Spatial Distribution of Non-Immune Cells Expressing Glycoprotein A Repetitions Predominant in Human and Murine Metastatic Lymph Nodes.

Several types of cancer spread through the lymphatic system via the sentinel lymph nodes (LNs). Such LN-draining primary tumors, modified by tumor factors, lead to the formation of a metastatic niche associated with an increased number of Foxp3+ regulatory T cells (Tregs). These cells are expected to contribute to the elaboration of an immune-suppressive environment. Activated Tregs express glycoprotein A repetitions predominant (GARP), which binds and presents latent transforming growth factor beta 1 (TGF-ß1) at their surface. GARP is also expressed by other non-immune cell types poorly described in LNs. Here, we mapped GARP expression in non-immune cells in human and mouse metastatic LNs. The mining of available (human and murine) scRNA-Seq datasets revealed GARP expression by blood (BEC)/lymphatic (LEC) endothelial, fibroblastic, and perivascular cells. Consistently, through immunostaining and in situ RNA hybridization approaches, GARP was detected in and around blood and lymphatic vessels, in (αSMA+) fibroblasts, and in perivascular cells associated with an abundant matrix. Strikingly, GARP was detected in LECs forming the subcapsular sinus and high endothelial venules (HEVs), two vascular structures localized at the interface between LNs and the afferent lymphatic and blood vessels. Altogether, we here provide the first distribution maps for GARP in human and murine LNs.

Comparison of Estetrol Exposure between Women and Mice to Model Preclinical Experiments and Anticipate Human Treatment.

Estetrol (E4) is a natural estrogen with promising therapeutic applications in humans. The European Medicines Agency and the Food and Drug Administration have approved the use of 15 mg E4/3 mg drospirenone for contraceptive indication. Phase III clinical trials with 15-20 mg E4 for the relief of climacteric complaints are currently running. Relevant data from preclinical animal models are needed to characterize the molecular mechanisms and the pharmacological effects of E4 and possibly to reveal new therapeutic applications and to anticipate potential adverse effects. Therefore, it is important to design experimental procedures in rodents that closely mimic or anticipate human E4 exposure. In this study, we compared the effects of E4 exposure after acute or chronic administration in women and mice. Women who received chronic E4 treatment per os at a dose of 15 mg once daily reached a steady state within 6 to 8 days, with a mean plasma concentration of 3.20 ng/mL. Importantly, with subcutaneous, intraperitoneal or oral administration of E4 in mice, a stable concentration over time that would mimic human pharmacokinetics could not be achieved. The use of osmotic minipumps continuously releasing E4 for several weeks provided an exposure profile mimicking chronic oral administration in women. Measurements of the circulating concentration of E4 in mice revealed that the mouse equivalent dose necessary to mimic human treatment does not fit with the allometric prediction. In conclusion, this study highlights the importance of precise definition of the most appropriate dose and route of administration to utilize when developing predictive preclinical animal models to mimic or anticipate specific human treatment.

Molecular Mechanisms Driven by MT4-MMP in Cancer Progression.

MT4-MMP (or MMP-17) belongs to the membrane-type matrix metalloproteinases (MT-MMPs), a distinct subset of the MMP family that is anchored to the cell surface, in this case by a glycosylphosphatidylinositol (GPI) motif. Its expression in a variety of cancers is well documented. However, the molecular mechanisms by which MT4-MMP contributes to tumor development need further investigation. In this review, we aim to summarize the contribution of MT4-MMP in tumorigenesis, focusing on the molecular mechanisms triggered by the enzyme in tumor cell migration, invasiveness, and proliferation, in the tumor vasculature and microenvironment, as well as during metastasis. In particular, we highlight the putative substrates processed and signaling cascades activated by MT4-MMP that may underlie these malignancy processes and compare this with what is known about its role during embryonic development. Finally, MT4-MMP is a relevant biomarker of malignancy that can be used for monitoring cancer progression in patients as well as a potential target for future therapeutic drug development.

Design, Synthesis, and Evaluation of Novel Pyruvate Dehydrogenase Kinase Inhibitors.

AIMS: The present work describes the synthesis and the biological evaluation of novel compounds acting as pyruvate dehydrogenase kinase (PDK) inhibitors. These drugs should become a new therapeutic approach for the treatment of pathologies improved by the control of the blood lactate level. METHODS: Four series of compounds belonging to N-(4-(N-alkyl/aralkylsulfamoyl)phenyl)-2- methylpropanamides and 1,2,4-benzothiadiazine 1,1-dioxides were prepared and evaluated as PDK inhibitors. RESULTS: The newly synthesized N-(4-(N-alkyl/aralkylsulfamoyl)phenyl)-2-methylpropanamides structurally related to previously reported reference compounds 4 and 5 were found to be potent PDK inhibitors (i.e. 10d: IC50 = 41 nM). 1,2,4-Benzothiadiazine 1,1-dioxides carrying a (methyl/ trifluoromethyl)-propanamide moiety at the 6-position were also designed as conformationally restricted ring-closed analogues of N-(4-(N-alkyl/aralkylsulfamoyl)phenyl)-2-hydroxy-2-methylpropanamides. Most of them were found to be less potent than their ring-opened analogues. Interestingly, the best choice of hydrocarbon side chain at the 4-position was the benzyl chain, providing 11c (IC50 = 3.6 µM) belonging to "unsaturated" 1,2,4-benzothiadiazine 1,1-dioxides, and 12c (IC50 = 0.5 µM) belonging to "saturated' 1,2,4-benzothiadiazine 1,1-dioxides. CONCLUSION: This work showed that ring-closed analogues of N-(4-(N-alkyl/aralkylsulfamoyl) phenyl)- 2-hydroxy-2-methylpropanamides were less active as PDK inhibitors than their corresponding ring-opened analogues. However, the introduction of a bulkier substituent at the 4-position of the 1,2,4-benzothiadiazine 1,1-dioxide core structure, such as a benzyl or a phenethyl side chain, was allowed, opening the way to the design of new inhibitors with improved PDK inhibitory activity.

Comparison of Morphological and Digital-Assisted Analysis for BCL6 Endometrial Expression in Women with Endometriosis.

BCL6 (B-cell lymphoma 6) is a proto-oncogene and transcriptional repressor initially described as being involved in B-cell lymphoma. Recently, this factor has been identified as a promising tissue biomarker which could be used to diagnose women affected by endometriosis. Previous studies used HSCORE for BCL6 staining quantification in the endometrium. However, this semi-quantitative technique of analysis has some limitations, including a lack of objectivity, robustness, and reproducibility that may lead to intra- and inter-observer variability. Our main goal was to develop an original computer-assisted method to quantify BCL6 staining from whole-slide images reliably. In order to test the efficiency of our new digital method of quantification, we compared endometrial BCL6 expression between fertile and infertile women without or with different stages of endometriosis by using the widely used HSCORE analysis and our new automatic digital image analysis. We find a higher expression of BCL6 in the endometrium of infertile women with endometriosis and women with stage IV endometriosis. Furthermore, we demonstrate a significant correlation between the two types of independent measurements, indicating the robustness of results and also the reliability of our computer-assisted method for BCL6 quantification. In conclusion, our work, by using this original computer-assisted method, enables BCL6 quantification more objectively, reliably, robustly, and promptly compared to HSCORE analysis.

Kidney-targeted irradiation triggers renal ischemic preconditioning in mice.

Renal ischemia-reperfusion (I/R) causes acute kidney injury (AKI). Ischemic preconditioning (IPC) attenuates I/R-associated AKI. Whole body irradiation induces renal IPC in mice. Still, the mechanisms remain largely unknown. Furthermore, the impact of kidney-centered irradiation on renal resistance against I/R has not been studied. Renal irradiation (8.5 Gy) was done in male 8- to 12-wk-old C57bl/6 mice using a small animal radiation therapy device. Left renal I/R was performed by clamping the renal pedicles for 30 min, with simultaneous right nephrectomy, at 7, 14, and 28 days postirradiation. The renal reperfusion lasted 48 h. Following I/R, blood urea nitrogen (BUN) and serum creatinine (SCr) levels were lower in preirradiated mice compared with controls; so was the histological Jablonski score of AKI. The metabolomics signature of renal I/R was attenuated in preirradiated mice. The numbers of proliferating cell nuclear antigen (PCNA)-, cluster of differentiation molecule 11b (CD11b)-, and cell surface glycoprotein F4/80-positive cells in the renal parenchyma post-I/R were reduced in preirradiated versus control groups. Such IPC was significantly observed as early as day 14 postirradiation. RNA sequencing showed an upregulation of angiogenesis- and stress response-related signaling pathways in irradiated nonischemic kidneys on day 28. Qualitative RT-PCR confirmed the increased expression of vascular endothelial growth factor (VEGF), activin receptor-like kinase 5 (ALK5), heme oxygenase-1 (HO1), platelet endothelial cell adhesion molecule-1 (PECAM1), NADPH oxidase 2 (NOX2), and heat shock proteins 70 and 27 (HSP70 and HSP27, respectively) in irradiated kidneys compared with controls. In addition, irradiated kidneys showed an increased CD31-positive vascular area compared with controls. A 14-day gavage of irradiated mice with the antiangiogenic drug sunitinib before I/R abrogated the irradiation-induced IPC at both functional and structural levels. Our observations suggest that kidney-centered irradiation activates proangiogenic pathways and induces IPC, with preserved renal function and attenuated inflammation post-I/R.NEW & NOTEWORTHY This study based on a mouse model of renal ischemia-reperfusion (I/R) aimed to 1) test whether and how irradiation strictly centered on the kidney protects against the I/R injury and 2) determine the shortest efficient delay of kidney irradiation to achieve such nephroprotection. Kidney irradiation increased the vascular surface in the renal parenchyma and conferred resistance against renal I/R damage, which highlights novel putative strategies in the field of ischemic acute kidney injury.

Regulation of Tissue Factor by CD44 Supports Coagulant Activity in Breast Tumor Cells.

Previous work identified Tissue Factor (TF), a key activator of the coagulation cascade, as a gene induced in cellular contexts of Epithelial-Mesenchymal Transitions (EMTs), providing EMT+ Circulating Tumor Cells (CTCs) with coagulant properties that facilitate their metastatic seeding. Deciphering further molecular aspects of TF regulation in tumor cells, we report here that CD44 and TF coexpress in EMT contexts, and that CD44 acts as a regulator of TF expression supporting procoagulant properties and metastatic seeding. A transcriptional regulatory mechanism bridging CD44 to TF expression was further evidenced. Comparing different TF -promoter luciferase reporter constructs, we indeed found that the shortest -111 pb TF promoter fragment harboring three Specificity Protein 1 (Sp1) binding sites is still responsive to CD44 silencing. The observation that (i) mutation within Sp1 binding sites decreased the basal activity of the -111 pb TF promoter construct, (ii) CD44 silencing decreased Sp1 protein and mRNA levels and (iii) Sp1 silencing diminished TF expression further points to Sp1 as a key mediator linking CD44 to TF regulation. All together, these data thus report a transcriptional regulatory mechanism of TF expression by CD44 supporting procoagulant activity and metastatic competence of CTCs.

Periostin in lymph node pre-metastatic niches governs lymphatic endothelial cell functions and metastatic colonization.

Although lymph node (LN) metastasis is an important prognostic parameter in cervical cancer, the tissue remodeling at a pre-metastatic state is poorly documented in LNs. We here identified periostin (POSTN) as a component of non-metastatic LNs by applying proteomic analyses and computerized image quantifications on LNs of patients with cervical cancer. We provide evidence for remarkable modifications of POSTN and lymphatic vessel distributions and densities in non-metastatic sentinel and metastatic human LNs, when compared to distant non-metastatic LNs. POSTN deposition at a pre-metastatic stage was demonstrated in a pre-clinical murine model (the ear sponge assay). Its expression by fibroblastic LN cells was assessed by in situ hybridization and in vitro cultures. In vitro, POSTN promoted lymphatic endothelial cell functions and tumor cell proliferation. Accordingly, the in vivo injection of recombinant POSTN together with VEGF-C boosted the lymphangiogenic response, while the metastatic potential of tumor cells was drastically reduced using a POSTN blocking antibody. This translational study also supports the existence of an unprecedented dialog "in cascade", between the primary tumor and the first pelvic nodal relay in early cervical cancer, and subsequently from pelvic LN to para-aortic LNs in locally advanced cervical cancers. Collectively, this work highlights the association of POSTN deposition with lymphangiogenesis in LNs, and provides evidence for a key contribution of POSTN in promoting VEGF-C driven lymphangiogenesis and the seeding of metastatic cells.

Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis.

Autophagy has vasculoprotective roles, but whether and how it regulates lymphatic endothelial cells (LEC) homeostasis and lymphangiogenesis is unknown. Here, we show that genetic deficiency of autophagy in LEC impairs responses to VEGF-C and injury-driven corneal lymphangiogenesis. Autophagy loss in LEC compromises the expression of main effectors of LEC identity, like VEGFR3, affects mitochondrial dynamics and causes an accumulation of lipid droplets (LDs) in vitro and in vivo. When lipophagy is impaired, mitochondrial ATP production, fatty acid oxidation, acetyl-CoA/CoA ratio and expression of lymphangiogenic PROX1 target genes are dwindled. Enforcing mitochondria fusion by silencing dynamin-related-protein 1 (DRP1) in autophagy-deficient LEC fails to restore LDs turnover and lymphatic gene expression, whereas supplementing the fatty acid precursor acetate rescues VEGFR3 levels and signaling, and lymphangiogenesis in LEC-Atg5-/- mice. Our findings reveal that lipophagy in LEC by supporting FAO, preserves a mitochondrial-PROX1 gene expression circuit that safeguards LEC responsiveness to lymphangiogenic mediators and lymphangiogenesis.

uPARAP/Endo180: a multifaceted protein of mesenchymal cells.

The urokinase plasminogen activator receptor-associated protein (uPARAP/Endo180) is already known to be a key collagen receptor involved in collagen internalization and degradation in mesenchymal cells and some macrophages. It is one of the four members of the mannose receptor family along with a macrophage mannose receptor (MMR), a phospholipase lipase receptor (PLA2R), and a dendritic receptor (DEC-205). As a clathrin-dependent endocytic receptor for collagen or large collagen fragments as well as through its association with urokinase (uPA) and its receptor (uPAR), uPARAP/Endo180 takes part in extracellular matrix (ECM) remodeling, cell chemotaxis and migration under physiological (tissue homeostasis and repair) and pathological (fibrosis, cancer) conditions. Recent advances that have shown an expanded contribution of this multifunctional protein across a broader range of biological processes, including vascular biology and innate immunity, are summarized in this paper. It has previously been demonstrated that uPARAP/Endo180 assists in lymphangiogenesis through its capacity to regulate the heterodimerization of vascular endothelial growth factor receptors (VEGFR-2 and VEGFR-3). Moreover, recent findings have demonstrated that it is also involved in the clearance of collectins and the regulation of the immune system, something which is currently being studied as a biomarker and a therapeutic target in a number of cancers.

In Vitro, In Vivo, and In Silico Models of Lymphangiogenesis in Solid Malignancies.

Lymphangiogenesis (LA) is the formation of new lymphatic vessels by lymphatic endothelial cells (LECs) sprouting from pre-existing lymphatic vessels. It is increasingly recognized as being involved in many diseases, such as in cancer and secondary lymphedema, which most often results from cancer treatments. For some cancers, excessive LA is associated with cancer progression and metastatic dissemination to the lymph nodes (LNs) through lymphatic vessels. The study of LA through in vitro, in vivo, and, more recently, in silico models is of paramount importance in providing novel insights and identifying the key molecular actors in the biological dysregulation of this process under pathological conditions. In this review, the different biological (in vitro and in vivo) models of LA, especially in a cancer context, are explained and discussed, highlighting their principal modeled features as well as their advantages and drawbacks. Imaging techniques of the lymphatics, complementary or even essential to in vivo models, are also clarified and allow the establishment of the link with computational approaches. In silico models are introduced, theoretically described, and illustrated with examples specific to the lymphatic system and the LA. Together, these models constitute a toolbox allowing the LA research to be brought to the next level.

ADAMTS2 and ADAMTS14 can substitute for ADAMTS3 in adults for pro-VEGFC activation and lymphatic homeostasis.

The capacity of ADAMTS3 to cleave pro-VEGFC into active VEGFC able to bind its receptors and to stimulate lymphangiogenesis has been clearly established during embryonic life. However, this function of ADAMTS3 is unlikely to persist in adulthood because of its restricted expression pattern after birth. Because ADAMTS2 and ADAMTS14 are closely related to ADAMTS3 and are mainly expressed in connective tissues where the lymphatic network extends, we hypothesized that they could substitute for ADAMTS3 during adulthood in mammals allowing proteolytic activation of pro-VEGFC. Here, we demonstrated that ADAMTS2 and ADAMTS14 are able to process pro-VEGFC into active VEGFC as efficiently as ADAMTS3. In vivo, adult mice lacking Adamts2 developed skin lymphedema due to a reduction of the density and diameter of lymphatic vessels, leading to a decrease of lymphatic functionality, while genetic ablation of Adamts14 had no impact. In a model of thermal cauterization of cornea, lymphangiogenesis was significantly reduced in Adamts2- and Adamts14-KO mice and further repressed in Adamts2/Adamts14 double-KO mice. In summary, we have demonstrated that ADAMTS2 and ADAMTS14 are as efficient as ADAMTS3 in activation of pro-VEGFC and are involved in the homeostasis of the lymphatic vasculature in adulthood, both in physiological and pathological processes.

Evaluation of an alternative heterotopic transplantation model for ovarian tissue to test pharmaceuticals improvements for fertility restoration.

BACKGROUND: Ovarian tissue cryopreservation and transplantation (OTCTP) is currently the main option available to preserve fertility in prepubertal patients undergoing aggressive cancer therapy treatments. However, a major limitation of OTCTP is follicle loss after transplantation. The mouse is a model of choice for studying ovarian function and follicle development after ovarian tissue grafting in vivo. In these mouse models, ovarian tissue or ovaries can be transplanted to different sites. Our aim was to evaluate a new alternative to heterotopic transplantation models that could be useful to test pharmaceutical improvement for ovarian grafts after OTCTP. METHODS: Slow frozen murine whole ovaries were transplanted into the mouse ears (between the external ear skin layer and the cartilage). Ovarian transplants were recovered after 3, 14 or 21 days. Grafts were analyzed by immunohistochemistry and follicle density analyses were performed. RESULTS: An increase of ovarian vascularization (CD31 and Dextran-FITC positive staining), as well as cellular proliferation (Ki67 staining) were observed 3 weeks after transplantation in comparison to 3 days. Fibrosis density, evaluated after Van Gieson staining, decreased 3 weeks after transplantation. Furthermore, transplantation of cryopreserved ovaries into ovariectomized mice favored follicle activation compared to transplantation into non-ovariectomized mice. CONCLUSION: The present study indicates that surgical tissue insertion in the highly vascularized murine ear is an effective model for ovarian grafting. This model could be helpful in research to test pharmaceutical strategies to improve the function and survival of cryopreserved and transplanted ovarian tissue.

Remodelling of the fibre-aggregate structure of collagen gels by cancer-associated fibroblasts: A time-resolved grey-tone image analysis based on stochastic modelling.

Introduction: Solid tumors consist of tumor cells associated with stromal and immune cells, secreted factors and extracellular matrix (ECM), which together constitute the tumor microenvironment. Among stromal cells, activated fibroblasts, known as cancer-associated fibroblasts (CAFs) are of particular interest. CAFs secrete a plethora of ECM components including collagen and modulate the architecture of the ECM, thereby influencing cancer cell migration. The characterization of the collagen fibre network and its space and time-dependent microstructural modifications is key to investigating the interactions between cells and the ECM. Developing image analysis tools for that purpose is still a challenge because the structural complexity of the collagen network calls for specific statistical descriptors. Moreover, the low signal-to-noise ratio of imaging techniques available for time-resolved studies rules out standard methods based on image segmentation. Methods: In this work, we develop a novel approach based on the stochastic modelling of the gel structure and on grey-tone image analysis. The method is then used to study the remodelling of a collagen matrix by migrating breast cancer-derived CAFs in a three-dimensional spheroid model of cellular invasion imaged by time-lapse confocal microscopy. Results: The structure of the collagen at the scale of a few microns consists in regions with high fibre density separated by depleted regions, which can be thought of as aggregates and pores. The approach developped captures this two-scale structure with a clipped Gaussian field model to describe the aggregates-and-pores large-scale structure, and a homogeneous Boolean model to describe the small-scale fibre network within the aggregates. The model parameters are identified by fitting the grey-tone histograms and correlation functions of the images. The method applies to unprocessed grey-tone images, and it can therefore be used with low magnification, noisy time-lapse reflectance images. When applied to the CAF spheroid time-resolved images, the method reveals different matrix densification mechanisms for the matrix in direct contact or far from the cells. Conclusion: We developed a novel and multidisciplinary image analysis approach to investigate the remodelling of fibrillar collagen in a 3D spheroid model of cellular invasion. The specificity of the method is that it applies to the unprocessed grey-tone images, and it can therefore be used with noisy time-lapse reflectance images of non-fluorescent collagen. When applied to the CAF spheroid time-resolved images, the method reveals different matrix densification mechanisms for the matrix in direct contact or far from the cells.

Role for the metalloproteinase ADAM28 in the control of airway inflammation, remodelling and responsiveness in asthma.

Background: Asthma is characterized by morphological modifications of the airways (inflammation and remodelling) and bronchial hyperresponsiveness. Mechanisms linking these two key features of asthma are still poorly understood. ADAM28 (a disintegrin and metalloproteinase 28) might play a role in asthma pathophysiology. ADAM28 exists as membrane-bound and soluble forms and is mainly expressed by lymphocytes and epithelial cells. Methods: ADAM28-/- mice and ADAM28+/+ counterparts were sensitized and exposed to ovalbumin (OVA). Airway responsiveness was measured using the flexiVent® system. After sacrifice, bronchoalveolar lavage (BAL) was performed and lungs were collected for analysis of airway inflammation and remodelling. Results: The expression of the soluble form of ADAM28 was lower in the lungs of OVA-exposed mice (as compared to PBS-exposed mice) and progressively increased in correlation with the duration of allergen exposure. In lungs of ADAM28-/- mice exposed to allergens, the proportion of Th2 cells among CD 4 + cells and the number of B cells were decreased. Bronchial responsiveness was lower in ADAM28-/- mice exposed to allergens and similar to the responsiveness of sham-challenged mice. Similarly, features of airway remodelling (collagen deposition, smooth muscle hyperplasia, mucous hyperplasia) were significantly less developed in OVA-exposed ADAM28-/- animals in sharp contrasts to ADAM28+/+. In addition, we report the first evidence of ADAM28 RNA expression by lung fibroblasts and we unveil a decreased capacity of lung fibroblasts extracted from OVA-exposed ADAM28-/- mice to proliferate as compared to those extracted from OVA-exposed ADAM28+/+ suggesting a direct contribution of this enzyme to the modulation of airway remodelling. Conclusion: These results suggest that ADAM28 might be a key contributor to the pathophysiology of asthma.

ZO-1 Intracellular Localization Organizes Immune Response in Non-Small Cell Lung Cancer.

Delocalization of zonula occludens-1 (ZO-1) from tight junctions plays a substantial role in epithelial cell plasticity observed during tumor progression. In vitro, we reported an impact of ZO-1 cyto-nuclear content in modulating the secretion of several pro-inflammatory chemokines. In vivo, we demonstrated that it promotes the recruitment of immune cells in mouse ear sponge assays. Examining lung cancers, we showed that a high density of CD8 cytotoxic T cells and Foxp3 immunosuppressive regulatory T cells in the tumor microenvironment correlated with a cyto-nuclear expression of ZO-1. Taken together, our results support that, by affecting tumor cell secretome, the cyto-nuclear ZO-1 pool may recruit immune cells, which could be permissive for tumor progression.

Combined Blockade of GARP:TGF-ß1 and PD-1 Increases Infiltration of T Cells and Density of Pericyte-Covered GARP+ Blood Vessels in Mouse MC38 Tumors.

When combined with anti-PD-1, monoclonal antibodies (mAbs) against GARP:TGF-ß1 complexes induced more frequent immune-mediated rejections of CT26 and MC38 murine tumors than anti-PD-1 alone. In both types of tumors, the activity of anti-GARP:TGF-ß1 mAbs resulted from blocking active TGF-ß1 production and immunosuppression by GARP-expressing regulatory T cells. In CT26 tumors, combined GARP:TGF-ß1/PD-1 blockade did not augment the infiltration of T cells, but did increase the effector functions of already present anti-tumor T cells. Here we show that, in contrast, in MC38, combined GARP:TGF-ß1/PD-1 blockade increased infiltration of T cells, as a result of increased extravasation of T cells from blood vessels. Unexpectedly, combined GARP:TGF-ß1/PD-1 blockade also increased the density of GARP+ blood vessels covered by pericytes in MC38, but not in CT26 tumors. This appears to occur because anti-GARP:TGF-ß1, by blocking TGF-ß1 signals, favors the proliferation of and expression of adhesion molecules such as E-selectin by blood endothelial cells. The resulting densification of intratumoral blood vasculature probably contributes to increased T cell infiltration and to the therapeutic efficacy of GARP:TGF-ß1/PD-1 blockade in MC38. We conclude from these distinct observations in MC38 and CT26, that the combined blockades of GARP:TGF-ß1 and PD-1 can exert anti-tumor activity via multiple mechanisms, including the densification and normalization of intratumoral blood vasculature, the increase of T cell infiltration into the tumor and the increase of the effector functions of intratumoral tumor-specific T cells. This may prove important for the selection of cancer patients who could benefit from combined GARP:TGF-ß1/PD-1 blockade in the clinics.

Multi- and Transgenerational Outcomes of an Exposure to a Mixture of Endocrine-Disrupting Chemicals (EDCs) on Puberty and Maternal Behavior in the Female Rat.

BACKGROUND: The effects of endocrine-disrupting chemicals (EDCs) on fertility and reproductive development represent a rising concern in modern societies. Although the neuroendocrine control of sexual maturation is a major target of EDCs, little is known about the potential role of the hypothalamus in puberty and ovulation disruption transmitted across generations. OBJECTIVES: We hypothesized that developmental exposure to an environmentally relevant dose of EDC mixture could induce multi- and/or transgenerational alterations of sexual maturation and maternal care in female rats through epigenetic reprograming of the hypothalamus. We investigated the transmission of a disrupted reproductive phenotype via the maternal germline or via nongenomic mechanisms involving maternal care. METHODS: Adult female Wistar rats were exposed prior to and during gestation and until the end of lactation to a mixture of the following 13 EDCs: di-n-butyl phthalate (DnBP), di(2-ethylhexyl) phthalate (DEHP), bisphenol A (BPA), vinclozolin, prochloraz, procymidone, linuron, epoxynaxole, dichlorodiphenyldichloroethylene, octyl methoxynimmate, 4-methylbenzylidene camphor (4-MBC), butylparaben, and acetaminophen. Perinatally exposed offspring (F1) were mated with unexposed males to generate germ cell (F2) and transgenerationally exposed (F3 and F4) females. Sexual maturation, maternal behavior, and hypothalamic targets of exposure were studied across generations. RESULTS: Germ cell (F2) and transgenerationally (F3) EDC-exposed females, but not F1, displayed delayed pubertal onset and altered folliculogenesis. We reported a transgenerational alteration of key hypothalamic genes controlling puberty and ovulation (Kiss1, Esr1, and Oxt), and we identified the hypothalamic polycomb group of epigenetic repressors as actors of this mechanism. Furthermore, we found a multigenerational reduction of maternal behavior (F1-F3) induced by a loss in hypothalamic dopaminergic signaling. Using a cross-fostering paradigm, we identified that the reduction in maternal phenotype was normalized in EDC-exposed pups raised by unexposed dams, but no reversal of the pubertal phenotype was achieved. DISCUSSION: Rats developmentally exposed to an EDC mixture exhibited multi- and transgenerational disruption of sexual maturation and maternal care via hypothalamic epigenetic reprogramming. These results raise concerns about the impact of EDC mixtures on future generations. https://doi.org/10.1289/EHP8795.