PD-L1 blockade potentiates the antitumor effects of ALA-PDT and optimizes the tumor microenvironment in cutaneous squamous cell carcinoma

Abstract

Immune checkpoint blockade (ICB) is a powerful oncologic treatment modality for a wide variety of human malignancies, but the patient response rate to this treatment remains low, especially in patients with cutaneous squamous cell carcinoma (cSCC). 5-Aminoleuvulinic acid-photodynamic therapy (ALA-PDT) is widely used to treat cancerous and precancerous skin diseases, but the value of ALA-PDT in the treatment of invasive cSCC is debatable. Our previous studies have shown that ALA-PDT can induce antitumor immune responses by promoting the immunogenic death of tumor cells. However, it is unclear whether ALA-PDT exerts synergistic effects with ICB in cSCC. Here, we report that PD-L1 blockade potentiates the antitumor effects of ALA-PDT both on primary and distant tumors, and optimizes the tumor microenvironment in cSCC. In this study, we first detected PD-L1 expression in patients with different grades of cSCC. Then we found the combination of anti-PD-L1 monoclonal antibody (mAb) and ALA-PDT killed tumor cells by apoptosis- and/or ferroptosis-mediated immunogenic cell death (ICD) and stimulated systemic immune response, as well as building the immunological memory response to prevent tumor recurrence. Furthermore, we found that combination therapy can be used to recruit tertiary lymphoid structure (TLS)-like intratumoral lymphoid aggregates, which may promote tumor-infiltrating lymphocyte (TIL)-mediated antitumor immunity. In summary, our work demonstrates that ICB treatment with an anti-PD-L1 antibody is a promising strategy that may potentiate the antitumor effects of ALA-PDT in cSCC.

Keywords: 5-aminolevulinic acid photodynamic therapy; PD-L1; cutaneous squamous cell carcinoma.

Figure 1.

PD-L1 expression is correlated with the progression of cSCC. (a) PD-L1 expression as indicated in the GEO and Oncomine databases. (b) PD-L1 expression in cSCC patient tumor tissues, determined by IHC. The tumor proportion score (TPS) represents the proportion of PD-L1-positive expression. (c) PD-L1 expression grade of different differentiated cSCC tissues (n = 28). (d) PD-L1 expression in UV-induced cSCC mouse tissues as determined by IHC. (e) Histological and dermoscopic images of normal skin and cSCC skin during UV-induced cSCC mouse model construction. *p < 0.05, **p < 0.01; the scale bar is 600 μm for the 4X images, 200 μm for the 20X images and 100 μm for HE staining.

Figure 2.

Synergistic antitumor effects of an anti-PD-L1 mAb and ALA-PDT in an implanted cSCC mouse model. (a) Diagram of the experimental design of ALA-PDT and an anti-PD-L1 mAb applied to the cSCC mouse model. (b) Representative tumor growth in each group. (c) Primary tumor growth curve of each group (n = 5 for each group); (d) distant tumor growth curve of each group (n = 5 for each group). (e) Mouse weight curve of each group. (f) Representative HE and Ki67 staining of distant tumors in each group. *p < 0.05, **p < 0.01; the scale bar is 200 μm.

Figure 3.

Combined anti-PD-L1 mAb and ALA-PDT therapies are associated with an increased number of tumor-infiltrating lymphocytes. (a-b) Relative mRNA expression was detected by qPCR (n = 5 for each group). (c-d) Tumors were collected from each treatment group, and single-cell suspensions were prepared and then stained for specific antibodies against immune cell surface markers (n = 3 for each group). The average percentage of positive surface markers for each group was calculated using flow cytometry. *p < 0.05, **p < 0.01.

Figure 4.

Synergistic antitumor effects of an anti-PD-L1 mAb and ALA-PDT in a UV-induced cSCC mouse model. (a) Representative tumor growth of the UV-induced cSCC mouse model. ALA-PDT was applied to tumors in the red circle, and anti-PD-L1 mAb was intraperitoneally injected. (b) Tumor count curve of the UV-induced cSCC mouse model (n = 3 for each group). (c) Largest tumor growth curve of the UV-induced cSCC mouse model (n = 3 for each group). (d) OCT analyses of mouse skin structure, left panel. Dermoscopic images of mouse skin, right panel. (e) After 24 h of treatment, immunofluorescence of tumor tissue in the UV-induced cSCC mouse model was detected. (f) After 24 h of treatment, tumor tissue lysates of the UV-induced cSCC mouse model (left panel) and implanted cSCC mouse model (right panel) were subjected to Western blotting with the indicated antibodies. The blots were probed with GAPDH as the loading control. (g) After 6 h of treatment, A431 cell membrane lysates were subjected to Western blotting using the indicated antibodies. The blots were probed with Na-K-ATPase as the loading control. (h) Tumor growth curves of rechallenged tumors inoculated 30 days post-elimination of their first tumors by either surgery or combined treatment with anti-PD-L1 mAb and ALA-PDT (n = 3 for each group). (i) Cytokine levels of IFN-γ and TNF-α in sera from mice isolated 7 days after mice were rechallenged with secondary tumors. *p < 0.05, **p < 0.01.

Figure 5.

ALA-PDT suppresses tumor growth by altering the TLS-mediated tumor microenvironment in cSCC patients. (a) Tumor tissues from cSCC patients were subjected to immunohistochemistry staining. Tumor tissues (48 h after ALA-PDT) were randomly selected from 2 sites. The yellow circles show TLSs, the yellow arrows indicate the enlarged area in the image below. (b) Tumor tissues (24 h after ALA-PDT) were randomly selected from 2 sites. The yellow circles show TLSs. The scale bar is 200 μm (white) and 50 μm (yellow).

Figure 6.

The combined treatment with an anti-PD-L1 mAb and ALA-PDT suppresses tumor growth by altering the TLS-mediated tumor microenvironment in UV-induced cSCC mouse model. (a) After 24 h of treatment, tumor tissues of the UV-induced cSCC mouse model were collected, and relative mRNA expression was detected by qPCR. (b) Tumor tissues (before or 24 h after ALA-PDT and anti-PD-L1) from UV-induce cSCC mouse were subjected to immunohistochemistry staining of consecutive sections. (c) Tumor tissues of mice (before or 24 h after ALA-PDT and anti-PD-L1) were subjected to immunohistochemistry staining and randomly selected from 2 sites. The yellow circles show TLSs. *p < 0.05, **p < 0.01; the scale bar is 100 μm.