Multiplexed immunofluorescence reveals potential PD-1/PD-L1 pathway vulnerabilities in craniopharyngioma

Abstract

Background: Craniopharyngiomas are neoplasms of the sellar/parasellar region that are classified into adamantinomatous craniopharyngioma (ACP) and papillary craniopharyngioma (PCP) subtypes. Surgical resection of craniopharyngiomas is challenging, and recurrence is common, frequently leading to profound morbidity. BRAF V600E mutations render PCP susceptible to BRAF/MEK inhibitors, but effective targeted therapies are needed for ACP. We explored the feasibility of targeting the programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) immune checkpoint pathway in ACP and PCP.

Methods: We mapped and quantified PD-L1 and PD-1 expression in ACP and PCP resections using immunohistochemistry, immunofluorescence, and RNA in situ hybridization. We used tissue-based cyclic immunofluorescence to map the spatial distribution of immune cells and characterize cell cycle and signaling pathways in ACP tumor cells which intrinsically express PD-1.

Results: All ACP (15 ± 14% of cells, n = 23, average ± SD) and PCP (35 ± 22% of cells, n = 18) resections expressed PD-L1. In ACP, PD-L1 was predominantly expressed by tumor cells comprising the cyst lining. In PCP, PD-L1 was highly expressed by tumor cells surrounding the stromal fibrovascular cores. ACP also exhibited tumor cell-intrinsic PD-1 expression in whorled epithelial cells with nuclear-localized beta-catenin. These cells exhibited evidence of elevated mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) signaling. Profiling of immune populations in ACP and PCP showed a modest density of CD8+ T cells.

Conclusions: ACP exhibit PD-L1 expression in the tumor cyst lining and intrinsic PD-1 expression in cells proposed to comprise an oncogenic stem-like population. In PCP, proliferative tumor cells express PD-L1 in a continuous band at the stromal-epithelial interface. Targeting PD-L1 and/or PD-1 in both subtypes of craniopharyngioma might therefore be an effective therapeutic strategy.

Fig. 1

PD-L1 expression in ACP is predominantly localized to well-keratinized cyst-lining epithelium and regions adjacent to wet-keratin and ghost cells (A; PD-L1, red; 4′,6′-diamidino-2-phenylindole [DAPI], blue). Hematoxylin and eosin (H&E) (B) and PD-L1 IHC (C, D) of cyst-lining epithelium. Single cells are occasionally positive in the basaloid epithelium (D, arrowhead). H&E (E) and PD-L1 IHC (F, G) show PD-L1 expression in cells near “wet-keratin/ghost cells.” H&E (H) and PD-L1 IHC (I) show basaloid epithelium with a cluster of PD-L1 expressing cells. Scattered immune cells show PD-L1 expression (J). Transcriptomic analysis of PD-L1 mRNA in a separate cohort of 27 ACPs and 77 other brain tumors and non-neoplastic brain specimens including choroid plexus papillomas (CPP) and medulloblastomas (MED) (K). Values expressed as log2 gene expression. Scale bars 20 μm (B–D, F–L), 50 μm (E).

Fig. 2

PD-L1 expressing cells in the basaloid epithelium of ACP (A) showed membranous (inactive) beta-catenin (B) and were typically keratin positive (C). Whorls of epithelium with nuclear beta-catenin do not express PD-L1, but are weakly positive for keratin (D–F). Regions of cyst lining epithelium with PD-L1 expression have membranous beta-catenin (G–I). PD-L1 expressing cells near “wet keratin” were keratin expressing epithelial cells with membranous beta-catenin (J–L). Scale bars 20 µm.

Fig. 3

PD-1 IHC in ACP showed tumor cell–intrinsic PD-1 expression in whorls of tumor epithelium (A, B). In situ hybridization showed expression of PD-1 mRNA in epithelial whorls (C). Multiplexed cyclic immunofluorescence (t-CyCIF) showed co-localization of PD-1 (D, E) using multiple antibodies, and nuclear beta-catenin (F, G) in the whorled cells. PD-L2 positive cells were observed closely associated with whorls (H) (different whorl pictured than D–L). Whorls showed elevated phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (I), phospho-S6 (Ser235/236) (J), and (Ser240/244) (K). PD-1 expressing whorls had diffuse nuclear p21^Cip1/Waf1 but MIB-1/Ki-67 staining was not typically present (L). Quantitative analysis (heatmaps of 5 ACP resections depicted with 0.0 [low] to 1.0 [high] expression by normalized mean intensity) (M) showed significantly increased expression (P < 0.025, red dots indicate significance) (N) of PD-1, pS6, and pERK in whorls compared with adjacent non-whorled epithelium. Multiple PD-1 antibodies showed high pixel-by-pixel correlation coefficients (O). Scale bar 100 µm (C); scale bars 20 µm (D–L).

Fig. 4

PD-L1 is diffusely expressed in papillary and flat epithelium in PCP (A). Hematoxylin and eosin (B) and PD-L1 IHC (C, D) in PCP show strong membranous expression in basal cells circumferentially surrounding fibrovascular stroma. Continuous expression was also present in basal cells in regions of flat epithelium (E, F). Scattered immune cells with PD-L1 expression were present in each case (G). ACP and PCP each show significantly increased PD-L1 expression, with higher levels in PCP (H). In situ hybridization shows PD-L1 mRNA expression in PCP (I, J) and ACP (K, L) epithelium in the same distribution as the protein expression observed by IHC. Scale bars 20 μm (D, F, G, I, K, L), 50 μm (B, C, H, J).

Fig. 5

Tissue-based CyCIF was used to profile, quantify, and generate spatial representations of tumor and immune cell populations in whole slide sections (A, hematoxylin and eosin; B, representative “dot-plot”). Each immune population was quantified in the PD-L1 expressing tumor epithelium, PD-L1 negative epithelium, and stroma (C, representative data from PCP-3). The density of immune cells was quantified in ACP (n = 3) (D) and PCP (n = 3) (E) (mean ± SEM). The percent of stromal immune cells (CD45+) with PD-L1 expression was quantified in ACP (n = 3) and PCP (n = 3) (F).