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The TGFβ receptor inhibitor galunisertib showed promising efficacy in patients with pancreatic ductal adenocarcinoma (PDAC) in the phase 2 H9H-MC-JBAJ study. Identifying biomarkers for this treatment remains essential. Baseline plasma levels of chemokine CCL3 were integrated with clinical outcomes in PDAC patients treated with galunisertib plus gemcitabine (n = 104) or placebo plus gemcitabine (n = 52). High CCL3 was a poor prognostic factor in the placebo group (mOS 3.6 vs. 10.1 months; p < 0.01) but a positive predictor for galunisertib (mOS 9.2 vs. 3.6 months; p < 0.01). Mechanistically, tumor-derived CCL3 activates Tgfβ signaling in macrophages, inducing their M2 phenotype and Lif secretion, sustaining a mesenchymal/basal-like ecotype. TGFβ inhibition redirects macrophage polarization to M1, reducing Lif and shifting PDAC cells to a more epithelial/classical phenotype, improving gemcitabine sensitivity. This study supports exploring TGFβ-targeting agents in PDAC with a mesenchymal/basal-like ecotype driven by high CCL3 levels., (© 2024. The Author(s).)

Background: Pancreatic cancer (PC) is characterised by late diagnosis, tumour heterogeneity, and a peculiar immunosuppressive microenvironment, leading to poor clinical outcomes. Local ablative techniques have been proposed to treat unresectable PC patients, although their impact on activating the host immune system and overcoming resistance to immunotherapy remains elusive., Methods: We dissected the immune-modulatory abilities triggered by local ablation in mouse and human PC models and human specimens, integrating phenotypic and molecular technologies with functional assays., Results: Local ablation treatment performed in mice bearing orthotopic syngeneic PC tumours triggered tumour necrosis and a short-term inflammatory process characterised by the prompt increase of HMGB1 plasma levels, coupled with an enhanced amount of circulating and tumour infiltrating myeloid cells and increased MHCII expression in splenic myeloid antigen-presenting cells. Local ablation synergised with immunotherapy to restrict tumour progression and improved the survival of PC-bearing mice by evoking a T lymphocyte-dependent anti-tumour immune response. By integrating spatial transcriptomics with histological techniques, we pinpointed how combination therapy could reshape TME towards an anti-tumour milieu characterised by the preferential entrance and colocalization of activated T lymphocytes and myeloid cells endowed with antigen presentation features instead of T regulatory lymphocytes and CD206-expressing tumour-associated macrophages. In addition, treatment-dependent TME repolarization extended to neoplastic cells, promoting a shift from squamous to a more differentiated classical phenotype. Finally, we validated the immune regulatory properties induced by local ablation in PC patients and identified an association of the short-term treatment-dependent increase of neutrophils, NLR and HMGB1 with a longer time to progression., Conclusion: Therefore, local ablation might overcome the current limitations of immunotherapy in PC., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: VB reports personal fees from Codiak BioSciences and IO Biotech ApS outside of the submitted work. SP receives consultancy honoraria from AlphaTau Medical. The other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: The dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of the axon guidance cue SEMA3A in supporting PDAC progression., Design: We integrated bulk and single-cell transcriptomic datasets of human PDAC with in situ hybridisation analyses of patients' tissues to evaluate SEMA3A expression in molecular subtypes of PDAC. Gain and loss of function experiments in PDAC cell lines and organoids were performed to dissect how SEMA3A contributes to define a biologically aggressive phenotype., Results: In PDAC tissues, SEMA3A is expressed by stromal elements and selectively enriched in basal-like/squamous epithelial cells. Accordingly, expression of SEMA3A in PDAC cells is induced by both cell-intrinsic and cell-extrinsic determinants of the basal-like phenotype. In vitro , SEMA3A promotes cell migration as well as anoikis resistance. At the molecular level, these phenotypes are associated with increased focal adhesion kinase signalling through canonical SEMA3A-NRP1 axis. SEMA3A provides mouse PDAC cells with greater metastatic competence and favours intratumoural infiltration of tumour-associated macrophages and reduced density of T cells. Mechanistically, SEMA3A functions as chemoattractant for macrophages and skews their polarisation towards an M2-like phenotype. In SEMA3A high tumours, depletion of macrophages results in greater intratumour infiltration by CD8+T cells and better control of the disease from antitumour treatment., Conclusions: Here, we show that SEMA3A is a stress-sensitive locus that promotes the malignant phenotype of basal-like PDAC through both cell-intrinsic and cell-extrinsic mechanisms., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Richter's syndrome (RS) is the transformation of chronic lymphocytic leukemia (CLL) into a high-grade B-cell malignancy. Molecular and functional studies have pointed out that CLL cells are close to the apoptotic threshold and dependent on BCL-2 for survival. However, it remains undefined how evasion from apoptosis evolves during disease transformation. Here, we employed functional and static approaches to compare the regulation of mitochondrial apoptosis in CLL and RS. BH3 profiling of 17 CLL and 9 RS samples demonstrated that RS cells had reduced apoptotic priming and lower BCL-2 dependence than CLL cells. While a subset of RS was dependent on alternative anti-apoptotic proteins and was sensitive to specific BH3 mimetics, other RS cases harbored no specific anti-apoptotic addiction. Transcriptomics of paired CLL/RS samples revealed downregulation of pro-apoptotic sensitizers during disease transformation. Albeit expressed, effector and activator members were less likely to colocalize with mitochondria in RS compared to CLL. Electron microscopy highlighted reduced cristae width in RS mitochondria, a condition further promoting apoptosis resistance. Collectively, our data suggest that RS cells evolve multiple mechanisms that lower the apoptotic priming and shift the anti-apoptotic dependencies away from BCL-2, making direct targeting of mitochondrial apoptosis more challenging after disease transformation., (© 2024. The Author(s).)

Background: Cancer is still among the leading causes of death all over the world. Improving chemotherapy and minimizing associated toxicities are major unmet medical needs. Recently, we provided a preliminary preclinical evaluation of a human ferritin (HFt)-based drug carrier (The-0504) that selectively delivers the wide-spectrum topoisomerase I inhibitor Genz-644282 to CD71-expressing tumors. The-0504 has so far been evaluated on four different human tumor xenotransplant models (breast, colorectal, pancreatic and liver cancers)., Methods: Herein, we extend our studies, by: (a) testing DNA damage in vitro, (b) treating eight additional tumor xenograft models in vivo with The-0504; (c) performing pharmacokinetic (PK) studies in rats; and (d) evaluating The-0504 anti-tumor xenotransplant efficacy by optimizing its administration schedule based on PK considerations., Results: Immunofluorescence demonstrated that The-0504 induces foci expressing the DNA double-strand break marker γH2AX. Expression increases up to 4-fold and is more persistent as compared to free Genz-644282. In vivo studies confirmed a remarkable anti-tumor activity of The-0504, resulting in tumor eradication in most murine xenograft models, regardless of embryological origin (e.g. epithelial, mesenchymal or neuroendocrine), and molecular subtypes. PK studies demonstrated a long persistence of The-0504 in rat serum (half-life of about 40 h as compared to 15 h of the free drug), with a 400-fold increase in peak concentrations as compared to the free drug. On this basis, we reduced The-0504 administration frequency from twice to once per week, with no appreciable loss in therapeutic efficacy in mice., Conclusion: The results presented here confirm that The-0504 is highly active against several human tumor xenotransplants, even when administered less frequently than previously reported. The-0504 may be a good candidate for further clinical development in a tumor histotype-agnostic setting., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Pierpaolo Ceci and Elisabetta Falvo are inventors on patent application EP3186192B1 held by Thena Biotech that covers fusion proteins based on human ferritins and methods of use thereof. Pierpaolo Ceci is a member (free of charge) of the Scientific Advisory Committee of Thena Biotech. All the authors declare no conflict of interest., (© 2023 The Authors.)

Cancer cells favor the generation of myeloid cells with immunosuppressive and inflammatory features, including myeloid-derived suppressor cells (MDSCs), which support tumor progression. The anti-apoptotic molecule, cellular FLICE (FADD-like interleukin-1β-converting enzyme)-inhibitory protein (c-FLIP), which acts as an important modulator of caspase-8, is required for the development and function of monocytic (M)-MDSCs. Here, we assessed the effect of immune checkpoint inhibitor (ICI) therapy on systemic immunological landscape, including FLIP-expressing MDSCs, in non-small cell lung cancer (NSCLC) patients. Longitudinal changes in peripheral immunological parameters were correlated with patients' outcome. In detail, 34 NSCLC patients were enrolled and classified as progressors (P) or non-progressors (NP), according to the RECIST evaluation. We demonstrated a reduction in pro-inflammatory cytokines such as IL-8, IL-6, and IL-1β in only NP patients after ICI treatment. Moreover, using t -distributed stochastic neighbor embedding ( t -SNE) and cluster analysis, we characterized in NP patients a significant increase in the amount of lymphocytes and a slight contraction of myeloid cells such as neutrophils and monocytes. Despite this moderate ICI-associated alteration in myeloid cells, we identified a distinctive reduction of c-FLIP expression in M-MDSCs from NP patients concurrently with the first clinical evaluation (T1), even though NP and P patients showed the same level of expression at baseline (T0). In agreement with the c-FLIP expression, monocytes isolated from both P and NP patients displayed similar immunosuppressive functions at T0; however, this pro-tumor activity was negatively influenced at T1 in the NP patient cohort exclusively. Hence, ICI therapy can mitigate systemic inflammation and impair MDSC-dependent immunosuppression., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The p140Cap adaptor protein is a tumor suppressor in breast cancer associated with a favorable prognosis. Here we highlight a function of p140Cap in orchestrating local and systemic tumor-extrinsic events that eventually result in inhibition of the polymorphonuclear myeloid-derived suppressor cell function in creating an immunosuppressive tumor-promoting environment in the primary tumor, and premetastatic niches at distant sites. Integrative transcriptomic and preclinical studies unravel that p140Cap controls an epistatic axis where, through the upstream inhibition of β-Catenin, it restricts tumorigenicity and self-renewal of tumor-initiating cells limiting the release of the inflammatory cytokine G-CSF, required for polymorphonuclear myeloid-derived suppressor cells to exert their local and systemic tumor conducive function. Mechanistically, p140Cap inhibition of β-Catenin depends on its ability to localize in and stabilize the β-Catenin destruction complex, promoting enhanced β-Catenin inactivation. Clinical studies in women show that low p140Cap expression correlates with reduced presence of tumor-infiltrating lymphocytes and more aggressive tumor types in a large cohort of real-life female breast cancer patients, highlighting the potential of p140Cap as a biomarker for therapeutic intervention targeting the β-Catenin/ Tumor-initiating cells /G-CSF/ polymorphonuclear myeloid-derived suppressor cell axis to restore an efficient anti-tumor immune response., (© 2023. The Author(s).)

Inflammatory responses rapidly detect pathogen invasion and mount a regulated reaction. However, dysregulated anti-pathogen immune responses can provoke life-threatening inflammatory pathologies collectively known as cytokine release syndrome (CRS), exemplified by key clinical phenotypes unearthed during the SARS-CoV-2 pandemic. The underlying pathophysiology of CRS remains elusive. We found that FLIP, a protein that controls caspase-8 death pathways, was highly expressed in myeloid cells of COVID-19 lungs. FLIP controlled CRS by fueling a STAT3-dependent inflammatory program. Indeed, constitutive expression of a viral FLIP homolog in myeloid cells triggered a STAT3-linked, progressive, and fatal inflammatory syndrome in mice, characterized by elevated cytokine output, lymphopenia, lung injury, and multiple organ dysfunctions that mimicked human CRS. As STAT3-targeting approaches relieved inflammation, immune disorders, and organ failures in these mice, targeted intervention towards this pathway could suppress the lethal CRS inflammatory state., (© 2021. The Author(s).)

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors owing to its robust desmoplasia, low immunogenicity, and recruitment of cancer-conditioned, immunoregulatory myeloid cells. These features strongly limit the success of immunotherapy as a single agent, thereby suggesting the need for the development of a multitargeted approach. The goal is to foster T lymphocyte infiltration within the tumor landscape and neutralize cancer-triggered immune suppression, to enhance the therapeutic effectiveness of immune-based treatments, such as anticancer adoptive cell therapy (ACT)., Methods: We examined the contribution of immunosuppressive myeloid cells expressing arginase 1 and nitric oxide synthase 2 in building up a reactive nitrogen species (RNS)-dependent chemical barrier and shaping the PDAC immune landscape. We examined the impact of pharmacological RNS interference on overcoming the recruitment and immunosuppressive activity of tumor-expanded myeloid cells, which render pancreatic cancers resistant to immunotherapy., Results: PDAC progression is marked by a stepwise infiltration of myeloid cells, which enforces a highly immunosuppressive microenvironment through the uncontrolled metabolism of L-arginine by arginase 1 and inducible nitric oxide synthase activity, resulting in the production of large amounts of reactive oxygen and nitrogen species. The extensive accumulation of myeloid suppressing cells and nitrated tyrosines (nitrotyrosine, N-Ty) establishes an RNS-dependent chemical barrier that impairs tumor infiltration by T lymphocytes and restricts the efficacy of adoptive immunotherapy. A pharmacological treatment with AT38 ([3-(aminocarbonyl)furoxan-4-yl]methyl salicylate) reprograms the tumor microenvironment from protumoral to antitumoral, which supports T lymphocyte entrance within the tumor core and aids the efficacy of ACT with telomerase-specific cytotoxic T lymphocytes., Conclusions: Tumor microenvironment reprogramming by ablating aberrant RNS production bypasses the current limits of immunotherapy in PDAC by overcoming immune resistance., Competing Interests: Competing interests: AF, SU, and VB hold proprietary rights on the patent applications about engineered cells for inducing tolerance by BioNTech (Mainz, Germany). SU and VB hold proprietary rights on the patent applications about TERT engineering T cells by University of Verona (Verona, Italy). VB holds proprietary rights on the patent applications about nitric oxide furoxan derivative compounds by Humanitas Mirasole (Milan, Italy). MF reports grants from Astellas Pharma, QED Therapeutics, Macrophage Pharma and advisory roles in Astellas Pharma, Tesaro, GlaxoSmithKline, Diaceutics, Roche. VB reports personal fees from Codiak BioSciences and IO Biotech ApS outside the submitted work. No potential conflicts of interest were disclosed by the other authors., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Expression of the L-arginine catabolizing enzyme arginase 1 (ARG1) is a central immunosuppressive mechanism mediated by tumor-educated myeloid cells. Increased activity of ARG1 promotes the formation of an immunosuppressive microenvironment and leads to a more aggressive phenotype in many cancers. Intrinsic T-cell immunity against ARG1-derived epitopes in the peripheral blood of cancer patients and healthy subjects has previously been demonstrated. To evaluate the antitumor efficacy of ARG1-derived peptide vaccines as a monotherapy and as a combinational therapy with checkpoint blockade, different in vivo syngeneic mouse tumor models were utilized. To evaluate the antitumor effects, flow cytometry analysis and IHC were performed on tumors, and ELISPOT assays were performed to characterize immune responses. We show that ARG1-targeting therapeutic vaccines were able to activate endogenous antitumor immunity in several in vivo syngeneic mouse tumor models and to modulate the cell composition of the tumor microenvironment without causing any associated side effects or systemic toxicity. ARG1-targeting vaccines in combination with anti-PD-1 also resulted in increased T-cell infiltration, decreased ARG1 expression, reduced suppressive function of tumor-educated myeloid cells, and a shift in the M1/M2 ratio of tumor-infiltrating macrophages. These results indicated that the induced shift toward a more proinflammatory microenvironment by ARG1-targeting immunotherapy favors effective tumor control when combined with anti-PD-1 checkpoint blockade. Our data illustrate the ability of ARG1-based immune modulatory vaccination to elicit antigen-specific immunosurveillance and imply the feasibility of this novel immunotherapeutic approach for clinical translation., (©2021 American Association for Cancer Research.)

Plasticity and adaptation to environmental stress are the main features that tumor and immune system share. Except for intrinsic and high-defined properties, cancer and immune cells need to overcome the opponent's defenses by activating more effective signaling networks, based on common elements such as transcriptional factors, protein-based complexes and receptors. Interestingly, growing evidence point to an increasing number of proteins capable of performing diverse and unpredictable functions. These multifunctional proteins are defined as moonlighting proteins. During cancer progression, several moonlighting proteins are involved in promoting an immunosuppressive microenvironment by reprogramming immune cells to support tumor growth and metastatic spread. Conversely, other moonlighting proteins support tumor antigen presentation and lymphocytes activation, leading to several anti-cancer immunological responses. In this light, moonlighting proteins could be used as promising new potential targets for improving current cancer therapies. In this review, we describe in details 12 unprecedented moonlighting proteins that during cancer progression play a decisive role in guiding cancer-associated immunomodulation by shaping innate or adaptive immune response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Adamo, Frusteri, Pallotta, Pirali, Sartoris and Ugel.)

Background: IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (T R 1) cells but is also produced by CD25 + regulatory T (Treg) cells., Objective: We aimed to identify and characterize human intestinal T R 1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs)., Methods: CD4 + T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4 + T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1β and IL-23 responsiveness was assessed., Results: Intestinal T R 1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5 + PD-1 + T R 1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ + T R 1 cells, but not IL-7 receptor-positive T H cells or CD25 + Treg cells, showed lower IL-10 expression in patients with IBDs. T R 1 cells were responsive to IL-23, and IFN-γ + T R 1 cells downregulated IL-10 with IL-1β and IL-23. Conversely, CD25 + Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression., Conclusions: We provide the first ex vivo characterization of human intestinal T R 1 cells. Selective downregulation of IL-10 by IFN-γ + T R 1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)