Protein palmitoylation and immune regulation in pancreatic ductal adenocarcinoma: Integrating insights from cross-cancer studies.

Cancer immunotherapy is considered to be one of the biggest breakthroughs in cancer therapy in the last decade. However, the success of immunotherapy has so far been limited to a few solid malignancies including melanoma, renal cell carcinoma, non-small-cell lung cancer (NSCLC) and a few hematologic malignancies. In 2011, ipilimumab, a therapeutic monoclonal antibody that blocks CTLA-4, the bona fide immune checkpoint, was US FDA-approved for advanced melanoma [1]. Subsequently, other checkpoint inhibitors including anti-PD-1 and anti-PD-L1 blockade antibodies were also demonstrated to yield an objective response in approximately 20–30% of patients of these malignant diseases; and among the patients who had an objective response, many had a durable response [2–4]. One of anti-PD-1 antibodies (pembrolizumab) was most recently approved by US FDA for unresectable or metastatic melanoma. In addition, sipuleucel-T, a dendritic cell vaccine, has been shown to improve the overall survival of metastatic prostate cancer and subsequently gained FDA approval [5]. Nevertheless, significant objective response and durable responses were not seen in sipuleucel-T-treated pancreatic cancer patients. Melanoma, renal cell carcinoma and NSCLC were unique in their high infiltration of effector lymphocytes in tumor microenvironment (TME) [6]. By contrast, many other solid malignancies including pancreatic cancer are characterized by a highly immunosuppressive TME [7]. Immune tolerance mechanisms within the TME are a major obstacle to effective treatment of these cancers with immunotherapy. Pancreatic cancer and many other malignancies are thus considered ‘nonimmunogenic’ neoplasms. This notion has drastically slowed the development and application of immune-based therapies for these diseases.

Periampullary cancers constitute about 5% of gastrointestinal malignancies. They are comprised of tumors of diverse origins and are generally subdivided into pancreatic and non-pancreatic carcinomas. Immune checkpoint regulators, cytotoxic T-lymphocyte antigen 4 (CTLA-4), and the programmed cell death ligand-1 (PDL-1) have emerged as promising new targets for cancer therapeutics. Aim: This study aims to determine the possible role of immune checkpoint inhibitors PDL-1 and CTLA-4 in periampullary carcinoma of pancreatic and non-pancreatic adenocarcinoma subtypes, in an attempt to investigate the possible introduction of their related immunotherapy in the management of these tumors. Materials and Methods: Expression of immune inhibitory molecules was examined by immunohistochemistry in 40 cases including (20) pancreatic adenocarcinoma and (20) non-pancreatic adenocarcinoma. The association between markers and clinicopathological parameters was evaluated. Results: Statistically significant differences in the immunoexpression of both CTLA-4 and PDL-1 in the two studied groups were noticed with higher expression in non-pancreatic adenocarcinoma in relation to pancreatic adenocarcinoma (P=0.004, P=0.008) respectively. PDL-1 expression was positive in 15% and 55% of pancreatic and non-pancreatic adenocarcinoma cases, respectively with a significant correlation with lymph nodes metastasis in non-pancreatic adenocarcinoma cases. CTLA-4 was positive in 20% of pancreatic carcinoma with a significant correlation with lymph node metastasis, perineural invasion and T stage. In non-pancreatic periampullary adenocarcinoma, CTLA-4 was positive in 65% of cases with a significant association with lymph nodes metastasis and T stage. Conclusions: Immunotherapy using anti-PDL-1 and CTLA-4 are proposed as a novel promising management tool in non-pancreatic periampullary adenocarcinoma not in pancreatic adenocarcinomas

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.

PAK1 inhibition increases TRIM21-induced PD-L1 degradation and enhances responses to anti-PD-1 therapy in pancreatic cancer

Pancreatic cancer is one of the deadliest malignancies with an average five-year survival of mere 2-9%. Emerging evidence suggests that the presence of polymorphonuclear (PMN) myeloid derived suppressor cells (MDSCs) with neutrophil-like phenotypes and morphologies play a major role in adverse clinical outcomes due in part to their contribution in establishing immunosuppressive tumor microenvironment (TME). Despite recent advances in our understanding of MDSC phenotypes and characteristics in the TME, the underlying molecular mechanisms by which classical neutrophils differentiate into MDSC within the TME remails largely unknown. To investigate the microenvironmental cues that regulate PMN-MDSC differentiation at the tumor site, we established several in vitro co-culture systems using pancreatic cancer cells and 3D spheroid conditions. With live cell migration microscopy, we first showed that neutrophils actively swarmed into the pancreatic cancer spheroids and stayed active for an extended time period in the tumor. We further found that cancer cell culture supernatant significantly extended neutrophil survival and successfully differentiated neutrophils into MDSCs that exhibited decreased type 1 Interferon levels, increased arginase and ROS expression, and had the ability to suppress CD8 cytotoxicity. Additionally, when compared to other forms of neutrophil activators such as LPS and TNF, only tumor conditioned media enhanced neutrophil survival and MDSC differentiation. Our data suggest the presence of novel molecular processes that support MDSC differentiation at the tumor site. To identify the molecular regulators of MDSC formation, we have developed a CRISPR-Cas9 pooled library screen using the immortalized granulocyte-monocyte progenitor cell line, HoxB8. This study will further our understanding of MDSC differentiation and may help to develop therapeutics to limit MDSC development in the TME of pancreatic and other solid cancers. Citation Format: Ankit Dahal, Yeonsun Hong, Minsoo Kim. Elucidating the molecular mechanism of Myeloid Derived Suppressor Cell (MDSC) divergence in the tumor microenvironment (TME) of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B21.

BackgroundV-domain immunoglobulin suppressor of T cell activation (VISTA) is a B7 family inhibitory immune checkpoint protein and is highly expressed on myeloid cells and T cells.1 VISTA acts as both...

Pancreatic ductal adenocarcinoma (PDA) is associated with an immunosuppressive tumor‐microenvironment (TME) that supports the growth of tumors and mediates tumors enabling evasion of the immune system. Expression of programmed cell death ligand 1 (PD‐L1) and loss of human leukocyte antigen (HLA) class I on tumor cells are methods by which tumors escape immunosurveillance. We examined immune cell infiltration, the expression of PD‐L1 and HLA class I by PDA cells, and the correlation between these immunological factors and clinical prognosis. PDA samples from 36 patients were analyzed for HLA class I, HLA‐DR, PD‐L1, PD‐1, CD4, CD8, CD56, CD68, and FoxP3 expression by immunohistochemistry. The correlations between the expression of HLA class I, HLA‐DR, PD‐L1 or PD‐1 and the pattern of tumor infiltrating immune cells or the patients’ prognosis were assessed. PD‐L1 expression correlated with tumor infiltration by CD68+ and FoxP3+ cells. Low HLA class I expression was an only risk factor for poor survival. PD‐L1 negative and HLA class I high‐expressing PDA was significantly associated with higher numbers of infiltrating CD8+ T cells in the TME, and a better prognosis. Evaluation of both PD‐L1 and HLA class I expression by PDA may be a good predictor of prognosis for patients. HLA class I expression by tumor cells should be evaluated when selecting PDA patients who may be eligible for treatment with PD‐1/PD‐L1 immune checkpoint blockade therapies.

MYd88 Blockade Caused Reduction of PDL1 on Tumor Infiltrating MDSCs in a Murine Model of Melanoma

2620 Background: Programmed death ligand-1 (PD-L1) contributes to immune suppression in the tumor microenvironment (TME) by interacting with programmed cell death-1 (PD-1) on infiltrating T lymphocytes leading to tumor immune escape. Application of omics technologies has shed light on the relevance of the TME for response to immunotherapies and development of novel treatment options. Here we have applied a multi-plex immunofluorescence/multi-tumor tissue microarray (TMA) approach to examine the immunobiology of different TMEs with respect to the contribution by tumor cells and macrophages to overall PD-L1 expression. Methods: A TMA comprising 11 tumor types and a total of 144 different donors, each represented by two cores [1mm; 1 from invasive margin (IM) and 1 from tumor center (TC)], was stained using Ultivue’s Immuno8 FixVUE panel (CD3, CD4, CD8, FOXP3, CD68, PD-1, PD-L1, pan-CK/SOX10). Whole slide images from two rounds of imaging (x20 magnification; four markers in each round) were aligned using Ultivue's UltiStacker software based on the nuclear counterstains from the two imaging rounds, to provide precise marker colocalization data. Cell phenotype data for each core was generated using Visiopharm software. Results: Overall PD-L1 positivity was greatest for NSCLC, SCLC, TNBC and gastric cancer ranging from approximately 600-1000 PD-L1+ cells/mm2/core, compared with CRC, breast, pancreatic, liver, and gastric esophageal junction (GOJ) cancers (approx. 50-300 PD-L1+ cells/mm2/core). Contribution by macrophages to overall PD-L1 expression (dual CD68+/PD-L1+) varied by tumor type representing 25-35% for NSCLC (SCC and ADC) and gastric cancer, whereas a converse pattern was apparent for SCLC, TNBC, breast (ER+ and Her2+) and pancreatic cancers where PD-L1+ macrophages accounted for a large proportion (approx. 60-85%) of overall tumor PD-L1 expression. Interestingly, as a proportion of total macrophage infiltration, approximately 65% of CD68+ macrophages were PD-L1+ for SCLC and TNBC, compared with less than 10% for pancreatic and liver cancer. Spatial analysis revealed PD-L1+ macrophage infiltration to be generally higher for IM versus TC, and the distribution between tumor (CK+) and stroma (CK-) remained similar despite exclusion of CD3+/CD8+ cytotoxic T cells from CK+ tumor regions for pancreatic and liver cancer. Conclusions: Taken together, these data illustrate the benefits of combining multiplexed immunofluorescence staining, with digital analysis of cell phenotypes within well characterized tumor samples to better understand the relative immunobiology of different TMEs. Here we demonstrate that the relative contribution of macrophages to the overall PD-L1 microenvironment varies between tumor types, which may help guide options for successful immunotherapy strategies.

BackgroundNeural crest-associated genes play pivotal roles in tumor initiation, progression, and the intricate dynamics of the tumor microenvironment (TME). Myeloid-derived suppressor cells (MDSC) within the TME are important in dampening...

Although it is now widely appreciated that antitumor immunity is critical to impede tumor growth and progression, there remain significant gaps in knowledge about the mechanisms used by tumors to escape immune control. In tumor cells, we hypothesized that one mechanism of immune escape used by tumors involves the synthesis and extracellular shedding of gangliosides, a class of biologically active cell surface glycosphingolipids with known immunosuppressive properties. In this study, we report that tumor cells engineered to be ganglioside deficient exhibit impaired tumorigenicity, supporting a link between ganglioside-dependent immune escape and tumor outgrowth. Notably, we documented a dramatic reduction in the numbers and function of tumor-infiltrating myeloid-derived suppressor cells (MDSC) in ganglioside-deficient tumors, in contrast with the large MDSC infiltrates seen in ganglioside-rich littermate control tumors. Transient ganglioside reconstitution of the tumor cell inoculum was sufficient to increase MDSC infiltration, supporting a direct connection between ganglioside production by tumor cells and the recruitment of immunosuppressive MDSC into the tumor microenvironment. Our results reveal a novel mechanism of immune escape that supports tumor growth, with broad implications given that many human tumors produce and shed high levels of gangliosides.

BackgroundInterferon regulatory factor 4 (IRF4) is a crucial member of the IRF family of transcription factors and is pivotal in orchestrating the body’s defense against tumors and infections by modulating the differentiation and functionality of immune cells. The role of IRF4 in mice during Schistosoma japonicum infection, as well as the effects of IRF4 deficiency on myeloid-derived suppressor cells (MDSCs), remains inadequately understood.MethodsHematoxylin and eosin staining was used to evaluate the pathological damage in different organs of mice following infection with S. japonicum. Flow cytometry was employed to study the effect of IRF4 on the proliferation and function of myeloid-derived suppressor cells (MDSCs) in S. japonicum-infected mice.ResultsKnockout of IRF4 in myeloid cells significantly mitigated pathological damage to the liver and lungs in mice infected with S. japonicum. Knockout of IRF4 in myeloid cells also inhibited the expansion and functionality of MDSCs by downregulating programmed death ligand 2 (PD-L2) expression and interleukin-1 alpha (IL-1α) secretion in mice infected with S. japonicum. Mechanistic studies revealed that IRF4 deficiency inhibited the expansion and function of MDSCs and that this inhibition was mediated by the STAT3 and AKT signaling pathways. Also, IRF4 myeloid knockout promoted the expansion of T cells in S. japonicum-infected mice, but had no significant effect on B cell aggregation.ConclusionsOverall, these findings highlight the importance of IRF4 in regulating MDSCs and their impact on tissue damage during S. japonicum infection, providing valuable insights into potential therapeutic targets for managing the pathological consequences of this parasitic infection.Graphical

Multiple studies have explored the potential role of programmed death-ligand 1 (PD-L1) as a mediator of Myeloid-derived suppressor cells (MDSCs) effects in various cancers. However, the role PD-L1 expression in MDSCs on autoimmune disease is still largely unknown.This study was undertaken to whether MDSC expressing PD-L1 have more potent immunoregulatory activity and control autoimmunity more effectively in two murine models of lupus (MRL/lpr mice and Roquinsan/san mice). The populations of MDSC were increased in peripheral blood of lupus patients. The mRNA levels of immunosuppressive molecules were profoundly decreased in MDSCs from lupus patients and mice. Co-culture with splenocytes showed that PD-L1 expressing MDSCs from control mice expand both Treg cells and regulatory B cells more potently. Infusion of PD-L1 expressing MDSCs reduced autoantibody levels and degree of proteinuria and improved renal pathology of two animal models of lupus. Moreover, PD-L1 expressing MDSCs therapy can suppress double negative (CD4-CD8-CD3+) T cells, the major pathogenic immune cells and follicular helper T cells in MRL/lpr mice, and podocyte damage. Our results indicate PD-L1 expressing MDSCs have more potent immunoregualtory activity and ameliorate autoimmunity more profoundly. These findings suggest PD-L1 expressing MDSCs be a promising therapeutic strategy targeting systemic autoimmune diseases.

Pancreatic cancer is the fourth most common cause of cancer-related death in the United States with many patients that are diagnosed dying within one year. Pancreatic ductal adenocarcinoma is an inflammatory disease and includes several stromal elements such as lymphocytes and other immune cells, fibroblasts, and stellate cells. Pancreatic stellate cells (PSC), also known as cancer associated fibroblasts, can provide pro-survival signals to tumor cells, however their interactions with immune cells within the tumor microenvironment have not been explored in detail. We hypothesized that factors produced by human PSC could enhance myeloid-derived suppressor cell (MDSC) differentiation and function, which promotes immunosuppression in the tumor microenvironment. Primary PSC were harvested from human specimens and morphology was confirmed via immunofluorescent microscopy and staining for vimentin, alpha-smooth muscle actin (α SMA), and glial fibrillary acidic protein (GFAP). We analyzed soluble factors from PSC and human fetal primary pancreatic fibroblast cell line (HPPFC; negative controls) using a Luminex assay. Normal donor peripheral blood mononuclear cells (PBMC, n=3 donors) were cultured with 5 and 10% PSC supernatants, 10% HPPFC supernatants (negative control), or IL-6/GM-CSF (positive control) for 7 days, and assessed for MDSC phenotype by flow cytometry. The FLLL32 STAT3 inhibitor was used to determine whether PSC-mediated MDSC differentiation or PSC viability was STAT3-dependent. Stellate cell lines (n=7) were generated from patients and validated. Luminex analysis indicated that PSC produced cytokines involved in MDSC differentiation (IL-6, VEGF, MCSF) and chemotaxis (SDF-1, MCP-1). Culture with PSC supernatants for 7 days promoted PBMC differentiation into an MDSC (CD11b+CD33+) phenotype (p<0.01) and a sub-population of polymorphonuclear CD11b+CD33+CD15+ cells (p<0.05). Supernatants from a HPPFC were used as a negative control, which when cultured with PBMC did not induce an MDSC phenotype. The resulting CD11b+CD33+ cells were functional as they suppressed autologous T cell proliferation (p<0.05). Culture of normal PBMCs with PSC supernatants led to STAT3 but not STAT1 or STAT5 phosphorylation. Finally, the FLLL32 STAT3 inhibitor abrogated PSC-mediated MDSC differentiation, PSC viability, and reduced autocrine IL-6 production indicating these processes are STAT3 dependent (p<0.01). These data demonstrate a novel role for PSC in supporting immunosuppression associated with cancer and suggest that STAT3 within both stromal and immunosuppressive cells represents a therapeutic target. Citation Format: Thomas A. Mace, Zeenath Ameen, Wendy Frankel, Amy Collins, Sylwia Wojcik, Markus Mair, Gregory S. Young, James R. Fuchs, Tim D. Eubank, Tanios Bekaii-Saab, Mark Bloomston, Gregory B. Lesinski. Pancreatic cancer associated stellate cells promote differentiation of myeloid-derived suppressor cells in a STAT3-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2874. doi:10.1158/1538-7445.AM2013-2874

Chronic stress is an established risk factor for pancreatic ductal adenocarcinoma (PDAC). Although the autonomic nervous system (ANS), a key component of the tumor microenvironment (TME), has been closely associated with PDAC pathogenesis and prognosis, the underlying mechanisms remain incompletely understood. This study reveals that chronic stress-induced sympathetic nervous system (SNS) activation upregulates the hepatic complement system, leading to the recruitment of myeloid-derived suppressor cells (MDSCs) that promotes tumor progression. Specifically, chronic stress-triggered neurotransmitter norepinephrine (NE) enters the liver via systemic circulation and binds to upregulated β1 adrenergic receptor (ADRB1) on hepatocytes, enhancing the expression of complement components C3, C5, and CFH. In murine chronic stress-model, elevated systemic and intratumoral complement component 5a (C5a) levels correlated positively with MDSC expansion in both the spleen and tumor tissues. The effect was abrogated by the C5aR1 antagonist PMX-53, identifying C5a as a critical mediator of MDSC accumulation. MDSCs infiltrating the TME exhibited elevated expression of programmed death-ligand 1 (PD-L1) and transforming growth factor-β (TGF-β), contributing to an immunosuppressive niche. Intervention with the ADRB1 antagonist atenolol in PDAC mice significantly reduced serum/tumoral C5a levels, diminished splenic/intratumoral MDSCs proportions, and attenuated tumor growth. Our findings propose that targeting the SNS-driven complement-MDSCs axis represents a promising strategy for PDAC patients.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-025-04249-z.