Deficiencies in T cells are a major limitation for cancer immunotherapy. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) are key mediators of immune suppression, contributing to poor clinical outcomes and resistance to checkpoint inhibitors (CPI). Excessive peroxidation of polyunsaturated phospholipids, especially phosphatidylethanolamines (PE), is a critical stage of the ferroptotic cell death process. PMN-MDSC are undergoing ferroptosis in tumors. Here, we show that initiation of ferroptosis causes PMN-MDSCs to release oxidized PE (oxPE) via extracellular vesicles, shielding themselves from immediate death while triggering fatal injury to neighboring T cells. The accumulation of oxPE and oxidized bis-monoacylglycetophosphates (oxBMP) in the endo-lysosomes of T cells increases lysosomal membrane permeability (LMP), resulting in T cell death. In vivo inhibition of ferroptosis in PMN-MDSCs reduces LMP, increases intratumoral T cell numbers, and promotes antitumor effects across multiple mouse models. Targeting ferroptosis reverses CPI resistance in mice. Analysis of tumor samples from cancer patients revealed association of T cell depletion with ferroptotic PMN-MDSC and T-cell depletion in regions enriched for PMN-MDSCs, with T cell LMP closely associated with PMN-MDSC proximity. This translated to worse clinical outcome in patients with high ferroptosis signature. These findings indicate that PMN-MDSC ferroptosis drives T cell loss, highlighting a potential therapeutic target for cancers with high PMN-MDSC infiltration.

Background. Antibody drug conjugates (ADCs) represent a transformative class of cancer therapeutics, yet the mechanisms underlying their synergy with immunotherapy remain poorly understood. We investigated the mechanistic basis of ADC combinations with T cell engagers (TCEs) and checkpoint inhibitors (CPI). Methods ADC/TCE and ADC/CPI combinations were evaluated in vitro using co culture cytotoxicity assays and synergy analyses, and in vivo in humanized mouse tumor models. Mechanistic studies employed TNF and cytokine blocking antibodies, receptor knockout cell lines, autophagy inhibitors, and siRNA silencing. Results ADC/TCE combinations produced synergistic antitumor activity independent of target antigen and payload, persisting despite ADC induced T cell loss. ADC treatment induced autophagy, upregulating TNF receptors (TNFR1/2) and mannose 6 phosphate receptor (M6PR) on tumor cell surfaces. In ADC/TCE combinations, TCE derived TNF; acting on ADC upregulated TNFRs was the primary mediator of enhanced cytotoxicity, confirmed in vivo by TNF blockade. In contrast, combinations with CPI expanded antigen specific T cells operated through a TNF independent, M6PR dependent pathway involving enhanced granzyme B uptake. Conclusions ADC induced autophagy is a unifying, target- and payload-agnostic mechanism sensitizing tumor cells to T cell mediated killing. TCEs exploit a TNF/TNFR axis, whereas antigen specific T cells leverage granzyme B/M6PR uptake. This mechanistic framework enables rational selection and design of ADC immunotherapy combination strategies.

Tumor-associated macrophages (TAMs) are key players in tumor progression, yet their role in this process remains only partially understood. In this issue of Cancer Cell, Sheban et al. demonstrate that zinc finger E-box-binding homeobox 2 (ZEB2) acts as a master regulator that reprograms TAMs toward a pro-tumor phenotype and that therapeutic targeting of ZEB2 exhibits anti-tumor activity.

Co-occupied and unique target genes shared between Creb and RNApol-II in chromatin immunoprecipitation and sequencing (ChIP-seq) experiments performed in murine Kras-Trp53 cooperative KPC pancreatic cancer cells

Plasticity of myeloid cells, characterized by their ability to undergo reprogramming in response to environmental cues, is a fundamental feature enabling their versatile functions during immune responses. Macrophages and neutrophils, the primary myeloid cell types, exhibit distinct polarization states. Classical polarization states of macrophages and neutrophils are associated with antimicrobial activity, inflammation promotion, and tissue remodeling. Pathological polarization, observed in chronic inflammation, cancer, and other conditions, is marked by enhanced immune-suppressive activity, aberrant enzymatic activity, and atypical cytokine production, diverging from their classical functions. This review delves into the most up-to-date characterization of those polarization states, the transcriptional and epigenetic factors, and the metabolic pathways governing myeloid cell reprogramming, highlighting the influence of cytokines and tissue-specific conditions, such as hypoxia in tumors, on this process. Understanding the mechanisms underlying the pathological polarization of myeloid cells offers a promising avenue to modulate their activity for targeted therapeutic interventions.

Neutrophils have a pivotal role in safeguarding the host against pathogens and facilitating tissue remodeling. They possess a large array of tools essential for executing these functions. Neutrophils have a critical role in cancer, where they are largely associated with negative clinical outcome and resistance to therapy. However, the specific role of neutrophils in cancer is complex and controversial, owing to their high functional diversity and acute sensitivity to the microenvironment. In this Perspective, we discuss the accumulated evidence that suggests that the functional diversity of neutrophils can be ascribed to two principal functional states, each with distinct characteristics: classically activated neutrophils and pathologically activated immunosuppressive myeloid-derived suppressor cells. We discuss how the antimicrobial factors in neutrophils can contribute to tumor progression and the fundamental mechanisms that govern the pathologically activated myeloid-derived suppressor cells. These functional states play divergent roles in cancer and thus require separate consideration in therapeutic targeting. Gabrilovich and colleagues discuss how the functional diversity of neutrophils in cancer can be ascribed to two functional states: classically activated neutrophils and pathologically activated myeloid-derived suppressor cells.

Neutrophils, previously considered a homogeneous immune cell population, exhibit substantial heterogeneity. Their diverse phenotypic and functional states are shaped by tissue microenvironments and disease-specific signals. However, the lack of robust fate-mapping methods and standardized classification criteria has led to overlapping and ambiguous descriptions of neutrophil heterogeneity. The growing number of neutrophil subpopulations reported in recent years highlights the need for a standardized framework to report how they might relate to each other. Here, we propose a framework that integrates maturation, tissue localization, and functional adaptations. This standardized system aims to harmonize research efforts, foster clearer cross-disciplinary communication, and accelerate both fundamental discoveries in neutrophil biology and the development of targeted therapies.

Differentially expressed pathways comparing transcriptomes in KRAS-TP53 co-altered (n=23) and KRAS-altered/TP53WT (n=5) derived from Cancer Cell Line Encyclopedia

The Endoplasmic Reticulum (ER) is a critical immunomodulatory hub governing multiple processes relating to anti-tumor immunity. TMEM33 is an ER-resident membrane protein implicated in various ER-associated functions including cholesterol metabolism, calcium oscillations, and proteostasis. We previously reported TMEM33 as a negative modulator of the innate immune signaling protein STING (STimulator of INterferon Genes). Here we identify a central role of TMEM33 in tumor immunology where its deficiency profoundly impacts infiltrating immune populations and T cell phenotype. STING-mediated ISG (interferon-stimulated gene) expression was augmented in Tmem33-/- bone marrow-derived macrophages and TMEM33-depleted Flp-In T-REx 293 cells. Strikingly, Tmem33-/- mice exhibited significantly attenuated B16F10-OVA tumor growth and enhanced CD8+ T cell tumor infiltration compared to wild-type (WT) controls, while immunosuppressive immune populations including Treg cells, monocytic myeloid-derived suppressor cells and M2-like macrophages were diminished. CD8+ T cell enrichment was also observed in MC38 tumor-bearing Tmem33-/- hosts. Unchallenged mice displayed no overt phenotypic or immunological differences compared to WT, however. High dimensional spectral flow cytometry was leveraged to further examine effects of Tmem33-/- on T cell compartments in B16F10-OVA tumors and draining lymph nodes (DLNs). Progenitor exhausted (Tpex) TCF-1+PD-1+ tumor infiltrating lymphocytes (TILs) were significantly enriched in Tmem33-/- hosts, notable given that Tpex are responsive to checkpoint blockade therapy and associate with improved patient prognosis. Compared to WT counterparts, OVA-specific Tmem33-/- TILs exhibited increased cytotoxicity and effector function, expressing elevated granzyme-B and TNF-α. Moreover, EOMESloT-bethi TILs were more frequent among TCF-1+PD-1+ and TCF-1+ populations in Tmem33-/- hosts further suggestive of a more persistent CD8+ precursor pool, while inhibitory receptor (LAG-3, TIGIT) abundance was blunted, indicating reduced exhaustion. In DLNs of Tmem33-/- mice, antigen-specific CD8+ populations demonstrated enhanced effector memory (CD44+CD62L-) phenotypes alongside elevated CXCR3 expression compared to WT, suggesting increased tumor-migratory potential. In summary, here we identify TMEM33 as a novel immunoregulatory factor, the loss of which in hosts majorly reshapes the tumor-immune landscape and ameliorates multiple aspects of CD8+ T cell fitness including stemness, exhaustion, memory, cytotoxicity and migratory capacity. Further analysis of T cell populations from tumor-bearing mice and ex vivo analysis will enable mechanistic refinement of the role of TMEM33. Moreover, our study supports the importance of ER-resident proteins in fine-tuning immune cell function, underlining their potential as promising targets for therapeutic intervention. Matthew T. Jackson, Tianming Zhao, Isabela Pedroza-Pacheco, Amit Grover, Dmitry Gabrilovich, David Withers, Jan Rehwinkel, Eric Honore, John C. Christianson, Eileen E. Parkes. TMEM33 loss improves CD8+ T cell fitness and tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4859.

The Ataxia telangiectasia and Rad3-related (ATR) inhibitor ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. Here we show that modelling of intermittent ceralasertib treatment in mouse tumor models reveals CD8 + T-cell dependent antitumor activity, which is separate from the effects on tumor cells. Ceralasertib suppresses proliferating CD8 + T-cells on treatment which is rapidly reversed off-treatment. Ceralasertib causes up-regulation of type I interferon (IFNI) pathway in cancer patients and in tumor-bearing mice. IFNI is experimentally found to be a major mediator of antitumor activity of ceralasertib in combination with PD-L1 antibody. Improvement of T-cell function after ceralasertib treatment is linked to changes in myeloid cells in the tumor microenvironment. IFNI also promotes anti-proliferative effects of ceralasertib on tumor cells. Here, we report that broad immunomodulatory changes following intermittent ATR inhibition underpins the clinical therapeutic benefit and indicates its wider impact on antitumor immunity.

Abstract BACKGROUND: Circulating polymorphonuclear neutrophils (PMN) traffic to pancreatic cancer (PDAC)-derived cues, where they acquire specialization as myeloid-derived suppressor cells (MDSC) to exert immune tolerance and stromal inflammation. We sought to dissect the developmental heterogeneity of PMN/MDSCs during PDAC tumorigenesis, molecular networks underlying these fate transitions, and their correlation with oncologic outcomes in PDAC patients. METHODS:Pancreatic tumors & spleens from Ptf1a Cre/+;LSL-Kras G12D/+;Tgfbr2 fl/fl (PKT) mice at 4 and 6 wks of age, along with pancreata/spleens from age-matched littermates, were subjected to single-cell RNA sequencing (scRNAseq). Clustering and splicing kinetics identified distinct populations of circulating and tissue-resident PMN/MDSCs. Ingenuity Pathway Analysis (IPA) nominated unique surface markers for each subset, which was validated by flow cytometry. Paired RNA-ATAC sequencing and functional studied were performed in marker- sorted PMNs. RESULTS: scRNAseq of PKT tumors/spleens revealed striking temporal and phenotypic heterogeneity in PMN subclusters with PDAC tumorigenesis. RNA velocity analysis identified distinct terminal-state (TS) populations in tumors and circulation. The intratumoral-TS fate of PMNs resembled MDSC-like specialization, with enrichment of gene programs indicating suppressive function (Tnf,Il1b,Cd14). Conversely, spleen-TS PMNs showed imprinting of trafficking programs (Cxcr2,Il1rap,Gab2). Intriguingly, a discrete PMN cluster in both spleen and tumors displayed upregulated MHC-II and co-stimulatory molecule genes (H2-Ab,Cd79a,Cd80). IPA of differentially expressed genes with transmembrane localization nominated Cd170, Cd162, and MHC-II as surface markers for tumor-TS, spleen-TS, and MHC-II PMN subsets, which were validated by flow cytometry. RNAseq of Cd170/SiglecF+ intratumoral MDSCs revealed upregulation in inflammatory and pro-survival signaling pathways (Akt/mTor-PID, Il1-KEGG, Myc targets-HALLMARK), with TNF as top predicted upstream regulator by IPA. Enrichment in fibrosis-related genes were supported by Smad4 motif enrichment in Cd170+ RNA/ATAC-seq, suggesting novel profibrotic function. Transcription factor (TF) inference in MHC-II+ PMNs revealed high activity scores for TFs (Rora,Irf1) regulating antigen presentation pathways. Ex vivo co-culture of T-cells with marker-sorted PMNs showed reduced T-cell proliferation/activation preferentially with Cd170+ MDSCs, which were partially and completely reversed upon Cd162+ and MHC-II+ PMN co-culture, respectively. In human PDAC scRNAseq data, increased ratio of CD170:MHC-II PMN correlated with chemoresistance and poor survival. CONCLUSIONS: Phenotypic plasticity of PMNs during PDAC progression drive functional divergence and terminal fate determinism in distinct tissue niches. Relative dosage of MDSCs to MHC-II+ PMN may have prognostic and predictive value. Mechanisms governing fate transitions in PMN subsets could pave the way for future therapeutic targets to overcome immunotherapy resistance in PDAC. Citation Format: Anna Bianchi, Manan Patel, Da Yin, Haleh Amirian, Karthik Rajkumar, Andrew Mark Adams, Erin Dickey, Harper Margaret Marsh, Nagaraj Nagathihalli, Nipun Merchant, Dmitry Gabrilovich, Jashodeep Datta. Phenotypic plasticity and functional divergence of neutrophilic MDSCs in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C068.

Abstract This study is aimed to investigate the effect of ATR inhibitor (ATRi) ceralasertib on tumor microenvironment (TME). Ceralasertib has demonstrated encouraging clinical benefits in patients with non-small cell lung cancer who were resistant to PD1/PDL1 treatment. Using different pre-clinical mouse tumor models, we found that antitumor effect of ceralasertib was dependent on CD8 T cells. In fact, depletion of CD8 cells abrogated therapeutic effect of the ATRi. Ceralasertib treatment caused pleotropic effect in TME. It improved the function of T cells, depleted tumor associated macrophages and monocytic myeloid-derived-suppressor cells (MDSC) and inactivated suppressive activity of PMN-MDSC. Ceralasertib treatment resulted in activation of dendritic cells (DC) in tumor-bearing mice. DCs demonstrated increased ability to stimulate T cells, that resulted in enhanced antigen-specific T cell responses in mice treated with ceralasertib in combination with anti-PDL1. Gene expression profile showed significant up-regulation of type I interferon (IFN1) pathway in mice and patients treated with ceralasertib. Using BM chimera mice reconstituted with IFANR1KO for or IFNAR1SA bone marrow we demonstrated a cancelation of antitumor effect, or an enhancement of antitumor effect respectively induced by ceralasertib or combination therapy. DC cells isolated from IFNAR1KO mice treated with Ceralasertib failed to show an activated phenotype and to increase T cell activation demonstrating the pivotal role of type I IFN in ceralasertib-mediated immune activation. In conclusion, our finding showed that ATR inhibitor ceralasertib has a second mechanism of action necessary for its efficacy: modulating the tumor immune microenvironment and increasing sensitivity to anti-PDL1 treatment. Citation Format: Emilio Sanseviero, Devon Taylor, Sehmus Tohumeken, Mimi Mai, Ali Mostafa, Marta Milo, Kathy Mulgrew, Serge Fuchs, Alan Lau, Simon Barry, Mark Cobbold, Dmitry Gabrilovich. Ceralasertib enhance efficacy of anti-PDL1 treatment by modulating the tumor microenvironment in a IFNAR I dependent manner [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B006.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

Phenotype of cells used for the analysis of tumour tissues

Demographic and baseline characteristics

Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3, Supplementary Figure 4, Supplementary Figure 5, Supplementary Figure 6, Supplementary Figure 7

Most frequently reported drug related adverse event