Inorganic polyphosphate as a novel fecal biomarker for the early diagnosis of colorectal cancer

Abstract

Based on experimental data, observed i n the Personalized Medicine Laboratory of the requesting institute, which reveal an increase in the inorganic polyphosphate (iPolyp) in fecal samples from subjects affected by colorectal cancer compared to samples from individuals not affected by the same disease, inorganic polyphosphate is suggested as a novel non-invasive biomarker to be detected in fecal samples for the early diagnosis of colorectal cancer, to be associated with other screening methods such as fecal occult blood, in order to provide greater predictivity, i.e., the probability that a subject with a positive diagnostic test is actually affected by colorectal cancer.

Description

[0001] INORGANIC POLYPHOSPHATE AS A NOVEL FECAL BIOMARKER FOR THE EARLY DIAGNOSIS OF COLORECTAL CANCER

[0002] DESCRIPTION

[0003] Summary of the invention inorganic polyphosphate is suggested as a novel non-invasive biomarker to be detected in fecal samples for the early diagnosis of colorectal cancer, to be associated with other screening methods such as fecal occult blood, in order to provide a greater predictivity, i.e. , the probability that a subject with a positive diagnostic test is actually affected by colorectal cancer.

[0004] Recent research of the requesting institute identified inorganic polyphosphate (i Polyp) as a new key molecule in the tumor progression and growth of colorectal cancer, inorganic polyphosphate (i Polyp) is a high energy linear polymer consisting of hundreds of repeating units of orthophosphate and is involved in a series of pathophysiological processes, including cellular proliferation.

[0005] For such a reason, the requesting institute carried out screening tests on tumor and normal biopsy tissues from patients affected by colorectal cancer; such an analysis highlighted a significantly greater concentration of inorganic polyphosphate in tumor biopsy tissues compared to the normal counterpart.

[0006] Following this result, the possibility that the concentration of inorganic polyphosphate observed in biopsy tissues simultaneously reflects the presence thereof in feces was investigated . The experimental data, observed in the

[0007] Personalized Medicine Laboratory of the requesting institute, which are reported below and reveal an increase in inorganic polyphosphate (i Polyp) in fecal samples from subjects suffering from colorectal cancer, compared to samples from individuals not affected by the same disease, confirm such a hypothesis, whereby said inorganic polyphosphate is claimed for use as a fecal marker for the early diagnosis of colorectal cancer. Field of the invention

[0008] Colorectal cancer is the third most commonly diagnosed neoplasm and the second leading cause of cancer death globally.

[0009] Adenocarcinomas, which develop from the glandular epithelial cells of the colon and rectum, form the most commonly identified colorectal tumors (>90%).

[0010] There are three main types of CRC: sporadic, hereditary and associated with colitis.

[0011] About 60 / 65% of CRC cases arise sporadically, i.e., in individuals without a family history of CRC or hereditary genetic mutations that increase the risk of onset.

[0012] Sporadic CRC arises as a result of acquired somatic genetic and epigenetic aberrations, attributable for the most part to potentially modifiable risk factors.

[0013] About 25% of CRC cases have a family history without any apparent hereditary tumor syndrome.

[0014] Only 5% is assigned to hereditary tumor syndromes such as hereditary non-polyposi s colorectal cancer (HNPCC, also known as Lynch syndrome) or familial adenomatous polyposis (FAP), caused by inherited germline mutations in rare but high-penetrance genes (for example, MLHI and APC, respectively).

[0015] The natural history of CRC can be divided into four main stages: initiation, promotion, progression and metastasis.

[0016] CRC develops when epithelial cells acquire a series of genetic or epigenetic alterations that render them hype rprol i fe rati ve . These rapidly developing cells form a benign adenoma, which can evolve into cancer and metastasize.

[0017] The preliminary screening test for colorectal cancer includes the examination of occult blood in the feces; however, given the considerable number of diseases and clinical situations that can induce positive occult blood, it should be underlined that there is no direct correlation between positivity and neoplastic pathology of the colon - rectum, therefore occult blood in the feces does not establish a diagnosis, but reveals a condition attributable to multiple conditions, for example: peptic ulcers, chronic inflammatory bowel diseases, intestinal polyps, angiodysplasias, aspirin and antiinflammatories, parasitosis, Meckel’s disease, vasculopathies, portal vein thrombosis, gingivitis, periodontal diseases, oral traumas.

[0018] The diagnostic screening tests for colorectal cancer include more invasive examinations, including:

[0019] 1. research of blood tumor biomarkers, such as CEA (carci no-embryonic antigen) and CA19-9 (carci no- embryonic antigen 19.9 or Gica), although an increase thereof can be detected in other types of pathologies as well, both neoplastic and non neoplastic;

[0020] 2. colonoscopy.

[0021] To date, it is estimated that 2 / 10% of patients with positive occult blood have colorectal cancer, while about 30% have adenomatous polyps, (https : / / www.1 orenzobe rtani . i t / i 1 -sangue-occul to- nelle-feci / ) .

[0022] The clinical / experimental need to identify new biomarkers associated with colorectal cancer by means of the use of less invasive methods derives therefrom, such as the detection of occult blood in the feces, which, however, provide a greater percentage of correlation between test positivity and the diagnosis thereof.

[0023] Task of the invention

[0024] Therefore, it is the task of the present invention to suggest a biomarker to be detected in fecal samples for the early diagnosis of colorectal cancer, to be associated with other screening methods such as fecal occult blood, in order to provide a greater predictivity, i.e., the probability that a subject with a positive diagnostic test is actually affected by colorectal cancer.

[0025] Starting from our recent research that identified inorganic polyphosphate (i Polyp) as a new key molecule in the progression and tumor growth of colorectal cancer, in tumor and non-tumor tissues (histologically confirmed) taken from patients affected by colorectal cancer who underwent surgical intervention, the experimental data obtained in our laboratory are given below, which reveal an increase in inorganic polyphosphate (i Polyp) in fecal samples from subjects affected by colorectal cancer, compared to samples from individuals not affected by the same disease. it is another task of the present invention to provide a method for identifying patients at risk of having developed colorectal cancer, during population screening processes for early diagnosis (similarly to what is currently done with testing for occult blood in feces) by determining the amount of inorganic polyphosphate present in at least one fecal sample from said subject.

[0026] The phrase identifying patients at risk relates to the main object of all screening programs carried out on the population in order to select those who will need further examinations, e.g., colonoscopy.

[0027] Such an assessment, although certainly preferable, could usually be incorrect for 100% of the subjects under investigation. The term, however, requires that a statistically significant portion of subjects can be correctly assessed and then diagnosed, whether a portion is statistically significant can be determined without further delay by those skilled in the art using various well-known statistical assessment tools, for example determination of confidence intervals, Student’s t-test of the p-value, Mann-whitney test, etc. Details are found in Dowdy and wearden, statistics for Research, John Wiley & Sons, New York 1983. The preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80% at least 90% or at least 95%. The p-values (degree of sample significance), are preferably 0.2, 0.1, or 0.05. This step requires determination of the test “sensitivity” and “specificity” thresholds and after special construction of an ROC curve with determination of the area under the curve.

[0028] As used below, the terms "preferably", "more preferable" "particularly", "more particularly", “specifically", “more specifically” or similar terms are used together with optional features, without limiting alternative possibilities.

[0029] The term “subject” as used herein relates to a primate and more preferably to a human being.

[0030] Preferably the subject is an apparently healthy subject. Preferably the subject is a subject at risk of suffering from colorectal cancer. The suspicion that the subject can suffer from colorectal cancer preferably derives from at least one clinical symptom known to those skilled in the art as associated with colorectal cancer.

[0031] The term “compare” means determining whether the established value of the diagnostic biomarkers is essentially identical to a reference or differs therefrom. Based on the above comparison, it can be assessed whether a subject is at risk or not of being affected by a colorectal tumor. The comparison is preferably assisted by automation. For example, a suitable computer program can be used comprising algorithms for the comparison of two different data sets (data set comprising the values of the features). Such computer programs and algorithms are well known in the art, notwithstanding the above, the comparison can also be performed manually.

[0032] The description will be more readily understood with reference to the appended drawings which show the results obtained by way of non-limiting example. in the figures:

[0033] Fig. 1 is a box plot of the distribution of ipolyp, stratified by peritumoral or tumoral patients. A statistical difference was observed (median value of 373,097.70 ± 210,216.20 pmol / mg for the tumor tissue with respect to 166,102.70 ± 124,618.80 pmol / mg for the peritumoral counterpart, **** p < 0.0001). The analysis was performed by means of the Mann-whitney rank-sum test;

[0034] Figures 2A and 2B show that CRC exhibits a high level of PCNA. A. in particular:

[0035] Fig. 2A shows the cell extracts from 50 human biopsies, in which the tumor sample (T) was compared with the peritumoral counterpart (P) of the same patient (Pt), analyzed by immunoblotting for the level of PCNA expression. Actin was used as loading control for normalization;

[0036] Fig. 2B shows the Spearman Correlation between ipolyp and PCNA in the total cohort denoting a strong positive correlation (**** p <0.0001). Fold changes with respect to Peritumoral (P), normalized to 1;

[0037] Figs. 3A and 3B show that CRC exhibits a high level of the TRPM8 receptor, in particular:

[0038] Fig. 3A shows the cell extracts from 50 human biopsies, in which the tumor sample (T) was compared with the peritumoral counterpart (P) of the same patient (Pt), analyzed by immunoblotting for the level of TRPM8 expression. Actin was used as loading control for normalization.

[0039] Fig. 3B shows the Spearman Correlation between i Polyp and TRPM8 in the total cohort denoting a strong positive correlation (**** p <0.0001). Fold changes with respect to Peritumoral (P), normalized to 1;.

[0040] Figs. 4A ,4B, and 4C, show how i Polyp improves the expression of PCNA and promotes the proliferation of Caco-2 colorectal cancer, in particular: in Fig. 4A the cell extracts of the WT and siRNA- mediated TRPM 8 knockdown Caco-2 cell lines were analyzed by immunoblotting for the PCNA expression level, GAPDH was used as loading control; in Fig. 4B, representative micrographs of the crystal violet assay performed on WT and siRNA- mediated TRPM 8 knockdown Caco-2 cell lines after treatment for 96 hours with i Polyp, with TRPM8 inhibitor or with both. Scale bar 10 pm. The images are representative of three independent experiments; in Fig. 4C, the statistical analysis of the crystal violet assay by Student’s t-test is reported, respectively for panel C, (*** p < 0.001 and **** p < 0.0001). Fold changes with respect to the control, untreated (UT), normalized to 1. The data are given as mean ± SD for triplicate wells from three independent experiments ;

[0041] Figures 5A, 5B, 5C and 5D, show that i Polyp promotes patient-derived colorectal cancer organoids and 3D spheroids derived from Caco-2 and SW620. in parti cular:

[0042] Figs. 5A and 5B illustrate two independent experiments of patient-derived CRC organoids, evaluated by optical microscopy, in the absence (UT) or incubated for 10 days in the presence of i Polyp. Scale bar 100 pm. Fold changes with respect to the control, untreated (UT), normalized to 1. Statistical analysis performed by Student's t-test (*** p <0.001);

[0043] Fig. 5C shows representative bright field images of spheroid formation induced at 24 hours and 96 hours derived respectively from the HCEC-ICT, Caco-2 and SW620 cell lines, after treating with iPolyP, TRPM8 inhibitor or both for 96 hours. Scale bar 100 pm. The images are representative of three independent experiments .

[0044] Fig. 5D reports the quantification relative to panel C. Fold changes with respect to the control, untreated (UT). statistical analysis performed by Student t test (**** p < 0.0001). The data are given as mean ± DS for triplicate wells from three independent experiments.

[0045] Figs. 6A, 6B, 6C and 6D show how iPolyP guides the cells into the G2 / M phase, in particular:

[0046] Fig. 6A shows the real time PCR on the Caco-2 cell line treated with iPolyP for 72 hours on cyclins involved in the different phases of the cell cycle; the untreated samples, UT, were normalized to 1;

[0047] Fig. 6B is a diagram showing cycl in-dependent cell cycle regulation, consisting of Gap 1 (Gl) , synthesis (S) , Gap 2 (G2) , and mitosis (M) . The figure was created with BioRender.

[0048] Fig. 6C reports representative micrographs of the cell cycle analysis on the Caco-2 cell line (upper panel) and SW620 (lower panel) treated for 72 hours with iPolyP, TRPM8 inhibitor or both. Scale bar = 10 pm. The images are representative of three independent experiments .

[0049] Fig. 6D shows the percentage of cells in G2 / M phase. Statistical analysis performed using student's t-test (*** p < 0.001). Fold changes with respect to the control, untreated (UT). The data are given as mean ± DS for triplicate wells from three independent experiments .

[0050] Fig. 7 is a diagram of the role of iPolyP derived from platelets and from the microbiota on macrophages and on CRC tumor cells. The iPolyP-TRPM8 axis stimulates the expansion of colorectal cancer, improving the level of the ccnbl gene, which coordinates the M step of the cell cycle, together with the expression of two authentic proliferative markers, PCNA and Ki 67.

[0051] Fig. 8 shows the increase in inorganic polyphosphate (iPolyP) observed in fecal samples from 3 subjects affected by colorectal cancer with respect to fecal samples from 3 individuals not affected by the same disease.

[0052] Fig. 9 shows the results of an analysis performed on n=82 healthy subjects and subjects affected by CRC, where the CRC fecal samples exhibit a statistically significant higher level of the inflammatory molecule inorganic polyphosphate (i Polyp) compared to the healthy individuals (***p=0.0002) .

[0053] Fig. 10 shows the results of a further analysis on the same number of subjects (82) aimed at confirming the hypothesis that i Polyp can act as a biomarker for the early diagnosis of CRC, based on the TNM staging system (Tumor, Nodes, Metastases), patients affected by CRC were subdivided into two main groups: early-stage CRC including adenoma

[0054] (precancerous) , and advanced-stage CRC. The concentration of iPolyP in early stage CRC (n=8) exhibits significantly high iPolyP levels similar to those observed in advanced stage CRC (n=33) and both are substantially upregulated with respect to healthy fecal samples (n=82) .

[0055] Fig. 11 shows that, surprisingly, the concentration of iPolyP is statistically significant in patients affected by CRC, also for those for whom the fecal occult blood test produced a negative result (estimated at about 36.5% of all patients affected by CRC worldwide (n=30) with respect to healthy fecal samples (N=82).

[0056] DETAILED DESCRIPTION OF THE INVENTION inorganic polyphosphate, a molecule consisting of a range from a few to several hundred orthophosphates, is involved in different pathological processes, including the development and progression of cancer. in a preliminary study, analyzing biopsies derived from 50 subjects affected by colorectal cancer collected by the Histopathology unit of our institute, we discovered a significant discrepancy in the level of inorganic polyphosphate between the tumor counterpart and the peritumoral counterpart, the former exhibiting a higher concentration. Furthermore, using in vitro and ex vivo approaches, we demonstrated an involvement of inorganic polyphosphate in the proliferation of colorectal cancer cells. Furthermore, we identified the TRPM8 calcium channel as the receptor of the inorganic polyphosphate responsible for the propagation of the downstream signal and, ultimately, for the enhancement of the expression of proliferative markers. Therefore, our results identify inorganic polyphosphate as a novel fundamental biomarker associated with the growth of colorectal cancer.

[0057] Colorectal cancer (CRC) is characterized by a proi nfl ammatory microenvironment and exhibits high- energy molecules which ensure the tumor growth. An as yet underestimated macromolecule is inorganic Polyphosphate (i Polyp), a high energy linear polymer ubiquitous in all forms of life. Consisting of hundreds of repeated orthophosphate units, iPolyP is essential for a wide variety of functions in mammalian cells, including the regulation of proliferative signaling pathways. Some evidence has suggested the involvement thereof in carcinogenesis, although further studies are needed, in this study, we tested the role of iPolyP on the proliferation of CRC using in vitro and ex vivo approaches, in order to evaluate the effect thereof on tumor growth, we found that i Polyp was significantly increased in tumor tissues with respect to the corresponding peritumoral counterparts. Furthermore, iPolyP signaling occurs through the TRPM8 receptor, promoting the proliferation of CRC cells. Experiments of TRPM8 pharmacological inhibition or RNA interference showed that the involvement of TRPM8 is essential, greater than that of the other two known iPolyP receptors, P2Y1 and RAGE. The presence of iPolyP drives tumor cells toward the mitotic step of the cell cycle by improving the expression of ccnbl, which encodes the Cyclin B protein. The 2D and 3D in vitro data reflected the ex vivo results, obtained from the generation of organoids derived from CRC, which increased in size. These results indicate that iPolyP can be considered a new and unexpected early biomarker which supports the proliferation of colorectal cancer cel 1 s .

[0058] Colorectal cancer (CRC) remains the third most lethal cancer diagnosed worldwide and shows an increasing incidence in both developing and developed countries [1]. individuals with CRC show backgrounds covering a wide range of genetic or epigenetic alterations [2, 3]. Sporadic or induced mutations in the CRC scenario often fall within a discrete set of tumor suppressor genes, such as adenomatous polyposis coli (APC) , SMAD family member 4 (SMAD4) , or those traditionally involved in the regulation of cell proliferation and the cell cycle, such as p53 [4]. Furthermore, mutations in oncogenes, in particular involving genes such as Kirsten rat sarcoma virus (KRAS), phosphatidylinositol 4.5-bisphosphate 3-kinase catalytic subunit-oc (PIK3CA) , or the proto-oncogene b- raf (BRAF) , confer hyperprol iterative traits to intestinal epithelial cells, which promote a scenario of genetic hypermutability [5]. Lifestyle, environmental mutagens or, more recently, dysbiotic metabolites, are emerging as key ingredients in the onset and development of CRC [6], although from a mechanistic point of view, a series of questions remains unresolved and new potential candidates must be disclosed. The inorganic polyphosphate food additive (iPolyP) is a biological polymer, structurally consisting of a range of from 3 to over 1000 orthophosphates linked to each other by ATP-like bonds [7], and can spontaneously derive from non-living matter or be enzymatically produced by living organisms, including bacteria and eukaryotes [8]. while the synthesis and degradation of iPolyP in bacterial cells have been well studied, the corresponding metabolic pathways in the eukaryotic counterpart are still poorly known [9]. Mammalian iPolyP has been found in the nuclei, mitochondria, lysosomes, platelet dense granules, and granules derived from mast cells and basophils [10-13]. The iPolyP architecture makes it an obvious participant in energy metabolism, indeed, nowadays it is considered a phosphate donor for ATP synthesis

[0014] . Therefore, considered an optimal source of energy, in recent decades the emerging literature has begun to associate iPolyP with various pathophysiological processes, including inflammation-guided diseases [15-20], tumori genesis [9], tumor metastasis [21, 22] and cell proliferation [23, 24] . It has been demonstrated that ipolyp mediates cell proliferation by selectively upregulating the mechanistic target of rapamycin

[0059] (mTOR) phosphorylation at Ser2481

[0023] . Three iPolyP- binding receptors have been identified: namely the receptor for advanced glycation end products (RAGE), the purinergic receptor P2Y1 and the transient receptor potential cation channel subfamily M (melastatin) member 8 (TRPM8) [25,26]. Preliminary evidence has highlighted a novel pathway involving the iPolyP / RAGE receptor linked to the wingless (wnt) related integration si te / 0-catenin signaling axis, of crucial importance for the regulation of key pathophysiological processes in tumor cells

[0027] .

[0060] However, in addition to limited evidence on ipolyp mediated cell proliferation, few studies have yet reported associations between ipolyp, its receptor and cancer

[0028] , perhaps due to difficulties in quantifying ipolyp and the lack of comparative data for the neoplastic counterpart and the corresponding normal counterpart, in parallel, recent literature has reported an overexpression of the TRPM8 receptor in CRC samples, which is correlated with poorer survival

[0029] . TRPM8s belong to the superfamily of transient receptor potential (TRP) cation channels, melastatin (M) subfamily, member 8 (TRPM8) , also known as cold and menthol receptor 1 (CMR1)

[0030] . However, the clinical relevance thereof remains largely fragmented, in our study we tested the amount of ipolyp in tumor and peritumoral tissues from subjects suffering from colorectal cancer, showing a significant discrepancy. Furthermore, we determined the role of the TRPM8 receptor as a mediator of iPolyP signaling, which, ultimately, leads to the activation of the proliferation marker in CRC. Overall, these results can add iPolyP to the list of early CRC biomarkers, together with DNA, RNA and proteins, which have been shown to prolong overall patient survival

[0031] , thereby potentially paving the way for the development of new anti -cancer agents as adjuvants to conventional chemotherapy.

[0061] 1. Materials and methods

[0062] 1.1. Patient samples in this retrospective study samples derived from 50 patients with CRC, candidates for surgical treatment, were studied. The patients provided written informed consent to the collection of blood samples for biomarker analysis in accordance with Prot. No. 397 / C. E. dd. 9 / 16 / 2020 of the Local Ethics Committee “Gabriella Serio” IRCCS “Giovanni Paolo II” Cancer institute, Bari, Italy. Biopsy tissue samples were provided by the Histopathology unit of the IRCCS “S. de Bellis”. The analyses were carried out by comparing two different sections of histological sample originating from the same patients, so as to identify a healthy portion (Normal or Peri tumoral) and a diseased portion (Pathological or Tumoral).

[0063] 1.2. Tissue preparation

[0064] The tissues of the patients (Pt), partitioned into two counterparts, Peri tumoral (PT) and Tumoral (T), were assayed for the amount of iPolyP, by fluorometric assay, and for the level of PCNA and TRPM8 by immunoblotting. The tissues were lysed using a tissue homogenizer (TissueLyser II) , (QIAGEN, Hilden, Germany; Cat. no. 85300) in Buffer m / Poly P Extraction Buffer, for the detection of i Polyp, described in detail in the section related to inorganic polyphosphate detection assay, or in the T- PERTM tissue protein extraction reagent supplemented with the single-use protease and phosphatase inhibitor cocktail Halt™, the immunoblotting section.

[0065] 1.3. inorganic polyphosphate detection assay inorganic polyphosphate was detected in tumor and peri tumoral tissues using the inorganic polyphosphate (fluorometric) assay kit (Abeam, Cambridge Biomedical Campus, Cambridge, united Kingdom; Cat. No.: ab284528) following the manufacturer’s related recommendations, in brief, the homogenized tissues were centrifuged at 10,000 x g for 15 minutes at 4°c and the clear supernatant was collected and treated with positive RNAsi / RNasi control, DNasi and proteinase K. 100 pl of the standard solution of polyphosphate standard / i Polyp standard were mixed with 900 pl of water to prepare a 10 pM polyphosphate / i Polyp standard solution used for analyzing and plotting the polyphosphate standard curve, in order to test 5 pl of each sample in triplicate, 96 well plates were prepared, adjusting the volume of each well to 50 pl using the polyphosphate assay buffer / i Polyp assay buffer. To each well, including standards and samples, a 50 pi_ reaction mixture was added, including 47 pi_ of polyphosphate assay buffer and 3 pi_ of polyphosphate dye. The plates were incubated for 10 minutes at ambient temperature and the fluorescence of all wells was measured at Ex / Em = 415 / 550 nm at ambient temperature in endpoint mode. The calculation of the amount of inorganic polyphosphate was performed using the following equation: Amount of i Polyp (pmol / mg) = A x D / w x vt / va, where: A = The amount of i Polyp was calculated from the standard curve of polyphosphate / i Polyp (pmol), D = sample dilution factor (D = 1, for undiluted samples), w = weight of the tissue used (in mg) or amount of proteins determined by the protein assay (mg), vt = total volume of the sample, va = volume of the sample measured in the well. The amount of proteins (in mg) was determined using the Bio-Rad protein assay dye reagent (Bio-Rad Laboratories, Hercules, California, U.S.A.; Cat. no.: Hercules, California, U.S.A.; Cat. no.: 5000006EDU) .

[0066] All cell lines were maintained in a humidified atmosphere at 37 °C with 5% CO2. The cells were passaged and the medium was changed every other day. 1.4. Cell cultures and reagents

[0067] Caco-2 and SW620 human colorectal adenocarcinoma cells were purchased from the American Tissue Culture Collection (ATCC, Manassas, Virginia, U.S.A.; Cat. no. HTB-37 and CCL-227, respectively). Human colon epithelial cells 1 transduced with CDK4 and telomerase HCEC-ICT were purchased from Evercyte GmbH (Vienna, Austria; cat. no. CkHT-039-0229) . Caco-2 and SW620 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) , (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no.: 11965092), supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, Massachusetts , U.S.A.; Cat. no.: A5256701), 1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no.: 11360039), 25 mM HEPES (Thermo Fisher

[0068] Scientific, Waltham, Massachusetts, U.S.A.; Cat. no.: 15630056) and 100 U / mL antibiotic-antimycotic (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no.: 15240062). The epithelial cells of the human colon 1 transduced with CDK4 and telomerase (HCEC-ICT) were cultured in ready-to-use Coloup medium (Evercyte GmbH, Vienna, Austria; Cat. no.: MHT-039) , supplemented with 100 u / mi_ antibiotic-antimycotic.

[0069] 1.5. Cellular treatments

[0070] The HCEC-ICT, Caco-2 and sw-620 cell lines were seeded in 6 well plates (Corning, New York, New York, U.S.A.; Cat. no. 3516) at a density of 0.5 x 106 cells / well in 2 mi_ of complete cell culture medium. The seeded cells were treated with 0.5 pM of sodium phosphate glass (i Polyp) (Sigma-Aldrich, st. Louis, Missouri, U.S.A.; Cat. no.: S4379-500mg) or with 10 pM of the TRPM8 receptor inhibitor N- (3-Ami nopropyl )-2- [(3-methyl phenyl ) methoxy] -N- (2-thi enyl methyl ) benzamide hydrochloride (AMTB) , (Santa Cruz Biotechnology, Dallas, Texas, U.S.A.; Cat. no.: 926023-82-7), or in combination for 72 hours. Di methyl sulfoxide (DMSO) , (Sigma-Aldrich, st. Louis, Missouri, U.S.A.; Cat. no. D8418-100mL) was added to the control cells. The pharmacological inhibition of the iPolyP / TRPM8 axis was performed by adding AMTB to the HCEC-ICT, Caco-2 and SW620 cell lines. The pharmacological inhibition of the P2Y1 receptor was performed with adenosine 2 5 '-diphosphate sodium salt (Santa Cruz Biotechnology, Dallas, Texas, U.S.A.; Cat. no.: SC- 214495); The inhibition of the RAGE receptor was performed with (Abeam, Cambridge Biomedical Campus, Cambridge, United Kingdom; Cat. no. ab235552). The cells were centrifuged at 1100 revolutions per minute for 5 minutes at 4°c and washed with lx Dulbecco's phosphate-buffered sterile saline solution 1 (lx DPBS) (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no. 14190-094) two times. The dried pellets were frozen at -80°C or resuspended and lysed in 200 pL of T-PERTM Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; cat. no.: 78510) supplemented with Halt™ Protease and Phosphatase inhibitor single -use Cocktail, EDTA-Free (lOOx) (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no. 78443) for immunoblotting analysis.

[0071] 1.6. immunoblot

[0072] Both tissue and cell lysates were incubated on ice for 30 minutes and stirred on a vortex mixer every 10 minutes. The samples were then centrifuged at 16,000 rev / min at 4°C for 20 minutes to clarify and precipitate insoluble debris. The extracted total proteins were analyzed to measure the concentrations using Bio-Rad Protein Assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, California, U.S.A.; Cat. No: 5000006EDU) . Then the proteins were mixed with 4 x Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, California, U.S.A.; Cat. No: 1610747) and 10% of (3-mercaptoethanol (Sigma-Aldrich, st. Louis, Missouri, U.S.A.; Cat No: M6250-100mL) and denatured at 95°C for 5 minutes. 25 pg of proteins were loaded onto 4-20% pre-cast polyacrylamide gels (Bio-Rad Laboratories, Hercules, California, U.S.A.; Cat. No: 4568094), then blotted onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules , California, U.S.A.; Cat. No.: 1704156) using the turbo trans-blot transfer system (Bio-Rad Laboratories, Hercules, California, U.S.A.; Cat. No. 1704150). The membranes were blocked using Pierce™ Protein-Free Blocking Buffer (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. No: 37571) for 1 hour and stained overnight with primary antibodies. The following day, the membranes were washed three times with lx Tris Buffered saline solution (lx TBS), (BioRad Laboratories, Hercules, California, U.S.A.; Cat. No: 1706435 diluted in ddH20 to reach 1X) / T EEN20 (Sigma-Aldrich, st. Louis, Missouri, U.S.A.; no.: P9416-100 mL) incubated for 1 hour with the respective secondary antibodies conjugated with horseradish peroxidase.

[0073] The proteins were detected using the Clarity Max western ECL substrate (Bio-Rad Laboratories, Hercules, California, U.S.A.; Cat. No: 1705062) and the signals were obtained using the chemidoc MP imaging System (Bio-Rad Laboratories, Hercules, California, U.S.A. ; Cat. 1708280). The following primary antibodies were used: anti-PCNA, 1:1000 (Santa Cruz Biotechnology, Dallas, Texas, U.S.A.; Cat. No. SC-56); anti-TRPM8, 1:1000 (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. No. MA5-35474); anti- GAPDH, 1:1000 (Santa Cruz Biotechnology, Dallas, Texas, U.S.A.; Cat. No. SC-47724) ; anti -0-acti n , 1:1000 (Cell Signaling Technology, Danvers, Massachusetts, U.S.A.; Cat. No. 4970S); anti-P2Yl-R, 1:1000 (Abeam, Cambridge Biomedical Campus, Cambridge, United Kingdom; Cat. No. abl68918) ; anti-RAGE-R, 1:1000 (Abeam, Cambridge Biomedical Campus, Cambridge, United Kingdom; Cat. No. ab216329) . GAPDH and p-Actin were used as loading controls. The following secondary antibodies were employed: Anti-rabbit igG, HRP-linked Antibody, 1:2000 (Cell signaling Technology, Danvers, Massachusetts, U.S.A.; Cat. no. 7074s) ; Goat antimouse igG (H+L)-HRP conjugate (Bio-Rad Laboratories, Hercules, California, U.S.A.; Cat. no.: 1706516).

[0074] 1.7. TRPM8 knockdown with siRNA

[0075] For TRPM8 knockdown experiments using siRNA, HCEC-ICT, Caco-2 and SW620 were electroporated with two TRPM8 Silencer siRNA (Thermo Fisher Scientific, Cat. no. 4392420, ID: S35489 and ID: S35490, respectively) or with silencer Negative Control siRNA (Thermo Fisher Scientific, Cat. no. 4390843). Electroporation was performed using the Neon™ NXT electroporation system (Thermo Fisher Scientific, cat. no. NEON1SK) , according to the manufacturer's recommendati ons .

[0076] 1.8. Crystal violet analysis

[0077] HCEC-ICT, Caco-2 and SW620 cells were seeded into 96 well plates (Corning, New York, New York, U.S.A.; Cat. No. 3599) at a density of 2 x 103 cells / well in 100 pL of complete medium and left overnight to allow complete attachment. On the following day (considered t = 0 hour), the medium was removed and replaced with 100 pL of fresh serum-free medium to allow synchronization of the cell cycle. The cells seeded in triplicate wells were treated and maintained under different conditions: 0.5 pM of iPolyP, or 10 pM of AMTB, or both in combination, for 96 hours. The controls were cells treated with DMSO. After 96 hours, the cells were fixed by adding 100 pL / well of 4% paraformaldehyde (PFA), (Sigma-Aldrich, st. Louis, Missouri, U.S.A.; Cat. no. P6148-500G), pH 7.6 at ambient temperature for 30 minutes to the medium (obtaining a final concentration of PFA of 2%). Each well was then colored with 0.02% crystal violet solution (Sigma-Aldrich, st. Louis, Missouri, U.S.A.; Cat. no. HT90132-1L) for 10 minutes and washed with cold water to remove the excess staining, images were acquired with an inverted laser scanning confocal microscope Nikon Ti2, equipped with a 10X objective (numerical aperture 0.25), as described in the immunofluorescence and confocal microscopy section, in bright field and analyzed with Nis-Elements software (version 5.11.01 ) and lmageJ2 (version 2.14.0 / 1.54f) prior to complete solubilization. Finally, the crystal violet staining bound to the cells was redissolved in an aqueous solution containing 1% sodium dodecyl sulfate (SDS) , (Sigma-Aldrich, st. Louis, Missouri, U.S.A.; Cat. no. L3771-100G) at ambient temperature for 30 minutes and the absorbance was measured at X = 595 nm with the iMarkTM microplate absorbance reader (Bio-Rad Laboratories, Hercules, California, U.S.A.; Cat. No. 168-1130).

[0078] 1.9. immunofluorescence microscopy

[0079] 2 cells at 105 / mL were cultured in 35 mm Petri dishes, no. 1.5 coverslip (MatTek, Ashland, Massachusetts, U.S.A.; cat. no. P35G-1, 5-14-C) . Fixative, permeabilizing and blocking buffers were prepared in lx DPBS. The cells were fixed with 4% PFA for 30 minutes at ambient temperature and then washed twice using lx DPBS. Permeabilization was performed for 5 minutes at ambient temperature using 0.15% Triton x-100 (in DPBS lx), washing was performed to remove the permeabilization buffer. The cells were then blocked for 1 hour at ambient temperature using blocking buffer (3% bovine serum albumin (BSA) , (Sigma-Aldrich, st. Louis, Missouri, U.S.A.; Cat. no. A7030-100G) / lXDPBS) . The cells were incubated with the primary antibody overnight. The nuclei were stained using PureBlu DAPI (Bio-Rad Laboratories, Hercules, California, U.S.A.; Cat. no. 1351303). Extensive washing steps were performed to remove the unbound stain. The anti-Ki67 antibody was used as the primary antibody (ready-to-use , Abeam, Cambridge Biomedical Campus, Cambridge, United Kingdom; cat. no. abl6667) . The images were acquired with an inverted laser scanning confocal microscope Nikon Ti2 provided with Plan Apo 20x (numerical aperture 0.75) in bright field and analyzed with the software Nis-Elements (version 5.11.01) and image! 2 (version 2.14.0 / 1.54f) . All fluorescence images were acquired with a Yokogawa spinning-disk confocal on an inverted Nikon Ti microscope provided with a Plan Fluor 20x lens (numerical opening 0.75); bright field micrographs related to CRC organoids were acquired with Plan Fluor 60x (numerical opening 0.85); bright field micrographs related to spheroids were acquired with Plan Fluor 40x

[0080] (numerical opening 0.60) and 20x. The images were acquired with a Hamamatsu ORCA ER cooled CCD camera controlled with the Nis-Elements software (version

[0081] 5.11.01). The images were acquired using an exposure time of 700 milliseconds. Gamma, brightness and contrast were adjusted on the displayed images

[0082] (identically for the comparative image sets) using the NIS-E! ements software (version 5.11.01). The Perfect Focus system was kept in operation for the continuous focusing maintenance. During the image acquisition, DMEM without phenol red was used (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no. 21063-045).

[0083] 1.10. CRC tumor organoids

[0084] CRC samples originating from surgically resected tumor tissue were washed three times with lx DPBS and digested with a collagenase / hyaluronidase mixture (Stemcell Technologies, Cat. no. 07912), diluted in HBSS solution (with CaC12 and MgC12, Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no. 14025-050) for 5 hours under gentle oscillation at 37°c. A single-cell suspension was obtained and the cells were incorporated into the basement membrane of the matrigel matrix (Corning, New York, New York, U.S.A.; Cat. no. 356231) and cultured in intestiCult- SF medium (ICT-SF) (stemcell Technologies, Vancouver , British Columbia, Canada; Cat. no. 100-0340) medium diluted 1:2 in Advanced DMEM / F-12 (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no.

[0085] 12634-010), supplemented with N- 2 (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no.

[0086] 17502-048), supplement B27 (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no. 12587-010), 0.01% bovine serum albumin, antibiotic / antimycotic and HEPES. The medium was renewed every two days until complete development of the organoids (7-10 days). The mature organoids were divided using the dissociation agent TrypLE Select (Thermo Fisher Scientific, waitham, Massachusetts, U.S.A.; Cat. no. A12177-01) according to

[0032] and the obtained suspended cells were re-cultured at a lower density in mat ri gel.

[0087] 1.11. CRC tumor organoid growth test

[0088] The mature CRC tumor organoids were divided to obtain single cell suspensions. The cells were counted and 2,000 cells were embedded in 40 pl of matrigel (with a matrigel density of 65%) in 96 well plates, cultured in ICT-SF medium and allowed to grow for 10 days in the presence or absence of i Polyp at a concentration of 0.5 pM. Medium and iPolyP were replaced each 2 days. At the end of the experiment, microscopic images of the organoids were acquired in bright field using the Nikon Eclipse Ti2 confocal microscope. The growth rate of the organoids was determined using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega Corporation, Fitchburg, Wisconsin, U.S.A.; Cat. no. G3580) , according to the manufacturer's recommendations.

[0089] 1.12. Spheroids derived from cell lines

[0090] 1 x 103 Caco-2, SW620 and HCEC-1CT cells were seeded in 96 well 3D low attachment cell culture plates (Corning, New York, New York, U.S.A.; Cat. no. 4520) to obtain 3D cell line spheroids with 100 p 1 of growth medium in each well. The cells were maintained in culture under the conditions mentioned above. After seeding, all cell lines were treated with i Polyp and / or AMTB, while untreated cells were used as controls (untreated, UT). The spheroid cultures were observed at 24 and 96 hours and maintained in a humidified incubator set to 37°C and 5% CO2. After 96 hours, UT and treated cells were fixed with a 4% paraformaldehyde solution in PBS lx and used for microscopic analysis.

[0091] 1.13. Gene expression analysis by reverse transcription quantitative real time PCR

[0092] Caco-2 cells were seeded in 6 well plates at a density of 0.5 x 106 cells / well in 2 ml_ of complete cell culture medium. The seeded cells were treated with 0.5 pM iPolyP, for 72 hours; untreated cells were used as control. Caco-2 cells were seeded in 6 well plates at a density of 0.5 x 106 cells / well in 2 mi_ of complete cell culture medium. The seeded cells were treated with 0.5 pM iPolyP, for 72 hours; untreated cells were used as control, untreated cells were used as control. RNA extraction was performed from frozen cell pellets using the RNeasy Mini Kit (QIAGEN, Hilden, Germany; Cat. no.: 74104) according to the manufacturer's recommendations. The RNA concentration was measured using a NanoDrop 2000c (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no. ND-2000) and 2 pg of total RNA were reverse- transcribed into CDNA using a high-capacity CDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, Massachusetts, U.S.A.; Cat. no. 4368814) following the corresponding protocol. The reactions were initiated using iTaq universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, California, U.S.A.; Cat. no.: 1725124) and the validated human primers purchased from Bio-Rad (Hercules, California, U.S.A.), with the following assay ID Numbers: CCNA1, qHsaClD0008934; CCNB1, qHsaClD0010571; CCND1, qHsaClDOO13833 ; GAPDH, qHsaCED0038674. Real-time PCR analysis was performed with the CFX96 Touch Deep well real-time PCR detection system (Bio-Rad Laboratories, Hercules, California, U.S.A.; Cat. no. 3600037) and the experiments were conducted three times in triplicate. The relative expression was calculated using the 2-AACt method. 1.14. Cell cycle analysis

[0093] Caco-2 and SW620 cells were seeded in 24-well plates (Corning, New York, New York, U.S.A.; Cat. no. 3524) at a density of 2 x 104 cells / well in 100 pL of complete medium and left overnight to allow complete attachment. The following day the medium was replaced with fresh serum-free medium to allow synchronization of the cell cycle. The cells were treated with 0.5 pM ipolyp or with 10 pM AMTB hydrochloride or both in combination for 72 hours. The controls were cells treated with DMSO. After the treatments, 150 pi_ of cell clock dye, from the Biocolor Cell-ClockTM Cell Cycle Assay kit (ilex Life Science, Candler, North Carolina, U.S.A.; Cat. no. C1000) , were added to the center of each well and incubated for 1 hour at 37°c, according to the manufacturer's recommendations. The culture medium supplemented with the reagent was gently discarded and replaced with 200 pL of fresh medium. Live-cell micrographs were acquired using a Nikon Eclipse Ti2 confocal microscope in bright field provided with a Plan Fluor 20x lens (numerical aperture 0.75) and all experiments were conducted in triplicate. The calculation of the phase percentages was obtained by means of analyses performed with the imageJ2 software. The Redox dye used in this test is absorbed by live cells and the result is a cell cycle phase defined by color; each color was associated with cells in G0-G1 (yellow staining), S (green staining), G2 and M (dark green / blue phases).

[0094] 1.15. Statistical analysis

[0095] The patients’ features are reported as mean and standard deviation (M ± DS) and as frequencies and percentages (%) per category, in order to compare the ipolyp values between the groups, the Mann-whitney rank test was used for continuous variables. The Spearman rank correlation coefficient was used to test the strength and direction of the associations between ipolyp and TRPM8 / PCNA. During the test of the null hypothesis of no association, the two tailed probability error level was set at 0.05. All statistical calculations were performed using StataCorp. (2021) stata Statistical Software: Release 17 (StataCorp LLC, College Station, Texas, U.S.A.), while RStudio (“Chocolate Cosmos” Release, Posit PBC, Massachusetts, U.S.A.) was used for the charts.

[0096] 2. Results

[0097] 2.1. Human colorectal tumor tissue shows increased levels of iPolyP which are correlated with the proliferation marker PCNA.

[0098] To estimate iPolyP levels in patients with colorectal cancer (CRC) , we enrolled 50 patients undergoing surgery. The males (m) had a greater prevalence of CRC (52.00%) than females (f) (48.00%) with a mean average age of 71.53 ± 11.19 years.

[0099] Tumor-node-metastasis (TNM) and classification of the classification of each subject are shown in Table 1. we collected the peritumoral portion and the tumor counterpart portion from each subject to perform the detection of the iPolyP level. Curiously, the concentration of iPolyP was higher in the tumoral tissue than in the peritumoral section, with a statistically significant difference (373,097.70 ± 210,216.20 pmol / mg vs 166,102.70 ± 124,618.80 pmol / mg, respectively), suggesting a putative role of iPolyP in promoting the tumorigeni ci ty of CRC (the Figure 1) . Furthermore, in the same analyzed samples, the tumoral tissue showed high levels of the PCNA protein (proliferating cell nuclear antigen), a known marker of DNA replication and cell proliferation, with respect to the matched peritumoral tissue (Figure 2A). Furthermore, a strong positive correlation was found between i Polyp and the expression of PCNA (p = 0.44, **** p <0.0001) (Figure 2B), which further suggests a synergistic pro-tumori genic contribution of i Polyp to the development of CRC.

[0100] 2.2. The i PolyP-TRPM8 signaling axis supports the proliferation of colorectal cancer cells:

[0101] To determine whether iPolyP has pro-neoplastic properties and, if so, which receptor-binding partner signals to the colorectal cancer environment, we initially examined biopsies from the same cohort of enrolled subjects, for the P2Y1 receptor (P2Y1-R), the RAGE receptor (RAGE-R) and the TRPM8 channel receptor, all wel 1 -recognized iPolyP receptors. Among these, only TRPM8 appeared to be overexpressed in the tumor fraction with respect to the peri tumoral fraction (Figure 3A) in all samples, while the levels of P2Y1-R and RAGE-R were not different. Furthermore, similarly to PCNA, we found a strong positive correlation between the concentration of iPolyP within tumor tissues and the expression of the TRPM8 receptor (p = 0.48, **** p < 0.0001), (Figure 3B), supporting the hypothesis that iPolyP can transduce primarily through the TRPM8 receptor in the CRC context. Following these results, we initially explored whether the presence of iPolyP could in some manner promote the proliferation of colorectal cancer cells by interacting with the TRPM8 receptor. using an in vitro approach, we demonstrated that iPolyP administration induces the expression of PCNA in Caco-2 (Figure 4A) and in SW620 , non metastatic and metastatic colorectal cancer cell lines, respectively, without altering the TRPM8 Levels (Figure 4A). Surprisingly, no detectable effect of i Polyp at the level of PCNA was observed in HCEC-ICT cells, the cytogenetically normal and non-tumoral colon-derived cell line. Following the pharmacological inhibition or the genetic abrogation of the TRPM8 receptor, we were able to restore the expression of PCNA to levels comparable to those observed in the untreated samples, in both colorectal cancer cell lines examined (Figure while no difference was observed in HCEC Cells -1CT after ipolyp challenge. Direct evidence of cell proliferation mediated by the iPolyP-TRPM8 axis was obtained with crystal violet staining, a quantitative assay which discriminates live cells from dead ones based on the DNA- intercalating dye, proportional to the number of adherent cells. After 96 hours, ipolyp revealed a marked propensity to promote the proliferation of Caco-2 and SW620. Furthermore, as expected, by antagonizing or knocking down the TRPM8 receptor, we flattened the proliferation rate (Figure 4B, c). This significant highly proliferative phenotype is not distinguishable in HCEC-ICT cells after ipolyp treatment, whose division rate remains unchanged (Supplementary figure S3B, c) . i Polyp does not appear to influence the expression levels of the P2Y1 and RAGE receptors, either in HCEC-1CT or in Caco-2 or SW620 cells (Supplementary figure S4A,B), thus excluding a positive feedback mechanism mediated by ipolyp underlying the molecule's regulation of the receptors thereof. The proliferation assays performed on Caco-2 and SW620 with the P2Y1 or RAGE inhibitor demonstrated that i Polyp primarily triggers the expansion of colorectal cancer cells by binding to TRPM8, since the blockade of these pathways did not affect iPolyP induced proliferation, in order to corroborate the proliferative propensity of iPolyP, we performed fluorescence microscopy experiments directed to the Kiel 67 antigen, known as Ki -67, a second common marker for proliferation, in our model cell lines, the result of which revealed alterations in PCNA. in particular, after administration of iPolyP, Caco-2 and SW620 cells, but not HCEC-ICT, exhibited an increased level of Ki-67, which was suppressed following inhibition of TRPM8 or when silenced. Furthermore, SW620 cells exhibited self-organization into spheroids, the morphology of which appeared enlarged in the presence of iPolyP. Overall, these data provide an important message regarding the ability of iPolyP to promote the growth of colorectal tumor cells by engaging the TRPM8 receptor.

[0102] 3.3. iPolyP promotes the growth of patient-derived CRC organoids and the formation of spheroids derived from Caco-2 and SW620 cells

[0103] Based on the above results, we recapitulated the proliferative role of iPolyP with growth assays of patient-derived CRC organoids, we generated organoids from two CRC subjects and incubated them with iPolyP. As shown in Figure 5 panel A-B, the growth rate of the organoids in the presence of iPolyP was significantly higher than those untreated, for both independent experiments (Figure 5A, B) . Furthermore, these data reflected the evidence observed with spheroids derived from Caco-2 and SW620 cells; in particular, after 96 hours of incubation with iPolyP, the diameter of the spheroids was double compared to those untreated. The presence of the TRPM8 inhibitor abrogated the proliferative effect due to iPolyP. Finally, we were not able to observe any change in HCEC-ICT cells (Figure 5C, D) , confirming our conclusions regarding the role of iPolyP in CRC.

[0104] 3.4. iPolyP induces the expression of ccnbl in the Caco-2 cell line and drives the cells into the M phase by means of the TRPM8 receptor

[0105] To further explore the molecular mechanism through which inorganic polyphosphate drives the expansion of colorectal tumor cells, we performed quantitative real-time reverse transcription PCR experiments on RNA derived from Caco-2 cells, 72 hours after treatment with iPolyP, probing genes involved in different steps of the cell cycle. Although we noted a considerable increase in terms of the number of copies of transcripts related to genes such as ccndl (cyclin Dl) and ccnal (cyclin Al), mainly involved in the phases G1 and S / G2, respectively, a significant amplification of the signal was observed for the ccnbl transcript, which encodes the mitotic protein cyclin B, a principal regulator of the phase G2 / M (Figure 6A). Figure 6B summarizes the most relevant cyclins associated with the different phases of the cell cycle, in order to carry out a cellular interpretation of the gene expression data, we performed a "cell clock" cycle assay on Caco-2 and SW620 cells exposed to i Polyp for 72 hours. This assay consists of detecting live cells employed to monitor the four principal phases of the mammalian cell cycle by means of a redox dye. After absorption of the dye, a marked change in color occurs within the cells, which denotes the specific Gl, S, G2 and M phases of the cycle, in detail, it becomes yellow in the phase Gl, green in S / G2 and blue in the phase M. A significantly high percentage of blue cells was identified after administration of iPolyP, compared to the control sample, the latter being comparable to the cells treated with TRPM8 inhibitors or iPolyP + TRPM8 inhibitors (Figure 6C-D) . Overall, these data strengthen our interpretation of a pro-tumorigeni c role of iPolyP in colorectal tumor cells. in this context, we investigated whether iPolyP, of bacterial or human origin, could in some way promote the pathogenesis of CRC. By applying in vitro and ex vivo approaches, we have shown for the first time that iPolyP facilitates the proliferation of CRC cells by considerably engaging the channel of the TRPM8 receptor. This conclusion is based on the following evidence, summarized in Figure 7A: (i) tumor tissues isolated from CRC subjects showed high levels of iPolyP, proportional to the amount of the proliferation marker PCNA; (ii) iPolyP governs the proliferation of CRC cell lines by interacting with the TRPM8 receptor, a result positively correlated with the concentrations of iPolyP within tumor tissues; (ii'i) ex vivo experiments, performed on organoids derived from CRC patients and 3D in vitro cell cultures, showed a significantly higher growth rate in the presence of iPolyP than untreated samples or samples in which TRPM8 was antagonized, (iv) the engagement of the iPolyP-TRPM8 axis triggers the expression of ccnbl, the corresponding protein product of which is cyclin B, which drives cells into the mitotic phase of the cell cycle. Comprising the source of iPolyP is becoming decisive to direct the proliferative path. Several studies in the field of microbiology today are focusing on pathogenic bacteria, such as Salmonella enterica, with particular emphasis on the mechanism employed by iPolyP in the onset of different mammalian diseases [47, 48, 49]. interestingly, a recent article conducted by Boyineni and colleagues demonstrated that tumor cells are capable of endogenously producing iPolyP which promotes metabolism during the starvation period, due to the insufficient vascular supply to the tumor

[0050] .

[0106] Therefore, the ongoing mass spectrometry experiments in our laboratory primarily aim to clarify the source of iPolyP in the CRC context, directing the research towards appropriate in vivo models targeted to the bacterial or human kinase responsible for the biosynthesis of inorganic polyphosphate. Although the molecular pathway has yet to be fully interpreted and further experimental evidence is needed to confirm our hypothesis, these results reveal a new functional axis in the CRC background, shedding light on new directions of study and paving the way for the development of new therapeutic strategies for patients with CRC.

[0107] 5. Conclusions

[0108] The results on the involvement of the iPolyP- TRPM8 axis can offer new therapeutic options in the fight against CRC, in combination with the conventional chemotherapeutic protocols applied today.

[0109] An early diagnosis of CRC requires a rather invasive method, which generally involves the use of biopsy samples taken at determined time intervals. Liquid biopsy offers a valid alternative, without the use of invasive procedures, to monitor the status of the disease, enabling detection of the disease and monitoring the progression or the response to therapy. in order to test the efficacy of i Polyp as a new diagnostic and prognostic biomarker, we performed screening tests on tumor and normal biopsy tissues derived from patients affected by colorectal cancer; as shown in Fig. 8.

[0110] Such an analysis highlighted a significantly greater concentration of inorganic polyphosphate in tumor biopsy tissues than the normal counterpart.

[0111] Following this data, it was verified that the concentration of inorganic polyphosphate observed in the biopsy tissues simultaneously reflects the presence thereof in the feces of the same patient.

[0112] A) Detection of inorganic polyphosphate in feces for diagnostic colorectal cancer (CRC) screening.

[0113] Tumor cells base the energy metabolism thereof on alternative sources useful for the survival and proliferation thereof, inorganic polyphosphate is well suited to the role of energy supplier, by virtue of the phospho-anhydride bonds thereof similar to those of ATP. indeed, our recent investigations performed in vitro have demonstrated the involvement of inorganic polyphosphate in tumor proliferation and progression, as well as an increase in the concentration thereof in pathological biopsy tissues compared to normal tissues (as previously described).

[0114] Taking into account that inorganic polyphosphate is produced not only by human cells but also by certain bacterial strains forming part of the microbiota, the researchers of the Histopathology unit of the requesting institute hypothesized a colonization of the tissue by fecal microbiota, which could have a role in the pathogenesis of the disease. indeed, since a close relationship exists between the epithelial cells of the colon and the feces transiting therein, it was investigated whether the concentration of inorganic polyphosphate observed in biopsy tissues simultaneously reflected the presence thereof in the feces.

[0115] This could have paved the way for the identification of inorganic polyphosphate as a new non-invasive biomarker useful in the early diagnosis of colorectal cancer.

[0116] Following the assay of inorganic polyphosphate in the feces of 3 healthy subjects (N) and 3 patients with colorectal cancer (p), the preliminary data collected showed the expected results (for the methodological part, reference is made to the preceding section “1.3. inorganic polyphosphate detection assay”). indeed, as observed in Figure 8, the concentration of inorganic polyphosphate in the feces of patients with colorectal cancer is from 2 to 8 times greater than that observed in the feces of healthy subjects.

[0117] These preliminary data, observed in the Applicant’s laboratory, reveal an undeniable and significant increase in inorganic polyphosphate (i Polyp) in fecal samples from subjects suffering from colorectal cancer, with respect to samples from individuals not affected by the same disease.

[0118] This allows us to claim the use of inorganic polyphosphate as a new non-invasive biomarker to be detected in fecal samples for early diagnosis, which can be associated with other screening methods such as fecal occult blood, in order to provide greater predictivity, i.e., the probability that a subject with a positive diagnostic test is actually affected by colorectal cancer.

[0119] Following this preliminary analysis, a more extensive investigation conducted on n = 82 healthy subjects and 82 affected by CRC unequivocally confirmed, as seen in Fig. 9, that CRC fecal samples exhibit a statistically significant higher level of the inflammatory molecule inorganic polyphosphate (i Polyp), compared to healthy individuals (***p = 0.0002).

[0120] Therefore, the hypothesis that the presence of high levels of iPolyP in subjects suffering from CRC could serve as an early-stage marker for CRC was also fully confirmed.

[0121] The research was carried out as follows:

[0122] According to the TNM staging system (Tumor, Node, Metastasis), the extent and severity of the cancer are classified into five main stages, conventionally indicated from stage 0 to Stage iv (T0-T4) . Therefore, the Applicant initially divided the patients affected by CRC into two main groups: early-stage CRC and advanced-stage CRC. The early-stage group included the adenoma (precancerous) and stages TO, T1 and T2, while the advanced-stage group comprised stages T3 and T4. The concentration of iPolyP was then determined in the fecal samples from patients stratified according to the TNM system, it is completely apparent, as seen in Fig. 10, that early stage CRC (n=8) exhibits significantly high iPolyP levels, similar to those observed in advanced stage CRC (n=33), and both are widely upregulated with respect to healthy fecal samples (n=82) (early stage vs. healthy: ***p = 0.0003; advanced stage vs. healthy: ****p = 0.00006).

[0123] Therefore, these results indicate that iPolyP can serve as a biomarker for the early diagnosis of CRC, enabling timely intervention and significantly improving the patient’s survival prospects.

[0124] B) inorganic polyphosphate (iPolyp) as a biomarker for the early diagnosis of colorectal cancer and use thereof with the fecal occult blood test (FOBT)

[0125] Although the fecal occult blood test (FOBT) is a valuable, low cost and non-invasive tool for the early diagnosis of colorectal cancer, it also has various important limitations.

[0126] One of the main disadvantages is the risk of false positives. The test can detect blood in the feces not caused by cancer or precancerous polyps, but rather by benign conditions such as hemorrhoids or i nfl ammati ons .

[0127] Furthermore, cancerous polyps could bleed at the time of the test, causing false negatives and missed diagnoses.

[0128] Therefore, the researchers of the requesting institute wondered whether the determination of iPolyP could compensate for these cases of false negatives. Surprisingly, as seen in Fig. 11, the concentration of iPolyP was statistically significant in patients affected by CRC, including those for whom FOBT produced a negative result (estimated at about 36.5% of all patients affected by CRC worldwide) (n=30) , with respect to healthy fecal samples (n=82) , (**p = 0.002), (FOBT, n = 52 + vs. healthy, n = 82: ***p = 0.0002)

[0129] These data demonstrate that the measurement of iPolyP, in combination with FOBT, can provide a better assessment than FOBT alone for the diagnosis of CRC.

Claims

CLAIMS1) use of inorganic polyphosphate (i Polyp) as a non-invasive biomarker in fecal samples for the screening and early diagnosis of colorectal cancer (CRC) .2) use of inorganic polyphosphate (i Polyp) as a non-invasive biomarker in feces, to be associated with the fecal occult blood test (FOBT) in order to improve predictivity, specificity, and sensitivity for CRC diagnosis, thus contributing to the reduction of false negatives compared to FOBT alone.3) Use of inorganic polyphosphate (i Polyp) as a non-invasive biomarker in feces, in the management and follow-up of CRC patients after (surgical and / or medical) therapy alone or in association with other tumor markers or indicators of disease activity.

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