Immune Checkpoint Ligand Research Articles

Microsatellite Instability and Loss of Heterozygosity as Prognostic Markers in Oral Squamous Cell Carcinoma: Molecular Mechanisms, Detection Techniques, and Therapeutic Strategies.

The aim of this study was to conduct a systematic review of research investigating the potential role of microsatellite instability (MSI) and loss of heterozygosity (LOH) in oral squamous cell carcinoma (OSCC), with a focus on molecular mechanisms, detection methods, and therapeutic approaches. Search for articles involved the PubMed and Scopus. Previous retrospective and prospective studies identified variations between oral cancers that exhibit microsatellite stability and LOH. In this search, 294 articles were initially retrieved. Of these, 70 were excluded due to duplication, 106 were identified as ineligible by automated tools, and 24 were excluded as they were published in languages other than English. An additional 94 articles were excluded, 32 of which focused on head and neck cancers broadly, and 8 could not be accessed due to withdrawal. Ultimately, a systematic review was conducted based on 54 selected articles. Among the chromosomes analyzed for MSI and LOH, the highest frequency of LOH was observed on chromosome 9p. The MSI subtype is particularly susceptible to immunotherapeutic methods, such as the use of anti-PD-L1 and anti-CTLA4 antibodies, owing to its strong immunogenicity and ubiquitous expression of immune checkpoint ligands. Given the distinct characteristics and clinical behavior of oral cancer with MSI compared to microsatellite stable disease, it is advisable to incorporate MSI testing into the diagnostic process for all stages of tumor development. This ensured that each patient had received precise and effective treatment.

Effect of plasma-induced oxidation on NK cell immune checkpoint ligands: A computational-experimental approach

Non-thermal plasma (NTP) shows promise as a potent anti-cancer therapy with both cytotoxic and immunomodulatory effects. In this study, we investigate the chemical and biological effects of NTP-induced oxidation on several key, determinant immune checkpoints of natural killer (NK) cell function. We used molecular dynamics (MD) and umbrella sampling simulations to investigate the effect of NTP-induced oxidative changes on the MHC-I complexes HLA-Cw4 and HLA-E. Our simulations indicate that these chemical alterations do not significantly affect the binding affinity of these markers to their corresponding NK cell receptor, which is supported with experimental read-outs of ligand expression on human head and neck squamous cell carcinoma cells after NTP application. Broadening our scope to other key ligands for NK cell reactivity, we demonstrate rapid reduction in CD155 and CD112, target ligands of the inhibitory TIGIT axis, and in immune checkpoint CD73 immediately after treatment. Besides these transient chemical alterations, the reactive species in NTP cause a cascade of downstream cellular reactions. This is underlined by the upregulation of the stress proteins MICA/B, potent ligands for NK cell activation, 24 h post treatment. Taken together, this work corroborates the immunomodulatory potential of NTP, and sheds light on the interaction mechanisms between NTP and cancer cells.

Multi-omics landscape of Interferon-stimulated gene OASL reveals a potential biomarker in pan-cancer: from prognosis to tumor microenvironment.

OASL (Oligoadenylate Synthetase-Like), an interferon-induced protein in the OAS family, plays a significant role in anti-viral response. Studies have demonstrated its association with prognosis of certain tumors. However, the mechanism through which OASL affects tumors is unclear. A systemic pan-cancer study of OASL needs to be illustrated. Analysis of OASL expression across 33 tumors was conducted utilizing TCGA, GTEx and CPTAC databases. COX and Log-Rank regressions were employed to calculate the prognosis. We validated the impact of OASL on apoptosis, migration, and invasion in pancreatic cancer cell lines. Moreover, we employed seven algorithms in bulk data to investigate the association of OASL expression and immune cell infiltration within tumor immune microenvironment (TIME) and ultimately validated at single-cell transcriptome level. We discovered elevated expression of OASL and its genetic heterogeneity in certain tumors, which link closely to prognosis. Validation experiments were conducted in PAAD and confirmed these findings. Additionally, OASL regulates immune checkpoint ligand such as programmed death ligand 1 (PD-L1), through IFN-γ/STAT1 and IL-6/JAK/STAT3 pathways in tumor cells. Meanwhile, OASL affects macrophages infiltration in TIME. By these mechanisms OASL could cause dysfunction of cytotoxic T lymphocytes (CTLs) in tumors. Multi-omics analysis reveals OASL as a prognostic and immunological biomarker in pan-cancer.

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.

IFNγ-Induced Bcl3, PD-L1 and IL-8 Signaling in Ovarian Cancer: Mechanisms and Clinical Significance.

IFNγ, a pleiotropic cytokine produced not only by activated lymphocytes but also in response to cancer immunotherapies, has both antitumor and tumor-promoting functions. In ovarian cancer (OC) cells, the tumor-promoting functions of IFNγ are mediated by IFNγ-induced expression of Bcl3, PD-L1 and IL-8/CXCL8, which have long been known to have critical cellular functions as a proto-oncogene, an immune checkpoint ligand and a chemoattractant, respectively. However, overwhelming evidence has demonstrated that these three genes have tumor-promoting roles far beyond their originally identified functions. These tumor-promoting mechanisms include increased cancer cell proliferation, invasion, angiogenesis, metastasis, resistance to chemotherapy and immune escape. Recent studies have shown that IFNγ-induced Bcl3, PD-L1 and IL-8 expression is regulated by the same JAK1/STAT1 signaling pathway: IFNγ induces the expression of Bcl3, which then promotes the expression of PD-L1 and IL-8 in OC cells, resulting in their increased proliferation and migration. In this review, we summarize the recent findings on how IFNγ affects the tumor microenvironment and promotes tumor progression, with a special focus on ovarian cancer and on Bcl3, PD-L1 and IL-8/CXCL8 signaling. We also discuss promising novel combinatorial strategies in clinical trials targeting Bcl3, PD-L1 and IL-8 to increase the effectiveness of cancer immunotherapies.

Cancer cell plasticity defines response to immunotherapy in cutaneous squamous cell carcinoma

Immune checkpoint blockade (ICB) approaches have changed the therapeutic landscape for many tumor types. However, half of cutaneous squamous cell carcinoma (cSCC) patients remain unresponsive or develop resistance. Here, we show that, during cSCC progression in male mice, cancer cells acquire epithelial/mesenchymal plasticity and change their immune checkpoint (IC) ligand profile according to their features, dictating the IC pathways involved in immune evasion. Epithelial cancer cells, through the PD-1/PD-L1 pathway, and mesenchymal cancer cells, through the CTLA-4/CD80 and TIGIT/CD155 pathways, differentially block antitumor immune responses and determine the response to ICB therapies. Accordingly, the anti-PD-L1/TIGIT combination is the most effective strategy for blocking the growth of cSCCs that contain both epithelial and mesenchymal cancer cells. The expression of E-cadherin/Vimentin/CD80/CD155 proteins in cSCC, HNSCC and melanoma patient samples predicts response to anti-PD-1/PD-L1 therapy. Collectively, our findings indicate that the selection of ICB therapies should take into account the epithelial/mesenchymal features of cancer cells.

The expression of Galectin-9 correlates with mTOR and AMPK in murine colony-forming erythroid progenitors.

Galectin-9 (Gal-9) is an immune checkpoint ligand for T-cell immunoglobulin and mucin domain 3. Although the roles of Gal-9 in regulating immune responses have been well investigated, their biological roles have yet to be fully documented. This study aimed to analyse the expression of Gal-9 bone marrow (BM) cells in C57BL/6J (B6) mice. Furthermore, the co-expression of Gal-9 with the mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) was investigated. The BM cells in adult C57BL/6J (B6) mice were collected and analysed in vitro. In a flow cytometric analysis of BM cells, Gal-9 was highly expressed in c-KithiSca-1-CD34-CD71+ erythroid progenitors (EPs), whereas it was downregulated in more differentiated c-KitloCD71+TER119+ cells. Subsequently, a negative selection of CD3-B220-Sca-1-CD34-CD41-CD16/32- EPs was performed. This resulted in substantial enrichment of KithiCD71+Gal-9+ cells and erythroid colony-forming units (CFU-Es), suggesting that the colony-forming subset of EPs are included in the KithiCD71+Gal-9+ population. Furthermore, we found that EPs had lower mTOR and AMPK expression levels in Gal-9 knockout B6 mice than in wild-type B6 mice. These results may stimulate further investigation of the role of Gal-9 in haematopoiesis.

Mathematical modeling and analysis of cancer treatment with radiation and anti-PD-L1

In cancer treatment, radiation therapy (RT) induces direct tumor cell death due to DNA damage, but it also enhances the deaths of radiosensitive immune cells and is followed by local relapse and up-regulation of immune checkpoint ligand PD-L1. Since the binding between PD-1 and PD-L1 curtails anti-tumor immunities, combining RT and PD-L1 inhibitor, anti-PD-L1, is a potential method to improve the treatment efficacy by RT. Some experiments support this hypothesis by showing that the combination of ionizing irradiation (IR) and anti-PD-L1 improves tumor reduction comparing to the monotherapy of IR or anti-PD-L1. In this work, we create a simplified ODE model to study the order of tumor growths under treatments of IR and anti-PD-L1. Our synergy analysis indicates that both IR and anti-PD-L1 improve the tumor reduction of each other, when IR and anti-PD-L1 are given simultaneously. When giving IR and anti-PD-L1 separately, a high dosage of IR should be given first to efficiently reduce tumor load and then followed by anti-PD-L1 with strong efficacy to maintain the tumor reduction and slow down the relapse. Increasing the duration of anti-PD-L1 improves the tumor reduction, but it cannot prolong the duration that tumor relapses to the level of the control case. Under some simplification, we also prove that the model has an unstable tumor free equilibrium and a locally asymptotically stable tumor persistent equilibrium. Our bifurcation diagram reveals a transition from tumor elimination to tumor persistence, as the tumor growth rate increases. In the tumor persistent case, both anti-PD-L1 and IR can reduce tumor amount in the long term.

GAS-Luc2 Reporter Cell Lines for Immune Checkpoint Drug Screening in Solid Tumors.

Recent studies highlight the integral role of the interferon gamma receptor (IFNγR) pathway in T cell-mediated cytotoxicity against solid but not liquid tumors. IFNγ not only directly facilitates tumor cell death by T cells but also indirectly promotes cytotoxicity via myeloid phagocytosis in the tumor microenvironment. Meanwhile, full human ex vivo immune checkpoint drug screening remains challenging. We hypothesized that an engineered gamma interferon activation site response element luciferase reporter (GAS-Luc2) can be utilized for immune checkpoint drug screening in diverse ex vivo T cell-solid tumor cell co-culture systems. We comprehensively profiled cell surface proteins in ATCC's extensive collection of human tumor and immune cell lines, identifying those with endogenously high expression of established and novel immune checkpoint molecules and binding ligands. We then engineered three GAS-Luc2 reporter tumor cell lines expressing immune checkpoints PD-L1, CD155, or B7-H3/CD276. Luciferase expression was suppressed upon relevant immune checkpoint-ligand engagement. In the presence of an immune checkpoint inhibitor, T cells released IFNγ, activating the JAK-STAT pathway in GAS-Luc2 cells, and generating a quantifiable bioluminescent signal for inhibitor evaluation. These reporter lines also detected paracrine IFNγ signaling for immune checkpoint-targeted ADCC drug screening. Further development into an artificial antigen-presenting cell line (aAPC) significantly enhanced T cell signaling for superior performance in these ex vivo immune checkpoint drug screening platforms.

Bioengineered Artificial Extracellular Vesicles Presenting PD-L1 and Gal-9 Ameliorate New-Onset Type 1 Diabetes.

An important factor in the development of Type 1 diabetes (T1D) is the deficiency of inhibitory immune checkpoint ligands, specifically programmed cell death ligand 1 (PD-L1) and Galectin-9 (Gal-9), in β-cells. Hence, modulation of the pancreas infiltrated T lymphocytes by exogenous PD-L1 or Gal-9 is an ideal approach for treating the new-onset T1D. Herein, we genetic engineered the macrophage cells to generate artificial extracellular vesicles (aEVs) overexpressing PD-L1 and Gal-9, which could restrict the islets autoreactive T lymphocytes and protect β-cells from destruction. Intriguingly, overexpressing Gal-9 spurred macrophage polarization to M2 phenotype with immune suppressive attribute. Alternatively, both of PD-L1 and Gal-9 presenting aEVs (PD-L1-Gal-9 aEVs) favorably adhere to T cells via the interaction of programmed cell death protein 1 (PD-1)/PD-L1 or T cell immunoglobulin mucin 3 (TIM-3)/Gal-9. Moreover, PD-L1-Gal-9 aEVs prominently promoted effector T cell apoptosis and splenic regulatory T cells (Treg) cells differentiation in vitro. Virtually, PD-L1-Gal-9 aEVs efficaciously reversed the new-onset hyperglycemia in the NOD mice, prevented T1D progress, and declined the proportion and activation of CD4+ and CD8+ T cells infiltrating the pancreas notably, which together contributed to preserving the residual β-cells survival and mitigating the hyperglycemia.

Activity of pyridyl-pyrazolone derivatives against Trypanosoma cruzi

New affordable drugs are needed for the treatment of infection with the protozoan parasite Trypanosoma cruzi responsible for the Chagas disease (CD). Only two old drugs are currently available, nifurtimox and benznidazole (Bz) but they exhibit unwanted side effects and display a weak activity in the late chronic phase of the disease. In this context, we evaluated the activity of a series of aryl-pyrazolone derivatives against T cruzi, using both bloodstream trypomastigote and intracellular amastigote forms of the parasite. The test compounds originate from a series of anticancer agents targeting the immune checkpoint ligand PD-L1 and bear an analogy with known anti-trypanosomal pyrazolones. A first group of 6 phenyl-pyrazolones was tested, revealing the activity of a single pyridyl-pyrazolone derivative. Then a second group of 8 compounds with a common pyridyl-pyrazolone core was evaluated. The in vitro testing process led to the identification of two non-cytotoxic and highly potent molecules against the intracellular form of T. cruzi, with an activity comparable to Bz. Moreover, one compound revealed an activity largely superior to that of Bz against bloodstream trypomastigotes, while being non-cytotoxic (selectivity index >1000). Unfortunately, the compound showed little activity in vivo, most likely due to its very limited plasma stability. However, the study opens novel perspectives for the design of new anti-trypanosomal products and the mechanism of action of the compounds is discussed.

Abstract PO3-24-03: Cisplatin synergizes immune checkpoint blocking through RNFT1 mediated PD-L1 recycling in Triple Negative Breast Cancer

Abstract Since chemotherapy showed synergies with PD-1/PD-L1 blockage therapy in solid tumors, the efficacy in triple negative breast cancer (TNBC) varies greatly and only a subset of patients achieves durable responses. To increase their clinical efficacy, it is important to explore the underlying mechanisms of immune checkpoint ligand PD-L1. Here, we found cisplatin influenced the controlled intracellular transport of PDL1 by inducing the RNFT1-mediated endoplasmic reticulum autophagy. Overexpression of RNFT1 increases the endocytosis-recycling rate of the plasm membrane PD-L1, which is regulated by K63-polyubiquitination and endoplasmic reticulum autophagy degradation of PD-L1, thereby having negative impact on antitumor immunotherapy, such as reduced effective drug concentration and avoiding immune surveillance. Genetically or pharmacologically modulating RNFT1-induced K63 polyubiquitination blocks endocytosis of PD-L1, consequently enhances the antitumor response to PD-L1 blockade. Thus, our results suggest that cisplatin treatment is involved in RNFT1-induced K63 polyubiquitination of PD-L1 endocytosis-recycling pathway that governs immune response, and blocking RNFT1 might enhance the efficacy of PD-1/PD-L1 blockade. These studies have high application and translational potential in triple-negative breast cancer. Citation Format: Jiahui Chu, Yifan Zhang, Yijia Hua, Jue Gong, Jie Mei, Ziyi Fu, Yongmei Yi. Cisplatin synergizes immune checkpoint blocking through RNFT1 mediated PD-L1 recycling in Triple Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-03.

Immunological Characteristics of Hepatic Dendritic Cells in Patients and Mouse Model with Liver Echinococcus multilocularis Infection.

The cestode Echinococcus multilocularis, which mainly dwells in the liver, leads to a serious parasitic liver disease called alveolar echinococcosis (AE). Despite the increased attention drawn to the immunosuppressive microenvironment formed by hepatic AE tissue, the immunological characteristics of hepatic dendritic cells (DCs) in the AE liver microenvironment have not been fully elucidated. Here, we profiled the immunophenotypic characteristics of hepatic DC subsets in both clinical AE patients and a mouse model. Single-cell RNA sequencing (scRNA-Seq) analysis of four AE patient specimens revealed that greater DC numbers were present within perilesional liver tissues and that the distributions of cDC and pDC subsets in the liver and periphery were different. cDCs highly expressed the costimulatory molecule CD86, the immune checkpoint molecule CD244, LAG3, CTLA4, and the checkpoint ligand CD48, while pDCs expressed these genes at low frequencies. Flow cytometric analysis of hepatic DC subsets in an E. multilocularis infection mouse model demonstrated that the number of cDCs significantly increased after parasite infection, and a tolerogenic phenotype characterized by a decrease in CD40 and CD80 expression levels was observed at an early stage, whereas an activated phenotype characterized by an increase in CD86 expression levels was observed at a late stage. Moreover, the expression profiles of major immune checkpoint molecules (CD244 and LAG3) and ligands (CD48) on hepatic DC subsets in a mouse model exhibited the same pattern as those in AE patients. Notably, the cDC and pDC subsets in the E. multilocularis infection group exhibited higher expression levels of PD-L1 and CD155 than those in the control group, suggesting the potential of these subsets to impair T cell function. These findings may provide valuable information for investigating the role of hepatic DC subsets in the AE microenvironment and guiding DC targeting treatments for AE.

Abstract 3189: A novel monoclonal antibody targeting TM4SF4 enhances antitumor activity through regulation of cellular levels of immune checkpoint ligands and antibody dependent cellular cytotoxicity

Abstract Transmembrane 4 super family member 4(TM4SF4) protein has been shown to be involved in EMT-associated stemness through autocrine of insulin-like growth factor 1(IGF1) and osteopontin(OPN) in non-small cell lung cancer(NSCLC) cells. However, its potential as a therapeutic target has not been evaluated. In this study, anti-human TM4SF4 monoclonal antibodies(anti-hTM4SF4 mAbs) were prepared in mouse as part of a strategy for developing anticancer drug using humanized antibody. A synthetic peptide(15 mer, 126-140th AA) derived from the extracellular loop(ECL) 2 of human TM4SF4 exposed on outer cell surface was used as an immunization antigen. Among the selected anti-hTM4SF4 mAbs, ECL-2B7 and ECL-12A8 showed the best affinity for the antigen(Kd), approximately 2.66 nM and 8.34 nM, as determined by surface plasmon resonance analysis. Using mouse xenograft of NSCLC cell line, it was confirmed that ECL-2B7 was superior to ECL-12A8 in producing humanized antibody. ECL-2B7 inhibited EMT-associated stemness of TM4SF4 positive cancer cells by blocking cellular signaling events such as IGF1Rβ and CD44 and their downstream signals, PI3K/AKT/GSK3β/NF-κB and JAK2/STAT3 pathways. ECL-2B7 also enhanced antitumor activity by downregulating cellular and exosomal level of PD-L1 and B7-H4, immune-checkpoint ligands that elicit immunosuppression of T cell, and by mediating antibody-dependent cellular cytotoxicity. Treatment with OPN- and IGF1-neutralizing antibodies suppressed intracellular PD-L1 and B7-H4 level. It means that the regulation of PD-L1 and B7-H4 levels by TM4SF4 is closely related to the autocrine action of OPN and IGF1 induced by TM4SF4. Using patient-derived xenograft model, it was confirmed that the ECL-2B7 has excellent antitumor activity that effectively inhibits tumor progress. These results strongly suggest that it is necessary to construct humanized antibody based on ECL-2B7 to confirm its potential as a novel anticancer drug. Citation Format: Rae Kwon Kim, Chang-Kyu Heo, Yeon-jee Kahm, Uhee Jung, Byung-Chul Shin, Won-Yong Kim, Bum-Jin Kim, Sung-Chul Kim, Chun-jeih Ryu, Eun-Wie Cho, In-Gyu Kim. A novel monoclonal antibody targeting TM4SF4 enhances antitumor activity through regulation of cellular levels of immune checkpoint ligands and antibody dependent cellular cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3189.

Abstract 1407: Investigating the effect of an aged liver microenvironment on immune surveillance and predisposition to cancer​

Abstract The incidence of liver cancer is increasing and there is an urgent need for new therapies and preventative strategies. Age is a major risk factor for liver cancer, the reasons for which are not well defined. One of the hallmarks of aging is immune dysfunction which affects liver homeostasis potentially making it prone to cancer. To understand the role of immune dysfunction in aging liver, we analyzed immune cells from young and old livers. As is reported before, in aged livers we observed an increase in CD101+PD1+CD8+ T cell population as well as an increase in an IFNγ+TNFα+ population suggesting an increase in T cell response in an aging liver. We next sought to understand the cause of T cell exhaustion in aged livers. Cell cluster analysis of the young (4-month-old) and old (22 month old) livers by scRNA seq revealed a cell population unique to the aged livers, showing increased expression of immune checkpoint ligands as well as tumor-initiating cell markers. We validated this data by flow cytometry. We next functionally probed the immune environment of aged liver, by testing the effect of cognate antigen expression in young and old hepatocytes on adoptively transferred activated P14 CD8+ T cells isolated from a young P14 mouse. Analysis of the ex vivo activated P14 CD8+ T cells from young and old antigen-expressing livers by flow cytometry and scRNA sequencing revealed increased exhaustion and cytokine-deficiency in old mice as compared to the young mice. This study explores the age-associated alterations in the immune cells in the liver, aiming to alter liver-resident immune cells and their metabolic states in a way that promotes anti-tumor immunity in an aging liver. Citation Format: Armin Aabish Gandhi, Filipe Araujo Hoffmann, Michael LaPorte II, Aaron Havas, Adarsh Rajesh, Andrew Davis, Marcos G. Teneche, Jessica Proulx, Susan Kaech, Peter D. Adams. Investigating the effect of an aged liver microenvironment on immune surveillance and predisposition to cancer​ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1407.

Abstract 6212: A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly cancer with a low tumor mutational burden that leads to the generation of few neo-epitopes derived from mutated genes that can be recognized by cytotoxic T cells. The immunosuppressive microenvironment of PDAC comprises suppressive cell populations that include myeloid cells, cancer associated fibroblast subpopulations, and regulatory T cells that reduce T cell infiltration and cytotoxic function. We have developed a murine personalized cancer vaccine to enhance cytotoxic T cell function against PDAC, PancVAX (Kinkead et al, JCI Insight 2018). Survival benefit with PancVAX was dependent on the addition of immune checkpoint inhibitor (ICI) therapy to increase the number of cytotoxic CD8 T cells infiltrating tumors and maintain their cytotoxic function (Huff et al, JCI Insight 2023). Yet, the impact of cell-cell communication on effector T cell function arising from the immunosuppressive microenvironment of PDAC remains unknown. We previously developed a ligand-receptor inference method called Domino that infers inter- and intra-cellular signaling networks in single-cell RNA-seq data (Cherry et al, Nat Biomed Eng 2021). However, this technique is limited to data from single conditions. Understanding how this combination immunotherapy alters cellular signaling networks requires new computational tools that identify changes in regulatory networks between treatment conditions. We developed capabilities for assessment of differential signaling with Domino founded on a basis of differential signal receipt. Active receptors are identified by receptor expression in recipient cells that is correlated with downstream transcription factors in the intracellular signaling cascade. Identification of causative ligands for receptor activation is assessed by a probability of interaction for ligand-receptor interactions between cell types. This probability metric considers mean expression of ligand and receptor genes as well as the number of sender cells for comparison across treatment groups. Comparing communication networks in PancVAX alone to PancVAX with ICI, PancVAX with ICI enhances cell-cell adhesion and interferon gamma signaling in cytotoxic CD8 T cells towards exhausted CD8 T cells. Macrophages in PancVAX with ICI enhance expression of immune checkpoint ligands towards CD8 T cells, despite the presence of blocking ICI antibodies. Intracellular signaling downstream of PD-1 in exhausted T cells shows diminished numbers of transcription factors linked to PD-1 signaling. This software informs prioritization of inhibitory signals for modulation in future immunotherapy regimens and can be applied generally to other tumor subtypes and treatment contexts. Citation Format: Jacob T. Mitchell, Atul Deshpande, Amanda L. Huff, Chris Cherry, Sushma Nagaraj, Kavita Krishnan, Dmitrijs Lvovs, Jennifer H. Elisseeff, Elizabeth M. Jaffee, Neeha Zaidi, Elana J. Fertig. A differential ligand-receptor network inference method to identify alterations in communication between myeloid cells and CD8 T cells in response to PancVAX neo-epitope peptide vaccine, anti-CTLA-4 and anti-PD-1 antibodies in a murine model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6212.

Immune checkpoint ligands expressed on mature high endothelial venules predict poor prognosis of NSCLC: have a relationship with CD8+ T lymphocytes infiltration.

An insufficient number of intratumoral CD8+ T lymphocytes is a major barrier to antitumor immunity and immunotherapy. High endothelial venules (HEVs) are the major sites through which lymphocytes enter tumors; however, the molecular mechanism through which HEVs mediate CD8+ T lymphocyte infiltration remains poorly understood. Forty-two patients with stage IIIA lung adenocarcinoma, who underwent surgery, were recruited. Multiplex immunohistochemical staining was conducted on tumor tissues to detect the immune checkpoint ligands (ICLs) expressed in the HEVs, blood vessels, and lymphatics. A new ICL score model was constructed to evaluate ligand expression. The relationship between ICL score, tumor-infiltrating CD8+ T cell frequency, and survival of patients was investigated. Mature HEVs, but not blood vessels or lymphatics, mediated CD8+ T cell infiltration. However, the ICLs expressed on mature HEVs could negatively regulate CD8+ T cell entry into tertiary lymphoid structures (TLSs). In addition, according to the results obtained using our ICLtotal score model, the expression of ICLs on HEVs was observed to be a predictor of both CD8+ T cell infiltration and survival, in which a high ICLtotal score > 1 represent a weak CD8+ T cell infiltration and a high ICLtotal score > 2 predicts poor survival. Using the ICL score model, we discovered that ICLs expressed on HEVs are indicative of CD8+ T cell subset infiltration in TLSs, as well as of patient survival with lung cancer.

Abstract C014: CXCR4 inhibition enhances anti-tumor immune response in an ex vivo autologous patient-derived organoids and PBMC model system of pancreatic ductal adenocarcinoma

Abstract Introduction Immune checkpoint blockade either alone or in combination with chemotherapy has been ineffective in pancreatic ductal adenocarcinoma (PDAC) likely due to underlying immunosuppressive pathways. The CXC chemokine receptor type 4 (CXCR4)/chemokine (CXC motif) ligand 12 (CXCL12) axis is one key immunosuppressive pathway. CXCL12 (released by cancer-associated fibroblasts (CAF)) binds to CXCR4 on CD8+ T cells (CTL) and decreases its infiltration into the tumor. Encouraging preclinical studies from murine PDAC models and human PDAC tumor sections have demonstrated infiltration of T cells with tumors after co-inhibition of CXCR4 and PD1/PDL1, however with limited tumor regressions in mouse model. We established an innovative ex vivo autologous patient-derived immune/organoid (PDIO) co-culture system that allows studying the interactions between distinct immune cells and tumor cells. Methods Patient-derived tumor organoids (PDTOs) generated from tumors resected from patients diagnosed with pancreas cancer were grown to confluence in Matrigel domes. We conducted ex vivo migration assays with autologous peripheral blood mononuclear cells (PBMCs) which were placed in the surrounding liquid phase of the matrigels. We separately treated PBMCs and PDTOs with AMD3100, an inhibitor of CXCR4, and tested the role of CXCR4 inhibition on PBMCs and PDTOs in T cell exclusion and immune cell activation. Results Pre-treatment of PBMCs with AMD3100 increased CD8+ T cell mobilization towards autologous PDTOs in matrigel dome migration assays by up to 11-fold. After co-culture in the liquid interphase of PDTOs matrigel domes, AMD3100 pre-treated PBMCs exhibit a more robust proliferative response compared to untreated PBMCs and a significant increase in the number of reactive T cells migrated inside the PDTO matrigel dome and expressing activation markers (p=0.02). Nearly 50% of T cells in proximity of tumor cells produce the cytotoxic factor Granzyme B (p=0.002), with AMD3100 treatment of PBMC increasing it by up to 3-fold compared to untreated PBMC. CXCR4 inhibition of PDTOs resulted in modulation of immune checkpoint ligands involved in the tumor immune response and tumor cell immunogenicity. Combination treatment of PBMCs and PDTOs with AMD3100 and anti-PD1 antibody ultimately resulted in increased tumor cell death with up to 45% of PDTOs exhibiting apoptosis markers after coculture with AMD3100-treated PBMC, implicating a role of this combination in the clinic. Conclusions We demonstrate that inhibition of CXCR4, not only increases migration of autologous T cells towards PDTOs, but also results in proliferation of PBMCs and activation of T cells. Furthermore, CXCR4 inhibition of PDTOs increases antigen presentation and tumor cell death. Inhibition of CXCR4 is an attractive therapeutic target as it enhances anti-tumor immune response via dual targeting of immune and neoplastic compartments. We are testing this combination in a multicenter investigator-initiated clinical trial in treatment-naïve metastatic PDAC patients. Citation Format: Ilenia Pellicciotta. CXCR4 inhibition enhances anti-tumor immune response in an ex vivo autologous patient-derived organoids and PBMC model system of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C014.

Uncovering immune checkpoint heterogeneity in oral squamous cell carcinoma using single cell RNA-sequencing data highlights three subgroups of patients with distinct immune phenotypes

ObjectivesIn head and neck squamous cell carcinoma (HNSCC), PD-1/PD-L1 inhibitors remain inefficient in most patients, which points to the need for better characterization of immune checkpoint (ICP) molecule expression. Material and methodsWe evaluated the expression of 22 ICP ligands (ICPL) in 2,176 malignant cells from 10 patients in a public single-cell RNA-sequencing dataset and in two cohorts of HNSCC patients for which gene expression data are available. ResultsBased on ICPL expression, malignant cells formed three distinct clusters characterized either by a strong expression of ICPL together with an immune phenotype linked to IFN-γ response (cluster 1) or by a weak ICPL expression and little response to IFN-γ (clusters 2 and 3). Malignant cells from cluster 3 showed a high PD-L1 expression associated with NRF2 signature. The relevance of 3 groups of patients, i.e “high ICPL/high IFN-γ”, “low ICPL/low IFN-γ” or “low ICPL/low IFN-γ/high PD-L1” was confirmed in a cohort of 259 OSCC whole tumor samples from TCGA and in the CLB-IHN cohort including patients treated with PD1/PD-L1 inhibitors. The heterogeneous expression of ICPL among patients’ malignant cells was associated with immunologically distinct microenvironments, evaluated with the “hot/cold” and the Tumor microenvironment (TME) classification. Finally, the “low ICPL/low IFN-γ/high PD-L1” group 3 displayed a poor prognosis in the TCGA cohort. ConclusionHence, the global picture of ICPL gene expression in malignant cells from HNSCC patients may contribute to the broader issue of improving immunotherapy strategies though a better stratification of patients and the design of new treatment combinations.

Gender differences, environmental pressures, tumor characteristics, and death rate in a lung cancer cohort: a seven-years Bayesian survival analysis using cancer registry data from a contaminated area in Italy.

In Taranto, Southern Italy, adverse impacts on the environment and human health due to industrial installations have been studied. In the literature, associations have been reported between gender, environmental factors, and lung cancer mortality in women and men. The aim of this study was to investigate the relationships between gender, residence in areas with high environmental pressures, bronchus/lung cancer characteristics, and death rate. Data from the Taranto Cancer Registry were used, including all women and men with invasive bronchus/lung cancer diagnosed between 1 January 2016 and 31 December 2020 and with follow-up to 31 December 2022. Bayesian mixed effects logistic and Cox regression models were fitted with the approach of integrated nested Laplace approximation, adjusting for patients and disease characteristics. A total of 2,535 person-years were observed. Male gender was associated with a higher prevalence of histological grade 3 (OR 2.45, 95% CrI 1.35-4.43) and lung squamous-cell carcinoma (OR 3.04, 95% CrI 1.97-4.69). Variables associated with higher death rate were male gender (HR 1.24, 95% CrI 1.07-1.43), pathological/clinical stage II (HR 2.49, 95% CrI 1.63-3.79), III (HR 3.40, 95% CrI 2.33-4.97), and IV (HR 8.21, 95% CrI 5.95-11.34), histological grade 3 (HR 1.80, 95% CrI 1.25-2.59), lung squamous-cell carcinoma (HR 1.18, 95% CrI 1.00-1.39), and small-cell lung cancer (HR 1.62, 95% CrI 1.31-1.99). Variables associated with lower death rate were other-type lung cancer (HR 0.65, 95% CrI 0.44-0.95), high immune checkpoint ligand expression (HR 0.75, 95% CrI 0.59-0.95), lung localization (HR 0.73, 95% CrI 0.62-0.86), and left localization (HR 0.85, 95% CrI 0.75-0.95). The results among patients with lung cancer did not show an association between residence in the contaminated site of national interest (SIN) and the prevalence of the above mentioned prognostic factors, nor between residence in SIN and death rate. The findings confirmed the independent prognostic values of different lung cancer characteristics. Even after adjusting for patients and disease characteristics, male gender appeared to be associated with a higher prevalence of poorly differentiated cancer and squamous-cell carcinoma, and with an increased death rate.