Pericyte stem cells induce Ly6G+ cell accumulation and immunotherapy resistance in pancreatic cancer.

We report the identification of a cell population that shares pericyte, stromal and stemness features, does not harbor the KrasG12D mutation and drives tumoral growth in vitro and in vivo. We term these cells pericyte stem cells (PeSCs) and define them as CD45- EPCAM- CD29+ CD106+ CD24+ CD44+ cells. We perform studies with p48-Cre;KrasG12D (KC), pdx1-Cre;KrasG12D ;Ink4a/Arffl/fl (KIC) and pdx1-Cre;KrasG12D ;p53R172H (KPC) and tumor tissues from PDAC and chronic pancreatitis patients. We also perform single-cell RNAseq analysis and reveal a unique signature of PeSC. Under steady-state conditions, PeSCs are barely detectable in the pancreas but present in the neoplastic microenvironment both in humans and mice. The coinjection of PeSCs and tumor epithelial cells leads to increased tumor growth, differentiation of Ly6G+ myeloid-derived suppressor cells, and a decreased amount of F4/80+ macrophages and CD11c+ dendritic cells. This population induces resistance to anti-PD-1 immunotherapy when coinjected with epithelial tumor cells. Our data reveal the existence of a cell population that instructs immunosuppressive myeloid cell responses to bypass PD-1 targeting and thus suggest potential new approaches for overcoming resistance to immunotherapy in clinical settings.

Immune checkpoint inhibitors reverse tolerogenic mechanisms induced by melanoma targeted radionuclide therapy.

In line with the ongoing phase I trial (NCT03784625) dedicated to melanoma targeted radionuclide therapy (TRT), we explore the interplay between immune system and the melanin ligand [131I]ICF01012 alone or combined with immunotherapy (immune checkpoint inhibitors, ICI) in preclinical models. Here we demonstrate that [131I]ICF01012 induces immunogenic cell death, characterized by a significant increase in cell surface-exposed annexin A1 and calreticulin. Additionally, [131I]ICF01012 increases survival in immunocompetent mice, compared to immunocompromised (29 vs. 24 days, p = 0.0374). Flow cytometry and RT-qPCR analyses highlight that [131I]ICF01012 induces adaptive and innate immune cell recruitment in the tumor microenvironment. [131I]ICF01012 combination with ICIs (anti-CTLA-4, anti-PD-1, anti-PD-L1) has shown that tolerance is a main immune escape mechanism, whereas exhaustion is not present after TRT. Furthermore, [131I]ICF01012 and ICI combination has systematically resulted in a prolonged survival (p < 0.0001) compared to TRT alone. Specifically, [131I]ICF01012 + anti-CTLA-4 combination significantly increases survival compared to anti-CTLA-4 alone (41 vs. 26 days; p = 0.0011), without toxicity. This work represents the first global characterization of TRT-induced modifications of the antitumor immune response, demonstrating that tolerance is a main immune escape mechanism and that combining TRT and ICI is promising.

Stromal protein ßig-h3 reprogrammes tumour microenvironment in pancreatic cancer.

OBJECTIVE: Pancreatic cancer is associated with an abundant stromal reaction leading to immune escape and tumour growth. This massive stroma drives the immune escape in the tumour. We aimed to study the impact of ßig-h3 stromal protein in the modulation of the antitumoural immune response in pancreatic cancer. DESIGN: We performed studies with p48-Cre;KrasG12D, pdx1-Cre;KrasG12D;Ink4a/Arffl/fl, pdx1-Cre;KrasG12D; p53R172H mice and tumour tissues from patients with pancreatic ductal adenocarcinoma (PDA). Some transgenic mice were given injections of anti-ßig-h3, anti-CD8, anti-PD1 depleting antibodies. Tumour growth as well as modifications in the activation of local immune cells were analysed by flow cytometry, immunohistochemistry and immunofluorescence. Tissue stiffness was measured by atomic force microscopy. RESULTS: We identified ßig-h3 stromal-derived protein as a key actor of the immune paracrine interaction mechanism that drives pancreatic cancer. We found that ßig-h3 is highly produced by cancer-associated fibroblasts in the stroma of human and mouse. This protein acts directly on tumour-specific CD8+ T cells and F4/80 macrophages. Depleting ßig-h3 in vivo reduced tumour growth by enhancing the number of activated CD8+ T cell within the tumour and subsequent apoptotic tumour cells. Furthermore, we found that targeting ßig-h3 in established lesions released the tissue tension and functionally reprogrammed F4/80 macrophages in the tumour microenvironment. CONCLUSIONS: Our data indicate that targeting stromal extracellular matrix protein ßig-h3 improves the antitumoural response and consequently reduces tumour weight. Our findings present ßig-h3 as a novel immunological target in pancreatic cancer.

Natural killer cells and T cells induce different types of skin reactions during recall responses to haptens.

The role of T cells in contact hypersensitivity (CHS) to haptens has been well described. However, recent reports demonstrated that CHS-like reactions to experimental haptens could be induced in mice deficient in T cells and B cells, as a result of adaptive-like features of NK cells. Here, we compared hapten-specific inflammatory reactions induced by memory T cells or NK cells. Classical CHS protocols were applied to WT or T- and B-cell deficient mice. Adoptive transfers of hapten-specific T cells and NK cells were also performed. Liver NK cells from hapten-primed mice induced specific recall responses to haptens upon transfer in CD3ε-deficient mice, thus confirming the existence of "memory" NK cells in the liver. We investigated the nature of the inflammation generated in these transfer conditions and found that hapten-induced skin inflammation mediated by CD8(+) T cells or "memory" NK cells are different. Indeed, ear swelling induced by memory NK cells was transient and not associated with cellular infiltrate and inflammation markers, characteristic for T-cell-mediated responses. Thus, NK cells and T cells mediate distinct forms of skin inflammation. NK cell-mediated pathogenesis does not rely on cellular infiltrate and could be involved in atypical forms of adverse drug reactions.

Stiffness-induced cancer-associated fibroblasts are responsible for immunosuppression in a platelet-derived growth factor ligand-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is associated with a vast stromal reaction that arises mainly from cancer-associated fibroblasts (CAFs) and promotes both immune escape and tumor growth. Here, we used a mouse model with deletion of the activin A receptor ALK4 in the context of the KrasG12D mutation, which strongly drives collagen deposition that leads to tissue stiffness. By ligand-receptor analysis of single-cell RNA-sequencing data, we identified that, in stiff conditions, neoplastic ductal cells instructed CAFs through sustained platelet-derived growth factor (PDGF) signaling. Tumor-associated tissue rigidity resulted in the emergence of stiffness-induced CAFs (siCAFs) in vitro and in vivo. Similar results were confirmed in human data. siCAFs were able to strongly inhibit CD8+ T-cell responses in vitro and in vivo, promoting local immunosuppression. More importantly, targeting PDGF signaling led to diminished siCAF and reduced tumor growth. Our data show for the first time that early paracrine signaling leads to profound changes in tissue mechanics, impacting immune responses and tumor progression. Our study highlights that PDGF ligand neutralization can normalize the tissue architecture independent of the genetic background, indicating that finely tuned stromal therapy may open new therapeutic avenues in pancreatic cancer.

Histamine receptor H1 signaling on dendritic cells plays a key role in the IFN-γ/IL-17 balance in T cell-mediated skin inflammation.

BACKGROUND: The diverse effects of histamine on immune regulation are a result of the differential expression and regulation of 4 histamine receptors. Many of the immediate allergic and inflammatory actions of histamine are mediated via the type 1 receptor (H1R). OBJECTIVES: We hypothesized that H1R was involved in the fine-tuning of the initiation of T cell-mediated skin pathology-that is, dermatitis. METHODS: The impact of the H1R invalidation on the development of skin inflammation was analyzed in a mouse model of atopic dermatitis. RESULTS: We show that H1r(-)/(-) mice developed reduced allergen-specific skin lesions. Lack of H1R expression on dendritic cells (DCs) led to diminished IL-12, upregulated IL-23, and IL-6 production upon allergen stimulation. H1R engagement on dendritic cells was necessary for DC activation and subsequent priming of effector IFN-γ(+)CD8(+) T cells. We demonstrate here that H1R blockade on DCs promotes generation of noneffector IL-17(+)CD8(+) T cells that are unable to initiate the skin inflammation. CONCLUSION: Our data identify that histamine signaling through the H1R on DCs is an important early event conditioning the quality of the skin effector immune response.

ßig-h3-structured collagen alters macrophage phenotype and function in pancreatic cancer.

Macrophages play an important role in immune and matrix regulation during pancreatic adenocarcinoma (PDAC). Collagen deposition massively contributes to the physical and functional changes of the tissue during pathogenesis. We investigated the impact of thick collagen fibers on the phenotype and function of macrophages. We recently demonstrated that the extracellular protein ßig-h3/TGFßi (Transforming growth factor-ß-induced protein) plays an important role in modulating the stiffness of the pancreatic stroma. By using atomic force microscopy, we show that ßig-h3 binds to type I collagen and establishes thicker fibers. Macrophages cultured on ßig-h3-structured collagen layers display a different morphology and a pro-tumoral M2 phenotype and function compared to those cultured on non-structured collagen layers. In vivo injection of those instructed CD206+CD163+ macrophages was able to suppress T cell responses. These results reveal for the first time that the collagen structure impacts the phenotype and function of macrophages by potentiating their immunosuppressive features.

Intratumoural spatial distribution of S100B + folliculostellate cells is associated with proliferation and expression of FSH and ERα in gonadotroph tumours.

Folliculostellate cells are S100B-expressing cells with numerous functions in the normal anterior pituitary. These cells have also been identified in pituitary neuroendocrine tumours (PitNETs), where their precise role remains elusive. Here, we aimed to build a refined cartography of S100B-expressing cells to characterise their interpatient and intratumoural spatial distribution, and to start identifying their potential functions in PitNETs. High-throughput histological analysis of S100B-stained tumour sections of 54 PitNETs revealed a significant decrease in S100B + cells in PitNETs compared to the normal anterior pituitary. A Ki67 index ≥ 3, a mitosis count > 2/10 per high power fields, and a proliferative status, were all associated with fewer S100B + cells in gonadotroph tumours. Gonadotroph tumours also showed interpatient and intratumoural heterogeneity in the spatial distribution of S100B + cells. The existence of an intratumoural heterogeneity was further confirmed by the incorporation to our spatial analysis of additional markers: Ki67, FSH, LH, ERα and SSTR2. The tumour areas with fewer S100B + cells displayed a higher percentage of Ki67 + cells, whereas strong positive correlations were observed between S100B + , FSH + , and ERα + cells. Such spatial associations suggest that S100B + folliculostellate cells could play a role in gonadotroph tumorigenesis, and may contribute to the maintenance of tumour cells in a low proliferating, FSH + /ERα + differentiated state. Albeit, further in-depth functional studies are required to decipher the mechanisms underlying these spatial associations and to potentially identify a therapeutic use.

Confined migration promotes cancer metastasis through resistance to anoikis and increased invasiveness.

Mechanical stress is known to fuel several hallmarks of cancer, ranging from genome instability to uncontrolled proliferation or invasion. Cancer cells are constantly challenged by mechanical stresses not only in the primary tumour but also during metastasis. However, this latter has seldom been studied with regards to mechanobiology, in particular resistance to anoikis, a cell death programme triggered by loss of cell adhesion. Here, we show in vitro that migrating breast cancer cells develop resistance to anoikis following their passage through microporous membranes mimicking confined migration (CM), a mechanical constriction that cancer cells encounter during metastasis. This CM-induced resistance was mediated by Inhibitory of Apoptosis Proteins, and sensitivity to anoikis could be restored after their inhibition using second mitochondria-derived activator of caspase (SMAC) mimetics. Anoikis-resistant mechanically stressed cancer cells displayed enhanced cell motility and evasion from natural killer cell-mediated immune surveillance, as well as a marked advantage to form lung metastatic lesions in mice. Our findings reveal that CM increases the metastatic potential of breast cancer cells.

A Promising Biomarker and Therapeutic Target in Patients with Advanced PDAC: The Stromal Protein ßig-h3.

With an overall survival rate of 2-9% at 5 years, pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related deaths in the industrialized world and is predicted to become the second by 2030. Owing to often late diagnosis and rare actionable molecular alterations, PDAC has not yet benefited from the recent therapeutic advances that immune checkpoint inhibitors (ICI) have provided in other cancer types, except in specific subgroups of patients presenting with tumors with high mutational burden (TMB) or microsatellite instability (MSI). The tumor microenvironment (TME) plays a substantial role in therapeutic resistance by facilitating immune evasion. An extracellular stromal protein, ßig-h3/TGFßi, is involved in the pathogenesis of PDAC by hampering T cell activation and promoting stiffness of the TME. The study BIGHPANC included 41 patients with metastatic PDAC, and analyzed ßig-h3 levels in serum and tumor samples to assess the ßig-h3 prognostic value. ßig-h3 serum levels are significantly associated with overall survival (HR 2.05, 95%CI 1.07-3.93; p = 0.0301). Our results suggest that ßig-h3 serum levels may be considered a prognostic biomarker in patients with metastatic PDAC.

Stromal Protein-Mediated Immune Regulation in Digestive Cancers.

The stromal tumor microenvironment (TME) consists of immune cells, vascular and neural structures, cancer-associated fibroblasts (CAFs), as well as extracellular matrix (ECM), and favors immune escape mechanisms promoting the initiation and progression of digestive cancers. Numerous ECM proteins released by stromal and tumor cells are crucial in providing physical rigidity to the TME, though they are also key regulators of the immune response against cancer cells by interacting directly with immune cells or engaging with immune regulatory molecules. Here, we discuss current knowledge of stromal proteins in digestive cancers including pancreatic cancer, colorectal cancer, and gastric cancer, focusing on their functions in inhibiting tumor immunity and enabling drug resistance. Moreover, we will discuss the implication of stromal proteins as therapeutic targets to unleash efficient immunotherapy-based treatments.

Stroma Involvement in Pancreatic Ductal Adenocarcinoma: An Overview Focusing on Extracellular Matrix Proteins.

Pancreatic cancer is the seventh leading cause of cancer-related deaths worldwide and is predicted to become second in 2030 in industrialized countries if no therapeutic progress is made. Among the different types of pancreatic cancers, Pancreatic Ductal Adenocarcinoma (PDAC) is by far the most represented one with an occurrence of more than 90%. This specific cancer is a devastating malignancy with an extremely poor prognosis, as shown by the 5-years survival rate of 2-9%, ranking firmly last amongst all cancer sites in terms of prognostic outcomes for patients. Pancreatic tumors progress with few specific symptoms and are thus at an advanced stage at diagnosis in most patients. This malignancy is characterized by an extremely dense stroma deposition around lesions, accompanied by tissue hypovascularization and a profound immune suppression. Altogether, these combined features make access to cancer cells almost impossible for conventional chemotherapeutics and new immunotherapeutic agents, thus contributing to the fatal outcomes of the disease. Initially ignored, the Tumor MicroEnvironment (TME) is now the subject of intensive research related to PDAC treatment and could contain new therapeutic targets. In this review, we will summarize the current state of knowledge in the field by focusing on TME composition to understand how this specific compartment could influence tumor progression and resistance to therapies. Attention will be paid to Tenascin-C, a matrix glycoprotein commonly upregulated during cancer that participates to PDAC progression and thus contributes to poor prognosis.

TGFBI Production by Macrophages Contributes to an Immunosuppressive Microenvironment in Ovarian Cancer.

The tumor microenvironment evolves during malignant progression, with major changes in nonmalignant cells, cytokine networks, and the extracellular matrix (ECM). In this study, we aimed to understand how the ECM changes during neoplastic transformation of serous tubal intraepithelial carcinoma lesions (STIC) into high-grade serous ovarian cancers (HGSOC). Analysis of the mechanical properties of human fallopian tubes (FT) and ovaries revealed that normal FT and fimbria had a lower tissue modulus, a measure of stiffness, than normal or diseased ovaries. Proteomic analysis of the matrisome fraction between FT, fimbria, and ovaries showed significant differences in the ECM protein TGF beta induced (TGFBI, also known as ßig-h3). STIC lesions in the fimbria expressed high levels of TGFBI, which was predominantly produced by CD163-positive macrophages proximal to STIC epithelial cells. In vitro stimulation of macrophages with TGFß and IL4 induced secretion of TGFBI, whereas IFNγ/LPS downregulated macrophage TGFBI expression. Immortalized FT secretory epithelial cells carrying clinically relevant TP53 mutations stimulated macrophages to secrete TGFBI and upregulated integrin αvß3, a putative TGFBI receptor. Transcriptomic HGSOC datasets showed a significant correlation between TGFBI expression and alternatively activated macrophage signatures. Fibroblasts in HGSOC metastases expressed TGFBI and stimulated macrophage TGFBI production in vitro. Treatment of orthotopic mouse HGSOC tumors with an anti-TGFBI antibody reduced peritoneal tumor size, increased tumor monocytes, and activated ß3-expressing unconventional T cells. In conclusion, TGFBI may favor an immunosuppressive microenvironment in STICs that persists in advanced HGSOC. Furthermore, TGFBI may be an effector of the tumor-promoting actions of TGFß and a potential therapeutic target. SIGNIFICANCE: Analysis of ECM changes during neoplastic transformation reveals a role for TGFBI secreted by macrophages in immunosuppression in early ovarian cancer.

Immune Landscape of Pituitary Tumors Reveals Association Between Macrophages and Gonadotroph Tumor Invasion.

PURPOSE: Pituitary neuroendocrine tumors (PitNETs) are frequent intracranial neoplasms that present heterogenic characteristics. Little is known about the immune cell network that exists in PitNETs and its contribution to their aggressive behavior. METHODS: Here we combined flow cytometry, t-SNE analysis, and histological approaches to define the immune landscape of surgically resected PitNETs. Xenografts of rodent pituitary tumor cells and resected PitNETs were performed in Rag2KO mice, in combination with in vitro analysis aimed at dissecting the role of pituitary tumor-cells in monocyte recruitment. RESULTS: We report that gonadotroph PitNETs present an increased CD68+ macrophage signature compared to somatotroph, lactotroph, and corticotroph PitNETs. Transcriptomic and histological characterizations confirmed gonadotroph infiltrating macrophages expressed CD163, MRC-1, ARG1, and CSF1R M2 macrophage markers. Use of growth hormone (GH)3/GH4 somatotroph and LßT2/αT3.1 gonadotroph cells drove THP1 macrophage migration through respective expression of CCL5 or CSF1. Although both LßT2 and GH3 cells recruited F4/80 macrophages following their engraftment in mice, only LßT2 gonadotroph cells showed a capacity for M2-like polarization. Similar observations were performed on patient-derived xenografts from somatotroph and gonadotroph tumors. Analysis of clinical data further demonstrated a significant correlation between the percentage of CD68+ and CD163+ infiltrating macrophages and the invasive character of gonadotroph tumors. CONCLUSIONS: Gonadotroph tumor drive the recruitment of macrophages and their subsequent polarization to an M2-like phenotype. More importantly, the association between infiltrating CD68+/CD163+ macrophages and the invasiveness of gonadotroph tumors points to macrophage-targeted immunotherapies being a potent strategy to limit the progression of gonadotroph PitNETs.

Oncogene-Induced Senescence Limits the Progression of Pancreatic Neoplasia through Production of Activin A.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly and aggressive cancer. Understanding mechanisms that drive preneoplastic pancreatic lesions is necessary to improve early diagnostic and therapeutic strategies. Mutations and inactivation of activin-like kinase (ALK4) have been demonstrated to favor PDAC onset. Surprisingly, little is known regarding the ligands that drive ALK4 signaling in pancreatic cancer or how this signaling pathway limits the initiation of neoplastic lesions. In this study, data mining and histologic analyses performed on human and mouse tumor tissues revealed that activin A is the major ALK4 ligand that drives PDAC initiation. Activin A, which is absent in normal acinar cells, was strongly induced during acinar-to-ductal metaplasia (ADM), which was promoted by pancreatitis or the activation of KrasG12D in mice. Activin A expression during ADM was associated with the cellular senescence program that is induced in precursor lesions. Blocking activin A signaling through the use of a soluble form of activin receptor IIB (sActRIIB-Fc) and ALK4 knockout in mice expressing KrasG12D resulted in reduced senescence associated with decreased expression of p21, reduced phosphorylation of H2A histone family member X (H2AX), and increased proliferation. Thus, this study indicates that activin A acts as a protective senescence-associated secretory phenotype factor produced by Kras-induced senescent cells during ADM, which limits the expansion and proliferation of pancreatic neoplastic lesions. SIGNIFICANCE: This study identifies activin A to be a beneficial, senescence-secreted factor induced in pancreatic preneoplastic lesions, which limits their proliferation and ultimately slows progression into pancreatic cancers.

Immunoprotection by polyethylene glycol in organ preservation solutions is not due to an immunomasking effect.

BACKGROUND: New organ preservation solutions that contain soluble polyethylene glycol (sPEG) molecules have been associated with reduction of acute rejection episodes. METHODS: In the present manuscript we tested in vitro whether sPEG molecules were able to mask donor alloantigens and reduce graft immunogenicity. RESULTS: Immunomasking effect was only evidenced when PEG molecules were covalently bound to donor cell surface. CONCLUSION: We concluded that sPEG in preservation solution are unlikely to display 'immunocamouflage' property.

Menin regulates Inhbb expression through an Akt/Ezh2-mediated H3K27 histone modification.

Although Men1 is a well-known tumour suppressor gene, little is known about the functions of Menin, the protein it encodes for. Since few years, numerous publications support a major role of Menin in the control of epigenetics gene regulation. While Menin interaction with MLL complex favours transcriptional activation of target genes through H3K4me3 marks, Menin also represses gene expression via mechanisms involving the Polycomb repressing complex (PRC). Interestingly, Ezh2, the PRC-methyltransferase that catalyses H3K27me3 repressive marks and Menin have been shown to co-occupy a large number of promoters. However, lack of binding between Menin and Ezh2 suggests that another member of the PRC complex is mediating this indirect interaction. Having found that ActivinB - a TGFß superfamily member encoded by the Inhbb gene - is upregulated in insulinoma tumours caused by Men1 invalidation, we hypothesize that Menin could directly participate in the epigenetic-repression of Inhbb gene expression. Using Animal model and cell lines, we report that loss of Menin is directly associated with ActivinB-induced expression both in vivo and in vitro. Our work further reveals that ActivinB expression is mediated through a direct modulation of H3K27me3 marks on the Inhbb locus in Menin-KO cell lines. More importantly, we show that Menin binds on the promoter of Inhbb gene where it favours the recruitment of Ezh2 via an indirect mechanism involving Akt-phosphorylation. Our data suggests therefore that Menin could take an important part to the Ezh2-epigenetic repressive landscape in many cells and tissues through its capacity to modulate Akt phosphorylation.

Pathophysiology of urticaria.

Urticaria is dermal edema resulting from vascular dilatation and leakage of fluid into the skin in response to molecules released from mast cells. The major preformed mediator histamine produces a prototypic, short-lived urticaria. However, the clinical spectrum and pattern of lesions indicate that other molecules, including prostaglandins, leukotrienes, cytokines, and chemokines, produced at different times after mast cell activation contribute to the polymorphism of this symptom and the variable evolution of this disease. It is a common practice to distinguish immunological and nonimmunological urticaria. Immunological urticaria is a hypersensitivity reaction mediated by antibodies and/or T-cells that results in mast cell activation. Although immunoglobulin (Ig)E-mediated type I hypersensitivity (HS) was long postulated to be the major immunological pathway associated with mast cell activation, interaction between IgE-bound mast cells and allergens is unlikely to be the mechanism by which urticaria develops in most patients. It is now well established that urticaria may result from the binding of IgG auto-antibodies to IgE and/or to the receptor for IgE molecules on mast cells, thus corresponding to a type II HS reaction. These auto-immune urticarias represent up to 50% of patients with chronic urticaria. Mast cell activation can also result from type III HS through the binding of circulating immune complexes to mast cell-expressing Fc receptors for IgG and IgM. Finally, under certain circumstances, T-cells can induce activation of mast cells, as well as histamine release (type IV HS). Nonimmunological urticarias result from mast cell activation through membrane receptors involved in innate immunity (e.g., complement, Toll-like, cytokine/chemokine, opioid) or by direct toxicity of xenobiotics (haptens, drugs). In conclusion, urticaria may result from different pathophysiological mechanisms that explain the great heterogeneity of clinical symptoms and the variable responses to treatment.