Myeloid-derived Suppressor Cells Populations Research Articles

Viral nanoparticle vaccines against S100A9 reduce lung tumor seeding and metastasis

Metastatic cancer accounts for 90% of all cancer-related deaths and continues to be one of the toughest challenges in cancer treatment. A growing body of data indicates that S100A9, a major regulator of inflammation, plays a central role in cancer progression and metastasis, particularly in the lungs, where S100A9 forms a premetastatic niche. Thus, we developed a vaccine against S100A9 derived from plant viruses and virus-like particles. Using multiple tumor mouse models, we demonstrate the effectiveness of the S100A9 vaccine candidates in preventing tumor seeding within the lungs and outgrowth of metastatic disease. The elicited antibodies showed high specificity toward S100A9 without cross-reactivity toward S100A8, another member of the S100A family. When tested in metastatic mouse models of breast cancer and melanoma, the vaccines significantly reduced lung tumor nodules after intravenous challenge or postsurgical removal of the primary tumor. Mechanistically, the vaccines reduce the levels of S100A9 within the lungs and sera, thereby increasing the expression of immunostimulatory cytokines with antitumor function [(interleukin) IL-12 and interferonγ] while reducing levels of immunosuppressive cytokines (IL-10 and transforming growth factorβ). This also correlated with decreased myeloid-derived suppressor cell populations within the lungs. This work has wide-ranging impact, as S100A9 is overexpressed in multiple cancers and linked with poor prognosis in cancer patients. The data presented lay the foundation for the development of therapies and vaccines targeting S100A9 to prevent metastasis.

Effect of External Beam Radiation Therapy (EBRT) and Brachytherapy (BT) on Circulating MDSC Populations in Patients Treated Definitively for Cervical Cancer

The immunosuppressive function of myeloid derived suppressor cells (MDSCs) has been implicated in the regulation of immune responses against cancer. MDSCs have been associated with progression and poor response to therapy in various types of cancer, including cervical cancer. Radiation treatment (RT) alters immune cell populations within the tumor and is thought to augment antitumor responses. However, whether RT also recruits immunosuppressive MDSC populations is not well understood. Here, we investigate how circulating MDSC populations change in response to RT and if changes in MDSC frequency or subsets is predictive of RT responses in cervical cancer patients. Newly diagnosed, treatment-naïve pts with locally advanced carcinoma of the cervix will be enrolled from July 2022 to July 2023. EBRT to the pelvis was delivered at a dose of 45 Gy in 25 fractions with a simultaneous integrated boost of 52-55 Gy to involved regional lymph nodes and parametria and concurrent weekly cisplatin. Gross tumor was boosted via interstitial or tandem ring BT (22.5-27.5 Gy) after completion of EBRT. Serial blood samples were collected prior to initiating therapy (T0), post-EBRT and pre-BT (T1), and one-month post-BT (T2). Treatment response was determined based on pre-treatment MRI compared to MRI post-EBRT. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using density gradient centrifugation and stained for analysis via flow cytometry. MDSC populations were identified by Live/Dead-CD11b+CD33+HLA-DR- staining. MDSC subsets were further subdivided into granulocytic (G-, CD15+CD14-), monocytic (M-, CD15-CD14+), or early-MDSCs (e-, CD15-CD14-). Blood samples were collected at indicated time points for four patients (FIGO stage IIA-IIB). Three had partial responses to chemoradiotherapy (CRT), while one had a complete response. All three patients with partial response had an increase in total frequency of circulating MDSCs in response to EBRT/BT (mean %fx MDSC 16.6 at T0 to 35.9 at T2), and an increase in total MDSCs in two of these patients occurred with EBRT alone. Interestingly, the patient that had a complete response had fewer MDSCs at T2 relative to T0 (35.8% at T0 to 27% at T2). Proportion of MDSC subsets varied considerably among the patients, and all had altered distribution of subsets in response to RT. G-MDSCs expanded the most to RT while M-MDSCs and e-MDSCs were less affected (mean fold change from T0 to T2 G-MDSC 4.75, M-MDSC 1.27, e-MDSC 0.942). In this cohort of patients, an increase in MDSC frequency occurred after RT and altered subset distribution. Only the patient with a complete response had fewer total MDSCs following completion of CRT, suggesting further studies are needed to determine if circulating MDSCs could be a biomarker for treatment response to RT in cervical cancer.

Abstract PO-088: TRPV1+ sensory neurons provide a tumor supportive environment through recruitment of MDSCs

Abstract Cancer neuroscience has become an increasing area of focus in cancer research as our understanding of the presence and function of intra-tumoral neurons continues to emerge, with studies indicating that tumor infiltrating neurons impact components of the tumor microenvironment promoting cancer initiation and progression. We have previously published that head and neck squamous cell carcinomas are innervated. We now focus on defining the contributions of intra-tumoral neurons to disease progression in HNSCC. Tumor implantation in a double transgenic mouse (TRPV1cre::DTAfl/wt) lacking the cancer-recruited TRPV1-expressing sensory neurons allows us to study the consequences of this neuronal loss on the local immune response. Importantly, comparison between wild type and TRPV1cre::DTAfl/wt mice demonstrated no significant immune population differences at baseline. Thus, wildtype and TRPV1cre::DTAfl/wt mice were orthotopically implanted with tumor and tumoral immune cell populations identified by flow cytometry. Cytokine and peptide concentrations were measured by ELISA. In vitro assays were preformed to molecularly define the neuronal signals and immune responses. Here, co-culture experiments of immortalized head and neck cancer cell lines and dorsal root ganglia (DRG) from wildtype mice were used to model tumor innervation in vitro. The media generated was applied to bone marrow derived (BMD) immune cells and the impact on immune cell phenotypes assayed by flow cytometry. Our orthotopic model demonstrates that the presence of HNSCC promotes a nerve-injury phenotype in ipsilateral trigeminal ganglion, demonstrated by increased expression of activating transcription factor 3 (ATF3). Utilizing a 12-color flow cytometry panel we show that tumors from TRPV1cre::DTAfl/wt mice harbor a significantly smaller population of myeloid derived suppressor cells (MDSCs) compared to those from wildtype animals. Further analysis demonstrates this population shift occurs primarily due to a decrease in the CD11b+ Ly6G+/Ly6Clo population. In vitro studies using BMD immune cells show that tumor cell-DRG co-culture conditioned media induces an expansion of the MDSC population compared to condition media from DRG or tumor cells alone. Additionally, transwell migration assays show that co-culture condition media also increases cellular migration compared to condition media from DRG or tumor cells alone. Moreover, ELISA analysis indicates that co-culture of tumor cells and DRG increases neuronal release of the neuropeptide, Substance P (SP), potentiating IL-6 release from tumor cells. In summary, we identified several mechanisms by which TRPV1-expressing intra-tumoral neurons promote MDSC infiltration at the tumor bed and promote disease progression. These mechanisms define targets for therapeutic intervention including SP and IL-6 signaling. Future studies will test the clinical utility of these targets. Together, our data suggests that intra-tumoral neurons contribute to cancer progression through generation of a tumor supportive microenvironment. Citation Format: Anthony C. Restaino, Maryam Ahmadi, Sebastien Talbot, Paola D. Vermeer. TRPV1+ sensory neurons provide a tumor supportive environment through recruitment of MDSCs [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-088.

The Cxcr2+ subset of the S100a8+ gastric granylocytic myeloid-derived suppressor cell population (G-MDSC) regulates gastric pathology.

Gastric myeloid-derived suppressor cells (MDSCs) are a prominent population that expands during gastric pre-neoplastic and neoplastic development in humans and mice. However, the heterogeneity of this population has circumvented the ability to study these cells or understand their functions. Aside from Schlafen-4+ (Slfn-4+) MDSCs in mouse studies, which constitute a subset of this population, limitations exist in characterizing the heterogeneity of the gastric CD11b+Ly6G+ population and targeting its different subsets. Here we identify S100a8 as a pan-specific marker for this population and utilize it to study the role of the S100a8+Cxcr2+ subset. We profiled gastric CD11b+Ly6G+ versus CD11b+Ly6G- myeloid cells by transcriptomic and single-cell RNA sequencing. We identified S100a8 as a pan-specific marker of the gastric granulocytic MDSC (G-MDSC) population, and generated S100a8CreCxcr2flox/flox to study the effects of Cxcr2 knockdown. Following 6-months of Helicobacter felis infection, gastric CD11b+Ly6G+ G-MDSCs were highly enriched for the expression of S100a8, S100a9, Slfn4, Cxcr2, Irg1, Il1f9, Hcar2, Retnlg, Wfdc21, Trem1, Csf3R, Nlrp3, and Il1b. The expression of these distinct genes following 6mo H. felis infection marked heterogeneous subpopulations, but they all represented a subset of S100a8+ cells. S100a8 was identified as a pan-marker for CD11b+Ly6G+ cells arising in chronic inflammation, but not neutrophils recruited during acute gut infection. 6mo Helicobacter felis-infected S100a8CreCxcr2flox/flox mice exhibited worsened gastric metaplastic pathology than Cxcr2flox/flox mice, which was associated with dysregulated lipid metabolism and peroxidation. S100a8 is a pan-specific marker that can be used to target gastric G-MDSC subpopulations, of which the Cxcr2+ subset regulates gastric immunopathology and associates with the regulation of lipid peroxidation.

Arachidonic acid activates NLRP3 inflammasome in MDSCs via FATP2 to promote post-transplant tumour recurrence in steatotic liver grafts

The steatotic grafts have been applied in liver transplantation frequently owing to the high incidence of non-alcoholic fatty liver disease. However, fatty livers are vulnerable to graft injury. Myeloid-derived suppressor cell (MDSC) recruitment during liver graft injury promotes tumour recurrence. Lipid metabolism exerts the immunological influence on MDSCs in tumour progression. Here, we aimed to explore the role and mechanism of inflammasome activation in MDSCs induced by lipid metabolism during fatty liver graft injury and the subsequent effects on tumour recurrence. MDSC populations and nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome levels were investigated in a clinical cohort and a rat liver transplantation model. The mechanism of NLRP3 activation by specific fatty acids was explored in mouse hepatic ischaemia/reperfusion injury (IRI) with tumour recurrence model and invitro studies. MDSC populations and NLRP3 levels were increased with higher tumour recurrent rate in patients using steatotic grafts. NLRP3 was upregulated in MDSCs with lipid accumulation post mouse fatty liver IRI. Mechanistically, arachidonic acid was discovered to activate NLRP3 inflammasome in MDSCs through fatty acid transport protein 2 (FATP2), which was identified by screening lipid uptake receptors. The mitochondrial dysfunction with enhanced reactive oxygen species bridged arachidonic acid uptake and NLRP3 activation in MDSCs, which subsequently stimulated CD4+ T cells producing more IL-17 in fatty liver IRI. Blockade of FATP2 inhibited NLRP3 activation in MDSCs, IL-17 production in CD4+ T cells, and the tumour recurrence post fatty liver IRI. During fatty liver graft injury, arachidonic acid activated NLRP3 inflammasome in MDSCs through FATP2, which subsequently stimulated CD4+ T cells producing IL-17 to promote tumour recurrence post transplantation. The high incidence of non-alcoholic fatty liver disease resulted in the frequent application of steatotic donors in liver transplantation. Our data showed that the patients who underwent liver transplantation using fatty grafts experienced higher tumour recurrence. We found that arachidonic acid activated NLRP3 inflammasome in MDSCs through FATP2 during fatty liver graft injury, which led to more IL-17 secretion of CD4+ T cells and promoted tumour recurrence post transplantation. The inflammasome activation by aberrant fatty acid metabolism in MDSCs bridged the acute-phase fatty liver graft injury and liver tumour recurrence.

Content of myeloid-derived suppressor cells in autoimmune diseases in children

Myeloid-derived suppressor cells (MDSCs) play an important role in regulation of immune response. An increase in their number in adult patients with autoimmune diseases has been reported. G-MDSCs, M-MDSCs, and MDSCs(M-G-) at different stages of autoimmune disease may both activate T cell proliferation, leading to disease progression, or inhibit it, thus promoting Treg differentiation. Arginase-1 (Arg- 1) is an enzyme in MDSCs that reduces the concentration of arginine required for T lymphocyte proliferation. Our aim was to evaluate the content of MDSCs populations and functional activity of MDSCs in children with autoimmune diseases. 75 children with inflammatory bowel diseases (IBD), 60 children with multiple sclerosis (MS), 69 children with psoriasis (PS), 62 healthy age-matched children were included into the study. The content of MDSCs ((CD3, CD19, CD56, HLA-DR)-, CD11b+ and CD33+), subpopulations of MDSCs (M-MDSCs, G-MDSCs expressing CD14 and CD15), assessment of Arg-1 activity were performed by flow cytometry techniques. The content of MDSCs in patients with IBD, MS and PS was significantly higher than in the comparison group and depended on the state of exacerbation/remission. In exacerbation and remission of IBD, MS and PS, a significant increase of MDSCs was revealed when compared with healthy children; the highest values were found in children in exacerbation of MS (Me-3.5 (2.5-5.6) % MNC against Me-1.6 (0.9-2.5) % MNC, p 0.001). In patients with MS, the content of G-MDSC, M-MDSC was significantly higher, and MDSC(M-G-) was lower than in healthy children. An increase in absolute amounts of G-MDSCs was shown in MS exacerbation compared to the disease remission state (p = 0.022). For patients with IBD, a significant increase in percentage of MDSCs and M-MDSCs (p = 0.014 and p = 0.045, respectively) was obtained in exacerbation of the disease relative to remission state. In patients with IBD, MS, and PS, a significant increase in Arg-1 activity in MDSCs was found, with a decreased number of MDSCs in patients in remission compared to exacerbation phase of the disease. In children with autoimmune diseases, an increase in the MDSC populations was found. The activity of arginase-1 in MDSCs is increased in remission, along with a decrease in their numbers.

Role of myeloid-derived suppressor cells in prediction of the effectiveness of biologics in children with psoriasis

Psoriasis is a chronic inflammatory skin disease characterized by increased proliferation of epidermal cells, impaired keratinization, and an inflammatory reaction in the dermis due to activation of T-lymphocytes and synthesis of proinflammatory cytokines. Pathophysiology of psoriasis is associated not only with activation of proinflammatory reactions, but also with decreased anti-inflammatory functions of immunosuppressive cells. It is known that Treg, Breg and MDSCs do not perform their classical homeostatic functions in psoriasis. In recent years, there have been more and more cases of developing resistance to ongoing therapy with genetically engineered biological drugs (GEBD) in childhood, requiring replacement or discontinuation of the drug. The aim of our work was to estimate the content of MDSCs subpopulations and their functional activity in the peripheral blood of children with psoriasis at different effectiveness of biotherapeutic drugs. We examined 110 children with psoriasis vulgaris before the appointment of biologics, at 16 and 52 weeks of therapy with adalimumab, etanercept and ustikinumab, aged 6 to 18 years. Сomparison group consisted of 34 healthy children. The effectiveness of therapy was assessed by the achievement of PASI 75 by one year of therapy. Contents of myeloid-derived suppressor cells (MDSCs) and their subpopulations, and the activity of arginase-1 were assessed by multicolor flow cytometry. An increased content of MDSCs was found in children with psoriasis against the comparison group (p = 0.000). Analysis of the effectiveness of biologics in children with psoriasis, according to PASI, showed a significant reduction in disease severity in the group of patients with good effect, both at week 16 of therapy (p = 0.000) and by one year (p = 0.017). In the group of patients with good effect of biological therapy, percentage of total MDSCs population was higher, both before start of treatment and by 52nd week of therapy (p 0.01). Children with psoriasis showed increased immunosuppressive function of MDSCs by arginase-1 activity versus the comparison group (p = 0.000). The arginase-1 activity in patients with psoriasis at the stage of disease regression (PASI 10) was significantly increased relative to children in progressive stage of psoriasis (PASI10; p = 0.001). Thus, the content of MDSCs and their suppressive activity in children with psoriasis is an informative efficiency predictor of the biological drugs. Fading of biotherapy effect after the induction course is accompanied by decreased number of MDSCs and their functional activity.

Recruitment of myeloid‑derived suppressor cells and regulatory T‑cells is associated with the occurrence of acute myocardial infarction.

The roles of myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs) in acute myocardial infarction (AMI) remain elusive. The present study aimed to analyze the proportions of the granulocytic and monocytic populations of MDSCs (G-MDSCs and M-MDSCs, respectively), and Tregs in the peripheral blood mononuclear cells (PBMCs) of patients with AMI. The present study recruited 34 patients with AMI and 37 healthy controls without clinical signs of myocardial ischemia. PBMCs were isolated from the peripheral blood samples of patients with AMI within 24 h following admission to the hospital and from those of the healthy controls during a physical examination. Two subsets of MDSCs, G-MDSCs (CD15+CD33+CD11b+CD14-HLA-DRlow) and M-MDSCs (CD14+CD15-CD11b+HLA-DRlow), and Tregs (CD3+CD4+CD25highCD127low T-cells) in the PBMCs derived from the patients with AMI and healthy controls were analyzed using flow cytometry. The effects of MDSCs derived from patients with AMI on naïve CD4+ T-cells were examined in the co-culture system. The results revealed that the proportions of G-MDSCs and M-MDSCs were higher in the peripheral blood of patients with AMI than in that of the healthy controls. The patients with AMI had significantly higher numbers of programmed death-ligand (PD-L)1- and PD-L2-positive G-MDSCs and M-MDSCs compared with the healthy controls (P<0.05). The MDSCs could acquire a granulocytic phenotype following AMI, and the G-MDSCs and M-MDSCs would be more likely to express PD-L2 and PD-L1, respectively. The ratios of Tregs to CD4+ T-cells and PD-1+ Tregs in the peripheral blood of patients with AMI were significantly higher than those in the healthy controls (P<0.05). The results of flow cytometry demonstrated an increase in the numbers of inducible Tregs in the co-culture system with the G-MDSCs derived from patients with AMI compared with the G-MDSCs derived from the healthy controls (P<0.01). On the whole, the findings presented herein demonstrate the accumulation of MDSCs, and the upregulation of PD-L1 and PD-L2 expression on the surface of MDSCs in patients with AMI. MDSCs can induce the expansion of Tregs by binding PD-1 on the surface of Tregs, thus playing a crucial role in AMI.

YTHDF2 inhibition potentiates radiotherapy antitumor efficacy.

RNA N6-methyladenosine (m6A) modification is implicated in cancer progression. However, the impact of m6A on the antitumor effects of radiotherapy and the related mechanisms are unknown. Here we show that ionizing radiation (IR) induces immunosuppressive myeloid-derived suppressor cell (MDSC) expansion and YTHDF2 expression in both murine models and humans. Following IR, loss of Ythdf2 in myeloid cells augments antitumor immunity and overcomes tumor radioresistance by altering MDSC differentiation and inhibiting MDSC infiltration and suppressive function. The remodeling of the landscape of MDSC populations by local IR is reversed by Ythdf2 deficiency. IR-induced YTHDF2 expression relies on NF-κB signaling; YTHDF2 in turn leads to NF-κB activation by directly binding and degrading transcripts encoding negative regulators of NF-κB signaling, resulting in an IR-YTHDF2-NF-κB circuit. Pharmacological inhibition of YTHDF2 overcomes MDSC-induced immunosuppression and improves combined IR and/or anti-PD-L1 treatment. Thus, YTHDF2 is a promising target to improve radiotherapy (RT) and RT/immunotherapy combinations.

Androgen receptor blockade resistance with enzalutamide in prostate cancer results in immunosuppressive alterations in the tumor immune microenvironment

BackgroundEmerging data suggest that patients with enzalutamide-treated prostate cancer with increased programmed death-ligand 1 (PD-L1) expression may benefit from anti-PD-L1 treatment. Unfortunately, the Phase III IMbassador250 clinical trial revealed that...

Tim-3 regulates the immunosuppressive function of decidual MDSCs via the Fyn-STAT3-C/EBPβ pathway during Toxoplasma gondii infection.

Myeloid-derived suppressor cells (MDSCs) play a key role in maintaining maternal-fetal tolerance for a successful pregnancy, but the role of MDSCs in abnormal pregnancy caused by Toxoplasma gondii infection is unknown. Herein, we revealed a distinct mechanism by which T-cell immunoglobulin domain and mucin domain containing protein-3 (Tim-3), an immune checkpoint receptor that balances maternal-fetal tolerance during pregnancy, contributes to the immunosuppressive function of MDSCs during T. gondii infection. The expression of Tim-3 in decidual MDSCs was significantly downregulated following T. gondii infection. The proportion of monocytic MDSCs population, the inhibitory effect of MDSCs on T-cell proliferation, the levels of STAT3 phosphorylation, and the expression of functional molecules (Arg-1 and IL-10) in MDSCs were all decreased in T. gondii-infected pregnant Tim-3 gene knockout (Tim-3KO) mice compared with infected pregnant WT mice. After treatment with Tim-3-neutralizing Ab in vitro, the expression levels of Arg-1, IL-10, C/EBPβ, and p-STAT3 were decreased, the interaction between Fyn and Tim-3 or between Fyn and STAT3 was weakened, and the binding ability of C/EBPβ to the promoters of ARG1 and IL10 was decreased in human decidual MDSCs with T. gondii infection, while opposite results were observed following treatment with galectin-9 (a ligand for Tim-3). Inhibitors of Fyn and STAT3 also downregulated the expression of Arg-1 and IL-10 in decidual MDSCs and exacerbated adverse pregnancy outcomes caused by T. gondii infection in mice. Therefore, our studies discovered that the decrease of Tim-3 after T. gondii infection could downregulate the functional molecules of Arg-1 and IL-10 expression in decidual MDSCs through the Fyn-STAT3-C/EBPβ signaling pathway and weaken their immunosuppressive function, which eventually contribute to the development of adverse pregnancy outcomes.

Modulation of T-cell function by myeloid-derived suppressor cells in hematological malignancies.

Myeloid-derived suppressor cells (MDSCs) are pathologically activated neutrophils and monocytes that negatively regulate the immune response to cancer and chronic infections. Abnormal myelopoiesis and pathological activation of myeloid cells generate this heterogeneous population of myeloid-derived suppressor cells. They are characterized by their distinct transcription, phenotypic, biochemical, and functional features. In the tumor microenvironment (TME), myeloid-derived suppressor cells represent an important class of immunosuppressive cells that correlate with tumor burden, stage, and a poor prognosis. Myeloid-derived suppressor cells exert a strong immunosuppressive effect on T-cells (and a broad range of other immune cells), by blocking lymphocyte homing, increasing production of reactive oxygen and nitrogen species, promoting secretion of various cytokines, chemokines, and immune regulatory molecules, stimulation of other immunosuppressive cells, depletion of various metabolites, and upregulation of immune checkpoint molecules. Additionally, the heterogeneity of myeloid-derived suppressor cells in cancer makes their identification challenging. Overall, they serve as a major obstacle for many cancer immunotherapies and targeting them could be a favorable strategy to improve the effectiveness of immunotherapeutic interventions. However, in hematological malignancies, particularly B-cell malignancies, the clinical outcomes of targeting these myeloid-derived suppressor cells is a field that is still to be explored. This review summarizes the complex biology of myeloid-derived suppressor cells with an emphasis on the immunosuppressive pathways used by myeloid-derived suppressor cells to modulate T-cell function in hematological malignancies. In addition, we describe the challenges, therapeutic strategies, and clinical relevance of targeting myeloid-derived suppressor cells in these diseases.

Abstract 5493: Novel strategy to make KRAS targeted therapies more effective for PDAC treatment

Abstract Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) patients exhibit extremely poor prognosis. KRAS mutation on codon-12 is present in 70-95% of PDAC cases and it drives PDAC growth and progression. Galectin-1 (Gal-1) is present in both PDAC and stromal cells, being involved in tumor microenvironment, immune cell activation and metastasis. Therefore, this study discusses the efficiency of combined inhibition of mutated KRASG12D and Gal-1 inhibition to effectively suppress PDAC growth and progression. For this we have delivered KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: SPION nano-formulation was used to deliver siKRASG12D and investigate in conjunction with Gal-1 inhibitor for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated. Anti-cancer efficacy was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, tumor spheroid formation, and in a KrasG12D;LSL-Trp53R172H syngeneic mouse model. Results: Our results demonstrate that SP-siKRAS efficiently internalized in PDAC cells and suppressed KRASG12D as well as its downstream targets, YAP and PDL-1. Combined targeting of siKRAS and Gal-1 inhibited cell proliferation, clonogenicity, migration, and invasion of PDAC cells and tumor spheroid growth in 3D cell models, which recapitulate the heterogeneity and pathophysiology of PDAC. We have used -KrasG12D;LSL-Trp53R172H syngeneic mouse model of PDAC for investigating efficacy of combined SP-siKRAS formulation and galectin-1 inhibitor. Our results showed that the combination treatment inhibited the fibrotic tumor growth and increased survival rate. The combined treatment increased infiltration of total T cell population and CD8+T cells, reduced the population of myeloid-derived suppressor cells (MDSCs) by 50% (CD45+, CD3-, CD11b+, Ly6C high, Ly6G-) and T-Regulatory cells (Treg) by 57% (FoxP3+CD25+CD45+CD3+) and increased memory T cells by 34% in mice. Conclusion: This gene therapy targeting KRAS G12D mutation with a Gal-1 inhibition has a potential to modulate the oncogenic network and tumor microenvironment resulting in the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This study will develop a novel sustainable therapeutic approach to target PDAC growth and improve patient survivability. Citation Format: Ana I. Bulnes, Orlando Garcia, Poornima Shaji, Swathi Holla, Nirnoy Dan, Anupam Dhasmana, Shabnam Malik, Murali Yallapu, Stephen Behrman, Subhash Chauhan, Sheema S. Khan. Novel strategy to make KRAS targeted therapies more effective for PDAC treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5493.

Abstract 5151: Enzalutamide resistance results in immunosuppressive alterations in prostate tumor immune microenvironment

Abstract Background: Emerging data suggested that enzalutamide-treated prostate cancer patients with increased programmed death-ligand 1 (PD-L1) expression may benefit from anti-PD-L1 treatment. Unfortunately, the Phase III IMbassador250 trial revealed that the combination of atezolizumab (PD-L1 inhibitor) and enzalutamide failed to extend overall survival in patients with castration-resistant prostate cancer (CRPC). The mechanisms underlying treatment failure remain unknown. In this study, we investigated the regulation of immunosuppressive signaling in enzalutamide resistant prostate cancer and characterized the immune infiltrating cells in murine prostate tumors. Methods: The expression of interferon gamma-related genes was determined using qRT-PCR and/or western blotting. Androgen receptor (AR) and AR-V7 levels were downregulated using specific siRNA. Myc-CaP cells were chronically exposed to increasing concentrations of enzalutamide (5-50 μM) for &amp;gt;12 months, and the cells resistant to enzalutamide were referred to as Myc-CaP MDVR. The gene-regulating mechanisms in drug-resistant prostate cancer cells were determined by RNA sequencing analyses. Myc-CaP and Myc-CaP MDVR xenograft tumors were established in FVB mice, and tumor-infiltrating lymphocytes were isolated using Ficoll. The stained immune cells were determined by flow cytometry, and the data were analyzed using Flowjo. Results: Immune-related signaling pathways (interferon alpha/gamma response, T cell activation, and cell chemotaxis pathways) were suppressed in C4-2B MDVR cells. However, PD-L1 expression was highly upregulated and negatively regulated by AR in C4-2B MDVR cells. Enzalutamide treatment decreased CD8+ T cell but increased monocytic myeloid-derived suppressor cell (M-MDSC) population and PD-L1 expression in murine Myc-CaP tumors. Consistently, chemotaxis and immune response-regulating signaling pathways were suppressed, and PD-L1 expression was increased in enzalutamide-resistant Myc-CaP MDVR cells. Notably, MDSC populations were significantly increased in Myc-CaP MDVR orthotopic tumors compared with those in Myc-CaP parental tumors. Co-culturing bone marrow cells with Myc-CaP MDVR cells significantly promoted MDSC differentiation and polarized macrophages from the M1 to M2 phase. Conclusions: Immunosuppressive signaling is activated in enzalutamide resistant prostate cancer cells. The activated immune-related signatures involved in T cells, MDSC, and macrophages may reduce the efficacy of immune checkpoint inhibitors in enzalutamide-resistant prostate cancer. Grant Support: This work was supported in part by grants NIH/NCI R37CA249108 (C, Liu) and R01CA251253 (C, Liu). Citation Format: Pengfei Xu, Joy C. Yang, Bo Chen, Christopher Nip, Jonathan E. Van Dyke, Christopher P. Evans, William J. Murphy, Chengfei Liu. Enzalutamide resistance results in immunosuppressive alterations in prostate tumor immune microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5151.

Abstract 5167: Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling

Abstract Metastatic breast cancer remains one of the leading causes of global cancer incidence in women, despite the benefit of immune checkpoint inhibitors (ICIs) in managing various cancers and improving patient quality of life. Metastatic breast cancer is characterized by extensive infiltration of the tumor microenvironment (TME) with immunosuppressive cells, such as myeloid derived suppressor cells (MDSCs), that inhibit anti-tumoral immune cells and prevent effective activation of the adaptive immune system by ICIs. Previously, our group has shown in the breast TME that epigenetic reprogramming of MDSCs by entinostat, a histone deacetylase inhibitor, decreases MDSC immunosuppressive function and enhances response to ICIs, in part mediated by altered signaling within the Signal transducer and activator of transcription 3 (STAT3) and Nuclear factor kappa B (NFκB) axis. Next, we sought to examine the effects of entinostat on MDSC reprogramming in a distant metastatic TME. Using the syngeneic NT2.5LM NeuN mouse model of metastatic breast cancer, we established spontaneous lung metastases and treated with either vehicle or entinostat (5 mg/kg by oral gavage, 5x/week) for 3 weeks. Single cell RNA sequencing (scRNAseq) of macro-dissected lung metastases revealed a large MDSC population, and unsupervised pathways analysis of the MDSC cluster showed differential regulation of the IL6-JAK-STAT3 and TNFα-signaling-via-NFκB pathways upon entinostat treatment. In line with reports of crosstalk among STAT3, NFκB, and AP-1 pathways regulating inflammation in breast cancer, we also found through scRNAseq differential expression of AP-1 subunits JunB and FOSL1 upon entinostat treatment. At the protein level, western blot analysis of isolated intratumoral MDSCs from lung metastases revealed decreased STAT3 phosphorylation upon entinostat treatment. Using J774M cells, an MDSC-like cell line, we found decreased JunB phosphorylation and decreased FOSL1 protein upon entinostat treatment. Furthermore, preliminary evaluation of imaging mass cytometry (IMC) from tumor biopsies in selected patients with metastatic breast cancer treated with entinostat during the clinical trial NCI-9844 confirmed decreased STAT3 phosphorylation in MDSCs. Taken together, we provide new evidence in the metastatic lung TME that implicates a STAT3-NFκB-AP-1 mediated mechanism leading to decreased MDSC suppression. Evaluating this mechanism in MDSCs taken directly from treated lung metastases represents the most biologically relevant mechanistic study to date, and preliminary translation of findings in patients suggests that planned studies will lead to identification of the mechanism driving response. Citation Format: Aaron G. Baugh, Edgar Gonzalez, Valerie H. Narumi, Sofi Castanon, Jesse Kreger, James Leatherman, Won Jin Ho, Ashley Cimino-Mathews, Vered Stearns, Roisin M. Connolly, Elizabeth M. Jaffee, Adam L. MacLean, Evanthia T. Roussos Torres. Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5167.

Dioscin modulates macrophages polarization and MDSCs differentiation to inhibit tumorigenesis of colitis-associated colorectal cancer.

It has been reported that colitis is one of risk factors in colorectal cancer (CRC). Intervention of intestinal inflammation and in the early stage of tumorigenesis is of great significance to control the incidence and mortality of CRC. In recent years, natural active products of traditional Chinese medicine have been confirmed that they had made great progress in disease prevention. Here, we showed that Dioscin, a natural active product of Dioscorea nipponica Makino, inhibited initiation and tumorigenesis of AOM/DSS-induced colitis-associated colon cancer (CAC), including alleviating colonic inflammation, improving intestinal barrier function and decreasing tumor burden. In addition, we also explored the immunoregulatory effect of Dioscin on mice. The results showed that Dioscin modulated M1/M2 macrophages phenotype in spleen and decreased monocytic myeloid-derived suppressor cells (M-MDSCs) population in blood and spleen of mice. The in vitro assay demonstrated that Dioscin promoted M1 as well as inhibited M2 macrophages phenotype in LPS- or IL-4-induced bone marrow-derived macrophages (BMDMs) model. Based on the plasticity of MDSCs and its ability to differentiate into M1/M2 macrophages, we here found that Dioscin increased M1- and decreased M2-like phenotype during the process of MDSCs differentiation in vitro, suggesting Dioscin promoted MDSCs differentiate into M1 as well as inhibited its differentiation into M2 macrophages. Taken together, our study indicated that Dioscin had the inhibitory effect on the initial of tumorigenesis at early stage of CAC via the ant-inflammatory effect, which provided a natural active candidate for effective prevention of CAC.

MP20-11 ENZALUTAMIDE RESISTANCE RESULTS IN IMMUNOSUPPRESSIVE ALTERATIONS IN PROSTATE TUMOR IMMUNE MICROENVIRONMENT

You have accessJournal of UrologyCME1 Apr 2023MP20-11 ENZALUTAMIDE RESISTANCE RESULTS IN IMMUNOSUPPRESSIVE ALTERATIONS IN PROSTATE TUMOR IMMUNE MICROENVIRONMENT Pengfei Xu, Joy C. Yang, Bo Chen, Christopher Nip, Jonathan E Van Dyke, Christopher P. Evans, William J. Murphy, and Chengfei Liu Pengfei XuPengfei Xu More articles by this author , Joy C. YangJoy C. Yang More articles by this author , Bo ChenBo Chen More articles by this author , Christopher NipChristopher Nip More articles by this author , Jonathan E Van DykeJonathan E Van Dyke More articles by this author , Christopher P. EvansChristopher P. Evans More articles by this author , William J. MurphyWilliam J. Murphy More articles by this author , and Chengfei LiuChengfei Liu More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.11AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Emerging data suggested that enzalutamide-treated prostate cancer patients with increased programmed death-ligand 1 (PD-L1) expression may benefit from anti-PD-L1 treatment. Unfortunately, the Phase III IMbassador250 trial revealed that the combination of atezolizumab (PD-L1 inhibitor) and enzalutamide failed to extend overall survival in patients with castration-resistant prostate cancer (CRPC). The mechanisms underlying treatment failure remain unknown. In this study, we investigated the regulation of immunosuppressive signaling in enzalutamide resistant prostate cancer and characterized the immune infiltrating cells in murine prostate tumors. METHODS: The expression of interferon gamma-related genes was determined using qRT-PCR and/or western blotting. Androgen receptor (AR) and AR-V7 levels were downregulated using specific siRNA. Myc-CaP cells were chronically exposed to increasing concentrations of enzalutamide (5-50 μM) for >12 months, and the cells resistant to enzalutamide were referred to as Myc-CaP MDVR. The gene-regulating mechanisms in drug-resistant prostate cancer cells were determined by RNA sequencing analyses. Myc-CaP and Myc-CaP MDVR xenograft tumors were established in FVB mice, and tumor-infiltrating lymphocytes were isolated using Ficoll. The stained immune cells were determined by flow cytometry, and the data were analyzed using Flowjo. RESULTS: Immune-related signaling pathways (interferon alpha/gamma response, T cell activation, and cell chemotaxis pathways) were suppressed in C4-2B MDVR cells (p<0.001). However, PD-L1 expression was highly upregulated and negatively regulated by AR in C4-2B MDVR cells. Enzalutamide treatment decreased CD8+ T cell (14.8% vs. 10.3%, p<0.05) but increased monocytic myeloid-derived suppressor cell (M-MDSC) population (23.0% vs. 34.2%, p<0.05) and PD-L1 expression in murine Myc-CaP tumors. Consistently, chemotaxis and immune response-regulating signaling pathways were suppressed, and PD-L1 expression was increased in enzalutamide-resistant Myc-CaP MDVR cells. Notably, MDSC populations were significantly increased in Myc-CaP MDVR orthotopic tumors compared with those in Myc-CaP parental tumors (4.0% vs. 1.6%, p<0.01). Co-culturing bone marrow cells with Myc-CaP MDVR cells significantly promoted MDSC differentiation and polarized macrophages from the M1 to M2 phase. CONCLUSIONS: Immunosuppressive signaling is activated in enzalutamide resistant prostate cancer cells. The activated immune-related signatures involved in T cells, MDSC, and macrophages may reduce the efficacy of immune checkpoint inhibitors in enzalutamide-resistant prostate cancer. Source of Funding: This work was supported in part by grants NIH/NCI R37CA249108 (C, Liu) and R01CA251253 (C, Liu) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e279 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Pengfei Xu More articles by this author Joy C. Yang More articles by this author Bo Chen More articles by this author Christopher Nip More articles by this author Jonathan E Van Dyke More articles by this author Christopher P. Evans More articles by this author William J. Murphy More articles by this author Chengfei Liu More articles by this author Expand All Advertisement PDF downloadLoading ...

THE EFFECTS OF BIOLOGIC AND SMALL MOLECULE INHIBITOR THERAPY ON THE MUCOSAL IMMUNE SYSTEM IN ULCERATIVE COLITIS

Ulcerative colitis (UC) is a subtype of inflammatory bowel disease (IBD) that causes relapsing and remitting inflammation in the colon and rectum. Numerous treatment options are available for patients with UC including biologics and small molecule inhibitors that target the immune system. Although these therapies have dramatically improved options for patients with IBD, there remains a modest ceiling for their overall efficacy. Currently there are no biomarkers that can guide treatment decisions in patients with UC. Some studies have reported certain molecules or immune populations that are associated with treatment resistance or as predictors of treatment response. However, their clinical utility have yet to be fully determined because there is a paucity of longitudinal studies examining the direct effects of current biologics and small molecule inhibitors approved for UC on the immune biology of the colon mucosa. In this study, we perform longitudinal immune phenotyping using flow cytometry in patients with UC before and after therapy. Colon biopsy tissue was taken prior to treatment from 10 UC patients who were then followed and repeat biopsies were obtained from the same patients after 6-12 months of treatment. Tissue was analyzed using high dimensional flow cytometry with a panel of 24 markers of both lymphoid and myeloid lineages. Pre-treatment samples with inflammation correlated with higher populations of CD4+ T cells and CD4+ FOXP3 regulatory T cells also positive for TIGIT and GITR. Inflammation also corresponded with the presence of a CD33+ HLA-DR- population classically known as myeloid-derived suppressor cells (MDSC). Overall, therapy significantly decreased immune cell infiltration in the colon, most notably in B and T cell populations. However, certain treatments increased the MDSC population along with the stromal compartment of non-immune cells. A comprehensive analysis of the populations present during active inflammation and the treatment-induced changes in mucosal immunity will help identify biomarkers to predict response to therapy. In addition, examining the interplay between immune cells and the surrounding stroma can may lead to important findings that improve our understanding of each drug’s mechanism and efficacy. Future experiments with single cell RNA and T cell receptor sequencing on the same patients will identify effects of biologic therapy on the T cell repertoire and interactions between immune, epithelial, and stromal cells in the colonic mucosa. With the advent of novel sequencing and immune profiling assays, we now have the ability to harness the power of precision medicine to make more informed therapeutic decisions for IBD patients that improve clinical outcomes.

Abstract P1-11-12: Soluble TNFα blockade enhances trastuzumab deruxtecan antitumor effect in HER2-positive breast cancer model

Abstract Background. Clinical trials have demonstrated that trastuzumab deruxtecan (T-DXd) provides a durable responses for patients with HER2-positive and HER2 low metastatic breast cancer (BC). With T-DXd treatment, approximately 50% of patients with HER2+ metastatic BC were still alive and progression-free at 24 months (DESTINY-Breast03). We previously shown that mucin 4 (MUC4) expression is an independent predictor of poor response to trastuzumab in HER2-positive BC patients. We also showed that MUC4 is upregulated by soluble TNFα (sTNFα) secreted by the tumor, which confers primary trastuzumab resistance since it hides trastuzumab epitope on the HER2 molecule, reducing its binding and diminishing its therapeutic effects. In preclinical models of de novo trastuzumab-resistant tumors, we proved that administration of the sTNFα blocking agent INB03 (DN) together with trastuzumab inhibited tumor growth and induced an innate immune response in the tumor microenvironment (TME). DN is a dominant-negative inhibitor of sTNFα that is not immunosuppressive because it does not affect transmembrane TNFα. Our goal is to study whether neutralizing sTNFα can improve T-DXd effects in a multiple HER2-targeted therapy-resistant model. Methods JIMT-1 is a HER2-positive BC cell line resistant to trastuzumab, pertuzumab and lapatinib, which expresses MUC4. JIMT-1 tumor-bearing nude mice were treated with (1) IgG 5 mg/kg, (2) T-DXd 5 mg/kg, (3) T-DXd 2.5 mg/kg, (4) T-DXd 1.25 mg/kg, (5) DN 10 mg/kg, (6) T-DXd 5 mg/kg +DN, (7) T-DXd 2.5 mg/kg +DN and (8) T-DXd 1.25 mg/kg +DN. T-DXd and IgG were administered i.v. on days 0, 7 and 14. DN was administered i.p. twice a week for 3 weeks. Tumor growth was monitored regularly. Tumor-infiltrating macrophages, NK cells and myeloid-derived suppressor cells (MDSCs) were evaluated by immunofluorescence and flow cytometry. Results The dose-response curve of T-DXd exhibited tumor growth inhibitions of 83% (5 mg/kg), 61% (2.5 mg/kg) and 37% (1.25 mg/kg) vs IgG-treated tumors. DN alone had no antitumor effect. Combination of T-DXd with DN resulted in a reinforced antitumor effect, as the tumor growth inhibition escalated to 98% (T-DXd 5 mg/kg+ DN), 81% (T-DXd 2.5 mg/kg+DN) and 73% (T-DXd 1.25 mg/kg+DN). Moreover, we observed that addition of DN to T-DXd 1.25 and 5 mg/kg enhances the infiltration of resident macrophages (p &amp;lt; 0.05 and p&amp;lt;0.01, respectively) and promotes polarization to the M1-like phenotype (p &amp;lt; 0.05 and p&amp;lt;0.001, respectively). While T-DXd 2.5 and 5 mg/kg alone achieved a decrease in M2-like macrophages (p &amp;lt; 0.05), combination of T-DXd 1.25mg/kg+DN impaired M2-like polarization at similar levels of that observed with 2.5 and 5 mg/kg T-DXd (p &amp;lt; 0.05). Notably, the M1/M2 ratio escalated from 10.9% to 51.5% when DN was added to the lowest T-DXd dose (p &amp;lt; 0.01). In addition, T-DXd 2.5 and 5 mg/kg induce an increase in the proportion of NK cells in TME (p &amp;lt; 0.001 and p&amp;lt;0.05, respectively), which T-DXd 1.25 mg/kg+ DN treatment mimicked (p &amp;lt; 0.05). Although an increase in NK cell activation was observed with 1.25 and 2.5 mg/kg treatments (p &amp;lt; 0.05 and p&amp;lt;0.001, respectively), adding DN did not further improved this effect. Only the highest dose of T-DXd in combination with DN was able to increase NK cell degranulation (p &amp;lt; 0.05), compared to T-DXd alone. Finally, the percentage of MDSCs population decreases with the addition of DN to the T-DXd 2.5 and 5 mg/kg. Conclusions Our results suggest that sTNFα blockade is able to enhance T-DXd effect in a multiple HER2-targeted therapy resistant model. Notably, the administration of T-DXd 1.25 mg/kg+DN achieved a similar antitumor effect to T-DXd 5 mg/kg alone and also transforms the TME to an antitumor one with a reinforced immune response. This finding highlights that sTNFα and MUC4 expression are important variables in the response to T-DXd. Neutralization of sTNFα may open new therapeutic strategies for treatment of patients who present with MUC4 expression or have progression on T-DXd therapy. Citation Format: Sofia Bruni, Florencia Mauro, Cecilia Proietti, Rosalia Cordo-Russo, Mercogliano María Florencia, Roxana Schillaci. Soluble TNFα blockade enhances trastuzumab deruxtecan antitumor effect in HER2-positive breast cancer model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-11-12.

Effect of Direct Acting Antivirals (DAAs) on Myeloid-Derived Suppressor Cells Population in Egyptian Chronic Hepatitis C Virus Patients: A Potential Immunomodulatory Role of DAAs.

Chronic hepatitis C is a major health concern with high morbidity and mortality rates. The introduction of direct acting antivirals (DAAs) as a first-line treatment for hepatitis C virus (HCV) has significantly enhanced HCV eradication. However, DAA therapy is facing rising concerns regarding long-term safety, viral resistance, and reinfection. HCV is associated with different immune alteration mechanisms that can evade immunity and establish persistent infection. One of these suggested mechanisms is the accumulation of myeloid-derived suppressor cells (MDSCs), which is known to accumulate in chronic inflammatory conditions. Moreover, the role of DAA in restoring immunity after successful viral eradication is still unclear and needs further investigations. Thus, we aimed to investigate the role of MDSCs in chronic HCV Egyptian patients and its response to DAA in treated compared with untreated patients. Fifty untreated chronic hepatitis C (CHC) patients, 50 DAA-treated CHC patients, and 30 healthy individuals were recruited. We used flow cytometer analysis to measure MDSCs frequency and enzyme-linked immunosorbent assay analysis to evaluate the serum level of interferon (IFN)-γ. We found a significant elevation in MDSC% among the untreated group (34.5 ± 12.4%) compared with the DAA-treated group (18.3 ± 6.7%), while the control group had a mean of (3.8 ± 1.6%). IFN-γ concentration was higher in treated patients compared with untreated. We also found a significant negative correlation (rs -0.662) (p < 0.001) between MDSC% and IFN-γ concentration among treated HCV patients. Our results revealed important evidence of MDSCs accumulation in CHC patients and partial retrieval of the immune system regulatory function after DAA therapy.