PART 2. Effects of extremely low frequency electromagnetic fields on the tumor cell inhibition and the possible mechanism
Spontaneous aggregation of suspended tumor cells was retained, and magnetic inhibition
appeared. In the earlier experiment on Raji lymphoma cells, the spontaneous aggregation of suspended
tumor cells was destroyed and magnetic inhibition disappeared. We demonstrated that the 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 the effect of magnetic inhibition on tumor growth. Aggregated Raji cells
in clusters were studied in a 9-day continuous-exposure experiment. The cells were transferred to a larger con
tainer, while the culture medium was changed every 3 days. "Agglomerate" group: No procedures were per
formed except for medium replacement every 3 days and transfer to larger containers. "Dispersed" group: Prior
to daily exposure, the cells were blown with a pipette gun to separate the cells in suspension and destroy the
agglomerative structure.
The initial number of all cells was 2× 105 . Figure 3a shows that the number of Raji cells under suspension
aggregation in the magnetic field was significantly lower than that in the unexposed control group, whereas
the number of Raji cells under suspension separation in the magnetic field showed no significant differences
compared with the unexposed control. Figure 3b shows the difference in the magnetic field inhibition rate
Figure 2. Cell growth curve of freely suspended Raji tumor cells in the infusion and change groups. (a) Cell
inhibition curves under different degrees of the freshness of the culture medium. Under the condition of cell
dispersion, no significant difference was found between the number of cells exposed to the magnetic field and
those in the unexposed control group. (b) Inhibition rate curves under the condition of cell dispersion; the
tumor cells were not inhibited on magnetic field exposure under different degrees of the freshness of the culture
medium. (c) Spontaneous cell clustering (right) and artificial separation (left).
between the two groups of cells (suspension aggregation and suspension separation). It indicated that Raji cells
were inhibited by the magnetic field under the condition of suspension aggregation. Meanwhile, Raji cells were
not inhibited by the magnetic field under the suspension separation condition. Raji cells were less sensitive to
magnetic inhibition than adherent cells, and the inhibition rate of Raji cells on day 6 was similar to that of A549
cells on day 3. In the absence of the magnetic field, the number of Raji cells in the suspended aggregation group
was significantly higher than that in the suspended separation group. In contrast, on exposure to the magnetic
field, the number of Raji cells in the suspended separation group was significantly higher than that in the sus
pended aggregation group. However, on day 9, the magnetic inhibition decreased or disappeared. We repeated
the experiment to determine the reasons for the disappearance of the inhibition rate on day 9. We took the area
at the bottom of the container as the control to investigate whether the differences in cell cluster density caused
the reduction of magnetic inhibition (less contact between cells), and exposed the cells in the clustered state
to a 5-mT, 20 Hz magnetic field for 9 days. The conditioned medium was changed every 3 days, and the large
container was replaced. The operation remained unchanged for the first 6 days. On the sixth day of transfer, the
cells were divided into containers with different base sizes [10-cm Petri (55-cm2 ) dish and 25-cm2 culture flask].
The only change was that the cells were transferred on day 6, using the 10-cm Petri dish (55 cm2 ) and 25-cm2
flask (under different basal areas, the cell masses were more concentrated) as controls. Surprisingly, the inhibition
rate of Raji cells in the 25-cm2 flask was as high as 36% on day 9. The inhibition of Raji cells in the 10-cm Petri
dish (55 cm2 ) disappeared on day 9 (Fig. 3c). The results were consistent with previous findings. The reason for
this result should be related to the closeness of the cells (Fig. 3d).
Figure 3. Agglomerate and dispersed inhibition of suspended Raji tumor cells. (a) Growth curve of suspended
cells with different contact structures showed that the number of cells in the group exposed to the magnetic
field was significantly different from that in the group not exposed to the magnetic field under the condition of
cell aggregation. However, no significant difference was found between the group exposed to the magnetic field
and the group not exposed to the magnetic field under the condition of cell dispersion. (b) Structure difference
inhibition curve: The clustered cells had significant inhibition, but the dispersed cells had no inhibition. (c) On
day 9, under the same volume and different basal areas, the number of cells in the group with smaller basal areas
significantly reduced (* P<0.05, vs the no-exposure control group; * P<0.05, vs the group with different bottle
area) (d) As shown in the figure, the cells clustered more closely in the culture flask, while the cells in the Petri
dish were scattered at the bottom due to the low liquid level. (e) Differential growth curve of the bottom area of
the culture vessel on day 9.
Membrane potential was related to magnetic field inhibition. The experiments on suspended
tumor cells showed that the inhibitory effect of magnetic field on cancer cells was accomplished through contact
and communication between cells, having implications for signal transmission between cells and ionic changes
in the cell microenvironment. Calcium, sodium, potassium, and pH kits Calbryte 520 AM, SBFI AM, PBFI AM,
and BCECF AM were used to observe the changes in intracellular free ions of four kinds of cells after 3 days of
magnetic exposure so as to investigate whether the magnetic field suppression was associated with ionic signal
ing. No changes in intracellular sodium and potassium ion concentrations were observed in normal or tumor
cells (Fig. 4a and b). Also, no significant difference was found in pH fluorescence intensity in all groups of cells
except A549 (Fig. 4c). Normal 293 T cells showed a significant decrease in the intracellular free calcium ion con
centration. The solid tumor cells showed no significant change, while the suspended tumor cells showed a slight
increase in the calcium ion concentration (Fig. 4d).
A change in the calcium ionic concentration is usually reflected by a change in membrane potential. The
membrane potential kit DiBAC4 (3) was used to observe the exposed cells on day 3 with ΔIIF correspond
ing to ration of the intracellular ion fluorescence for Day 3 and initially. The adherent cells showed significant
hyperpolarization (ΔIIF 293 T cells: −25%, ΔIIF Hepg2 cells: −20% and ΔIIF A549 cells: −13%). The tumor cell
agglomerates showed significant depolarization (ΔIIF Raji cells:+9%). The free suspended tumor cells showed
no significant depolarization (Fig. 4e). Figures S1–S3 show flow cytometry data.
Figure 4. Differences in ionic strength and membrane potential of cells under 3-day 5-mT 20-Hz magnetic
field exposure. (a) No significant difference was found in the fluorescence intensity of sodium ions after 3 days
of exposure. (b) No significant difference was found in the fluorescence intensity of potassium ions after sodium
ion exposure for 3 days. (c) After 3 days of exposure, no significant difference was found in the fluorescence
intensity in all groups of cells except A549. (d) Calcium ion concentration in the 293 T cells decreased, while
no significant difference in the calcium ion concentration was observed in the other groups. (e) Adherent cells
showed significant hyperpolarization, tumor cell agglomerates showed significant depolarization, and the freely
suspended tumor cells showed no significant depolarization. These changes corresponded to the changes in
intracellular calcium ion concentrations (* P<0.05, vs the no-exposure control group).
Cells secreted substances in the conditioned medium, which interacted with the magnetic
field to inhibit tumor cells; the substance had universal expression and was tumor spe-
cific. A549 and Raji cells in agglomerates were exposed independently to the 5-mT magnetic field for 3 h to
determine whether the magnetic field inhibition was related to a change in the conditioned medium in the state
of cell aggregation. The conditioned medium was then transferred to feed unexposed cells of the same species for
2 days. The number of cells in the A549 "transfer" group was 4× 105 . The number of cells in the Raji "transfer"
group was 8× 105 . The number of cells in the A549 "be transferred" group was 2× 105 . The number of cells in
the Raji "be transferred" group was 4× 105 . Under these conditions, the cells in the A549 "be transferred" group
were significantly inhibited at a rate of approximately 10%, which was nearly half of that of the cells with direct
exposure on day 1 in the fluid infusion group. The cells in the Raji "be transferred" group showed no inhibition,
but the cells in the transfer groups were inhibited compared with those in the unexposed control group (Fig. 5a
and b).
In addition, A549 and 293 T cells were exposed to the 5-mT magnetic field for 3 h to determine whether this
conditioned medium was unique to tumor cells and inhibited normal cells. The exposed culture media were then
transferred to feed unexposed cells of the different species for 2 days. The results showed that A549 cells were
significantly inhibited in 293 T culture media. The cells in the 293 T transfer group showed no inhibition, but the
cells in all transfer groups were inhibited compared with those in the unexposed control group (Fig. 5c and d).
Figure 5. Conditioned medium inhibited tumor cell growth. (a) After A549 cells were exposed to the magnetic
field, the conditioned medium was filtered and replaced with the unexposed conditioned medium. The
exposed conditioned medium A549 had an inhibitory effect on the unexposed A549 cells. (b) After Raji cells
were exposed to the magnetic field, the conditioned medium was filtered and replaced with the unexposed
conditioned medium. No difference was found in the number of cells between the change groups, but the
number of cells in the change group was significantly lower than that in the untreated control group. (c) Number
of A549 cells transferred from the exposed 293 T cell conditioned medium significantly reduced compared with
that from the untreated control and unexposed conditioned medium. (d) No significant difference was found
in the number of 293 T cells in the conditioned medium of A549 cells after exposure compared with that in the
untreated and unexposed groups (* P<0.05, vs the no-exposure control group; * P<0.05,vs the no-transfer control
group).
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