Bacterial targets, therapeutics, compositions, kits and methods of use in treating and / or preventing cancer

Abstract

This invention is directed to methods of reducing incidence or reducing severity or preventing progression of or preventing or treating or preventing pathogenesis of a cancer in a subject, wherein said subject is predisposed to, showing early indications of or suffering from cancer, or methods of reducing intracellular bacterial infection in subjects at risk for or suffering from carcinoma, or from pre-neoplastic or metaplastic tissue changes, comprising contacting at least one cell in an affected tissue in said subject with an agent reducing intracellular bacterial infection in affected tissue, reducing localization of intracellular bacteria proximally to nuclei in cells of affected tissue, or a combination thereof. This invention is further directed to methods of personalized optimized diagnosis and therapy in a subject, comprising screening a subject predisposed to, or at risk for cancer and identifying whether the pre-cancerous or cancerous tissue in the subject is intracellularly infected with bacteria, optionally assessing the intracellular localization of the bacteria-containing compartment to determine a distance of same from a nucleus in the cell; optionally assessing the oncogene profile expression in the subject, and further scoring the subject in terms of the prognosis and potential for therapy and then treating the subject to reduce the intracellular bacterial burden, promote re-localization of the bacterial compartment to be at a further distance from the nucleus, successfully eradicate intracellular bacterial infection, prevent or reduce cell-to-cell spread of the bacteria or any combination thereof. Compositions and kits for effecting the methods of this invention are also provided. This invention still further provides a system for identifying patients who would benefit from adjunctive antimicrobial therapies, enhancing treatment outcomes by providing comprehensive microbial feature characterization, including spatial localization of key microbes associated with treatment resistance in an integrated platform with advanced machine learning algorithms, ultimately providing a personalized patient assessment, enhancing treatment outcomes and optimizing therapeutic strategies.

Description

BACTERIAL TARGETS, THERAPEUTICS, COMPOSITIONS, KITS AND METHODS OF USE IN TREATING AND / OR PREVENTING CANCERINTRODUCTION

[0001] Pancreatic ductal adenocarcinoma (PDAC) carries a dismal prognosis. There is an urgent need to find and understand modifiable factors that predispose to PDAC, since most cases are sporadic and arise in individuals without known risk factors. Multiple epidemiological studies link periodontal disease, a chronic inflammatory condition of microbial origin that affects the soft and hard tissues of the oral cavity, to PDAC risk. Bacterial species associated with periodontal disease, such as Fusobacterium micleatum (F. nucleatum) and Porphyromonas gingivalis (P. gingivalis) are detected in human PDAC, and oral bacterial species are found in the fluid of cystic precursors to invasive pancreatic cancer.

[0002] P. gingivalis is an assacharolytic and proteolytic anaerobic bacterium that thrives in the inflamed milieu of periodontal disease, where there is a steady supply of tissue break-down products essential for its survival. High-titer antibodies to P. gingivalis are associated with increased PDAC risk, and salivary carriage of P. gingivalis, even years prior to PDAC diagnosis, predisposes to PDAC development.

[0003] Intracellular bacteria have recently been described as a component of most human cancers, including PDAC. We demonstrated that P. gingivalis survives in human PDAC cell lines, and promotes their proliferation, an effect that is enhanced in hypoxia, a prominent characteristic of PDAC lesions. We now hypothesized that P. gingivalis translocates from the oral cavity to the exocrine pancreas, where it can cooperate with oncogenic mutations to accelerate PDAC development. Mutations in the KRAS gene are hallmarks of PDAC, and considered drivers of tumor initiation and maintenance.

[0004] Indeed, genetically engineered mice (GEM) that express mutant Kras in the acinar cells of the pancreas, develop spontaneous pancreatic intraepithelial neoplasia (PanIN), the most common precursor lesion to PDAC, and low grade PanIN lesions bearing Kras mutations are also found in most adults.

[0005] However, progression to PDAC requires additional environmental or oncogenic insults despite the presence of oncogenic Kras. In humans, PanIN to PDAC progression follows a stepwise accumulation of oncogenic mutations. In mice, combining P53 inactivation with mutant Kras, or induction of pancreatitis in the presence of acinar Kras mutation, drives PanIN to PDAC development.

[0006] While an association between PDAC and P. gingivalis is suggested, the potential for P. gingivalis to promote development of PDAC and whether or not PDAC development and / or pathogenesis in affected individuals can be addressed remains to be seen.SUMMARY OF THE INVENTION

[0007] This invention uniquely links between P. gingivalis chronic persistence in the oral cavity and the development of acinar cell-to-ductal metaplasia in the pancreas, in addition to changes in the pancreatic microbiome.

[0008] This invention also demonstrates that oral cavity application of P. gingivalis accelerated progression of pancreatic intraepithelial neoplasia (PanIN) to pancreatic ductal adenocarcinoma (PDAC) in mice expressing inducible acinar cell oncogenic KRAS, while oncogenic KRAS enabled intracellular survival of P. gingivalis, and intracellular bacteria promoted PDAC cell survival in hypoxic and nutrient-depleted conditions, demonstrating the causal role for P. gingivalis in pancreatic cancer development.

[0009] This invention provides, in some embodiments, therapeutic compounds or combinations, compositions and kits and methods of reducing intracellular bacterial infection in patients at risk for or suffering from carcinoma, or from pre-neoplastic or metaplastic tissue changes, wherein the bacteria are derived from the oral cavity.

[0010] In some embodiments, the therapeutic compounds or combinations, compositions and kits and methods of this invention reduce intracellular P. gingivalis infection. In some embodiments, the therapeutic compounds or combinations, compositions and kits reduce intracellular T. forsythia or F. nucleatum infection. In some embodiments, the therapeutic compounds or combinations, compositions and kits and methods of this invention reduce intracellular bacterial infection with a combination of oral cavity bacterial species, or other deleterious infection, in patients at risk for or suffering from carcinoma, or from pre -neoplastic or metaplastic tissue changes.

[0011] According to this aspect and in some embodiments, the therapeutic compounds or combinations, compositions and kits and methods of this invention reduce intracellular bacterial infection as herein described which thereby prevents, reduces the incidence of, treats, delays onset of, reduces severity of, prevents progression of or otherwise positively impacts patients at risk for or suffering from carcinoma, or from pre -neoplastic or metaplastic tissue changes associated therewith.

[0012] According to this aspect and in some embodiments, the pre-neoplastic or metaplastic tissue changes are in any affected tissue, for example, in any tissue susceptible to precancerous or cancerous events in carcinoma pathogenesis. In some embodiments, the tissue is an epithelial mucosal tissue, which in some embodiments, specifically relates to pancreatic tissue.

[0013] In some embodiments, according to this aspect, the therapeutic compounds or combinations, compositions and kits and methods of this invention specifically contemplate making use of antibiotics that specifically treat, reduce, or positively impact intracellular bacterial infection, in particular, intracellular bacterial infection in precancerous or cancerous cells.

[0014] According to this aspect and in some embodiments, the antibiotic may comprise moxifloxacin, gentamicin, metronidazole or a combination thereof.

[0015] In still other embodiments, according to this aspect, the therapeutic compounds or combinations, compositions and kits and methods of this invention specifically contemplate making use of supplying a chemotherapeutic agent along with the antibiotic therapy, to further enhance the positive effects on precancerous or cancerous affected tissue.

[0016] In still other embodiments, according to this aspect, the therapeutic compounds or combinations, compositions and kits and methods of this invention specifically contemplate making use of supplying an antibiotic as part of a liposome composition, further comprising a cellular targeting ligand, such as, for example, a receptor promoting uptake and / or intracellular trafficking of the bacterial agent infecting the precancerous or cancerous tissue, so as to enhance or otherwise positively impact bacterial clearance, reduced replication, alter intracellular trafficking, reduce intracellular localization proximal to cellular nuclei or any combination thereof. According to this aspect and in some embodiments, the targenting ligand may specifically target the CXCR4 receptor.

[0017] In still other embodiments, according to this aspect, the therapeutic compounds or combinations, compositions and kits and methods of this invention specifically contemplate making use of an inhibitor of intracellular uptake and / or intracellular trafficking of the bacterial agent infecting the precancerous or cancerous tissue.

[0018] In still other embodiments, according to this aspect, the therapeutic compounds or combinations, compositions and kits and methods of this invention specifically contemplate making use of prebiotics or alternate microbiome components to bias the tissue microbiome in the tissue of origin of the cancer to reduce population levels of bacterial components that ultimately localize intracellularly within precancerous or cancerous cells in the tissue.

[0019] This invention provides, in some embodiments, therapeutic compounds or combinations, compositions and kits and methods of specific targeting and / or killing of intracellular bacterial infection in carcinomatous tissue or precancerous tissue manifestations of same, which prevents, reduces the incidence and / or severity of, delays progression of, alters pathogenesis of or otherwise treats cancer pathogenesis and / or progression in a subject.

[0020] According to this aspect and in some embodiments, the therapeutic compounds or combinations, compositions and kits and methods make use of antibiotics, targeted antibiotics,inhbitors or receptors mediating bacterial entry and other therapeutic compounds in a subject with with a malignancy, or cancerous tissue or precancerous tissue, marked by intracellular infection; as well.

[0021] In still other embodiments, this invention provides kits for treating carcinoma, including an antibiotic as herein described, a liposome composition comprising same, optionally wherein the liposomes comprise targeting moieties, such as, for example, an agent specifically interacting with cellular receptors utilized by bacterial species for intracellular localization within cells in affected tissue, optionally further comprising agents that inhibit intracellular penetration by bacterial species that would otherwise infect cells in the affected tissue, or any combination thereof.

[0022] In still other embodiments, this invention provides compositions comprising an antibiotic as herein described, liposomes comprising same, optionally wherein the liposomes comprise targeting moieties, such as, for example, an agent specifically interacting with cellular receptors utilized by bacterial species for intracellular localization within cells in affected tissue, optionally further comprising agents that inhibit intracellular penetration by bacterial species that would otherwise infect cells in the affected tissue, or any combination thereof.

[0023] In still other embodiments, according to this aspect, the subject is further assessed for oncogene expression, and the chemotherapeutic compound may in still other embodiments, be adjusted or otherwise tailored to suit the particular oncogene profile of the subject. In still further embodiments, the aggressiveness of the therapeutic regimen chosen for a particular subject may reflect a consideration of the oncogene profile in the subject.

[0024] In some embodiments, this invention provides compounds or combinations, compositions and kits and methods of use for personalized optimized diagnosis and therary in a subject, the method comprising the step of screening a subject predisposed to, or at risk for cancer and identifying whether the pre-cancerous or cancerous tissue in the subject is intracellularly infected with bacteria, optionally assessing the intracellular localization of the bacteria-containing compartment to determine a distance of same from a nucleus in the cell; optionally assessing the oncogene profile expression in the subject, and further comprising the step of scoring the subject in terms of the prognosis and potential for therapy and then treating the subject to reduce the intracellular bacterial burden, promote relocalization of the bacterial compartment to be at a further distance from the nucleus, successfully eradicate intracellular bacterial infection, prevent or reduce cell-to-cell spread of the bacteria or any combination thereof.

[0025] According tot his aspect, and in still other embodiments, any of the therapeutic approaches described hereinabove for affecting a precancerous lesion or cancerous tissue in a subject or for asubject susceptible to or suffering from preneoplasia or cancer, can also be considered as applicable for use in the personalized optimized diagnosis and therary method described herein.

[0026] In some embodiments, such therapeutic approaches may include administration of any therapeutic, such as an antibiotic, receptor inhibitor as described herein, or microbiome maniputation method as herein described.

[0027] In some embodiments, this invention provides for biasing of the tissue microbiome in tissue susceptible to or marked by pre-neoplasia, metaplasia or cancer pathogenesis, the method comprising contacting an affected tissue in the subject directly or indirectly with a prebiotic material that promotes growth of a healthy microbiome component or in some embodiments, that reduces growth of a bacteria which is associated with intracellular infection in precancerous or cancerous tissue, or in some embodiments, contacting affected tissue in the subject with microbiome strains that capable of colonizing the affected tissue region and establishing growth at such region, and optionally providing additional supplementation to favor the growth of these exogenously supplied microbiome strain.

[0028] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.DESCRIPTION OF THE DRAWINGS:

[0029] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0030] In the drawings:

[0031] Figure 1: P. gingivalis (PG) translocates to the pancreas. (A) Schematic representation of the experimental design: mice were administered 3 oral lavages of 109CFU of PG or carboxymethylcellulose (CMC) carrier and sacrificed three days after the last administration. (B) DNA was extracted from the pancreas, and PG copy number was determined by matching Cq values with a standard curve prepared from serial dilutions of cultured PG DNA. (C) qPCR measurement of PG-specific ISPG1 gene in pancreata of control and PG-infected mice. (D) Quantification of viable PG colonies recovered from the pancreata of control and PG-infected mice. (E) Species-specific 16S rRNA FISH staining of PG (red) in pancreata of control and PG-infected mice, and universal 16S FISH staining (green) showing the total bacterial content. Magnification of PG positive area is shown. (F) qPCR of the bacterial load in the PG-administered group compared to control. Data are presented as mean ± SEM for n=4-5 mice per group. Scale bars indicate 100 pm. Statistical significance is denoted by ***p<0.001. ns, non-significant. (G) Human pancreatic tissue co-stained for PG (red), T. forsythia (purple), and a universal 16S bacterial rRNA FISH probe (green). Magnifications of PG positive areas are shown.

[0032] Figure 2. Chronic exposure to P. gingivalis (PG) induces pancreatic acinar-to-ductal metaplasia (ADM) in WT mice. (A) Schematic representation of the experimental design for chronic PG exposure. Mice were administered PG to the oral cavity 3 times per week for 12 weeks. (B) Representative images of hematoxylin and eosin (H&E) staining of pancreata from mice exposed to PG or carboxymethyl cellulose (CMC) carrier, evaluated by a blinded pathologist (n=10 / group). (C) Alcian blue staining (top) and Sox9 immunofluorescence (bottom) of pancreata from CMC and PG groups. The graphs show the measurement of the number of alcian blue positive ducts or SOX9 positive nuclei per slide (n=10 / group). (D) PG-specific 16S rRNA FISH staining (red) showing PG (white arrows) in areas of ADM. (E) Immunohistochemistry showing PG (brown) in the pancreata of mice administered PG. (F) Universal and PG-specific 16S rRNA FISH of control and PG-infected mice. Magnification of PG positive area is shown. (G) Quantification of the bacterial load in the PG- administered group compared to control (n=4 / group; ns, non-significant). C-F, scale bars 100 pm. **p<0.01. Data are presented as mean ± SEM.

[0033] Figure 3: P. gingivalis (PG) promotes PDAC development in iKC mice. (A) Schematic representation of the experimental design. KrasG12Dwas induced at 8 weeks of age and then PG was administered to the oral cavity three times weekly for 12 weeks. (B) Percentage of acinar area for PG- infected and non-infected mice calculated by image analysis (n=8 iKC, n=9 iKC+PG) (C) Percentage of alcian blue staining area of pancreata of iKC mice and iKC+PG mice measured by image analysis (n=8-9 / group). B-C, Data are presented as mean ± SEM (**p<0.01, ***p<0.001). (D) H&E sections from uninfected iKC mice and PG-infected iKC mice. Representative images of two mice per group are shown for PanIN 1 / 2 (iKC mice) and PDAC (iKC+PG mice). (E) Mice were scored by a pathologist blinded to the group identity for the most severe pathology present (n=8 iKC, n=9 iKC+PG, 2-3 sections were examined per mouse). (F) The percentage of lobular area scored as ADM / PanIN 1 / 2 or PanIN3 / PDAC out of the total area in pancreata of iKC and iKC+PG mice (n=8 iKC, n=9 iKC+PG).(G-H) Representative images of 16S FISH and IHC staining for PG in PanIN and PDAC regions of iKC+PG mice.

[0034] Figure 4: Chronic P. gingivalis (PG) administration alters the pancreatic microbiome in mice. (A) Alpha diversity (Simpson, Shannon and Fisher) of the intrapancreatic microbiome in CMC and PG-administered WT mice. (B) Principal Coordinate Analysis (PCoA) using the Bray-Curtis metric distances to assess the intrapancreatic microbiome beta diversity of WT mice administered PG vs. CMC. (C) Rank correlation feature of different microbial families between PG and CMC administered WT mice. (D) PCoA using Bray-Curtis metric distances to assess beta diversity in PG vs. CMC administered iKC mice. (E) Rank correlation feature of different microbial families between PG and CMC administered iKC mice. (F) Log transformed counts of 3 bacterial families measured from pancreata of WT and iKC mice.

[0035] Figure 5: Intracellular PG survival and reduced ROS in KrasG12D266-6 cells. (A, left) 266-6 cells were infected with PG MOI 100 under normoxic conditions, and proliferation was measured 48 hours after infection (MTS). (A, right) Live cell imaging was used under hypoxic conditions to count 266-6 cells with and without PG MOI 100 infection using a Holomonitor M4 microscope over 48 hours (data not shown). (B) SOX9 and CK19 mRNA levels 24 h after infection of 266-6 cells with PG MOI 100 vs. no infection measured by qRT-PCR. (C) PITC-labeled PG was used to infect 266-6 or PANC02 cells. 24 hours after infection, cells were fixed and nuclei were stained with DAPI prior to visualization using a NIKON confocal microscope at 60X magnification. Inset shows magnification of a PANC02 cell with multiple bacteria. (D) 266-6 KrasWTand 266-6 KrasG12Dcells (doxycycline induced) were infected with PG MOI 100 for 1 h, and then treated with gent / met for 1 hour. 24 h later PG was detected by species-specific 16s rRNA PISH. White arrows indicate intracellular PG. (E) PG Intracellular survival assay of 266-6 cells with or without KrasG12Dactivation. (P) 266-6 KrasWTand 266-6 KrasG12Dcells were infected with PG MOI 100 for 1 h and then either treated (right) or untreated (left) with gent / met. Cell viability was tested 48 hours after infection. (G) 266-6 KrasWTand 266-6 KrasG12Dcells were infected with PG MOI 100 for 1 h and then treated with gent / met. ROS levels were determined using DHR123 48h after infection. All experiments were repeated at least 3 times.

[0036] Figure 6: Intracellular P. gingivalis (PG) protects PDAC cells from nutrient stress- induced reactive oxygen species (ROS) production and cell death. (A) PDAC cells (PANC-1, MIA PaCa-2, BxPC-3, and ASPC1) were cultured in hypoxia and infected with PG (MOI 10, vs. no infection) in full medium 24 hours before medium exchange to DMEM containing glucose (Glue) and glutamine (Glut), or glutamine alone, as indicated. ROS levels (DHR123) were measured after 24 hours. (B) PDAC cells (infected vs. control) were cultured 24 hours after infection in media with orwithout glucose or glutamine as indicated, and cell viability was determined by MTS assay after 6 days. (C) 24 hours after infection with PG (vs. control) PDAC cell lines were cultured in media devoid of glucose and containing limiting concentrations of glutamine. Cell viability was measured by MTS assay at 5 days after transfer to stress medium. (D) Clonogenic assay was performed on surviving cells of 2 different cell lines after growing the surviving cells in replenished nutrient medium. Representative wells are shown and the measurement of crystal violet staining in 10% acetic acid was measured at 590nm absorbance. (E) Live cell imaging of two PDAC cell lines in nutrient stress medium as in D (supp movies 3-4). (F) PG detection by 16S rRNA FISH in PDAC cells at the endpoint of nutrient stress when there is no survival in non-infected cells (PANC-1, 10 days; PANC02, 3 days; BXPC-3, 6 days). (G) PANC-1 cells were infected with PG (MOI 10) for one hour and washed to remove extracellular bacteria but then treated with either gentamicin and metronidazole (gent / met) as in other intracellular survival experiments, or with moxifloxacin (mox) for one hour. Uninfected control cells received either gent / met or mox. Cells were washed and incubated overnight in full medium. The following day medium was changed to ImM glutamine without glucose for 3 more days. Surviving clones of cells were stained with crystal violet. All experiments were performed in hypoxia and repeated at least 3 times. Statistical significance was calculated using Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as mean ± SD.

[0037] Figure 7: P. gingivalis (PG) enhances antioxidant capacity in glucose -deprived PDAC cells. (A) Cells were infected with PG for 24 hours in full medium and then transferred to medium containing 2 mM Glut without glucose. Cells were treated with or without hydrogen peroxide (0.1 mM / 0.01 mM). After 6 hours cell viability was tested using crystal violet. Representative photographs of the wells are shown prior to acidic elution of the crystal violet. (B) Representative images of nutrient- stressed PDAC cells exposed to hydrogen peroxide, with or without FITC- labeled PG infection, counter-stained with DAPI. (C) PANC-1 cells were infected with PG MOI 10 for 1 hour (and then washed and treated with antibiotics to eliminate extracellular bacteria), or treated with 100 mM Trolox (not removed). Cells were then exposed to nutrient starvation (1 mM glut, no glucose) and incubated for 96 hours in hypoxia. Cell viability was measured by crystal violet staining. (D) Canonical and non- canonical glutamine metabolism pathways in PDAC cells. (E) qRT-PCR analysis of gene expression in PDAC cells 48 hours following PG infection vs. control in PANC-1 and MIA-PaCa-2 cells. (F) Measurement of reduced glutathione levels in PDAC cells cultured in medium containing physiological levels of glucose and 2mM glutamine or 2mM glutamine in the absence of glucose, with or without PG infection. (G) PANC-1 cells were plated in 24 well microplates and divided to 3 groups: Control, PG infected cells, or cells were exposed to heat-killed PG. Oxygen consumption rate was measured by seahorse analyzer. (H) qRT-PCR analysis of gene expression in the pancreata of iKC mice chronicallyinfected with PG. (I) Immunofluorescence staining of SLC1A5 expression in PanIN areas of control and PG-infected iKC mice. All experiments were repeated at least two times. Data are presented as mean ± SEM, n=3-5. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (J) Kaplan-Meier survival curve of PDAC patients stratified by SLC1A5 expression levels using the online Kaplan-Meier plotter based on data from the GEO, EGA and TCGA databases.(71)

[0038] Figure 8: The role of CXCR4 in intracellular colonization of PDAC cells by P. gingivalis (PG). (A) CXCR4 expression on PDAC cell lines with or without PG intracellular infection. Expression was measured by qRT-PCR normalized to GAPDH in 3 PDAC cell lines at 48 hours after infection with PG. (B) Immunofluorescence staining of CXCR4 (red) in PANC-1 cells 48 after intracellular infection with PG. (C) FITC-labeled PG (green) co-localization with CXCR4 (red) in PANC-1 cells. The time points shown are 3h and 24h. Nuclei are stained with DAPI. (D) PANC-1 cells were pre -treated with 1 pg / ml of AMD3100 vs non-treated cells prior to infection with PG MOI 10. Cells were treated with gent / met to eradicate extracellular bacteria. Cell lysates were plated on blood agar at 24 hours after infection and CFU were enumerated. (E) Survival of cells treated or untreated with AMD3100 (Ipg / ml) for 30 min before infection with PG MOI 10 for 24h in full medium. The day after infection, cells were exposed to nutrient-deprived medium (ImM Glut, no glucose), and cell viability was measured using crystal violet after 72h. (F-G) Relative gene expression of CXCR4 (F), and immunofluorescent staining of PanIN areas (G) in the pancreata of iKC mice after chronic exposure to PG as in figure 3 A. (H) Relative CXCR4 expression in PDAC cells treated with 1 pg / ml gemcitabine (Gem) for 24 hours. (I) Intracellular PG CFUs from cells untreated or treated with 1 pg / ml Gem for 24 hours before infection with PG. Cells were lysed 24 hours after infection and CFUs were determined. (J) PANC-1 cell survival in nutrient stress. Cells were pretreated overnight with 1 pg / ml of Gem prior to intracellular infection with PG MOI 10. 24 hours after infection the medium was changed to ImM glutamine without glucose for 72 hours. Cell viability was measured by crystal violet. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0039] Supplementary Figures:

[0040] Supplementary Figure 1: Oral inflammation does not impact on P. gingivalis (PG)

[0041] translocation to the pancreas. (A) Representative images of the jaw alveolar bone loss induced by ligature placement for 10 days vs. no ligature control. The vertical line marks the distance from the cemento-enamel junction of the tooth to the alveolar bone crest. (B) Schematic representation of the experimental setting. A silk ligature was placed around the second maxillary molar of mice to induce oral inflammation, and PG vs. CMC was orally lavaged three times during a period of 10 days. Mice were sacrificed three days after the last lavage. (C) PG copy number in the pancreas of mice with ligature-induced oral inflammation lavaged with PG vs. CMC. (D) PG copy number in pancreata ofmice administered PG 3 times as in (B) with or without ligature-induced oral inflammation. Data are presented as mean ± SEM for n=4-5 mice per group, and statistical analysis was performed using unpaired two-tailed t-test.

[0042] Supplementary figure 2: Orally administered T. forsythia (TF) does not translocate efficiently to the pancreas and does not induce pancreatic ADM. (A) Schematic representation of the repetitive administration of 109 CFU TF in CMC vs. TF to WT mice, three times per week over 12 weeks. (B) Quantitative PCR (qPCR) measurement of TF 16S gene abundance in pancreata from CMC and TF-administered mice. Data are presented as mean ± SEM. (ns, non-significant). (C) TF-specific 16S FISH staining in pancreata sections of mice administered CMC vs. TF. Nuclei were stained with DAPI. (D) Hematoxylin and eosin (H&E) staining of pancreata sections from TF-administered mice and CMC controls assessed by a pathologist blinded to the group identity (n=4 / group).

[0043] Supplementary figure 3: Low and high grade PanIN lesions in iKC mice. Representative H&E images of low grade PanIN (PanIN 1 / 2) and high grade PanIN (PanIN3) observed in iKC mice administered P. gingivalis.

[0044] Supplementary Figure 4: Functional pathways differentially enriched by P. gingivalis. The functional profiles of the intrapancreatic microbiomes of control (CMC) vs. P. gingivalis (PG) administered mice were analyzed using Tax4Fun2 based on the 16S rRNA gene sequencing data. Average relative abundance was calculated per treatment and KEGG pathways differentially regulated (FDR corrected p-value<0.02) with reported relevance were displayed in a bar plot using the R package ggplot2 v.3.4.2.

[0045] Supplementary figure 5: P. gingivalis (PG) location in the acinar / ductal cells of wild-type (WT) and iKC mice. Individual distances from labeled PG (double positive for universal and speciesspecific 16S FISH) to nearest nucleus (border of DAPI-stained nucleus) were measured by QuPath (n=4 mice per group, and 4 acinar / ductal fields were measured per mouse). (A) Example of measurements in QuPath. (B) Distribution of distances for each group (mean distance for WT=4.65 pm, and for iKC = 2.44 pm). ****p<0.0001.

[0046] Supplementary Figure 6: Moxifloxacin eliminates intracellular P. gingivalis (PG) in PDAC cells. Panc-1 cells were seeded and subjected to hypoxia for 4 hours, followed by infection with PG at MOI 10. Cells were then treated with either gentamicin / metronidazole (gent / met), or moxifloxacin (mox) for Ih and then washed extensively. After 48 hours, cells were lysed in sterile water and plated on blood agar and intracellular PG colonies were enumerated. The data are represented as the mean ± standard error of the mean (SEM).

[0047] Supplementary Figure 7 : Effect of bacterial co-culture on PDAC cell viability. (A) PANC 1 and MIA PaCa-2 cells were infected with P. gingivalis (PG) strains 33277, W83, or a PG clinicalisolate, treated with gent / met to eliminate extracellular bacteria, and then subjected to nutrient stress (ImM glutamine, no glucose) in hypoxic conditions. Cell viability was measured via crystal violet staining at 96 hours. (B) PANC-1 or MIA PaCa-2 cells were infected with PG 381, F. nucleatum (Fn), or T. forsythia (TF), treated with gent / met, and subjected to nutrient stress conditions as in (A) in hypoxia. Viability was measured by crystal violet after 96 hours. (C) PANC-1 or MIA PaCA-2 cells were co-cultured with A. actinomycetemcomitans (AA) or a clinical isolate of S. oralis, in full medium. Viability was measured at 96 hours. Results in A-C are presented as mean ± SEM and are representative of at least three independent experiments. Statistical analysis was performed using unpaired two-tailed t-test (A and C) and one-way ANOVA (B). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to control (untreated) cells.

[0048] Supplementary Figure 8: Toll-like receptor 2 (TLR2) and CXCR-4 expression across cell lines. (A) PDAC cell lines (as indicated in figure), and 266-6 cells, were analyzed by flow cytometry for TLR2 expression. (B) PDAC cell lines as indicated were analyzed for CXCR4 expression by flow cytometry.

[0049] Supplementary Figure 9: Blocking CXCR-4 reduces intracellular survival of P. gingivalis (PG). (A) Representative images showing BxPC-3 cells (KRASWT) infected with FITC-labeled PG (green) in the presence or absence of AMD3100 treatment. Percentage of PG-positive cells per field was established by counting FITC-positive cells per total cells across 5 high power fields. (B) Survival analysis of cells treated or untreated with AMD3100 (Ipg / ml) for 30 minutes prior to infection with PG (MOI 10) for 24 hours in full medium. Following infection, cells were exposed to nutrient-deprived medium (ImM glutamine, no glucose), and cell viability was assessed after 72 hours using crystal violet staining. Results, presented as mean ± standard error of the mean (SEM), are representative of at least three independent experiments. Statistical analysis was performed using unpaired two-tailed t- test. *p<0.05, ***p<0.001.

[0050] Supplementary Table 1 : Main indication for surgical resection in each of the seven subjects studied.

[0051] Supplementary Table 2: Statistical comparison of feature-level alpha diversity (Shannon, Simpson, and Fisher), and rank correlation of bacterial families associated with the intrapancreatic microbiome of control (CMC) and P. gingivalis (PG) administered mice.

[0052] Figure 9 describes the bacterial localization measurement in pancreata of wild-type (WT) and iKC mice. Individual distances from each bacteria to nearest nucleus were measured in QuPath. Bacteria were labeled using a universal 16S fluorescently-labeled probe and nuclei were stained by DAPI. Sample images are shown in (A) and the distribution of distances is shown in (B). Mean distance of the WT group is 7.3 micron and the iKC group is 5.3 micron..

[0053] Figure 10 describes Bacteria cultured from oral squamous cell carcinoma (OSCC) tumors. (A) Isolation and identification of tumor-associated bacteria. (B) Intracellular CFU recovered in aerobic and anaerobic conditions. (C) Species cultured from different patient tumors (71 total unique species recovered). Red arrow indicates that 9 species were recovered from 6 patients (of 29).

[0054] Figure 11 describes Bacterial load and distance signify tumor vs. adjacent tissue. The universal 16S probe was used to detect bacteria in OSCC tumors. Bacterial load was significantly increased in tumor vs. adjacent epithelium (B-C). The distance between bacteria and nucleus is significantly shortened in tumors vs. adjacent (D, violin plot of pooled measurements of four patients). For each patient the distance is shortened in tumor vs. adjacent tissue (E, lines show matched samples). The distribution of relative frequency of distances, loglO transformed, is shown in (F) for adjacent and tumor tissue.

[0055] Figure 12 describes Bacterial load correlates with de-differentiation. The universal 16S probe was used to detect bacteria in OSCC tumors demonstrating areas with keratin pearls and areas of de- differentiation evidenced by loss of keratin production and greater dysplasia. Bacterial load was significantly increased in areas lacking evidence of keratin production.

[0056] Figure 13 describes Validation of phyla-specific probes. Fluorescently-labeled probes to Bacteroidota, Fusobacteriota, and Actinomycetota, and a universal 16S probe were tested on representative bacterial species from each phylum. Pure cultures of each species were adhered to glass slides and labeled with each probe. Each phyla-specific probe labeled only the representative species of that phylum.

[0057] Figure 14 describes Multi-color phyla-specific 16S FISH of OSCC. (A) Co-fluorescence FISH with universal, and phyla-specific probes. Overlay indicates Fusobacteriota (white arrows), Bacteroidota (yellow arrows), and bacteria belonging to another phyla (red arrows). (B) Scanned OSCC slide labeled for Fusobacteriota (red) and Bacteroidota (green). Magnified regions of interest (RO I) show areas dominated by each of the phyla. Nuclei are counterstained with DAPI.

[0058] Figure 15 describes Phyla specific shifts in distance between tumor and adjacent tissue. The distribution of relative frequency of distances, log 10 transformed, is shown for the phylum Fusobacteriota (A-B) and Firmicutes (C-D) for adjacent and tumor tissue.

[0059] Figure 16 describes a functional study of tumor-derviced bacteria. Colonies from tumor intracellular culture were identified. Bacteria were subcultured and used to infect SCC25 cells. Gentamicin and metronidazole (Gent / Met) were used to eliminiate extracellular bacteria. Non-infected vs. infected cells were irradiated (8 Gy) and then counted and plated at low density. Colony formation was assessed by crystal violet staining at two weeks (A). In (B) 1 hr after irradiation cells were stainedfor yHSAX immunofluorescence to measure DNA DSBs. Nuclei were counterstained with DAPI (blue).

[0060] Figure 17 describes Moxifloxacin reversal of radiation resistance. Tumor isolates (P. gingivalis and C. acnes) were used to infect CAL27 OSCC cells (MOI 10). After 1 hour cells were treated with either Gentamicin and metronidazole (Gent / Met) or moxifloxacin for one hour. Noninfected vs. infected cells were irradiated (8 Gy) and then counted and plated at low density. Colony formation was assessed by crystal violet staining after two weeks (A). Colony number was counted using QuPath (B).DETAILED DESCRIPTION OF THE INVENTION

[0061] This invention provides in some embodiments, compositions, kits, combination therapies, uses and methods for treating, reducing the severity of, improving the outcome of, improving symptoms in, reducing mortality associated with, preventing and / or positively impacting the clinical condition and / or setting of a patient at risk for or suffering from a cancer, such as carcinoma, in particular pancreatic cancer.

[0062] This invention provides in some embodiments, compositions, kits, combination therapies, uses and methods for better diagnosis and / or treatment of a patient at risk for or suffering from a cancer, such as carcinoma.

[0063] As described and exemplified herein, P. gingivalis migrated from the oral cavity to the pancreas in mice and was detected in human PanIN lesions. Repetitive P. gingivalis administration to wild-type mice induced pancreatic acinar-to-ductal metaplasia (ADM), and altered the composition of the intra- pancreatic microbiome.

[0064] Moreover, in iKC mice, P. gingivalis accelerated PanIN to PDAC progression. In vitro, P. gingivalis infection induced acinar cell ADM markers SOX9 and CK19, and intracellular bacteria protected PDAC cells from reactive oxygen species-mediated cell death resulting from nutrient stress. These results demonstrate, inter alia, a causal role for intracellular P. gingivalis in pancreatic cancer development and pathogenesis.

[0065] In certain aspects of the invention and as described and demonstrated herein the association of periodontal disease with pancreatic cancer risk highlights direct bacterial effects as an important factor underlying the association, further indicating that the methods, compositions, kits of this invention that target intracellular bacteria reduces the risk for pancreatic cancer and / or improves anticancer therapies.

[0066] This invention provides, in some embodiments, therapeutic compounds or combinations, compositions and kits and methods of reducing intracellular bacterial infection in patients at risk for orsuffering from carcinoma, or from pre-neoplastic or metaplastic tissue changes, wherein the bacteria are derived from the oral cavity.

[0067] In some embodiments, the therapeutic compounds or combinations, compositions and kits and methods of this invention reduce intracellular P. gingivalis infection. In some embodiments, the therapeutic compounds or combinations, compositions and kits reduce intracellular T. forsythia or F. nucleatum infection. In some embodiments, the therapeutic compounds or combinations, compositions and kits and methods of this invention reduce intracellular bacterial infection with a combination of oral cavity bacterial species, or other deleterious infection, in patients at risk for or suffering from carcinoma, or from pre-neoplastic or metaplastic tissue changes.

[0068] According to this aspect and in some embodiments, the therapeutic compounds or combinations, compositions and kits and methods of this invention reduce intracellular bacterial infection as herein described which thereby prevents, reduces the incidence of, treats, delays onset of, reduces severity of, prevents progression of or otherwise positively impacts patients at risk for or suffering from carcinoma, or from pre-neoplastic or metaplastic tissue changes associated therewith.

[0069] According to this aspect and in some embodiments, the pre-neoplastic or metaplastic tissue changes are in any affected tissue, for example, in any tissue susceptible to precancerous or cancerous events in carcinoma pathogenesis. In some embodiments, the tissue is an epithelial mucosal tissue, which in some embodiments, specifically relates to pancreatic tissue.

[0070] In some embodiments, according to this aspect, the therapeutic compounds or combinations, compositions and kits and methods of this invention specifically contemplate making use of antibiotics that specifically treat, reduce, or positively impact intracellular bacterial infection, in particular, intracellular bacterial infection in precancerous or cancerous cells.

[0071] In some embodiments, this invention provides a method of reducing incidence or reducing severity or preventing progression of or preventing or treating or preventing pathogenesis of a cancer in a subject, wherein said subject is predisposed to, showing early indications of or suffering from cancer, said method comprising reducing intracellular bacterial infection in affected tissue in a subject, reducing localization of intracellular bacteria proximally to nuclei in cells of affected tissue in said subject, or a combination thereof.

[0072] As used herein the phrase "inhibiting" or "treating" refers to reducing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of hyperplasia, or other precancerous or cancer promoting activities or cancer pathogenesis.

[0073] In some aspects, when referring to the prevention of hyperplasia, or other precancerous or cancer promoting activities or cancer pathogenesis, such reference may be with regard to reduction of incidence of the disease on a population level. In some aspects, such reference may be with regard toa patient suffering from a repeat or relapsing disease, where failure to develop full symptomatology, pathogenesis or severity of the disease as previously occurred in such patient, may serve as an indication of true prevention.

[0074] In some aspects, when referring to the prevention of necrosis on a cell or tissue level, same may refer to obvious reduction in classic markers or histopathologic evidence or secreted signals typically associated with necrosis.

[0075] According to one embodiment, treating or inhibiting hyperplasia, or other precancerous or cancer promoting activities or cancer pathogenesis, of a cell or tissue may reduce same by at least about 10 %, by at least about 20 %, by at least about 30 %, by at least about 40 %, by at least about 50 %, by at least about 60 %, by at least about 70 %, by at least about 80 %, by at least about 90 % or by at least about 100 %, as compared to a control cell / tissue of the same type which has not been treated with the agents of the present invention but otherwise has been subjected to the same hyperplastic, or other precancerous or cancer promoting activities or cancer pathogenesis signals as in the treated cells / tissues.

[0076] Referring, for example, to supplementary Figure 6, moxifloxacin clears intracellular P. gingivalis, and Figure 8D, as well as supplementary Figure 9 show that AMD3100 (plerixafor) blocks P. gingivalis intracellular survival, specifically in cancer cells. Furthermore, supplementary Figure 5 shows the distance mapping between the 16S FISH signal of P gingivalis and the border of the nearest nucleus, all of which findings support the described therapeutic compounds or combinations, compositions and kits and methods of this invention.

[0077] This invention provides therapeutic compounds or combinations, compositions and kits and methods of this invention specifically contemplate making use of supplying a chemotherapeutic agent along with the antibiotic therapy, to further enhance the positive effects on precancerous or cancerous affected tissue in a subject.

[0078] As used herein, the term “subject” refers to an animal, preferably a mammal, most preferably a human being, including both young and old human beings of both genders who suffer from or are predisposed to a precancerous or cancerous related disorder or condition.

[0079] This invention also provides compounds, compositions, kits and methods for reducing intracellular bacterial infection in subjects at risk for or suffering from carcinoma, or from pre- neoplastic or metaplastic tissue changes, said method comprising contacting at least one cell in an affected tissue in said subject with an agent reducing intracellular bacterial infection in affected tissue, reducing localization of intracellular bacteria proximally to nuclei in cells of affected tissue, or a combination thereof.

[0080] In any of the uses of the compounds, compositions or kits of the invention, or for any of the methods of the invention representing contemplated embodiments herein, in reference to a precancerous or cancerous affected cell or tissue, in some aspects the pre-cancerous or cancerous tissue is an intraepithelial neoplasia, pre-neoplasia or carcinoma, and in some embodiments, the affected tissue is mucosal tissue. In some embodied aspects, the cancer is pancreatic cancer.

[0081] In other embodiments, the intracellular bacteria referred to herein are derived from the oral cavity of the subject and in some embodiments, the intracellular bacteria are P. gingivalis, T. forsythia or F. micleatum.

[0082] In some embodiments, any of the compounds, compositions, kits and methods as herein described, when referring to reducing intracellular bacterial infection in affected tissue, same may involve contacting at least one cell in said affected tissue with an antibiotic, which in some embodiments, may comprise moxifloxacin, gentamicin, metronidazole or a combination thereof.

[0083] It will be clear to the skilled artisan that antibiotics are commercially available, and numerous options exist that will be more specifically suited for use in eradicating intracellular bacteria, depending on the species of bacteria localized within the affected cell or tissue and any appropriate choice in antibiotic agent is to be considered a contemplated, embodied aspect of the invention.

[0084] In some embodiments, such antibiotic is provided as part of a liposome composition and in still further embodiments, the liposome further comprises a targeting moiety to specifically target said liposome to infected cells in said affected tissue. According to this aspect, and in some embodiments, the targeting moiety specifically targets a receptor enabling bacterial intracellular entry or cell-to-cell spread, for example, and in some embodiments, where reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an agent that reduces the ability of said bacteria to access an intracellular compartment. According to this aspects and in some embodiments, the agent prevents said bacteria from undergoing receptor mediated endocytosis, or in some embodiments, the agent prevents bacteria from interacting with a cell-surface receptor.

[0085] In still other embodiments, reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an agent that alters the traditional intracellular compartment or intracellular trafficking of a compartment in which said bacteria is localized.

[0086] In still other embodiments, the compounds, compositions, kits for use and the methods of this invention contemplate further providing a chemotherapeutic agent to the subject. In some embodimetns, the agents, compositions, etc. further enhances or otherwise positively impacts bacterial clearance, reduces bacterial replication, alters bacterial intracellular trafficking, or reduces bacterial intracellular localization proximal to nuclei or any combination thereof in affected tissue.

[0087] In still other embodimens, the methods of this invention still further provide a prebiotic compound or composition or at least one positive microbiome component species or a combination thereof, which biases the tissue microbiome in the tissue of origin of the cancer to reduce population levels of bacterial components that ultimately localize intracellularly within precancerous or cancerous cells in affected tissue.

[0088] It will be appreciated that each of the compounds as described herein for use in this invention can be administered to the individual per se or as part of a pharmaceutical composition which also includes a physiologically acceptable carrier, or in some aspects, as part of a conjugate, charged particle, liposome or any known carrier in the art, which in turn may, in some aspects, be further formulated as part of a pharmaceutical composition. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism.

[0089] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0090] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0091] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0092] Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0093] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0094] According to an embodiment of the present invention, the pharmaceutical composition is formulated for penetrating a cell membrane. Thus, for example, the pharmaceutical composition may comprise a lipid vesicle.Y1

[0095] Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient (e.g. precancerous or cancerous tissue).

[0096] Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, drageemaking, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0097] Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0098] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0099] For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.[000100] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.[000101] Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.[000102] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.[000103] For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.[000104] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.[000105] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.[000106] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.[000107] The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.[000108] Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of a disorder or prolong the survival of the subject being treated.[000109] The term "pharmaceutically acceptable salt" may refer herein to salts that retain the biological effectiveness and properties of the compounds of this invention and, which are not biologically or otherwise undesirable. In many cases, the compounds of this invention are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.[000110] Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like ; particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.[000111] The term "solvate" herein may refer to a compound in physical association with one or more molecules of a pharmaceutically acceptable solvent. The term “solvate” will be understood to encompass the stated compound, a pharmaceutically acceptable salt of the compound, a solvate of the compound, and a solvate of a pharmaceutically acceptable salt of the compound.[000112] It will be understood that phrases such as “a compound as herein described” or “a compound for use as described” is to be considered to encompass a compound or any pharmaceutically acceptable salt, isomer, N-oxide, isomer or solvate thereof.[000113] Prodrugs and solvates of the compounds of the invention are also contemplated herein. A discussion of prodrugs is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems (1987) 14 of the A. C. S. Symposium Series, and in Bioreversible Carriers in Drug Design, (1987)Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press. The term "prodrug" means a compound (e.g., a drug precursor) that is transformed in v vo to yield a compound of Formula (I) -• (IV) or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (e.g., by metabolic or chemical processes), such as, for example, through hydrolysis in blood. A discussion of the use of prodrugs is provided by T. Higuchi and W. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of die A. C. S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.[000114] In some aspects, the compounds / compositions of this invention comprise liposomes, which in some aspects, specifically target active agents to an intracellular compartment, which in still other embodiments, specifically impacts an intracellularly localized bacterium associated with disease progression, as herein described.[000115] According to this aspect, and in some embodiments, any method known in the art can be used to incorporate a therapeutic agent of some embodiments of the invention into the liposome. For example, the compounds of this invention may be encapsulated within the liposome. Alternatively, it may be adsorbed on the liposome's surface. Other methods that may be used to incorporate a pharmaceutical agent into a liposome of the present invention are those described by Alfonso et al., [The science and practice of pharmacy, Mack Publishing, Easton Pa 19thed., (1995)] and those described by Kulkarni et al.,[ J. Microencapsul.1995, 12 (3) 229-46].[000116] The liposomes used in the methods of the present invention preferably access cellular targets for preneoplastic, hyperplastic or neoplastic pathogenesis. Thus, the liposomes of the present invention may comprise a targeting moiety, including peptide or polysaccharide moieties in their membrane portion. Examples of such peptides include but are not limited to a chemokine such as CXCR4, a cell surface receptor used for bacterial invasion, a transferrin, insulin, IGF- 1 , IGF-2 anti-transferrin receptor antibody, anti-insulin receptor antibody, anti-IGF-1 receptor antibody and anti-IGF-2 receptor antibody.[000117] In order to determine liposomes that are especially suitable in accordance with the present invention a screening assay can be performed such as the assays described in U.S. Pat. Appl. No. 20040266734 and U.S. Pat. Appl. No. 20040266734; and in Danenberg et al., Journal of cardiovascular pharmacology 2003, 42:671-9; Circulation 2002, 106:599-605; Circulation 2003, 108:2798-804.[000118] In some embodiments, the methods of this invention, including those related to prevention, inhibition, treatment, etc., may be effected in-vitro, in-vivo or ex-vivo.[000119] In other aspects of the invention, it is noted that compositions / methods / kits / combinations of the invention contemplate that a similar outcome occurs with multiple cancers, where bacteria associatedwith tumorss are located more intracellularly in the areas of tumor as opposed to healthy tissue, and this is not a hallmark exclusively to pancreatic cancer.[000120] In some embodiments, the cancers may include colon cancer, breast cancer, or prostate cancer. In some embodiments, the cancer may include any carcinoma.[000121] In some embodiments, this invention provides a method of reducing incidence or reducing severity or preventing progression of or preventing or treating or preventing pathogenesis of a cancer in a subject, wherein said subject is predisposed to, showing early indications of or suffering from cancer, said method comprising reducing intracellular bacterial infection in affected tissue in a subject, reducing localization of intracellular bacteria proximally to nuclei in cells of affected tissue in said subject, or a combination thereof.[000122] In other embodiments, the invention provides a method of reducing intracellular bacterial infection in subjects at risk for or suffering from carcinoma, or from pre -neoplastic or metaplastic tissue changes, said method comprising contacting at least one cell in an affected tissue in said subject with an agent reducing intracellular bacterial infection in affected tissue, reducing localization of intracellular bacteria proximally to nuclei in cells of affected tissue, or a combination thereof.[000123] According to these aspects and in some embodiments, the cancer is a carcinoma and in some embodiments, the affected tissue is mucosal tissue, while in some embodiments, the cancer is pancreatic cancer.[000124] According to other asepcts and in some embodiments, the intracellular bacteria are derived from the oral cavity of the subject and in some embodiments, the intracellular bacteria are P. gingivalis, T. forsythia or F. nucleatum. In some embodiments, n reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an antibiotic, while in some embodiments, the antibiotic may comprise moxifloxacin, gentamicin, metronidazole or a combination thereof and in some embodiments, the antibiotic is provided as part of a liposome composition and in other embodiments, the liposome further comprises a targeting moiety to specifically target said liposome to infected cells in said affected tissue. In some embodiments, the targeting moiety specifically targets a receptor enabling bacterial intracellular entry or cell-to-cell spread.[000125] According to other asepcts and in some embodiments, reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an agent that reduces the ability of said bacteria to access an intracellular compartment.[000126] In some embodiments, the agent prevents said bacteria from undergoing receptor mediated endocytosis and in some embodimens, the agent prevents bacteria from interacting with a cell-surface receptor.[000127] In some embodiments, reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an agent that alters the traditional intracellular compartment or intracellular trafficking of a compartment in which said bacteria is localized.[000128] In some embodiments, the methods of this invention as described herein further comprise providing a chemotherapeutic agent to said subject and in other embodiments of the methods of this invention, such method further comprises exposing the affected cells or tissue to radiation.[000129] In still other embodiments, according to these aspects, the agent enhances or otherwise positively impacts bacterial clearance, reduces bacterial replication, alters bacterial intracellular trafficking, or reduces bacterial intracellular localization proximal to nuclei or any combination thereof in affected tissue.[000130] In still other embodiments, according to these aspects, the method provides a prebiotic compound or composition or at least one positive microbiome component species or a combination thereof, which biases the tissue microbiome in the tissue of origin of the cancer to reduce population levels of bacterial components that ultimately localize intracellularly within precancerous or cancerous cells in affected tissue.[000131] In some embodiments, this invention further provides a method of personalized optimized diagnosis and therapy in a subject, said method comprising the step of: screening a subject predisposed to, or at risk for cancer and identifying whether the pre-cancerous or cancerous tissue in the subject is intracellularly infected with bacteria, optionally assessing the intracellular localization of the bacteria- containing compartment to determine a distance of same from a nucleus in the cell; optionally assessing the oncogene profile expression in the subject, and further comprising the step of scoring the subject in terms of the prognosis and potential for therapy and then treating the subject to reduce the intracellular bacterial burden, promote re-localization of the bacterial compartment to be at a further distance from the nucleus, successfully eradicate intracellular bacterial infection, prevent or reduce cell-to-cell spread of the bacteria or any combination thereof.[000132] In some embodiments, the methods as herein described may comprise any embodied aspect of any method as described herein.[000133] In other embodiments, the method further comprises assessing intracellular infection and intracellular localization of the bacteria-containing compartment as an indicator of cancerous versus non-cancerous tissue in situ and in some embodiments, the method further comprises assessing intracellular infection and intracellular localization of the bacteria-containing compartment as an indicator of aggressivity of cancerous tissue.[000134] In still other embodied aspects, this invention provides a method of personalized optimized diagnosis and therapy in a subject, said method comprising the step of: screening a subject predisposedto, or at risk for cancer and identifying whether the pre-cancerous or cancerous tissue in the subject is intracellularly infected with bacteria and assessing the intracellular localization of the bacteria- containing compartment to determine a distance of same from a nucleus in the cell, wherein said distance calculated represents a mean distance value of a threshold percentage of the bacteria within a nearest nuclear boundary of a cell in a plurality of cells to establish said distance value.[000135] According to this aspect and in some embodiments, the method further comprises correlating the distance calculated with histopathologic features indicative of cancer and in some embodiments, the method further comprises establishing a score reflective of the distance calculated and histopathologic feature severity, which score indicates tumor pathogenesis severity. In still other embodiments, the score is further refined using machine learning to arrive at more reliable values obtained for the distance calculated, histopathologic features identified or a combination thereof.[000136] In other embodied aspects, the method refines the determination of the of the cancerous tissue area versus adjacent non-cancerous tissue, or in some embodiments, the score reflects a likelihood of cancer recurrence in a subject. In some embodiments, the score reflects a likelihood of response to an immunotherapy in a subject. In still other embodiments, the score reflects a likelihood of a need for adjunctive therapy for the subject. In still other embodiments, the score improves assessment of a treatment plan for a given subject.[000137] In some embodiments of the methods of this invention, the methods may comprise assessment of intracellular bacteria enablement of cancer cells to resist treatment modalities such as chemotherapy, immunotherapy, and ionizing radiation.[000138] In other embodiments, a bacterial localization signal (BLS) is defined through the methods / kits / compositions herein, as relating to the distance measurement of bacteria to nearest nuclear border across a tissue or cell preparation. According to this aspect, the BLS distance can be log transformed to address non-normality in the continuous data and enable statistical analyses, to enhance the reliability for example of the methods of the invention.[000139] Further according to this aspect, the mean distance across taxa and cutoff values of % bacteria within a specific distance for each taxa (e.g. 3 microns) can be used to define tumor characteristics. This information is integrated, for example, with number of particular bacteria within specific distance per number of cells in that region [e.g. score of x#bacteria at <3 microns / thousand cells, to differentiate pathology states.][000140] Thus, some embodied aspects of the invention will comprise the use of a scoring system which in turn may correlate with histopathologic features, which can be improved, for example, via implementation of machine learning / Al algorithms as informatic tool for the pathologist and the treating clinician, as will be appreciated by the skilled artisan.[000141] According to this aspect, and in some embodiments, such scoring system may be useful as a diagnostic aid for pathologist to define areas of tumor in cases of indecision, or in some embodiments, such scoring system may be useful as a prognostic tool to determine likelihood of recurrence and therefore need for follow-up plan including surgery. In other embodiments, such scoring system may be useful as part of arriving at a personalized treatment plan for a patient / subject, and in some embodiments, such scoring system may be useful as part of predicting likelihood of response to immunotherapy, and in some embodiments, such scoring system may be useful as part of arriving at a determination, which patients should be treated with adjunctive treatments that target the tumor microbiome and specifically intracellular bacteria.[000142] It will be appreciated that regarding any of the methods or uses as described herein or kits and combinations and compositions for addressing same, that the subject may be prediscposed to, at risk for or suffering from any cancer and in particular any embodiment as described herein with regard to the compounds, compositions, kits, therapies, etc. is applicable for every method and use as described herein in any embodiment or combination of embodiments as appropriate.[000143] In still other embodiments, the methods may comprise the step of assessing the number of microbial taxa that can be tested in the same slide, which is not to be limited, for example, by using techniques that are available for multiple rounds of hybridization - imaging - probe removal - rehybridization with new set of probes - imaging, as will be appreciated by the skilled artisan. This can be done as sequential FISH or using a combinatorial approach. Therefore, all bacterial taxa of interest or other microbes such as fungi can be determined in the tumor section.[000144] According to this aspect, and in some embodiments, certain considerations may be effected regarding probe design, the implementation of which will be readily known to the skilled argument. For example, and representing embodied aspects, the FISH technique was demonstrated in the Examples section herein, using probes targeting the 16S rRNA, but it will be appreciated that probes can be designed to any DNA or RNA target, and specificity of detection of particular taxa can be increased by designing multiple probes to the same taxa.[000145] Further according to this aspect and representing some embodiments, probe chemistry can be enhanced by using locked nucleic acids (LNA) or other techniques known to those in the art.[000146] In some embodiments, techniques making use of formalin-fixed and paraffin embedded (FFPE) sections were demonstrated / exemplified herein, the technique can be applied to tissue sections prepared using other methods (e.g. fresh frozen), or to cytology samples such as brush biopsies or cell mounts from biological fluids (blood, urine, etc.).[000147] This invention also provides a kit for reducing incidence or reducing severity or preventing progression of or preventing or treating or preventing pathogenesis of a cancer in a subject, whereinsaid subject is predisposed to, showing early indications of or suffering from cancer, said kit comprising an antibiotic agent or a liposome composition comprising an antibiotic agent that specifically enhances or otherwise positively impacts intracellular bacterial clearance, reduces intracellular bacterial replication, alters bacterial intracellular trafficking, or reduces bacterial intracellular localization proximal to nuclei or any combination thereof.[000148] In some embodiments, according to this aspect, the liposome further comprises a targeting moiety to target said liposome to a cell in an affected tissue infected with intracellular bacteria and in some embodiments, the kit further comprises a chemotherapeutic agent.[000149] In some embodiments, the antibiotic comprises moxifloxacin, gentamicin, metronidazole or a combination thereof.[000150] In still further embodiments, the composition for reducing incidence or reducing severity or preventing progression of or preventing or treating or preventing pathogenesis of a cancer in a subject, wherein said subject is predisposed to, showing early indications of or suffering from cancer, said composition comprising a liposome comprising an antibiotic agent that specifically enhances or otherwise positively impacts intracellular bacterial clearance, reduces intracellular bacterial replication, alters bacterial intracellular trafficking, or reduces bacterial intracellular localization proximal to nuclei or any combination thereof, wherein said liposome further comprises a targeting moiety to target said liposome to a cell in an affected tissue infected with intracellular bacteria.[000151] In some embodiments, the antibiotic comprises moxifloxacin, gentamicin, metronidazole or a combination thereof.[000152] In some embodiments, the methods of this invention may be uniquely carried out by sophisticated automated systems, as will be appreciated by the skilled artisan.[000153] For example and in some embodimens, for assessing tumor intracellular microbiota, a system may be constructed, such as a Tumor Intracellular Microbiota Scanner (TIMScan) system.[000154] In some embodiments, such system may comprise:• A chamber sized to accommodate a glass slide or coverslip onto which cells or tissue may be deposited for assay, which cells or tissue suspected of comprising intracellular bacteria of interest and at risk for pre-neoplasia or neoplasia;• A robotics assembly, which assembly can operationally connect and optionally re-connect said chamber to a labelling module, a washing module and a scanning module;• A labelling module which can apply reagent to said chamber to label specific components of said cells or tissue;A washing module which can apply wash solution to said chamber to remove excess and unbound label;• A scanning module, which comprises a microscope;• A processor;• A non-transitory computer-readable storage medium having computer executable instructions stored thereon, which computer executable instructions when executed by the processor case the processor to perform: o Acquiring and storing data collected by said scanning module; o Calculating a BLS distance from the acquired data in said scanning module in cells or tissue suspected of comprising intracellular bacteria of interest and at risk for pre-neoplasia or neoplasia; and o Scoring the cell or tissue sample based on the BLS distance and histopathologic analysis from the acquired data, and optionally providing as an output a treatment recommendations based on said scoring.[000155] In some embodiments, the labelling module and washing module may be provided as part of a microfluidics chamber operationally connected to the robotics assembly and scanning module.[000156] As will be appreciated by the skilled artisan, there are numerous ways in which a chamber housing a glass slide or coverslip onto which cells or tissue may be deposited for assay, can be processed, via automated methodology. For example, the chamber may be self-contained, such as in microfluidic assay chambers, where reagents may be applied to the chamber to for example, label certain components of the cells or tissue be assessed. For example, and in some embodiments, as the cells or tissue being assessed in certain aspects of this invention will comprise intracellular bacteria, as herein described, in particular, for example P. gingivalis, T. forsythia or F. nucleatum , or for example, Bacteroidota and / or Fusobacteriota, reagents as described herein can be readily applied to the chamber, and wash solution may be applied, so that unbound / unreacted label may be removed, and such reagent and wash solution applications may be conducted in series, repeating applications as needed or desired. In some embodied aspects, multiple wash steps may be undertaken, as well.[000157] In some embodiments, the labelling module applies a fluorescence in situ hybridization (FISH) probe or probes as described herein.[000158] In some embodiments, the cell or tissue being evaluated is derived from a sample from a subject suspected to be at risk for pre-neoplasia or neoplasia. According to this aspect and in some embodiments, the cells or tissue may therefore also be labelled and assessed for cyto / histopathologic changes consistent with pre-neoplasia or neoplasia. According to this aspect, for example, the cells ortissue may be probed using common H&E staining, if desired, and in some embodiments, the systems of the invention may process separately two successive sections from cell or tissue samples isolated from the subject so that the successive sections can be probed in parallel using labels in a first section for fluorescence microscopy, and the successive section is probed for light microscopy cyto / histopathologic analysis, which image collection by the scanning module and system analysis can then in some aspects still score the sample, in consideration of the collected data from the successive assayed cells or tissue.[000159] As will be appreciated therefore the scanning module may be equipped to assess via light micrscopy, flurescence microscoy, including confocal microscopy, as desired.[000160] Referring now to Figure 18, a non-limiting example of elements of the systems of this invention is described, whereby the sample is applied to a microfluidic chamber operationally connected to a fluorescent microscope scanner, where the labelling module applies FISH probes of a first set, and the washing module enables washing and de-hybridization conditions, so that signal acquired by the scanning module can then be removed, whereby a further FISH second probe set may be applied, by the labelling module, scanned, and then the washing module enables washing and de-hybridization conditions, so that second signal acquired by the scanning module can then be removed, as well, to allow for multiple signal acquiring by the scanning module, which through the analysis can then be compiled to provide data for scoring as described herein.[000161] It will be appreciated that Figure 18 represents one embodied step-wise description for the application of the systems of this invention, however, other steps and analysis procedures may be conducted, as well.[000162] In some embodiments, Patients undergoing radiation therapy, chemotherapy, and immune checkpoint inhibitors frequently develop resistance to treatment. Accurate prediction of resistance to specific therapies would enable tailoring personalized treatment regimens, thereby minimizing toxicity and maximizing efficacy. This approach not only enhances the therapeutic advantage but also fosters a deeper understanding of the mechanisms behind resistance. Such insights would empower the integration of combination therapies designed to prevent or mitigate the development of resistance, ultimately leading to improved patient outcomes and advancing the field of oncology.[000163] In some embodiments, advanced characterization of tumor microbial features is achievable using the systems of this invention, which may further leverage machine learning to address critical unmet needs in oncology. According to this aspect and in some embodiments, the system assesses resistance phenotypes across various treatment modalities, empowering clinicians to make more informed therapeutic decisions. Furthermore, and representing further embodied aspects of the invention, the system serves as a robust platform for identifying patients who would benefit fromadjunctive antimicrobial therapies, enhancing treatment outcomes. Still further the systems of the invention and methods of use thereof provide for a transformative approach in personalized cancer care, potentially improving patient outcomes and revolutionizing oncology treatment strategies.[000164] According to this aspect and in some embodiments, the systems of the invention enable comprehensive microbial feature characterization, including the spatial localization of key microbes associated with treatment resistance. Still further embodied aspects of the systems and uses thereof integrate tumor-type specific bacterial targets with advanced machine learning algorithms, so that the systems of the invention can provide an output that essentially is a personalized patient assessment. In still further embodied aspects, therefore, the systems and uses of same as described herein empower clinicians to make informed decisions at crucial points in patient care, ultimately enhancing treatment outcomes and optimizing therapeutic strategies.[000165] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".[000166] The term “consisting of' means “including and limited to”.[000167] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.[000168] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.[000169] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.[000170] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a secondindicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.[000171] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. [000172] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.[000173] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES:[000174] Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion.[000175] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freemanand Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.Materials and methods[000176] Cell culture: Human pancreatic ductal adenocarcinoma (PDAC) cell lines Panc-1, MIA-PaCa-2, AsPC-1, BxPC-3, and murine PDAC cell line Panc02, were cultured in Dulbecco's Modified Eagle Medium (DMEM) or RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 U / mL penicillin and 100 pg / mL streptomycin. For nutrient starvation assays, the cells were cultured in DMEM or RPMI-1640 media lacking glucose and glutamine and supplemented with 10% dialyzed FCS, penicillin, streptomycin, and varying concentrations of glutamine or glucose as indicated. 266-6 cells, and 266-6 KrasG12DTet on / off murine acinar cells were previously described. (Husanie H, Abu-Remaileh M, Maroun K, Abu- Tair L, Safadi H, Atlan K, et al. Loss of tumor suppressor WWOX accelerates pancreatic cancer development through promotion of TGFbeta / BMP2 signaling. Cell Death Dis. 2022;13(12):1074.) To culture 266-6 cells, plates were coated with 0.1% gelatin type A and incubated at 4°C for 10 minutes. The plates were then washed, and 266-6 cells were seeded in DMEM medium supplemented with 10% FCS, 1% glutamine, 1% non-essential amino acids, 1% penicillin / streptomycin, and 1% sodium pyruvate. Other cell culture reagents (antibiotics, AMD3100, gemcitabine) were from Sigma. To induce KRAS mutation in 266-6 KrasG12Dcells, doxycycline was added to the medium at a concentration of 2 pg / mL and incubated over-night.For cell viability experiments, 1000-4000 cells were plated in 96-well plates and allowed to adhere overnight at 37°C and 5% CO2. The next day, the cells were incubated under hypoxic conditions for 4 hours before infection with bacteria at the indicated MOI for 1 hour. After infection, the cells were treated with gentamycin and metronidazole (gent / met) for 1 hour to eradicate extracellular bacteria. The cells were then washed and incubated under hypoxic conditions in medium containing penicillin and streptomycin, but without gent / met. After the required days of the experimental setting, cell viability was measured using the MTS assay (Promega CellTiter 96® AQueous One Solution Cell Proliferation Assay, Cat No. G5430, Promega, Madison, WI, USA) according to the manufacturer's instructions. Alternatively, survival was assessed using crystal violet staining. At the assay endpoint, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Next, cells were washed twice with phosphate-buffered saline (PBS) and stained with 0.1% crystal violet for 30 minutes. The excess crystal violet was removed by washing the wells with distilled water, and images of the wells were captured using a Nikon camera. To assess cell number, the crystal violet was solubilized with 10% acetic acid for 30 minutes at room temperature, and absorbance was measured at 590 nm.[000177] Bacterial strains and growth conditions: Unless otherwise stated, experiments with Porphyromonas gingivalis were conducted using ATCC strain 381. All P. gingivalis strains (381, 33277, W83, and clinical isolate), and Fusobacterium nucleatum strain 23726, were grown in reduced Wilkins broth (Oxoid, Basingstoke, UK) under anaerobic conditions (AnaeroJarTM, Oxoid) at 37 °C. The P. gingivalis and Streptococcus oralis clinical isolates were obtained from the oral cavity of a patient with oral squamous cell carcinoma (under Hadassah Medical Organization approval number HMO 0629-20). Tanerella forsythia (T. forsythia) strain 43037 was grown in brain heart infusion (BHI) medium supplemented with 0.5% peptone, 0.5% yeast extract, 5% fetal calf serum, 5pg / ml Hemin, 0.5pg / ml vitamin K, and lOpg / ml N-acetyl muramic acid. Aggregatibacter actinomycetemcomitans strain 275253 was grown in BHI medium supplemented with 0.6% yeast extract, 0.8% dextrose, and 0.4% sodium bicarbonate and was grown under aerobic conditions. To label bacteria with FITC, P. gingivalis was incubated with 0.1 mg / mL FITC (Sigma) in carbonate buffer (pH 9.5) for 20 min at room temperature, and then extensively washed.[000178] P. gingivalis intracellular survival: PDAC cells (5 x 10A5 - l x 10A6) were seeded onto a 6-well plate and incubated in hypoxic conditions (1% 02, 5% CO2) for 4 hours. The cells were then infected with P. gingivalis at a multiplicity of infection (MOI) of 10 for 1 hour. To eradicate extracellular bacteria, the cells were treated with gentamicin (0.3 mg / mL) andmetronidazole (0.22 mg / mL) for 1 hour. After treatment, the cells were washed with phosphate - buffered saline (PBS) and incubated in medium supplemented with penicillin / streptomycin (P / S) without gent / met for the indicated time. To lyse the infected cells, sterile ice-cold ddthO was added to the wells and the plate was incubated for 20 minutes. The lysate was then collected and plated on blood agar plates and incubated in anaerobic conditions for 2 weeks. Colonyforming units (CFUs) were then enumerated to determine the intracellular survival (ICS) of P. gingivalis.[000179] Fluorescence in situ hybridization: Fluorescence in situ hybridization (FISH) was performed to detect bacterial species in tissue samples or cells. Tissue sections were deparaffinized in xylene and rehydrated through a series of ethanol washes. Slides were then washed with phosphate -buffered saline (PBS) and incubated with fluorescently labeled probes. In this study three probes were used: a universal 16S rRNA probe labeled with Cy3 (EUB 338), a P. gingivalis -specific probe (POGI) labeled with Cy5, and a T. forsythia-specific probe labeled with Cy2.(65) All probes were diluted to a final concentration of 1:100 in pre-warmed hybridization solution (0.9 mM NaCl, 20 mM Tris / HCl, pH 7.3, 0.01% SDS) and applied to the tissue sections, which were then covered and incubated in a humid chamber at 46 °C for 3.5 hours. After hybridization, the slides were washed in sterile water, stained with DAPI, and washed again three times with PBS. A non-specific 16S rRNA probe was used as a negative control. Images were captured using a Nikon TL microscope. To visualize P. gingivalis in vitro, cells were fixed in 4% formaldehyde and FISH probe was applied at a concentration of 5 pg / mL in hybridization solution (0.9 mMNaCl, 20 mM Tris / HCl, pH 7.3, and 0.01% SDS). The cells were incubated in a humid chamber at 46°C for 3.5 hours in the dark. After hybridization, the cells were washed for 15 minutes in wash buffer (0.1 M Tris-HCl [pH 7.2], 0.18 M NaCl, 0.05 M EDTA, and 0.005% SDS [wt / vol]) and then stained with DAPI at a concentration of 1 pg / mL for 5 minutes. The cells were then washed for 10 minutes in wash buffer and examined under a Nikon-TL microscope. QuPath was used to quantify the distance of the P. gingivalis FISH signal from the nuclear signal. A script was developed within QuPath to automate the measurement of the distance from the center of bacteria co-labeled with the universal EUB 338 probe and the P. gingivalis-specific POGI probe, to the border of the nearest DAPI-stained nucleus. Negative values were assigned when the bacterial signal overlapped with the nuclear signal.[000180] Mice: C57BL / 6 female mice were from Envigo (Rehovot, Israel) and were used for all experiments involving wild-type (WT) mice. / <ra.v+ / LSL-G I 2D; Ptfla-CreER mice (iKC, backcrossed to C57BL / 6 background) were a gift from the laboratory of Prof. Ittai Ben-Porathand were maintained by breeding an iKC female mouse with a C57BL / 6 wild-type male mouse. All mice were housed at the specific -pathogen-free unit of Hebrew University (ALAAC approved) in the same room with identical handling (co-caging was avoided to prevent P. gingivalis transfer between groups). The institutional animal care and use committee of the Hebrew University of Jerusalem approved all experiments (approval #MD- 19- 15985-3) which complied with the guidelines of the National Research Council Guide for the Care and Use of Laboratory Animals (NIH Publication no. 85-23, revised 1996).[000181] P. gingivalis oral application: P. gingivalis strain 381 was grown overnight in reduced Wilkins broth under anaerobic conditions, and washed extensively in PBS. CFU were determined based on optical density (OD 0.1 equivalent to IO10CFU). For all oral applications of bacteria, mice were administered 109CFU P. gingivalis prepared in 100 microliters of 2% carboxymethylcellulose (CMC):PBS. The inoculum was delivered to the oral cavity via a 22G round-tipped feeding needle.[000182] Human samples: FFPE sections of pancreatic tissue were obtained from patients who underwent surgical resection of intrapancreatic mucinous neoplastic lesions. Approval for the study was obtained from the Hadassah Medical Center Helsinki committee (#HMO-0150-21 and #HMO-0845-20).[000183] Reactive oxygen species (ROS) detection: Cells were seeded in Greiner 96 well Black plates at a density of 25,000 cells per well and allowed to attach overnight. The following day, cells were treated as indicated, and then incubated with 10 p M DHR123 for 30 minutes at 37°C in the dark. After washing with PBS, fluorescence intensity was measured using a Tecan plate reader with excitation and emission wavelengths of 500 nm and 536 nm, respectively.[000184] Identification of P. gingivalis in pancreatic tissue: Following euthanization, the entire skin surface was sterilized with 70% ethanol and 2% chlorhexidine gluconate. Pancreata were isolated aseptically and pancreatic DNA was extracted and purified using the Qiagen Blood and Tissue Kit according to the manufacturer's instructions. Briefly, pancreatic tissue was homogenized in lysis buffer and DNA was extracted using spin columns. DNA purity and concentration were determined using a NanoDrop spectrophotometer. The P. gingivalis copy number was determined using a quantitative PCR (qPCR) assay targeting the P. gingivalis- specific ISPG1 gene.(Hajishengallis G, Liang S, Payne MA, Hashim A, Jotwani R, Eskan MA, et al. Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement. Cell Host Microbe. 2011;10(5):497-506.) The PCR reaction was performed using the following primers: F-GGCTCCCCCTTGATTTTCTC and R-AAGGAGGTGGAGGGAAGAGGA. A standard curve was generated from serialdilutions of cultured P. gingivalis DNA and used to calculate the copy number of P. gingivalis DNA in the pancreas samples. To grow bacteria from the pancreas, aseptically collected pancreata were homogenized in a bullet blender tissue homogenizer (Next Advance) in PBS / TWEEN 20. The homogenate was plated on blood agar plates and incubated in anaerobic conditions for 2 weeks. Plates were assessed for black-pigmented colonies (a characteristic of P. gingivalis) which were determined to be P. gingivalis by colony PCR and Sanger sequencing. Briefly, colonies were picked and boiled in sterile water to extract DNA. Following DNA extraction, PCR amplification was conducted using P. gingivalis-specific 16S primers (F-ACCCTTTAAACCCAATAAATC and R-ACGAGTATTGCATTGAATG).[000185] Ligature-induced periodontitis: 8-12 week old C57BL / 6 female mice (Envigo) were anesthetized with Ketamine (80 mg / kg) and Xylazine (10 mg / kg) for the placement of 0.5 mm silk ligatures around the second maxillary molar tooth (Roboz Surgical Instrument Co., MD, USA). Ligatures were tied gently and left in position for the entire experimental period. Mice were euthanized on day 10 after ligature placement. Maxillae were extracted, autoclaved in water at 15 psi for 10 min and brushing was used to remove any remaining soft tissue. Samples were then stained with 1 % methylene blue for 1 min to allow for visualization of the periodontal bone. Periodontal bone heights were assessed under a Nikon SMZ800 stereomicroscope using a 40 x objective and bone heights were measured using NIS -Elements software (Nikon Instruments, Inc.). Measurements were taken at three different points around the second molar measuring the distance between the alveolar bone crest to the cementoenamel junction, and the mean value was calculated for each mouse.[000186] RNA extraction and qPCR: Tissues and cells were homogenized in Trizol (Sigma) and RNA was purified according to the manufacturer’s instructions. cDNA was synthesized using the Maxima H Minus RT kit (Thermo Scientific). qPCR was performed in a 10 pL reaction volume containing SYBR Green PCR mix (Thermo Scientific), 500 nM of each genespecific primer, and 1 pL of cDNA template. The reaction conditions used were as follows: 10 min at 95°C, 40 cycles of 15 s at 95°C, and 60 s at 60°C. Reactions were run in triplicate on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). Gene expression levels were normalized to the housekeeping genes 18S or GAPDH using the comparative threshold cycle (Ct) method.[000187] iKC mouse model : Male and female iKC mice were randomly assigned to CMC or P. gingivalis groups. Recombinase activation to induce the KRAS mutation was performed at age 6-8 weeks by tamoxifen administration (400 mg / kg s.c. in corn oil) twice over consecutivedays. The tamoxifen was first dissolved in corn oil at a concentration of 20 mg / mL, and then the solution was filtered using a 0.22 gm filter before being injected.[000188] Live cell imaging: A HoloMonitor M4 digital holographic cytometer (DHC) from Phase Holographic Imaging (PHI, Lund, Sweden) was placed in a Don Whitley H35 Hypoxystation (don whitley Scientific) maintained at 94%N2 / 5%CO2 / 1%O2 and 37°C. Timelapse phase imaging, as well as image processing, segmentation, and analyses, were performed using the Hstudio software package (PHI). Cells were plated in 96 well plates and each well was imaged in 3-5 areas every 60-90 minutes over 48-72 hours. The Cell Count module in HStudio was used to segment the phase images per well, followed by the object identification and cell counting modules in HStudio. Multiple images of the same gap and multiple wells were analyzed to assess reproducibility.[000189] P.gingivalis immunohistochemistry: FFPE slides were stained with the P. gingivalis- specific gingipain antibody clone 61BG1.3 developed by R. Gmuer, and obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Sections were deparaffinized and rehydrated and antigen retrieval was performed by microwave heating the tissue sections in a EDTA buffer (pH=8). The sections were blocked and then incubated with the primary antibody overnight at 4°C. After washing, sections were incubated with HRP- conjugated anti-mouse antibody for 2 hours. Enzyme detection was achieved using the DAB peroxidase kit (Thermo) and slides were counterstained with hematoxylin.[000190] Histology immunofluorescence and immunohistochemistry: Immunofluorescence was used to study the expression of CXCR4, SOX9, and SLC1A5. FFPE sections were deparaffinized and rehydrated. Antigen retrieval was then performed by microwave heating of the slides. Non-specific binding was blocked using 5% bovine serum albumin (BSA) and 10% FCS in phosphate-buffered saline (PBS). The tissue sections were then incubated with primary antibodies against each antigen overnight at 4°C. After washing, the sections were incubated with secondary antibodies conjugated to fluorophores and counterstained with DAPI. For Alcian blue staining, slides were incubated with alcian blue (Abeam) for 30 min at room temperature and then counterstained with hematoxylin. Alcian blue and H&E stained slides were scanned using a 3D Histech scanner (for H&E slides) or an Aperio AT2 scanner (Leica, Alcian blue stained slides), and ADM / PanIN / PDAC lesions, acinar or alcian blue areas were measured with Qupath image analysis. (Bankhead P, Loughrey MB, Fernandez JA, Dombrowski Y, McArt DG, Dunne PD, et al. QuPath: Open source software for digital pathology image analysis. Sci Rep. 2017;7(l): 16878. ) To assess ADM, PanIN and PDACpathologies in mice, H&E stained slides were evaluated by a pathologist blinded to the group assignment.[000191] Live cell metabolic profiling: PANC-1 cells were plated in 24 well plates and infected with P. gingivalis MOI 10, treated with gent / met, and then incubated in hypoxic conditions for 24 h. Profiling was immediately performed in a Seahorse XF24 analyzer (Agilent) using the Seahorse XF Cell Mito Stress Test. The results were normalized for the cell number per well by measuring protein concentration (Bio-Rad protein assay) after lysis in RIPA buffer containing protease and phosphatase inhibitors.[000192] Flow cytometry: 0.5 to 1 x 106PDAC cells were plated in 6-well plates. Cells were incubated in full medium in hypoxic or normoxic conditions for 24 h. The cells were collected by enzymatic detachment with accutase solution (PromoCell GmbH). For total CXCR4 detection, cells were fixed with 2% PFA at 37°C for 10 min, washed with PBS, and permeabilized for 30 min on ice with permeabilization buffer (BD Phosflow™ Perm Buffer III, BD Biosciences). Cells were then washed at 500g with FACS buffer (PBS containing 2% FCS). For non-permeabilized samples accutase was deactivated by full medium and samples were resuspended in FACS buffer. FC-blocker (FcR Blocking Reagent, human, Miltenyi Biotec) was applied for 20 min on ice prior to incubation with PE / Cyanine5 anti -human CD 184 (CXCR-4, Clone 12G5, Biolegend) or FITC anti-mouse / human CD282 (TLR2, Clone T2.5, Biolegend) for 40 min. Cells were washed twice with FACS buffer and processed in a CytoFLEX cytometer (Beckman Coulter). Data was analyzed with Flow Jo software 10.8.1(BD Biosciences).[000193] Statistical analysis: All results except for the microbiome results were analyzed using Prism v9.3.0 (GraphPad Software Inc. San Diego, USA). Significant mean differences were assessed using the unpaired Student's t-test for comparisons among two groups and one- or two- way ANOVA analysis for comparisons of three or more groups. Asterisks were used for data that reached a significance of P < 0.05 (*), P < 0.01 (**), and P < 0.005 (***). Bars show mean and standard deviation (SD).[000194] Microbiome analysis: Pyrosequencing was performed by PCR amplification of the V4 region of the 16S rRNA gene. Following addition of adaptor and index sequences, samples were sequenced using 500-bp paired-end sequencing (Illumina MiSeq), generating 2 fastq files per sample. The raw sequence files were deposited in NCBI under BioProject ID 1005478. (Saba E, Farhat M, Plaschkes I, Nussbaum G. Data from: Oral bacteria accelerate pancreatic cancer development in mice. BioProject - NCB 12023.) Bioinformatics analyses were performed with QIIME 2.(Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019;37(8):852-7). Briefly, de-multiplexed fastq files were processed for adapter sequence removal using the Cutadapt trim-paired plugin followed by denoising with DADA2 (via q2-dada2) to identify all observed amplicon sequence variants (AS Vs). AS Vs were aligned with mafft (via q2-alignment) and used to construct a phylogeny with fasttree2 (via q2 -phylogeny). Taxonomy was assigned to ASVs using the q2-feature- classifier classify-sklearn naive Bayes taxonomy classifier which was pre-trained against sequences extracted from Greengenes 13_8 99% OTUs sequences using primers set 515F- 806R. Qiime2 output files were used as inputs to “MicrobiomeAnalyst - comprehensive statistical, visual and meta-analysis of microbiome data” online tool,( Chong J, Liu P, Zhou G, Xia J. Using MicrobiomeAnalyst for comprehensive statistical, functional, and meta-analysis of microbiome data. Nat Protoc. 2020;15(3):799-821.) after minor manual format editing. The R package Tax4Fun2 was used to predict functional profiles of prokaryotic communities from 16S rRNA gene sequences against the tool’s default reference data Ref99NR.(Wemheuer F, Taylor JA, Daniel R, Johnston E, Meinicke P, Thomas T, et al. Tax4Fun2: prediction of habitatspecific functional profiles and functional redundancy based on 16S rRNA gene sequences. Environ Microbiome. 2020; 15(1):11.) Each of the Pathways relative abundance predictions per sample were subjected to Kruskal-Wallis test (Adamska A, Domenichini A, Falasca M. Pancreatic Ductal Adenocarcinoma: Current and Evolving Therapies. Int J Mol Sci. 2017;l 8(7).) using the R package HybridMTest v. 1.40.0 .(Thomas RM, Jobin C. Microbiota in pancreatic health and disease: the next frontier in microbiome research. Nat Rev Gastroenterol Hepatol. 2020; 17( 1 ):53-64.) Pathways with Benjamini Hochberg FDR corrected p-value < 0.02 were kept (full list- not shown). Significant pathways with biological relevance were selected for display. Average relative abundance was calculated per treatment and displayed in a bar plot using the R package ggplot2 v.3.4.2.EXAMPLE 1:P. gingivalis translocates from the oral cavity to the pancreas[000195] To determine if oral P. gingivalis can migrate to the pancreas in an experimental setting, we coated the oral cavity of 8-10 week old C57BL / 6 female mice with 109colony-forming units (CFU) of bacteria (P. gingivalis strain 381), suspended in carboxymethylcellulose (CMC), three times at two-day intervals. On day 10, three days after the last administration, mice were sacrificed, and compared to control mice administered CMC alone. P. gingivalis DNA was detected in the pancreata of all infected mice (and none of the controls) by qPCR of the P.gingivalis-specific ISPgl gene, and calculation of genome equivalents did not show significant differences between mice (figure 1A-C). Furthermore, viable P. gingivalis colonies were recovered from the pancreata of all infected mice (figure ID). P. gingivalis was also detected in the pancreas by species-specific 16S rRNA FISH (figure IE). As reported by others, (23) bacteria were detected in the pancreata of all mice using a universal 16S rRNA FISH probe. The amount of bacterial DNA in the pancreas was equivalent between control and P. gingivalis- infected mice, and universal 16S FISH staining was similar (figure 1E-F). Thus, in this acute setting in mice, and in the absence of oral inflammation, P. gingivalis translocates from the oral cavity to the pancreas, and remains viable. To address the role of tissue destruction in P. gingivalis migration to the pancreas, silk ligatures were tied around the second molar tooth of mice, a procedure that leads to localized bone resorption within 5-10 days due to bacterial accumulation on the ligature and a subsequent host inflammatory response (supplementary figure 1A).(24, 25) P. gingivalis was administered to the oral cavity of mice following ligature placement (supplementary figure IB), and translocation to the pancreas was measured (supplementary figure 1C). Localized oral inflammation and tissue damage did not prevent, or potentiate, P. gingivalis migration to the pancreas (supplementary figure ID). Thus, the presence of P. gingivalis, rather than inflammation per say, is critical for bacterial translocation from the oral mucosa to the pancreas in mice.[000196] To evaluate if P. gingivalis can colonize the human pancreas, we tested seven samples of pancreata obtained from surgeries to remove intraductal papillary mucinous neoplasia (IPMN), a precursor lesion that infrequently develops to PDAC. Although subjects with IPMN are usually treated conservatively, surgical resection is recommended in some cases based on characteristics of the cyst, or clinical sequelae attributable to it.(26) In addition to the IPMN, the samples we analyzed contained healthy areas, and areas of pancreas intraepithelial neoplasia (PanIN), another precursor lesion to invasive PDAC that is often found concurrently with IPMN.(27) We found cell-associated P. gingivalis in PanIN areas and areas of healthy pancreas in six of seven subjects (supplementary table 1 lists the indications for surgery), and did not observe any staining for Tanerella for sy thia (T. for sy thia), a species also strongly associated with periodontal disease but not identified in epidemiological studies as associated with PDAC risk. (12) Co-staining with a universal 16S bacterial rRNA FISH probe revealed P. gingivalis to be a minor component of the pancreatic bacterial community (figure 1G), which can explain why it is not reported in previous studies of the microbiome of cystic precursor lesions to PDAC that are based on next-generation sequencing (NGS).(9) These findings demonstrate that P. gingivalis can be identified in the pre-neoplastic pancreas in humans.EXAMPLE 2:Chronic P. gingivalis administration induces pancreatic acinar-to-ductal metaplasia (ADM)[000197] To model the effects of long-term exposure to P. gingivalis on the pancreas, as would be expected for individuals with periodontal disease, the oral cavity of adult wild-type (WT) C57BL / 6 mice was coated with P. gingivalis (109CFU per administration vs. CMC carrier) three times a week over a twelve-week period, at which point mice were sacrificed (figure 2A). In contrast to controls, the pancreata of the majority of mice chronically exposed to P. gingivalis developed areas of acinar-to-ductal metaplasia (ADM) (figure 2B). ADM areas demonstrated enhanced mucin production by Alcian blue staining, and increased expression of the transcription factor Sox9, considered hallmarks of the ADM process (figure 2C).(28) P. gingivalis was detected by 16S FISH in areas of ADM (figure 2D), and bacterial protease production was detected by immunohistochemistry (figure 2E). Nevertheless, despite significant histologic alterations, P. gingivalis remained a minor component of the total pancreatic microbial community also in this setting of chronic exposure, and the bacterial load in the pancreas was not affected by P. gingivalis administration (figure 2F-G). Administration of T. forsythia using the same inoculum load (109CFU per administration), and dosing schedule, did not lead to histologic changes in the pancreas, and T. forsythia DNA was detectable in the pancreas of only one mouse (supplementary figure 2). Therefore, although P. gingivalis may not be unique, not all oral bacterial species translocate efficiently to the pancreas and induce ADM.EXAMPLE 3:Chronic P. gingivalis administration accelerates PDAC development in iKC mice[000198] Oncogenic or tumor suppressor mutations can transform ADM to pancreatic intraepithelial neoplasia (PanIN), a precursor lesion to PDAC. To test if oral application of P. gingivalis cooperates with oncogenic Kras to promote PDAC, P. gingivalis was administered to mice expressing tamoxifen-inducible Cre recombinase under the acinar cell promoter Ptfl a (Ptfla-CreER), and G12D mutant Kras (KrasG12D) held in the off-state by a loxP-flanked stop codon (iKC mice). Acinar expression of mutant Kras was induced at 8 weeks of age, and then mice were administered P. gingivalis to the oral cavity three times weekly for 12 weeks, at which point the mice were sacrificed (figure 3A). Compared with uninfected iKC mice, mice administered P. gingivalis demonstrated loss of acinar areas and enhanced alcian blue positive ductal areas (figure 3B-C). Pancreas H&E sections were scored for the most severe pathologypresent per mouse (figure 3D, supplementary figure 3 provides examples of PanIN 1 / 2 vs. PanIN 3). iKC mice developed ADM and low-grade PanIN lesions (PanIN 1 / 2), but no highgrade PanIN (PanIN 3) or PDAC lesions were observed, as expected for this age group. (29) In contrast, the majority of mice administered P. gingivalis developed PanIN 3 or full-blown PDAC (figure 3E). The lobular area affected by ADM / PanIN 1 / 2 (changes expected in iKC mice at this age), and the area affected by PanIN 3 / PDAC, was significantly increased in the mice infected by P. gingivalis (figure 3F). P. gingivalis was detected in the PanIN and PDAC regions using 16S FISH and IHC (figure 3G-H). Thus, oral application of P. gingivalis cooperates with acinar cell KrasG12Dto accelerate PDAC development.EXAMPLE 4:P. gingivalis -induces a shift in the pancreatic microbial community[000199] In models of periodontal disease, P. gingivalis administration effects a shift in the composition of the oral microbiome, although P. gingivalis itself remains a minor member of the new disease-associated microbial community.(30) To determine if P. gingivalis translocation to the pancreas influences the local diversity of microbes, we analyzed the pancreatic microbiome in mice administered P. gingivalis over 12 weeks. In WT mice, P. gingivalis administration to the oral cavity increased the alpha diversity of the intrapancreatic microbiome (figure 4A), and shifted the composition of species present in the pancreas, leading to separate clustering of the groups analyzed by PCoA (figure 4B). Pattern search analysis using Spearman rank correlation demonstrated several bacterial families that correlated with P. gingivalis treatment (figure 4C, supplementary table 2 shows FDR-corrected p- values). We also detected enrichment of differential functional pathways between control vs. P. gingivalis infected mice (supplementary figure 4, and supplementary table 3) using Tax4Fun2, a tool that predicts the functional profiles of bacterial communities based on 16S rRNA sequencing data.(31) In iKC mice, chronic P. gingivalis administration did not increase alpha diversity of the pancreatic microbiome (data not shown), however the composition was distinct between the infected and uninfected iKC mice (figure 4D). Pattern analysis showed some of the same bacterial family correlations in P. gingivalis-txeated iKC mice as in WT mice (figure 4E, supplementary table 2). Of note, P. gingivalis infection in both WT and iKC mice increased pancreatic Mycoplasmatacea, Helicobacteraceae, and Paraprevotellaceae (log -transformed counts) (figure 4F). Therefore, chronic exposure to P. gingivalis alters the pancreatic tissuemicrobiome, leading to increased relative abundance of bacterial families previously associated with PDAC development in mice.EXAMPLE 5:Kras mutation enhances P. gingivalis intracellular survival in acinar cells[000200] We previously found that P. gingivalis survives inside human PDAC cells, and that intracellular bacteria promote cell proliferation. (14) To determine if P. gingivalis promotes acinar cell proliferation in a similar manner to its effects on PDAC cells, we challenged the murine 266-6 acinar cell line with P. gingivalis. P. gingivalis induced acinar cell proliferation in both normoxic and hypoxic conditions (figure 5 A and Supplmovl-2), and infection led to increased acinar cell expression of the ADM markers SOX9 and CK19 (figure 5B). To distinguish effects due to intracellular vs. extracellular bacteria, following a one hour infection period we treated the cell culture with gentamicin and metronidazole (gent / met), antibiotics that effectively eliminate extracellular bacteria but do not kill intracellular bacteria.(33) We were unable to detect labeled P. gingivalis in acinar cells following gent / met, in contrast to PDAC cells (figure 5C). We next induced KrasG12Dmutation in the acinar cells using a Tet-on system.(34) Remarkably, KrasG12Dactivation enabled P. gingivalis to survive inside acinar cells (where they are protected from gent / met, figure 5D-E). Although P. gingivalis enhanced KrasWTand KrasG12Dacinar cell proliferation, these effects could be distinguished since gent / met treatment to select for intracellular bacteria eliminated the effect on KrasWTcells, whereas the effect on KrasG12Dacinar cells remained intact (figure 5F). As a byproduct of KrasG12D-enhanced proliferation, cellular levels of reactive oxygen species (ROS) increase, which ultimately compromise cell survival. (35) Intracellular P. gingivalis, however, mitigated the increase in ROS levels that resulted from KrasG12Dinduction (figure 5G). Thus, oncogenic KRAS enabled intracellular bacterial survival, and in turn intracellular P. gingivalis reduced ROS production, an effect expected to benefit both the intracellular bacteria and the host cell. We next hypothesized that KrasG12Dinduction in vivo promotes P. gingivalis intracellular localization, resulting in more intracellular P. gingivalis in infected iKC mice compared to WT mice. We mapped the distance between the pixels of the P. gingivalis 16S FISH signal to the nearest nuclear signal, stained with DAPI, in the acinar and ductal areas of pancreatic sections from WT and iKC mice infected with P. gingivalis. In iKC mice, most P. gingivalis were located within 3 pm of the cell nuclei (indicating intracellular location), (36) in contrast to WT mice (mean distance 4.65 pm for WT mice and 2.44 pm for iKC mice, supplementary figure5). Therefore, Kras induction in vivo correlates with a significant shift to P. gingivalis intracellular localization.EXAMPLE 6:Intracellular P. gingivalis protects PDAC cells from glucose deprivation[000201] We now tested if the mitigation of ROS production by intracellular P. gingivalis in KrasG12Dmutant acinar cells is true for PDAC cell lines that harbor multiple and varied oncogenic mutations (including two KRASWTcell lines, BxPC-3 and Panc02), and whether intracellular bacteria protect PDAC cells from ROS -mediated cell death. Since PDAC lesions are subject to extreme nutrient deprivation, a known trigger of ROS production and cell death, (37) we asked if intracellular P. gingivalis can protect cells in these conditions. The absence of glucose strongly induced ROS production across PDAC cell lines (figure 6A). Intracellular P. gingivalis blocked ROS induction triggered by the absence of glucose in all cell lines (independent of activating KRAS mutations), and also reduced ROS levels in two of the cell lines (MIA PaCa and Panc02) cultured in medium containing physiologic levels of glucose and glutamine (figure 6A). Consistent with reduced ROS, bacterial infection enabled some cells to survive in the prolonged absence of glucose (6 days, figure 6B). However, in the absence of glutamine P. gingivalis did not protect cells and even led to increased cell death in one cell line (BxPC-3, figure 6B). To further simulate the extreme nutrient stress of the PDAC environment, naive and infected cells were exposed to medium devoid of glucose and with glutamine at decreasing concentrations. Intracellular P. gingivalis favored cell survival in these conditions of extreme nutrient stress that led to complete cell death in uninfected cells (figure 6C-D). Live cell imaging of cells subjected to hypoxia and nutrient stress demonstrated cell death in the uninfected cells, in contrast to the P. gingivalis infected cells where there was a population of hypermobile cells, and evidence for stress responses such as entosis (figure 6E and Supplmov3- 4, Panc02; Supplmov5-6, Panc-1). Many of the surviving cells in the infected wells harbored intracellular P. gingivalis (figure 6F). To confirm that intracellular bacterial survival is necessary for PDAC cells to survive nutrient stress, we treated cells with moxifloxacin, a cellpenetrating antibiotic(38) that clears P. gingivalis from PDAC cells, vs. treatment with gent / met (supplementary figure 6). Moxifloxacin treatment, but not gent / met treatment, reversed the effect of P. gingivalis on PDAC cell survival in nutrient stress (figure 6G). Strains of P. gingivalis that differ in their interactions with host cells compared to strain 381,(39) and a fresh clinical isolate, behaved similarly (supplementary figure 7A), however P. gingivalis was notunique since PDAC cell infection with T. forsythia or F. nucleatum, bacteria that survive inside cancer cells, (40) also enabled cells to withstand harsh nutrient conditions (supplementary figure 7B). In contrast, co-culture with Aggregatibacter actinomycetemcomitans, or with a clinical isolate of Streptococcos oralis, was cytotoxic (supplementary figure 7C). Thus, intracellular survival of oral bacteria in PDAC cells confers protection from ROS-mediated cell death resulting from glucose deprivation.EXAMPLE 7:Intracellular bacteria enhance PDAC non-canonical glutamine metabolism[000202] Reduced ROS levels in nutrient-stressed cells harboring intracellular bacteria could stem from lowered ROS production, or from increased anti-oxidant capacity. Consistent with the latter possibility, P. gingivalis infection protected PDAC cells from 6h exposure to H2O2, and P. gingivalis was observed in the surviving cells (figure 7A-B). To demonstrate that antioxidant capacity protects PDAC cells from nutrient stress-induced cytotoxicity, we compared P. gingivalis infection to exogenous anti-oxidant treatment with a vitamin E analog (Trolox). Both Trolox and infection with P. gingivalis protected cells from nutrient stress-induced cell death to a similar degree (figure 7C). Since the protective effects of intracellular P. gingivalis are glutamine-dependent, and glutamine metabolism can influence antioxidant levels (figure 7D and),(41) the effect of infection on glutamine transport and metabolism were next examined. Intracellular P. gingivalis enhanced expression of the glutamine transporter SLC1A5, but not SLC2A1 (also known as Glutl), the main transporter for glucose in PDAC cells (figure 7E).(42) Enzymes that regulate non-canonical glutamine metabolism (GOT1, GOT2, MEI) were upregulated by intracellular bacteria, whereas the canonical pathway enzyme GLUD1 that converts glutamine to > keto-glutarate for entry to the TCA cycle, and the upstream enzyme GLS shared by canonical and non-canonical pathways, were unaffected (figure 7E). Glutamine metabolism through the non-canonical pathway produces pyruvate, with NADPH production as a by-product (figure 7D). NADPH conversion to NADP is coupled to reduction of glutathione, a major component of cellular anti-oxidant capacity.(43) In line with the enhanced expression of non-canonical glutamine pathway enzymes, intracellular P. gingivalis increased the levels of reduced glutathione in cells cultured in the absence of glucose (figure 7F). Therefore, intracellular P. gingivalis enhances glutamine transport and non-canonical metabolism leading to production of anti-oxidant compounds. Consistent with glutamine shuttling away from the TCA cycle we also found that P. gingivalis intracellular infectionreduced cellular oxygen consumption measured by metabolic flux analysis (figure 7G). Similar effects on gene expression were induced by P. gingivalis in vivo. Chronic infection of iKC mice induced SLC1A5, G0T1, and GOT2 gene expression in the pancreas, but did not affect GLS expression (figure 7H). Increased SLC1A5 expression was also detected by immunofluorescence in the PanIN areas (figure 71). Of note, SLC1A5 expression in PDAC strongly correlates with patient survival (figure 7J). Together, these findings implicate intracellular bacteria in the promotion of cancer cell metabolic rewiring that contributes to cell survival in nutrient stress.EXAMPLE 8:CXCR4 is the PDAC cell receptor for P. gingivalis[000203] We and others have shown that TLR2 plays a major role in the response to P. gingivalis on innate immune cells and oral squamous cell carcinoma cells. (44, 45) Although 266-6 acinar cells express TLR2, we were unable to detect TLR2 expression on PDAC cell lines (supplementary figure 8A and). (14) The absence of TLR2 expression prompted us to examine the role of CXCR4, a chemokine receptor previously shown to bind P. gingivalis fimbriae on macrophages. (46) In contrast to TLR2, PDAC cell lines expressed CXCR4 constitutively, regardless of KRAS mutational status (supplementary figure 8B and). (47) Intracellular P. gingivalis enhanced CXCR4 expression at 48h (figure 8A-B). P. gingivalis co-localized with CXCR4 on the cell membrane of PDAC cells, and, following infection, CXCR4 was internalized and continued to co-localize with the bacteria (figure 8C). Blocking CXCR4 with AMD3100 (plerixafor) prevented P. gingivalis intracellular survival (figure 8D and supplementary figure 9A), and in turn P. gingivalis infection did not protect PDAC cells from nutrient stress when CXCR4 was blocked (figure 8E, and supplementary figure 9B). In mice, chronic P. gingivalis application to the oral cavity of iKC mice induced pancreatic CXCR4 expression detected by RT-PCR, and CXCR4 expression was upregulated in PanIN areas (figure 8F-G). Finally, cancer cells are known to regulate CXCR4 expression in response to environmental stimuli such as hypoxia and chemotherapy, and CXCR4 expression increases with PanIN advancement.(48-50) PDAC cell treatment with gemcitabine led to enhanced CXCR4 expression (figure 8H). In turn, pre-treatment with gemcitabine (at below cytotoxicity) enhanced P. gingivalis intracellular colonization of PDAC cells (figure 81). Consistent with greater intracellular load, PDAC cells treated with gemcitabine prior to P. gingivalis infectionwere better able to withstand nutrient starvation (figure 8J). These findings demonstrate an interplay between environmental stress, oncogenic mutations (KRAS and others), and bacterial intracellular infection, that enables cancer cell persistence in harsh nutrient conditions.[000204] Most PanINs never progress to PDAC, despite harboring cells with mutations in oncogenic driver genes. (19) The latency period between the initial somatic mutation (almost always in KRAS) that gives rise to PanIN development, and the appearance of a metastatic tumor, estimated at decades, provides ample time for environmental factors to influence PDAC development. (51) Our study sheds light on how periodontal disease, a condition that affects at least one third of adults and disproportionately afflicts minorities and individuals of lower socioeconomic status, (52, 53) can impact PDAC progression during this window of opportunity. P. gingivalis is found in the subgingival plaque of the majority of individuals with periodontal disease, and its numbers increase incrementally with disease severity. (54) Previously, salivary carriage of P. gingivalis, even years prior to PDAC diagnosis, was shown to raise the risk of developing PDAC.(12) Our findings provide biological plausibility for this association and establish causality in a preclinical model. P. gingivalis was shown by others to translocate from the oral cavity to islet beta cells of the pancreas. (55) We found that P. gingivalis translocated from the oral cavity to the exocrine pancreas where it triggered ADM, and a shift in the pancreatic tissue microbiome. In cooperation with the induction of acinar cell KrasG12Dexpression, P. gingivalis accelerated tumorigenesis, with the majority of mice developing full-blown PDAC at 20 weeks of age. This observation is remarkable since mutations in Kras, when they are induced in the pancreatic acini of adult mice, cause mice to be refractory to PDAC development, and even pancreatitis induction in iKC mice does not lead to invasive disease at this time point.(56, 57) Similarly, autopsy studies have shown that most adults have PanINs containing cells with mutations in KRAS without developing invasive disease, further supporting the notion that oncogenic KRAS does not induce PDAC efficiently.(19) We observed two processes in the pancreata of WT mice exposed to P. gingivalis that can synergize with oncogenic KRAS. The first, ADM, is an adaptation to environmental stress that increases PanIN development when Kras mutations are present. (58) The second is a strong shift in the bacterial microbiome of the pancreas. Bacterial communities, both in the gut and in the pancreas, were shown to differ between healthy vs. PDAC patients, between early and advanced PDAC, and between short-term and long-term PDAC survivors. (59-61) Oral bacterial species are also enriched in cystic precursors to invasive PDAC.(9) Similarly, in mice the pancreatic microbiome shifts during PDAC development. (32) The increases in relative abundance of taxa induced by P. gingivalis (figure 4), and themetabolic pathways predicted to be induced (supplementary figure 2), echo these shifts described for tumor development in the pancreas in both patients and mice. These data suggest that particular bacterial species such as P. gingivalis can play a pivotal role in driving community-wide changes toward microbes shown to be adapted to a tumor microenviroment.(32) Processes induced by P. gingivalis can explain the abundance and advancement of PanINs when P. gingivalis administration was combined with oncogenic KrasG12Dmutation. However, Kras activation strongly increases ROS, leading to oncogene- induced senescence that halts PanIN progression to PDAC in the absence of additional oncogenic or tumor suppressor mutations. (35, 62) Therefore, although increasing the number and stage of PanINs provides opportunity for some to progress to PDAC, bacterial infection must overcome the ROS barrier.(35) Our finding that acinar cell KrasG12Dmutation permits P. gingivalis intracellular survival, and that intracellular bacteria tempered oncogene -induced ROS production (whether driven by KrasG12Dor other oncogenic mutations), provides a potential mechanistic explanation. Heightened ROS production is also a by-product of the nutrient-deprived PDAC microenvironment, an outcome of the highly desmoplastic nature of this cancer.(22) In the setting of hypoxia and glucose deprivation, we found that intracellular P. gingivalis rescues PDAC cells by increasing antioxidant capacity via non-canonical glutamine metabolism.(41) These are mutually -beneficial effects, since access to glutamine and reduced ROS make the intracellular microenvironment well-suited for asaccharolytic anaerobic bacteria such as P. gingivalis. Cooperation between intracellular bacteria and cancer cells in settings of extreme stress is an additional molecular mechanism by which the tumor microbiome can promote the pathogenesis of cancer.(63) Finally, as shown for other infectious agents, (64) we found that the chemokine receptor CXCR4 mediates P. gingivalis entry to PDAC cells. Upregulation of CXCR4 in response to environmental stress(49) or chemotherapy(48) represents a mechanism by which cancer cells can acquire intracellular bacteria that contribute to their survival. Targeting the pathways bacteria use to enter cancer cells, or antibiotic strategies aimed at eradicating intracellular bacteria, are novel approaches that may improve PDAC outcomes.EXAMPLE 9:TARGETED LIPOSOMES COMPRISING ANTIBIOTIC THERAPEUTICS[000205] This invention provides, in some aspects, targeted antibiotic containing liposomes that can serve as a therapeutic in cancer. CXCR4-targeted antibiotic (moxifloxacin) containing liposomes are prepared, and assessed to support this aspect of the invention. Mice carryinginducible pancreatic acinar cell expression of mutant Kras together with inducible loss of the tumor suppressor P53 (iKPC mice) are administered P. gingivalis as described hereinabove. Mice are either treated with liposomes, or not (serving as controls) prior to, or after, P. gingivalis administration, and colonization of P. gingivalis in developing and established PDAC lesions is determined by 16S rRNA FISH on pancreatic sections. Treatment is expected to lower P. gingivalis colonization of the pancreatic lesions. Since P. gingivalis colonization also alters the endogenous pancreatic tissue microbiome, the effect of treatment on all bacteria in the pancreas will be determined by next generation sequencing.[000206] This will be further extended to assess liposomal treatment on the intracellular localization of P. gingivalis and other bacteria as determined using species specific versus phyla- specific, and universal, 16S rRNA FISH probes through measurement of the distance between the bacterial signal and the nearest nuclear signal as described in Example 8.[000207] Liposomal antibiotic delivery will be assessed for its effect on survival in iKPC mice administered P. gingivalis. iKPC mice will be split to groups, administered P. gingivalis for several weeks and compared to non-infected mice. Infected and non-infected mice will be followed for survival, as well as being provided with liposomal therapy and followed for treatment response. P. gingivalis infection accelerates cancer-related mortality and the liposome therapy will reverse this effect.[000208] The effect of liposomal antibiotic delivery on gemcitabine efficacy can also be assessed in iKPC mice administered P. gingivalis. iKPC mice will be administered P. gingivalis for several weeks, compared to uninfected mice, and followed for survival. Additional infected and non-infected mice will be administered gemcitabine chemotherapy for several weeks and then followed for treatment response. P. gingivalis infection reduces the efficacy of gemcitabine chemotherapy and the liposomes reverse this effect.[000209] Example 8 demonstrated that P. gingivalis infection is mediated / promoted via CXCR4. In some aspects, CXCR4 inhbitors, for example, AMD3100, can be supplied in parallel to or following the liposomal therapy, and in some asepcts, the best results are achieved with the combination treatment of the antibiotic-containing liposomes and following administration of the CXCR4 inhibitors.EXAMPLE 10:INTRACELLULAR BACTERIAL LOCALIZATION REFLECTS ONCOGENICMUTATION[000210] This invention provides, in some aspects, methods, materials and kits for reducing incidence or reducing severity or preventing progression of or preventing or treating or preventing pathogenesis of a cancer in a subject, wherein said subject is predisposed to, showing early indications of or suffering from cancer, said method comprising reducing localization of intracellular bacteria proximally to nuclei in cells of affected tissue in said subject.[000211] Referring to Figure 9, intracellular bacterial localization reflecting oncogenic mutation is shown. Individual distances from each bacteria to nearest nucleus were measured in the pancreata of wild-type (WT) mice and mice in whom a mutation in KRAS (G12D) was induced in the pancreatic acinar cells (iKC mice). A 16S rRNA universal probe (EUB338) was used for FISH analysis. Induction of oncogenic mutation in the pancreas correlated with significant shortening of the average distance of bacteria from the nucleus, a signal of intracellular localization.[000212] Figure 10 shows that oral cancers contain cultivable bacteria that are anaerobic and intracellular. Culture of tumor bacteria was performed by first preparing single cell suspensions from tumor tissue (and adjacent when available), then dividing the sample and treating one group of cells with an antibiotic cocktail to eliminate extracellular bacteria. Samples were plated on blood agar in aerobic and anaerobic conditions, and colonies were identified by Sanger sequencing of the 16S rRNA gene (or MALDI-TOF MS in some cases). The experimental workflow is shown in Fig. 10A. We found that cultivable bacteria are intracellular, and mostly anaerobic (Fig. 10B). Extensive laboratory controls are conducted to rule out laboratory contamination. Bacterial growth from a negative control, and tumor for which tissue from the margin free of tumor was available (“adjacent”) is shown in Fig. 10A, demonstrating higher numbers of intracellular cultivable anaerobic bacteria from the tumor cells. Twenty-nine patients have been tested in this manner (adjacent tissue free of tumor available on a subset), and representative colonies of each morphology were sequenced. Multiple anaerobic species associated with periodontitis were recovered, and some species were recovered from multiple patients (Fig. 10C). Species recovered from at least 6 patients (9 thus far, of 29 tumors) were the focus for functional studies. Recurring species from the culturomics were the focus for identification of co-occurring species combinations in the microbiome data of these cohorts in order to probe bacterial combinatorial or community effects, and compensate for missing species due to unculturability in these conditions.[000213] Figure 11 extends these studies further to show that bacterial load and intracellular bacterial localization by distance measurement reflects tumor versus adjacent tissue. Tumorversus adjacent tissue was defined for cases of squamous cell carcinomas (A), and universal bacterial 16S FISH performed (B). Tumors are marked by increased bacterial load (C) and significant shortening of average distance to nucleus (D-E), demonstrating that these parameters can define tumor areas.[000214] Furthermore, within tumors, bacterial density increased in the poorly differentiated areas, as seen in Figure 12. Bacterial density clearly increased in poorly differentiated areas, that are absent of keratin pearls (Figure 12B) demonstrating the utility of a bacterial load and intracellular localization score for defining areas of aggressive nature in the tumor specimen.[000215] Multicolor taxa- specific 16S FISH was used as shown in Figure 13-14, to further characterize intratumoral bacteria. Phyla-specific 16S rRNA probes to dominant phyla (Firmicutes, Fusobacteriodota, Bacteroidota, and Actinobacteria) were designed and validated that were identified by cultivation from tumor tissue (Figure 13). These probes were used together with the universal bacterial 16S probe for confirmation to study the distribution of bacterial phyla in oral squamous cell carcinoma (OSCC) tumors. An example of phyla identification in tumor tissue is shown in Figure 14. The distribution of distances for each phyla differs and some phyla, such as Fusobacteiota for OSCC demonstrated more significant shortening whereas others such as Firmicutes shortened to a much lesser extent.[000216] In addition to phyla-specific probes, 16S FISH probes specific to critical species identified, such as P. gingivalis are used to track the spatial distribution of these species (Figure 15). Bacterial distribution by taxa-specific probe, in regions of interest or across the entire scanned section, is calculated using detection functions in QuPath (as we showed previously for the universal probe22). Bacterial distribution by taxa-specific labeling correlated to histopathologic features and specific immune cell types by combining the mTSF with spatial phenotyping techniques. Using the multiplex immunofluorescence images generated, cell-type classifiers were trained in QuPath or other analysis software, and distances between specific bacterial taxa tracked by FISH to each cell type used for machine learning and Al algorithms.[000217] Figure 16 expands the above by demonstrating tumor isolated bacteria protected OSCC cells from DNA damage. We infected the OSCC cell line SCC-25 (MOI 10) with strains of periodontal pathogens isolated from OSCC tumors and eliminated extracellular bacteria. Infected vs. non-infected cells were then irradiated (8 Gy) and functional effects of bacteria were assessed (Fig. 16A). Intracellular bacteria protected OSCC cells as measured by colony formation 2 weeks following radiation. P. gingivalis exerted a much stronger effect compared to F. nucleatum. Bacterially infected cells were significantly protected from DNA damage measured by yH2AX immunofluorescence 1 hour after irradiation (P. gingivalis-infected cells,Fig. 16B). Bacterially-infected cells also demonstrated reduced ROS levels (4HN E immunofluorescence, not shown). These findings demonstrated that bacteria isolated from tumors increased tumor resistance to ionizing radiation.[000218] Figure 17 further demonstrates that Moxifloxacin reversed P. gingivalis-conferred radiation resistance. CAL27 OSCC cells were infected with a strain of P. gingivalis or of C. acnes, each isolated from OSCC tumors. An hour following infection cells were treated either with gentamicin / metronidazole, or for comparison with moxifloxacin (in the case of P. gingivalis) for 1 hour. Cells were rested for 1 hour and then infected versus non-infected cells were irradiated (8 Gy) and radiation protection assessed by colony formation at two weeks. As above, P. gingivalis infection strongly protected cells from radiation cytotoxicity. In contrast, C. acnes represents a species recovered from tumors that does not enhance or inhibit radiation induced cytotoxicity. Moxifloxacin treatment cleared intracellular P. gingivalis (not shown) and fully reversed the radiation protection afforded by the intracellular bacteria.[000219] Taken together, the results support that during tumor development, there is a shift to greater bacterial load and more intracellular bacteria, as was shown in the results regarding the pancreas in mice where there was a significant shortening of the distance when oncogenic KRAS was induced in the acinar cells; and in the results regarding oral SCC comparing tumor to adjacent epithelium; and in the results showing colonic polyps comparing hyperplastic polyps to adenoma to adenocarcinoma (data not shown).[000220] The BLS shortening is more prominent for particular taxa and in areas of greater aggressive features of the tissue.[000221] Intracellular bacteria enable cancer cells to resist treatment modalities such as chemotherapy, immunotherapy, and ionizing radiation.[000222] The BLS distance can be log transformed to address non-normality in the continuous data and enable statistical analyses. Mean distance across taxa and cutoff values of % bacteria within a specific distance for each taxa (e.g. 3 microns) are used to define tumor characteristics. This information is integrated with number of particular bacteria within specific distance per number of cells in that region [e.g. score of x#bacteria at <3 microns / thousand cells, to differentiate pathology states.][000223] Thus, some embodied aspects of the invention will comprise the use of a scoring system which in turn may correlate with histopathologic features, which can be improved, for example, via implementation of machine learning / AI algorithms as informatic tool for the pathologist and the treating clinician.[000224] According to this aspect, and in some embodiments, such scoring system may be useful as a diagnostic aid for pathologist to define areas of tumor in cases of indecision, or in some embodiments, such scoring system may be useful as a prognostic tool to determine likelihood of recurrence and therefore need for follow-up plan including surgery. In other embodiments, such scoring system may be useful as part of arriving at a personalized treatment plan for a patient / subject, and in some embodiments, such scoring system may be useful as part of predicting likelihood of response to immunotherapy, and in some embodiments, such scoring system may be useful as part of arriving at a determination, which patients should be treated with adjunctive treatments that target the tumor microbiome and specifically intracellular bacteria.[000225] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.[000226] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

[000227] WHAT IS CLAIMED IS:

1. A method of personalized optimized diagnosis and therapy in a subject, said method comprising the step of: screening a subject predisposed to, or at risk for cancer and identifying whether the pre- cancerous or cancerous tissue in the subject is intracellularly infected with bacteria, optionally assessing the intracellular localization of the bacteria-containing compartment to determine a distance of same from a nucleus in the cell; optionally assessing the oncogene profile expression in the subject, and further comprising the step of scoring the subject in terms of the prognosis and potential for therapy and then treating the subject to reduce the intracellular bacterial burden, promote re-localization of the bacterial compartment to be at a further distance from the nucleus, successfully eradicate intracellular bacterial infection, prevent or reduce cell-to-cell spread of the bacteria or any combination thereof. The method of claim 1 , wherein said cancer is a carcinoma.3 The method of claim 1, wherein said affected tissue is mucosal tissue. The method of claim 1 , wherein said cancer is pancreatic cancer.5 The method of claim 1, wherein said intracellular bacteria are derived from the oral cavity of the subject.6 The method of claim 1 , wherein said intracellular bacteria are Fusobacteriodota. The method of claim 1, wherein said intracellular bacteria are P. gingivalis, T. forsythia or F. micleatum.8 The method of claim 1, wherein reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an antibiotic. The method of claim 8, wherein said antibiotic may comprise moxifloxacin, gentamicin, metronidazole or a combination thereof.10 The method of claim 8, wherein said antibiotic is provided as part of a liposome composition.11 The method of claim 8, wherein said liposome further comprises a targeting moiety to specifically target said liposome to infected cells in said affected tissue.12 The method of claim 11, wherein said targeting moiety specifically targets a receptor enabling bacterial intracellular entry or cell-to-cell spread.13 The method of claim 1, wherein reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an agent that reduces the ability of said bacteria to access an intracellular compartment.

14. The method of claim 13, wherein said agent prevents said bacteria from undergoing receptor mediated endocytosis.

15. The method of claim 13, wherein said agent prevents bacteria from interacting with a cell-surface receptor.

16. The method of claim 1, wherein reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an agent that alters the traditional intracellular compartment or intracellular trafficking of a compartment in which said bacteria is localized.

17. .The method of claim 1, further comprising providing a chemotherapeutic agent to said subject.

18. The method of claim 1, further comprising providing radiation therapy to said subject.

19. The method of claim 1, wherein said agent enhances or otherwise positively impacts bacterial clearance, reduces bacterial replication, alters bacterial intracellular trafficking, or reduces bacterial intracellular localization proximal to nuclei or any combination thereof in affected tissue.

20. The method of claim 1, wherein said method provides a prebiotic compound or composition or at least one positive microbiome component species or a combination thereof, which biases the tissue microbiome in the tissue of origin of the cancer to reduce population levels of bacterial components that ultimately localize intracellularly within precancerous or cancerous cells in affected tissue.

21. The method of claim 1, further comprising assessing intracellular infection and intracellular localization of the bacteria-containing compartment as an indicator of cancerous versus non-cancerous tissue in situ.

22. The method of claim 1, further comprising assessing intracellular infection and intracellular localization of the bacteria-containing compartment as an indicator of aggressivity of cancerous tissue.

23. A method of personalized optimized diagnosis and therapy in a subject, said method comprising the step of: screening a subject predisposed to, or at risk for cancer and identifying whether the precancerous or cancerous tissue in the subject is intracellularly infected with bacteria and assessing the intracellular localization of the bacteria-containing compartment to determine a distance of same from a nucleus in the cell, wherein said distance calculated represents a mean distance value of a threshold percentage of the bacteria within a nearest nuclear boundary of a cell in a plurality of cells to establish said distance value.

24. The method of claim 23, comprising further correlating the distance calculated with histopathologic features indicative of cancer.

25. The method of claim 23, comprising establishing a score reflective of the distance calculated and histopathologic feature severity, which score indicates tumor pathogenesis severity.

26. The method of claim 23, wherein said score is further refined using machine learning to arrive at more reliable values obtained for the distance calculated, histopathologic features identified or a combination thereof.

27. The method of any one of claims 23-26, wherein said method refines the determination of the of the cancerous tissue area versus adjacent non-cancerous tissue.

28. The method of any one of claims 23-26, wherein said score reflects a likelihood of cancer recurrence in a subject.

29. The method of any one of claims 23-26, wherein said score reflects a likelihood of response to an immunotherapy in a subject.

30. The method of any one of claims 23-26, wherein said score reflects a likelihood of a need for adjunctive therapy for the subject.

31. The method of any one of claims 23-26, wherein said score improves assessment of a treatment plan for a given subject.

32. A Tumor Intracellular Microbiota Scanner (TIMScan) system, comprising• A chamber sized to accommodate a glass slide or coverslip onto which cells or tissue may be deposited for assay, which cells or tissue suspected of comprising intracellular bacteria of interest and at risk for pre-neoplasia or neoplasia;• A robotics assembly, which assembly can operationally connect and optionally re-connect said chamber to a labelling module, a washing module and a scanning module;• A labelling module which can apply reagent to said chamber to label specific components of said cells or tissue;• A washing module which can apply wash solution to said chamber to remove excess and unbound label;• A scanning module, which comprises a microscope;• A processor;• A non-transitory computer-readable storage medium having computer executable instructions stored thereon, which computer executable instructions when executed by the processor case the processor to perform: o Acquiring and storing data collected by said scanning module;o Calculating a BLS distance from the acquired data in said scanning module in cells or tissue suspected of comprising intracellular bacteria of interest and at risk for pre-neoplasia or neoplasia; and o Scoring the cell or tissue sample based on the BLS distance and histopathologic analysis from the acquired data, and optionally providing as an output at least one treatment recommendation based on said scoring.

33. The TIMScan system of claim 32, wherein said labelling module and said washing module may be provided as part of a microfluidics chamber operationally connected to said robotics assembly and said scanning module.

34. The TIMScan system of claim 32, wherein said scanning module comprise a microscope scanner.

35. The TIMScan system of claim 35, wherein said microscope scanner is a fluorescent microscope scanner.

36. The TIMScan system of claim 32, wherein said labelling module applies a fluorescence in situ hybridization (FISH) probe or probes.

37. The TIMScan system of claim 36, wherein a plurality of FISH probes may be applied in a desired sequence.

38. A method of reducing incidence or reducing severity or preventing progression of or preventing or treating or preventing pathogenesis of a cancer in a subject, wherein said subject is predisposed to, showing early indications of or suffering from cancer, said method comprising reducing intracellular bacterial infection in affected tissue in a subject, reducing localization of intracellular bacteria proximally to nuclei in cells of affected tissue in said subject, or a combination thereof.

39. The method of claim 38, wherein said cancer is a carcinoma.

40. The method of claim 38, wherein said affected tissue is mucosal tissue.

41. The method of claim 38, wherein said cancer is pancreatic cancer.

42. The method of claim 38, wherein said intracellular bacteria are derived from the oral cavity of the subject.

43. The method of claim 38, wherein said intracellular bacteria are P. gingivalis, T. forsythia or F. micleatum.

44. The method of claim 38, wherein reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an antibiotic.

45. The method of claim 44, wherein said antibiotic may comprise moxifloxacin, gentamicin, metronidazole or a combination thereof.

46. The method of claim 44, wherein said antibiotic is provided as part of a liposome composition.

47. The method of claim 44, wherein said liposome further comprises a targeting moiety to specifically target said liposome to infected cells in said affected tissue.

48. The method of claim 47, wherein said targeting moiety specifically targets a receptor enabling bacterial intracellular entry or cell-to-cell spread.

49. The method of claim 38, wherein reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an agent that reduces the ability of said bacteria to access an intracellular compartment.

50. The method of claim 49, wherein said agent prevents said bacteria from undergoing receptor mediated endocytosis.

51. The method of claim 49, wherein said agent prevents bacteria from interacting with a cell-surface receptor.

52. The method of claim 38, wherein reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an agent that alters the traditional intracellular compartment or intracellular trafficking of a compartment in which said bacteria is localized.

53. .The method of claim 38, further comprising providing a chemotherapeutic agent to said subject.

54. The method of claim 38, further comprising exposing the affected cells or tissue to radiation.

55. The method of claim 38, wherein said agent enhances or otherwise positively impacts bacterial clearance, reduces bacterial replication, alters bacterial intracellular trafficking, or reduces bacterial intracellular localization proximal to nuclei or any combination thereof in affected tissue.

56. The method of claim 38, wherein said method provides a prebiotic compound or composition or at least one positive microbiome component species or a combination thereof, which biases the tissue microbiome in the tissue of origin of the cancer to reduce population levels of bacterial components that ultimately localize intracellularly within precancerous or cancerous cells in affected tissue.

57. A method of reducing intracellular bacterial infection in subjects at risk for or suffering from carcinoma, or from pre-neoplastic or metaplastic tissue changes, said method comprising contacting at least one cell in an affected tissue in said subject with an agent reducing intracellular bacterial infection in affected tissue, reducing localization of intracellular bacteria proximally to nuclei in cells of affected tissue, or a combination thereof.

58. The method of claim 57, wherein said cancer is a carcinoma.

59. The method of claim 57, wherein said affected tissue is mucosal tissue.

60. The method of claim 57, wherein said cancer is pancreatic cancer.

61. The method of claim 57, wherein said intracellular bacteria are derived from the oral cavity of the subject.

62. The method of claim 57, wherein said intracellular bacteria are P. gingivalis, T. forsythia or F. micleatum.

63. The method of claim 57, wherein reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an antibiotic.

64. The method of claim 63, wherein said antibiotic may comprise moxifloxacin, gentamicin, metronidazole or a combination thereof.

65. The method of claim 63, wherein said antibiotic is provided as part of a liposome composition.

66. The method of claim 63, wherein said liposome further comprises a targeting moiety to specifically target said liposome to infected cells in said affected tissue.

67. The method of claim 66, wherein said targeting moiety specifically targets a receptor enabling bacterial intracellular entry or cell-to-cell spread.

68. The method of claim 63, wherein reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an agent that reduces the ability of said bacteria to access an intracellular compartment.

69. The method of claim 68, wherein said agent prevents said bacteria from undergoing receptor mediated endocytosis.

70. The method of claim 68, wherein said agent prevents bacteria from interacting with a cell-surface receptor.

71. The method of claim 57, wherein reducing intracellular bacterial infection in affected tissue comprises contacting at least one cell in said affected tissue with an agent that alters the traditional intracellular compartment or intracellular trafficking of a compartment in which said bacteria is localized.

72. .The method of claim 57, further comprising providing a chemotherapeutic agent to said subject.

73. The method of claim 72, further comprising assessing oncogene expression in said subject and adjusting chemotherapeutic choice in consideration of said oncogene expression profile in said subject.

74. The method of claim 57, wherein said agent enhances or otherwise positively impacts bacterial clearance, reduces bacterial replication, alters bacterial intracellular trafficking, or reduces bacterial intracellular localization proximal to nuclei or any combination thereof in affected tissue.

75. The method of claim 57, wherein said method provides a prebiotic compound or composition or at least one positive microbiome component species or a combination thereof, which biases the tissue microbiome in the tissue of origin of the cancer to reduce population levels of bacterial components that ultimately localize intracellularly within precancerous or cancerous cells in affected tissue.

76. A kit for reducing incidence or reducing severity or preventing progression of or preventing or treating or preventing pathogenesis of a cancer in a subject, wherein said subject is predisposed to, showing early indications of or suffering from cancer, said kit comprising an antibiotic agent or a liposome composition comprising an antibiotic agent that specifically enhances or otherwise positively impacts intracellular bacterial clearance, reduces intracellular bacterial replication, alters bacterial intracellular trafficking, or reduces bacterial intracellular localization proximal to nuclei or any combination thereof.

77. The kit of claim 76, wherein said liposome further comprises a targeting moiety to target said liposome to a cell in an affected tissue infected with intracellular bacteria.

78. The kit of claim 76, wherein said kit further comprises a chemotherapeutic agent.

79. The kit of claim 76, wherein said intracellular bacteria are P. gingivalis, T. forsythia or F. nucleatum.

80. The kit of claim 76, wherein said antibiotic comprises moxifloxacin, gentamicin, metronidazole or a combination thereof.

81. A composition for reducing incidence or reducing severity or preventing progression of or preventing or treating or preventing pathogenesis of a cancer in a subject, wherein said subject is predisposed to, showing early indications of or suffering from cancer, said composition comprising a liposome comprising an antibiotic agent that specifically enhances or otherwise positively impacts intracellular bacterial clearance, reduces intracellular bacterial replication, alters bacterial intracellular trafficking, or reduces bacterial intracellular localization proximal to nuclei or any combination thereof, wherein said liposome further comprises a targeting moiety to target said liposome to a cell in an affected tissue infected with intracellular bacteria.

82. The composition of claim 81, wherein said antibiotic comprises moxifloxacin, gentamicin, metronidazole or a combination thereof.

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