Tumor-associated neutrophils upregulate Nectin2 expression, creating the immunosuppressive microenvironment in pancreatic ductal adenocarcinoma.

BACKGROUND: Tumor-associated neutrophils (TANs) constitute an abundant component among tumor-infiltrating immune cells and have recently emerged as a critical player in pancreatic ductal adenocarcinoma (PDAC) progression. This study aimed to elucidate the pro-tumor mechanisms of TAN and identify a novel target for effective immunotherapy against PDAC. METHODS: Microarray and cytokine array analyses were performed to identify the mechanisms underlying the function of TANs. Human and mouse TANs were obtained from differentiated HL-60 cells and orthotopically transplanted PDAC tumors, respectively. The interactions of TANs with cancer and cytotoxic T-cells were evaluated through in vitro co-culture and in vivo orthotopic or subcutaneous models. Single-cell transcriptomes from patients with PDAC were analyzed to validate the cellular findings. RESULTS: Increased neutrophil infiltration in the tumor microenvironment was associated with poor survival in patients with PDAC. TANs secreted abundant amounts of chemokine ligand 5 (CCL5), subsequently enhancing cancer cell migration and invasion. TANs subpopulations negatively correlated with cytotoxic CD8+ T-cell infiltration in PDAC and promoted T-cell dysfunction. TANs upregulated the membranous expression of Nectin2, which contributed to CD8+ T-cell exhaustion. Blocking Nectin2 improved CD8+ T-cell function and suppressed tumor progression in the mouse model. Single-cell analysis of human PDAC revealed two immunosuppressive TANs phenotypes: Nectin2+ TANs and OLR1+ TANs. Endoplasmic reticulum stress regulated the protumor activities in TANs. CONCLUSIONS: TANs enhance PDAC progression by secreting CCL5 and upregulating Nectin2. Targeting the immune checkpoint Nectin2 could represent a novel strategy to enhance immunotherapy efficacy in PDAC.

Pan-cancer analysis of immune checkpoint receptors and ligands in various cells in the tumor immune microenvironment.

Drugs that target immune checkpoint have become the most popular weapon in cancer immunotherapy, yet only have practical benefits for a small percentage of patients. Tumor cells constantly interact with their microenvironment, which is made up of a variety of immune cells as well as endothelial cells and fibroblasts. Immune checkpoint expression and blocked signaling of immune cells in the tumor microenvironment (TME) are key to tumor progression. In this study, we perform deliberation convolution on the TCGA database for human lung, breast, and colorectal cancer to infer crosstalk between immune checkpoint receptors (ICRs) and ligands (ICLs) in TME of pan-carcinogenic solid tumor types, validated by flow cytometry. Analysis of immune checkpoints showed that there was little variation between different tumor types. It showed that CD160, LAG3, TIGIT were found to be highly expressed in CD8+ T cells instead of CD4+ T cells, PD-L1, PD-L2, CD86, LGALS9, TNFRSF14, LILRB4 and other ligands were highly expressed on macrophages, FVR, NECTIN2, FGL1 were highly expressed on Epithelial cells, CD200 was highly expressed in Endothelial cells, and CD80 was highly expressed in CD8 High expression on T cells. Overall, our study provides a new resource for the expression of immune checkpoints in TME on various types of cells. Significance: This study provides immune checkpoint expression of immune cells of multiple cancer types to infer immune mechanisms in the tumor microenvironment and provide ideas for the development of new immune checkpoint-blocking drugs.

The Nectin family ligands, PVRL2 and PVR, in cancer immunology and immunotherapy.

In recent years, immunotherapy has emerged as a crucial component of cancer treatment. However, its efficacy remains limited across various cancer types, highlighting unmet needs. Poliovirus receptor-related 2 (PVRL2) and Poliovirus receptor (PVR) are members of the Nectin and Nectin-like Molecules family, known for their role as cell-cell adhesion molecules. With the development of immunotherapy, their involvement in tumor immune mechanisms as immune checkpoint factors has garnered significant attention. PVRL2 and PVR are predominantly expressed on tumor cells and antigen-presenting cells, binding to PVRIG and TIGIT, respectively, which are primarily found on T and NK cells, thereby suppressing antitumor immunity. Notably, gynecological cancers such as ovarian and endometrial cancers exhibit high expression levels of PVRL2 and PVR, with similar trends observed in various other solid and hematologic tumors. Targeting these immune checkpoint pathways offers a promising therapeutic avenue, potentially in combination with existing treatments. However, the immunomodulatory mechanism involving these bindings, known as the DNAM-1 axis, is complex, underscoring the importance of understanding it for developing novel therapies. This article comprehensively reviews the immunomodulatory mechanisms centered on PVRL2 and PVR, elucidating their implications for various cancer types.

Identification of Poliovirus Receptor-like 3 Protein as a Prognostic Factor in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents an aggressive subtype of breast cancer, with a bad prognosis and lack of targeted therapeutic options. Characterized by the absence of estrogen receptors, progesterone receptors, and HER2 expression, TNBC is often associated with a significantly lower survival rate compared to other breast cancer subtypes. Our study aimed to explore the prognostic significance of 83 immune-related genes, by using transcriptomic data from the TCGA database. Our analysis identified the Poliovirus Receptor-Like 3 protein (PVRL3) as a critical negative prognostic marker in TNBC patients. Furthermore, we found that the Enhancer of Zeste Homolog 2 (EZH2), a well-known epigenetic regulator, plays a pivotal role in modulating PVRL3 levels in TNBC cancer cell lines expressing EZH2 along with high levels of PVRL3. The elucidation of the EZH2-PVRL3 regulatory axis provides valuable insights into the molecular mechanisms underlying TNBC aggressiveness and opens up potential pathways for personalized therapeutic intervention.

A cutting-edge 68Ga-labeled bicyclic peptide PET molecular probe for noninvasive assessment of Nectin4 expression.

The diagnosis and treatment of triple negative breast cancer (TNBC) are huge challenges due to the lack of identifiable molecular targets. The high expression of Nectin4 in a variety of tumors, including TNBC, is associated with the occurrence, invasion, progression and poor prognosis of tumors. Therefore, Nectin4 is an emerging biomarker for the diagnosis and treatment of TNBC. A PET imaging method to non-invasively quantify Nectin4 expression levels may aid in TNBC diagnosis and classification. In this study, a novel bicyclic peptide molecular probe [68Ga]Ga-DN68 was used to evaluate the expression of Nectin4 in tumors. The radiolabeling rate of [68Ga]Ga-DN68 was over 97 %, while maintaining more than 99 % radiochemical purity. In vitro experiments showed that [68Ga]Ga-DN68 could effectively target Nectin4 in tumor cells, and the cellular uptake of MC38-Nectin4 cells (Nectin4+) was significantly higher than that of MC38 cells (Nectin4-). Biodistribution and PET imaging studies consistently showed that [68Ga]Ga-DN68 was specifically accumulated in MC38-Nectin4 and MDA-MB-468 tumors, which was significantly higher than that of MC38. When co-injected with cold DN68, the specific accumulation could block the tumor uptake of MDA-MB-468. Notably, the signal-to-noise ratio at the tumor site gradually increased over time, reaching a peak at 1 h. These results strongly suggest that [68Ga]Ga-DN68 has broad application prospects as a PET tracer in TNBC imaging.

Nectin-4-directed antibody-drug conjugates (ADCs): Spotlight on preclinical and clinical evidence.

Nectin-4 (Nectin cell adhesion molecule 4), a type I transmembrane cell adhesion protein, was demonstrated to be overexpressed in a variety of tumors, making it an attractive antigen for targeted therapies such as antibody-drug conjugates (ADCs). Of great note, the US Food and Drug Administration (FDA)-approval of the first Nectin-4-directed ADC, enfortumab vedotin (EV), in urothelial cancer (UC) not only introduced Nectin-4 as a clinically validated and reliable target antigen but also confirmed the evolving role of Nectin-4-directed ADCs as novel and promising cancer therapeutics. In addition to EV, there have been or are currently being seven and eleven Nectin-4-directed ADCs, respectively, in various stages of clinical trials and preclinical development, offering a promising future for the treatment of Nectin-4-positive cancer patients. This study reviewed clinical- and preclinical-stage Nectin-4-directed ADCs.

Increased PVR Expression on Bone Marrow Macrophages May Promote Resistance to TIGIT Blockade in Multiple Myeloma.

PURPOSE: TIGIT blockade in our ex vivo model of bone marrow (BM) reduced the number of malignant plasma cells (PC) in only half of patients with multiple myeloma. Here, we wanted to investigate whether increased expression of TIGIT ligands may inhibit T-cell immune response promoting resistance to TIGIT blockade. EXPERIMENTAL DESIGN: We first characterized the number and phenotype of BM macrophages in different stages of the disease by multiparameter flow cytometry. We assessed the effect of TIGIT ligands on PC survival by performing experiments in the ex vivo BM model and analyzed changes in gene expression by using NanoString technology and real-time PCR. RESULTS: The frequency of BM macrophages was significantly decreased in multiple myeloma, which was accompanied by changes in their immunophenotype. Moreover, we found a higher number of malignant PC in ex vivo BM cells cultured onto the poliovirus receptor (PVR) and nectin-2 compared with control, suggesting that both ligands may support PC survival. In addition, the presence of PVR, but not nectin-2, overcame the therapeutic effect of TIGIT blockade or exogenous IL2. Furthermore, exogenous IL2 increased TIGIT expression on both CD4+ and CD8+ T cells and, indirectly, PVR on BM macrophages. Consistently, PVR reduced the number of cytotoxic T cells and promoted a gene signature with reduced effector molecules. CONCLUSIONS: IL2 induced TIGIT on T cells in the BM, in which increased PVR expression resulted in cytotoxic T-cell inhibition, promoting PC survival and resistance to TIGIT blockade.

Effects of PEGylation on Imaging Contrast of 68Ga-Labeled Bicyclic Peptide PET Probes Targeting Nectin-4.

Nectin cell adhesion molecule 4 (Nectin-4) is overexpressed in various malignant tumors and has emerged as a promising target for tumor imaging. Bicyclic peptides, known for their conformational rigidity, metabolic stability, and membrane permeability, are ideal tracers for positron emission tomography (PET) imaging. In this study, we evaluated the feasibility of visualizing Nectin-4-positive tumors using radiolabeled bicyclic peptide derivatives and optimized the pharmacokinetics of radiotracers by introducing PEG chains of different lengths. Five PEGylated radiotracers radiolabeled with 68Ga3+ exhibited high radiochemical purity and stability. As the chain length increased, the Log D values decreased from -2.32 ± 0.13 to -2.50 ± 0.16, indicating a gradual increase in the hydrophilicity of the radiotracers. In vitro cell-binding assay results showed that the PEGylated bicyclic peptide exhibits nanomolar affinity, and blocking experiments confirmed the specific binding of the tracers to the Nectin-4 receptor. In vivo PET imaging and biodistribution studies in SW780 and 5637 xenograft mice showed that [68Ga]Ga-NOTA-PEG12-BP demonstrated optimal pharmacokinetics, characterized by rapid and good tumor uptake, faster background clearance, and improved tumor-to-tissue contrast. Finally, compared with 18F-FDG, PET imaging, in vivo blocking assays of [68Ga]Ga-NOTA-PEG12-BP and histological staining confirmed that specific tumor uptake was mediated by Nectin-4 receptors. The results indicated that [68Ga]Ga-NOTA-PEG12-BP was a promising PET radiotracer for Nectin-4 targeting, with applications for clinical translation.

Exploring membranous NECTIN-4 expression patterns and enfortumab vedotin response in prostate cancer.

Antibody-drug conjugates (ADCs) represent a novel type of targeted cancer therapy combining the specificity of monoclonal antibodies with the cytotoxicity of conventional chemotherapy. Recently, ADCs have demonstrated practice-changing efficacy across diverse solid cancers. The anti-NECTIN-4 ADC enfortumab vedotin (EV) has just been approved for patients with urothelial cancer and is currently under investigation for patients with castration-resistant prostate cancer (CRPC e.g. Phase II ENCORE trial). Our objective was to evaluate the efficacy of EV in established prostate cancer (PCa) cell lines and to examine the membranous NECTIN-4 expression in primary tumours (PRIM) and distant metastases (MET). NECTIN-4 was heterogeneously expressed in the panel of PCa cell lines. EV led to growth inhibition in NECTIN-4 expressing PCa cells (22Rv1 and LNCaP), whereas the NECTIN-4-negative PC-3 cells were significantly less responsive to EV, emphasizing the dependence of EV response on its target expression. Immunohistochemical staining revealed moderate membranous NECTIN-4 expression only in a small subgroup of CRPC patients with lung and peritoneal MET [n = 3/22 with H-score ≥100, median H-score 140 (IQR 130-150)], while 100% of PRIM (n = 48/48) and 86.4% of common MET sites (n = 19/22), including lymph node, bone and liver MET, were NECTIN-4 negative. In summary, EV may be effective in NECTIN-4-positive PCa. However, our findings demonstrate that the tumoural NECTIN-4 expression is predominantly low in metastatic PCa, which suggests that EV may only be effective in a biomarker-stratified subgroup.

Preclinical evidence for the use of anti-Trop-2 antibody-drug conjugate Sacituzumab govitecan in cerebral metastasized castration-resistant prostate cancer.

PURPOSE: Improved survival rates have been observed in castration-resistant prostate cancer (CRPC) due to advancements in treatment options. However, individuals with brain metastases still have limited therapeutic options and an unfavorable prognosis. Therefore, there is an urgent need to explore new therapeutic avenues, such as antibody-drug conjugates (ADCs), which have demonstrated significant clinical activity against active brain metastases in solid tumors. Our objective was to determine the expression levels of the ADC targets Trop-2 and NECTIN-4 in cerebral metastasized CRPC (mCRPC). METHODS: Immunohistochemical staining of Trop-2 and NECTIN-4 with evaluation of H-score was performed in CRPC brain metastases (n = 31). Additionally, we examined Trop-2 protein expression in prostate cancer cell lines and studied their responsiveness to the anti-Trop-2 ADC Sacituzumab govitecan (SG) in vitro. RESULTS: Our analysis revealed that most patients exhibited moderate to strong Trop-2 expression [n = 27/31 with H-score ≥100, median H-score 220 (IQR 180-280)], while NECTIN-4 was absent in all cerebral metastases. Mechanistically, we demonstrated that the efficacy of SG depends on Trop-2 expression levels in vitro. Overexpression of Trop-2 in Trop-2-negative PC-3 cells led to sensitization to SG, whereas CRISPR-Cas9-mediated knockdown of Trop-2 in Trop-2-expressing DU-145 cells conferred resistance to SG. CONCLUSION: The substantial expression of Trop-2 in cerebral metastases, along with our preclinical in vitro results, supports the efficacy of SG in treating cerebral mCRPC. Thus, our results extend the understanding of the potential of ADCs in prostate cancer treatment and provide an additional treatment strategy for the challenging subset of patients with cerebral metastases.