Assay Method

Abstract

Methods for detecting or predicting cancer and / or metastasis are provided. Tumor samples can be used to determine the presence of mutations in the sodium leak channel (NALCN). A risk score for cancer and / or metastasis can be determined based on whether the mutation causes a decrease in the pore size of the NALCN. Computational models, compositions, and kits are also provided.

Description

[Technical field]

[0001] The present invention relates to a method for detecting or predicting cancer and / or metastasis, the method comprising the step of detecting a mutation in and / or a decrease in the activity of a sodium leak channel (NALCN). [Background technology]

[0002] Many patients with cancer die as a result of metastasis (Dillekas et al., 2019), the process by which cancer cells spread from the primary tumor to other organs in the body (Ganesh, K. & Massague, J. 2021). Cancer cells can spread throughout the body through various mechanisms, and some can form new tumors in other parts of the body. Metastatic cancer cells can also remain inactive for long periods at distant sites before they begin to grow again. Blocking metastasis could significantly improve the survival of patients with cancer; however, how this process is triggered within the complex cascade of tumorigenesis remains unclear (Massague, J. & Obenauf, AC, 2016).

[0003] Since metastasis is considered a completely abnormal process restricted to malignant tissues, the focus has been on identifying genetic mutations that arise as drivers of cancer metastasis. This work has revealed mutations that promote metastasis in mouse models and humans, including various ion channels that induce changes in gene transcription while altering transmembrane voltage, thus inducing a metastatic-like phenotype (House, CD et al., 2010; Sheth, M. and Esfandiari, L., 2022; Wang T et al., 2020), but so far no recurrent metastasis-specific mutations have been identified (Ganesh, K. and Massague, J, 2021; Massague, J. & Obenauf, AC, 2016; Nguyen, B. et al., 2022).

[0004] Other cellular functions involved in the metastatic cascade include “stem cell-like” pluripotency and plasticity. Metastatic cancer cells have been attributed stem cell potential because of their ability to reconstitute heterogeneous malignant cell populations such as metastatic tumors (Ganesh, K. et al., 2020, and Laughney, AM et al., 2020). Epithelial-mesenchymal transition (EMT), a type of cellular plasticity that appears during normal cystogenesis and tissue healing (Ganesh, K. & Massague, J, 2021), is also a well-established feature of the metastatic cascade (Ganesh, K. & Massague, J, 2021, and Pastushenko, I. et al., 2018). It remains unclear how cancer “hijacks” such normal cellular functions to enable metastasis.

[0005] Thus, there is a need to develop methods to detect metastasis and cancer. In this application, we identify the single ion channel NALCN as a key regulator of epithelial cell migration to distant tissues. NALCN is involved in the background sodium leak conductance that maintains the resting membrane potential. NALCN controls important functions in excitable tissues, such as respiration and circadian rhythms (Chua, HC et al., 2020; Kschonsak, M. et al., 2020; and Lu, B. et al., 2007), and gain-of-function mutations in the gene are associated with neurological disorders (Bend EG et al., 2016). However, little is known about the role of NALCN in non-excitable tissues. The present invention demonstrates that NALCN controls the release of malignant and normal epithelial cells into the blood and their migration to distant sites, where they form metastatic cancer or apparently normal tissue, respectively. Hereby, we demonstrate that the metastatic cascade can be initiated and operated independently of tumor formation. Such observations have important implications for our understanding of epithelial cell migration in health and disease and identify novel targets for antimetastatic therapies. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012) [Non-Patent Document 2] Smart et al., HOLE: A program for the analysis of the pore dimensions of Ion Channel Structural models. Journal of molecular Graphics, doi:10.1016 / S0263-7855(97)00009-X (1996) [Non-Patent Document 3] Klesse et al., CHAP: A Versatile tool for the Structural and Functional Annotation of Ion Channel Pores. J Mol Biol. 2019;431(17):3353–3365 [Non-Patent Document 4] Chovancova et al., CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput Biol. 2012;8(10):e1002708. [Non-Patent Document 5] Sehnal et al., MOLE 2.0: advanced approach for analysis of biomacromolecular channels. J Cheminform. 2013;5(1):39 [Non-Patent Document 6] Rahrmann et al., The NALCN channel regulates metastasis and nonmalignant cell dissemination. Nature Genetics, doi.org / 10.1038 / s41588-022-01182-0, 2022 [Non-Patent Document 7] https: / / doi.org / 10.1038 / s41588-022-01182-0 Summary of the Invention [Problem to be solved by the invention]

[0007] We identify the single ion channel NALCN as a key regulator of both malignant and non-malignant cell metastasis, providing important insights into the metastatic process and novel targets for anti-metastatic therapy. In 10,022 human cancers, NALCN loss-of-function mutations were selectively enriched in advanced gastric and colorectal cancers. Deletion of Nalcn from mice susceptible to developing gastric, intestinal, or pancreatic adenocarcinomas did not alter the incidence of these tumors, but significantly increased the levels of circulating tumor cells (CTCs) and metastatic spread in the peritoneum, kidney, liver, and lungs. Treatment of such mice with gadolinium, a Nalcn channel blocker, similarly increased CTCs and metastases. Remarkably, deleting Nalcn from mice that lack oncogenic mutations and will not develop cancer caused a similar dissemination of cells into the peripheral blood at levels comparable to those observed in tumor-bearing animals. These cells migrated to distant organs, where they formed nontumor but apparently normal structures, including kidney glomeruli and tubules. The transcriptomes of these circulating cells in tumor- and nontumor-bearing mice were indistinguishable but were also closely related to those of human CTCs. Thus, NALCN controls cell dispersal from solid tissues independently of cancer, uncoupling this process from tumorigenesis and revealing NALCN as a critical mediator of metastasis. [Means for solving the problem]

[0008] One aspect of the present invention is a method for detecting or predicting cancer and / or metastasis, comprising the steps of: Analysing a tumor sample obtained from the subject; determining the presence of at least one mutation in a sodium leak channel (NALCN) in the tumor sample relative to a reference sample; determining whether the at least one mutation causes a decrease in the pore size of the NALCN; If the mutation causes a decrease in the pore size of the NALCN, the decrease in pore size is used to determine a risk score for cancer and / or metastasis. The present invention relates to a method comprising the steps of:

[0009] One aspect of the present invention is a method for detecting or predicting cancer and / or metastasis, comprising the steps of: Analyzing a biological sample obtained from the subject to assess sodium leak channel (NALCN) activity; providing a cancer and / or metastasis risk score based on the level of NALCN activity; The present invention relates to a method comprising the steps of:

[0010] One aspect of the present invention is a method for detecting or predicting cancer and / or metastasis, comprising the steps of: Analyzing the biological sample to detect the presence of one or more mutations corresponding to reduced function of NALCN; providing a cancer and / or metastasis risk score based on the presence of one or more mutations corresponding to reduced function of NALCN; The present invention relates to a method comprising the steps of:

[0011] One aspect of the invention is a method for determining the activity of NALCN, comprising the steps of: Analysing the biological sample to detect one or more of the mutations identified in Table 2; The present invention relates to a method, wherein the presence of one or more mutations identified in Table 2 is indicative of reduced activity of NALCN.

[0012] One aspect of the invention relates to kits comprising reagents for detecting one or more mutations in NALCN identified in Table 2, and optionally instructions for use.

[0013] One aspect of the invention relates to a composition comprising a reagent for detecting one or more mutations in NALCN identified in Table 2.

[0014] One aspect of the invention is a computer-implemented method for determining a cancer and / or metastasis risk score, comprising: obtaining data indicative of the presence of at least one mutation in the sodium leak channel NALCN in the tumor sample; inputting the data into a computational model of NALCN that simulates the effect of mutations on NALCN; Using a computational model to determine whether at least one mutation causes a decrease in the pore size of the NALCN; If at least one mutation is determined to cause a decrease in the pore size of the NALCN, outputting a risk score for cancer and / or metastasis. The present invention relates to a method comprising the steps of: [Brief description of the drawings]

[0015] [Figure 1]Figure 1: NALCN loss of function characterizes aggressive intestinal cancer. (a) Volcano plot of differential gene expression between Prom1+ cells isolated from mouse gastric epithelium and P1KP-GAC: highlighting downregulated ion channels. (b) t-distributed stochastic neighbor embedding plot of 10,022 samples from 32 human cancer types: highlighting NALCN mutant samples and cancer types enriched for NALCN mutations (p-values, dN / dS shown). (c) Mutant residues were significantly enriched within the pore turret and voltage-sensitive domain of NALCN. (d) The effect of 196 different NALCN mutations on pore radius was determined by HOLE analysis. (e) Mutations caused closure of the NALCN pore in several different cancer stages (*=p<0.05, Mann-Whitney). [Diagram 2]Figure 1: Loss of function of NALCN increases tumor metastasis. (a) Unsupervised hierarchical clustering of 77 primary and metastatic P1KP-GAC, V1KP-IAC, Pdx1KP-PAC tumors and 4 P1;PtenFlx / Flx;Tp53Flx / Flx (P1PtP) primary hepatocellular carcinomas. The lower heatmap reports the enrichment of the indicated primary tumor transcriptome in each metastatic tumor. (b) Macroscopic images of specimen ZsGreen+ (ZSG) metastatic tumors ([met] outlined). (c) Photomicrographs of metastases in (b) (hematoxylin and eosin on the left, immunohistochemistry / fluorescence on the right). Scale bar 50 μm. (d) Left side shows cumulative total number of metastases per mouse necropsied for the indicated genotypes at the indicated times after tamoxifen induction (age of Pdx1KP mice; p=Mann-Whitney for total tumor burden in Nalcn-deficient and wild-type mice, see also Tables 5 and 6 in the supplementary material). Right side shows organ heatmap for total metastases per mouse for the indicated genotypes. Values ​​for male:female (M:F) and P3:P60 induced mice are shown. (e) Cumulative metastatic burden and organ metastasis heatmap plots of V1KP-IAC in gadolinium or control treated Nalcn+ / + mice. (*=p<0.05, **=p<0.005, Mann-Whitney). [Diagram 3]Figure 1: Loss of function of NALCN increases dissemination of circulating tumor cells. (a) Scatter plots of CZCs identified in peripheral blood of indicated mice treated or not with gadolinium (expressed as a percentage of total peripheral blood cells) (ns=not significant, *=p<0.05, **=p<0.005, Mann-Whitney). (b) Uniform manifold approximation and projection (UMAP) of single-cell RNA-seq profiles for CZCs and mouse peripheral blood mononuclear cells. (c) Heatmap reporting gene set enrichment analysis in the UMAP clusters identified in (b). Test gene sets were derived from 2,086 different tissues and cell types, including bulk RNAseq of mouse normal tissues and tumors, huCTC signatures, and normal PBMCs (Methods). (d) Sample co-immunofluorescence staining of CZC and PBMC in peripheral blood smears from P1KP (top) and V1KP (bottom) mice (ZsGreen [ZSG], scale bar = 10 μm). (e) Top left, macroscopic direct green fluorescence imaging of whole mouse lung samples showing Pdx1KP-PAC CZC metastases in immunocompromised recipient mice. Other images show sample hematoxylin and eosin staining or co-immunofluorescence staining of P1KP-GAC or V1KP-IAC CZC metastases in immunocompromised recipient mice (scale bar = 10 μm). (f) Organ heatmap of total metastases per mouse identified in recipient mice injected with the indicated CZCs. [Figure 4]Figure 2. Loss of NALCN function increases the dissemination of circulating non-tumor cells. (a) Scatter plot of CZCs identified in peripheral blood of the indicated non-tumor-bearing mice, expressed as a percentage of total cells (***=p<0.0005, Mann-Whitney). (b) Uniform manifold approximation and projection (UMAP) of 201,183 single-cell RNA sequencing profiles (SCS) for PBMCs and tumor-bearing (t) and non-tumor-bearing (nt) CZCs, as well as cells derived from the indicated normal and malignant mouse tissues. (c) Sample co-immunofluorescence staining of CZCs and PBMCs in peripheral blood smears of P1RNalcnFlx / Flx mice (ZsGreen [ZSG], scale bar=10 μm). (d) Organ heatmap of the total number of CZC cell clusters per mouse identified in organs of recipient mice injected with the indicated P1RNalcnFlx / Flx CZCs. (e) Immunofluorescence staining of specimens of P1RNalcnFlx / Flx CZC (arrow) integrated in the kidney of a recipient mouse (arrow indicates ZSG+ cell, scale bar = 50 μm). (f) Confocal laser scanning microscope image of P1RNalcnFlx / Flx CZC integrated in the renal cortex of a recipient mouse (scale bar = 100 μm). [Diagram 5]Figure 1: Loss of Nalcn does not affect the incidence, tumor-free survival, or growth rate of P1KP, V1KP, or Pdx1KP primary tumors. a-c Tumors and representative photomicrographs for lineage tracing markers (ZSG), epithelial markers (CK7, CK20), and EMT markers (CDH2, CDH1) for P1KP-GAC (a), V1KP-IAC (b), and Pdx1KP-PAC (c) are shown (H&E from all tumors (left; Table 9 in Supplementary Material) and dual immunofluorescence staining (right) from each of five independent tumors). Scale bar, 50 μm. d-g Top: Organ heatmaps of tumor incidence within P1KP at P3 and V1KP in mice of each Nalcn genotype recombined at P3 (d, e) or P60 (f, g). Bottom: Survival curves of mice within each cohort. Male to female ratios (M:F) are shown. P1KP P3, P = 0.6912; P1KP P60, P = 0.3897; V1KP P3, P = 0.1900; and V1KP P60, P = 0.8301. Mantel-Cox test. h, Organ primary tumor heatmaps and survival curves of Pdx1KP mice (P = 0.1095). Mantel-Cox test. Raw data for d-h are presented in Table 9 in the supplementary material. i, Growth rates of P1KP-GAC (n = 38), V1KP-IAC (n = 57), and Pdx1KP-PAC (n = 28) tumors are shown. Two-tailed Mann-Whitney U tests revealed no significant differences in proliferation rates between tumors with Nalcn genotype P1KP-GAC: Nalcn+ / + (n=11) and Nalcn+ / Flx (n=18; P=0.912), Nalcn+ / + (n=11) and NalcnFlx / Flx (n=9; P=0.7103), and V1KP-IAC: Nalcn+ / + (n=16) and Nalcn+ / Flx (n=25; P=0.5169), Nalcn+ / + (n=16) and NalcnFlx / Flx (n=16; P=0.7309). Pdx1KP-PAC: Nalcn+ / + (n=10) and Nalcn+ / Flx (n=13; P=0.7844), Nalcn+ / + (n=10) and NalcnFlx / Flx (n=5; P=0.1292). Bars represent medians.The raw data are presented in Supplementary Material, Table 10. j, Gene set enrichment analysis of the transcriptomes of Nalcn+ / Flx and NalcnFlx / Flx P1KP-GAC, V1KP-IAC, and Pdx1KP-PAC versus Nalcn+ / + tumors. [Figure 6]Figure 1: Loss of Nalcn does not affect cell proliferation, apoptosis, immune infiltration, vasculature, or ASMA expression in primary tumors in P1KP, V1KP, or Pdx1KP mice. (a) HALO quantification of Nalcn mRNA transcripts per cell detected by RNAscope analysis in mouse gastric adenocarcinomas (GAC), intestinal adenocarcinomas (IAC), and pancreatic adenocarcinomas (PAC) with the indicated Nalcn genotypes (bars = median; *p=0.0294; ***p=0.0004; ****=p<0.0001, two-tailed Mann-Whitney test). Data are tumor fields (5–8 images per tumor) from n=5 tumors for each Nalcn genotype in P1KP-GAC, V1KP-IAC, and Pdx1KP-PAC mice (total n=45 unique tumors, 289 unique tumor fields). (b) Representative photomicrographs of Nalcn RNA in situ hybridization in GAC with the indicated Nalcn genotypes are shown (n=15 biologically distinct tumors, 100 tumor fields) (scale=50 μm). (c) On the left are HALO quantifications (data are means ± SD) of immunohistochemically detected tumor cell expression of MKI67 (proliferation), cleaved caspase 3 (CC3; apoptosis), CD45 (immune cell infiltration), CD31 (endothelial cells), and alpha smooth muscle actin ASMA (corneal stroma) in five complete biologically independent tumor fields (total n=45 unique tumors) obtained for each Nalcn genotype in P1KP-GAC, V1KP-IAC, and Pdx1KP-PAC mice, respectively. Two-tailed Mann-Whitney U tests revealed no significant differences in such markers between tumors with different Nalcn genotypes.p values ​​for GAC, IAC, and PAC for Nalcn+ / + and Nalcn+ / Flx, and Nalcn+ / + and NalcnFlx / Flx, respectively: KI67 (0.4206, 0.4206, 0.4206, 0.5476, 0.2222, 0.5476), CC3 (0.9999, 0.5476, 0.0952, 0.5476, 0.9999, 0.2222), CD45 (0.6905, 0.8413, 0.1508, 0.3095, 0.6905, 0.8413), ASMA (0.0556, 0.8413, 0.3095, 0.0556, 0.2222, 0.1508), and CD31 (0.9999, 0.0952, 0.0952, 0.4206, 0.8413, 0.1508). On the right are specimen photomicrographs of the indicated markers in the indicated tumor types, respectively (scale = 50 μm). [Figure 7]Figure 1: Loss of function of NALCN increases tumor metastasis. a, Unsupervised hierarchical clustering of P1KP (GAC, n=10; lung adenocarcinoma, n=6; prostate adenocarcinoma, n=2), V1KP (IAC, n=19), Pdx1KP (PAC, n=13), and P1;PtenFlx / Flx;Trp53Flx / Fl (P1PtP) (hepatobiliary, n=3; lung adenocarcinoma, n=1) primary tumors, and metastatic (liver, n=2; peritoneum, n=11; kidney, n=1; thoracic, n=4; lung, n=1; lymph node, n=2) tumors. Heatmap reports enrichment of primary tumor transcriptome in metastatic tumors. b, Sample ZSG+ metastatic tumors are shown (met, outlined). Scale bar, 0.5 cm. c, Micrographs of metastases in b (H&E (left) and immunohistochemistry / fluorescence (right)). Scale bar, 50 μm. All counted metastases were evaluated using H&E (full list presented in Rahrmann et al., 2022 - Table 9 in Supplementary Material; n=7,076 metastases); n=59 metastases were evaluated by ZSG for IHC, and n=20 metastases were evaluated by immunofluorescence staining. Single channel images are shown in Fig. 15. d, Left: Cumulative total number of adenocarcinoma metastases per mouse after Cre recombination (two-tailed Mann-Whitney U test, total tumor burden in Nalcn-deficient and wild-type mice; Rahrmann et al., 2022 - Table 9 in Supplementary Material). Right: Total metastases per mouse within anatomical regions. Male / female (M:F) and P3 / P60 mice are shown. V1KP IAC for individual organs: liver, *P=0.0371 (NalcnFlx / Flx); kidney, *P=0.0229 (NalcnFlx / Flx); and peritoneum, *P=0.0492 (Nalcn+ / Flx) and **P=0.0015 (NalcnFlx / Flx). Pdx1KP PAC individual organs: lung, *P=0.0328 (Nalcn+ / Flx); and peritoneum, **P=0.0050 (Nalcn+ / Flx). P1KP GAC and IAC individual organs: lung, **P=0.0085 (Nalcn+ / Flx) and **P=0.0048 (NalcnFlx / Flx). e, Metastatic burden and organ metastases in V1KP-IAC gadolinium or control treated mice.**P = 0.0090, two-tailed Mann-Whitney U test. [Figure 8] Figure 1: P1KP-GAC, V1KP-IAC, and Pdx1KP-PAC metastases. Photomicrographs of (a) P1KP-GAC, (b) V1KP-IAC, and (c) Pdx1KP-PAC metastases for the indicated tissues are shown. The top row shows immunohistochemistry with ZsGreen staining, respectively. The bottom row shows hematoxylin and eosin (H&E) staining, respectively (scale = 100 μm). All counted metastases were assessed by H&E (full list, Rahrmann et al., 2022 - Table 7 in Supplementary Material; n = 7,076 metastases), and n = 59 metastases were assessed by ZSG for IHC. [Figure 9]Figure 13 shows that loss of function of NALCN increases nucleated CZCs in P1KP, V1KP, and Pdx1KP mice. a, FACS profiles gating CZCs in blood samples of P1KP Nalcn+ / + and NalcnFlx / Flx mice (% of nucleated cells). Gating strategy is shown in Figure 16. b, Scatter plots of CZCs (% of total nucleated blood cells) in Prom1CreERT2 / LacZ (n=397), Villin-1CreERT2 (n=162), or Pdx1Cre (n=40) mice that did or did not contain primary tumors. Data are biologically independent peripheral blood samples. Bars represent medians. V1-Cre: *P=0.0499, ****P<0.0001; Pdx1-Cre: not significant (NS) P=0.0513, **P=0.0033; P1-Cre: **P=0.0033, ****P<0.0001; Mann-Whitney two-tailed U test. The original data are available in Rahrmann et al., 2022 - Table 13 in the Supplementary Material. c, Scatter plot of CZC according to genotype and gadolinium treatment in tumor-bearing animals. Data are biologically independent peripheral blood samples. Bars represent medians. P1KP (n=112): *P=0.02, NS P=0.1204; V1KP (n=64): **P=0.0088, ***P=0.0004, NS P=0.4213; Pdx1KP (n=34): *P=0.0499, **P=0.0027; two-tailed Mann-Whitney U test. Original data are available in Rahrmann et al., 2022 - Table 13 in the Supplementary Material. d, Representative photomicrographs of ZSG immunohistochemistry of mouse bone marrow with the indicated genotypes at least 100 days after Cre recombination. Scale, 100 μm. Three mice were evaluated for each Cre line. e, f, FACS quantification of CZCs in P1KP (Nalcn+ / +, n = 11; Nalcn+ / Flx, n = 4; NalcnFlx / Flx, n = 6) mice (e) and V1KP (Nalcn+ / +, n = 9; Nalcn+ / Flx, n = 4; NalcnFlx / Flx, n = 4) mice (f) 1 week after tamoxifen induction (mean ± sem).The original data are presented in Rahrmann et al., 2022 - Table 13 in the Supplementary Material. [Figure 10] Nucleated CZCs in P1KP, V1KP, and Pdx1KP mice are CTCs. a, UMAP of SCS profiles of CZCs (n=1,820) and PBMCs (n=559). b, Gene set enrichment from 2,086 gene sets present in the UMAP clusters in a. c, Simultaneous immunofluorescence staining of CZCs and PBMCs in P1KP (top) and V1KP (bottom) mice (ZSG; scale bar, 10 μm). Representative photomicrographs of 22 cells identified across n=20 blood films assessed from n=5 tumor-bearing animals. d, Autofluorescence of Pdx1KP-PAC CZC metastases in whole lungs of recipient immunodeficient mice (top left; scale bar, 0.5 cm). Other images show H&E (representative image of 3,061 metastases evaluated) or simultaneous immunofluorescence staining (representative image of 28 metastases evaluated) of P1KP-GAC or V1KP-IAC CZC metastases in recipient mice (scale bar, 50 μm). Single channel images are shown in FIG. 15. e, Total metastases per organ in recipient mice injected with 25,000 CZCs. P1KP Nalcn+ / Flx PAC, n=5 mice; P1KP Nalcn+ / + GAC, n=2 mice; P1KP Nalcn+ / Flx GAC, n=3 mice; V1KP Nalcn+ / Flx IAC, n=2 mice; V1KP Nalcn+ / ++GdCl3 IAC, n=5 mice. Original data are presented in Rahrmann et al., 2022 - Table 19 in the supplementary material. f, Metastasis-free survival of immunodeficient NOD scid gamma recipient mice injected with different numbers (10,000, 1,000, 100, or 10) of P1KP GAC or Pdx1KP PAC CZC (n=3 mice per condition). ***P=0.0002 Mantel-Cox statistics. Original data are available in Rahrmann et al., 2022 - Table 19 in the Supplementary Material. [Figure 11]Figure 3 shows human circulating tumor cells (CTCs) and peripheral blood mononuclear cells (PBMCs). (a) UMAPs of single cell RNA sequencing (SCS) profiles of human CTCs and PBMCs (see main text for SCS sources). Gene sets enriched within the indicated SCS clusters are shown with adjusted p-values ​​for enrichment (in brackets). (b) Heatmaps of indicated gene expression from associated gene sets enriched in each cell from each cluster in (a). (c) Feature plots of specimen genes enriched in human CTCs in (a). (d) Mouse orthologues of human genes in (c) mapped onto the UMAP of mouse CZCs and PBMCs in main figure 3b. (e) UMAPs of SCS profiles of shared orthologues expressed in human CTCs and mouse tCZCs. (f) Enrichment of hemoglobin gene expression in the UMAP shown in (e). (g) Gene set enrichment in the central cluster, enclosed by the dotted line, is reported in (e) compared to other SCS profiles. [Figure 12]Figure 2: Loss of function of NALCN increases the dissemination of ntCZC. a, ntCZC identified in individual non-tumor-bearing P1RNalcn+ / + (n=87), P1RNalcn+ / Flx (n=50), and P1RNalcnFlx / Flx (n=37) mice. Bars represent medians. ****P<0.0001, two-tailed Mann-Whitney U test. Original data are available in Rahrmann et al., 2022 - Table 13 in the Supplementary Material. b, UMAP of 201,183 SCS profiles for PBMCs, tCZCs, and ntCZCs, as well as cells derived from the indicated normal and malignant mouse tissues. c, Simultaneous immunofluorescence staining of ntCZCs and PBMCs contained within peripheral blood smears of P1RNalcnFlx / Flx mice (ZSG; scale bar, 10 μm). Representative micrographs of 11 cells identified in n=20 blood films from n=4 mice are shown. Single channel images are shown in Fig. 15. d-f, in lungs (no Cre, n=2 mice, 5 lobes; Nalcn+ / +, n=3 mice, 9 lobes; Nalcn+ / Flx, n=3 mice, 8 lobes; NalcnFlx / Flx, n=5 mice, 12 lobes; NS P=0.1312, *P=0.0168, two-tailed Mann-Whitney U test) (e) and in kidneys (no Cre, n=2 mice, 4 kidney sections; Nalcn+ / +, n=3 mice, 11 kidney sections; Nalcn+ / Flx, n=3 mice, 10 kidney sections; NalcnFlx / Flx, n=5 mice, 18 kidney sections; ****P<0.0001, two-tailed Mann-Whitney U test) (f) Direct ZSG immunofluorescence micrographs (scale bar, 50 μm) of ZSG+ cells in lung and kidney (d) counted by odor. g) Organ heatmap of total number of ZSG+ cell clusters per mouse identified in organs of recipient mice injected with P1RNalcnFlx / Flx ntCZC. h) Co-immunofluorescence staining of P1RNalcnFlx / Flx ntCZC (arrows) integrated in kidney of recipient mice (arrows represent ZSG+ cells; scale bar, 50 μm). Representative micrographs of n=5 ZSG rests identified in one tissue field from n=5 mice are shown.Single channel images are shown in FIG. 15. GLO, glomerulus. i, Confocal laser scanning microscope images of P1RNalcnFlx / Flx CZC integrated into the renal cortex of recipient mice. Scale bar, 100 μm. Representative images of n=2 mouse kidneys evaluated are shown. [Figure 13] Figure 1: NALCN loss-of-function circulating non-tumor cells (ntCZCs) resemble human and mouse CTCs and are embedded in distant organs. (a) Test gene sets were derived from 2,086 different tissues and cell types, including bulk RNAseq of mouse normal tissues and tumors, huCTC signatures, and mouse and human intestinal stem cell and mature cell signatures (see Methods). (b) ZSG immunohistochemistry of lung bronchioles from aged Pdx1RNalcn+ / + mice (top left) and Pdx1RNalcnFlx / Flx mice (bottom left) (scale = 100 μm). On the right, the number of intrapulmonary ZSG+ cells / bronchioles of Pdx1RNalcn+ / + (n=2 mice, 6 lobes, 121 bronchiole) and Pdx1RNalcnFlx / Flx (n=1 mouse, 4 lobes, 57 bronchiole) is shown (bar=median; **p=0.0051 Mann-Whitney two-tailed U test). (c) Two-photon direct ZSG+ cell clusters detected in whole lung sections of Pdx1RNalcnFlx / Flx mice. (d) Co-immunofluorescence staining of tail vein injected P1RNalcnFlx / Flx ntCZC (arrow) incorporated into organs of recipient mice (arrow indicates ZSG+ cell, scale bar=50 μm). [Figure 14]FIG. 1: Transient deletion of Nalcn at P3 in P1R mice leads to organ fibrosis. Fibrosis-free survival for all organs (a) or indicated organs (b-i) is shown. P values ​​are reported for log-rank statistics (Mantel-Cox). Numbers of animals of each genotype are shown. p-values ​​for each graph compare P1RNalcn+ / + and P1RNalcn+ / Flx and P1RNalcn+ / + and P1RNalcnFlx / Flx, respectively: total organs (0.0664, 0.0035), kidney (0.0037, 0.0022), skin (0.1195, 0.0569), lung (0.3791, 0.1000), liver (0.8846, 0.7250), stomach (0.4938, 0.4225), small intestine (>0.9999, 0.2348), large intestine (0.1312, 0.2655), pancreas (0.4764, 0.7571). (j) Micrographs of hematoxylin and eosin (H&E) and picrosirius red staining, and simultaneous immunofluorescence staining of ZsGreen, alpha smooth muscle actin (ASMA), and Dapi in kidneys from P1RNalcn+ / + and P1RNalcnFlx / Flx mice over 400 days of age. Special note in P1RNalcnFlx / Flx kidney: below dotted line (H&E) indicates extensive fibrosis; prominent picrosirius red staining indicates extensive fibrosis; ZsGreen recombination, gross distortion of normal kidney architecture, and extensive alpha SMA expression. (k) Micrographs of H&E stained skin from P1RNalcn+ / + and P1RNalcnFlx / Flx mice over 400 days of age. Special notes in P1RNalcnFlx / Flx skin: ulceration and thickening of the keratinized layer, marked thickening of the squamous cell layer, and fibrosis of the dermal layer (scale 100 μm). [Figure 15] Single channel images of immunofluorescence studies in the indicated panels. All scale bars = 50 μm, except for FIG. 7c which is 10 μm. [Figure 16]Standard curves generated by two "spike-in" control techniques. (a) Normal peripheral blood was collected from adult P1-KP mice bearing gastric adenocarcinoma. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. ZSG+ cells were counted manually and spiked into fresh peripheral blood to obtain the final number / ml of actual ZSG+ cells (x-axis). This sample was then subjected to the same FACS protocol used in all blood isolation studies to provide the observed (y-axis) quantification. The vertical dotted lines represent the 25th and 75th percentiles of observed CZCs / ml recorded in Table 11 of Rahrmann et al., 2022-Supplementary Material. (b) The normal PBMCs used in (a) were also spiked into buffered saline at the indicated percentage of total PBMCs and quantified as in (a). In both graphs, the black dotted lines represent the ideal curve where the expected and observed results are identical. [Figure 17] Figure 1 shows a specimen FACS gating strategy for isolating ZSG+ cells from peripheral blood samples: (a) An example of a tumor-bearing animal that did not have ZSG+ cells in its peripheral blood; (b) An example of a tumor-bearing animal that had ZSG+ cells in its peripheral blood. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The embodiments of the present invention will now be further described. In the following passages, different embodiments are described. Each aspect defined can be combined with any other aspect or aspects unless a contrary example is expressly stated.

[0017] In general, the nomenclature used in connection with and in the techniques of cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein is well known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. See, for example, Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012).

[0018] Ion channels are crucial components of cell excitability and are involved in many diseases. The present inventors have demonstrated herein that NALCN plays an important role in both malignant and non-malignant cell metastasis. NALCN is a non-selective monovalent cation channel, but it is the only member of the distinctive branch of voltage-gated sodium and calcium channels that controls resting membrane potential and neuronal excitability. NALCN is most abundantly expressed in the nervous system and conducts a sustained sodium leak current that contributes to sustained neuronal excitability. The sequence of NALCN is known and may include sequences presented in ENSG00000102452 (ensemble), 259232 (NCBI Entrez Gene), 19082 (HGNC), Q8IZF0 (UniProtKB / Swiss-Prot), or 611549 (OMIM). In one embodiment, the sequence of NALCN comprises SEQ ID NO:1. NALCN exists in multiple splice variants, and the present invention extends to these variants. NALCN forms a polypeptide chain consisting of 24 transmembrane helices (TM) that form four homologous functional repeats (also called α subunits) connected by an intracellular linker. Each functional repeat contains a voltage-sensitive domain, a pore helix, and an ion-selective filter.

[0019] It has been shown herein that loss or reduction of NALCN function contributes to increased circulating tumor cell (CTC) levels and metastatic spread. Thus, by identifying mutations in NALCN that correlate with loss of NALCN function, it is possible to detect and / or predict subjects who may exhibit metastasis.

[0020] Thus, in one aspect, the present invention provides a method for detecting or predicting cancer and / or metastasis, comprising: Analysing a tumor sample obtained from the subject; determining the presence of at least one mutation in a sodium leak channel (NALCN) in the tumor sample relative to a reference sample; determining whether the at least one mutation causes a decrease in the pore size of the NALCN; If the mutation causes a decrease in the pore size of the NALCN, the decrease in pore size is used to determine a risk score for cancer and / or metastasis. The present invention relates to a method comprising the steps of:

[0021] To identify the presence of a mutation in NALCN present in a tumor sample, a reference sample is used to compare said tumor sample. The reference sample may be a sample obtained from a healthy subject or a sample derived from a subject. When the reference sample is obtained from a subject or a healthy subject, the reference sample may comprise germline DNA. The germline DNA sample may be obtained by any reasonable means. The reference sample may be obtained from a blood sample, tissue sample, saliva sample of a healthy subject. The reference sample may be obtained from a blood sample, tissue sample, saliva sample of said subject. In one embodiment, the reference sample is a sample of germline DNA obtained from said subject or a sample of germline DNA obtained from a healthy subject.

[0022] The comparison of the NALCN sequence in tumor sample and reference sample can be performed by sequencing.Sequencing can be performed using whole genome, whole exome, targeted exome, transcriptome and methylome sequencing.Several techniques are known in the art for comparing sequences to identify the presence of mutations, for example, sequence alignment can be used.

[0023] Once the presence of at least one mutation in NALCN in a tumor sample has been identified, computational modeling can be used to determine whether the at least one mutation causes a decrease in the pore size of the NALCN. The term "pore size" as used herein refers to the ion-conducting pore of NALCN. "Pore size" can be measured in terms of the ion selectivity filter radius and / or gate radius. The selectivity filter radius refers to the region of the protein that confers sodium ion specificity. It is a rigid portion of the structure that is shaped to allow only sodium ions to pass easily. The ion selectivity filter is the narrowest portion of the ion channel where the amino acids lining the filter (NALCN: EEKE, EKEE, or EEEE) directly interact and discriminate between ion species. In human NALCN, the selectivity filter is specifically residues E280, E554, K1115, and E1389. These residues form a ring within the channel domain of the protein that constricts the channel to the correct radius for sodium ions. Gates within the channel pore control ion permeation and refer to regions at the end of each S6 helix that constrict closed when the protein's channel is in its depolarized state. These helices can slide open like an iris when polarized to open the channel and allow ions to pass through.

[0024] Computational modeling can be performed by generating a model of NALCN in a lipid membrane and then simulating the effect of the identified mutations on the NALCN model. Several techniques and software for generating computational models of NALCN are known in the art, for example, using available X-ray crystallography or cryo-EM structures of NALCN (such structures are accessible through databases, such as the PDB (Protein Data Bank)). To clarify the effect of mutations on the pore size of NALCN, suitable programs are available, such as HOLE (Smart et al., HOLE: A program for the analysis of the pore dimensions of ion channel structural models. Journal of Molecular Graphics, doi:10.1016 / S0263-7855(97)00009-X (1996)), a program that allows the analysis and visualization of pore dimensions through the molecular structure of ion channels. There are also additional tools that can be used to calculate pore properties, including CHAP (Klesse et al., CHAP: A Versatile Tool for the Structural and Functional Annotation of Ion Channel Pores. J Mol Biol. 2019;431(17):3353-3365), CAVER (Chovancova et al., CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput Biol. 2012;8(10):e1002708.), and MOLE (Sehnal et al., MOLE 2.0: advanced approach for analysis of biomacromolecular channels. J Cheminform. 2013;5(1):39).

[0025] In one aspect, the present invention provides a computer-implemented method for determining a risk score for cancer and / or metastasis, comprising: obtaining data indicative of the presence of at least one mutation in the sodium leak channel NALCN in the tumor sample; inputting the data into a computational model of NALCN that simulates the effect of mutations on NALCN; Using a computational model to determine whether at least one mutation causes a decrease in the pore size of the NALCN; If at least one mutation is determined to cause a decrease in the pore size of the NALCN, outputting a risk score for cancer and / or metastasis. The present invention relates to a method comprising the steps of:

[0026] There may also be a computing device comprising at least one processor coupled to a memory and configured to perform the computer-implemented methods described herein. There may also be a computer-readable storage medium that contains instructions that, when executed by the processor, cause the processor to perform the acquisition, input, decision, and output steps of the computer-implemented methods described herein. The computer-readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any suitable combination of the above.

[0027] In one aspect, the present invention provides a method for detecting or predicting cancer and / or metastasis, comprising: Analyzing a biological sample obtained from the subject to assess sodium leak channel (NALCN) activity; providing a cancer and / or metastasis risk score based on the level of NALCN activity; The present invention relates to a method comprising the steps of:

[0028] The biological sample can be a tumor sample, a blood sample, or a tissue sample. The tumor or tissue sample can be obtained via biopsy.

[0029] Since NALCN is an ion channel involved in the resting Na+ permeability of cells, the activity of NALCN can be assessed using a variety of techniques: activity can be assessed by whole cell electrophysiology, fluorescence assays, membrane potential sensitive dyes, and / or ion flux assays.

[0030] To determine whether the activity of NALCN in the biological sample is altered, the method may further include comparing the level of NALCN activity in the biological sample to a reference value, which may be a NALCN activity measurement obtained from a healthy subject.

[0031] As used herein, a "healthy subject" is defined as a subject who has no diagnosable cancer disease state.

[0032] In one aspect, the present invention provides a method for detecting or predicting cancer and / or metastasis, comprising: Analyzing the biological sample to detect the presence of one or more mutations corresponding to reduced function of NALCN; providing a cancer and / or metastasis risk score based on the presence of one or more mutations corresponding to reduced function of NALCN; The present invention relates to a method comprising the steps of:

[0033] The NALCN protein comprises multiple domains, and thus the method may detect mutations in one of the following domains; the pore turret domain, the voltage sensitive domain, or the linker domain of NALCN. The linker domain may be a linker domain that extends either extracellularly or intracellularly. The method may detect mutations in one or more of the domains comprising any one of the amino acid sequences represented by SEQ ID NOs: 2-23. The domains of NALCN and their sequences are set forth in the table below: [Table 1A] [Table 1B] [Table 1C] [Table 1D] [Table 1E] [Table 1F]

[0034] NALCN protein forms a channelosome complex in the cell membrane. The channelosome includes various proteins associated with NALCN, including G protein-coupled receptor, UNC-79, UNC-80, SLO2.1, NCA localization factor-1, FAM155A, and src family tyrosine kinase. Thus, the methods described herein may further include detecting mutations in one or more of the proteins associated with NALCN. Proteins associated with NALCN in which mutations can be detected include M3 muscarinic receptor (M3R), UNC80, UNC79, FAM155A, Fam155B, SLO2.1, NCA localization factor-1, src family tyrosine kinase. When a mutation is identified in a protein associated with NALCN, the mutation can be identified by determining the presence of a known mutation or by determining the presence of at least one mutation in a protein associated with NALCN compared to a reference sample.

[0035] The methods of the invention may detect specific mutations in NALCN. The methods may detect one or more specific mutations that correlate with reduced activity of NALCN or reduced pore size of NALCN. One or more of the mutations in NALCN are L588M, P573, R855, K1213, T71, P225, D1527, D416, C1348, R297, V1386, A1091, V1229, D134, T272, R43, A1157, V1036, M520, R1500, V320, V53, W1085, E1458, N1274, V1542, Y1300, R1174, H1523, F332, Q549, L999, F540, A1421, R1384, H569, A1435, M55, R1 495, C245, F110, V510, C970, E454, V273, R1556, S174, S1068, V385, S384, A401, S902, R1495, A276, R1540, L517, R295, R382, H876, F300, R 164, E257, R995, G1526, D291, V1239, E1552, N1475, M55, L1553, Y1349, E323, A1044, T1281, V1007, L253, L564, F1427, V949, Q279, T539, R1 59, K452, R1127, V1490, G555, E62, L1461, L942, R166, P65, D952, I322, F154, K1163, L305, R152, W1085, R143, A1444, R989, R143, R1193, D 1466, M520, V1285, S52, I51, E1518, E532, L1279, V1329, T57, A1378, S121, K498, R1094, V120, A88, A401, L1548, G1303, M150, D1277, E432, L1442, P1082, T1165, G1316, R1273, E128, E906, F1311, R1481, T204, T552, F389, D1527, P908, A1166, I577, G954, G1013, P65, E1016, N107 0, S980, A1217, V1503, T1320, A223, A310, R1127, D1504, D1277, E128, K1491, Q553, V511, F1250, S1374, D211, T1149, D1099, M1425, M1003,The mutations may be present at positions selected from, but are not limited to, P467, R43, L222, V400, M1244, A424, F1410, G193, H39, W219, F1018, R1193, K1069, V50, R1498, K1230, S403, S1264, R995, Q238, I1433, P66, L428, D1171, A1107, S1033, I1017, K1259, M986. It has been demonstrated herein that mutations at each of these positions can cause the closure of the NALCN pore, i.e., a reduction in the size of the NALCN pore, and thus a reduction in NALCN activity. It is hypothesized that these amino acid residues may be involved in controlling the opening of the NALCN pore, and therefore mutations at one or more of these positions may result in a reduction in the pore diameter and therefore in a reduction in NALCN activity.

[0036] In one embodiment, the method may detect one or more mutations selected from those identified in Table 2. Table 6 provides further details on the mutations listed in Table 2 and how metastatic risk was assessed. [Table 2A] [Table 2B] [Table 2C] [Table 2D]

[0037] In one embodiment, the method includes the further step of identifying a stage of the cancer based on the identified mutation(s). The method may include the further step of identifying a risk of metastasis based on the identified mutation(s).

[0038] In one embodiment, the method comprises the further step of determining / selecting a treatment. Thus, we also describe a method for determining a treatment for a subject, comprising one or more of the above-mentioned methods, and further comprising the further step of determining a treatment. The treatment may be selected from any suitable anti-cancer and / or anti-metastatic treatment. The treatment may be selected from chemotherapy, hormonal therapy, immunotherapy, radiation therapy, stem cell therapy, surgery, or targeted therapy, such as small molecule therapy, antibody therapy, checkpoint inhibitors, or CAR-T therapy. Such treatments are known in the art. It is recognized that there are various types of immunotherapy, such as immune checkpoint inhibitors, oncolytic virus therapy, T-cell therapy, and cancer vaccines. The appropriate therapy may be selected.

[0039] The method of the present invention allows for the detection or prediction of cancer. In one embodiment, the cancer is gastric cancer, gastric adenocarcinoma, colorectal cancer, lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bone cancer, pancreatic cancer, colon cancer, colorectal cancer, skin cancer, head or neck cancer, head and neck squamous cell carcinoma, melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, The cancer is selected from testicular cancer, breast cancer, brain cancer, hepatocellular carcinoma, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, renal cancer, soft tissue sarcoma, urethral cancer, bladder cancer, kidney cancer, thymoma, urothelial carcinoma, leukemia, prostate cancer, prostate adenocarcinoma, mesothelioma, adrenocortical carcinoma, lymphoma, such as Hodgkin's disease, non-Hodgkin's disease, and multiple myeloma. In one embodiment, the cancer is selected from gastric cancer, intestinal cancer, or pancreatic cancer.

[0040] The method for detecting or predicting cancer and / or metastasis includes determining a risk score for cancer / metastasis. The risk score can be based on determining a reduction in NALCN pore size due to a mutation identified in NALCN. The inventors herein have demonstrated that it is possible to determine the reduction in pore size caused by a mutation through computational modeling. A greater reduction in NALCN pore size correlates with a greater reduction in NALCN activity and thus a higher risk of cancer and / or metastasis. The reduction in NALCN pore size can be calculated by determining the difference in size of the ion-selective filter radius in a NALCN variant containing a mutation compared to a wild-type NALCN filter radius. The reduction in NALCN pore size can be calculated by determining the difference in size of the gate radius in a NALCN variant containing a mutation compared to a wild-type NALCN gate radius. The risk score can also be determined based on the presence of a particular mutation identified, where a risk of cancer and / or metastasis is associated with the particular mutation. When the method for predicting cancer and / or metastasis includes determining a risk score based on the activity of NALCN, a higher risk score is associated with a greater decrease in the activity of NALCN.

[0041] As an example, the risk score may be calculated using the following steps: - Taking the wild type NALCN structure and mutagenizing it so that the amino acid changes are observed in the subject. This mutagenesis process can be carried out multiple times, for example three times. - The mutant structures are minimized using small molecular dynamics simulations, where Newtonian physics is applied to the structure so that the atoms are adaptable to the induced changes. If more than one is used, this same process is applied to each of the mutant structures. - Calculate the pore radius for the minimized structure. If multiple structures are used, calculate and average the pore radii to provide an estimate for pore closure. Calculate the pore radius using a program such as HOLE, or another suitable program. As an example, the program HOLE calculates the pore radius by making a sphere larger and larger along the channel axis until it touches the protein, providing an estimate for the cross section of the channel.

[0042] The cancer risk score can then be used to determine the likelihood of a cancer or metastatic disease state, by which is meant that there is about a 50% or greater probability, e.g., 60%, 70%, 80%, or 90%, that a cancer disease state is present in a subject sample.

[0043] "Prognosis" refers, for example, to overall survival, long-term mortality, and disease-free survival. In one embodiment, long-term mortality refers to death within five years after a lung cancer diagnosis.

[0044] In one aspect, the present invention relates to a method for determining the activity of NALCN, comprising analyzing a biological sample to detect one or more mutations identified in Table 2, wherein the presence of one or more mutations identified in Table 2 indicates reduced activity of NALCN.

[0045] In one embodiment, the method of the invention may comprise analyzing a biological sample to detect one or more mutations identified in any one of Tables 3, 4, or 5.

[0046] The method of the present invention includes detecting mutations in the NALCN. In one embodiment, the mutations are detected via whole genome, whole genome, whole exome, targeted exome, transcriptome, and methylome sequencing. In one embodiment, the mutation may be detected using one or more techniques selected from allele-specific polymerase chain reaction (PCR), high-resolution melting curve analysis, genomic sequencing fluorescence in situ hybridization (FISH); comparative genomic hybridization (CGH), restriction fragment length polymorphism (RELP), amplification refractory mutation system (ARMS), reverse transcriptase PCR (RT-PCR), real-time PCR, multiplex ligation-dependent probe amplification (MLPA), denaturing gradient gel electrophoresis (DGGE), single strand conformational polymorphism (SSCP), chemical cleavage of mismatches (CCM), protein truncation test (PTT), or oligonucleotide ligation assay (OLA).

[0047] The method for detecting or predicting cancer and / or metastasis, or for determining NALCN activity, is generally carried out in vitro or ex vivo. The method requires a biological sample obtained from a subject, which is then analyzed. Thus, the step of analysis is carried out outside the human body, i.e., in vitro or ex vivo. The biological sample can be a tumor sample. The sample can be obtained from the subject via biopsy or during surgery to remove the tumor. The biological sample may be processed after removal from the subject, for example, the sample can be cryopreserved.

[0048] The subject from which the biological sample is obtained may be a subject that contains a somatic mutation, for example the sample may be a tissue sample or a tumor sample.

[0049] In one aspect, the invention relates to a kit comprising reagents for detecting one or more mutations in NALCN, and optionally instructions for use, wherein the mutations correlate with decreased activity of NALCN and / or decreased pore size of NALCN. In one embodiment, the kit comprises reagents for detecting one or more mutations in NALCN identified in Table 2. In one embodiment, the kit comprises reagents for detecting one or more mutations in NALCN identified in Table 6, identified by either amino acid changes or nucleotide changes.

[0050] In one aspect, the invention relates to a composition comprising reagents for detecting one or more mutations in NALCN, which mutations correlate with decreased activity of NALCN and / or decreased pore size of NALCN. In one embodiment, the composition comprises reagents for detecting one or more mutations in NALCN identified in Table 2. In one embodiment, the composition comprises reagents for detecting one or more mutations in NALCN identified in Table 6, identified by either amino acid changes or nucleotide changes.

[0051] In one embodiment, the kit or composition of the present invention comprises reagents suitable for performing whole genome, whole exome, targeted exome, transcriptome, and methylome sequencing.Preferably, the reagents are suitable for performing allele-specific polymerase chain reaction (PCR), high-resolution melting curve analysis, genome sequencing fluorescence in situ hybridization (FISH); comparative genomic hybridization (CGH), restriction fragment length polymorphism (RELP), amplification refractory mutation system (ARMS), reverse transcriptase PCR (RT-PCR), real-time PCR, multiplex ligation-dependent probe amplification (MLPA), denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism analysis (SSCP), chemical cleavage of mismatches (CCM), protein truncation test (PTT), or oligonucleotide ligation assay (OLA).

[0052] In one embodiment, the kit comprises reagents for detecting one or more of the mutations in NALCN identified as high risk mutations for metastasis. NALCN mutations identified as high risk for metastasis are listed in the table below: [Table 3A] [Table 3B]

[0053] In one embodiment, the kit comprises reagents for detecting one or more of the mutations in NALCN identified as medium risk mutations for metastasis. NALCN mutations identified as medium risk for metastasis are listed in the table below: [Table 4A] [Table 4B]

[0054] In one embodiment, the kit comprises reagents for detecting one or more of the mutations in NALCN identified as low risk mutations for metastasis. NALCN mutations identified as low risk metastasis are listed in the table below: [Table 5]

[0055] The kit of the invention may be provided as a panel of reagents designed to detect one or more of the NALCN mutations listed in Table 2. The panel of reagents may be designed to detect one or more of the mutations identified as indicative of a high risk of metastasis as listed in Table 3. The panel of reagents may be designed to detect one or more of the mutations identified as indicative of a medium risk of metastasis as listed in Table 4. The panel of reagents may be designed to detect one or more of the mutations identified as indicative of a low risk of metastasis as listed in Table 5.

[0056] In one embodiment, the tumor sample or biological sample is obtained from a subject, such as a mammal, preferably a human.

[0057] Unless otherwise specified herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. The above disclosure provides a general description of the subject matter encompassed within the scope of this disclosure, including how to realize and use the disclosure, and the best mode thereof, but the following examples are provided to further enable those skilled in the art to practice the disclosure. However, those skilled in the art will recognize that the specific details of such examples should not be construed as limitations on the present disclosure, and the scope should be understood from the claims appended to this disclosure and their equivalents. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in light of the present disclosure.

[0058] All documents mentioned herein, including gene accession numbers, scientific publication references, and patent publication references, are hereby incorporated by reference in their entirety.

[0059] "And / or," as used herein, is understood as a specific disclosure of each of the two specified features or elements, without the other. For example, "A and / or B" is understood as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein. Unless the context dictates otherwise, the descriptions and definitions of features set forth above are not limited to any particular aspect or embodiment of the present invention, and apply equally to all aspects and embodiments described.

[0060] The terms "comprising" or "comprises," as used herein, mean including the elements specified, but not excluding the presence of other elements. The terms "consisting essentially of" or "consisting essentially of" mean including the elements specified, but excluding other elements (except for materials present as impurities, unavoidable materials present as a result of the process used to provide the elements, etc.).

[0061] The terms "consisting of" or "consisting of" mean the inclusion of the specified elements and the exclusion of other elements.

[0062] Wherever applicable, and depending on the context, use of the terms "comprise" or "comprising" shall be understood to include the meaning "consisting essentially of" or "consisting essentially of" and also the meaning "consisting of" or "consisting of".

[0063] The optional features described in this specification may be used individually or in combination with each other, where applicable, in particular in the combinations described in the accompanying claims. The optional features for each aspect or exemplary embodiment of the present invention as described in this specification also apply to all other aspects or exemplary embodiments of the present invention, where applicable. In other words, a person skilled in the art reading this specification should consider the optional features for each aspect or exemplary embodiment of the present invention as interchangeable and combinable between different aspects and exemplary embodiments.

[0064] The invention is further illustrated in the following non-limiting examples. EXAMPLES

[0065] Intestinal cancer, including stomach cancer, is thought to originate from stem cells. 7~9 However, it remains unclear how oncogenic mutations transform intestinal stem cells to generate invasive cancers. We show that prominin 1 (Prom1) is characteristic of basal stem cells in the gastric pyloric gland and that their lineage is related to mutant-Kras G12D Expression of Trp53 7 After the loss of Prom1 CreERT2 / LacZ Kras G12D ;Trp53 Flx / Flx (P1 KP We have previously shown that P1 forms adenocarcinomas in mice. KP Gastric adenocarcinoma (P1 KP -GAC) + Cell(Prom1 - Prom1 cells (but not Prom1 cells) readily propagate such tumors as allografts in immunocompromised mice. + P1 KP -GAC cells are suggested to be the malignant counterpart of pyloric basal stem cells.

[0066] Example 1 Loss of Nalcn function is a hallmark of advanced cancer. To better understand how pyloric basal stem cells are disrupted during transformation, we analyzed their transcriptomes using Prom1 + P1 KP The ion channels and solute transporters in Prom1 cells were compared with those in Prom1-GAC cells. + P1 KP -Enriched in genes downregulated in GAC cells (adjusted p-value = 1.7e -3 ; Fig. 1a). A review of 10,022 human cancers in The Cancer Genome Atlas revealed that nonsynonymous mutations within NALCN were enriched in gastric adenocarcinomas (n=43 / 422; dN / dS ratio, p=0.007) and colorectal adenocarcinomas (n=45 / 528, p=0.04; Fig. 1b). 5, 6 We mapped these mutated residues on the cryo-EM structure of NALCN, embedded and relaxed in silico in a 575-POPC lipid bilayer. 3, 10, 11 We found significant spatial clustering within the pore turret and voltage-sensitive domains that control channel opening (p = 0.03; Fig. 1c). We performed HOLE analysis on end frames of equilibrium molecular dynamics simulations of membrane-embedded NALCN. 12 (which estimates the ion channel pore radius size), the analysis predicts that 76% (n=224 / 295) of such mutations block the NALCN selectivity filter and thus block the channel. 2, 3 (Fig. 1d). In 221 patients for whom both disease stage and NALCN mutation status were available, 13, 14 , NALCN mutations predicted to cause maximal pore closure were enriched in the most advanced cancers (Fig. 1e). Furthermore, human GACs with NALCN mutations upregulated genes expressed during epithelial-mesenchymal transition (EMT, p-value = 1.26e -9 )-Characteristics of invasive cancer 15 .

[0067] As a first step to test whether Nalcn regulates cancer progression, we investigated whether Nalcn regulates cancer progression by using genetic (Nalcn-shRNA and NALCN-cDNA lentiviral transduction) or chemical (Nalcn channel blocker gadolinium chloride [GdCl3] 4 ) while using P1 KP -GAC cells. KP Whole-cell membrane voltage clamp analysis of -GAC cells revealed GdCl3-sensitive currents that were linear to voltage steps within the range of ±80 mV, as previously reported. 4 This current is NALCN shRNA Transduction P1 KP -GAC cells. KP Decreased Nalcn function in -GAC cells increased their proliferation in vitro and altered the EMT morphology and transcriptome on orthotopic tumor allografts of such cells (adjusted p-value = 5.29e -6 ) 16 On the other hand, P1 KP Increased NALCN expression in -GAC cells increased GdCl3-sensitive currents, reduced proliferation, and produced a prominent hyperepithelialized morphology in allogeneic transplants.

[0068] Example 2 Loss of Nalcn promotes cancer metastasis. To test how loss of function of Nalcn affects cancer initiation and progression in disease-free tissues, we generated mice harboring a provisional Nalcn allele in which exons 5 and 6 of the gene are flanked by loxP sites (Nalcn Flx ; ). This mouse, P1 KP , Villin 1-Cre ERT2 Kras G12D ;Trp53 Flx / Flx (V1 KP ), or Pdx1-Cre;Kras G12D ;Trp53 Flx / + (Pdx1 KP ) mice to obtain Nalcn-wild type (Nalcn+ / + ), Nalcn + / Flx , or Nalcn Flx / Flx We generated equal numbers of male and female mice that were either Rosa26-ZsGreen (Rosa26 ZSG ) lineage-tracing allele. KP and Pdx1 KP Cancer in mice was induced by Cre expression 17, 18 and pancreas 19, 20 Each is limited to Prom1. CreERT2 / LacZ is expressed by a variety of stem / progenitor cells and P1 KP Induces tumors of the small intestine, liver, lung, salivary gland, prostate, uterus, skin, and stomach in mice 7, 8 Because tissues may exhibit age-dependent susceptibility to transformation 7 The inventors have used tamoxifen on postnatal day (P) 3 or P60 to KP and V1 KP Cre recombination was activated in mice. Mice showing signs of tumor development were sacrificed and subjected to macroscopic and microscopic whole body necropsy. As expected, V1 KP Mice (n=127 / 141) and Pdx1 KP Mice (n=55 / 55) developed intestinal and pancreatic tumors, respectively. P1 KP Mice developed tumors in the stomach (n=49 / 269), small intestine (n=59 / 269), and other sites (n=108 / 269). 7, 18, 20 : P1 KP 99% (n=212 / 214) of mice developed a single primary cancer. Neither age of induction, sex, nor Nalcn status significantly altered the site, type, size, or incidence of primary tumors, or tumor-free survival in these mouse models. Thus, Nalcn function does not appear to affect the tissue transformation capacity of Kras and Trp53 oncogenic mutations.

[0069] However, heterozygous or homozygous deficiency of Nalcn results in P1 KP , V1 KP , and Pdx1KP In mice, V1 dramatically increased tumor metastasis to the peritoneum, retroperitoneum, liver, lymph nodes, lungs, and / or kidneys (Figures 2a-c). Metastatic and primary tumors were readily distinguished from each other by expert pathologists, blinded histology review; co-isolation of "matched" primary and secondary tumor transcriptomes by unsupervised hierarchical clustering; and highly selective enrichment within metastatic tumor transcriptomes of histology-predicted primary tumor gene sets (Figures 2a and 2c). KP Nalcn + / + Intestinal adenocarcinoma (IAC) and Pdx1 in mice (n=27 mice) KP Nalcn + / + Pancreatic adenocarcinoma (PAC) in mice (n=19 mice) generated 2.8±4.9SE and 5.5±4.0SE metastases / mouse, respectively (FIG. 2d). In stark contrast, V1 KP Nalcn + / Flx (n=51), V1 KP Nalcn Flx / Flx (n=26), Pdx1 KP Nalcn + / Flx (n=23), and Pdx1 KP Nalcn Flx / Flx These same tumors in mice (n=13) were 16.2±5.7 SE (Mann-Whitney, Nalcn + / + p=0.03), 26.0±10.18SE (p=0.0009), 15.0±3.62SE (p=0.007), and 13.5±5.01SE (p=0.02) more metastases / mouse compared to V1. KP Loss of Nalcn from the -IAC ​​increased metastasis, particularly to the peritoneum, kidney, and liver, whereas loss of Pdx1 KP Deletion of Nalcn from -PAC increased metastasis to the peritoneum and lungs (Fig. 2d). A similar pattern of IAC and GAC metastatic burden was observed in P1 KP Observed in 80 mice: P1 KP Nalcn + / + (11.6±3.45SE metastases / mouse), P1 KP Nalcn+ / Flx (42.2±11.23 SE metastases / mouse), and P1 KP Nalcn Flx / Flx (40.24.0±15.51 SE metastases / mouse): Loss of Nalcn significantly increased metastases to the lungs and peritoneum in these mice (FIG. 2d).

[0070] To further validate the loss of function of Nalcn as a driver of cancer metastasis, we investigated the function of V1 KP Nalcn + / + (n=37), V1 KP Nalcn Flx / + (n=17), and V1 KP Nalcn Flx / Flx An additional cohort of mice (n=8) was treated with the Nalcn channel blocker gadolinium chloride (2 μg / kg / week for up to 30 weeks). KP Nalcn + / + IAC developing in mice (n = 28) generated 18.3 ± 5.94 SE metastases / mouse compared with 2.8 ± 4.9 SE in controls (p = 0.02; Fig. 2e). In particular, gadolinium enhanced the V1 KP Nalcn Flx / + Mouse or V1 KP Nalcn Flx / Flx It did not increase IAC metastasis in any of the mice, confirming that the drug induced metastasis by blocking Nalcn-mediated currents.

[0071] Example 3 Loss of Nalcn increases the numbers of circulating tumor cells Because loss of Nalcn function increased metastasis and enriched primary tumor transcriptomes with a set of genes expressed by human circulating tumor cells (CTCs), we reasoned that loss of Nalcn function might increase the release of CTCs from primary tumors into the peripheral blood, where CTCs shed from tumors as precursors of metastatic disease. 21 ROSA ZSG Alleles and carcinogenesis and NalcnFlx Prom1 carrying various combinations of alleles CreERT2 / LacZ (n=337 mice), Villin-1 CreER (n=121 mice), or Pdx1 Cre (n=40 mice) Nucleated circulating ZSG was isolated from the peripheral blood of mice. + Cells (CZC) were quantified by fluorescence-activated cell sorting (FACS). After blood sampling, all mice underwent total body necropsy. The mean was 4.5e 3 ±1.1SE CZCs / ml of blood (0.078% ±0.02SE relative to total cells) were isolated from all mice on average 296 ±9.8SE days after Cre recombination (). Across all three Cre lines, the number of CZCs correlated significantly with both the presence of primary tumors and the total number of metastases, independent of the sex of the mice or age at induction (multiple linear regression, T=10.43, p<0.0001;). Deleting Nalcn or treating with gadolinium reduced the number of tumor-bearing P1 KP , V1 KP , and Pdx1 KP The levels of CZC were significantly increased in mice (Fig. 3a). CreERT2 / LacZ , Villin-1 CreERT2 , or Pdx1 Cre Because neither of these cells recombine hematopoietic cells in the bone marrow, these data strongly suggest that CZCs are CTCs that migrate from the primary tumor through a process regulated by Nalcn.

[0072] To gain a deeper understanding of the origin of the CZC, we investigated the P1 KP -GAC (n=1,701 cells) or V1 KP We generated single-cell RNA-sequencing (SCS) profiles of CZCs isolated from mice with -IAC ​​(n=119) and peripheral blood mononuclear cells (PBMCs, n=559) and compared these to published SCS profiles of human breast, lung, pancreatic, and prostate CTCs (n=360) and PBMCs (n=500). 22~27Human CTCs were easily isolated from PBMCs and consisted of three overlapping clusters: “huCTC1” (epithelial cell gene set [adjusted p-value = 1.0e -26 ] and dendritic cell gene set [adjusted p-value=0.003]); huCTC3 (CD71 + Erythroid cell-enriched [adjusted p-value = 1.9e- 43 ]); and huCTC2 (which shares the profile of huCTC1 and 3). huCTC1-3 are survival factors for β-globin (HBB)-human CTCs. 24 Mouse CZCs expressed huCTC1 (mCZC2-5), huCTC2 (mCZC2-7), and huCTC3 (mCZC6 and 7), as well as HBA1, HBA2, and HBD. Mouse CZCs formed seven clusters with well-matched transcriptomes, including huCTC1 (mCZC2-5), huCTC2 (mCZC2-7), and huCTC3 (mCZC6 and 7), and contained orthologs of HBA1, HBA2 (Hba-a1, Hba-a2), HBB (Hbb-bs, Hbb-bt), ANXA2, and LGALS3, as well as genes expressed in normal and malignant stomach and small intestine (Fig. 3c). KP -IAC and P1 KP Simultaneous immunofluorescence staining of peripheral blood smears from mice with -GAC confirmed CZC expression of Hba-a2, Lgals3, and epithelial cell markers (Krt80, Cdh1), as well as Cdx2, characteristic of the intestinal epithelium (Fig. 3d). PBMCs did not express these markers, but did express PBMC markers, such as Cd45.

[0073] To directly test whether CZCs are CTCs, we analyzed Pdx1 KP -PAC, P1 KP -GAC or V1 KP Individual aliquots of 25,000 CZCs isolated from mice with -IAC ​​were injected into the tail vein of eight immunocompromised mice. Within 75 days, all mice developed respiratory distress and numerous ZSGs were found in the lungs, liver, kidneys, and peritoneum. +Thus, CZCs contain CTCs that recapitulate the transcriptome of human CTCs and are released into the peripheral blood through a process regulated by Nalcn.

[0074] Example 4 Nalcn regulates proliferation of solid tissue cells in a cancer-independent manner Preventing CTC dispersal into the peripheral blood may halt metastasis, but disentangling this process from the complex cascade of tumorigenesis has proven challenging. To test whether Nalcn controls cell dispersal from solid tissues independently of tumorigenesis, we transformed Prom1, a mouse model of tumorigenesis that lacks the oncogenic allele and never develops tumors, into a mouse model of tumorigenesis that is characterized by a lack of the oncogenic allele. CreERT2 / LacZ ;Rosa26 ZSG ;Nalcn + / + (P1 R Nalcn + / + n=87), P1 R Nalcn + / Flx (n=48), and P1 R Nalcn Flx / Flx We probed for CZCs in the peripheral blood of mice (n=37) (). Notably, CZCs were easily isolated from the peripheral blood of these mice, and Nalcn deficiency significantly increased the number of these cells to a degree similar to that observed in tumor-bearing animals (Fig. 3a and Fig. 4a). The SCS profile of CZCs isolated from non-tumor-bearing (ntCZC) mice co-clustered with CZCs from tumor-bearing (tCZC) animals, as well as IAC and GAC metastatic SCS (Fig. 4b). The majority of tCZC SCSs and ntCZC SCSs did not cluster with profiles generated from primary IAC, GAC, or normal lung, liver, small intestine, stomach, kidney, uterus, or epididymal cells (Fig. 4b). Similar to human CTCs, 1The SCS profiles of tCZC and ntCZC were highly enriched for gene sets expressed by gastric and small intestinal stem / progenitor cells (tCZC1 nt / tCZC1-4), huCTC-1 (tCZC1, nt / tCZC8 and 9), huCTC-2 (nt / tCZC4-9), and huCTC-3 (nt / tCZC8 and 9). Simultaneous immunofluorescence staining of blood smears confirmed that both ntCZC and tCZC shared markers of huCTCs, including Hba-a1 (Figures 3d and 4c).

[0075] To understand the ultimate outcome of CZC in non-tumor-bearing mice, we performed R Nalcn Flx / Flx Individual aliquots of 25,000 CZCs isolated from mice were injected into the tail vein of six immunodeficient mice. All recipient mice remained in good clinical condition after an average of 100 days, but contained numerous ZSGs in their lungs, livers, kidneys, and peritoneum. + / Cdh1 + / Icam1 + The donor cell clusters were contained at a frequency similar to that of metastatic tumors formed by tail vein injection of tCZC (Fig. 3f, Fig. 4d-f). ntCZC appears to incorporate and / or form components of apparently normal recipient organs (the most extreme example being its incorporation into the glomeruli, blood vessels, and / or tubules of the kidney) (Fig. 4d-f). Thus, Nalcn regulates cell dispersal from solid tissues independently of cancer, uncoupling this process from tumor formation and revealing an oncogene-independent metastatic pathway.

[0076] Example 5 Blockade of Nalcn causes gadolinium-induced systemic fibrosis P1 R Nalcn + / Flx (n=118) and P1 R Nalcn Flx / Flx (n=112) Mice did not develop tumors, but whole-body necropsy of these mice revealed tumors in the kidneys and skin (P1 RNalcn + / + (n=65) mice compared with Prom1 CreERT2 / LacZ This is the site where driven recombination occurs. 7 Nalcn deficiency was found to increase fibrosis in the liver, lungs, pancreas, stomach, or intestine. Nalcn deficiency did not increase fibrosis in the liver, lungs, pancreas, stomach, or intestine. This pathology occurred over 400 days and reproduced the pathology of gadolinium-induced systemic fibrosis (GISF, previously called nephrogenic systemic fibrosis), a debilitating condition manifested by the development of severe cutaneous and systemic fibrosis following administration of gadolinium-based contrast agents (GBCAs). 28 Thus, our data directly relate to gadolinium blockade of NALCN as the mechanism underlying GISF.

[0077] Consideration Many patients with cancer die as a result of metastasis, the process by which cancer cells spread from the primary tumor to other organs in the body. The current understanding of metastasis is based on the concept that oncogenic mutations drive a cascade of events that lead to stem cell-like cancer cells breaking away from the primary tumor, entering the bloodstream, and traveling to distant sites where they form new malignant growths. 1, 29 If correct, this model requires the presence of a primary tumor at some stage in the disease history and assumes that this process is abnormal and specific to malignant tumors. By demonstrating that a single ion channel, NALCN, controls cell trafficking from both non-malignant and malignant tissues to distant organs, we provide important new insights into the metastatic process and a possible explanation for a long-standing and puzzling observation.

[0078] Developing inhibitory therapies has proven difficult because potential therapeutic targets within primary tumors that drive metastasis, such as mutant oncoproteins, have proven hard to find. 1By uncoupling the process of CTC dispersal from "upstream" tumorigenesis, our data reveal Nalcn function and thus manipulation of resting membrane potential (depolarization) as a promising novel approach to block metastasis. Gadolinium blockade of Nalcn increased the abundance of tCZC in our mice; thus, drugs capable of reopening the channel could be effective anti-metastatic drugs. A precursor to this approach is offered by drugs that open chloride ion channels mutated in the disease cystic fibrosis. 30 .

[0079] The model in which metastases always originate from the primary tumor is supported by the observation that metastases can appear many years after the localized cancer has been removed. 31 , and up to 5% of patients with metastases apparently lack a primary tumor. 32 This finding is difficult to reconcile with the observation that loss of Nalcn function in our mice caused abundant and persistent dissemination of cells embedded in distant organs, even in the absence of primary tumors. Because human epithelial tissues contain regions of phenotypically normal cells that harbor oncogenic mutations, 33, 34 Loss of NALCN function in such cells may provide a source of CTCs that form metastases in the absence of a primary tumor or long after the primary tumor has been removed from within the region of the mutant cells. Such cells would likely need to acquire additional mutations in order to form tumors at metastatic sites, consistent with the relative rarity of such an event. Our data may also explain why CTCs were found in the bone marrow of patients lacking metastases. Although such cells may represent "quiescent" CTCs as previously suggested, 29 Similar to ntCZC in our mice, such cells can emanate from non-transformed epithelia that have lost NALCN function but have not acquired the ability to form metastatic tumors.

[0080] Our observations raise the question: "How does loss of Nalcn function promote cell dispersion?" and we show that P1 R Nalcn + / + Since we observed CZC in mice, albeit at lower levels than in NALCN-deficient animals, it also raises an important question: "Are epithelial cells carrying normal phenomena that are being disrupted in cancer?" Since loss of function of Nalcn promoted EMT phenotypes and transcriptomes in tumors and CTCs in our mice, Nalcn is a calcium ion channel (calcium pump PMCA4) that has been reported to regulate EMT transcriptomes in gastric cancer cells. 36 ) may regulate gene transcription in a manner similar to that of 35 Further studies will elucidate the role of epithelial cell transport in normal tissue maintenance and other disease states.

[0081] Our observation that Nalcn deficiency recapitulates GISF in the kidney and skin of aged animals points to blockade of Nalcn channels as a possible mechanism underlying this debilitating condition. KP Mouse, P1 R The mice died of cancer before organ fibrosis developed, and R Nalcn deficiency in mice leads to gastric, intestinal, pancreatic, pulmonary, or hepatic fibrosis (P1 KP Fibrosis is unlikely to contribute to metastasis in NALCN-deficient mice, because NALCN-deficient mice did not induce GISF (the main site of primary and metastatic tumors in mice). However, even limited exposure to gadolinium can induce GISF in humans, raising concerns that gadolinium-enhanced imaging of cancer patients may accelerate metastasis. References for Examples 1 to 5 (References)

[0082] [Table 6A] [Table 6B] [Table 6C] [Table 6D] [Table 6E] [Table 6F] [Table 6G] [Table 6H] [Table 6I] [Table 6J] [Table 6K] [Table 6L] [Table 6M]

[0083] Examples 6-10 are related to the inventors' published literature, Rahrmann et al., The NALCN channel regulates metastasis and nonmalignant cell dissemination. Nature Genetics, doi.org / 10.1038 / s41588-022-01182-0, 2022. Extended data is available at https: / / doi.org / 10.1038 / s41588-022-01182-0. Supplementary information is available at https: / / doi.org / 10.1038 / s41588-022-01182-0.

[0084] Example 6 Loss of NALCN function in cancer In related studies to Examples 1-5, NALCN channels were demonstrated to control metastasis and non-malignant cell dissemination.

[0085] To investigate how nonsynonymous mutations affect NALCN function in cancer, we performed HOLE analysis. 21 used to in silico predict the effect of NALCN embedded in the 575-lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer and in its relaxed state on the ion channel pore radius. 12, 22, 23 This model is a gain-of-function 12 Each of the 22 mutations known to result in loss of function and opening of the NALCN channel 11 We correctly predicted channel closure for every two mutations that cause AF (Rahrmann et al., 2022 - Table 3 in the supplemental material (reproduced below as Table 6)).

[0086] [Table 7]

[0087] Nonsynonymous cancer-associated mutations were clustered within the pore turret and voltage-sensitive domains that control NALCN channel opening 11, 12 75% (n=147 / 196) of these mutations were predicted to close the channel (Fig. 1c, 1d and Rahrmann et al., 2022 - Supplementary Table 4). Mutations predicted to cause maximal pore closure were enriched in the most advanced cancers (Fig. 1e). Furthermore, human GAC with NALCN mutations were more likely to undergo EMT. 24 , upregulated genes associated with metastasis, and cell migration ( Rahrmann et al., 2022 - Tables 5 and 6 in the supplementary material).

[0088] As a first step to test whether Nalcn regulates cancer progression, we investigated the P1 KP -GAC cells. KP Whole-cell membrane voltage clamp analysis of P1-GAC cells revealed GdCl3-sensitive currents that were linear to voltage steps within the range of ±80 mV, as previously reported13. KP Decreased Nalcn expression in -GAC cells abolished NALCN currents, increased cell proliferation, and downregulated P1 KP -GAC orthotopic grafts resulted in EMT morphology and transcriptome (Rahrmann et al., 2022 - Tables 7 and 8 in Supplementary Material). Conversely, increased NALCN expression in P1 KP This resulted in increased GdCl3-sensitive currents in -GAC cells, decreased cell proliferation, and a hyperepithelialized morphology in allografts.

[0089] Example 7 Loss of Nalcn promotes cancer metastasis To test how loss of function of Nalcn affects cancer initiation and progression in disease-free tissues, we generated mice harboring a provisional Nalcn allele (Nalcn Flx ). This mouse is KP , Villin 1-Cre ERT2 Kras G12D ;Trp53 Flx / Flx (V1 KP ), or Pdx1-Cre;Kras G12D ;Trp53 Flx / + (Pdx1 KP ) mice to obtain Nalcn-wild type (Nalcn + / + ), Nalcn + / Flx , or Nalcn Flx / FlxWe generated equal numbers of male and female mice (total n = 551; Rahrmann et al., 2022 - Table 9 in the Supplementary Material) that were either Rosa26-ZsGreen (Rosa26 ZSG ) lineage-tracing allele. KP and Pdx1 KP Cancer in mice was induced by Cre expression 25, 26 and pancreas 27, 28 Each is limited to Prom1. CreERT2 / LacZ is expressed by a variety of stem / progenitor cells and P1 KP Induces tumors of the small intestine, liver, lung, salivary gland, prostate, uterus, skin, and stomach in mice 15, 29 Because tissues may exhibit age-dependent susceptibility to transformation 15 The inventors have used tamoxifen on postnatal day (P) 3 or P60 to KP and V1 KP Cre recombination was activated in mice. As expected, V1 KP (n=127 / 141) and Pdx1 KP (n=55 / 55) mice developed intestinal and pancreatic tumors, respectively, while P1 KP Mice developed tumors in the stomach (n=49 / 269), small intestine (n=59 / 269), and other sites (n=108 / 269). 15、26、28 : P1 KP Ninety-nine percent (n=212 / 214) of tumors in mice arose as single primary tumors (Fig. 5a-g and Table 9 in Supplementary Material). Detailed macroscopic and microscopic analysis of tumors revealed that age at induction, sex, and / or Nalcn status had no significant effect on tumor incidence, type, tumor-free survival, tumor growth rate, immune cell infiltration, proliferation, or other key primary tumor characteristics (Fig. 5, Fig. 6a-c, and Tables 13-15). However, P1 KP -GAC and Pdx1 KP Pancreatic adenocarcinoma (Pdx1 KP -PAC) transcriptomes were enriched for genes associated with human CTCs and EMT (Figure).

[0090] Given these transcriptome changes, Nalcn deficiency may be responsible for the P1 KP , V1 KP and Pdx1 KP In mice, NALCN dramatically increased cancer metastasis (Figures 7a-d and 8, and Rahrmann et al., 2022 - Table 12 in Supplementary Material). Metastatic and primary tumors were distinguished from each other by combined histology review, co-isolation of "matched" primary and secondary tumor transcriptomes by unsupervised hierarchical clustering, and enrichment of histology-predicted primary tumor gene sets within metastatic tumor transcriptomes (Figures 7a, 7c, and 8). + / + V1 in the mouse KP Intestinal adenocarcinoma (V1 KP -IAC, n = 27 mice) and Pdx1 KP -PAC (n=19 mice) generated 2.82±4.88 (mean±sem) and 5.53±4.02 metastases per mouse, respectively (Fig. 7d and Rahrmann et al., 2022 - Tables 9 and 12 in Supplementary Material). In stark contrast, V1 KP Nalcn + / Flx (n=51), V1 KP Nalcn Flx / Flx (n=26), Pdx1 KP Nalcn + / Flx (n=23), and Pdx1 KP Nalcn Flx / Flx (n=13) These same tumors in mice numbered 16.82±5.69 per mouse (two-tailed Mann-Whitney U test, Nalcn + / + V1 generated 26.04 ± 10.18 (P = 0.0009), 15.04 ± 3.62 (P = 0.007), and 13.46 ± 5.01 (P = 0.02) more metastases than V2, respectively. KP -Deletion of Nalcn from IACs increased metastasis, particularly to the peritoneum, kidney, and liver: Pdx1 KP Deletion of Nalcn from P1-PAC increased metastasis to the peritoneum and lungs (Fig. 7d). KP ;Nalcn + / +From 11.60 ± 3.45 metastases per mouse, P1 KP ;Nalcn + / Flx 42.21 ± 11.23 metastases per mouse, and P1 KP ;Nalcn Flx / Flx Up to 40.24.0 ± 15.51 metastases per mouse, P1 KP It also increased IAC and GAC metastasis in mice (n = 80) (Fig. 7d and Rahrmann et al. 2022 -Tables 9 and 12 in the supplementary material).

[0091] To further validate the loss of function of Nalcn as a driver of cancer metastasis, we investigated the function of V1 KP ;Nalcn + / + (n=37), V1 KP ;Nalcn Flx / + (n=17), and V1 KP ;Nalcn Flx / Flx An additional cohort of mice (n=8) was treated with GdCl3 (2 μg per kg body weight per week). GdCl3-treated V1 KP ;Nalcn + / + IAC in mice (n = 28) generated 18.32 ± 5.95 metastases per mouse compared with only 2.82 ± 4.88 in controls (P = 0.02; Fig. 7e and Rahrmann et al., 2022 - Table 12 in Supplementary Material). However, GdCl3 did not significantly increase the number of metastases in V1 KP ;Nalcn + / Flx Mouse or V1 KP ;Nalcn Flx / Flx It did not increase metastasis in any of the mice, confirming that the agent specifically phenocopied the metastatic phenotype of Nalcn deficiency.

[0092] Example 8 NALCN regulates CTCs Because Nalcn deficiency increased tumor metastasis and expression by GACs, IACs, and PACs of genes enriched in the human CTC transcriptome (Fig. 5j), we reasoned that Nalcn may regulate the egress of CTCs from primary tumors: CTCs shed from tumors into the blood as precursors of metastasis. 30 To test this, we transfected the corresponding epithelium with Rosa26 ZSG Nucleated GAC, IAC, and PAC cells genetically tagged by recombining lineage-tracing alleles were isolated from whole blood and quantified using ZsGreen (ZSG) fluorescence-activated cell sorting (FACS). ZSG Alleles, oncogenic alleles and Nalcn Flx Prom1 carrying various combinations of alleles CreERT2 / LacZ (n=397), Villin-1 CreERT2 (n=162), or Pdx1 Cre Serial samples from peripheral blood taken from (n=40) mice were analyzed (Rahrmann et al., 2022 - Table 13 in Supplementary Material). A mean (±sem) of 3.8 × 10 3 ±0.9×10 3 Individual Circulation ZSG + Cells (CZCs) (0.066% ± 0.02% of total cells) were isolated from all mice on average 254 ± 9.1 days after Cre recombination (Figures 9a-c and Rahrmann et al., 2022 - Table 13 in Supplementary Material). Across all three Cre lines, the number of CZCs correlated significantly with both the presence of primary tumors (Figure 9b) and the total number of metastases, independent of the sex of the mice or the age of induction (multiple linear regression, T = 10.43, P = 0.000043; Rahrmann et al., 2022 - Table 13 in Supplementary Material). Deletion of Nalcn or treatment with GdCl3 reduced the number of tumor-bearing P1 KP , V1 KP , and Pdx1 KP The levels of CZC in mice were significantly increased (Fig. 9c). CreERT2 / LacZ , Villin-1 CreERT2 , or Pdx1 CreSince neither of these recombined hematopoietic cells in the bone marrow (Fig. 9d), these data strongly suggest that CZCs are CTCs that have spread from the primary tumor through a process regulated by Nalcn. + / + , Nalcn + / Flx , or Nalcn Flx / Flx P1 KP and V1 KP Similar levels of circulating CZCs were observed in mice, suggesting that Nalcn controls cell dispersal as a late event (Figure 9e, Figure 9f, and Rahrmann et al., 2022 - Table 13 in Supplementary Material); however, CZC numbers may be underestimated at early time points in our mice, as lineage tracing takes time to reach steady state.

[0093] To gain a deeper understanding of the origin of the CZC, we investigated the P1 KP -GAC (n=1,701 cells) or V1 KP We generated single-cell RNA-seq profiles of CZCs isolated from mice with -IAC ​​(n=119) and peripheral blood mononuclear cells (PBMCs, n=559; Fig. 10a) and compared these to published single-cell RNA-seq profiles of human breast, lung, pancreatic, and prostate CTCs (n=360), and PBMCs (n=500). 31, 32, 33, 34, 35, 36Human CTCs comprised three overlapping clusters (Figure ​(Figure11a–c and Rahrmann et al., 2022 - Tables 14 and 15 in Supplementary Material): "huCTC1" (enriched in cancer metastasis, EMT, and epithelial gene sets); huCTC3 (enriched in early erythroid and EMT gene sets); and huCTC2 (sharing the profile of huCTC1 and huCTC3). huCTC1–3 expressed β-globin (HBB) - a survival factor for human CTC33 - as well as HBA1, HBA2, and HBD. Mouse CZCs formed seven clusters with transcriptomes significantly concordant with huCTC1 (mCZC2-7), huCTC2 (mCZC2, 3, 5-7), and huCTC3 (mCZC2-7), and also contained orthologs of HBA1, HBA2 (Hba-a1, Hba-a2), HBB (Hbb-bs, Hbb-bt), ANXA2, and LGALS3, as well as genes expressed in normal and malignant stomach and small intestine (Figures 10a, 10b, and 11c-g, and Rahrmann et al., 2022 - Tables 16 and 17 in the Supplementary Material). Normalization and uniform manifold approximation and projection (UMAP) of all single-cell RNA-seq profiles also revealed significant overlap in the mouse CZC and human CTC transcriptomes, particularly enriched for CD71+ erythroid genes (Figures ​(Figure11e–g, and Rahrmann et al., 2022 - Table 18 in Supplementary Material). V1 KP -IAC and P1 KP Concurrent immunofluorescence staining of peripheral blood smears from mice with -GAC confirmed CZC expression of HBA-A1, LGALS3, and epithelial cell markers (KRT80, CDH1), as well as CDX2, characteristic of the intestinal epithelium (Fig. 10c). PBMCs did not express these markers, but did express PBMC markers (e.g., CD45).

[0094] To directly test whether CZCs possess metastatic potential, we investigated the role of Pdx1 KP -PAC, P1 KP -GAC or V1 KPIndividual aliquots of 25,000 CZCs isolated from mice with -IAC ​​were injected into the tail vein of immunocompromised mice. Within 75 days, all mice had numerous ZSGs in the lungs, liver, kidneys, and / or peritoneum. + CZCs developed metastases (Fig. 10d, Fig. 10e, and Table 19 in Supplementary Material in Rahrmann et al., 2022). Similar studies with increasing dilutions of the cells revealed that only 10 CZCs were required to generate metastases (Fig. 10f, and Table 19 in Supplementary Material in Rahrmann et al., 2022). Thus, CZCs are highly enriched in CTCs that recapitulate the transcriptome of human CTCs and are dissipated into the peripheral blood through a process regulated by Nalcn.

[0095] Example 9 NALCN and circulating non-cancer cells Preventing CTC dissemination into the peripheral blood may halt metastasis, but distinguishing this process from the complex cascade of tumorigenesis has proven challenging. Flx / Flx Deletion of Nalcn from gastric stem cells rapidly upregulated genes associated with invasion (e.g., Mmp7, Mmp9, Mmp10, and Mmp19) and gastric EMT (e.g., Zeb1, Fstl1, Sparc, Sfrp4, Cdh6, and Timp3; Rahrmann et al., 2022 - Tables 20 and 21 in Supplementary Material), suggesting that NALCN may regulate cell dispersal from solid tissues independently of transformation. To test this, we transformed gastric stem cells, which lack the oncogenic allele and never develop tumors, into Prom1 CreERT2 / LacZ ;Rosa26 ZSG ;Nalcn + / + (P1 R Nalcn + / + n=87), P1 R Nalcn + / Flx (n=50), and P1 R Nalcn Flx / FlxCZCs were searched for in the peripheral blood of mice (n=37) (Rahrmann et al., 2022 - Table 13 in Supplementary Material). Notably, deficiency of Nalcn increased the number of CZCs in such mice to levels similar to those observed in tumor-bearing animals (Fig. 12b, Fig. 12c, and Fig. 15a). Single-cell RNA sequencing (SCS) profiles of CZCs isolated from non-tumor-bearing (ntCZC) mice co-clustered with CZCs from tumor-bearing animals (tCZC; Fig. 13b). The majority of tCZC and ntCZC SCSs did not cluster with primary IAC, GAC, or normal tissues, but did cluster with the SCS profile of metastases (Fig. 13b and Rahrmann et al., 2022 - Table 22 in Supplementary Material). The SCS profiles of both tCZC and ntCZC were consistent with those of human CTCs and expressed genes associated with stem and progenitor cells similar to human CTC2; however, tCZC was relatively more enriched for metastasis- and invasion-related gene sets (Figure 13a and Rahrmann et al., 2022 - Tables 23 and 24 in Supplementary Material). Concurrent immunofluorescence staining of blood smears confirmed that both ntCZC and tCZC shared markers of huCTCs, including HBA-A1 (Figure 13c and Figure 15c).

[0096] To understand the ultimate outcome of ntCZC, we investigated the function of aged V1 R and Pdx1 R Nalcn + / + , Nalcn + / Flx , and / or Nalcn Flx / Flx ZSG in mouse lungs and kidneys + Notably, ZSG + Cell clusters were readily detected in these organs in Nalcn-deficient animals, but NALCN + / + In mice, ntCZC was absent or detected at significantly lower levels, suggesting that it migrates to and is embedded in distant organs (Fig. 12d-f and Fig. 13b, c). To test this more directly, we performed a 3D CT scan of P1R Nalcn Flx / Flx Individual aliquots of 25,000 ntCZC isolated from mice were injected into the tail vein of six immunodeficient mice. All recipient mice remained in clinically good condition after an average of 100 days, but contained numerous ZSGs in their lungs, livers, kidneys, and peritoneum. + / Cdh1 + / Icam1 + The metastatic tumors formed by tail vein injection of tCZC contained donor cell clusters at a frequency similar to that of the metastatic tumors formed by tail vein injection of tCZC (Fig. S10e, S12g-i, and S10d). The translocated ntCZC formed apparently normal structures within the target organs, the most extreme being the glomeruli and tubules of the kidney (Fig. S12h, S12i). Thus, NALCN regulates cell dispersal from solid tissues independently of cancer, uncoupling this process from tumor formation and revealing an oncogene-independent metastatic pathway.

[0097] Example 10 Blockade of NALCN leads to systemic fibrosis P1 R Nalcn + / Flx (n=118) and P1 R Nalcn Flx / Flx (n=112) mice did not develop cancer, but whole-body necropsy of these mice revealed that P1 R Nalcn + / + Compared to mice with gliomas (n=65), mice with gliomas showed severe kidney and skin fibrosis (Rahrmann et al., 2022 - Table 25 and Figure 14 in the Supplementary Material). This pathology occurred over 400 days and reproduced the pathology of gadolinium-induced systemic fibrosis (GISF), a debilitating condition manifested by severe organ fibrosis following administration of gadolinium-based contrast agents. 37 How gadolinium-based contrast agents cause GISF is unclear, but suggested mechanisms include tissue retention of gadolinium-based contrast agents and recruitment and induction of bone marrow-derived fibrocytes. 38Our data strongly suggest that gadolinium-mediated blockade of NALCN channels recruits epithelial cells into various epithelial tissues, migrates to the kidney and other organs, and ultimately induces a fibrotic response, leading to GISF.

[0098] Developing anti-metastatic therapies has proven challenging because finding targets within the primary tumor that drive metastasis has proven difficult. 2 By uncoupling the process of CTC dispersal from "upstream" tumor formation, our data highlight the manipulation of NALCN function as a promising novel approach to block metastasis. In particular, drugs capable of reopening NALCN channels could be effective anti-metastatic therapies. A precursor to this approach is offered by drugs that open chloride ion channels mutated in cystic fibrosis. 39 If successful, such agents may also be useful in treating GISF.

[0099] It is important to point out that our observations are based on deleting Nalcn from mouse tissues, whereas NALCN in human cancers is primarily affected by nonsynonymous mutations. Our in silico modeling strongly suggests that such cancer-associated mutations close the NALCN channel, but it will be important to functionally demonstrate this by modeling nonsynonymous Nalcn mutations in vivo. Such studies should also include testing in patient-derived xenografts of gastric, colon, and other cancers to confirm that NALCN controls human as well as mouse cell migration.

[0100] Loss-of-function mutations in NALCN may also help explain a variety of puzzling features of human cancer. Metastases may occur many years after removal of the localized cancer. 40 or in the absence of a primary tumor 41Loss of NALCN function in our mice caused abundant and persistent dissemination of cells embedded in distant organs, even in the absence of primary tumors. Because human epithelial tissues contain regions of phenotypically normal cells that harbor oncogenic mutations, 42, 43 Loss of NALCN function in such cells may provide a source of CTCs that form metastases in the absence of a primary tumor or long after the primary tumor has been removed. Such cells would likely need to acquire additional mutations to form tumors at metastatic sites, consistent with the relative rarity of such an event. Our data may also explain why CTCs were found in the bone marrow of patients lacking metastases. Although such cells may represent "quiescent" CTCs as previously suggested, 3 Similar to our ntCZC in mice, such cells can sprout from nontransformed epithelia that have lost NALCN function but have not acquired the ability to form metastatic tumors. A series of analyses of CZC in mice suggests that cellular sprouts after loss of NALCN function are a late rather than an early event; however, NALCN mutations can promote both linear and parallel progression models of cancer. 44 .

[0101] Our data also provide insight into how NALCN may regulate epithelial cell proliferation. We observed upregulation of genes associated with EMT and invasion within 72 hours of depleting Nalcn from normal gastric stem cells, suggesting that this channel may regulate gene transcription in a manner similar to that reported for calcium ion channels. 6, 45 Our electrophysiological studies demonstrate that GAC cells possess NALCN-mediated currents. However, more detailed electrophysiological studies are required to define the exact mechanism by which NALCN controls gene expression and cell efferent potential, and whether this is related to the maintenance of the resting membrane potential.

[0102] Aged Nalcn-deficient mice develop kidney and skin fibrosis reminiscent of GISF, pointing to NALCN channel blockade as a possible cause of this debilitating condition. KP Mouse, P1 R Mice died of cancer well before organ fibrosis developed, and P1 R Nalcn deficiency in mice leads to gastric, intestinal, pulmonary, pancreatic, or hepatic fibrosis (P1 KP GISF was not induced in Nalcn-deficient mice (the main site of primary and metastatic tumors in mice). Thus, it is unlikely that fibrosis contributed to metastasis in Nalcn-deficient mice. However, even limited exposure to gadolinium can induce GISF in humans, raising concerns that gadolinium-enhanced imaging of cancer patients may accelerate metastasis.

[0103] method Gastric stem cell culture Gastric glands were isolated by perfusing mice with 30 mM EDTA / PBS, removing the stomach, and scraping the pyloric mucosa into 10 mM EDTA / PBS at 4°C.46 Dissociated, filtered, and resuspended cells were cultured in Matrigel (cat. no. 354230, BD Biosciences) and culture medium: growth factors (50 ng ml -1 of EGF (PeproTech), 1mg ml -1 R-spondin1 (Cat. no. 120-38, PeproTech), 100ng ml -1 Noggin (Cat. No. 250-38, PeproTech), 100 ng ml -1The cells were placed in Advanced DMEM / F12 (catalog no. 31330038, Thermo Fisher Scientific), B27 (catalog no. 12587010, Thermo Fisher Scientific), N2 (catalog no. A1370701, Thermo Fisher Scientific), N-acetylcysteine ​​(catalog no. A9165, Sigma-Aldrich), and 10 nM gastrin (catalog no. G9145, Sigma-Aldrich) containing 10 nM FGF10 (catalog no. 100-26, PeproTech), and Wnt3A conditioned medium (L Wnt-3A, catalog no. ATCC-CRL-2647, American Type Culture Collection). The gastric spheres were subjected to dispase (catalog no. D4818, Sigma-Aldrich) digestion and dissociated into single cells (StemPro Accutase, Life technologies). Gadolinium (catalog number 439770, Sigma-Aldrich) was diluted in culture medium and layered onto cells embedded in Matrigel (Rahrmann et al., 2022 - Tables 26 and 27 in the Supplementary Material).

[0104] Lentivirus generation and transduction Nalcn-shRNA lentivirus was generated as previously described. 47 Three shRNAs per target (two open reading frames and one 3'-untranslated region) were cloned into pFUGWH1-RFPTurbo, and the plasmids pVSV-G and pCMVd8.9 were co-transfected into 293FT (Thermo Fisher Scientific, Catalog No. R70007) cells. NALCN cDNA (NM_052867) was obtained from OriGene (Cat. No. RC217074). A total of 2 × 10 4 Gastric cells were mixed with lentivirus (20 particles per cell) seeded in matrigel. + (shRNA) or green fluorescent +(cDNA) Cells were sorted using a Becton Dickinson Aria II Cell Sorter (Rahrmann et al., 2022 -Tables 26 and 28 in the supplementary material).

[0105] Whole-cell electrophysiology NALCN channel currents were measured as reported. 48 Whole-cell recordings were obtained from tumor cells on 12-mm coverslips coated with Matrigel at a density of 25,000 cells per ml and perfused (2–3 ml min) with warmed (30–32°C) recording solution containing 120 mM NaCl, 5 mM CsCl, 2.5 mM KCl, 2 mM CaCl, 2 mM MgCl, 1.25 mM NaHPO, 26 mM NaHCO, 20 mM glucose, and 1 M tetrodotoxin (300–310 mOsm) with 95% O / 5% CO. -1Patch pipettes (open pipette resistance, 3–4 MΩ) were filled with an internal solution containing 125 mM CsMeSO3, 2 mM CsCl, 10 mM HEPES, 0.1 mM EGTA, 4 mM MgATP, 0.3 mM NaGTP, 10 mM disodium creatine phosphate, 5 mM QX-314, and 5 mM tetraethylammonium chloride (pH 7.4, adjusted with CsOH, 290–295 mOsm). The inclusion of tetrodotoxin and QX-314 blocked voltage-sensitive sodium channels in the cells being recorded from, whereas cesium and tetraethylammonium chloride blocked voltage-sensitive potassium channels. Voltage-clamp recordings were made using a Multiclamp 700B (Molecular Devices), digitized (10 kHz; DigiData 1322A, Molecular Devices), and recorded using pCLAMP v.10.0 software (Molecular Devices). In all experiments, membrane potential was corrected for a liquid junction potential of -10 mV. After forming a gigaseal on the cell and rupturing the cell membrane, the tumor cell membrane potential was maintained at -70 mV. Cell membrane capacitance, membrane resistance, and pipette access resistance were then measured using the pCLAMP cell membrane test function. Recordings were excluded if the pipette access resistance was higher than 20 MΩ or if the access resistance changed by more than 20% during the experimental period. After the cell membrane resistance had stabilized, the membrane potential was then increased in a series of 250 ms voltage steps from -80 mV to +80 mV in 20 mV increments after 100 ms at 0 mV, and the current responses to these voltage steps were recorded. GdCl3 (100 μM) was then applied to the bath solution to eliminate voltage-independent "leak" currents associated with Nalcn. Calculation of Nalcn currents was performed offline by subtracting the current response in GdCl3 from the preceding GdCl3-free current recording. Tumor cell Nalcn current density was determined by dividing the Nalcn current by the cell membrane capacitance. RFP + (Nalcn shRNA ) or GFP + (NALCNcDNA To verify the expression efficacy of the constructs, they were imaged using a two-photon laser scanning microscope (Prairie Technologies) using a Ti:Sapphire Chameleon Ultra femtosecond pulsed laser (Coherent) and a ×60 (0.9NA) water immersion infrared objective (Olympus). Red fluorescent protein was visualized using an excitation wavelength of 1030 nM, while green fluorescent protein (GFP) was visualized using an excitation wavelength of 820 nM (Rahrmann et al., 2022 - Tables 26 and 28 in the Supplementary Material).

[0106] Gastric adenocarcinoma allograft The P1 mice were treated in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC-SJ) of St. Jude Children's Research Hospital. KP Orthotopic and ventral allografts of CD-Foxn1-GAC were generated. For orthotopic grafts, a longitudinal abdominal incision was made to separate the CD-Foxn1 NU The pyloric valve of the mouse was exposed and 2 × 10 5 Freshly dissociated P1 KP -GAC cells were injected into the pyloric gastric epithelium. The wound was closed and the mice were monitored daily for tumor development. Animals that reached a humane endpoint were promptly euthanized and a full necropsy was completed, based on veterinary guidelines and procedures approved by the IACμC-SJ (Rahrmann et al., 2022 - Tables 26 and 29 in the Supplementary Material).

[0107] Nalcn Flx Generation of alleles Mice were derived from targeted embryonic stem cells (ESCs) (UCDAVIS KOMP Repository Knockout mice Project clone EPD0383_5_C01). ESCs were screened using the KOMP PCR strategy for Nalcntm1a (KOMP)Wstsi. ESCs were transplanted into recipient C57 / Bl6 mice according to a protocol approved by the IACUC-SJ. Wild-type Nalcn and Nalcn Fl Alleles were detected using standard PCR and primers (UCDAVIS KOMP Repository Knockout Mouse Project clone EPD0383_5_C01). Nalcn RNA expression was quantified by quantitative PCR (qPCR) using reverse transcription and a Bio-Rad CFX96 Touch Real-Time PCR Detection System using primers (for details on animals and oligonucleotide sequences, see Tables 26 and 29-31 in Rahrmann et al., 2022 - Supplementary Material).

[0108] Tumor formation and surveillance All animal studies in the United Kingdom (UK) were performed in accordance with UK Home Office licenses (Project License 70-8823, P47AE7E47, PP7834816) and in accordance with the Animals (Scientific Procedures) Act 1986 approved by the Cancer Research UK (CRUK) Cambridge Institute Animal Welfare and Ethical Review Board. Mice were housed in individual air-conditioned cages with wood chip bedding and environmentally enriched (cardboard play tunnels and tube block) nestlets at 21±2°C and 55%±10% humidity under a 12-hour light / dark light cycle. Diet was irradiated LabDiet 5R58 with water ad libitum. Animals carrying the modified Nalcn allele were crossed with RosaFLPe expressing mice to remove the LacZ and Neo cassettes. Animals that completed recombination were crossed with Prom1C-L29; Nestin-cre49; Rosa-CreERT50; Villin-CreER25; Pdx1-cre28; RosaZSG51; and KrasG12D / +52, Trp53flx53. Cre recombination was activated by administering 1 mg tamoxifen per 40 g (body weight) at P3 or 8 mg tamoxifen per 40 g (body weight) at P60. Mice were maintained for 2 years and at the humane endpoint or at the time of indication (whichever comes first), a full-body necropsy was performed as described4. All tissues were examined for macroscopic tumors using direct green fluorescence detection. Tissues were formalin-fixed, paraffin-embedded, some were also flash-frozen, or used for tissue dissection for sequencing (Rahrmann et al., 2022-Tables 26 and 29 in the Supplementary Material).

[0109] histology Hematoxylin and eosin (H&E) staining was performed using standard procedures (catalog numbers 7221, 7111, Thermo Fisher Scientific). Fibrosis was assessed using modified Masson's trichrome and picrosirius red staining. Immunohistochemistry was performed using standard procedures and primary antibodies: Ki67 (Cat. No. IHC-00375, Bethyl Laboratories, 1:1,000), ZSG (Cat. No. 632474, Clontech, 1:2,000), pancytokeratin (AE1 / AE3) (Cat. No. 901-011-091620, BioCare Medical, 1:100), CK5 (Cat. No. ab52635, Abcam, 1:100), vimentin (Cat. No. 5741S, Cell Signaling Technology, 1:200), cleaved caspase 3 (Cat. No. 9664, Cell Signaling Technology, 1:200), CD31 (Cat. No. 77699, Cell Signaling Technology, 1:100), α-smooth muscle actin (Cat. No. ab5694, Abcam, 1:100). 1:500), CD45 (Cat. no. ab25386, Abcam, 5μg ml -1 ) was used. Secondary antibodies were anti-rabbit poly-horseradish peroxidase-IgG (included in the kit) or rabbit anti-rat (catalog no. A110-322A, Bethyl Laboratories, 1:250). Digital images of entire tissue sections were captured using a Leica Aperio AT2 digital scanner (×40, 0.25 μM resolution per pixel), viewed using Leica Aperio Image Scope v.12.3.2.8013, and quantified by HALO (Indica Labs) image analysis (Rahrmann et al., 2022 - Tables 28 and 33 in the Supplementary Material).

[0110] For immunofluorescence, tissue sections were incubated with primary antibodies: rhodamine-conjugated DBA (catalog no. RL-1032, Vector Laboratories, 1:100), rhodamine-conjugated UEA I (catalog no. RL-1062, Vector Laboratories, 1:100), ZSG (catalog no. TA180002, Origene, 1:1,000), CK7 (catalog no. ab181598, Abcam, 1:200), CK20 (catalog no. ab97511, Abcam, 1:200), E-cadherin (catalog no. AF748, R&D Systems, 1:100), N-cadherin (catalog no. 13116, Cell Signaling Technology, 1:100), Icam1 (catalog no. ab179707, Abcam, 1:100), and Cdx2. (cat# ab76541, Abcam, 1:100), Krt80 (cat# 16835-1-AP, Protein Tech, 1:100), Hba-a1 (cat# ab92492, Abcam, 1:100), Lgals3 (cat# ab209344, Abcam, 1:200), CD45 (cat# ab10558, Abcam, 1:200). Secondary antibodies included Alexa 488, 594, and 647 (cat# A-11055, A-21207, and -31571, Thermo Fisher Scientific, 1:500). Sections were counterstained (4,6-diamidino-2-phenylindole (DAPI); Cat. No. 4083, Cell Signaling Technology, 1:10,000) and images were captured at ×40 magnification using a Zeiss ImagerM2 and Apotome microscope, or a Zeiss Axioscan.Z1 (Zeiss), and processed using ZEN2.3 (Zeiss) software (Rahrmann et al., 2022 - Tables 28 and 33 in the Supplementary Material).Nalcn RNA expression was detected in formalin-fixed, paraffin-embedded sections using Advanced Cell Diagnostics (ACD) RNAscope 2.5 LS Reagent Kit-RED (ACD, Cat. No. 322150) and RNAscope 2.5 LS Mm Nalcn (ACD, Cat. No. 415168). Probe hybridization and signal amplification were performed according to the manufacturer's instructions. Fast Red detection of mouse Nalcn was performed on a Bond Rx using the Bond Polymer Refine Red Detection Kit (Leica Biosystems, Cat. No. DS9390) according to the manufacturer's protocol. Whole tissue sections were imaged on an Aperio AT2 (Leica Biosystems) and analyzed for immunohistochemistry using HALO (Indica Labs) imaging analysis software. β-galactosidase staining was performed as described. 4 (Rahrmann et al., 2022 - Tables 26, 28, and 30 in the Supplementary Material).

[0111] Histological review and classification of primary and metastatic tumors were performed by expert pathologists (P. Vogel and B. Mahler-Araujo) blinded to mouse genotype and clinical history. ZSG + The number of cell clusters or metastases was counted in each organ in each mouse. Tissue fibrosis was assessed by expert pathologist R. Nazarian using sections stained with H&E, Masson's trichrome, and picrosirius red.

[0112] Whole tissue imaging Kidneys were exsanguinated and perfused at 37°C and 80 rpm with PBS and 4% PFA, followed by a PBS wash and immersion reagent 1a (150 g ultrapure water, 20 g Triton X-100 (catalog no. 10254583, Thermo Fisher Scientific), 10 g N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine (catalog no. 122262, Sigma) 100% solution, 20 g urea (catalog no. 140750010, ACROS Organics), 1 ml 5M NaCl) containing 10 μM DAPI (catalog no. 4083; Cell Signaling Technology). Solutions were changed every 2 days until the tissue was clear. The cleared tissues were washed and soaked in 50% PBS / 50% Reagent 2 (15 g ultrapure water, 50 g sucrose (Cat. No. 220900010, ACROS Organics), 25 g urea (Cat. No. 140750010, ACROS Organics), 10 g 2,2,2-nitrilotriethanol (Cat. No. 90279, Sigma)) for 6 hours (room temperature with gentle shaking), followed by 1 day (room temperature) in 100% Reagent 2 (10 ml). Tissues were mounted and scanned for endogenous expression of DAPI and ZSG using a ×10 objective on a TCS SP5 confocal laser scanning microscope (Leica). Images were processed using Imaris x64 v.9.3.0 software (Oxford Instruments) (Rahrmann et al., 2022 -Tables 26, 28, and 30 in the Supplementary Material).

[0113] Sequential two-photon tomographic imaging was performed on a TissueCyte 1000 instrument (Tissue Vision), where a series of mosaic two-dimensional images are acquired from the tissue, followed by a subsequent round of physical sectioning and imaging using a vibratome. This continues in an automated fashion to generate 15 μm consecutive two-photon tomographic sections that can be mounted on standard microscope slides and imaged by Axioscan fluorescent scanning (Zeiss) for section identification and relocation. Fiducial GFP-labeled agarose marker beads are distributed throughout the embedding medium to aid in relocation of the sample for eventual use (Rahrmann et al., 2022 - Tables 26, 28, and 30 in the Supplementary Material).

[0114] Harvesting and injection of circulating ZSG cells Peripheral blood (500 μl to 1 ml) was collected from mice at necropsy in 10 μl of 0.5 M EDTA diluted in PBS and assessed for ZSG expression by MACSQuant Analyzer (Miltenyi Biotech Inc.) at 525 / 50 nm (FITC) and 614 / 50 nm (propidium iodide). Cells for SCS and tail vein injections were sorted using excitation at 525 / 50 nm (FITC) and 614 / 50 nm (propidium iodide) on a BD FACSAria II Cell Sorter (BD Biosciences). Non-tamoxifen-induced mouse peripheral blood served as a negative control for the set gate parameters (Figures 16 and 17). 25,000 ZSG + A portion of the cells were sorted, injected into recipient NOD SCID gamma mice (Charles River) and timed. For serial dilution assessment of tCZC metastasis initiation, tCZC were isolated from donor tumor-bearing animals via FACS based on ZSG expression and placed in culture medium. The culture medium was as follows: growth factors (50 ng ml -1 Epidermal growth factor (PeproTech), 100ng ml -1The cells were cultured in Advanced DMEM / F12 (catalog no. 31330038, Thermo Fisher Scientific), 2 mM L-glutamine (catalog no. 25030024, Thermo Fisher Scientific), B27 (catalog no. 12587010, Thermo Fisher Scientific), and N2 (catalog no. A1370701, Thermo Fisher Scientific) containing basic fibroblast growth factor (catalog no. 100-18c, PeproTech), and 1% FBS (catalog no. 10500064, Thermo Fisher Scientific). Cells were grown at 37°C in 5% CO2. Recipient NOD SCID gamma mice (Charles River) were injected with either 10, 100, 1,000, or 10,000 tCZCs via tail vein injection and timed. Complete necropsy and tissue collection were performed as described above. Full necropsy and tissue collection were performed as described above (Rahrmann et al., 2022 -Tables 26, 28, and 29 in the Supplementary Material).

[0115] Bulk RNA sequencing Total RNA was extracted from tissues using the Maxwell RSC miRNA Tissue Kit (cat. no. AS1460, Promega). RNA quality was assessed using the TapeStation System (cat. no. 5067-5579, Agilent). RNA libraries and downstream sequencing were performed as previously described54. The Illumina TruSeq stranded messenger RNA kit (cat. no. 20020595, Illumina) was used to prepare RNA libraries, RNA quality was confirmed using TapeStation (Agilent) and quantified using the KAPA qPCR Library Quantification Kit for Illumina Platform (cat. no. KK4873, KAPA Biosystems). Samples were normalized using Agilent Bravo, pooled, and sequenced on an Illumina NovaSeq SP flow cell to generate single-end 50 bp reads at 20 million reads per sample.

[0116] Single-end 50 bp RNA reads were aligned to GRCm38 using HISAT2 (using default parameters). Each sample was sequenced across several lanes; BAM files per lane were merged into a BAM file per sample. Quality control metrics were collected for each file, including overlap statistics and number of reads assigned to genes. Reads were counted based on annotated features using subread featureCounts to obtain "total", "aligned to genome", and "assigned to genes" (i.e., included in analysis) counts. The percentage of aligned bases was computed for several categories: coding, untranslated regions, introns, and intergenic. Other quality control metrics were the percentage of reads on the correct strand, median coefficient of variation of coverage, median 5' bias, median 3' bias, and the ratio of 5' coverage to 3' coverage. Quality control also included expression heatmaps drawn using log2-transformed counts. Log2-transformed counts were generated from the standardized counts using the log2 function in the R language and the counts function in DEseq2. Genes were considered to show differential expression between sample cohorts if they showed ≥1 or ≤-1 log(fold difference) in expression levels and an adjusted P value ≤0.05 (Rahrmann et al., 2022 -Tables 26, 28, and 30 in the Supplementary Material).

[0117] Single-cell RNA sequencing The animals were perfused with PBS, followed by 100 mL of CaCl2 containing 3 mM CaCl2. 2+ and Mg 2+ 100 U ml -1 The organs were dissected, dissociated, and placed in 2 ml of the appropriate dissociation buffer: lungs and stomach were perfused with 100 ml of collagenase type IV medium containing 3 mM CaCl2; 2+ and Mg 2+200μml of HBSS (Life Technologies) containing -1 of collagenase type IV (Sigma) and 100 μgμl -1 The liver was dissociated using DNAse I (Roche) medium containing 3 mM CaCl2; 2+ and Mg 2+ Collagenase type I (100 U ml) dissolved in HBSS (Life Technologies) containing -1 ), Dispase (2.4Uml -1 ), DNAse I (100 μg ml -1 ) medium; the kidneys were dissociated using papain (20 U ml -1 ) and DNAse I (100 mg ml -1 ), 2 mM L-glutamine (Life Technologies) containing 1x Pen-Strep (penicillin-streptomycin), and 10% FBS; the uterus and epididymis were dissociated in CaCl2 containing 3 mM CaCl2. 2+ and Mg 2+ Collagenase type I (100 U ml) dissolved in HBSS (Life Technologies) containing -1 ) and DNAse I (100 mg ml -1 The cells were dissociated using HBSS without calcium and magnesium and the cell suspension was filtered, washed with HBSS without calcium and magnesium, and centrifuged at 300 g for 5 min at 4° C.

[0118] Single cell suspensions of solid tissues were multiplexed and labeled with cell hashing conjugates: 0301-0315 anti-mouse hashtag (BioLegend) prior to sequencing. Nucleated cells isolated from peripheral blood and ZSG +None of the cells were multiplexed and placed into the 10x Genomics pipeline. SCS libraries were prepared using the Chromium Single Cell 3' Library & Gel Bead Kit v.3, Chromium Chip B Kit, and Chromium Single Cell 3' Reagent Kits v.3 User Guide (Manual CG000183 Rev A; 10x Genomics). The cell suspension was loaded onto the Chromium instrument with the expectation that a gel-bead emulsion containing single cells would be collected. RNA from barcoded cells per sample was then reverse transcribed in a C1000 Touch thermal cycler (Bio-Rad), and all subsequent steps to generate single cell libraries were performed according to the manufacturer's protocol without modification (for most samples, 12 cycles were used for the cDNA amplification method, and 16 cycles for samples with very low cell concentrations). After measuring the quality and quantity of cDNA using an Agilent Tapetation 4200 (High Sensitivity D5000 ScreenTape), 25% of the material was used for the preparation of gene expression libraries. Library quality was confirmed using an Agilent Tapetation 4200 (High Sensitivity D1000 ScreenTape to assess library size) and a Qubit 4.0 Fluorometer (Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) to assess double-stranded DNA quantity). Samples were normalized and pooled at equimolar concentrations. To confirm the concentration, pools were subjected to qPCR using the KAPA Library Quantification Kit on a QuantStudio 6 Flex prior to sequencing. Pools were sequenced on an Illumina NovaSeq6000 sequencer using the following parameters: 28bp, read 1; 8bp, i7 index; and 91bp, read 2.

[0119] Raw RNA reads were processed with cellranger using mm10 from 10x as the reference genome to create a filtered gene expression matrix. For hashtagged sequence data (solid organs), cell barcodes detected by cellranger were used as input for CITESeq to generate a count matrix containing cell barcodes and hashtag oligo sequences for each cell. Seurat's HTODemux function was then used to identify clusters and classify cells based on cell barcodes, including negative and doublet cells. Quality control metrics were generated using Scater, after which single cell objects were converted to Seurat objects, objects were merged, and analysis was performed using the standard Seurat pipeline (Rahrmann et al., 2022 - Tables 26, 28, and 30 in the Supplementary Material).

[0120] SCS profiles of 500 cells obtained from Illumina 10x corresponding to human CTCs (GSE75367; GSE74639; GSE60407; GSE67980; GSE114704; GSE144494) and human PBMC raw counts were merged in python v.3.7.3 using the pandas library. Only genes common between the datasets were analyzed. Seurat objects were created from PBMCs and CTCs. Following this step, the data was analyzed using the standard Seurat pipeline (Rahrmann et al., 2022 - Table 33 in the Supplementary Material).

[0121] In a direct comparison of human CTCs and mouse tCZCs, 15,328 orthologs were identified and profiles were processed through the standard Seurat workflow, including cell-by-cell normalization of each gene expression count. Expression enrichment of hemoglobin genes was performed in UCell and enrichment scores were generated using the Mann-Whitney two-tailed U statistic. References for Examples 6-10 (References) TIFF2024544018000031.tif241152TIFF2024544018000032.tif241155TIFF2024544018000033.tif241153TIFF2024544018000034.tif29154

Claims

1. 1. A method for detecting or predicting cancer and / or metastasis, comprising: analyzing a tumor sample obtained from the subject; determining the presence of at least one mutation in a sodium leak channel (NALCN) in the tumor sample relative to a reference sample; determining whether the at least one mutation causes a decrease in the pore size of the NALCN; if the mutation causes a decrease in NALCN pore size, using the decrease in pore size to determine a risk score for cancer and / or metastasis. A method comprising:

2. 2. The method of claim 1, wherein the reference sample is a sample of germline DNA obtained from the subject or a sample of germline DNA obtained from a healthy subject.

3. The method of claim 1 or 2, wherein computational modeling is used to determine whether the at least one mutation causes a decrease in the pore size of the NALCN, and optionally, the computational modeling is performed using HOLE, CHAP, CAVER, or MOLE.

4. The method of claim 3, wherein the reduction in NALCN pore size is calculated by determining the difference in size of the ion-selective filter radius in a NALCN variant containing the mutation compared to the wild-type NALCN filter radius.

5. The method of claim 3, wherein the reduction in NALCN pore size is calculated by determining the difference in size of the gate radius in a NALCN variant containing the mutation compared to the wild-type NALCN gate radius.

6. 1. A method for detecting or predicting cancer and / or metastasis, comprising: analyzing a biological sample obtained from the subject to assess sodium leak channel (NALCN) activity; providing a cancer and / or metastasis risk score based on the level of NALCN activity; A method comprising:

7. The method of claim 6, wherein the activity of the NALCN is assessed by whole-cell electrophysiology, a fluorescence assay, a membrane potential-sensitive dye, and / or an ion flux assay.

8. The method of claim 6, further comprising comparing the activity level of NALCN in the biological sample with a reference value.

9. 1. A method for detecting or predicting cancer and / or metastasis, comprising: analyzing the biological sample to detect the presence of one or more mutations corresponding to reduced function of NALCN; providing a risk score for cancer and / or metastasis based on the presence of one or more mutations corresponding to reduced function of NALCN; A method comprising:

10. 10. The method of any one of claims 1, 2 or 9, wherein the one or more mutations are located in the pore turret domain or voltage-sensitive domain of NALCN.

11. 10. The method of any one of claims 1, 2 or 9, wherein the one or more mutations are selected from the mutations identified in Table 2.

12. The method of claim 9, wherein computational modeling is used to determine whether one or more mutations corresponding to reduced function of NALCN cause a reduction in the pore size of NALCN, and optionally, the computational modeling is performed using HOLE, CHAP, CAVER, or MOLE.

13. The method described in claim 9, wherein computational modeling is used to determine whether one or more mutations corresponding to a decrease in NALCN function cause a decrease in the pore size of the NALCN, and the decrease in NALCN pore size is calculated by determining the difference in size of the ion-selective filter radius in a NALCN variant containing the mutation compared to the wild-type NALCN filter radius.

14. The method described in claim 9, wherein computational modeling is used to determine whether one or more mutations corresponding to a decrease in NALCN function cause a decrease in the pore size of the NALCN, and the decrease in NALCN pore size is calculated by determining the difference in the size of the gate radius in a NALCN variant containing the mutation compared to the wild-type NALCN gate radius.

15. 10. The method of claim 9, comprising the further step of identifying a stage of the cancer based on the identified mutation(s).

16. 10. The method of claim 1, 2, 6, or 9, comprising the further step of selecting a treatment.

17. The cancer is selected from the group consisting of gastric cancer, gastric adenocarcinoma, colorectal cancer, lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bone cancer, pancreatic cancer, colon cancer, colorectal cancer, skin cancer, head or neck cancer, head and neck squamous cell carcinoma, melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer 10. The method of claim 1, 2, 6, or 9, wherein the cancer is selected from testicular cancer, breast cancer, brain cancer, hepatocellular carcinoma, cancer of the fallopian tubes, cancer of the endometrium, cancer of the cervix, cancer of the vagina, cancer of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, renal cancer, soft tissue sarcoma, cancer of the urethra, cancer of the bladder, kidney cancer, thymoma, urothelial carcinoma, leukemia, prostate cancer, prostate adenocarcinoma, mesothelioma, adrenocortical carcinoma, lymphoma, such as Hodgkin's disease, non-Hodgkin's disease, and multiple myeloma.

18. A method for determining the activity of NALCN, comprising analyzing a biological sample to detect one or more mutations identified in Table 2, wherein the presence of one or more mutations identified in Table 2 indicates decreased activity of NALCN.

19. 20. The method of claim 1, 2, 6, 9, or 18, wherein the mutation is detected via allele-specific polymerase chain reaction (PCR), high-resolution melting curve analysis, genomic sequencing fluorescence in situ hybridization (FISH); comparative genomic hybridization (CGH), restriction fragment length polymorphism (RELP), amplification refractory mutation system (ARMS), reverse transcriptase PCR (RT-PCR), real-time PCR, multiplex ligation-dependent probe amplification (MLPA), denaturing gradient gel electrophoresis (DGGE), single-strand conformational polymorphism (SSCP), chemical cleavage of mismatches (CCM), protein truncation test (PTT), or oligonucleotide ligation assay (OLA).

20. 20. The method of claim 1, 2, 6, 9, or 18, wherein the biological sample is analyzed in vitro or ex vivo.

21. 20. The method of claim 1, 2, 6, 9, or 18, wherein the biological sample is a tissue sample or a tumor sample.

22. A kit comprising reagents for detecting one or more mutations in NALCN, and optionally instructions for use, wherein the mutations correlate with decreased activity of NALCN and / or decreased pore size of NALCN.

23. A composition comprising a reagent for detecting one or more mutations in NALCN, wherein the mutations correlate with decreased activity of NALCN and / or decreased pore size of NALCN.

24. 24. The kit of claim 22 or the composition of claim 23, wherein the mutation is selected from one or more of the mutations listed in Table 2.

25. 24. The kit of claim 22 or the composition of claim 23, wherein the reagents are suitable for performing allele-specific polymerase chain reaction (PCR), high-resolution melting curve analysis, genomic sequencing fluorescence in situ hybridization (FISH); comparative genomic hybridization (CGH), restriction fragment length polymorphism (RELP), amplification refractory mutation system (ARMS), reverse transcriptase PCR (RT-PCR), real-time PCR, multiplex ligation-dependent probe amplification (MLPA), denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism analysis (SSCP), chemical cleavage of mismatches (CCM), protein truncation test (PTT), or oligonucleotide ligation assay (OLA).

26. 1. A computer-implemented method for determining a cancer and / or metastasis risk score, comprising: obtaining data indicating the presence of at least one mutation in the sodium leak channel NALCN in the tumor sample; inputting the data into a computational model of NALCN that simulates the effect of mutations on NALCN; using a computational model to determine whether the at least one mutation causes a decrease in the pore size of the NALCN; If the at least one mutation is determined to cause a decrease in the pore size of the NALCN, outputting a risk score for cancer and / or metastasis. A method comprising:

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