An Orthogonal Nucleic Acid/Peptide Amplification Circuit Enables Protease-Triggered, Cancer Cell-Selective PD-L1 Imaging.

Molecular imaging offers a powerful approach for in situ detection of programmed death‐ligand 1 (PD‐L1), however, achieving cancer cell‐selective imaging that discriminates PD‐L1 expression on malignant versus normal cells remains a challenge. Here, we present an orthogonal nucleic acid/peptide amplification circuit that integrates protease‐activated hybridization chain reaction (HCR) with aptamer‐mediated target recognition for cancer‐selective PD‐L1 imaging. In the design, PD‐L1 aptamer is coupled with an HCR initiator for targeting PD‐L1, while PNA is employed as a bridge scaffold to engineer the initiator with protease‐responsive peptide substrate and thus block the HCR. Within the tumor microenvironment, protease‐mediated peptide cleavage liberates the initiator, thereby triggering localized HCR amplification at PD‐L1 sites. In contrast, in normal tissues lacking the relevant proteases, the initiator remains inactive, yielding markedly improved spatial selectivity for cancer cell‐specific PD‐L1 imaging. Using mouse models, we further demonstrate that this strategy allows for non‐invasive assessment of tumor responses to immune checkpoint blockade therapy. This methodology will build a bridge between DNA nanobiotechnology and peptide‐based biochemistry for diverse biomedical applications.

PD-L1 promotes myofibroblastic activation of hepatic stellate cells by distinct mechanisms selective for TGF-β receptor I versus II.

BackgroundThe quantification of PD-L1 (programmed cell death ligand 1) has been used to predict patient’s survival, to characterize the tumor immune microenvironment, and to predict response to immune checkpoint therapies....

Introduction: The quantification of PD-L1 (programmed cell death ligand 1) has been used to predict patient's survival, to characterize the tumor immune microenvironment, and to predict response to immune checkpoint therapies. However, a framework to assess the PD-L1 status with a high interobserver reproducibility on tumor cells and different types of immune cells has yet to be established. Methods: To study the impact of PD-L1 expression on the tumor immune microenvironment and patient outcome, a framework for fully automated PD-L1 quantification on tumor cells and immune cells was established and validated. Automated PD-L1 quantification was facilitated by incorporating three different deep learning steps for the analysis of more than 80 different neoplasms from more than 10'000 tumor specimens using a bleach & stain 15-marker multiplex fluorescence immunohistochemistry panel (i.e., PD-L1, PD-1, CTLA-4, panCK, CD68, CD163, CD11c, iNOS, CD3, CD8, CD4, FOXP3, CD20, Ki67, CD31). Clinicopathological parameter were available for more than 30 tumor entities and overall survival data were available for 1517 breast cancer specimens. Results: Comparing the automated deep-learning based PD-L1 quantification with conventional brightfield PD-L1 data revealed a high concordance in tumor cells (p<0.0001) as well as immune cells (p<0.0001) and an accuracy of the automated PD-L1 quantification ranging from 90% to 95.2%. Across all tumor entities, the PD-L1 expression level was significantly higher in distinct macrophage/dendritic cell (DC) subsets (identified by CD68, CD163, CD11c, iNOS; p<000.1) and in macrophages/DCs located in the Stroma (p<0.0001) as compared to intratumoral macrophages/DC subsets. Across all different tumor entities, the PD-L1 expression was highly variable and distinct PD-L1 driven immune phenotypes were identified based on the PD-L1 intensity on both tumor and immune cells, the distance between non-exhausted T-cell subsets (i.e. PD-1 and CTLA-4 expression on CD3+CD8+ cytotoxic T-cells, CD3+CD4+ T-helper cells, CD3+CD4+FOXP3+ regulatory T-cells) and tumor cells as well as macrophage/(DC) subtypes. In breast cancer, the PD-L1 fluorescence intensity on tumor cells showed a significantly higher predictive performance for overall survival with an area under receiver operating curves (AUC) of 0.72 (p<0.0001) than the percentage of PD-L1+ tumor cells (AUC: 0.54). In PD-L1 positive as well as negative breast cancers a close spatial relationship between T- cell subsets (CD3+CD4±CD8±FOXP3±PD-1±CTLA-4±) and Macrophage/DC subsets (CD68±CD163±CD11c±iNOS) was found prognostic relevant (p<0.0001). Conclusion: In conclusion, multiplex immunofluorescence PD-L1 assessment provides cutoff-free/continuous PD-L1 data which are superior to the conventional percentage of PD-L1+ tumor cells and of high prognostic relevance. The combined analysis of spatial PD-L1/PD-1 data and more than 20 different immune cell subtypes of the immune tumor microenvironment revealed distinct PD-L1 immune phenotypes. Citation Format: Niclas C. Blessin, Elena Bady, Tim Mandelkow, Cheng Yang, Jonas B. Raedler, Ronald Simon, Christoph Fraune, Maximilian Lennartz, Sarah Minner, Eike Burandt, Doris Höflmayer, Guido Sauter, Katharina Möller, Sören A. Weidemann. Automated cell type specific PD-L1 quantification by artificial intelligence using high throughput bleach & stain 15-marker multiplex fluorescence immunohistochemistry in human cancers [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P068.

Novel Mechanism of Post-Transcriptional Regulation of PD-L1 Expression By 3'-UTR Binding Proteins

Background: Studies pointed out that the tumor-infiltrating lymphocytes (TILs) have considerable importance in canine mammary tumor (CMT). On the other hand, cancer cells sometimes find ways to use immune checkpoint proteins as a shield to avoid being identified and attacked by the immune system as programmed death 1 ligand 1 (PD-L1). In this study, it was investigated the relationship between PD-L1 expression, stromal tumor-infiltrating lymphocytes (TILs) in canine mammary tumor (CMT), and the association with clinical and pathological characteristics of the tumors.Materials, Methods & Results: PD-L1 expression and TILs were assessed in 23 female dogs with CMT. The tumors were grouped into simple carcinoma (CA, n = 8) and complex carcinoma (CC, n = 15). Stromal TILs were assessed using two thresholds as TILs-Low representing < 50% of infiltrate within stromal area and TILs-High representing ≥ 50% of stromal area. Clinicopathological data of CMT was characterized according to key parameters, as well as survival rates. TILs evaluation within tumor stroma revealed that 65.2% (n = 15) of tumors had TILs-Low. PD-L1 expression and stromal TILs were significantly associated (P = 0.009). PD-L1 expression was observed in 39% (n = 9) of all tumors of which 17.4% (n = 4) were from CA group and 21.7% (n = 5) were from CC group. PD-L1 expression within TILs was observed in 39% (n = 9) of the tumors. PD-L1 in malignant epithelium was present in all lymph node metastasis (n = 5). PD-L1 was associated with involvement of regional lymph nodes (P = 0.034). Survival curves demonstrated TILs-Low had higher (P = 0.010) overall survival (OS) compared with TILs-High, and PD-L1+ and PD-L1– (P = 0.06) did not differed. The clinicopathological variables significantly correlated with OS by univariate analysis were the histological grade (P = 0.009), lymph node involvement (P = 0.004), stromal TILs (P = 0.016), and PD-L1+/TILs-High vs. PD-L1–/TILs-Low (P = 0.010). Multivariate analysis revealed that group of tumors with grade II-III was independent and negative prognostic factors for OS.Discussion: In this study, PD-L1 was differently expressed according to the histologic subtypes of TMC. Currently, has been showed the presence of PD-L1 in several canine cancer. Nevertheless, only a few studies have described PD-L1 protein expression in dog tumors and showed PD-L1 was constitutively expressed on canine tumor cell lines, although the levels of basal expression were very variable. This expression can be modulated by IFN-γ exposure. In the present study, it was found a strong PD-L1 expression on TILs. The increase in PD-L1 cell surface expression by tumor cells can lead to decreased T-cell proliferation and increased apoptosis. In human breast cancer (BC) the PD-L1 expression was expressed in TILs and tumor epithelium. It has been reported the association of stromal TILs and PD-L1 expression with aggressive types and stages of BC. In this study, it was detected PD-L1 expression in malignant epithelium in all lymph node metastasis. PD-L1 overexpression was significantly associated with a series of clinicopathological parameters. It was demonstrated that PD-L1+/TILs-High had higher risk of overall survival (OS) than another group of interaction. High PD-L1 expression may be a prognostic indicator for reduced OS, while tumor PD-L1+ was associated with poorer disease-free survival. The presence of TILs has shown to be potentially predictive and a prognostic factor in BC subtypes. In CMT, it has been reported that a high proportion of TILs was correlated to several malignancy characteristics. In relation to PD-L1, further research is necessary to clarify this immune checkpoint as a potential therapeutic target and its application in clinical practice in CMT.

IntroductionCombined radiotherapy and immune checkpoint inhibition is a potential treatment option for head and neck squamous cell carcinoma (HNSCC). Immunocompetent mouse models can help to successfully develop radio- immunotherapy combinations and to increase our understanding of the effects of radiotherapy on the tumor microenvironment for future clinical translation. Therefore, the aim of this study was to develop a homogeneous, reproducible HNSCC model originating from the Mouse Oral Cancer 1 (MOC1) HNSCC cell line, and to explore the radiotherapy-induced changes in its tumor microenvironment, using flow cytometry and PD-L1 microSPECT/CT imaging.Materials and MethodsIn vivo growing tumors originating from the parental MOC1 line were used to generate single cell derived clones. These clones were screened in vitro for their ability to induce programmed cell death ligand 1 (PD-L1) and major histocompatibility complex class I (MHC-I) following IFNγ exposure. Clones with different IFNγ sensitivity were inoculated in C57BL/6 mice and assessed for tumor outgrowth. The composition of the tumor microenvironment of a stably growing (non)irradiated MOC1-derived clone was assessed by immunohistochemistry, flow cytometry and PD-L1 microSPECT/CT.ResultsLow in vitro inducibility of MHC-I and PD-L1 by IFNγ was associated with increased tumor outgrowth of MOC1 clones in vivo. Flow cytometry analysis of cells derived from a stable in vivo growing MOC1 clone MOC1.3D5low showed expression of MHC-I and PD-L1 on several cell populations within the tumor. Upon irradiation, MHC-I and PD-L1 increased on leukocytes (CD45.2+) and cancer associated fibroblasts (CD45.2−/EpCAM−/CD90.1+). Furthermore, PD-L1 microSPECT/CT showed increased tumor uptake of radiolabeled PD-L1 antibodies with a heterogeneous spatial distribution of the radio signal, which co-localized with PD-L1+ and CD45.2+ areas.DiscussionPD-L1 and MHC-I inducibility by IFNγ in vitro is associated with tumor outgrowth of MOC1 clones in vivo. In tumors originating from a stably growing MOC1-derived clone, expression of these immune-related markers was induced by irradiation shown by flow cytometry on several cell populations within the tumor microenvironment such as immune cells and cancer associated fibroblasts. PD-L1 microSPECT/CT showed increased tumor uptake following radiotherapy, and autoradiography showed correlation of uptake with areas that are heavily infiltrated by immune cells. Knowledge of radiotherapy-induced effects on the tumor microenvironment in this model can help optimize timing and dosage for radio- immunotherapy combination strategies in future research.

PD-L1 (B7-H1) expression and the immune tumor microenvironment in primary and metastatic breast carcinomas

Placental trophoblasts contribute to regulatory T (Treg) function via the programmed cell death-1 (PD-1)/PD-1 ligand 1 (PD-L1) pathway during normal pregnancy. Decreased expression of PD-L1 in trophoblasts was closely associated with Treg deficiency in the development of pregnancy failure. Thus, targeting PD-L1 might be a novel therapy to prevent pregnancy loss. However, the mechanisms for modulating the expression of PD-L1 in trophoblasts are an enigma. The proportion of decidual Treg cells, and the profile of decidual macrophages (DMs) sampled from women with normal pregnancy (NP) and recurrent miscarriage (RM) were evaluated by flow cytometry. The expression of Yin and Yang 1 protein (YY1) and PD-L1 in human villous were measured by Immunohistochemistry (IHC), qRT-PCR and western blot. The determination of soluble PD-L1 (sPD-L1) in serum from NP and RM, and trophoblast conditioned media (TCM) was performed by the PD-L1 SimpleStep ELISA kit. Knockdown of YY1 was processed in the human trophoblast derived cell lines, HTR-8 and Bewo, with siYY1 transfection. Peripheral naïve CD4+ T cells were isolated from women with NP for the in vitro culture. The percentages of Treg cells differentiated from peripheral naïve CD4+ T cells were measured by flow cytometry. The interaction between YY1 and CD274 was proved by CHIP. The expression of inducible nitric oxide synthase (iNOS) in decidua was evaluated by IHC. The level of NO in serum from women with NP and RM was determined by the Griess reagent system. The effects of NO on YY1 were determined by the in vitro culture of HTR-8 cells with the NO donor, SNAP. The in vivo model comprising twelve pregnant mice and underwent different treatment. The percentages of Treg cells in murine uterus were measured by flow cytometry. Similarly, Western blot and IHC were performed to determine the expression of YY1 and PD-L1 in murine placenta. Decreased expression of YY1 and PD-L1 in trophoblasts and lower proportion of decidual Treg cells were observed in patients with RM. Knockdown of YY1 contributes to a lower expression of YY1 and PD-L1. Soluble PD-L1 in the supernatant from HTR-8 cells was also decreased with siYY1 administration. Lower Treg differentiation was observed in the presence of supernatant from HTR-8 cells treated with siYY1. CHIP analysis revealed that endogenous YY1 directly occupied the promoter region of the CD274 (PD-L1) gene. Accompanied with increased M1 DMs, higher NO was observed in serum sampled from patients with RM. In the presence of Reduced expression of YY1 and PD-L1 was observed in HTR-8 cells with the treatment of SNAP. Furthermore, less Treg differentiation was observed with SNAP treated TCM. Moreover, our in vivo data found that YY1 deficiency was associated with decreased PD-L1, which further resulting in less Treg differentiation and Treg deficiency at the maternal-fetal interface and increased embryo loss. Our work found the modulatory capacity of YY1 on PD-L1 in trophoblasts during early pregnancy. Furthermore, reduced YY1 was supposed resulting from higher levels of NO produced from the M1 DMs in RM.

Recurrent PDL1 expression and PDL1 (CD274) copy number alterations in breast implant–associated anaplastic large cell lymphomas

Triple-negative breast cancers (TNBC) have been particularly challenging to manage due to their lack of intrinsic cellular receptors and a consequent lack of targetable therapy. Recently, the programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) immune checkpoint pathway has become the focus of immunotherapy in general, and especially for TNBCs. This study aimed to determine the pattern of expression of PD-L1 in TNBC cases in Benin City. Formalin-fixed, Paraffin-embedded tissue blocks of TNBC cases diagnosed in the Department of Anatomical Pathology, University of Benin Teaching Hospital, Benin City, Nigeria from 1st January, 2017 to 31st December, 2019 were re-sectioned for PD-L1 immunohistochemistry. Ninety-two cases of TNBCs were tested for PD-L1 expression. Thirteen (14.1%) of the TNBC cases were PD-L1 positive of varying degrees in tumour cells. Diffuse tumoural PD-L1 staining was seen in four (30.8%) of the PD-L1 positive cases. PD-L1 expression was significantly associated with increasing age up to the fifth decade (p =0.030). All the PD-L1 positive TNBCs were invasive breast carcinomas of no special type and mostly grade 2 tumours; however, there was no significant association between PD-L1 expression and histological subtype or grade. PD-L1 expression was shown to occur at a relatively lower rate among TNBC cases in this southern region of Nigeria, and was significantly associated with increasing age. About 14.1% (1 in 7) of our TNBC patients could potentially benefit from immune checkpoint inhibitor therapy. We therefore recommend further PD-L1 immunohistochemistry assay for TBNC cases and the use of appropriate immune therapy when indicated.

Programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) pathway blockade has emerged as a promising strategy for cancer therapy. Extranodal natural killer/T cell lymphoma (ENKTL) is an aggressive disease characterized by a strong association with Epstein-Barr virus (EBV), and chronic EBV infection is known to induce PD-L1 expression. However, the PD-1/PD-L1 pathway status in ENKTL remains elusive. Thus, the expression pattern of PD-1 and PD-L1 was investigated in 73 ENKTL cases, and its clinicopathological features and prognostic significance were analyzed. Most ENKTLs had few PD-1+ lymphocytes in the tumor microenvironment. PD-L1 was positive in 56% (n=41/73) with a cutoff value of ≥10% of tumor cells and in 62% (n=45/73) with a cutoff value of ≥10% of total cells including malignant and non-malignant cells. PD-L1 expression on tumor cells was mostly correlated with PD-L1 expression on non-malignant cells. PD-L1 positivity showed no significant relationship with clinicopathological features. However, patients with PD-L1+ ENKTL exhibited better 5-year overall survival (OS) and a trend for longer 5-year progression-free survival. Moreover, in the subgroups with clinically advanced parameters including late stage III/IV, higher International Prognostic Index scores of 2-5 or non-upper aerodigestive tract involvement PD-L1 positivity was also associated with favorable OS. PD-L1 expression was the only significant independent predictor for longer OS in patients with advanced stage (III/IV) ENKTL. These results suggest that PD-L1 might be used as a novel prognostic marker.

Objective: To explore the expression of programmed cell death ligand 1 (PD-L1) in patients with locally advanced and non-EGFR-mutated non-small cell lung cancer (LA-NSCLC) undergoing concurrent chemoradiotherapy (cCRT) and its association with clinical outcome of patients. Methods: The basic clinical information of 19 patients with unresectable, non-EGFR mutated LA-NSCLC receiving radical cCRT in Cancer Hospital Chinese Academy of Medical Sciences from January 2016 to December 2017 was retrospectively analyzed. The rabbit monoclonal antibody SP263 was used for immunohistochemical analysis to detect the expression of PD-L1 in LA-NSCLC tissues and the tumor proportion score (TPS) equal to or greater than 1% was defined as PD-L1 positive. The associations between PD-L1 ≥1% and PD-L1 ≥25% with the clinical characteristics and clinical outcome of LA-NSCLC patients were evaluated respectively. Results: Among 19 LA-NSCLC patients, 13 had PD-L1 positive expression, and 4 had PD-L1 expression greater than or equal to 25%. No significant difference was observed between patients with PD-L1 positive and negative expressions regarding the distribution of age, smoking history, pathological classification, and TNM staging (P>0.05). A total of 15 patients could be evaluated for therapeutic effect, including 7 patients with partial response (PR), 7 patients with stable disease (SD), and 1 patient with progressive disease (PD). In the group with PD-L1 expression<1%, 3 patients were in objective response, and 4 patients were in disease control. In the group with PD-L1 expression ≥1%, 4 patients were in objective response, and 10 patients were in disease control. When the PD-L1 expression was less than 25%, 6 patients gained the objective response, and 11 patients gained the disease control. When the PD-L1 expression was greater than or equal to 25%, 1 patient gained the objective response, and 3 patients gained the disease control. The median overall survival (OS) was 35 (95%CI: 12.7-57.3) months for patients with PD-L1 ≥1% and 40 (95%CI: not reaching the end point) months for patients with PD-L1<1% (P=0.284). Patients with PD-L1 ≥25% had a median survival time of 12 (95%CI:0.0-34.5) months, and patients with PD-L1<25% had a median survival time of 40 (95%CI: 27.4-52.6) months (P=0.241). Conclusions: The prognosis of LA-NSCLC patients with PD-L1 positive and no-EGFR mutation receiving concurrent chemoradiation has a trend of poor prognosis. A larger sample size study is warranted to explore the prognostic value of PD-L1 expression in inoperable LA-NSCLC patients and to further explore the effect of immunotherapy on patients with different PD-L1 expression levels.

How low can you go? PD-L1 expression as a biomarker in trials of cancer immunotherapy

Differential expression of PD-L1 and IDO1 in association with the immune microenvironment in resected lung adenocarcinomas