Eight strains of Salmonella Enterica serovar Typhimurium were used throughout the experiments (Table 1). The control strain (Sal) was based on an attenuated therapeutic strain of Salmonella (VNP20009) that has three deletions, ΔmsbB, ΔpurI, and Δxyl, that eliminate most toxicities in vivo. The background strain was transformed with a plasmid containing two gene circuits, P _lac /DsRed and P _SSEJ /GFP, that constitutively express DsRed and express GFP after intracellular invasion (Table 1; Additional file 1: Figure S1-A). The constitutive lac DsRed gene circuit was created by adding the wild-type lac promoter and a ribosomal binding site (AAGGAG) to the 5’ end of the forward DsRed primer. The SSEJ promoter was copied by PCR from VNP20009 genomic DNA using the following primers: forward-ACATGTCACATAAAACACTAGCACTTTAGC and reverse- TCTAGACCTCCTTACTTTATTAAACACGCT. The second strain, F-Sal, was transformed with a plasmid that contains a third gene circuit that enables induction of flhDC with arabinose (Table 1; Additional file 1: Figure S1-B). PCR was used to amplify the flhDC genes from Salmonella genomic DNA using the following primers: forward-AAAAAACCATGGGTTAATAAAAGGAGGAATATATATGCATACATCCGAGTTGCTAAAACA and reverse- AAAAAACTCGAGAAAAATTAAACAGCCTGTTCGATCTGTTCAT. The PCR product and PBAD-his-myc plasmid (Invitrogen, Carlsbad, CA) were digested with NcoI and XhoI and ligated with T4 DNA ligase. The flhDC expression cassette, which includes the AraC regulator and PBAD controlled flhDC, was amplified with PCR and combined with a plasmid containing SSEJ-GFP and Lac-DsRed using Gibson Assembly. Both S-Sal, which has a sipB deletion, and the ΔflgE strain were generated using lambda red recombination [53]. When the flagellar hook (flgE) is deleted, Salmonella are unable to produce functional flagella and are non-motile [54]. The S-Sal strain (strain three) was transformed with the plasmid containing P _lac /DsRed and P _SSEJ /GFP (Table 1; Additional file 1: Figure S1-A). The fourth strain, FS-Sal, was transformed with a plasmid that contains inducible flhDC (P _BAD /flhDC), constitutive DsRed expression (P _lac /DsRed) and intracellular GFP expression (P _SSEJ /GFP) in a ΔsipB background (Table 1; Additional file 1: Figure S1-B). A second control Salmonella strain (strain five) was transformed with a plasmid containing P _lac /GFP to constitutively express GFP (Table 1; Additional file 1: Figure S1-C). The constitutive lac GFP gene circuit was created similarly to the lac DsRed circuit, by adding the wild-type lac promoter and a ribosomal binding site (AAGGAG) to the 5’ end of the forward GFP primer. The sixth strain, Salmonella+pflhDC, expresses GFP constitutively and flhDC upon induction with arabinose (Table 1; Additional file 1: Figure S1-D). The seventh strain, ΔflgE, is non-motile and expresses GFP constitutively (Table 1; Additional file 1: Figure S1-C). The eighth strain, ΔflgE+pflhDC, expresses GFP constitutively and flhDC upon induction with arabinose (Table 1; Additional file 1: Figure S1-D). All cloning was performed with DH5α E. Coli (New England Biolabs, Ipswich, MA) and all plasmids contained a ColE1 origin and either chloramphenicol or ampicillin resistance (Additional file 1: Figure S1). Salmonella were transformed by electroporation. All cloning reagents, buffer reagents, and primers were from New England Biolabs, Fisher Scientific (Hampton, NH), and Invitrogen, (Carlsbad, CA), respectively, unless otherwise noted.Tab1

Salmonella strains and plasmids

MCF7 breast carcinoma cells and LS174T colorectal carcinoma cells (ATCC, Manassas, VA) were maintained in DMEM (Dulbecco's Modified Eagle Medium; Sigma Aldrich, St. Louis, MO) with 1 g/L glucose, 3.7 g/L sodium bicarbonate (pH 7.4) and 10% FBS using standard cell culture techniques. Between passages of LS174T cells, single cell suspensions were transferred to PMMA coated cell culture flasks (2 g/L PMMA in 100% ethanol, dried before use) in order to produce spheroids.

Photolithography was used to make silicon wafer masters as previously described [55]. Two silicon wafers were made: One silicon wafer was used to make the pneumatic valve layer (layer 1). The other wafer was to make the media perfusion layer (layer 2). The fabrication of multi-layer tumor-on-a-chip devices was based on a previous method [56]. The microfluidic device was fabricated in two parts. Layer 1 was created by mixing 9 parts of Sylgard 184 PDMS (Ellsworth Adhesives, Wilmington, MA) with 1 part of curing agent and poured onto the pneumatic valve layer silicon master wafer. Layer 2 was created by mixing 15 parts of PDMS with 1 part (weight by mass) of curing agent and spun coat onto the media perfusion silicon wafer to a height of 200 μm. Both layers of PDMS were cured at 65 °C for 1.5 h and layer 1 was aligned on top of layer 2. Both layers were cured together at 95 °C for 1 h. Holes were punched in the PDMS layers to receive fluidic and control tubing. The PDMS layers were bonded to a glass slide by plasma treatment (Harrick Plasma Cleaner). The valves were pneumatically actuated before bonding to prevent the valve from sealing. Devices were taped to a microscope stage adaptor and inlet and outlet tubes were inserted. A 10% bleach solution was perfused at 3 μl/min throughout the device for 2 h followed by 70% ethanol for 1 h. The device was prepared for spheroid loading by perfusing for 1 h with DMEM with 1 g/L glucose, 20 mM HEPES (pH 7.4), 10% FBS and 33 μg/ml chloramphenicol (henceforth referred to as DMEM-HEPES-chlor). For all experiments, ~300 μm diameter LS174T spheroids were positioned into a microfluidic device and equilibrated in DMEM-HEPES-chlor for 6 h at a flow rate of 3 μl/min. Some spheroids were damaged in the insertion process and these cell masses were not included in the image analysis.

Four experiments were performed with tumor-on-a-chip device to quantify colonization and intracellular accumulation for (1) induced F-Sal compared to Sal, (2) FS-Sal compared to S-Sal, (3) S-Sal compared to Sal, and (4) for intratumoral induction of F-Sal compared to Sal. Salmonella strains were grown in LB with chloramphenicol (33 μg/ml) to a density of approximately 250 million CFU/ml. Bacteria were resuspended in DMEM-HEPES-chlor at a density of 10 million CFU/ml. The bacterial suspension was perfused into the tumor-on-a-chip device for 1h at a flowrate of 3 μl/min followed by bacteria-free DMEM-HEPES-chlor at the same flowrate for 48 h. In experiments one and two, the F-Sal and FS-Sal conditions contained 0.4% arabinose to induce flhDC. Flowing bacteria-fee medium prevents over growth in the flow channel and mimics clearance in vivo. For experiment four, the procedure was the same (bacterial perfusion for 1 h, followed by perfusion with bacteria-free medium), except that after 11 h, medium containing 0.4% arabinose was perfused into the device to induce flhDC intratumorally.

Transmitted and fluorescent images (480/525 excitation/emission for GFPmut3 and 525/590 for DsRed) of tumor masses were acquired every hour with an Olympus IX71 or a Zeiss Axio Observer Z.1 microscope. Time lapse microscopy images of each tumor mass were cropped using the rectangular cropping tool in ImageJ and were analyzed in Matlab. Each image was background subtracted. Fluorescent intensities of ten spatially equal sections of each tumor mass were averaged to quantify bacterial profiles for each time point. Overall bacterial density as a function of time was determined by averaging fluorescent intensities for entire tumor masses per time point. Red fluorescence was used to calculate total bacterial colonization and green fluorescence was used to calculate intracellular bacterial density. Each experiment was normalized by dividing every calculated average fluorescence intensity by the highest fluorescent intensity observed, which occurred during the last time point.

Aqueous motility was determined by growing flhDC inducible Salmonella in 0.4% arabinose. Twenty microliters of 400 million CFU/ml of either flhDC induced or control Salmonella was placed between a coverslip and a glass slide. Transmitted light microscopy images were taken every 0.68 seconds for approximately 30 seconds. The automated particle tracking plugin in ImageJ, Trackmate, was used to analyze bacterial swimming velocity. Aqueous velocity histograms were generated by binning the fraction of total bacteria into three velocity categories: 0-15 μm/s, 15-30 μm/s and >30 μm/s. Motility assays were performed in triplicate.

Intracellular invasion of Salmonella was quantified by growing in LB and adding to monolayer cultures of MCF7 cancer cells. Four strains were used to quantify the dependence on flhDC expression and flagella formation: control Salmonella, Salmonella+pflhDC, ΔflgE, ΔflgE+pflhDC. Two strains were used to show the intracellular specificity of the P _SSEJ promoter and the dependence on T3SS: Sal and S-Sal, using a modified gentamycin protection assay. Each strain was grown in LB to a density of 5 × 10⁸ CFU/ml and added to 6-well plates of MCF7 cells at a density of 5 × 10⁶ CFU/ml. After two hours of incubation, each well was washed ten times with one milliliter of phosphate buffered saline. DMEM with 20 mM HEPES and 40 μg/ml gentamycin was added to each well to remove residual extracellular bacteria. For two hours following the addition of gentamycin, the cultures were observed microscopically to assess the effectiveness of the PBS washes to remove extracellular bacteria. The few remaining extracellular bacteria were observed over this period to ensure that they were eliminated by the gentamycin treatment. After two hours, intracellular Salmonella were imaged over time at 10X magnification with fluorescence microscopy. After 18 hours, bacterial invasion was quantified by randomly identifying 20 cells in each culture and counting the fraction of cells that contained intracellular Salmonella, as indicated by GFP fluorescence.

A similar invasion protocol was used to calculate the intracellular growth rate of Salmonella. Both control Salmonella and Salmonella+pflhDC constitutively expressed GFP (Table 1). Time lapse fluorescence microscopy was used to quantify the fluorescence from P _lac /GFP Salmonella inside MCF7 cells over time. Salmonella density was determined by multiplying the average intensity by the area of all intracellular bacteria within a cell, as a function of time. It was assumed that the amount of GFP produced per bacterium was constant over time. Only MCF7 cells containing bacteria and that did not divide for a six hour interval were used. Intracellular growth rate was calculated by fitting an exponential growth function to the intracellular bacterial density.

A mathematical model was created to interpret the spatiotemporal dynamics of bacterial dispersion, growth and invasion in tumor masses. This model was based on a previous model of bacterial growth in tumor tissue [57]. 1 ∂cex∂t=D∂2cex∂x2+∂∂xkaffdcchemdxcex+μgcex−μinvcexθ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \frac{{\partial c}_{ex}}{\partial t}=D\frac{\partial^2{c}_{ex}}{{\partial x}^2}+\frac{\partial }{\partial x}\left({k}_{aff}\frac{d{c}_{chem}}{dx}{c}_{ex}\right)+{\mu}_g{c}_{ex}-{\mu}_{inv}{c}_{ex}\theta $$\end{document} 2 ∂cin∂t=μg,incin+μinvcexθ\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \frac{{\partial c}_{in}}{\partial t}={\mu}_{g, in}{c}_{in}+{\mu}_{in v}{c}_{ex}\theta $$\end{document} 3 cex,in|t=0=0,∂cex∂t|x=0=F0V(cex,0−cex)+AVD∂cex∂x|x=0,dcex∂x|x=1=0\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \operatorname{}{c}_{ex, in}{\left|{}_{t=0}=0\kern0.50em ,\operatorname{}\frac{{\partial c}_{ex}}{\partial t}\right|}_{x=0}=\frac{F_0}{V}\left({c}_{ex,0}-{c}_{ex}\right)+\frac{A}{V}D\operatorname{}\frac{{\partial c}_{ex}}{\partial x}{\left|{}_{x=0},\operatorname{}\frac{d{c}_{ex}}{\partial x}\right|}_{x=1}=0 $$\end{document}

The coupled PDE model incorporated a balance on extracellular (eq. 1) and intracellular (eq. 2) bacteria. The balance for extracellular bacteria includes the effects of dispersion [ D∂2cex∂x2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ D\frac{\partial^2{c}_{ex}}{{\partial x}^2} $$\end{document} ], chemotaxis [ ∂∂xkaffdcchemdxcex\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \frac{\partial }{\partial x}\left({k}_{aff}\frac{d{c}_{chem}}{dx}{c}_{ex}\right) $$\end{document} ], growth [μ _g c _ex ], and invasion [μ _inv c _ex θ]. The intracellular balance includes the effects of intracellular growth [μ _{g, in} c _in ] and invasion [μ _inv c _ex θ]. The initial and boundary conditions (eq. 3) state that (1) there were no intracellular or extracellular bacteria initially within the tumor mass; (2) the flux into or out of the tumor mass was equal to the flux in the supply channel; and (3) there was no flux at the distal (x = 1) boundary. The supply of extracellular bacteria (C _ex,0 ) is a stepwise function that was set to match experimental conditions: 10⁷ CFU/ml of bacteria were administered for 2 h, followed by perfusion of bacteria-free media for the remaining time.

The variables in the model are as follows: C _ex and C _in are the normalized extracellular and intracellular densities (a value of one corresponds to 1x10¹⁰ CFU/ml), D is the dispersion coefficient, μ _g and μ _g,in are the extracellular and intracellular growth rates, μ _inv is the intracellular invasion rate, θ is the fraction of viable tumor cells, K _aff is the chemotactic affinity to chemokines in the tumor mass, C _chem is the normalized chemokine concentration, C _ex,0 is the normalized density of bacteria that was perfused into the microfluidic device as a function of time (1x10⁷ CFU/ml for t ≤ 2 h and 0 for t > 2 h), F ₀ is the media flow rate in the perfusion channel, V is the volume of the section of the perfusion channel in front of the tumor chamber, and A is the cross-sectional area of the tumor chamber. All intracellular and total bacterial fluorescence values were normalized to the highest cross sectional fluorescence intensity that occurred during the experiment.

Equations were discretized in space and solved in Matlab (The MathWorks, Inc., Natick, MA) using a finite difference method. The spatially discretized coupled ordinary differential equations were solved with the built-in ode15s function in Matlab for all spatial (discretized in ten points in space) and temporal points between 0 and 40 hours in 1 hour intervals. The fraction of viable cancer cells within the tumor mass (θ) was calculated based on previous data [9]. The extracellular growth rate was calculated based on the growth rate in liquid culture.

Two datasets (F-Sal vs. Sal and S-Sal vs. Sal) were used for modelling and normalized to one another to match control (Sal) conditions. The bacterial dispersion coefficient was calculated by fitting the model (eq. 1-3) to the tumor-on-a-chip experimental data of GFP for all spatial and temporal points up to 40 hours. The fminsearch function in Matlab was used to minimize the sum of least squares error between the experimental data and model by adjusting (and calculating) the rates of intracellular invasion and dispersion for both Sal datasets. The intracellular invasion rate of S-Sal was calculated by fixing the dispersion coefficient to be the same as Sal. The dispersion coefficient and intracellular invasion rate of F-Sal were calculated by bounding the dispersion coefficient such that it could not be lower than that of Sal. The intracellular accumulation rate was determined by quantifying the total change in intracellular density between 47 and 48 h.

Image and statistical analysis was performed in Matlab software. Unpaired t-tests with unequal variance were used to determine statistical significance with a level of P < 0.05.

Overexpressing flhDC in Salmonella increased intratumoral dispersion and colonization (Fig. 1). When administered to a tumor-on-a-chip device (Fig. 1A), F-Sal (induced flhDC) colonized tumor masses more than Sal (control) Salmonella (Fig. 1B). Both strains contained P _lac /DsRed and expressed DsRed constitutively. In these images, red fluorescence indicates overall bacterial density. At 30 h, the size of the colony formed by F-Sal (white arrows) was considerably larger than the one formed by Sal (black arrows, Fig. 1B). The area of both colonies increased in size from 30 to 48 h after bacterial administration. Both colonies were located deep into the tissue, away from the perfusion channel (see Fig. 1A), indicating that both strains actively penetrated the tumor masses as we have described previously [5, 6]. Across multiple cell masses (n = 3 for Sal and n = 5 for F-Sal), the average density of F-Sal was significantly greater than Sal within entire tumor masses between 29 and 45 hours of bacterial colonization (P < 0.05; Fig. 1C). After 48 hours of bacterial colonization, F-Sal colonized both proximal (x ≤ 0.5) and distal (x = 0.9) tumor tissue more than Sal (P < 0.05; Fig. 1D). The density of F-Sal was greater than Sal throughout the middle of tumor masses (0.6 ≤ x 0.8), but was not significant (0.05 < P < 0.08) because of heterogeneous localization of colonies between cell masses (Fig. 1D). Overall, F-Sal colonized tumor tissue five-fold more than Sal (P < 0.05, Fig. 1E).Fig1Fig. 1

Inducing Salmonella with flhDC increase bacterial tumor colonization and dispersion. a) The microfluidic device contained a media perfusion channel and a chamber that holds tumor cell masses. The perfusion channel mimics tumor vasculature. Masses are formed as spheroids and inserted through tubing and control valves. Prior to insertion, spheroids are approximately 300 μm in diameter. b) Colonization of control Sal (black arrows) and flhDC-induced F-Sal (white arrows) was measured by with red fluorescence (red). Tumor cell masses are shown in the transmitted images under the fluorescence images. Images were background subtracted and shown with the maximum red intensity at the greatest observed value. Scale bar is 100 um. c) Salmonella with induced flhDC (F-Sal) colonized tumors significantly more than Salmonella (Sal) from 29 to 45 hours after bacterial administration (*, P<0.05, n = 3 for Sal and n = 5 for F-Sal). d) F-Sal colonized proximal (x≤0.5) tissue more than control Salmonella (Sal; *, P<0.05). The density was ten-fold greater for F-Sal in distal tumor tissue. e) At 48 hours after administration, F-Sal colonized tumors five-fold more control Sal (*, P<0.05).

Upregulating flhDC in Salmonella increased intracellular accumulation in cells and tumor masses (Fig. 2). After induction with 0.2% arabinose, Salmonella motility increased by 25% (P<0.05, Fig. 2A). The non-motile fraction of bacteria (<15 μm/s) decreased seven-fold (P<0.01) and the motile fraction (>15 μm/s) increased two-fold (P<0.01, Fig. 2B).Fig2Fig. 2

Induction of flhDC increases intracellular accumulation. a) After flhDC induction, Salmonella (F-Sal) were 33% more motile in aqueous solution than control Salmonella (Sal). b) In aqueous solution, the motile fraction of Salmonella (15-30 μm/s) increased while the non-motile fraction (0-15 um/s) decreased (*, P < 0.05). c) In monolayer culture, Salmonella (green) invaded into MCF7 cells. Salmonella with flagella (control and pflhDC) invaded cells more than non-motile (ΔflgE and ΔflgE+pflhDC) Salmonella. Some ΔflgE+pflhDC Salmonella invaded cells. All Salmonella constitutively expressed GFP. Scale bar is 100 μm. d) Salmonella overexpressing flhDC invaded cells 1.25 times more than control Salmonella (***, P < 0.001). Salmonella with intact flagella (control and pflhDC) invaded cells significantly more than non-flagellated (ΔflgE and ΔflgE+pflhDC) Salmonella (***, P < 0.001). Non-motile ΔflgE+pflhDC Salmonella invaded cells more than ΔflgE Salmonella (**, P <0.01). e) Four strains of Salmonella were transformed with P _SSEJ /GFP and P _lac /DsRed to identify extracellular (red only) and intracellular (green and red) bacteria. f) The P _SSEJ promoter is intracellularly activated. At an early time after invasion (2 hours), Salmonella only express DsRed (top left) and do not express GFP (bottom left). After 18 hours of incubation, intracellular Salmonella express both GFP (bottom right) and DsRed (top right). Scale bar is 100 μm. g) In tumor masses, many of the colonized Salmonella were intracellular. Scale bar is 100 μm. h) Overexpression of flhDC (F-Sal) increased the density of intracellular Salmonella in tumor masses 2.5 fold more than control Salmonella (Sal) at times greater than 29 hours after bacterial administration (*, P < 0.05). i) The average intracellular density of flhDC induced Salmonella was 2.5 fold greater than control Salmonella (*, P < 0.05). j) Induction of flhDC increased intracellular accumulation of F-Sal in medial (0.5 ≤ x ≤ 0.6) and distal (x ≥ 0.8) tumor tissue compared to controls (Sal; *, P < 0.05).

To investigate the effect of flhDC induction in the absence of T3SS-based invasion, ∆sipB Salmonella (S-Sal) were administered to a tumor-on-a-chip device (Fig. 3). No difference was seen in the colonization pattern of extracellular (red) or intracellular (green) Salmonella (Fig. 3A). Across multiple tumor cell masses (n = 3), no differences were observed in the location of Salmonella colonization after flhDC induction, based on DsRed expression (Fig. 3B), and there was no effect on total bacterial density (Fig. 3C). Similarly, flhDC induction did not affect the location of intracellular Salmonella based on GFP expression (Fig. 3D) or overall density of intracellular Salmonella (Fig. 3E). The lack of difference between FS-Sal and S-Sal indicates that flhDC-mediated intracellular accumulation requires a functional T3SS-I.Fig3Fig. 3

Induction of flhDC does not increase tumor colonization in the absence of T3SS1. a) In the absence of T3SS, extracellular (red only) and intracellular colonization (green and red) was minimal and uneven for flhDC-induced (FS-Sal) and control (S-Sal) Salmonella. Images were acquired 36 h after bacterial administration. Scale bar is 100 μm. b-e) When compared to control ΔsipB Salmonella (S-Sal), flhDC-induced ΔsipB Salmonella (FS-Sal) did not affect (b) the location of colonization, (c) the overall bacterial density, (d) the location of intracellular invasion, or (e) the overall extent of intracellular accumulation. Data (n = 3) were acquired 36 h after bacterial administration.

Minimally invasive, ΔsipB Salmonella (S-Sal) colonized tumor tissue less than control Salmonella (Sal, Fig. 4). Both S-Sal and control Sal expressed GFP after intracellular invasion and constitutively expressed DsRed (Table 1). Without sipB, Salmonella invaded cancer cells considerably less than controls, as indicated by diminished GFP fluorescence (Fig. 4A). S-Sal invaded MCF-7 cells six-fold less than the Sal control (P < 0.05, Fig. 4B). When, S-Sal were administered to tumor-on-a-chip devices the amount of intracellular bacteria (green) was considerably less than for control Sal (Fig. 4C). The number of intracellular Sal increased from 30 to 48 hours as indicated by the increase in GFP intensity, but little increase was observed for S-Sal (Fig. 4C). Over multiple devices (n = 6), S-Sal accumulated within tumor masses 2.5 fold less than the Sal control (P<0.05, Fig. 4D) and the rate of GFP fluorescence increase of S-Sal was four fold less than Sal (P<0.05; Fig. 4E). Total tumor colonization was quantified through constitutive DsRed fluorescence. Thirty hours after administration, more control Sal bacteria were present in devices than S-Sal (Fig. 4F). The difference between Sal and S-Sal was due to the increase in intracellular invasion because knocking out sipB did not affect the growth rates of the strains (Additional file 3: Figure S3-A). Over multiple masses, S-Sal colonized tumor tissue four fold less (P<0.05, Fig. 4G) and grew four fold slower than the Sal control (P<0.05; Fig. 4H). Sal visibly grew between 30 and 48 hours after bacterial administration, while the S-Sal density remained relatively unchanged during the same time period (Fig. 4F). These results demonstrated that intracellular accumulation is an essential component of Salmonella tumor colonization in vitro.Fig4Fig. 4

Tumor colonization of Salmonella depends on intracellular accumulation in tumor masses. a) Control Salmonella (Sal) intracellularly invaded MCF7 cells more than the minimally invasive ΔsipB Salmonella (S-Sal). Green fluorescence indicates induction of GFP expression by the P _SSEJ promoter, which is activated intracellularly. Scale bar is 100 μm. b) The ΔsipB mutant (S-Sal) intracellularly invaded tumor cells ten-fold less than control Salmonella in monolayer (*, P<0.05). c) The sipB knockout reduced the amount of intracellular Salmonella (green) in devices at 30 and 48 h after administration. Scale bar is 100 μm. d, e) Compared to control Sal, S-Sal (d) accumulated in tumor cells in devices 2.5 fold less (*, P<0.05, n = 6) and (e) had a four-fold slower rate of fluorescence increase (*, P<0.05). f) The sipB knockout also reduced the total density of colonized Salmonella (red) in devices at 30 and 48 h after administration. Scale bar is 100 μm. g, h) Compared to control (Sal), S-Sal (G) colonized tumors 2.5 fold less (*, P<0.05) and (h) grew in tumors four-fold slower (*, P<0.05).

To determine if flhDC could be induced intratumorally, F-Sal was grown without arabinose and administered to tumor-on-a-chip devices. After induction with arabinose, F-Sal were 1.2 times faster in aqueous media compared to uninduced F-Sal (P<0.05; Fig. 5A). To test intratumoral induction, F-Sal were administered to devices for one hour in arabinose free medium (Fig. 5B). Twelve hours after administration, 0.4% arabinose added to the medium delivered in the flow channel to induce flhDC (Fig. 5B). Twelve hours was chosen as the time to induce, because this was the time when bacterial colonies could first be seen in the tumor cell masses (red arrows, Fig. 5C). At 47 h after administration, colonies grew in both uninduced and induced devices, but the induced colonies were visibly larger and located farther from the flow channel (Fig. 5C). Over multiple devices (n = 5 for uninduced and n = 6 for induced), intratumorally induced F-Sal colonized distal tumor tissue (0.8 ≤ x ≤ 1) five-fold more than the Sal control after 47 hours (P<0.05, Fig. 5D). The total amount of intratumorally induced F-Sal was two-fold greater than Sal (P <0.05, Fig. 5E).Fig5Fig. 5

Intratumoral flhDC induction increases colonization, dispersion and intracellular accumulation of Salmonella. a) When flhDC was induced in Salmonella, aqueous motility increased by 18% compared to uninducedSalmonella containing the same pBAD-flhDC construct (*, P<0.05). b) Graphical depiction of the dosing scheme. One hour after tumors were placed into devices, Salmonella was administered for 1 hour. Eleven hours after bacterial administration, media with 0.4% (w/v) arabinose was administered to the devices to induce bacterial flhDC expression. c) When F-Sal was administered to devices, bacteria colonies (red arrows) were first detected at 12 hours. At 47 h, colonies formed by F-Sal with intratumorally induced flhDC were larger than control Salmonella (Sal). Scale bar is 100 μm. d) Spatial distribution of intratumoral bacteria. Intratumoral induction of flhDC increased the level of distal bacterial colonization in tumor masses after 47 hours (*, P<0.05). e) Intratumoral induction of flhDC increased overall tumor colonization (*, P<0.05). f) Intratumorally induction of flhDC increased the number of intracellular Salmonella (green). Scale bar is 100 μm. g) Intratumoral flhDC expression increased intracellular accumulation in the distal region (0.6 < x < 1) of tumor masses (*, P < 0.05). h) Induction of flhDC increased intracellular accumulation within entire tumor masses after 36 hours (*, P < 0.05).

A model of bacterial dispersion, growth and intracellular invasion was used to determine how modulating intracellular accumulation affected tumor colonization. The model includes balances on extracellular and intracellular bacteria (eq. 1-2). Extracellular bacteria (eq. 1) could accumulate, disperse, chemotax, invade cells, or be convectively transferred into the perfusion channel at the x = 0 boundary (eq. 3 middle). The number of intracellular bacteria increase because of either growth or cell invasion (eq. 2).

The model was used to calculate rates of intracellular accumulation and the bacterial dispersion coefficient in tumor masses. The model was fit to the spatiotemporal profiles of intracellular bacterial density for S-Sal, Sal and F-Sal (Fig. 6A-C). The dispersion coefficient (D) was calculated to be 23.5 μm²/s, by fitting to the Sal data set. The dispersion coefficient did not increase when the mathematical model was fit to the F-Sal dataset. The rate of intracellular accumulation for F-Sal was 4.47 times greater than Sal, and the accumulation rate of S-Sal was 2.39 times less than Sal (Table 2).Fig6Fig. 6

Intracellular accumulation increases retention of bacteria by preventing flux out of tumors. a-c) The mathematical model of intratumoral dispersion and invasion (eq 1-3) was fit to (a) ΔsipB Salmonella (S-Sal), (b) Salmonella (Sal), and (c) pflhDC+Salmonella (F-Sal) to determine the intracellular accumulation rate of the three strains. The model was fit to all time points; images show the data and model fit at 31 h. d) The mathematical model fits experimental data and predicts that increasing intracellular accumulation would increase overall tumor colonization. e) The model predicts that increasing the rate of intracellular accumulation would increase overall tumor colonization, especially in intermediate tumor tissue (0.4 < x < 0.7). f, g) When the extracellular bacteria density is higher (compare S-Sal to F-Sal), there is a larger gradient at the front edge of the tumor (f), which causes more bacteria to leak out of tumors (g).