Tumor-specific inhibition with magnetic field

doi:10.21203/rs.3.rs-1313249/v1

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Tumor-specific inhibition with magnetic field

https://doi.org/10.21203/rs.3.rs-1313249/v1

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Low frequency magnetic field exerted a significant inhibitory effect on tumor growth and has been developed as a therapeutic modality. Our study demonstrated that it increased intracellular calcium ions leading to membrane depolarization that interrupted intercellular signaling. Inhibition was observed in exposed tumor adherent cells, agglomerates, but not in freely tumor suspended cells and is therefore more pronounced on solid tumors. Our study provides evidence that exposure to magnetic field is broadly applicable to treating a variety of solid tumors.

magnetic field

intercellular signaling

agglomerates

solid tumors

Low-frequency magnetic field exert non-invasive, non-ionizing and non-thermal effects on cells and tissues. It enhances cell oxidative stress response and regulates apoptosis signaling pathway, changing intracellular Ca²⁺ concentration to induce apoptosis^1-3. It is widely used in the treatment of tumors, neuropsychiatric and bone diseases. In clinical studies, it was shown to have inhibited the proliferation of tumor cells in vivo, and have lengthened the lifespan of the subjects^4-5. Combining its use with paclitaxel in treating mouse cancer, magnetic field increased the execution lethality of paclitaxel⁶. At an extremely low frequency magnetic field of 10 mT and in combined usage with cisplatin, cell membrane permeability was altered and the therapeutic effect of cisplatin was significantly enhanced⁷. Ren Jing et al. found that magnetic field reduced the number of F4/80 positive macrophages and significantly increased the number of dendritic cells in liver cancer. It also reduced the proportion of MDSCs in tumor tissues and weakened the inhibitory function of MDSCs, thus enhancing the anti-tumor effect⁸. Studies have confirmed that magnetic field inhibited tumor cell proliferation and induced apoptosis and cell cycle arrest mainly through ROS², but the specific mechanism remains unclear. In the development of antineoplastic therapies, the inhibitory effect of magnetic field on tumor growth is a significant attribute to the clinical performance of many existing technologies.

Differential inhibitory effect of magnetic field on adherent cells

Normal human renal epithelial cells 293T, human liver cancer cells Hepg2, and human non-small cell lung cancer cells A549, processed independently through culture medium “infusion” or “change”, were exposed to 5 mT intensity of magnetic field for 2h each day for a total of 3 days. No significant difference was found between the exposed 293T cell count versus the control group without magnetic exposure on the third day of exposure. However, exposed Hepg2, A549 cell counts were significantly lower than the unexposed control groups (the highest inhibition rate of Hepg2 was about 18%, and the highest inhibition rate of A549 was about 30%). “Infusion” tumor cells showed inhibition on the first day whereas “fluid change” showed no significant inhibition. The inhibition trend of the “infusion” group was significantly stronger than that of the “fluid change” group (Figure 1).

Cell aggregation and growth inhibition correlation

Lymphoma Raji cells in culture aggregated spontaneously. These cells were dispersed to be freely suspended on day 3 using the processes of 1) fluid change or 2) infusion coupled with a blow and disperse operation. Both processes bypassed the detrimental effect of trypsin, thus removing the possibility that trypsin was responsible for tumor growth inhibition⁹. Cell growth was sustained with either process regardless of the freshness of culture media. Under these conditions, neither group showed significant inhibition to magnetic field during the six days of exposure at 5mT (Figure 2).

Cell contact in compact clustering produces inhibition

In the earlier experiment of Raji lymphoma cells, we demonstrated that magnetic field did not exert any inhibitory effect on freely suspended cancer cells. In this group of experiments, we examined whether cell contact played a role in magnetic inhibition on tumor growth. Aggregated Raji cells in clusters were studied in a nine-day continuous exposure experiment. Cells were transferred to a larger container while the culture medium was changed every three days. It was found that Raji cells responded to the magnetic field exposure pertaining to the concentration of suspended cell agglomerates, but their sensitivity to magnetic inhibition was weaker than that exhibited by adherent cells. The inhibition rate of Raji cells on the sixth day was similar to that of A549 cells on the third day. Interestingly, the inhibition rate of Raji cells decreased or even disappeared on the ninth day with the volume of the culture medium increased from 4 ml to 10 ml after the cells being transferred from 6-well plate to 10cm dish. In another similar experiment in which the volume of the was 15 ml instead of 10 ml, Raji inhibition rate decreased by about 50%. In order to determine the reasons for the disappearance of inhibition rate on the 9th day, we repeated the experiment, and the only change was that the cells were transferred on the 6th day, using the 10cm petri dish and 25cm² flask as controls. Surprisingly, the inhibition rate of Raji cells in 25cm² was as high as 36% on the 9th day. The inhibition rate of Raji cells in the 10 cm dish disappeared on day 9 (Figure 3).

Intracellular ions are not the cause of magnetic inhibition, calcium ions are associated with changes in membrane potential

Through experiments on suspended tumor cells, we found that the inhibition of magnetic field on cancer cells was accomplished through contact and possibly communication between cells, having implications of signal transmission between cells and ionic changes in cell microenvironment. To investigate whether magnetic field suppression is associated with ionic signaling, calcium, sodium, potassium, pH kit Calbryte ™ 520 AM, SBFI AM, PBFI AM, BCECF AM were used to observe the changes in intracellular free ions of four kinds of cells after 3 days of magnetic exposure. No change in intracellular sodium and potassium ions were observed in normal or tumor cells (Figure 4 a, b). There was no significant difference in fluorescence intensity in all groups except A549 (Figure 4 c, d). Normal cells 293T showed a significant decrease in intracellular free calcium ion, whereas solid tumor cells showed no significant change, while suspended tumor showed a slight increase in calcium ion (Figure 4 e, f).

A change in calcium ionic concentration is usually reflected in a change of membrane potential. The membrane potential kit DiBAC4 (3) was used to observe the exposed cells on the third day. Adherent cells showed significant hyperpolarization; tumor cell agglomerates showed significant depolarization; the freely tumor suspended cells showed no significant depolarization (Figure 4 g, h).

Inhibitory factors were universal and tumor specific

To determine whether magnetic field inhibition is related to a change of ionic microenvironment, A549 and Raji cells in agglomerate independently were exposed to the magnetic field with the strongest inhibition efficiency for 3h. The exposed culture media were then transferred to feed unexposed cells of the same species for two days. Under these conditions, A549 cells were significantly inhibited at approximately 10% rate, nearly half of that of cells exposed on the first day with direct exposure in the fluid infusion group. The Raji transfer group showed no inhibition, but all transfer groups were also inhibited compared to the unexposed control groups (Figure 5 a,b).

In addition, To determine whether this microenvironmental production is unique to tumor cells and inhibits normal cells, A549 and 293T were exposed to the magnetic field with the strongest inhibition efficiency for 3h. The exposed culture media were then transferred to feed unexposed cells of the different species for two days.The results showed that A549 was significantly inhibited in 293T culture media. The 293T transfer group showed no inhibition, but all transfer groups were also inhibited compared to the unexposed control groups(Figure 5 c,d).

Magnetic field exposure has potential to be an advanced strategy in cancer management. Reactive oxygen species (ROS) produced by cells using this method appears to be the key to tumor growth inhibition. We demonstrated that the primary mechanism is not only ROS related, but that exposure to magnetic field changed the signal communication between tumor cells, thus strengthening or restoring the inhibition of tumor cell contact. Magnetic field inhibition of tumor cells has four key properties. First, the inhibition of magnetic field requires close contact between cells. Second, the magnetic field inhibition rate depends on the stability of the environment - changing the medium - that is, changing the original environment - the inhibition efficiency is lower than that of the supplement medium; Third, the cell signaling system is more stereoscopic - suspension cells are already in close contact, but too much distance between cell clusters reduces the inhibition rate. Fourth, cell signals generated by magnetic fields are universal and tumor broad spectrum. This cellular signal that enhances or restores cell contact inhibition under a magnetic field is only effective on tumor cells.

Through the magnetic field inhibition experiment of Raji cells, we demonstrated that close contact between cells directly determined whether the inhibition effect existed. Therefore, dispersed cells were basically not inhibited by magnetic field, which might even accelerated growth. In addition, in the solid tumor exposure experiment, the inhibition effect of supplemented medium was different from that of replacement medium. The original environmental conditions of supplementary media were retained, and the inhibition effect of magnetic field was more stable, which perfectly matched the two properties of tumor inhibition by magnetic field. These two points were consistent with the previous results that the inhibition rate of magnetic field exposure decreased after digestion of adherent tumor cells⁹. Moreover, in the experiment of Raji cells, the same volume, the same number of cells, the same cell contact, and different basal area resulted in different inhibition rates. There was only a difference in stereoscopic height, and cell inhibition disappeared in the 10cm petri dish, the cell inhibition rate increased in 25cm² culture flask on the ninth day of magnetic field exposure. Although this condition is also related to the first property, it is more closely related to the communication between the cell clusters. We speculate that this signal only moves around the cell in the extracellular environment, so we define this condition as the third property. Solid tumors have all three properties, so it's reasonable to assume that they apply to all tumor cells that respond to magnetic fields.

We demonstrated that intercellular signaling was the crucial cause of magnetic suppression. Among homotype tumor cells, inhibition was observed when the medium environment was transferred to unexposed cells after magnetic field exposure, demonstrating the presence of inhibition signals still in the medium.With the xenogeneic cell study in which the culture media of normal cells 293T and solid tumor A549 were exchanged and transferred to unexposed cells after magnetic field exposure, tumor cells also showed inhibition, while normal cells did not showed significant inhibition, indicating that the inhibition signal is a kind of universal intercellular signal, which is sensitive to tumor cells. In combination with the result that the inhibition rate of suspended tumor cells decreases after deviating from the third property, it is speculated that the inhibition signal is similar to auxin, ---- promotes growth at low concentration and inhibits growth at high concentration.

The specific effect of magnetic field on signaling in the cellular environment is still unclear. The signaling pathway involved in magnetic field may be related to contact inhibition and epigenetic inheritance. Prompted by cellular signals ^3,10-11, we think of the electrical signals between cells and calcium ions, the second messenger associated with cell division. Under several fluorescent probe experiment, we found that the intracellular calcium signal and the membrane potential in the change of the magnetic field is always more synchronization, but under different experiment time, the results of calcium ion in cells and the membrane potential are different from the last experiment, we hypothesized that each time in the magnetic field exposure, calcium ion and the membrane potential has been in oscillating, Synchronous real-time detection is needed to clarify the specific relationship. At present, the relationship between calcium ion and membrane potential and inhibition signal of magnetic field is not clear. Moreover, changes in the status of ca₂⁺ and membrane potential also indicate changes in intracellular signals, so it is speculated that ca₂⁺ and membrane potential can be used as a new means to verify the inhibition effect of magnetic field on tumor cells.

In addition, Yong Zhou's team's discovering¹⁰ provide an explanation for magnetic suppression. The cell growth rate increases during depolarization, and the depolarization involves the nanoaggregation of k-Ras switches on the membrane surface. We demonstrated that in solid tumors, membrane potential is always hyperpolarized. When cells receive inhibition signals under the magnetic field, the duration of depolarization is shortened to achieve inhibition effect. This signal possibly related to the hypermethylation of the tumor - associated calcium signaling network¹².We will further analyze the environmental components to research the existence of inhibitory signals.

Combining the four key properties of cell inhibition of magnetic field will make it a good adjuvant anticancer modality. Recent clinical effective treatments have proved the effectiveness of magnetic field in treating tumors^4-5_.Moreover, the universality of cell signals with magnetic field exposure and the broad spectrum of tumors render it an excellent methodology for treatment and prognosis. Advances have indicated that magnetic field can reduce neoplastic macrophages and inhibit the generation of myeloid inhibitory cells MDSC⁸. In the future, magnetic field therapy will become one of the mainstream treatment by virtue of its advantages of low adverse effects, improved tumor microenvironment and good solid tumor treatment effect.

Acknowledgements We thank Professor Peter K. Law from The Cell Therapy Institute,Wuhan,China for technical assistance. This work was supported by grants from the Special fund for cancer prevention and treatment of Shanghai Science and Technology Development Foundation(CT20200517A) and the private foundation from The Zhejiang Huayi Health Industry Development Co., Ltd,Zhejiang,China.

Author contributions J.S. designed the study, performed most of the experiments, analysed data and wrote the manuscript.H.W. advised on experiment design and helped with experiments. Y.J.,X.Y.,Y.CandY.Y.T. participated in study design and performed experiments.W.Y.J. commented on the manuscript. G.H.Y. designed and supervised the study, interpreted data and wrote the manuscript.

Competing interests The authors declare no competing interests.

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Test kit

Calbryte 520 AM calcium probe kit (article No. 20650) was purchased from AATBioquest, USA. Hanks' Buffer with 20 mM Hepes (Item No. 20011) DiBAC4(3) membrane potential fluorescent probe (item no. 21411) purchased from AATBioquest, USA; Sodium ion fluorescent probe SBFI (article No. 18764) was purchased from Cayman Company of America; Potassium ion fluorescent probe PBFI (article no. 21602) was purchased from Cayman Company in the United States; Pluronic® F-127 (ST501-10G) was purchased from Beyotime Biotechnology

Cell culture

293T cells, Hepg2 cells and A549 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cell lines were cultured in DMEM medium (Biological Industries, Israel) with 10% fetal bovine serum (FBS, ExCell Bio, China) and 1% penicillin-streptomycin (P/S, Industries, Israel). The cells were incubated at 37°C in 5% CO2. All operations were conducted inside the vertical flow clean bench .

Raji cells were cultured in RPMI1640 medium (Biological Industries, Israel) with 10% fetal bovine serum (FBS, ExCell Bio, China) and 1% penicillin-streptomycin (P/S, Beyotime,Industries, Israel).The cells were incubated at 37°C in 5% CO2. All operations were conducted inside the vertical flow clean bench.

p^Hvalue detection

The membrane-permeable fluorescence dye Calbryte™ 520 AM (AAT Bioquest, USA) pH-sensitive fluorescent probe 2’,7’-bis-(2-carboxyethyl)-5-carboxyfluorescein (BCECF AM, Beyotime, China) was used to assess intracellular p^H value. The cells were cultured in 96-well plates and exposed to 5 mT, 20Hz magnetic field for 2h on the first two days. On the third day, the cells were resuspended in 100ul of 25uM BCECF AM in 0.04%Pluronic® F-127 working solution and incubated in CO₂ incubator at 37℃ for 1h. The supernatant was replaced with HHBS buffer (AAT Bioquest, USA), and the cells were subjected to magnetic field exposure for 2h. The supernatant was then replaced with PBS, and the p^Hvalues were determined using FITC flow cytometry (FACS Calibur, BD).

Intracellular calcium concentration assays

The membrane-permeable fluorescence dye Calbryte™ 520 AM (AAT Bioquest, USA) was used to assess intracellular calcium concentration. The cells were cultured in 96-well plates and exposed to 5 mT, 20Hz magnetic field for 2h on the first two days. On the third day, the cells were resuspended in 100ul of 5uM Calbryte™ 520 AM in 0.04%Pluronic® F-127 working solution and incubated in CO₂incubator at 37℃ for 1h. The supernatant was replaced with HHBS buffer (AAT Bioquest, USA), and the cells were subjected to magnetic field exposure for 2h. The supernatant was then replaced with PBS, and the intracellular calcium concentrationvalues were determined using FITC flow cytometry (FACS Calibur, BD).

Determination of membrane potential of cells

Membrane potential was determined using the potential-sensitive fluorescence dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC4 (3) (AAT Bioquest, USA). In depolarized cells with high membrane potential, the fluorescent dye DiBAC4 (3) permeates into the cells, leading to intracellular fluorescence intensity increase. In hyperpolarized cells DiBAC4(3) discharges from the cells, and intracellular fluorescence intensity decreases. The cells were cultured in 96-well plates and exposed for 2h in a 5mT, 20Hz magnetic field for the first 2 days. The cells were cultured in 100ul HHBS. On the third day, the cells were resuspended in 100ul of 10uM DIBAC4 (3) AM in 0.04%Pluronic® F-127 working solution and incubated in CO₂ incubator at 37℃ for 1h. The supernatant was replaced with HHBS buffer (AAT Bioquest, USA) and the cells were subjected to magnetic field exposure for 2h. The supernatant was then replaced with PBS, and the cell membrane potentials were determined using FITC flow cytometry (FACS Calibur, BD).

Sodium ion detection

The cells were cultured in 96-well plates and exposed for 2h in a 5mT, 20Hz magnetic field for the first 2 days. On the third day, the medium was replaced with 100ul HHBS and 100ul 10uM SBFI AM (Cayman Chemical,USA) in 0.04%Pluronic® F-127 working solution. After 4h incubation in CO₂incubator at 37 ° C, the supernatant was replaced with HHBS buffer (AAT Bioquest, USA), and the cells were subjected to magnetic field exposure for 2h. Sodium ion determination was conducted at 330/80 excitation and emission of 528/20 using a microplate tester.

Potassium ion detection

The cells were cultured in 96-well plates and exposed for 2h in a 5 mT, 20Hz magnetic field for the first 2 days. On the third day, the medium was replaced with 100ul HHBS and 100ul 10uM PBFI AM (Cayman Chemical, USA) in 0.04%Pluronic® F-127 working solution. After 4h incubation in CO₂incubator at 37 ° C, the supernatant was replaced with HHBS buffer (AAT Bioquest, USA), and the cells were subjected to magnetic field exposure for 2h. Potassium ion determination was conducted at 330/80 excitation and emission of 528/20 using a microplate tester.

Trypan blue dyeing

293T, Hepg2 and A549 cells in logarithmic growth phase were trypsin digested and harvested. Raji cells were collected directly. Following centrifugation, the cells were stained with the vital dye trypan blue. Live cells were counted under an inverted light microscope. Cell growth inhibition rates were determined and representative curves plotted. Inhibition rate (%) (number of cells in control group - number of cells in magnetic field group)/number of cells in control group *100%.

Statistical method

All data were expressed as mean ± S.E.M, and one-way ANOVA was used for comparison between multiple groups. Student’s t-test was used for comparison of the treated versus untreated groups, and P<0.05 was considered as significant different. Confirmatory results at P < 0.05 were obtained from repeating studies at least 2 times.

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Tumor-specific inhibition with magnetic field

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