<i>In Vivo</i> Gene Expression Dynamics of Tumor-Targeted Bacteria

The engineering of bacteria to controllably deliver therapeutics is an attractive application for synthetic biology. While most synthetic gene networks have been explored within microbes, there is a need for further characterization of <i>in vivo</i> circuit behavior in the context of applications where the host microbes are actively being investigated for efficacy and safety, such as tumor drug delivery. One major hurdle is that culture-based selective pressures are absent <i>in vivo</i>, leading to strain-dependent instability of plasmid-based networks over time. Here, we experimentally characterize the dynamics of <i>in vivo</i> plasmid instability using attenuated strains of <i>S. typhimurium</i> and real-time monitoring of luminescent reporters. Computational modeling described the effects of growth rate and dosage on live-imaging signals generated by internal bacterial populations. This understanding will allow us to harness the transient nature of plasmid-based networks to create tunable temporal release profiles that reduce dosage requirements and increase the safety of bacterial therapies

Correlation coefficients and <i>p</i>-values between the ratiometric analysis and the Monte Carlo analysis.

<p>Correlation coefficients and <i>p</i>-values between the ratiometric analysis and the Monte Carlo analysis.</p

Results for the <i>in vivo</i> cervix study.

<p>Boxplots for (A) full spectral Monte Carlo extracted and (B) ratiometrically (584/545) extracted [THb]. Unpaired two-sided student <i>t</i>-tests were performed between normal, CIN1, and CIN2+. Significant <i>p</i>-values are shown.</p

Wavelengths used for extraction of [THb] and SO₂.

<p>Wavelengths used for extraction of [THb] and SO₂.</p

Correlation between Monte Carlo analysis and ratiometric analysis.

<p>(A) Correlation coefficients between the full spectral Monte Carlo and ratiometrically extracted [THb] as a function of the average full spectral Monte Carlo extracted [THb] for the 9 tissue groups in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082977#pone-0082977-t004" target="_blank">Table 4</a>. (B) Correlation coefficients between the full spectral Monte Carlo and ratiometrically extracted SO₂ as a function of the average full spectral Monte Carlo extracted [THb] for the 9 tissue groups in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082977#pone-0082977-t004" target="_blank">Table 4</a>.</p

Results for the <i>in vivo</i> head and neck study.

<p>Boxplots for SO₂ at malignant and normal sites extracted with the (A) full spectral Monte Carlo analysis and the (B) ratiometric analysis. Wilcoxon rank-sum tests were performed between normal and SCC sites for each tissue type. Significant <i>p</i>-values from are shown. (SCC: squamous cell carcinoma).</p

The area under the receiver operating curves computed from the logistic regression model built base on different optical biomarkers from the full spectral MC and the ratiometric analyses for lymphoid tissues.

<p>(A) Full spectral MC and the ratiometrically extracted SO₂, log([THb]) were used for building the MC and the ratiometric logistic regression models respectively. (B) SO₂, log([THb]), log(μ<i>_s</i>') were used to build the logistic regression model for the full spectral MC analysis and the ratiometric ROC curve was built based on the SO₂, log([THb]).</p

Optical properties for simulated and experimental tissue-mimicking phantoms.

<p>Optical properties for simulated and experimental tissue-mimicking phantoms.</p

Flow chart illustrating the ratiometric analysis for [THb] and SO₂ estimation.

<p>Flow chart illustrating the ratiometric analysis for [THb] and SO₂ estimation.</p

Optical properties used for testing the ratiometric analysis with various scattering powers.

<p>Optical properties used for testing the ratiometric analysis with various scattering powers.</p