Integrated regulation of cell migration

Abstract

The present invention provides systems and methods for reducing cell metastatic potential and / or modulating solid biological function by effecting changes in at least one biophysical (e.g., bioelectric) characteristic, at least one structural characteristic, and at least one physiological characteristic of a cell. The systems and methods can include administration of biofield therapy, and in some embodiments, altering expression of genes associated with changes in bioelectric, structural, and / or physiological characteristics.

Description

[0001] This invention relates to the comprehensive effects, assessment, and mechanisms for regulating cellular structure and function to address carcinogenic states and / or functional problems, particularly effects and mechanisms based on bioelectricity, energy, and / or information, and their applications. Background of the Invention Cancer remains a leading cause of death, second only to heart disease, and is now the leading cause of death for people under 85. In the United States, one in six deaths is caused by cancer. Despite decades of research, the five-year survival rate for lung, liver, esophageal, and pancreatic cancer remains below 25%. Pancreatic cancer is currently the third leading cause of cancer-related deaths in the United States, with a particularly poor prognosis; the average survival from diagnosis to death is less than six months.

[0003] Cancer cells typically grow faster than normal cells and have a greater potential for metastasis, which leads to the spread of cancer within the patient's body. Cancer can be understood as the loss of the ability of the surrounding environment to regulate the behavior of isolated cells (Wang IC, et al., Molecular and Cellular Biology. 2005;25(24):10875-10894; Myatt SS, et al., Nature Reviews Cancer.2007;7(11):847-859; Anders L, et al., Cancer Cell.2011;20(5):620-634), and / or dysregulation of cellular pathways, cell communication, and cellular states. For example, some cancer cell types exhibit depolarized cell voltage potentials. Some cancer cell types, including pancreatic cancer cells, may show elevated expression of certain proteins that drive metastasis.

[0004] Most currently approved cancer treatments are toxic to patients and typically fail to restore cancer cells to a non-cancerous state and / or non-cancerous function, nor do they maintain the homeostasis of potential cancer cells. These approved treatments usually target only one or two abnormal proteins or pathways, have limited biological scope, and may lead to treatment resistance. Invention Abstract

[0005] This invention provides systems and methods for modulating the biological functions of cells by directly or indirectly inducing various biophysical, structural, and physiological changes in some or all of the cells. The cells may be in vitro or in vivo. The systems and methods can be used to treat cancerous cell activity (cancer) in an organism, and / or to evaluate treatment methods and / or therapists.

[0006] In one embodiment, the system and method can induce biophysical changes, such as changes in bioelectrical characteristics, in cells (e.g., a first subset of cells). Such biophysical changes (e.g., changes in bioelectrical characteristics) can cause structural and / or physiological changes in the same cells (the first subset of cells) or other cells (a second subset of cells). In one embodiment, the first subset of cells includes immune cells. In one embodiment, the second subset of cells includes cancer cells or tumor cells. Typically, the biophysical changes, the structural changes, and the physiological changes each cause changes in one or more aspects of one or more other categories of cells, for example, changes among themselves. The biophysical changes, the structural changes, and the physiological changes each can be associated with one or more other categories of cells. In some embodiments, the system and method further include altering the expression of genes associated with changes in the bioelectrical characteristics, the structural characteristics, and / or the physiological characteristics. The altered gene expression may include epigenetic effects, and changes in the transcriptomic signature and / or proteomic signature of the bioelectrical characteristics, the structural characteristics, and / or the physiological characteristics.

[0007] In some embodiments, the system and method may reduce the metastatic potential of cells (including, but not limited to, reducing the incidence and / or likelihood of metastasis, wherein metastasis includes and / or is characterized by, but not limited to, cell invasion and / or migration) and / or reduce tumor growth or size. In some embodiments, one, two, or each of the biophysical changes, the structural changes, and the physiological changes is each associated with at least one, two, three, four, five, or all of the following: restriction of cell cycle progression or cell cycle “arrest” (e.g., longer cell residence time in the G0 / G1 and / or G2 / M phases of the cell cycle) and / or expression of cell cycle regulatory proteins; reduction of cell-free DNA or endorphin; reduction of cell growth and / or metabolism; reduction of cell migration; inhibition of stemness; and reduction of cell invasion.

[0008] Making biophysical changes or altering biophysical aspects may include at least one, two, three, or all of the following: altering the intracellular ion levels of the cell (e.g., altering, for example, decreasing, the intracellular calcium ion levels of the cell); altering the cell membrane potential of the cell (e.g., decreasing the cell membrane potential and / or hyperpolarizing the cell); altering the membrane potential of the mitochondria (e.g., decreasing the mitochondrial membrane potential); and altering the expression of ion channel genes (including, but not limited to, genes of proteins associated with voltage-gated ion channels, sodium channels, potassium channels, and / or calcium-regulated channels (e.g., Nav1.5, Kv1.3, and / or KCa3.1)).

[0009] Structural changes or alterations in structural aspects may include at least one, two, three, four, or all of the following: altering the structural characteristics of the mitochondria (e.g., size, swelling, cristae organization); altering the cytoskeleton organization of the cell (e.g., reducing cytoskeleton structure, regulating the activity of actin (including F-actin and / or β-actin) and / or tubulin); altering the cell's membrane ion channels (e.g., voltage-gated ion (e.g., calcium and / or potassium) channels on the cell membrane, for example, altering membrane ion proteins (e.g., CFTR, GABARP, and / or Ryanodine receptor 3), for example, altering (e.g., reducing) cholinergic receptor anions). This includes altering (e.g., reducing) the expression of subchannel proteins (e.g., CFTR), GABA receptor proteins (e.g., GABRP), and / or altering (e.g., reducing) calcium release proteins (e.g., Ryanodine receptor 3), and / or altering the expression of genes encoding one or more membrane ion proteins (e.g., CFTR, GABARP, and / or RYR3); altering the expression of cytoskeleton genes (e.g., activating cytoskeleton-related genes such as actin, pectin, spectrin, TRIOBP, kinases, and / or PDZ); and altering the expression of genes associated with said cytoskeleton communication (e.g., altering cytoskeleton communication factors (PDZ)).

[0010] Physiological changes or alterations in physiological aspects may include one, two, three, four, five, six, seven, or all of the following: altering one or more EMT marker proteins in the cells (e.g., increasing the epithelial marker E-cadherin and / or decreasing the mesenchymal marker N-cadherin and / or Twist). 1) Regulate FOXM1 in the cells (e.g., reduce FOXM1 levels or activity); alter mitochondrial energy metabolism in the cells (e.g., alter mitochondrial function, expression of protein kinases and / or ATP-binding genes, and / or glucose and glutamine metabolism); alter the expression of one or more EMT genes (e.g., reduce N-cadherin expression, reduce CD44 expression, and / or increase E-cadherin expression); alter the expression of one or more FOXM1-related protein genes (e.g., reduce pAKT, ALKBH5, cyclin B1, and / or CDK1 expression; and / or increase P21 expression); alter the expression of one or more serine / threonine kinase genes; alter the expression of cell junction genes; and alter the expression of extracellular matrix genes.

[0011] In some embodiments, the system and method further include additional modifications to biophysical, structural, and / or physiological aspects. These additional modifications may include one, two, three, four, five, or all of the following: modulating hypoxia and / or anoxic conditions in the cells (e.g., reducing protein expression of one or more hypoxia markers HIF-1 and / or PDK1); altering the expression of genes associated with the Ras pathway, extracellular matrix, serine / threonine kinases, and / or cell connectivity; altering cell signal transduction (e.g., altering microtubule and / or cytoskeleton markers); and modulating the PI3K / mTOR pathway in the cells (e.g., reducing pAkt levels and / or the ratio of pAkt to total Ak). The ratio of t); and altering the levels of immune cells (e.g., B cells and / or T cells, e.g., reducing B cells and / or increasing T cells); reducing nodules (e.g., reducing the number or visibility of nodules) and / or metastases when cells are part of a cancerous organism, e.g., reducing liver nodules and / or metastases to said liver; and altering the tumor microenvironment (TME) when cells are part of a tumor-bearing organism (e.g., reducing the percentage of B lymphocytes, increasing the percentage of cytotoxic T cells, and / or reducing M2 tumor-associated macrophages).

[0012] In some embodiments, the system and method include the application of biofield therapy, such as the Bengston Energy Healing Method. The biofield therapy may directly or indirectly induce or induce biophysical changes (e.g., changes in bioelectrical characteristics), structural changes, and / or physiological changes. In some embodiments, the application of the biofield therapy is associated with at least one, at least two, or all of the following: the biophysical changes, the structural changes, and the physiological changes. In some embodiments, it includes, for example, the application of biofield therapy to an organism, where the biophysical changes, the structural changes, and / or the physiological changes are each associated with a reduction in migration and / or invasion (e.g., a reduction in cell migration and / or invasion and / or a reduction in tumor size or burden). The reduction in cell migration and / or invasion can be achieved without toxicity to the cells or the host of the cells. In some embodiments, the biofield therapy or a portion thereof is applied for approximately 15 minutes or approximately 30 minutes daily. In some embodiments, the biofield therapy or a portion thereof is applied for at least 2 days or at least 3 days per week. The biofield therapy may be applied, in whole or in part, locally or remotely. In some implementations, the biofield therapy is administered by more than one therapist. A portion (including some or all) of the biofield therapy may be administered by a group of therapists.

[0013] The application (e.g., duration and / or frequency) and / or the user of the biofield therapy may be determined based on the efficacy indicators of the person administering the biofield therapy and / or the implementation of the biofield therapy. The efficacy indicators of the biofield therapist may be based on biophysical, structural, or physiological changes in one or more treated entities, including combinations and embodiments of the various effects described in this invention (e.g., as stated above and in the filed claims). The efficacy indicators of the biofield therapist may be based on or reflect measurements of the therapist's electroencephalogram (EEG). The efficacy indicators of the biofield therapist may be based on or reflect the degree of association between changes in the therapist's EEG (e.g., EEG) and any effects and / or integration of effects described in this invention, including combinations and embodiments of the various effects described in this invention (e.g., as stated above and in the filed claims). In one embodiment, calcium signaling and / or β-actin in the treated cells are correlated with changes in the therapist's EEG (electroencephalogram).

[0014] The system and method may include the application of biofield therapy, as well as chemical agents and / or radiotherapy. In such embodiments, the application of biofield therapy may mitigate the adverse effects of the application of the chemical agents and / or radiotherapy. In such embodiments, the application of biofield therapy may enhance the efficacy of the chemical agents and / or radiotherapy.

[0015] The present invention also provides systems and methods for improving cell function, for example, by reducing the growth and / or metastatic potential of cells and / or cancer cells through at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the following: altering membrane potential (e.g., decreased membrane potential and / or hyperpolarization of cells); altering cytoskeleton organization (e.g., reducing cytoskeleton structure, regulating actin and / or tubulin activity); regulating energy metabolism (e.g., altering mitochondrial function, expression of protein kinases and / or ATP-binding genes, and / or glucose and glutamine metabolism); altering mitochondrial morphology (e.g., size, swelling, cristae tissue degree); and restricting the progression through the cell cycle. (e.g., cells remain in the G0 / G1 and / or G2 / M phases of the cell cycle for a longer period); regulate hypoxia and / or anoxic conditions (e.g., reduce protein expression of one or more hypoxia markers HIF-1 and / or PDK1); regulate the FOXM1 and / or PI3K / mTOR pathways (e.g., reduce the level or activity of FOXM1 and / or PI3K / mTOR); regulate epigenetic processes (e.g., alter the expression of genes in the cells, including genes related to the Ras pathway, extracellular matrix, cell junctions, and cytoskeleton); and alter the expression of EMT markers in the cancer cells (e.g., increase the epithelial marker E-cadherin and / or decrease the mesenchymal marker N-cadherin and / or Twist 1). The methods may include the administration of biofield therapy, and the effects may be a result of the administration of said biofield therapy.

[0016] In some implementation schemes, reducing metastatic potential includes one, two, three, four, or all of the following: reducing cell growth and / or metabolism; reducing the size of the tumor containing the cells; reducing the likelihood of metastasis; reducing the migration or other metastatic behavior of cells or their progeny; and reducing the penetration or other metastatic ability of cells or their progeny.

[0017] In some embodiments, the system and method comprise two or more of the following: altering bioelectricity (e.g., altering the membrane potential of the cell or mitochondria within the cell, e.g., reducing membrane potential and / or hyperpolarizing the cell); altering the cytoskeleton organization of the cell (e.g., reducing cytoskeleton structure, modulating the activity of actin (including β-actin and / or F-actin), and / or tubulin); and modulating FOXM1 in the cell (e.g., reducing FOXM1 levels or activity). For example, the system and method comprise modulating FOXM1 in the cell and altering the cytoskeleton organization of the cell. In one embodiment, FOXM1 modulation is achieved without toxicity to the cell and / or the host of the cell. In one embodiment, the method comprises administering a biofield therapy and reducing FOXM1 in the cell. In one embodiment, the reduction of FOXM1 in the cell is associated with a decrease in cell migration and / or invasiveness.

[0018] The cells in the systems and methods of the present invention may comprise cells within or derived from organoids, such as patient-derived organoids and / or pancreatic cancer organoids. The cells may comprise pancreatic ductal adenocarcinoma (PDAC) cells. The cells may comprise cells derived from cell lines (e.g., one or more of PANC-1, Panc02, MiaPaca, Capan-2, KPCY, COLO357, and L3.7 cell lines). In some embodiments, the cells are human cells. In some embodiments, the cells are mouse cells. In some embodiments, the cells are a component of an organism (e.g., human or mouse) (present as part of the organism).

[0019] The systems and methods provided by this invention also include methods for determining therapeutic efficacy, including therapeutic efficacy and / or the administerer or treatment device for administering the therapy, such as therapy administration (e.g., dosage or treatment regimen), biofield therapy technology, therapist, or device for administering biofield therapy or its equivalent, for example, based on efficacy indicators. Efficacy indicators may include and / or be based on the effects and effects integration described in this invention, including but not limited to combinations and embodiments of the effects described in this invention (e.g., the foregoing and filed claims). For example, efficacy indicators may include and / or be based on measurements indicating a decrease or downregulation of FOXM1, for example, relative to a standard or previous measurement. Efficacy indicators may include and / or be based on measurements indicating at least one, two, three, four, five, six, seven, or all of the following: changes in membrane voltage potential, changes in cytoskeleton organization, regulation of energy metabolism, restriction of cell cycle progression, hypoxia and / or anaerobic regulation, regulation of the PI3K / mTOR pathway, regulation of epigenetic processes, and changes in the expression of EMT markers in the cancer cells. The efficacy indicators may include and / or be based on the determination of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of the following: membrane ion channels, cell membrane voltage potential, membrane potential of the mitochondria, expression of voltage-gated ion channel genes, structure of the mitochondria, cytoskeleton organization of the cells, endosmosis of the cells, expression of cytoskeleton genes, EMT markers, FOXM1, energy metabolism of the mitochondria of the cells, expression of one or more EMT genes, and expression of one or more FOXM1-related genes, including those listed above and in this invention.

[0020] The systems and methodologies described in this invention can be used to treat tumor cells and / or potentially metastatic cells (including, but not limited to, pancreatic cancer cells), and / or to design or evaluate treatment regimens for tumor cells and / or potentially metastatic cells (including, but not limited to, pancreatic cancer cells) (e.g., assessing and / or comparing the potential effectiveness of various treatment regimens). The systems and methodologies can also be used to treat, for example, cancers and / or cancer cells or cell lines, and / or to design or evaluate treatment regimens for, for example, cancers and / or cancer cells or cell lines (e.g., assessing and / or comparing the potential effectiveness of various treatment regimens). In some embodiments, the cancers or cell lines have high FOXM1 expression, such as BRCA, CESC, LUAD, STAD, and THCA tumor tissues. The use of the systems and methodologies in treating and / or designing or evaluating treatment regimens may include and / or be based on any effects and / or integration of effects described in this invention, including but not limited to combinations and embodiments of the effects described in this invention (e.g., the foregoing and the filed claims).

[0021] Brief description of the attached figures Figure 1 Factors that may be involved in or influenced by mechanisms of cell homeostasis and / or maintenance or restoration of non-cancerous cell states, including factors that can be characterized as structural, physiological and / or biophysical.

[0022] Figure 2 An exemplary comparison of the effects of three treatment modalities (drug therapy, radiation therapy, and biofield therapy) on cellular structural characteristics (e.g., cytoskeleton), physiological characteristics (e.g., protein pathways and epigenetic regulation), and biophysical characteristics (e.g., membrane potential). Biofield therapy affects these three aspects of the cell in a comprehensive manner, thereby rebalancing cellular structure and function, whereas drug therapy and radiation therapy may target only one or more aspects, damaging others.

[0023] Figure 3 An overview of the mechanisms of action of BT on the structure, physiology, and biophysics of cells, and its corresponding effects on certain characteristics and indicators of cancer, including electrical and molecular mechanisms of action and their interrelationships.

[0024] Figure 4 (A) Effects of biofield therapy (BT) on the growth of pancreatic ductal adenocarcinoma cells, including human PANC-1 (a and b) and COLO357 cells (c), mouse Panc02 cells, and immortalized human pancreatic ductal epithelial cells (HPDE-1 / E6E7; d) treated by Biofield Therapist 1 (Therapist 2), and PANC-1 (f), COLO357 (g), L3.7 (h), and HPDE cells (i) treated by Biofield Therapist 2 (Therapist 2), compared with sham control (SC) and incubator control (IC). (B) Effects of BT on the growth of human patient-derived pancreatic cancer organoids (PDO, AM67): (a) mean number of PDO AM67 cells 11 days after BT (15 min and 30 min) or SC treatment; (b) size of PDO AM67 cells after BT treatment (15 min) or SC treatment. All data are expressed as mean ± SD (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n>4).

[0025] Figure 5 The effects of biofield therapy (BT) on the growth of PANC-1 cells (AC) in five different experimental groups and Panc02 cells (DF) in three different experimental groups were investigated 15 minutes and 1 hour after treatment, and compared with the sham control (SC) and incubator control (IC). All data are expressed as mean ± SD (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n>4).

[0026] Figure 6 The effect of biofield therapy (BT) on the growth of MiaPaCa-2 (A) and KPCY cells (B) compared with the sham control (SC) and incubator control (IC). Data are presented as mean ± SD (*p<0.05; **p<0.01; ***p<0.001; n>4).

[0027] Figure 7 The effect of biofield therapy (BT) on the growth of PANC-1 cells treated by a third therapist (therapist 3) compared with the sham control (SC) and incubator control (IC). Data are presented as mean ± SD (*p<0.05; ***p<0.001; n>4).

[0028] Figure 8 Transmission electron microscopy (TEM) images showing morphological changes in PANC-1 cells from incubator control groups (A and D), sham control groups (B and E), and biofield treatment groups (C and F) 15 minutes after exposure to treatment (samples were collected immediately after treatment). Note that mitochondria appear normal in the incubator control and sham control groups (D and E) (red arrows), while mitochondria are swollen in the biofield treatment group (yellow arrow, F). Images AC are magnified 5000x; image DF is magnified 25000x.

[0029] Figure 9 Cell cycle analysis of PANC-1 cells. (AC) PANC-1 cells were seeded in 6-well plates overnight and then exposed to biofield therapy (BT), sham control (SC), or incubator control (IC) for 15 minutes, respectively. Cells were collected immediately after treatment. Cell cycle analysis was performed using propidium iodide staining and flow cytometry. (A) Histograms of the cell cycle populations of PANC-1 cells in the IC, SC, and BT groups after 15 minutes of treatment. (B and C) Quantitative analysis of PANC-1 cells at different cell cycle phases after 15 minutes of BT, SC, or IC treatment in two independent experiments. (D) PANC-1 cells were seeded in 6-well plates, cultured for 48 hours, and then treated with BT (15 minutes). (E and F) L3.7 cells and COLO357 cells were seeded overnight and then exposed to BT for 30 minutes. All data are expressed as mean ± SD (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n=3).

[0030] Figure 10(A and B) Effects of biofield therapy (BT), sham control (SC), or incubator control (IC) on the cell cycle of PANC-1 cells in two separate studies. (C and D) Cell cycle of PANC-1 cells 15 minutes after receiving BT treatment from a second therapist (therapist 2) (C) and a third therapist (therapist 3) (D). All data are presented as mean ± SD (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; n>4).

[0031] Figure 11 Cell membrane voltage potentials of pancreatic ductal adenocarcinoma cells. (A) Representative fluorescence imaging of DiBAC4 and Hoechst in PANC-1 cells under IC, SC, and BT (15 min) conditions. (B) Quantitative analysis of cell voltage in PANC-1 cells 15 min after BT treatment. (C) Quantitative analysis of cell voltage 15 min after BT treatment for two consecutive days. (D) Cell voltage of MiaPaCa-2 cells measured 1 hour after BT treatment. (E) Cell voltage of KPCY cells measured 1 hour after BT treatment. All data are expressed as mean ± SD (*p<0.05; **p<0.01; ***p<0.001; n>4).

[0032] Figure 12 Cell membrane voltage potentials of (A) MiaPaCa-2 cells and (B) KPCY cells immediately after treatment with biofield therapy (BT), sham control (SC), or incubator control (IC). Data are expressed as mean ± SD (**p<0.01; ***p<0.001; n>6).

[0033] Figure 13 Compared with the sham control group (SC) and the incubator control group (IC), biofield therapy (BT) significantly inhibited the invasiveness and migration of pancreatic ductal adenocarcinoma (PDAC) cells. (A) Invasiveness of PDAC cells: PANC-1(a) cells, L3.7(b) cells, COLO357(c) cells, KPCY(d) cells; and migration of PANC-1(e) cells and L3.7(f) cells. (B) Migration of MiaPaCa-2 cells as detected by scratch assay, all data are from the 48-hour study. (C) Invasiveness of PANC-1(a) cells, COLO357(b) cells, and L3.7(c) cells treated with biofield therapy BT, and migration of PANC-1(d) cells and L3.7(e) cells. All data are expressed as mean ± SD (*p<0.05; ***p<0.001; ****p<0.0001).

[0034] Figure 14 15 or 30 minutes after treatment by Therapist 1, biofield therapy (BT) significantly inhibited the invasiveness of PANC-1 cells (AD) and L3.7 cells (E) compared with the sham control group (SC). (F) Invasiveness of PANC-1 cells cultured in SC or BT incubators. Data are presented as mean ± SD (**p<0.01; ***p<0.001; ****p<0.0001).

[0035] Figure 15 The expression of proteins regulating cell cycle and cell signal transduction in PANC-1 cells 15 minutes after exposure to biofield therapy (BT) was compared with that of a sham control group (SC) and an incubator control group (IC). (A) Heatmap of cell cycle regulatory proteins and cell signal transduction proteins in PANC-1 cells by reversed-phase protein microarray (RPPA) analysis. According to the Wilcoxon rank-sum test, the proteins included in the heatmap were statistically significant between different treatment groups, with p-values ​​below the Benjamini-Hochberg false discovery rate threshold (<0.05). (B) Expression of cell cycle regulatory proteins and epigenetic modification proteins (e.g., CDK1, cyclin B1, FOXM1, and ALKBH5) in PANC-1 cells after BT treatment, compared with the SC group, by RPPA detection (n=7). (C and D) Western blot analysis of FOXM1 (C) and p21 (D) in PANC-1 cells treated with BT for 15 minutes. All data are expressed as mean ± SD (*p<0.05; ***p<0.001; n=7).

[0036] Figure 16 Compared with the sham control group (SC), the expression of proteins regulating cell cycle and cell signal transduction in PANC-1 cells 30 minutes after exposure to biofield therapy (BT) was compared. (A) Heatmap of cell cycle regulatory proteins and cell signal transduction proteins in PANC-1 cells as detected by reversed-phase protein microarray (RPPA). According to the Wilcoxon rank-sum test, the proteins included in the heatmap were statistically significant across different treatment groups, with p-values ​​below the Benjamini-Hochberg false discovery rate threshold (<0.05). (B) Expression of cell cycle regulatory proteins and epigenetic modification proteins (e.g., cyclin B1, FOXM1, and TRIM24) in PANC-1 cells after BT treatment, compared with the SC group, as detected by RPPA (n=7).

[0037] Figure 17The expression of FOXM1 (A) and p21 (B) proteins in L3.7 cells exposed to biofield therapy (BT) was detected by Jess and compared with the sham control group (SC) and the incubator control group (IC). All data are expressed as mean ± SD (*p<0.05; ***p<0.001).

[0038] Figure 18 The effect of biofield therapy (BT) on epithelial-mesenchymal transition (EMT) in PANC-1 cells compared with the sham control group (SC) and the incubator control group (IC). Immunoblot analysis was performed on E-cadherin (A), N-cadherin (B), and CD44 (C) in PANC-1 cells immediately 15 minutes after BT treatment. Data are presented as mean ± SD (*p<0.05; **p<0.01; ****p<0.0001).

[0039] Figure 19 The percentage of FOXM1 inhibition by BT and the percentage of PDAC cell invasiveness inhibition by BT, and their correlation.

[0040] Figure 20 Modification of FOXM1 in PANC-1 cells altered the cell cycle, and biofield therapy (BT) induced resistance to invasion in PDAC cells compared to the sham control (SC). (A) Western blot analysis of FOXM1 in wild-type PANC-1 cells, FOXM1 stably knocked-down PANC-1 cells, and FOXM1 overexpressing (OE) PANC-1 cells. (B) Cell cycle progression of FOXM1 knocked-down PANC-1 cells under SC, IC, and BT treatment compared to parental (“WT”) PANC-1 cells. (C) Invasivity of parental PANC-1 cells and FOXM1 knocked-down (KD) PANC-1 cells exposed to BT. (D) Invasivity of wild-type (WT) L3.7 cells and FOXM1 knocked-down L3.7 cells exposed to BT. (E) Invasivity of wild-type (WT) PANC-1 cells and FOXM1 knockdown PANC-1 cells exposed to BT. (F) Invasivity of wild-type (WT) COLO357 cells and FOXM1-overexpressing COLO357 cells exposed to BT. (G) Invasivity of COLO357 cells and FOXM1-conditionally overexpressing COLO357 cells exposed to BT. All data are expressed as mean ± SD (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

[0041] Figure 21In a human PANC-1 cell orthotopic mouse model, biofield therapy (BT) inhibited the growth of primary pancreatic tumors and significantly reduced liver metastases (i.e., cell metastasis to the liver) compared to the sham control (SC) and colony control (CC). (A) Terminal tumor weight of PANC-1 tumors after 3 weeks of treatment with BT, control, or gemcitabine, followed by a 7-week observation period. (B) Percentage of PANC-1 tumor-bearing mice with liver metastases at the end of the study. (C) Mean number of liver nodules in PANC-1 tumor-bearing mice with liver metastases. (D) Representative images of liver tissue from mice in the CC, SC, and BT groups. Red arrows indicate liver nodules. (E) Representative hematoxylin and eosin-stained images of liver tissue from mice in the SC and BT groups bearing PANC-1 orthotopic tumors. (F) Percentage of PANC-1 tumor-bearing mice with liver metastases at the end of the repeat study. (G) Mean liver tumor burden in PANC-1-bearing mice with liver metastases in repeated studies. Data are presented as mean ± SE (*p<0.05; n>12 mice per group).

[0042] Figure 22 Jess analysis was used to determine the expression of FOXM1 protein in L3.7 wild-type and L3.7 CRISPR-Cas9 FOXM1 knockdown clones.

[0043] Figure 23 Effect of FOXM1 overexpression on the anti-invasiveness of biofield therapy (BT) induced PANC-1 cells. (A) Invasivity of control PANC-1 cells and stable FOXM1-overexpressing PANC-1 cells after exposure to sham control (SC) or BT. (B) Invasivity of control PANC-1 cells and Tet-induced FOXM1-overexpressing PANC-1 cells after exposure to SC or BT. Data are presented as mean ± SD (** p < 0.01; *** p < 0.001; **** p < 0.0001).

[0044] Figure 24 Effects of biofield therapy (BT) on epithelial-mesenchymal transition markers and endocrine infiltration in PANC-1 tumor-bearing mice. (A) Gene expression of Twist1 in PANC-1 cells 15 minutes after BT treatment. (B) Cell-free DNA content in plasma of PANC-1 tumor-bearing mice in the SC and BT groups. Data are presented as mean ± SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0045] Figure 25A and Figure 25B Transmission electron microscopy (TEM) images showing morphological changes in PANC-1 cells at 15 minutes (A) and 30 minutes (B) after treatment in the IC group (a and d), sham control group (b and e), and biofield treatment group (c and f) (samples were collected immediately after treatment). Note that the mitochondria in the incubator control and sham control groups (d and e) appear normal (red arrows), while the mitochondria in the biofield treatment group are swollen (yellow arrow, f). Image ac is magnified 5000x; image df is magnified 25000x.

[0046] Figure 26 The role of FOXM1 in BT-induced resistance to invasion in PDAC cells. Western blot analysis was performed on FOXM1 in WT, KD (stable knockdown), and OE (overexpression) PANC-1 cells. Data are presented as mean ± SD (**p<0.01; ***p<0.001; ****p<0.0001).

[0047] Figure 27 Effects of BT on voltage-gated potassium channels in PANC-1 cells and on cell voltage in PANC-1 and FOXM1 KO PANC-1 cells. (A) PANC-1 cells were collected immediately 15 minutes after BT treatment to detect the expression of voltage-gated potassium channel (VGKC) and voltage-gated sodium channel genes in PANC-1 cells after treatment. The expression of SCN5A (Nav1.5, B), KCNA3 (Kv1.3, C), and KCNN4 (KCa3.1, D) genes in PANC-1 cells treated for 15 and 30 minutes was detected by real-time quantitative PCR (qPCR). (E) Cell membrane voltage potentials of PANC-1 and FOXM1 KO PANC-1 cells were detected by DiABC4 staining. All data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0048] Figure 28In human PANC-1 cell orthotopic mouse models and KPCY 6419 homologous mouse models, biofield therapy (BT) significantly reduced liver metastasis compared with the sham control group (SC) and the feeder control group (CC). (A) Terminal tumor weight of PANC-1 tumors after 3 weeks of treatment with BT, control group, or gemcitabine, followed by a 7-week observation period. (B) Percentage of PANC-1 tumor-bearing mice with liver metastasis at the end of the study. (C) Mean liver tumor burden of PANC-1 tumor-bearing mice with liver metastasis. (D) Representative images of liver tissue from mice in the CC, SC, and BT groups. Red arrows indicate liver nodules. (E) Percentage of liver metastasis visible in the KPCY 6419 mouse model after treatment with CC, SC, BT, and gemcitabine. (F) Quantification of liver metastasis in the KPCY 6419 mouse model by imaging YFP+ cells in liver tissue. (G) Representative images of liver tissue from WT, SC, and BT mice. Blue arrows indicate liver nodules. All data are expressed as mean ± SE (*p<0.05; n>12 mice per group).

[0049] Figure 29 Immunological characteristics of KPCY tumors after BT treatment. A) Percentage of CD19+ B cells to total parental cells; B) Percentage of CD4+ T cells to total parental cells; C) Percentage of CD8+ T cells relative to CD45+ T cells; and E) Percentage of CD4+ / FOXP3+ T-reg cells to CD45+ T cells. ** p < 0.01, compared with the SC control group.

[0050] Figure 30 Effects of Faraday cages on cell cycle and cell invasion of BT-treated PDAC cells. A) Faraday cage study setup: Cells were placed on an experimental table (right) or in a Faraday cage chamber (left). B) Human PDAC PANC-1 cells were placed in an incubator (IC), on an experimental table (a) or in a Faraday cage (b), and treated with either a sham control group (SC) or a BT treatment group for cell cycle analysis. Cells used for cell cycle analysis were collected immediately 15 minutes after treatment. C) Invasion of PANC-1 cells placed on an experimental table or in a Faraday cage. D) Invasion of human pancreatic cancer L3.7 cells placed on an experimental table or in a Faraday cage. Invasion assays were performed 48 hours after 15 minutes of treatment. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0051] Figure 31Effects of aluminum foil on cell cycle and cell invasion of pancreatic ductal adenocarcinoma (PDAC) cells treated with biofield therapy (BT). A) Experimental setup for the aluminum foil study: Cells were placed in either an unfoiled blue box (left) or a foil-covered box (right). B) Cell cycle of PDAC PANC-1 cells: PDAC PANC-1 cells were placed in an incubator (IC), an unfoiled experimental bench box (a), or a foil-covered box, and treated with either a sham control group (SC) or biofield therapy (BT, b). Cells used for cell cycle analysis were collected immediately 15 minutes after treatment. C) Invasion of PANC-1 cells placed in unfoiled or foil-covered experimental bench boxes. D) Invasion of human pancreatic cancer L3.7 cells placed in unfoiled or foil-covered experimental bench boxes. Invasion assays were performed 48 hours after 15 minutes of treatment. Data are presented as mean ± SD. *p<0.05, **p<0.01, ****p<0.0001.

[0052] Figure 32 Effects of remote biotherapy (BT) on the invasiveness of Panc-1 cells (A) and L3.7 cells (B). Therapist 2 treated Panc-1 and L3.7 cells for 15 minutes each at their laboratory office (a), their Houston apartment (b), and their California home (c). Therapists could observe the cell-containing culture plates using a magnifying glass. Immediately after treatment, cells were transferred to an incubator and cultured for 48 hours before cell invasion assays were performed. Data are presented as mean ± SD. *** p < 0.001, **** p < 0.0001.

[0053] Figure 33 Experimental setup for the entanglement study. A BT therapist administers treatment to human pancreatic cancer cells. Cells designated for tubulin assays are located on the left in an incubator equipped with a CytoSmart Lux microscope. To the BT therapist's right, an EVOS M7000 microscope is used to acquire images of changes in Ca2+ or β-actin. An electroencephalogram (EEG) amplifier is located behind the BT therapist. The cells are enclosed within the CytoSmart microscope and temperature-controlled by it, so the BT therapist's proximity is unlikely to affect the experimental results, and he remains unaware of the cell type. The BT therapist's hands are sometimes on the table and sometimes raised. When hands are on the table, the BT therapist's hands never touch the cell assay equipment.

[0054] Figure 34The significance of cellular assays (tubulin, Ca2+, and β-actin) under BT and sham treatment control conditions (A) and the Granger causality between brain EEG signals at different frequency bands (θ, α, β, γ) and cellular data (tubulin and Ca2+) (B). Each assay value represents the change from baseline obtained one minute before treatment (A). Logarithmic scale indicates significance of multiple comparisons after FDR correction (red electrode below 0.05 is statistically significant, B).

[0055] Figure 35 Following treatment by a second biofield therapist, the invasiveness of PANC-1 cells (A) and liver metastasis in the PANC-1 mouse orthotopic model (B) were compared. (A) Invasiveness of PANC-1 (a) cells, COLO357 (b) cells, and L3.7 (c) cells, and migration of PANC-1 (d) cells and L3.7 (e) cells. (B) Percentage of liver nodules in mice bearing PANC-1 tumors (a); percentage of liver metastases (i.e., metastatic or metastatic cancer) in mice bearing tumors of different sizes (b); and mean tumor weight of PANC-1 tumors without liver metastases in the SC and BT groups. All data are expressed as mean ± SD. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0056] Figure 36 ATACseq analysis of chromatin modification in PANC-1 cells 15 minutes after treatment.

[0057] Figure 37 . Figure 16 Mitochondrial membrane potential of PANC-1 cells after BT treatment for 15 minutes (A) or 30 minutes (B). Mitochondrial membrane potential was measured using JC-1 dye. A decrease in the red to green ratio of JC-1 dye staining indicates mitochondrial depolarization.

[0058] Figure 38 Transmission electron microscopy (TEM) images showing morphological changes in L3.7 cells from IC (A and D), SC (B and E), and BT (C and F) groups 15 minutes after treatment (samples were collected immediately after treatment). Note the presence of cytoskeletal components (red arrows) in IC and SC groups (D and E), while similar cytoskeletal structures appear only in small amounts in BT group (F). Images AC are magnified 5000x; image DF is magnified 25000x.

[0059] Figure 39The effect of BT on the invasiveness of PANC-1 cells after treatment with single therapy (J), therapist group therapy (Group), or BT-treated medium under remote treatment conditions. Data are expressed as mean ± SD (*p<0.05; **p<0.01; ****p<0.0001). Invention Details This invention describes systems and methodologies for maintaining the health and / or homeostatic function of an individual or specific biological entity (e.g., an organism (e.g., a mouse, a human)), including its populations and components (e.g., cells, tissues, organs, organelles, organoids), such as individuals or biological entities in potentially harmful cellular states or functions (e.g., cancer and / or cell metastasis). The systems and methods may include interactions and / or synergistic effects or modalities, such as influences and / or changes on the entity or its interior (e.g., on the entity's state or kinetic energy), and / or influences and / or changes on the entity's exterior or non-entity (e.g., on its microenvironment), which may result in a range of biological effects.

[0061] The systems and methodologies described herein can influence (e.g., modulate, alter, or adjust) one or more aspects of the biophysical and / or bioelectrical aspects of an entity (e.g., a patient, tissue, and / or cell line) that is experiencing or may experience dysfunction, poor health, and / or dysregulation (e.g., a carcinogenic state or cellular activity or a characteristic or indicator of cancer). The systems and methodologies may also, or optionally, influence or cause the modulation or adjustment of the structure and / or physiology of an entity (e.g., a patient, tissue, and / or cell line) that is experiencing or may experience dysfunction, poor health, and / or dysregulation (e.g., a carcinogenic state or cellular activity or a characteristic or indicator of cancer). The systems and methods can alter system-level patterns of biological function, for example, by generating epigenetic changes (as shown, e.g., through RNA methylation and / or histone modifications), altering protein and / or gene expression levels, and / or reprogramming cellular signal transduction between entities.

[0062] The systems and methodologies described in this invention enable the entire biological system (e.g., tissue, organ, cell population, or individual) to benefit, relying on synergistic effects rather than methods that may target or act only on specific molecular sites, responses, or pathways (or sets thereof). The systems and methodologies described in this invention maintain the sustained or regulated function and / or homeostasis of such systems. The systems and methodologies also encompass forces, factors, and / or conditions of synergistic and / or external influences on such biological systems (e.g., cell populations), including environmental (e.g., culture medium), fields (e.g., electromagnetic fields), and / or proximity effects (e.g., signal transduction and network effects), all of which may affect and / or constitute an integral part of their function. The biological system may comprise multiple biological entities, and the systems and methodologies may, for example, directly influence or act on one or more biological entities by causing changes within or on the entities themselves; or indirectly influence or act on one or more biological entities by causing changes within or on the biological entities themselves (e.g., in the environment of another entity and / or the entity, e.g., the culture medium used to culture cells).

[0063] The systems and methodologies described in this invention can be used to treat entities exhibiting dysfunction, poor health, and / or disorder (e.g., organisms and / or cells undergoing cancerous growth and / or metastasis (e.g., cancer patients, cancer cells, or cell lines)), or to design and / or evaluate treatment options for entities exhibiting dysfunction, poor health, and / or disorder (e.g., organisms and / or cells undergoing cancerous growth and / or metastasis (e.g., cancer patients, cancer cells, or cell lines)). This includes, for example, evaluating and / or comparing the potential effectiveness of treatment options or the administerers of treatment options, including but not limited to evaluating or comparing individual therapists or groups of therapists, drug treatment options, radiation therapy options, and treatment devices. In one embodiment, the cells are human or mouse cells, such as human or mouse cancer cells. The systems and methodologies described herein can be used to treat cancer patients and / or cancer cells or cell lines, or to design and / or evaluate treatment regimens for cancer patients and / or cancer cells or cell lines (e.g., to assess and / or compare the potential effectiveness of treatment regimens), such as pancreatic cancer patients and / or cells or cell lines, such as pancreatic ductal adenocarcinoma (PDAC) cells, including but not limited to cells derived from human cell lines (e.g., PANC-1, MiaPaca, COLO357, L3.7, and Capan2 cell lines) and cells derived from murine cell lines (e.g., Panc02 and KPCY). The systems and methodologies may, for example, promote the healthy function and / or well-being of organelles, organoids, cells, tissues, and / or organisms (including populations thereof) by reducing the growth and / or metastasis of cells (e.g., cancer cells or cells within or near tumors).

[0064] Tumors are typically considered localized until metastasis occurs. However, mounting evidence suggests that multiple pre-existing distal and proximal systems within the tumor microenvironment influence tumorigenesis and development, including pre-tumor genome-wide epigenetic changes and inflammatory responses to stimuli, as discussed in Hill, W. et al., Nature 616:159-167 (2023), which is incorporated herein by reference. Cancer can be viewed as a deviation of cells from their correct target morphology—the normal pattern information field fails to coordinate the activities of individual cells, thus affecting the overall evolution of the organism. In short, cancer may be caused by impaired cellular information exchange and bioelectrical decoherence.

[0065] like Figure 1 As shown, the factors that may be involved in the methods and systems described in this invention include, but are not limited to, those characterized by their biophysical, structural, and / or physiological properties. For example, the cell membrane possesses both structural and biophysical characteristics. Changes in structure can be correlated with changes in biophysical properties, for example, they can cause changes in biophysical properties, and / or vice versa. For instance, an increase in membrane permeability can lead to a decrease in voltage potential. At the biophysical level, this includes (but is not limited to) [various aspects]. Figure 1 Factors identified under the "Biophysical" category appear to have a systematic, pre-existing, and dynamic influence on tumorigenesis. Factors characterized as physiological are also known to affect tumorigenesis, and may include structural and / or biophysical properties. Physiological changes can be correlated with structural changes, for example, causing structural changes, and / or vice versa.

[0066] The systems and methodologies described in this invention can have comprehensive and / or mutually beneficial effects on the biophysics, structure, and / or physiology of cells, including, for example, effects on the cellular microenvironment, structure, bioelectricity, and / or gene expression. For example, such as... Figure 2 As shown, biofield therapy can have beneficial effects on the structure, biophysics, and physiology of cells. In contrast, radiation therapy can improve the structural and biophysical properties of organs (e.g., the pancreas) by ablating and killing cancer cells (e.g., tumors), but it can also damage nearby healthy tissue. Similarly, while drug therapy can improve physiological function, it may not have a positive or beneficial effect on cell structure or biophysics.

[0067] In some implementations, the systems and methodologies can directly or indirectly influence (e.g., cause) changes in multiple factors that directly or indirectly promote or participate in the maintenance or restoration of cellular homeostasis and / or non-cancerous states, such as... Figure 1 The factors described herein may be components or characteristics of cell state and / or function. The systems and methodologies may have epigenetic effects, such as changes in gene expression and / or transcriptome. The systems and methodologies may affect the protein expression of FOXM1 or its associated proteins, membrane ion channel genes, cytoskeletal proteins, and / or proteins involved in cell migration and invasion, as well as infiltration.

[0068] In some embodiments, the systems and methodologies described in this invention may vary one, two, three, four, five, or all of the following: cellular energy (e.g., mitochondrial state and / or function; ATP levels); cell physiology (e.g., transcriptional regulators, FOXM1 and TRIM24, various protein kinases (including kinases associated with glucose and glutamine energy metabolism), PLK1, and / or Ras-related pathways); cell cycle (e.g., distribution or progression rate of cell cycle phases, p21, CDK, cyclins); cell structure (e.g., cell junctions and / or cytoskeleton organization); cell growth and / or metabolism (e.g., epidermal growth factor, EGF); cell biophysics (e.g., membrane voltage potential and / or cell polarization); extracellular matrix (e.g., cell signal transduction); intracellular communication networks (PDZ); and other aspects of cell or molecular biology. Changes in one or more factors (e.g., cell energy, physiology, cell cycle, structure, biophysical or bioelectrical properties, and extracellular matrix) that may be involved in the maintenance or restoration of cell homeostasis and / or non-cancerous cell state can cause changes in, for example, cell energy, physiology, cell cycle, structure, biophysical, bioelectrical properties, and / or extracellular matrix.

[0069] The systems and methodologies described in this invention can influence cell physiology (e.g., regulation of FOXM1, regulation of the PI3K / mTOR pathway, alteration of EMT marker expression, and delay of cell cycle progression and cell migration), cell structure (e.g., regulation of cytoskeleton organization by modulating F-actin, TRIO, and TRIOBP, as well as mitochondrial function and structure), and / or cell biophysics (e.g., regulation of membrane voltage potential, prevention or reversal of cell depolarization, and other changes in cell bioelectric properties (e.g., causing beneficial changes in cell physiology, cell structure, and / or cell biophysics)). The systems and methodologies described in this invention can also influence the physiology of organoids, organs, tissues, or organisms (e.g., causing physiological changes). For example, the systems and methodologies can influence the function of the autonomic nervous system (parasympathetic and sympathetic), the immune system, the brain, and / or the heart (e.g., as measured by indicators such as heart rate, heart rate variability, high-frequency heart rate variability, ECG, brain waves, EEG, skin electrical activity, and pupillary dilation). The systems and methodologies described may affect physiology and / or cell structure due to changes in cellular biophysics (e.g., causing changes in physiology and / or cell structure). These systems and methodologies may also affect cellular biophysics due to changes in physiology and / or cell structure (e.g., causing changes in cellular biophysics). Changes in cellular physiology, structure, and / or biophysics can reduce cell growth and / or metastasis (e.g., cancer cells).

[0070] The definition is as follows: The term "living entity" or "biological entity" as used in this invention broadly refers to any living individual and any group or component thereof, including, for example, cells, single-celled and multicellular organisms (such as animals, mammals, mice, and humans), organoids, organelles, embryos, tissues, organs, tumors, and proliferative organisms in any form (including in vitro and in vivo forms).

[0071] The term "growth" as used in this invention refers broadly to the growth of an individual and / or a group, such as an increase in the mass of a single cell or tissue, and / or an increase in cell viability, proliferation and / or reproduction.

[0072] The terms “adjustment” and / or “regulation” used in this invention refer to adjusting, maintaining or controlling a process or state, including but not limited to preventing or reducing deviations from an ideal state.

[0073] The term "homeostasis" as used in this invention refers to any self-regulating process by which a biological system maintains stability, such as the tendency to move toward a relatively stable state or process.

[0074] The terms “metastasis / metastatic foci” and “metastatic potential” used in this invention refer to the actual or potential development of the ability of cancer cells to separate, migrate and / or invade (e.g., other tissues or organs), such ability, for example, can contribute to the formation of secondary malignant growths in locations distant from the primary site of cancer.

[0075] As used in this invention, the terms "energy therapy" and "energy healing" refer to any therapy aimed at altering the energy balance or state of a living organism, including, for example, changes in the balance or state of bioelectricity, biophysics, and / or metabolism. Energy therapy / healing includes (but is not limited to) techniques involving the so-called "channeling" of energy (e.g., healing energy), i.e., the transfer of energy into the entity by a practitioner. Such techniques can be used alone or in combination with other medical means (e.g., administration of medication and / or radiation therapy) to treat a variety of conditions and health problems. In general, energy therapy aims to restore or establish a beneficial state and / or promote healthier functioning of the entity receiving the therapy.

[0076] The term "biofield therapy" or "BT" as used in this invention is an energy therapy, broadly referring to any and all treatment methods that utilize the body's energy field (i.e., "biofield") to achieve therapeutic effects; the term encompasses related devices, such as those capable of simulating the fields and / or information emitted or exchanged by people (e.g., therapists and / or patients) during the application or receipt of biofield therapy. Currently, biofield therapy is used by the U.S. Veterans Administration and dozens of large hospitals, with tens of thousands of practitioners in the United States alone; research has shown that this therapy can induce changes in the biophysical state and biochemical signal transduction of treated cells.

[0077] The terms "treatment" and "treatment" as used in this invention, unless otherwise stated, are not limited to the treatment of a disease or condition. For clarity, "treatment" or "treatment" includes, but is not limited to, utilizing the effects and mechanisms of action described herein to eliminate or mitigate the effects of a disease or condition, restore health and / or homeostatic function, promote overall health and well-being, and / or for the prevention or control of disease; and the terms cover situations in which the effects and mechanisms of action are utilized in the presence or absence of a disease state or condition diagnosis.

[0078] therapeutic indicators The systems and methodologies described in this invention can provide indicators of therapeutic efficacy, including, for example, but not limited to, a particular form or manner of treatment, the treatment administerer or type of administerer, biofield therapy, pharmacological therapy, radiation therapy, or any combination thereof. These efficacy indicators can be used, for example, to diagnose the effect or effectiveness of biofield therapy, biofield therapists, or therapy teams. These efficacy indicators can also be used to determine whether an entity (e.g., an entity characterized by cancer (e.g., pancreatic cancer and / or cancer characterized by FOXM1 overexpression)) is likely to benefit from or respond to a particular treatment (e.g., biofield therapy and / or the administerer of biofield therapy).

[0079] The indicators may include or be based on, for example, one or more measurements indicating physiological changes or no changes in the treatment user (such as a BT practitioner). For example, the indicators may be based on one or more measurements of the autonomic nervous system (parasympathetic and sympathetic), brain, and / or heart (e.g., heart rate, heart rate variability, high-frequency heart rate variability, ECG, brain wave frequency or spectrum, EEG, skin electrical activity, pupillary dilation), as described, for example, in Cohen, L., et al., Examining the effects of biofield therapy through simultaneous assessment of electrophysiological and cellular outcomes, Nature Scientific Reports 14:29221 (2024) (https: / / doi.org / 10.1038 / s41598-024-79617-3) (“Cohen et al.2024”), the entire text of which is incorporated herein by reference.

[0080] The indicator may include or be based on, for example, one or more assays that indicate a change or lack thereof in the treatment recipient, and the indicator may include one or more factors that contribute to and / or are involved in the maintenance or restoration of cellular homeostasis and / or non-cancerous cell status, such as... Figure 1 Those shown. For example, the indicators may include or be based on measurements of the physiological, biological function, physical structure, and / or biophysical state of the therapeutic entity, including, for example, those described in the 2024 literature by Cohen et al. The indicators may be selected or determined based on the initial state of the therapeutic entity or the measurements performed on it.

[0081] The indicators may be based on one or more correlations with the physiology of the administerer (as previously described) or the treatment recipient (as previously described).

[0082] In one embodiment, the efficacy endpoint comprises or is at least partially based on the measurement of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more proteins, for example, measurements of one or more of the following: calcium, FOXM1, pAKT, ALKBH5, cyclin B1, CDK1, PI3K / mTOR, P21, TRIM24, EMT marker expression, E-cadherin, N-cadherin, Twist, and / or CD44. In one embodiment, the efficacy endpoint comprises or is based on at least one or at least two measurements of FOXM1. For example, the efficacy endpoint may comprise or be based on measurements of FOXM1, and may be selected based on pre-treatment or initial FOXM1 measurements indicating FOXM1 overexpression and a decrease in FOXM1 levels after treatment. Any endpoint or measurement result regarding FOXM1 expression may be used as a efficacy endpoint.

[0083] In one embodiment, the efficacy indicator comprises or is at least partially based on measurements of the cell cycle, such as the number or proportion of cells in a certain phase of the cell cycle (e.g., G0 / G1 and / or G2 / M phase). In a preferred embodiment, the efficacy indicator comprises or is based on at least one or more measurements of the proportion of cells in G0 and / or G1 phases. In one embodiment, the efficacy indicator comprises or is at least partially based on measurements of cell structure, such as measurements of the state of the cytoskeleton (e.g., tubulin), cytoskeletal proteins, mitochondria, and / or F-actin, TRIO, and TRIOBP. In one embodiment, the efficacy indicator comprises or is at least based on at least one or more measurements of mitochondrial state, such as measurements of morphology (e.g., shape, swelling), orderliness, membrane (e.g., voltage potential, polarization), and energy (e.g., ATP, utilization). In one embodiment, the efficacy indicator comprises or is at least partially based on at least one or more measurements of TRIOBP.

[0084] In one embodiment, the efficacy indicator comprises or is at least partially based on measurements of biophysical or bioelectrical states, such as measurements of the voltage potential or polarization of membranes (e.g., cell membrane, nuclear membrane, and / or mitochondrial membrane). In a preferred embodiment, the efficacy indicator comprises or is based on at least one or at least two measurements of cell membrane voltage potential.

[0085] The efficacy indicators may include or be at least partially based on one or more of two, three, four, five, six, seven, eight, nine, ten or more aspects of an entity (e.g., protein levels or expression, cell cycle, cell structure, and / or biophysical state, as described in the accompanying drawings and embodiments provided in this invention).

[0086] Measurements can be performed, or the measurements can indicate the post-treatment state. These two measurements may include, for example, a first measurement performed before treatment or indicating the pre-treatment state, and a second measurement performed after treatment or indicating the post-treatment state. In one embodiment, the first and second measurements both target the same aspect, characteristic, indicator, or factor, such as the same protein, the same cell cycle indicator, the same cell structure measurement, and / or the same biophysical state.

[0087] Biofield therapy Compared to radiation therapy and / or drug therapy, biofield therapy methods and / or related devices produce fewer toxic or harmful effects. Therefore, as a treatment approach, it may be more acceptable and / or tolerable than radiation therapy and / or drug therapy. Furthermore, given its lower toxicity and fewer safety concerns, it may also have an advantage in terms of faster approval.

[0088] The systems and methodologies described in this invention can integrate and / or relate scientific aspects from multiple research fields, including: physics (covering biophysics and / or bioelectricity), consciousness (covering healing), biology (covering quantum biology and pharmacology), and medicine / health (covering conventional and unconventional therapies). The systems and methodologies can originate from the interaction and / or exchange between conscious human intentions and energy (e.g., electric fields, light fields, biophoton fields, and / or magnetic fields), information, and / or biology. Their effects and / or mechanisms may be generated by one or more fields (e.g., information fields and / or energy fields) existing in a variety of environments, including but not limited to chemical and / or physical environments. In one embodiment, the one or more fields include: a dynamic spatial field containing information. The "field" can be a massless field (possibly an electromagnetic field, but not necessarily) that surrounds, permeates, and / or influences biological entities. The field may have self-organizing properties and / or may contain or provide templates or blueprints. Fields can influence biological entities and / or characteristics or aspects of said biological entities (e.g., tumors, the autonomic nervous system (parasympathetic and sympathetic nervous systems), the brain, brain function, the heart, heart rate, metastatic processes, metabolism, gene and protein function, etc.), prompting them to evolve towards a more ordered and / or more stable state. Energy and / or information can be transmitted through one or more of the following mechanisms: quantum entanglement; diffusion or propagation of extracellular chemicals; frequency, resonance, and / or vibration (e.g., longitudinal waves); electromagnetism (EMF); and sound, light, and / or dark matter. Fields, energy, and / or information can be referred to as, for example, healing energy, subtle energy, elan vital, prana, qi / chi, vital force, and / or biofield.

[0089] These changes may originate from the actions or intentions of an individual (e.g., a BT therapist or therapy group, or a patient). These changes may also originate from the operation of a device (e.g., a field or information generator). The field or information generator may be, for example, a device that generates or emits electromagnetic energy, light, and / or sound. The actions, intentions, and / or device operation may facilitate the exchange or flow of energy or information between entities such as one or more organs, organelles, organoids (e.g., pancreatic cancer organoids), cells, tissues, and / or organisms (particularly mice and / or humans), where such entities are, for example, in pairs, groups, or collectives. The actions, intentions, and / or device operation may influence or manipulate various fields (e.g., energy fields or information fields). The actions, intentions, and / or device operation may occur near the entity. The actions, intentions, and / or device operation may occur before the corresponding effect is produced.

[0090] The effects and mechanisms of the BT can be demonstrated by correlations between the practitioner's or device's behavior, intentions, and / or operations and the observed changes, outcomes, or states exhibited by one or more entities (e.g., see Cohen et al., 2024). The entity can be any biological entity, such as an organism (e.g., a mouse, a human), and / or may include cells, organs, organelles, organoids, tissues, tumors, aberrant growths, etc., or groups thereof. In one embodiment, the treatment method comprises actions, intentions, and / or device operations associated with observed changes, outcomes, or states in relation to the subject entity. The method can be transpersonal, for example, conducted between a therapist (or a team of therapists) and the subject entity. In one embodiment, the method is a form of treatment mediated by transpersonal consciousness. In one embodiment, the method consists of the application of some form of energy therapy or biofield therapy (e.g., Bengston Healing, Healing Touch, Reiki, Therapeutic Touch, or External Qigong). In one implementation, the method includes applying some form of energy therapy or biofield therapy, such as Bengston healing, Healing Touch, Reiki, Therapeutic Touch, or External Qigong.

[0091] On the one hand, the effects of energy therapy administered by a person or device can be assessed, quantified, evaluated, or otherwise indicated based on one or more efficacy indicators. For example, FOXM1 levels in the cells of an entity in a cancerous state can be measured before and after the administration of biofield therapy. If the pre-treatment FOXM1 level indicates FOXM1 overexpression, one or more measurements of FOXM1 expression after treatment can be used as efficacy indicators. For instance, if the post-treatment FOXM1 measurement is significantly lower than the pre-treatment measurement, the treatment (e.g., the person or device administering the treatment) can be considered effective.

[0092] Comprehensive effect Overall, changes in cell structure, biophysical properties, and physiology can have a combined effect on cell health, especially on cell carcinogenesis and / or cell status.

[0093] From a physiological perspective, including (but not limited to) Figure 2 Factors categorized as "physiological" are widely considered an important background for understanding cancer. For example, EMT markers are known to serve as indicators of cancer and cancer metastasis. At the structural level, this includes (but is not limited to) [the following]. Figure 1 Factors categorized as "structure" are known to participate in the regulation of the cell cycle, cell growth, and cell metastasis (a recognized characteristic of cancer). At the biophysical level, this includes (but is not limited to) [the following]. Figure 1 The factors categorized under "biophysics" encompass characteristics of cell polarization. Cancer cells typically exhibit a depolarized state.

[0094] Related structural, biophysical, and physiological changes in cells, and the systems and methods utilizing these changes, may offer particular benefits for the treatment of cancers (e.g., pancreatic cancer). Cancer may be a reversible physiological state of a multicellular dynamic system. Therefore, a comprehensive consideration of multiple aspects is crucial for understanding carcinogenesis. Cancer may be caused by the combined effects of structural, biophysical, and physiological factors leading to dysfunction of the cellular and tissue microenvironment. Several such combinations are discussed here.

[0095] Cellular voltage and cancer Cancer cells are known to possess unique bioelectrical properties. The endogenous spatiotemporal differences in resting membrane potential and bioelevation are a crucial aspect contributing to the dysregulation of intercellular interactions in cancer (Chernet B, Levin M., J ClinExp Oncol. 2013; Suppl 1). Membrane voltage potential (Vm), the transmembrane voltage resulting from the presence of various ion channels / transporters with specific ion selectivity and permeability, is closely related to cancer cell proliferation and metastasis. Furthermore, aberrant depolarization of the resting membrane voltage potential (Vm) is considered a convenient marker of tumorigenesis and can activate genetically normal cells to exhibit a metastatic phenotype in vivo. Changes in Vm can trigger long-distance metastasis, a process driven by transcriptional and epigenetic effects mediated by electro-signal-regulated serotonin and butyrate flows. See Yang M, Brackenbury WJ. Front Physiol. 2013;4:185; Chernet BT, Levin M., Dis ModelMech. May 2013;6(3):595-607; Levin M., Mol BiolCell. Dec 1 2014;25(24):3835-50.

[0096] Cell membrane potential is regulated by differences in ion concentration and ion channels, factors that play important roles in tumorigenesis (Becchetti A., Am J Physiol Cell Physiol. Aug 2011;301(2):C255-65). Electrophysiological analyses of various cancer cell types show that depolarization of the membrane potential (Vm) is beneficial to cell proliferation and migration. Proliferating cells, including tumor cells and electrically excitable normal cells, typically have a Vm range higher than -50 mV, while terminally differentiated cells such as neurons and smooth muscle cells have a Vm range of around -90 mV, indicating that Vm has a functional guiding role in cell development. The Vm of human breast cancer MCF-7 cells fluctuates between -9 and -30 mV (the specific value depends on the cell cycle phase), a range still higher than that of immortalized MCF-10A cells (-40 to -58 mV). However, transient hyperpolarization promotes the migration and metastasis of breast cancer cells, a process mainly mediated by voltage-gated potassium channels. See Yang M, et al., FrontPhysiol. 2013;4:185; Levin M., Trends Cell Biol. Jun 2007; 17(6):261-70; Yang M, et al. Breast Cancer Res TreatJul 2012; 134(2):603-15; Marino AA, et al. Tumour Biol. 1994;15(3):147-52; Fraser SP, et al. Clinicalcancer research. Aug1 2005;11(15):5381-9; Payne SL, et al. EBioMedicine. Jan 2022;75:103767; HofschroerV, et al. Front Pharmacol. 2020;11:586599.

[0097] Ion channels and transfer Ion channels are an important class of membrane proteins that can sense and alter the properties of the tumor microenvironment. They can transduce signal cascades triggered by components of this microenvironment, both by altering cell membrane conductivity and through non-conductive mechanisms. Ion channels are expressed in various cell types and play cell-specific roles and maintain basic cellular physiological activities (i.e., "housekeeping" functions). These include generating and regulating membrane potential, which is a prerequisite for many processes, such as Ca2+ signaling, mitochondrial function, and the interaction between tumor cells and stromal cells in the tumor microenvironment (Djamgoz MB, et al. Philos Trans R Soc Lond B Biol Sci. Mar 19 2014;369(1638):20130092). Among various ion channels, voltage-gated sodium channels (Nav) and potassium channels (Kv) are known regulators of cell membrane voltage and are involved in the proliferation and metastasis of cancer cells (Djamgoz MBA, et al. Cancers(Basel). Oct 28 2019;11(11); Zuniga L, et al., Onco Targets Ther. 2022;15:783-797). Activation of potassium channels can cause hyperpolarization, while opening of sodium or chloride channels can cause depolarization. Nav1.5 has been reported to be associated with enhanced metastatic potential of breast cancer cells.51 The Kv channel family comprises 12 subfamilies and 40 genes, playing a crucial role in the repolarization of the resting membrane potential in excitatory cells (Wulff H, et al. Naturereviews Drug discovery. Dec2009;8(12):982-1001; Feske S, et al. Annu RevImmunol. 2015;33:291-353). In non-excitatory proliferative cells, Kv channels play important roles in cell proliferation, Ca2+ signaling, migration, and cell volume regulation (Chesnokov MS, et al., Front Oncol. 2021;11:696532). Kv1.3 channels are overexpressed in various pancreatic ductal adenocarcinoma (PDAC) cell lines, and their overexpression is closely related to increased cell proliferation and cancer cell survival.

[0098] Targeting PDAC cells with the Kv1.3 inhibitor (clofazimine) induces apoptosis and slows the growth of orthotopically transplanted PDAC cells (Lobikin M, et al., Phys Biol. 2012;9(6):065002). Kv1.3 is also expressed in mitochondria (i.e., mitoKv1.3), which regulates apoptosis in PDAC cells (Leanza L, et al., Cancer cell. Apr 102017;31(4):516-531 e10). In addition, KCa3.1, a calcium-activated potassium channel, is functionally expressed in the pancreatic duct and participates in transepithelial ion and fluid transport mechanisms (Hayashi M, et al. Am J Physiol Cell Physiol. Jul 15 2012;303(2):C151-9; Wang J, et al, Am JPhysiol Cell Physiol. Apr 1 2013;304(7):C673-84). KCa3.1, as the first K+ channel, is overexpressed in primary pancreatic cancer samples and has functional activity in pancreatic cancer cell lines (Zaccagnino A, et al., Eur Biophys J. Oct 2016;45(7):749-763; Jager H, et al., MolPharmacol. Mar2004;65(3):630-8; Jiang S, et al., Biochem Biophys Res Commun. Dec 9 2017;494(1-2):113-119; JiangSH, et al., Gut. Nov 2019;68(11):1994-2006). High expression of KCa3.1 channels is associated with poorer survival outcomes in pancreatic ductal adenocarcinoma (PDAC) and other cancer types (Faouzi M, et al., Oncotarget. Jun 14 2016;7(24):36419-36435; Bulk E, et al., IntJCancer. Sep 15 2015;137(6):1306-17). KCa3.1-mediated K+ efflux is crucial for cell volume dynamics during cell cycle and migration.63 Therefore, KCa3.1 can promote tumor progression by regulating cell proliferation, migration, and invasion. Finally, similar to Kv1.3, KCa3.1 channels are also expressed on the inner mitochondrial membrane (IMM) of normal cells and PDAC cells,64 and regulate mitochondrial metabolic activity, possibly through modulation of mitochondrial membrane potential.

[0099] As described in the "Examples" below, BT treatment significantly reduced the invasion of Panc-1, L3.7, COLO357 (human), and KPC (mouse) cells, and regulated the expression of Na+ and K+ channel genes. Figure 6 Data indicate that BT treatment modulates the expression of Nav1.5 and Kv1.3 genes in a time-dependent manner, while the expression of KCa3.1 gene is downregulated in the BT-treated group. This regulatory effect may play an important role in BT-induced anti-metastasis activity.

[0100] Cell volume regulation, morphology and metastasis Cancer metastasis, the process by which cancer cells spread from the primary site to distant sites or organs, is a highly coordinated, multi-step process involving the stroma, blood vessels, and cytoskeleton. Successful metastasis depends on invasion, migration, extravasation, anoikis, and angiogenesis. Invasion is a local step that occurs at the tumor-host border, during which tumor cells and stromal cells exchange enzymes and cytokines, thereby regulating the local extracellular membrane (ECM) and promoting cell migration (de Visser KE, Joyce JA. Cancer cell. Mar 13 2023;41(3):374-403).

[0101] Cytoskeleton and morphological remodeling is a key event in tumor metastasis, enabling cells to successfully complete invasion and extravasation. Changes in cell morphology (e.g., glioma cell shrinkage) promote glioma cell migration and extravasation (Sontheimer H. Exp Biol Med (Maywood). Jul 2008;233(7):779-91). Cell shrinkage requires a reduction in cytoplasmic water content, a process likely mediated by electrochemical forces of K+ and Cl-. Hypertonic cell shrinkage may potentially inhibit local volume expansion, thus favoring cell migration.67 The outflow of Cl- and K+ through ion channels or transporters provides the driving force for water expulsion from the cell, a mechanism presumably occurring in migrating cells.

[0102] There is ample evidence that cell migration is regulated by water and ions (Schwab A, et al., Physiol Rev. Oct 2012;92(4):1865-913). Ca2+-sensitive potassium channels (especially KCa3.1) are locally activated in the posterior region of migrating MDCK-F cells, which may reflect the role of these channels in the reduction of cell volume due to posterior retraction during cell migration (Schwab A, et al., Pflugers Arch. Sep 1995;430(5):802-7). Changes in cell volume can be observed through cytoskeleton deformation. The cytoskeleton plays a regulatory role both by altering the availability of signal transduction devices and as an integral component of the signal transduction system. In G6 glial cells, a decrease in extracellular osmotic pressure (hypotonic environment) can cause actin to convert to its polymerized form, F-actin (Persinger MA, et al., Int J Yoga. Jul 2012;5(2):140-5). Ion channel-mediated bioelectrical changes can lead to osmotic pressure alterations, which in turn alter the cytoskeleton and affect the cell's migration potential.

[0103] Furthermore, changes in cytoskeleton markers suggest that microtubules may represent a biophysical signal transduction system. These ferromagnetic lattices align parallel to strong magnetic fields, attributed to the presence of a single unpaired electron in each tubulin molecule, which participates in intracellular transport and chromosome segregation during mitosis and meiosis. Microtubules are also nonlinear oscillatory structures capable of generating electromagnetic fields within living cells. Structural and functional properties of microtubules (e.g., their role in cell division and / or quantum vibrations) may influence cell communication. Microtubules may be associated with cancer because disruption of quantum coherence and / or quantum entanglement between microtubule-based mitotic spindles and centrioles can lead to abnormal chromosome distribution, abnormal cell differentiation, and uncontrolled cell growth. β-actin (cytoskeleton) fibers can also generate spatially distributed endogenous bioelectric fields, further illustrating how structural properties influence biophysical properties. Cytoskeleton activity is closely related to various healing therapies, as are the activities reflected by Viafluo (targeting tubulin, the cytoskeleton) and Fluo4 (targeting voltage).

[0104] Mitochondria, Metabolism and Transfer Mitochondria play a vital role in providing energy to cells by producing ATP and regulate intracellular energy metabolism via oxidative phosphorylation (OXPHOS). Mitochondrial ion transport mechanisms also play a key role in regulating mitochondrial bioenergetics by altering the Ca2+ level in the matrix, thereby affecting mitochondrial respiration and ATP production under physiological conditions (Kaufman RJ, et al., Biochim Biophys Acta. Oct 2014;1843(10):2233-9).

[0105] Under physiological conditions, ion transport (i.e., Na+, K+, Mg2+, H+, and Ca2+) across the inner mitochondrial membrane (IMM) regulates mitochondrial matrix volume. The mitochondrial K+ / H+ exchanger protein (KHE), an IMM protein, plays a crucial role in regulating K+ transport and may thereby modulate mitochondrial volume and metabolism. Studies have shown that K+ and Ca2+ are the main drivers of changes in mitochondrial matrix volume. The primary mechanism of mitochondrial swelling involves the opening of non-selective channels (called permeability transition pores (PTPs)) on the inner mitochondrial membrane under both low-conductivity (physiological) and high-conductivity (pathological) modes. Depolarization of the mitochondrial potential can induce mitochondrial swelling. Excessive increase in matrix volume or matrix swelling impairs mitochondrial metabolism and function and can lead to cell death. See Douglas MG, et al., J Biol Chem. Sep 10 1974;249(17):5464-71; Halestrap AP, et al., Biochem J. Jun 15 1986;236(3):779-87; Chapa-Dubocq XR, et al., Antioxidants (Basel). Jul 28 2023;12(8); Kwong JQ, et al., Cellmetabolism. Feb 32015;21(2):206-214.

[0106] Immunity and Cancer The tumor microenvironment (TME) plays a crucial role in tumor development, progression, and treatment response. Immunotherapy (e.g., using immune checkpoint inhibitors to treat various malignancies) highlights the critical role of the immune system in cancer and its treatment (Shalapour S, et al., The Journal of Clinical Investigation. Sep 2015;125(9):3347-55). Crosstalk between cancer cells and immune cells is one of the main characteristics of cancer (Hanahan D, et al., Cell. Mar 4 2011;144(5):646-74).

[0107] Immune cells, due to their high diversity and plasticity, exert multiple effects in tumor-bearing hosts, and different types of immune cells play different roles. The composition and function of immune cells within tumors vary significantly between different tumor types and within tumor types. The immunosuppressive tumor microenvironment (TME) hinders the infiltration, activation, and clearance of tumor cells by effector T cells, which is considered a major obstacle to the success of cancer treatment, including immunotherapy, and is particularly prominent in pancreatic ductal adenocarcinoma (PDAC) (Dudley ME, et al., Journal of clinical oncology. Jun 10 2013;31(17):2152-9; Schumacher TN, et al., Cancer cell. Jan 12 2015;27(1):12-4). Based on the relative proportion of CD3+ and CD8+ cells in the tumor to the total number of cells, PDAC is often listed as one of the "coldest" human tumors (Galon J,Bruni D. Nature reviews Drug discovery. Mar 2019;18(3):197-218; Maleki Vareki S. J Immunother Cancer. Dec 27 2018;6(1):157).

[0108] Targeting CTLA-4 can enhance anti-tumor immune responses and lead to tumor regression in various cancers, including PDAC (Babamohamadi M, et al., Cell Death Dis. Jan 8 2024;15(1):17; Lisi L, et al., PharmacolRes. Jan 2022;175:105997; Ni R, et al., Biomed Pharmacother. Oct2024;179:117430). Furthermore, inhibiting B cell infiltration into tumors by blocking the chemokine CXCL13, suppressing B cell activity using Bruton's tyrosine kinase (BTK) inhibitors, or clearing B cells using specific mAbs can all significantly slow tumor progression (ChenSS, et al., Leukemia. Apr 2016;30(4):833-43). Studies on PDAC cell lines derived from transgenic KrasG12D;Pdx Cre mice carrying p16Ink4a or Trp53 deletions have shown that BTK can regulate T cell suppression mediated by B cells and macrophages during PDAC development (Ireland L, et al., Cancer research. Dec 1 2016;76(23):6851-6863), indicating that tumor-infiltrating B cells also play an important role in PDAC.

[0109] As illustrated in the embodiments, the data from this invention demonstrate that BT treatment can significantly reduce B cells and increase CD8+ T cells (…). Figure 8 This indicates that BT can modulate the immune tumor microenvironment (TME) and may help BT exert its anti-metastatic potential in PDAC.

[0110] Metabolism of cancer cells and immune cells Cancer cells exhibit metabolic characteristics distinctly different from resting cells or tissues (Warburg Effect). Even under aerobic conditions, cancer cells reprogram their glucose metabolism and energy production processes by primarily limiting their energy metabolism to glycolysis (Liberti MV, et al., Trends in biochemicalsciences. Mar 2016;41(3):211-218). Studies have shown that glycolysis for energy is closely related to the activation of oncogenes such as RAS and MYC, as well as the mutant tumor suppressor gene (TP53) (Jones RG, et al., Genes & development. Mar 12009;23(5):537-48; Wise DR, et al., Proc Natl Acad Sci USA. Dec 2, 2008;105(48):18782-7), thereby promoting cell proliferation, evading cell growth inhibition regulation, and reducing apoptosis. Energy metabolism reprogramming has been recognized as an emerging hallmark of cancer (Hanahan D, et al., Cell. Mar 4 2011;144(5):646-74).

[0111] Reconnected cellular metabolism is not only a hallmark of tumors but also a characteristic of normally proliferating immune cells, such as T cells (Andrejeva G, et al., Cell metabolism. Jul 5 2017;26(1):49-70). For example, activated T cells and leukemic T cells both exhibit significantly higher glucose metabolism rates (Kishton RJ, et al., Cellmetabolism. Apr 12 2016;23(4):649-62). T cell tumor transport and T cell cytotoxicity are impaired in glycolytic tumors, and inhibition of glycolysis enhances the antitumor activity of tumor-reactive T cells (Cascone T, et al., Cell metabolism. May 1 2018;27(5):977-987.e4), suggesting that tumor metabolic programming may potentially influence immune cell function.

[0112] Brief summary based on the examples As further demonstrated and illustrated by the embodiments provided in this invention, systems and methods having the physiological, structural, and biophysical effects and mechanisms described in this invention (e.g., biofield therapy) can inhibit the growth of primary tumors and reduce the metastatic potential of cancer cells.

[0113] As described in Example 1, BT exhibited antitumor and antimetastatic effects in various pancreatic cancer cell lines and two PDAC models.

[0114] FOXM1 is one of the mechanisms responsible for BT-induced anti-metastatic activity. The method described above can reduce FOXM1 levels, for example, by decreasing its expression in PANC-1 and L3.7 cells. By downregulating FOXM1, this method can alter the metabolic activity, growth, and invasiveness of PDAC cells. Downregulating FOXM1 can reduce the invasiveness of cancer cells (e.g., PDAC cells). Downregulation of FOXM1 is associated with reduced invasiveness of cancer cells (e.g., PDAC cells). The effect of BT on FOXM1 is highly positively correlated with the effect of BT on invasiveness. This means that when the reduction in FOXM1 by BT is stronger, the reduction in invasiveness is also greater, compared to a smaller reduction in FOXM1 induced by BT.

[0115] The method described above can alter cell membrane potential. Biofield therapy (BT) alters cell membrane voltage potential and modulates mitochondrial ultrastructure. Furthermore, BT treatment significantly alters gene expression of voltage-gated sodium (Na+) and potassium (K+) channels, which are key regulators of cell membrane voltage potential. Biofield therapy can inhibit the growth and / or invasiveness of cancer cells (e.g., pancreatic cancer cells), and this inhibition of growth and / or invasiveness is partly mediated by altering the cell cycle, changing cell voltage potential, downregulating the PI3K / Akt pathway, and / or downregulating (reducing) FOXM1.

[0116] As described in Example 2, this invention utilizes various in vitro cell models and in vivo PDAC models to evaluate the various stages of the transfer process in which BT plays a role. This invention identifies several potential interactions between bioelectricity and cell behavior (such as...). Figure 3 As shown), this includes cell and mitochondrial membrane potential, mitochondrial volume, voltage-gated ion channels, FOXM1 signal transduction, and cytoskeleton changes (e.g., Figure 1 (The changes shown). Furthermore, this invention also determined the effects of BT on the tumor microenvironment (TME).

[0117] Multiple repeat studies have shown that BT treatment can significantly inhibit the invasion and migration of various human and mouse PDAC cells. Figure 13 BT affects the EMT and infiltration processes. Figure 18 , Figure 24 This effect is mediated at least in part by the FOXM1 cell signaling pathway. Figure 19 , Figure 20 , Figure 26Furthermore, BT treatment also resulted in significant ultrastructural changes in PANC-1 cells, characterized by mitochondrial swelling and cristae disorganization. Figure 8 , Figure 25A , Figure 25B Furthermore, compared to the sham control (SC) group, the BT treatment group showed significant changes in gene expression of voltage-gated ion channels, including Nav1.5, Kv1.3, and KCa3.1, in PANC-1 cells. Figure 27 Furthermore, the cell membrane voltage potential appears to be related to the FOXM1 gene. Similarly, intracellular calcium levels are also significantly affected by BT ( ). Figure 33 In replicate studies, compared with the SC group and the feeder control (CC) group, PANC-1 tumor-bearing mice treated with BT showed a significant reduction in liver metastases. Figure 28 Furthermore, the anti-metastasis potential of BT was further confirmed in the KPCY mouse orthotopic model.

[0118] These studies indicate that BT has a therapeutic effect in the treatment of cancer, especially pancreatic cancer.

[0119] This invention also utilizes the well-known EMF shielding device, the Faraday cage, to investigate whether EMF is a rational mechanism for BT therapy. Regardless of whether cells were placed inside or outside the Faraday cage, BT-induced G1 phase arrest and anti-invasive effects were similar, indicating that EMF does not directly participate in the therapeutic effect of BT on pancreatic ductal adenocarcinoma (PDAC) cells. Figure 30 Therefore, it is clear that the mechanism by which BT functions does not depend on EMF.

[0120] In summary, BT exhibits antitumor activity, particularly in pancreatic cancer cells (in vitro) and their corresponding in situ models, demonstrating anti-invasive and anti-metastatic effects. BT can alter bioelectricity, cell membrane and mitochondrial membrane potential, and also changes the cytoskeleton. Epigenetic data show that among the top 10 genes altered by BT, 7 (including actin, plecktrin, spectrin, TRIO, and PDZ) are associated with the cytoskeleton and / or intracellular communication networks involved in the cytoskeleton. Figure 36 In vitro experimental data also showed that BT can inhibit cell migration and invasion of PDAC cells, an effect mediated at least in part by the FOXM1 gene. In vivo data from other animal models also confirmed the potential of BT to reduce liver metastasis of pancreatic cancer. Finally, this invention found that BT can alter the immune TME (transmissible endothelial growth factor). Figure 29 ).

[0121] Example 1 This study aimed to evaluate the potential role of BT in the anti-invasive properties of human pancreatic ductal adenocarcinoma (PDAC) cells and an animal model. Results showed that PANC-1 cells treated with BT (for 15 minutes) exhibited a 55% reduction in the number of invasive cells compared to the untreated incubator control and sham control groups (P<0.001). Similar results were observed in human PDAC L3.7 cells and mouse pancreatic cancer KPCY cells. Furthermore, the study assessed cell migration ability using Transwell migration and scratch assays. Consistent with the cell invasion assays, BT treatment significantly reduced the migration of PANC-1 and L3.7 cells by 34% and 41%, respectively, compared to the sham control group.

[0122] Two other therapists independently replicated the study of the anti-invasion and anti-migration effects of BT on these PDAC cells. This study demonstrated reproducibility between the results and the therapists. Furthermore, compared to the sham control group, E-cadherin expression was significantly upregulated in BT-treated PANC-1 cells, while the expression of the mesenchymal cell marker N-cadherin was significantly downregulated. Researchers also examined other regulatory factors related to migration and invasion (such as MMP2, MMP9, VEGF, and uPA), finding that BT treatment had only a moderate effect on these markers, suggesting a slowdown in the endocrine process (i.e., EMT transformation) in PANC-1 cells.

[0123] In a repeated study of the PANC-1 mouse orthotopic model, only 28% of mice in the BT treatment group developed grossly visible liver nodules, while 55% and 60% of mice in the untreated feeder control and sham-operated control groups, respectively, showed liver metastases. Furthermore, compared to the sham control group, BT treatment significantly reduced the liver tumor burden by 73% (p<0.05), a result similar to previous studies. Similar to previous PANC-1 mouse model studies, BT treatment had only a moderate effect on the growth of primary pancreatic tumors. In summary, these results indicate that in animal models, BT treatment can inhibit cell invasion and migration processes and significantly reduce pancreatic tumor metastasis.

[0124] Biofield therapy inhibits the growth and metastasis of pancreatic cancer cells through the FOXM1 signaling pathway. Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, currently ranking third among cancer-related deaths in the United States.1 It is projected that by 2030, PDAC will surpass colorectal cancer to become the second leading cause of cancer-related deaths. Despite advancements in surgical techniques, systemic therapies, and targeted therapies, the prognosis for PDAC patients remains extremely poor, with severe cytotoxic effects during conventional chemoradiotherapy.2 The 5-year overall survival rate is less than 13%, and the average survival after diagnosis is less than 6 months, making it the worst-prognostic cancer among all epithelial-derived cancers.3 Most existing therapies, including immunotherapy, have limited efficacy against pancreatic cancer, primarily due to two factors: the presence of excessive desmoplastic stroma, which limits drug penetration; and the presence of an immunosuppressive tumor microenvironment.4,5 Cancer-related deaths in PDAC are mainly attributed to early metastasis and treatment resistance or recurrence; therefore, achieving early diagnosis and more effective treatment of PDAC is a critical medical need that urgently needs to be addressed. In light of this, efforts are being made to explore new alternative treatments that are less toxic and more effective.

[0125] Biofield therapy (BT) can serve as a beneficial complement to conventional treatments for pancreatic cancer (PDAC). The National Center for Complementary and Integrative Health classifies BT as an "energy therapy." Preclinical evidence suggests that BT can alter cellular function and influence tumor growth (Gronowicz G, et al., Evid Based Complement Alternat Med. 2015;2015:926565). However, no studies have yet explored whether BT affects the occurrence and progression of pancreatic cancer, nor have their underlying mechanisms been investigated.

[0126] There is growing interest in the view that cancer is essentially a progressive loss of the body's ability to organize, rather than merely a heterogeneous behavior exhibited by isolated cells (Bissell MJ, et al., Nature reviews Cancer. 2001;1(1):46-54; Ingber DE. Semin Cancer Biol. 2008;18(5):356-364). Extensive research has explored the chemical and biomechanical factors mediating cancer suppression and malignant transformation. Building on this, it is now recognized that endogenous spatiotemporal differences in resting cell membrane potential and bioelevation constitute a crucial aspect leading to dysregulation of intercellular interactions, thereby triggering cancer. Recent research data indicates that cell membrane voltage potential (Vm), the transmembrane voltage generated by various ion channels / transporters with specific ion selectivity and permeability, plays a vital role in the proliferation and metastasis of cancer cells. See Chernet B, et al., J Clin Exp Oncol. 2013;Suppl 1; Yang M, et al., Front Physiol. 2013;4:185; Chernet BT, Levin M. Dis Model Mech.2013;6(3):595-607.

[0127] This invention has conducted a series of studies to explore the effects of BT in different PDAC in vitro and in vivo cancer models (the research protocol has been pre-registered: https: / / osf.io / qtbvf / wiki / home / ). In addition to repeating relevant experiments for cancers derived from other organ sites, this invention has conducted in-depth evaluations to determine the effects of BT on: 1) the proliferation and invasion of PDAC cells and human PDAC-derived organoids, and the associated molecular mechanisms; 2) visible changes in cell morphology; 3) cellular resting volume (Vm) as a mechanistic link between BT and cancer phenotype; and 4) an orthotopic pancreatic cancer mouse model. Furthermore, this invention has invited three biofield therapists to attempt to repeat key in vitro experimental studies.

[0128] Materials and Methods The methodology and materials described in Example 3 (more details are provided here) were used in the study, and the insights and results described below were obtained. For details, please refer to [link to relevant documentation]. Figures 4 to 22 .

[0129] Cell lines and patient-derived organoids (PDOs) The normal human pancreatic ductal epithelial cells HPDE-1 / E6E7 and mouse pancreatic cancer Panc02 cells used were as described in previous literature (Huang C, et al., Cancer research. 2012;72(3):655-665). The routine culture method for Panc02 cells was the same as that used for PANC-1 and other cells in Example 3. HPDE-1 / E6E7 cells were cultured in Gibco keratinocyte serum-free medium (Keratinocyte SFM, catalog number 17-005-042) supplemented with bovine pituitary extract (25 mg / 500 ml) and EGF (2.5 μg / 500 ml, Invitrogen). All experiments were performed using mycoplasma-free cells.

[0130] Pancreatic cancer PDO (AM67) cells were purchased from Dr. Maitra at The University of Texas MD Anderson Cancer Center. Organoids were detached by treating with 2 mg / mL neutral dispase dissolved in phosphate-buffered saline (PBS) for 15 minutes, followed by washing with PBS at 1400 rpm for 2 minutes, and then dissociating using Triple E at 37°C for 5 minutes. Triple E was quenched with wash media, and the cells were centrifuged again for 5 minutes (125 × g). The dissociated cells were counted and then resuspended in 350–700 µL (depending on cell clump size) of Geltrex LDEV-free, hESC-suitable grade, low-growth factor basement membrane matrix (Gibco), and then seeded in dome shapes on Nunclon Delta surface-treated 12-well plates (ThermoFisher). Matrigel was cured at 37°C for up to 45 minutes. Then, approximately 1 mL of PaTOM 68 medium supplemented with Y-27632 dihydrochloride (Tocris) was added to each well of a 12-well plate to ensure complete submersion of the organoid dome. The organoids were cultured for at least 7 days after inoculation before the first passage.

[0131] When testing or evaluating animals, neither laboratory members nor pathologists were aware of the grouping process.

[0132] In vitro cell proliferation and growth Human or mouse pancreatic cancer cells (1 × 10⁴) were seeded in triplicate in 96-well plates. After culturing for 16–24 hours, the cells were exposed to BT for treatment. Immediately after treatment, cell growth was assessed using the PrestoBlue assay (Yang P, et al. Scientific reports. 2019;9(1):6428).

[0133] To assess the growth of PDO (patient-derived organoids), acid-soluble rat tail collagen type I (3 mg / ml, Corning, USA) was used. First, it was neutralized with 375 µl of 10-fold DMEM (containing phenol red) and 1N NaOH (100 µl). The neutralized collagen type I (25 µl) was seeded at the bottom of a 12-well plate (NUNC, Life Technology) as the substrate and polymerized on ice for two hours until the substrate became turbid. Then, it was transferred to 37°C and incubated for at least 30 minutes to 1 hour before cell seeding. Approximately 500 pancreatic organoid single cells were mixed with the cold-neutralized collagen (Padmanaban V, et al., Nat Protoc. 2020;15(8):2413-2442) and gently seeded into the seeded wells (placed on a 37°C heated plate). The culture plates were incubated at 37°C and 5% CO2 for 1 hour to complete gelation, followed by the addition of organoid culture medium. The cells were then subjected to BT treatment. Tumor organoid cells continued to grow in the incubator for 3 to 6 days. The growth of AM67 was monitored using an EVOS M7000 imaging system (Thermo Fisher Scientific), and quantitative analysis was performed using ImageJ software.

[0134] Cell voltage characterization In short, cells (5-10 × 10³) were seeded the day before treatment. After treatment, the plates were incubated for 5 days in an incubator at 37°C, 5% CO₂, and 95% humidified air. DiBAC4(3), a slow-response potentiometric probe, was used to stain and measure cell depolarization. The DiBAC4(3) working solution (1 mg / mL) was prepared by mixing the stock solution with Dulbecco modified Eagle medium (without fetal bovine serum) at a ratio of 1:4000. Cells were washed with PBS and the DiBAC4(3) working solution, along with 1 drop of NucBlue Live Ready Probes reagent, and incubated at 37°C for 15 minutes. Images of green fluorescent protein and DAPI were captured using an EVOS M5000 imaging system (AMF5000) or an EVOS M7000 imaging system (AMF7000). For images captured by EVOS M5000, fluorescence intensity was quantified using ImageJ; for images captured by EVOS M7000, fluorescence intensity was quantified using Cellslate software.

[0135] Reverse protein microarray (RPPA) Following BT treatment for 15 or 30 minutes, PANC-1 cells were immediately lysed in lysis buffer provided by the MD Anderson Functional Proteomics Core Facility. Cell lysates were then analyzed by reverse-phase protein microarray (RPPA) analysis via the MD Anderson Functional Proteomics Core Facility as previously described (Yang P, et al. Scientificreports. 2019;9(1):6428).

[0136] Animal models of pancreatic cancer Procedure: Two independent animal studies were conducted. Mice with tumors ranging from 1 to 10 mm² were divided into four groups, as described in Example 2. For Study #1, n = 11-16 mice per group; for Study #2, n = 25-30 mice per group.

[0137] Transmission electron microscope Immediately after biofield therapy (BT), PANC-1 cells were washed with phosphate-buffered saline (Ca++ / Mg++-free) and then fixed with EM fixative in 0.1 M diarsethate buffer (pH 7.3) containing 3% glutaraldehyde and 2% paraformaldehyde. Cells were then washed in 0.1 M sodium diarsethate buffer, treated with 0.1% Millipore-filtered diarsethate-buffered tannins, post-fixed with 1% buffered osmium, stained with 0.1% Millipore-filtered uranyl acetate, dehydrated with increased concentrations of ethanol, and then infiltrated and embedded in LX-112 medium. Samples were then polymerized in a 60°C oven for approximately 3 days. Ultrathin sections were cut using a Leica Ultracut microtome (Leica, Deerfield, IL) and stained with uranyl acetate and lead citrate in a Leica EM staining system. Samples were examined using a JEM 1010 transmission electron microscope (JEOL USA, Inc., Peabody, MA) with an accelerating voltage of 80 kV. Digital images were obtained using an AMT imaging system (Advanced Microscopy Techniques Corp., Danvers, MA).

[0138] For the scratch assay, a Culture-Insert 2 Well (dual-well culture insert) (Ibidi, Fitchburg, WI) was used with a µ-Dish 35 mm culture dish. The dish is equipped with a culture insert to ensure reproducible scratch gap widths. MiaPaca-2 cells (6 × 10⁵ cells) were seeded into two wells of the culture insert and cultured for 24 hours. Subsequently, the culture insert was carefully removed using sterile forceps, and 1 mL of culture medium was added to each culture dish as an aliquot. The culture dishes were placed under two CytoSmart FL microscopes (Axion Biosystem, Atlanta, GA) in separate incubators, exposing the cell culture dishes to either a sham control or BT treatment. Images were acquired every 30 minutes over 48 hours and analyzed using the CytoSmart scratch assay online program.

[0139] Cell cycle analysis For cell cycle analysis, cells (2.5 × 10⁵) cultured in 6-well plates were exposed to BT or a sham control for 15 minutes. Cells were immediately trypsinized and centrifuged. Cell clumps were resuspended, washed with 1X PBS, and fixed overnight in 70% ethanol at 4°C. Fixed cells were further washed with 1X PBS and resuspended in PBTB staining solution containing PBS, propidium iodide (5 μg / mL), and DNase-free RNase A (10 μg / mL). Cells were incubated in the dark at room temperature for 15 minutes, then placed on ice until data were acquired using a BD FACS Calibre flow cytometer (BD Biosciences, San Jose, CA). The percentage of cells in each phase of the cell cycle was determined based on DNA histogram content.

[0140] Immunoblotting analysis Immunoblot analysis was performed using an automated Western blotting system: the JessSimple Western system (ProteinSimple, San Jose CA, USA) automatically separates and immunodetects proteins by the size of its capillaries. To quantify the expression of target proteins in treated cells, this study followed standard procedures using the 12-230 kDa Jess separation module (SM-W004). Final protein concentrations (0.4 mg / mL to 1 mg / mL) of tissue lysates were obtained by mixing the assayed cell lysate (0.6 mg / mL to 2 mg / mL) with 0.1x sample buffer and a Fluorescent 5X Master mix (ProteinSimple). The resulting mixture was denatured at 95°C for 5 minutes. The Results section lists the target primary antibody; β-actin or tubulin were prepared by mixing antibody dilutions at ratios of 1:10 to 1:50. Secondary antibodies were prepared by mixing chemiluminescent and fluorescent antibodies. Subsequently, cell lysate samples were loaded into Ladder plates (12–230 kDa), and antibody diluent, primary antibody, secondary antibody, luminol-S, and peroxide solution (for chemiluminescence detection) were added sequentially according to the manufacturer's instructions, followed by washing buffer. Chemiluminescence and fluorescence digital images of the target protein were acquired using Compass Simple Western blotting software (version 4.1.0, ProteinSimple), and protein quantification was performed based on signal area.

[0141] result BT reduced the growth of human and mouse pancreatic cancer cells and PDO in cell cultures. First, the proliferation of human (PANC-1 and MiaPaCa-2) and mouse (Panc02 and KPCY) pancreatic cancer cells was examined using the PrestoBlue assay after 5, 15, or 30 minutes of BT treatment. Figure 4 As shown in Figure Aa, compared with the IC and SC groups, the growth of PANC-1 cells treated with BT for 15 minutes and 30 minutes was moderately but significantly inhibited 1 hour after BT treatment. Cell growth in the SC group (i.e., cells placed on the same table as the BT group cells) was also reduced, but the reduction was greater in the BT group compared to the other two groups. Given that 5 minutes of BT treatment did not cause significant changes in PANC-1 cell growth, and that the effects of 15 minutes and 30 minutes of BT treatment on PANC-1 cell growth were similar, this study repeated the experiment more than 12 times using the 15-minute dose. Figure 4 The four duplicate samples shown in Ab; Figure 5 Five replicates are shown in AC; all experiments were performed on different days. Following exposure to BT treatment, human pancreatic cancer COLO357 cells ( Figure 4 Ac) and MiaPaCa-2 cells ( Figure 6 Similar activity was also observed in Panc02 cells (A) and mouse pancreatic cancer cells. Compared to PANC-1 cells, BT treatment at a 30-minute dose appeared to show the greatest decrease in cell growth in Panc02 cells compared to the IC and SC groups. Figure 4 Ad). Then, in three independent experiments, Panc02 cells were treated with BT for 30 minutes, and it was observed that BT consistently and significantly inhibited the growth of Panc02 cells compared with the IC and SC groups. Figure 5 DF). After 15 minutes of exposure, BT also significantly reduced the growth of KPCY (mouse pancreatic cancer) cells. Figure 6 B). To further investigate whether the inhibitory effect of BT on cell growth is specific to cancer cells, this study exposed immortalized human pancreatic ductal epithelial cells (HDPE-1 / E6E7) to BT treatment for 15 minutes. Figure 4 As shown in Figure Ae, the growth rate of HPDE-1 / E6E7 cells exposed to BT was similar to that of the IC group, but slightly higher than that of the SC group, indicating that BT may reduce the growth capacity of PDAC cells in vitro.

[0142] The experiment was repeated using PANC-1, COLO357, and L3.7 cells by a second biofield therapist (therapist 2). Figure 4As shown in Af-h, compared to the IC and SC groups, 15-minute BT treatment resulted in sustained and significant slowing of growth in all three cell lines. Although the growth rate of HPDE-1 / E6E7 cells in the SC group was significantly lower than that in the IC group, the growth rate in the BT group was similar to that in the SC group. Figure 4 This is consistent with the results observed in HPDE-1 / E6E7 cells treated by the first biofield therapist. PANC-1 cells treated by the third therapist (therapist 3) for 15 and 30 minutes exhibited moderate growth rates, but significantly lower than those observed in the IC and SC groups. Figure 7 Therefore, the results of treatment with three different biofield therapists consistently showed that BT could reduce the growth of human PDAC cells. Given that patient-derived organoids (PDOs) can reflect the genetic and epigenetic landscape of each PDAC patient and are a reliable model for studying cancer biology and predicting treatment efficacy, this study further investigated the growth of AM67 PDOs after BT treatment. The results showed that 11 days after BT treatment, the number of PDOs in the BT group was significantly less than that in the SC group (…). Figure 4 Ba). Similarly, compared to the SC group, BT also reduces the size of AM67 PDO ( Figure 4 This further supports the potential role of BT in suppressing PDAC growth.

[0143] BT alters the ultrastructure of PANC-1 cells Organelles exist within highly dynamic cellular networks, performing essential functions through communication with each other. This study used transmission electron microscopy (TEM) to examine the ultrastructural changes of organelles in PANC-1 cells immediately after BT treatment. BT treatment resulted in significant changes in the ultrastructure of mitochondria. Figure 8 As shown, IC group ( Figure 8 A and Figure 8 D) and SC group ( Figure 8 B and Figure 8 In PANC-1 cells treated with BT, the mitochondria were all elongated and had well-developed cristae. In contrast, in PANC-1 cells treated with BT, the mitochondrial cristae were disordered, and the mitochondria showed obvious expansion and swelling. Figure 8 C and Figure 8 F). This significant and objective change in the ultrastructure of mitochondria not only confirms the biological effects of BT on important cellular functions, but also provides a new perspective for understanding this effect.

[0144] BT arrests the cell cycle of human PDAC cells in the G0 / G1 phase. To further understand the biological changes in pancreatic cancer cells after BT treatment, this study performed cell cycle analysis on human PDAC cells. Figure 9 As shown, compared with the IC group and the SC group, BT significantly increased the proportion of PANC-1 cells in the G0 / G1 phase by 1.5 times. Figure 9 A and Figure 9 B). Subsequently, the study was repeated three more times, and the same changes in cell cycle patterns were observed in all experiments. Figure 9 C and Figure 10 A and Figure 10 B). To confirm that the cell cycle changes observed in PANC-1 cells after BT treatment were independent of baseline cell growth, PANC-1 cells were inoculated 2 days before treatment, BT-treated, and cells were collected immediately after treatment. Figure 10 As shown in D, although with Figure 9 B and Figure 9 Compared to the results shown in C, the proportion of cells in the G0 / G1 phase was lower in the IC and SC groups, while the proportion of cells in the G2 / M phase almost doubled. However, PANC-1 cells treated with BT were still arrested in the G0 / G1 phase of the cell cycle. Similarly, BT also affected other PDAC cells (including L3.7 cells). Figure 9 E) and COLO357 cells ( Figure 9 F) showed significant G0 / G1 phase arrest. Furthermore, PANC-1 cells treated for 15 minutes by two other biofield therapists also showed a significantly higher proportion of cells in the G0 / G1 phase compared to the SC or IC groups. Figure 10 C and Figure 10 D).

[0145] BT reduces the membrane potential of pancreatic cancer cells, causing hyperpolarization in various PDAC cells. Given the known role of resting Vm in maintaining and suppressing cancer phenotypes (Chernet B, Levin M. J ClinExp Oncol. 2013;Suppl 1; Chernet BT, Levin M. Oncotarget. 2014;5(10):3287-3306), this study investigated whether resting Vm is part of the mechanism by which BT exerts its effects. Figure 11 As shown, after 5 days of exposure to BT, SC, or IC, the DiBAC4 fluorescence intensity of PANC-1 cells in the IC and SC groups was similar, while the staining intensity of PANC-1 cells exposed to BT was significantly lower than that in the other two groups. Figure 11 A and Figure 11B). When PANC-1 cells were exposed to BT for two consecutive days, their Vm decreased by nearly 50% compared to the other two groups (p<0.05 and p<0.01, respectively). Figure 11 C). Immediately after or 1 hour after BT treatment (15 or 30 minutes), in MiaPaCa-2 cells ( Figure 11 D、 Figure 12 A) and KPCY cells ( Figure 11 E, Figure 12 In B), the effect of BT in reducing Vm was also observed. In summary, these results indicate that BT can alter Vm, which reflects the hyperpolarized state of pancreatic cancer cells, and this hyperpolarization can contribute to a slowdown in cell growth.

[0146] BT inhibits the migration and invasion of PDAC cells The effect of BT on the invasiveness of PDAC cells was investigated. Figure 13 As shown in Figure Aa, compared with the IC and SC groups, the invasiveness of cells treated with BT was reduced by 55% (p<0.0001), while no difference was observed between the IC and SC groups. Given the lack of difference between the IC and SC groups, the experiment was repeated four times on different dates for both the BT and SC groups. The results consistently showed that BT significantly inhibited the invasiveness of PANC-1 cells. Figure 14 AD). This study further investigated the anti-invasive effects of BT on human PDAC cells (COLO357) with relatively low FOXM1 expression and human PDAC cells (L3.7, a metastatic clone of COLO357 cells) with high FOXM1 expression (Huang C, et al., Clinical cancer research. 2014;20(6):1477-1488) and mouse pancreatic tumor KPCY cells. After BT treatment, L3.7 cells showed a reduction in invasiveness similar to that of PANC-1 cells (AD). Figure 13 Ab and Figure 14 E), while the reduction in invasiveness of COLO357 cells treated with BT (22%) was less than that of PANC-1 cells ( Figure 13 Ac). Furthermore, the reduction in KPCY cell invasiveness by BT was similar to that observed in PANC-1 cells (Ac). Figure 13 Ad).

[0147] Consistent with the results of the cell invasion assay, BT treatment also significantly reduced the migration of PANC-1 cells and L3.7 cells compared with the SC group, with reductions of 34% and 41%, respectively (p<0.001). Figure 13 Ae and Figure 13Af). The inhibitory effect of BT on PDAC cell migration was also confirmed in MiaPaca-2 cells using scratch-migration assays. Figure 13 B). The anti-invasion and anti-migration effects of BT in these PDAC cells were tested by a second therapist (therapist 2), and the results were similar to those observed when the first therapist (therapist 1) performed the test. Figure 13 (C) indicates that these results are reproducible across different studies and therapists.

[0148] To confirm that the BT-induced resistance to invasion in PANC-1 cells was not due to differences in the incubators used for culture, the invasiveness of untreated PANC-1 cells cultured in the SC and BT incubators was tested after treatment and culture of cells in the SC and BT groups. Figure 14 As shown in F, PANC-1 cells exhibited similar invasive capabilities in both incubators.

[0149] BT alters key cell signaling proteins (especially FOXM1). To further explore the potential mechanism of action of BT-induced effects, RPPA analysis was performed on PANC-1 cells, and the results were compared with changes observed in the SC group. The analysis was conducted blinded, meaning that staff at the Core Facility were unaware of the experimental design or group assignments.

[0150] like Figure 15 A and Figure 15 As shown in B, both BT treatment for 15 minutes and 30 minutes altered the expression of several proteins. Although BT upregulated some proteins, 15 minutes of BT treatment significantly downregulated the expression of ALKBH5 (a known m6A demethylase and epigenetic transcriptional regulator of FOXM1) by 41%. FOXM1 expression was downregulated by 21%, and the expression of cyclin B1 and pCDK1 was each downregulated by 15% (p values ​​for all proteins <0.0005). Figure 15 B). In PANC-1 cells, a similar decrease in the expression of cyclin B1 and FOXM1 was observed 30 minutes after BT treatment, while the expression of TRIM24, TFRC, and PAI-1M also decreased. Figure 16 Automated immunoblotting analysis (Western Blot analysis) (Jess; Figure 15 C) This further confirmed that BT (treatment for 15 minutes) significantly downregulated the level of FOXM1 protein in PANC-1 cells. Given that p21 can arrest cells in G1 and G2 / M phases, it was investigated whether BT also affected p21 protein. Figure 15As shown in Figure D, BT did indeed upregulate p21 expression in PANC-1 cells. Similar results were also observed in BT-treated L3.7 cells. Figure 17 These results suggest that BT can potentially influence the cell cycle of PANC-1 cells by regulating cell cycle transcription factors or cell cycle protein kinases such as FOXM1 and p21.

[0151] BT alters markers associated with epithelial-mesenchymal transition in PANC-1 cells. To investigate how BT inhibits the invasiveness of PDAC cells, expression markers related to epithelial-mesenchymal transition and invasion in PANC-1 cells were detected. Figure 18 As shown, in BT-treated PANC-1 cells, the expression level of the epithelial cell marker E-cadherin was significantly higher than that in the IC and SC groups. Figure 18 A). In contrast, the expression level of the mesenchymal cell marker N-cadherin was significantly lower in the BT group than in the IC and SC groups (p<0.05). Figure 18 B).

[0152] Compared with the IC group, BT treatment also significantly reduced the expression of CD44 protein. Figure 18 (C) This indicates that BT can slow down the endosmotic process, namely the epithelial-mesenchymal transition (EMT) of PANC-1 cells. CD44 is one of the important markers of PDAC stemness. Knockdown or overexpression of CD44 affects PDAC stemness, which can be confirmed by the spheroid formation assay (Yan Y, et al. Cancer Res. 2016 Apr 15;76(8):2419-31). BT treatment significantly downregulated the expression of CD44 protein in PANC-1 cells, suggesting that BT may potentially inhibit the stemness of PDAC cells.

[0153] Modification of the FOXM1 gene alters the response of PANC-1 cells to BT. To determine whether FOXM1 is a potential molecular target mediating BT-induced changes, the correlation between the ability of BT to downregulate FOXM1 protein expression and the inhibitory effect of BT on PDAC cell invasion was first examined. Figure 19As shown, the two were highly correlated (R² = 0.85), indicating that the stronger the inhibitory effect of BT on FOXM1 expression, the more significant its inhibitory effect on cell invasion. BT had the strongest inhibitory effect on FOXM1 in PANC-1 cells (>40%), and the corresponding cell invasion inhibition rate was also the highest (54%); while in COLO357 cells, BT failed to inhibit FOXM1 expression and had the lowest invasion inhibition rate (17.3%). This study suggests that the BT-induced anti-invasion effect of PDAC cells may be mediated at least in part by its ability to downregulate FOXM1 protein. Subsequently, the cell cycle and invasiveness of parental PANC-1 cells and FOXM1-modified PANC-1 cells after BT or SC treatment were examined. In FOXM1 stably knocked-down PANC-1 cells, FOXM1 expression was reduced by nearly 90% compared to parental PANC-1 cells ( Figure 20 A). When parental PANC-1 cells and FOXM1 stably knocked-down PANC-1 cells were treated with BT, the parental PANC-1 cells showed a higher proportion of G0 / G1 phase cell population compared to the SC group, as did the FOXM1 stably knocked-down PANC-1 cells. Figure 20 B). When parental PANC-1 cells were treated with BT (15 minutes), the invasiveness of these cells was significantly reduced compared to the SC group (p<0.001). Figure 20 C), and it was also significantly lower than in previous experiments (C). Figure 13 Aa and Figure 14 AD. FOXM1 knockdown reduced the invasiveness of PANC-1 cells by nearly 50%, an effect similar to that of the BT group, and there was no significant difference in invasiveness between the BT and SC groups. Figure 20 C). Although BT treatment significantly reduced the invasiveness of unmodified L3.7 cells by 62.2% compared to the SC group ( Figure 20 D、 Figure 13 Ab and Figure 14 However, after knocking out FOXM1 in L3.7 cells, their cell invasiveness was significantly reduced by 61% compared to parental L3.7 cells, and there was no longer a difference between the BT group and the SC group. Figure 20 D). Similarly, compared to parental PANC-1 cells, FOXM1 knockdown (KO) PANC-1 cells exhibited a significantly reduced number of invasive cells; furthermore, BT-induced inhibition of PANC-1 cell invasion was again reduced to its lowest level after FOXM1 knockout. Figure 20 E), this result is consistent with the experimental results of FOXM1 KO L3.7 cells ( Figure 20D). Conversely, overexpression of FOXM1 in COLO357 cells significantly increased the number of invaded cells by 19% compared to parental COLO357 cells. Furthermore, BT significantly reduced the invasiveness of parental COLO357 cells by 33.6%; while in COLO357 cells overexpressing FOXM1, invasiveness was also significantly reduced, with a decrease of approximately 59.5% compared to the SC group. Figure 20 F). Furthermore, using the Tet-on tetracycline (or doxycycline) induction system, the anti-invasive effect of BT was investigated in COLO357 and PANC-1 cells conditionally overexpressing the FOXM1 gene. Similarly, compared to parental COLO357 cells, conditional overexpression of FOXM1 significantly increased the number of invasive COLO357 cells by 65.6%. BT significantly reduced the invasiveness of parental COLO357 cells by 57.4%; and in FOXM1-overexpressing COLO357 cells, compared to the SC group, its invasiveness was also significantly reduced by approximately 78.4% (p<0.0001). Figure 20 G). These data suggest that FOXM1 may be at least partially responsible for BT activity, as it is associated with changes in cell cycle and cell invasiveness in PDAC cells.

[0154] BT inhibits liver metastasis of PANC-1 cells in an orthotopic mouse model. To further evaluate the antitumor effect of BT on pancreatic cancer, an antitumor efficacy study was conducted in a human PANC-1 mouse orthotopic model. Figure 21 As shown in Figure A, the mean terminal tumor weight of mice treated with BT was 36% and 26% lower than that of the CC and SC groups, respectively. However, this difference did not reach statistical significance. Compared with the CC group, gemcitabine (one-quarter of the maximum tolerated dose, i.e., 40 mg / kg) significantly reduced the growth of PANC-1 in situ tumors by 35% (p<0.05), with no difference observed between the gemcitabine group and the BT group. Figure 21 A). It was also observed that 30% of mice in the BT group developed liver metastases, a proportion that was 43% lower than that in the CC group (53.8% liver metastases) and 17% lower than that in the SC group. Figure 21 B). Macroscopic observation of the number of liver nodules in mice with liver metastases revealed that the average number of liver nodules per liver in mice treated with BT was 1.6 ± 0.9, a 75% reduction compared to the CC group (6.6 ± 3.6) and the SC group (6.4 ± 3.0) (p<0.05). Mice treated with gemcitabine also showed a 39% reduction in the number of liver nodules compared to the CC group, but this difference was not statistically significant. Figure 21 C). Although the number and size of liver nodules were greater in both the CC and SC groups, the livers of the BT group mice appeared normal. Figure 21D).

[0155] Histopathological examination of liver nodules ( Figure 21 E) showed that the average number of liver nodules in the CC group was 16.3, while that in the BT group was 8.7, with the number of nodules in the BT group also being nearly 50% less than that in the SC group. In a study repeated by the same therapist using a similar PANC-1 mouse orthotopic model, only 28% of the mice in the BT group had visible liver nodules, while the proportions of liver metastases observed in the CC group and SC group were 55% and 60%, respectively. Figure 21 F). Furthermore, compared to the SC group, the liver tumor burden in the BT group was significantly reduced by 73% (p<0.05); Figure 8 (G), this result is similar to the observations made in the first study of this invention.

[0156] discuss This invention provides multifaceted evidence demonstrating the antitumor activity of BT in cell culture and in an orthotopic mouse model of human pancreatic cancer PANC-1. BT persistently inhibited the growth and invasiveness of various human and mouse PDAC and PDO cells in vitro, accompanied by significant changes in mitochondrial morphology, significant induction of cell cycle arrest, and reduced expression of epithelial-mesenchymal transition markers. These findings were replicated in multiple experiments using rigorous controls (including incubator / feeding room controls and sham controls) involving three different biofield therapists. BT also significantly reduced the cell membrane voltage potential of PANC-1 cells. In two independent orthotopic mouse model studies, although BT only moderately inhibited the growth of the primary tumor, it significantly reduced liver metastasis of PANC-1 cells, indicating that BT not only slows the growth of PANC-1 tumors but, more importantly, effectively inhibits metastatic growth to the liver. The results from the animal models are consistent with the in vitro experimental results, both demonstrating the effectiveness of BT in inhibiting invasiveness (see...). Figure 15 Its effect on delaying PDAC cell growth is stronger than its effect on (see) Figure 4 ).

[0157] This study explored the cellular mechanism of BT's anti-PDAC effect through multiple experiments. The results showed that BT significantly reduced the expression of FOXM1 and its signaling circuit-related proteins in PANC-1 cells. Upregulation, knockdown, or elimination of FOXM1 expression all significantly altered the effect of BT on PDAC cell invasiveness. Combining these in vitro and in vivo data, this study is the first to demonstrate that BT can alter the metabolic activity, growth, and invasiveness of PDAC cells by downregulating FOXM1 (an important regulator of pancreatic cancer cell cycle, metastasis, and stemness). Therefore, BT holds promise as an important treatment option for pancreatic cancer patients. Furthermore, BT did not affect the growth of normal cells, indicating that BT has limited toxicity and a good safety profile.

[0158] PDAC is known to be accompanied by a variety of genetic and epigenetic alterations, including activation of the KRas oncogene mutation and inactivation of the CDKN2A, TP53, and SMAD4 tumor suppressor genes (Murphy SJ, et al., Gastroenterology. 2013;145(5):1098-1109.e1091; Su GH, Kern SE. Currentopinion in gastroenterology.2000;16(5):419-425). To date, therapeutic interventions targeting these alterations have not been effective. As an alternative strategy, researchers are working to identify and target key downstream effectors of these oncogenic signaling pathways. FOXM1 transcription factor has been identified as one of these effectors because signals from multiple key oncogenic signaling pathways converge here, jointly activating the expression and function of FOXM1 (Huang C, et al. Biochimica et biophysica acta. 2014;1845(2):104-116;Li L, et al., Gastroenterology. 2014;147(2):485-497.e418;Pandit B, et al.,Cellcycle (Georgetown, Tex). 2009;8(20):3425-3427;Park HJ, et al.,The EMBOjournal. 2009;28(19):2908-2918;Ma RY, et al.,Journal of cell science.2005;118(Pt 4):795-806), and FOXM1 is the master regulator of cell cycle progression.FOXM1 protein levels also change dynamically with cell cycle progression, peaking in S and G2 / M phases (Wang X, et al., Proceedings of the National Academy of Sciences of the United States of America. 2002;99(26):16881-16886; Wang IC, et al., Molecular and cellular biology.2005;25(24):10875-10894). The functional activation, nuclear translocation, and stability of FOXM1 protein all depend on its phosphorylation and are regulated by the cyclin / CDK and KRAS-MAPK / AKT signaling pathways (Ma RY, et al., Journal of cellscience. 2005;118(Pt 4):795-806; Myatt SS, et al., Naturereviews Cancer. 2007;7(11):847-859; Anders L, et al., Cancer cell.). 2011;20(5):620-634). FOXM1 is a transcription factor overexpressed in a variety of human cancers, including pancreatic cancer, and its functions involve promoting cell proliferation and driving tumor cell invasion, metastasis and stemness (Huang C, et al., Cancer research.2012;72(3):655-665; Bella L, et al., Semin Cancer Biol. 2014;29:32-39; Quan M, et al., MolCancer.2013;12:159; Kalathil D, et al., Front Oncol. 2020;10:626836).Elevated expression of FOXM1 protein is associated with various human malignancies, including pancreatic cancer, and studies have shown that FOXM1 is an important predictor of poor prognosis in 39 types of human tumors (Huang C, et al., Cancer research.2012;72(3):655-665; Huang C, et al., Clinical cancer research 2014;20(6):1477-1488; Huang C, et al., Biochimica et biophysica acta. 2014;1845(2):104-116; Gentles AJ, et al., Nature medicine. 2015;21(8):938-945; Kong X, et al., Cancerresearch. 2013;73(13):3987-3996; Pilarsky C, et al., Neoplasia (NewYork, NY). 2004;6(6):744-750; Wei G, et al., Aging.2022;14(22):9128-9148). In summary, these data establish FOXM1 as a highly promising target for cancer treatment (Borhani S, et al., Expert opinion on therapeutic targets. 2020;24(3):205-217), and inhibitors targeting FOXM1 are under development for clinical application (Gormally MV, et al., Nature communications.2014;5:5165; Shukla S, et al., Mol Cancer Ther. 2019;18(7):1217-1229).

[0159] This study, through blinded RPPA analysis, is the first to demonstrate that BT significantly affects the protein expression of FOXM1 and its related signaling circuit genes (including ALKBH5, cyclin B1, and CDK1) in PANC-1 cells. The effects of knockdown of FOXM1 expression on the G0 / G1 phase and cell invasion were similar to those observed with BT, while overexpression of FOXM1 attenuated BT activity. These results confirm that FOXM1 plays a crucial role in mediating the antitumor activity of BT. The specific mechanisms by which BT regulates FOXM1 expression and function require further elucidation. FOXM1 expression and activity are tightly regulated and coordinated by transcription factors (activators) and epigenetic modifications (including transcriptional and translational regulation). Human AlkB homolog H5 (ALKBH5) is an important m6A demethylase that plays a vital role in various biological processes during the development and progression of multiple tumors, including cell proliferation, invasion, and metastasis. Previous studies have shown that in glioblastoma, ALKBH5 can demethylate newly generated FOXM1 transcripts and increase FOXM1 expression. Given that BT treatment for only a short period of time (15-30 minutes) can significantly downregulate FOXM1 protein expression, it is highly likely that this is due to post-transcriptional modifications at the FOXM1 mRNA and / or protein level, such as ALKBH5-mediated m6A methylation of FOXM1 mRNA or pAKT-mediated phosphorylation of FOXM1 protein, which can contribute to the rapid change in FOXM1 protein expression after BT treatment (Chesnokov MS, et al., Front Oncol. 2021;11:696532; Zhang S, et al., Cancer cell. 2017;31(4):591-606 e596). Emerging evidence suggests that FOXM1 may mutually promote AKT activation, forming a positive feedback loop (Borhani S, et al., Expert opinion on therapeutic targets. 2020;24(3):205-217; Chesnokov MS, et al., Front Oncol.2021;11:696532). Previous studies have shown that BT treatment can downregulate pAKT levels (Yang P, et al., Integr Cancer Ther. 2019;18:1534735419840797). Therefore, BT may potentially affect the regulatory network of the FOXM1 and AKT pathways in PDAC cells.

[0160] As is well known, cancer cells possess unique bioelectrical properties. Abnormal depolarization of the resting membrane potential (Vm) is considered a convenient marker of tumor formation and can activate the metastatic phenotype of genetically normal cells in vivo (Chernet BT, et al., Dis Model Mech. 2013;6(3):595-607; Levin M. et al., Mol BiolCell.2014;25(24):3835-3850; Fraser SP, et al., Clinical cancer research. 2005;11(15):5381-5389; Prevarskaya N, et al., Physiol Rev. 2018;98(2):559-621). Changes in Vm can trigger metastasis over considerable distances, a process mediated by transcriptional and epigenetic effects induced by electrochemically regulated serotonin and butyrate flows (Chernet BT, Levin M. et al., Dis Model Mech. 2013;6(3):595-607). Cell membrane voltage potential is mediated by varying concentrations of ions and ion channels, which are now considered to play important roles in tumorigenesis (Prevarskaya N, et al., Physiol Rev. 2018;98(2):559-621). Electrophysiological analyses of various cancer cell types have shown that depolarized Vm promotes cell proliferation and migration. Hyperpolarization of CHO cells can induce mitotic arrest and cell division, and hyperpolarization can inhibit tumorigenesis induced by oncogenes (Chernet B, Levin M. et al., J Clin Exp Oncol. 2013;Suppl 1; one; CD, Jr., J Theor Biol. 1971;30(1):151-181). Using a well-defined molecule, DiBAC4, to stain cell membrane potential, it was observed that BT significantly reduced the cell membrane potential of PANC-1, MiaPaCa-2 (human), and KPCY (mouse) cells. It is well known that cell migration is regulated by water and ions, and Vm is considered an indirect influencing factor that can affect cell migration by regulating the Ca2+ electrodynamic force mediated by TRP channels (Schwab A, et al., Physiol Rev. 2012;92(4):1865-1913; Lobikin M, et al., Phys Biol. 2012;9(6):065002).Several members of the TRP channel family (such as TRPM8) play a crucial role in the proliferation and metastasis of pancreatic cancer cells, and TRPM8 expression is associated with poorer overall survival in PDAC patients (Yee NS, et al., Cells.2014;3(2):500-516; Du JD, et al., Med Sci Monit. 2018;24:3720-3725; Liu J, et al., Pancreatology. 2018;18(8):935-944; Mesquita G, et al., Cells. 2021;10(5)). Therefore, it is necessary to further investigate how BT reduces the cellular Vm and causes hyperpolarization in PDAC cells, thereby exerting an anti-metastatic effect; at the same time, it is also necessary to deepen the understanding of cell polarization and carcinogenesis.

[0161] The physical mechanisms by which biophoton emission (BT) affects cellular processes remain unclear. Possible mechanisms include biophoton emission and electromagnetic fields (EMF) (Roll WG, et al., Int J Neurosci. 2002;112(2):197-224; Persinger MA, et al., Int J Yoga. 2012;5(2):140-145; Karbowski LM, et al., Neurosci Lett. 2012;523(2):131-134; Buckner CA, et al., PLoS One. 2015;10(4):e0124136; Hu JH, et al., Int J Radiat Biol. 2010;86(2):79-88). It is well known that the human body emits EMFs, and these electromagnetic fields play intricate roles in maintaining overall homeostasis and the function of many specific organ systems. Electrocardiography (ECG) and electroencephalography (EEG) are used to measure the electromagnetic field (EMF) generated by the heart and brain, respectively. Other techniques, such as detecting biophotons emitted by the human body, can also be used to measure the EMF in the human body.64 The EMF can be measured in any living organism (Zhou SA, Uesaka M. Int J Eng Sci. 2006;44(1-2):67-92). Studies have shown that the EMF can modulate tumor growth, angiogenesis, and tumor necrosis factor levels in animals (Crocetti S, et al., PLoSOne. 2013;8(9):e72944), including inhibiting the growth of EpH4-MEK-Bcl2 mammary tumors in mice by inducing apoptosis (Tatarov I, et al., Comp Med. 2011;61(4):339-345). The EMF has begun to be used in routine cancer treatment. For example, the U.S. Food and Drug Administration (FDA) has approved the use of low-frequency alternating electric fields (i.e., "tumor therapy electric fields") to treat refractory glioblastoma multiforme and other cancers (Stupp R, et al., JAMA.2017;318(23):2306-2316; Rick J, et al., J Neurooncol. 2018;137(3):447-453).

[0162] This study investigated dose-dependent cell biological changes in PDAC cells regarding cell proliferation at the early stages of the study. Although two independent experiments conducted by two different biofield therapists showed that 5 minutes of BT treatment had a negligible effect on PANC-1 cell growth (…),… Figure 4 A and Figure 7 However, 15-minute and 30-minute doses of BT treatment administered by two different therapists appeared to have similar inhibitory effects on PANC-1 cell growth, with the 15-minute dose potentially showing stronger inhibition than the 30-minute dose. In fact, compared to 30-minute BT treatment, 15-minute BT treatment exhibited stronger inhibitory activity on the cell growth of various PDAC cell types, including PANC-1, L3.7, and COLO357 cells. Figure 4 Aa、 Figure 4 Af、 Figure 4 Ag and Figure 4 The results indicate a nonlinear relationship between treatment duration and PDAC cell growth. This phenomenon is similar to those observed in studies using low-intensity phototherapy (Huang YY, et al., Dose Response. 2009;7(4):358-383). The mechanism of this nonlinear dose-response relationship between BT and PDAC cells remains unclear and requires further investigation.

[0163] In summary, the research data of this invention demonstrate that BT exhibits antitumor activity against cultured pancreatic cancer cells, PDO, and related orthotopic xenograft models. In vitro studies show that BT can regulate key cell cycle regulators such as FOXM1 and p21, thereby leading to cell cycle arrest and cell hyperpolarization. Furthermore, this invention is the first to demonstrate that BT treatment can alter mitochondrial ultrastructure and has the potential to reduce pancreatic cancer metastasis.

[0164] Example 2 The following research was conducted using the methodology and materials described in Example 3, and corresponding insights and results were obtained (see details). Figure 23-38 In addition, other methods described below are also employed.

[0165] For the KPCY model, pathogen-free female C57BL / 6 mice (6 to 8 weeks old, weighing 22 ± 5 g) purchased from Jackson Laboratory were used. Each mouse received an intrapancreatic injection of KPCY cells at a dose of 1 × 10⁵ cells per 50 µL of Hank balanced salt solution. Ten to fourteen days post-injection, all animals that received the orthotopic tumor cell injection were imaged using a magnetic resonance imaging (MRI) scanner at the Small Animal Imaging Facility of MD Anderson Cancer Center, following standard protocols for mouse pancreatic cancer imaging.

[0166] Mice with tumors ranging from 1 to 10 mm² were divided into four groups (n = 16 mice per group): 1) the BT treatment group, receiving treatment for 30 minutes daily, three times a week, for 3 weeks; 2) the SC group, receiving the same treatment as the BT group, but with a dummy therapist mimicking the actions and distance of a biofield therapist; 3) the housing control (CC) group, where animals remained in the housing facility throughout the treatment; and 4) the gemcitabine positive control group, receiving gemcitabine treatment and also remaining in the housing facility throughout the treatment. The BT and SC groups (3-5 mice per group) were transferred from the housing to the laboratory along with their original cages and treated directly in their cages on the experimental table. For the gemcitabine positive control group, mice received intraperitoneal injections of gemcitabine (40 mg / kg, saline solution) three times a week in the original housing facility. Similarly, mice in the CC group received intraperitoneal injections of an equal volume of saline three times a week and remained in the original housing facility.

[0167] The weight of each mouse was measured weekly. Three days after the end of treatment, the mice were euthanized according to standard procedures. Primary tumors were collected and weighed, and the final tumor weight was recorded. Nodules in the liver tissue were observed macroscopically, and the mice were imaged using an IVIX Spectrum imaging system at the MDACC small animal facility to determine the intensity of YFP+ images in the liver. Tumors and liver tissues were either flash-frozen or fixed with 10% formalin for subsequent analysis.

[0168] Cell-free DNA Cell-free DNA (cfDNA) was measured using the NucleoSpin cfDNA XS kit (Macherey-Nagel, Allenton, PA). In short, 240 μL of plasma or other cell-free body fluid was transferred to a microcentrifuge tube. For smaller biological samples, binding buffer was used for conditioning. An aliquot of 20 μL proteinase K was added to the plasma, and the mixture was incubated at 37°C for 10 minutes. After incubation, 360 μL of binding buffer was added. The sample was then mixed by inverting the tube three times and then vortexing briefly (3 seconds). The resulting mixture was loaded into a NucleoSpin cfDNA XM column with a collection tube and centrifuged at 2000 xg for 30 seconds. The column was washed twice, first with 500 μL and then with 250 μL of washing buffer. The sample was centrifuged at 11,000 xg for 30 seconds each time. The cfDNA was eluted with elution buffer (20–30 μL). The eluent was incubated at 90°C for 8 minutes to remove residual ethanol. The absorbance was measured at 260 nm / 280 nm using a microplate reader (Molecular Devices, San Jose, CA).

[0169] Expression of human voltage-gated ion channel genes in PANC-1 cells First, the expression of voltage-gated ion channel genes was detected using a human voltage-gated ion channel TaqMan array (catalog number: 4418744, Thermo Fisher), and then validated using specific primers for each gene.

[0170] In short, the 96-well plates were pre-configured / pre-coated with expression primers targeting human ion channel genes from all four classes of voltage-gated ion channels: 1) voltage-gated potassium channels, 2) sodium channels, 3) voltage-dependent calcium channels, and 4) cyclic nucleotide-gated potassium channels. RNA was extracted from PANC-1 cells using an RNA micro-extraction kit (Life Technologies, catalog number #12183018A). Subsequently, voltage-gated ion channel genes were detected using the TaqMan array human voltage-gated ion channel detection plate (#4414099, Thermo Fisher) according to the manufacturer's instructions (https: / / www.thermofisher.com / order / catalog / product / 4414099). Real-time quantitative PCR was performed on a QuantStudio 3 real-time quantitative PCR system (Applied Biosystems, Waltham, MA). Control group and BT treatment group samples were run on two separate detection plates.

[0171] To further validate the changes in voltage-gated ion channel genes, individual primer sets were purchased from Applied Biosystems / Life Technologies for the selected ion channel genes, including: Hs00240655_m1: CACNA1A; Hs00184168_m1: CACNA1I; Hs00185764_m1: KCNAB1; Hs00428197_m1: KCNC1; Hs00158410_m1: KCNG1; Hs00608142_m1: KCNH1; Hs00158470_m1: KCNN4; Hs00704943_s1: KCNA3 (kv.1.3); and Hs00165693_m1: SCNA5. Detailed information about these primer sets is available on the supplier's website (https: / / www.thermofisher.com / us / en / home / brands / applied-biosystems.html). Real-time PCR was performed using each primer set individually, RNA samples (20 ng / well, triple replicates), and TaqMan Fast Virus 1-StepMaster Mix (Applied Biosystems).

[0172] BT inhibits the migration and invasion of PDAC cells As described in Example 1 and Figure 13 As shown, the effect of BT on the invasiveness of PDAC cells was investigated. Further research showed that, compared with the SC group, BT also significantly reduced the migration of both PANC-1 and L3.7 cells, with reductions of 34% and 41%, respectively (p<0.001). Figure 13 ).

[0173] BT alters EMT-related markers in PANC-1 cells and reduces cell-free DNA in a PANC-1 tumor model. To investigate how BT inhibits the invasiveness of PDAC cells, the expression of EMT-related markers in PANC-1 cells was detected. As described in Example 1 and... Figure 18 As shown, in PANC-1 cells treated with BT, the protein expression level of the epithelial cell marker E-cadherin was significantly higher than that in the IC group and the SC group. Figure 18 Aa). In contrast, the protein expression levels of N-cadherin, a marker of mesenchymal cells, and the expression levels of the Twist gene in the BT group were significantly lower than those in the IC and SC groups (p<0.05); Figure 18 Ab and Figure 24Furthermore, compared with SC group mice bearing PANC-1 in situ tumors, BT-treated mice showed a significant decrease in cell-free tumor DNA levels. Figure 24 (C) indicates that BT has the potential to mitigate the intravasation process, a result consistent with the findings of BT's inhibitory effect on the migration and invasion of PDAC cells.

[0174] BT alters the ultrastructure of PANC-1 and L3.7 cells. This study investigated the effects of BT on the ultrastructure of PANC-1 and L3.7 cells. Transmission electron microscopy (TEM) was used to measure changes in organelle ultrastructure immediately after BT treatment of PANC-1 and L3.7 cells. As described in Example 1 and... Figure 8 As shown, the mitochondria in PANC-1 cells from both the IC and SC groups were elongated and had well-developed cristae. In contrast, PANC-1 cells treated with BT exhibited disordered mitochondrial cristae arrangement and significant mitochondrial swelling. In a repeat study of PANC-1 cells, after 15 minutes of BT treatment ( Figure 25A , Figure 25B ) and 30 minutes ( Figure 25B Following this, similar mitochondrial changes were observed in all groups, namely loss of mitochondrial cristae integrity and significant mitochondrial swelling. Furthermore, compared to the IC and SC groups, the cytoskeleton structure of L3.7 cells in the BT treatment group was significantly less pronounced (data not shown). These significant and intuitive morphological changes in the ultrastructure of mitochondria and the cytoskeleton not only confirm the biological role of BT in important cellular functions but also provide new insights.

[0175] For example, such as Figure 37 As shown, compared with the sham control group, BT treatment reduced the ratio of mitochondria stained with red and green fluorescent JC-1 dye, indicating that BT may potentially reduce the mitochondrial membrane potential of PANC-1 cells. Figure 36 As shown, epigenetic testing revealed that BT treatment altered chromatin openness in PANC-1 cells related to cytoskeleton structure, serine / threonine kinases, cell junctions, extracellular matrix, and cell cycle, indicating that BT treatment can regulate these genes at the transcriptional level. Figure 38 As shown, transmission electron microscopy (TEM) revealed that BT treatment reduced the cytoskeleton filamentous structure in human pancreatic cancer L3.7 cells compared to the IC and SC groups, indicating that BT treatment can alter the mechanical properties of cells.

[0176] Regulating FOXM1 expression can alter the PDAC cell response to BT. As described in Example 1 and Figure 19As shown, the stronger the inhibitory effect of BT on FOXM1 expression, the more significant its inhibitory effect on cell invasion. This suggests that the anti-invasion effect of BT-induced PDAC cells may be mediated, at least in part, through its ability to downregulate FOXM1 protein.

[0177] Subsequently, the cell cycle and invasiveness of control and FOXM1-modified PDAC cells after BT or SC treatment were examined. In FOXM1 stably knocked-down (KD) PANC-1 cells, FOXM1 expression was reduced by nearly 90% compared to parental PANC-1 cells. Figure 19 , Figure 20 and Figure 26 In FOXM1 conditionally overexpressing PANC-1 cells, although the parental PANC-1 cells in the BT treatment group showed 60.5% lower invasiveness than those in the SC group, BT had a stronger inhibitory effect on the invasiveness of FOXM1 conditionally overexpressing PANC-1 cells compared to the SC group (inhibition rate 77.6%, p<0.001). Figure 23 B). Similar changes were also observed in BT-treated FOXM1-stably overexpressing PANC-1 cells. Figure 23 A).

[0178] BT treatment alters voltage-gated sodium and potassium channels, and FOXM1 appears to be involved in cellular voltage potential. Given the consistent observation that BT alters cellular voltage potential, and considering that voltage-gated ion channels (VGICs) are key regulators of resting cellular voltage potential, this study investigated the gene expression of VGICs (including sodium channels (VGSCs), potassium channels (VGKCs), and calcium channels (VGCCs)) in PANC-1 cells treated with BT. Figure 27 As shown, compared with the SC group, the expression of more than 10 VGKC-related genes was downregulated in PANC-1 cells of the BT treatment group, and the expression levels of two VGSC genes (including Nav1.1 and Nav1.5) were also lower in PANC-1 cells of the BT treatment group. Figure 27 A). Only VGCC gene expression was moderately affected (data not shown). Real-time quantitative PCR results confirmed that, compared with the SC group, Nav1.5 gene expression was significantly increased in PANC-1 cells treated with BT for 15 minutes, while it was significantly decreased in PANC-1 cells treated with BT for 30 minutes. Figure 27 B). In contrast, compared with the SC group, the Kv1.3 gene level in PANC-1 cells 15 minutes after BT treatment was moderately but significantly reduced, but significantly increased at 30 minutes after BT treatment. Figure 6C). Given that Nav1.5 is primarily responsible for Na+ influx, while Kv1.3 regulates K+ influx, these data suggest that BT treatment leads to cell depolarization, which may subsequently cause hyperpolarization in PANC-1 cells.

[0179] Furthermore, it was observed that BT significantly downregulated the gene expression of Kca3.1 in PANC-1 cells. Figure 27 D). Given that BT-induced anti-invasion activity appears to be partially mediated by FOXM1, the question of whether FOXM1 is involved in cell membrane voltage potential was investigated. DiBAC4 staining was used to measure the resting membrane voltage potential of PANC-1 cells and FOXM1 CRISPR FOXM1 KO PANC-1 cells 15 minutes after BT treatment. Interestingly, compared to parental PANC-1 cells, FOXM1 KO PANC-1 cells exhibited a significantly higher resting cell membrane voltage potential (…). Figure 27 E), indicating that decreased FOXM1 expression is associated with depolarization of PANC-1 cells, and this phenomenon is consistent with the changes in cell membrane voltage potential observed in PDAC cells treated with BT for 15 minutes.

[0180] The antitumor activity of BT was confirmed in the PANC-1 mouse orthotopic model and the KPCY mouse syngeneic model. To further confirm the antitumor effect of BT on pancreatic cancer, especially its anti-metastatic potential, this invention replicated the previous antitumor efficacy study conducted in the human PANC-1 mouse orthotopic model and further evaluated its antitumor efficacy in the KPCY mouse syngeneic model. The experimental procedure was similar to the in vivo experimental method previously used in the PANC-1 mouse orthotopic model.

[0181] The mean terminal tumor weight to body weight ratio was similar in all treatment groups. Figure 28 A), which differs slightly from the results of the first study using the PANC-1 in-situ model. In contrast, and compared with those described in Example 1 and Figure 21 The results were similar, with 28% of mice in the BT group showing liver metastases, which was 51%, 55%, and 53% lower than the CC, SC, and Gem groups, respectively. Figure 28 B). Notably, in the gross assessment of liver tumor burden in mice with liver metastases, the mean tumor burden in BT-treated mice was 8.1 ± 5.4 mm3, which was 73% lower than that in the SC group (26.0 ± 6.2 mm3) (p<0.05). Compared with the CC group, mice treated with gemcitabine alone showed only a moderate reduction in liver tumor burden ( Figure 28 C). Numerous and large nodules were observed in the livers of mice in both the CC and SC groups, while the livers of mice in the BT treatment group appeared normal. Figure 28 D). Similarly, compared with the CC group (metastasis rate 92%) and the SC group (metastasis rate 79%), BT treatment significantly reduced visible liver metastases (metastasis rate 35%) in mice bearing tumors of the KPCY T cell-low expression clone (6419C5) (p<0.01). Figure 28 E). When imaging YFP+ 6419C5 cells in liver tissue using an IVIS imaging system, it was noted that the signal intensity of YFP+ cells in the liver tissue of the BT treatment group was statistically significantly lower than that in the SC group ( Figure 7 F). Liver tissue images showed that, compared with the BT-treated group, the SC group had higher YFP+ signal intensity in liver metastases (F). Figure 28 (G). In summary, these data strongly demonstrate that BT can effectively inhibit liver metastasis in both PANC-1 and KPCY, two different orthotopic pancreatic cancer models.

[0182] BT treatment alters the immune cell characteristics in pancreatic tumors in KPCY mice. Although no significant survival benefit was observed with BT in mice bearing tumors derived from the mouse pancreatic KPCY high T cell clone (7160C2), this invention still conducted a pilot study to investigate the effects of BT on the tumor microenvironment (TME). Figure 29 As shown, compared with the SC control group, BT treatment significantly reduced the infiltration of CD19+ B cells in KPCY 7160C2 derived tumors. Figure 29 A), and increased CD3+ T cell infiltration ( Figure 29 B). In assessing T cell subtypes, the number of CD8+ cytotoxic T cells in the tumors of the BT treatment group was found to be 2.74 times and 1.58 times higher than that of the CC and SC groups, respectively. Figure 29 C). In contrast, the mean percentage of the FOXP3+CD25+ regulatory T cell population in tumors in the BT treatment group was similar to that in the CC group and slightly lower than that in the SC group ( Figure 29 D). Given that CD8+ cytotoxic T cells have the function of killing cancer cells, while regulatory T (T-Reg) cells promote tumor growth, these preliminary data provide a strong basis for in-depth evaluation of the overall immune cell atlas of pancreatic tumors after BT treatment.

[0183] Faraday cages, aluminum foil, and distance all failed to block the effects of BT on the cell cycle and cell invasion of PDAC cells. To further explore the possible mechanism of action of BT, this invention investigated the cell cycle and invasion behavior of PDAC cells when placed in an electromagnetic field (EMF) shielding device such as a Faraday cage or wrapped in aluminum foil. For the Faraday cage test, PDAC cells were placed on an experimental table or in a Faraday cage (Holland Shielding System BV, Netherlands). Figure 30 A) Inside. Before placing the cells into the Faraday cage, we calibrated the EMF shielding effect by blocking cell phone signals. This study also tested the effect of aluminum foil on BT-induced resistance to invasion, aiming to assess whether the length of conventionally produced aluminum foil in industrial production could attenuate or block this effect. The aluminum foil manufacturing process is said to induce ordered molecular spin alignment, thereby forming a directionally aligned structure. For the aluminum foil test, PDAC cells, as a sham control, were placed in a regular shoebox on the lab bench, or a similar box lined with two layers of aluminum foil inside and outside, placed at a 90-degree angle with the shiny sides facing back to back. Figure 31 A), designed to form a polarizer to block any signal that may have spin or chiral characteristics.

[0184] like Figure 30 As shown, BT can induce G1 phase arrest, as evidenced by the significantly higher proportion of PANC-1 cells in the G0 / G1 phase (44.5%) in the BT-treated group compared to the IC group (38.67%) and the SC group (39.26%) (p values ​​<0.001 and <0.01, respectively). Figure 30 Aa). Similar results were also observed in cells placed in Faraday cages (Aa). Figure 30 Ab). When measuring cell invasion of PANC-1 and L3.7 cells placed on an experimental table or in a Faraday cage, it was found that BT had a similar inhibitory effect on cell invasion of PANC-1 and L3.7 cells in the Faraday cage as it did on cell invasion on the experimental table. Figure 29 B and Figure 29 C). On the experimental table, similar changes in cell cycle and resistance to invasion were observed whether BT-treated PANC-1 and L3.7 cells were placed in boxes wrapped with or without aluminum foil. Figure 31 Furthermore, this invention also determined the effect of BT administered via Zoom on cell invasion of PANC-1 cells. The procedure was performed at three different distances from the experimental table where the cells were placed: 1) a laboratory office 150 feet away; 2) 1.5 miles away; and 3) from the San Francisco Bay Area. This series of studies was conducted by two different therapists, with control samples simultaneously evaluated in another area of ​​the laboratory. Figure 32As shown, distance did not alter the effect of BT. These data strongly suggest that electromagnetic fields (EMF) are not directly involved in BT-induced antiproliferative and antimetastatic potential of PDAC cells, and that non-EMF mechanisms may be involved.

[0185] BT alters intracellular calcium signaling and beta-actin, which is correlated with changes in the therapist's EEG. To explore the mechanism of action of BT, this invention conducted another exploratory study, analyzing changes in cellular processes and therapist physiological characteristics simultaneously. This study included multiple treatment and control (non-treatment) phases (n=60), with therapists blinded to the specific experimental conditions. The experimental setup is as follows: Figure 33 As shown. During the treatment phase, therapists attempted to alter the experimental results of in vitro cells; this was alternated with the rest phase, during which no such attempts were made. This study used three biomarkers: two cytoskeletal changes (tubulin and β-actin) and Ca2+ uptake to assess the activity of human pancreatic cancer cells. Given the changes observed in therapists' electroencephalograms (EEGs) during treatment in the first phase of the study, this study aimed to investigate changes in physiological parameters (EEGs) and heart rate measurements obtained by therapists when treating two groups of subjects: 1) live cells; and 2) dead cells or cell-free / culture medium only (control group); to explore changes in cellular outcomes and analyze whether there was a correlation between therapists' physiological parameters and cellular outcomes. The experimental setup used a 2×2 design to compare cell types (live cells vs. control cells) and experimental phase types (treatment vs. non-treatment). Changes in cellular parameters over time were detected in parallel sham-treated control cells, and a cell invasion assay was used as a positive control. Throughout the experimental phase, therapists' physiological data (including 64-channel EEG and heart rate) were continuously monitored.

[0186] This study observed significant spectral changes in therapists' electroencephalograms (EEGs) across all target frequency bands and in heart rate variability (HRV) (RMSSD; p<0.01) during BT treatment (p<0.01). Furthermore, significant differences were observed in EEG and HRV (pNN50) at both the β and γ bands when therapists treated live cells instead of control cells (p=0.02). However, no interaction was observed between treatment modality and cell type (live cells vs. dead cells / culture medium). We observed that Ca2+ uptake increased over time during both BT and sham treatments, but the increase was significantly smaller in the BT group compared to the sham treatment control group (p=0.03). Figure 34A). When using Granger causality to assess the causal orientation between cellular markers and therapist-obtained physiological parameters, the results showed a significant bidirectional causal effect between EEG measurements and cellular indicators (particularly β-actin and Ca2+ uptake) (p<0.000001). Figure 34 B). Although the phenomenon of quantum entanglement could not be observed due to technical challenges (EEG measurement accuracy is in the millisecond range, while cellular indicator measurement is only in the minute range), these results indicate that there is a complex relationship between physiological responses and cellular effects during BT treatment, which warrants further research.

[0187] The second biofield therapy (BT) confirmed that BT has anti-metastatic effects. To verify the hypothesis that "a second BT has similar therapeutic effects in in vitro experiments and in vivo animal models," this invention conducted related research to examine whether the invasion and migration of PDAC cells and human PANC-1 mouse orthotopic models had similar therapeutic outcomes when BT was administered by a second biofield therapist. Figure 35 As shown in Figure A, in these PDAC cells treated by the second therapist (therapist 2), BT exhibited similar anti-invasive and anti-migration effects as observed when treated by the first therapist (therapist 1). Compared to in vitro results, in the PANC-1 mouse orthotopic model, mice treated with BT showed only a moderate reduction in liver metastasis compared to the SC group. Figure 35 Ba). However, when comparing the primary tumor weight and liver metastasis rate in tumor-bearing mice, it was found that mice with larger tumors in the BT treatment group had 27% fewer liver nodules than those in the SC group ( Figure 35 Bb). In fact, in mice that did not develop liver metastases in the BT treatment group, the size of the primary tumor was significantly larger than that in the SC group ( Figure 35 The results (Bc) indicate that BT treatment did indeed delay liver metastasis in the PANC-1 animal model. In summary, these data suggest that the above findings are reproducible across different studies and therapists.

[0188] Example 3 (Materials and Methods) Reagents and Antibodies. All procedures were performed in accordance with the guidelines, rules, and regulations of The University of Texas MD Anderson Cancer Center. PrestoBlue was purchased from ThermoFisher; the invasion kit and cell migration assay kit were purchased from CellBiolabs, Inc.; DiBAC4 (bis-[1,3-dibutylbarbituric acid]trimethyloxacyanine) (B438) was purchased from ThermoFisher; and gemcitabine was purchased from MD Anderson Pharmacy. Antibodies against FOXM1 (5436), p21 (2947), N-cadherin (13116), and E-cadherin (14472) were purchased from Cell Signaling Technology (Danvers, MA), and anti-actin antibody (MAB8929) was purchased from R&D Systems. The 12-230 kDa separation module, 8 × 25 capillary cartridge (SM-W004), anti-rabbit detection module (DM-001), and anti-mouse near-infrared (NIR) detection module (DM-009) used to run the Jess system were all purchased from ProteinSimple (CA, USA). The Invitrogen NucBlue LiveReadyProbes reagent (Hoechst 33342, R37605) for nuclear viable cell staining was purchased from ThermoFisher.

[0189] Cell lines. Human pancreatic cancer cells (PANC-1 and MiaPaCa-2) were purchased from the American Type Culture Collection (ATCC, Manassas, VA); the use of human pancreatic cancer cells COLO357 and L3.7 was in accordance with previous literature (Huang C, etla., Cancer research. 2012;72(3):655-665). Mouse pancreatic cancer KPCY cells were purchased from Kerafast (Boston, MA). All cancer cells were maintained in a humid environment at 37°C and 5% carbon dioxide. PANC-1, COLO357, and L3.7 cells were routinely cultured in high-glucose Dulbecco modified Eagle medium (DMEM) or RPMI 1640 medium (Invitrogen Corp, Grand Island, NY) containing 10% fetal bovine serum (Hyclone Laboratories Inc, Logan, UT), supplemented with 50 IU / mL penicillin, 50 µg / mL streptomycin, and 2 mM L-glutamine (all purchased from GIBCO / Invitrogen). All human cell lines were identified within the past 6 months by short tandem repeat (STR) profiling (available upon request).

[0190] BT procedures for in vitro studies. Most experiments are performed by one therapist, while some cell experiments are replicated by one or two other therapists (labeled "Therapist 2" and / or "Therapist 3" in the accompanying figures). In one experiment, BT therapy was administered remotely by a team of therapists; as shown... Figure 39 As shown, the results revealed that PANC-1 cells treated by either a single therapist or a team of therapists exhibited reduced invasiveness compared to the sham control group, with similar results in both groups. This finding supports the hypothesis that BT does indeed have the potential to inhibit PANC-1 cell invasion.

[0191] Preliminary cell experiments explored potential dose-response relationships, examining BT exposure times of 5, 15, and 30 minutes, and 60 and 120 minutes (data not shown). No clear dose-response relationship was observed, and the most significant effects were observed at 15 and 30 minutes of exposure, depending on the cell line; therefore, these two treatment durations were used in most experiments. Unless otherwise stated, the treatment duration was 15 minutes. Each study included two or three groups: 1) the BT treatment group, where cells were treated either on a tabletop or in an incubator; 2) the sham tabletop control group (SC), where cells were seeded and placed on a counter for the same duration as the BT group, with personnel mimicking the BT group's manipulation and cell distance; and 3) in some experiments, the seeded cells were kept in an incubator (incubator control group [IC]). Pathologists were unaware of the groupings when performing tests or assessing the animals.

[0192] The BT protocol. The BT protocol incorporates various techniques, including the Bengston Healing Energy Technique. The protocol involves creating a series of images, which the therapist displays in rotation as quickly as possible while performing a specific manipulation. Detailed descriptions of these rapid imaging and manipulation techniques have been previously documented (Bengston VF, Kinsley, D. Journal of Scientific Exploration. 2000;14(3):353-364; Bengston WF, Moga MJ Altern Complement Med. 2007;13(3):317-327). In short, the therapist sits in front of the target subject (petri dish or animal cage) while displaying these images in rotation. The therapist remains relaxed, with hands approximately 6 to 24 inches away from the target subject, making slow movements but never touching the petri dish / cage / animal. In experiments using sham therapy, the sham therapist mimics the manipulations and distances used by a biofield therapist on the cells / animal.

[0193] Transmission electron microscopy (TEM) was used to examine the fine details of intracellular organelle structures at the High Resolution Electron Microscopy Facility (HREMF) of MD Anderson Cancer Center. PANC-1 cells (1 × 10⁵ cells) were seeded in 12-well plates and then exposed to BT treatment for 15 minutes. Immediately after treatment, the cells were washed with PBS (without Ca++ / Mg++) and then fixed with electron microscopy (EM) fixative containing 3% glutaraldehyde and 2% paraformaldehyde in 0.1 M dimethylarsine buffer (pH 7.3). The samples were then further processed according to HREMF standard operating procedures and previously published procedures (Yang P, et al., Prostate. 2003;55(4):281-291).

[0194] Cell migration and invasion. Colorimetric cell invasion assays were performed using Cytoselect and a 24-well basement membrane kit (Cell Biolabs, #CBA-110). Briefly, PANC-1 cell suspension (1.0 x 10⁶ cells, 300 µL) was seeded into polycarbonate inserts / upper chambers containing a basement membrane (8 µm pore size) in FBS-free DMEM, while the lower chamber contained 500 µL of FBS-containing DMEM. Control and treatment cells were seeded in two different culture plates. After BT treatment (15 or 30 minutes), cells were incubated at 37°C with 5% CO₂ for 48 hours. After 48 hours, the culture medium was carefully aspirated from the inserts, and the inserts were transferred to clean wells containing 400 µL of the kit's accompanying staining solution. Staining was performed for 10 minutes, followed by gentle rinsing with water. Finally, the insert was air-dried, and the invading cells were imaged using the EVOS M7000 imaging system (Thermo Fisher Scientific).

[0195] Cell migration assays were performed in 24-well plates using the Cytoselect Cell Migration Detection Kit (Cell Biolabs, USA). 300 µL of serum-free culture medium containing 1.0 × 10⁶ cells was seeded into each insert (6 inserts / set). Aliquots of 500 µL of culture medium containing 10% fetal bovine serum or the desired chemokine were added to the lower wells of the migration plate. The plates were then subjected to BT treatment (15 min) and incubated in a humidified CO₂ incubator for 24 h. Inserts were processed according to the manufacturer's instructions. Similarly, cell invasion assays were performed using the CytoSelect Cell Invasion Detection Kit (24-well plate) from CellBiolabs, which uses basement membrane-coated inserts to detect the invasive characteristics of tumor cells. Endpoint indicators were monitored 48 h after BT treatment. Fluorescence intensity was measured at 480 nm / 520 nm using a microplate reader (Molecular Device, San Jose, CA).

[0196] For the scratch assay, a Culture-Insert 2 Well (dual-well culture insert) was used with a µ-Dish 35 mm culture dish (Ibidi, Fitchburg, WI). This dish is equipped with a culture insert to ensure reproducible scratch gap widths. MiaPaca-2 cells (6 × 10⁵ cells) were seeded into two wells of the culture insert and cultured for 24 hours. Subsequently, the culture insert was carefully removed using sterile forceps, and 1 mL of culture medium was added to each culture dish as an aliquot. The culture dishes were then placed under two CytoSmart FL microscopes (Axion Biosystem, Atlanta, GA) in separate incubators, exposing the cell culture dishes to either a sham control or BT treatment. Images were acquired every 30 minutes over 48 hours and analyzed using the CytoSmart scratch assay online program.

[0197] Cell voltage characterization. Briefly, cells (5-10 × 10³) were seeded one day prior to treatment. After treatment, plates were incubated for 5 days in an incubator at 37°C, 5% CO₂, and 95% humidified air. DiBAC4(3), a slow-response potential-sensitive probe, was used to stain and measure cell depolarization. The DiBAC4(3) working solution (1 mg / mL) was prepared by mixing the stock solution with Dulbecco modified Eagle medium (without fetal bovine serum) at a ratio of 1:4000. Cells were washed with PBS and the DiBAC4(3) working solution, along with 1 drop of NucBlue Live Ready Probes reagent, and incubated at 37°C for 15 minutes. Images of green fluorescent protein and DAPI were captured using an EVOS M5000 imaging system (AMF5000) or an EVOS M7000 imaging system (AMF7000). For images captured by the EVOS M5000, fluorescence intensity was quantified using ImageJ; for images captured by the EVOS M7000, fluorescence intensity was quantified using Cellslate software.

[0198] Immunoblot analysis. Immunoblot analysis was performed using an automated Western blotting system: the Jess Simple Western blotting system (ProteinSimple, San Jose CA, USA), which automatically separates and immunodetects proteins by the size of its capillaries. To quantify the expression of the target protein in treated cells, this study followed standard procedures using the 12-230 kDa Jess separation module (SM-W004). The final protein concentration of the tissue lysate (0.4 mg / mL to 1 mg / mL) was obtained by mixing the assayed cell lysate (0.6 mg / mL to 2 mg / mL) with 0.1x sample buffer and a Fluorescent 5X Master mix (ProteinSimple). The resulting mixture was denatured at 95°C for 5 min. The target primary antibody is listed in the "Results" section; β-actin or tubulin was prepared by mixing the antibody dilution at a ratio of 1:10 to 1:50. Secondary antibodies are prepared by mixing chemiluminescent and fluorescent antibodies. Cell lysate samples are then loaded into Ladder plates (12–230 kDa), and antibody diluent, primary antibody, secondary antibody, luminol-S, and peroxide solution (for chemiluminescence detection) are added sequentially according to the manufacturer's instructions, followed by washing buffer. Chemiluminescence and fluorescence digital images of the target protein are acquired using Compass Simple Western blotting software (version 4.1.0, Protein Simple), and protein quantification is performed based on signal area.

[0199] FOXM1 gene modification. To construct FOXM1-overexpressing PANC-1 cells, hFOXM1 human ORF clone lentiviral particles purchased from OriGene (USA) were used to overexpress the FOXM1 gene in PANC-1 cells. The lentiviral particles were thawed on ice and directly added to 6-well plates containing culture medium (500 µL) and polybrene (final concentration 8 µg / mL). The lentiviral particles were then incubated at 37°C in a humidified CO2 incubator for 20 hours. To construct a stable cell line, transduced cells were seeded at low density in fresh 6-well plates, and clones were selected using puromycin (50 µg / mL). Single clones were then picked for further amplification culture and identified by Jess / Western blot.

[0200] To construct a stable FOXM1 knockdown PANC-1 cell line, three groups of human FOXM1 hCMV-TurboRFP shRNA lentiviruses (Horizon Discovery Bioscience) were transfected into PANC-1 cells. Forty-eight hours after transfection, colonies were screened using puromycin (10 µg / mL) for at least two weeks. Colonies exhibiting downregulated FOXM1 expression were then cultured in medium containing 5 ng / mL puromycin for all subsequent experiments. The knockdown effect of hFOXM1 was confirmed by Western blot (Jess analysis), and PANC-1 cells with the highest FOXM1 knockdown efficiency were selected for experimental use.

[0201] In some experiments, FOXM1 knockdown L3.7 cells constructed via CRISPR-Cas9 transfection were used. In short, L3.7 cells were seeded in 6-well plates and allowed to adhere overnight. Subsequently, following the manufacturer's instructions, transfection was performed using the TurboFectin transfection reagent (OriGene, catalog number TF81001) via the CRISPR-Cas9 system with either the disordered control vector or the FOXM1 knockdown gene kit (OriGene, Rockville, MD, catalog number KN402246). In short, 1 μg of one of the gRNA vectors (gRNA1 or gRNA2) was added to 250 μL of Opti-MEM I (LifeTechnologies) in separate tubes and gently vortexed to mix. Then, 1 μg of donor DNA was added to this 250 μL Opti-MEM I solution. Add 6 μL of Turbofectin 8.0 aliquot to the diluted DNA solution and gently pipette to mix thoroughly. Incubate the mixture at room temperature for 15 minutes. Then, add the mixture to cells seeded in 6-well plates. Gently shake the plate back and forth and side to side to distribute the complex evenly. Incubate the cells in a 5% CO2 incubator. 48 hours after transfection, passage the cells at a 1:10 ratio and continue culturing for 3 days, then passage again at a 1:10 ratio. This procedure was repeated a total of 7 times. After the 7th passage, puromycin was added, and expression was detected using the Jess system. Stable knockdown colonies of FOXM1 (g2-1) were selected by puromycin selection and significant reduction in FOXM1 expression levels. Figure 22 ).

[0202] To construct PANC-1 cells capable of inducibly expressing FOXM1, tetracycline-inducible pCW57.1HA-FOXM1b was purchased from Addgene. Using X-tremeGENE9 DNA (Roche) transfection reagent, and strictly following the manufacturer's instructions, the inducible pCW57.1HA-FOXM1b (5 mg), along with helper plasmid psPAX2 (6 mg) and pMD2G (2 mg; Addgene: 12260), was transiently transfected into HEK293T cells cultured in 10 cm plates. Forty-eight hours after transfection, the viral supernatant was collected and filtered through a 0.45 mm Millipore filter. The viral supernatant was then serially diluted and transduced into PANC-1 cells in 10 cm plates in the presence of polybrene (4 μg / ml, Sigma). Forty-eight hours after transfection, 0.5 μg / ml puromycin was added to the culture plates for treatment, and the puromycin selection process lasted for 5 days. After 5 days, the puromycin was removed, and the cells were cultured for another week. To detect the expression of doxycycline (Sigma)-induced human FOXM1 in PANC1 cells, cells were seeded in 6-well plates and treated with or without doxycycline (250 ng, 500 ng, and 1000 ng). The culture medium with or without doxycycline was changed every 24 hours. Seventy-two hours after doxycycline treatment, the expression of induced FOXM1 was monitored using an automated Western blot system (Jess, Protein Simple).

[0203] Animal model of pancreatic cancer. Animal studies were conducted in accordance with the regulations of the Animal Care and Use Committee of MD Anderson Cancer Center (IACUC Protocol No.: 00001212-RN02). Animals were housed in facilities accredited by the International Committee for Evaluation and Accreditation of Laboratory Animals (ICAUC) and in accordance with current regulations and standards of the U.S. Department of Agriculture and the Department of Health and Human Services. To induce tumorigenesis of human PANC-1 cells, pathogen-free female athymic BALB / c nude mice (6 to 8 weeks old, weighing 22 ± 5 g) purchased from Jackson Laboratory were used. Each mouse received an intrapancreatic injection of PANC-1 cells at a dose of 0.5 × 10⁶ cells / 50 µL of Hank balanced salt solution. Ten days post-injection, all animals that received in situ tumor cell injections were imaged using a magnetic resonance imaging (MRI) scanner at the Small Animal Imaging Facility of MD Anderson Cancer Center, following standard protocols for mouse pancreatic cancer imaging.

[0204] Mice with tumors ranging from 1 to 10 mm² were divided into four groups (n = 25–30 mice per group): 1) the BT treatment group, receiving treatment for 30 minutes daily, three times a week, for 3 weeks; 2) the SC group, receiving the same treatment as the BT group, but with a dummy therapist mimicking the actions and distance of a biofield therapist; 3) the cage control (CC) group, where animals remained in the animal housing facility throughout the treatment; and 4) the gemcitabine positive control group, receiving gemcitabine treatment and also remaining in the animal housing facility throughout the treatment. Mice in the BT and SC groups (3–5 mice per group) were transferred from the animal housing facility to the laboratory along with their original cages and treated directly in their cages on the experimental table. For the gemcitabine positive control group, mice received intraperitoneal injections of gemcitabine (40 mg / kg, saline solution) three times a week in the original housing facility. Similarly, mice in the CC group received intraperitoneal injections of an equal volume of saline three times a week and remained in the original housing facility.

[0205] Mice received treatment for 3 weeks, followed by a 7-week observation period. The weight of each mouse was measured weekly. At the end of the study, mice were euthanized according to standard procedures, and blood, tumor / pancreas, spleen, liver, and lung samples were collected. These samples were either flash-frozen or fixed in 10% formalin for subsequent analysis.

[0206] Immunohistochemical analysis. Liver tissue samples were obtained from mice during necropsy. The tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin using the standard protocol of the MD Anderson Histopathology Core Facility. The stained sections were scanned using an Aperio AT2 brightfield slide scanner and analyzed blinded by a certified pathologist.

[0207] Statistical analysis. Statistical analyses (t-tests or ANOVA) were performed using GraphPad Prism software in both in vitro and in vivo studies. For RPPA data, statistical analyses were performed using R statistical software version 4.1.1 (R Foundation for Statistical Computing, Vienna, Austria) and the Wilcoxon rank-sum test. For these analyses, a p-value <0.05 was considered statistically significant. Unless otherwise stated, data are expressed as mean ± SD.

[0208] Although specific embodiments of the invention have been shown and described, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the broader aspects of the invention. Therefore, the appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the invention. All publications mentioned in this application are incorporated herein by reference to the same extent that each individual publication expressly and individually designates its inclusion by reference.

Claims

1. A method for reducing the transfer potential of mitochondrial cells, the method comprising: The biophysical changes include at least one of the following: Altering the intracellular ion levels of the cells. Change the cell membrane potential of the cells, Changing the membrane potential of the mitochondria, and Altering the expression of ion channel genes; The structural change includes at least one of the following: Altering the structural characteristics of the mitochondria, Altering the cytoskeleton organization of the cells, Altering the membrane ion channels of the cells Altering the expression of cytoskeleton genes, and Altering the expression of genes associated with cytoskeleton communication; The physiological changes include at least one of the following: Alteration of one or more EMT marker proteins in the cells, Regulate FOXM1 in the cells, Altering the energy metabolism of the cells' mitochondria Altering the expression of one or more EMT genes, Altering the expression of genes containing one or more FOXM1-related proteins. Altering the expression of serine / threonine kinase genes, Altering the expression of genes involved in cell junctions, and Altering the expression of genes in the extracellular matrix; in, The biophysical changes, the structural changes, and / or the physiological changes are each associated with at least one of the following: The expression of cell cycle arrest and / or cell cycle regulatory proteins in the cells. Cell-free DNA from the cells, or reduced endosmosis, The cell's growth or metabolism is reduced. The migration of the cells is reduced. Stemness is suppressed, and The invasion of the cells is reduced.

2. The method according to claim 1, wherein, The biophysical changes include at least two, at least three, or all of the following: Altering the intracellular ion levels of the cells. Change the cell membrane potential of the cells, Changing the membrane potential of the mitochondria, and Altering the expression of ion channel genes; The structural changes include at least two, at least three, or all of the following: Altering the structural characteristics of the mitochondria, Altering the cytoskeleton organization of the cells, Altering the membrane ion channels of the cells Altering the expression of cytoskeleton genes, and Altering the expression of genes associated with cytoskeleton communication; The physiological changes include at least two, at least three, at least four, at least five, at least six, at least seven, or all of the following: Alteration of one or more EMT marker proteins in the cells, Regulate FOXM1 in the cells, Altering the energy metabolism of the mitochondria in the cells Altering the expression of one or more EMT genes, Altering the expression of genes containing one or more FOXM1-related proteins. Altering the expression of serine / threonine kinase genes, Altering the expression of genes involved in cell connections, and Altering the expression of genes in the extracellular matrix.

3. The method according to claim 1 or 2, wherein, The biophysical changes, the structural changes, and / or the physiological changes are each related to at least two, at least three, at least four, or all of the following: The expression of cell cycle arrest and / or cell cycle regulatory proteins in the cells. Cellular DNA from the cells, or reduced endosmosis, The cell's growth or metabolism is reduced. The migration of the cells is reduced. Dryness is suppressed, and The invasion of the cells is reduced.

4. The method according to any one of claims 1-3, wherein, The biophysical changes, the structural changes, and the physiological changes are each associated with at least one, at least two, at least three, at least four, or all of the following: The expression of cell cycle arrest and / or cell cycle regulatory proteins in the cells. Cellular DNA from the cells, or reduced endosmosis, The cell's growth or metabolism is reduced. The migration of the cells is reduced. Dryness is suppressed, and The invasion of the cells is reduced.

5. The method according to any one of claims 1-4, further comprising: Applying biofield therapy, in, The application of the biofield therapy is related to at least one, at least two, or all of the following: the biophysical changes, the structural changes, and the physiological changes.

6. The method according to claim 5, wherein, The biofield therapy mentioned is the Bengston Energy Healing Method.

7. The method according to any one of claims 1-6, wherein, The biophysical changes, the structural changes, and / or the physiological changes are each associated with a reduction in the migration and / or invasion of the cells.

8. The method according to claim 7, wherein, To achieve reduced cell migration and / or invasion without causing toxicity to the cells or their host.

9. The method according to any one of claims 1-8, further comprising: Administering chemical agents and / or radiation therapy.

10. The method according to any one of claims 5-9, wherein, Apply biofield therapy so that: (i) To mitigate the harmful effects of the application of the chemical agents and / or the radiotherapy, or (ii) To enhance the efficacy of the chemical agents and / or the radiotherapy.

11. The method according to any one of claims 5-10, wherein, The biofield therapy is applied for approximately 15 minutes or 30 minutes daily.

12. The method according to any one of claims 5-11, wherein, The biofield therapy is administered for at least 2 or 3 days per week.

13. The method according to any one of claims 5-12, wherein, The biofield therapy is administered by more than one therapist.

14. The method according to any one of claims 5-13, wherein, Part of the biofield therapy is administered by a group of therapists.

15. The method according to any one of claims 5-14, wherein, The biofield therapy can be administered locally or remotely.

16. The method according to any one of claims 5-15, wherein, The application of the biofield therapy is determined based on the efficacy indicators of the person implementing the biofield therapy or the implementation method of the biofield therapy.

17. The method according to claim 16, wherein, The efficacy indicators are based on the measurement of biophysical changes, structural changes and / or physiological changes, or electroencephalogram (EEG) of the person undergoing the biofield therapy.

18. The method according to claim 17, wherein, The changes in the brain electrical activity were correlated with the cellular calcium signaling and / or β-actin.

19. The method according to any one of claims 1-18, further comprising: Additional changes are made, including at least one of the following: Regulate the hypoxia and / or anaerobicness of the cells; Altering the expression of genes associated with the Ras pathway, extracellular matrix, serine / threonine kinases, and / or cell connectivity; Altering cell signal transduction; Regulation of the PI3K / mTOR pathway in these cells; and It alters immune cells.

20. The method according to claim 19, wherein, Regulating hypoxia and / or anoxic conditions in the cells involves reducing the protein expression of hypoxia markers HIF-1α and / or PDK1.

21. The method according to claim 19 or 20, wherein, Regulation of the PI3K / mTOR pathway in these cells involves reducing pAkt levels and / or the ratio of pAkt to total Akt.

22. The method according to any one of claims 1-21, wherein, Cell cycle arrest includes arresting the cells in the G0 / G1 phase and / or G2 / M phase of the cell cycle.

23. The method according to any one of claims 19-22, wherein, Altering immune cells involves reducing B cells and increasing T cells.

24. The method according to any one of claims 19-23, wherein, Altering cell signal transduction involves altering microtubule and / or cytoskeleton markers.

25. The method according to any one of claims 1-24, wherein, Changing the intracellular ion levels of the cells includes changing the intracellular calcium ion levels of the cells.

26. The method according to any one of claims 1-25, wherein, Changing the cell membrane potential includes lowering the cell membrane potential and / or hyperpolarizing the cell.

27. The method according to any one of claims 1-26, wherein, Changing the membrane potential of the mitochondria includes lowering the mitochondrial membrane potential.

28. The method according to any one of claims 1-27, wherein, Altering the expression of ion channel genes involves altering the expression of one or more genes among voltage-gated channel proteins, sodium channel proteins, potassium channel proteins, and calcium-regulated channel proteins.

29. The method according to any one of claims 1-28, wherein, Altering the expression of ion channel genes includes altering the expression of one, two, or all of the following genes: voltage-gated sodium channel (e.g., Nav1.5) protein, voltage-gated potassium channel (e.g., Kv1.3) protein, and calcium-regulated potassium channel (e.g., KCa3.1) protein.

30. The method according to any one of claims 1-29, wherein, Altering the structural characteristics of the mitochondria includes increasing the size of the mitochondria and / or disrupting the cristae structure.

31. The method according to any one of claims 1-30, wherein, Altering the cytoskeleton organization of the cells includes reducing cytoskeleton structure and / or regulating the activity of actin (e.g., F-actin, β-actin) and / or tubulin.

32. The method according to any one of claims 1-31, wherein, Altering the cell's membrane ion channels includes: altering the cholinergic receptor anion channel CFTR, altering the GABA receptor GABRP, and / or altering the calcitonin Ryanodine receptor 3.

33. The method according to any one of claims 1-32, wherein, Altering the expression of cytoskeleton genes involves activating cytoskeleton-related genes, including actin, pectin, spectrin, TRIOBP, kinases, and / or PDZ.

34. The method according to any one of claims 1-33, wherein, Alterations in one or more EMT marker proteins in the cells include at least one, at least two, or all of the following: an increase in the epithelial marker E-cadherin; a decrease in the mesenchymal marker N-cadherin; and a decrease in Twist 1.

35. The method according to any one of claims 1-34, wherein, Regulating FOXM1 in the cells involves reducing the level of FOXM1.

36. The method according to any one of claims 1-35, wherein, Regulating the energy metabolism of the cells includes at least one, at least two, or all of the following: altering mitochondrial function; altering the expression of protein kinases and / or ATP-binding genes; and altering the metabolism of glucose and glutamine.

37. The method according to any one of claims 1-36, wherein, Altering the expression of one or more EMT genes includes: reducing the expression of N-cadherin, reducing the expression of CD44, and / or increasing the expression of E-cadherin.

38. The method according to any one of claims 1-37, wherein, Altering the expression of one or more FOXM1-related genes includes: decreasing the expression of pAKT, ALKBH5, cyclin B1, and / or CDK1; and / or increasing the expression of P21.

39. The method according to any one of claims 1-38, wherein, The cell is a component of an organism.

40. The method according to claim 39, wherein, The reduction of intracellular or cell-free DNA from the cells includes reducing cell-free DNA in the plasma of the organism.

41. The method according to claim 39 or 40, wherein, The cells include components of a tumor in an organism.

42. The method according to claim 41, wherein, The reduction in cell growth or metabolism corresponds to a decrease in tumor size or burden.

43. The method according to any one of claims 1-42, further comprising: Altering the tumor microenvironment (TME).

44. The method according to claim 43, wherein, Altering the tumor microenvironment (TME) includes: reducing the percentage of B lymphocytes, increasing the percentage of cytotoxic T cells, and / or reducing M2 tumor-associated macrophages.

45. The method according to any one of claims 39-44, further comprising: Reduce nodules and / or metastases in the organism.

46. ​​The method according to claim 45, wherein, Reducing the number or visibility of nodules in the organism includes reducing the number or visibility of liver nodules and / or metastasis to the liver.

47. Methods for regulating the biological functions of an entity, including: This induces changes in the bioelectrical characteristics of the first portion of cells and / or mitochondria within the first portion of cells of the entity, wherein, The induction effect causes: The second part of the cell in the entity undergoes structural changes; The second part of the cells undergoes physiological changes; and Altering the expression of genes associated with changes in the bioelectrical characteristics, changes in the structural characteristics, and / or changes in the physiological characteristics.

48. The method according to claim 47, wherein, The second part of the cells includes the first part of the cells or is the same as the first part of the cells.

49. The method according to claim 47 or 48, wherein, The first group of cells includes immune cells.

50. The method according to any one of claims 47-49, wherein, The second part of the cells includes cancer cells.

51. The method according to any one of claims 47-50, wherein, The structural aspect includes at least one of the following: The structural features of the mitochondria The cytoskeleton of the cells The cell's membrane ion channels; and The physiological aspect includes at least one of the following: EMT markers in the cells The energy metabolism of the mitochondria in the cell, and The cells contain FOXM1 or FOXM1-regulated proteins.

52. The method according to any one of claims 47-51, wherein, At least one, at least two, at least three, or all of the following: (a) a change in the bioelectrical characteristics, (b) a change in the structural aspects, (c) a change in the physiological aspects, and (d) a change in the expression of the gene, are associated with at least one of the following: The expression of cell cycle arrest and / or cell cycle regulatory proteins in the cells. Cellular DNA from the cells, or reduced endosmosis, The cell's growth or metabolism is reduced. The migration of the cells decreased, and The invasion of the cells is reduced.

53. The method according to any one of claims 47-52, wherein, Inducing changes in the bioelectrical characteristics involves the application of biofield therapy.

54. The method according to any one of claims 47-53, wherein, Altering gene expression includes at least one of the following: epigenetic effects; changes in transcriptomic signature; and changes in proteomic signature.

55. Methods for improving cell function that include two or all of the following: Change the membrane potential of the cell; Alteration of the cytoskeleton organization in the cells; and Regulate FOXM1 in the cells.

56. The method according to claim 55, wherein, The cell membrane potential decreases.

57. The method according to claim 55 or 56, further comprising: Regulates the cellular energy metabolism of the cells.

58. The method according to any one of claims 55-57, further comprising: Restricting the cell's progression through the cell cycle.

59. The method according to any one of claims 55-58, further comprising: Regulate the hypoxia and / or anaerobicness of the cells.

60. The method according to any one of claims 55-59, further comprising: This causes changes in protein expression in the cells.

61. The method according to any one of claims 55-60, wherein, The method comprises: modulating FOXM1 in the cells, and achieving the modulation of FOXM1 without producing toxic effects on the cells or the host of the cells.

62. The method according to any one of claims 55-61, wherein, The method comprises: administering biofield therapy, and reducing FOXM1 in the cells.

63. The method according to any one of claims 55-62, wherein, The reduction of FOXM1 in the cells was associated with a decrease in the invasiveness of the cells.

64. The method according to any one of claims 55-63, wherein, The efficacy of the method is determined by the reduction of FOXM1 in the cells.

65. The method according to any one of claims 1-64, wherein, The cells include organoids, patient-derived organoids, or cells from pancreatic cancer organoids.

66. The method according to any one of claims 1-65, wherein, The morphology of mitochondria in the cells is altered.

67. The method according to any one of claims 1-66, wherein, The method reduces the metastatic potential of the cells and / or cancer cells.

68. The method according to claim 67, wherein, Reduced transfer potential includes one or more of the following: Reduce the growth of the cells and / or cancer cells; Reduce the size of tumors containing cancer cells; Reduce the likelihood of metastasis; Reduce the migration or other metastatic behavior of the cancer cells or their progeny; and Reduce the ability of the cancer cells or their progeny to penetrate or metastasize.

69. The method according to any one of claims 1-68, wherein, The cells are human or mouse cells.

70. The method according to any one of claims 1-69, wherein, The cells are components of a human or mouse (which exist in the body).

71. The method according to any one of claims 1-68, wherein, The cells include pancreatic ductal adenocarcinoma (PDAC) cells.

72. The method according to any one of claims 1-68, wherein, The cells include cells derived from one or more of the following cell lines: PANC-1, Panc02, MiaPaca, Capan-2, KPCY, COLO357, and L3.7.

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