The motility regulator flhDC drives intracellular accumulation and tumor colonization of Salmonella

Abstract

Background

Salmonella have potential as anticancer therapeutic because of their innate tumor specificity. In clinical studies, this specificity has been hampered by heterogeneous responses. Understanding the mechanisms that control tumor colonization would enable the design of more robust therapeutic strains. Two mechanisms that could affect tumor colonization are intracellular accumulation and intratumoral motility. Both of these mechanisms have elements that are controlled by the master motility regulator flhDC. We hypothesized that 1) overexpressing flhDC in Salmonella increases intracellular bacterial accumulation in tumor cell masses, and 2) intracellular accumulation of Salmonella drives tumor colonization in vitro.

Methods

To test these hypotheses, we transformed Salmonella with genetic circuits that induce flhDC and express green fluorescent protein after intracellular invasion. The genetically modified Salmonella was perfused into an in vitro tumor-on-a-chip device. Time-lapse fluorescence microscopy was used to quantify intracellular and colonization dynamics within tumor masses. A mathematical model was used to determine how these mechanisms are related to each other.

Results

Overexpression of flhDC increased intracellular accumulation and tumor colonization 2.5 and 5 times more than control Salmonella, respectively (P < 0.05). Non-motile Salmonella accumulated in cancer cells 26 times less than controls (P < 0.001). Minimally invasive, ΔsipB, Salmonella colonized tumor masses 2.5 times less than controls (P < 0.05). When flhDC was selectively induced after penetration into tumor masses, Salmonella both accumulated intracellularly and colonized tumor masses 2 times more than controls (P < 0.05). Mathematical modeling of tumor colonization dynamics demonstrated that intracellular accumulation increased retention of Salmonella in tumors by effectively causing the bacteria to bind to cancer cells and preventing leakage out of the tumors. These results demonstrated that increasing intracellular bacterial density increased overall tumor colonization and that flhDC could be used to control both.

Conclusions

This study demonstrates a mechanistic link between motility, intracellular accumulation and tumor colonization. Based on our results, we envision that therapeutic strains of Salmonella could use inducible flhDC to drive tumor colonization. More intratumoral bacteria would enable delivery of higher therapeutic payloads into tumors and would improve treatment efficacy.

Electronic supplementary material

The online version of this article (10.1186/s40425-018-0490-z) contains supplementary material, which is available to authorized users.

Introduction

Effective tumor colonization is essential for bacterial anti-cancer therapy. With poor colonization, insufficient treatment is delivered and the tumor response is hampered. For bacterial therapy, the tumor density is controlled more by the rate of colonization than the administered dose [1]. However, the mechanisms that control colonization are poorly understood. It has been well established that after intravenous injection into mice, Salmonella colonizes tumor tissue at ratios greater than 10,000:1 compared to other organs in the body [2]. It is this tumor specificity that makes Salmonella-based therapy particularly attractive as a targeted delivery agent [3]. Unfortunately, clinical trials showed that tumor colonization in humans was not sufficient to induce a lasting response [4]. Therefore, understanding and controlling the mechanisms that drive bacterial tumor colonization could greatly improve bacterial tumor therapy.

Two mechanisms that could affect tumor colonization are intratumoral motility and intracellular accumulation. We have previously shown that bacterial motility plays a critical role in the accumulation of Salmonella in tumors [5–7]. Upregulating motility by swim-plate selection increases distal tumor colonization of the bacteria [6, 8] and altering chemotactic sensing increases bacterial penetration into tumor masses [7–9]. Salmonella motility is controlled by the master regulator flhDC [10–12]. The flhDC protein complex regulates expression of the functional flagellar components [13]. This regulator is one of the most tightly regulated transcription factors within bacteria [14–19]. Flagella-dependent motility is downregulated under nutrient deprivation in Salmonella, which helps Salmonella survive intracellularly where there is limited availability of nutrients [20].

Intracellular invasion and growth are important mechanisms that could also affect Salmonella colonization of tumors. Salmonella have two type three secretion systems, T3SS1 and T3SS2, that promote invasion, survival, and growth inside epithelial cells [21]. Other Salmonella invasion systems include the Rck system, which invades cells by binding to epidermal growth factor receptor [22]. In the gut, Salmonella use these systems to invade and grow inside intestinal cells [23]. Disabling T3SS2 limits the ability of Salmonella to inhibit tumor growth [24]. When T3SS2 genes are deleted by transposon insertion, bacterial accumulation in the spleen is reduced [25]. After serial passaging in mice, Salmonella with increased intracellular invasion had enhanced persistence [26]. We have seen similar effects in tumor cell masses in vitro. Compared to K-12 E. Coli that is T3SS deficient, Salmonella had considerably greater colonization [5].

The two Salmonella secretions systems have distinct functions. T3SS1 initiates invasion into epithelial cells and T3SS2 enables intracellular growth and survival [21]. Both systems are composed of a needle apparatus that spans the inner and outer membranes, and the peptidoglycan layer [27]. Effector proteins are injected into the mammalian cells through the T3SS1 [27, 28]. Secretion of T3SS1 effectors into mammalian cell cytoplasm is required for T3SS dependent intracellular invasion of Salmonella [29]. Once injected, these effectors cause a rearrangement of the mammalian actin cytoskeleton and endocytosis of Salmonella [30, 31]. One essential effector protein is sipB. When knocked out, Salmonella cannot invade using T3SS1 [32]. When Salmonella have internalized, the bacteria modify the endocytic vacuole by secreting T3SS2 effectors [33–35]. These modifications confer protection to the bacteria and enable intracellular growth and survival [36, 37]. The T3SS-dependent intracellular invasion and survival of Salmonella confers protection against extracellular clearance mechanisms, like compliment and attack by macrophages and neutrophils [23, 38]. A non-functional T3SS2 apparatus impairs in vivo colonization and anti-tumor efficacy of Salmonella [24, 25], indicating the importance of intracellular growth for survival of bacteria in vivo.

Flagella-dependent motility and intracellular invasion are not regulated independently. Rather, both of these systems are intertwined and there is a complex feedback between them [39, 40]. Increasing bacterial motility also increases intracellular invasion [41]. The flhDC transcriptional complex controls elements of both motility and cellular invasion. In addition to controlling expression of motility genes, it directly controls the expression of the dual regulatory element, fliZ. FliZ controls both flagellar hook assembly and upregulates the transcription factor hilD [39–41]. HilD expression directly upregulates T3SS1 expression and intracellular invasion [39, 40]. The systems are further connected because flagella can act as physical cell surface sensors to determine the optimal extracellular location to initiate invasion [42]. These systems are connected in part because the T3SS evolved from the flagellar type three secretion system (fT3SS), which is used to assemble functional flagella [43, 44]. The co-regulation of motility and intracellular invasion further supports the idea that both of these phenomena are important for bacterial tumor colonization.

In addition to affecting intracellular invasion, flagella-dependent motility also affects the intracellular lifestyle of Salmonella. Immediately after invasion the majority of Salmonella reside in intracellular vacuoles. A small but significant fraction of the intracellular bacteria escape from the vacuoles into the cytosol [45–47]. Some cytosolic bacteria are degraded by host ubiquitination machinery [48–52]. Those that escape degradation replicate rapidly and are extruded from the cell [45]. The T3SS1 system and functional flagella play important roles in the escape from the vacuole and the hyper-replication [45–47]. After extrusion, the bacteria are primed for reinvasion because of the expression of flagella and SPI-I invasion genes [45, 46].

The goal of this study was to measure the effect of intracellular accumulation on bacterial tumor colonization and quantify the interplay between intracellular accumulation and motility. The interaction of these mechanisms has not been previously studied in relation to using bacteria for cancer therapy. We hypothesized that 1) overexpressing flhDC in Salmonella increases intracellular accumulation in tumor cell masses, and 2) intracellular accumulation of Salmonella drives tumor colonization in vitro. To test these hypotheses, Salmonella were transformed with genetic circuits that induce flhDC and express green fluorescent protein (GFP) after cell invasion. Genetically modified Salmonella were perfused into a microfluidic tumor-on-a-chip device to assess colonization and invasion using time-lapse fluorescence microscopy. The potential to use flhDC as a bispecific switch to increase tumor colonization was determined by inducing expression after initial penetration. A mathematical model was used to investigate why intracellular invasion and growth improved tumor colonization of Salmonella. Controlling Salmonella invasion into cells will increase overall tumor colonization and has the potential to make these therapeutic bacteria more effective in the clinic.

Materials and Methods

Bacterial Strains and Plasmid Construction

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.

Table 1.

Salmonella strains and plasmids

Strain	Background	Genetic circuits	Description
1. Sal	ΔmsbB, ΔpurI, Δxyl (VNP20009)	P lac /DsRed P SSEJ /GFP	Constitutive DsRed Intracellularly inducible GFP Additional file 1: Figure S1-A
2. F-Sal	Sal	P lac /DsRed P SSEJ /GFP P BAD /flhDC	Arabinose Inducible flhDC Constitutive DsRed Intracellularly inducible GFP Additional file 1: Figure S1-B
3. S-Sal	ΔsipB Sal	P lac /DsRed P SSEJ /GFP	Minimally Intracellularly Invasive Constitutive DsRed Intracellularly inducible GFP Additional file 1: Figure S1-A
4. FS-Sal	ΔsipB Sal	P lac /DsRed P SSEJ /GFP P BAD /flhDC	Arabinose Inducible flhDC Minimally Intracellularly Invasive Constitutive DsRed Intracellularly inducible GFP Additional file 1: Figure S1-B
5. Salmonella (control)	ΔmsbB, ΔpurI, Δxyl	P lac /GFP	Constitutive GFP Additional file 1: Figure S1-C
6. Salmonella + pflhDC	ΔmsbB, ΔpurI, Δxyl	P lac /GFP P BAD /flhDC	Arabinose inducible flhDC Constitutive GFP Additional file 1: Figure S1-D
7. ΔflgE	ΔmsbB, ΔpurI, Δxyl ΔflgE	P lac /GFP	Non-motile Constitutive GFP Additional file 1: Figure S1-C
8. ΔflgE+pflhDC	ΔmsbB, ΔpurI, Δxyl ΔflgE	P lac /GFP P BAD /flhDC	Arabinose inducible flhDC Non-motile Constitutive GFP Additional file 1: Figure S1-D

Cell Culture

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.

Fabrication and Operation of Microfluidic Devices

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.

Quantifying Intracellular Invasion and Colonization of Salmonella in a Tumor-on-a-chip

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.

Quantifying Aqueous Motility of Salmonella

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.

Quantifying Intracellular Invasion and Growth inside MCF7 Cells in Monolayer

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.

Mathematical Modeling

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].

$$\frac{{\partial c}_{\mathit{ex}}}{\partial t}=D\frac{{\partial }^2{c}_{\mathit{ex}}}{{\partial x}^2}+\frac{\partial }{\partial x}\left(k_{\mathit{aff}}\frac{d{c}_{\mathit{\text{chem}}}}{\mathit{dx}}{c}_{\mathit{ex}}\right)+{\mu }_g{c}_{\mathit{ex}}-{\mu }_{\mathit{inv}}{c}_{\mathit{ex}}\theta$$ 1

$$\frac{{\partial c}_{\mathit{\text{in}}}}{\partial t}={\mu }_{g,\mathit{\text{in}}}{c}_{\mathit{\text{in}}}+{\mu }_{\mathit{inv}}{c}_{\mathit{ex}}\theta$$ 2

$${c_{ex,in}|}_{t=0}=0,{\frac{{\mathrm{\partial }c}_{ex}}{\mathrm{\partial }t}|}_{x=0}=\frac{F_0}{V}(c_{ex,0}-c_{ex})+\frac{A}{V}D{\frac{{\mathrm{\partial }c}_{ex}}{\mathrm{\partial }x}|}_{x=0},{\frac{d{c}_{ex}}{\mathrm{\partial }x}|}_{x=1}=0$$ 3

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 [$D\frac{{\partial }^2{c}_{\mathit{ex}}}{{\partial x}^2}$], chemotaxis [$\frac{\partial }{\partial x}\left(k_{\mathit{aff}}\frac{d{c}_{\mathit{\text{chem}}}}{\mathit{dx}}{c}_{\mathit{ex}}\right)$], growth [μ_gc_ex], and invasion [μ_invc_exθ]. The intracellular balance includes the effects of intracellular growth [μ_{g, in}c_in] and invasion [μ_invc_exθ]. 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), F₀ 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.

Data and Statistical Analysis

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.

Results

Induction of flhDC increases tumor colonization of Salmonella

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).

Fig. 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).

Overexpression of flhDC increases intracellular accumulation of Salmonella

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).

Fig. 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).

In monolayer culture, Salmonella invaded into MCF7 cells and the extent of invasion was dependent on flagella (Fig. 2C). Overexpression of flhDC increased invasion 1.25 times compared to control Salmonella (P < 0.001, Fig. 2D). Invasion was highly dependent on functional flagella. Control Salmonella invaded cells 26-fold more than non-motile ΔflgE Salmonella (P < 0.001; Fig. 2D). Similarly, functional flagella had a large effect on cell invasion for Salmonella overexpressing flhDC; pflhDC Salmonella invaded 7.2 times more than ΔflgE+pflhDC Salmonella (P < 0.001). Flagella-independent invasion was increased 4.6 times by overexpression of flhDC (P < 0.01).

Four of the Salmonella strains (Sal, F-Sal, S-Sal and FS-Sal; Table 1) were transformed with P_SSEJ/GFP (intracellular GFP) and P_lac/DsRed (constitutive DsRed) to identify and differentiate total (red only) and intracellular (red and green) Salmonella (Fig. 2E). This genetic circuit is necessary in tumor cell masses, because constitutive fluorescence would not differentiate intracellular and extracellular bacteria. A gentamycin protection assay was used to show that P_SSEJ is a specific intracellular promoter. After applying control Salmonella (Sal) to a monolayer of cancer cells, all extracellular bacteria were removed with gentamycin. At early time points (2 h after gentamycin addition), GFP had yet to be translated (Fig. 2F, lower left) and all bacteria expressed DsRed (Fig. 2F, upper left). By 18 h, all intracellular bacteria (Fig. 2F, upper right) expressed both DsRed (Fig. 2F, upper right) and GFP (Fig. 2F, lower right), showing that the genetic circuits functioned as expected. In tumor-on-a-chip devices, overexpressing flhDC increased intracellular bacterial density (green, Fig. 2G). The high expression of GFP throughout the tumor masses (Fig. 2G) indicates that many of the Salmonella (both Sal and F-Sal) were intracellular (Additional file 2: Figure S2). Across all cell masses, the intracellular density of flhDC-induced F-Sal was significantly greater than control Sal from 29 to 45 h after administration (P < 0.05; Fig. 2H). Forty-eight hours after bacterial administration, the intracellular colonization of F-Sal was 2.5 fold more than Sal (P<0.05, Fig. 2I). In the middle of cell masses (0.5 < x < 0.6), induced F-Sal accumulated in cells 2.5 times more than control Sal (P < 0.05, Fig. 2J). Highly motile F-Sal also accumulated in distal tumor tissue (x ≥ 0.8) ten-fold more than Sal (P<0.05, Fig. 2J). These results demonstrate that flhDC induced Salmonella to accumulate in tumor cells.

Induction of flhDC does not increase tumor colonization in the absence of intracellular accumulation

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.

Fig. 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.

Intracellular accumulation of Salmonella increases tumor colonization in vitro

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.

Fig. 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).

Intratumoral induction of flhDC improves colonization and intracellular accumulation of Salmonella

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).

Fig. 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).

Similar to overall density, induction increased the amount of intracellular F-Sal (Fig. 5F). Intracellular accumulation of intratumorally induced F-Sal was five-fold greater (P< 0.5) in intermediate tumor tissue (0.6 ≤ x ≤ 0.7) and two-fold greater (P< 0.5) in distal tumor tissue (0.8 ≤ x ≤ 1) compared to Sal (Fig. 5G). Total intracellular colonization of F-Sal was 1.8 fold greater than Sal after 30 hours (P <0.05, Fig. 5H). Intratumoral flhDC induction in Salmonella improved both distal colonization and intracellular accumulation when compared to Salmonella control, demonstrating that flhDC could be induced within tumors.

Intracellular accumulation improves bacterial retention in tumors

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).

Fig. 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).

Table 2.

Calculated Intracellular accumulation rates

Strain	Intracellular Accumulation Rate
S-Sal	1.8x107 CFU ∗ hr−1
Sal	4.3x107 CFU ∗ hr−1
F-Sal	19.2x107 CFU ∗ hr−1

The model prediction of overall colonization as a function of the intracellular accumulation closely matched experimental data (Fig. 6D). When intracellular accumulation increased, overall tumor colonization increased. Theoretically extrapolating to bacteria that neither invade nor grow intracellularly suggests that they would not colonize tumors (Fig. 6D). Based on the model, the increase in bacterial density with higher rates of intracellular accumulation occurred primarily in intermediate regions of the cell masses (0.4 ≤ x ≤ 0.6; Fig. 6E). The calculated amounts of extracellular bacteria was greater for bacteria with lower rates of intracellular accumulation (i.e. S-Sal and Sal compared to F-Sal; Fig. 6F). Based on the model, this higher extracellular density (Fig. 6F) lead to greater leakage from the tumor and a lower overall density (Fig. 6G).

Discussion

The results of this study demonstrate key mechanisms that control Salmonella colonization of tumors. Using in vitro tumors that can be monitored for bacterial infiltration and proliferation in real time, we demonstrated that overexpressing the master motility regulator, flhDC, increased tumor colonization (Fig. 1). As expected, induction of flhDC increased the motility of Salmonella, but it also increased the accumulation inside cancer cells (Fig. 2). In Salmonella with impaired invasiveness, flhDC induction did not affect colonization (Fig. 3) showing that flhDC enhances colonization by increasing the number of intracellular bacteria. Similarly, when Salmonella were modified to impair their invasiveness, tumor colonization was dramatically reduced (Fig. 4), showing that intracellular invasion and growth is important for Salmonella colonization of tumors, independent of flhDC overexpression. Integrating the spatial and temporal tumor penetration data into a mathematical model enabled calculation of the intracellular accumulation rate and showed that invasion promotes colonization by increasing bacterial retention in tumors (Fig. 6). These mechanisms could be used to improve therapeutic efficacy by enhancing bacterial tumor colonization. When flhDC was induced after initial penetration, intracellular accumulation and tumor colonization both increased (Fig. 5).

Overexpression of flhDC increased intracellular accumulation through a T3SS-dependent mechanism. When flhDC was upregulated in T3SS-deficient Salmonella (FS-Sal), neither intracellular accumulation nor colonization increased (Fig. 3B-E). Induction of flhDC increased T3SS-dependent intracellular accumulation primarily through flagella production and moderately through increased synthesis of T3SS components (Figs. 2 and 3). Salmonella that were incapable of producing flagella (ΔflgE and ΔflgE+pflhDC) accumulated significantly less than those able to assemble flagella (Fig. 2C, D). Overexpressing flhDC in ΔflgE Salmonella only marginally improved intracellular accumulation (Fig. 2D). The difference between these effects shows that the major contribution of flhDC was to produce flagella, which in turn improved accumulation. The increase in accumulation of non-motile ΔflgE+pflhDC Salmonella, however, shows that flhDC control of T3SS synthesis does play a role in controlling accumulation.

Two primary mechanisms could have increased intracellular accumulation after flhDC induction: cell invasion and intracellular growth. The T3SS1 system and functional flagella are important for both. The injection of T3SS1 effectors into mammalian cells is critical for cell invasion [29]. Similarly, T3SS1 plays an important role in the escape of Salmonella from intracellular vacuoles and hyper-replication in the cellular cytoplasm [45–47]. In addition to T3SS, invasion could have been mediated by alternate mechanisms, such as the EGFR-dependent Rck system. The contribution of alternate mechanisms was considerably less than the T3SS system (Fig. 4B). T3SS-deficient Salmonella (S-Sal) colonized tumor masses three-fold less than T3SS-competent control bacteria (Sal; Fig. 4G), although residual intracellular accumulation (Fig. 4D) and colonization (Fig. 4G) was observed.

The intracellular niche provides Salmonella with an environment to proliferate (Additional file 3: Figure S3B-C) and that is protected from convective clearance (Fig. 6G). In MCF7 cells in monolayers, Salmonella grew with a doubling time of 3.6 h (Additional file 3: Figure S3C), which is considerably faster than the doubling time within tumors in mice (16.8 h) [58]. Overexpressing flhDC increased bacterial density inside cells (Fig. 2D) and in distal tumor tissue (Fig. 1D). The fact that T3SS-deficient Salmonella accumulated far less in tumor masses than control Salmonella (Sal, Fig. 4F, G) suggests that intracellular and distal tumor tissue are protected from convective clearance (Fig. 6E, F).

The mathematical model of bacterial invasion and colonization shows how intracellular accumulation would improve bacterial retention (Fig. 6). Convection continuously clears bacteria from tumor tissue located near the perfusion channel (Fig. 6F). This mechanism is analogous to convective clearance of bacteria from tumors by the bloodstream. By invading tumor cells, fewer bacteria would reside extracellularly (Fig. 6F) and fewer would be cleared (Fig. 6G). As the rate of intracellular accumulation increases, more bacteria are retained within the tumor (Fig. 6D), a mechanism similar to the ‘binding’ of small-molecule drugs to cancer cells [59]. With small molecule drugs, it has been shown that drug/receptor binding improves retention within tumors once the drug clears from the blood [59]. By ‘binding’ to cancer cells, the model suggested that Salmonella with higher rates of intracellular accumulation are less prone to leaking out of tumors (Fig. 6G).

A distally located reservoir of extracellular bacteria could serve as a continuous source for intracellular invasion and colonization of tumors. Within in vitro tumor masses, there is a considerable amount of bacterial colonization in necrotic and quiescent tissue, which is located between necrotic and actively dividing tumor tissue [7]. Of the total population of colonized bacteria, the majority of extracellular bacteria were located in necrosis (Fig. 6F). Neither intracellular nor extracellular bacteria resided in tissue near the channel because of the high rate of convective clearance (Fig. 6E, F). Due to the high dispersion coefficient, extracellular bacteria would rapidly clear out of proximal tissue close to the perfusion channel. However, extracellular bacteria residing in necrosis could grow faster than the rate of dispersion (Fig. 6F) allowing for high regional accumulation and migration to viable tissue to invade cells.

Controlling intracellular accumulation by inducing flhDC would increase tumor colonization. It would be beneficial to suppress flagellar expression outside of tumors. Flagella biosynthesis is an energetically costly process and can consume as much as 2% of bacterial energy [10, 60]. In addition , Salmonella flagellin is an immunogenic agonist that facilitates accelerated bacterial clearance [61]. Inducing flhDC selectively after initial penetration into tumors would improve fitness prior to administration, while promoting invasion and colonization within tumors (Fig. 7).

Fig. 7.

Graphical depiction of how intracellular accumulation could mechanistically improve tumor colonization. a) When Salmonella have a low intracellular accumulation rate, the rate of dispersion back into tumor vasculature is high, thus reducing bacterial tumor colonization due to a lack of “binding” to cancer cells. b) When Salmonella have a high intracellular accumulation rate, more bacteria would be retained in the tumor and not leak back into tumor microvasculature, thus increasing overall tumor colonization.

Conclusion

This study demonstrates that overexpressing flhDC increases intracellular accumulation within tumor cell masses, which drives tumor colonization. Robust tumor colonization is necessary for Salmonella to be an effective drug delivery vehicle. Intracellular accumulation increased colonization by causing Salmonella to ‘bind’ to tumor cells. This binding prevented bacteria from being convectively cleared from tumor masses. Selectively inducing flhDC expression within tumor masses would promote fitness prior to administration and enhance colonization after initial penetration. We envision that therapeutic strains of Salmonella will utilize inducible flhDC to drive colonization in human tumors. After intravenous administration and a period of initial penetration, an inducer would be provided to activate the flhDC regulator. Intracellular invasion enables Salmonella to deliver a wide range therapies directly into the intracellular space of tumors. Measuring the mechanisms of intracellular bacterial accumulation and tumor colonization has identified a key regulator, flhDC, that could be used to amplify colonization and make Salmonella an effective anticancer therapeutic.

List of abbreviations

flhDC

Salmonella master motility regulator

T3SS1

Type three secretion system-1

T3SS2

Type three secretion system-2

fT3SS

Flagellar type three secretion system

sipB

Type three secretion system cap protein

GFP

Green fluorescent protein

DsRed

A red fluorescent protein

SSEJ-GFP

Intracellular GFP expression genetic circuit

Lac-DsRed

Constitutive red fluorescent protein expression

F-Sal

Salmonella transformed with SSEJ-GFP and Lac-DsRed

Sal

Salmonella transformed with SSEJ-GFP and Lac-DsRed

S-Sal

ΔsipB Salmonella transformed with Lac-DsRed

FS-Sal

ΔsipB Salmonella transformed with SSEJ-GFP

Lac

DsRed and PBAD-flhDC

DMEM

Dulbecco’s minimal eagle medium

FBS

Fetal bovine serum

PMMA

Poly-(methyl)-methacrylate

PDMS

Poly-(dimethyl)-siloxane

HEPES

(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)

CFU

Colony forming unit

LB

Luria Bertani broth

Authors’ contributions

VR designed, performed all experiments and wrote the manuscript. NVD and OMO assisted in performing experiments. NSF designed the experiments and wrote the manuscript.

Ethics approval and consent to participate

The research (protocol 2015-004) was approved by the UMass Institutional Animal Care and Use Committee on August 26, 2017.

Consent for publication

All listed authors consent to the publication of this research article.

Competing interests

Vishnu Raman and Owen O’Connor do not have any competing interests. Neil Forbes and Nele Van Dessel share ownership of Ernest Pharmaceuticals.

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