Patent Application
20230123148
A computer-readable form (CRF) sequence listing having file name GHE0049PA-SEQlisting.txt (3548 bytes), created Sep. 26, 2022, is incorporated herein by reference. The nucleic acid sequences and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations as defined in 37 C.F.R. § 1.822.
The present disclosure relates to quorum-sensing peptides as diagnostic biomarkers, and to quorum-sensing peptide inhibiting substances for use in the treatment and/or prevention of metastasis of colorectal cancer in a subject; in particular a human subject.
Growing evidence obtained in the last decade suggests that the gut microbiota influences the health of the host. For example, by comparing faeces from healthy persons and patients, higher abundances of Enterococcus, Escherichia and Fusobacterium species were observed in multiple intestinal disorders, including colorectal cancer (CRC) and Crohn's disease. However, the causative factors for diseases development or progression are not well understood and current research is mainly limited to bacterial-derived short-chain fatty acids and amino-acid derived amines.
Quorum sensing peptides are traditionally regarded as intra- and inter-bacterial communication molecules, but given their wide structural variety and co-evolution, we anticipate that these molecules may also interact with the host. Up till now, however, quorum sensing peptides have not yet been unambiguously demonstrated to be present in biofluids. Only an indirect indication of the in vivo presence of an unidentified quorum sensing peptide was described in the stool of patients suffering from Clostridium difficile infection. Previously, the researchers of the present disclosure focused on Enterococcus faecium, one of the most abundant species in the human intestinal microbiota, which synthesizes the enterocin induction factor, i.e. the propeptide of the EntF quorum sensing peptide (AGTKPQGKPASNLVECVFSLFKKCN; SEQ ID NO:2). This peptide serves as a communication signal, regulating the production of enterocin A and B toxins, which are produced to inhibit the growth of similar or closely related bacterial strains. Standard protein BLAST searches thereby indicate that EntF-producing E. faecium strains indeed are present in different human faeces samples (Table 1). On the other hand, our experimental data indicated that not all E. faecium strains have this EntF gene in their genome (Table 2), indicative for the inter-individual variation in the (gut) microbial community.
Previously, it was demonstrated that metabolization of EntF in faeces and colonic tissue homogenate yields a 15-mer peptide EntF* (SNLVECVFSLFKKCN; SEQ ID NO:1). This quorum sensing peptide was found to promote angiogenesis and tumor cell invasion in in vitro experiments using HCT-8 CRC cells (Wynendaele et al., Peptides 2015, 64: 40-48).
In the present disclosure, the inventors show that the EntF* quorum sensing peptide can be detected and targeted in the gastro-intestinal tract and/or the circulation of a subject. More specifically, the inventors demonstrate for the first time that quorum sensing peptides, and in particular the quorum-sensing peptide EntF*, promotes in vivo colorectal cancer metastasis, and has pro-metastatic properties in vivo.
Based on the finding that quorum-sensing peptides, and in particular the pro-metastatic EntF* quorum-sensing peptide, promote cancer-metastasis, the present application relates to the use of quorum-sensing targeting substances for the treatment and/or prevention of metastasis of colorectal cancer. Said quorum-sensing targeting substances can be peptide inhibitors, peptide antagonists, molecules that degrade quorum-sensing peptides, or pre-, pro- or synbiotics, such as e.g. (engineered) micro-organisms, that are unable to synthesize the metastasis-promoting quorum sensing peptides and that reduce or inhibit the functionality of the targeted quorum-sensing peptide or the production of the quorum-sensing peptide by gut microbiota.
The present disclosure is thus directed to a quorum-sensing inhibiting substance, in particular a microorganism, for use in the treatment, reduction and/or prevention of metastasis of colorectal cancer in a subject. Typical for various embodiments is that said quorum-sensing inhibiting substance reduces and/or inhibits the activity and/or production of one or more pro-metastatic quorum-sensing peptides and/or their metabolites in the gastro-intestinal tract and/or blood of said subject. In a particular aspect, the pro-metastatic quorum-sensing peptide is derived from enterocin induction factor (SEQ ID NO:3, MEEKNRLNAKQCSDQELKKIKGGAGTKPQGKPASNLVECVFSLFKKCN). More specifically, the pro-metastatic quorum-sensing peptide is the peptide EntF (SEQ ID NO:2, AGTKPQGKPASNLVECVFSLFKKCN) and/or the EntF metabolite EntF* (SEQ ID NO:1, SNLVECVFSLFKKCN). Since it was shown for the first time in the present disclosure that quorum-sensing peptides are able to promote in vivo colorectal cancer metastasis, inhibition or degradation of said quorum-sensing peptides is a novel treatment strategy to prevent and/or reduce the presence and/or development of colorectal cancer metastasis.
In one aspect, the present disclosure is thus directed to a quorum-sensing inhibiting substance for use in the treatment and/or prevention of metastasis of colorectal cancer, wherein said quorum-sensing inhibiting substance reduces and/or inhibits the activity of a quorum-sensing peptide and/or its metabolites in the gastro-intestinal tract and/or blood of the subject. In one embodiment, the quorum-sensing inhibiting substance is a microorganism as provided herein.
In a further embodiment, the quorum-sensing inhibiting substance according to the present disclosure is a product or compound that reduces, inhibits or blocks the production and/or activity of the one or more quorum-sensing (pro)peptide(s) in the gastro-intestinal tract and/or the blood or serum of the subject. In an even more preferred embodiment, said product is a compound or peptide having antagonistic activity to the pro-metastatic quorum-sensing peptide. In still a further embodiment, the quorum-sensing inhibitor of the present disclosure targets the peptide EntF (SEQ ID NO:2) and/or the peptide EntF* (SEQ ID NO:1), in particular EntF*. In an even further embodiment, the quorum-sensing inhibiting substance targets the first, second and/or tenth amino acid of the quorum-sensing peptide EntF* (SEQ ID NO:1).
In another aspect, the present disclosure is directed to a quorum-sensing inhibiting substance for use in the treatment and/or prevention of metastasis of colorectal cancer, wherein said quorum-sensing inhibiting substance is a quorum-quenching agent that degrades a quorum-sensing peptide; in particular that degrades the quorum-sensing peptide EntF*. In a further embodiment, said quorum-sensing inhibiting substance is an enzyme that degrades the quorum-sensing peptide, such as pepsin, trypsin, chymotrypsin, papain, bromelain, serrapeptase, pancrelipase. In the alternative, said quorum-sensing inhibiting substance is an enzyme activator that selectively activates endogenous proteases and/or peptidases. These agents can be used as such or as a targeted-delivery formulation.
In still another aspect, the present disclosure is directed to a quorum-sensing inhibiting substance for use in the treatment and/or prevention of metastasis of colorectal cancer in a subject, wherein the quorum-sensing inhibiting substance is a microorganism, in particular a bacterial strain that does not produce the enterocin induction factor, and in particular the quorum-sensing peptide EntF (SEQ ID No: 1) and/or its metabolite EntF* (SEQ ID No: 2), e.g. as determined by qPCR and/or UHPLC-MS/MS and/or any other established technique. More specific, the bacterial strain is lacking the EntF gene or is genetically modified, e.g. by mutating or deletion of part or all of the EntF gene. In a further embodiment, said bacterial strain is selected from the order lactic acid bacteria (Lactobacillales), and more specific from the genus Enterococcus; preferably said bacterial strain is E. faecium LMG15710, NCIMB10415(SF68), W54, LMG S-28935, THT020101, or any other strain where absence of EntF and/or EntF* has been proven by qPCR and/or UHPLC-MS/MS and/or any other established technique. In still a further embodiment, the bacterial strain that does not produce the quorum-sensing peptide EntF and its metabolite EntF*, is administered to the subject and reduces the production of the quorum-sensing peptide EntF by other bacterial strains in the gastro-intestinal tract e.g. by altering the bacterial flora and/or suppressing the levels of the EntF/EntF* producing strains. In other words, in one aspect of the present disclosure, administration of the quorum-sensing inhibiting substance reduces the production of the quorum-sensing peptide EntF by other bacterial strains in the gastro-intestinal tract.
In a further aspect, the presence of bacterial strains producing the quorum-sensing peptide EntF is reduced or prevented when the quorum-sensing inhibiting substance, in particular the bacterial strain that does not produce the quorum-sensing peptide EntF, is administered to the subject, e.g. as (part of) a probiotic or medicine.
In the different embodiments of the present disclosure, a quorum-sensing targeting or inhibiting substance, such as a microorganism, for use in the treatment and/or prevention of metastasis of colorectal cancer is administered to a subject. In a further embodiment, said subject is a mammal. In an even further embodiment, the subject is a human subject. In yet another embodiment, the subject is a human subject diagnosed with a colorectal primary tumour. In still another embodiment, the subject is a human subject diagnosed with or at risk of colorectal cancer metastasis.
In another aspect, the present disclosure discloses a method of treating, reducing and/or preventing metastasis of colorectal cancer in a subject, wherein a therapeutically effective amount of a quorum-sensing inhibiting substance is administered to the subject and wherein said quorum-sensing inhibiting substance reduces and/or inhibits the activity and/or production of a pro-metastatic quorum-sensing peptide and/or its metabolites in the gastro-intestinal tract and/or blood of said subject. The quorum-sensing inhibiting substance in said method is a quorum-sensing inhibiting substance according to all the different embodiments as described herein. In a particular embodiment, the quorum-sensing inhibiting substance is a microorganism, such as e.g. a bacterial strain as provided herein.
The methods according to all the different embodiments of the present disclosure may further comprise a step of adjusting the diet of the subject to prevent and/or reduce the uptake and/or presence of Enterococcus faecium producing a quorum-sensing peptide; in particular a quorum-sensing peptide that promotes colorectal cancer metastasis. In a further embodiment, the methods may comprise a step of adjusting the diet of the subject to prevent and/or reduce the uptake and/or presence of Enterococcus faecium producing the EntF quorum-sensing peptide.
In still another aspect of the present disclosure, a method of preventing, treating and/or reducing metastasis of colorectal cancer in a subject is disclosed, wherein the method comprises the following step: detecting the level of a quorum-sensing peptide in a sample derived from a subject, and based on the outcome, adjusting the diet of said subject to prevent and/or reduce the uptake and/or presence of Enterococcus faecium producing the quorum-sensing peptide.
In a further embodiment, a method for preventing, treating and/or reducing metastasis of colorectal cancer in a subject is disclosed wherein the method comprises the steps of detecting the level of the quorum-sensing peptide EntF (SEQ ID NO:2) in a sample derived from the subject, and based on the outcome adjusting the diet of said subject to prevent and/or reduce the uptake and/or presence of Enterococcus faecium producing the quorum-sensing peptide EntF. In another aspect, a method for preventing, treating and/or reducing metastasis of colorectal cancer in a subject is disclosed wherein the method comprises the steps of detecting the level of the EntF metabolite EntF* (SEQ ID NO:1) in a sample derived from the subject, and based on the outcome adjusting the diet of said subject to prevent and/or reduce the uptake and/or presence of Enterococcus faecium producing the quorum-sensing peptide EntF.
In all said methods of the present disclosure, the level of a quorum-sensing peptide is detected in (a sample of) a subject; preferably a mammal; even more preferably a human subject.
In a further aspect, the present disclosure is also directed to a composition, including its use as a medicament, comprising an EntF*-antagonistic peptide, a molecule derived from an EntF*-antagonistic peptide, an Enterococcus strain not producing EntF/EntF*, or a genetically modified Enterococcus strain wherein part or all of the gene producing EntF/EntF* has been deleted or mutated, and a pharmaceutically acceptable carrier. In one embodiment, the Enterococcus strain is an Enterococcus faecium strain.
In another embodiment, the present disclosure is directed to a diagnostic method for analyzing the presence of quorum-sensing peptides in a sample of a subject to assess the presence of such peptides that influence tumor metastasis in a subject having colorectal cancer.
Screening can be done by analyzing feces and/or blood or serum, other body-fluids, and/or other samples.
With specific reference now to the figures, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the different embodiments of the present disclosure only. They are presented in the cause of providing what is believed to be the most useful and readily description of the principles and conceptual aspects of the present disclosure. In this regard no attempt is made to show structural details of the present disclosure in more detail than is necessary for a fundamental understanding of the present disclosure. The description taken with the drawings making apparent to those skilled in the art how the several forms of the present disclosure may be embodied in practice.
In the present disclosure, the inventors show that the EntF* quorum sensing peptide can be detected and targeted in the gastro-intestinal tract and/or the circulation of a subject. More specifically, the inventors demonstrate for the first time that quorum sensing peptides, and in particular the quorum-sensing peptide EntF*, promote in vivo colorectal cancer metastasis, and has pro-metastatic properties in vivo.
In a first aspect, a quorum-sensing inhibiting substance for use in the treatment and/or prevention of metastasis of colorectal cancer is thus provided. Said quorum-sensing inhibiting substance reduces and/or inhibits the activity and/or production of enterocin induction factor in bacteria, and more specific of one or more pro-metastatic quorum sensing peptides or metabolites thereof. As used herein, “pro-metastatic” means that said peptides or metabolites promote or induce metastasis of cancer cells from the primary tumor in vivo. A representative in vivo model to determine pro-metastatic activity is provided herein. Said pro-metastatic peptides are for example the EntF peptide (SEQ ID No: 2) and the metabolite thereof, i.e. EntF* peptide (SEQ ID No: 1). The “inhibiting substances” or “inhibitor” can be found by commonly applied screening techniques known to the skilled person, and include:
In one embodiment, the quorum-sensing inhibiting substance is thus an antagonistic compound to the pro-metastatic peptide or metabolites thereof. As used herein, an “antagonistic compound” “antagonist” refers to any chemical compound or biological molecule has antagonistic activity and/or that impairs, in particular decreases, the ability of such targeted quorum-sensing peptide as defined herein, to respond or bind to their respective ligand or receptor such as e.g. the CX chemokine receptor 4 (CXCR4); or to impair its functionality such as the epithelial-mesenchymal promoting (EMT) activity or angiogenesis.
In one aspect, the quorum-sensing inhibiting substance is a product or compound that specifically binds the peptide EntF* and decreases its activity or functionality. For example, the quorum-sensing inhibiting substance is a compound that targets the EntF* peptide, for example a compound that targets or specifically binds the first, second and/or tenth amino acid of the quorum-sensing peptide EntF*.
In one embodiment, said inhibitor includes antibodies and antigen-binding fragments thereof. In the alternative, EntF or EntF* binding moieties or antagonists can be used which include a variety of different types of molecules including those that specifically bind resp. EntF or EntF*, in particular the first, second and tenth amino acid of the peptide EntF* which have been shown to be involved in the EMT activity of the peptide. Such ligands include small molecules, polypeptides (e.g. a fusion protein), antibodies, or nucleic acids, and the like. The term “antibody” refers to polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, a human engineered antibody, a human antibody, as well as antigen binding antibody fragments, a domain antibody (dAb), heavy chain antibodies (hcAb), minibodies, a variable domain of camelid heavy chain antibody (VHH or Nanobody®), a variable domain of the new antigen receptor (VNAR) and engineered CH2 domains (nanoantibodies), and molecules having antigen binding functionality. EntF* antibodies and methods of making the same are well known in the art. As demonstrated herein, the peptide Nef-M1 blocks the effect of EntF* on the CXCR4 receptor and is thus an example of a quorum-sensing inhibiting substance.
The effect on EMT activity of EntF* (also referred herein as QSP76) was modulated by alanine-scans. The results show the importance of certain amino acid residues for the biological activity on EMT, in particular residues on position one, two and ten of EntF*. Changing amino acids is a flexible way of increasing as well as of decreasing the activity, and said method can be used to design antagonistic peptides. In one embodiment, the antagonistic peptide is characterized by the following sequence: X1X2LVECVFSX3FKKCN (SEQ ID NO:4), wherein is X1 is an amino acid other than S, and/or wherein X2 is an amino acid other than N and/or X3 is an amino acid other than L.
Hence in one embodiment, the antagonist is an oligopeptide analogous to the quorum sensing peptide EntF* but different at positions one, two and/or ten, in particular at position one. As demonstrated herein, the EntF* homologues peptide QSP76S1A (ANLVECVFSLFKKCN, SEQ ID NO:5) goes in competition with EntF* on the CXCR4 receptor and is thus an example of a EntF* antagonist.
In a further embodiment, the quorum-sensing inhibiting substance is a quorum-quenching agent that degrades a quorum-sensing (pro)peptide, and as such inhibits or prevents its activity. For example this quorum-quenching agent is an enzyme, for example pepsin, trypsin, chymotrypsin, papain, bromelain, serrapeptase, pancrelipase. On the other hand, the quorum-sensing inhibiting substances can also be an enzyme activator that selectively activates endogenous proteases and/or peptidases.
In another aspect, the quorum-sensing inhibiting substance can also be a microorganism, in particular a bacterial strain that does not produce the quorum-sensing peptide EntF*. Said bacterial strain can for example be genetically modified to lack the EntF family enterocin induction factor gene as e.g. defined by accession no NC_021994.1 or as provided in
A bacterial strain not producing a functional EntF or EntF* peptide and/or lacking the EntF gene can for example be the E. faecium LMG15710 (ATCC 6569), NCIMB10415 (E. faecium SF68; DSM 10663 commercially available—EU authorized feed additive for animals), W54 (Strasser et al., Nutrients 2016; 8(11)), LMG S-28935 (Chisari et al. Curr Clin Pharmacol 2017; 12(2): 99-105), THT020101, or any other (Enterococcus) strain where absence of EntF and/or EntF* has been proven by qPCR and/or UHPLC-MS/MS and/or any other established technique, and in particular by Liquid chromatography-mass spectrometry (LC-MS) under the same conditions as demonstrated herein.
As shown for the first time in the present disclosure, said bacterial strains are able to alter the bacterial flora of the subject and/or to overgrow or to suppress the levels of the EntF* producing strains in the subject.
The bacterial strains can be administered to the subject orally, in all possible forms such as formulated in a powder, tablet, capsule, or any other way known to the skilled person. In one embodiment, the bacterial strain is administered as part of a probiotic composition, for example via the daily food or via a specific pharmaceutical composition.
In a further aspect, the diet of the subject can be adjusted so that it comprises a quorum-sensing inhibiting substance that prevents and/or reduces the uptake and/or presence of quorum-sensing (pro)peptides in the subject. For example, the substance can prevent and/or reduce the uptake and/or presence of Enterococcus faecium producing the quorum-sensing (pro)peptides in the subject.
In the methods provided herein, the quorum sensing inhibiting substance is used or administered to the subject in an effective amount. The terms “effective amount” or “effective dose” are used interchangeably herein and denote an amount of the pharmaceutical active inhibiting substance of the present disclosure which has a prophylactically or therapeutically relevant action on cancer metastasis, in particular on colorectal cancer metastasis. The spread of cancer from its tissue of origin and its subsequent growth in other organs is the most life-threatening aspect of the disease. This process is called metastasis, and requires cancer cells to survive and proliferate outside their tissue of origin. The first crucial step in this process is the invasion of cancer cells into tissue surrounding the tumour and the vasculature.
The quorum sensing inhibiting substance of the present disclosure is capable to prevent and/or reduce cancer cell dissemination and metastasis formation. As such, the present disclosure relates to the quorum sensing inhibiting substance described herein for use in treating cancer by inhibiting the invasion of tumour cells into surrounding or adjacent tissue, cells and/or their entry into the circulatory system. Hence the substances or compounds of the present disclosure are especially useful for inhibiting or stopping tumour spread or cancer metastasis, in particular colorectal cancer i.e. the development of cancer from the colon or rectum (parts of the large intestine, including rectal cancer, colon cancer and bowel cancer). In a specific embodiment, the quorum sensing inhibiting substance prevents or reduces colon cancer metastasis. In a further embodiment, the tumour or cancer is a CXCR4 positive tumour or cancer, i.e. whereby the cancer cells express CXCR4 as e.g. can be determined using different techniques known to the skilled person, such as flow cytometry, qPCR analysis and/or western blotting. C-X-C chemokine receptor type 4 (CXCR-4) also known as fusin or CD184 (cluster of differentiation 184) is a protein that in humans is encoded by the CXCR4 gene. While CXCR4's expression is low or absent in many healthy tissues, it was demonstrated to be expressed in over 23 types of cancer, including breast cancer, ovarian cancer, melanoma, and prostate cancer. Expression of this receptor in cancer cells has been linked to metastasis to tissues containing a high concentration of CXCL12, such as lungs, liver and bone marrow.
Generally, for pharmaceutical use, the quorum sensing inhibiting substance of the present disclosure may be formulated as a pharmaceutical preparation or pharmaceutical composition comprising at least one quorum sensing inhibiting substance of the present disclosure and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active compounds. Such a formulation may be in a form suitable for oral administration, for administration by suppository, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for intranasal, transdermal, transmucosal, rectal or pulmonary administration.
Examples of such formulations include tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, creams, lotions, soft and hard gelatin capsules, suppositories, eye drops, sterile injectable solutions and sterile packaged powders (which are usually reconstituted prior to use) for administration as a bolus and/or for continuous administration. Carriers, excipients, and diluents that are suitable for such formulations are e.g. lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, (sterile) water, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate, edible oils, vegetable oils and mineral oils or suitable mixtures thereof.
This present disclosure further provides a pharmaceutical composition comprising a quorum sensing inhibiting substance according to this present disclosure and a pharmaceutically acceptable carrier, diluent and/or excipient, and the uses thereof as provided herein.
The present disclosure also provides an in vitro method for the detection of a quorum-sensing peptide or metabolite thereof in a sample from a subject, in particular a subject diagnosed with cancer, more in particular diagnosed with colorectal cancer. In one embodiment, this method comprises (1) preparing the sample by using C18 or hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction, preferably HILIC-amide solid-phase extraction, (2) performing liquid chromatography on the sample prepared in step (1) wherein the gradient conditions are such that loss of the analyte, e.g. by adsorption, is minimized, such as for example by the choice of a suitable start mobile phase and/or gradient slope, and (3) detection of the peptide or metabolite using a suitable detection method, such as a MS-based method. In one embodiment, the mobile phase consists of water:acetonitrile:DMSO (V/V) containing 0.05%-1% of an acid, in particular formic acid. The flow rate is set to 0.1-1 mL/min, in particular 0.5 mL/min. In a further embodiment the gradient program starts with 70%-95% of the mobile phase, in particular 75-85%, for about 0.5 to 1.5 min, followed by a linear gradient to 30-50%, in particular 35-45% of the mobile phase for 2.5-4 minutes, in particular 3-4 minutes. After that the gradient is decreased to 10-20% mobile phase, in particular about 15%, for 4-6 min.
In another aspect of the present disclosure, the detection of a quorum-sensing peptide or metabolite thereof, in particular EntF and/or EntF*, can be used in the diagnosis of colorectal metastasis in a subject.
More specific, the present disclosure is directed to a diagnostic method for analyzing the presence of quorum-sensing peptides in a sample of subject to assess the presence of such peptides that influence tumor metastasis in a subject having colorectal cancer.
Screening can be done by analyzing feces and/or blood, other body-fluids, and/or other samples.
The present disclosure furthermore relates to the use of the knowledge obtained by the diagnostic method in order to aid in the differential patient classification and in prognosis, as well as to influence the progression of cancer metastasis, by for example providing beneficial bacteria, providing antagonists for harmful quorum sensing peptides and other measures as provided herein.
The present disclosure furthermore relates to a method of detecting a patient at risk of colorectal cancer metastasis, said method comprising detecting the presence and/or amount of the quorum-sensing peptide EntF (e.g. in faeces) and/or EntF* (e.g. in plasma) in a sample of said subject.
In a further aspect, the current disclosure relates to the use of the knowledge obtained by the diagnostic method in order to reduce or prevent cancer metastasis in a patient diagnosed with colorectal cancer, by for example providing bacteria that produce non-harmful quorum sensing peptides and/or providing antagonists for harmful quorum-sensing peptides.
The present disclosure in particular relates to microorganisms, having neutral quorum sensing peptides to replace in the gut the strains that have quorum sensing peptides shown herein to negatively influence metastasis, such as the EntF* peptides. In one embodiment, these microorganisms are probiotica (bacterial strains that are considered healthy for the gut).
More specific, the present disclosure provides a method of preventing, risk-evaluation, diagnosis, treating and/or reducing metastasis of colorectal cancer in a subject, said method comprising the steps of:
Homogenate preparation. Krebs-Henseleit (KH) buffer (pH 7.4) (Sigma-Aldrich, Belgium) was prepared by dissolving the powdered medium in 900 mL water while stirring. To this solution, 0.3790 g CaCl2×2H2O and 2.098 g NaHCO3 are subsequently added while stirring. NaOH or HCl was used to adjust to pH 7.4. This solution was then further diluted to 1000 mL using ultrapure water.
For the preparation of colon tissue homogenate, two colons were collected from two C57BL/6 female mice after cervical dislocation. After cleaning and rinsing the organs using ice-cold KH buffer, the colons were cut in little pieces and transferred into a 15 mL tube to which 5 mL ice-cold KH buffer was added. The colons were then homogenized for 1 minute. After the larger particles were allowed to settle for about 30 minutes at 5° C., approximately 2 mL of the middle layer was dispensed into a 2 mL Eppendorf tube, and stored at −35° C. until use. Just before use, the homogenate was diluted to a protein concentration of 0.6 mg/mL.
Faeces homogenate was prepared by collecting faeces from two C57BL/6 female mice after cervical dislocation. The same procedures as with the colon tissue were performed. Just before use, the homogenate was diluted to a protein concentration of 0.6 mg/mL.
Peptide adsorption. Due to adsorption of EntF* to different kinds of plastic, all plastic tubes and containers were coated before use with a BSA-based anti-adsorption solution.
Metabolization kinetics. 500 μL of homogenate and 400 μL of KH buffer were mixed, together with 100 μL of KH buffer (blank) or 1 mg/mL EntF* peptide solution (test), all equilibrated and incubated at 37° C. After 0, 5, 10, 30, 60, 120 and 180 minutes, 100 μL aliquots were taken and immediately mixed with 100 μL of 1% V/V trifluoroacetic acid solution in water, heated for 5 min at 95° C., and cooled for 30 min in an ice-bath. After centrifugation at 16,000 g for 30 min at 5° C., supernatants were analysed by LC1-MS1 for EntF* quantification.
Intestinal permeability. Caco-2 cells were seeded on Transwell polycarbonate membrane filters (0.4 μm pore size) (Corning, Germany) at a density of 2.6×105 cells/cm2 and the permeability study performed as described by Hubatsch et al.26. Cells were filled with Hank's Balanced Salt Solution (HBSS) and the TER values measured before and after the experiment. Peptide solution (1 μM) was added to the apical chamber and 300 μL aliquots taken after 30, 60, 90 and 120 min of incubation. Samples were analysed using LC1-MS1. Linear curve fitting was used to calculate the apparent permeability coefficient (Papp).
Standard protein BLAST. The amino acid sequence of the EntF* peptide was blasted against the NCBI non-redundant (nr) database by Basic Local Alignment Search Tool protein (BLASTp). This blast search was performed with the organism limited to bacteria (taxid:2). Only alignment hits with a 100% coverage and 100% identity were retained.
PCR on E. faecium strains. E. faecium was grown on MRS agar. DNA was extracted using alkaline lysis after which the EntF* fragment was amplified using 2× Biomix (Bioline, Belgium) in a Mastercycler PCR system (Eppendorf, Belgium). Each reaction was performed in a 10 μL total reaction mixture using 1 μL of the DNA sample and 0.5 μM final primer concentration (EntF*-PCR primers). The PCR conditions used: 1 cycle of 95° C. for 5 min, followed by 30 cycles of 94° C. for 30 sec, 55° C. for 30 sec and 72° C. for 1 min. Final elongation was performed at 72° C. for 10 min, after which the PCR product was hold at 4° C. The PCR amplification products were visualized on 1.5% agarose gel.
Sample collection and preservation. Mice (C57BL/6) were euthanized by cervical dislocation and the blood collected. After standing for 30 min on ice, blood was centrifuged at 1,000 g for 10 min (room temperature). The supernatant (serum) was then transferred and stored at −35° C. until use.
After defaecation, two droppings of faeces were immediately collected and put in liquid nitrogen for max. 1 h. The samples were then stored at −80° C. until use.
Sample preparation. 50 μL of mice serum was mixed with 150 μL of 0.5% formic acid in acetonitrile. After sonication for 5 min and vortexing for 5 sec, the mixture was heated for 30 sec at 100° C. The solution was again vortexed and centrifuged for 20 min at 20,000 g (4° C.). The supernatant was then further purified using solid phase extraction (SPE) on HyperSep C18 plates (Thermo Fisher Scientific, Belgium), which were previously conditioned with acetonitrile and equilibrated with 75% acetonitrile in water, containing 0.375% formic acid. After loading 150 μL of the samples, 120 μL eluent was collected and the organic solvents evaporated using nitrogen for 5 minutes. The resulting solutions were then further diluted with 30 μL of BSA-based anti-adsorption solution, followed by LC1-MS1 analysis.
LC1-MS1 analysis. EntF* was detected and quantified on a Waters Acquity UPLC H-class system, connected to a Waters Xevo™ TQ-S triple quadrupole mass spectrometer with electrospray ionization (operated in positive ionization mode). Autosampler tray and column oven were thermostated at 10° C.±5° C. and 60° C.±5° C., respectively. Chromatographic separation was achieved on a Waters Acquity® UPLC BEH Peptide C18 column (300 Å, 1.7 μm, 2.1 mm×100 mm). The mobile phases consisted of 93:2:5 water:acetonitrile:DMSO (V/V) containing 0.1% formic acid (i.e. mobile phase A) and 2:93:5 water:acetonitrile:DMSO (V/V) containing 0.1% formic acid (i.e. mobile phase B), and the flow rate was set to 0.5 mL/min. From the samples, a 10 mL aliquot was injected. The gradient program started with 80% of mobile phase A for 1 minute, followed by a linear gradient to 40% of mobile phase A for 3.5 minutes. Gradient was then changed to 14.2% mobile phase A at 5 min, followed by a 1 min equilibration, before starting conditions were applied. EntF* showed retention at 4.25-4.45 min.
An optimised capillary voltage of 3.00 kV, a cone voltage of 20.00 V and a source offset of 50.0 V was used. Acquisition was done in the multiple reaction monitoring (MRM) mode. The selected precursor ion for EntF* was m/z 865.7 with two selected product ions at m/z 202.08 (36 V) as quantifier and m/z 315.17 (31 V) as qualifier.
A sample was considered positive for the presence of EntF* when following criteria were met: correct retention time, quantifier/qualifier peak area ratio's between 2.0 and 4.0, both quantifier (b2 fragment) and qualifier (b3 fragment) with a signal-to-noise ratio above 3.0 and a concentration above the LOQ of 100 pM.
LC1-MS2 analysis. Chromatographic separation was achieved on a Waters Acquity® UPLC HSS T3 Column (100 Å, 1.8 μm, 2.1 mm×100 mm), with detection using the Waters SYNAPT G2-Si High Definition Mass Spectrometry with electrospray ionization (operated in the positive ionization mode). Gradient composition and UPLC-MS settings were the same as with the LC1-MS1 method; a TOF-MS/MS mode was applied with a fixed mass on the quadrupole of 865.157, a fixed trap collision energy of 30 V and an acquired MS/MS over the range of 100-1450 m/z (scan time 1 second). EntF* retention was observed between 3.40-3.50 min. When at least four daughter ions (m/z±0.05) of EntF* were detected at the expected retention time and at least three of the most abundant isotope parent peaks (m/z±0.05) were detected, the sample was considered to contain the EntF* peptide.
LC1-MS3 analysis. While the UPLC separation system was the same as with the LC1-MS1 method, the third detection system consisted of a Thermo Fisher Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer. The mass spectrometer was operated using a heated electrospray ionization source with the following setting: capillary temperature set at 300° C., S-Lens RF level set at 50, spray voltage set at 3.00 kV and auxiliary gas flow set at 20.
A full MS/MS mode was applied with a fixed mass on the quadrupole of 865.157, a fixed trap collision energy of 30 V and 35 V and an acquired MS/MS over the range of 100-1800 m/z. EntF* retention was observed at 4.13-4.16 min. When at least two daughter ions (m/z±0.005) of EntF* were detected at the expected retention time and at least four of the most abundant isotope parent peaks (m/z±0.005) were detected, the sample was considered to be positive for the presence of EntF*.
LC2-MS1 analysis. Chromatographic separation was achieved on a Waters Acquity® UPLC BEH Amide Column (130 Å, 1.7 μm, 2.1 mm×100 mm). Mobile phase composition, sample volume, flow rate and MS settings were the same as described for the LC1-MS1 method. The gradient program started with 10% of mobile phase A for 2 minutes, followed by a linear gradient to 40% of mobile phase A for 3.0 minutes. Gradient was then changed to 85% mobile phase A at 6 min, followed by a 1 min equilibration, before starting conditions were applied. EntF* showed retention at 4.85-4.95 min. A sample was considered positive for the presence of EntF* when following criteria were met: correct retention time, both daughter fragments (i.e. b2 and b3) with a signal-to-noise ratio above 3.0 and quantifier/qualifier peak area ratio's between 2.0 and 4.0.
DNA extraction of faeces. To 20-40 mg faeces, 500 mg of unwashed glass beads, 0.5 mL CTAB buffer (hexadecyltrimethylammonium bromide 5% (w/v), 0.35 M NaCl, 120 mM K2HPO4) and 0.5 mL phenol-chloroform-isoamyl alcohol mixture (25:24:1) were added. The mixture was homogenized two times for 1.5 min at 22.5 Hz using a TissueLyser II (Qiagen, Belgium). The mixture was centrifuged for 10 minutes at 8,000 rpm and 300 μL of the supernatant was transferred to a new Eppendorf tube. For a second time, 0.25 mL of CTAB buffer was added to the original DNA sample, which was again homogenized in the TissueLyser and centrifuged for 10 minutes at 8,000 rpm. Of this supernatant, 300 μL was added to the first 300 μL supernatant. The phenol was removed by adding an equal volume of chloroform-isoamyl alcohol (24:1) followed by centrifugation at 16,000 g for 10 sec. The aqueous phase was transferred to a new tube. Nucleic acids were precipitated with 2 volumes PEG-6000 solution (polyethyleenglycol 30% (w/v), 1.6 M NaCl) for 2 h at room temperature. The pellet was obtained by centrifugation at 13,000 g for 20 min and washed with 1 mL of ice-cold 70% (v/v) ethanol. After centrifugation at 13,000 g for 20 min, the pellet was dried and resuspended in 50 μL de-ionized water. The quality and the concentration of the DNA was examined spectrophotometrically.
qPCR on faeces. qPCR was performed using SYBR-green 2× master mix in a Bio-Rad CFX-384 system. Each reaction was done in sixfold in a 12 μL total reaction mixture using 2 μL of the DNA sample and 0.5 μM final qPCR primer concentration. The qPCR conditions used: 1 cycle of 95° C. for 10 min, followed by 40 cycles of 95° C. for 30 sec, 60° C. for 30 sec, and stepwise increase of the temperature from 65° to 95° C. (at 10 sec/0.5° C.). Melting curve data were analysed to confirm the specificity of the reaction. Samples with a specific melting peaks were discarded from further analyses. After purification and determination of the DNA concentration, the concentration of the linear dsDNA standard was adjusted from 107 to 101 copies (EntF*). The copy numbers of samples were determined by reading of the standard series with the Ct values of the samples. The standard curves were extrapolated for samples containing less than 101 copies. Because the Cq values of the EntF* qPCR analyses were around the limit of detection (LOD), with a notable amount of left-truncated data (data below LOD), a maximum likelihood (ML) approach was used to find the best estimation of mean and standard deviation for each sample.
Cell culture. Caco-2 and luciferase transfected HCT-8/E11 cells were grown in DMEM medium supplied with 10% foetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U/mL) solution. The cells were cultured in an incubator set at 37° C. and 5% CO2. When confluent, cells were detached using 0.25% trypsin-EDTA.
Orthotopic colorectal cancer mouse model. All in vivo experiments were performed according to the Ethical Committee principles of laboratory animal welfare and approved by our institute (Ghent University, Faculty of Medicine and Health Sciences, approval number ECD 17-90). Mice were maintained in a sterile environment with light, humidity and temperature control (light-dark cycle with light from 7:00 h to 17:00 h, temperature 21-25° C. and humidity 45-65%). Before the experiment, mice were allowed to acclimatize for a minimum of seven days.
Six-weeks old female athymic nude mice (Swiss nu/nu) were anesthetized and a small midline laparotomy executed to localize the caecum. The caecum was then gently exteriorized and luciferase transfected HCT-8/E11 cells (1×106 cells) in a volume of 20 μL serum-free DMEM medium with matrigel (1:1) injected into the caecal wall. Cells were previously treated with EntF* (10 nM, 100 nM or 1 μM), Phr0662 (100 nM) or with the vehicle (PBS) or positive control (Transforming Growth Factor α (TGFα), 0.1 μg mL−1) solution for 5 days before they were implanted in the mice. The caecum was then carefully returned to the abdominal cavity and the laparotomy closed in two layers by sutures of PDS 6/0. Starting from the day after tumour cell injection, mice were daily treated by i.p. injection with vehicle (PBS, n=15), EntF* (10 nmol kg−1, n=12; 100 nmol kg−1, n=18; 1 μmol kg−1, n=8), Phr0662 (100 nmol kg−1, n=5) or positive control (Epidermal Growth Factor (EGF), 100 μg kg−1, n=18) for 6 weeks. Once a week, mice were investigated for tumour growth and metastases using bioluminescent imaging with the IVIS Lumina II (Perkin Elmer, Belgium) after i.p. injection with 200 μL luciferin (150 mg kg−1). After 6 weeks, mice were euthanized using cervical dislocation, followed by macroscopic evaluation of the liver, diaphragm, lungs, caecum, duodenum and peritoneum for the presence of tumour nodules. Liver and lung tissues were then fixed in formalin during 24 h and stored in 70% ethanol for max. 3 days before embedding in paraffin. Afterwards, a haematoxylin & eosin (H&E) staining was performed on 8 μm sections and 3 sections were visualized per mouse using microscopy. The slides of all tumour-bearing mice were scored by two blinded, independent investigators using a scoring system. In the case of a difference in scoring, the slide was scored again by a third blinded, independent investigator for consensus.
Daily peptide exposures were calculated after i.p. injection of 25 μL of a 100 μM EntF* solution into female Swiss nu/nu mice (n=14), followed by LC1-MS1 analyses of mice serum at different time points after injection. A distribution and early exponential phase (α, 0-30 min), followed by a terminal elimination phase (β, 30-180 min) could be distinguished. The exposure was determined for 24 hours (x) as follows: Exposure (nM×min)=∫030Ae−αt+∫30xBe−βt.
Western Blot analyses. 1×106 luciferase transfected HCT-8/E11 cells were seeded in each well of a 6-well plate. 24 hours post-seeding, cells were treated with EntF* and its synthesised alanine-derived analogues (100 nM) or placebo, or with Nef-M1/QSP76S1A (10 μM) or mixtures (10M Nef-M1/QSP76S1A with 5 μM EntF*) to demonstrate one of the targets, i.e. CXCR4. After 24 hours, cells were detached from the surface and lysed with Thermo Scientific RIPA-buffer. The protein concentration was then determined using the modified Lowry protein assay kit, according to the manufacturer's instructions. All samples were diluted to the same concentration (i.e. 4 μg/μL) using water and diluted 1:1 using 2× Laemmli buffer. Next, the samples were boiled for 5 min at 95° C. for denaturation, after which centrifugation for 5 min at 16,000 g was performed; the supernatant was then used for Western blot analyses. Therefore, proteins (20 μg) were separated using a Bio-Rad Any kD gel (SDS-PAGE) and transferred to a PVDF membrane. Before the membranes were incubated with antibodies, non-specific binding sites were blocked using 5% skimmed milk solution (1 hour). Western blot was performed using an anti-E-cadherin (1/1000) antibody and incubated overnight at 4° C. Signal intensity was normalized against the total protein content in the lanes. Anti-rabbit-HRP antibody was used for detection (1/2000) (1 hour). Finally, the substrate (5 min) was added and the results were analyzed using the Bio-Rad ChemiDoc EZ imager and Image Lab software. TBS buffer with 0.05% Tween 20 was used for washing between the different steps.
Sample collection and preservation of human plasma. Blood was collected in a conventional EDTA tube and centrifuged at 3,000 g for 10 min (room temperature). The supernatant (plasma) was then transferred and immediately processed.
Sample preparation of human plasma. 900 μL of human plasma was mixed with 2.1 mL of 2% hydrochloric acid in a h (97/3) acetonitrile:DMSO solution. After sonication and vortexing for 1 min, the mixture was heated for 1 minute at 100° C. The solution was again vortexed and centrifuged for 1 min at 3,000 g. Subsequently, the supernatant (2 mL) was lyophilized and dissolved in 970 μL acetonitrile containing 0.1% formic acid. This solution was then further purified using AMIDE solid phase extraction (SPE). After the SPE column is conditioned with (90/10/0.1) water:acetonitrile:NH4OH and equilibrated with (97/3/0.1) acetonitrile:DMSO:Formic acid, the sample is loaded and eluted with a (75/20/5) water:acetonitrile:DMSO:Formic acid solution in a BSA-based anti-adsorption diluent coated vial. The resulting solution was analysed using LC-MS analysis.
Statistical analyses. The Kolmogorov-Smirnov test was used to assess if data obtained were normally distributed. For sample sizes of n<10, non-parametric tests (Mann-Whitney U test) were performed directly. Slope comparison was based on linear regression analysis. Bootstrapped medians and Hedges G-values were used to calculate the effect size.
Quorum sensing peptides are traditionally regarded as intra- and inter-bacterial communication molecules, but given their wide structural variety and co-evolution, we anticipate that these molecules may also interact with the host. Up till now, however, quorum sensing peptides have not yet been unambiguously demonstrated to be present in biofluids. Only an indirect indication of the in vivo presence of an unidentified quorum sensing peptide was described in the stool of patients suffering from a Clostridium difficile infection12. We have previously focussed on Enterococcus faecium, one of the most abundant species in the human intestinal microbiota, which synthesizes the enterocin induction factor, i.e. the EntF quorum sensing peptide (AGTKPQGKPASNLVECVFSLFKKCN, SEQ ID NO:2,
E. faecium LIM714,
E. faecium 2014-VREF-41,
E. faecium VRE-1402237,
E. faecium 2014-VREF-63,
E. faecium U-1313438,
E. faecium 2014-VREF-26,
E. faecium E1071,
E. faecium F1129F 09,
E. faecium ERV165,
E. faecium F1213D 01,
E. faecium GMD5E,
E. faecium 24,
E. faecium GMD4E,
E. faecium Hp_24-3,
E. faecium GMD3E,
E. faecium Hp_5-10,
E. faecium GMD2E,
E. faecium Hp_6-10,
E. faecium GMD1E,
E. faecium Hp_5-7,
E. faecium 6E6,
E. faecium 8_Efcm_HA-DE,
E. faecium MXVK29,
E. faecium 11_Efcm_HA-DE,
E. faecium S-1001508,
E. faecium FDAARGOS_326,
E. faecium VRE-1402513,
E. faecium 4278,
E. faecium VRE-1402563,
E. faecium 1584,
E. faecium VRE-1406033,
E. faecium IHC105,
E. faecium VRE-1504220,
E. faecium IHC116,
E. faecium LMG 8148,
E. faecium IHC106,
E. faecium Isolate 30,
E. faecium IHC113,
E. faecium VRE16,
E. faecium IHC115,
E. faecium XH877,
E. faecium IHC130,
E. faecium HMSC063H10,
E. faecium IHC117,
E. faecium HMSC069A01,
E. faecium IHC121,
E. faecium HMSC056C08,
E. faecium IHC118,
E. faecium 97-7_S6,
E. faecium IHC127,
E. faecium LIM1546,
E. faecium IHC108,
E. faecium LIM1547,
E. faecium IHC120,
E. faecium LIM1759,
E. faecium IHC123
On the other hand, our experimental data indicated that not all E. faecium strains have this EntF gene in their genome (Table 2), indicative for the inter-individual variation in the (gut) microbial community.
E. faecium LMG 20720
E. faecium LMG 23236
E. faecium LMG 15710
E. faecium ATCC 8459
E. faecium T110
E. faecium NCIMB 10415 (SF68)
We demonstrated that metabolization of EntF in faeces and colonic tissue homogenate yielded a 15-mer peptide EntF* (SNLVECVFSLFKKCN, SEQ ID NO:1) (
Mice serum is mixed (1/4) with of 0.5% formic acid in acetonitrile. After sonication and vortexing, the mixture is boiled. The solution is again vortexed and centrifuged. The supernatant is then further purified using solid phase extraction (SPE). After loading the samples, the eluent is collected in a BSA-based anti-adsorption diluent coated vial and the organic solvents evaporated using nitrogen. The resulting solutions are then further diluted with BSA-based anti-adsorption solution, followed by LC-MS analysis.
Plasma is mixed (3/10) with (94/3/3) acetonitrile:DMSO:Formic acid. After sonication and vortexing, the mixture was boiled and subsequently put on ice. The solution was centrifuged and the supernatant is diluted (1/9) with (97/3/0.1) acetonitrile:DMSO:Formic acid. The supernatant is then further purified using AMIDE solid phase extraction (SPE). After the SPE column is conditioned with (90/10/0.1) water:acetonitrile:NH4OH and equilibrated with (97/3/0.1) acetonitrile:DMSO:Formic acid, the sample is loaded and eluted with a (75/20/5) water:acetonitrile:DMSO:Formic acid solution in a BSA-based anti-adsorption diluent coated vial. The resulting solution get analysed using LC-MS analysis.
Feces are mixed (1/1) with acidified water. After sonication and vortexing, the solution is diluted (1/1) with (97/3/0.1) acetonitrile:DMSO:Formic acid. The solution is again vortexed and sonicated and is subsequently boiled and centrifuged. The supernatant is diluted (1/9) with (97/3/0.1) acetonitrile:DMSO:Formic acid. The supernatant is then further purified using AMIDE solid phase extraction (SPE). After the SPE column is conditioned with (90/10/0.1) water:acetonitrile:NH4OH and equilibrated with (97/3/0.1) acetonitrile:DMSO:Formic acid, the sample is loaded and eluted with a (75/20/5) water:acetonitrile:DMSO:Formic acid solution in a BSA-based anti-adsorption diluent coated vial. The resulting solution get analysed using LC-MS analysis.
Chromatographic separation was achieved on C18 reverse phase column. The mobile phases consisted of 93:2:5 water:acetonitrile:DMSO (V/V) acidified with formic acid (i.e. mobile phase A) and 2:93:5 water:acetonitrile:DMSO (V/V) acidified with formic acid (i.e. mobile phase B), and the flow rate was set to 0.5 mL/min. From the samples, a 10 μL aliquot was injected. The gradient program started with 80% of mobile phase A, followed by a linear gradient to 40% of mobile phase A. Gradient was then changed to 14.2% mobile phase A, followed by an equilibration, before starting conditions were applied. Serum samples of 35 healthy mice (C57BL/6 mice, aged 5-18 months) were collected and analysed for the presence of EntF* (
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Following these findings, further proof was obtained by subjecting a selected set of samples to three additional methods: HILIC-UPLC-TQ-MS (
Having demonstrated the presence of EntF* in vivo both directly and indirectly, we evaluated its in vivo metastasis-promoting activities using an orthotopic colorectal cancer mouse model20,21. Before the luciferase transfected HCT-8 cells were implanted into the wall of the caecum of the mice, cells were treated daily for five days with EntF* (100 nM), phosphate-buffered saline (PBS) vehicle or Transforming Growth Factor α (TGFα, positive control) (0.1 μg mL−1). On the sixth day, 5-weeks-old female Swiss nu/nu mice were orthotopically injected with 1×106 luciferase transfected colorectal cancer cells, followed by a once-daily i.p. treatment of EntF* (100 nmol kg−1), PBS vehicle or Epidermal Growth Factor (EGF, positive control) (100 μg kg−1) (
Histopathological data further showed a significant higher number of tumours in both lungs and liver (
Nef-M1 blocks the effect of QSP76 on the CXCR4 receptor by bringing the reduced relative E-cadherin expression from 66.0% (SEM=12.1%) caused by QSP76, back to 93.7% (SEM=7.0%). This significant normalisation in E-cadherin expression (one-sided student's t test: p=0.0365; very large Cohen's d effect size of 1.22), together with the similar E-cadherin expression of Nef-M1 (99.3%±13.5%) compared to PBS (100%), proves that Nef-M1 can serve as a competitive antagonist by blocking the binding of QSP76 on the CXCR4 receptor. QSP76S1A goes in competition with QSP76 on the CXCR4 receptor by bringing the reduced relative E-cadherin expression from 66.0% (SEM=12.1%) caused by QSP76, back to 91.8% (SEM=15.7%) (one-sided student's t test: p=0.1420; medium Cohen's d effect size of 0.77).
Collectively, our findings demonstrate that EntF*, a quorum sensing peptide metabolite, is present in vivo in biofluids of mice and promotes the metastasis of CRC in an orthotopic animal model, with a potency comparable to that of the well-established human colorectal cancer growth factor, EGF. Our findings indicate that quorum sensing peptides are an additional factor influencing microbiota-host interactions during CRC metastasis. This offers new possibilities in disease prevention, diagnosis and therapy by selective modulation of the gut microbiome as provided herein.
Human plasma as well as feces samples of healthy volunteers were investigated for the presence of EntF*. An additional lyophilisation step was used in addition to the analytical methodology as provided herein, in order to further improve the detection limits, e.g. to 0.1 pM for plasma. In the majority of the feces samples, EntF* was found with concentration levels ranging between 20 to 800 pM, while in some plasma samples, levels of around 3 pM were found. These findings highlight further the human clinical relevance of our findings, and evidence that plasma and feces detection of EntF* is feasible, such as in the context of cancer metastasis risk monitoring.
Western Blot analyses. 106 HCT-8 (n=15), HCT-116 (n=12), CaCo-2 (n=6) and HT-29 (n=12) cells were seeded in each well of a 6-well plate. 24 hours post-seeding, cells were treated with EntF* (all cells with 1 μM), CXCL12 (10 ng/ml) or placebo. After 24 hours, cells were detached from the surface and lysed with Thermo Scientific RIPA-buffer. The protein concentration was then determined using the modified Lowry protein assay kit, according to the manufacturer's instructions. All samples were diluted to the same concentration (i.e. 4 μg/μL) using water and diluted 1:1 using 2× Laemmli buffer. Next, the samples were boiled for 5 min at 95° C. for denaturation, after which centrifugation for 5 min at 16,000 g was performed; the supernatant was then used for Western blot analyses. Therefore, proteins (20 μg) were separated using a Bio-Rad Any kD gel (SDS-PAGE) and transferred to a PVDF membrane. Before the membranes were incubated with antibodies, non-specific binding sites were blocked using 5% skimmed milk solution (1 hour). Western blot was performed using an anti-E-cadherin (1/1000) antibody and incubated overnight at 4° C. Signal intensity was normalized against the total protein content in the lanes. Anti-rabbit-HRP antibody was used for detection (1/2000) (1 hour). Finally, the substrate (5 min) was added and the results were analyzed using the Bio-Rad ChemiDoc EZ imager and Image Lab software. TBS buffer with 0.05% Tween 20 was used for washing between the different steps.
Result Western blot. Treatment of CXCR4 positive cell lines (HCT-8, HT-29 and CaCo-2) with EntF* results in a statistically significant (p<0.0001) decreased expression of E-cadherin (decrease of 26%, 60% and 38% respectively). Alternatively, when a CXCR4 negative cell line (HCT-116) is treated with EntF*, no decrease was observed (
Gnotobiotic mouse model. Eight male gnotobiotic C57BL6 mice (housed randomly in three different cages) were treated once with a mixture of three EntF positive E. faecium (i.e. E. faecium LMG23236, T-110 and ATCC 8459) at a concentration of 108 CFU/300 μL per strain. Three days after this pre-treatment, the mice are equally divided into two treatment groups, kept separately per group in separate cages. The following treatment groups were applied (each treatment applied during five consecutive days): (1) The placebo group was treated five consecutive days with the cell medium (BHI). (2) The test group received a mixture of three EntF negative E. faecium strains (108 CFU) suspended in 300 μl of a BHI broth for five consecutive days (i.e E. faecium NCIMB 10415, W54 and LMG S-28935). After the five day treatment, the amount of EntF-producing strains in the different groups were monitored using qPCR for another week without treatment. The entire experiment was performed under gnotobiotic conditions. The faeces (2 droplets/day) of each mice was collected at day 3, 4, 8 and 14, immediately put in suitable, pre-labelled recipients, put in liquid nitrogen and stored at −80° C. Later, the faeces samples were analysed for DNA presence of EntF*. At day 14, the faecal droplets were collected, the mice were sacrificed and the blood and the whole colon (including the content) were obtained. Blood samples were immediately transferred to a 1.5 ml Eppendorf, put on ice for 10 to 30 min and centrifuged at 1,000 g for 10 min per group. The serum was stored at −80° C. and analysed for the presence of EntF* using UHPLC-MS2. Colon contents were immediately transferred to a 15 ml tube, put on dry ice for 1 to 3 hours and stored at −80° C. The contents were removed and analysed for the presence of EntF using UHPLC-MS2. An overview of the experiment is given in
Sample preparation serum. 50 μL of mice serum was mixed with 150 μL of 0.5% formic acid in acetonitrile. After sonication for 5 min and vortexing for 5 sec, the mixture was heated for 30 sec at 100° C. The solution was again vortexed and centrifuged for 20 min at 20,000 g (4° C.). The supernatant was then further purified using solid phase extraction (SPE) on HyperSep C18 plates (Thermo Fisher Scientific, Belgium), which were previously conditioned with acetonitrile and equilibrated with 75% acetonitrile in water, containing 0.375% formic acid. After loading 150 μL of the samples, 120 μL eluent was collected and the organic solvents evaporated using nitrogen for 5 minutes. The resulting solutions were then further diluted with 30 μL of BSA-based anti-adsorption solution, followed by LC-MS analysis.
Sample preparation colon. For EntF analysis in colon content, 50 mg of colon content is suspended in 100 μL of a 2% hydrochloric acid solution which is vortexed and sonicated for 30 seconds. The suspension is again centrifuged for 1 min at 3,000 g and 50 μL of supernatant was heated for 1 min at 100° C. and cooled down for 1 min on ice. This solution was centrifuged again for 1 min at 10,000 g and too 30 μL of supernatant, 900 μL of a 3% DMSO solution in acetonitrile acidified by adding 0.1% formic acid was added (=equilibration solution). After a previously conditioning step with acetonitrile and an equilibration step with the equilibration solution, 900 μL of the diluted sample is loaded on a HILIC amide SPE MonoSpin column. The sample was eluted using a mixture of 75/20/5 (V/V/V) H2O/acetonitrile/DMSO acidified by adding 0.1% formic acid, followed by LC-MS analysis.
LC-MS analysis. EntF and EntF* were detected and quantified on a Waters Acquity UPLC H-class system, connected to a Waters Xevo™ TQ-S triple quadrupole mass spectrometer with electrospray ionization (operated in positive ionization mode). Autosampler tray and column oven were thermostated at 10° C.±5° C. and 60° C.±5° C., respectively. Chromatographic separation was achieved on a Waters Acquity® UPLC BEH Peptide C18 column (300 Å, 1.7 μm, 2.1 mm×100 mm) for serum and colon content samples. The mobile phases consisted of 93:2:5 water:acetonitrile:DMSO (V/V/V) containing 0.1% formic acid (i.e. mobile phase A) and 2:93:5 water:acetonitrile:DMSO (V/V/V) containing 0.10% formic acid (i.e. mobile phase B), and the flow rate was set to 0.4 mL/min. From the samples, a 10 μL aliquot was injected. The gradient program started with 80% of mobile phase A for 30 sec, followed by a linear gradient to 40% of mobile phase A for 7 minutes. Gradient was then changed to 14.2% mobile phase A at 7.5 min, followed by a 30 sec equilibration, before starting conditions were applied. An optimised capillary voltage of 3.00 kV, a cone voltage of 20.00 V and a source offset of 50.0 V was used. Acquisition was done in the multiple reaction monitoring (MRM) mode. The selected precursor ion for EntF was m/z 667.10 with two selected product ions at m/z 949.39 (CE: 22 eV, Y17 fragment) as quantifier and m/z 129.07 (CE: 30 eV, b2 fragment) as qualifier. The selected precursor ion for EntF* was m/z 865.18 with two selected product ions at m/z 202.08 (CE: 36 eV, b2 fragment) as quantifier and m/z 315.15 (CE: 31 eV, b3 fragment) as qualifier.
Statistical analyses. To determine statistical difference in EntF*-copies between the placebo and the test group during the live biotherapeutic potential test of E. faecium, a two-way ANOVA was used. Significant differences (p<0.05; with treatments (column) and time (row) as the two factors; and H0: the average parameter value for the different factor-levels are equal, and no interaction) between the treatments and time were evaluated. As a post-hoc analysis, a bonferroni's multiple comparison test (within each row, compare columns and within each column, compare rows) was applied. Graphpad was used to display the data, determine the statistical differences and calculate the 95% confidence interval mean difference between the test and placebo groups over time. The statistically significant differences of EntF concentrations in the colon and EntF* concentrations in serum were determined using a two-tailed unpaired t test (H0: the average peptide content in the different groups are equal) in Graphpad.
Result gnotobiotic mice experiment. After one day of treatment, a non-significant reduction of 10% EntF*-copies was determined in the test group compared to placebo. However, after five days of treatment (1 week in
In the colon content of the placebo group, that did not receive any treatment, an average concentration of 820 pmol/g (SEM=134 pmol/g) EntF was determined. However, the level of EntF decreased significantly with 59%, to 335 pmol/g (SEM=56 pmol/g), when mice were treated with the test mixture. In
In the serum of the placebo group an average concentration of 96 pM (SEM=8 pM) EntF-metabolite (EntF*) was determined. However, the level of EntF* decreased significantly with 37%, to 61 pM (SEM=11 pM), when mice were treated with the test mixture. In
| Number | Date | Country | Kind |
|---|---|---|---|
| 20166415.8 | Mar 2020 | EP | regional |
| 21150360.2 | Jan 2021 | EP | regional |
This application is a national-stage application under 35 U.S.C. § 371 of International Application No. PCT/EP2021/057976, filed Mar. 26, 2021, which International Application claims benefit of priority to European Application No. 20166415.8, filed Mar. 27, 2020 and European Application No. 21150360.2, filed Jan. 6, 2021.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/057976 | 3/26/2021 | WO |
