Detection of cell cystic canals in tissue for cancer metastasis analysis

Abstract

The present disclosure provides methods of detecting cell cystic canals in tissue of a subject for cancer diagnosis, screening, and metastasis prediction.

Description

[0001] Related application data

[0002] This application claims priority to U.S. Provisional Application No. 63 / 386,248, filed on December 6, 2022, and U.S. Provisional Application No. 63 / 604,962, filed on December 1, 2023, both of which are hereby incorporated by reference in their entirety for all purposes. Technical Field

[0003] The present invention relates to the field of detection of cell encapsulation in tissue for use in cancer metastasis analysis. Background Art

[0004] Cancer is the leading cause of all deaths. Treating cancer is one of the most pressing needs of human healthcare (Cassetta L, Pollard J, A timeline of tumor-associated macrophage biology. Nat Rev Cancer. 23, 238-257 (2023);Chaffer C, Weinberg RA, A Performance on Cancer Cell Metastasis. Science. 331, 1559-64 (2011);Hanahan D, Weinberg RA, Hallmarks of cancer: the next generation. Cell. 144, 646-74 (2011)). In 2020 alone, there were nearly 10 million cancer deaths and approximately 19.3 million new cancer cases worldwide (Sung H et al., Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. Online ahead of print (2021)). The number of new cancer cases and cancer deaths worldwide is increasing year by year. Cancer metastasis is the leading cause of cancer death. Cancer metastasis is the process by which cancer cells migrate from their primary site of origin to secondary sites in adjacent and distant tissues and organs (Friedl P, Wolf K, Tumor-cell invasion and migration: Diversity and escape mechanisms. Nat Rev Cancer. 3, 362–374 (2003); Barbolina MV et al., Microenvironmental regulation of ovarian cancer metastasis. Cancer Treat Res. 149, 319–334 (2009)).

[0005] Cancer is a general term for a class of related diseases that involve abnormal cell growth and have the potential to invade or spread to other parts of the body. Cancer includes malignant tumors and malignant neoplasia, which are characterized by uncontrolled cell proliferation and metastasis. Benign tumors are neoplasias with uncontrolled cell proliferation but no metastasis. Based on various classification methods and standards around the world, there are hundreds of different types and subtypes of cancer. Most of the different types and subtypes of cancer are classified based on the tissue site or cell of origin, tumor grade, cancer stage and molecular markers and their combinations (Thiery JP, Acloque H, Huang RY, Nieto MA, Epithelial-mesenchymal transitions in development and disease. Cell. 139, 871–890 (2009)).

[0006] Many aspects of cancer progression have been intensively studied over the past decades. These aspects include subclonality, cellularity, cellular plasticity, genetics, genomics, proteomics, signaling, metabolism, and tumor microenvironment topics (Chiang A, Massagué J, Molecular basis of metastasis. N Engl J Med. 359, 2814-23 (2008); Dienstmann R et al., Personalizing colon cancer adjuvant therapy: selecting optimal treatments for individual patients. J Clin Oncol. 33, 1787-96 (2015); Mody K, Bekaii-Saab T, Clinical Trials and Progress in metastatic Colon Cancer. Surg Oncol Clin N Am. 27, 349-365 (2018); Patras L et al., Immune determinants of the pre-metastatic niche. Cancer Cell. 41, 546-572 (2023); Turajlic S, Swanton C, Metastasis as an evolutionary process.Science.352,169-75(2016); Vendramin R et al., Cancerevolution: Darwin and beyond.EMBO J.40,e108389(2021); Valastyan S, Weinberg RA, Tumor metastasis: molecular insights and evolving paradigms.Cell.147,275-292(2011)).Several models have been postulated for tumor progression that invoke different hierarchies of drivers, including genetic alterations, genomic aberrations (inconsistent inheritance, DNA macroalterations), oncoprotein promotion, signaling pathways, cellular plasticity, intercellular responses, and the microenvironment (Erler J et al., Lysyl oxidase is essential for hypoxia-induced metastasis. Nature. 440, 1222-6 (2006); Kannarkatt J et al., Adjuvant Chemotherapy for Stage II Colon Cancer: A Clinical Dilemma. J Oncol Pract. 13, 233-241 (2017); Koo M et al., Conceptual Framework to Guide Early Diagnosis Programs for Symptomatic Cancer as Part of Global Cancer Control, JCO Glob Oncol. 7, 35-45 (2021); Nowell P, The clonal evolution of tumor cell populations. Science. 194, 23-28 (1976); Ring et al., Cancer Cell Biology. 198, 23-28 (2008); A et al., Biology, vulnerabilities and clinical applications of circulating tumor cells. Nat Rev Cancer. 23, 95-111 (2023); Seferbekova Z et al., Spatial biology of cancer evolution. Nat Rev Genet. 24, 295-313 (2023)).The biological, biochemical, biophysical and metabolic characteristics of tumors are used as targets for the development of diagnostic and therapeutic approaches for various types of cancer (Friedl P, Wolf K, Tumor-cell invasion and migration: Diversity and escape mechanisms. Nat Rev Cancer. 3, 362-374 (2003); Huerta S et al., Colon cancer and apoptosis. Am J Surg. 191, 517-26 (2006); Labianca R et al., Colon cancer. Crit Rev Oncol Hematol. 74, 106-33 (2010); Visser K et al., The evolving tumor microenvironment: From cancer initiation to metastatic outgrowth. Cancer Cell. 41, 374-403 (2023)).

[0007] The result of cancer treatment and patient survival rate depend on the accurate diagnosis, prognosis, metastasis prediction and evaluation of cancer.However, in clinical pathology determination, lack the unified and accurate prediction of cancer metastasis grade (the key feature of cancer and the main cause of cancer death).In addition, current clinical cancer treatment results show that the potential mechanism of cancer development and progression in vivo is not yet fully resolved (Gerstberger S et al., Metastasis.Cell.186,564-1579 (2023);Iqbal A,George TJ,Randomized Clinical Trials in Colon and Rectal Cancer.Surg Oncol Clin N Am.26,689-704 (2017);Schiffman J et al., Early detection of cancer:past,present,and future.Am Soc Clin Oncol Educ Book.57-65 (2015)).

[0008] There remains a continuing need in the art for methods that can achieve accurate diagnosis, prognosis, and metastasis prediction and assessment of cancer. Summary of the Invention

[0009] The present disclosure addresses this need and is based on the surprising discovery that endogenous plasma membrane Ca 2+-ATPase (PMCA) is highly expressed in the membrane of the cell cysts in cancer tissues. Using anti-PMCA antibodies, cell cysts were detected in cancer tissues, but not in normal or benign tissues.

[0010] According to one aspect, the present disclosure provides a method for detecting cell cysts in a tissue of a subject. In one embodiment, the method comprises obtaining a tissue from a subject and isolating the tissue by contacting the tissue with an anti-plasma membrane Ca 2+ -ATPase (PMCA) antibody contact was used to detect the presence of cell cysts in the tissue.

[0011] According to another aspect, the present disclosure provides a method for predicting the grade of cancer metastasis in a subject. In one embodiment, the method comprises obtaining tissue from the subject and contacting the tissue with an anti-plasma membrane Ca 2+ The presence of cystic tubes in the tissue is detected by contacting the tissue with an antibody to a poly(vinylidene phosphatase) (PMCA). In one embodiment, the presence of cystic tubes in the tissue is associated with metastatic cancer. In certain embodiments, the density and morphology of cystic tubes detected in the tissue are used to predict the grade of cancer metastasis in the subject. In one embodiment, an increased density of cystic tubes is associated with a more advanced grade of cancer metastasis. In another embodiment, a thinner and more cloudy cystic tube morphology and greater degradation are associated with a more advanced grade of cancer metastasis.

[0012] According to yet another aspect, the present disclosure provides a method for screening a subject for cancer. In one embodiment, the method comprises obtaining a tissue from the subject and 2+ -ATPase (PMCA) antibody contact to detect the presence of cell cysts in the tissue. In one embodiment, the presence of cell cysts in the tissue indicates that the tissue is cancerous. In another embodiment, the absence of cell cysts in the tissue indicates that the tissue is healthy or benign.

[0013] According to one aspect, the present disclosure provides a method for detecting cell cysts in a tissue sample of a subject. In one embodiment, the method comprises contacting the tissue sample with an antibody, wherein the antibody binds to a plasma membrane Ca in the cell capsule of the cell cyst. 2+ -ATPase (PMCA) binding, and detecting the presence of cell cysts in the tissue sample by detecting the antibody. In another embodiment, the method includes detecting cell cysts, cell cystic vesicles, cell cystic cancerous cells, cell cyst tumor spheres, and cell cyst tumor sphere network systems in the tissue sample. The presence of cell cysts and / or cell cysts in the tissue sample indicates the presence of cancer or the risk of cancer (i.e., the potential for developing cancer) in the subject.

[0014] According to another aspect, the present disclosure provides a method for detecting cell capsules in vitro. In one embodiment, the method comprises contacting cell capsules in a 3D matrix culture with an antibody, wherein the antibody binds to the plasma membrane Ca in the cell capsule membrane of the cell capsule. 2+ -ATPase (PMCA) binding, and detecting the presence of cell capsule tubes in 3D matrix culture by detecting the antibody. In another embodiment, the method includes detecting cell capsules, cell capsule vesicles, cell capsule cancer cells, cell capsule tumor spheres and cell capsule tumor sphere network systems in 3D matrix culture.

[0015] Note that in this disclosure, particularly in the claims and / or paragraphs, terms such as “comprises, comprised, comprising” and the like may have the meanings ascribed to them in U.S. patent law; for example, they may mean “includes, included, including” and the like; and terms such as “consisting essentially of and consists essentially of” have the meanings ascribed to them in U.S. patent law, for example, they allow for elements not expressly recited but exclude elements found in the prior art or elements that affect the basic or novel characteristics of the invention.

[0016] These and other embodiments will be disclosed or apparent from, and encompassed by, the following Detailed Description. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing and other features and advantages of embodiments of the present invention will be more fully understood from the following detailed description of exemplary embodiments provided in conjunction with the accompanying drawings, in which:

[0018] Figures 1A to 1F Depicts clinically normal breast tissue ( Figure 1A ), benign breast tumor tissue ( Figure 1B ) and breast cancer tissue ( Figure 1C ), and clinical normal breast tissue ( Figure 1D ), benign breast tumor tissue ( Figure 1E ) and breast cancer tissue ( Figure 1F) Representative immunohistochemistry (IHC) fluorescence staining images, which were performed with anti-PMCA2 rabbit polyclonal antibodies, anti-γ-actin antibodies and DAPI (nuclei). Anti-PMCA2 rabbit polyclonal antibodies showed that PMCA2 expression increased in the cell capsule tube (CCT, white arrow) of clinical malignant breast cancer tissue ( Figure 1F ). Breast cancer cells in malignant breast cancer tissue ( Figure 1F ) produced a large number of elongated cell cysts (tubular morphology) with enhanced green fluorescence, while normal breast tissue cells ( Figure 1D ) or benign breast tumor tissue ( Figure 1E ) This phenomenon was not observed. Scale bar: 10 μm.

[0019] Figures 2A to 2B Depicted are representative H&E-stained images of stage II malignant breast cancer ( Figure 2A 、 Figure 2A 'and Figure 2A ”). Many cancer cells (yellow arrows, spindle-shaped in the slice and dark brown in the exposed part) can be observed distributed in the curved but smeared tissue background. The black box area is magnified and shown in the right figure ( Figure 2A ”), showing several sections and exposed migrating cancer cells (yellow arrows, in dark brown). Figure 2B Representative immunohistochemistry (IHC) fluorescence staining images of stage II breast cancer are depicted, and the IHC fluorescence staining was performed with anti-PMCA2 rabbit polyclonal antibody, anti-γ-actin antibody, and DAPI (cell nucleus). Breast cancer cells in malignant breast cancer tissue were observed to produce a large number of elongated cell capsules (tubular morphology) with enhanced green fluorescence. Many curved cell capsules (CCT, white arrows) are arranged together to form bundles, and cancer cells (red arrows, spindle-shaped in the section, with exposed nuclei in blue) migrate inside the CCT. The white box area is magnified and shown in the right figure ( Figure 2B ), which shows several sections and exposed cancer cells (red arrows, in blue) migrating inside CCT (white arrows). Scale bar: 10 μm.

[0020] Figures 3A to 3B Depicts representative H&E-stained images of clinically malignant breast cancer at stage III ( Figure 3A ) and immunohistochemistry (IHC) fluorescence staining images ( Figure 3B ). This IHC fluorescent staining was performed using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). Anti-PMCA2 rabbit polyclonal antibody demonstrates abundant PMCA2 expression in cystic ducts (CCTs, white arrows) of clinical malignant breast cancer. Breast cancer cells in malignant breast cancer tissue produce numerous long, curved cystic ducts and form CCT masses. Scale bar: 10 μm.

[0021] Figures 4A to 4E Representative H&E staining images are depicted ( Figure 4A ) and immunohistochemistry (IHC) staining images, the IHC fluorescent staining was used to identify ER (estrogen receptor, Figure 4B ), PR (progesterone receptor, Figure 4C ) and HER2 (human epidermal growth factor receptor 2, Figure 4D ) antibodies. In breast cancer tissue, staining was performed as PR+ (dark brown cancer cells) / HER2+ (brown cancer cells) / ER-. Anti-PMCA2 rabbit polyclonal antibody, anti-γ-actin antibody and DAPI (nucleus) in IHC ( Figure 4E ) showed high levels of PMCA-2 protein expression in cell capsule tubes (CCT, curved tubular morphology with a diameter / width of 3 μm to 6 μm, white arrows, red box area). These CCT morphologies will provide additional metastatic information to pathologists. The samples in the above analysis were five serial section samples (thickness 5 μm, Figures 4A to 4E ). Scale bar: 10 μm.

[0022] Figures 5A to 5B Depicted are representative H&E-stained images of clinically malignant prostate cancer ( Figure 5A ) and immunohistochemistry (IHC) fluorescence staining images ( Figure 5B ), IHC fluorescence staining was performed using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). Anti-PMCA2 rabbit polyclonal antibody shows abundant PMCA2 expression in cytocystic tubes (CCTs, white arrows) of clinically malignant prostate cancer. Prostate cancer cells in malignant prostate tumors produce long, curved cytocystic tubes that can form large, irregular structures. Many CCTs exhibit a thinner, filamentous morphology, corresponding to more degraded CCTs (CTSs, orange arrows). Scale bar: 10 μm.

[0023] 6A to 6D Describes clinically malignant adrenal carcinoma ( Figure 6A ), appendiceal cancer ( Figure 6B ), bladder cancer( Figure 6C ) and bone marrow cancer ( Figure 6D Representative immunohistochemistry (IHC) fluorescence staining images of 100 cells (Figure 5A) using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). The anti-PMCA2 rabbit polyclonal antibody showed abundant PMCA2 expression in cellular cystic tubes (CCTs, white arrows). Many CCTs exhibited a thinner, filamentous morphology, corresponding to more degraded CCTs (CTSs, orange arrows). Scale bar: 10 μm.

[0024] 7A to 7D Describes clinical brain cancer ( Figure 7A ), cervical cancer ( Figure 7B ), colon cancer( Figure 7C ) and endocrine cancer ( Figure 7D Representative immunohistochemistry (IHC) fluorescence staining images of 100 cells (Figure 5A) using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). The anti-PMCA2 rabbit polyclonal antibody showed abundant PMCA2 expression in cellular cystic tubes (CCTs, white arrows). Many CCTs exhibited a thinner, filamentous morphology, corresponding to more degraded CCTs (CTSs, orange arrows). Scale bar: 10 μm.

[0025] Figures 8A to 8D Describes clinical esophageal cancer ( Figure 8A ), head / neck cancer( Figure 8B ), heart cancer( Figure 8C ) and colorectal cancer ( Figure 8D Representative immunohistochemistry (IHC) fluorescence staining images of 100 cells (Figure 5A) using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). The anti-PMCA2 rabbit polyclonal antibody showed abundant PMCA2 expression in cellular cystic tubes (CCTs, white arrows). Many CCTs exhibited a thinner, filamentous morphology, corresponding to more degraded CCTs (CTSs, orange arrows). Scale bar: 10 μm.

[0026] 9A to 9D Describes clinical renal cancer ( Figure 9A ), liver cancer( Figure 9B ), lung cancer( Figure 9C ) and lymphoid tissue cancer ( Figure 9D Representative immunohistochemistry (IHC) fluorescence staining images of 100 cells (Figure 5A) using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). The anti-PMCA2 rabbit polyclonal antibody showed abundant PMCA2 expression in cellular cystic tubes (CCTs, white arrows). Many CCTs exhibited a thinner, filamentous morphology, corresponding to more degraded CCTs (CTSs, orange arrows). Scale bar: 10 μm.

[0027] 10A to 10D Describes clinical melanoma cancer ( Figure 10A ), mesothelioma ( Figure 10B ), nasopharyngeal carcinoma ( Figure 10C ) and laryngeal and oral cancers ( Figure 10DRepresentative immunohistochemistry (IHC) fluorescence staining images of 100 cells (Figure 5A) using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). The anti-PMCA2 rabbit polyclonal antibody showed abundant PMCA2 expression in cellular cystic tubes (CCTs, white arrows). Many CCTs exhibited a thinner, filamentous morphology, corresponding to more degraded CCTs (CTSs, orange arrows). Scale bar: 10 μm.

[0028] 11A to 11D Describes clinical ovarian cancer ( Figure 11A ), pancreatic cancer ( Figure 11B ), penile cancer( Figure 11C ) and bile duct cancer ( Figure 11D Representative immunohistochemistry (IHC) fluorescence staining images of 100 cells (Figure 5A) using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). The anti-PMCA2 rabbit polyclonal antibody showed abundant PMCA2 expression in cellular cystic tubes (CCTs, white arrows). Many CCTs exhibited a thinner, filamentous morphology, corresponding to more degraded CCTs (CTSs, orange arrows). Scale bar: 10 μm.

[0029] 12A to 12D Describes clinical rectal cancer ( Figure 12A ), skin cancer( Figure 12B ), soft tissue (smooth muscle) cancer ( Figure 12C ) and gastric cancer ( Figure 12D Representative immunohistochemistry (IHC) fluorescence staining images of 100 cells (Figure 5A) using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). Anti-PMCA2 rabbit polyclonal antibody showed abundant PMCA2 expression in cellular cystic tubes (CCTs, white arrows). Many CCTs exhibited a thinner, filamentous morphology, corresponding to more degraded CCTs (CTSs, orange arrows). Scale bar: 10 μm.

[0030] 13A to 13D Describes clinical testicular cancer ( Figure 13A ), thyroid cancer( Figure 13B ), uterine cancer ( Figure 13C ) and vulvar cancer ( Figure 13D Representative immunohistochemistry (IHC) fluorescence staining images of 100 μm (Fig. 2 ). Anti-PMCA2 rabbit polyclonal antibody shows abundant PMCA2 expression in cellular cystic tubes (CCTs, white arrows). Some CCTs exhibit a thinner, filamentous morphology, corresponding to more degraded CCTs (CTSs, orange arrows). Scale bar: 10 μm.

[0031] Figure 14Depicted are representative immunohistochemistry (IHC) fluorescence staining images of breast cancer paraneoplastic tissue, using anti-PMCA2 rabbit polyclonal antibodies, anti-γ-actin antibodies, and DAPI (nuclei). Anti-PMCA2 rabbit polyclonal antibodies show abundant PMCA2 expression in cell cystic tubules (CCT, white arrows). Scale bar: 10 μm.

[0032] Figure 15 Representative immunohistochemistry (IHC) fluorescence staining images of a clinically metastatic gastric cancer that migrated to the lymph node via CCTs were depicted. The IHC fluorescence staining was performed using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). Numerous cellular cysts (CCTs, white arrows) were observed encapsulating and invading the lymph node. Scale bar: 10 μm.

[0033] Figure 16 Representative immunohistochemical (IHC) fluorescence staining images of a clinically metastatic colon cancer that has migrated to the liver via CCTs are depicted. The IHC staining was performed using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). Numerous cellular cysts (CCTs, white arrows) are observed tangled together to form CCT masses that have invaded the liver. As a result of CCT invasion, liver tissue has largely disappeared. Scale bar: 10 μm.

[0034] Figure 17 Depicted are representative immunohistochemical (IHC) fluorescent staining images of a clinically metastatic adenocarcinoma originating from the ovary and migrating into the omentum via CCT. The IHC fluorescent staining was performed using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). Numerous curved cellular capsules (CCT, white arrows) were observed, forming bundle-like structures. Scale bar: 10 μm.

[0035] Figure 18 Representative immunohistochemical (IHC) fluorescence staining images of a clinically metastatic mucinous adenocarcinoma originating from an unknown site and migrating to the omentum via CCTs are depicted. The IHC fluorescence staining was performed using an anti-PMCA2 rabbit polyclonal antibody, an anti-γ-actin antibody, and DAPI (nuclei). Numerous cellular tubes (CCTs, white arrows) were observed within the omentum, surrounding and filling the spaces between the cell clusters. Scale bar: 10 μm.

[0036] Figure 19Depicted are representative immunohistochemistry (IHC) fluorescence staining images of clinically metastatic rectal cancer that migrated to lymph nodes via CCTs. The IHC fluorescence staining was performed using anti-PMCA2 rabbit polyclonal antibodies, anti-γ-actin antibodies, and DAPI (nuclei). Numerous cellular cystic tubes (CCTs, white arrows) were observed forming curved bundles in the lymph node. Scale bar: 10 μm.

[0037] Figure 20 A to Figure 20 E depicts a representative immunohistochemistry (IHC) fluorescent staining image of a clinical needle biopsy of breast cancer. In an elongated clinical tissue sample of malignant breast tumor tissue obtained by needle biopsy, a fluorescent immunohistochemistry staining assay was performed using anti-PMCA2 rabbit polyclonal antibody, anti-γ-actin antibody, and DAPI (nucleus). Fluorescent images were taken using a high-quality fluorescence microscope. No cell capsule was detected at site 1 or site 2, many curved and winding cell capsules (CCT, white arrows) were detected at site 3, and many thinner, for example, filamentous cell capsules were detected at sites 4 and 5, corresponding to cell capsules in the degradation process (CTS, orange arrows). At site 3, many breast cancer cells (blue nuclei) were observed migrating in the CCT. Scale bar 10 μm.

[0038] Figures 21A to 21C Depicted are images of immunohistochemistry (IHC) fluorescent staining of a clinically malignant breast cancer using anti-PMCA1 ( Figure 21A ), anti-PMCA3( Figure 21B ) and anti-PMCA4 ( Figure 21C ) rabbit polyclonal antibodies. Each antibody recognizes its corresponding target protein and shows cell capsule duct staining (CCT, white arrows) in clinical malignant breast cancer tissue. Scale bar: 10 μm.

[0039] Figures 22A to 22F Depicted are schematic diagrams and microscopic images illustrating the cell life cycle of cystic carcinoma cells in vitro and in vivo in human tissue. Figure 22A Schematic diagram depicting the proteomic analysis of cancer cell cysts and the identification of molecular markers for cystic vesicles (CCs) and cystic tubes (CCTs). Figure 22B Depicted are representative bright field (BF) and immunohistochemistry (IHC) fluorescence microscopy images of pancreatic cancer Bxpc3 cells with cell vesicles (CCs) in a 3D CC / CCT culture kit matrix. Shown are cancer cells with CCs (white arrows), decellularized cell vesicles (ECC / ECs, orange arrows), and cancer cell proliferation in CCs (purple arrows). Figure 22CRepresentative IHC fluorescence microscopy images of primary breast cancers with cystic carcinomatous cells (white arrows) are depicted. Intracellular cystic carcinomatous cells (red arrows), decellularized cysts (ECCs, purple arrows), cell capsule membranes (CCMs, orange arrows), proliferation of carcinomatous cells in the CC (red asterisks), and collapsed CCs (white asterisks) are shown. Figure 22D Schematic diagram depicting a cystic carcinomatous cell with its vesicles, cystic bodies, and nanoprotrusions.

[0040] Figure 22E Depicted are representative fluorescence microscopy images showing that during the initiation of CCT regeneration at the acellular amyloid carcinomatous cluster-CC / CCT complex (AMCC) stage, a single breast cancer cell generates multiple CCTs pointing in different directions during in vivo metastasis. Seven CCTs (individually numbered) and multiple sectioned CCT fragments (SCFs, purple arrows) are interconnected at CCT nodes (yellow arrows) and exhibit a radial morphology. Figure 22F Representative fluorescence microscopy images of individual pancreatic cancer cells producing elongated and highly curved CCTs in primary pancreatic cancer tissue are depicted. Scale bar, 10 μm.

[0041] 23A to 23D Depicted are schematic diagrams and microscopic images illustrating the detection of cystic cells and their life cycle. Figure 23A Depicted are representative bright field (BF) and immunohistochemistry (IHC) microscopy images of cell capsule tubes (CCTs) in an in vitro 3D CC / CCT culture kit matrix. A single migrating Bxpc3 cancer cell is depicted within the CCT. The CCT membrane edge is shown (orange arrow). Figure 23B Representative immunohistochemical microscopy images of cell-encapsulated tumor spheres (CT, purple arrows) in a 3D CC / CCT culture kit matrix in vitro are depicted. Cell vesicles (CC, white arrows), decellularized CC (ECC, orange arrows), and ECC with open pores (white asterisks) are shown. Figure 23C Representative immunohistochemical microscopy images of normal, benign, and cancerous human tissues are depicted, with CC / CCT detection performed using anti-PMCA2 and anti-γ-actin antibodies. Terminal ducts (TDs), cystic ducts (CCTs, white arrows), and CCT filaments (CTSs, orange arrows) are shown. Figure 23DSchematic diagram depicting the life cycle of a cystic cell: Transformed (cancerous) cells undergo CC generation and undergo cyst-driven vesicle formation. The vesicle encapsulates the cancerous cell and forms a cystic cell. (1) The cystic cell proliferates and grows into a cystic tumor. (2) The cystic cell proliferates, and the vesicle elongates and develops into a CCT. (3) Decellularization of the cystic cell produces a cell-free vesicle and a cell-free cystic cell. The cell-free vesicle and CCT undergo auto-degradation, decomposition, and disappearance. Scale bar 10 μm.

[0042] 24A to 24D Depicted are microscopic images showing the detection of CCT by IHC fluorescent staining with anti-PMCA2 antibody. Figure 24A and Figure 24B Depicted are H&E staining of two consecutive sections of adjacent colon cancer tissue samples ( Figure 24A ) and IHC fluorescence staining using anti-PMCA2 antibody ( Figure 24B ). Plate 1 is a microscopic image of an entire colon cancer core. The boxed area in Plate 1 is magnified and shown in Plate 2. The boxed area in Plate 2 is magnified and shown in Plate 3. Figure 24C and Figure 24D Depicted are H&E staining of two consecutive sections of adjacent lung cancer tissue samples ( Figure 24C ) and IHC fluorescence staining using anti-PMCA2 antibody ( Figure 24D ). CCT in colon and lung cancer tissues is not visible using H&E techniques ( Figure 24A and Figure 24C ), but it is clearly shown in the image of IHC fluorescent staining with anti-PMCA2 antibody ( Figure 24B and Figure 24D ). Cell capsule tube (CCT, white arrow) and CCT filament (CTS, orange arrow). Scale bar 10 μm.

[0043] Figures 25A to 25C Depicted are schematics and microscopic images illustrating the detection of CCTs, CCT formation, and the cellular cyst tumor life cycle. Figure 25A Representative images of three serially sectioned colon cancer tissue samples are depicted, showing H&E staining (Plate 1), IHC staining with an antibody recognizing the colon cancer molecular marker MSH-2 (Plate 2), and IHC staining with an anti-PMCA2 antibody (Plate 3). The numerous CCTs (white arrows) presented in Plate 3 are not detectable in Plates 1 and 2. Figure 25BRepresentative bright field (BF, Plate 1) and fluorescence microscopy (Plate 2) images of the same imaging area are depicted, showing a slender, straight, and stretched CCT (white arrow) connecting the two CCs of two cytocystic tumor spheres (CTs), with open ends at both connection sites. The figure shows a cytocystic tube (CCT, white arrow), the cytocysts of two CTs (CC1 and CC2), and the open ends of the CCTs (cyan arrows). Plate 3 shows a schematic diagram of two cytocysts connected to each other by a CCT with two open ends. Figure 25C Schematic diagram depicting the life cycle of cystic tumors: (1) Generation of cystic cells; (2) Cystic cells proliferate and grow into pre-cystic tumors (PCTs) without CCTs. Intracellular cystic cells may undergo CC generation and generate independent CCs in the PCT cyst cavity. (3) PCT develops into CTs with CCTs. CCTs provide a membrane-enclosed high-speed channel for the metastasis of intracellular cystic cells. (4) CCs and CCTs of cystic tumors degrade to form cell-free cystic cell clusters of the NT. (5) Some cell-free cystic cells undergo CC generation in the stress microenvironment of the NT and generate cystic cells, generate CCTs, and form cell-free cystic cell clusters-CT / CCT complexes (AMCCs). Cell-free cystic cells surrounding the CCT invade the CCT by allogeneic entry and undergo cancer metastasis. Scale bar 10 μm.

[0044] 26A to 26I Schematic diagram and microscopy images depicting the life cycle of cellular cystic tumors in human tissue and in vitro 3D matrix cultures. Figure 26A Representative bright field (BF) and fluorescence microscopy images of Bxpc3 pancreatic cancer cell spheroids in a 3D CC / CCT matrix culture kit are depicted. The cell spheroids (white arrows) and cell capsules (orange arrows) are shown. Figure 26B Depicted are representative IHC fluorescence microscopy images of procystic tumors (PCTs, diameter / width <20 μm), the initiation stage in early breast cancer. Figure 26C and Figure 26C Depicts early developmental stages ( Figure 26C , diameter / width<30μm) and mid-stage development ( Figure 26D Representative IHC fluorescence microscopy images of PCT (diameter / width <40 μm) are shown. Cell vesicles (CC, white arrows) and decellularized CC (ECC, purple arrows) are shown. Figure 26E Depicted are representative IHC fluorescence microscopy images of early-stage cellular cystic tumors (CTs), in which cellular cystic ducts (CCTs, orange arrows) extend from the CTs. Figure 26FRepresentative IHC fluorescence microscopy images of breast cancer tissue with high PCT and CT density are depicted. Multiple CTs are merged into a larger CT. The figure shows a CT with three CT branches (CTBs), a cellular cyst (CC, white arrows), and a cellular cystic duct (CCT, orange arrows). Figure 26G Representative IHC fluorescence microscopy images of developing cystic tumors (CTs) are depicted, with a thick CCT layer enveloping the exterior of the CT. The images show the CT's cysts (CCs, white arrows), cystic membranes (CCMs, red arrows), cystic tubes (CCTs, purple arrows), intracellular cystic carcinomatous cells (cyan arrows), and CCT filaments (CTSs, orange arrows). Figure 26H Schematic representation of a cellular cystic tumor with its CCT, nanoprotrusions, and intracellular cystic carcinomatous cells with or without isolated CCs is depicted. Figure 26I Representative images of a cell-free cystic carcinomatous cluster-CC / CCT complex (AMCC) are depicted. Nuperphase tumor (NT), CCT (orange arrows), and degraded CCT filaments (CTS, pink arrows) are shown. Scale bar: 10 μm.

[0045] Figures 27A to 27M The distribution of cellular cystic duct tumors in 35 human normal, benign, and cancerous tissues was described. Figure 27A This is a statistical table showing the presence of CCT in 35 types of human tissues and organs tested. Figures 27B to 27M Representative IHC fluorescence microscopy images of CCT (white arrows) in 35 human tissues and organs are depicted. Degraded CCT filaments (CTS, orange arrows) are shown. Scale bar 10 μm.

[0046] FIG. 28A to FIG. 28B Depicted are schematics and microscope images showing that cell cysts drive cell vesicle growth. Figure 28ADepicted is the real-time analysis of cyst-driven cell vesicle growth using bright-field phase-contrast microscopy. Images are taken from recorded videos. Cyst activity process: (1) Cysts (CSs) are produced and released by cancerous cells and attached to the outer cytoplasmic membrane (cyan arrows); (2) CSs spontaneously detach from the surface of cancerous cells (orange arrows in panels 5 and 6); (3) The detached CSs move randomly in the cystic fluid (yellow arrows in panels 4, 8, 9, 10, and 11); (4) CSs reach the inner side of the CT's cystic membrane and contact and integrate into the CT's cystic (CC) membrane, increasing the area of ​​the CC membrane (red arrows in panels 8 and 9). In cystic tumors with a surface of cancerous cell clusters tightly packed with cysts, contact and integration of cysts with the CC membrane will be faster and more efficient, without the need for random and long-distance movement in the cystic fluid. The figure shows intracellular cystic tumorspheres (ICTs), cystic vesicles (CCs, white arrows), cystic membranes (CCMs, green arrows), cystic spikes (purple arrows), and cystic bodies (CSs; yellow arrows, CSs move randomly in the cystic fluid; orange arrows, CSs detach from the surface of cancerous cells; and red arrows, CSs reach and fuse into the CC membrane; cyan arrows: CSs attach to the surface of cancerous cells). Figure 28B Schematic diagram of a cytospheroid tumorsphere, depicting numerous cytospheric bodies and spike-like structures in its lumen, with cytospheric bodies detaching from the cancerous cell surface, migrating, and fusing into an enlarged CC supporting CC growth. Scale bar, 10 μm.

[0047] Figures 29A to 29C Depicted are schematic diagrams, graphs, and microscope images showing a large number of cell vesicles that promote cell vesicle growth. Figure 29A Depicts real-time analysis of cystic growth driven by cystic bodies using brightfield phase contrast microscopy. Images are taken from recorded videos. Shown are intracellular cystic tumor spheres (ICTs), cystic bodies (CCs, black arrows), cystic membranes (CCMs, orange arrows), cystic bodies (CSs, red and green arrows), decellularized cysts (ECCs), and cystic tubes (CCTs, within the lumen of the enlarged CC). Figure 29B Depicted are graphs showing the quantification of cellular cysts in cellular cyst tumor spheres. Figure 29C Schematic depiction of a cytocystic tumor sphere that generates a large CC with numerous cytocysts in the lumen. Intracellular cytocystic carcinomatous cell clusters generate secondary large "L"-shaped CCTs in the lumen of the large CC. Two intervening acellular CCs are located within the lumen of the large CC (ECC). Scale bar 10 μm.

[0048] 30A to 30D Depicted are schematics, graphs, tables and microscope images showing the characterization of cell cysts and their life cycle. Figure 30ARepresentative images of cystic bodies (CS, white arrows) in CC-initiating gastric cancer cells during the generation of cystic carcinoma cells in vivo are depicted. Numerous CS are present in the cytoplasm. Figure 30B Depicted are graphs showing the quantification of cellular cysts in gastric cancer cells and breast cancer cells having cellular cysts in vivo. Figure 30C Depicted is a table showing comparative differences and similarities between cytosomes and four other extracellular vesicles. Figure 30D Schematic diagram depicting the cyst life cycle: (1) CS is produced in the cytoplasm of CC-initiating cancerous cells, (2) CS is released to the outside of the cytoplasmic membrane and attaches to the cell membrane surface, (3) Multiple CS on the cell membrane contact and integrate into the cyst fragment, (4) The cyst fragment grows as more CS are integrated, (5) The cyst envelops the entire single cell and produces cystic cancerous cells, separating them from the ECM, (6) CS reaches and integrates into the cyst and increases the cyst area, (7) The cyst grows and produces an enlarged cyst or elongates and develops into a cyst tube. Scale bar 10 μm.

[0049] Figures 31A to 31I Depicted are schematic diagrams, graphs, and microscopic images showing cell tube formation and extension and cell migration of CCT in vitro and in vivo. Figure 31A Depicts real-time analysis of cyst tube initiation, generation, and extension using MCF-7 cells cultured in a 3D matrix CC / CCT in vitro culture kit. Images are taken from recorded videos. The image shows cyst tubes (CCTs, red arrows), cells within CCTs (white arrows), vesicles (cyan arrows), and cystic spikes (CSPs, yellow arrows). Figure 31B Depicted are graphs showing the quantification of cell tube extension velocity in CC / CCT culture kit matrix in vitro. Figure 31C Depicted are representative images of initiating cell ducts (ICs, white arrows) in breast cancer tissue in vivo. Figure 31D Schematic diagram depicting the extension of the cell cyst tube: (1) Intracellular cystic cells produce and release cell cysts, and drive the increase of cell cyst membrane area, (2) Cell cystic cells produce many vesicles in all directions, sense the microenvironment, and select and decide the direction of movement, (3) Cell cysts continuously drive the cell vesicle (CC) membrane to increase in the area with many vesicles in the CC lumen, and (4) Cell cystic cells move forward, extend the length of the CCT, and produce a long CCT. Figure 31E Representative images of cell migration in CCT in vitro are depicted. Figure 31FDepicts real-time analysis of intracellular cystic carcinoma cell migration within a CCT. Images were taken from a recorded video. The image shows the cystic tube (CCT, red arrow), migrating intracellular cystic carcinoma cells (white arrow), the direction of migration (pink arrow), the reverse direction of migration (yellow arrow), cells with lamellipodia at the leading edge (red asterisk), and cells transitioning in the CCT's migratory direction (blue asterisk). Figure 31G Depicted are graphs showing quantification of Bxpc3 pancreatic cancer cell migration in CCT in vitro. Figure 31H Representative images of migrating colon cancer cells in colon CCT are depicted. Colon cancer intracellular cystic carcinoma cells in CCT have a thin, elongated, and spindle-shaped morphology. The figure shows a cell cyst tube (CCT, red arrow) and colon cancer cells migrating in CCT (white arrow). Figure 31I Schematic diagram of a cellular cystic tube tumor is depicted. Scale bar 10 μm.

[0050] Figures 32A to 32C Depicted are microscopic images showing in vitro integrated cytocystic oncogenic cells, cytocystic tumorspheres, CCT networks, and cytocystic tumorsphere network systems. Figure 32A Representative images of a cytocystic tumor sphere network system (CTNS) integrating primary and secondary cytocystic tumor spheres in vitro are depicted. First, primary cytocystic tumor spheres (black asterisks) are interconnected via the CCT network. Disseminated intracellular cytocystic carcinoma cells aggregate at CCT network nodes and grow into secondary cytocystic tumor spheres (red asterisks). Secondary cytocystic tumor spheres are interconnected with primary cytocystic tumor spheres via CCTs, generating a combined primary and secondary cytocystic tumor sphere network system in a CC / CCT culture kit (6-well plates). Figure 32B Representative images depicting cytocystic tumorspheres of CCTs with open ends on both sides of the CCTs connecting to each other and intracellular cystic tumorigenic cell migration in CCTs and CTNSs. Figure 32C Representative images of membrane-enclosed cystic tumor cells, cystic tumor spheres, CCT networks, and integrated CTNS are depicted. After decellularization, cell-free CC and CCTs show interconnected CC and CCT membrane systems that integrate and interconnect primary (white asterisks) and secondary (red asterisks) CT network systems (CTNS). The image shows cystic tubes (CCTs, white arrows), cystic vesicles (CCs, yellow arrows), open ends on both sides of CCTs (cyan arrows), and intracellular cystic tumor cell migration within CCTs (red arrows). Scale bar 10 μm.

[0051] Figures 33A to 33L Depicted are microscopic images illustrating the life cycle of a primary cellular cystic tumor network system in vivo. Figure 33ARepresentative images of a dense precellular cystic tumor (PCT, white arrows) cluster in a primary invasive ductal breast cancer are depicted. Degradation of cell-free cellular cysts (CCs) results in the formation of cloud-like CC filaments (orange arrows). Boxed areas 1 and 2 are magnified and shown in Figure 3. Figure 34A and Figure 34B middle. Figure 33B Representative images of a primary invasive ductal breast cancer with dense cellular cystic tumor (CT) and CT network system (CTNS) are depicted. The white dashed box area is magnified and shown in Figure 33C middle. Figure 33C Depicts Figure 33B The magnified box area of ​​CT is shown. CT, cell vesicles (CC, purple arrows), cell capsule membrane (CCM, orange arrows), CCT (orange arrows) and degraded CCT filaments (CTS, pink arrows) are shown. Figure 33D Representative images depicting CT encapsulated by an outer thick CCT layer. Shown are the CCT outside the CC (white arrow), the CCT inside the CC (red arrow), the CC (purple arrow), and the CC membrane (CCM, orange arrow). Figure 33E Representative images depicting the CCT (white arrows) network of primary CTNS in primary colon cancer. Figure 33F Representative images of early and late-stage tumors are depicted. CT cellular vesicles are degraded, while CC fragments (orange arrows) are retained. CCT is degraded into CCT filaments (CTS, white arrows). Figure 33G Representative images of advanced-stage tumors are depicted. Dense acellular carcinomatous cells without CCTs are present (AO, cyan arrows). Figure 33H Representative images of acellular carcinomatous clusters (AMCCs) in primary breast cancer are depicted. Many new CCTs (white arrows) are regenerated from some acellular carcinomatous cells. Figure 33I Representative images of CCT degradation in AMCC are depicted. Cell-free cystic carcinomatous cells (AO, cyan arrows) and CCT (white arrows) are shown. Figure 33J Representative images showing numerous cell-free cystic carcinomatous cells invading CCTs (white arrows) via xenograft entry, metastasizing, and escaping are depicted. Localized cancerous cell density is very low. Many CCTs degrade into CCT filaments (CTS, orange arrows). Figure 33K Depicted are representative images of severe CCT degradation showing numerous CCT fragments and filaments (CTS, orange arrows). Figure 33L Depicted are representative images showing CCT degradation and apoptosis of cancerous cells in the cystic tumor cavity. The figure shows cellular vesicles (CC, purple arrows), CCT (yellow arrows), cystic tube filaments (CTS, orange arrows), and apoptotic cancerous cells in CC (cyan arrows). Figures 33J to 33LFigure 5 shows apoptosis of normal tissue, cell-free cystic carcinoma metastasis by CCT or apoptosis, and cavities caused by CCT degradation (black areas, tissue liquefaction, orange asterisks). Scale bar: 10 μm.

[0052] Figures 34A to 34F Depicted are microscopic images and graphs showing pre-CT, CT, and CCT networks in vivo. Figure 34A Depicts Figure 33A Magnified image area 1 shows an early procystic tumor (EPCT, size <50 μm and number of intracellular cystic carcinomatous cells <20; white arrows). Figure 34B Depicted are representative images showing dense PCT and decellularized cell vesicles (ECC, purple arrows) in the enlarged CC lumen. Figure 34C Depicted are graphs showing quantification of PCT density in breast, colon, and prostate cancers. Figure 34D Depicted are graphs showing quantification of CT density in breast, colon, and prostate cancers. Figure 34E Depicted are representative images showing CCT networks composed of straight CCTs in primary breast cancer. Figure 34F Depicted are representative images showing a CCT network composed of curved and coiled CCTs in a primary breast cancer. Scale bar 10 μm.

[0053] Figures 35A to 35D Depicted are microscopic images and tables showing the widespread presence of CCT in normal tissue (NAT) adjacent to tumors. Figure 35A Depicted are representative images of NAT in primary breast cancer showing numerous CCT networks with high density. Figure 35B Depicted are representative images showing NAT in primary plasma cell myeloma bone marrow, where bundles of CCT (white arrows) colocalize with immune cells. Figure 35C Depicted are representative images showing trabecular bone NAT in primary plasma cell myeloma. CCT (white arrows) invade the hard tissue of trabecular bone. There is no PMCA2 signal in the bone matrix. Figure 35D Depicted is a table showing the quantification of CCT density in 14 human cancer tissues NAT. Scale bar 10 μm.

[0054] Figures 36A to 36J Depicted are microscopic images showing cystic carcinomatous metastasis of cells in primary CTNS in the primary cancer niche. Figure 36A Representative images of CCT in loose soft tissue in lung cancer are depicted. Figure 36B and Figure 36C Describes thyroid cancer ( Figure 36B ) and oral cancer ( Figure 36C ) Representative images of CCT in dense soft tissue. Figure 36DRepresentative images of CCT ultrastructure in pancreatic cancer are depicted. Figure 36E Representative images depicting a large-scale curved CCT network invading a dense acellular cystic carcinomatous mass to form AMCC. Figure 36F Depicted are representative images of dense primary prostate cancer tissue showing dense CCT network masses invading AMCC. Figure 36G Representative images of CCT tracts in primary breast cancer CTNS are depicted. Figure 36H Representative images of CCT clusters in primary breast cancer CTNS are depicted, with most acellular cystic carcinoma cells exiting through the CCTs. (I) Representative images of highly tortuous and coiled CCTs in primary pancreatic cancer CTNS, with many acellular cystic carcinoma cells exiting through the CCTs. Figure 36J Representative images of complex CCT ultrastructure in primary CTNS are depicted. CCT (white arrows) and CCT filaments (CTS, orange arrows) are shown. Figures 36A to 36J Middle. Scale bar 10 μm.

[0055] Figures 37A to 37F Depicted are schematics, tables, and microscopy images showing that cellular cystic networks direct cancer metastasis in vivo. Figure 37A Representative images of CCTs and blood vessels in primary prostate cancer tissue are depicted. The figure shows CCTs (white arrows) and migrating intracellular cystic carcinoma cells (purple arrows) within CCTs. Figure 37B Depicted is a table showing a quantitative comparison of cellular vasculature and humoral vessels in the primary cancer niche, tumor-adjacent normal tissue (NAT), and secondary cancer niches. Figure 37C and Figure 37D Representative images depicting microvessels encapsulated by CCTs, CCT invasion of blood vessels, and the release of intracellular encapsulated oncocytic cells into the blood as a source of circulating tumor cells (CTCs). Figure 37C The boxed area in the Figure 37D middle. Figure 37D Depicts Figure 37C The image shows intracellular cystic carcinomatous cells (ICO, yellow arrows), CCT (white arrows), CCT filaments (CTS, orange arrows), and red blood cells (RBS, purple arrows) entering the blood. Figure 37E Representative images of advanced cancer stages are depicted, in which many red blood cells are distributed in the tissue due to CCT invading damaged blood vessels. CCT (white arrows), CCT filaments (CTS, orange arrows) and red blood cells (RBS, purple arrows) are shown. Figure 37F Schematic diagram depicting CTC invasion, CTC origin, microvascular damage, and erythrocyte distribution in extravascular tissue. Scale bar 10 μm.

[0056] Figures 38A to 38B Depicted are microscopic images showing that a massive network of cellular cysts, outside of bodily fluid vessels, dominates the pathways of cancer metastasis. Figure 38A Depicted is a representative image of a clinical stage IIb prostate cancer, in which a large number of CCTs and CCT networks occupy most of the space outside the body fluid vessels (blood vessels, lymphatic vessels) and the prostate cavity, and dominate the cancer cell metastasis pathway. Most prostate cancer cells have already left through the CCT network. The white dashed box area is Figure 38B The image is enlarged in Figure 1. Microvessels (MBV, orange arrows) are shown. Figure 38B Depicts Figure 38A Magnified image of the middle framed area. Numerous CCTs (white arrows) and CCT networks are present outside the integrated microvasculature (MBV). Most prostate cancer cells have already exited through the CCT network. At this stage of cancer, the MBV is intact and undamaged. Degraded CCT filaments (CTS, red arrows) are shown.

[0057] Figures 39A to 39L Depicted are microscopic images illustrating the life cycle of a secondary cellular cystic tumor network system in vivo. Figure 39A Representative images depict thin (1) and thick (2) fasciculations of metastatic breast cancer CCT (white arrows) encapsulating and invading lymph nodes. Figure 39B Representative images depicting 3D CCT (white arrows) networks of metastatic cervical squamous cell carcinoma invading and distributing to most lymph node regions. White dashed box regions 1, 2, and 3 are shown in Figure 2. Figure 40A 、 Figure 39C and Figure 40B is magnified. Figure 39C Depicts Figure 39B The enlarged white dashed box area in Figure 2. Metastatic cervical squamous cell carcinoma (CCT) invades the lymph node, develops into a CCT mass, and occupies space within the lymph node. The figure shows a cross-section of the CCT (orange arrow). Figure 39D Depicts the growth of a metastatic breast CCT that invaded the lymph nodes into a multifocal cystic tumor (CT). Enlarged cellular cysts (CC, purple arrows), CC membranes (CCM, yellow arrows), and CT cavities (white asterisks) are shown. Figure 39E Depicts representative images of secondary breast CTNS with high CT density in lymph nodes. Breast CCT invades lymph nodes, and intracellular cystic carcinomatous cells proliferate and grow into many cystic tumors. A large number of secondary breast cystic tumors in lymph nodes are interconnected by CCT networks and form dense CTNS in lymph nodes. Cellular cystic tumors (CT, yellow arrows), cellular vesicles (CC, white arrows), CCT (orange arrows), CCT filaments (CTS, purple arrows) and cystic cavities (white asterisks) are shown in the figure. Figure 39FDepicted are representative images showing degradation of the CC of secondary CT, degradation of the CCT, and uncontrolled proliferation of acellular cystic ovarian cancerous cells, leading to enlargement of acellular cystic ovarian cancerous cell masses in secondary CTNS in the omentum. Figure 39G Depicts some acellular cystic ovarian cancerous cells regenerating CCT and forming AMCC in secondary CTNS of the omentum. The figure shows CCT (white arrow), acellular cystic cancerous cells (AO, cyan arrow) and migrating cancerous cells in CCT (purple arrow). Figure 39H Representative images of secondary cervical CTNS in lymph nodes are depicted, with numerous cervical CCTs in the lymph nodes. The images show CCTs (white arrows) and migrated cancerous cells within the CCTs (purple arrows). Figure 39I Representative images of the entire tissue core of secondary rectal CTNS in the mesentery with high CCT (white arrow) density are depicted. White box areas 1, 2, and 3 are magnified and shown in Figure 40E 、 Figure 39J and Figure 40F middle. Figure 39J Depicts Figure 39I Magnified area of ​​​​the ​​tumor to show the high density of CCTs. The figure shows CCTs (white arrows) and cancerous cells migrating within CCTs (purple arrows). Figure 39K Depicted are representative images of secondary colon CTNS showing severe CCT degradation in the liver. The figure shows CCT (white arrows) and migrating cancerous cells in CCT (purple arrows). Figure 39L Depicted are representative images showing secondary hepatocellular carcinoma CTNS with severe CCT degradation, severe vascular damage and severe red blood cell leakage, metastasis of the main acellular cystic carcinoma cells through CCT (white arrows) and low cell density in the local brain. Red blood cells (RBC, red arrows) and CCT filaments (CTS, orange arrows) randomly distributed outside the blood vessels and in the tissue are shown. Figures 39K to 39L Figure 5 shows apoptosis of normal tissue cells, cell-free cystic carcinoma cell metastasis by CCT or apoptosis, and cavities caused by CCT degradation (black space, tissue liquefaction, orange asterisks). Scale bar: 10 μm.

[0058] Figures 40A to 40F Depicted are microscopic images and graphs showing metastatic CCT, secondary CT density, and secondary AMCC in vivo. Figure 40A Depicts Figure 39B Magnified image of the middle white dashed box area 1. 3D metastatic cervical squamous cell carcinoma CCT (white arrow) invades the lymph node and forms highly tortuous / coiled CCT bundles and CCT clusters, which coexist with immune cells in the lymph node. Figure 40B Depicts Figure 39B Magnified image of the middle white dashed box area 3. 3D metastatic cervical squamous cell carcinoma (CCT) (white arrows) randomly invades the lymph nodes, coexists with immune cells in the lymph nodes, and occupies space in the lymph nodes. Figure 40C Depicted are diagrams showing quantitative analysis of secondary breast CT in the bladder, liver, and lymph nodes. Figure 40D Depicted are representative images showing degradation of the CC of secondary CT, degradation of CCT, and uncontrolled proliferation of acellular cystic ovarian cancerous cells, leading to the enlargement of acellular cystic ovarian cancerous cell clusters in secondary CTNS in the omentum. A cluster of acellular cystic cancerous cells (AO, cyan arrows) is shown in the figure. Figure 40E Depicts Figure 39I Magnified image of the white dashed box area 1. Very dense and numerous CCT network bundles are present in the secondary CTNs of mesenteric midrectal cancer. Figure 40F Depicts Figure 39I Magnified image of the white dashed box area 3. Very dense CCT network clusters are present in the secondary CTNs of rectal cancer in the mesentery.

[0059] Figure 41A schematic diagram of the evolutionary life cycle of cystic cells in vivo is depicted. The simplified diagram of the evolutionary life cycle of cystic cells in vivo includes 10 steps: (1) Normal cells are transformed into abnormal and cell-free cystic cells, which are caused by accumulated genetic mutations and chemical and physical stimuli from the extracellular microenvironment. (2) Through unknown molecular mechanisms, some cell-free cystic cells undergo CC generation: cell cysts and cell vesicles are generated to seal cells and isolate cells from the stress microenvironment. The additional extracellular protective cell vesicles of cystic cells enhance survival in the stress microenvironment. (3) Intracellular cystic cells proliferate in the cell cyst cavity and give rise to pre-cystic tumors (PCTs) and cystic tumors (CTs). (4) CTs in the primary niche give rise to CCTs, CCT networks, and cell-free cystic cell clusters-CC / CCT complexes (AMCCs). (5) All CTs in the primary niche are interconnected through the CCT network and form the primary cystic tumor network system (CTNS). (6) The primary CCT network expands and invades adjacent and distant tissues and organs. (7) In the secondary niche, CCT branch morphogenesis, new CCT network formation, and new CT formation and growth occur. (8) All CTs in the secondary niche are interconnected with the CCT network and form secondary CTNS. AMCCs are formed in the secondary niche. (9) The primary and secondary CTNS are interconnected with the CCT metastatic CCT network and form integrated primary and secondary CTNS. (10) A series of activities and responses of CT, CCT, CCT network, and CTNS under various conditions form a dynamic integrated CTNS. The expansion and invasion of CCT, CT, CTNS, and AMCC in normal tissues and organs lead to normal cell apoptosis, normal tissue biological function failure, and structural destruction. Normal cell apoptosis, apoptosis of acellular cystic carcinoma cells or escape through the CCT network, and CCT degradation lead to the local formation of cavities filled with intercellular fluid but lacking tissue (called tissue liquefaction), and trigger the biological function failure of tissues / organs. DETAILED DESCRIPTION

[0060] Embodiments of the present disclosure are based on the surprising discovery that endogenous plasma membrane Ca 2+-ATPase (PMCA) is highly expressed in the membrane of cell cysts (CCTs) in cancer tissues. Early studies reported that single human cells can produce new types of membranous organelles - cell vesicles and cell cysts in an optimal 3D environment (Tingfang Yi and Gerhard Wagner, Cytocapsular Tubes Conduct Cell Translocation. Proc Natl Acad Sci USA. 2018 Feb 6; 115(6): E1137-E1146). Reports show that in xenograft mouse models, cell cysts undergo cell migration and translocation in tissues in vitro and in vivo. However, due to the lack of markers for these new membranous organelles, it is difficult to detect cell vesicles and cell cysts. The inventors of the present disclosure were surprised to find that endogenous plasma membrane Ca2+-ATPase (PMCA) is highly expressed in the membrane of cell vesicles and cell cysts (i.e., cell capsule) in cancerous tissues in vivo. However, PMCA expression or CCT was not detected in healthy or benign tumor tissues. The study also found that the density and morphology of cell cysts detected in cancer tissues can be used to predict the extent of cancer progression and the grade of cancer metastasis, providing new standards for clinical cancer analysis (including diagnosis, prognosis and treatment evaluation).

[0061] Currently, cancer treatment mainly includes surgery, radiotherapy, chemotherapy, immunotherapy and hormone therapy. Early detection and accurate diagnosis are the key to successful treatment. Although tumor markers have been identified for some types of cancer, in many other cancer types, no tumor markers useful for cancer diagnosis have been found. Therefore, not all cancers can be easily detected or diagnosed with specific markers. In addition, since most of the currently known tumor markers are present in very small amounts in cancer cells or tissues, detection requires highly sensitive measurement methods and special techniques. The embodiments of the present disclosure relate to novel and simple methods that allow all cancers to be detected with high sensitivity and accuracy. According to some embodiments, tissue can be obtained from anywhere in the subject. According to some embodiments, the tissue can be healthy, benign or cancerous. According to other embodiments, cancerous tissue includes the primary site / niche where the tumor cells originate or the secondary site / niche where the tumor cells spread to.

[0062] It is expected that with the availability of the new method described herein, the detection and diagnosis of all cancers will be opened. In the case where the tumor is not large enough to be confirmed by the naked eye (found by ultrasound, computed tomography or MRI (magnetic resonance imaging)), these "invisible" tumors that are difficult to find by conventional means can be detected by the method described herein. Since the invisible part of cancer can be diagnosed by this method, this method can be used for early detection of cancer metastasis when the tumor is small but highly malignant.

[0063] A hallmark of cancer is the ability of cancer cells to metastasize. Embodiments of the present disclosure relate to detecting cancer tissue at a primary site. Embodiments of the present disclosure also relate to detecting secondary sites of metastatic cancer tissue. Therefore, the present methods can be used to detect and diagnose cancer arising in non-visible areas, diagnose cancer progression, diagnose cancer malignancy, diagnose progression after surgery and treatment, diagnose recurrence, diagnose metastasis, and the like.

[0064] Cancer diagnosis involves determining the extent of cancer progression. There are different ways to classify the extent based on the extent of spread from the primary site of the tumor and whether there is metastasis to regional lymph nodes or distant organs. For example, there are clinical and pathological stages of cancer progression, and the extent of cancer progression also depends on grade, location, organ, tumor markers, and genetics. In contrast to, for example, blood cancers, most cancers involving tumors (solid tumor cancers) are classified into five broad clinical stages. Other types (such as blood cancers, lymphomas, and brain cancers) have their own staging systems. For clinical cancer stages, stage 0 means there is no cancer, only abnormal cells that have the potential to become cancer. This is also called carcinoma in situ. Stage I means that the cancer is small and only in one area. This is also called early-stage cancer. Stages II and III mean that the cancer is larger and has grown into nearby tissues or lymph nodes. Stage IV means that the cancer has spread to other parts of the subject's body. Stage IV is also called advanced or metastatic cancer. These broad groupings are based on a more detailed system that contains specific information about the tumor and its impact on other parts of the body. Another common cancer staging system is the TNM system, which stands for tumor, node, and metastasis. Embodiments of the present disclosure relate to a method that provides a unified approach for detecting, predicting, and determining the stage of cancer metastasis based on the detection of cystic ducts, which are ubiquitous in all cancerous tissues. In one embodiment, the method comprises detecting PMCA expression in cystic ducts in a tissue of a subject. In some embodiments, the methods described herein detect PMCA-1, PMCA-2, PMCA-3, and PMCA-4 expression in a tissue and / or cystic ducts within the tissue.

[0065] Predicting or determining the stage of cancer metastasis is important for selecting cancer treatments. For example, early-stage cancers may require surgery, while late-stage cancers may require chemotherapy. The outlook for recovery also depends in part on how early the cancer is detected. When the extent of cancer progression is known, in addition to determining the appropriate treatment plan, the effectiveness of anticancer drugs can also be determined. Specific examples of treatment decisions can vary depending on the type of cancer. For example, prostate cancer is often less aggressive and may be treated differently than bone metastasis, which often results in death and pain for the patient. Treatments such as hormone therapy and surgical resection have side effects, making it necessary to appropriately determine the treatment approach for each cancer. Furthermore, if the appropriate choice of anticancer agent and when to discontinue administration can be appropriately determined, the physical and financial burden on patients can be reduced. Embodiments of the present method provide a unified way to determine the stage of cancer metastasis across all solid tumor cancer types, thereby introducing a new standard for cancer detection, diagnosis, treatment, and prognosis. Furthermore, according to embodiments of the present disclosure, the extent of cancer progression, such as tumor growth, infiltration into surrounding tissues, and metastasis to lymph nodes and distant organs, can be diagnosed.

[0066] Embodiments of the present disclosure relate to detecting cellular ducts in so-called "normal adjacent tissue" (NAT) surrounding tumor tissue. During surgical resection of a tumor, even if the tumor is small, if the malignancy is high, it is often necessary to leave a wider resection margin when resecting the tumor, or to carefully observe a large area of ​​surrounding tissue. When postoperative diagnosis, including recurrence and metastasis, is possible, the present method can be used to detect the presence of cellular ducts in tissue surrounding the tumor to diagnose whether the tumor was completely removed by surgery and the risk of residual NAT recurrence and metastasis. If resection is incomplete, recurrence is possible, and the current method of detecting CT in surrounding tissue can be used as a basis for follow-up at shorter intervals and, in some cases, as a basis for early reoperation during cancer treatment. In addition, if the tumor recurs, CT is likely to be detected early using the methods described herein. If distant metastasis has occurred, the present method can serve as a basis for expanding the scope of examination beyond the resection site and its surroundings.

[0067] Embodiments of the present disclosure relate to methods that allow for early-stage detection of cancer metastasis, as the method detects cellular cysts in tissue, independent of tumor size. Therefore, even if a tumor is invisible, it can be detected if it is metastatic. This increases the effectiveness and accuracy of cancer diagnosis. Furthermore, the present method significantly contributes to regular health checkups, cancer screening, preoperative diagnosis, and treatment decision-making.

[0068] Aspects of the present disclosure are based on the hitherto unknown observation that endogenous PMCA is highly expressed in the membranes of cystic tubes in cancerous tissue. As observed, cystic tubes are present in cancerous tissue but not in normal or benign tissue. In the case of benign tumors, surgery can be performed even if the tumor is large. Postoperative care of the resected site is sufficient, eliminating the need for expensive anticancer drug treatment or follow-up. Because PMCA expression and cystic tubes are not detected in benign tumors using this method, this method provides a convenient and simple cancer detection method that can be used for rapid screening and treatment selection decisions in humans and animals.

[0069] According to certain aspects, the detection of cellular cysts in cancer tissue is in vitro, in vivo, or ex vivo.

[0070] In some embodiments, the expression of PMCA is measured in the tissue sample separated, derived or obtained from the experimenter.The method for obtaining the tissue for cancer detection and diagnosis is well known to those skilled in the art.According to one embodiment, this tissue or tissue sample is obtained from the experimenter by biopsy.The method for preparing the biopsy tissue sample (comprising tissue collection, fixation and sectioning) is well known in the art.In some embodiments, biopsy comprises clinical biopsy, bone marrow puncture and biopsy, heart biopsy, core needle biopsy, endometrial biopsy, endoscopic biopsy, excisional biopsy and incisional biopsy, fine needle aspiration biopsy, lymph node biopsy, needle biopsy, open biopsy, punch biopsy, sentinel lymph node biopsy, cutting biopsy, skin biopsy etc.

[0071] According to certain embodiments, the present method can be used to detect and diagnose all kinds of cancer tissues of primary origin and secondary metastasis. Tissue from any part of the subject's body can be obtained for detection and screening by the present method. For example, primary cancers and secondary cancers that can be detected by the present method include but are not limited to the following and their related types and subtypes: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adolescent cancer, adrenocortical carcinoma, AIDS-related cancers, Kaposi's sarcoma (soft tissue sarcoma), AIDS-related lymphoma (lymphoma), primary CNS lymphoma (lymphoma), anal canal cancer, appendix cancer, astrocytoma, children (brain cancer), atypical teratoid / rhabdoid tumors, children's central nervous system (brain cancer), skin basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer (including Ewing's sarcoma and osteosarcoma and malignant fibrous histiocytoma), brain Tumors, breast cancer, bronchogenic carcinoma (lung cancer), Burkitt's lymphoma, carcinoid tumor (gastrointestinal tract), cancer of unknown primary site, childhood cardiac (heart) tumor, central nervous system tumor, medulloblastoma and other CNS embryonal tumors, childhood (brain cancer), germ cell tumor, childhood (brain cancer), primary CNS lymphoma, cervical cancer, bile duct cancer, chordoma, childhood (bone cancer), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, childhood (brain cancer), cutaneous T-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumor, endometrial cancer (uterine cancer), ependymoma, childhood (brain cancer) ), esophageal cancer, esthesia neuroblastoma (head and neck cancer), extracranial germ cell tumor, childhood, extragonadal germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, malignant fibrous histiocytoma and osteosarcoma of bone, gallbladder cancer, stomach (gastric) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST) (soft tissue sarcoma), gastrointestinal lymphoma, gastrointestinal lymphoma, germ cell tumor, childhood extracranial germ cell tumor, ovarian germ cell tumor, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, childhood heart tumor, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer (head and neck cancer), intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, kidney (kidney) Cell) cancer, Langerhans cell histiocytosis, laryngeal cancer (head and neck cancer), leukemia, liver cancer, lung cancer (non-small cell lung cancer, small cell lung cancer, thoracic pulmonary blastoma and tracheobronchial tumors), lung adenocarcinoma or uterine adenocarcinoma, lymphoma, male breast cancer, melanoma, intraocular melanoma, Merkel cell carcinoma (skin cancer), malignant mesothelioma, malignant mixed tumors, metastatic cancer, cryptic primary metastatic squamous cell neck cancer, median duct cancer with NUT gene mutation, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides (lymphoma), myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, chronic myeloproliferative neoplasms, gingival tumors,Perianal adenocarcinoma, anal sac cyst, anal sac apocrine gland carcinoma, Sertoli cell tumor, vaginal vestibule cancer, sebaceous gland carcinoma, sebaceous epithelioma, sebaceous adenoma, sweat gland carcinoma, intranasal gland carcinoma, nasal gland carcinoma, colon cancer, bronchial adenocarcinoma, adenocarcinoma, ductal carcinoma, breast cancer, combined breast cancer, malignant mixed breast tumor, intraductal papillary adenocarcinoma, fibrosarcoma, perivascular osteoma, chondrosarcoma, histiocytic sarcoma, myxoid sarcoma, anaplastic sarcoma, mast cell tumor, skin flat Leiomyoma, intraperitoneal leiomyoma, leiomyoma, chronic lymphocytic leukemia, lymphoma, small to medium cell lymphoma, adrenal medullary tumor, granular cell tumor, cyst tumor, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cancer, lip and oral cavity cancer and oropharyngeal cancer, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumor (islet cell tumor), papillomatosis (laryngeal papilloma in children), paraganglioma, parathyroid cancer, penile cancer, Pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, pregnancy-related breast cancer, primary central nervous system (CNS) lymphoma, primary peritoneal cancer, prostate cancer, rare childhood cancers, rectal cancer, recurrent cancer, retinoblastoma, salivary gland cancer, sarcoma, childhood rhabdomyosarcoma (soft tissue sarcoma), childhood vascular tumor (soft tissue sarcoma), soft tissue sarcoma, uterine sarcoma, Sézary syndrome (lymphoma), skin cancer, small intestine cancer, esophageal squamous cell carcinoma, cutaneous squamous cell carcinoma, cryptogenic metastatic squamous cell neck cancer, cutaneous T-cell lymphoma, pharyngeal cancer, hypopharyngeal cancer, thymoma and thymic cancer, thyroid cancer, tracheobronchial tumor (lung cancer), transitional cell carcinoma of the renal pelvis and ureter (kidney cancer), cancer of unknown primary, urethral cancer, endometrial cancer, uterine sarcoma, vaginal cancer, vascular tumor (soft tissue sarcoma), vulvar cancer, Wilms' tumor and other childhood kidney tumors, and adolescent cancers.

[0072] Plasma membrane Ca 2+ ATPase (PMCA) is a transport protein in the plasma membrane. One of the functions of PMCA is to remove calcium (Ca) from the cell. 2+ ), hence also called plasma membrane Ca 2+ Pump (PMCA). Known Ca 2+ Regulation of transport is crucial in physiological processes including lactation, proliferation, and apoptosis. 2+ The PMCA pump (PMCA) is a family of P-type ATPases that includes four isoforms (PMCA1-4) encoded by different genes, with additional variants generated by alternative splicing (Jason I E Bruce, Metabolic Regulation of the PMCA: Role in Cell Death and Survival. Calcium 2018: 69: 28-36). Plasma membrane Ca 2+PMCA2 isoform, a calcium efflux pump, is the first to be identified as a Ca2+ pump that regulates Ca2+ production during lactation in mice. 2+ A protein crucial for the transport of ions into milk. In the mammary glands of rats and mice from lactating animals, PMCA2 is the predominant isoform (Peters A et al., The Calcium Pump Plasma Membrane Ca( 2+ )-ATPase 2 (PMCA2) Regulates Breast Cancer Cell Proliferation and Sensitivity to Doxorubicin. Sci Rep 2016 May 5; 6: 25505). The different distribution of these isoforms suggests that they play different roles. PMCA isoforms 1 and 4 are ubiquitously expressed, including in brain, skeletal muscle, and breast tissue, while PMCA2 and PMCA3 show a more limited distribution, with PMCA2 expression found in the brain, heart, and breast. Emerging evidence supports the view that changes in PMCA expression or activity are associated with many pathological conditions (Jeong J et al., PMCA2 Regulates HER2 Protein Kinase Localization and Signaling and Promotes HER2-mediated Breast Cancer. Pro Natl Acad Sci USA 2016; 113 (3): E282-90). However, it is unclear whether endogenous PMCA (PMCA1 to 4) is expressed in the membranes of cell ducts in vivo or in vitro.

[0073] According to some embodiments, the present method detects the endogenous expression of PMCA1 to 4 in the membranes (cystic membranes) of cell cysts, cell cysts, cell cystic body vesicles, cell cystic carcinomatous cells, cell cystic tumor spheres, and cell cystic tumor sphere network systems from cancer tissue using anti-PMCA1 to 4 antibodies raised in rabbits against PMCA antigens. These antibodies bind to any one of PMCA1, PMCA2, PMCA3, or PMCA4. According to some embodiments, the antibodies that bind to any one of PMCA1 to 4 are produced in animal species commonly used for polyclonal antibody preparation in the laboratory according to methods known to those skilled in the art, including but not limited to rabbits, rats, mice, guinea pigs, hamsters, goats, chickens, sheep, and llamas.

[0074] The method for measuring or detecting protein expression in tissue is well known to those skilled in the art. Based on immunological methods (such as quantitative enzyme-linked immunosorbent assay (ELISA), Western blotting and dot blot) is very common and sensitive mensuration for protein detection, and they use the antibody that specifically reacts with whole protein or specific epitope (such as fusion tag) after tissue or cell lysis. According to some embodiments, the immunoassay using anti-PMCA antibodies is used to detect and measure the expression of PMCA in the tissue of experimenter. Detection method is not limited to immunoassay. The mRNA of PMCA also can be detected by method known to those skilled in the art. Any method known in the art can be used for detecting and measuring the PMCA expression in the membrane of cell capsule tube in the tissue sample from experimenter. As used herein, term " measurement " comprises detection, qualitative, quantitative and semi-quantitative.

[0075] According to some embodiments, the measurement of the antibody bound to the antigen can be carried out by immunoassay by antigen-antibody reaction well known to those skilled in the art. The immunoassay method itself is a conventional method well known to those skilled in the art. As specifically described in the examples herein, the antibody produced for human PMCA can detect human PMCA. If the homology degree of PMCA between humans and animal species is high, the antibody produced for human PMCA can be used for detecting human and animal PMCA in immunoassay.

[0076] According to one embodiment, immunostaining includes immunohistochemistry (IHC), which is a process for selectively identifying antigens (PMCA) by antibodies that specifically bind to PMCA in tissue samples. IHC is a method well known to those skilled in the art. The visualization of antibody-antigen interactions can be accomplished in a variety of ways, such as chromogenic immunohistochemistry (CIH), in which antibodies are conjugated to enzymes that catalyze color-producing reactions, such as peroxidase (this combination is called immunoperoxidase); or immunofluorescence, in which antibodies are labeled with fluorophores, such as fluorescein or rhodamine. In immunohistochemical detection methods, antibodies are divided into primary and secondary antibodies. A primary antibody is produced against the target antigen and is typically unconjugated (unlabeled), while a secondary antibody is produced against an immunoglobulin of the same type as the primary antibody. The secondary antibody is typically conjugated to a linker molecule, such as biotin, and then a reporter molecule is recruited, or the secondary antibody itself is directly bound to the reporter molecule. According to some embodiments, the secondary antibody for an anti-γ actin primary antibody is conjugated to a red dye, while the secondary antibody for an anti-PMCA primary antibody is conjugated to a green dye. The embodiments of the present disclosure provide methods for direct and indirect immunohistochemical staining using antibodies for any one of PMCA1 to 4. The direct method of immunohistochemical staining uses a labeled antibody that directly binds the PMCA antigen being detected. The indirect method of immunohistochemical staining uses a first antibody (primary antibody) for the antigen PMCA being detected and a second antibody (secondary antibody) for the labeling of the first antibody. Antibodies or antigen-binding fragments of the present disclosure can be coupled with other parts such as detectable labels, i.e., covalently or non-covalently connected. In some embodiments, a primary antibody and / or secondary antibody are coupled with at least one detectable label. Detectable labels include any enzymatic molecules, chromogenic molecules and / or fluorescent molecules for labeling antibodies for detection known to those skilled in the art.

[0077] According to some embodiments, the antibody for specific detection comprises polyclonal or monoclonal antibodies to PMCA or its peptide fragments. The preparation method of polyclonal antibodies is by injecting PMCA or its peptide fragments into animals, and after stimulating the secondary immune response, separating the antibodies from whole serum. Polyclonal antibodies are normally heterogeneous mixtures of antibodies that recognize several epitopes. The preparation method of monoclonal antibodies is that the antigen is injected into animals, then a specific immune tissue sample is taken, the parental cells are separated, and the immortalized cell line obtained is utilized to produce antibodies. Monoclonal antibodies show specificity to a single epitope.

[0078] Aspects of the present disclosure provide methods for detecting cystic ducts in tissue of a subject. In one embodiment, the method comprises obtaining tissue from a subject and contacting the tissue with an anti-plasma membrane Ca 2+-ATPase (PMCA) antibody contact to detect the presence of cell cysts in the tissue. In one embodiment, the subject is a mammal. In one embodiment, the subject is a human or an animal. In another embodiment, the subject is healthy or sick. In another embodiment, the subject has cancer or is suspected of having cancer. In certain embodiments, tissue is obtained from any location in the subject's body. In other embodiments, tissue includes healthy, benign or cancerous tissue. In other embodiments, cancerous tissue includes any tumor tissue comprising the primary site of tumor origin or the secondary site of tumor metastasis. In another embodiment, tissue adjacent to cancerous tissue is obtained. Tissue adjacent to cancerous tissue is also referred to by those skilled in the art as "normal adjacent tissue" (NAT). In one embodiment, the tissue is obtained by biopsy. In certain embodiments, the four isoforms of PMCA include PMCA-1, PMCA-2, PMCA-3 and PMCA-4 and their natural variants. In some embodiments, anti-PMCA antibodies include anti-PMCA-1 antibodies, anti-PMCA-2 antibodies, anti-PMCA-3 antibodies and anti-PMCA-4 antibodies, as well as antibodies against their variants.

[0079] Certain aspects of the present disclosure provide a method for predicting the level of cancer metastasis in a subject. In one embodiment, the method comprises obtaining tissue from the subject and detecting the presence of cystic tubes in the tissue. In one embodiment, the tissue is treated with an anti-plasma membrane Ca 2+ -ATPase (PMCA) antibody contact is used to detect cell capsules in tissues. In certain embodiments, antibodies against four isoforms of PMCA (including PMCA-1, PMCA-2, PMCA-3 and PMCA-4 and their natural variants) are used for detection. In one embodiment, the presence of cell capsules in the tissue is associated with metastatic cancer. In certain embodiments, the density and morphology of the cell capsules detected in the tissue are used to predict the cancer metastasis grade of the subject. In one embodiment, an increase in the density of cell capsules is associated with a more advanced grade of cancer metastasis. In another embodiment, thinner and cloud-like cell capsule morphology and more degradation are associated with a more advanced grade of cancer metastasis. In one embodiment, if cell capsules (CCT) are not detected and CCT degradation is not detected, it is predicted to be grade 0 cancer metastasis (CM). In another embodiment, if the density of CCT is detected at 1 / mm 2 Up to 10 / mm 2 In one embodiment, if the density of CCT is detected at 11 / mm 2 Up to 40 / mm 2In another embodiment, if the density of CCT is detected at 41 / mm 2 Up to 80 / mm 2 In one embodiment, if the density of CCT is >81 / mm 2 If the CCT is within the range of , and the CCT degrades into a cloud-like morphology or completely decomposes, it is predicted to be a grade 4 cancer metastasis (CM). In certain embodiments, the subject is a mammal, a human, or an animal. In some embodiments, the subject is healthy or sick. In other embodiments, the subject has cancer or is suspected of having cancer. In some embodiments, tissue is obtained from any location in the subject's body. In other embodiments, the tissue includes healthy, benign, or cancerous tissue. In some embodiments, cancerous tissue includes any tumor tissue comprising a primary site of tumor origin or a secondary site of tumor metastasis.

[0080] Other aspects of the present disclosure provide methods for screening a subject for cancer. In one embodiment, the method comprises obtaining a tissue from a subject and contacting the tissue with an anti-plasma membrane Ca 2+ -ATPase (PMCA) antibody contact is used to detect the presence of cell capsules in the tissue. In certain embodiments, antibodies against four isoforms of PMCA (including PMCA-1, PMCA-2, PMCA-3 and PMCA-4 and their natural variants) are used for detection. In one embodiment, the presence of cell capsules in the tissue indicates that the tissue is cancerous. In another embodiment, the absence of cell capsules in the tissue indicates that the tissue is healthy or benign. In certain embodiments, the subject is a mammal, a human or an animal. In some embodiments, the subject is healthy or sick. In other embodiments, the subject suffers from cancer or is suspected of having cancer. In some embodiments, the subject has been screened by other methods and has been determined to have or not have cancer. The methods provided herein can assist cancer screening by other methods and provide more accurate cancer diagnosis, detection or additional information that contributes to cancer screening and diagnosis. In some embodiments, tissue is obtained from any position in the subject's body. In other embodiments, tissue includes healthy, benign or cancerous tissue. In some embodiments, cancerous tissue includes any tumor tissue comprising the primary site of tumor origin or the secondary site of tumor metastasis.

[0081] Embodiments of the present disclosure relate to methods for detecting cellular cysts (CCs) and cellular cyst tubes (CCTs) in a tissue sample of a subject or in a 3D matrix culture in vitro. As used herein, the terms "cytocapsule" and "cytocapsulas" are used interchangeably to refer to a plurality of cellular cysts. The terms "subject," "individual," or "patient" are used interchangeably and refer to vertebrates, preferably mammals. For example, in the context of the present disclosure, mammals are humans, non-human primates, domesticated animals (such as dogs, cats, sheep, cattle, goats, pigs, horses, etc.), experimental animals (such as mice, rats, rabbits, guinea pigs, etc.), and captive animals (such as zoo animals). The term "animal" as used herein also includes humans. The term "subject" may also include patients, i.e., animals, preferably humans suffering from a disease, preferably a disease such as cancer as described herein.

[0082] The term "normal," such as used in the term "normal tissue" or "normal cells," refers to healthy tissue or cells in a healthy subject, ie, a non-pathological condition, while "healthy" used in the term "healthy subject" means non-cancerous.

[0083] The term "tumor" as used in the term "tumor cell" or "tumor tissue" refers to a swelling or lesion formed by the abnormal / uncontrolled growth of cells (called neoplastic cells or tumor cells). Tumors can be benign or malignant. Benign tumors refer to tumors that lack all three malignant characteristics of cancer, i.e., benign tumors do not grow in an unlimited, aggressive manner, do not invade surrounding tissues, and do not spread to non-adjacent tissues (metastasis). Malignant tumors refer to tumors that exhibit all three malignant characteristics of cancer. Malignant tumors grow in an unlimited, aggressive manner, invade surrounding tissues, and spread to non-adjacent tissues (metastasis). Cancerous cells exhibit uncontrolled growth (division beyond normal limits), invasion (invasion and destruction of adjacent tissues), and sometimes metastasis (spreading to other parts of the body through lymph or blood). Most cancers form tumors, but some cancers (such as leukemias) do not form tumors.

[0084] The terms "malignant tumor progression," "cancer progression," and "cancer metastasis" are used interchangeably herein and refer to the stage of uncontrolled tumor cell growth, invasion of surrounding tissues, and spread to secondary, non-adjacent tissues. As used herein, "tumor growth" relates to the tendency of a tumor to increase its size and / or the tendency of tumor cells to proliferate.

[0085] The inventors of the present disclosure have discovered that during malignant transformation, invasive cancerous cells produce a second membrane outside the plasma membrane to form cell vesicles (CCs) and cell cyst tubes (CCTs), constituting cell cyst-cancer cells, which have pleiotropic biological functions in cancer patient tissues in vivo and in vitro. These cancerous cells, invasive cancerous cells, cancerous cells, and malignant tumor cells are believed to have undergone malignant transformation and are able to produce organelles lined with a second membrane in vivo, such as cell vesicles (CCs) and cell cyst tubes (CCTs). Cell vesicles (CCs) and cell cyst tubes (CCTs) are lined with a second membrane, i.e., a cell capsule membrane that is different from the plasma membrane. Proteomic analysis revealed that the PMCA2 calcium pump is highly upregulated in the CC and CCT membranes of malignant tumors, but not in normal tissues, thus identifying unique cancer biomarkers and targets for cancer treatment. Cell vesicles (CCs) and cell cyst tubes (CCTs) are organelles lined with a second membrane.

[0086] The inventors of the present disclosure have discovered that cystic carcinoma cells are prevalent in solid cancers and appear in blood cancers of immune organs. As used herein, the term "cystic carcinoma cell" refers to a previously unidentified single cancerous cell that is surrounded / encapsulated in a second extracellular membrane of a cellular vesicle or cyst tube in vivo and in vitro.

[0087] The inventors of the present disclosure have also discovered that multicellular malignant tumors are also wrapped by a protective CC membrane (i.e., cell capsule) both in vivo and in vitro. These multicellular malignant tumors surrounded / wrapped by cell capsules are interchangeably referred to herein as "cell capsule tumors" or "cell capsule tumor spheres." It has been observed that these cell capsule tumors (CTs) produce many CCTs, forming high-speed channels for cancer cells to metastasize to adjacent and distant sites. The entire CT network (CTN) dominates the physical pathway of cancer metastasis in vivo in cancer patients. Subsequently, CCTs invade microvessels and release cell capsule cancer cells into the blood, providing a source of circulating malignant tumor cells. CTNs interconnect cell capsule tumors in primary and secondary cancer niches to produce a larger cell capsule tumor network system (CTNS). Primary and secondary CTNSs are interconnected and form a dynamic and integrated CTNS. Therefore, interconnected cell capsule cancer cells, CTNs, and CTNSs coordinate cancer progression in an integrated cell capsule system.

[0088] Embodiments of the present disclosure provide methods for detecting cell cysts in a tissue sample of a subject. In one embodiment, the method comprises contacting the tissue sample with an antibody, wherein the antibody binds to plasma membrane Ca in the cell capsule of the cell cyst. 2+-ATPase (PMCA) binding, and detecting the presence of cell cysts in the tissue sample by detecting the antibody. In another embodiment, the method includes detecting cell cysts, cell cyst vesicles (cell cyst bodies), cell cystic cancerous cells, cell cyst tumor spheres (cell cyst tumors), and cell cyst tumor sphere network systems in the tissue sample. The presence of cell cyst tubes and / or cell cysts in the tissue sample indicates the presence of a cancer disease or a risk of a cancer disease (i.e., the potential for developing a cancer disease) in the subject.

[0089] Embodiments of the present disclosure provide a method for detecting cell capsules in vitro. In one embodiment, the method comprises contacting cell capsules in a 3D matrix culture with an antibody, wherein the antibody binds to a plasma membrane Ca in the cell capsule membrane of the cell capsule. 2+ -ATPase (PMCA) binding, and the presence of cell capsule tubes in 3D matrix culture is detected by detecting the antibody. In another embodiment, the method includes detecting cell capsules, cell capsule vesicles (cell capsules), cell cystic cancer cells, cell capsule tumor spheres (cell capsule tumors) and cell capsule tumor sphere network systems in 3D matrix culture. The method for producing cell vesicles and cell capsule tubes in in vitro 3D matrix culture has been previously described (Tingfang Yi and Gerhard Wagner, Cytocapsular Tubes Conduct Cell Translocation. Proc Natl Acad Sci US A. 2018 Feb 6; 115 (6): E1137-E1146). Cell capsules, cell capsule tubes, cell capsule vesicles (cell capsules), cell cystic cancer cells, cell capsule tumor spheres (cell capsule tumors) and cell capsule tumor sphere network systems all have cell capsule membranes and are collectively referred to as cell capsule membrane-related structures.

[0090] The present disclosure provides a kit for use in the methods described herein. The kit comprises an antibody or antigen-binding fragment as described herein, wherein the antibody or antigen-binding fragment is bound to PMCA 1 to 4 as described herein. The kit can be used in the methods of the present disclosure, such as methods for detecting cell encapsulation in vivo or in vitro. The kit can optionally comprise a detectable label, such as an indicator enzyme, a radiolabel, a fluorophore, biotin, colored latex beads, gold nanoparticles, or a metal tag. The kit can include an informational brochure, for example, a brochure that provides instructions for using the reagents to practice the methods of the present disclosure.

[0091] Malignant tumor progression, cancer metastasis, cancer recurrence, pan-cancer drug resistance and immunotherapy escape are unmet challenges in cancer treatment. The underlying mechanisms of cancer metastasis and progression in vivo are still unclear. The inventors of the present disclosure have discovered that cancerous cells produce cell vesicles and cell capsules outside the cytoplasmic membrane and form a protective membrane channel system to shield cancer cell proliferation, metastasis and tumor growth. The membrane that encapsulates or surrounds the cell vesicles and cell capsules produced by cancerous cells is referred to as the cell capsule herein, which is not the cytoplasmic membrane of cancerous cells. It has been observed that in immune organs, cell cysts are shielded and coexist with immune cells. A single intracellular cell cyst or a single malignant tumor cell can proliferate in the CC cavity and form a small cancerous cell cluster, which is composed of several cancerous cells. At this stage, these small cancerous cell clusters have not yet developed CCT. As used herein, the term "pre-cystic tumor" (PCT) refers to a small cancerous cell cluster enclosed in the CC cavity, wherein these small cancerous cell clusters have not yet developed CCT. In the later stages, the CC encapsulating these cancerous cell clusters enlarges and generates CCTs, which extend to adjacent and distant tissues, enabling the metastasis of cancerous cells shielded and protected by the CCT membrane. As used herein, the terms "cystic tumor" and "cystic tumor sphere" (CT) refer to cancerous cell clusters enclosed in the CC cavity, wherein CCTs appear in these cancerous cell clusters.

[0092] The in vivo cancer progression map shows that a 3D cell capsule network interconnects cell capsule tumors in primary and secondary niches, generating a dynamic and complex cell capsule tumor network system. The findings described in this disclosure reveal that the cell capsule system membrane-encloses and protects cancer progression in vivo.

[0093] In a previous report, it was found that in vitro 3D matrix culture, invasive cancer cells produce a second membrane outside the plasma membrane of cytocapsular vesicles (CC) or cytocapsular tubes (CCT) (Yi T, Wagner G, Cytocapsular tubes conduct cell translocation. Proc Natl Acad Sci USA. 115, E1137-E1146 (2018)). The present disclosure provides an in-depth study of the biology of these previously unrecognized organelles and their connection to cancer progression in vivo. We identified a biomarker for CC / CCT membranes, Ca 2+-ATPase 2 (PMCA2 or ATP2B2), which enabled us to reveal the life cycle of cystic carcinoma cells in vitro and in vivo (Jeong J et al., PMCA2 regulates HER2 protein kinase localization and signaling and promotes HER2-mediated breast cancer. Proc Natl Acad Sci USA. 113, E282-90 (2016); Street A et al., Mutations in aplasma membrane Ca2+-ATPase gene cause deafness in deafwaddler mice. Nat Genet. 19, 390-4 (1998); Van Houten J et al., PMCA2 regulates apoptosis during mammary gland involution and predicts outcome in breast cancer. Proc Natl Acad Sci USA. 107, 11405-11410 (2010)). This disclosure describes a series of previously unreported cancer phenomena, such as: pre-cystic tumors (PCTs), cystic tumors (CTs), late tumors (NTs), acellular cancerous cell clusters - CC / CCT complexes (AMCCs), cystic bodies, cystic tumor network systems (CTNSs), and integrated primary and secondary CTNSs. The results and observations of this disclosure expand our understanding of cancer progression in vivo as a systemic system of cancerous cell degeneration and evolution, shielded by a network of cystic membranes.

[0094] Example

[0095] The following examples are given to illustrate various embodiments of the present invention and are not intended to limit the present invention in any way. The examples and methods described herein represent presently preferred embodiments and are illustrative and are not intended to limit the scope of the present invention. Variations and other uses encompassed by the spirit of the invention as defined by the claims will occur to those skilled in the art.

[0096] Example 1

[0097] Endogenous PMCA-2 expression in cystic ducts of normal, benign and primary cancer tissues was detected.

[0098] Embodiments of the present disclosure relate to methods for detecting endogenous PMCA-2 expression of cell cysts in tissues. In some embodiments, whether normal cells, benign tumor cells, and cancer cells in tissues obtained from human subjects can generate cell cysts in vivo is studied. In some embodiments, H&E staining and immunohistochemistry (IHC) fluorescence staining analysis are performed using clinical normal breast tissue, benign breast tissue, and breast cancer tissue (5 samples from 5 different patients of each tissue, 1 sample per patient; for 5 breast cancer patients, 1 is in stage I, 2 are in stage II, 1 is in stage III, and 1 is in stage IV, surgical biopsy), and the IHC fluorescence staining is performed using a rabbit polyclonal antibody against PMCA2 (green) and a mouse monoclonal antibody against γ-actin (red). Figures 1A to 1F No cystic ducts were detected in normal breast tissue or benign breast tumor tissue (Table 1). However, a large number of cystic ducts (CCTs, with diameters of 3 μm to 6 μm, lengths of 300 μm to 4000 μm, and a CCT density of 60 CT / mm in the sectioned samples) were detected in all tested stage I to IV breast cancer tissues. 2 Up to 115CT / mm 2 These observations demonstrate that breast cancer cells, but not normal breast cells or benign breast tumor cells, produce CCTs in breast cancer tissue in vivo. Interestingly, H&E staining assays did not show CCTs in the same tissue samples ( Figures 1A to 1F These data indicate that although the presence of CCTs is not visible / detectable by H&E staining, immunohistochemistry (IHC) fluorescent staining with an antibody against PMCA2 can detect PMCA2 protein expression in CCT membranes and is a sensitive marker specific for CCTs in cancer tissues.

[0099] Next, we examined whether cancer cells migrated within CCTs in cancer tissues. Clinical breast cancer tissues (five samples from five different stage II breast cancer patients, one sample per patient, surgical biopsies) were subjected to H&E staining and immunohistochemistry (IHC) fluorescent staining analysis using a rabbit polyclonal antibody against PMCA2 (green) and a mouse monoclonal antibody against γ-actin (red). Figures 2A to 2B As expected, there are many CTs in clinical stage II breast cancer tissue (CCT density is 60 CCT / mm 2 Up to 110 CCT / mm 2 ), and many cancer cells in CCT showed long and narrow spindle-shaped morphologies when exposed in sectioned CCT, suggesting that these cancer cells migrate in CCT and use CCT as membrane-enclosed tubular high-speed channels as a means of metastasis in dense and crowded cancer tissues ( Figures 2A to 2B).

[0100] Next, we investigated the density and morphology of CCTs in clinical breast cancer tissues at later clinical stages (5 samples from 5 different patients with stage III breast cancer, 1 sample per patient, surgical biopsies) using a rabbit polyclonal antibody against PMCA2 (green) and a mouse monoclonal antibody against γ-actin (red). Figures 3A to 3B There are a lot of (CCT density of 75CCT / mm) in stage III breast cancer tissue. 2 Up to 126 CCT / mm 2 ) Long and winding CCT ( Figures 3A to 3B The above results (Figures 1 to 3 and Table 2) show that cancer cells in late-stage clinical cancer tissues produce an increased number of long, membrane-enclosed CCTs. The increase in the number / density of CCTs appears to be correlated with the degree of cancer metastasis in these cancer tissues.

[0101] To compare the sensitivity of breast cancer tumor markers, five consecutive sections of clinical stage III breast cancer tissue were subjected to conventional H&E staining, IHC using antibodies recognizing breast cancer molecular markers ER, PR, and HER-2, and IHC fluorescence using anti-PMCA2 antibody recognizing PMCA2 protein ( Figures 4A to 4E ). Only the IHC fluorescence assay performed with anti-PMCA2 antibody showed the detection of CCT in clinical breast cancer tissue. Conventional H&E staining assay did not show any CCT in breast cancer tissue. IHC staining performed with antibodies that recognize breast cancer molecular markers ER, PR, and HER-2 showed that these breast cancers were positive or negative, and their respective markers ER, PR, and HER-2 had different signal intensities, and they failed to show CCT ( Figures 4B to 4D These data (Figures 1 to 4) strongly suggest that, compared with conventional H&E staining assays and IHC assays using antibodies recognizing the breast cancer molecular markers ER, PR, and HER-2, IHC fluorescence assays using anti-PMCA2 antibodies have higher sensitivity in detecting breast cancer and can provide higher sensitivity and accuracy for the diagnosis, prognosis, and monitoring of breast cancer by detecting CCT in breast cancer tissues.

[0102] Table 1. List of clinical normal human tissue samples showing that CCT was not detected in these tissues.

[0103] organize Number of positive CCT staining Total number of tests breast 0 3 liver 0 2 colon 0 2 Stomach 0 3 lung 0 3 esophagus 0 2 prostate 0 1

[0104] Table 2. Summary of breast cancer cell cyst tube analysis in clinical patient breast cancer tissues by immunohistochemistry (IHC) using anti-PMCA2, anti-ER, anti-PR, and anti-HER2 antibodies, as well as anti-γ-actin antibodies. The table shows the number of cyst tubes (CCTs), degradation status, and cancer metastasis grade.

[0105]

[0106]

[0107]

[0108] (Note: F, female; “+”, detected; “−”, not detected; T1, tumor invades the submucosa; T2, tumor invades the muscularis propria; T3, tumor invades the submucosa or nonperitonealized paracolic or pararectal tissue through the muscularis propria; T4, tumor directly invades other organs or structures and / or perforated visceral peritoneum. N0, no regional lymph node metastasis; N1, 1 to 3 regional lymph node metastasis; N2, 4 or more regional lymph node metastasis. M0, no distant metastasis; M1, distant metastasis. CT / CCT, cytocystic tubing).

[0109] Prostate cancer is one of the top 5 cancers in men. Next, CCT was investigated in clinical prostate cancer tissues (5 samples from 5 different prostate cancer patients, 2 at stage I, 1 at stage II, 1 at stage III, and 1 at stage IV; 1 sample per patient, surgical biopsy) by H&E staining and IHC fluorescence assay using a rabbit polyclonal antibody against PMCA2 (green) and a mouse monoclonal antibody against γ-actin (red). Figures 5A to 5B IHC fluorescence assay using anti-PMCA2 antibody (instead of H&E staining) showed that there were a large number of CCTs (CCT density of 55 CCT / mm) in stage II clinical prostate cancer tissue. 2 Up to 123 CCT / mm 2 ) Long and winding CCT ( Figures 5A to 5B These data suggest that IHC fluorescence assay using anti-PMCA2 antibody to detect CCT in prostate cancer tissues can serve as a sensitive method for the diagnosis, prognosis, and monitoring of prostate cancer.

[0110] Example II.

[0111] Detection of endogenous PMCA-2 expression in the cystic ducts of primary cancer tissues.

[0112] Cancer can originate from any part of the subject's body. The detection of CCT in breast and prostate cancer tissues strongly suggests that CCT can be produced by cells in other types of cancer. Next, the IHC fluorescence assay method performed with anti-PMCA2 antibodies studied CCT in another 32 types of primary cancers from various organs and tissues. Shown here are the results of IHC fluorescence assay studies of adrenal cancer, appendix cancer, bladder cancer, and bone marrow cancer (5 samples from 5 different cancer patients in each type of cancer, 2 in stage I, 1 in stage II, 1 in stage III, and 1 in stage IV; 1 standard sample per patient, surgical biopsy). Many different densities of CCT have been detected in all these cancer tissues studied (CCT density is 45CCT / mm 2 Up to 118 CCT / mm 2 As metastasis progresses to later clinical stages, some CCTs in cancerous tissues exhibit a degenerating morphology, with a progression from thick to thin and very fine filaments throughout the CCT life cycle ( 6A to 6D Some degraded CCTs exhibit a cloud-like morphology at a very late stage (clinical stage IV). This cloud-like morphology corresponds to the end of the CCT life cycle.

[0113] The study of CCTs was subsequently extended to other cancer types, including brain, cervical, colon, and endocrine cancers, by fluorescence IHC assays using anti-PMCA2 antibodies (five samples from five different cancer patients per cancer type, one at stage I, one at stage II, two at stage III, and one at stage IV; one sample per patient, surgical biopsy). Many CCTs of varying densities were detected in all these cancer types (CCT density was 56 CCTs / mm 2 Up to 121 CCT / mm 2 As metastasis progresses to later clinical stages, some CCTs in cancerous tissues exhibit a degenerating morphology, with a progression from thick to thin and very fine filaments throughout the CCT life cycle ( 7A to 7D ).

[0114] Table 3. Summary of colon cancer tissues of different cancer types or subtypes, showing the analysis of cell cystic tubes in colon cancer tissues of clinical patients by immunohistochemistry (IHC) using anti-PMCA2 antibody. The table shows the number of cell cystic tubes (CCT / CT), degradation status and cancer metastasis grade.

[0115]

[0116]

[0117] (Note: F, female; M, male; “+”, detected; “−”, not detected; T1, tumor invades the submucosa; T2, tumor invades the muscularis propria; T3, tumor invades the submucosa or nonperitonealized paracolic or pararectal tissue through the muscularis propria; T4, tumor directly invades other organs or structures and / or perforates the visceral peritoneum. N0, no regional lymph node metastasis; N1, 1 to 3 regional lymph node metastasis; N2, 4 or more regional lymph node metastasis. M0, no distant metastasis; M1, distant metastasis. CT / CCT, cytocystic tubing).

[0118] The study of CCT was further extended to oesophageal, head / neck, cardiac, and intestinal cancers (5 samples from 5 different cancer patients for each type of cancer, 1 at stage I, 2 at stage II, 1 at stage III, and 1 at stage IV; 1 sample per patient, surgical biopsy) by IHC fluorescence assay with anti-PMCA2 antibodies. Many CCTs of varying densities have been detected in all these cancer types (CCT density of 54 CCT / mm 2 Up to 118 CCT / mm 2 As metastasis progresses to later clinical stages, some CCTs in cancerous tissues exhibit a degenerating morphology, with a progression from thick to thin and very fine filaments throughout the CCT life cycle ( Figures 8A to 8D ).

[0119] The study of CCTs was further extended to renal, liver, lung, and lymphoid cancers (5 samples from 5 different cancer patients for each type of cancer, 1 at stage I, 1 at stage II, 2 at stage III, and 1 at stage IV; 1 sample per patient, surgical biopsy) by IHC fluorescence assay using anti-PMCA2 antibodies. Many CCTs of varying densities have been detected in all these cancer types (CCT density of 48 CCT / mm 2 Up to 127 CCT / mm 2 As metastasis progresses to later clinical stages, some CCTs in cancerous tissues exhibit a degenerating morphology, with a progression from thick to thin and very fine filaments throughout the CCT life cycle ( 9A to 9D ).

[0120] The study of CCTs was further extended to melanoma, mesothelioma, nasopharyngeal and laryngeal carcinoma, and oral cancer (5 samples from 5 different cancer patients for each type of cancer, 1 at stage I, 1 at stage II, 1 at stage III, and 2 at stage IV; 1 sample per patient, surgical biopsy) by IHC fluorescence assay with anti-PMCA2 antibodies. Many CCTs of varying densities have been detected in all these cancer types (CCT density of 54 CCT / mm 2 Up to 108 CCT / mm2 As metastasis progresses to later clinical stages, some CCTs in cancerous tissues exhibit a degenerating morphology, with a progression from thick to thin and very fine filaments throughout the CCT life cycle ( 10A to 10D ).

[0121] The study of CCTs was further extended to ovarian, pancreatic, penile, and bile duct cancers by fluorescence IHC assay using anti-PMCA2 antibodies (five samples from five different cancer patients for each type of cancer, one at stage I, one at stage II, one at stage III, and two at stage IV; one sample per patient, surgical biopsy). Numerous curved and intertwined CCTs of varying densities were detected in all these cancer types (CCT density of 47 CCT / mm). 2 Up to 107 CCT / mm 2 As metastasis progresses to late clinical stages, some CCTs in cancer tissues exhibit a degenerating morphology, progressing from thick to fine and very fine filaments throughout the CCT life cycle, with cloud-like morphology corresponding to the end of the CCT life cycle ( 11A to 11D ).

[0122] The study of CCT was further extended to colorectal cancer, skin cancer, soft tissue (smooth muscle) cancer, and gastric cancer (5 samples from 5 different cancer patients for each type of cancer, 1 at stage I, 2 at stage II, 1 at stage III, and 1 at stage IV; 1 sample per patient, surgical biopsy) by IHC fluorescence assay with anti-PMCA2 antibody. Many CCTs of varying densities have been detected in all these cancer types (CCT density of 44 CCT / mm 2 Up to 118 CCT / mm 2 As metastasis progresses to late clinical stages, some CCTs in cancer tissues exhibit a degenerating morphology, progressing from thick to fine and very fine filaments throughout the CCT life cycle, with cloud-like morphology corresponding to the end of the CCT life cycle ( 12A to 12D ).

[0123] The study of CCT was further extended to testicular, thyroid, uterine, and vulvar cancers (5 samples from 5 different cancer patients for each cancer type, 1 at stage I, 1 at stage II, 2 at stage III, and 1 at stage IV; 1 sample per patient, surgical biopsy) by IHC fluorescence assay using anti-PMCA2 antibodies. 2 Up to 108 CCT / mm 2As metastasis progresses to late clinical stages, some CCTs in cancerous tissues exhibit a degenerative morphology, progressing from thick to fine and very fine filaments throughout the CT life cycle, with cloud-like morphology of CT corresponding to the end of the CCT life cycle ( 13A to 13D ).

[0124] Example III

[0125] CT detection in paraneoplastic tissue

[0126] Paraneoplastic tissue is often referred to as normal adjacent tissue (NAT). The presence of CCTs in paraneoplastic tissue was then investigated. Paraneoplastic tissue from breast cancer was examined by fluorescence immunohistochemistry (IHC) using an anti-PMCA2 antibody (5 samples from 5 different cancer patients, 1 at stage I, 1 at stage II, 2 at stage III, and 1 at stage IV; 1 sample per patient, surgical biopsy). Surprisingly, a large number of long, curved, and intertwined CCTs (CCT density of 95 CCTs / mm) were detected in these tissues from all stages of breast cancer examined. 2 Up to 128 CCT / mm 2 )( Figure 14 (A NAT of a stage II breast cancer is shown). These data demonstrate that conventional NAT may not actually be normal tissue, as it still harbors a large number of CCTs, presumably extended from adjacent cancerous tissue. The observation that paraneoplastic tissue contains a large number of CCTs is consistent with the fact that CCTs extend through paraneoplastic tissue and serve to transport cancer cells from the primary site to adjacent and distant organs and tissues. This observation may be used to reassess areas adjacent to cancerous tissue to be removed during cancer surveillance and treatment, as well as to reassess the extent of cancer spread during metastasis.

[0127] Example IV

[0128] CT Detection of Secondary Cancers

[0129] Cancer can be transferred from the primary site of origin to the secondary site of metastasis. Cancer metastasis causes cancer cells to spread to adjacent and distant tissues and organs, causing multi-organ biological function failure and ultimately death. Therefore, by IHC fluorescence determination with anti-PMCA2 antibodies, CCT was studied in a variety of metastatic cancers in distant tissues and organs (5 samples from 5 different cancer patients in metastatic cancer, 3 in stage III and 2 in stage IV; 1 sample per patient, surgical biopsy). These metastatic cancers are metastatic gastric cancers that metastasize to lymph nodes via CCT ( Figure 15 ), metastatic colon cancer to the liver via CCT ( Figure 16), metastatic adenocarcinoma from the ovary to the omentum via CCT ( Figure 17 ), metastatic mucinous adenocarcinoma from an unknown site to the omentum via CCT ( Figure 18 ) and metastatic rectal cancer that metastasizes to lymph nodes via CCT ( Figure 19 As expected, many CTs were detected in metastatic cancers in distant organs and tissues in all tested samples (CCT density was 92 CCT / mm 2 Up to 124 CCT / mm 2 The above consistent data (Figure 1 to Figure 19 and Tables 1 to 4) demonstrate that, in all types of clinical solid cancers, cancer cells produce CCTs for cancer cell migration, spread, and cancer metastasis, and that the method of IHC fluorescence assay using an anti-PMCA2 antibody is reliable and suitable for detecting CCTs in cancer tissues, and that this method is suitable for predicting cancer metastasis with high sensitivity and accuracy, and can be used for all types of solid tumor cancers.

[0130] Embodiments of the present disclosure relate to using this method to study CCT in a variety of cancer types and subtypes. Table 4 provides a partial list of the cancer tissues tested. Almost all cancer tissue samples tested stained positive for PMCA-2 and were also positive for CCT. The data demonstrate that this method for detecting PMCA-2 and CCT in cancer tissues is applicable to all types of cancer.

[0131] Table 4. List of cancer tissues with cellular capsule tubes (CCTs) detected by IHC using anti-PMCA2 antibody.

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] Example V

[0139] CT scans of breast cancer tissue using needle biopsy

[0140] Needle biopsy is a powerful tool for cancer prognosis and diagnosis with minimal tissue damage. Next, needle biopsy samples of clinical breast cancer tissue were tested by IHC fluorescence assay using anti-PMCA2 antibody (5 samples from 5 different cancer patients in breast cancer, 1 in stage I, 2 in stage II, 1 in stage III, and 1 in stage IV; 1 sample per patient, needle biopsy). Scanning from the top to the bottom of the needle biopsy sample, CCT ( Figure 20 A to Figure 20 E), and obtain the number and density of CCTs distributed along the depth of the tissue (CCT density is 0CCT / mm 2 Up to 112 CCT / mm 2 These results suggest that the method of detecting CCT by IHC fluorescence assay using anti-PMCA2 antibody can be applied to clinical needle biopsy samples.

[0141] Example VI

[0142] CT testing using anti-PMCA1 to 4 antibodies

[0143] PMCA has four homologues, PMCA1 to 4. Next, we studied whether other PMCAs such as PMCA1, PMCA3 and PMCA4 can be detected in CCT membranes and used for CCT detection in cancer analysis. We used clinical breast cancer tissue (5 samples from 5 different cancer patients in stage III breast cancer; 1 sample per patient, surgical biopsy) and performed IHC fluorescence determination with antibodies against PMCA1, PMCA3 and PMCA4. In fact, antibodies against PMCA1, PMCA3 and PMCA4 showed many different densities of CCT in clinical breast cancer tissue and at different stages of progression throughout the CCT life cycle (CCT density was 35 CCT / mm 2 Up to 112 CCT / mm 2 )( Figures 21A to 21C ). Therefore, these data demonstrate that IHC fluorescence assays using anti-PMCA1, anti-PMCA2, anti-PMCA3, and anti-PMCA4 antibodies can be used as various embodiments of the present method to detect CCTs in all types of cancer tissue (such as all solid tumor cancers). The present method can be used for cancer prognosis, diagnosis, and monitoring in humans and animals.

[0144] The membrane proteins PMCA1, PMCA2, PMCA3, and PMCA4 are highly expressed in the membranes of cellular cysts, compared to relatively low abundance in the membranes of cancerous and normal cells. Studies have found that the relative expression ratios are as follows: PMCA1 in CCTs versus cancer / normal cells is approximately 20:1; PMCA2 in CTs versus cancer / normal cells is approximately 60:1; PMCA3 in CCTs versus cancer / normal cells is approximately 15:1; and PMCA4 in CCTs versus cancer / normal cells is approximately 12:1. The high abundance of PMCAs in CCTs facilitates the detection of CCTs with high sensitivity above background in analyses of various clinical cancer tissues, which facilitates fluorescent IHC assays using anti-PMCA antibodies, compared to conventional H&E staining and IHC staining with other cancer cell markers.

[0145] Example VII

[0146] Proposing a cancer metastasis grade classification system using CCT analysis

[0147] Based on the density and morphology of CCTs observed in all cancer tissues examined and their association with cancer tissue stage, it was found that CCTs with lower density and less or no degradation were associated with earlier cancer stages of the examined tissues, while CCTs with higher density and presence of degradation were associated with more advanced cancer tissue stages of the examined tissues. Therefore, a new classification system for predicting the grade of cancer metastasis (CM) on a scale of 0 to 4 was proposed based on the density and morphology / degradation of CCTs in cancer tissues. For example, if no cellular cystic ducts (CCTs) were detected and no CCT degradation was detected, a grade of cancer metastasis (CM) of 0 was predicted; if the density of CCTs detected was 1 / mm 2 Up to 10 / mm 2 If the density of CCT detected is within 11 / mm, the cancer metastasis (CM) of grade 1 is predicted. 2 Up to 40 / mm 2 If the density of CCT detected is within the range of 41 / mm, and CCT is degraded into filaments and filaments, it is predicted to be grade 2 cancer metastasis (CM); if the density of CCT detected is 41 / mm 2 Up to 80 / mm 2 If the CCT is degraded into filaments, fine filaments and filament-like structures, it is predicted to be grade 3 cancer metastasis (CM); and if the density of the detected CCT is >81 / mm 2 If the CCT is within the range of 4 and degraded into a cloud-like morphology or completely decomposed, it is predicted to be cancer metastasis (CM) grade 4. The relationship between the proposed CM grade and conventional cancer staging is shown in Table 5.

[0148] Table 5. Comparison of clinical cancer stages and proposed cancer metastasis (CM) grades based on data described in this disclosure:

[0149] Clinical cancer staging Cancer metastasis (CM) grade 0 0,1,2,3,4 I 1,2,3,4 II 2,3,4 III 3,4 IV 4

[0150] Conventional cancer diagnostics cannot detect CCTs (due to their diameter / width being approximately 3 μm to 6 μm, making them difficult to find). For example, using conventional cancer diagnostics, a supposedly "normal" tissue at stage 0 may actually contain CCTs of varying densities and stages of degradation, which can be detected using the methods described herein. Thus, clinical cancer stage 0 may correspond to a CM grade of 0 to 4. If no CCTs are detected in the tissue, the methods of the present disclosure can predict true healthy tissue, thus providing a more sensitive and accurate prediction of cancer metastasis grade compared to conventional cancer diagnostic methods.

[0151] Cancer metastasis, a hallmark of malignant tumors, is the main cause of cancer mortality, while the underlying mechanisms of cancer spread have long been unclear (Sporn, MB (1996). The war on cancer. Lancet 347, 1377–1381; Kinzler, KW, and Vogelstein, B. (1998). Landscaping the cancer terrain. Science 280, 1036–1037; Chambers, AF, and Matrisian, LM (1997). Changing views of the role of matrix metalloproteinases in metastasis. J. Natl. Cancer Inst. 89, 1260–1270; Johnson, JP (1991). Cell adhesion molecules of the immunoglobulin supergene family and their role in malignant transformation and progression to metastatic disease. Cancer Metastasis Rev. 10, 11–22). The embodiments of the present disclosure are based on the following surprising discovery: endogenous PMCA is highly expressed in the membranes that form cell capsules in cancer tissues. Antibodies produced against PMCA (including anti-PMCA1, anti-PMCA2, anti-PMCA3 and anti-PMCA4 antibodies) have been shown to detect their respective PMCA1 to 4 proteins in the CCT membranes of cancer tissues using IHC fluorescence assays. CCTs were not detected in healthy or benign tissues. The data show that cancer cells in cancer tissues produce these membrane-enclosed cell capsules and use these CCTs as cancer cell metastasis high-speed channels for cancer migration and spread. Hundreds of cancer types and subtypes have been studied by this method. Cell capsules were detected in all clinical cancers studied, and images of 32 representative solid tumor tissues and organs were shown using both surgical biopsy samples and needle biopsy samples. These data demonstrate that this method provides accurate and quantitative analysis of cellular tubules that can be used to accurately predict the extent of cancer metastasis in cancer prognosis, diagnosis, and monitoring during cancer treatment and management of all types of solid tumors in humans and animals, and that the IHC fluorescence assay using anti-PMCA1, anti-PMCA2, anti-PMCA3, and anti-PMCA4 antibodies can be reliably used to detect cellular tubules in cancer, paracancerous, and metastatic tissues and organs.

[0152] Accurate cancer metastasis analysis was previously impossible due to the lack of clear mechanisms underlying cancer metastasis. The data described in this article provide strong evidence that CCTs are membrane-enclosed high-speed pathways for cancer cells to spread and migrate to adjacent and distant tissues and organs in all types of solid tumor cancers. This method provides a way to accurately measure cancer during prognosis, diagnosis, and monitoring before and after treatment / intervention / management. CCT analysis will be a powerful tool for accurate quantitative analysis of human cancers and prediction of cancer metastasis during the treatment of clinical cancer patients. Because cancer cells rely on CCTs for cancer cell spread and migration, CCTs will undoubtedly become targets for the development of drugs and treatments to effectively inhibit cancer metastasis.

[0153] Embodiments of the present disclosure also relate to grading cancer metastasis by the density and morphology of CCTs detected in cancer tissue. The proposed cancer metastasis (CM) grade will facilitate accurate and quantitative analysis of cancer metastasis status and thus enhance the effectiveness and efficiency of cancer treatment and management. CCTs in cancer always present a complex appearance, with curved, winding, rounded, dense, and large structures with irregular structures and shapes, which makes it difficult to accurately count the number / density of CCTs. Therefore, the number and density of CCTs can only be estimated. In addition, in the same sample tissue, CCTs with or without degradation and filamentous morphology of varying thickness can often be detected simultaneously, which also increases the difficulty of accurately quantifying the grade of cancer metastasis based solely on CCT density and morphology. However, the CCT grade proposed herein is based on an analysis of >7,000 cancer patients (from 5 countries and the United States, Asia, and Europe) covering >260 types and subtypes of human cancer. With the improvement of detection methods and statistical data, this grade is a powerful tool for relatively accurate and quantitative cancer analysis. Embodiments of the present disclosure involve detecting cellular tubes using fluorescence immunohistochemistry (IHC) assays with antibodies against PMCA (PMCA 1 to 4) to measure tube density and morphology. Data indicate that CCTs are formed by cancerous cells in cancer tissue and undergo various degradation stages throughout the CCT lifecycle. Initially, cellular tubes appear as fewer, shorter, and thicker structures. CCTs then grow into longer, larger, and thinner tubes. In later stages of degradation, CCTs further degrade into thinner, filamentous and cloud-like morphologies, ultimately completely disintegrating.

[0154] Furthermore, it has been found that so-called normal adjacent tissue (NAT) is associated with a high number of CCTs in CCT analysis. These findings will help correct for false "NAT" as abnormal paracancerous tissue, thereby helping clinicians obtain more accurate samples for the correct diagnosis, prognosis, and assessment of cancer, and select the best treatment options for cancer patients, reducing cancer recurrence caused by the spread of cancer from previously "normal adjacent tissue."

[0155] Example VIII

[0156] Materials and methods

[0157] Reagents and antibodies.

[0158] Rabbit polyclonal antibodies against PMCA 1 to 4 (1:200 dilution) were generated by immunizing rabbits with each of the PMCA 1 to 4 antigens. DAPI (1:1,000 dilution in immunofluorescence assays) was ordered from Sera Care. The following antibodies were ordered from Abcam (all at 1:1,000 dilution): For immunofluorescence assays, anti-γ-actin (γ-actin, monoclonal, ab123034).

[0159] H&E staining assay.

[0160] Clinical formalin-fixed paraffin-embedded (FFPE) tissue samples (normal tissue, benign tumor tissue, and cancer tissue) were ordered from USBiomax, Inc. or obtained from hospitals in Boston, Chicago, and Florida, USA. H&E staining was performed as previously described (Barbolina MV et al. (2009) Microenvironmental regulation of ovarian cancer metastasis. Cancer Treat Res 149:319–334.)

[0161] Immunohistochemistry (IHC)-fluorescence assay.

[0162] Clinical FFPE tissue (normal tissue, benign tumor tissue, and cancer tissue) samples were ordered from USBiomax, Inc. or obtained from hospitals in the United States, Ukraine, Vietnam, and China.

[0163] Tissue slides were deparaffinized and rehydrated, and antigen retrieval (0.6 mM sodium citrate, boiled for 30 minutes) was performed. The slides were cooled for 30 minutes, treated with 0.3% H₂O₂ for 15 minutes, and blocked for 30 minutes with 1% BSA in 1× TBST buffer. The slides were then incubated with a primary antibody against PMCA2 (1:200 dilution) for 40 minutes in the dark, followed by three washes with TBST buffer. A secondary antibody, goat anti-rabbit IgG (Alexa Fluor 488 (green)) antibody (1:1000 dilution) was added and incubated for 30 minutes. The slides were then washed three times with TBST buffer and blocked for 30 minutes in 1× TBST buffer with 1% FBS. The slides were incubated with a primary antibody against γ-actin (1:200 dilution) for 40 minutes in the dark, followed by a secondary antibody, goat anti-mouse IgG (Alexa Fluor 568 (red) antibody (1:1000 dilution) for 30 minutes. The slides were then stained with DAPI (1:1000 dilution) in TBST buffer, and washed three times for 5 minutes. Immunofluorescence-stained slides were then fixed and imaged using a fluorescence microscope to analyze the cell cysts of breast cancer cells for the identification and assessment of breast cancer metastasis.

[0164] Quantitative and statistical analysis

[0165] ImageJ was used to measure the diameter, width, length, number, and density of cell capsule tubes (CCTs). For clinical surgical biopsy samples, the number of CCTs at five sites (left, top, right, bottom, and center) was counted, and the CCT density (CT / mm 2 The average CCT density of the five sites was used as the CCT density of one sample.

[0166] Example IX

[0167] Identification of PMCA2 as a biomarker for cellular vesicles and cystic tubes

[0168] To explore the structure and protein composition of previously discovered cell vesicle (CC) membranes, we established conditions for the detachment of cancer cells from cell vesicles in in vitro 3D matrix culture to obtain the proteome of cell-free cell vesicles. Using the CC / CCT 3D matrix culture kit and Unipick, we cultured and collected cell-free SILAC-labeled cell vesicles (CCs) from Bxpc3 pancreatic cancer cells, MCF-7 breast cancer cells, and SK-CO-1 colon cancer cells for CC proteomic analysis ( Figure 22A ). Research has found that an important protein is the calcium pump plasma membrane Ca 2+ -ATPase 2 (PMCA2), which is always present in the plasma membrane of closed cancer cells ( Figure 22BHowever, the abundance of PMCA2 was much higher in CC membranes encapsulating single or multiple cancer cells (n=601) or in membranes of decellularized CC in vitro (n=514). No PMCA2 signal was found in CC / CCT culture kit matrices other than CC or CCT. Figure 22B ). The study found that PMCA2 and γ-actin were constantly co-localized in the plasma membrane of cancer cells (n=601) and in the membrane of completely decellularized CC (n=514) in vitro ( Figure 22B Consistently, PMCA2 showed high abundance and co-localized with γ-actin in the cell capsule tube (CCT) membrane surrounding Bxpc3 cancer cells (n=106, Figure 23A ). Enlarged CC membrane around tumor spheres (n=546, Figure 23B ) and enlarged CC membranes of decellularized tumor spheres (n=127, Figure 23B ). These observations suggest that PMCA2 is an in vitro molecular biomarker for CC and CCT. Next, we analyzed human normal, benign, and malignant (cancer) tissues using anti-PMCA2 antibodies. In clinically normal patients (n = 14 patients, 1 tissue / patient) ( Figure 23C , Plate 1) or benign tumor tissue (n = 126 patients, 1 tissue / patient) ( Figure 23C , Plate 2), the PMCA2 signal is very low and there is no CC / CCT, indicating that normal tissues and benign tumor tissues do not produce CC / CCT, and PMCA2 expression is strictly controlled and maintained at a low abundance in normal human tissues or benign tumors ( Figure 23C , Plate 1 and Plate 2). In contrast, in clinical breast cancer tissue (n = 685 patients, Figure 23C , Plate 3) and pancreatic cancer tissue (n = 310 patients, Figure 23C , Plate 4), there are many long and curved CCTs and high abundance of PMCA2 in the CCT membrane ( Figure 23C , Plates 3 and 4). In addition, in vivo, PMCA2 signals are absent in the extracellular matrix (ECM) of normal, benign, and cancerous tissues ( Figure 23C The above observations indicate that PMCA2 is a molecular marker for CC / CCT in vitro and in vivo.

[0169] Example X

[0170] Characterization and life cycle of cystic carcinoma cells: in vitro experiments and in vivo studies of human cancer tissues

[0171] Using the cell vesicle (CC) / cell cyst tube (CCT) biomarker PMCA2, we tested whether cancerous cells produce CC in vivo. In fact, in the early stages of breast cancer, studies have found that many cancerous breast cells are enclosed by CC. These CCs shield the cancerous cells inside and isolate them from the microenvironment outside the cell capsule, thereby protecting them ( Figure 22C CCs showed diversity in morphology, size, membrane tension or folding state, and presence / absence of decellularization, autodegradation, and autolysis. Some CCs (n=135) appeared with irregular morphology, with highly folded membranes enveloping breast cancer cells, whereas other CCs (n=268) tightly and smoothly enveloped breast cancer cells ( Figure 22C Some breast cancer cells escaped from the CC (n=1023), leaving behind a cell-free CC ( Figure 22C , ECC). These acellular CCs are arranged together to form a cell-free CC cluster ( Figure 22C Some acellular CCs appeared in round or oval morphology (n=576), and many CCs were irregular in shape (n=1217) ( Figure 22C Some cell-free CC membranes appeared taut and tight (n=107, Figure 22C The phenomenon of CC decellularization and the morphology of cell-free CC observed in vivo ( Figure 22C ) is consistent with the in vitro observation results ( Figure 22B and Figure 23B Importantly, cancer cells proliferate in the CC lumen and migrate within the CCT lumen in vitro and in vivo ( Figures 22B to 22C and FIG. 23A to FIG. 23B These observations suggest that the cell capsule not only shields and protects the cancerous cells inside, but also allows them to perform various cellular behaviors and activities within the CC / CCT cavity. We termed this previously unidentified single cancerous cell enclosed in the extracellular second membrane of the CC or CCT "cell encapsulated cancer cells" ( Figure 22D ). The cystic cancer cells carry out cellular activities in the cell cyst cavity.

[0172] Next, we investigated the activity and behavior of single-cell cystic carcinomatous cells in human cancer tissues in vitro and in vivo. We found that single-cell cystic carcinomatous cells produced long CCTs, which allowed these cancerous cells to migrate in vitro. The CCT membrane tightly wrapped around the cancerous cells and appeared bulging ( Figure 23AThe width / diameter of the stretched and contracted CCT ranged from 2 μm to 3 μm, while the width of the enlarged, bulging CCT segments caused by the internal cancerous cells ranged from 5 μm to 10 μm, increasing their width by 2.5 to 5 times and their calculated circumference by 7.85 to 15.7 times (circumference = 3.14 × diameter of a circle). These observations suggest that the CCT membrane possesses effective elasticity, allowing for the dynamic migration of cancerous cells of varying sizes within it. Figure 23A In dense breast cancer in vivo, a single mammary cystic carcinomatous cell can invasively generate multiple CCTs in different directions, but connected by nodes ( Figure 22E Single pancreatic cystic cells can strongly generate very long and highly curved CCTs in pancreatic cancer tissue ( Figure 22F These observations strongly suggest that single cystic carcinoma cells have the ability to generate multiple, elongated, elastic, and robust CCTs for CCT membrane-enclosed protected high-speed channels and directed cell migration. Cytocystic carcinoma cells proliferate in the cystic cavity and grow into cystic tumor spheres in vitro ( Figure 22B and Figure 23B ) and initiate tumor formation in vivo ( Figure 22C Sometimes, decellularization of cystic cells produces cell-free cystic cells and cell-free cysts, which then automatically degrade into cystic filaments ( Figure 22C Briefly, the life cycle of cystic carcinoma cells consists of three sequential processes: 1) intracellular cystic carcinoma cells proliferate and grow into cystic tumors enclosed in enlarged cystic vesicles, 2) cystic vesicles elongate and generate cystic tubes with migrating cancerous cells inside, 3) cystic decellularization produces cell-free cystic carcinoma cells and cell-free cystic vesicles (CCs), followed by CC degradation ( Figures 1A to 1F and Figure 23D ).

[0173] Next, we examined why CCs and CCTs had not previously been identified by conventional methods. In adjacent samples of serial sections from the same site of the same cancer specimen, hematoxylin and eosin (H&E) staining did not reveal the characteristics of a large number of CCTs in cancer due to poor staining of CCT membranes by eosin (n = 352 patients, Figure 24A and Figure 24C Antibodies that recognize clinical breast cancer cell marker proteins ER, PR, and HER-2 do not recognize CCT marker proteins in these breast cancer samples (n=213 patients, Figures 4A to 4E In addition, the colon cancer cell marker MSH-2 did not show the characteristics of CCT in colon cancer (n=86 patients, Figure 25AIndeed, CC and CCT were only detected after we observed the production of a second membrane outside the cell membrane in the CC / CCT 3D matrix culture kit, managed to separate the second membrane from the cancer cells, obtained the CC proteome, and compared the relative abundance of markers in approximately 10,000 clinically annotated cancer / normal samples ( Figures 22A to 22F 、 24A to 24D 、 Figures 4A to 4E 、 Figure 25A ).

[0174] Example XI

[0175] Cytocystic tumor progression and life cycle

[0176] Next, we investigated whether and how single cystic carcinoma cells grow into tumors. Indeed, at 48 and 72 hours, in the CC / CCT culture kit matrix, cystic Bxpc3 carcinoma cells proliferated in CCs and grew into tumor spheres in vitro, and small CCs developed into enlarged CCs with increasing size, enclosing large tumor spheres inside (n = 458, Figure 26A and Figure 23B In our experiments, the width / diameter of the CC increased from 8 to 10 μm in single cystic tumor cells to large tumor spheres up to 40 μm in vitro, with the width / diameter increasing by up to 5 times. In vitro, the CC volume of large cell cystic tumor spheres was calculated (according to the sphere volume formula v = 4 / 3πr 3 These observations suggest that intracellular cystic carcinoma cells in CCs have the effective ability to support / drive the growth of small CCs into large CCs, which have an increased volume to allow the cancerous cells to proliferate and grow into large tumor spheres ( Figure 26A and Figure 23B Similarly, as with the decellularization of single-cell cystic carcinoma cells, some tumor spheres in large CCs underwent spontaneous decellularization (n = 23 tumor spheres, Figure 23B In enlarged CC, decellularization of single cancerous cells of tumor spheres resulted in large, reclosed and reunited, cell-free, shrunken and concave CC discs (n=35, Figure 23B In large CCs, decellularization of tumorspheres resulted in large, non-reclosed / non-reunited, cell-free, shrunken CCs with large openings (n=74, Figure 23B These observations demonstrate that solid cell cystic carcinoma cells grow into cystic tumorspheres within enlarged CCs in vitro.

[0177] We then investigated how cystic carcinoma cells progress to malignant tumors in vivo in human tissues. Initially, in early clinical breast cancer tissues, the diameter / width of the extracellular vesicles of single cystic carcinoma cells was slightly larger than 15 μm. Single intracellular carcinoma cells proliferated and formed small cancerous cell clusters (n=675, 100 cells) consisting of several cancerous cells (n≥2 cancerous cells) in the CC cavity. Figure 26B At this stage, no CCTs emerged from these small cancerous cell clusters (n=675, Figure 26B We named this early stage "pre-cystic tumor (PCT)" ( Figure 26B Subsequently, the CC of PCT grew and increased in diameter / width, and the mammary intracellular carcinomatous cells continued to proliferate and form larger and denser mammary carcinomatous cell clusters enclosed in the enlarged CC (n=312, Figure 26C In the examined breast cancers (n=25), the width / diameter of PCT ranged from 15 μm to 50 μm, and the calculated CC size of PCT (estimated as spherical morphology) was 1,432.7 μm. 3 to 49,062.5μm 3 within the volume range (n=89, Figures 26B to 26D Sometimes, cell-free vesicles (n=161) appeared in the breast PCT cavity ( Figure 26D ), indicating that PCT intracellular cystic cells generate secondary independent cell vesicles, and these secondary cystic cells can undergo decellularization and generate decellularized cell vesicles. Subsequently, these decellularized cell vesicles in PCT automatically degrade into filaments, then disintegrate and disappear, leaving behind fluid-filled cavities (n=78, Figure 26D In the enlarged CC, breast cancer cells can extend part of the CC membrane, increase the area of ​​the CC membrane, deform the CC membrane, and form a tubular CCT, thereby providing a membrane-shielded high-speed channel for intracellular cystic cancer cells, allowing them to diffuse out of the CC of the dense tumor. These CCT membranes are the CC membranes of PCT in vitro (n=65, Figure 25B ) and in vivo (n=117, Figure 26E We termed this previously unrecognized single malignancy "cellular cyst tumor, CT," which is encapsulated in an enlarged CC and generates CCTs extending into adjacent and distant tissues for CCT membrane-shielded and protected cancerous cell metastasis ( Figure 26E ).

[0178] Subsequently, uncontrolled proliferation of breast intracellular cystic carcinoma cells generates more carcinoma cells, forming tubular structures in the CT lumen and growing into larger malignant tumors in the enlarged CC lumen (n=117, Figure 26ETypically, in the early stages, two or more adjacent small mammary PCTs, CTs, or PCTs and CTs merge into larger PCTs (or CTs) through CC membrane contact, integration, degradation, and the formation of open junctions, forming long or irregularly shaped cell capsule tumor spheres enclosed in longer and larger CCs (n = 145, Figure 26F Some newly merged CTs had three or more tumor branches (n=23, Figure 26F The coexistence of PCTs with large size differences reflects the heterogeneity of PCTs ( Figure 26F Subsequently, with the uncontrolled proliferation of cancerous cells, the merged CTs with tumor branches were reshaped and transformed into spherical, elliptical, or irregularly shaped and dense CTs (n=132, Figure 26G The width of the CT in the breast cancers examined (n=51) ranged widely from 50 μm to 400 μm, suggesting that the CC membrane can shield and protect the enlarging CT until it reaches a larger size. Subsequently, the breast CT generates a large number of CCTs outside the CT, preparing for the metastasis of cancerous cells. Many long CCTs surround the cellular cyst tumor (CT) and form a thick CCT layer that encapsulates the CT. The CCT layer of the CT measured a thickness of 22 μm to 510 μm (n=132 CTs, Figure 26G CTs in the primary niche are interconnected through a dense CCT network and form a cellular cystic tumor network system (CTNS, Figure 26G CT showed great heterogeneity in terms of regular or irregular morphology, size, number of CCTs, thickness of the encapsulated CCT layer, and density of cancerous cells in the CC cavity ( Figure 26G The above observations showed that cystic tumors with intracellular cystic carcinomatous cell clusters and CCTs ( Figure 26H ), providing two intrinsic physical and structural drivers for the two main characteristics of malignant tumors (cancer) observed in clinical practice: uncontrolled proliferation and metastasis ( Figures 26G to 26H ).

[0179] Subsequently, in the advanced cancer stage, the CC membrane of CT degrades, disintegrates, and disappears, leaving behind a dense, acellular, cystic carcinomatous mass without CC (n=814, Figure 26I We named the tumors at this stage “late-stage tumors” (“late” means “late stage” in Latin). The late-stage tumors consisted of acellular cystic carcinomatous cell clusters after CC degradation, without enlarged CC encapsulating the tumor. Some acellular cystic carcinomatous cells in late-stage tumors (NT) regenerated many new, long and highly curved CCTs (n=1021NT). NT cancerous cells can invade these CCTs and spread by allogeneic entry, resulting in a decrease in the density of in situ cancerous cells (n=25, Figure 26INT acellular cystic carcinomatous cell clusters and newly generated CCTs formed large acellular cystic carcinomatous cell clusters-CT / CCT complexes (AMCC) (n=825, Figure 26I In summary, the life cycle of cystic tumors includes four consecutive processes: 1) a single cystic tumor produces a pre-cystic tumor (PCT) without CCT, 2) a cystic tumor (CT) with many CCTs for cancer metastasis, 3) CC degradation, forming a late tumor (NT) without CC enclosing cancerous cell clusters, 4) some acellular cystic tumor cells in the NT regenerate new CC / CCT or new small CT and form AMCC ( Figure 25C ).

[0180] Example XII

[0181] Distribution of cystic carcinoma cells and tumors in human tissues and organs

[0182] It is well known that cancer commonly occurs in most human organs and tissues (Sung H et al., Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. Online ahead of print (2021)). Using PMCA2 as a CC / CCT biomarker, we studied the distribution of cystic carcinoma cells and tumors in 34 human organs and tissues. In the six normal human organ tissues tested from breast, colon, liver, lung, prostate and stomach, we did not find detectable amounts of cystic carcinoma cells / tumors ( Figure 27A , n = 14 patients). Among the 38 benign tumor subtypes tested ( Figure 27A , n = 126 patients), most benign tumors did not show cellular cystic carcinoma cells / tumor, while 13.4% of benign tumors showed cellular cystic carcinoma cells, indicating that these clinically annotated benign tumors may have undergone transformation and appeared cellular cystic carcinoma cells. Among the 290 types / subtypes of cancer in 34 human organs / tissues tested (excluding blood cancer in the blood, number of patients, n = 9,770; number of samples, n = 9,958), 100% of the tested cancers showed cellular cystic carcinoma cells ( Figure 27A These observations indicate that normal and benign tumor tissues do not produce cystic carcinoma cells, and that human cancers commonly produce cystic carcinoma cells. Hematologic cystic carcinoma cells appear in immune organs / tissues such as the bone marrow, lymph nodes, spleen, and thymus ( Figures 27B to 27M(Table 6). Numerous cell-free cystic cells were located outside the CC and CCT, indicating that cell-free cystic cells coexist with cellular cystic cells in vivo. Cellular cystic cells exhibited three prominent characteristics: 1) high abundance of PMCA2 in CC / CCT membranes, 2) CCTs with diameters / widths ranging from 3 μm to 10 μm and lengths up to >3000 μm in sectioned samples, and 3) localization within the CC or migration within the CCT ( Figures 27B to 27M (Table 6). Across 283 solid cancer subtypes tested, CCTs of cystic cells exhibited a great diversity. This diversity included CCT density, morphology, ultrastructure, degradation, interconnectivity, intracellular cystic cell migration within CCTs, bundles, and intermixing with cell-free cystic cells, reflecting the considerable heterogeneity of cystic cells in solid cancers ( Figures 27B to 27M Cytocystic tumors are commonly found in human solid cancers, but not in hematologic cancers in the blood (Table 6). The above observations indicate that cytocystic tumor cells are commonly found in solid cancers and hematologic cancers in the bone marrow, lymph nodes, spleen, and thymus, but not in normal or benign tumor tissues.

[0183] Table 6. Characterization of cancerous cells, cell-free cystic cancerous cells, cell-cystic cancerous cells, cell-cystic tumors, and cell-cystic tumor network systems in 293 types / subtypes of cancer in human organs and tissues.

[0184]

[0185]

[0186]

[0187]

[0188] Example XIII

[0189] Mechanisms of vesicle growth, generation, and extension, as well as migration of cystic carcinomatous cells in CCTs

[0190] Next, we investigated the mechanism of vesicle growth around cultured cancerous cells in vitro. Using an inverted bright-field microscope, we recorded videos of Bxpc3 tumor spheres in large CCs, which contained large gaps between the tumor sphere edge and the CC membrane. Multiple spike-like structures (0.2 μm to 0.9 μm in diameter / width and 4 μm to 9 μm in length) were present within the CC cavity (called vesicle spikes, CSs). Figure 28A and Figure 28BCytocystic spikes (CC examined / detected, n=26; mean: n=4 to 10 CS per cystic tumor) interconnected the tumor sphere surface with the inner surface of the CC membrane. These cystic spikes pointed in multiple 3D directions, supporting the enlarged CC membrane and maintaining the CC spherical / irregular morphology, possibly preventing collapse ( Figure 28A and Figure 28B ).

[0191] Furthermore, we observed numerous tiny (0.1 μm to 0.5 μm in diameter / width) spherical membrane-enclosed particles (subsequently identified as PMCA2-containing vesicles) attached to and covering the surface of the tumor spheres, indicating that the oncogenes of the tumor spheres produced a large number of such vesicles (n = 12 cell-encapsulated tumor spheres, Figure 28A and Figure 28B These vesicles detach from the surface of cancerous cells and move freely and randomly in the intracellular fluid ( FIG. 28A to FIG. 28B 、 Figures 29A to 29C Ultimately, these vesicles contact, fuse, and integrate into the CC membrane, increasing the CC membrane area and size and promoting CC growth ( FIG. 28A to FIG. 28B 、 Figures 29A to 29C ).

[0192] Consistently, in vivo, in early gastric cancer tissues, there are numerous tiny membrane-enclosed vesicles with high PMCA2 abundance, which are located inside and outside the cancer cell cytoplasm ( Figure 30A These vesicles then fuse together to create cellular vesicles that enclose the cancerous cell inside, creating cellular vesicles that encapsulate the cancerous cell ( Figure 30A These vesicles that support CC production / growth exist in vitro and in vivo (Figures 28 to 30). We named these tiny, membrane-enclosed vesicles with high PMCA2 abundance "cellular vesicles," which serve as cell membrane building materials to deliver cargo and support CC production and growth ( 30A to 30D ).

[0193] Initially, cysts were generated in the cytoplasm of cell-free cyst-transformed cells (n = 56 samples, Figure 30A ). Subsequently, the cell cysts were released outside the cytoplasmic membrane (n=87). These external cell cysts fused together and formed small cell capsule fragments, which then grew into cell vesicles that encapsulated the entire cancerous cell and produced cell cyst cancerous cells (n=62 cancer samples, Figure 30A In gastric cancer and breast cancer, the average number of detectable cystic bodies (CS) per cystic cell in vivo was 34±8 CS / cell (n=65) and 28±7 CS / cell (n=120), respectively ( Figure 30BThe above observations indicate that cells without cell cyst transformation initially produce cell cysts in the cytoplasm. Subsequently, the cell cysts are released outside the cell membrane, randomly diffuse in the lumen and attach to the CC membrane. Then, the cell cysts gradually merge together to form cell vesicles and produce cell cyst cancer cells. Cell cysts act as cell cyst material cargo delivery vehicles to drive cell vesicle growth ( Figure 30D ).

[0194] Next, we evaluated the mechanism of CCT generation and extension in cystic carcinoma cells. Using time-lapse DIC microscopy, we studied the generation and extension of CCT in cystic carcinoma cells in vitro. Initially, single MCF-7 mammary carcinoma cells generate CCs whose membranes tightly wrap around the carcinoma cells. Using vesicle-based motility, single cystic carcinoma cells in the CC push the CC membrane forward, deforming the CC membrane shape into a tubular morphology and generating short CCT segments ( Figure 31A , Plates 1 to 3). The initial CCT segments were well anchored in the sticky CC / CCT culture kit matrix and maintained a wide tubular shape without stretching and shrinking into a thin and long CCT tail shape ( Figure 31A , Plates 2 to 3). Cytocystic cancer cells continuously produce cell cysts, which merge and integrate into the front of the CC membrane. These cell cysts maintain close contact with the cancerous cell membrane and increase the CC membrane area. Cytocystic cancer cells continuously and dynamically produce many transient vesicles of different sizes. In the CC, vesicles continuously protrude and retract outward, sense the extracellular microenvironment in many 3D directions, and select or determine the direction of movement. After cell vesicle formation, cancerous cells migrate backward in the CCT they produce. These cancerous cells produce several short cell cyst spikes in the CCT cavity, which connect the cancerous cells and the rear of the CCT membrane ( Figure 31A Subsequently, when individual cystic cells migrate backward within the established CCT, they switch to a lamellipodia-based motility mode ( Figure 31A The average CCT extension rate of MCF-7 cystic carcinoma cells in the CC / CCT culture kit matrix was 1.25±0.3 μm / min (n=3 cells, Figure 31B ).

[0195] Next, we investigated the generation and extension of CCTs in vivo. Figure 31C ), there are many initial cystic carcinoma cells with long and thin CCT tails (width of 0.1μm to 1μm, IC). In the initiation of CCT regeneration at the AMCC stage ( Figure 22E), single acellular mammary cystic carcinoma cells generate multiple CCTs with thin tails (width 0.2 μm to 2 μm), and during cancer metastasis, these CCTs attempt multiple different migration directions. This suggests that when the initial CCT fragments move forward, they are stretched and contracted by single cell mammary cystic carcinoma cells, and the initial CCT fragments are not well anchored to the ECM ( Figure 22E and Figure 31C Subsequently, when CCT was well anchored in the ECM via the extended CCT nanoprotrusions, the diameter / width of CCT in solid cancers was consistently maintained at 3 μm to 6 μm ( Figure 31C The above in vitro and in vivo observations indicate that single cystic cells in the CC adopt a vesicle-based sensing and motility form. This suggests a possible mechanism: how vesicles influence direction and promote forward movement, produce vesicles to support the increase in CC membrane size, push and change the CC membrane to form a tubular morphology, and generate and extend the CCT ( Figure 31D ). In vivo, the initial CCT fragment usually has a long and thin CCT tail.

[0196] Next, we investigated the mechanism of cell cystic migration in CCTs. Single Bxpc3 pancreatic cell cystic cells migrating in long CCTs (3 μm to 6 μm in diameter) appeared to be squeezed by the stretching and contracting CCT membrane and molded into a long, thin, spindle-shaped morphology. In CCTs, single cell cystic cells typically appear as single-cell mesenchymal migration ( Figure 31E ). In addition, using time-lapse DIC technology, we studied the dynamic cell migration activities in CCT, in which multiple MCF-7 breast cystic cells migrated in long CCTs. In most cases, multiple cystic cells in CCTs were in a single epithelial migration form, rather than a collective migration mode. Polarized, thin and long cystic cells in CCTs migrated forward by periodic protrusion and retraction of leading lamellipodia and movement of blunt cell rears. Sometimes, the long lamellipodia retracted completely, and the cell transiently appeared in a spherical morphology in the CCT. The CCT membrane was always tightly wrapped and adhered to the cancer cell plasma membrane and locally dynamically increased / decreased the CCT diameter / width, showing considerable CCT membrane elasticity ( Figure 31F Polarized cystic cells in the CCT can switch their migration direction freely and can therefore migrate bidirectionally in the CCT. Here, multiple cystic cells migrate freely and bidirectionally in the long CCT, with a dynamic cell morphology. The elastic CCT membrane shields the obstacles in the external heterogeneous extracellular matrix and provides a membrane-enclosed and protected high-speed channel for the migration of cystic cells inside ( Figure 31E and Figure 31FThe average cell migration speed of multiple single Bxpc3 pancreatic carcinoma cells in CCT in vitro was 2.7±0.5μm / min (n=10 cells) ( Figure 31G Consistently, in colon cancer tissues, long (straight or curved) cystic colon carcinoma cells (CCTs) (3 μm to 6 μm in diameter) migrated within CCTs, often presenting a single, long, thin, and spindle-shaped morphology in vivo (CCTs, n = 256; tissues, n = 34; Figure 31H This suggests that migrating cystic carcinoma cells in CCT in vivo use a single mesenchymal migration pattern ( Figure 31H The above observations indicate that, in vitro and in vivo, cystic carcinomatous cells can migrate freely, dynamically, and bidirectionally in a single mesenchymal migration form as well as in thin and elongated forms within the membrane-enclosed and protected CCT. CCTs with multiple or a large number of cancerous cells inside are elongated and tubular cystic tumors in terms of topology and biological function, and are therefore called "cystic tube tumors" ( Figures 31H to 31I ).

[0197] Example XIV

[0198] Progress of the Primary Cytocystic Tumor Network

[0199] Primary malignancies are the origin of metastatic (secondary) cancers. Therefore, we asked how primary cystic tumors progress within the primary niche. We evaluated cystic tumor sphere metastasis and secondary cystic tumor sphere growth in vitro. In CC / CCT culture kit matrix, single Bxpc3 cystic tumorigenic cells proliferated and grew into large primary cystic tumor spheres (CT) within 68 hours in CC. Figure 32A Two or more primary CTs merge together through CC membrane contact, contact interface degradation, and the formation of open junctions, forming a long, irregularly shaped CT enclosed in a long, large CC ( Figure 32A The enlarged CC cells in the cystic carcinoma push the CC membrane, extend and generate CCTs. CCTs connect to each other and form a CCT network ( Figure 32A The CCT network interconnects all primary and secondary CTs and forms a cell-encapsulated tumor sphere network system (CTNS) in the wells of a 6-well plate ( Figure 32A Cell-free cystic carcinoma cells can enter the CCT through allogeneic entry. Intracellular cystic carcinoma cells undergo spontaneous decellularization and become cell-free cystic carcinoma cells outside the CC / CCT ( Figure 32A Decellularized, cell-free cystic carcinoma cells can regenerate new cell vesicles and CCTs ( Figure 32B Intracellular cystic carcinoma cells metastasize through the CCT network ( Figure 32BFurthermore, cystic carcinomatous cells metastasized and resided in interconnected nodes of CCTs, proliferated, and grew into secondary cystic tumor spheres that were integrated into established CTNS (n = 38 secondary cystic tumor spheres examined) ( FIG. 32A to FIG. 32B Intracellular cystic carcinoma cells migrate and translocate within the CTNS through the CCT network ( Figure 32B Spontaneous decellularization of cytocystic tumorspheres yielded acellular CT cytocystic fractions and acellular CCT segments, allowing for visualization of open CCT connections between CTs ( Figure 32B At 78 h, all primary and secondary CTs in the wells of a 6-well plate were interconnected and covered by an integrated cell capsule system consisting of a network of cell capsule tumorspheres (CCs) and CCTs (number of CTNSs, n = 55, Figure 32C These observations suggest that: (1) primary CTs metastasize, generating CCTs, CCT networks, and primary CTNSs; (2) metastasis of cytocystic carcinomatous cells develops into secondary cytocystic tumorspheres and forms secondary CTNSs; (3) decellularization and allograft entry enable carcinomatous cells to bidirectionally migrate out of and into CC / CCTs; and (4) primary and secondary CTNSs integrate through CCT networks and form dynamically integrated primary and secondary CTNSs enclosed in an in vitro cell capsule system.

[0200] Next, we investigated the primary cellular cystic tumor network system (CTNS) in vivo, followed by Figure 25C The subsequent stages of development are shown.

[0201] Precursor CT (PCT) formation: In early primary breast cancer, there are many different sizes (diameter / width 25μm to 120μm, Figure 26F 、 Figure 33A and Figures 34A to 34B spherical or irregularly shaped pre-cystic tumors (PCTs). The average PCT density in early-stage cancers was 202 ± 59 PCTs / mm in breast, colon, and prostate cancers, respectively. 2 , 125±32 PCT / mm 2 and 173±26 PCT / mm 2 (samples, n=3 to 6, 1 sample / patient, 1 to 2 subtypes / cancer type, Figure 34C ).

[0202] PCT to CT: Subsequently, PCT develops into different sizes (diameter / width 50 μm to 320 μm, Figures 33B to 33C ) spherical or irregularly shaped cellular cystic tumors (CT). The average CT density of early-stage cancers in breast cancer, colon cancer, and prostate cancer was 176 ± 38 CT / mm, respectively.2 、87±34 CT / mm 2 and 158±28 CT / mm 2 (samples, n=5 to 12, 1 sample / patient, 3 to 4 subtypes / cancer type) Figure 34D These data indicate that the PCT and CT densities in early-stage primary solid cancers are statistically high. Consistent with the in vitro cell-tumor sphere merging, two or more small PCT / CTs can merge into medium or large PCT / CTs in vivo ( Figure 26F and Figure 33A CT with diameter / width>50μm begins to generate some cellular cystic tubes (CCT) on the outside. Cellular cystic tumors with diameter / width>70μm generate many CCTs on the outside and form a thick surrounding CCT layer that envelops the cell cystic tumor ( Figure 26G and Figure 33D The dense CCT layer has different thicknesses (30 μm to 803 μm) ( Figure 33D Straight, curved, or coiled CCTs centrally interconnect and form a 3D CCT network that extensively interconnects primary CTs in the primary cancer niche ( Figure 33E and Figures 34E to 34F These observations suggest that cellular cystic tumors in the primary niche are physically interconnected through 3D CCT networks and form a primary cellular cystic tumor network system (CTNS) in vivo.

[0203] CC degradation: Subsequently, the CC and CCT of cystic tumors with diameter / width > 800 μm degrade into filaments and then disappear, resulting in acellular cystic malignancies / cancerous cell masses with high cell density and no CC or CCT ( Figure 33F ).

[0204] Late-stage tumors: Uncontrolled proliferation of acellular cystic carcinomatous cells produced large, irregularly shaped acellular cystic malignant tumor / cancer cell masses (up to >2 cm in width) in the examined biopsies ( Figure 33G ).

[0205] AMCC: The cell-free cystic state of these large malignant tumors / cancerized cell clusters is transient. Subsequently, some cell-free cystic cancerous cells regenerate some or many new straight, curved, or coiled CCTs and form CCT network bundles and clusters that invade and disperse in the dense cell-free cystic cancerous cell clusters, forming a complex that is a mixture of dense cancerous cell clusters and CCT networks, but without the enlarged CC that surrounds them ( Figure 33HCCT bundles and clusters generate numerous CCT cluster cavities (cross-section: circular, elliptical, or irregular, with a diameter / width of 30 μm to 200 μm; longitudinal section: straight, curved, or irregular cylindrical, with a length of 80 μm to 850 μm), which are interconnected and form CCT cluster-cavity complexes ( Figure 23C , Plate 3 and Figure 33H The cell-free cystic carcinomatous cells surrounding the CCT enter the CCT and spread through the CCT ( Figure 23C , Plate 3 and Figure 33H ).

[0206] AMCC with CCT degradation: Subsequently, CCT degraded and disappeared, leaving behind the CCT mass cavity filled with intercellular fluid ( Figure 33I At the same time, some acellular cystic carcinoma cells regenerate new CCTs ( Figure 33I CCT degradation and regeneration coexist in the cell-free cystic carcinoma cell cluster-CCT network complex (AMCC) in vivo ( Figure 33I ).

[0207] Late AMCC stage: Subsequently, after many cancerous cells enter and leave the CCT, only a few cancerous cells and many CCTs ( Figure 33J ). Then, the CCT degrades, breaks down, and disappears ( Figure 33K On the other hand, CTNS in the primary niche at advanced cancer stages still persist ( Figure 33L Some small or medium-sized CTs have cancerous cell apoptosis and CCT degradation inside ( Figure 33L These observations suggest that: 1) primary CTs develop into primary CTNSs in vivo; 2) CC / CCT degradation, proliferation of acellular cystic carcinomatous cells, and CC / CCT regeneration generate AMCCs; and 3) primary CTNSs are a dynamic system that includes CCT allograft entry, decellularization, CC / CCT generation, degradation, and regeneration, as well as cystic carcinomatous cell spread through CCTs.

[0208] Normal tissue adjacent to the tumor (NAT) is the site that CCT must pass through when it expands outward. In fact, in breast cancer tissue, a large number of CCTs invasively invade and pass through the NAT in one or more 3D directions. Figure 35A ), there are many CCTs in the bone marrow NAT of plasma cell myeloma ( Figure 35B Even in the hard tissue NAT of trabecular bone in plasma cell myeloma, a small amount of CCT is present ( Figure 35C Of the NATs across 68 cancer subtypes examined, 100% of these NATs had numerous CCTs and networks in which cystic cells migrated within them ( Figure 35D). The CCT density in soft tissue cancer can be as high as 114 CCT / mm 2 Even in hard tissue (bone) cancers, the CCT density can be as high as 10 CCT / mm 2 ( Figure 35D The above observation results (Table 6 and Figures 35A to 35D ) showed that cystic tumor progression in the primary niche includes seven major sequential stages: 1) generation of transformed acellular cystic cells, 2) regression of acellular cystic cells and generation of cellular cystic cells, 3) generation of precystic tumors and cystic tumors, 4) formation of CCT networks and primary CTNS, 5) metastasis of cystic cells through CCTs and across the NAT, 6) CC / CCT degradation and AMCC formation, 7) CCT regeneration, xenograft entry, decellularization and dynamic CTNS formation, accompanied by continuous cystic tumor metastasis and CTNS regeneration.

[0209] Example XV

[0210] Cystic metastasis of cancer cells in human tissues in vivo

[0211] Most cancer deaths are due to metastasis (Gerstberger S et al., Metastasis. Cell. 186, 564-1579 (2023)). Therefore, we evaluated how cystic carcinoma cells in CCTs spread to different tissues and organs in vivo. Single CCTs not only invade and spread to loose ( Figure 36A ) and dense ( Figures 36B to 36C ) tissue, and also invades and spreads into the hard tissue of trabecular bone ( Figures 35B to 35C ), indicating that cystic carcinoma cells in a single CCT can invade and pass through various tissues with different cell densities and ECM / matrix stiffness. A single CCT can be highly curved and very long ( Figure 36D ). 3D CCT networks enhance collective cellular cystic carcinomatosis and metastasis in and across various tissues ( Figures 33A to 33L and Figures 36E to 36F The large number and density of CCT network bundles increase the density of metastatic highways and improve the diffusion efficiency of cystic carcinoma cells ( Figures 36G to 36I The ultra-large structure of the CCT network promotes the entry of cell-free cystic carcinoma cells in dense tumor / cancerous cell clusters into the CCT and their diffusion through the CCT ( Figure 36J ).

[0212] In the bone marrow ( Figure 27B 、 Figure 35BThe presence of cystic carcinoma cells and CCT networks in the lymph nodes (Table 6), spleen, and thymus (Table 6) suggests that CC / CCT membranes can effectively shield immune cells and their attack from the outside, thereby protecting the cystic carcinoma cells inside. CCT networks are present in 290 cancer subtypes and 34 human tissues ( Figure 27A and Figure 27B , Table 6, Figure 35 and Figure 36), indicating that CCT effectively penetrates all kinds of human tissues and organs for cell cyst tumor metastasis. A large number of CCTs were found outside the body fluid vessels ( Figure 37A and Figures 38A to 38B Furthermore, the average ratio of CCT density to fluid vascular density was as high as 289 ± 6 times in the primary niche, NAT, and secondary niche ( Figure 37B The above observations indicate that cell cyst tumor metastasis through the CCT high-speed channel system dominates tumor metastasis in vivo. Sometimes, a very low percentage of CCTs invade microvessels and release cancerous cells into the circulatory system ( Figures 37C to 37F ), indicating that the release of cancerous cells caused by CCT invasion of humoral blood vessels is a source of circulating tumor cells in the blood. In addition, we tested the metastasis of cystic cancer cells in 35 types of cancer (1 to 10 secondary niches per type) and observed that cystic tumors widely spread to multiple secondary niches (Table 6), which is consistent with the clinical observation that primary tumors always metastasize to multiple tissues and organs.

[0213] Example XVI

[0214] Progress of the Secondary Cyst Tumor Network

[0215] Cancer metastasis and secondary tumor-induced tissue / organ biological failure are the main causes of cancer death (Gerstberger S et al., Metastasis. Cell. 186, 564-1579 (2023); Hebert D et al., Dissecting metastasis using preclinical models and methods. Nat Rev Cancer. 23, 391-407 (2023)). Therefore, we evaluated whether the metastatic cystic carcinoma cells in the secondary niche generate secondary cystic tumors and CTNS in vivo. In fact, after the mammary cystic carcinoma cell CCT network metastasizes and reaches the lymph nodes, they initially form a thin CCT layer that wraps around the lymph nodes ( Figure 39A , Plate 1). Subsequently, as CCT branching morphogenesis occurs, more mammary cell cystic carcinomatous cells CCT are generated, and a thicker CCT layer is formed, encapsulating the lymph node ( Figure 39A, Plate 2). Cervical cystic carcinoma cells (CCTs) invaded lymph nodes with dense lymphocytes in large numbers, and many metastatic cervical cystic carcinoma cells spread to dense lymph nodes ( Figures 39A to 39C and FIG. 40A to FIG. 40B Subsequently, in the lymph nodes, the metastatic mammary cystic carcinomatous cells in the CCT proliferate and give rise to many small secondary mammary cystic tumors in the enlarged CC with high CT density ( Figure 39D In the early stages, secondary cystic breast tumors exhibit C-shaped or irregularly shaped CCT luminal spaces (up to 30 μm in width) between the surface of the cystic carcinomatous cell mass and the CC membrane ( Figures 39D to 39E ). The mammary cystic carcinoma cells in CCT that metastasize to the lymph nodes grow into a large number of secondary mammary cystic tumors, which occupy the space of normal lymph node cells, and many normal lymph node cells disappear ( Figure 39F These secondary mammary cystic tumors are interconnected through the CCT network and form secondary mammary CTNS in the secondary niche ( Figure 39F The mean density of secondary mammary cell cystic tumors in the bladder, liver, and lymph nodes examined was 163 ± 71 CT / mm 2 、160±68CT / mm 2 and 170±56CT / mm 2 (patients, n=4 to 6) Figure 40C The above observations ( Figures 39D to 39F ) showed that primary CTNS are physically interconnected with secondary CTNS through a network of CCTs, forming an integrated primary and secondary CTNS. Small nasopharyngeal secondary cystic tumors grow and give rise to many CCTs ( Figure 39F The CC and CCT of ovarian secondary cystic tumors in the omentum degrade and produce large acellular cystic ovarian tumors / cancerous cell masses without CC / CCT (up to 2 cm or more in diameter / width) ( Figure 39G and Figure 40D Subsequently, some cancerous cells in the acellular cystic ovarian tumor in the omentum regenerate new CCTs, CTs, and CNTSs ( Figure 39G Consistently, some cervical carcinomatous cells of acellular cystic cervical tumors in lymph nodes generate numerous CCTs in lymph nodes ( Figure 39H ), and rectal cancer cells in acellular cystic rectal tumors in the mesentery generate numerous new CCTs and networks in the mesentery ( Figures 39I to 39J and Figures 40E to 40F Cell-free cystic carcinoma cells invade the regenerated CCT through allogeneic entry and escape ( Figures 39G to 39JMany secondary colonic acellular cystic carcinomatous cells in the liver spread through the regenerating colon CCT, leaving many spaces without cells and filled only with intercellular fluid ( Figure 39K ).

[0216] In late cancer stages, after secondary spread of metastatic cystic tumors through the CCT network, CCTs are degraded and the CCT network disintegrates ( Figures 39K to 39L Sometimes, many red blood cells are randomly distributed in a large area in secondary hepatocellular carcinoma of the brain, indicating that some (micro)vessels are ruptured and leaking due to CCT invasion and (micro)vascular breakdown, and red blood cells are released ( Figure 39L ). The above observations indicate that the progression of metastatic cystic tumors in the secondary niche includes six major sequential stages: 1) arrival and invasion of metastatic cystic tumor cells in CCTs in the secondary niche, 2) generation of secondary cystic tumors, 3) formation of secondary CTNS, 4) formation of dynamically integrated primary and secondary CTNSs through the CCT network, 5) degradation of CC / CCT and formation of AMCC, 6) generation of new CCTs for the next metastasis of cancerous cells (Figures 39 and 40). These observations (Table 6, Figures 37 and 39, and Figures 35, 36, 38, and 40) indicate that metastatic cystic tumor cells have the ability to generate a large number of small / medium-sized secondary cystic tumors and dense CTNS in secondary niches (in adjacent or distant organs / tissues), and lead to the disappearance of a large number of normal cells, which are subsequently affected, damaged, or even lose biological function in tissues / organs associated with the secondary niche. In conclusion, our results suggest that cystic carcinomatosis, cystic tumors, and integrated primary and secondary CTNs orchestrate membrane-shrouded cancer progression. Figure 41 and Table 7 ).

[0217] Table 7. Advantages and disadvantages of CC / CCT, CT, and CTNS compared to other organelles, compartments, and cellular activities outside of CC / CCT in vivo.

[0218]

[0219]

[0220] This disclosure provides observations and results showing that CC and CCT are ubiquitous in cancers in vivo. The PMCA2 calcium pump was identified as the most highly upregulated factor in malignant tumors, but found at very low abundance in healthy tissues. This conclusion was based on an analysis of over 10,000 samples from cancer tissue banks worldwide.

[0221] The present disclosure provides novel mechanistic insights, listed below, that greatly broaden our understanding of cancer malignancies.

[0222] (i) Discovery of cystic carcinomatous cells, cystic tumors, cystic bodies, cystic tumor network systems (CTNS), and integrated primary and secondary CTNS.

[0223] (ii) All malignant tumors produce a second outer membrane, similar to ancient life forms such as mitochondria, chloroplasts, and Gram-negative bacteria. The presence of the PMCA2 calcium pump in this second outer membrane is essential for the malignant properties of all known cancers. The absence of high levels of PMCA2 defines tissue as benign, non-malignant, and normal. This is a powerful tool for cancer diagnosis and treatment.

[0224] (iii) PMCA2 has been validated by the FDA as a marker for malignant cancer (available on the FDA website, 2020).

[0225] (iv) PMCA2-containing cytosomal vesicles appear to promote CCT extension, aiding tumor progression and even invasion into solid tissues or trabecular bone.

[0226] (v) Multicellular malignant tumor surrounded by CC membrane.

[0227] (vi) Xenograft entry enables acellular cystic carcinoma cells to invade CCTs and subsequently metastasize to CCTs; decellularization of large CCs generates acellular scaffold structures.

[0228] (vii) CCT-mediated clearance of tumor and normal cells creates empty, fluid-filled pores, rendering the tissue nonfunctional ( FIG. 34 ).

[0229] (viii) Initial metastasis involves the transport of tumor cells through CCTs, which then penetrate only the body fluid vessels to allow cancer spread via previously known pathways.

[0230] (ix) Secondary cell cyst network system using CCT for the spread of multicellular cancer metastasis.

[0231] (x) Cytocystinization cells, cystic tumors, cystic tumor metastasis in CCT networks and cystic tumor network systems in cancer development and progression, which can promote the research of effective therapies for cancer.

[0232] The discovery of cystic carcinoma cells and the progression of CT, CTNS, and integrated primary and secondary CTNS in human organs / tissues described in this disclosure can promote further cancer research, early screening, prognosis, diagnosis, drug development, therapy, and treatment. In-depth analysis of the proteome and metabolome of CCT in various cancer types may provide more insights into cancer mechanisms and bring new targets for cancer diagnosis and therapy (in progress). The time course and causal relationship of cancer progression proposed in this article are mainly based on comparisons of static snapshots of a large number of tissue samples, and these comparisons are consistent with the mechanism of progression.

[0233] The discovery of cystic carcinomatous cells and the progression of CT, CTNS, and integrated primary and secondary CTNS in human organs / tissues described in this disclosure may facilitate further cancer research, early screening, prognosis, diagnosis, drug development, therapy, and treatment.

[0234] Example XVII

[0235] Additional Materials and Methods

[0236] Reagents, antibodies, and equipment

[0237] The CC / CCT culture kit (Celldevi, available from the Celldevi website www.celldevi.com, catalog number CD0104, 6-well plate; catalog number CD0105, 12-well plate; and catalog number CD 0106, 24-well plate) and a kit having a glass coverslip at the bottom of the well and embedded with a CC / CCT culture substrate layer (catalog number CD 0112, 6-well plate) were ordered from Celldevi Inc., and the CC / CCT culture kit fixation kit (catalog number CD0201) was ordered from Celldevi Inc. Cancer cell lines and cell culture media for pancreatic cancer cell Bxpc3, breast cancer cell MCF-7, and colon cancer cell SK-CO-1 were ordered from ATCC. 13 C6, 15 N2-L-lysine (Cat. No. 88209) and 13 C6, 15 N4-L-arginine (Cat. No. 89990) was ordered from Thermo Fisher Scientific. Unipick TMCapillary units were ordered from NeuroInDx Inc. Rabbit anti-PMCA2 antibody (polyclonal, ab3529; 1:200 dilution) and mouse anti-γ-actin antibody (monoclonal, ab123034; 1:200 dilution) for immunofluorescence assays were ordered from Abcam. DAPI (1:1,000 dilution for immunofluorescence assays) was ordered from KPL. Human normal and cancer tissue samples were ordered from TissueArray and USBiolab (or donated by local hospitals).

[0238] Stable SILAC-labeled CC / CCT cultures and their collection for CC / CCT proteome analysis

[0239] According to the instructions of CC / CCT culture kit, stably SILAC-labeled Bxpc3, MCF-7 and SK-CO-1 cancer cells (labeled with 13 C6, 15 N2-L-lysine and 13 C6, 15 N4-L-arginine) implants containing 13 C6, 15 N2-L-lysine and 13 C6, 15 N4-L-arginine 1 The CC / CCT culture kit (6-well plate) was cultured in culture medium containing 500 μg / mL of culture medium. Stably SILAC-labeled cancer cells produced stably SILAC-labeled cell vesicles (CC) and cell cyst tubes (CCT). Sometimes, some intracellular cystic cancer cells spontaneously moved out of CC and CCT in vitro. After decellularization, the cells were washed three times with 1× PBS to remove the removed cancer cells. Cell-free CC / CCT (ECC / ECCT) was collected by Unipick and kept on ice and then stored at -80°C. More than 400,000 stably SILAC-labeled cell-free CC / CCT were collected over >4 years for each CC / CCT proteome analysis sample.

[0240] In vitro growth, immunohistochemical staining, and imaging of cytospheroids and vesicles

[0241] According to the kit manual, pancreatic cancer Bxpc3 cells were implanted into the CC / CCT culture kit (Cat. No. CD 0112, Celldevi). At 36 hours, Bxpc3 cells produced cell vesicles (CC). Some cell cystic carcinoma cells were decellularized. The cell cystic carcinoma cells and the decellularized CC were fixed with a kit and immunohistochemically stained. At different times of 48 hours, 72 hours, 68 hours, 74 hours, 78 hours, 84 hours, 96 hours, and 108 hours after cell implantation, Bxpc3 cancer cells produced cell cystic carcinoma cells and grew into cell cyst tumor spheres of different sizes, in which CC tightly wrapped cancerous cell clusters or these tumor spheres had wide cell cyst cavities, and the cell cyst tumor spheres were decellularized. These cell cyst tumor spheres and decellularized cell cyst tumor spheres were fixed in 6-well plates using the CC / CCT fixation kit (Celldevi, CD0201) from Celldevi Inc., then taken out and placed on a slide for immunohistochemical (IHC) staining.

[0242] Liquid chromatography tandem mass spectrometry CC / CCT proteome analysis

[0243] The stable SILAC-labeled ( 13 C6, 15 N2-L-lysine and 13 C6, 15N4-L-arginine) in the ECC / CCT to extract and purify protein. After Coomassie brilliant blue staining, the SDS gel strip of a sample was cut into 4 to 5 gel slices. After in-gel digestion of 12.5ng / μL trypsin, the digested peptides were extracted and enriched. The enriched peptides were used for liquid chromatography tandem mass spectrometry (LC-MS / MS) analysis, as previously described (Everley A et al., Quantitative cancer proteomics:stable isotope labeling with amino acids in cell culture (SILAC) as a tool for prostate cancer research. Mol Cell Proteomics.3,729-35 (2004)). The enriched peptide fractions were analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS) on a LTQ Orbitrap Velos mass spectrometer (Thermo Scientific), which was equipped with a Thermo Fisher Scientific nanospray source, an Agilent 1100 series binary HPLC pump, and a Famos autosampler. Peptides were separated on a 0.125 mm × 180 mm fused silica microcapillary column with an internal tip (prepared in-house) and an inner diameter of approximately 5 μm. The silica microcapillary column was packed with magic C18AQ C18 reversed phase resin (5 μm particle size, A pore size (100 nm; Michrom Bioresources). Separation was performed using a 57-minute gradient elution from 7% to 28% acetonitrile, with 0.125% formic acid in the eluent. The mass spectrometer was operated under default settings: full MS (automatic gain control (AGC), 1×106; resolution, 6×104; m / z range, 375 to 1,800; maximum ion time, 1,000 ms) and MS / MS (AGC, 5×103; maximum ion time, 120 ms; minimum signal threshold, 4×103; dynamic exclusion time setting, 30 s; charged ions and ions of uncertain charge state were excluded from MS / MS selection). Three independent experiments were performed.

[0244] Database searching, data filtering, validation of protein detection rates, and proteomic analysis.

[0245] Spectral data are obtained using the trademark SEQUEST TMThe tandem mass spectrometry data analysis program sold by the company was searched against a database containing a human protein sequence database (available from the ensembl.org website) and its reverse complement sequence. The LC-MS / MS identification results were filtered to a false discovery rate (FDR) of 0.98% for proteins and a FDR of 0.1% for peptides. Peptide quantification and phosphorylation site mapping were analyzed using in-house software and Ascore, as previously described (Yi T et al., Quantitativephosphoproteomic analysis reveals system-wide signaling pathways downstream of SDF-1 / CXCR4 in breast cancer stem cells. Proc Natl Acad Sci USA. 111, E2182-90 (2014)).

[0246] In vitro growth, immunohistochemical staining, and imaging of cytospheroids and vesicles

[0247] IHC staining was performed using the following antibodies and staining reagents: rabbit anti-PMCA2 polyclonal primary antibody (1:200 dilution), mouse anti-γ-actin monoclonal primary antibody (1:200 dilution), goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody Alexa Fluor Plus 555 (Thermo Fisher) and goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody Alexa Fluor Plus 488 (Thermo Fisher), and DAPI staining (1:000 dilution). Fluorescence images were captured using a Nikon 80i upright microscope with a 20× or 40× lens. All images were acquired using MetaMorph image acquisition software and analyzed using ImageJ software.

[0248] CCT histological and immunohistochemical staining analysis

[0249] Immunohistochemistry and hematoxylin and eosin (H&E) staining were performed on 9972 formalin-fixed, paraffin-embedded (FFPE) human cancer tissue samples (4 to 5 μm thickness) from 9784 cancer patients, 14 FFPE human normal tissue samples from 14 patients, and 126 FFPE human benign tumor tissue samples from 126 patients. Fluorescent immunohistochemistry was used to stain the cystic tubules using the following antibodies and staining reagents: rabbit anti-PMCA2 polyclonal primary antibody (1:200 dilution), mouse anti-γ-actin monoclonal primary antibody (1:200 dilution), goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibodies Alexa Fluor Plus 555 (Thermo Fisher) and goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibodies Alexa Fluor Plus 488 (Thermo Fisher), and DAPI staining (1:000 dilution). Fluorescence images were captured using a Nikon 80i upright microscope with a 20× or 40× lens. All images were acquired using MetaMorph image acquisition software and analyzed using ImageJ software.

[0250] Time-lapse DIC microscopy and video

[0251] Time-lapse DIC microscopy of vesicle extension and cell migration was performed using a Nikon Ti motorized inverted microscope and a digital Hamamatsu ORCA-ER cooled CCD camera with a 20× lens. The time-lapse microscope was equipped with DIC, phase contrast, and epifluorescence optics, a Prior ProScan III motorized stage and shutter, a perfect focus system, and an Okolab 37°C, 5% CO2 cage microscope incubator (Okolab). Images were taken every 30 seconds for approximately 10 to 36 hours. All images were acquired using MetaMorph software. The tracks formed by vesicle extension for 2 hours were acquired using MetaMorph and ImageJ software. The extension velocity of vesicles was also calculated by length and time measurements. Movies were prepared using images collected using time-lapse and Meta-Morph software (15 frames / s).

[0252] Brightfield microscopy and video

[0253] Bright field microscopy analysis of vesicle growth in cells with cystic activity was performed using a Nikon Eclipse TS2 inverted conventional microscope equipped with a DS-FI3 microscope camera with a 20× phase contrast lens. Videos were captured using NIS-Elements software (25 fps).

[0254] Imaging acquisition

[0255] DIC and fluorescence images of fixed cells (with or without vesicles) were acquired using an 80i upright microscope and a digital Hamamatsu ORCA-ER cooled CCD camera with a 20× or 40× lens. Brightfield phase contrast images were acquired using a Nikon digital camera. The ratio of vesicle initiation per high-power field (HPF, 200×) and the number of elongated vesicles per HPF were quantified. All images were acquired using MetaMorph image acquisition software and analyzed using ImageJ software.

[0256] Data Collection

[0257] Undegraded cellular cystic tubes (CT, unsectioned, longitudinally sectioned, and transversely sectioned) (measuring 3 μm to 6 μm in diameter) were counted using fluorescence microscopy and ImageJ. Without quantification, studies reported the presence of CT that degraded into three morphologies: thick filaments (measuring 1 μm to 2 μm in diameter), thin filaments (measuring 0.2 μm to 1 μm in diameter), or a disintegrated state. Patients who provided formalin-fixed, paraffin-embedded tissue samples provided written informed consent, indicating their understanding that biopsies (needle or surgical biopsies, from USBiomax) would be used for in vitro research purposes only. De-identified control samples of normal, benign, carcinoma in situ, carcinoma, paraneoplastic, and metastatic tissues with cancer stage identified according to the TNM staging system (cancer stage: 0, I, II, III, IV) for tumor (T), node (N), and metastasis (M). Cancerous, paraneoplastic, and metastatic cancer tissues were identified by the hospital pathology laboratory and obtained from archival materials, where the original cancer niche was identified using designated cancer-specific molecular markers. Postmortem CC / CCT degradation is extensive in autopsy tissue samples and is not quantitatively analyzed. Biopsy samples derived from FFPE (formulated plastic prepolymer) provide high-fidelity CC / CCT status compared to fresh tissue, and CC / CCT is quantified and reported.

[0258] Additional quantitative and statistical analyses

[0259] The statistical methods used for comparison are shown in the relevant figure legends and the following sections. The diameter, width and length of the cell vesicles and cell cyst tubes were measured using MetaMorph or ImageJ. The time from the generation of cell vesicles to the decomposition of cell-free cell vesicles (or cell cyst tubes) was timed. For the determination of the lifespan of cell cystic cells and cell cyst tumor spheres, at least 20 cell cystic cells or cell cyst tumor spheres were measured for each condition, and a two-tailed Student's test was used to determine statistical significance. The time of shooting the video was marked in the format of hours: minutes: seconds (in Figure 28A and Figure 29A ), while the time after cell implantation is 96 hours in FIG28 and 108 hours in FIG29. Figure 31A In the video, the time of shooting was just after the cell implantation. The curve graph is the mean ± SD. In the CCT analysis of cancer types / subtypes, at least 3 samples of each cancer subtype were examined. In the cell capsule quantification assay, for each sample, the number of complete cell capsules was counted in 5 areas (0.35 mm × 0.35 mm, length × width) of the sample (top, bottom, left, right and center), and the cell capsule density (CCT / mm) of each area was calculated and determined. 2 The average CCT density of the five sites was considered the overall CCT density of the sample and rounded up. The quantification of body fluid vascular density was performed using a method similar to that used for CCT quantification.

[0260] Other embodiments will be apparent to those skilled in the art. It should be understood that the foregoing description is provided for clarity only and is exemplary only. The spirit and scope of the present invention are not limited to the above embodiments, but are encompassed by the appended claims. All publications and patent applications cited above are incorporated herein by reference in their entirety for all purposes, to the same extent as if each individual publication or patent application was specifically indicated to be so incorporated by reference.

Claims

1. A method for detecting cell cysts in a tissue of a subject, comprising: a) obtaining tissue from the subject, and b) by contacting the tissue with anti-plasma membrane Ca 2+ -ATPase (PMCA) antibody contact to detect the presence of cell cysts in the tissue.

2. The method according to claim 1, wherein The subject is a mammal.

3. The method according to claim 1, wherein The subject is a human or an animal.

4. The method according to claim 1, wherein The subject is healthy or diseased.

5. The method according to claim 1, wherein The subject has cancer or is suspected of having cancer.

6. The method of claim 1, wherein: The tissue is obtained from any location within the subject.

7. The method of claim 1, wherein: The tissue includes healthy, benign or cancerous tissue.

8. The method of claim 7, wherein: The cancerous tissue includes any tumor tissue including the primary site of tumor origin or the secondary site of tumor metastasis.

9. The method of claim 7, wherein: Tissue adjacent to the cancerous tissue is obtained.

10. The method of claim 1, wherein: The tissue is obtained by biopsy.

11. The method according to claim 10, wherein: The biopsy includes clinical biopsy, bone marrow puncture and biopsy, cardiac biopsy, hollow needle biopsy, endometrial biopsy, endoscopic biopsy, excisional biopsy and incisional biopsy, fine needle aspiration biopsy, lymph node biopsy, needle biopsy, open biopsy, punch biopsy, sentinel lymph node biopsy, cutting biopsy, skin biopsy, etc.

12. The method of claim 1, wherein: The PMCA includes PMCA-1, PMCA-2, PMCA-3 and PMCA-4.

13. The method of claim 1, wherein: The antibodies include anti-PMCA-1 antibody, anti-PMCA-2 antibody, anti-PMCA-3 antibody and anti-PMCA-4 antibody.

14. A method for predicting the grade of cancer metastasis in a subject, comprising: a) obtaining tissue from a subject, and b) by contacting the tissue with anti-plasma membrane Ca 2+ -ATPase (PMCA) antibody contact to detect the presence of cell cysts in the tissue.

15. The method of claim 14, wherein: The presence of cellular ducts in the tissue is associated with metastatic cancer.

16. The method of claim 14, wherein: The density and morphology of the cell cysts detected in the tissue are used to predict the grade of cancer metastasis in the subject.

17. The method of claim 14, wherein: Increased density of cellular cysts correlated with more advanced stages of cancer metastasis.

18. The method of claim 14, wherein: Thinner and cloudier cell cyst morphology and more degradation were associated with more advanced grades of cancer metastasis.

19. The method of claim 14, wherein: If no cell capsule tube (CCT) was detected and no CCT degradation was detected, grade 0 cancer metastasis (CM) was predicted.

20. The method of claim 14, wherein: If the density of CCT is detected at 1 / mm 2 Up to 10 / mm 2 If the expression of CCT is within the range of 0.05 and no CCT degradation is detected, it is predicted to be grade 1 cancer metastasis (CM).

21. The method of claim 14, wherein: If the density of CCT is detected at 11 / mm 2 Up to 40 / mm 2 If the CCT is within the range of 0.05 and CCT is degraded into filaments and filaments, it is predicted to be grade 2 cancer metastasis (CM).

22. The method of claim 14, wherein: If the density of CCT is detected at 41 / mm 2 Up to 80 / mm 2 If the CCT is within the range of 0.05 and CCT is degraded into filaments, thin filaments and filament-like structures, it is predicted to be grade 3 cancer metastasis (CM).

23. The method of claim 14, wherein: If the density of CCT is detected to be >81 / mm 2 If the CCT is within the range of 0.05, and the CCT is degraded into a cloud-like morphology or completely decomposed, it is predicted to be grade 4 cancer metastasis (CM).

24. The method of claim 14, wherein: The subject is a mammal.

25. The method of claim 14, wherein: The subject is a human or an animal.

26. The method of claim 14, wherein: The subject is healthy or diseased.

27. The method of claim 14, wherein: The subject has cancer or is suspected of having cancer.

28. The method of claim 14, wherein: The tissue is obtained from any location within the subject.

29. The method of claim 14, wherein: The tissue includes healthy, benign or cancerous tissue.

30. The method of claim 29, wherein: The cancerous tissue includes any tumor tissue including the primary site of tumor origin or the secondary site of tumor metastasis.

31. The method of claim 29, wherein: Tissue adjacent to the cancerous tissue is obtained.

32. The method of claim 14, wherein: The tissue is obtained by biopsy.

33. The method of claim 32, wherein: The biopsy includes clinical biopsy, bone marrow puncture and biopsy, cardiac biopsy, hollow needle biopsy, endometrial biopsy, endoscopic biopsy, excisional biopsy and incisional biopsy, fine needle aspiration biopsy, lymph node biopsy, needle biopsy, open biopsy, punch biopsy, sentinel lymph node biopsy, cutting biopsy, skin biopsy, etc.

34. The method of claim 14, wherein: The PMCA includes PMCA-1, PMCA-2, PMCA-3 and PMCA-4.

35. The method of claim 14, wherein: The antibodies include anti-PMCA-1 antibody, anti-PMCA-2 antibody, anti-PMCA-3 antibody and anti-PMCA-4 antibody.

36. A method of screening a subject for cancer, comprising a) obtaining tissue from a subject, and b) by contacting the tissue with anti-plasma membrane Ca 2+ -ATPase (PMCA) antibody contact to detect the presence of cell cysts in the tissue.

37. The method of claim 36, wherein: The presence of the cellular cysts in the tissue indicates that the tissue is cancerous.

38. The method of claim 36, wherein: The absence of the cellular capsule in the tissue indicates that the tissue is healthy or benign.

39. The method of claim 36, wherein: The subject is a mammal.

40. The method of claim 36, wherein The subject is a human or an animal.

41. The method of claim 36, wherein: The subject is healthy or diseased.

42. The method of claim 36, wherein: The subject has cancer or is suspected of having cancer.

43. The method of claim 36, wherein: The tissue is obtained from any location within the subject.

44. The method of claim 36, wherein: The tissue includes healthy, benign or cancerous tissue.

45. The method of claim 44, wherein: The cancerous tissue includes any tumor tissue including the primary site of tumor origin or the secondary site of tumor metastasis.

46. ​​The method of claim 44, wherein Tissue adjacent to the cancerous tissue is obtained.

47. The method of claim 36, wherein: The tissue is obtained by biopsy.

48. The method of claim 47, wherein The biopsy includes clinical biopsy, bone marrow puncture and biopsy, cardiac biopsy, hollow needle biopsy, endometrial biopsy, endoscopic biopsy, excisional biopsy and incisional biopsy, fine needle aspiration biopsy, lymph node biopsy, needle biopsy, open biopsy, punch biopsy, sentinel lymph node biopsy, cutting biopsy, skin biopsy, etc.

49. The method of claim 36, wherein The PMCA includes PMCA-1, PMCA-2, PMCA-3 and PMCA-4.

50. The method of claim 36, wherein The antibodies include anti-PMCA-1 antibody, anti-PMCA-2 antibody, anti-PMCA-3 antibody and anti-PMCA-4 antibody.

51. A method for detecting cell cysts in a tissue sample of a subject, comprising: a) contacting the tissue sample with an antibody, wherein the antibody binds to the plasma membrane Ca in the cell capsule of the cell capsule 2+ -ATPase (PMCA) binding, and b) detecting the presence of cell cysts in the tissue sample by detecting the antibody.

52. The method of claim 51, wherein The subject is a mammal.

53. The method of claim 51, wherein The subject is a human or an animal.

54. The method of claim 51, wherein The subject is healthy or diseased.

55. The method of claim 51, wherein The subject has cancer or is suspected of having cancer.

56. The method of claim 51, wherein The tissue sample is obtained from any location within the subject.

57. The method of claim 51, wherein The tissue sample may include healthy, benign or cancerous tissue.

58. The method of claim 57, wherein The cancerous tissue includes any tumor tissue including the primary site of tumor origin or the secondary site of tumor metastasis.

59. The method of claim 57, wherein The tissue sample includes tissue adjacent to the cancerous tissue.

60. The method of claim 51, wherein The tissue sample is obtained by biopsy.

61. The method of claim 60, wherein The biopsy includes clinical biopsy, bone marrow puncture and biopsy, cardiac biopsy, hollow needle biopsy, endometrial biopsy, endoscopic biopsy, excisional biopsy and incisional biopsy, fine needle aspiration biopsy, lymph node biopsy, needle biopsy, open biopsy, punch biopsy, sentinel lymph node biopsy, cutting biopsy, skin biopsy, etc.

62. The method of claim 51, wherein The PMCA includes PMCA-1, PMCA-2, PMCA-3 and PMCA-4.

63. The method of claim 51, wherein The antibodies include anti-PMCA-1 antibody, anti-PMCA-2 antibody, anti-PMCA-3 antibody and anti-PMCA-4 antibody.

64. The method of claim 51, wherein The antibody comprises a detectable label, and the detecting comprises detecting the detectable label.

65. The method of claim 51, wherein The antibody is a primary antibody, and wherein the first antibody is detected by a secondary antibody that binds to the first antibody.

66. The method of claim 51, wherein The antibody is a polyclonal or monoclonal antibody.

67. The method of claim 51, further comprising detecting cell cysts, cell cystic vesicles, cell cystic cancerous cells, cell cystic tumor spheres, and cell cystic tumor sphere network systems.

68. The method of claim 51, wherein The presence of cellular tubes in the tissue sample is indicative of cancer metastasis.

69. A method for detecting cell cysts in vitro, comprising a) contacting the cell capsule in the 3D matrix culture with an antibody, wherein the antibody binds to the plasma membrane Ca in the cell capsule membrane of the cell capsule 2+ -ATPase (PMCA) binding, and b) detecting the presence of the cell capsule in the 3D matrix culture by detecting the antibody.

70. The method of claim 69, wherein The PMCA includes PMCA-1, PMCA-2, PMCA-3 and PMCA-4.

71. The method of claim 69, wherein The antibodies include anti-PMCA-1 antibody, anti-PMCA-2 antibody, anti-PMCA-3 antibody and anti-PMCA-4 antibody.

72. The method of claim 69, wherein The antibody comprises a detectable label, and the detecting comprises detecting the detectable label.

73. The method of claim 69, wherein The antibody is a primary antibody, and wherein the first antibody is detected by a secondary antibody that binds to the first antibody.

74. The method of claim 69, wherein The antibody is a polyclonal or monoclonal antibody.

75. The method of claim 69, wherein It also includes the detection of cell cysts, cell cystic vesicles, cell cystic cancerous cells, cell cystic tumor spheres and cell cystic tumor sphere network systems.

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