Low-dose photodynamic therapy promotes vascular E-selectin expression in chest malignancies, improving immune infiltration and tumor control

Abstract

Background: Chest malignancies such as non-small cell lung cancer (NSCLC) or pleural mesothelioma (PM) have an ominous prognosis. Photodynamic therapy (PDT) of NSCLC and PM improves patient survival, but the precise underlying mechanism remains unknown. Here, we hypothesized that low-dose PDT (L-PDT) alters the expression of tumor endothelial cell adhesion molecules favoring immune cell recruitment and tumor control. We explored this hypothesis in two mouse models of NSCLC and PM. We validated our findings in 82 PM patient samples.

Methods: We assessed, in C56BL/6 mice bearing 344SQ-NSCLC and in BALB/c mice bearing AB12-PM, how L-PDT (400 μg/kg Visudyne administered intravenously, irradiance: 50 mW/cm², light dose: 10 J/cm²) affected tumor growth, modulated the tumor immune microenvironment and the expression of endothelial selectin cell adhesion molecule (E-selectin) using real-time multiphoton imaging, immunofluorescence staining and flow cytometry. We then blocked E-selectin, canonical nuclear factor kappa B (NF-κB) pathway or selectively depleted CD8+ lymphocytes with dedicated peptides/antibodies in mouse models to evaluate the effect of L-PDT on tumors. Finally, we assessed in 82 PM patient samples the correlation between vascular E-selectin and CD8+ lymphocyte content by immunofluorescence staining of tissue sections and their association with patient survival.

Results: L-PDT induced vascular E-selectin in both NSCLC and PM, which enhanced granzyme B+/CD3+/CD8+ lymphocyte infiltration and improved tumor control. Blockade of E-selectin or immunodepletion of CD8+ lymphocytes abrogated the L-PDT-mediated cancer regression. Moreover, canonical NF-κB pathway blockade impaired enhanced vascular E-selectin expression and CD8+ T cells infiltration in tumors following L-PDT. In human malignant pleural mesothelioma samples, we found a correlation between vascular E-selectin and CD8+ T cell infiltration, which was associated with improved patient outcome.

Conclusion: L-PDT remodels the vasculature of chest tumors and favors a cytotoxic immune microenvironment promoting tumor control. This approach could complement current immunotherapy approaches in these malignancies.

Keywords: immune checkpoint inhibitor; immunotherapy; lung cancer; mesothelioma; surgery.

Figure 1. Low-dose photodynamic therapy (L-PDT) enhances tumor vascular E-selectin expression and CD8+ tumor infiltrating lymphocytes in orthotopic non-small cell lung cancer (NSCLC) and pleural mesothelioma (PM) mouse models. (A) Illustrative immunofluorescence images of E-selectin (green), VE-cadherin (red) and their colocalization signal (white) in AB12 PM and 344SQ NSCLC under untreated (NT) or L-PDT conditions. Scale bar: 100 µm (B) Quantification of E-selectin and VE-cadherin colocalization expressed as percentage of AB12 tumor area (AB12 PM: n=6–8/group; 344SQ NSCLC: n=4–8/group). (C) Quantification of VE-cadherin signal expressed as percentage of tumor area (n=6/8 per group). (D) Illustrative immunofluorescence images showing CD3+ (red), CD8+ (green) and granzyme B+ (cyan) cells infiltrating AB12 PM and 344SQ NSCLC. Colocalization of CD3+/CD8+ with granzyme B+ appears in white. Scale bar: 100 µm. (E) Quantification of CD3/CD8+ colocalization expressed as percentage of tumor area (AB12 PM: n=6–8/group; NSCLC: n=4–8/group). (F) Quantification of CD3/CD8+/granzyme B colocalization expressed as percentage of CD3+CD8+ T cells (AB12 PM: n=6–8/group; NSCLC: n=4–8/group). (G–H) Flow cytometry analysis and representative histograms of (G) CD8+ T cells among CD45+ cells and (H) PD1+ CD8+ T cells among CD45+ cells in AB12 PM and 344SQ NSCLC. A two-tailed unpaired t-test was used to compare treatment groups. *p ≤ 0.05.

Figure 2. Functional blockade of E-selectin impedes low-dose photodynamic therapy (L-PDT)-induced CD8+ T lymphocytes infiltration into pleural mesothelioma (PM) and non-small cell lung cancer (NSCLC). (A) Schematic overview of the E-selectin blockade experiment in AB12 PM. Scale bar: 100 µm. (B) Illustrative immunofluorescence images of CD3+ (red), CD8+ (green) cells and their colocalization signal (white) in AB12 PM under the different treatment conditions (untreated (NT), L-PDT, L-PDT+E-selectin ab, L-PDT+IgG ctrl ab). (C) Quantification of CD3/CD8 colocalization signal shown in B, expressed as percentage of tumor area (n=5–7/group). (D) Left: schematic overview of intravital imaging experiment in 344SQ NSCLC. Right: illustrative representation of the imaging device with a thoracic window over a lung tumor and under a 2-photon microscope objective lens. (E) Longitudinal observation of CD2+ cells (white) and cancer cells (green) in a tumor region of interest (ROI). Acquisition began 3 days before treatment and continued for 9 days after treatment with L-PDT in the presence or absence of a specific anti-E-selectin antibody. Scale bar: 50 µm. (F) Median curves of CD2+ cell counts in the L-PDT+ IgG control group (n=4) vs L-PDT+anti-E-selectin antibody treatment group (n=5). Every dot corresponds to the mean of three ROI per animal. (G) Median curves of GFP+ cell count in the L-PDT+IgG control group versus L-PDT+E-selectin antibody group. *p ≤ 0.05.

Figure 3. E-selectin expression is essential for tumor control following low-dose photodynamic therapy (L-PDT). (A) Schematic overview of the experimental design. (B) Kaplan-Meier survival curves of AB12 pleural mesothelioma (PM)-bearing mice (n=5–6/group). (C) Kaplan-Meier survival of 344SQ non-small cell lung cancer (NSCLC)-bearing mice (n=10/group). (D) Occurrence of metastases in 344SQ NSCLC-bearing mice. Treatment groups B–D: untreated (NT), L-PDT+IgG isotype control and L-PDT+E-selectin antibody. (E) Kaplan-Meier survival curves of NT or L-PDT-treated athymic BALB/c mice bearing AB12 PM (n=10/group). (F) Kaplan-Meier survival curves of NT AB12 PM-bearing mice or treated with L-PDT in the presence of an anti-CD8+ or IgG control antibody. Untreated (n=6), treated with L-PDT (n=5), and treated with L-PDT+anti-CD8 (n=6). Survival curves were compared by using the log-rank (Mantel-Cox) test. *p ≤ 0.05.

Figure 4. Canonical nuclear factor kappa B (NF-κB) pathway mediates low-dose photodynamic therapy (L-PDT)-induced E-selectin expression in ECRF-24 endothelial cells. (A) Illustrative immunofluorescence images of NF-κB p65 subunit localization in untreated or L-PDT-treated ECRF-24 cells in the presence of control (ctrl) small interfering RNA (siRNA) or NEMO siRNAs (siRNA1 and siRNA2). p65 is shown in red, nuclei are stained with DAPI (blue) and the colocalization appears in white. Scale bar: 100 µm. (B) Quantification of nuclear p65 normalized to DAPI. (C) Western blot analysis of E-selectin and NEMO expression in ECRF-24 cells either untreated or subjected to L-PDT or tumor necrosis factor (TNF)-α (20 ng/mL) treatments in the presence of ctrl or NEMO siRNAs (siRNA1 and siRNA2). (D) Densitometric quantification of five independent experiments. (E) Representative greyscale images of immunofluorescence staining of E-selectin in untreated ECRF-24 cells or treated with Visudyne (50 ng/mL), Light (0.15 J/cm²), L-PDT or TNF-α 20 ng/mL with quantification in F (n=5). Scale bar: 50 µm. (G) Representative greyscale images of immunofluorescence staining of E-selectin in ECRF-24 cells treated or not with L-PDT in the presence of NEMO siRNA with quantification (H). Scale bar: 50 µm. Statistical analysis was performed using one-way analysis of varianc followed by Dunnett’s multiple comparisons test or two-tailed unpaired t-tests with Bonferroni correction. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 5. Peptide-based inhibition of IκB kinase (IKK) complex formation in vivo abrogates the expression of tumor endothelial E-selectin following low-dose photodynamic therapy (L-PDT) in pleural mesothelioma (PM) and non-small cell lung cancer (NSCLC). (A) Schematic overview of the experimental design. (B) Illustrative immunofluorescence images of E-selectin (green), VE-cadherin (red) expression in PM tumors. Colocalization signal is shown in white. Scale bar: 100 µm. (C) Quantification of VE-cadherin and E-selectin colocalization signal expressed as percentage of tumor area. (D) Illustrative immunofluorescence images of CD3+ (red) CD8+ (green) and granzyme B+ (cyan) cells. Colocalization of CD3+/CD8+ with granzyme B+ appears in white. Scale bar: 100 µm. (E) Quantification of CD3/CD8 colocalization signal expressed as percentage of tumor area. (F) Quantification of CD3/CD8/granzyme B colocalization signal expressed as percentage of tumor area. NBD, NEMO binding domain peptide; NT, non-treated. Statistical analysis was performed using one-way analysis of variance followed by Dunnett’s multiple comparisons test, n=4–7/group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Figure 6. Vascular E-selectin expression is associated with better survival in patients with malignant pleural mesothelioma. (A) Representative images of H&E staining (left), and vascular E-selectin and VE-cadherin expression with their colocalization signal in yellow (right). (B–C) Kaplan-Meier survival curves analysis for patients with an increase (red) or a decrease (blue) in vascular E-selectin expression after treatment including (B) all histological subtypes, (C) epithelioid tumors only. (D–E) Kaplan-Meier survival curves analysis for patients with vascular E-selectin expression above (red) or below (blue) the median expression value considering all histological subtypes (D) or epithelioid malignant pleural mesothelioma (MPM) only (E). (F) Representative images of H&E staining (left), and CD3+CD8+ expression with colocalization signal in yellow (right). (G–I) Kaplan-Meier survival curves analysis for patients with CD3+CD8+ cells infiltration above (red) or below (blue) the median value in (G) all histological subtypes, (H) epithelioid tumors only or (I) biphasic and sarcomatoid tumors. (J) Pearson’s correlation between vascular E-selectin and CD3+CD8+ cells infiltration in patients with CD3+CD8+ cells infiltration above the median value.