Fractional laser exposure induces neutrophil infiltration (N1 phenotype) into the tumor and stimulates systemic anti-tumor immune response

Background: Ablative fractional photothermolysis (aFP) using a CO2 laser generates multiple small diameter tissue lesions within the irradiation field. aFP is commonly used for a wide variety of dermatological indications, including treatment of photodamaged skin and dyschromia, drug delivery and modification of scars due to acne, surgical procedures and burns. In this study we explore the utility of aFP for treating oncological indications, including induction of local tumor regression and inducing anti-tumor immunity, which is in marked contrast to current indications of aFP. Methodology/Principal findings We used a fractional CO2 laser to treat a tumor established by BALB/c colon carcinoma cell line (CT26.CL25), which expressed a tumor antigen, beta-galactosidase (beta-gal). aFP treated tumors grew significantly slower as compared to untreated controls. Complete remission after a single aFP treatment was observed in 47% of the mice. All survival mice from the tumor inoculation rejected re-inoculation of the CT26.CL25 colon carcinoma cells and moreover 80% of the survival mice rejected CT26 wild type colon carcinoma cells, which are parental cells of CT26.CL25 cells. Histologic section of the FP-treated tumors showed infiltrating neutrophil in the tumor early after aFP treatment. Flow cytometric analysis of tumor-infiltrating lymphocytes showed aFP treatment abrogated the increase in regulatory T lymphocyte (Treg), which suppresses anti-tumor immunity and elicited the expansion of epitope-specific CD8+ T lymphocytes, which were required to mediate the tumor-suppressing effect of aFP. Conclusion: We have demonstrated that aFP is able to induce a systemic anti-tumor adaptive immunity preventing tumor recurrence in a murine colon carcinoma in a mouse model. This study demonstrates a potential role of aFP treatments in oncology and further studies should be performed

Cellular and Vascular effects of the photodynamic agent temocene are modulated by the delivery vehicle

The effects of the drug delivery system on the PDT activity, localization, and tumor accumulation of the novel photosensitizer temocene (the porphycene analogue of temoporfin or m-tetrahydroxyphenyl chlorin) were investigated against the P815 tumor, both in vitro and in DBA/2 tumor bearing mice. Temocene was administered either free (dissolved in PEG400/EtOH mixture), or encapsulated in Cremophor EL micelles or in DPPC/ DMPG liposomes, chosen as model delivery vehicles. The maximum cell accumulation and photodynamic activity in vitro was achieved with the free photosensitizer, while temocene in Cremophor micelles hardly entered the cells. Notwithstanding, the micellar formulation showed the best in vivo response when used in a vascular regimen (short drug light interval), whereas liposomes were found to be an efficient drug delivery system for a tumor cell targeting strategy (long drug-light interval). PEG/EtOH formulation was discarded for further in vivo experiments as it provoked lethal toxic effects caused by photosensitizer aggregation. These results demonstrate that drug delivery systems modulate the vascular and cellular outcomes of photodynamic treatments with temocene. © 2012 Elsevier B.V. All rights reserved

Fractional Laser Releases Tumor-Associated Antigens in Poorly Immunogenic Tumor and Induces Systemic Immunity

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

Ablative fractional photothermolysis.

<p>(A) photo of skin surface appearance immediately after ablative fractional photothermolysis (100 mJ pulse energy). Black arrow indicates one of the laser-generated holes formed by tissue vaporization/ablation. There is an absence of graying or blistering immediately after laser exposure, which also suggests an absence of major thermal injury. (B) H&E-stained CT26.CL25 tumor immediately after the ablative fractional photothermolysis (aFP) laser treatment with a pulse energy of 100mJ. White arrows indicate an ablated hole which is characteristic of aFP procedures. The ablated hole appeared to be collapsed and distorted within the tumor tissue. (C) CT26.CL25 tumor immediately after aFP, stained by NBTC staining that shows vital cells as a blue color. White arrows indicate dead cells caused by physical effects of the laser treatment. Most of the tissue within the aFP volumes exhibited a blue staining, indicating an absence of widespread thermal injury or tissue bulk heating.</p

Immunohistochemical staining for apoptotic tumor cells.

<p>(A and B) Immunohistochemical staining for apoptotic cells in the tumor 1 day after aFP in the control group and aFP group respectively. Representative images are shown. Cells stained as red color, which are indicated by white arrow heads are apoptotic cells.</p

aFP treatment in absence of CD8⁺ T lymphocytes.

<p>(A) tumor volume curves of mice in the control group (no treatment), anti-CD8+aFP and aFP group after tumor inoculation. ** <i>P</i> < 0.001 comparing control to anti-CD8+aFP or aFP group. * <i>P</i> < 0.05 comparing anti-CD8+aFP to aFP group. The bars represent SD. (B) Kaplan-Meier survival curves of mice receiving tumor inoculation. The significance values for the difference between the survival curves are: control or anti-CD8+aFP vs. FP group (<i>p</i> < 0.01).</p

Tumor volume and survival curves after aFP treatment.

<p>(A) tumor volume curves of mice in the control group and aFP group after tumor inoculation. *** <i>P</i> < 0.0001. The bars represent SD. (B) tumor volume curve of mice in the control group, and tumor volume curves of cured mice and non-cured mice which are split from original curve in aFP group. * <i>P</i> < 0.01, ** <i>P</i> < 0.005 comparing control to aFP-non cured group. The bars represent SD. (C) Kaplan-Meier survival curves of mice receiving tumor inoculation. The significance values for the difference between the survival curves are: control vs. FP (<i>p</i> < 0.05).</p

Immunochemical staining and flow cytometric analysis for tumor infiltrating neutrophils.

<p>(A) proportion of neutrophil compared with CD45⁺CD3^- leukocytes in the tumor of flow cytometric analysis. (B and C) representative images of flow cytometry for neutrophil on day 1 after aFP in the control group and aFP group respectively. The number in the figures represents proportion of neutrophil compared with CD45⁺CD3^- leukocytes. (D and E) immunohistochemical staining for neutrophil in the tumor 1 days after aFP in the control group and aFP group respectively. Cells stained as red color are neutrophils. Inset shows multi nucleated neutrophils as the dominant immune infiltrate seen in H&E-stained aFP-treated tumor. (F) magnified image of neutrophil in figure (4D, G and H) immunohistochemical staining for CD206-expressing neutrophil in the tumor 1 days after aFP in the control group and aFP group respectively. Cells stained as yellow color are neutrophils expressing CD206.</p

Flow cytometric analysis for tumor infiltrating lymphocytes.

<p>(A) proportion of CD8⁺ T lymphocytes compared with CD3⁺ T lymphocytes in the tumor. (B) representative images of flow cytometry for CD8⁺ T lymphocytes on day 5 after aFP in the control group and aFP group respectively. The number in the figures represents proportion of CD8⁺ T lymphocytes compared with CD3⁺ T lymphocytes. (C and D) proportion of Treg compared with CD3 and CD4⁺ T lymphocytes in the tumor respectively. (E) representative images of flow cytometry for Treg on day 5 after aFP in the control group and aFP group respectively. The number in the figures represents proportion of CD4⁺ Foxp3⁺ Treg compared with CD4⁺ T lymphocytes. (F) proportion of beta-gal epitope specific CD8⁺ T lymphocytes compared with total CD8⁺ T lymphocytes in the tumor. (G) proportion of CD8⁺ T lymphocytes compared with Treg in the tumor.</p

Cytotoxicity assay and Treg function examination.

<p>(A) the number in the figures represents % specific lysis of sorted CD8⁺ T lymphocytes from TILs against CT26.CL25 cells. Average specific lysis against CT26.CL25 cells in the aFP group was significantly higher than in the control group (<i>P</i> < 0.05). (B) percentage of specific lysis CT26.CL25 by sorted CD8⁺ T lymphocytes from TILs with and without CD4⁺CD25⁺ T lymphocytes (sorted from tumor drainage lymph node).</p