Neil S. Forbes

Tuning payload delivery in tumour cylindroids using gold nanoparticles.

Nanoparticles have great potential as controllable drug delivery vehicles because of their size and modular functionality. Timing and location are important parameters when optimizing nanoparticles for delivery of chemotherapeutics. Here we show that positively- and negatively-charged gold nanoparticles carrying either fluorescein or doxorubicin molecules move and localize differently in an in vitro three dimensional model of tumour tissue. Fluorescence microcopy and mathematical modelling showed that uptake, and not diffusion, is the dominant mechanism in particle delivery. Our results suggest that positive particles may be more effective for drug delivery because they are more significantly taken up by proliferating cells. Negative particles, which diffused faster, may perform better when delivering drugs deep into the tissues. An understanding of how surface charge can control tissue penetration and drug release may overcome some of the current limitations in drug delivery.

Efficient Gene Delivery Vectors by Tuning the Surface Charge Density of Amino Acid-Functionalized Gold Nanoparticles

Gold colloids functionalized with amino acids provide a scaffold for effective DNA binding with subsequent condensation. Particles with lysine and lysine dendron functionality formed particularly compact complexes and provided highly efficient gene delivery without any observed cytotoxicity. Nanoparticles functionalized with first generation lysine dendrons (NP-LysG1) were approximately 28-fold superior to polylysine in reporter gene expression. These amino acid-based nanoparticles were responsive to intracellular glutathione levels, providing a tool for controlled release and concomitant expression of DNA.

Salmonella typhimurium Lacking Ribose Chemoreceptors Localize in Tumor Quiescence and Induce Apoptosis

The effectiveness of most chemotherapeutics is limited by their inability to penetrate deep into tumor tissue and their ineffectiveness against quiescent cells. Motile Salmonella typhimurium, which are specifically attracted to compounds produced by quiescent cancer cells, could overcome this therapeutic barrier. We hypothesized that individual chemoreceptors target S. typhimurium to specific tumor microenvironments. To test this hypothesis, we used time-lapse fluorescent microscopy and tumor cylindroids to quantify the accumulation of chemotaxis machinery knockouts, including strains lacking individual cell surface chemoreceptors, chemotaxis signal transduction pathway enzymes, and the flagella and motor assemblies. To measure the extent of apoptosis induced by individual bacterial strains, caspase-3 activity was measured as a function of time. Our results showed how chemoreceptors directed bacterial chemotaxis within cylindroids: the aspartate receptor initiated chemotaxis toward cylindroids, the serine receptor initiated penetration, and the ribose/galactose receptor directed S. typhimurium toward necrosis. In addition, strains lacking proper flagella constructs, signal transduction proteins, or active motor function did not chemotax toward tumor cylindroids, indicating that directed chemotaxis is necessary to promote accumulation in tumors. By deleting the ribose/galactose receptor, bacterial accumulation localized to tumor quiescence and had a greater individual effect on inducing apoptosis than wild-type S. typhimurium. This new understanding of the mechanisms of Salmonella migration in tumors will allow for the development of bacterial therapies with improved targeting to therapeutically inaccessible regions of tumors.

Can engineered bacteria help control cancer

Hypoxia and anoxia are pathophysiologic characteristics of most solid tumors (1, 2). For nearly 150 years, nonpathogenic, anaerobic bacteria that preferentially localize and proliferate in the hypoxic regions of tumors have been investigated as treatments for experimental and human tumors with mixed success (Table 1). In recent years, there has been a renewed interest in using these bacteria as innovative delivery vehicles for gene therapy (Table 1). Now, as described in this issue of PNAS, Vogelstein and coworkers (11) have created a new strain of anaerobic bacteria, devoid of its toxic genes, that leads to dramatic and prolonged regression of subcutaneous tumors when systematically administered with conventional drugs. This strategy, referred to as combination bacteriolytic therapy (COBALT), adds a new weapon in the war against cancer. However, there are still obstacles that need to be overcome before it can be used safely in the clinic. Ironically, a tumors metabolically compromised microenvironment provides a haven for a number of anaerobic bacteria.

Tumour-targeted delivery of TRAIL using Salmonella typhimurium enhances breast cancer survival in mice

Background:An effective cancer therapeutic must selectively target tumours with minimal systemic toxicity. Expression of a cytotoxic protein using Salmonella typhimurium would enable spatial and temporal control of delivery because these bacteria preferentially target tumours over normal tissue.Methods:We engineered non-pathogenic S. typhimurium to secrete murine TNF-related apoptosis-inducing ligand (TRAIL) under the control of the prokaryotic radiation-inducible RecA promoter. The response of the RecA promoter to radiation was measured using fluorometry and immunoblotting. TRAIL toxicity was determined using flow cytometry and by measuring caspase-3 activation. A syngeneic murine tumour model was used to determine bacterial accumulation and the response to expressed TRAIL.Results:After irradiation, engineered S. typhimurium secreted TRAIL, which caused caspase-3-mediated apoptosis and death in 4T1 mammary carcinoma cells in culture. Systemic injection of Salmonella and induction of TRAIL expression using 2 Gy γ-irradiation caused a significant delay in mammary tumour growth and reduced the risk of death by 76% when compared with irradiated controls. Repeated dosing with TRAIL-bearing Salmonella in conjunction with radiation improved the 30-day survival from 0 to 100%.Conclusion:These results show the pre-clinical utility of S. typhimurium as a TRAIL expression vector that effectively reduces tumour growth and extends host survival.

A multipurpose microfluidic device designed to mimic microenvironment gradients and develop targeted cancer therapeutics.

The heterogeneity of cellular microenvironments in tumors severely limits the efficacy of most cancer therapies. We have designed a microfluidic device that mimics the microenvironment gradients present in tumors that will enable the development of more effective cancer therapies. Tumor cell masses were formed within micron-scale chambers exposed to medium perfusion on one side to create linear nutrient gradients. The optical accessibility of the PDMS and glass device enables quantitative transmitted and fluorescence microscopy of all regions of the cell masses. Time-lapse microscopy was used to measure the growth rate and show that the device can be used for long-term efficacy studies. Fluorescence microscopy was used to demonstrate that the cell mass contained viable, apoptotic, and acidic regions similar to in vivo tumors. The diffusion coefficient of doxorubicin was accurately measured, and the accumulation of therapeutic bacteria was quantified. The device is simple to construct, and it can easily be reproduced to create an array of in vitro tumors. Because microenvironment gradients and penetration play critical roles controlling drug efficacy, we believe that this microfluidic device will be vital for understanding the behavior of common cancer drugs in solid tumors and designing novel intratumorally targeted therapeutics.

In tumors Salmonella migrate away from vasculature toward the transition zone and induce apoptosis

Motile bacteria can overcome diffusion resistances that substantially reduce the efficacy of standard cancer therapies. Many reports have also recently described the ability of Salmonella to deliver therapeutic molecules to tumors. Despite this potential, little is known about the spatiotemporal dynamics of bacterial accumulation in solid tumors. Ultimately this timing will affect how these microbes are used therapeutically. To determine how bacteria localize, we intravenously injected Salmonella typhimurium into BALB/c mice with 4T1 mammary carcinoma and measured the average bacterial content as a function of time. Immunohistochemistry was used to measure the extent of apoptosis, the average distance of bacteria from tumor vasculature and the location of bacteria in four different regions: the core, transition, body and edge. Bacteria accumulation was also measured in pulmonary and hepatic metastases. The doubling time of bacterial colonies in tumors was measured to be 16.8 h, and colonization was determined to delay tumor growth by 48 h. From 12 and 48 h after injection, the average distance between bacterial colonies and functional vasculature significantly increased from 130 to 310 μm. After 48 h, bacteria migrated away from the tumor edge toward the central core and induced apoptosis. After 96 h, bacteria began to marginate to the tumor transition zone. All observed metastases contained Salmonella and the extent of bacterial colocalization with metastatic tissue was 44% compared with 0.5% with normal liver parenchyma. These results demonstrate that Salmonella can penetrate tumor tissue and can selectively target metastases, two critical characteristics of a targeted cancer therapeutic.

Integrating Cell Cycle Progression, Drug Penetration and Energy Metabolism to Identify Improved Cancer Therapeutic Strategies

The effectiveness of chemotherapeutic drugs in tumors is reduced by multiple effects including drug diffusion and variable susceptibility of local cell populations. We hypothesized that quantifying the interactions between drugs and tumor microenvironments could be used to identify more effective anti-cancer strategies. To test this hypothesis we created a mathematical model that integrated intracellular metabolism, nutrient and drug diffusion, cell-cycle progression, cellular drug effects, and drug pharmacokinetics. To our knowledge, this is the first model that combines these elements and has coupled them to experimentally derived parameters. Drug cytotoxicity was assumed to be cell-cycle phase specific, and progression through the cell cycle was assumed to be dependent on ATP generation. The model consisted of a coupled set of nonlinear partial differential, ordinary differential and algebraic equations with an outer free boundary, which was solved using orthogonal collocation on a moving grid of finite elements. Model simulations showed the existence of an optimum drug diffusion coefficient: a low diffusivity prevents effective penetration before the drug is cleared from the blood and a high diffusivity limits drug retention. This result suggests that increasing the molecular weight of the anti-cancer drug paclitaxel from 854 to approximately 20,000 by nanoparticle conjugation would improve its efficacy. The simulations also showed that fast growing tumors are less responsive to therapy than are slower tumors with more quiescent cells, demonstrating the competing effects of regrowth and cytotoxicity. The therapeutic implications of the simulation results are that (1) monolayer cultures are inadequate for accurately determining therapeutic effects in vitro, (2) decreasing the diffusivity of paclitaxel could increase its efficacy, and (3) measuring the proliferation fraction in tumors could enhance the prediction of therapeutic efficacy.

Bacterial therapies: completing the cancer treatment toolbox

Current cancer therapies have limited efficacy because they are highly toxic, ineffectively target tumors, and poorly penetrate tumor tissue. Engineered bacteria have the unique potential to overcome these limitations by actively targeting all tumor regions and delivering therapeutic payloads. Examples of transport mechanisms include specific chemotaxis, preferred growth, and hypoxic germination. Deleting the ribose/galactose chemoreceptor has been shown to cause bacterial accumulation in therapeutically resistant tumor regions. Recent advances in engineered therapeutic delivery include temporal control of cytotoxin release, enzymatic activation of pro-drugs, and secretion of physiologically active biomolecules. Bacteria have been engineered to express tumor-necrosis-factor-alpha, hypoxia-inducible-factor-1-alpha antibodies, interleukin-2, and cytosine deaminase. Combining these emerging targeting and therapeutic delivery mechanisms will yield a complete treatment toolbox and increase patient survival.

Quorum-sensing Salmonella selectively trigger protein expression within tumors

Significance Nonpathogenic Salmonella localize to tumors and can be engineered to secrete anticancer proteins, but tumor-specific expression is essential to prevent systemic toxicity. To reduce unwanted side effects in healthy tissue, we integrated Salmonella with a quorum-sensing (QS) switch that only initiates drug expression in the tightly packed colonies present within tumors. Using an in vitro 3D-tumor-on-a-chip device and in vivo mouse models, we show that QS Salmonella specifically initiates protein expression within cancerous tissue while remaining uninduced in livers. Protein expression was triggered when inducer molecules from enough close neighbors reached a critical concentration. Because of these selective qualities, QS Salmonella are a promising tool for tumor-specific delivery of therapeutic proteins. Salmonella that secrete anticancer proteins have the potential to eliminate tumors, but nonspecific expression causes damage to healthy tissue. We hypothesize that Salmonella, integrated with a density-dependent switch, would only express proteins in tightly packed colonies within tumors. To test this hypothesis, we cloned the lux quorum-sensing (QS) system and a GFP reporter into nonpathogenic Salmonella. Fluorescence and bacterial density were measured in culture and in a tumor-on-a-chip device to determine the critical density necessary to initiate expression. QS Salmonella were injected into 4T1 tumor-bearing mice to quantify GFP expression in vivo using immunofluorescence. At densities below 0.6 × 1010 cfu/g in tumors, less than 3% of QS Salmonella expressed GFP. Above densities of 4.2 × 1010 cfu/g, QS Salmonella had similar expression levels to constitutive controls. GFP expression by QS colonies was dependent upon the distance to neighboring bacteria. No colonies expressed GFP when the average distance to neighbors was greater than 155 µm. Calculations of autoinducer concentrations showed that expression was sigmoidally dependent on density and inversely dependent on average radial distance. Based on bacterial counts from excised tissue, the liver density (0.0079 × 1010 cfu/g) was less than the critical density (0.11 × 1010 cfu/g) necessary to initiate expression. QS Salmonella are a promising tool for cancer treatment that will target drugs to tumors while preventing damage to healthy tissue.