Multimodal Imaging Techniques to Evaluate the Anticancer Effect of Cold Atmospheric Pressure Plasma

Abstract

Background: Skin cancer is the most frequent cancer worldwide and is divided into non-melanoma skin cancer, including basal cell carcinoma, as well as squamous cell carcinoma (SCC) and malignant melanoma (MM).

Methods: This study evaluates the effects of cold atmospheric pressure plasma (CAP) on SCC and MM in vivo, employing a comprehensive approach using multimodal imaging techniques. Longitudinal MR and PET/CT imaging were performed to determine the anatomic and metabolic tumour volume over three-weeks in vivo. Additionally, the formation of reactive species after CAP treatment was assessed by non-invasive chemiluminescence imaging of L-012. Histological analysis and immunohistochemical staining for Ki-67, ApopTag^®, F4/80, CAE, and CD31, as well as protein expression of PCNA, caspase-3 and cleaved-caspase-3, were performed to study proliferation, apoptosis, inflammation, and angiogenesis in CAP-treated tumours.

Results: As the main result, multimodal in vivo imaging revealed a substantial reduction in tumour growth and an increase in reactive species after CAP treatment, in comparison to untreated tumours. In contrast, neither the markers for apoptosis, nor the metabolic activity of both tumour entities was affected by CAP.

Conclusions: These findings propose CAP as a potential adjuvant therapy option to established standard therapies of skin cancer.

Keywords: kINPen™; malignant melanoma; plasma medicine; reactive oxygen and nitrogen species; skin cancer; squamous cell carcinoma.

Figure 1

Representative MR (T2 weighted, axial, week three) and PET/CT images ([¹⁸F]FDG; summed images, axial slices, week three) (A; SCC and B; MM) as well as histological and immunohistochemical images of H&E, Ki-67, ApopTag^® and CD31 staining (C; SCC and D; MM) for characterization of tumour biology. In NSG mice 1 × 10⁶ tumour cells of either squamous cell carcinoma (SCC) or malignant melanoma (MM) were injected s.c. into the left and right hind flank. Over the three-weeks observation time, anatomical tumour volumes were measured by MRI, and metabolic tumour volumes were determined by PET/CT. Measurements were performed one, two and three weeks after tumour cell implantation. After in vivo characterization, the tumours were excised and prepared for histological analysis. Asterisks mark central tumour necrosis. Arrows point to CD31-positive vessels.

Figure 2

Representative MR images (A,C; T2 weighted, axial) and quantitative assessment of tumour volumes (B,D). NSG mice with s.c. flank tumours, either squamous cell carcinoma (SCC) or malignant melanoma (MM), were treated with or without cold atmospheric pressure plasma (CAP) over three weeks. CAP treatment started four days after tumour cell injection and was repeated every four days. CAP treatment caused a significant reduction in both SCC and MM growth over the three-week observation time. Empty symbols represent untreated tumours, and filled symbols represent CAP-treated tumours. Data are presented as mean ± SD (n = 20–24 samples per time point); two-way ANOVA followed by Bonferroni correction for multiple comparison. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001 vs. untreated SCC or MM, respectively. Dotted line mark right tumour volume.

Figure 3

Representative PET/CT images from [¹⁸F]FDG (A,C; summed images, axial slices) and quantitative assessment of metabolic tumour volumes (B,D). NSG mice with two s.c. flank tumours, either squamous cell carcinoma (SCC) or malignant melanoma (MM), were treated with or without cold atmospheric pressure plasma (CAP) over three weeks. CAP treatment started four days after tumour cell injection and was repeated every four days. Data are presented as mean ± SD (n = 6–20 samples per time point); mixed-effects analysis, followed by Bonferroni correction. Large, dotted line mark manually placed volume of interest. Short, dotted line mark right metabolic tumour volume.

Figure 4

Representative luminescence images (A,C; luminescence probe: L-012) and quantitative assessment of relative luminescence intensity (B,D). NSG mice with two s.c. flank tumours, either squamous cell carcinoma (SCC) or malignant melanoma (MM), were treated with or without cold atmospheric pressure plasma (CAP) over three weeks. CAP treatment started four days after tumour cell injection and was repeated every four days. L-012 was injected i.p. and the luminescence intensity was measured before and after CAP treatment. The measurement was performed two hours before and immediately after the CAP treatment. Data are presented as mean ± SD (n = 10–15); one-way ANOVA followed by Tukey’s multiple comparison. *** p ≤ 0.001 vs. untreated SCC or MM, respectively or before CAP treatment of SCC or MM, respectively.

Figure 5

Quantitative immunohistochemical assessment of Ki-67, ApopTag^®, CD31, CAE and F4/80 expression (A,B) as well as representative images (C,D) and quantitative western blot analysis of PCNA, caspase-3 and cleaved-caspase-3 (E,F) in SCC and MM, respectively. NSG mice with two s.c. flank tumours, either squamous cell carcinoma (SCC) or malignant melanoma (MM), were treated with or without cold atmospheric pressure plasma (CAP) over three weeks. CAP treatment started four days after tumour cell injection and was repeated every four days. After three weeks, tumours were excised and prepared for immunohistochemical and molecular biological analysis. Data are presented as mean ± SD (immunohistochemistry: n = 8–14; molecular biology: n = 5–8); unpaired two-tailed t-test. ** p ≤ 0.01 vs. untreated SCC.