Bacterial particles retard tumor growth as a novel vascular disrupting agent

Biomedicine & Pharmacotherapy 122 (2020) 109757

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Bacterial particles retard tumor growth as a novel vascular disrupting agent Fengzhu Guo

a,b,1

, Gaili Ji

a,1

a,1

, Qiqi Li

a

a

a

T

a,

, Yun Yang , Lin Shui , Yuge Shen , Hanshuo Yang *

a

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biotherapy, Chengdu 610041, Sichuan, China Lung Cancer Center, West China Hospital of Sichuan University, Chengdu 610041, Sichuan, China

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A R T I C LE I N FO

A B S T R A C T

Keywords: Bacterial particle Vascular disrupting agent Tumor microenvironments Inflammatory cytokines

Due to hypoxia and poor circulation in the tumor interior, malignant cells in solid tumors are resistant to traditional therapies. In the present study, we reported that bacterial particles (BactPs) functioned effectively in retarding tumor growth as a novel vascular disrupting agent. The BactPs were inactivated intact bacteria. Intravenous administration of BactPs extensively disrupted vessels in the tumor interior, but not in normal organs, and resulted in tumor hemorrhage and necrosis in six hours. We revealed that the extensive disruption of tumor vasculature was due to drastic changes in the inflammatory factors in mice sera and the tumor microenvironments, indicating the critical role of the host immune response to the BactPs. Furthermore, we showed that a combination of six inflammatory cytokines was capable of inducing tumor hemorrhage and necrosis, similar to the effects of the BactPs. Together, these results suggest that BactPs are a novel kind of tumor vascular disruptor with a promising potential for solid tumor treatment.

1. Introduction Solid tumor therapy remains a big challenge. Substantial emerging strategies for antineoplastic management, including antiangiogenic agents, are still under active investigation. Tumor vasculature differs from normal vessels, both in morphology and biological behavior [1]. Only a fraction of the tumor vessels is responsible for transporting blood, leading to the heterogeneity of blood supply spatially and temporally and reduced blood perfusion, together with hypoxia, in most parts of the tumor entity [2,3]. Hypoxia is a predominant feature of solid tumors and induces complex intrinsic responses in tumor cells, conferring resistance to traditional antitumor treatment, such as chemotherapy and radiotherapy, and even targeted therapy [2–5]. Additionally, the failure of efficient drug delivery into hypoxic tumor cells further contributes to the ineffectiveness of systemically administered medication [2,4–6]. Therefore, malignant cells in hypoxic niches are the primary elements resistant to present tumor therapies. Angiogenesis-inhibiting agents (AIAs) hinder tumor progression by targeting tumor angiogenic endothelial cells. AIAs have proven to be effective in treating various kinds of tumors, such as non-small cell lung cancer, renal cell carcinoma, colorectal cancer, and hepatocellular carcinoma [7,8]. Clinical antiangiogenic approaches include monoclonal antibodies targeting vascular endothelial growth factor (VEGF,

bevacizumab) or vascular endothelial growth factor receptor 2 (VEGFR2, ramucirumab), VEGFR ligand traps (e.g., aflibercept) or multi-target tyrosine kinase inhibitors (e.g., sorafenib and sunitinib) [8]. AIAs have been used in combination with chemotherapy for more than ten years, leading to an increase in the overall survival of patients with types of tumors, but clinical benefits for cancer patients are limited, and even promote cancer invasion and metastasis [9,10]. A wealth of evidence has revealed the inherent limitations of AIAs to provide clinical benefits to tumor patients that cannot be overlooked [10–12]. Mechanistically, AIAs target newly growing tumor vasculatures, particularly functioning in the peritumoral regions where newly progressing tumors undergo robust tumor angiogenesis. Thus, AIAs, in theory, are more apt to stabilize than cause the regression of established tumors [13,14], suggesting the need for novel antitumor strategies targeting existing tumor vasculatures. Vascular disrupting agents (VDAs) represent an alternative vasculartargeted therapy of established solid tumors, which are distinct from conventional AIAs. [14,15]. Conventional VDAs include small molecules and ligand-directed VDAs [16]. The majority of small-molecule VADs are tubulin-binding agents, such as combretastatin A4-phosphate (CA4P), BNC105P, and ombrabulin [17]. Ligand-directed or biological VDAs deliver toxins, procoagulants, or proapoptotic effectors to tumorassociated blood vessels by antibodies, peptides or growth factors [18].

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Corresponding author. E-mail address: [email protected] (H. Yang). 1 These authors have contributed equally and share first authorship. https://doi.org/10.1016/j.biopha.2019.109757 Received 15 September 2019; Received in revised form 7 November 2019; Accepted 29 November 2019 0753-3322/ © 2019 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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eight-week-old female BALB/c or C57BL/6N mice were injected with 1 × 106 CT26 colon cancer cells or LL2 Lewis lung cancer cells to establish subcutaneous tumors on the right lower flanks. Tumor volume (V, mm3) was determined by measuring the length (L, mm) and width (W, mm) using electronic calipers every two days and calculated as V = 0.52 × L × W2 to monitor the dynamic development of tumors. To explore the antitumor capability of the BactPs, mice with tumors (diameter = 8−10 mm) were treated intravenously with 2.5 × 108 BactPs or normal saline (NS) as a control.

Currently, VDA nanodrugs have been identified as another subtype. Numerous VDA nanoparticles target the tumor vascular system more specifically, including combretastatin A4 nanodrugs, poly(L-glutamic acid)-combretastatin A4 conjugates, and PEGylated poly(alpha-lipoic acid) graft combretastatin A4 nanoparticles with glutathione stimulus responsive ability [19–24]. The synergistic regimens of nanomedicine and various antineoplastic agents, such as doxorubicin, sorafenib, and tumor-associated enzyme-activated prodrugs, also exhibit superior antitumor efficacy and alleviate systemic toxicity via different mechanisms [19–22,25]. VDAs do not only act on tumor neovascularization, but rapidly and selectively destroy the established tumor vascular network and irreversibly cause a pronounced shutdown of blood flow to tumor tissues. Therefore, VDAs result in tumor vascular collapse, massive tumor necrosis due to anoxia, and subsequent tumor regression [6,26]. Importantly, these activities take place in the tumor interior where the tumor microenvironment is hypoxic, immature vessels are abundant, and tumor cells are resistant to traditional chemotherapy or radiation [17,27]. Thus, VDAs offer an attractive opportunity to conquer the resistance of hypoxic tumor cells to conventional therapies [28]. The fundamental distinction between neoplastic vasculature and that of non-malignant tissues enables these vascular-directed agents to specifically decrease circulation to the tumor with little effect on healthy tissues [27]. Moreover, studies also have shown enhanced efficacy and less toxicity when VADs are used in combination with chemotherapy or AIAs [29,30]. VDAs have been regarded as a promising strategy for solid tumor therapy [31]. Here, we report that fixed bacteria (bacterial particles, BactPs) are a novel and effective VDA that disrupts established tumor vascular vessels and produces extensive cell death in the central areas of tumors by triggering a robust innate response to the tumors.

2.5. Detection of hemorrhage in the tumors To determine the hemoglobin content in the tumors, the mice with subcutaneous tumors were intravenously administered NS or different doses of BactPs. Six hours later, the tumors were dissected out and homogenized in 1 mL ice-cold, sterile PBS following the mice were sacrificed. To lyse the erythrocytes, 500 μL of Ammonium-ChloridePotassium (ACK) lysing buffer (Quality Biological) was added to the homogenate and incubated for 10 min at room temperature. After centrifuging at 13,000 rpm (16,200 g) for 10 min, 300 μL of supernatant was mixed with 1 mL of Drabkin’s reagent (Sigma-Aldrich, St. Louis, MO, USA). Then the samples were incubated for 30 min and the optical density was measured at 540 nm. 2.6. Evaluation of the hemorrhage effect of conditioned sera To test the effect of conditioned sera on bleeding in the CT26 tumors, we designed the following two groups of experiments as diagramed in Fig. 5A. In the first group, we collected sera from tumorbearing mice (donors) treated with or without BactPs, then intratumorally injected these two conditioned sera into other tumorbearing mice (recipients) with the same baseline. In the second group, the protocol only differed in conditioned sera that were harvested from tumor-free mice (donors). Six hours later, mice were sacrificed, and tumors were removed followed by gross photography and histological examination.

2. Materials and methods 2.1. Generation of the BactPs Escherichia coli (E. coli, TOP10), E. coli (TOP10) expressing mCherry, the double attenuated (aroA− and dam-) Salmonella typhimurium (S. typhimurium) strain RE88, and Streptococcus thermophilus (S. thermophilus) were used to prepare the BactPs. Briefly, 5 × 108 bacteria (100 μL) were mixed with 1 mL paraformaldehyde (PFA, final concentration, 4 %) at room temperature. One hour later, the bacteria were washed with phosphate-buffered saline (PBS) three times. After filtering with 800 mesh strainers to remove the big particles, BactPs were suspended with PBS (pH 7.4) for use. To examine the BactPs closer, samples were prepared using the standard protocol for transmission electron microscopy (TEM; HITACHI H-600, Japan).

2.7. Detection of inflammatory cytokines To evaluate the BactP-induced changes in inflammatory factors/ cytokines in mice serum and tumor, blood and tumors (10 mice per group) were collected and prepared as suggested by the manufacturer. Non-cross-reacting murine multiplex cytokine kits (MILLIPLEX MAP Mouse Cytokine/Chemokine-Pre-mixed 32, Plex catalog no. MPXMCYTO-70K-PMX32, Millipore Corporation, Billerica, MA, USA) were used for this assay. The concentration of each cytokine was quantified using the Luminex 100 (Luminex Corporation, Austin, TX, USA).

2.2. Measurements of granulocytes and lymphocytes

2.8. Evaluation of the hemorrhage effect of inflammatory cytokines

Blood samples from mice were drawn to sodium heparin-containing tubes at different time points following the administration of BactPs. Granulocyte and lymphocyte counts were determined using a Coulter LH 780 Hematology Analyzer (Beckman Coulter Inc., Brea, CA, USA).

Mice with tumors (diameter = 8 ± 2 mm) were randomly assigned into four groups and received intravenously different combinations of cytokines. The four groups of mice were administrated with NS, the combination “TKMGIP10IL6” composed of six inflammatory cytokines (0.05 μg tumor necrosis factor alpha (TNFα), 2.5 μg keratinocyte-derived chemokine (KC, mouse C-X-C motif chemokine ligand 1, mCXCL1), 2.5 μg monocyte chemoattractant protein 1 (MCP-1, C-C motif chemokine ligand 2, CCL2), 0.4 μg granulocyte-colony-stimulating factor (G-CSF), 0.5 μg interferon-inducible protein 10 (IP-10, C-XC motif chemokine ligand 10, CXCL10), 0.5 μg interleukin 6 (IL-6)), the combination “KMGIP10IL6” composed of five inflammatory cytokines (2.5 μg KC, 2.5 μg MCP-1, 0.4 μg G-CSF, 0.5 μg IP-10, 0.5 μg IL-6), or TNFα alone (0.05 μg). Six hours later, the homografts were removed and dissected from the sacrificed mice followed by the subsequent histological along with quantitative analysis.

2.3. Safety testing of the BactPs Two groups of six- to eight-week-old female mice were intravenously injected with 1 × 109 BactPs and live bacteria, respectively. Then, the survival status was observed and recorded. 2.4. Tumor growth in mice All animal experiments were performed according to the guidelines of the Animal Care and Use Committee of Sichuan University and approved by the Institutional Review Board of the Medical Faculty at West China Hospital, Sichuan University. After an acclimation period, six- to 2

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Fig. 1. Characteristics of the BactPs. (A) Representative image of BactPs. Scale bar, 2 μm. (B) The average size of the BactPs. (C, D) The proportion of granulocytes and lymphocytes determined at different time points after the intravenous administration of BactPs to mice. (E) Kaplan-Meier survival curve of mice receiving BactPs or live bacteria. *p < 0.05, **p < 0.01.

overnight at 4 ℃. Goat anti-rat IgG/TRITC was used to visualize the primary antibody reactions. The nuclei were counterstained with 4’-6diamidino-2-phenylindole (DAPI, Sigma). The fluorescent staining was analyzed using a Leica TCS SP5 II Confocal Microscope (Leica Microsystems, Germany).

2.9. Histology analysis Paraffin sections from each group were stained with hematoxylineosin (H&E) in strict accordance with the standard protocol. Hypoxia in the tumors was determined using a Hypoxyprobe-1 kit according to the manufacturer’s guidelines. For apoptosis detection using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, dewaxed and rehydrated paraffin tumor tissue sections were incubated with proteinase K for 15 min at 37 ℃, rinsed twice with PBS, and the TUNEL Assay In Situ Cell Death Detection Kit was used to assess the apoptotic cells according to the manufacturer’s instructions. Positive TUNEL staining was visualized by confocal microscopy and the apoptotic index was calculated as the ratio of the apoptotic cell numbers to the total tumor cell numbers in each microscopic field.

2.12. Tube formation assay The human umbilical vein endothelial cell (HUVEC) tube formation assay was performed as described previously [32]. A 50 μL aliquot of Matrigel (10 mg/mL, BD Biosciences) was applied to a chilled 96-well plate, followed by polymerization at 37 ℃ for 30 min. After coating, the HUVECs were seeded onto the solidified Matrigel at a density of 2 × 104 cells/well with conditioned medium containing or without the six cytokines (scheme 1: 123 ng/mL IL-6, 53 ng/mL IP-10, 1.8 ng/mL TNFα, 37.5 ng/mL MCP-1, 743 ng/mL G-CSF, and 368 ng/mL IL-8; scheme 2: 61.5 ng/mL IL-6, 26.5 ng/mL IP-10, 0.9 ng/mL TNFα, 19 ng/ mL MCP-1, 372 ng/mL G-CSF, 184 ng/mL IL-8). Subsequently, the plates were incubated at 37 ℃ in a 5 % CO2 incubator. Tube formation was analyzed in the first 12 and 24 h by observing changes in the cellular morphology and the tubular structure using an inverted microscope (Zeiss, Germany), together with the quantitative measurement of total mesh area, total tube length, and master junctions by Image J software (NIH, Bethesda, MD, USA).

2.10. Assessment of tissue vasculature To visualize functional vessels in the tumor and organs, the mice were injected intraperitoneally with a lethal dose of anesthesia comprised of 1 mL ketamine (100 mg/mL; Ketaset III, Fort Dodge, IA, USA) plus 0.1 mL xylazine (100 mg/mL; Lloyd Laboratories, Shenandoah, IA, USA) in 8.9 mL PBS. The thoracic cavity was opened to expose the heart before a vacutainer blood collection needle was inserted into the left ventricle and the right atrium was clipped with Vannas scissors. NS was delivered into the blood flow via the heart, followed by PFA mounting and Fluorescein isothiocyanate (FITC)-dextran dye (Sigma, 100 mg, 2 million WM) to label the blood vasculatures. Tumors and normal organs were extracted and observed under a stereomicroscope. Frozen sections of the tissues were cut with a microtome-cryostat and imaged using confocal microscopy.

2.13. Statistical analyses The data are presented as means ± SD. Differences between the two groups were analyzed by unpaired student’s t-tests and comparisons among the groups were determined by one-way ANOVA using GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA). A level of P < 0.05 was regarded as statistically significant.

2.11. Immunofluorescent staining assay

3. Results

Tumors were removed from the sacrificed mice and snap-frozen in Tissue-Tek OCT Compound (Sakura Finetek). The frozen sections (5–6 μm) were air-dried at room temperature overnight and fixed in acetone at 4 ℃ for 20 min. The slides were rehydrated in PBS, blocked with 5 % BSA for 30 min, and incubated with rat anti-mouse CD31 monoclonal antibody or PierceTM PEPTIDOGLYCAN antibody

3.1. Characteristics of the BactPs We prepared BactPs by fixing intact bacteria with 4 % PFA. The BactPs had typical bacillus morphology with an average size of 3

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from either gram-negative (E. coli and attenuated S. typhimurium) or gram-positive (S. thermophilus) bacteria both induced overt tumor hemorrhage and necrosis (Fig. 2E).

approximately 1300 nm in length (Fig. 1A, B). The systemic administration of BactPs to mice produced a transient infection-like hemogram. Granulocytes started to increase three hours after injection accompanied by a relative decrease in the proportion of lymphocytes (Fig. 1C, D), suggesting the activation of innate immunity against the BactPs. At 72 h post-BactP treatment, both granulocytes and lymphocytes recovered to normal (Fig. 1C, D), indicating the complete clearance of BactPs from the mice. The mice survived the intravenous injection of 1 × 109 BactPs for more than 100 days, whereas all control mice treated with the same dose of live bacteria died within 25 days (Fig. 1E). The biochemical analysis of hepatic and renal function also showed that these major organs were not damaged (Fig. S1). Body weight of the mice slightly decreased within two days after BactP treatment due to a slight increase in body temperature and decrease in appetite induced by BactPs but both recovered in six days (Fig. S2) when the BactPs were completely cleared (Fig. 1C, D).

3.3. Antitumor effects of BactPs To determine the antitumor efficacy of BactPs, we constructed two different tumor-bearing mouse models using CT26 colon cancer cells in BalB/c mice and LL2 lung cancer cells in C57BL/6 mice. Meanwhile, we prepared three types of BactPs made of E. coli, attenuated S. typhimurium, and S. thermophilus, respectively. Subsequently, the volume of the subcutaneous tumors that developed were monitored for at least six days but no more than 12 days immediately after the injection of BactPs through the tail vein, since after 12 days, the tumor cells remaining around the tumor necrosis area grew and proliferated, causing the tumor to “re-grow”. At this time, the volume calculated by measuring the tumor diameter would not represent the true size of the tumor. BactP (E. coli) produced marked suppressing effects on both the CT26 and LL2 transplanted tumors (Fig. 3A, B). Furthermore, BactPs derived from attenuated S. typhimurium, and S. thermophilus also inhibited CT26 tumor growth (Fig. 3A, C, D). Together, these results demonstrated the general anti-tumor effect of BactPs derived from different bacterial strains on solid tumors in different strains of mice.

3.2. BactPs led to hemorrhage in the tumors Next, we investigated the effect of BactPs on established solid tumors in mice. The subcutaneous tumors (CT26 colon cancer in BalB/C mice, diameter = 8−10 mm) became dark red three hours post-injection (hpi) of BactPs (2.5 × 108/mouse) through the tail vein and the color change was more evident at 6 hpi, indicating hemorrhage in the tumors (Fig. 2A). By 24 h, the tumors were scabbed and had shrunk dramatically due to extensive cell death in the tumor interior (Fig. 2A). BactPs also produced similar effects in spontaneous breast cancer in MMTVneu transgenic mice (Fig. 2B). The severity of the tumor hemorrhage was positively correlated with the quantity of injected BactPs (Fig. 2C, D) but was not related to the bacterial species. BactPs made

3.4. Disruption of tumor vasculature induced by BactPs Tumor hemorrhage and widespread cell death in the central tumor area are manifestations of the vascular shutdown caused by VDAs [17,33]. Therefore, we examined the ability of BactPs to disrupt tumor vessels. We first investigated functional tumor vessels by intravenously

Fig. 2. BactPs led to hemorrhage in the tumors. (A, B) Representative gross images and histological images (H&E) of tumor hemorrhage and cell death in CT26 tumors (A) and spontaneous breast cancer (B) at different time points after intravenous injection of BactPs (gram-negative bacteria). (C, D) Representative images and quantitative analysis of the hemoglobin content in the tumors of mice that received different doses of BactPs. (E) Representative gross and histological images of tumor hemorrhage and necrosis in CT26 tumors at different times post-BactP (gram-positive bacteria) administration. **p < 0.01, ***p < 0.001. 4

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Fig. 3. Antitumor effects of BactPs. Tumor growth curve of mice bearing CT26 colon cancer or LL2 Lewis lung cancer after injection with BactPs derived from different kinds of bacteria, including E.coli (A, B), Streptococcus thermophiles (C), and attenuated Salmonella typhimurium (D). *p < 0.05.

from the secretion of chemokines and the recruitment of inflammatory cells by malignant cells [35]. Moreover, VDAs have been described to damage or distort vascular endothelial cells through the activation of tumor-associated macrophages and dendritic cells and generation of cytotoxic T lymphocytes [36,37]. Thereby, we speculated that BactPtriggered changes in the host immune responses might result in the disruption of tumor vasculatures. To test this hypothesis, first, we collected sera from tumor-bearing mice treated with or without BactPs, then intratumorally injected these two conditioned sera into other tumor-bearing mice with the same baseline (Fig. 5A). In the BactPtreated group, examination of the gross images and histological sections revealed apparent hemorrhage and necrosis in the tumors (Fig. 5B, C) similar to the effects of BactPs on the tumors (Fig. 2). Next, to discriminate whether the tumor-burden participated in the conditioned sera-induced tumor hemorrhage and necrosis, we tested conditioned sera harvested from tumor-free mice administered BactPs or no BactPs and observed similar hemorrhage and necrosis in the tumors (Fig. 5D, E). Together, these results suggested that the host immune response, unrelated to the existence of tumors, played a pivotal role in the BactP-induced tumor vascular damage.

administering high molecular weight FITC-dextran (two million daltons). Observation of whole tumor frozen sections using fluorescent stereoscopic microscopy showed that the majority of the BactP-treated tumors were dark, demonstrating the severe lack of blood circulation but only a small decrease in blood perfusion was noted in the control tumors (Fig. 4A, B). Confocal microscopy imaging further validated that there were fewer functional vasculatures in the BactP-treated tumors than in the control group (Fig. 4A, B). Given that both functional and nonfunctional vessels coexisted in the tumor, we examined all tumor vessels by staining with CD31. The results also demonstrated that the vasculatures in the BactP-treated tumors were substantially reduced compared to the control (Fig. 4C, D). Consequently, cell hypoxia (Fig. 4E, F) and apoptosis (Fig. 4G, H) were markedly increased in the tumors. Importantly, BactPs did not affect the vascular system in normal tissues (Fig. S3) and no observable pathological changes were seen in several vital organs, such as the heart, liver, spleen, lung, and kidneys (Fig. S4). 3.5. Conditioned sera resulted in hemorrhage in the tumors Abundant but abnormal vasculatures are a hallmark of malignant tumor-induced angiogenesis, attributing to the imbalance of angiogenic stimulators and inhibitors present in the tumor environment, especially the significant increase in angiogenic stimulators [34]. The tumor niche in which vascularization occurs is suffused with inflammatory activity

3.6. Alternations of inflammatory factors in the sera and the tumors BactPs induced drastic immune responses in the mice (Fig. 1). To probe alternations of BactP-triggered immune responses to the tumor 5

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Fig. 4. Disruption of tumor vasculature induced by BactPs. (A, B) Stereoscopic and confocal microscopic images and quantitative analysis of the functional vessels in tumors after the intravenous injection of FITC-dextran (two million daltons). (C, D) Representative fluorescence microscopy images and quantitative analysis of CD31 staining in tumors representing the microvessel density. (E, F) Determination of tumor hypoxia and quantitative analysis using Hypoxyprobe staining. (G, H) Representative images and quantitative analysis of apoptosis in tumors by TUNEL staining. *p < 0.05.

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Fig. 5. Conditioned sera resulted in hemorrhage in the tumors. (A) The schema for evaluating the effect of host immune responses and tumor-burden on the disruption of tumor vasculatures. (B–E) Representative gross and histological (H&E) images of hemorrhagic necrosis (B, D) and necrosis severity analysis represented by necrosis-total area ratios (C, E) in the tumors from tumor-bearing mice injected intratumorally with conditioned sera. **p < 0.01.

to disorder angiogenesis. In the assays treated with a combination of cytokines, the cells were arranged together in a fragmented tube-like pattern in a dose- and time-dependent manner (Fig. 7C–F). In contrast, complete tube structures were identified more frequently in the absence of cytokines (Fig. 7C–F). The incomplete tube pattern in the treated cells demonstrated that the cytokine combination disturbed tube formation, which might produce bleeding in the tumors.

microenvironments, we quantitatively examined the concentration of inflammatory cytokines in mice sera and tumor tissues. Cytokines were detected three hours after injection with BactPs when tumor hemorrhage was very obvious. A panel of 18 cytokines was assayed, of which KC, MCP-1, monokine induced by gamma interferon (MIG), G-CSF, IL-6, and IP-10 displayed the most substantial rise in mice sera (Fig. 6A). Interleukin 10 (IL-10), macrophage colony-stimulating factor (M-CSF), and TNFα were moderately, but significantly, increased (Fig. 6A). In tumor tissues, KC, G-CSF, and IL-6 were increased the most (> 30 folds), followed by IL-1α, IL-1β, and TNFα (Fig. 6B). The inflammatory cytokines that were increased synchronously in both the sera and the tumor issues and the increasing amplitude of which ranked high were responsible for bleeding. We identified six core inflammatory cytokines (TNFα, KC, MCP-1, G-CSF, IP-10, and IL-6) that met these criteria.

4. Discussion Therapy for established bulky tumors remains challenging. In this study, we found that the systemic administration of BactPs triggered a powerful but transient innate immune response by phagocytosis and a subsequent release of inflammatory cytokines to perturb vascular endothelial cells, resulting in the disruption of vessels and extensive cell death in the tumor microenvironment, which further attenuated the development of existing tumors in mice. Additionally, we validated that a combination of six inflammatory cytokines caused the tumor vasculature shutdown. Bacteria are potent stimulators of the innate immune response. Phagocytes, such as neutrophils, monocytes, and macrophages, engulf bacteria depending on the interaction between the pattern recognition receptor (PRR) of immune cells and the pathogen associated molecular pattern (PAMP) of bacteria, the latter of which is expressed as part of the bacterial cell wall, in the absence of the host [38]. During the process of producing the BactPs, the fixation treatment kept antigens (PAMP) involved in stimulating phagocytes on the surface of the BactPs, ensuring the potential to activate the immune system. BactPs derived from different strains all exerted significant antitumor activity and this effect applied to various tumors, indicating broad efficacy. Moreover, BactPs, as inactivated bacteria, did not confer an increased

3.7. A combination of inflammatory cytokines disrupted vessels Tumor vascular system highly depends on tumor microenvironments. To test whether the cytokines directly led to hemorrhage in the CT26 tumors, six cytokines were intravenously injected into tumorbearing mice. Indeed, six cytokines were sufficient to induce an influx of blood from the tumor vessels into the stroma. H&E-stained paraffin sections confirmed the presence of a plethora of erythrocytes within the tumors 6 hpi (Fig. 7A). However, the amount of bleeding decreased in the absence of TNFα. Strangely, injection of TNFα alone at the same dose failed to cause an influx of blood into the tumors (Fig. 7B). This suggested that the anti-tumor effect triggered by BactPs relied on a combination of miscellaneous cytokines, instead of just one single cytokine. Subsequently, we carried out tube formation assays using HUVECs to further estimate the integrated effect of six factors mentioned above 7

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symptoms resulting from BactP injection that recovered approximately 72 h later did not deteriorate into uncontrollable consequences. BactPs are a new type of VDA with multiple similarities to previous VDAs. For instance, BactPs induce massive cell death in the tumor mass interior, providing a promising option for overcoming the resistance of hypoxic cells to conventional therapies. Furthermore, differences between malignant and normal vasculatures led to the selective disruption of established tumor vessels by BactPs but not healthy tissues, offering the opportunity to inhibit locally advanced tumors in a precise manner. More than 100 years ago, William Coley found that acute bacterial infections inhibited tumor growth. Some tumors showed complete regression, some cancers shrank, and patients lived longer than expected after bacterial treatment with Coley’s toxin [4,39]. Today, bacillus Calmette–Guérin (BCG) treatment has achieved responses in greater than 60 % of the bladder cancer patients [40,41]. The intratumoral injection of Clostridium novyi-NT spores induced tumor-suppressing responses in dogs and human patients [42]. Bacteria engineered with a cytotoxic gene or with a synthetic genetic circuit also have shown impressive effects in animals [43,44]. However, the use of live bacteria for anticancer therapy is associated with a high risk of infection and toxicity, especially for immunocompromised patients and those with latestage malignant tumors. In contrast, BactPs have an excellent safety profile without any risk of causing unexpected infections. Compared to previous bacterial therapeutics that utilized live bacteria and required bacterial propagation in the tumor, a unique merit of BactPs is their tumor-suppressing effect that does not require bacterial reproduction. Moreover, clinical trials have revealed that live bacteria do not propagate in human tumors as expected [45]. Furthermore, the preparation of BactPs is relatively convenient, time-saving and cost-effective. Thus, BactPs represent a novel VDA for the treatment of established solid tumors. There are still unanswered questions about the mechanisms involved, including the downstream signaling pathways activated in vascular endothelial cells by a combination of six inflammatory cytokines. According to previous studies, the effect of these inflammatory factors on vascular endothelial cells during angiogenesis is inconsistent.

Fig. 6. Alternations of inflammatory factors in the sera and the tumors. (A, B) Changes in inflammatory cytokines profiles in sera (A) and tumors (B) of mice three hours after tail vein injection of BactPs.

risk of severe infection, which has been confirmed in animal studies. BactPs work without amplification, making it almost impossible to predispose the host to infections. The temporary infection-like

Fig. 7. A combination of inflammatory cytokines disrupted vessels. (A, B) Representative H&E stains and statistical analysis of hemorrhage in CT26 tumors injected intravenously with different inflammatory cytokines. (C) Representative images of the HUVEC tube formation assay captured by light microscopy after an incubation period of 12 h (top) or 24 h (bottom) with or without six cytokines (IL-6/IP-10/TNFα/MCP-1/G-CSF/IL-8). Bar scale, 100 μm. (D–F) Quantitative analysis of the degree of tube formation measured as total mesh area, total tube length, and master junctions. *p < 0.05, **p < 0.01, ***p < 0.001. 8

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Appendix A. Supplementary data

For instance, TNFα has been proposed as a dual-role angiogenic factor, stimulating angiogenesis at a low dose (0.01−1 ng), with a maximum effect at 0.1 ng, but causing the opposite effects at high levels (1, 5 μg) [46]. 5,6-Dimethylxanthenone-4-acetic acid (also known as DMXAA), a VDA of flavonoids, induce the localized release of TNFα by tumor-associated macrophages, contributing to hemorrhagic necrosis and tumoricidal effects [36]. IP-10/CXCL10 is a competent angiogenic inhibitor [47]. Previous work reported that a broad spectrum of mechanisms mediated anti-vessel activity, including binding to the C-XC motif chemokine receptor 3, interplay with the heparan sulfate site on the cell surface shared with platelet factor 4, and disruption of the proangiogenic effect of basic fibroblast growth factor and interleukin 8 (IL8) [48,49]. In contrast, IL-6, G-CSF, KC/mCXCL1, and MCP-1/CCL2 were reported to promote angiogenesis [50–53]. Feurino et al. showed that IL6 upregulated VEGF165 and neuropilin 1 (NRP-1) expression in pancreatic cancer cells [54]. VEGF produced by tumors is the most predominant secreted pro-angiogenic factor and NRP-1 serves as the receptor for VEGF [55]. A prior study dissecting ovarian cancer demonstrated that the neutralizing anti-IL-6 antibody siltuximab led to a decline in plasma concentrations of CCL2, CXCL12, and VEGF, accompanied by a reduction in angiogenesis [51]. G-CFS is considered a pro-oncogenic cytokine that is overexpressed in several tumors, facilitating tumor vascularization and metastasis via a diverse array of molecular events, such as the mobilization of granulocytes and endothelial precursor cells [52]. KC/mCXCL1 is the mouse functional ortholog of human IL-8 [56]. IL-8 has been described as a pro-tumor factor, increasing endothelial cell angiogenesis. However, another study of ovarian cancer suggested that IL-8 inhibited tumorigenicity by increasing the number of neutrophils at the tumor lesion [56]. Like VEGF, MCP-1/CCL2 is also involved in macrophage-mediated angiogenesis [53]. As discussed above, the angiogenic activity of some inflammatory cytokines may be paradoxically separate, but the combination of these cytokines has substantial effects on the rupture of vessels and cessation of blood flow. In summary, our findings demonstrated that a novel and efficient VDA, BactPs, prevented tumor growth by extensive abrogation of tumor vessels and the subsequent hemorrhagic necrosis in the tumor interior. Furthermore, this study demonstrated a combinatory effect of six inflammatory cytokines regulated by the host immune response that led to the disruption of tumor vasculatures. BactPs preserve the benefits of VDAs and compensate for the shortcomings of previous antitumor therapy based on live bacteria. The obtained principal data on the efficacy and safety lay the foundation for the development of BactPs as an option for cancer treatment, especially for bulky tumors.

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Authors’ contribution HSY conceived and designed the study. All authors performed the experiments and conducted the data analysis. FZG, GLJ, and QQL drafted the text and conceived of and generated the figures. HSY was a significant contributor to the manuscript revision regarding the important intellectual content. All authors approved the final manuscript and take responsibility for publishing this original article.

Declaration of Competing Interest The authors declare that there are no conflicts of interest.

Acknowledgement This work was supported by the National Natural Science Foundation of China [grant numbers 81272216, 81171956, 81572402]. 9

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