Endoscopic non-ablative fractional laser therapy in an orthotopic colon tumour model

Abstract

Colorectal cancer is one of the leading causes of cancer-related deaths. Although several therapeutic management strategies are available at the early colon cancer stages, such as endoscopic mucosal or submucosal dissection, associated complications often include bleeding or bowel perforations. As an alternative approach, we investigated endoscopic non-ablative fractional laser (eNAFL) irradiation as a minimally invasive therapeutic modality for the treatment of early-stage colorectal cancer. By implanting SL4-DsRed colon cancer cells into the colons of the C57BL/6 mice, we developed an orthotopic colon tumour mouse model and demonstrated the early-stage tumour growth delay following the eNAFL irradiation. Additionally, we evaluated the temperature changes in the eNAFL-irradiated area using numerical simulations, and induced inflammation using histological analysis. Our results indicate a minimal thermal damage confined to the irradiated spot, sparing the adjacent tissue and alteration in the tumour microenvironment. eNAFL irradiation may be clinically useful as a minimally invasive therapeutic intervention at the early stage of tumourigenesis. In future, an optimal eNAFL therapeutic dose should be determined, in order to increase the efficacy of this approach.

Figure 1

Development of the orthotopic mouse colon tumour model. (A) Endoscopic non-ablative fractional laser and endoscopic needle system. (B) Eight-inch needle (white arrow) was inserted thorough the instrument channel of endoscopic system. (C) Endoscopic implantation of colon cancer cells into the colonic wall using the needle (white arrow) in vivo. (D) Haematoxylin & eosin staining of the implanted cancer cells. Tumour cells (T) grew in the submucosal (SM) space. Mucosa (M), submucosa (SM, black arrow), and muscularis propria (MP, white arrow) are presented.

Figure 2

Therapeutic efficacy of the endoscopic non-ablative fractional laser (eNAFL) irradiation. (A) Endoscopic images of the orthotopic tumour models after eNAFL irradiation. Representative white light and fluorescence images of the control and eNAFL-irradiated mice are presented. (B) Blinded visual grading of the tumours in the orthotopic tumour model mice. eNAFL-irradiated group (n = 5) was shown to have lower average tumour grades than the control group (n = 4). *p < 0.05 (Mann-Whitney U test). All results are presented as mean ± standard deviation (SD).

Figure 3

Numerical simulation of the endoscopic non-ablative fractional laser (eNAFL) irradiation of a colon. (A) Left: 3D image of the tissue model used for numerical simulation. Right: Cross-cut image of the 3D tissue model with the α-α′ plane. (B) Temporal analysis of temperature changes in four representative spots. (C and D) Spatial temperature distribution during heating (C) and cooling (D) periods in the tissue model. (E–G) Temperature distributions at different time points during and after laser irradiation. Cross sections were made through the centre of the tissue model.

Figure 4

Histological changes induced by the endoscopic non-ablative fractional laser (eNAFL) irradiation in the normal mouse colons. Haematoxylin & eosin (H&E) staining images were obtained with at 100× (A,D,G), 200× (B,E,H), and 400 × (C,F,I) magnification. (A–C) Tissue images before the eNAFL irradiation, with the normal glandular structure. (D–F) Tissues at 6 h after eNAFL irradiation. Black arrows, damaged nuclei; red arrowheads, immune cell infiltration. (G–I) Tissue images at 48 h after eNAFL irradiation. Black arrows, diffusely erosive mucosal layer.

Figure 5

Histological changes in the orthotopic colon tumour mouse tissues after endoscopic non-ablative fractional laser (eNAFL) irradiation. Haematoxylin & eosin (H&E) staining images were obtained at 200× magnification. (A) Tumour tissue before the eNAFL irradiation. Black arrows, tumour cells located in the submucosal space; red arrowheads, immune cell infiltrations. (B) Colon tissue at 6 h after the eNAFL irradiation. Red arrowheads, many infiltrated immune cells; black arrows, tumour cells. (C) Colon tissue, at 48 h after the eNAFL irradiation. Black arrows, tumour cells invading mucosal tissue; red arrowheads, peripherally accumulated immune cells.

Figure 6

An endoscopic non-ablative fractional laser (eNAFL) system for the treatment of orthotopic mouse colon tumours. (A) Schematic illustration of the eNAFL system. (B) Images of the endoscope. (C) eNAFL delivering multimode fibre was inserted via the instrument channel of the endoscope. (D) Schematic representation of the experiment. Tumour-bearing mice were divided into the irradiated and control groups. eNAFL (70 mJ/spot) was applied in the irradiated group. Serial tumour volume was measured to estimate the therapeutic efficacy of the eNAFL irradiation for 1 week.