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. 2017 Jun 13;7(1):3437.
doi: 10.1038/s41598-017-03040-0.

Pancreatic adenocarcinoma response to chemotherapy enhanced with non-invasive radio frequency evaluated via an integrated experimental/computational approach

Matthew J Ware  1 , Louis T Curtis  2 , Min Wu  3 , Jason C Ho  1 , Stuart J Corr  1   4   5 , Steven A Curley  1 , Biana Godin  6 , Hermann B Frieboes  7   8
Affiliations

Pancreatic adenocarcinoma response to chemotherapy enhanced with non-invasive radio frequency evaluated via an integrated experimental/computational approach

Matthew J Ware et al. Sci Rep. .

Abstract

Although chemotherapy combined with radiofrequency exposure has shown promise in cancer treatment by coupling drug cytotoxicity with thermal ablation or thermally-induced cytotoxicity, limited access of the drug to tumor loci in hypo-vascularized lesions has hampered clinical application. We recently showed that high-intensity short-wave capacitively coupled radiofrequency (RF) electric-fields may reach inaccessible targets in vivo. This non-invasive RF combined with gemcitabine (Gem) chemotherapy enhanced drug uptake and effect in pancreatic adenocarcinoma (PDAC), notorious for having poor response and limited therapeutic options, but without inducing thermal injury. We hypothesize that the enhanced cytotoxicity derives from RF-facilitated drug transport in the tumor microenvironment. We propose an integrated experimental/computational approach to evaluate chemotherapeutic response combined with RF-induced phenotypic changes in tissue with impaired transport. Results show that RF facilitates diffusive transport in 3D cell cultures representing hypo-vascularized lesions, enhancing drug uptake and effect. Computational modeling evaluates drug vascular extravasation and diffusive transport as key RF-modulated parameters, with transport being dominant. Assessment of hypothetical schedules following current clinical protocol for Stage-IV PDAC suggests that unresponsive lesions may be growth-restrained when exposed to Gem plus RF. Comparison of these projections to experiments in vivo indicates that synergy may result from RF-induced cell phenotypic changes enhancing drug transport and cytotoxicity, thus providing a potential baseline for clinically-focused evaluation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Combination treatment with Gem and non-invasive RF field exposure yields mildly enhanced cytotoxicity and cytostaticity as compared to Gem alone in PDAC monolayer cell culture. (a) Percent viability of PANC-1 cells treated with various combinations of RF and Gem; (b) Percent viability of PANC-1 cells treated with Gem alone and Gem+RF at 48 h; (c) Percent cytostaticity of PANC-1 cells treated with Gem alone and Gem+RF at 48 h. In all cases Gem was administered immediately after RF exposure (mean ± SD, n = 6). Statistical significance determined using two-tailed Student’s t-test with significance level 0.05 (*) or 0.01 (**).
Figure 2
Figure 2
Exposure to non-invasive RF field increases diffusive penetration of molecules in 3D tissue. Left: Brightfield image of untreated Capan-1 spheroid; right: DAPI wavelength image. (a) Spheroid untreated with RF and incubated with DAPI for 24 h; (b) Spheroid treated with RF and incubated with DAPI for 1 h; (c) Spheroid treated with RF and incubated with DAPI for 24 h. Spheroid in (b) and (c) is the same lesion. Magnification: 10x.
Figure 3
Figure 3
Evaluation of transport in PDAC tumors in mice. (a) Representative intravital microscopy images of FITC penetration in tumor tissue following systemic administration before and after RF exposure. (b) Sample image analysis to quantify percent area fraction of FITC-positive regions with and without RF, highlighting a large region in the upper right corner (with RF). (c) Fraction of tissue area stained for FITC. (d) Quantification of distance of FITC-positive regions from the surrounding vessels.
Figure 4
Figure 4
Effect of Gem treatment in non-invasive RF pretreated Capan-1 spheroids. Left to right: Brightfield, DAPI and DRAQ7 channels: (a) Untreated spheroid; (b) Spheroid exposed to RF only; (c) Spheroid exposed to 100 μM Gem only for 24 h; (d) Spheroid pretreated with RF and exposed to 100 μM Gem for 24 h.
Figure 5
Figure 5
DRAQ7 signals denoting cell death induced by Gem through RF pretreated Capan-1 spheroids. (a) A representative spheroid image is shown. For the analysis, the center of the inner circle (enclosing the “core”) was positioned in the center of the spheroid (enclosed by the larger circle), with a radius half that of the whole spheroid. (a) Pixel intensity was quantified for whole spheroid (within outer circle) and for spheroid core (within inner circle). The regions of interest (ROI) were randomly selected. (b) Percent increase in DRAQ7 intensity for the whole spheroid and for the core, normalized to pixel intensity in the untreated group. Error bars represent standard deviation (n = 3). Statistical significance (*) determined using two-tailed Student’s t-test with significance level of 0.05.
Figure 6
Figure 6
Simulation of combination RF and Gem treatment. (a) Gem with no RF pre-treatment; (b) Gem with RF pre-treatment. For each panel, vascular flow is from bottom left to upper right. Tumor tissue includes proliferating (red), hypoxic (blue) and necrotic (brown) regions. Existing capillary network is denoted by regularly spaced grid (brown), with vessels induced by angiogenesis shown as irregular lines growing towards the hypoxic tumor regions acting as a source of angiogenic stimuli. At 1.9 hr post injection, the drug concentration is high. Over the course of 3.6 hr, most of the drug washes out. By 72 hr, tumor lesion has visibly shrunk due to the drug effect.
Figure 7
Figure 7
Analysis of treatment simulations. (a) Change in simulated tumor lesion radius based on different therapy scenarios. (b) Minimal tumor size achieved for each simulated treatment. Dotted boxes enclose cases with like diffusive penetration. A trend of minimal size decreasing in time emerges, which highlights the relative contribution to regression of RF modulation of drug diffusive penetration and vascular extravasation transfer rate. Middle (triangle) indicates Gem penetration and transfer rate increasing by 75% following non-invasive RF exposure (calibrated from experimental data). TR: drug vascular extravasation transfer rate (for O2, TR = 5); D: drug diffusive penetration (for O2, D = 1.00).
Figure 8
Figure 8
Simulation of weekly repetitive treatment for 7w with Gem or combination of Gem and non-invasive RF. (a) For Gem only case (topmost curve, large dash), lesion increases by 166% by the end of 6w compared to the initial size, while for Gem+RF assuming a 75% increase in drug diffusive penetration and vascular extravasation transfer rate (middle curve, short dash; calibrated from experimental data), the lesion is restrained after the 4th treatment. TR: drug extravasation vascular extravasation transfer rate (for O2, TR = 5); D: drug diffusive penetration (for O2, D = 1.00). (b) Summary of tumor regression as fraction of untreated control predicted by the simulations assuming a 75% increase in drug penetration and transfer rate for Gem+RF. (c) Tumor regression measured as fraction of untreated control for PANC-1 ectopic tumors in mice in vivo (data from ref. 7). Mean ± SEM. Statistical significance (*)determined using two-tailed Student’s t-test with significance level 0.05.
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