Tissue tolerable plasma (TTP) induces apoptosis in pancreatic cancer cells in vitro and in vivo

Abstract

Background: The rate of microscopic incomplete resections of gastrointestinal cancers including pancreatic cancer has not changed considerably over the past years. Future intra-operative applications of tissue tolerable plasmas (TTP) could help to address this problem. Plasma is generated by feeding energy, like electrical discharges, to gases. The development of non-thermal atmospheric plasmas displaying spectra of temperature within or just above physiological ranges allows biological or medical applications of plasmas.

Methods: We have investigated the effects of tissue tolerable plasmas (TTP) on the human pancreatic cancer cell line Colo-357 and PaTu8988T and the murine cell line 6606PDA in vitro (Annexin-V-FITC/DAPI-Assay and propidium iodide DNA staining assay) as well as in the in vivo tumour chorio-allantoic membrane (TUM-CAM) assay using Colo-357.

Results: TTP of 20 seconds (s) induced a mild elevation of an experimental surface temperature of 23.7 degree Celsius up to 26.63+/-0.40 degree Celsius. In vitro TTP significantly (p=0.0003) decreased cell viability showing the strongest effects after 20s TTP. Also, TTP effects increased over time levelling off after 72 hours (30.1+/-4.4% of dead cells (untreated control) versus 78.0+/-9.6% (20s TTP)). However, analyzing these cells for apoptosis 10s TTP revealed the largest proportion of apoptotic cells (34.8+/-7.2%, p=0.0009 versus 12.3+/-6.6%, 20s TTP) suggesting non-apoptotic cell death in the majority of cells after 20s TTP. Using solid Colo-357 tumours in the TUM-CAM model TUNEL-staining showed TTP-induced apoptosis up to a depth of tissue penetration (DETiP) of 48.8+/-12.3μm (20s TTP, p<0.0001). This was mirrored by a significant (p<0.0001) reduction of Ki-67+ proliferating cells (80.9+/-13.2% versus 37.7+/-14.6%, p<0.0001) in the top cell layers as well as typical changes on HE specimens. The bottom cell layers were not affected by TTP.

Conclusions: Our data suggest possible future intra-operative applications of TTP to reduce microscopic residual disease in pancreatic cancer resections. Further promising applications include other malignancies (central liver/lung tumours) as well as synergistic effects combining TTP with chemotherapies. Yet, adaptations of plasma sources as well as of the composition of effective components of TTP are required to optimize their synergistic apoptotic actions.

Figure 1

Plasma jet kINPen09 (Neoplas GmbH, Greifswald, Germany) - application of TTP in vitro.

Figure 2

TUM-CAM (Colo-357) before and 48h after 10s TTP treatment (asterisk: tumour; black arrow: supplying blood vessel; white arrow: silicone ring).

Figure 3

Plasma induced cell death in pancreatic cancer cells in vitro as shown by Annexin-V-FITC/DAPI-staining A: after 1h; B: after 24 h; C: after 48h; D: 72 h after TTP treatment.

Figure 4

Histology using HE-, Ki-67-, and TUNEL-staining of the TUM-CAM model after plasma treatment. Plasma treatment of 10 seconds and 20 seconds (not shown) led to cell death in the upper 3–5 cell layers (above the dotted line), accompanied by a complete absence of viable proliferating cells (Ki-67-staining) and a strong increase in apoptotic activity (TUNEL staining). However, the bottom cell layers were not affected. One mitotic figure (arrow) and a spontaneous apoptotic body (arrowhead) of the unaffected deep layers of a plasma treated tumour as well as an untreated control tumour were marked. The lower edge of HE- and Ki-67-stains measure 250μm (original magnification 400x). Original magnification of TUNEL staining: 200x.

Figure 5

Impact of TTP in the TUM-CAM model on tumour proliferation and apoptosis A: Using the proliferation marker Ki-67 there were not significant differences in proliferation of total tumour specimens (Figure5A, p= 0.9355).B: Analyzing the upper 100 μm of tumour tissue argon gas had no significant effect on proliferation. 10s TTP highly significantly decreased proliferation (p< 0.0001), with 20s TTP showing no additional effects (10s versus 20s p= 0.9359). C: TTP induced highly significant apoptosis after 10 seconds (p< 0.0001), and after 20 seconds (p= 0.0006) in the upper 100 μm of the tumour. D: Depth of effective tissue penetration (DETiP) of 20s TTP was up to about 60μm (p<0.0001), with 10s TTP not being significantly different (p=0.1428).