Anti-tumor Phenotype Research Articles

Abstract B149: Anti-tumor and immunostimulatory properties of ST316, a peptide antagonist of beta-catenin for treatment of cancers with aberrant Wnt pathway activity

Abstract Beta-catenin represents a significant challenge as a therapeutic target due to its multifaceted role in tumor development as well as normal cell homeostasis. Dysregulation of the Wnt/beta-catenin signaling pathway is implicated in various cancers, including colorectal, hepatocellular, breast, ovarian, and pancreatic cancers. We developed ST316 as a peptide antagonist of the interaction of beta-catenin with BCL9, a co-activator implicated in oncogenic beta-catenin signaling. ST316 is currently being evaluated in a Phase 1-2 study (NCT05848739) that is currently enrolling patients with selected advanced solid tumors likely to harbor abnormalities of the Wnt/beta-catenin signaling pathway. In vitro studies indicate that ST316 selectively attenuates Wnt transcriptional activity in beta-catenin-dependent but not -independent cell lines. Gene expression profiling using RNA sequencing in beta-catenin mutant HCT116 cells and APC mutant Colo320DM cells demonstrates a significant downregulation of Wnt pathway target genes, oncogenic signature genes and pro-tumor immunologic gene sets within 24 hours of ST316 treatment. Consequently, ST316 induced a dose-dependent decrease in viability of beta-catenin-dependent cells while beta-catenin-independent cells were resistant, indicating that the anticancer effects of ST316 are mediated by inhibition of the Wnt/beta-catenin pathway. To evaluate the impact of ST316 in vivo, a 4T1 triple negative breast cancer orthotopic tumor model was employed. Once weekly injection of ST316 (5mg/kg SC) over eight weeks suppressed expression of Wnt target genes CDK4 and cMyc and resulted in a significant 84.3% inhibition of tumor growth compared to the control group. In addition to direct anti-tumor activity, ST316 triggers a pro-inflammatory immune microenvironment. ST316 induces polarization of human macrophages derived from peripheral blood mononuclear cells, resulting in >100-fold shift from immunosuppressive M2 macrophages towards the anti-tumor M1 phenotype. Co-cultures of M2 macrophages with T cells treated with ST316 demonstrate a significant three-fold increase in T-cell activation, as indicated by intracellular IFN-γ staining. Finally, ST316 enhances the anti-tumor activity of anti-PD-1 therapy in a syngeneic orthotopic 4T1 mouse model, where combination ST316 and anti-PD-1 antibody results in enhanced tumor growth inhibition compared to either single agent alone (p<0.01). These findings demonstrate the antitumor and immunostimulatory effects of ST316 and highlight its potential as a therapeutic agent for targeting cancers with aberrantly activated Wnt signaling pathways. Citation Format: Lila Ghamsari, Claudio Scuoppo, Erin Gallagher, Siok Leong, Mark Koester, Rick Ramirez, Zachary Mattes, Jerel Gonzales, Gene Merutka, Barry Kappel, Abi Vainstein, Jim Rotolo. Anti-tumor and immunostimulatory properties of ST316, a peptide antagonist of beta-catenin for treatment of cancers with aberrant Wnt pathway activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B149.

Influenza Training of Granulopoiesis Promotes Long-Term Anti-Leukemia Activity

Abstract Influenza viral infections have the ability to reprogram the immune system of the host and alter its functions to fight infections. However, the exact mechanisms and functions of virus-induced innate immunity in the context of leukemia are not yet fully understood. Our previous research has shown that a patient with refractory acute myeloid leukemia (AML) achieved complete remission (CR) after being infected with the Influenza A virus (IAV, H1N1 subtype), and this was functionally validated in two different animal disease relevant models. Our recent study demonstrates that mice pre-trained with the IAV promotes a long-term anti-leukemia effect. The anti-leukemia activity of IAV-induced trained immunity is due to the transcriptomic rewiring of hematopoietic stem and progenitor cells (HSPCs), resulting in a sustained enhancement of myeloid cells reprogramming toward an antitumor phenotype. This mechanism involves the upregulation of type I interferons (IFNs) and subsequent downregulation of inflammatory proteins. Our findings shed light on the dynamic changes in innate immune cells following viral infections and indicate a novel anti-leukemia facet of appropriate rewiring of granulopoiesis via upregulation of type I IFNs. Statement of significance The study profiles the dynamic changes in immune cells during exposure to the Influenza virus and sheds light on the role of Influenza trained innate immunity in anti-leukemia effect. The study also explores a novel anti-leukemia facet of appropriate rewiring of granulopoiesis via upregulation of the type I IFN pathway. These findings suggest that innate immune training could be a promising adjuvant therapy for cancer and warrant further investigation. Legend for Figure 5 . H1N1 reprogrammed granulopoiesis via up-regulation of type I interferon pathway. (A-C) FCM analyses and absolute counts of BM progenitor cells on day 7 after training with H1N1 or PBS. FCM, flow cytometry; BM, bone marrow; LK, Lin-c-kit+Sac1-; LSK, Lin-c-kit+Sac1+; GMP, granulocyte-monocyte progenitors (Lin-c-kit+Sac1-CD16/32 hiCD34+); CMP, common myeloid progenitors (Lin-c-kit+Sac1-CD16/32 intCD34+); MEP, megakaryocyte-erythroid progenitor cell (Lin-c-kit+Sac1-CD16/32-CD34-). (D) T-SNE plot of top 7 progenitor cells population changes. t-SNE, t-distributed stochastic neighbor embedding. (E) Heatmap visualization of the expression of different genes in HSPCs. The upregulated genes related to the type I IFNs pathway are listed on the right. HSPCs and hematopoietic stem/progenitor cells. (F) The top enriched GO terms of biological processes for upregulated genes in H1N1 trained group. GO, gene ontology. (G-H) Heatmap and t-SNE plot of type I IFN-associated upregulation genes in progenitor cells of HSPCs, GMP, CMP, and MEP. t-SNE, t-distributed stochastic neighbor embedding.

Somatic TET2 Mutations Prime the Immune System for Response to Immune Checkpoint Blockade

The driver mutations causing Hematopoietic Stem Cell (HSC) expansion in Clonal Hematopoiesis (CH) are inherited by HSC progeny and in some settings alter the function of mutant immune cells. It is unclear how CH mutations impact immune cell function in response to immune checkpoint blockade (ICB), which is a critical cancer therapy for many solid tumours and hematological cancers. Understanding how CH influences ICB could provide a novel biomarker for patient stratification or personalized therapeutics and may offer insight into immunotherapy mechanisms, yielding novel drug targets to synergistically improve patient outcomes. Somatic mutations inactivating TET2, which is responsible for 5-methylcytosine hydroxylation and demethylation, are one of the most frequent drivers of CH. TET2 inactivation can enhance inflammation from monocytes/macrophages, CD4 + and CD8 + T cells, but its impact on ICB is unknown. Here we sought to define the impact of TET2-mutant CH (TET2-CH) on ICB response. First, we modelled TET2-CH by rescuing lethally irradiated C57BL6/j mice with Tet2-knockout (-KO), Tet2-heterozygous null (-het), or wild type (WT) bone marrow. Mice were subsequently implanted with the syngeneic colorectal adenocarcinoma line MC38 and treated with Programmed Cell Death Protein-1 (PD-1) targeted ICB or Isotype Control (control) antibodies. PD-1 ICB, but not control, treated tumors were significantly smaller in mice with Tet2-het or Tet2-KO versus WT hematopoiesis (Figure 1). There was a trend towards a dose-responsive effect, with more Tet2-het mice responding to PD-1 ICB than wt, and more Tet2-KO mice responding than Tet2-het or wt mice. Therefore, Tet2-mutant leukocytes promote response to PD-1 blockade. PD-1 ICB with targeted cell depletion revealed that the enhanced response in Tet2-het mice requires CD4 + T Cells, CD8 + T Cells and macrophages, but not NK cells. To understand the mechanism through which Tet2-inactivation promotes ICB responses we performed 10X single cell RNA-sequencing on 38,994 CD45 + tumor-infiltrating leukocytes (TILs) from Tet2-het and wt mice treated with PD-1 ICB or control. Tet2-het mutations dramatically changed the TIL landscape with PD-1 blockade by promoting the acquisition of cell fates that drive durable immunotherapy responses including CD8 + effector T cells, CD8 + memory T cells, CD4 + interferon-responsive cells, and macrophages expressing an inflammatory anti-tumour gene signature (Figure 2). In contrast, wt TILs from PD-1 treated tumours were significantly enriched for exhausted CD8 + T cells, CD4 + regulatory T cells, and immunosuppressive macrophages that can promote tumor growth (Figure 2). TIL DNA methylation analysis using Infinium Beadchip arrays revealed promoter hypermethylation in genes defining the immunosuppressive macrophage state. Collectively, this suggests that Tet2-CH reshapes TIL fate following ICB by promoting anti-tumor phenotypes and protecting cells from immunosuppressive states. To investigate the potential clinical relevance of our findings, we retrospectively tested for an association between TET2-CH and immunotherapy outcome in 569 ICB-treated solid tumor patients. CH mutations were detected in publicly-available exome-sequencing data at VAF >0.02 using Mutect2. While CH as a composite of all driver mutations did not correlate with outcome, in multivariate analysis TET2-CH was associated with 6-times greater odds of achieving 6 month clinical benefit in n=205 melanoma patients (95% CI 1.01-66.6). To explore whether TET2-CH might identify patients primed for ICB response, we screened for CH in the prospectively collected baseline PBMCs from 90 solid tumour patients treated in a Phase 2 trial of the PD-1 inhibitor Pembrolizumab (NCT02644369). While underpowered to draw conclusions, we noted that one patient, treated for the skin cancer Merkel Cell Carcinoma, was positive for TET2-CH and responded to ICB, and also survived longer than the cohort median and Merkel Cell Carcinoma median. In summary, our results show that TET2-CH promotes immunotherapy response by shifting TILs from immunosuppressive to anti-tumor fates. Mechanistically, this occurs through lineage-specific promoter hypermethylation and gene silencing. Further study of TET2-CH as a potential biomarker of ICB response is warranted and targeting TET2-regulated pathways may yield novel strategies to improve immunotherapy outcomes.

Regulatory mechanism of macrophage polarization based on Hippo pathway.

Macrophages are found to infiltrate and migrate in a large number of Tumor-associated macrophages (TMEs) and other macrophages in the microenvironment of tumors and related diseases, and undergo phenotypic changes in response to a variety of cytokines, mainly including the primary phenotype M2 and the anti-tumor phenotype M1. The Hippo signaling pathway affects the development of cancer and other diseases through various biological processes, such as inhibition of cell growth. In this review, we focus on immune cells within the microenvironment of tumors and other diseases, and the role of the Hippo pathway in tumors on macrophage polarization in the tumor microenvironment (TME) and other diseases.

Tuning the Innate Immune Multiple Myeloma Microenvironment By Modulating IRF4

The tumor microenvironment (TME) has emerged as a key contributor to cancer progression in numerous solid tumors as well as blood cancers that evolve in the bone marrow (BM). In the context of multiple myeloma (MM), survival of malignant plasma cells is supported by interactions with the BM microenvironment, which is comprised of various components including cells, extracellular matrix, and soluble factors. Therefore, the development of strategies that specifically target the MM BM microenvironment is crucial for improving current disease prognosis. Within the BM niche, M2-like myeloma-associated macrophages (MAMs) play a significant role in cancer cell survival and progression and have been implicated in producing an immune-suppressive TME by generating inflammatory mediators, growth factors, cytokines, chemokines, etc. Macrophages exhibit plasticity with regard to their polarization state, which enable them to dynamically respond and adapt to different environmental stimuli in order to fulfill important immune functions. Thus, the reprogramming of M2-like, pro-tumoral MAMs toward a M1-like, anti-tumoral phenotype represents a promising therapeutic strategy. In MM, inflammatory and anti-viral pathways promote disease progression. Interferon regulatory factor 4 (IRF4) is a transcription factor that has previously been shown to promote MM progression through enhanced survival of malignant plasma cells. Under physiological conditions, IRF4 also regulates macrophage polarization, where its expression drives alternative activation of macrophages primarily to M2-like (anti-inflammatory) phenotypes. However, the extent to which IRF4 activation or direct inhibition governs the polarization status and pro-tumoral activity of macrophages in the MM microenvironment has not been fully elucidated. In this study, we sought to further explore the role of IRF4 in macrophage polarization and investigated the potential of modulating IRF4 expression to reprogram myeloma-associated macrophages (MAMs) as a novel therapeutic strategy. First, we developed myeloma-tailored macrophage polarization models using primary human peripheral blood-derived monocytes and the THP1 monocyte cell line. Our M1 and M2 polarization models utilize a customized cocktail of myeloid growth factors and cytokines, supplemented with interleukin (IL)-6 to mimic the IL-6-enriched MM microenvironment. Characterization of M1 (CD80, TNFα, and CXCL10) and M2 (MRC1, FN1, and CCL22) markers confirmed the presence of distinct macrophage subpopulations, as well as upregulated expression of IRF4 in M2-like progeny and IRF5 in M1-like macrophages. To investigate the extent to which IRF4 activation or direct inhibition governs macrophage polarization and function, overexpression and knockdown experiments on macrophages were performed in vitro, along with whole transcriptome sequencing of IRF4-overexpressing THP1 monocytes compared to vector-transduced controls. Gene expression analyses revealed that genetic down-modulation of IRF4 in M2-like macrophages shifts the polarization status toward a hybrid phenotype that displays enhanced M1-like properties, while still retaining M2-like characteristics. This shift in polarization state was further amplified when monocytes were transduced to overexpress IRF4 prior to differentiation into M2-like macrophages and subsequent IRF4 knockdown, suggesting that IRF4 plays a key regulatory role in tuning macrophage polarization. Taken together, our preclinical findings provide groundwork for future studies on MAMs and suggest that an IRF4-mediated macrophage reprogramming strategy is a promising therapeutic approach for patients with MM and a variety of other malignancies typified by an anti-inflammatory TME.

Neutralizing tumor-related inflammation and reprogramming of cancer-associated fibroblasts by Curcumin in breast cancer therapy

Tumor-associated inflammation plays a vital role in cancer progression. Among the various stromal cells, cancer-associated fibroblasts are promising targets for cancer therapy. Several reports have indicated potent anti-inflammatory effects attributed to Curcumin. This study aimed to investigate whether inhibiting the inflammatory function of cancer-associated fibroblasts (CAFs) with Curcumin can restore anticancer immune responses. CAFs were isolated from breast cancer tissues, treated with Curcumin, and co-cultured with patients' PBMCs to evaluate gene expression and cytokine production alterations. Blood and breast tumor tissue samples were obtained from 12 breast cancer patients with stage II/III invasive ductal carcinoma. Fibroblast Activation Protein (FAP) + CAFs were extracted from tumor tissue, treated with 10 μM Curcumin, and co-cultured with corresponding PBMCs. The expression of smooth muscle actin-alpha (α-SMA), Cyclooxygenase-2(COX-2), production of PGE2, and immune cell cytokines were evaluated using Real-Time PCR and ELISA, respectively. Analyzes showed that treatment with Curcumin decreased the expression of genes α-SMA and COX-2 and the production of PGE2 in CAFs. In PBMCs co-cultured with Curcumin-treated CAFs, the expression of FoxP3 decreased along with the production of TGF-β, IL-10, and IL-4. An increase in IFN-γ production was observed that followed by increased T-bet expression. According to our results, Curcumin could reprogram the pro-tumor phenotype of CAFs and increase the anti-tumor phenotype in PBMCs. Thus, CAFs, as a component of the tumor microenvironment, are a suitable target for combination immunotherapies of breast cancer.

Boosting chemotherapy of bladder cancer cells by ferroptosis using intelligent magnetic targeting nanoparticles

A versatile nano-delivery platform was reported to enhance the tumor suppression effect of chemotherapy by augmenting tumor cells' ferroptosis. The platform consists of pomegranate-like magnetic nanoparticles (rPAE@SPIONs) fabricated by encapsulating superparamagnetic iron oxide nanoparticles (SPIONs) within a reduced poly(β-amino ester)s-PEG amphiphilic copolymer (rPAE). The resulting platform exhibits several functionalities. Firstly, it promotes the doxorubicin (DOX) release by leveraging the mild hyperthermia generated by NIR irradiation. Secondly, it triggers ferroptosis in tumor cells, inducing their demise. Thirdly, it induces polarization of macrophages towards an anti-tumor M1 phenotype, contributing to ferroptosis of tumor cells and enhanced tumor cell suppression. This study effectively capitalizes on the versatility of SPIONs and offers a simple yet powerful strategy for developing a new nanosized ferroptosis-inducing agent, ultimately improving the inhibition of bladder cancer cells.

EXTH-44. ENHANCING GLIOBLASTOMA CLEARANCE: EMPOWERING ANTI-EGFRVIII CAR T CELLS WITH A PARACRINE SIRPΓ-DERIVED CD47 BLOCKER

Abstract A major challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is the immunosuppressive microenvironment (iTME), which is densely populated by protumoral glioma-associated microglia and macrophages (GAMs). Blockade of CD47, a “don’t-eat-me” signal overexpressed by GBM cells, disrupts the CD47-SIRPα axis, and regulates the phagocytic function of GAMs. However, systemic anti-CD47 monotherapy has been ineffective against human solid tumors to date. Here, we combined local CAR T therapy with paracrine GAM modulation to synergistically eliminate GBM. We engineered an armored CAR T cell against EGFRvIII that constitutively secretes a SIRPγ-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eliminated EGFRvIII+ GBM in vitro, and eradicated EGFRvIII-heterogeneous (EGFRvIII+ and EGFRvIII- mosaic) orthotopically xenografted tumors by locoregional application in vivo. This resulted in significant tumor-free long-term survival, followed by partial tumor control upon re-challenge. Combination of anti-CD47 antibodies with anti-EGFRvIII-CAR T cells failed to achieve synergy, pointing towards the importance of sustained paracrine GAM conversion. Paracrine SGRP enhanced CAR T infiltration, tumor clearance and GAM reconversion to an antitumor phenotype as shown by multidimensional brain immunofluorescence microscopy and in-depth spectral flow cytometry multiplexing. Moreover, the plasma of anti-EGFRvIII-SGRP CAR T-treated mice displayed a distinct signature of innate immune activation including TNFα and CCL3. Validation of the anti-EGFRvIII-SGRP CAR T in human explants is ongoing. Furthermore, armoring anti-CD19 CAR T cells with SGRP resulted in superior efficacy in a peripheral lymphoma mouse model compared to anti-CD19 CAR T treatment alone, most problably due to innate immune activation. Thus, local anti-EGFRvIII-SGRP CAR T cell therapy combines the potent antitumor effect of engineered T cells with the modulation of the surrounding innate iTME, inducing a synergistic clearance of GBM in a manner that overcomes known mechanisms of CAR T therapy evasion, such as tumor immune suppression and antigen escape.

KRASG12C inhibition using MRTX1257: a novel radio-sensitizing partner

BackgroundKRAS activating mutations are considered the most frequent oncogenic drivers and are correlated with radio-resistance in multiple cancers including non-small cell lung cancer (NSCLC) and colorectal cancer. Although KRAS was considered undruggable until recently, several KRAS inhibitors have recently reached clinical development. Among them, MRTX849 (Mirati Therapeutics) showed encouraging clinical outcomes for the treatment of selected patients with KRASG12C mutated NSCLC and colorectal cancers. In this work, we explore the ability of MRTX1257, a KRASG12C inhibitor analogous to MRTX849, to radio-sensitize KRASG12C+/+ mutated cell lines and tumors.MethodsBoth in vitro and in vivo models of radiotherapy (RT) in association with MRTX1257 were used, with different RAS mutational profiles. We assessed in vitro the radio-sensitizing effect of MRTX1257 in CT26 KRASG12C+/+, CT26 WT, LL2 WT and LL2 NRAS KO (LL2 NRAS−/−) cell lines. In vivo, we used syngeneic models of subcutaneous CT26 KRASG12C+/+ tumors in BALB/c mice and T cell deficient athymic nu/nu mice to assess both the radio-sensitizing effect of MRTX1257 and its immunological features.ResultsMRTX1257 was able to radio-sensitize CT26 KRASG12C+/+ cells in vitro in a time and dose dependent manner. Moreover, RT in association with MRTX1257 in BALB/c mice bearing CT26 KRASG12C+/+ subcutaneous tumors resulted in an observable cure rate of 20%. However, no durable response was observed with similar treatment in athymic nude mice. The analysis of the immune microenvironment of CT26 KRASG12C+/+ tumors following RT and MRTX1257 showed an increase in the proportion of various cell subtypes including conventional CD4 + T cells, dendritic cells type 2 (cDC2) and inflammatory monocytes. Furthermore, the expression of PD-L1 was dramatically down-regulated within both tumor and myeloid cells, thus illustrating the polarization of the tumor microenvironment towards a pro-inflammatory and anti-tumor phenotype following the combined treatment.ConclusionThis work is the first to demonstrate in vitro as in vivo the radio-sensitizing effect of MRTX1257, a potent KRASG12C inhibitor compatible with oral administration, in CT26 KRASG12C mutated cell lines and tumors. This is a first step towards the use of new combinatorial strategies using KRAS inhibitors and RT in KRASG12C mutated tumors, which are the most represented in NSCLC with 14% of patients harboring this mutational profile.

Ultrasound-Induced Gold Nanoparticle United with Acoustic Reprogramming of Macrophages for Enhanced Cancer Therapy.

Sonodynamic therapy (SDT) has considerable potential in cancer treatment and exhibits high tissue penetration with minimal damage to healthy tissues. The efficiency of SDT is constrained by the complex immunological environment and tumor treatment resistance. Herein, a specific acoustic-actuated tumor-targeted nanomachine is proposed to generate mechanical damage to lysosomes for cancer SDT. The hybrid nanomachine was assembled with gold nanoparticles (GNPs) as the core and encapsulated with macrophage exosomes modified by AS1411 aptamers (GNP@EXO-APs) to optimize the pharmacokinetics and tumor aggregation. GNP@EXO-APs could be specifically transferred to the lysosomes of tumor cells. After induction with ultrasound, GNP@EXO-APs generated strong mechanical stress to produce lysosomal-dependent cell death in cancer cells. Notably, tumor-associated macrophages were reprogrammed in the ultrasound environment to an antitumor phenotype. Enhanced mechanical destruction via GNP@EXO-APs and immunotherapy of cancer cells were verified both in vitro and in vivo under SDT. This study provides a new direction for inside-out killing effects on tumor cells for cancer treatment.

Enhanced Chemo-Immunotherapy Strategy Utilizing Injectable Thermosensitive Hydrogel for The Treatment of Diffuse Peritoneal Metastasis in Advanced Colorectal Cancer.

Patients with colorectal cancer (CRC) and diffuse peritoneal metastasis (PM) are not eligible for surgical intervention. Thus, palliative treatment remains the standard of care in clinical practice. Systemic chemotherapy fails to cause drug accumulation at the lesion sites, while intraperitoneal chemotherapy (IPC) is limited by high clearance rates and associated complications. Given the poor prognosis, a customized OxP/R848@PLEL hydrogel delivery system has been devised to improve the clinical benefit of advanced CRC with diffuse PM. This system is distinguished by its simplicity, security, and efficiency. Specifically, the PLEL hydrogel exhibits excellent injectability and thermosensitivity, enabling the formation of drug depots within the abdominal cavity, rendering it an optimal carrier for IPC. Oxaliplatin (OxP), a first-line drug for advanced CRC, is cytotoxic and enhances the immunogenicity of tumors by inducing immunogenic cell death. Furthermore, OxP and resiquimod (R848) synergistically enhance the maturation of dendritic cells, promote the expansion of cytotoxic T lymphocytes, and induce the formation of central memory T cells. Moreover, R848 domesticates macrophages to an anti-tumor phenotype. OxP/R848@PLEL effectively eradicates peritoneal metastases, completely inhibits ascites production, and significantly prolongs mice lifespan. As such, it provides a promising approach to managing diffuse PM in patients with CRC without surgical indications.

The invasive margin of early-stage human colon tumors is infiltrated with neutrophils of an antitumoral phenotype.

Neutrophils infiltrate several types of cancer; however, whether their presence is associated with disease progression remains controversial. Here, we show that colon tumors overexpress neutrophil chemoattractants compared to healthy tissues, leading to their recruitment to the invasive margin and the central part of colon tumors. Of note, tumor-associated neutrophils expressing tumor necrosis factor α, which usually represents an antitumoral phenotype, were predominantly located in the invasive margin. Tumor-associated neutrophils from the invasive margin displayed an antitumoral phenotype with higher ICAM-1 and CD95 expression than neutrophils from healthy adjacent tissues. A higher neutrophil/lymphocyte ratio was found at later stages compared to the early phases of colon cancer. A neutrophil/lymphocyte ratio ≤3.5 predicted tumor samples had significantly more neutrophils at the invasive margin and the central part. Moreover, tumor-associated neutrophils at the invasive margin of early-stage tumors showed higher ICAM-1 and CD95 expression. Coculture of colon cancer cell lines with primary neutrophils induced ICAM-1 and CD95 expression, confirming our in situ findings. Thus, our data demonstrate that tumor-associated neutrophils with an antitumoral phenotype characterized by high ICAM-1 and CD95 expression infiltrate the invasive margin of early-stage colon tumors, suggesting that these cells can combat the disease at its early courses. The presence of tumor-associated neutrophils with antitumoral phenotype could help predict outcomes of patients with colon cancer.

OR05-01 Myeloid Cell Intrinsic NR0B2 Inhibits Regulatory T Cell Expansion Within The Tumor Microenvironment: Potential Novel Strategy For Breast Cancer Immunotherapy

Abstract Disclosure: H. Vidana Gamage: None. S. Shahoei: None. S. Albright: None. T. Nguyen: None. R. Farmer: None. E. Weisser: None. R. Bautista Rivas: None. C.P. Schane: None. Y. Wang: None. A. Nelczyk: None. L. Ma: None. S. Tiwari: None. A. Das Gupta: None. S. Bendre: None. L. Apetoh: None. P. Hergenrother: None. E.R. Nelson: None. Breast cancer remains the second leading cause of cancer-related death in women. Immune-oncology interventions for the treatment of breast cancer have thus far been underwhelming, providing strong rationale for the development of drugs to increase their efficacy. A major obstacle to immunotherapy is the highly immunosuppressive tumor microenvironment. This phenomenon is strongly maintained by myeloid cells and regulatory T cells (Tregs), which are associated with immune escape, poor prognosis and treatment resistance. Importantly, myeloid cells are also critical for a robust anti-tumor response. Therefore, we require strategies to re-educate myeloid cells rather than eliminating them. Elevated cholesterol is a risk factor for breast cancer, while cholesterol-lowering drugs are associated with a good prognosis. Previous work has demonstrated that certain downstream metabolites of cholesterol, can promote immune escape and metastatic progression. The nuclear receptor NR0B2 is a critical negative regulator of cholesterol homeostasis. Mining clinical data, we have found that high tumoral expression of NR0B2 is associated with an increased time to recurrence. Single cell RNA-sequencing indicates that NR0B2 is expressed within the myeloid cells of normal breast tissue. Thus, we hypothesized that myeloid cell NR0B2 is involved in breast cancer pathophysiology and can be used as a druggable target. Intriguingly, overexpression of NR0B2 or activation with a small molecule agonist in murine bone marrow derived macrophages (BMDMs) or dendritic cells skewed T cell expansion away from Tregs. Conversely, Treg expansion increased when NR0B2 was knocked down. In order to investigate the role of NR0B2 in vivo, we monitored the growth of tumors grafted into mice that had NR0B2 genetically knocked out in the myeloid lineage (NR0B2fl/fl;LysMCre+) and respective control mice (NR0B2wt/wt;LysMCre+). Mice lacking NR0B2 exhibited significantly increased tumor growth and elevated tumor infiltrating Tregs. Additionally, human breast tumors samples indicated an inverse correlation between NR0B2 and FOXP3 (marker for Tregs). We further investigated the downstream targets of NR0B2 mediating this anti-tumor phenotype and identified that NLRP3 inflammasome-IL1β axis is a likely modulator in re-educating myeloid cell-Treg function. Importantly, a putative small molecule agonist decreased established metastatic lesions and increased the efficacy of αPD-L1 therapy. Subsequent medicinal chemistry was used to develop a novel NR0B2 agonist with strong anti-metastatic properties when used as a single agent in a preclinical mouse model. Collectively, our data implicates NR0B2 within myeloid cells as a modulator of Tregs, a cell population that has thus far been therapeutically intractable. Therefore, NR0B2 may prove to be a promising therapeutic strategy to improve breast cancer immunotherapy. Presentation: Thursday, June 15, 2023

Functional impact of oxygen-saturated zeolite nanoparticles on macrophages in the context of glioblastoma: an in vitro and in vivo study.

In the context of glioblastoma (GBM), hypoxia and inflammation are two main players of the tumor microenvironment. Hypoxia stimulates various features involves in tumor growth and also maintains a specific environment that favors protumor macrophages. Therefore, targeting hypoxia could potentially restore an anti-tumor M1 phenotype in macrophages. Besides, iron demonstrated its capacity to stimulate the polarization of macrophages towards an M1-like phenotype. In this paper we took advantages of microporous nanoparticles to co-deliver both oxygen and iron to bone marrow derived macrophages (BMDM) enabling the investigation of changes in polarization status and proteomic profiles. The nanoparticles were used in two in vivo models of glioblastoma, specifically, in both immunodeficient and immunocompetent settings. Our in vitro findings revealed that iron doped nanoparticles, saturated with oxygen were deemed safe for macrophages but did not demonstrate the capacity to change the M1 or M2 phenotypes. However, these nanoparticles induced some changes in proteomics pathways. The present study reports on in vivo experimentation that revealed the effects of nanoparticles on the hypoxic fraction, tumor volume, and macrophage phenotype in a GBM model. The findings indicated that the presence of nanoparticles led to a reduction in the hypoxic fraction in one of the GBM models, while no significant changes were observed in the tumor volume or macrophage phenotype. The present data showed that nanoparticles possess the capability of delivering both oxygen and iron to macrophages; though, they do not possess the ability to effectively repolarize M2 macrophages. Such strategies could be used in conjunction with other potent molecules to avoid M1 macrophages to inevitably differentiate to M2 macrophages.

Iron oxide polyaniline-coated nanoparticles modulate tumor microenvironment in breast cancer: an in vitro study on the reprogramming of tumor-associated macrophages

BackgroundBreast cancer is the neoplastic disease with the highest incidence and mortality in the female population worldwide. Treatment remains challenging due to various factors. Therefore, it is of great importance to develop new therapeutic strategies that promote the safe destruction of neoplastic cells without compromising patients' quality of life. Among advances in the treatment of breast cancer, immunotherapy stands out as a promising trend. Recent studies have demonstrated the potential of iron oxide nanoparticles in promoting the reprogramming of M2 macrophages (pro-tumor phenotype) into M1 macrophages (anti-tumor phenotype) within the tumor microenvironment, resulting in potent antitumor effects. In this study, the effect of polyaniline-coated iron oxide nanoparticles (Pani/y-Fe2O3) on macrophage polarization and breast cancer cell death was investigated.MethodsThe non-cytotoxic concentration of nanoparticles was determined using the MTT assay. For in vitro co-culture experiments, breast cancer cell lines MCF -7 and MDA-MB -231 and macrophages THP-1 were co-cultured in a Transwell system and then the effects of Pani/y-Fe2O3 on cell viability, gene expression, cytokine profile, and oxidative stress markers were investigated.ResultsThe results showed that Pani/y-Fe2O3 nanoparticles induced M2-to-M1 macrophage polarization in both cell lines through different pathways. In MCF -7 and THP-1 macrophage co-culture, the study showed a decrease in cytokine levels IL -1β, upregulation of M1-associated genes (IL-12, TNF-α) in macrophages, resulting in increased MCF -7 cell death by apoptosis (caspase 3/7+). In MDA-MB -231 co-cultures, increases in cytokines IL -6, IL -1β, and oxidative stress markers were observed, as well as upregulation of the inducible nitric oxide synthase (iNOS) gene in macrophages, leading to tumor cell death via apoptosis-independent pathways (Sytox+).ConclusionsThese findings highlight the potential of Pani/y-Fe2O3 as a promising therapeutic approach in the context of breast cancer treatment by effectively reprogramming M2 macrophages into an anti-tumor M1 phenotype, Pani/y-Fe2O3 nanoparticles demonstrated the ability to elicit antitumor effects in both MCF-7 and MDA-MB-231 breast cancer cell lines.

ILC1-derived IFN-γ regulates macrophage activation in colon cancer

BackgroundTumor-associated macrophages (TAMs) are an important subset of innate immune cells in the tumor microenvironment, and they are pivotal regulators of tumor-promoting inflammation and tumor progression. Evidence has proven that TAM numbers are substantially increased in cancers, and most of these TAMs are polarized toward the alternatively activated M2 phenotype; Thus, these TAMs strongly promote the progression of cancer diseases. Type 1 innate lymphocytes (ILC1s) are present in high numbers in intestinal tissues and are characterized by the expression of the transcription factor T-bet and the secretion of interferon (IFN)-γ, which can promote macrophages to polarize toward the classically activated antitumor M1 phenotype. However, the relationship between these two cell subsets in colon cancer remains unclear.MethodsFlow cytometry was used to determine the percentages of M1-like macrophages, M2-like macrophages and ILC1s in colon cancer tissues and paracancerous healthy colon tissues in the AOM/DSS-induced mouse model of colon cancer. Furthermore, ILC1s were isolated and bone marrow-derived macrophages were generated to analyze the crosstalk that occurred between these cells when cocultured in vitro. Moreover, ILC1s were adoptively transferred or inhibited in vivo to explore the effects of ILC1s on tumor-infiltrating macrophages and tumor growth.ResultsWe found that the percentages of M1-like macrophages and ILC1s were decreased in colon cancer tissues, and these populations were positively correlated. ILC1s promoted the polarization of macrophages toward the classically activated M1-like phenotype in vitro, and this effect could be blocked by an anti-IFN-γ antibody. The in vivo results showed that the administration of the Group 1 innate lymphocyte-blocking anti-NK1.1 antibody decreased the number of M1-like macrophages in the tumor tissues of MC38 tumor-bearing mice and promoted tumor growth, and adoptive transfer of ILC1s inhibited tumors and increased the percentage of M1-like macrophages in MC38 tumor-bearing mice.ConclusionsOur studies preliminarily prove for the first time that ILC1s promote the activation of M1-like macrophages by secreting IFN-γ and inhibit the progression of colon cancer, which may provide insight into immunotherapeutic approaches for colon cancer.

Phytochemical-derived tumor-associated macrophage remodeling strategy using Phoenix dactylifera L. boosted photodynamic therapy in melanoma via H19/iNOS/PD-L1 axis

BackgroundThe tumor microenvironment (TME) represents a barrier to PDT efficacy among melanoma patients. The aim of this study is to employ a novel muti-tactic TME-remodeling strategy via repolarization of tumor-associated macrophages (TAMs), the main TME immune cells in melanoma, from the pro-tumor M2 into the antitumor M1 phenotype using Phoenix dactylifera L. (date palm) in combination with PDT. MethodsScreening of different date cultivars was employed to choose extracts of selective toxicity to melanoma and TAMs, not normal macrophages. Potential extracts were then fractionated and characterized by gas chromatography-mass spectrometry (GC-MS). Finally, the efficacy and the potential molecular mechanism of the co-treatment were portrayed via quantitative real-time polymerase chain reaction (qRT-PCR) analysis. ResultsInitial screening resulted in the selection of the two Phoenix dactylifera L. cultivars Safawi and Sukkari methanolic extracts. Sukkari showed superior capacity to revert TAM phenotype into M1 as well as more prominent upregulation of M1 markers and repression of melanoma immunosuppressive markers relative to positive control (resiquimod). Molecularly, it was shown that PDT of melanoma cells in the presence of the secretome of repolarized TAMs surpassed the monotherapy via the modulation of the H19/iNOS/PD-L1immune-regulatory axis. ConclusionThis study highlights the potential utilization of nutraceuticals in combination with PDT in the treatment of melanoma to provide a dual activity through alleviating the immune suppressive TME and potentiating the anti-tumor responses.

Evaluation of the safety and immunological effects of Bacillus Calmette–Guérin in combination with checkpoint inhibitor therapy in a patient with neuroendocrine carcinoma: a case report

BackgroundImmune checkpoint inhibitors have revolutionized therapy of advanced and metastatic cancers. However, a significant proportion of patients do not respond to immune checkpoint inhibitors or develop resistance. Therefore, novel therapies or combinations of therapies that may act synergistically are needed. It has been suggested that induction of trained immunity may increase the response to immune checkpoint inhibitor therapy, through reprogramming myeloid cells toward an antitumor phenotype. On the other hand, activation of the immune system also carries the risk of potentially sustaining tumorgenicity and increasing immune- related toxicity.Case presentationWe report the case of a 37-year-old Dutch male suffering from gastric neuroendocrine carcinoma with liver metastases and high risk for an unfavorable outcome, who was treated with a combination of programmed cell death protein 1 inhibitor nivolumab and the trained immunity-inducer Bacillus Calmette–Guérin vaccine as a salvage therapy. Three doses of BCG vaccine were administered at 3-month intervals, in conjunction with the immune checkpoint inhibitor regimen. At a certain point, radiation therapy was added to the treatment regimen. During the combination of these therapies, the patient developed immune-mediated colitis, which necessitated discontinuation of all treatments. Bacillus Calmette–Guérin vaccination induced a trained immune response with elevated monocyte-derived interleukin-6 and interleukin-1β production capacity. From the first vaccination with Bacillus Calmette–Guérin until 3 months after the last vaccination with Bacillus Calmette–Guérin, the patient displayed only mild progression of the primary tumor and no progression of the metastases.ConclusionIn this study, we show the feasibility to combine checkpoint inhibitor therapy with inducers of trained immunity in a patient with an aggressive neuroendocrine tumor. Autoimmune side effects are common under programmed cell death protein 1 inhibitor therapy, which was considered the most likely cause of colitis, although an additive effect of Bacillus Calmette–Guérin vaccination or radiotherapy cannot be excluded. The patient displayed only mild progression during the combination therapy, but larger studies are warranted to fully explore the potential benefit of trained immunity inducers as an adjuvant to immune checkpoint inhibitor therapy.

Oroxylin a promoted apoptotic extracellular vesicles transfer of glycolytic kinases to remodel immune microenvironment in hepatocellular carcinoma model

Although oroxylin A, a natural flavonoid compound, suppressed progression of hepatocellular carcinoma, whether the tumor microenvironment especially the communication between cancer cells and immune cells was under its modulation remained obscure. Here we investigated the effect of extracellular vesicles from cancer cells elicited by oroxylin A on macrophages in vitro. The data shows oroxylin A elicits apoptosis-related extracellular vesicles through caspase-3-mediated activation of ROCK1in HCC cells, which regulates M1-like polarization of macrophage. Moreover, oroxylin A downregulates the population of M2-like macrophage and promotes T cells infiltration in tumor microenvironment, accompanied by suppression of HCC development and enhancement of immune checkpoint inhibitor treatment in mice model. Mechanistically, glycolytic proteins enriched in oroxylin A-elicited extracellular vesicles from HCC cells are transferred to macrophages where ROS-dependent NLRP3 inflammasome is activated, therefore contributing to anti-tumor phenotype of macrophage. Taken together, this study highlights that oroxylin A promotes metabolic shifts between tumor cells and immune cells, facilitates to inhibit tumor development, and improves immunotherapy response in HCC model.

Supermolecular nanovehicles co-delivering TLR7/8-agonist and anti-CD47 siRNA for enhanced tumor immunotherapy

Cancer immunotherapy is the most promising method for tumor therapy in recent years, among which the macrophages play a critical role in the antitumor immune response. However, tumor-associated macrophages (TAMs) usually display the tumor-promoting M2 phenotype rather than the tumor-killing M1 phenotype. Moreover, the over-expressed CD47 on tumor cells severely hinders the function of macrophages by blocking the CD47/SIRPα pathway. Herein, a nano-assembly system of CHTR/siRNA was constructed through the host-guest interaction of a hyperbranched amino-functionalized β-cyclodextrin and immune agonist imiquimod (R848), while CD47 siRNA was loaded inside through electrostatic interaction. The Toll-like receptor (TLR) 7/8 agonist R848 can “re-educate” macrophages from the protumoral M2 phenotype to antitumoral M1 phenotype, while CD47 siRNA can down-regulate the “don't eat me” CD47 signal on the surface of cancer cells and enhance the phagocytosis of cancer cells by macrophages. Through the dual regulation of TAMs, the immunosuppressive tumor microenvironment was relieved, and the host-guest drug-carrying system resulted in synergistic immunotherapy effect on tumors and inhibited tumor growth. The facile self-assembly of nanodrug offers a new strategy in co-delivery of multiple therapeutic agents for cascade cancer immunotherapy.