Currently ablative fractional photothermolysis (aFP) with CO2 laser is used for a wide variety of dermatological indications. This study presents and discusses the utility of aFP for treating oncological indications. We used a fractional CO2 laser and anti-PD-1 inhibitor to treat a tumor established unilaterally by the CT26 wild type (CT26WT) colon carcinoma cell line. Inoculated tumors grew significantly slower in aFP-treated groups (aFP and aFP + anti-PD-1 groups) and complete remission was observed in the aFP-treated groups. Flow cytometric analysis showed aFP treatment elicited an increase of CD3+, CD4+, CD8+ vand epitope specific CD8+ T cells. Moreover, the ratio of CD8+ T cells to Treg increased in the aFP-treated groups. Additionally, we established a bilateral CT26WT-inoculated mouse model, treating tumors on one-side and observing both tumors. Interestingly, tumors grew significantly slower in the aFP + anti-PD-1 groups and complete remission was observed for tumors on both aFP-treated and untreated sides. This study has demonstrated a potential role of aFP treatments in oncology.
Fractional photothermolysis (FP) can be characterized as laser-assisted treatment generating a pattern of microscopic treatment zones (MTZs) in biological tissue1. There are two FP modes, the non-ablative mode (nFP) and ablative (aFP) mode. nFP generates MTZs as small zones of thermally damaged tissue, whereas aFP additionally produces a central “hole” of physically-removed (ablated) tissue, surrounded by a small cuff of thermally-damaged tissue in MTZs2,3. In general, the width or diameter of the MTZs measures less than 0.5 mm. FP techniques typically expose only a small fraction of the tissue (often an areal fraction of approximately 5–27%), leaving the majority of tissue spared or unexposed4. A large range of dermatological indications, such as treatment of photodamaged skin, dyschromia, rhytides, and different kind of scars including acne, surgical and burn scars currently make use of FP1,5,6,7,8. However, FP is not used for the treatment of tumors and no studies to date have investigated production of systemic effects using FP methods.
Mroz et al. reported laser irradiation of photodynamic therapy (PDT) to treat mice subcutaneously inoculated with CT26.CL25 colon carcinoma cells inducing local remission and antigen-specific immune response systemically, promoting regression of a remote and untreated tumor9. However CT26.CL25 cells are artificially transduced with the lacZ gene to stably express a tumor antigen (beta-gal). One limitation of the study is that the tumor model used is not a clinically relevant tumor, and instead represents an artificially-induced cancerous state in the subject mice. The phenomenon promoting regression of a remote and untreated tumor was not observed when they employed CT26 wild type (CT26WT) undifferentiated colon carcinoma cells, which are beta-gal negative parental carcinoma of CT26.CL25 cells. This is surprising because many reports showed PDT could induce the immune response against locally inoculated CT26WT colon carcinoma cells10,11,12. These carcinoma cells are a clone of the N-nitoroso-N-methylurethan (NMU)-induced grade IV carcinoma, which is rapidly-growing and readily-metastasizing13. Therefore we decided to challenge and treat these poorly immunogenic CT26WT colon carcinoma cells with aFP and anti-PD-1 inhibitor, which boosts the function of CD8+ T cells by blocking the PD-1/ PD-L1 pathway14,15,16, to confirm if aFP can be used for induction of anti-tumor immunity and promote regression of a remote and untreated tumor in the clinical relevant situation, which is in marked contrast to the current applications of FP.
aFP laser irradiation was performed 8 days after tumor inoculation. The aFP significantly led to a reduction in tumor volume and growth rate of CT26WT tumors after the treatment (Fig. 1a). Tumors shrank in 6 mice of 11 mice in the aFP group and 8 mice of 11 mice in the aFP + anti-PD-1 group, but such shrinkage occurred in only 1 mouse (9%) from the anti-PD-1 group or does not occur in the control group (Fig. 1b). The significance value for the difference between the survival curves are: control vs. anti-PD-1 (p < 0.05), control vs. aFP (p < 0.0001), control vs. aFP + anti-PD-1 (p < 0.0001), anti-PD-1 vs. aFP (p < 0.005) and anti-PD-1 vs. aFP + anti-PD-1 (p < 0.0005).
Tumor Volume and Survival Curves after Treatment and Rechallenge Test in the One Tumor Mouse Model. Mice were inoculated unilaterally at the left leg with 3.5 × 105 CT26WT subcutaneously into the depilated thigh. aFP laser irradiation was performed 8 days after tumor inoculation, and then the growth of the tumors was observed. Anti-PD-1 blocking antibodies were administered intraperitoneally at a dose of 200 µg per mouse on days 8, 10, 12, 14, and 16 in the one tumor mouse model after tumor cell inoculation. (a) Tumor volume curves of mice in the control, anti-PD-1, aFP and aFP + anti-PD-1 groups after tumor inoculation. (b) Kaplan-Meier survival curves of mice receiving tumor inoculation. The significance values for the difference between the survival curves are: Control vs. anti-PD1: P < 0.05, Control vs. aFP: P < 0.0001, control vs. aFP + anti-PD1: P < 0.0001, anti-PD-1 vs. aFP: P < 0.05, anti-PD1 vs. aFP + anti-PD1: P < 0.0002. (c) Kaplan-Meier survival curves of mice receiving the rechallenge test with CT26WT cells. These mice were each inoculated with 3.5 × 105 CT26WT cells subcutaneously in the contralateral (right) thigh against previous inoculation. The significance values for the difference between the survival curves are: control mice vs. survival mice (p < 0.005), control vs. aFP + anti-PD1: P < 0.0001. *The bars represent SD. P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.
To assess the presence of any induced long term anti-tumor immunity, we performed a rechallenge experiment following the treatment. Six mice in the aFP group and eight mice in the aFP + anti-PD-1 group which survived for more than 90 days after the treatment, were subsequently inoculated subcutaneously with CT26WT cells in the contralateral (right) thigh. Eight age-matched naive mice were inoculated with the same number of CT26WT cells, respectively, in the right thigh as a control. The tumor on the naive mice in the control groups and one of the six mice in the aFP group progressed over time. Tumors on the rest of the survival mice in the aFP group and all survival mice in the aFP + anti- PD-1 group did not appear to progress (Fig. 1c). They remained tumor–free for at least another 60 days following the inoculation (Fig. 1c). The significance values for the differences between the survival curves are: control vs. aFP (p < 0.001), control vs. aFP + anti-PD-1 (p < 0.0001).
To investigate if the adaptive immune system was affected by aFP, we measured the number of CD3roup and all survival mice in, CD4roup and all survival mice in, and CD8+ T cells and Foxp3+ Tregs inside the tumor using flow cytometry 5 days after aFP treatment. We chose to measure on 5 days after aFP treatment because by this day the aFP-treated tumor in the aFP and aFP + anti-PD-1 groups showed significantly shrinkage. We found that CD3+, CD4+, CD8+ T cells increased per tumor weight in the aFP-treated groups (CD3/weight: control vs. aFP and control vs. aFP + anti-PD-1 (p < 0.005), anti-PD-1 vs. aFP and anti-PD-1 vs. aFP + anti-PD-1 (p < 0.05); CD4/weight: control vs. aFP, control vs. aFP + anti-PD-1, anti-PD-1 vs. aFP and anti-PD-1 vs. aFP + anti-PD-1 (p < 0.005); CD8/weight: control vs. aFP and control vs. aFP + anti-PD-1 (p < 0.005), anti-PD-1 vs. aFP and anti-PD-1 vs. aFP + anti-PD-1 (p < 0.05; Fig. 2a)). However, there is no significant difference regarding Treg in the all groups (Fig. 2a). Moreover, the ratio of CD8+ T cells compared to Treg also increased in the aFP-treated groups (control vs. aFP, control vs. aFP + anti-PD-1, anti-PD-1 vs. aFP and anti-PD-1 vs. aFP + anti-PD-1 (p < 0.005); Fig. 2b). Additionally, to investigate TAA specific CD8+ T cell numbers, we measured AH1 epitope specific CD8+ lymphocytes. We found that aFP led to a significant increase AH1 specific CD8+ T cells per tumor weight in the aFP-treated groups (control vs. aFP, control vs. aFP + anti-PD-1, anti-PD-1 vs. aFP and anti-PD-1 vs. aFP + anti-PD-1 (p < 0.005); Fig. 2c). However, the percentage of AH1 specific CD8+ T cells compared with total CD8+ T cells increased in only aFP + anti-PD-1 group (control vs. aFP + anti-PD-1 and anti-PD-1 vs. aFP + anti-PD-1 (p < 0.05); Fig. 2c).
Flow Cytometric Analysis for Tumor Infiltrating Lymphocytes 5 Days After aFP Treatment in the One Tumor Mouse Model. Flow cytometry analysis was performed to confirm whether CD3+ and CD8+ lymphocyte, and regulatory T cell (Treg) numbers were affected by aFP. (a) Proportion of CD3+, CD8+ and CD4+ T cells and Treg normalized to tumor weight. (b) Ratio of CD8+ T cells to Tregs (CD4+ Foxp3+). (c) Proportion of antigen specific CD8+ T cells normalized to tumor weight and the percentage of antigen specific CD8+ T cells normalized to total number of CD8+ T cells. The bars represent SD. * P < 0.05, **P < 0.005.
To investigate whether adaptive immunity is necessary to eradicate cancer cells after aFP, we repeated the experiment with anti-CD8 depletion antibody to ablate CD8+ T cells in the mouse. As shown in Fig. 3, treatment of aFP in absence of CD8+ T cells failed to prevent tumor growth and eventual euthanasia, while 4 out of 6 mice in control group, with no CD8 depletion, survived. (P < 0.005; Fig. 3). This observation indicates that adaptive immunity is necessary to eradicate cancer cells after aFP.
Tumor Survival Curves after aFP Treatment with Anti-CD8 Depletion Antibody. To investigate whether adaptive immunity is necessary to eradicate cancer cells after aFP, tumor inoculation was performed with anti-CD8 depletion antibody to ablate CD8+ T cells in the mouse in the one tumor model. Anti-CD8 depletion antibodies were administered intraperitoneally at a dose of 200 µg per mouse every 3 days from one day before tumor inoculation to removal of mice as endpoint. The graph shows Kaplan-Meier survival curves of mice receiving tumor inoculation and anti-CD8 depletion antibody. The significance values for the difference between the survival curves are: anti-CD8+ aFP vs. aFP: P < 0.005.
To investigate whether aFP treatment could induce systemic anti-tumor immunity, we established a two tumor mouse model, which has a tumor on both hind legs, with the aFP-treated tumor on the left leg and observed the growth of both tumors. aFP laser irradiation was performed 7 days after tumor inoculation. The aFP significantly led to a reduction in tumor volume and growth rate of aFP-treated tumors after the treatment (Fig. 4a and c). Regarding the untreated contralateral tumors, we found that the aFP + anti-PD-1 combination therapy led to a significant reduction in growth rate after the treatment (Fig. 4b and c). Moreover the untreated contralateral tumors shrank in 2 of the 6 mice in the aFP + anti-PD-1 group completely, but such shrinkage did not occur in the rest of the groups (Fig. 4c and d). The significance value for the difference between the survival curves are: control vs. aFP + anti-PD-1 (p < 0.005), anti-PD-1 vs. aFP + anti-PD-1 (p < 0.005), aFP vs. aFP + anti-PD-1 (p < 0.01).
Tumor Volume and Survival Curves after Treatment in the Two Tumor Mouse Model. To investigate whether aFP treatment could induce systemic anti-tumor immunity, we established a mouse model which has one tumor on each hind leg with the aFP-treated tumor on the left leg and observed the growth of both tumors. aFP laser irradiation was performed 7 days after tumor inoculation. Anti-PD-1 blocking antibodies were administered intraperitoneally at a dose of 200 µg per mouse on days 7, 9, 11, 13, and 15 after tumor cell inoculation. (a) Average of tumor volume curves on the treated legs of mice in the control, anti-PD-1, aFP and aFP + anti-PD-1 groups after tumor inoculation. (b) Average of tumor volume curves on the untreated contralateral legs of mice in the control, anti-PD-1, aFP and aFP + anti-PD-1 groups after tumor inoculation. (c) Individual tumor volume curves in the control, anti-PD-1, aFP and aFP + anti-PD-1 groups after tumor inoculation. (d) Kaplan-Meier survival curves of mice receiving tumor inoculation. The significance values for the difference between the survival curves are: Control vs. aFP + anti-PD1: P < 0.005, Anti-PD-1 vs. aFP + anti-PD1: P < 0.005, aFP vs. aFP + anti-PD1: P < 0.01. The bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.005.
aFP induces recruitment of CD3+, CD4+, CD8+ and epitope specific CD8+ T lymphocytes but increases infiltration of Treg into the untreated contralateral tumors in the two tumor mouse model
To investigate why the untreated contralateral tumor in the aFP group did not shrink even though epitope specific CD8+ T lymphocytes were developed in the aFP-treated tumor, we measured the number of CD3+, CD4+, CD8+, epitope specific CD8+ T cells, and Foxp3+ Tregs inside the untreated contralateral tumor using flow cytometry 12 days after aFP treatment. We chose to measure on 12 days after aFP treatment because on this day the untreated contralateral tumor in the aFP + anti-PD-1 group shrunk significantly (aFP-treated tumors in the two groups shrank within 5 days after aFP treatment). We found that CD4+, CD8+ and epitope specific CD8+ T cells increased per tumor weight in the aFP-treated groups (CD3/weight and CD8/weight: control vs. aFP, control vs. aFP + anti-PD-1 and anti-PD-1 vs. aFP + anti-PD-1 (p < 0.05); CD4/weight and epitope specific CD8+ T cells: control vs. aFP, control vs. aFP + anti-PD-1, anti-PD-1 vs. aFP and anti-PD-1 vs. aFP + anti-PD-1 (p < 0.05; Fig. 5a and c). Moreover the percentage of AH1 specific CD8+ T cells compared to total CD8+ T cells increased in the aFP-treated groups (control vs. aFP and control vs. aFP + anti-PD-1 (p <…