Cancer therapeutics through nervous system stimulation

Abstract

Methods for inhibiting, reducing, or stopping the growth of central nervous system cancers, such as brain tumors, or cancers influenced by the nervous system are described herein. The methods also include, in some instances, reducing or maintaining the size or volume of such cancers. Such methods are useful for treating and combatting aggressive brain cancers.

Description

[0001] CANCER THERAPEUTICS THROUGH NERVOUS SYSTEM STIMULATION

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] The application claims benefit of and priority to U.S. Provisional Application No. 63 / 553,455, filed February 14, 2024, which is hereby incorporated by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] This invention is in the field of stimulation methods which can be used for inhibiting the growth of central nervous system cancers, such as brain tumors or cancers influenced by the nervous system.

[0006] BACKGROUND OF THE INVENTION

[0007] Brain tumors remain a difficult challenge in the field of cancer research, characterized by their aggressive growth and resistance to conventional therapies. In recent years, research into brain cancers has shifted focus towards understanding the relationship between the tumor and its surrounding environment, such as healthy brain tissue. Notably, highly aggressive brain tumors have recently been observed to intricately integrate with the brain's neural circuitry, displaying electrical activity and interaction (Venkatesh, H. S. et al. Electrical and synaptic integration of glioma into neural circuits. Nature 573, 539-545 (2019); and Venkataramani, V. et al. Glutamatergic synaptic input to glioma cells drives brain tumour progression. Nature 573, 532- 538 (2019)). It has been demonstrated that neurons play a key role in the progression of brain tumors, particularly, peritumoral neurons that create functional excitant synapses with brain tumor cells (Tantillo, E. et al. Differential roles of pyramidal and fast-spiking, GABAergic neurons in the control of glioma cell proliferation. Neurobiology of disease 141, 104942 (2020)). Brain tumors influence neuronal excitability (Buckingham, S. C. et al. Glutamate release by primary brain tumors induces epileptic activity. Nature medicine 17, 1269-1274 (2011)) and, the discovery of direct synaptic connections between neurons and tumor cells, leads to brain tumor depolarization in response to neuronal activity and subsequent proliferation (Venkatesh, H. S. et al. Electrical and synaptic integration of glioma into neural circuits. Nature 573, 539-545 (2019); and Venkataramani, V. et al. Glutamatergic synaptic input to glioma cells drives brain tumour progression. Nature 573, 532-538 (2019)), is completely new and emerging. These electrochemical synapses formed directly between neurons and glioma cells shed light on a positive feedback mechanism. This mechanism amplifies neuronal excitation induced by brain cancers, such as gliomas through influencing mitosis and migration.

[0008] In addition to primary brain cancers, recent studies on metastatic organ colonization by non-neural tumor types, such as melanoma, lung, thyroid, and breast cancer, have unveiled a previously unrecognized facet of synaptic communication between neurons and cancer cells. This research not only expands the understanding of neuron-tumor interactions but also has implications for the progression of brain metastases and potential therapeutic interventions (Venkataramani, V. et al. Direct excitatory synapses between neurons and tumor cells drive brain metastatic seeding of breast cancer and melanoma. bioRxiv, 2024.2001. 2008.574608 (2024)). Such emerging evidence of direct synaptic connections between neurons-glioma cells, as well as between neurons and non-neural cancer cells, particularly in the context of brain metastases, represents a paradigm shift in the understanding of cancer progression and offers promising prospects for the development of therapeutic strategies.

[0009] Stimulation methods have emerged as a promising avenue for modulating various physiological processes, including those relevant to cancer progression. Several studies have demonstrated that various forms of neuronal stimulation can slow down or inhibit neuronal activities, contrary to the intuitive expectation that stimulation would always increase neural firing (Xiao, YiZi, et al. "Deep brain stimulation induces sparse distributions of locally modulated neuronal activity." Scientific reports 8.1 (2018): 2062., and Guidetti, Matteo, et al. "Clinical perspectives of adaptive deep brain stimulation." Brain Stimulation 14 (2021).). In addition, recent research has demonstrated that different forms of stimulation can influence not only neural activity but also immune function and tumor microenvironment, offering potential therapeutic applications for central nervous system cancers and those influenced by neural activity. Studies have shown that stimulation techniques can modulate immune cell behavior, including T and B lymphocytes, affecting their migration patterns and cytokine production and such immunomodulation can be obtained through various methods (Snigdha, R. B. et al. Electrical Stimulation for Immunomodulation. ACS Omega 9.1 (2023): 52-66). Furthermore, stimulation-driven immunomodulation or angiogenesis regulation has shown promise in inhibiting cancer progression (Das, Ritopa, et al. "Electrical stimulation for immune modulation in cancer treatments." Frontiers in bioengineering and biotechnology 9 (2022): 795300.).

[0010] Accordingly, there exists a need for methods to treat cancers which are associated with the nervous system. In particular, those that exploit interactions between neural tissue, immune cells, and tumor cells. Therefore, it is an object of the present invention to provide methods of treating cancers which are associated with the nervous system.

[0011] SUMMARY OF THE INVENTION

[0012] Stimulation-based approaches offer a novel and potentially powerful tool to address the need for methods to treat cancers that exploit interactions between neural tissue, immune cells, and tumor cells. The stimulation-based approaches described herein provide for non-invasive or minimally invasive means to modulate the tumor microenvironment, enhance immune responses, and disrupt / modulate pro-tumorigenic neural-cancer cell interactions.

[0013] Methods for applying a stimulation to a subject to inhibit, reduce, or halt the rate of cancer growth which can be used to treat these types of cancers where the stimulation applied is able to disrupt the communication between normal cells and the cancer cells are described herein. In one non-limiting instance, a method of inhibiting growth of a central nervous system cancer or a cancer influenced by the nervous system, includes the steps of:

[0014] (i) invasively, minimally invasively, or non-invasively applying a stimulation to a subject having a central nervous system cancer or a cancer influenced by the nervous system; wherein the stimulation inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the growth rate of the central nervous system cancer or the cancer influenced by the nervous system prior to the subject receiving the stimulation; and / or wherein the stimulation optionally inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the growth rate of the central nervous system cancer or the cancer influenced by the nervous system for a subject not receiving the stimulation; and / or wherein the stimulation reduces size or volume of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the size or volume prior to the subject receiving the stimulation; and / or wherein the stimulation optionally reduces or maintains size or volume of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the size or volume in the subject not receiving the stimulation.

[0015] In some instances, the stimulation inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the growth rate of the central nervous system cancer or the cancer influenced by the nervous system of a subject not receiving the stimulation. In some instances, the methods and treatments described herein can also be used in combination with additional therapies including but not limited to surgery, radiation, chemotherapy, immunotherapy. When applied in combination, each part can be applied simultaneously or one after the other in any sequence and at various intervals.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A depicts representative IVIS images capturing tumor dynamics of a comprehensive in vivo investigation of low-frequency electrical stimulation's impact on glioma progression.

[0018] Figure IB shows a line graph illustrating bioluminescence (total flux) over time (days) showing tumor growth curves for stimulated and non-stimulated mice with brain implants. The lines indicate differential tumor growth between the two cohorts.

[0019] Figure 2 shows a graph of total flux, as determined by in vivo imaging system (IVIS) bioluminescence measurements, on the inhibitory effect of tumor growth by low-frequency stimulation by invasive stimulation by implanted electrodes in the brain (frequency = 1 Hz, pulse width = 1 ms, amplitude = 100 pA). Out of six mice, two mice were not stimulated (controls), while two mice received continues stimulation, whereas two mice (represented by symbols -*■! and *♦”) received stimulation initially; however, the stimulation was discontinued from days 15 to 24 (white arrow) and days 21 to 27 (black arrow), respectively. Even though, the stimulation was discontinued, the tumor growth remained inhibited and did not grow.

[0020] Figure 3 shows a graph of total flux, as determined by in vivo imaging system (IVIS) bioluminescence measurements, on the inhibitory effect of tumor growth by non-invasive stimulation by auditory evocation (frequency = 1 Hz, pulse width = 1 ms, amplitude = 75 dB, tone frequency = 15 kHz).

[0021] Figure 4A shows MRI images of mouse brains showing a reduced / halted tumor growth in the mice with brain stimulation. Solid arrows indicate the dashed white circled region showing the tumors on day 11 (denoted “Dl l”, where DI is the day of tumor cell injection in the brain). DI 1 is the first day of stimulation for the experimental animals. A second row of MRI images indicate the tumors at day 30 (denoted “D30”).

[0022] Figure 4B shows a graph of tumor volume over time for the experimental animals, where tumors were reduced / halted in size and whereas significant growth of tumors was seen in the unstimulated control mouse

[0023] Figures 5A and 5B present a comprehensive analysis of RNA sequencing data derived from the glioma mouse model, providing insights into the molecular mechanisms underlying the observed tumor inhibition in electrically stimulated mice compared to unstimulated controls. Brain tissues from both treated and untreated mice were dissected to isolate tumor regions (of human origin) and surrounding mouse brain peripheries to compare differential gene expressions in mouse transcriptome (5 A) and human transcriptome (5B).

[0024] Figure 6A shows a confocal image of an untreated brain slice showing baseline mGluR8 expression in tumor and peripheral regions.

[0025] Figure 6B shows a confocal image of a treated brain slice demonstrating enhanced mGluR8 expression in tumor surrounding tissue.

[0026] Figure 6C shows a graph depicting notably higher mGluR8 expression levels in treated mice compared to control mice, quantified from IHC staining intensity.

[0027] Figure 6D shows a bar graph comparing tumor sizes (pm2) between treated and untreated mice, revealing significant tumor growth inhibition or halting in the treated group.

[0028] Figure 6E shows a bar graph showing the number of proliferative cells (Ki-67 positive nuclei) per field, with markedly higher counts in non-stimulated mice compared to stimulated ones.

[0029] Figure 6F shows a confocal image of a treated brain slice stained for Ki-67, demonstrating reduced proliferative activity.

[0030] Figure 6G shows a confocal image of an untreated brain slice stained for Ki- 67, showing elevated proliferative activity.

[0031] Figure 7 shows a non-limiting exemplar}' temporal pattern of a stimulation protocol having a Wl, pulse width; Pl, pulse period; W2, burst width; P2, burst period; W3, train width; P3, train period; W4, session width; On setup: the moment when a subject is set up with the stimulation system to receive stimulation but the stimulation may have not yet been applied; Off setup: the moment when the subject has finished a stimulation protocol and will disengage from the stimulation system afterward . The term “session” as used herein refers to the time period between on set up and off setup.

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] Methods for applying stimulation to a subject in order to inhibit, reduce, or stop the growth of central nervous system cancers, such as brain tumors, or cancers influenced by the nervous system are described herein.

[0034] I. Definitions

[0035] The term “subject,” as used herein, refers to a mammal, such as a human or animals, such as dogs and cats. “Invasive,” as used herein refers to delivering a stimulation to a subject which involves a surgical procedure that involves making incisions into the body of subject to access internal organs or tissues.

[0036] “Minimally invasive,” as used herein refers to delivering a stimulation to a subject which involves an invasive procedure or technique which does minimal disruption / modulation to surrounding structures to the body of a subject. Such procedures and techniques may be performed through a needle, small incisions or natural body openings.

[0037] “Non-invasive,” as used herein refers to delivering a stimulation to a subject which does not include or require the use of needles, surgical incisions or penetration of the skin or body tissues of the subject.

[0038] The terms “treatment” and “treating” refer to the medical management of a subject with the intent to cure, ameliorate, and / or stabilize a disease or symptoms thereof in the subject. This term includes active treatment toward the improvement of the disease, or palliative treatment designed for the relief of symptoms rather than intended for curing of the disease; preventative treatment is directed to minimizing or partially or completely inhibiting the development of a disease. It is understood that treatment, while intended to cure, ameliorate, and / or stabilize a disease or symptoms thereof, need not actually result in any particular degree of cure, amelioration, stabilization, and / or prevention.

[0039] The term “session” as used herein refers to the time period from the moment when a subject is set up with the stimulation system to receive a stimulation, but the stimulation may not yet have been applied, to the moment when the subject has finished a stimulation protocol and will disengage from the stimulation system afterward (see Fig. 7).

[0040] Numerical ranges include ranges of thicknesses, ranges of pressures, ranges of molecular weights, ranges of integers, ranges of times, ranges of electric current, ranges of length, ranges of diameters, etc. The ranges disclose individually each possible number that such a range could reasonably encompass, as well as any sub-ranges and combinations of sub-ranges encompassed therein. For example, an activating step may be carried out for a period of time in the range of about 1 min to 10 min, also refers to time values that can be selected independently from about 2, 3, 4, 5, 6, 7, 8, and 9 minutes, as well as any range between these numbers (for example, 3 min to 8 min), and any possible combination of ranges between these time values.

[0041] Use of the term "about" is intended to describe values either above or below the stated value, which the term “about” modifies, to be within a range of approximately + / - 10%. When the term "about" is used before a range of numbers (z.e., about 1-5) or before a series of numbers (i.e. about 1, 2, 3, 4, etc.) it is intended to modify both ends of the range of numbers and / or each of the numbers recited in the entire series, unless specified otherwise.

[0042] II. Methods of Stimulation for Treating Cancers

[0043] By targeting the complex interactions between neurons, immune cells, and tumor cells, the stimulation-based methods described herein offer a unique opportunity to disrupt / modulate the pro-tumorigenic microenvironment and potentially slow or halt tumor growth. In the context of brain tumors and cancers influenced by the nervous system, such stimulation techniques can provide a means to interfere with the newly discovered synaptic connections between neurons and cancer cells. By modulating neuronal activity or disrupting these aberrant synapses, it is possible to inhibit the proliferation and migration of tumor cells that rely on these connections for growth signals.

[0044] Thus, based on the connections between the nervous system and cancer cells reported for certain cancers, there is an implication that regulating the nervous system activity in, for example, the brain tumors of subjects / patients could potentially impede tumor growth and proliferation. Such an approach has the potential to extend patient survival and can be used to combat particularly aggressive cancers. Moreover, such interventions could not only impede primary brain cancer growth but could also inhibit other metastatic brain tumors, as well as cancers functionally influenced by the nervous system.

[0045] Described herein are methods for applying a stimulation to a subject to inhibit, reduce, or halt the rate of cancer growth which can be used to treat these types of cancers where the stimulation applied is able to disrupt / modulate the communication between normal cells and the cancer cells. In one non-limiting instance, a method of inhibiting growth of a central nervous system cancer or a cancer influenced by the nervous system, includes the steps of:

[0046] (i) invasively, minimally invasively, or non-invasively applying a stimulation to a subject having a central nervous system cancer or a cancer influenced by the nervous system; wherein the stimulation inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the growth rate of the central nervous system cancer or the cancer influenced by the nervous system prior to the subject receiving the stimulation; and / or wherein the stimulation optionally inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the growth rate of the central nervous system cancer or the cancer influenced by the nervous system for a subject not receiving the stimulation; and / or wherein the stimulation reduces size or volume of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the size or volume prior to the subject receiving the stimulation; and / or wherein the stimulation optionally reduces or maintains size or volume of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the size or volume in the subject not receiving the stimulation.

[0047] In some instances, the stimulation inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the growth rate of the central nervous system cancer or the cancer influenced by the nervous system of a subject not receiving the stimulation.

[0048] In some instances, the stimulation reduces size or volume of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the size or volume prior to the subject receiving the stimulation. In still other instances, the stimulation reduces size or volume of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the size or volume of a subject not receiving the stimulation.

[0049] In some instances, the stimulation keeps the size of the central nervous system cancer or the cancer influenced by the nervous system the same (in other words it maintains the original size or volume by application of the stimulation), as compared to the size or volume prior to the subject receiving the stimulation. In still other instances, the stimulation keeps the size of the central nervous system cancer or the cancer influenced by the nervous system the same, as compared to the size or volume of a subject not receiving the stimulation.

[0050] In some instances, the growth rate is inhibited or reduced. In some instances, the growth rate is stopped by the application of the stimulation. In some instances, the stimulation reduces or stops the growth rate of the central nervous system cancer, or the cancer influenced by the nervous system, for at least about 10-12 hours, or longer, after the stimulation is ceased. In some instances, the growth rate is inhibited or reduced by at least about 0.01 to about 99.99%, or individual values or sub-ranges contained within the aforementioned range. In some instances, the size or volume of the central nervous system cancer, or the cancer influenced by the nervous system, is reduced by at least about 0.01% or stays the same (is maintained). In some other instances, the size or volume of the central nervous system cancer, or the cancer influenced by the nervous system, is reduced by at least about 0.01% to 50%, or individual values or subranges contained within the aforementioned range. The skilled person is familiar with methods of measuring / determining the growth rates and size / volume of cancers (i.e., tumors). In some instances, the central nervous system cancer is a primary brain cancer. In some instances, the primary brain cancer is a glioma, glioneuronal, meningioma, medulloblastoma, schwannoma, pituitary adenoma, pineal region tumors, craniopharyngioma, primary central nervous system lymphoma, germ cell tumors, choroid plexus, mesenchymal, non- meningothelial, and haematolymphoid tumors. In some instances, the glioma is selected from glioblastoma multiforme, diffuse intrinsic pontine glioma, diffuse hemispheric glioma, astrocytoma, oligodendroglioma, oligoastrocytoma, ependymoma, or mixed gliomas.

[0051] In certain instances, the central nervous system cancer is due to a metastatic cancer originating from a non-neural tumor. In some instances, the non-neural tumor is from a cancer selected from melanoma, lung, thyroid, breast, renal cell carcinoma, colorectal, bladder, gastrointestinal tract, prostate, and ovarian cancer.

[0052] In some instances, the cancer influenced by the nervous system is selected from cancers in any organ or organ system in the subject’s body which is functionally connected to the central and / or peripheral nervous system.

[0053] In some instances, the stimulation is a neural stimulation. In some instances, the neural stimulation includes a neuronal stimulation, glial stimulation, synaptic stimulation, and / or combinations thereof. In some instances, the stimulation is selected from deep brain stimulation, nerve stimulation, spinal cord stimulation, transcranial stimulation, transcutaneous stimulation, sensory stimulation, peripheral nerves stimulation, and combinations thereof.

[0054] In some instances, the stimulation is an immune-cell stimulation. In some instances, the stimulation is a stimulation of vasculature cells optionally selected from endothelial cells and / or pericytes. In some instances, the stimulation is applied through any physical, chemical and / or biological stimulation which are known to regulate cell activities and / or combinations thereof. In certain instances, the physical stimulation is selected from the group consisting of electrical, electrochemical, mechanical, optical, optogenetic, thermal, magnetic, magneto-genetic, acoustic, sono-genetic stimulation, ultrasound-mediated stimulation, and combinations thereof, as are known in the art for nervous system stimulation. In some instances, the chemical stimulation includes drugs and / or chemogenetics, which are known in the art for nervous system stimulation. In some instances, the biological stimulation includes biomolecules, such as proteins, RNA, DNA, lipids, virus and / or bacteria, which are known in the art for nervous system stimulation. In some instances, the stimulation is selected from a brain stimulation, spinal cord stimulation, peripheral nerve stimulation, and combinations thereof. In some instances, the stimulation is selected from a stimulation through one or more implanted devices, body mounted devices, devices inside or on surface or outside body or combination thereof, wearable devices, transcranial stimulation, transcutaneous stimulation, sensory stimulation, peripheral nerves stimulation, and combinations thereof. In certain instances, the implanted device is selected from a brain penetrating electrode, subarachnoid electrode, electrocorticography electrode, epidural electrode, intra-spinal electrode, cuff electrode, intra-fascicular electrode, inter-fascicular electrode, implanted wireless devices, and nanoparticles. Such devices, electrodes, and nanoparticles are known in the art.

[0055] Among the transcranial stimulation paradigms, transcranial magnetic stimulation (“TMS”), an FDA-approved non-invasive neurostimulation technique, has unraveled insights into therapeutic interventions (Bliss TVP & Cooke SF. (2011) Clinics; Kricheldorff J, et al. (2022) Brain Sci). Both low-frequency rTMS (repetitive TMS) and cTBS paradigms have been shown to induce lasting depression of TEPs (TMS-evoked potential) or motor evoked potential (“MEP”) amplitude (Ziemann U. (2004) Rev Neurosci; Kricheldorff J, et al. (2022) Brain Sci) and have been applied as treatments in clinical trial studies for treatment-resistant depression (Bares M, et al. (2009) J Affect Disord), schizophrenia (Fitzgerald PB, et al. (2005) J Clin Psychopharmacol), and autism (Sokhadze E, et al. (2010) Appl Psychophys Biof). Besides TMS, transcranial electrical stimulation (TES) can also be employed. TES is a widely used non- invasive neurostimulation technique in humans, which has shown promise in fostering therapeutic interventions alongside low-frequency rTMS and cTBS that induced neuroplastic depression (Jamil A & Nitsche MA. (2017) Neuromodulation).

[0056] In some instances, the stimulation is a transcranial stimulation such as transcranial magnetic stimulation (TMS), transcranial electrical stimulation (TES), transcranial ultrasound stimulation (TUS), or a combination thereof.

[0057] In some instances, the TES is transcranial direct current stimulation (tDCS), transcranial pulsed current stimulation (tPCS), transcranial alternating current stimulation (tACS), transcranial oscillating direct current stimulation (toDCS), transcranial random noise stimulation (tRNS), galvanic vestibular stimulation (GVS), and electroconvulsive therapy (ECT). In addition, it may be selected from peripheral nerves stimulation, such as Vagnus Nerve Stimulation (VNS), and Transcutaneous Electrical Nerve Stimulation (TENS). The skilled person is familiar with these forms of stimulation which are routinely used in the field of neuroscience. The selection of conditions and parameters for providing such stimulation are also known to the skilled person.

[0058] Brain activities evoked by SST (sensory stimulation therapy) have also been demonstrated in both human and animal models (laccarino HF, el al. (2016) Nature; Pinto JO, el al. (2020) Expert Rev Neurother). Accordingly, in some instances, the stimulation is a sensory stimulation selected from auditory stimulation, visual stimulation, somatosensory stimulation, and combinations thereof. The somatosensory includes, but is not limited to, touch, pressure, pain, temperature, body position (proprioception), and vibration, which arise from receptors in the skin, joints, ligaments, muscles, fascia, and internal organs of a subject.

[0059] In some instances, the stimulation is applied continuously during step (i). In some other instances, the stimulation is applied non-continuously during step (i).

[0060] In some instances, the stimulation in step (i) includes a temporal pattern selected from the group consisting of continuous, randomized, adaptive, patterned phase-locked, dose-modulated, staircase, intermittent, rhythmic, pulse-burst, and combinations thereof. A non-limiting example of a temporal pattern is shown in Figure 7 of a stimulation protocol having a Wl, pulse width; Pl, pulse period; W2, burst width; P2, burst period; W3, train width; P3, train period; W4, session width; On setup: the moment when the subject is ready with the stimulation system; Off setup: the time point when the subject is prepared to disengage from the stimulation system. The time between on setup and off setup represent a session. In some instances, in a day, W = about O.lps to 24hr, P = about 0.01ms to 24hr. The pulse can be any shape (e.g. square, triangle, sine wave, random noise, combinations) or sign (i.e. positive, negative, zero, combinations). In some instances, the stimulation in step (i) includes one or more temporal patterns including any one, or any combination, or repetition of any one or combination, of the following which are applied during step (i), such as in a day, including: a pulse width (Wl) of about O.lps to 24hr, a pulse period (Pl) of about 0.01ms to 24hr, a burst width (W2) of about O.lps to 24hr, a burst period (Pl) of about 0.01ms to 24hr, a train width (W3) of about O.lps to 24hr, a train period (P3) of about 0.01ms to 24hr, and / or a session width (W4) of about O.lps to 24hr. In some instances, the temporal pattern includes a theta burst stimulation. In some instances, the temporal pattern comprises a low frequency stimulation of less than about 250 Hz or from about 0.01 to 250Hz. Individual values and sub-ranges contained within the aforementioned ranges are also disclosed herein.

[0061] In some instances, the stimulation in step (i) is applied for a total amount of time in a range of about 0.1 pico-second to 24 hours per day. In some instances, the stimulation in step (i) is applied over a period of time greater than one day and up to one or more years, optionally including one or more time gaps in the applications ) ranging from 0 days to the subject’s (patient) lifetime.

[0062] In some instances, the transcranial stimulation comprises one or more pulses having a pulse amplitude in a range of between 0% to about 150% of a resting motor threshold or between 0% to about 150% of an active motor threshold of the subject. In some instances, the stimulation is an auditory stimulation which includes a pulse amplitude of less than about 90 dB and / or a tone frequency of less than about 150 KHz.

[0063] In some instances, the stimulation is a visual stimulation including an illumination with less than about 25,000 lumen of visible light in a wavelength between from about 300 to 800 nm.

[0064] In some instances, the stimulation is a somatosensory stimulation which can be a mechanical stimulation. In some instances, the mechanical stimulation includes mechanical pulses ((e.g. air puffs, vibration, pressure, brushing)) of less than about 600, 500, 400, 300, 200, or 100 psi. In some other instances, the somatosensory stimulation is a thermal stimulation, where the thermal stimulation can include thermal pulses having a temperature between about - 10°C to about 65°C or about 20°C to about 65°C, as well as individual values or sub-ranges disclosed within the aforementioned range.

[0065] In some instances of the method, step (i) is repeated one or more times optionally including one or more time gaps ranging from 0 seconds to the subject’s (patient) lifetime.

[0066] In some instances, the stimulation applied induces or causes plasticity and / or modulates connections among nervous system cells of the central nervous system cancer or the cancer influenced by the nervous system and the cancer cells of the central nervous system cancer or the cancer influenced by the nervous system; and / or among the nervous system cells themselves; and / or among the cancer cells of the central nervous system cancer or the cancer influenced by the nervous system themselves; in order to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0067] In some instances, the stimulation applied induces or causes angiogenesis to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0068] In some instances, the stimulation applied induces or causes the subject’s immune system to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0069] In some instances, the stimulation applied induces or causes the disruption / modulation of sustained proliferative signaling to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0070] In some instances, the stimulation applied induces or causes the reactivation of growth suppressors to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0071] In some instances, the stimulation applied induces or causes programmed cell death (apoptosis) to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0072] In some instances, the stimulation applied induces or causes the limitation of replicative potential to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0073] In some instances, the stimulation applied induces or causes the inhibition of invasion and metastasis to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0074] In some instances, the stimulation applied induces or causes the alteration of cellular energetics to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0075] In some instances, the stimulation applied induces or causes the reduction of genomic instability and mutation to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0076] In some instances, the stimulation applied induces or causes the modulation of tumorpromoting inflammation to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0077] The methods and treatments described herein can also be used in combination with additional therapies including but not limited to surgery, radiation, chemotherapy, immunotherapy. When applied in combination, each part can be applied simultaneously or one after the other in any sequence and at various intervals.

[0078] The disclosed methods can be further understood through the following numbered paragraphs. Paragraph 1. A method of treating a central nervous system cancer or a cancer influenced by the nervous system, the method comprising the steps of:

[0079] (i) invasively, minimally invasively, or non-invasively applying a stimulation to a subject having a central nervous system cancer or a cancer influenced by the nervous system; wherein the stimulation inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the growth rate of the central nervous system cancer or the cancer influenced by the nervous system prior to the subject receiving the stimulation; and / or wherein the stimulation optionally inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the growth rate of the central nervous system cancer or the cancer influenced by the nervous system for a subject not receiving the stimulation; and / or wherein the stimulation reduces or maintains size or volume of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the size or volume prior to the subject receiving the stimulation; and / or wherein the stimulation optionally reduces or maintains size or volume of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the size or volume in the subject not receiving the stimulation.

[0080] Paragraph 2. The method of paragraph 1 , wherein the growth rate is inhibited or reduced by about 0.01 to about 99.99%; or the growth rate is stopped by the stimulation.

[0081] Paragraph 3. The method of paragraph 1 , wherein the size or volume of the central nervous system cancer or the cancer influenced by the nervous system is reduced by at least about 0.01%; or the size or volume is maintained.

[0082] Paragraph 4. The method of any one of paragraphs 2-3, wherein the stimulation inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system for at least 12 hours after the stimulation is ceased.

[0083] Paragraph 5. The method of any one of paragraphs 1-4, wherein the central nervous system cancer is a primary brain cancer.

[0084] Paragraph 6. The method of paragraph 5, wherein the primary brain cancer is selected from the group consisting of a glioma, glioneuronal, meningioma, medulloblastoma, schwannoma, pituitary adenoma, pineal region tumors, craniopharyngioma, primary central nervous system lymphoma, germ cell tumors, choroid plexus, mesenchymal, non- meningothelial, and haematolymphoid tumors. Paragraph 7. The method of paragraph 6, wherein the glioma is selected from the group consisting of glioblastoma multiforme, diffuse intrinsic pontine glioma, diffuse hemispheric gliomas, astrocytoma, oligodendroglioma, oligoastrocytoma, ependymoma, and mixed gliomas.

[0085] Paragraph 8. The method of any one of paragraphs 1-4, wherein the central nervous system cancer is a metastatic cancer from a non-neural tumor.

[0086] Paragraph 9. The method of paragraph 8, wherein the non-neural tumor is from a cancer selected from the group consisting of melanoma, lung, thyroid, breast, renal cell carcinoma, colorectal, bladder, gastrointestinal tract, prostate, and ovarian cancer.

[0087] Paragraph 10. The method of any one of paragraphs 1-4, wherein the cancer influenced by the nervous system is selected from cancers in any organ or organ system in the subject’s body which is functionally connected to the central and / or peripheral nervous system.

[0088] Paragraph 11 . The method of any one of paragraphs 1-10, wherein the stimulation is a neural stimulation.

[0089] Paragraph 12. The method of paragraph 11, wherein the neural stimulation is selected from the group consisting of neuronal stimulation, glial stimulation, synaptic stimulation, and combinations thereof.

[0090] Paragraph 13. The method of any one of paragraphs 1-10, wherein the stimulation is an immune-cell stimulation.

[0091] Paragraph 14. The method of any one of paragraphs 1-10, wherein the stimulation is a stimulation of vasculature cells optionally selected from endothelial cells and / or pericytes.

[0092] Paragraph 15. The method of any one of paragraphs 1-10, wherein the stimulation is selected from the group consisting of a physical stimulation, chemical stimulation, and biological stimulation that regulates cell activities; and combination thereof.

[0093] Paragraph 16. The method of paragraph 15, wherein the physical stimulation is selected from the group consisting of an electrical, electrochemical, mechanical, optical, optogenetic, thermal, magnetic, magneto-genetic, acoustic, sono-genetic stimulation, ultrasound-mediated stimulation, and combinations thereof .

[0094] Paragraph 17. The method of paragraph 15, wherein the chemical stimulation is selected from the group consisting of drugs, chemogenetics, and combinations thereof.

[0095] Paragraph 18. The method of paragraph 15, wherein the biological stimulation comprises biomolecules selected from the group consisting of proteins, RNA, DNA, lipids, viruses, bacteria, and combinations thereof. Paragraph 19. The method of any one of paragraphs 1-10, wherein the stimulation is selected from the group consisting of brain stimulation, spinal cord stimulation, peripheral nerve stimulation and combinations thereof.

[0096] Paragraph 20. The method of any one of paragraphs 1-10, wherein the stimulation is selected from the group consisting of a stimulation applied from one or more implanted devices, body mounted devices, devices inside or on surface or outside body or combination thereof, wearable devices, transcranial stimulation, transcutaneous stimulation, sensory stimulation, peripheral nerves stimulation, and combinations thereof.

[0097] Paragraph 21. The method of paragraph 20, wherein the implanted device is selected from the group consisting of but not limited to brain penetrating electrode, subarachnoid electrode, electrocorticography electrodes, epidural electrodes, intra-spinal electrodes, cuff electrodes, intra-fascicular electrode, inter-fascicular electrode, implanted wireless devices, and nanoparticles.

[0098] Paragraph 22. The method of any one of paragraphs 1-10, wherein the stimulation is a transcranial stimulation selected from the group consisting of transcranial magnetic stimulation (TMS), transcranial electrical stimulation (TES), transcranial ultrasound stimulation (TUS), and combinations thereof.

[0099] Paragraph 23. The method of paragraph 22, wherein the TES is selected from the group consisting of transcranial direct current stimulation (tDCS), transcranial pulsed current stimulation (tPCS), transcranial alternating current stimulation (tACS), transcranial oscillating direct current stimulation (toDCS), transcranial random noise stimulation (tRNS), galvanic vestibular stimulation (GVS), electroconvulsive therapy (ECT), and peripheral nerves stimulation (such as Vagnus Nerve Stimulation or Transcutaneous Electrical Nerve Stimulation (TENS)).

[0100] Paragraph 24. The method of any one of paragraphs 1-10, wherein the stimulation is a sensory stimulation selected from the group consisting of auditory stimulation, visual stimulation, somatosensory stimulation, and combinations thereof.

[0101] Paragraph 25. The method of any one of paragraphs 1-24, wherein the stimulation is applied continuously during step (i).

[0102] Paragraph 26. The method of any one of paragraphs 1-24, wherein the stimulation is applied non-continuously during step (i).

[0103] Paragraph 27. The method of any one of paragraphs 1-26, wherein the stimulation in step (i) comprises a temporal pattern selected from the group consisting of continuous, randomized, adaptive, patterned phase-locked, dose-modulated, staircase, intermittent, rhythmic, pulse-burst, and combinations thereof.

[0104] Paragraph 28. The method of any one of paragraphs 1-26, wherein the stimulation in step (i) comprises one or more temporal patterns comprising any one, or any combination, or a repetition of any one or combination of the following applied, in a day, during step (i): pulse width (Wl) of about 0. Ips to 24hr, a pulse period (Pl) of about 0.01ms to 24hr, a burst width (W2) of about O.lps to 24hr, a burst period (Pl) of about 0.01ms to 24hr, a train width (W3) of about O.lps to 24hr, a train period (P3) of about 0.01ms to 24hr, and / or a session width (W4) of about O.lps to 24hr.

[0105] Paragraph 29. The method of any one of paragraphs 1-28, wherein the stimulation in step (i) is applied for a total amount of time in a range of about 0.1 pico-second to 24 hours per day.

[0106] Paragraph 30. The method of any one of paragraphs 1-28, wherein the stimulation in step (i) is applied over a period of time greater than one day and up to one or more years optionally comprising one or more time gaps.

[0107] Paragraph 31. The method of paragraph 28, wherein the temporal pattern comprises a low frequency stimulation of less than about 250 Hz or from about 0.00001 to 250Hz.

[0108] Paragraph 32. The method of paragraph 28, wherein the temporal pattern comprises a theta burst stimulation.

[0109] Paragraph 33. The method of paragraph 22, wherein the transcranial stimulation comprises one or more pulses having a pulse amplitude in a range of 0% to about 150% of a resting motor threshold or 0% to about 150% of an active motor threshold.

[0110] Paragraph 34. The method of paragraph 24, wherein the auditory stimulation comprises a pulse amplitude of less than about 90 dB, and a tone frequency of less than about 150 KHz.

[0111] Paragraph 35. The method of paragraph 24, wherein the visual stimulation comprises illumination with less than about 25,000 lumen of visible light in a wavelength range from about 300 to 800 nm.

[0112] Paragraph 36. The method of paragraph 24, wherein the somatosensory stimulation is a mechanical stimulation.

[0113] Paragraph 37. The method of paragraph 36, wherein the mechanical stimulation comprises mechanical pulses of less than about 600 psi.

[0114] Paragraph 38. The method of paragraph 24, wherein the somatosensory stimulation is a thermal stimulation.

[0115] Paragraph 39. The method of paragraph 38, wherein the thermal stimulation comprises thermal pulses having a temperatures ranging from about 20°C to about 65°C. Paragraph 40. The method of paragraph 22, wherein the transcranial ultrasound stimulation is a transcranial focused ultrasound stimulation (tFUS) which comprises a fundamental frequency ranging from about 100 kHz to 650 kHz.

[0116] Paragraph 41. The method of paragraph 40, wherein the tFUS stimulation further comprises (a) a pulse repetition frequency (PRF) ranging from about 1 Hz to 4500 Hz; (b) a duty cycle (DC) ranging from about 1% to 100%; (c) a sonication duration (SD) ranging from about 10 ms to 500 ms; and / or (d) an intensity spatial-peak pulse-average (Isppa) ranging from about 0.69 W / cm2to 18.2 W / cm2.

[0117] Paragraph 42. The method of paragraph 41, wherein the tFUS stimulation parameters are adjusted to produce either excitatory or inhibitory effects on neuronal activity.

[0118] Paragraph 43. The method of any one of paragraphs 40-42, wherein the tFUS stimulation is repeated one or more times, optionally including one or more time gaps ranging from 0 seconds to the subject's lifetime.

[0119] Paragraph 44. The method of any one of paragraphs 1-43, wherein step (i) is repeated one or more times optionally including one or more time gaps ranging from 0 seconds to the subject’s lifetime.

[0120] Paragraph 45. The method of any one of paragraphs 1-44, wherein the stimulation applied induces plasticity and / or modulates connections among nervous system cells of the central nervous system cancer or the cancer influenced by the nervous system and the cancer cells of the central nervous system cancer or the cancer influenced by the nervous system; and / or among the nervous system cells themselves; and / or among the cancer cells of the central nervous system cancer or the cancer influenced by the nervous system themselves; in order to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system

[0121] Paragraph 46. The method of any one of paragraphs 1-44, wherein the stimulation applied induces angiogenesis to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0122] Paragraph 47. The method of any one of paragraphs 1-44, wherein the stimulation applied induces the subject’s immune system to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0123] Paragraph 48. The method of any one of paragraphs 1-44, wherein the stimulation applied induces or causes the disruption / modulation of sustained proliferative signaling to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0124] Paragraph 49. The method of any one of paragraphs 1-44, wherein the stimulation applied induces or causes the reactivation of growth suppressors to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0125] Paragraph 50. The method of any one of paragraphs 1-44, wherein the stimulation applied induces or causes programmed cell death (apoptosis) to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0126] Paragraph 51. The method of any one of paragraphs 1 -44, wherein the stimulation applied induces or causes the limitation of replicative potential to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system

[0127] Paragraph 52. The method of any one of paragraphs 1-44, wherein the stimulation applied induces or causes the inhibition of invasion and metastasis to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system

[0128] Paragraph 53. The method of any one of paragraphs 1-44, wherein the stimulation applied induces or causes the alteration of cellular energetics to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0129] Paragraph 54. The method of any one of paragraphs 1-44, wherein the stimulation applied induces or causes the reduction of genomic instability and mutation to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

[0130] Paragraph 55. The method of any one of paragraphs 1-44, wherein the stimulation applied induces or causes the modulation of tumor-promoting inflammation to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system. The present invention will be further understood by reference to the following nonlimiting example.

[0131] Examples:

[0132] Example 1: Invasive and Non-Invasive Stimulation of Brain Tumors

[0133] This example provides details of a study on the effect of brain stimulation through both invasive and non-invasive methodologies using a brain tumor model in mice, as described below.

[0134] Materials and Methods’.

[0135] Cell Culture". The cell lines used in this example included U87-MG. The cell lines were either grown in Eagle's Minimum Essential Medium (EMEM, 30-2003, ATCC) or DMEM / F-12 media (1: 1, Thermofisher, USA), supplemented with 10% fetal bovine serum (FBS, 30-2020, ATCC) and 1% penicillin / streptomycin (Sigma- Aldrich, MO, USA). All cell lines were maintained in a humidified 5% CO2 incubator at 37 °C. The cell lines were generally grown in a T75 cell culture flask (Corning, USA) prior harvesting by treatment with 0.25 % trypsin-EDTA solution (GIBCO, Invitrogen) for 3-5 min for the prospective intracranial tumor induction.

[0136] In addition, other cell lines used in this example included patient-derived xenograft (PDX) lines, that were established and maintained following protocols from the Mayo Clinic Brain Tumor Patient-Derived Xenograft National Resource. As per the Mayo Clinic Brain Tumor Patient- Derived Xenograft National Resource, the fresh tumor tissue samples were obtained from consenting patients undergoing surgical resection and immediately processed in a sterile environment. Tissues were mechanically dissociated and enzymatically digested to create a singlecell suspension. This suspension was then injected into the flanks or brains of immunodeficient mice (e.g., NOD-SCID) to establish the PDX lines. Once tumors reached a predetermined size, they were serially passaged in mice to maintain the line, with a portion cryopreserved at each passage. For in vitro culture, PDX tumors were dissociated into single-cell suspensions and cultured in DMEM / F-12 (1: 1) media (Thermofisher, USA) supplemented with 10% fetal bovine serum (Thermofisher, USA) and 1% Penicillin-Streptomycin (10,000 U / mL) (Thermofisher, USA). PDX lines were regularly characterized for genomic fidelity to the original patient tumor using immunohistochemistry and molecular profiling. For experimental use, PDX-derived cells were harvested using Accutase solution, counted, and resuspended in serum-free media for intracranial injection or other experimental procedures. All procedures were carried out in compliance with institutional guidelines for animal research and human tissue use. Tumor Induction: NCR-Nude mice were subjected to stereotactic surgery for glioma cells’ injection into the right hemisphere (injection coordinates: Bregma ML 1.7, AP -1.8, DV - 0.7). Total of 5 x 104cells suspended in 5 ul PBS were injected with an injection rate of 500 nl / minute. During the surgery, specially designed electrodes (8299-C Platinum Iridium Depth Electrode 2.5mm length - 1cm connector" obtained from Pinnacle Technology Inc.) were stereotactically implanted in the contralateral (left) hemisphere (electrode implant coordinates: Bregma ML -1.7, AP -1.8, DV -0.7) of the same mouse. Another electrode, which serves as a ground, was stereotactically implanted in the occipital region into the cerebellum with coordinates; Lambda ML -1.7, AP -3.0, DV -2.0. In another mouse group, electrodes were implanted on the ipsilateral side.

[0137] Invasive and Noninvasive Stimulation Protocol: The mice were allowed to develop tumors for 10 days, and tumor induction was confirmed using an in vivo imaging system (IVIS® Spectrum, Perkin Elmer). The implanted electrode was utilized to stimulate the brain at the following parameters: 1Hz frequency, lOOuA current, 1ms pulse-width, for 1800sec. For noninvasive stimulation, 1Hz auditory frequency was delivered from a computer system (MacBook Pro, 2018) using a MATLAB code, by generating 15kHz tone, for 0.1 sec duration, given every 1 sec for 1800sec in total.

[0138] Imaging Techniques: To follow up with the intracranial tumor growth, the tumor sizes were assessed using Magnetic Resonance Imaging (MRI) and IVIS, every 3 days. For the IVIS bioluminescence calculations, Luciferase activity, indicative of tumor growth, was measured using Living-Image® Software (IVIS® Spectrum, Perkin Elmer).

[0139] RNA sequencing: Brain tissues from orthotopic xenograft models were extracted and immediately placed in RNA-stabilizing solution. The tissues were then carefully dissected to separate tumor and tumor periphery regions, which were placed in separate vials. Total RNA was isolated from these tissue samples using the QIAGEN RNeasy Mini Kit, following the manufacturer's protocol for tissue samples. Briefly, tissues were homogenized in the provided lysis buffer using a rotor-stator homogenizer. The lysate was then processed through a series of spin columns and wash steps as per the kit instructions to purify the RNA. The quantity and quality of the extracted RNA were assessed using a NanoDrop micro-volume spectrophotometer (Thermofisher) and a fragment analyzer (Model, C), respectively. The fragment analyzer was used to determine the RNA integrity number (RIN) to ensure high-quality RNA suitable for downstream bulk RNA-seq analysis. This process allowed for the isolation and characterization of RNA from distinct regions of the tumor and its microenvironment, providing a comprehensive view of the transcriptomic landscape across different areas of the brain tumor model. Tissue Processing and Confocal Microscopy for Brain Slices: After the experimental period, the mice were anesthetized and transcardially perfused with 4% formalin solution for microtome, and / or perfused with PBS and PBS containing sucrose for cryotome. This step ensured fixation of brain tissues, preserving cellular structures and molecular details for subsequent analyses. Following perfusion, brains were carefully extracted to maintain anatomical integrity. The extracted brains underwent paraffin embedding to facilitate thin sectioning of 5-micron size for confocal microscopy. Tissues were dehydrated through a series of graded ethanol solutions, followed by infiltration with molten paraffin wax. The processed tissue sections were stained for immunohistochemistry using mGluR8 antibody (1:500, Thermofisher) and proliferative marker Ki67 (anti-Ki67 antibody, 1 :500, Abeam) and were subjected to confocal microscopy. The confocal images were subjected to quantitative analyses using NIS-software (Nikon) and ImageJ (Fiji, NIH).

[0140] Results:

[0141] Successful tumor induction and electrode implantation in the mice cohort was confirmed using MRI and IVIS imaging techniques. For invasive stimulation, such electrode-based stimulation can be translated in humans, as brain stimulation with electrodes is FDA approved and already used for humans in treating brain diseases.

[0142] For invasive stimulation, the effects of temporal pattern of a low frequency stimulation (<20Hz) on tumor growth were tested. Following treatment with the parameters 1Hz frequency with 1ms pulse-width for 1800 s, the tumor growth in the stimulated mice (IVIS ave. total flux (p / s) 6.86 x 107to 11.6 x 107) was observed to be halted / stopped, compared to the continuous tumor growth in control mice without the stimulation treatment (IVIS ave. total flux (p / s) 37.6 x 107to 79xl07), as shown in Figures 1A, IB, and 2. As shown in Figure 1 A, glioma tumors were successfully induced, and electrodes implanted in a cohort of 6 mice, with tumor establishment confirmed through bioluminescence imaging (IVIS). The mice were strategically randomized into two groups: an experimental group (n=3) receiving daily 1 Hz electrical stimulation and a control group (n=3) without stimulation. Over a 5 -week period, tumor progression was meticulously monitored via IVIS imaging performed twice weekly. Notably, the total bioluminescence flux revealed a significant divergence between groups, with non-stimulated mice demonstrating a marked increase in tumor growth compared to the electrically stimulated group. In addition, the electrical stimulation for two mice were discontinued between days 15 to 24 (mouse -*H) and days 21 to 27 (mouse “♦"), respectively. Remarkably, despite the cessation of stimulation, tumor growth remained inhibited and did not grow, as shown in Figure 2. A similar pattern of tumor size growth inhibition was observed using MRI-based analysis of stimulated versus unstimulated mice see MRI images in Figure 4A). Consistent with the results from IVIS, the MRI images revealed a static tumor growth in stimulated mice, while progressive size increases were observed in control mice (Figure 4B). Hence, the IVIS bioluminescence measurements and MRI imaging confirmed the inhibitory effect of brain stimulation on tumor growth in the stimulated mice.

[0143] In a similar manner, using a non-invasive auditory stimulation of 1Hz frequency, tumor growth was also observed to be halted / stopped from the start of stimulation, as shown in Figure 3.

[0144] In addition, the comprehensive RNA sequencing analysis of the glioma mouse model, revealed the molecular mechanisms behind tumor inhibition / halting in electrically stimulated mice. The analysis shows distinct gene expression profiles in both tumor and non-tumor regions between treated and untreated groups, as shown in Figures 5 A and 5B. Figure 5 A shows the transcriptome comparison of untreated and treated mouse tissues, while Figure 5B displays the transcriptome comparison of untreated and treated human-origin tumor tissue. Brain tissues from both treated and untreated mice were carefully dissected to isolate tumor regions (of human origin) and surrounding mouse brain peripheries. RNA sequencing revealed distinct differential gene expression profiles in both tumor and non-tumor regions between treated and untreated groups. In mouse-origin tissues, Gene Set Enrichment Analysis (GSEA) revealed significant downregulation (NES 0 to -3) of genes involved in cytokine responses, immune cell activation, and inflammatory processes across multiple Gene Ontology categories. In Biological Processes (BP), this included reduced expression of genes involved in cytokine responses, immune cell activation, and inflammatory processes. Cellular Component (CC) analysis revealed downregulation of genes associated with various immune-related complexes and cellular structures. Molecular Function (MF) analysis showed decreased expression of genes related to cytokine and chemokine activities, and ribosomal functions. Conversely, genes associated with neuronal function and synaptic transmission were upregulated (NES 0 to 3) in treated mice of genes associated with neuronal function and synaptic transmission across BP, CC, and MF categories. In human-origin tumor tissue, genes related to cell cycle progression, DNA replication, and metabolic processes were downregulated (NES 0 to -4.5), while genes involved in cell adhesion, synaptic assembly, and ion channel activity were upregulated (NES 0 to 2.5). These findings suggest that low-frequency electrical stimulation induces complex reprogramming of both the tumor microenvironment and the tumor itself, contributing to the observed tumor inhibition. Moreover, the Immunohistochemical (IHC) and immunofluorescence (IF) analyses revealed significant differences in mGluR8 expression between electrically stimulated and control mouse brains with glioma. Confocal imaging of untreated brain slices showed baseline mGluR8 expression in both tumor and peripheral regions (see Figure 6A). In contrast, treated brain slices exhibited markedly enhanced mGluR8 expression, particularly in the tissue surrounding the tumor peripheries (see Figure 6B). Quantification of IF staining intensity demonstrated significantly higher mGluR8 expression levels in treated mice compared to control mice (p < 0.05, see Figure 6C). This upregulation of mGluR8 in the tumor microenvironment of treated mice suggests a potential mechanism by which electrical stimulation may modulate the tumor-brain interface, possibly contributing to the observed tumor inhibition.

[0145] Furthermore, quantitative analysis of tumor characteristics revealed significant differences between electrically stimulated and control groups. Tumor size measurements showed a marked reduction in the treated group compared to untreated controls (see Figure 6D), indicating substantial tumor growth inhibition or halting in response to electrical stimulation. Assessment of cellular proliferation using Ki-67 staining demonstrated a significant decrease in proliferative activity in stimulated mice. The number of Ki-67 positive nuclei per field was markedly lower in treated mice compared to non-stimulated controls (see Figure 6E). Confocal imaging corroborated these findings, with treated brain slices exhibiting reduced Ki-67 staining (see Figure 6F) in contrast to the elevated proliferative activity observed in untreated brain slices (see Figure 6G). These results collectively support the anti-tumor effects of electrical stimulation, establishing a correlation between decreased pro-inflammatory cytokines expressions, increased mGluR8 expression, reduced tumor size, and decreased cellular proliferation in treated mice.

[0146] In summary, this example demonstrated that brain stimulation can effectively halt / stop the growth of brain tumors. Moreover, neuro-immunomodulation may be an underlying mechanism in this effect. The treatment appears to suppress pro-tumorigenic inflammatory responses in the surrounding tissue while also indirectly inhibiting tumor cell proliferation and altering their phenotype. This multi-faceted effect likely contributes to the observed tumor inhibition in treated mice, providing valuable insights into the molecular mechanisms underlying this novel therapeutic approach.

[0147] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A method of treating a central nervous system cancer or a cancer influenced by the nervous system, the method comprising the steps of:(i) invasively, minimally invasively, or non-invasively applying a stimulation to a subject having a central nervous system cancer or a cancer influenced by the nervous system; wherein the stimulation inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the growth rate of the central nervous system cancer or the cancer influenced by the nervous system prior to the subject receiving the stimulation; and / or wherein the stimulation optionally inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the growth rate of the central nervous system cancer or the cancer influenced by the nervous system for a subject not receiving the stimulation; and / or wherein the stimulation reduces or maintains size or volume of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the size or volume prior to the subject receiving the stimulation; and / or wherein the stimulation optionally reduces or maintains size or volume of the central nervous system cancer or the cancer influenced by the nervous system, as compared to the size or volume in the subject not receiving the stimulation.

2. The method of claim 1 , wherein the growth rate is inhibited or reduced by about 0.01 to about 99.99%; or the growth rate is stopped by the stimulation.

3. The method of claim 1 , wherein the size or volume of the central nervous system cancer or the cancer influenced by the nervous system is reduced by at least about 0.01%; or the size or volume is maintained.

4. The method of claim 2, wherein the stimulation inhibits, reduces, or stops the growth rate of the central nervous system cancer or the cancer influenced by the nervous system for at least 12 hours after the stimulation is ceased.

5. The method of claim 1, wherein the central nervous system cancer is a primary brain cancer.

6. The method of claim 5, wherein the primary brain cancer is selected from the group consisting of a glioma, glioneuronal, meningioma, medulloblastoma, schwannoma, pituitary adenoma, pineal region tumors, craniopharyngioma, primary central nervous system lymphoma, germ cell tumors, choroid plexus, mesenchymal, non-meningothelial, and haematolymphoid tumors.

7. The method of claim 6, wherein the glioma is selected from the group consisting of glioblastoma multiforme, diffuse intrinsic pontine glioma, diffuse hemispheric gliomas, astrocytoma, oligodendroglioma, oligoastrocytoma, ependymoma, and mixed gliomas.

8. The method of claim 1, wherein the central nervous system cancer is a metastatic cancer from a non-neural tumor.

9. The method of claim 8, wherein the non-neural tumor is from a cancer selected from the group consisting of melanoma, lung, thyroid, breast, renal cell carcinoma, colorectal, bladder, gastrointestinal tract, prostate, and ovarian cancer.

10. The method of claim 1, wherein the cancer influenced by the nervous system is selected from cancers in any organ or organ system in the subject’s body which is functionally connected to the central and / or peripheral nervous system.

11. The method of claim 1 , wherein the stimulation is a neural stimulation.

12. The method of any one of claim 11, wherein the neural stimulation is selected from the group consisting of neuronal stimulation, glial stimulation, synaptic stimulation, and combinations thereof.

13. The method of claim 1, wherein the stimulation is an immune-cell stimulation.

14. The method of claim 1, wherein the stimulation is a stimulation of vasculature cells optionally selected from endothelial cells and / or pericytes.

15. The method of claim 1, wherein the stimulation is selected from the group consisting of a physical stimulation, chemical stimulation, and biological stimulation that regulates cell activities; and combination thereof.

16. The method of claim 15, wherein the physical stimulation is selected from the group consisting of an electrical, electrochemical, mechanical, optical, optogenetic, thermal, magnetic, magneto-genetic, acoustic, sono-genetic stimulation, ultrasound-mediated stimulation, and combinations thereof .

17. The method of claim 15, wherein the chemical stimulation is selected from the group consisting of drugs, chemogenetics, and combinations thereof.

18. The method of claim 15, wherein the biological stimulation comprises biomolecules selected from the group consisting of proteins, RNA, DNA, lipids, viruses, bacteria, and combinations thereof.

19. The method of claim 1, wherein the stimulation is selected from the group consisting of brain stimulation, spinal cord stimulation, peripheral nerve stimulation and combinations thereof.

20. The method of claim 1, wherein the stimulation is selected from the group consisting of a stimulation applied from one or more implanted devices, body mounted devices, de vices inside or on surface or outside body or combination thereof, wearable devices, transcranial stimulation, transcutaneous stimulation, sensory stimulation, peripheral nerves stimulation, and combinations thereof.

21. The method of claim 20, wherein the implanted device is selected from the group consisting of but not limited to brain penetrating electrode, subarachnoid electrode, electrocorticography electrodes, epidural electrodes, intra-spinal electrodes, cuff electrodes, intra-fascicular electrode, inter-fascicular electrode, implanted wireless devices, and nanoparticles.

22. The method of claim 1, wherein the stimulation is a transcranial stimulation selected from the group consisting of transcranial magneticstimulation (TMS), transcranial electrical stimulation (TES), transcranial ultrasound stimulation (TUS), and combinations thereof.

23. The method of claim 22, wherein the TES is selected from the group consisting of transcranial direct current stimulation (tDCS), transcranial pulsed current stimulation (tPCS), transcranial alternating current stimulation (tACS), transcranial oscillating direct current stimulation (toDCS), transcranial random noise stimulation (tRNS), galvanic vestibular stimulation (GVS), electroconvulsive therapy (ECT), and peripheral nerves stimulation (such as Vagnus Nerve Stimulation or Transcutaneous Electrical Nerve Stimulation (TENS)).

24. The method of claim 1, wherein the stimulation is a sensory stimulation selected from the group consisting of auditory stimulation, visual stimulation, somatosensory stimulation, and combinations thereof.

25. The method of claim 1, wherein the stimulation is applied continuously during step (i).

26. The method of claim 1, wherein the stimulation is applied non- continuously during step (i).

27. The method of claim 1, wherein the stimulation in step (i) comprises a temporal pattern selected from the group consisting of continuous, randomized, adaptive, patterned phase-locked, dose-modulated, staircase, intermittent, rhythmic, pulse-burst, and combinations thereof.

28. The method of claim 1, wherein the stimulation in step (i) comprises one or more temporal patterns comprising any one, or any combination, or a repetition of any one or combination of the following applied, in a day, during step (i): pulse width (W 1 ) of about 0. Ips to 24hr, a pulse period (Pl) of about 0.01ms to 24hr, a burst width (W2) of about O.lps to 24hr, a burst period (Pl) of about 0.01ms to 24hr, a train width (W3) of about O.lps to 24hr, a train period (P3) of about 0.01ms to 24hr, and / or a session width (W4) of about O.lps to 24hr.

29. The method of claim 1, wherein the stimulation in step (i) is applied for a total amount of time in a range of about 0.1 pico-second to 24 hours per day.

30. The method of claim 1, wherein the stimulation in step (i) is applied over a period of time greater than one day and up to one or more years optionally comprising one or more time gaps.

31. The method of claim 28, wherein the temporal pattern comprises a low frequency stimulation of less than about 250 Hz or from about 0.00001 to 250Hz.

32. The method of claim 28, wherein the temporal pattern comprises a theta burst stimulation.

33. The method of claim 22, wherein the transcranial stimulation comprises one or more pulses having a pulse amplitude in a range of 0% to about 150% of a resting motor threshold or 0% to about 150% of an active motor threshold.

34. The method of claim 24, wherein the auditory stimulation comprises a pulse amplitude of less than about 90 dB, and a tone frequency of less than about 150 KHz.

35. The method of claim 24, wherein the visual stimulation comprises illumination with less than about 25,000 lumen of visible light in a wavelength range from about 300 to 800 nm.

36. The method of claim 24, wherein the somatosensory stimulation is a mechanical stimulation.

37. The method of claim 36, wherein the mechanical stimulation comprises mechanical pulses of less than about 600 psi.

38. The method of claim 24, wherein the somatosensory stimulation is a thermal stimulation.

39. The method of claim 38, wherein the thermal stimulation comprises thermal pulses having a temperatures ranging from about 20°C to about 65°C.

40. The method of claim 22, wherein the transcranial ultrasound stimulation is a transcranial focused ultrasound stimulation (tFUS) which comprises a fundamental frequency ranging from about 100 kHz to 650 kHz.

41. The method of claim 40, wherein the tFUS stimulation further comprises (a) a pulse repetition frequency (PRF) ranging from about 1 Hz to 4500 Hz; (b) aduty cycle (DC) ranging from about 1% to 100%; (c) a sonication duration (SD) ranging from about 10 ms to 500 ms; and / or (d) an intensity spatial-peak pulseaverage (Isppa) ranging from about 0.69 W / cm2to 18.2 W / cm2.

42. The method of claim 41, wherein the tFUS stimulation parameters are adjusted to produce either excitatory or inhibitory effects on neuronal activity.

43. The method of claim 40, wherein the tFUS stimulation is repeated one or more times, optionally including one or more time gaps ranging from 0 seconds to the subject's lifetime.

44. The method of claim 1, wherein step (i) is repeated one or more times optionally including one or more time gaps ranging from 0 seconds to the subject’s lifetime.

45. The method of claim 1, wherein the stimulation applied induces plasticity and / or modulates connections among nervous system cells of the central nervous system cancer or the cancer influenced by the nervous system and the cancer cells of the central nervous system cancer or the cancer influenced by the nervous system; and / or among the nervous system cells themselves; and / or among the cancer cells of the central nervous system cancer or the cancer influenced by the nervous system themselves; in order to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

46. The method of claim 1, wherein the stimulation applied induces angiogenesis to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

47. The method of claim 1, wherein the stimulation applied induces the subject’s immune system to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

48. The method of claim 1, wherein the stimulation applied induces or causes the disruption / modulation of sustained proliferative signaling to produce theinhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

49. The method of claim 1, wherein the stimulation applied induces or causes the reactivation of growth suppressors to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

50. The method of claim 1, wherein the stimulation applied induces or causes programmed cell death (apoptosis) to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

51. The method of claim 1 , wherein the stimulation applied induces or causes the limitation of replicative potential to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

52. The method of claim 1, wherein the stimulation applied induces or causes the inhibition of invasion and metastasis to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

53. The method of claim 1, wherein the stimulation applied induces or causes the alteration of cellular energetics to cause the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

54. The method of claim 1, wherein the stimulation applied induces or causes the reduction of genomic instability and mutation to produce the inhibition, reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

55. The method of claim 1, wherein the stimulation applied induces or causes the modulation of tumor-promoting inflammation to cause the inhibition,reduction, or the stop of the growth rate and / or reducing or maintaining the size or volume of the central nervous system cancer or cancer influenced by the nervous system.

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