Patent Application
20240066008
The invention relates to a device configured to block or inhibit the growth of neoplastic cells in a target region of a living organism, particularly the use of a device for blocking or inhibiting the growth of neoplastic cells in combination with an antitumor drug.
Neoplasms or tumors originate from alteration of cells that multiply and spread throughout the body, escaping regulatory mechanisms. There are numerous types of neoplasms that can affect substantially any organ. They are usually classified into benign or malignant according to the ability of the neoplastic cells to invade surrounding structures and more distant organs. In addition to tumors of solid organs, the term neoplasm includes also tumors of blood cells, such as lymphomas and leukaemias.
Neoplasms continue to be among the leading causes of death and deterioration in quality of life despite the major successes in the clinical field achieved in recent decades thanks to targeted prevention measures, early diagnosis and therapy.
Tumor therapy is essentially based on surgery, especially in localised forms, radiotherapy and drugs.
Glioblastoma multiforme (GBM) represents one of the most aggressive tumors for humans due to the ability of GBM neoplastic cells to infiltrate the parenchyma adjacent to the tumor lesion (DeAngelis et al, 2001).
GBM patients usually undergo resection, possibly complete, of the lesion by surgery, followed by radiotherapy and chemotherapy with the drug temozolomide, an alkylating antitumor drug (Alifieris et al, 2015).
However, despite a multi-pronged therapeutic approach, GBM is characterized by a high recurrence rate, drug resistance and devastating neurological deterioration (Wilson et al, 2014).
GBM is a particularly infiltrative tumor, and the performance of a complete surgical resection of the lesion is therefore substantially impossible, so that almost all patients develop a relapse and have a survival time of only 14 months (Becker et al, 2012). Additionally, 5 years after surgery, less than 5% of patients are still alive (Ostrom et al, 2014).
It is therefore clear how necessary it is to develop new therapeutic strategies to improve the efficacy of the drugs in use and to reduce the corresponding adverse effects, thus improving patients' quality of life.
In this sense, one of the most promising therapies comprises the use of non-ionizing electromagnetic fields (EMFs).
This therapy is particularly promising as it appears to have an excellent degree of safety, low toxicity and the possibility of combination with conventional drug therapies (Mattson et al, 2019).
In recent years, several EMFs technologies have been tested and have shown to have a good level of efficacy against different types of tumors, both when used alone and in combination with chemotherapy (Pasi et al, 2016).
It has been found that different frequency ranges trigger different response mechanisms in the cells that can lead to the reduction of cell proliferation of neoplastic cells by acting on the formation and stability of the mitotic spindle of these cells (Giladi et al, 2015).
In 2011, the American Food and Drug Administration approved the use of Tumor Treating Fields (TTFields) for the treatment of patients with recurrent GBM.
Several prior art documents have been published to this effect, such as, by way of example, documents US2020269041 and EP3439733.
The object of the present invention is to realize an alternative device and method to those of a known type, adapted to block or inhibit the growth of neoplastic cells in a target region of a living organism.
In particular, it is an object of the present invention that such a device and method may be used in the treatment of neoplasms, even of an aggressive type, such as glioblastoma multiforme.
Still, it is an object of the present invention that such method and use of the device in a patient may also be of the non-invasive type.
Furthermore, it is an object of the present invention to realize an antitumor drug which has greater therapeutic efficacy and a reduced toxicological profile, improving the patient's quality of life.
The objects mentioned above are achieved by an antitumor drug as described in the main claim.
The objects are also achieved by a device as set forth in claim 7.
Furthermore, the objects are also achieved by a method for treating neoplastic cells, according to what will be described below.
In particular, the present invention relates to an antitumor drug for use in a method for blocking or inhibiting the growth of neoplastic cells in a target region of a living organism, wherein said method comprises treating said neoplastic cells with the antitumor drug prior to, simultaneously with or subsequent to the application of electric current waves to the target region for a predefined period of time, and wherein the electric current waves have a waveform with a fundamental frequency higher than or equal to 2 MHz.
Preferably, such electric current waves have a waveform with a fundamental frequency ranging between 2 and 64 MHz, more preferably ranging between 2 and 20 MHz, even more preferably ranging between 4 and 20 MHz.
Much more preferably, such electric current waves have a waveform with a fundamental frequency of about 4 MHz.
It is specified that the application of such electric current waves will also be referred to in this document as Quantum Molecular Resonance or QMR.
Furthermore, according to an aspect of the present invention, such electric current waves have a sinusoidal waveform.
According to one aspect of the invention, this waveform is distorted by the presence of harmonics.
According to another aspect of the present invention, the sinusoidal waveform is distorted by the presence of at least second and third order harmonics.
According to one aspect of the invention, the treatment of neoplastic cells with the antitumor drug is carried out simultaneously with or subsequent to the application of the aforesaid electric current waves.
Preferably, according to one aspect of the invention, the treatment of the neoplastic cells with the antitumor drug is carried out simultaneously with the application of the electric current waves to the target region, while the neoplastic cells are treated with this antitumor drug.
According to one aspect of the invention, the aforesaid antitumor drug comprises temozolomide.
According to one aspect of the present invention, the neoplastic cells comprise glioblastoma cells.
In particular, the target region belongs to a living organism, preferably a human subject.
The aforesaid target region preferably comprises the central nervous system.
A device for blocking or inhibiting the growth of neoplastic cells in a target region of a living organism is also part of the present invention, wherein said device is configured to generate electric current waves having a waveform having a fundamental frequency higher than or equal to 2 MHz, preferably ranging between 2 and 64 MHz, more preferably ranging between 2 and 20 MHz, even more preferably ranging between 4 and 20 MHz, much more preferably about 4 MHz, and wherein said waveform is a sinusoidal waveform distorted by the presence of harmonics.
This device comprises one or more electrodes connected to at least one radiofrequency circuit adapted to supply each of these electrodes with a current wave having a fundamental frequency higher than or equal to 2 MHz.
Such one or more electrodes are configured so as to transmit said electric current waves to the aforesaid target region.
According to one aspect of the invention, the electrodes of the device are electrodes of the implantable type, adapted to be implanted at or near the aforesaid target region.
A method for blocking or inhibiting the growth of neoplastic cells in a target region is also part of the present invention.
This target region preferably belongs to a living organism.
According to one aspect of the method of the present invention, it comprises treating the aforesaid neoplastic cells by applying electric current waves to the aforesaid target region for a predefined period of time, wherein said electric current waves have a waveform with a fundamental frequency higher than or equal to 2 MHz, preferably ranging between 2 and 64 MHz, more preferably ranging between 2 and 20 MHz, even more preferably ranging between 4 and 20 MHz, much more preferably about 4 MHz.
Furthermore, according to one aspect of the method of the invention, such a waveform is a sinusoidal waveform.
Again, according to one aspect of the invention, this sinusoidal waveform is distorted by the presence of harmonics.
Again, according to another aspect of the method of the present invention, the sinusoidal waveform is distorted by the presence of at least second and third order harmonics.
Furthermore, according to one aspect of the method of the invention, the neoplastic cells comprise glioblastoma cells.
According to one aspect of the invention, the target region comprises the central nervous system.
According to a further aspect of the method of the invention, this method comprises the following steps:
Alternatively, according to another aspect of the method of the invention, this method comprises the following steps:
Preferably, the method of the present invention makes use of the device of the present invention.
Further features and advantages of the drug, device and method of the invention will be apparent to one skilled in the art from the following description of a preferred embodiment of the invention which is given by way of illustration, but not limitation.
As indicated above, the present invention relates to a method for blocking or inhibiting the growth of neoplastic cells in a target region of a living organism.
The term neoplastic cells is used in this document to refer to those cells that escape the proliferation control mechanisms and follow their own autonomous program of reproduction. They form a neoplasm, i.e., an abnormal mass of cells whose growth is excessive and uncoordinated compared to that of normal cells, and which progresses excessively even after the stimuli that evoked it have ceased.
The aforesaid neoplasm, and optionally the region of the body of the living organism where this tumor mass expands, also represents the target region mentioned above.
By way of example not to be considered limiting, this neoplasm may include lung, colorectal, mammary, prostate, pancreas, liver neoplasm, or haematological neoplasms of bone marrow cells, lymphatic system, immune system, etc.
In particular, the neoplastic cells of the present invention comprise glioblastoma (GBM) cells, a malignant astrocytic tumor, and the target region comprises the central nervous system.
The method of the invention provides for treating the aforesaid neoplastic cells by applying electric current waves to the aforesaid target region for a predefined period of time, wherein said electric current waves have a preferably sinusoidal waveform with a fundamental frequency higher than or equal to 2 MHz.
The application of current waves having a sinusoidal waveform with a fundamental frequency higher than or equal to 2 MHz transmits energy to the molecules to which these current waves are applied, which corresponds to the so-called “molecular resonance”, defined by the term Quantum Molecular Resonance (QMR).
It is known from document EP1087691 and from document Pozzato G et al, 2003, on behalf of the Applicant, that this QMR energy is just sufficient to break the bonds among the molecules hit by the passage of current, making it particularly useful when applied to a scalpel. This scalpel is in fact able to cut the regions of interest, without producing any effect of rupture, tear, necrosis, decrease or increase in the thickness of the tissues involved, alteration of the liquid content, coagulation or other degenerative effect around the cut.
However, it has now been surprisingly discovered that current waves having a waveform with a fundamental frequency higher than or equal to 2 MHz perform an antitumor action when applied to cells of the neoplastic type.
The Applicants have in fact discovered that the application of electric current waves, such as to transmit the QMR resonance energy to the neoplastic cells, allows to obtain the reduction of the motility and aggressiveness of the aforesaid cells, decreasing their ability to migrate through the matrices, for example, to give rise to new tumor lesions.
Experiments on BM-MSC cells have also highlighted that the application of such current waves appears to be harmless towards normal, non-neoplastic type cells, thus ensuring that the method of the invention is selective in blocking or inhibiting the growth of neoplastic cells only and not the growth of the supporting cells that are found around these neoplastic cells, as is the case, instead with standard type antitumor therapies.
Furthermore, it has been identified that the application of such current waves to glioblastoma cells causes a global alteration of their protein structure which ultimately leads to cell death.
Advantageously, instead, this protein alteration is quickly counteracted in healthy cells.
The use of fundamental frequency values ranging between 2 and 64 MHz, more preferably ranging between 2 and 20 MHz, even more preferably ranging between 4 and 20 MHz, has proved particularly advantageous in the method of the invention.
Particularly preferred is the use of electric current waves having a waveform with a fundamental frequency of about 4 MHz.
Preferably, such a waveform is a waveform of the sinusoidal type. Furthermore, according to one aspect of the method of the invention, such a sinusoidal waveform is distorted by the presence of harmonics.
Again, according to another aspect of the method of the present invention, the sinusoidal waveform is distorted by the presence of at least second and third order harmonics.
Preferably, the sinusoidal waveform is distorted by the presence of at least first, second and third order harmonics.
As shown by the results of the experiments described below, the application of the aforesaid electric current waves to neoplastic cells makes it possible to block or inhibit the growth of neoplastic cells with a high rate of success, leaving the healthy cells present in the target region substantially unharmed.
The effect of combining the application of such electric current waves with the treatment of neoplastic cells with antitumor drugs of known type was also tested.
The term antitumor drug refers to any molecule or combination of molecules, for pharmaceutical use, intended to inhibit or block the growth and spread of tumor formations.
The use of the antitumor drug temozolomide (TMZ) is particularly preferred.
It was surprisingly found that the combination of applying such electric current waves having a waveform with a fundamental frequency higher than or equal to 2 MHz, preferably ranging between 2 and 64 MHz, more preferably ranging between 2 and 20 MHz, even more preferably ranging between 4 and 20 MHz, much more preferably about 4 MHz, to neoplastic cells of the aforesaid target region for a predefined period of time, either previously, simultaneously or subsequent to the treatment with an antitumor drug, allows obtaining a greater antitumor efficacy of the antitumor drug itself.
In particular, the use of the antitumor drug in combination with QMR allows to obtain a greater reduction in the number of live neoplastic cells than the use of the antitumor drug alone on neoplastic cells.
It is therefore highlighted that the combination of the treatment with an antitumor drug with the application of the aforesaid electric current waves exerts a greater antitumor effect in the treated neoplastic cells than the use of the antitumor drug alone on these neoplastic cells, thus allowing, with equal antitumor effect, to decrease the dosage of the antitumor drug.
Still advantageously, thanks to this reduction in dosage achieved through the use of QMR, it is possible to achieve a substantial reduction in the side effects caused by treatment with the antitumor drug, for the same antitumor effect achieved.
This advantage is particularly evident when the antitumor drug is administered simultaneously with or after the treatment with QMR.
This advantage is even more evident when the drug is administered simultaneously with QMR treatment.
Still advantageously, the combination of treatment with antitumor drug and QMR allows to obtain an increase in the number of cells in early apoptosis.
It is clear that QMR can be used as an effective adjunct to standard chemotherapy therapies to enhance the effects of currently used antitumor drugs, without a corresponding increase in toxicity.
In addition to these antitumor drugs, QMR can also be applied in combination with other types of antitumor treatments.
Some examples, not to be considered limiting, of such antitumor treatments comprise surgical treatments, radiosurgery, ionizing radiation treatments, chemotherapy treatments with alkylating agents, antimetabolites, antitumor antibiotics, topoisomerase inhibitors, antifungal drugs, corticosteroids, biological drugs, differentiating agents, hormones, drugs that stimulate the immune system, monoclonal antibodies.
Therefore, an antitumor drug for use in a method for blocking or inhibiting the growth of neoplastic cells in a target region of a living organism also forms part of the present invention, wherein the method comprises treating the neoplastic cells with such antitumor drug simultaneously or after the application of electric current waves to the aforesaid target region for a predefined period of time. As stated above, such electric current waves have a waveform with a fundamental frequency higher than or equal to 2 MHz.
It is not excluded that, according to one aspect of the invention, the treatment of the neoplastic cells with the antitumor drug takes place prior to the application of the aforesaid electric current waves.
Preferably, this waveform has a fundamental frequency ranging between 2 and 64 MHz, preferably ranging between 2 and 20 MHz, more preferably ranging between 4 and 20 MHz.
More preferably, this waveform has a fundamental frequency of about 4 MHz.
Still preferably, the aforesaid waveform is a sinusoidal waveform.
Additionally, this sinusoidal waveform is distorted by the presence of harmonics.
Preferably, such a sinusoidal waveform is distorted by the presence of at least second and third order harmonics.
According to one aspect of the invention, the antitumor drug is used for use in a method comprising treating neoplastic cells with the antitumor drug simultaneously with the application of the aforesaid electric current waves to the target region of a living organism while the neoplastic cells are treated with the antitumor drug.
Returning to the method of the present invention, this method preferably comprises the following steps:
Preferably, by a living organism it is intended a human subject.
These electrodes are of a suitable shape and configuration to transmit the aforesaid electric current waves to the target region.
By way of example not to be considered limiting, such electrodes may be of a non-implantable type and, for example, may be in the form of a handpiece or in the form of a suction cup, or again may be essentially laminar in shape and applicable by adhesion to the skin of the subject, or such electrodes may be in the form of a probe.
When the electrodes used in the method of the invention have a laminar shape, they are essentially flexible so as to follow the shape of the surface of the body without difficulty and, moreover, they are optionally also equipped with an adhesive substance which eases keeping them in contact with the body during the application of the waveform generated by the device.
It is not excluded that one or more of the aforesaid electrodes may be of the implantable type.
A device for blocking or inhibiting the growth of neoplastic cells in a target region of a living organism therefore also forms part of the present invention.
Such a device is usable in the method of the invention.
In particular, the device of the invention is configured to generate electric current waves having a waveform with a fundamental frequency higher than or equal to 2 MHz.
The target region preferably belongs to a living organism, more preferably a human subject, as already indicated above.
According to a preferred embodiment of the device of the invention, it comprises a radiofrequency circuit connected to one or more electrodes and adapted to supply each of these electrodes with a current wave having a fundamental frequency higher than or equal to 2 MHz.
Preferably, the device further comprises a rectifier circuit powered by the mains voltage which supplies preferably continuous voltage, more preferably stabilised, to the aforesaid radiofrequency circuit.
Such a radiofrequency circuit preferably comprises at least one electronic switch which is powered by the voltage and driven by a driving circuit.
More in detail, the radiofrequency circuit presents at its output a current wave with a fundamental frequency higher than or equal to 2 MHz and is adapted to transmit this current wave to these electrodes. Preferably, this fundamental frequency is ranging between 2 and 64 MHz, more preferably ranging between 2 and 20 MHz, even more preferably ranging between 4 and 20 MHz.
More preferably, the fundamental frequency corresponds to about 4 MHz.
This current wave also has a sinusoidal shape.
This current wave is also advantageously distorted by the presence of harmonics.
These harmonics are preferably harmonics of at least the second and third order.
More preferably, the current wave has a sinusoidal shape distorted by the presence of at least first, second and third order harmonics.
This current wave also circulates in a preferably broadband resonant circuit on the frequency of the fundamental wave of the distorted sinusoidal shape.
According to the preferred embodiment of the invention, the device generates high frequency sinusoidal alternating electric current waves of 4 MHz with harmonics from 4 to 64 MHz.
According to one aspect of the device of the invention, the electrodes of said device are configured in such a way that they can transmit the aforesaid current wave to the target region.
It is specified that the electrodes of the device of the invention may have a shape and configuration corresponding to the shape and configuration of the electrodes described above for the method of the invention.
According to one aspect of the device of the invention, such electrodes are of the non-implantable type and are adapted to be applied to the surface of the body of the organism at or near which the target region comprising the neoplastic cells is located.
According to a variant embodiment of the device, it comprises a helmet configured to allow the housing of the aforesaid electrodes and to allow both their connection with the aforesaid radiofrequency circuit and their arrangement at or near the target region.
It should be noted that, according to a particular variant embodiment of the device comprising the aforesaid helmet, the latter is configured to also house the radiofrequency circuit.
According to a further variant of the device, it comprises a cap of the wearable type configured to house the aforesaid electrodes and allow them to be applied at or near the target region.
According to another variant embodiment of the device, it comprises at least one band provided with the aforesaid electrodes. Optionally, this band is configured to also house the device's radiofrequency circuit.
This band is also configured to be placed laterally and/or frontally and/or posteriorly to the head of the subject.
It is not excluded that such a band is configured to be placed at the person's waist and to be worn by the person as a belt.
Furthermore, it is not excluded that this band may be worn by the subject in a different way than indicated above.
According to a particular variant embodiment of the device of the invention, at least one or more of the electrodes of the device of the invention are electrodes of the implantable type.
The term “implantable” means an electrode intended to be implanted wholly or partially, by surgical or medical intervention, into the human body.
According to another variant embodiment of the device of the invention, it is configured so as to be completely implantable.
By way of example, the present implantable device has a configuration substantially similar to the configuration of an implantable pacemaker.
Other aspects and advantages of the present invention will appear when reading the following examples, which are to be considered as illustrative and non-limiting.
Glioblastoma cell lines A172, T98G and U87MG were used. A172 and T98G cells (Sigma-Aldrich, St. Louis, MO, USA) were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM/F12 GlutaMAX, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) added with 10% foetal bovine serum (FBS) (Qualified Australian, Gibco, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Sigma-Aldrich).
U87MG cells (courtesy of Prof. Massimo Dominici, Laboratory of Cellular Therapy, University Hospital of Modena and Reggio Emilia Modena, Italy) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with GlutaMAX (Gibco, Thermo Fisher Scientific) added with 10% FBS (Gibco, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Sigma-Aldrich).
Bone marrow-derived mesenchymal stromal cells (BM-MSC) were also used as non-tumor type control cells. BM-MSC cells were produced in the Advanced Cellular Therapies Laboratory of the Complex Operative Unit of Haematology of ULSS8 Berica as described previously (Sella et al, 2018). Briefly, MSC were isolated from cells obtained by washing discarded bone marrow collection bags and filters from healthy donors (Ethics Committee auth. No. 107/18 of Dec. 2, 2019). After washing, cells were centrifuged at 2000 rpm for 10 min and seeded at a density of 1×105 cells/cm 2 in DMEM with GlutaMAX (Gibco, Thermo Fisher Scientific,) added with 10% FBS (Gibco, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Sigma-Aldrich). Cell cultures were incubated at 37° C. in a humid atmosphere with 5% CO2. Cells that did not adhere were removed 72 hours after seeding and new medium was added. The medium was changed every 3-4 days. At 80% confluence, BM-MSC cells were re-seeded at a density of 2000 cells/cm2.
The cell lines were stimulated with QMR using a prototype QMR device from the company Telea (Telea Electronic Engineering, Sandrigo, VI, Italy). The device, suitably adapted for the present in vitro experiments, generated high-frequency sinusoidal alternating electric current waves of 4 MHz with harmonics from 4 to 64 MHz and had the following data: power supply 100÷230 V˜50/60 Hz; maximum output power, 5 W/400Ω.
QMR was applied to the cells using a pair of electrodes that were placed inside a 100 mm Petri dish, at the edge, and connected to the generator device. The transmission of electric fields to the medium of the cell cultures generated heat (Joule effect). The average temperature increase in the medium of the stimulated cells was 5° C. This value was measured by means of three independent measurements with a data-logger probe (iLog, Escort Scunthorpe, UK) placed in the culture medium. In order to compensate for the increased temperature of the medium of the stimulated cells and also to ensure an average temperature of 37° C. for the stimulated cells, the incubator used for the stimulated cells was set at 32° C. at 5% CO2 and the temperature of the laboratory was maintained between 20° C. and 25° C.
Based on the cell line used, a defined number of cells were seeded in 100 mm plates (Greiner Bio-One, Frickenhausen, DE) in order to be stimulated at 60% confluence. The cells were then exposed to QMR for 24 hours and analysed 0-24-48 hours after the end of the stimulation.
More specifically, the cells were washed with D-PBS (Sigma-Aldrich), detached with 1× TrypLE Select (Gibco, Thermo Fisher Scientific) and the aliquots obtained were used to investigate cell viability, apoptosis, cell cycle, karyotype and proteomics. A cell aliquot was re-seeded for evaluation of cell growth capacity in semi-solid medium (soft agar assay), while cell migration was monitored directly on QMR-stimulated plates before and after stimulation.
The effect of the combined use of QMR and the drug TMZ (Sigma-Aldrich) was also tested.
For these analyses, the cells were treated for 144 hours with 10-25 μM of TMZ administered at the same time as or after QMR stimulation. The TMZ stock solution was prepared in DMSO at a final concentration of 10 mM and maintained at 4° C. Subsequent dilutions were made in fresh culture medium and administered to the cells. Cell viability, apoptosis and the cell cycle were assessed after 144 hours.
After stimulation, the cells were harvested and suspended in a 1:1 ratio with a Trypan blue solution (Gibco, Thermo Fisher Scientific) in culture medium. The cells were counted using a Burker's chamber and the number of live cells was obtained by applying the following formula: [(number of cells×10,000×2×V)/9], where V is the total volume of cell solution obtained and 2 is the dilution factor of the solution with the dye (2×).
1.5 Apoptosis: Labelling with Annexin V/7-Aminoactinamycin D (7-AAD)
After stimulation, 1.5×105 cells were harvested and centrifuged at 400 g for six minutes. After washing with binding buffer, the cells were labelled with Annexin V/7-AAD according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). After dilution with binding buffer, the fluorescence of 2×104 cells/sample was detected using an FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). The cell population was separated into four groups: live cells with negative fluorescence for both Annexin V and 7-AAD; cells in early apoptosis positive for Annexin V and negative for 7-AAD (Annexin V+/7-AAD−); cells in late apoptosis with double positivity for Annexin V and 7-AAD (Annexin V+/7-AAD+) and dead cells negative for Annexin V and positive for 7-AAD (Annexin V−/7-AAD+).
After stimulation, 5×105 cells were harvested and centrifuged at 400 g for six minutes. The cells were then washed with D-PBS and fixed and permeabilised with acetone (80% solution in water). After one hour at 4° C., the acetone was removed by centrifugation, the cells were washed and labelled with 1 μg/mL with 7-AAD (Invitrogen) for 1 hour at room temperature. The fluorescence of 2.5×104 cells/sample was analysed using a FC500 flow cytometer (Beckman Coulter). EXPO 32 software (Coulter Systems, Fullerton, CA, USA) was used to calculate the percentage of cells in the different phases of the cell cycle. Diplode cycles were considered and corrected for cell clusters.
To assess whether QMR stimulation was safe and therefore did not cause chromosome alterations, BM-MSC mesenchymal stromal cells were stimulated for 24 hours with QMR and tested by G-Trypsin-Giemsa banding following standard techniques with 350/400 band resolution. 20 metaphases were analysed and 3 were karyotyped. Unstimulated BM-MSC cells were used as control cells.
The scratch test represents a 2D cell migration approach to semi-quantitatively detect cell motility. The purpose of scratching (opening) is to create a cell-free area (or opening) in order to induce the surrounding cells to migrate and close this opening. The cells were seeded in two-well silicone inserts (IBIDI GmbH, Grafelfing, DE) that achieve a 500 μm cell-free opening on the cell monolayer.
When 60% cell confluence was reached, the insert was removed and the cells were stimulated for 24 hours with QMR. Cell migration was monitored over time by image acquisition before stimulation and 0-24-48 hours after stimulation. Image acquisition was performed with an Axiovert 40 CFL inverted light microscope (Carl Zeiss, Oberkochen, DE). The percentage of closure of the opening was analysed with ImageJ software (National Institutes of Health, Bethesda, MD, USA) and the migratory capacity was assessed by means of a migration rate curve, which was calculated as shown below: Closure %=[(Area tx−Area ty)/Area tx]×100, where tx represents the acquisition time and ty the next time point.
The colony formation assay in semi-solid medium (soft agar) is a method used to monitor cell growth independently of cell anchoring to the substrate and which measures cell proliferation in semi-solid medium by optical colony counting. The rate of colony formation in soft agar changes depending on the cell line used. Therefore, the concentration of agarose, the number of cells seeded and the final day of the experiment were optimised for each cell line. The cell suspension was prepared in a 0.4% agarose solution in cell medium and seeded on a solidified layer of 0.6% agarose in complete medium. After 1 hour at room temperature, 160 μl of complete medium was added and a further 100 μl were added every week until the end of the experiment. The plates were transferred to an incubator at 37° C. at 5% CO2 for 21-24 days before being labelled with 100 μM calcein (Sigma-Aldrich) for 30 minutes. The colonies consisting of at least 50 cells were counted using an Axiovert 40 CFL inverted light microscope (Carl Zeiss).
1×106 cells were harvested and centrifuged at 400 g for 6 minutes, washed with D-PBS and lysed with lysis buffer on ice (Pierce™ RIPA buffer, Thermo Fisher Scientific) added with protease inhibitor cocktail (Cell Signalling, Danvers, MA, USA). After 30 minutes on ice, the cell lysates were centrifuged at 14000 g for 10 minutes at 4° C. and the protein supernatant was determined colorimetrically by BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific). Bovine serum albumin was used as the standard (Sigma Aldrich).
Liquid chromatography-mass spectrometry (LC/MS-MS) is an analytical technique that combines the separating capacity of liquid chromatography with the high sensitivity and selectivity of the mass analysis of the triple quadrupole mass spectrometry. For sample preparation, 50 μg of protein lysate were precipitated with acetone and the obtained protein pellet was dissolved in a 6M solution of urea and 100 mM ammonium bicarbonate (pH 8). Samples were reduced using 10 mM dithiothreitol for 1 hour at room temperature and alkylated with 20 mM iodoacetamide in the dark for 30 minutes at room temperature. Subsequently, the proteins were subjected to digestion with the endopeptidase enzyme Lys-C (Promega) at an enzyme/protein ratio of 1:100 (w/w) for 3 hours at room temperature. The proteins were then diluted 4 times in 50 mM ammonium bicarbonate and digested overnight with trypsin (Promega) 1:100 (w/w) at room temperature. Proteolysis was interrupted by the addition of 1% trifluoroacetic acid. Samples were then desalinated, vacuum dried and resuspended in 0.1% formic acid for LC-MS/MS analysis.
The samples were analysed using the Easy-nLC 1200 system (Thermo Fisher), coupled in-line with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher). A reversed-phase column (Acclaim PepMap RSLC C18, 2 μm particle size, 100 Å pore size, id 75 μm, Thermo Fisher Scientific) with a two-component mobile phase was used to separate the digested peptides: 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B). Peptides were eluted using a gradient from 5% to 25% for 52 minutes, followed by 25% to 40% for 8 minutes and finally by 40% to 98% for 10 minutes at a flow rate of 400 nL/min. The data-dependent acquisition (DDA) method is based on full scans performed at a resolution power of 120000 fwhm (full width at half maximum) (at 200 m/z), and a maximum injection time of 50 ms. The mass range of 350-1100 m/z was examined for the precursors, with the first mass set at 140 m/z for the fragments. Full scans were followed by a series of MS/MS (HCD) scans for a cycle time of 3 seconds, at a collision energy of 30% and detected in the ion trap with a maximum injection time of 150 ms. The dynamic exclusion time was set at 50 sec. Unprocessed data were searched in Proteome Discoverer 2.2 software (Thermo Fisher Scientific). Peptide searches were carried out using the Human protein FASTA file (uniprot). The proteins were identified with the MASCOT search engine (Matrix Science inc, Boston, MA, USA) using a precursor mass tolerance of 10 ppm and a product mass tolerance of 0.6 Da.
The different abundances of proteins expressed in the different experimental groups (at t0 and t24, respectively) were used to perform a hierarchical cluster analysis using the Clustvis tool (Metsalu T et al, 2015). Gene Ontology (GO) and Pathway annotation of protein IDs was performed using the EnrichR gene set enrichment analysis server (http://amp.pharm.mssm.edu/Enrichr/), by applying biological processes and Reactome categorisation with the significance threshold set at p<0.05. The network of protein interactions was constructed using the STRING interaction database, version 11.0 (https://string-db.org/) (Mering C et al, 2003).
All data were analysed using Graph Pad program (Graph Pad Software, San Diego, CA, USA) and were expressed as mean±standard error. The One-sample t-test was used for the analysis of the results expressed as a ratio/percentage of the control (e.g., trypan blue exclusion assay). The unpaired Student's t-test was used for all the other analyses. Data with p<0.05 were considered as significant.
The effects of QMR treatment on A172 glioblastoma cells were assessed by analysing cell morphology, proliferation rate, apoptosis and cell cycle of the cells immediately at the end of the treatment and 24 and 48 hours after the end of QMR stimulation. The compliance with these timeframes made it possible to obtain an assessment of the permanence of the effects obtained with the treatment and the ability of the treated cells to restore their cellular functions to pre-treatment levels.
It was surprisingly found that QMR treatment changes the morphology of
treated A172 cells compared to untreated control cells. QMR-treated cells appeared in fact more swollen and more granular than control cells, indicating that cell damage had occurred.
This damage was also found to be persistent over time, as shown in
The cell proliferation rate was also estimated by testing with Trypan Blue. 24 hours after treatment, QMR-treated cells showed a reduction in tumor growth by about 28%. Furthermore, the number of live cells tended to decrease even 24-48 hours after the treatment, suggesting an effect of QMR treatment on the cell cycle progression, as visible in
In support of this surprising finding, the cell cycle of A172 cells was also evaluated using flow cytometry experiments. The results, shown in
Further, the efficacy of treating cells with QMR was confirmed by the clear apoptotic activation in the treated cells. The number of live cells after QMR treatment was in fact significantly lower (p<0.01) than the number of control live cells not treated with QMR, as shown by the results in
2.2 QMR Treatment does not Cause Cytotoxic and Genotoxic Effects on BM-MSC Cells
The effects of QMR treatment on healthy BM-MSC cells were tested in order to verify the safety of QMR treatment on non-neoplastic type cells. At the end of QMR stimulation and 24 hours after the end of the treatment, cell morphology, proliferation rate, apoptosis, cell cycle and karyotype were evaluated in treated BM-MSC cells and in untreated control cells. Although a slight decrease in cell growth was displayed at the end of the QMR treatment, as shown in
Furthermore, BM-MSC cells treated with QMR showed no differences in cell morphology, cell cycle modulation and apoptosis, as visible in
Furthermore, time did not influence the final effect obtained, as demonstrated by the results obtained which are substantially superimposable at 0 and 24 hours after treatment.
The analysis of the karyotype of BM-MSC cells treated with QMR also showed that the QMR treatment advantageously causes no changes in the number of chromosomes or in their structure, as can be seen in
It is well known that neoplastic type cells are characterized by uncontrolled proliferation and migration. The ability of cells to grow in semi-solid medium and their mobility rate are therefore considered markers of tumor aggressiveness and invasiveness. Cell colony formation in soft agar and cell migration were therefore analysed under basal conditions and after QMR treatment. The results, shown in
The proteomic profile of A172 and BM-MSC cells treated with QMR was also investigated. The analysis was conducted at the end of the QMR stimulation (time 0) and 24 hours after the end of the treatment. Untreated A172 and BM-MSC cells were used as control groups.
A total of 5104 proteins were identified in the A172 cell line, of which 311 and 308 were found to be significantly altered after QMR treatment at time zero and 24 hours after the end of the treatment, respectively. Cluster analysis, not shown in the figures, highlighted two separate groups of proteins at both testing timeframes, demonstrating that QMR treatment interferes with neoplastic cell activity.
The proteins were found to be differentiated between treated and untreated cells. Specifically, at time zero, 111 proteins were found to be significantly up-regulated, while 200 proteins were found to be down-regulated. Instead, 24 hours after the end of the treatment, 103 up-regulated proteins and 205 down-regulated proteins were found.
More specific analyses of gene ontology and pathway enrichment showed that up-regulated proteins were strongly associated with stress response mechanisms, protein folding and extracellular matrix modelling, suggesting that QMR treatment also acts as a toxic-protein stimulus.
More specifically, QMR-treated cells, compared to untreated control cells, had undergone a significant down-regulation of key factors involved in protein translation, RNA processing and cell cycle involved pathways, leading to cell cycle arrest and death of treated cells.
Advantageously, the effects of the QMR stimulation on BM-MSC cells provided completely different results compared to A172 glioblastoma cells. At the end of QMR treatment, the activation of heat shock proteins was associated with a remodelling of the extracellular matrix. However, this alteration was found to be totally normalised 24 hours after treatment. Therefore, QMR treatment does not interfere with the proliferation rate of BM-MSC cells, supporting the selectivity of QMR treatment towards neoplastic type cells.
The experiments described above were also performed on glioblastoma cell lines that were more aggressive than the A172 cell line cells. These more aggressive cell lines comprise T98G and U87MG cells. Although the proliferation rate was found to be reduced in both cell lines following QMR treatment, there were no significant differences in apoptosis activation, cell cycle progression, clonogenic capacity and cell motility, as shown in
Cell viability was also tested after 48 and 72 hours of QMR treatment. Surprisingly, the cell proliferation rate for both A172 and U87MG cells was found to be significantly reduced over time.
Even a 15% increase in treatment power has been shown to affect the rate of cell proliferation. In fact, the cell viability of the QMR-treated cells at higher power was found to be decreased by up to 35%, as shown in the figures.
Standard therapies for patients with glioblastoma include, to date, total resection of the lesion, followed by radiotherapy and chemotherapy with the drug TMZ.
Disadvantageously, the efficacy of the drug is limited by the severity of the side effects caused by the drug itself and by the establishment of drug resistance mechanisms. It is therefore essential to find new therapeutic strategies to be used in combination with TMZ in order to lower the dosage thereof and improve the patient's quality of life.
The efficacy of the combined therapy of TMZ and QMR treatment on A172 glioblastoma cells was therefore tested. These cells were treated with 10-25 μM of TMZ at the same time as or after the QMR treatment. Cell viability, apoptosis and cell cycle values were analysed. The TMZ drug concentration was selected on the basis of its IC50 (
The results obtained have surprisingly shown that, when the TMZ drug at a concentration of 10 μM was used in combination with QMR treatment, cell viability was reduced by about 12% more than the reduction in cell viability obtained in cells treated with TMZ drug alone, as shown in
Furthermore, these data were found to be surprisingly correlated with the data on cell apoptosis. Indeed, the combined administration of TMZ and QMR showed that the reduction in the number of live cells is associated with an increase in the number of cells in early apoptosis, as shown in the graphs in
The simultaneous exposure of the cells to the drug TMZ 25 μM and to QMR activates a greater alteration in cell cycle progression, compared to cells treated with the TMZ drug alone.
These data surprisingly demonstrate that the combined therapeutic strategy of TMZ with QMR induces significant cell cycle arrest in the G2-M phase and reduces the percentage of cells in the G0-G1 phase, as shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 102021000000119 | Jan 2021 | IT | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2022/050042 | 1/4/2022 | WO |
