2.1.

Chemical Carcinogenesis

For our study, we chose a well-established in vivo model of chemically induced skin tumor on mice.¹⁸ We used 50 Swiss breed female mice, aged from 8 to 10 weeks, with a weight of 20 g. The animals were anesthetized with ketamine ($0.35\mathrm{ml}/\mathrm{kg}$) and xylazine ($0.20\mathrm{ml}/\mathrm{kg}$) during all stages of the protocol, which was approved by the ethics committee for research on animals of IPEN (No. 71/10-CEUA-IPEN/SP).

The mice were divided into four groups (Table 1). The chemical carcinogenesis consisted of two stages. The initiation phase was a topical application of 50-mg 7,12-dimethyl benzanthracene (DMBA) diluted in 100 mL of acetone on the shaved backs of the mice. The promotion phase begins 1 week after and consists of a biweekly application of 5 g of 12-O-tetradecanoyl-phorbol-13 acetate (TPA) diluted in 200 ml of acetone for 28 weeks. After 28 weeks, the animals obtained visible single or multiple tumor nodules with verrucous aspect (Fig. 1). The control group only received a topical application of acetone.

Table 1

Mice groups.

Fig. 1

Macroscopic view of the skin lesions.

A standard protocol was established to photograph the induced lesions at the different stages of the treatment in order to precisely follow their size evolution.

Considering the histopathological evaluation as the gold standard, we analyzed the histological profiles of healthy and tumorous mouse skin. We performed excisional biopsies of each papilomatous lesion with a size of about 0.5 to 1.0 cm. The collection was done immediately before PDT, at 10 days, and at 20 days after PDT. The histopathological analysis was performed by a pathologist. We can clearly see the differences between both tissues on Fig. 2. Healthy skin epidermis has three to four cell layers and a thin layer of keratin. The dermis is composed of dense connective tissue, clear hair follicles, and organized hypodermis. Neoplastic lesions show an intense proliferation of keratinocytes in an exophytic pattern [Fig. 2(b)]. The basal layer epithelium displays a moderate dysplasia and hyperchromatism. Hyperkeratosis and papillary projections were frequently observed. Twenty-five rats exhibited several lesions with a histological pattern clearly indicating SCC, with invasion of the neoplastic cells into the dermis, intense collagen deposition, and numerous blood vessels. In the remaining cases, neoplastic invasion was not evident, but the lesions were considered as potentially malignant tumors due to the dysplastic pattern present in all the cases. The lesions’ thicknesses vary from 200 to $500\mu \mathrm{m}$. The majority exhibited invasion in a depth restricted up to $300\mu \mathrm{m}$.

Fig. 2

Light microscopy of representative histological sections. (a) Healthy skin of control group, (b) neoplastic lesion with exophytic pattern e and intense keratinization k. Arrow—normal epithelium; head arrow—hair follicle; d—dermis; h—hypodermis (hematoxylin and eosin, original magnification with $4\times$ objective).

2.2.

Photodynamic Therapy

For PDT, we used homemade PS ointments. The base is prepared with lanolin and petrolatum, the active principle being either aminolevulinic acid (ALA) (20%) or aminolevulinic methyl ester (MEALA) (10%), and the other ingredients are kept confidential (patent pending PI N°0705591-9).¹⁹ The ointment was applied on the tumor lesions with a 5-mm additional margin, for each 30 min during a 5 h period, and the excess was removed with a gauze before PDT. Light irradiation was performed with a prototype of cluster of LEDs at 630 nm (power 180 mW, power density $5\mathrm{mW}/{\mathrm{cm}}^2$). We performed one session of PDT during which the lesions were irradiated for 40 min with an energy density of $12\mathrm{J}/{\mathrm{cm}}^2$.

Table 1 shows the different treatments applied to the four mice groups. Only 23 animals were further evaluated in our study due to a high death rate of 54%. This death rate is expected due to the toxicity of DMBA/TPA and is in agreement with other studies.¹⁸

2.3.

Optical Coherence Tomography Imaging

Analyses by optical coherence tomography were performed immediately before the PDT and at 10 and 20 days after PDT. OCT is a real-time technique so that the whole imaging protocol lasted less than 30 min. The commercial OCT system used in this study is an OCP930SR (Thorlabs Inc., USA; Fig. 3). It has a superluminescent diode source with a central wavelength of 930 nm and a bandwidth of 100 nm. Optical axial resolution was calculated at $4.38\mu \mathrm{m}$, after refraction index correction ($n=1.41$ at 930 nm), and a lateral resolution at $6.1\mu \mathrm{m}$. The images have dimensions of $1000\times 512\text{pixels}$, corresponding to a lateral field of 5.0 mm and a depth field of 1.5 mm.

Fig. 3

OCT imaging on anesthetized mouse.

2.4.

Optical Attenuation Coefficient Measurement

Two types of models exist for the OCT signal, depending on whether only single scattering or multiple scattering is considered.²⁰ In our case, we used a simple model of exponential decay of the backscattered light, according to the following equation:

Analysis was performed through a LabView software developed in our laboratory. The decay profile of each A-line $I(z)$ is fitted by an exponential curve to obtain a local estimation of the OAC. A global OAC for each image is obtained as the arithmetic mean over all selected A-lines. Indeed, the program allows selecting a region of interest in the lateral and axial dimensions, facilitating the location of tumoral epithelium and dermis and the exclusion of objects such as the hair follicles that could interfere with the optical signal.

3.1.

Clinical Evaluation of PDT Through Macroscopic Images and Histopathological Analysis

Clinical signs of the PDT effect were observed at 24 h after the treatment. The treated lesions exhibited a superficial necrosis and/or crust. At 10 and 20 days, the lesions’ sizes had diminished and the superficial crust had disappeared. The effect of PDT treatment on groups G3 and G4 was macroscopically assessed by the evolution of the area of each lesions, measured using ImageJ software. Figure 4 shows the frequency of the lesions depending on their percentage of area reduction. After 20 days, all lesions showed a positive response to PDT, ranking from a slight decrease (25%) to total disappearance (100%).

Fig. 4

Evolution of the area of each lesion. Number of lesions in each class (area reduction between 0% and 24%, 25% and 49%, 50% and 74%, or 75% and 100%) for G3 and G4, 10 and 20 days after PDT.

Globally, we can see a decrease in the total area of the lesions after PDT (Fig. 5). Normalization and variance homogenization of the data were performed using the Shapiro–Wilk and Bartlett’s test at a level of significance of 5%. Then statistical analysis of variance (ANOVA, 5%) and comparison of G3 and G4 using the Tukey–Kramer test (5%) demonstrated that there is no significant difference between G3 and G4 in any of the periods. The larger error bar in group G3 at day 20 after PDT is due to the loss of an animal during this time interval due to the aggressiveness of the tumor, which reduced the total number of evaluated lesions and might have influenced the outcome for group G3.

Fig. 5

Evolution of the total area of the neoplastic lesions for G3 (ALA-mediated PDT) and G4 (MEALA-mediated PDT), 10 and 20 days after PDT.

Representative samples from groups G3 and G4 were further subjected to histopathological analysis (Fig. 6). After 10 days of PDT, in G3 there was an evident reduction of the epithelium thickness [Fig. 6(a)], and in G4, ulceration and extensive epithelial necrosis were present [Fig. 6(c)]. At 20 days post-PDT, the papillary pattern was not visible in G3 [Fig. 6(b)] and the connective tissue exhibited intense reparative process. In G4, in the same experimental period, numerous fibroblasts and marked extracellular matrix deposition were observed accompanied with reduction of the epithelium thickness [Fig. 6(d)]. In some cases of G3 and G4 (about 15%), there was no sign of neoplastic cells. Total reepithelization and remodeling of the epithelial layers were evident. Fibrous connective tissue and regeneration of the hair follicles were observed in the dermis.

Fig. 6

Light microscopy of representative histological sections. (a) Reduction of epithelium thickness e at 10 days after PDT in G3 when compared with the initial aspect observed at Fig. 2(b). (b) Reduction of epithelium thickness e is marked at 20 days after PDT. The dermis d exhibits intense collagenization and hair follicles (arrow) are in regenerative process. (c) Ulceration u of epithelium e and necrosis of hair follicles (arrows) at 10 days after PDT in G4. (d) After 20 days in this group, reepithelization, reduction of exophytic pattern of the tumor e, and increase on cellularity and collagenization in dermis d are evident, indicating an advanced reparative process (hematoxylin and eosin, original magnification with $10\times$ objective).

Based on these clinical and histopathological evaluations, we can conclude that there is a significant reduction in neoplastic lesions after 20 days of PDT treatment.

3.2.

Optical Coherence Tomography Imaging

For each study group, we obtained OCT images and OAC estimation. Figure 7 shows the representative OCT images of healthy and neoplastic skins. The red arrows point to epithelium which is thin in healthy skin and becomes thick with cells in neoplastic tissue, suggesting epithelial hyperplasia.

Fig. 7

OCT images on skin of (a) healthy and (b) neoplastic tissues. The red arrows indicate the epithelium, which is thin in (a) and thick in (b). Scale bar $500\mu \mathrm{m}$.

3.3.

Optical Attenuation Coefficient Evolution

To evaluate the efficacy of PDT, we chose the OAC of healthy skin as the standard to achieve: the closer the OAC is to of that of healthy skin, the better the effect of PDT is.

The OAC measurement using the whole depth of OCT images, from 0 to 1 mm, did not show any difference between healthy and neoplastic tissues. This is due to the fact that the signal from this region is strongly influenced by deeper regions of the hypodermis which are not affected by tumor. Thus, we chose to analyze the data of the OCT images at restricted depths from 10 to $300\mu \mathrm{m}$. This depth range ignores the contribution of non-neoplastic signal and is the representative of changes associated with neoplastic lesions. It is also consistent with the depth of SCC lesions evaluated between 200 and $500\mu \mathrm{m}$ by histology.

Figure 8 shows the mean OAC of groups G2, G3, and G4, normalized by the OAC of control group G1.

Fig. 8

Optical attenuation coefficient (OAC) calculated for depths from 10 to $300\mu \mathrm{m}$. G1: normal skin; G2: skin with neoplastic lesions; G3: neoplastic lesions treated with ALA-mediated PDT; G4: neoplastic lesions treated with MEALA-induced PDT.

As before, normalization and variance homogenization of the data were performed using the Shapiro-Wilk and Bartlett’s test (5%), then statistical ANOVA (5%) and comparison using the Tukey–Kramer test (5%) demonstrated that there is significant difference between the groups.

The OAC of neoplastic tissue (G2) is greater than the OAC of healthy skin (G1) by a factor of around 1.4. The OAC of neoplastic tissue in G3 and G4 subjected to PDT tends to approach that of healthy tissue at 10 days after treatment, showing a positive response to treatment.

The OAC of G3 increases from 10 to 20 days after PDT with statistical significance, which is not in agreement with the clinical outcome. This increase is also observed for G4, but without statistical significance. This can be because the OAC measurement was influenced by the backscattering signal from hair follicles. The histological slides at day 20 showed the regeneration of hair follicles, which is greater in group G3 than G4 [Figs. 6(b) and 6(d)]. Group G4 presents fewer hair follicles due to more intense fibrosis that develops in the scar tissue of the dermis. This fibrosis could also be responsible for an increase in the OAC of group G4.