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. Author manuscript; available in PMC: 2024 May 1.
Published in final edited form as: Biotechnol Bioeng. 2023 Feb 12;120(5):1437–1448. doi: 10.1002/bit.28340

Bacterial Delivery of Therapeutic Proteins to the Nuclei of Cancer Cells

Shoshana M K Bloom 1, Nicholas O’Hare 1, Neil S Forbes 1,2,3,*
PMCID: PMC10101893  NIHMSID: NIHMS1870137  PMID: 36710503

Abstract

Targeting nucleic targets with therapeutic proteins would enhance the treatment of hard-to-treat cancers. However, exogenous proteins are excluded from the nucleus by both the cellular and nuclear membranes. We have recently developed Salmonella that deliver active proteins into the cytoplasm of cancer cells. Here, we hypothesized that bacterially delivered proteins accumulate within nuclei, nuclear localization sequences increase delivery, and bacterially delivered proteins kill cancer cells. To test this hypothesis, we developed intranuclear delivering (IND) Salmonella and quantified the delivery of three model proteins. IND Salmonella delivered both ovalbumin and GFP to nuclei of MCF7 cancer cells. The amount of protein in nuclei was linearly dependent on the amount delivered to the cytoplasm. The addition of a nuclear localization sequence increased both the amount of protein in each nucleus and the number of nuclei that received protein. Delivery of Omomyc, a protein inhibitor of the nuclear transcript factor, Myc, altered cell physiology and significantly induced cell death. These results show that IND Salmonella deliver functional proteins to the nucleus of cancerous cells. Extending this method to other transcription factors will increase the number of accessible targets for cancer therapy.

Introduction

Delivery of active proteins drugs directly into the nuclei of tumor cells would improve the treatment of cancer by targeting nuclear protein-protein interactions. Small molecular therapies have limited efficacy blocking these interactions due to their small size and flatness of the interacting protein surfaces (Bojadzic and Buchwald 2018; Mabonga and Kappo 2019). Because of this limitation, most (>98%) nuclear proteins and transcriptional complexes are considered “undruggable” (Bojadzic and Buchwald 2018). A larger protein molecule would better suited to prevent the formation of transcriptional complexes (Wan 2016) to prevent DNA transcription and cause cell death (Chen and Koehler 2020; Lambert et al. 2018). Unfortunately, most protein therapeutics are excluded from the nucleus by the cellular and nuclear membranes (Mattaj and Englmeier 1998). A nuclear acting anti-cancer therapy would open the range of protein targets and enable new therapies for hard-to-treat cancers (Lambert et al. 2018).

An effective nuclear-targeting therapy must (1) penetrate the cell membrane, (2) penetrate the nuclear membrane, and (3) be active against a nuclear protein or pathway (Figure 1A). The therapy would be more effective if it (4) is tumor specific and (5) penetrates the tumor interstitium. Several methods are currently used to deliver therapeutic agents to the nucleus, include antibodies and cell penetrating peptides (Cohen and Granek 2014; Hua et al. 2018; Slastnikova et al. 2018). Several classes of antibodies penetrate the cell membrane, including autoantibodies and bispecific antibodies (Slastnikova et al. 2018; Weisbart et al. 2012). Attachment of a nuclear localization sequence (NLS) enables antibodies to access the nucleus after crossing the cell membrane (Slastnikova et al. 2018). Similarly, cell penetrating peptides with an attached NLS have also been used to target the nucleus (Cohen and Granek 2014; Ngwa et al. 2017). Both antibodies and cell penetrating peptides have limited tumor specificity, which can lead to off-target effects and low tumor concentrations (Boohaker et al. 2012; Svoronos and Engelman 2021). Additionally, antibodies have poor tumor penetration (Kijanka et al. 2015; Van Audenhove and Gettemans 2016) and peptides have poor stability in the blood (Di 2015; Hossein-Nejad-Ariani et al. 2019; Powell 1993; Whitfield et al. 2017).

Figure 1. Bacterial delivery of proteins into the nucleus.

Figure 1.

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A) An effective nuclear-targeting therapy has three essential criteria. It must (1) penetrate the cell membrane, (2) penetrate the nuclear membrane, and (3) be active against a nuclear protein or pathway. Intranuclear-delivering (IND) Salmonella deliver proteins to the nucleus using a combination of engineered and innate properties. These bacteria lyse after invasion into cancer cells and release protein molecules into the cellular cytoplasm (Raman et al. 2019). Nuclear localization sequences (NLSs) on the cytoplasmic proteins promote binding to importins and transport through the nuclear pore complex (NPC). Cytoplasmic proteins also enter nuclei through non-facilitated, passive transport mechanisms. Within the nucleus, therapeutic proteins interact with target proteins to disrupt protein-protein interactions. B) To quantify nuclear delivery, we used microscopy, flow cytometry, and immunoblot to measure the (1) cellular distribution, (2) nuclear distribution, and (3) nuclear amount of the delivered protein. After administration of IND Salmonella (left panel), cancer cells contain different amounts of delivered protein (light blue) in the cytoplasm and the nucleus.

We have recently developed a strain of Salmonella that delivers proteins into the cytoplasm (Figure 1A) of cancer cells (Raman et al. 2021). These Salmonella invade cells by utilizing the type III secretion system (Raman et al. 2019). After invasion, the bacteria lyse and release their protein contents into Salmonella-containing vacuoles (SCVs) (Raman et al. 2021). With time, the vacuole membrane breaks down and delivered proteins fill the cellular cytoplasm (Raman et al. 2021). In addition to these intracellular delivery properties, engineered Salmonella selectively accumulate in tumors over other organs (Forbes et al. 2003; Ganai et al. 2011) and actively penetrate tumor tissue (Kasinskas and Forbes 2006; Zhang and Forbes 2015). The preferential accumulation of Salmonella in tumors prevents off-target effects and intracellular lysis prevents degradation in the blood after intravenous injection (Raman et al., 2021). This selective accumulation focuses therapeutic action specifically to tumors and is a critical component of a clinical bacterial strain (Forbes 2006; Van Dessel et al. 2015). By combining these characteristics, a nuclear delivering Salmonella strain could specifically target protein pathways in cancer cells and focus that effect only on malignant cells in tumors.

The oncogenic transcription factor, c-Myc, is a prominent nuclear target that is mutated in 40% of breast cancers (Chen and Olopade 2008; Whitfield et al. 2017; Xu et al. 2010). In the nucleus, c-Myc forms a dimeric complex with its partner Max that binds DNA and regulates gene transcription (Chen and Olopade 2008; Dang 2012). Because the Myc/Max complex does not have a conserved, three-dimensional structure it cannot be targeted with small molecules (Massó-Vallés and Soucek 2020). To overcome this limitation, the peptide Omomyc was developed to inhibit c-Myc (Soucek et al. 1998). Omomyc selectively binds to c-Myc over Max and represses transcription (Soucek et al. 2002). When expressed directly in cells, Omomyc induces apoptosis (Soucek et al. 2002) and inhibits self-renewal, growth, and migration of cells (Galardi et al. 2016). Omo C/S is an Omomyc derivative that has a cysteine-to-serine point mutation in its amino acid sequence, which was developed to enhance gene expression. When produced recombinantly, Omomyc retains its functionality (Beaulieu et al. 2019). Because of its vital role in tissue regeneration (Massó-Vallés and Soucek 2020) and cell cycle regulation (Bouvard et al. 2017; Dang 2012), a Myc-targeted therapy needs to be specific for cancerous cells.

Nuclear localization sequences (NLSs) are short peptide sequences that facilitate protein transport into the nucleus. These sequences interact with importins, soluble carrier proteins that bind to cargo in the cytoplasm and increase transport through the nuclear pore complex (NPC) (Freitas and Cunha 2009; Kitamura et al. 2015; Lange et al. 2007; Mattaj and Englmeier 1998). NPCs are multi-protein channels through the nuclear envelope that permit both passive and active molecular transport (Lange et al. 2007; Talcott and Moore 1999). Some proteins access the nucleus through passive diffusion, but many require an NLS and binding to importins (Freitas and Cunha 2009; Seibel et al. 2007). Because of their short length (e.g. the SV40 NLS, PKKKRKV, is seven amino acids), NLSs can be added to recombinant proteins to aid nuclear localization (Kijanka et al. 2015; Rodrigues et al. 2001).

The goal of this work was to develop a bacterial vector that delivers proteins to the nucleus of cancerous cells (Figure 1A). We hypothesized that (1) bacterially delivered proteins accumulate within the nucleus, (2) NLSs increase bacterial delivery to the nucleus, and (3) nuclear delivery of Omo C/S kills cancer cells. To test these hypotheses, we generated an intranuclear delivering (IND) strain of Salmonella and tested the delivery of three proteins: ovalbumin (OVA), green fluorescent protein (GFP), and Omo C/S. OVA and GFP were used as examples of large (45 kDa) and small (27 kDa) proteins. Omo C/S was used as a model peptide to demonstrate that nuclear-delivered proteins retain their functionality and induce cell death. We used microscopy, flow cytometry and immunoblot analysis to quantify the relationship between cytoplasmic and nuclear delivery, determine the fraction of nuclei with delivered proteins, and measure the effect of NLSs on the nuclear protein concentration (Figure 1B). The ability to deliver protein therapeutics to the nucleus with engineered Salmonella opens up a new range of potential targets for anti-cancer therapies that could be effective for a large population of cancer patients.

Materials and Methods

Bacterial Strains and Plasmid Construction

Three plasmids were created to deliver GFP, ovalbumin and Omo C/S. Plasmid pS5-OVA-NLS was generated by insertion of the OVA gene from pCDNA3-OVA (Diebold et al. 2001) into pBAD-flhDC / Plac-GFP (Raman et al. 2019). In this plasmid, the Plac promoter constitutively induces the expression of OVA. The Pssej-LysE gene circuit induces bacteria lysis after cell invasion and delivers the expressed OVA into the cytoplasm (Raman et al. 2021). OVA was tagged with the simian virus 40 (SV40) T-antigen NLS (Kalderon et al. 1984; Kosugi et al. 2009) and the c-Myc affinity tag to promote nuclear transport and facilitate quantification, respectively. A similar plasmid, pS5-GFP, was created to deliver GFP. It constitutively expresses GFP with Plac-GFP, contains Pssej-LysE, and has SV40 NLS and c-Myc tags on GFP. Plasmid pS5-OMO was constructed by inserting Omo-C/S from pET-3a-Omo-C/S (from the Soucek lab) into pBAD-flhDC / Plac-GFP (Raman et al. 2019). All plasmids were constructed using Gibson Assembly, have a ColE1 origin, and contain either ampicillin or chloramphenicol resistance. The three plasmids (PS5-OVA, PS5-GFP and PS5-OMO) were transformed into Salmonella Enterica serovar Typhimurium with three deletions (ΔmsbB, ΔpurI and Δxyl; VNP20009) to create three strains of protein-delivering Salmonella (IND-OVA, IND-GFP, and IND-OMO). Two control bacterial strains (IND-OVA-ctr and IND-GFP-ctr) were constructed with two similar plasmids (pS5-OVA-ctr and pS5-GFP-ctr) that delivered ovalbumin and GFP, but did not contain NLS tags. All bacteria were grown at 37°C in LB supplemented with antibiotics.

Mammalian Cell Culture

Human MCF7 breast carcinoma cells (ATCC, Manassas, VA) were maintained in low glucose Dulbecco’s Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO) supplemented with 3.7 g/L sodium bicarbonate (pH 7.4) and 10% fetal bovine serum (HyClone, Logan, UT) at 37°C and 5% CO2. For bacterial co-culture experiments (invasion assays), cells were grown on coverslips and maintained in DMEM was buffered with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Fisher Scientific, Hampton, NH) at 37°C.

Intracellular Invasion

Nuclear delivery was quantified by co-culturing IND Salmonella with MCF7 cancer cells. We have previously shown that after invasion into cancer cells, Salmonella with the PsseJ-LysE construct lyse and deliver their protein cargo in the cellular cytoplasm (Raman et al. 2021). To quantify the nuclear localization of this cargo, Salmonella strains were grown in LB supplemented with carbenicillin to a density of 5 × 108 CFU/ml and added to MCF7 cells at a density of 5 × 106 CFU/ml. At the time of bacterial addition, the cells on six-well plates were about 30% confluent and the multiplicity of infection (MOI) was about 25. After incubating for two hours, cells were washed with phosphate buffered saline (PBS). DMEM with 20 mM HEPES and 40 ug/ml gentamycin (Fisher Scientific, Hampton, NH) was added to each well or plate to remove remaining extracellular bacteria. After 24 hours of co-culture, three different techniques were used to measure nuclear localization: (1) cells were fixed and stained to identify nuclei and delivered proteins, (2) nuclei were extracted from the cells and analyzed with flow cytometry, and (3) the cellular contents were analyzed by immunoblot. For most delivery experiments, an NLS-containing strain (IND-OVA and IND-GFP) was compared to the similar control strain without an NLS (IND-OVA-ctr and IND-GFP-ctr). Experiments with IND-OVA and IND-GFP were repeated two or three times.

Microscopy Analysis of Nuclear Delivery

To quantify nuclear delivery, the location of ovalbumin was determined in cancer cells after administration of IND-OVA or IND-OVA-ctr. Both Salmonella strains were administered to MCF7 cells for two hours, and removed with gentamycin. After 24 hours of co-culture, the media was removed, and the cells were fixed with 4% paraformaldehyde in DMEM for 10 minutes and permeabilized with 0.5% Triton X-100 in PBS. The cells on coverslips were stained to identify myc-tagged ovalbumin and Salmonella with rat anti-myc (1:200 dilution, ChromoTek, Planegg-Martinsried, Germany) and FITC rabbit anti-Salmonella (1:200 dilution, Abcam, Cambridge, UK). The anti-myc nanobody from ChromoTek was designed to bind the EQKLISEEDL motif in recombinant proteins and does not bind endogenous c-myc. A secondary antibody (Alexa Fluor 568 goat anti-rat; 1:200 dilution, Life Technologies, Carlsbad, CA) was used to identify the anti-myc primary antibody. Three percent (w/v) bovine serum albumin (BSA) was used as the blocking agent. Coverslips were mounted on glass microscope slides a DAPI-containing mountant (ProLong Diamond Antifade, Invitrogen, Waltham, MA). All samples were imaged on a Zeiss Axio Observer Z.1 microscope. Fixed cells on coverslips were imaged with a 100x oil immersion objective (1.4 NA). Fluorescence images were acquired with either 480/525 or 525/590 excitation/emission filters. Staining intensities of the cytosol and nucleus were obtained at three random points in each cell and averaged.

Cells with distinct nuclear localization were identified in OVA-stained images. In the first step of this process, the edges of nuclei in DAPI-stained images were marked and saved as regions of interest (ROI). In ovalbumin images, the suspected locations of nuclei were also marked. If the ovalbumin boundary lined up with the nuclear boundary from the DAPI image, the cell was scored as having a distinct nuclear boundary. This procedure was also used to identify distinct nuclei after delivery of GFP with IND-GFP and IND-GFP-ctr.

Nuclear Extraction and Flow Cytometry

Nuclei were extracted from MCF7 cells 24 hours after administration of IND-OVA or IND-OVA-ctr. After removing the culture medium, cells were rinsed three times with ice-cold PBS. Cells were removed from culture plates via mechanical disruption and were pelleted. Pellets were suspended in lysis buffer (10 mM potassium chloride, 10 mM HEPES, 1.5 mM magnesium chloride, 340 mM sucrose, 10% glycerol) supplemented with 0.1% Triton X-100 and protease inhibitor (Thermo Scientific, Waltham, MA). Cells were incubated on ice in lysis buffer and pelleted at 4°C. Pellets were washed in lysis buffer and suspended in fixing buffer (1 mM EDTA, 2% formaldehyde in PBS, without magnesium or calcium) and incubated for 10 minutes. Fixing buffer was removed via centrifugation, and pellets were suspended in blocking buffer for 30 minutes.

Whole cells were prepared by rinsing with ice-cold PBS twice. Cells were detached from culture wells using trypsin, which was subsequentially quenched with DMEM. Cells were pelleted and re-suspended in fixing buffer and incubated for ten minutes. Fixing buffer was removed by centrifugation and the pellet was suspended in blocking/extracellular staining buffer (1% bovine serum albumin [BSA], 1 mM EDTA in PBS) and incubated for 30 minutes.

After blocking, both whole cells and extracted nuclei were treated identically. Samples were centrifuged at 1300 x g for seven minutes, and the supernatant was discarded. Pellets were suspended in intracellular staining buffer (1% BSA, 0.1% Tween-20 in PBS) supplemented with antibodies and incubated for 30 minutes. Cells and extracted nuclei were stained with PE/Dazzle 594 anti-mouse CD326 (1:1000 dilution, BioLegend, San Diego, CA), Alexa Fluor 647 mouse anti-nuclear pore complex (1:500 dilution, BioLegend, San Diego, CA), Alexa Fluor 594 mouse anti-myc tag (1:500 dilution, BioLegend, San Diego, CA), and DAPI (1:1000 dilution, Invitrogen, Waltham, MA). Whole cells and extracted nuclei were rinsed three times with intracellular and extracellular staining buffer, respectively. Samples were suspended in extracellular staining buffer for analysis by flow cytometer (Acea NovoCyte, Agilent, Santa Clara, CA). A gate to distinguish ovalbumin-containing nuclei was set at 1% of nuclei from uninfected cells. These nuclei do not contain ovalbumin and were identically stained with anti-myc antibody.

Immunoblotting

Immunoblotting was used to analyze the cellular contents of cells after administration of IND-GFP or IND-GFP-ctr. After 24 hours of bacterial co-culture, nuclei were extracted from the MCF7 cells as described above. Supernatants and pellets were retained from each centrifugation step to analyze the GFP content in (1) whole cell lysates (WC), (2) cytoplasmic fractions (C), (3) soluble nuclear fractions (SN), and (4) insoluble nuclear fractions (IN). Laemmli SDS 6x sample buffer (Alfa Aesar, Haverhill, MA) was added to each sample. Protein samples were loaded onto 4% to 12% Bis-Tris gels (Invitrogen, Waltham, MA) and separated. The IND-GFP and control fractions were loaded equally. After separation, proteins were transferred to poly-vinylidene difluoride membranes (MilliporeSigma, Burlington, MA) and probed using antibodies against the myc tag. Blots were developed using horseradish peroxidase-conjugated goat anti-mouse IgG. Bands were detected by enhanced chemiluminescence and compared to a bacterial loading control. The ratio of delivered GFP in the soluble and insoluble nuclear fractions of cells administered IND-GFP and control Salmonella was determine by division of the protein amounts (e.g. ININD-GFP / INcontrol).

Delivery of Omo C/S

To measure viability after intranuclear delivery of Omo C/S, cancer cells were co-cultured with IND-OMO. Cells were cultured without bacteria and with IND-GFP as controls. The same bacterial invasion procedure was used as described above: Salmonella strains were added to MCF7 cells at a density of 5 × 106 CFU/ml. After incubating for two hours, cells were washed with PBS and gentamycin to remove extracellular bacteria. Images of the cells in these cultures were acquired 2 and 24 hours after adding the bacteria. For this live cell imaging, the culture medium was supplemented with 1 μg/mL ethidium homodimer (EthD-1; Invitrogen, Waltham, MA). Eighteen images were acquired for each condition. The total number of viable cells, which did not stain with ethidium homodimer, were counted for each image. These experiments with Omo C/S were repeated twice.

Statistical Analysis

Results are shown as means ± standard error of mean (SEM). Hypothesis testing was performed with Student’s t-test or analysis of variance (ANOVA) followed by Tukey’s method. Differences were considered statistically significant when P < 0.05.

Results

Creation of Nuclear Targeting Salmonella

We constructed a bacterial system to specifically deliver proteins in the nucleus of cancer cells (Figure 2). This system contains the PsseJ-LysE genetic circuit, which induces Salmonella to lyse after cell invasion and deposit proteins in the cellular cytoplasm (Raman et al. 2021). Three strains of intranuclear delivering (IND) Salmonella were created that constitutively express ovalbumin (IND-OVA), GFP (IND-GFP), or Omo C/S (IND-OMO). Both ovalbumin and GFP were tagged with the simian virus 40 (SV40) T-antigen NLS (Kalderon et al. 1984; Kosugi et al. 2009). An NLS was not added to Omo C/S because it contains an innate NLS that is similar to the SV40 NLS (Dang and Lee 1988). Control strains (IND-OVA-ctr and IND-GFP-ctr) were created without NL sequences to test the effect on nuclear localization.

Figure 2. Intranuclear delivery of three proteins with IND Salmonella was controlled by three sets of genetic circuits.

Figure 2.

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A) Ovalbumin delivery by IND-OVA Salmonella was controlled by plasmid pS5-OVA. On this plasmid, the Plac-OVA circuit constitutively expresses ovalbumin (OVA), which was tagged with the SV40 T-antigen nuclear localization sequence (NLS) and c-Myc for protein detection. All three plasmids (A-C) contain the PsseJ-LysE gene circuit, which autonomously induces bacterial lysis after cell invasion to deliver proteins in to the cytoplasm of cancer cells. Each plasmid also contains the ColE1 origin of replication and either chloramphenicol (Chlorr) or ampicillin (Ampr) resistance to promote plasmid retention. The delivered proteins (OVA, GFP and OMO) were all tagged with SV40 NLS and a detection tag. B) The plasmid for nuclear delivery of GFP contains Plac-GFP tagged with the SV40 NLS and the c-Myc tag. C) The plasmid for delivery of Omomyc (Omo C/S) contains Plac-OMO tagged with the SV40 NLS and the Flag tag. These plasmids were transformed into IND Salmonella to create IND-OVA, IND-GFP, and IND-OMO. Two additional control strains (IND-OVA-ctr and IND-GFP-ctr) were created by transformation with plasmids identical to pS5-OVA and pS5-GFP, but without NLS tags on OVA and GFP.

Delivery of Ovalbumin to the Nucleus

IND Salmonella delivered ovalbumin into the cytoplasm and nuclei of cancer cells (Figure 3). After administration to a monolayer of cells, IND-OVA and control (IND-OVA-ctr) Salmonella invaded into the cells and delivered ovalbumin into the cytoplasm (Figure 3A). Twenty-four hours after invasion, ovalbumin was present in both the cytoplasm and nuclei of all cells with lysed bacteria (Figure 3A). For many cells, the nuclei were brighter than the cytoplasm (Figure 3A). In this experiment, cell invasion was close to 100% and almost all cells contained bacteria. In a few cells, the invaded bacteria had not lysed by the time the cells were fixed and stained (Figure S1). In these cells, ovalbumin was not delivered to the cytoplasm or nucleus (Figure S1). For the majority of IND-OVA cells in this experiment (Figure 3A), the ovalbumin concentration was greater in the nucleus than the cytoplasm (Figure 3B). On average, the addition of an NLS to the delivered ovalbumin in IND-OVA significantly increased the ratio of the nuclear to the cytoplasmic ovalbumin concentration (1.522) compared to controls (1.267, P = 0.043, Figure 3C). For IND-OVA, the nuclear to cytoplasmic ratio was significantly greater than one (P = 0.038, Figure 3C).

Figure 3. Delivery of ovalbumin to the nucleus.

Figure 3.

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A) IND-OVA and control (IND-OVA-ctr) Salmonella delivered ovalbumin to MCF7 human breast cancer cells. Bacteria were administered to monolayers of cancer cells for two hours. After 24 hours, cells were fixed and stained to identify nuclei (DAPI) and delivered ovalbumin (with the c-Myc tag; shown here in magenta). In these images, the nuclear and cytoplasmic boundaries are shown by white and blue dotted lines, respectively. For both IND-OVA and control Salmonella, ovalbumin (magenta) was present in both the cytoplasm (black arrows) and nucleus (white arrows). The scale bar is 10 μm. B) For most cells in the experiment in (A), the concentration of ovalbumin was greater in the nucleus than the cytoplasm. For both conditions, the extent of delivery to the cytoplasm affected delivery to the nucleus. These intensity values were measured at three random points in the nucleus and cytoplasm of each cell and averaged. The slope of nuclear to cytoplasmic fluorescence intensity was positive (IND-OVA, 1.049; control, 0.621) and significantly greater than zero (P < 0.0001) for both conditions. The vertical gray line is three times the minimum detected fluorescence intensity and separates cells with low and high cytoplasmic concentrations. C) The inclusion of an NLS (in IND-OVA cells) increased the ratio of nuclear to cytoplasmic ovalbumin compared to controls (P = 0.043). For IND-OVA cells, this ratio was significantly greater than one (P = 0.038). D) In cells with high cytoplasmic delivery (> three times the minimum), the nuclear concentration was greater in IND-OVA cells compared to controls (P = 0.0037). E) Some cells with delivered ovalbumin had a distinct boundary between the nucleus and the cytoplasm, indicating clear nuclear accumulation. In image (i), two cells have distinct nuclear accumulation (black arrows). The boundary is not as distinct for the third (white arrow). A systematic image-analysis technique was developed to identify these cells. First, the nuclear (ii) and ovalbumin (iii) boundaries were identified in DAPI and OVA images. If the boundaries overlapped (iv), then the cell had a distinct nucleus (marked ‘D’, or ‘-’). F) Distinct nuclei were present in cells administered IND-OVA. After staining for ovalbumin-myc (top row) three cells had distinct nuclei (black arrows). Cells were stained with DAPI to identify the nuclear boundaries (middle row). The three cells with distinct nuclear boundaries in the ovalbumin image (top row) had overlapping boundaries in the overlay image (bottom row, marked ‘D’). The punctate signals in these images are intact bacteria that have not lysed or that have recently lysed. G) More cells administered IND-OVA (n = 249 cells) had distinct nuclear boundaries than control cells (n = 231; P = 0.0005). This percentage is out of all cells, which all contained intracellular bacteria. For the data in (B), slopes were determined by linear regression and compared with Student’s t tests. Data in (C), (D) and (G) are shown as means ± SEM, where statistical comparisons are two-tailed, unpaired Student’s t tests, and asterisks indicates significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

For both conditions, the amount of ovalbumin in the nucleus was dependent on the amount in the cytoplasm (Figure 3B). As the concentration of ovalbumin in the cytoplasm increased, the concentration in the nucleus also increased (P < 0.0001). Across all measured cells, nuclear intensities varied between one and six times the dimmest cytoplasmic intensity (Figure 3B). For IND-OVA cells (with an NLS), the nuclear concentration increased proportionally to the cytoplasm (slope = 1.049). For control cells (without an NLS), the nuclear concentration increased less than IND-OVA cells (P = 0.024) and was not proportional to the cytoplasm (slope = 0.621). This difference is illustrated by cells with low (< three times minimum) and high (> three times minimum) cytoplasmic concentrations. At low cytoplasmic intensities, the nuclear intensity was similar to the cytoplasm for both conditions (Figure 3B). At high cytoplasmic concentrations (> three times minimum), the amount in the nucleus was significantly higher for IND-OVA cells compared to control cells (P = 0.0037, Figure 3D).

In the cells with delivered ovalbumin, there was a sub-population that had a distinct boundary between the nucleus and the cytoplasm (Figure 3E). In these cells, the ovalbumin intensity was visibly greater in the nucleus than the cytoplasm (Figure 3E–i). This difference indicates preferential accumulation of the delivered protein in the nucleus. In Figure 3E–i, two cells have distinct nuclear accumulation (black arrows). For the third cell, the nuclear accumulation is not as sharp (white arrow). We developed a method to systematically identify cells with distinct nuclei that is based on identifying the outer edge of ovalbumin staining (Figure 3E–ii) and nuclear (DAPI) staining (Figure 3E-iii). In cells with distinct nuclei, the ovalbumin edge aligns with the nuclear boundary (Figure 3E–iv). Few distinct nuclei were present in cells invaded with control Salmonella (Figure 3F, left). However, in cells invaded with IND-OVA, several contained distinct nuclei (Figure 3F, right). Three times more IND-OVA cells contained distinct nuclei compared to controls (P = 0.0005, Figure 3G).

An NLS Increased the Number of Nuclei with Ovalbumin

The addition of an NLS increased the fraction of nuclei with delivered ovalbumin (Figure 4). To distinguish nuclear delivery from cellular delivery, nuclei were extracted from cells. This physical separation eliminated the possibly confounding fluorescence signal from cytoplasm above nuclei in the microscopy images. After extraction, nuclei were intact (Figure 4A,B), did not contain components of the cell membrane (Figure 4A,C), and retained their DNA content (Figure 4B,D). Unlike whole cells, extracted nuclei did not stain with Ep-Cam, a cell membrane stain (Figure 4A,C). The Ep-CAM intensity of extracted nuclei was 16-fold less than whole cells (P < 0.01; Figure 4C, right). The presence of DNA indicates that the nuclei were not damaged during extraction (Figure 4B). As expected, the DNA content of extracted nuclei was comparable to whole cells (Figure 4D).

Figure 4. IND-OVA increased the fraction of nuclei cells with ovalbumin.

Figure 4.

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A) Nuclei were extracted from MCF7 cancer cells by lysis and centrifugation. When stained with epithelial cell adhesion molecule (Ep-CAM), whole cells contained cell membranes, whereas extracted nuclei (arrow) did not. Scale bars are 10 μm. B) Both whole cells and extracted nuclei contained DNA and stained with DAPI. C) After Ep-CAM staining, whole cells had a shift in fluorescence intensity (left, red to green). Comparatively, extracted nuclei did not have a fluorescence shift (left, black to blue). Whole cells (n = 4) had significantly greater Ep-CAM staining than extracted nuclei (n = 5; P = 0.0012). D) After DAPI staining, extracted nuclei and whole cells had similar shifts in intensity, indicating that the nuclei contained DNA and were not damaged during extraction. E) After bacterial administration to individual cultures of cancer cells, 2.12% of control nuclei and 9.56% of IND-OVA nuclei contained ovalbumin. The gate to distinguish ovalbumin-containing nuclei was set at 1% of nuclei from uninfected cells, which do not contain ovalbumin. F) On average (n = 3), the inclusion of the NLS in IND-OVA significantly increased the fraction of nuclei with delivered ovalbumin compared to controls (P = 0.0425). Data are shown as means ± SEM. The statistical comparisons in (C) and (F) are two-tailed, unpaired Student’s t tests, where the asterisks indicate significance (*, P < 0.05; **, P < 0.01).

When IND-OVA and control (IND-OVA-ctr) Salmonella where administered to cells, nuclei from both conditions contained ovalbumin (Figure 4E). The addition of an NLS increased the percentage of nuclei that constrained ovalbumin more than two fold (P = 0.0425; Figure 4F). The percentages of positive cells (Figure 4F) was similar to the percentage of cells with distinct nuclei in microscopy images (Figure 3G). Both with and without a NLS, bacterially delivered ovalbumin localized to the nucleus. However, incorporating a NLS increased the percentage of cells with nuclear ovalbumin.

Nuclear Delivery of GFP

To determine the efficacy of delivering GFP, IND-GFP Salmonella were administered to MCF7 cancer cells (Figure 5). The same procedures were used as delivery of ovalbumin (Figures 3–4). Similar to ovalbumin delivery, GFP was delivered to both the cytoplasm and nucleus of most cells (Figure 5A, left). Over all observed cells, the percentage with distinct nuclei (Figure 3A, right) was three times greater after administration of IND-GFP than control Salmonella (P < 0.0001, Figure 5B). Extraction of nuclei showed that, on average, the 81% of delivered GFP was in the nucleus (Figure 5C). In cells administered IND-GFP, there was 2.15 times more GFP in the soluble nuclear fraction and 1.67 times more GFP insoluble nuclear fractions than controls. These measurements show that, after delivery with IND Salmonella, an NLS was not necessary for GFP accumulation in the nucleus. However, inclusion of an NLS increased the number of cells with distinct nuclei and the overall amount of nuclear GFP.

Figure 5. Bacterial delivery of GFP to the nucleus.

Figure 5.

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A) IND-GFP and control (IND-GFP-ctr) Salmonella were administered to MCF7 human breast cancer cells for two hours. After 24 hours, cells were stained to identify GFP-myc (anti-myc nanobody) and nuclei (DAPI). GFP (green) was present in both the cytoplasm and nucleus for both IND-GFP and control Salmonella. Alignment of GFP and DAPI images (right) identified cells with distinct nuclear boundaries (arrows, left GFP image). Scale bar is 20 μm. B) Cells administered IND-GFP (n = 417 cells) had more distinct nuclear boundaries than control cells (n = 393, P < 0.0001). C) Nuclei were extracted from cells after administration of IND-GFP or control bacteria. IND-GFP had 2.15 times more protein in the soluble nuclear fraction (SN) and 1.67 times more protein in the insoluble nuclear fraction (IN) than controls. Equal loading was maintained between each condition (IND-GFP and control). The isolation process had different dilutions for each fraction. Cytoplasmic fractions (C), whole cell (WC) lysates, and pure IND-GFP Salmonella (S) are shown for comparison. Data are shown as means ± SEM. The statistical comparison in (B) is a two-tailed, unpaired Student’s t tests, where the asterisk indicates significance (****, P < 0.0001).

Delivery of Omo C/S induced cell death

Intranuclear delivery of Omo C/S with IND Salmonella killed cancer cells (Figure 6). Two hours after administration of IND-OMO or IND-GFP there was no difference in morphology compared to bacteria-free controls (Figure 6A, left). By 24 hours after bacterial administration, the morphology of cells treated with IND-OMO was considerably different from IND-GFP cells and bacteria-free controls (Figure 6A, middle). This time point was chosen because most intracellular IND bacteria lyse by 24 hours. Compared to IND-GFP cells (Figure 6A, inset i), IND-OMO cells were more rounded and less adherent to the culture surface (Figure 6A, inset ii). The number of viable cells (Figure 6B) matched these morphological differences. At two hours, there was no differences in cell numbers between any of the conditions. By 24 hours after bacterial administration, IND-OMO significantly reduced the number of viable cells compared to IND-GFP (P = 0.038). There was no statistical difference in viability between cells administered lysing IND-GFP controls and bacteria-free controls.

Figure 6. Nuclear delivery of Omo C/S killed cancer cells.

Figure 6.

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A) To determine the effect of delivering Omo C/S, IND-OMO and control (IND-GFP) Salmonella were administered to MCF7 human breast cancer cells. These treatments were also compared to bacteria-free controls. Two hours after adding bacteria, there was no morphological difference between the cells. By 24 hours, the cells treated with IND-OMO were more rounded and less adherent (arrows, inset ii) than untreated or IND-GFP-treated cells (inset i). Scale bars are 50 μm. B) Two hours after adding bacteria, there was no statistical difference in the number of viable cells per image (n = 18 images per condition). Viable cells were identified as cells that did not stain with ethidium homodimer. By 24 hours, there were fewer cells in cultures treated with IND-OMO (P = 0.0380). Data are shown as means ± SEM. The statistical comparison in (B) is ANOVA followed by Tukey’s method, where the asterisk indicates significance (*, P < 0.05).

Discussion

In this work, we have shown that IND Salmonella delivers proteins to the nucleus of cancer cells. For two proteins (OVA and GFP), the addition of an NLS increased the amount of protein delivered to the nucleus (Figures 3&5), and the number of nuclei with delivered protein (Figure 4). An NLS increased the fraction of nuclei with delivered protein above a basal level, from 3% to 11% of cells (Figure 3G). When delivered by Salmonella, the nuclear-acting peptide Omo C/S killed cells (Figure 6), showing that IND Salmonella deliver sufficient amounts of protein to be effective. The altered cell morphology after treatment with IND-OMO (Figure 6A) suggests that the effect was mediated by inhibition of c-Myc, which regulates both cell size and morphology (Grandori et al. 2005).

The nuclear transport of bacterially delivered proteins was mediated by both passive and active mechanisms. Both ovalbumin and GFP entered the nucleus without localization sequences (Figures 3–5), suggesting that some protein is passively transported across the nuclear membrane. The increased localization after addition of an NLS (Figure 3G, 4G, and 5B), suggests that importin-mediated transport was active after intracellular delivery with IND bacteria.

Active transport played a greater role in nuclear transport when more protein was available in the cytoplasm (Figure 3). On average, the amount of protein in the nucleus was greater when there was more protein in the cytoplasm (Figure 3B). The amount in the cytoplasm most likely varied because of differences the number of invaded bacteria per cell and/or protein expression by individual bacteria. The difference between the nucleus and the cytoplasm was greater when the protein had an NLS tag (Figure 3B–D). This difference was not present at lower concentrations of cytoplasmic protein (Figure 3B), suggesting that a basal amount of protein is transported by passive diffusion and it is not affected by the presence of an NLS. These observations further suggest that higher cytoplasmic concentrations increase the likelihood that delivered proteins encounter importin transporter molecules.

The detection of active nuclear transport enhances our understanding of the mechanisms of intracellular bacterial delivery. Previously, we showed that proteins delivered by bacteria are first released into Salmonella-containing vacuoles (SCVs) and then released into the cytoplasm (Raman et al., 2021). The significant effect of including an NLS suggests that delivered proteins are freely available in the cytoplasm outside the nuclear membrane where they can interact with importin molecules. This localization further suggests that, with the appropriate tags, it could be possible to selectively direct proteins to many intracellular targets.

By comparing two proteins, ovalbumin and GFP, we found that molecular weight had little effect on nuclear delivery. Both relatively small GFP (27 kDa) and larger ovalbumin (45 kDa) were transported into the nucleus by passive and active mechanisms. Without localization sequences, both proteins accumulated in nuclei by passive diffusion. Similarly, the addition of localization sequences increased the transport of both proteins.

We have developed a bacterial method to deliver proteins to the nucleus of cancerous cells. This technique satisfies three requirements for an effective nuclear targeting therapy (Figure 1A); it penetrates the cell membrane, delivers proteins across the nuclear membrane, and is active against nuclear targets. By directly carrying proteins across the cell membrane, IND Salmonella overcome the transport limitations that prevent delivery of proteins into cells. Because cargo is only released after cell invasion, delivered proteins would be protected from degratdation in the blood. The biggest potential of this technology is that it could disrupt protein-protein interactions that have previously been difficult to treat. By specifically accumulating in tumors, IND Salmonella would focus the effect of nuclear delivery on cancer cells. Clinically, IND Salmonella could deliver protein therapeutics that have been tailored to interfere with nuclear pathways that are essential for cancer cell growth and survival. By targeting the nucleus, this delivery approach will expand the number of treatable cellular mechanisms and has the potential to improve overall cancer therapy.

Supplementary Material

supinfo

Acknowledgements

We would like to thank Anthony Brouillard and Ashish Kulkarni in the Department of Chemical Engineering at the University of Massachusetts, Amherst for aiding in flow cytometry sample preparation. We would also like to thank Dr. Laura Soucek at Vall d’Hebron Institute of Oncology for sending us pET-3a-Omo C/S.

Funding statement

We gratefully acknowledge financial support from the National Cancer Institute of the National Institutes of Health, grants R01CA188382, R43CA250941, and R43CA9622551; the National Science Foundation, grants 1819794 and 2035560; the Department of Defense, grant W81XWH1910602; and the Manning/IALS Innovation Award from UMass Amherst.

Footnotes

Conflict of interest disclosure

Shoshana Bloom and Nicholas O’Hare have no competing interests. Neil Forbes is a co-founder of Ernest Pharmaceuticals, Inc.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

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Supplementary Materials

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NIHMS1870137-supplement-supinfo.docx (518.7KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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