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

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

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

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

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

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