Analysis of PD-L1 promoter methylation combined with immunogenic context in pancreatic ductal adenocarcinoma.

Despite the successful application of programmed cell death ligand 1 (PD-L1)-blocking strategies in some types of cancers and well-established prognostic indicators in pancreatic ductal adenocarcinoma (PDAC), the biological and clinical implications of the methylation status of PD-L1/PD-L2 in PDAC remain largely unknown. Therefore, this study aimed to explore the biological role of PD-L1/PD-L2 methylation and its association with clinicopathological features, clinical outcomes, and the immune microenvironment by analyzing the data on PD-L1/PD-L2 methylation and mRNA expression in PDAC cohorts obtained from the Cancer Genome Atlas and International Cancer Genome Consortium. The correlation between PD-L1 promoter methylation and PD-L1 expression and survival was further validated in an independent validation cohort (Peking Union Medical College Hospital [PUMCH] cohort) using pyrosequencing and immunohistochemistry. These results demonstrated that hypomethylation of the PD-L1 promoter was strongly associated with upregulated PD-L1 expression and shorter overall survival in PDAC. Multivariate Cox regression analyses revealed that the PD-L1 promoter methylation was an independent prognostic factor. PD-L1 promoter hypomethylation and high expression were related to aggressive clinical phenotypes. Moreover, both PD-L1 and PD-L2 methylation correlated with immune cell infiltration and the expression of immune checkpoint genes. PD-L1 promoter methylation status was further validated as an independent prognostic biomarker in patients with PDAC using the PUMCH cohort. The prognostic significance of PD-L1 promoter methylation was more discriminative in tumors with perineural/lymphovascular invasion and distant metastasis than in those without perineural/lymphovascular invasion and distant metastasis. In summary, the methylation status of the PD-L1 promoter is a promising biomarker for survival outcomes, immune infiltration, and the potential immune benefits of immunotherapy in PDAC.

Synergistic Immunoregulation: harnessing CircRNAs and PiRNAs to Amplify PD-1/PD-L1 Inhibition Therapy.

The utilization of PD-1/PD-L1 inhibitors marks a significant advancement in cancer therapy. However, the efficacy of monotherapy is still disappointing in a substantial subset of patients, necessitating the exploration of combinational strategies. Emerging from the promising results of the KEYNOTE-942 trial, RNA-based therapies, particularly circRNAs and piRNAs, have distinguished themselves as innovative sensitizers to immune checkpoint inhibitors (ICIs). These non-coding RNAs, notable for their stability and specificity, were once underrecognized but are now known for their crucial roles in regulating PD-L1 expression and bolstering anti-cancer immunity. Our manuscript offers a comprehensive analysis of selected circRNAs and piRNAs, elucidating their immunomodulatory effects and mechanisms, thus underscoring their potential as ICIs enhancers. In conjunction with the recent Nobel Prize-awarded advancements in mRNA vaccine technology, our review highlights the transformative implications of these findings for cancer treatment. We also discuss the prospects of circRNAs and piRNAs in future therapeutic applications and research. This study pioneers the synergistic application of circRNAs and piRNAs as novel sensitizers to augment PD-1/PD-L1 inhibition therapy, demonstrating their unique roles in regulating PD-L1 expression and modulating immune responses. Our findings offer a groundbreaking approach for enhancing the efficacy of cancer immunotherapy, opening new avenues for treatment strategies. This abstract aims to encapsulate the essence of our research and the burgeoning role of these non-coding RNAs in enhancing PD-1/PD-L1 inhibition therapy, encouraging further investigation into this promising field.

Effective delivery of anti-PD-L1 siRNA with human heavy chain ferritin (HFn) in acute myeloid leukemia cell lines.

Because of the high biocompatibility, self-assembly capability, and CD71-mediated endocytosis, using human heavy chain ferritin (HFn) as a nanocarrier would greatly increase therapeutic effectiveness and reduce possible adverse events. Anti-PD-L1 siRNA can downregulate the level of PD-L1 on tumor cells, resulting in the activation of effector T cells against leukemia. Therefore, this study aimed to produce the tumor-targeting siPD-L1/HFn nanocarrier. Briefly, the HFn coding sequence was cloned into a pET-28a, and the constructed expression plasmid was subsequently transformed into E. coli BL21. After induction of Isopropyl ß-D-1-thiogalactopyranoside (IPTG), HFn was purified with Ni-affinity chromatography and dialyzed against PBS. The protein characteristics were analyzed using SDS-PAGE, Western Blot, and Dynamic light scattering (DLS). The final concentration was assessed using the Bicinchoninic acid (BCA) assay. The encapsulation was performed using the standard pH system. The treatment effects of siPD-L1/HFn were carried out on HL-60 and K-562 cancer cell lines. The RT-PCR was used to determine the mRNA expression of PD-L1. The biocompatibility and excretion of siPD-L1/HFn have also been evaluated. The expression and purity of HFn were well verified through SDS-PAGE, WB, and DLS. RT-PCR analyses also showed significant siRNA-mediated PD-L1 silencing in both HL-60 and K-562 cells. Our study suggested a promising approach for siRNA delivery. This efficient delivery system can pave the way for the co-delivery of siRNAs and multiple chemotherapies to address the emerging needs of cancer combination therapy.

Determinants of gastric cancer immune escape identified from non-coding immune-landscape quantitative trait loci.

The landscape of non-coding mutations in cancer progression and immune evasion is largely unexplored. Here, we identify transcrptome-wide somatic and germline 3' untranslated region (3'-UTR) variants from 375 gastric cancer patients from The Cancer Genome Atlas. By performing gene expression quantitative trait loci (eQTL) and immune landscape QTL (ilQTL) analysis, we discover 3'-UTR variants with cis effects on expression and immune landscape phenotypes, such as immune cell infiltration and T cell receptor diversity. Using a massively parallel reporter assay, we distinguish between causal and correlative effects of 3'-UTR eQTLs in immune-related genes. Our approach identifies numerous 3'-UTR eQTLs and ilQTLs, providing a unique resource for the identification of immunotherapeutic targets and biomarkers. A prioritized ilQTL variant signature predicts response to immunotherapy better than standard-of-care PD-L1 expression in independent patient cohorts, showcasing the untapped potential of non-coding mutations in cancer.

RICTOR amplification is associated with Rictor membrane staining and does not correlate with PD-L1 expression in lung squamous cell carcinoma.

RICTOR gene, which encodes the scaffold protein of mTORC2, can be amplified in various tumor types, including squamous cell carcinoma (SCC) of the lung. RICTOR amplification can lead to hyperactivation of mTORC2 and may serve as a targetable genetic alteration, including in lung SCC patients with no PD-L1 expression who are not expected to benefit from immune checkpoint inhibitor therapy. This study aimed to compare RICTOR amplification detected by fluorescence in situ hybridization (FISH) with Rictor and PD-L1 protein expression detected by immunohistochemistry (IHC) in SCC of the lung. The study was complemented by analysis of the publicly available Lung Squamous Cell Carcinoma (TCGA, Firehose legacy) dataset. RICTOR amplification was observed in 20% of our cases and 16% of the lung SCC cases of the TCGA dataset. Rictor and PD-L1 expression was seen in 74% and 44% of the cases, respectively. Rictor IHC showed two staining patterns: membrane staining (16% of the cases) and cytoplasmic staining (58% of the cases). Rictor membrane staining predicted RICTOR amplification as detected by FISH with high specificity (95%) and sensitivity (70%). We did not find any correlation between RICTOR amplification and PD-L1 expression; RICTOR amplification was detected in 18% and 26% of PD-L1 positive and negative cases, respectively. The TCGA dataset analysis showed similar results; RICTOR copy number correlated with Rictor mRNA and protein expression but showed no association with PD-L1 mRNA and protein expression. In conclusion, the correlation between RICTOR amplification and Rictor membrane staining suggests that the latter can potentially be used as a surrogate marker to identify lung SCC cases with RICTOR amplification. Since a significant proportion of PD-L1 negative SCC cases harbor RICTOR amplification, analyzing PD-L1 negative tumors by RICTOR FISH or Rictor IHC can help select patients who may benefit from mTORC2 inhibitor therapy.

Next-generation anti-PD-L1/IL-15 immunocytokine elicits superior antitumor immunity in cold tumors with minimal toxicity.

The clinical applications of immunocytokines are severely restricted by dose-limiting toxicities. To address this challenge, here we propose a next-generation immunocytokine concept involving the design of LH05, a tumor-conditional anti-PD-L1/interleukin-15 (IL-15) prodrug. LH05 innovatively masks IL-15 with steric hindrance, mitigating the "cytokine sink" effect of IL-15 and reducing systemic toxicities associated with wild-type anti-PD-L1/IL-15. Moreover, upon specific proteolytic cleavage within the tumor microenvironment, LH05 releases an active IL-15 superagonist, exerting potent antitumor effects. Mechanistically, the antitumor efficacy of LH05 depends on the increased infiltration of CD8+ T and natural killer cells by stimulating the chemokines CXCL9 and CXCL10, thereby converting cold tumors into hot tumors. Additionally, the tumor-conditional anti-PD-L1/IL-15 can synergize with an oncolytic virus or checkpoint blockade in advanced and metastatic tumor models. Our findings provide a compelling proof of concept for the development of next-generation immunocytokines, contributing significantly to current knowledge and strategies of immunotherapy.

The effects of HIV and oncogenic human papillomavirus on the tumor immune microenvironment of penile squamous cell carcinoma.

Penile squamous cell carcinoma (PSCC) occurs more frequently in some developing countries compared to developed countries. Infection with HIV and/or high-risk human papillomavirus (hrHPV) are risk factors for penile cancer development. The tumor microenvironment of PSCC may predict prognosis and may inform on the best targets for immunotherapy. We evaluated the immune microenvironment of penile tumors histologically, and determined whether and/or how HIV and/or hrHPV infections affect this tumor microenvironment. We conducted a prospective analytical cross-sectional study in which penile cancer tumors from 35 patients presenting at the University Teaching Hospital in Lusaka, Zambia were histologically staged and assessed for presence of tumor infiltrating immune cells and expression of immune checkpoints. Immunohistochemistry was used to evaluate immune checkpoints and infiltrating immune cells, while multiplex real-time polymerase chain reaction was used for hrHPV genotyping. The median age of all participants was 55 years. About 24% had advanced histological stage, 83% were HIV+, and 63% had hrHPV detected in their tumors using multiplex real-time polymerase chain reaction. PDL1 expression was significantly higher in HIV- participants than HIV+ participants (p = 0.02). Tumors with multiple hrHPV infections had a significantly higher number of cells expressing TIM3 than those with one hrHPV (p = 0.04). High grade tumors had a significantly higher infiltrate of FoxP3+ cells (p = 0.02), CD68+ cells (p = 0.01), CD163+ cells (p = 0.01), LAG3+ cells (p = 0.01), PD1+ cells (p = 0.01) and TIM3+ cells (p = 0.03) when compared with low grade tumours. There was significant moderate to strong positive correlation of cells expressing PD1 and LAG3 (⍴ = 0.69; p = 0.0001), PD1 and TIM3 (⍴ = 0.49; p = 0.017) and TIM3 and LAG3 PDL1 (⍴ = 0.61; p = 0.001). In conclusion, the tumor microenvironment of penile squamous cell carcinoma seems to be affected by both HIV and HPV infections. TIM3 appears to be a potential therapeutic target in PSCC patients with hrHPV infections.

Upregulation of Immune checkpoint PD-L1 in Colon cancer cell lines and activation of T cells by Leuconostoc mesenteroides.

Globally colorectal cancer ranks as the third most widespread disease and the third leading cause of cancer-associated mortality. Immunotherapy treatments like PD-L1 blockade have been used to inhibit the PD-L1 legend, which boosts the activity of cytotoxic T lymphocytes. Recently, studies suggest that some probiotics could potentially enhance the effectiveness of immunotherapy treatments for cancer patients. We found that in Caco-2 and HT-29 cells, the live Leuconostoc mesenteroides treatment resulted an increase in the PD-L1 expression and this treatment stimulated interferon-gamma (IFN-γ) production in Jurkat T-cells. Due to the well-established ability of IFN-γ to enhance PD-L1 expression, the combination of IFN-γ and L. mesenteroides was used in colon cancer cell lines and a resulting remarkable increase of over tenfold in PD-L1 expression was obtained. Interestingly, when L. mesenteroides and IFN-γ are present, the blockage of PD-L1 using PD-L1 antibodies not only improved the viability of Jurkat T-cells but also significantly boosted the levels of IFN-γ and IL-2, the T-cells activation marker cytokines. In addition to upregulating PD-L1, L. mesenteroides also activated Toll-like receptors (TLRs) and NOD-like receptors (NODs) pathways, specifically through TLR2 and NOD2, while also exerting a suppressive effect on autophagy in colon cancer cell lines. In conclusion, our findings demonstrate a significant upregulation of PD-L1 expression in colon cancer cells upon co-culturing with L. mesenteroides. Moreover, the presence of PD-L1 antibodies during co-culturing activates Jurkat T cells. The observed enhancement in PD-L1 expression may be attributed to the inhibition of the Autophagy pathway or activation of the hippo pathway. KEY POINTS: Co-culturing L. mesenteroides increases PD-L1 gene and protein transaction in colon cancer. L. mesenteroides existing enhances T cells viability and activity. GPCR41/42 is a possible link between L. mesenteroides, YAP-1 and PD-L1.

Microsatellite Instability, Mismatch Repair, and Tumor Mutation Burden in Lung Cancer.

Since US Food and Drug Administration approval of programmed death ligand 1 (PD-L1) as the first companion diagnostic for immune checkpoint inhibitors (ICIs) in non-small cell lung cancer, many patients have experienced increased overall survival. To improve selection of ICI responders versus nonresponders, microsatellite instability/mismatch repair deficiency (MSI/MMR) and tumor mutation burden (TMB) came into play. Clinical data show PD-L1, MSI/MMR, and TMB are independent predictive immunotherapy biomarkers. Harmonization of testing methodologies, optimization of assay design, and results analysis are ongoing. Future algorithms to determine immunotherapy eligibility might involve complementary use of current and novel biomarkers. Artificial intelligence could facilitate algorithm implementation to convert complex genetic data into recommendations for specific ICIs.

circATAD2 mitigates CD8+ T cells antitumor immune surveillance in breast cancer via IGF2BP3/m6A/PD-L1 manner.

Immune surveillance and chemotherapy sensitivity play critical functions in the tumorigenesis of breast cancer (BC). Emerging findings have indicated that circular RNA (circRNA) and N6-methyladenosine (m6A) both participate in the BC tumorigenesis. Here, present study aimed to investigate the roles of m6A-modified circATAD2 on BC and explore better understanding for BC precision therapeutic. Results reported that m6A-modifid circRNA (m6A-circRNA) microarray revealed the m6A-circRNA landscape in BC. M6A-modifid circATAD2 upregulated in BC samples and was closely correlated to poor prognosis. Functionally, circATAD2 promoted the immune evasion of BC cells and reduced the CD8+ T cells' killing effect. Mechanistically, MeRIP-seq unveiled the m6A modification in the 3'-UTR of PD-L1 mRNA, which was bound by circATAD2 and recognized by m6A reader IGF2BP3 to enhance PD-L1 mRNA stability and expression. In summary, these findings revealed the circATAD2/m6A/IGF2BP3/PD-L1 axis in BC immune surveillance, suggesting the potential that circATAD2 as a potential target for PD-L1-mediated BC.

Proton pump inhibitors stabilize the expression of PD-L1 on cell membrane depending on the phosphorylation of GSK3ß.

BACKGROUND: Preclinical and clinical evidence indicates that proton pump inhibitors (PPIs) may indirectly diminish the microbiome diversity, thereby reducing the effectiveness of immune checkpoint inhibitors (ICIs). Conversely, recent publications have shown that PPIs could potentially enhance the response to ICIs. The precise mechanism through which PPIs modulate the ICIs remains unclear. In this study, we discovered a novel molecular function of PPIs in regulating immune invasion, specifically through inducing PD-L1 translocation in various tumor cells. METHODS: C57BL/6 mice subcutaneous transplantation model is used to verify the potential efficacy of PPIs and PD-L1 antibody. Western blotting analysis and phosphorylated chip are used to verify the alteration of PD-L1-related pathways after being treated with PPIs. The related gene expression is performed by qRT-PCR and luciferase reporter analysis. We also collected 60 clinical patients diagnosed with esophageal cancer or reflux esophagitis and then detected the expression of PD-L1 in the tissue samples by immunohistochemistry. RESULTS: We observed that the IC50 of tumor cells in response to PPIs was significantly higher than that of normal epithelial cells. PPIs significantly increased the expression of PD-L1 on cell membrane at clinically relevant concentrations. Furthermore, pre-treatment with PPIs appeared to synergize the efficiency of anti-PD-L1 antibodies in mouse models. However, PPI administration did not alter the transcription or total protein level of PD-L1 in multiple tumor cells. Using a phosphorylated protein chip, we identified that PPIs enhanced the phosphorylation of GSK3ß, then leading to PD-L1 protein translocation to the cell membranes. The capacity of PPIs to upregulate PD-L1 was negated following GSK3ß knockout. Furthermore, our clinical data showed that the PPIs use resulted in increased PD-L1 expression in esophageal cancer patients. CONCLUSION: We mainly address a significant and novel mechanism that the usage of PPIs could directly induce the expression of PD-L1 by inducing GSK3ß phosphorylation and facilitate primary tumor progression and metastasis.

Exploring novel immunotherapy biomarker candidates induced by cancer deformation.

Triple-negative breast cancer (TNBC) demands urgent attention for the development of effective treatment strategies due to its aggressiveness and limited therapeutic options [1]. This research is primarily focused on identifying new biomarkers vital for immunotherapy, with the aim of developing tailored treatments specifically for TNBC, such as those targeting the PD-1/PD-L1 pathway. To achieve this, the study places a strong emphasis on investigating Ig genes, a characteristic of immune checkpoint inhibitors, particularly genes expressing Ig-like domains with altered expression levels induced by "cancer deformation," a condition associated with cancer malignancy. Human cells can express approximately 800 Ig family genes, yet only a few Ig genes, including PD-1 and PD-L1, have been developed into immunotherapy drugs thus far. Therefore, we investigated the Ig genes that were either upregulated or downregulated by the artificial metastatic environment in TNBC cell line. As a result, we confirmed the upregulation of approximately 13 Ig genes and validated them using qPCR. In summary, our study proposes an approach for identifying new biomarkers applicable to future immunotherapies aimed at addressing challenging cases of TNBC where conventional treatments fall short.

Protein kinase CK2α is overexpressed in classical hodgkin lymphoma, regulates key signaling pathways, PD-L1 and may represent a new target for therapy.

Introduction: In classical Hodgkin lymphoma (cHL), the survival of neoplastic cells is mediated by the activation of NF-κB, JAK/STAT and PI3K/Akt signaling pathways. CK2 is a highly conserved serine/threonine kinase, consisting of two catalytic (α) and two regulatory (ß) subunits, which is involved in several cellular processes and both subunits were found overexpressed in solid tumors and hematologic malignancies. Methods and results: Biochemical analyses and in vitro assays showed an impaired expression of CK2 subunits in cHL, with CK2α being overexpressed and a decreased expression of CK2ß compared to normal B lymphocytes. Mechanistically, CK2ß was found to be ubiquitinated in all HL cell lines and consequently degraded by the proteasome pathway. Furthermore, at basal condition STAT3, NF-kB and AKT are phosphorylated in CK2-related targets, resulting in constitutive pathways activation. The inhibition of CK2 with CX-4945/silmitasertib triggered the de-phosphorylation of NF-κB-S529, STAT3-S727, AKT-S129 and -S473, leading to cHL cell lines apoptosis. Moreover, CX-4945/silmitasertib was able to decrease the expression of the immuno-checkpoint CD274/PD-L1 but not of CD30, and to synergize with monomethyl auristatin E (MMAE), the microtubule inhibitor of brentuximab vedotin. Conclusions: Our data point out a pivotal role of CK2 in the survival and the activation of key signaling pathways in cHL. The skewed expression between CK2α and CK2ß has never been reported in other lymphomas and might be specific for cHL. The effects of CK2 inhibition on PD-L1 expression and the synergistic combination of CX-4945/silmitasertib with MMAE pinpoints CK2 as a high-impact target for the development of new therapies for cHL.

GSG2 promotes progression of human endometrial cancer by regulating PD-1/PD-L1 expression via PI3K-AKT pathway.

Cell cycle dysregulation leading to uncontrolled growth is a primary characteristic of malignancy. GSG2, a mitosis-related kinase, affects the normal cell cycle by interfering with the normal dissociation of centromere cohesion, and its overexpression has been shown to play an important role in cancer cells. Here, we investigated the function of GSG2 as a tumor promoter in endometrial carcinoma and its relationship with the immunological microenvironment. We used immunohistochemistry to identify a correlation between the development and prognosis of GSG2 and endometrial cancer. Cell and animal experiments confirmed that GSG2 has a protumorigenic phenotype in endometrial cancer cell lines. Furthermore, using GeneChip analysis and a tumor-immune coculture model, we observed a link between GSG2 expression and the composition of the immune microenvironment. Therefore, we concluded that the activation of the PI3K/AKT pathway by GSG2 may impact DNA repair, disrupt the cell cycle, and regulate the immune response, all of which could increase the ability of EC cells to proliferate malignantly. Consequently, it is anticipated that GSG2 will be a viable therapeutic target in endometrial carcinoma.

PD-L1 knockdown suppresses vasculogenic mimicry of non-small cell lung cancer by modulating ZEB1-triggered EMT.

BACKGROUND: PD-L1 overexpression is commonly observed in various malignancies and is strongly correlated with poor prognoses for cancer patients. Moreover, PD-L1 has been shown to play a significant role in promoting angiogenesis and epithelial-mesenchymal transition (EMT) processes across different cancer types. METHODS: The relationship between PD-L1 and vasculogenic mimicry as well as epithelial-mesenchymal transition (EMT) was explored by bioinformatics approach and immunohistochemistry. The functions of PD-L1 in regulating the expression of ZEB1 and the EMT process were assessed by Western blotting and q-PCR assays. The impact of PD-L1 on the migratory and proliferative capabilities of A549 and H1299 cells was evaluated through wound healing, cell invasion, and CCK8 assays following siRNA-mediated PD-L1 knockdown. Tube formation assay was utilized to evaluate the presence of VM structures. RESULTS: In this study, increased PD-L1 expression was observed in A549 and H1299 cells compared to normal lung epithelial cells. Immunohistochemical analysis revealed a higher prevalence of VM structures in the PD-L1-positive group compared to the PD-L1-negative group. Additionally, high PD-L1 expression was also found to be significantly associated with advanced TNM stage and increased metastasis. Following PD-L1 knockdown, NSCLC cells exhibited a notable reduction in their ability to form tube-like structures. Moreover, the levels of key EMT and VM-related markers, including N-cadherin, MMP9, VE-cadherin, and VEGFA, were significantly decreased, while E-cadherin expression was upregulated. In addition, the migration and proliferation capacities of both cell lines were significantly inhibited after PD-L1 or ZEB1 knockdown. CONCLUSIONS: Knockdown PD-L1 can inhibit ZEB1-mediated EMT, thereby hindering the formation of VM in NSCLC.

Factors correlating the expression of PD-L1.

OBJECTIVE: PD-L1 was an important biomarker in lung adenocarcinoma. The study was to confirm the most important factor affecting the expression of PD-L1 remains undetermined. METHODS: The clinical records of 1045 lung adenocarcinoma patients were retrospectively reviewed. The High-Resolution Computed Tomography (HRCT) scanning images of all the participants were analyzed, and based on the CT characteristics, the adenocarcinomas were categorized according to CT textures. Furthermore, PD-L1 expression and Ki67 index were detected by immunohistochemistry. All patients underwent EGFR mutation detection. RESULTS: Multivariate logistic regression analysis revealed that smoking (OR: 1.73, 95% CI: 1.04-2.89, p = 0.004), EGFR wild (OR: 1.52, 95% CI: 1.11-2.07, p = 0.009), micropapillary subtypes (OR: 2.05, 95% CI: 1.46-2.89, p < 0.0001), and high expression of Ki67 (OR: 2.02, 95% CI: 1.44-2.82, p < 0.0001) were independent factors which influence PD-L1 expression. In univariate analysis, tumor size > 3 cm and CT textures of pSD showed a correlation with high expression of PD-L1. Further analysis revealed that smoking, micropapillary subtype, and EGFR wild type were also associated with high Ki67 expression. Moreover, high Ki67 expression was observed more frequently in tumors of size > 3 cm than in tumors with ≤ 3 cm size as well as in CT texture of pSD than lesions with GGO components. In addition, multivariate logistic regression analysis revealed that only lesions with micropapillary components correlated with pSD (OR: 3.96, 95% CI: 2.52-5.37, p < 0.0001). CONCLUSION: This study revealed that in lung adenocarcinoma high Ki67 expression significantly influenced PD-L1 expression, an important biomarker for immune checkpoint treatment.

Genomic characterization reveals distinct mutational landscapes and therapeutic implications between different molecular subtypes of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) has high heterogeneity, poor prognosis, and limited treatment success. Recently, an immunohistochemistry-based surrogate classification for the "Fudan University Shanghai Cancer Center (FUSCC) subtyping" has been developed and is considered more suitable for clinical application. Seventy-one paraffin-embedded sections of surgically resected TNBC were classified into four molecular subtypes using the IHC-based surrogate classification. Genomic analysis was performed by targeted next-generation sequencing and the specificity of the subtypes was explored by bioinformatics, including survival analysis, multivariate Cox regression, pathway enrichment, Pyclone analysis, mutational signature analysis and PHIAL analysis. AKT1 and BRCA1 mutations were identified as independent prognostic factors in TNBC. TNBC molecular subtypes encompass distinct genomic landscapes that show specific heterogeneities. The luminal androgen receptor (LAR) subtype was associated with mutations in PIK3CA and PI3K pathways, which are potentially sensitive to PI3K pathway inhibitors. The basal-like immune-suppressed (BLIS) subtype was characterized by high genomic instability and the specific possession of signature 19 while patients in the immunomodulatory (IM) subtype belonged to the PD-L1 ≥ 1% subgroup with enrichment in Notch signaling, suggesting a possible benefit of immune checkpoint inhibitors and Notch inhibitors. Moreover, mesenchymal-like (MES) tumors displayed enrichment in the receptor tyrosine kinase (RTK)-RAS pathway and potential sensitivity to RTK pathway inhibitors. The findings suggest potential treatment targets and prognostic factors, indicating the possibility of TNBC stratified therapy in the future.

Targeting PD-L1 in solid cancer with myeloid cells expressing a CAR-like immune receptor.

Introduction: Solid cancers Myeloid cells are prevalent in solid cancers, but they frequently exhibit an anti-inflammatory pro-tumor phenotype that contribute to the immunosuppressive tumor microenvironment (TME), which hinders the effectiveness of cancer immunotherapies. Myeloid cells' natural ability of tumor trafficking makes engineered myeloid cell therapy an intriguing approach to tackle the challenges posed by solid cancers, including tumor infiltration, tumor cell heterogenicity and the immunosuppressive TME. One such engineering approach is to target the checkpoint molecule PD-L1, which is often upregulated by solid cancers to evade immune responses. Method: Here we devised an adoptive cell therapy strategy based on myeloid cells expressing a Chimeric Antigen Receptor (CAR)-like immune receptor (CARIR). The extracellular domain of CARIR is derived from the natural inhibitory receptor PD-1, while the intracellular domain(s) are derived from CD40 and/or CD3ζ. To assess the efficacy of CARIR-engineered myeloid cells, we conducted proof-of-principle experiments using co-culture and flow cytometry-based phagocytosis assays in vitro. Additionally, we employed a fully immune-competent syngeneic tumor mouse model to evaluate the strategy's effectiveness in vivo. Result: Co-culturing CARIR-expressing human monocytic THP-1 cells with PD-L1 expressing target cells lead to upregulation of the costimulatory molecule CD86 along with expression of proinflammatory cytokines TNF-1α and IL-1ß. Moreover, CARIR expression significantly enhanced phagocytosis of multiple PD-L1 expressing cancer cell lines in vitro. Similar outcomes were observed with CARIR-expressing human primary macrophages. In experiments conducted in syngeneic BALB/c mice bearing 4T1 mammary tumors, infusing murine myeloid cells that express a murine version of CARIR significantly slowed tumor growth and prolonged survival. Conclusion: Taken together, these results demonstrate that adoptive transfer of PD-1 CARIR-engineered myeloid cells represents a promising strategy for treating PD-L1 positive solid cancers.

Comprehensive Analysis of CXCR4, JUNB, and PD-L1 Expression in Circulating Tumor Cells (CTCs) from Prostate Cancer Patients.

CXCR4, JUNB and PD-L1 are implicated in cancer progression and metastasis. The current study investigated these biomarkers in CTCs isolated from metastatic prostate cancer (mPCa) patients at the RNA and protein levels. CTCs were isolated from 48 mPCa patients using the Ficoll density gradient and ISET system (17 out of 48). The (CK/PD-L1/CD45) and (CK/CXCR4/JUNB) phenotypes were identified using two triple immunofluorescence stainings followed by VyCAP platform analysis. Molecular analysis was conducted with an EpCAM-dependent method for 25/48 patients. CK-8, CK-18, CK-19, JUNB, CXCR4, PD-L1, and B2M (reference gene) were analyzed with RT-qPCR. The (CK+/PD-L1+/CD45-) and the (CK+/CXCR4+/JUNB+) were the most frequent phenotypes (61.1% and 62.5%, respectively). Furthermore, the (CK+/CXCR4+/JUNB-) phenotype was correlated with poorer progression-free survival [(PFS), HR: 2.5, p = 0.049], while the (CK+/PD-L1+/CD45-) phenotype was linked to decreased overall survival [(OS), HR: 262.7, p = 0.007]. Molecular analysis revealed that 76.0% of the samples were positive for CK-8,18, and 19, while 28.0% were positive for JUNB, 44.0% for CXCR4, and 48.0% for PD-L1. Conclusively, CXCR4, JUNB, and PD-L1 were highly expressed in CTCs from mPCa patients. The CXCR4 protein expression was associated with poorer PFS, while PD-L1 was correlated with decreased OS, providing new biomarkers with potential clinical relevance.