused hyperthermia. Since, the toxicity properties of a new therapeutic delivery vehicle are crucial to its utility as an effective delivery vehicle, we evaluated the cytotoxicity of a thermally responsive Tat-ELP1 in various cell lines in response to hyperthermia. We report that Tat-ELP1 was not cytotoxic at 37 degrees C in SK-MEL-2, SKOV-3 and WI-38 cells, and only mildly toxic in the MCF-7 breast carcinoma cell line. Application of hyperthermia (42 degrees C) in combination with Tat-ELP1 resulted in cytotoxicity in all cell lines tested, and this toxicity was most prominent in the MCF-7 cell line, which was chosen to study the mechanism behind this increased toxicity. We found that Tat-ELP1 combined with hyperthermia caused membrane leakage and apoptosis, resulting in cell death, but no hemolytic effect was observed on murine erythrocytes.

Evaluation of cell penetrating peptides fused to elastin-like polypeptide for drug delivery.

Translocation through the plasma membrane is a major limiting step for the cellular delivery of macromolecules. Several cell penetrating peptides (CPP) have been demonstrated to efficiently internalize various molecular cargo to targets inside eukaryotic cells. In this study, the efficiency and mechanism of cellular uptake of the CPPs penetratin, Tat, and MTS fused to elastin-like polypeptides (CPP-ELP) were evaluated. Elastin-like polypeptides are biopolymers with features that make them useful as polymeric carriers for the delivery of therapeutics. Therefore, improving efficiency of cellular uptake by fusing ELPs to CPPs and understanding the mechanism of their cellular internalization could contribute to development of new therapeutic approaches. Flow cytometry and confocal fluorescence microscopy were used to elucidate the mechanism of CPP-ELP uptake. The internalization of all CPP-ELPs was impaired dramatically at 4 degrees C, under ATP depletion conditions, and with hyperosmolar sucrose, implicating involvement of an endocytic pathway for CPP-ELP internalization. Penetratin was identified as the most effective CPP for delivering ELP. Finally, in order to demonstrate the potential of CPP-ELP for drug delivery, a fusion polypeptide was made containing penetratin, ELP, and a peptide derived from the cyclin-dependent kinase inhibitor p21. This polypeptide was shown to inhibit proliferation of SKOV-3 and HeLa cells by slowing their growth rate.