Clinical parameters 137 pPET+ and pPET-

Intestinal lymphatic vessels are essential for dietary lipid absorption and immune cell trafficking. Villus lymphatic capillaries, lacteals, undergo continuous VEGF-C-dependent renewal to function in a hyperosmolar, inflammatory environment exposed to dietary and microbial by-products. To define mechanisms underlying this adaptation, we integrated new and published single-cell RNA-sequencing datasets of murine small intestinal lymphatic endothelial cells (LECs). Lacteal LECs resembled Ptx3+ immune-interacting LECs and were characterized by high expression of water channel AQP1. LEC-specific Aqp1 deletion reduced lacteal length, impaired lipid uptake, and limited weight gain on a high-fat diet, while mosaic deletion revealed a cell-autonomous requirement for AQP1 in LEC positioning at hyperosmolar tip regions. AQP1 promoted LEC migration under hyperosmotic stress by preserving cytoskeletal and junctional remodeling and alleviating osmotic stress-induced transcriptional programs. AQP1 was upregulated during inflammatory remodeling in lymphedema and lymphatic malformations, but not during embryonic lymphangiogenesis. These findings link lacteal regeneration to inflammatory lymphatic remodeling and highlight tissue osmolarity as a biophysical determinant of postnatal lymphangiogenesis.

The adaptation of lipid metabolism in cancer cells, driven by changes in the tumor microenvironment, presents major challenges for cancer therapy. Here, we addressed the problem of altered lipid metabolism and its role in cancer progression and therapeutic resistance. We demonstrate that hypoxia upregulates the key desaturase, stearoyl-CoA desaturase-1 (SCD1), and the lipid droplet (LD) protein PLIN2, thus promoting lipid metabolic adaptation, cell proliferation, migration, and tumor growth. We found that SCD1 and PLIN2 are essential and interdependent for LD formation. PLIN2 supports cell survival under hypoxic and metabolic stress, whereas SCD1 sustains cancer cell proliferation upon reoxygenation. In addition, we found that SCD1 expression in cancer cells affects nonhistone protein deacetylation, whereas PLIN2 expression enhances protein acetylation. Among these proteins, nucleophosmin(NPM1), a tumor suppressor and regulator of p53, was destabilized through SCD1-dependent deacetylation. In addition, SCD1 interacts with NPM1, influences its cellular localization, and recruits histone deacetylase-2 (HDAC2) to the complex. Notably, we observed that knockdown of SCD1 in vitro or its pharmacological inhibition in vivo enhances cancer cell sensitivity to HDAC inhibitors. Our findings underscore the role of SCD1 in reshaping the cellular acetylome and suggest that targeting SCD1 could sensitize cancer cells to HDAC inhibitors, highlighting a promising therapeutic strategy.

Clinical characteristics of patients included in statistical analyses

TUMOR UNIVARIATE and MULTIVARIATE ANALYSIS

Antigen processing and presentation (APP) is essential for adaptive immunosurveillance. We uncover a mechanism whereby activated T cell-derived extracellular vesicles (ATEVs) drive a positive feedback loop that enhances antigen presentation and immune responses in normal physiology and cancer. ATEV-induced immunogenicity relies on extracellular vesicular double-stranded DNA (EVDNA), which is notably abundant and primarily composed of genomic DNA enriched in immune-related genes, including those encoding APP machinery. Mechanistically, granzyme B (Gzmb) packaged by ATEVs disrupts the nuclear envelope of recipient cells, facilitating intranuclear transfer and subsequent transient expression of EVDNA encoding APP genes. DNase treatment removes most AT-EVDNA, abrogating APP upregulation and thus T cell activation and recruitment to tumors. Notably, ATEVs hold promise as an acellular immunotherapy, restoring APP and synergizing with checkpoint blockade in immunotherapy-refractory tumors. Collectively, our findings uncover a mechanism of transient, non-viral gene delivery by ATEVs that boosts APP and anti-tumor immunity while limiting autoimmunity.

Comparison of the model performance after inclusion of continuous PET-derived features

Illustrative IHC of studied PAoLN biological parmaeters

Lymphangiogenesis has gained considerable interest due to its established role in cancer progression and dissemination of metastatic cells through lymph nodes. Deciphering the molecular mechanisms that govern lymphangiogenesis within lymph nodes holds promise for revealing novel targetable molecules and pathways to inhibit metastasis. In this study, we revealed a previously unrecognized role of AXL, a tyrosine kinase receptor, in the lymphatic vessel formation. We first validated the expression of AXL in lymphatic endothelial cells (LECs), followed by functional studies using RNA interference and pharmacological inhibition with R428/Bemcentinib. These approaches provided compelling evidence that AXL promotes LEC migration in both 2D and 3D culture systems. Our findings demonstrated that AXL activation was induced by VEGF-C (Vascular Endothelial Growth Factor C) and further amplified downstream signaling via the AKT pathway. In vivo, the role of AXL in lymphatic vessel sprouting was demonstrated using R428 in a model of VEGF-C-induced lymphangiogenesis in lymph nodes. Interestingly, we discovered that AXL was predominantly expressed in MARCO+ LECs. Strikingly, under metastatic conditions, there was a notable increase in the density and penetration extent of these AXL-expressing LECs into the lymph node parenchyma. Collectively, our findings pinpoint AXL as a potent enhancer of lymphangiogenesis operating through the VEGF-C/AKT pathway. Furthermore, the identification of AXL expression within a distinct LEC subpopulation, particularly in the context of metastasis, underscores the intricate interplay between AXL signaling and lymphatic dynamics within the lymph node microenvironment.

Intestinal lymphatic vessels are essential for dietary lipid absorption and immune cell trafficking. Specialized villus lymphatic capillaries, lacteals, undergo constant VEGF-C-dependent renewal to maintain their function in a hyperosmolar, inflammatory microenvironment exposed to dietary by-products. The mechanisms of lacteal adaptation remain incompletely understood. We integrated new and published single-cell RNA-sequencing data to profile murine small-intestinal lymphatic endothelial cells (LECs) and identified three distinct subsets. Lacteal LECs display a transcriptional signature resembling Ptx3⁺ immune-interacting LECs characterized by high expression of Aqp1, encoding the aquaporin-1 water channel. LEC-specific deletion of Aqp1 reduced lacteal length, impaired lipid uptake, and limited weight gain on a high-fat diet, underscoring the importance of water homeostasis in lacteal maintenance. AQP1 also promoted VEGF-C-dependent LEC migration under osmotic stress and, uniquely, was upregulated during inflammatory remodelling in secondary lymphedema and lymphatic malformations, but not during embryonic lymphangiogenesis. These findings link lacteal regeneration to inflammatory lymphatic remodelling and highlight tissue osmolarity as a key biophysical factor in postnatal lymphangiogenesis. ### Competing Interest Statement The authors have declared no competing interest.

IntroductionThis study applies NMR-based metabolomics to investigate neovascular age-related macular degeneration (nAMD), addressing challenges in patient management, disease progression evaluation, and treatment response assessment. A two-year follow-up of 29 nAMD patients undergoing treatment provided 231 time points for analysis.MethodsOver the two-year period, 11 males and 18 females (aged 61–92 years) were monitored, yielding 231 time points. At each time point, blood samples for NMR metabolomics analysis, clinical measurements (e.g., lactate, glucose levels, HDL/LDL cholesterol, and blood pH), and optical coherence tomography (OCT) images were collected to evaluate the progression of choroidal neovascularization. 1H-NMR metabolomic analysis led to the quantification of over 60 metabolites and of the major lipoprotein fractions. Both multivariate and univariate statistical approaches tailored for longitudinal data were employed to identify biomarkers correlating metabolomic changes with ocular alterations during disease progression.Results and DiscussionDespite a rigorous analytical workflow enabling precise quantification of over 60 metabolites and the application of advanced statistical tools for longitudinal data, achieving consistent results across the cohort proved challenging. The dataset’s heterogeneity, reflecting real-world clinical practice, complicated the derivation of global conclusions. Personalized analyses on a patient-by-patient basis successfully identified individual correlation models, but a universal model remained elusive. This study highlights the inherent challenges of translating findings from controlled settings into clinical practice, where factors such as visit frequency, treatment variability, and disease heterogeneity limit data uniformity. We emphasize the importance of experimental design in longitudinal studies, particularly when dealing with incomplete and variable datasets. We are therefore confident that, considering both the challenges and difficulties identified in this work and the preliminary results presented here, it is possible to develop predictive and individualized models for monitoring patients with nAMD. Such models could greatly assist clinicians in providing better care for these patients.

Metastatic hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2−) breast cancer often develops resistance to first-line treatment, typically combining cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) with hormone therapy (HT) [1, 2]. After an initial response, most patients become resistant, and compensatory mechanisms are not fully uncovered [3]. To address this, we analyzed HR+ resistant CAMA1 and 747D cells using whole-exome and RNA sequencing, supplemented by proteomics and target validation with human samples. Additionally, we conducted combination therapy trials using xenografts and patient-derived xenografts (PDXs). Detailed study designs and methods are provided in the Supplementary file. In a cohort of 27 patients with metastatic breast cancer, we observed reduced progression-free survival in second- and third-line therapies following progression post palbociclib-HT treatment (Supplementary Figure S1A and Supplementary Table S1). Resistant tumors showed reduced estrogen receptor alpha (ERα) and progesterone receptor (PR) and increased proliferation rates (Supplementary Figure S1B-D). CAMA1 and T47D cells, treated with palbociclib and fulvestrant (PF) for 2 years, developed resistance (CAMA1-PFR and T47D-PFR) confirmed by proliferation assays and elevated half-maximal inhibitory concentrations. Resistant cells exhibited reduced levels of ERα and retinoblastoma protein (Supplementary Figure S2). Exome analysis revealed no drug resistance-related mutations (Supplementary Tables S2-S3), suggesting non-genetic factors. RNA sequencing of T47D cells treated with DMSO or PF for 20 days and T47D-PFR cells revealed 1,172 upregulated genes and 824 downregulated genes in the resistant cells (Supplementary Figure S3A). Gene set enrichment analysis indicated increased fatty acid localization (Supplementary Figure S3B), with a heatmap showing elevated fatty acid uptake and metabolism-related genes, such as fatty acid binding protein-6 (FABP6), FABP7, cluster of differentiation-36 (CD36), and proteasome proliferator-activated receptor-gamma (PPARγ) in T47D-PFR cells (Figure 1A). Lipid droplets accumulated in PF-treated parental and PF-resistant T47D and CAMA1 cells (Figure 1B and Supplementary Figure S3C). FABP6 levels were elevated in PF-treated parental and PF-resistant cells, with CD36 overexpression unique to T47D-PFR cells at both protein and mRNA levels (Figure 1C and Supplementary Figure S3D-E), suggesting that lipid uptake might be an adaptive response to oxidative stress [4, 5]. This was supported by elevated reactive oxygen species (ROS) levels in PF-treated parental cells (Figure 1D). Furthermore, proteomic analysis in human biopsies revealed a functional network of 11 oxidative stress-triggered proteasomes (Supplementary Figure S4A and Supplementary Tables S4-S5) as indicators of oxidative stress [6]. Immunohistochemistry validated increased proteasome subunit alpha type-7 (PSMA7) in resistant biopsies (Supplementary Figure S4B). Ferroptosis inducers overcome resistance of HR+HER2− breast cancer cells to palbociclib-hormone therapy. (A) Heatmap of upregulated genes (red) or downregulated genes (blue) during the treatment of T47D cells with PF combination for 20 days, and in PF-resistant cells (T47D-PFR) after continuous PF treatment for 2 years, compared to parental T47D cells treated with vehicle (DMSO). All represented genes had a P value < 0.05. (B) Lipid droplet detection by BODIPY 493/503 staining in parental CAMA1 and T47D cells after treatment with the PF combination for 20 days, and in CAMA1-PFR and T47D-PFR cells after continuous PF treatment for 2 years. DAPI staining was used to visualize the nuclei. (C) Western blot analysis of FABP6 and CD36 in parental CAMA1 and T47D cells after treatment with the PF combination for 20 days, and in CAMA1-PFR and T47D-PFR cells after continuous PF treatment for 2 years. (D) Measurement of ROS levels by DCFDA ROS assay in parental CAMA1 and T47D cells and in CAMA1-PFR and T47D-PFR cells treated for 5 days with DMSO or PF. Continuous PF treatment for resistant cells was discontinued 3 days prior to the assay. (E) Western blot analysis of GPX4 in parental CAMA1, T47D and ZR75.1 cells after treatment with PF combination for 20 days and in CAMA1-PFR, T47D-PFR and ZR75.1-PFR cells after continuous PF treatment for 2 years. (F) Immunohistochemistry analysis (images) and quantification (bar chart) of GPX4-positive cells in biopsies of patients taken before treatment with palbociclib-HT or after resistance (*P < 0.05). (G) Assessment of cell proliferation according to the fold change in confluence in parental and PF-resistant CAMA1 and T47D cells treated with DMSO, the ferroptosis inducer RSL3 (1 µmol/L), the ferroptosis inhibitor Trolox (10 µmol/L), or a combination of both for 7 days. Continuous PF treatment for resistant cells was discontinued 3 days prior to the assay. (H) Assessment of cell proliferation according to the fold change in confluence in parental and PF-resistant CAMA1 and T47D cells treated with different combinations of DMSO, palbociclib (P) (0.3 µmol/L), fulvestrant (F) (30 nmol/L), or eprenetapopt (Ep) (25 µmol/L) for 7 days. Continuous PF treatment for resistant cells was discontinued 3 days prior to the assay. (I, J) Tumor growth curves of parental CAMA1 (6 mice per group) (I) and CAMA1-PFR (8 mice per group) (J) xenografts treated with different combinations of palbociclib (P) 75 mg/kg daily, fulvestrant (F) 50 mg/kg once a week, and eprenetapopt (Ep) 150 mg/kg, 2 times/day or RSL3 100 mg/kg by intratumoral injection every two days for two weeks (***P < 0.001). (K) Growth curves of palbociclib-HT-naïve HBCx-124 PDXs (5 mice per group) treated with different combinations of palbociclib (P) 75 mg/kg daily, fulvestrant (F) 50 mg/kg once a week, and eprenetapopt (Ep) 150 mg/kg, 2 times/day (***P < 0.001). (L) Growth curves of palbociclib-HT-resistant HBCx-180 PDXs (8 mice per group) treated with different combinations of palbociclib (P) 75 mg/kg daily, fulvestrant (F) 50 mg/kg once a week, and eprenetapopt (Ep) 150 mg/kg, 2 times/day. The difference between the measured tumor volumes was significant between the "vehicle" group and the eprenetapopt + fulvestrant + palbociclib (FPE) group from Day 25 until the end of the experiment (P < 0.05). Tumor evolution formula is (Vf − V0 / V0) × 100%, where V0 is the initial volume and Vf is the final volume measured at each time point. Abbreviations: CD36, cluster of differentiation 36; DAPI, 4′,6-diamidino-2-phenylindole; DCFDA, 2′,7′-Dichlorofluorescin Diacetate; DMSO, dimethyl sulfoxide; Ep, eprenetapopt; FABP6, fatty acid binding protein 6; GPX4, Glutathione peroxidase 4; HR+HER2−, hormone receptor positive and human epidermal growth factor receptor-2 negative; HT, hormone therapy; PDX, patient-derived xenograft; PF, palbociclib-fulvestrant; PF RSL3, palbociclib-fulvestrant-RSL3; PFE, palbociclib-fulvestrant-eprenetapopt; ROS, reactive oxygen species, RSL3, ras-selective lethal small molecule 3; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. We investigated whether cell survival is affected by ferroptosis—a type of non-apoptotic cell death linked to lipid peroxidation. GPX4 protein, the main protector against ferroptosis, was overexpressed in parental cells after PF treatment and in PF-resistant cells, even after drug wash-out, with no changes in mRNA levels (Figure 1E and Supplementary Figure S5A-C). Silencing GPX4 expression reduced cell proliferation in parental and PF-resistant cells (Supplementary Figure S5D-F), indicating their reliance on GPX4. GPX4 overexpression was also observed in resistant human tumors (Figure 1F). Cells were treated with the GPX4 inhibitor RAS-selective lethal 3 (RSL3) and the antioxidant Trolox for 6 days. CAMA1 and T47D cells were insensitive to RSL3, whereas CAMA1-PFR and T47D-PFR cells showed high sensitivity to RSL3 (Figure 1G). Trolox reversed RSL3's effect in both cell lines (Figure 1G), highlighting the role of GPX4 in PF-resistant cell proliferation. Due to unverified safety of RLS3, we used eprenetapopt (Ep), a p53 activator and GSH depletory [7] proven safe in hematological cancer patients [8]. In vitro, CAMA1 and CAMA1-PFR cells were sensitive to Ep, while T47D cells were insensitive; T47D-PFR showed higher sensitivity (Figure 1H). To investigate the effect of pharmacologically induced ferroptosis on the palbociclib-fulvestrant response in vivo, we used nude mice implanted with estrogen pellets. Due to its low pharmacokinetics in mouse plasma [9], RSL3 was administered via intratumoral injection. Mice were treated with vehicle, PF, Ep, RSL3, a combination of palbociclib-fulvestrant and eprenetapopt (PFEp), or a combination of palbociclib-fulvestrant and RSL3 (PF-RSL3). PF effectively inhibited the growth of the CAMA1 xenografts, while CAMA1-PFR tumors were insensitive (Figure 1I and J). RSL3 alone did not affect CAMA1-PFR tumor growth, whereas PF-RSL3 demonstrated a strong antitumor effect (Figure 1J), suggesting that RSL3 sensitizes CAMA1-PFR cells to PF, or vice versa. Similarly, Ep alone did not inhibit CAMA1-PFR tumor growth, but its combination with PF completely abolished tumor growth (Figure 1J). To evaluate treatment effects on proliferation and cell death, we assessed Ki67, caspase-3 and hydroxynonenal (HNE) in tumors by immunohistochemistry. Parental CAMA1 tumors treated with PF showed a significant decrease in Ki67, with no effect on caspase-3, while CAMA1-PFR tumors exhibited no significant changes in either marker after treatment with PF, PFEp, or PF-RSL3 (Supplementary Figure S6A-D). HNE labeling revealed no difference between PF- and vehicle-treated CAMA1 tumors (Supplementary Figure S6E), but HNE increased moderately in CAMA1-PFR tumors treated with RSL3 and significantly with PFEp or PF-RSL3 (Supplementary Figure S6F), indicating potential cell death by ferroptosis. No treatments affected CD36 expression in either tumor type, although CAMA1-PFR xenografts showed significantly increased basal expression compared to human biopsies (Supplementary Figure S6G-J). To strengthen the translational impact of our findings, we used patient-derived xenografts (PDXs) from HR+HER2− breast cancer patients without p53 mutations (Supplementary Figure S7A) [10]. Consistent with human samples, GPX4 protein was upregulated in the palbociclib-HT-resistant PDX model (HBCx-180) compared to the palbociclib-HT-naïve PDX model (HBCx-124) (Supplementary Figure S7B). While GPX4 mRNA levels were unaffected in resistant cells, its expression was significantly higher in HBCx-180 (Supplementary Figure S7C), suggesting specific transcriptional/translational regulation differing between in vitro and in vivo contexts. In the HBCx-124 model, all the tumors (n = 5) responded well to PF, with no added benefit from Ep (PFEp), although a partial response to Ep monotherapy was observed (Figure 1K). Conversely, the HBCx-180 model showed no significant response to PF or Ep, with significant differences in tumor volumes between PFEp and vehicle and Ep groups from Day 25 onward (P < 0.05) (Figure 1L). These results confirm that adding Ep to PF in PF-resistant tumors produces a strong antitumor effect. Ki67 labeling decreased with PF in the HBCx-124 PDX but not in the HBCx-180 PDX (Supplementary Figure S8A-B). Aside from reduction in caspase-3 labeling in the HBCx-124 treated with PF, no significant changes were observed with Ep or PFEp, and none of the treatment conditions affected HBCx-180 (Supplementary Figure S8C-D), indicating that proliferation and apoptosis are not affected in HBCx-180. HNE labeling suggested that PF promoted ferroptosis in HBCx-124, while only tumors treated with PFEp in HBCx-180 exhibited significant HNE increase compared to vehicle (Supplementary Figure S8E-F). PF slightly increased HNE in HBCx-124 without significance but the only vulnerability of HBCx-180 is attributed to ferroptosis induced with PFEp. The p53-dependent antitumor effect of eprenetapopt was excluded, as p53 expression was similar in parental and PF-resistant cells and in PDXs, while slightly elevated in resistant human tumors (Supplementary Figure S9). Finally, the addition of Ep or RSL3 to PF did not induce significant renal, hepatic, or hematological toxicity in mice (Supplementary Figure S10). In conclusion, HR+HER2− tumors resistant to palbociclib-HT are vulnerable to ferroptosis inducers, highlighting the potential of collateral drug sensitivity and the promise of developing pro-ferroptosis agents for treating drug-resistant metastatic breast cancer (Supplementary Figure S11). Conception and design: CP and NES. Development of methodology: CP, LMR, NES. Acquisition of data: CP, LMR, RT, CW, RJ, AR, JC, CJ, SG, SB, AD, PD, LM and EM. Analysis and interpretation of data (e.g., statistical analysis, biostatistics): CP, LMR, DB, GM, RT, CJ and NES. Writing, review, and/or revision of the manuscript: CP, GJ, AN and NES. Study supervision: GJ and NES. The authors thank the Cell Imaging Core Facility of the GIGA institute for Incucyte and fluorescence microscopy experiments; Isabelle Dasoul, Emilie Feyereisen, Erika Konradowski and Nathalie Lefin for their technical support; Maud Piron for enrolling the cohort of patients; and Hélène Schroeder for correcting the file sent to the human ethics committee and the Biobank of Liège University for providing human samples. The authors also thank Latifa Karim and Manon Deckers from the GIGA Sequencing Platform (ULiège, Belgium) for their help and advice; Erik Maquoi for image acquisition with Nanolive and Louis Baudin (Animascience, Liège, Belgium) for the graphical abstract. The authors declare no competing interest except for Dr. Guy Jerusalem, who declares receiving grant support, paid to his institution, advisory board fees, lecture fees, travel support, and writing assistance from Novartis, Roche, and Pfizer. Disclosure is provided with the full text of this article. No other potential conflicts of interest relevant to this article were reported. This work was supported by grants from the National Fund for Scientific Research (NFSR-FNRS) Belgium (NES: PDR T.023020; CDR J.0178.22); the credit sectorial of the University of Liege (NES: FSR-S-SS-22/61; FSR-S-SS-22/64); and the Foundation Contre le Cancer, Belgium (NES and AN: FCC-2022-181). RNA sequencing data were deposited in the GEO-NCBI depository with the accession number: GSE270021 Exome sequencing data were registered in the BioProject database with the identification number: PRJNA1027140 Proteomic data were deposited in PRIDE with accession number: PXD053296. The data that support the findings of this study are available from the corresponding author upon reasonable request. A protocol was approved by the institutional Ethics Committee of the University Hospital of Liege (Liege, Belgium; file#2018/312) for the use of human samples in this study, and the ethical guidelines of the Declaration of Helsinki were followed. The ethical committee has authorized on May 15, 2019, to retrospectively use biopsies stored in the biobank of the University of Liège and anonymized clinical data associated with these biopsies. The committee noted that this non-interventional use has no impact on patients and is not within the scope of the law of May 7, 2004, on experiments on human beings and thus did not require informed consents. For the animal study, all procedures were performed according to the Federation of European Laboratory Animal Sciences Associations (FELASA) within the accredited GIGA animal facility (University of Liege, Liege, Belgium) (project authorization no. 2078). PDX experiments were performed at the Institute of Curie, in accordance with institutional guidelines and the rules of the French Ethics Committee (project authorization no. 02163.02). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Most cells produce latent transforming growth factor-beta 1 (TGF-β1), but only very few activate the cytokine via cell type-specific mechanisms. TGF-β1 favors cancer progression by suppressing anti-tumor T cell responses. Which cells produce this immunosuppressive TGF-β1 in human tumors is unknown. Putative sources include cells expressing the glycoprotein A repetitions predominant (GARP) protein, comprising mostly activated regulatory T cells (Tregs) (GARP+FOXP3+ cells) and blood endothelial cells (BECs). We performed multiplexed immunohistofluorescence and computerized image analyses on 186 tumor samples from 5 cancer types (colorectal, urothelial, lung and breast primary carcinomas and melanoma metastases), compared to patient-matched adjacent non-cancerous tissues. GARP+ Tregs were present in 29–75

BACKGROUND:ADAMTS2 is a metalloproteinase known to be implicated in collagen maturation and regulation of (lymph)angiogenesis. As these properties are likely to alter tumor progression, we aimed to assess the overall impact of ADAMTS2 on cancer development. METHODS AND RESULTS:Using publicly available human cancer datasets, we found that high expression of ADAMTS2 in primary tumors is associated with poor prognosis across various cancer types. Similar analyses were repeated, but this time using the ratio of ADAMTS2 on COL1A1 expression to take into account potential biases due to the involvement of ADAMTS2 in collagen fibril formation. Remarkably, these data indicate that patients with a high ADAMTS2/COL1A1 ratio exhibit an improved overall survival rate, suggesting that ADAMTS2 may inhibit cancer progression by a mechanism independent of collagen accumulation. This hypothesis was evaluated in vivo using ADAMTS2-KO mice and different tumor models characterized by the absence or presence of tumor collagen accumulation, as in MMTV-PyMT mice which develop spontaneous desmoplastic mammary tumors. In all the models, the growth of primary tumors was strongly increased in ADAMTS2-KO mice versus their wild type counterparts, confirming that ADAMTS2 displays anti-tumor properties. In stark contrast, the spread of lung metastases from mammary tumors was virtually prevented in ADAMTS2-KO mice, showing a dual role of ADAMTS2, either beneficial or detrimental, at different stages of cancer progression. Additional investigations, notably by FACS and single cell sequencing, showed that the effect of ADAMTS2 on primary tumors does not result from a direct effect on cancer cells, but rather from modifications in the intratumor innate immune system which becomes more immunosuppressive in the absence of ADAMTS2. CONCLUSION:We have shown that ADAMTS2 suppresses tumor growth by inhibiting the progressive establishment of an immunosuppressive microenvironment. Conversely, its presence allows efficient formation of lung metastases. These data identify ADAMTS2 as a cancer regulator with antagonistic functions, limiting initial progression but promoting efficient metastatic dissemination.

BACKGROUND:Lymphedema is an incurable disease associated with lymphatic dysfunction that causes tissue swelling and fibrosis. We investigated whether lymphedema could be attenuated by interfering with uPARAP (urokinase plasminogen activator receptor-associated protein; Mrc2 gene), an endocytic receptor involved in fibrosis and lymphangiogenesis. METHODS:We generated mice with lymphatic endothelial cell (LEC)-specific uparap deficiency and compared them with constitutive knockout mice by applying a preclinical model of secondary lymphedema (SL). Computerized methods were applied for 2-dimensional and 3-dimensional image quantifications. Cellular effects of uPARAP deletion on lymphatic permeability were assessed by small interfering RNA-mediated silencing in human dermal LECs and a pharmacologic treatment targeting ROCK (Rho-associated coiled coil containing kinase), an established regulator of cell junctions. The uPARAP and vascular endothelial cadherin partnership was investigated through proximity ligation assay, coimmunoprecipitation, and immunostaining. An in silico model was generated to analyze the fluid-absorbing function of the lymphatic vasculature. To interfere with uPARAP, its downregulation was achieved in vivo through a gapmer approach. RESULTS:uparap deficiency mitigated several key pathologic features of SL, including hindlimb swelling, epidermal thickening, and the accumulation and size of adipocytes. In both global and LEC-conditional uparap-deficient mice, induction of SL led to a distinctive labyrinthine vasculature, defined herein by twisted and hyperbranched vessels with overlapping cells. This topology, mainly composed of pre-collecting vessels, correlated with reduced SL, but not with change in fibrosis, highlighting the importance of uPARAP in regulating LEC functions in a lymphedematous context. In vitro, uPARAP knockdown in LECs impaired vascular endothelial growth factor C-mediated endosomal trafficking of vascular endothelial cadherin and induced overlapping cell junctions. The pharmacologic inhibition of ROCK recapitulated cell superimposition in vitro and the labyrinthine vasculature in vivo with attenuated SL. Computational modeling of labyrinthine lymphatic vasculature supported the observation on their improved fluid-absorbing function in comparison with a normal hierarchic network. These data provide proof of concept of inducing a labyrinthine topology to treat SL. For therapeutic purposes, we validated the use of an anti-uPARAP gapmer to induce a labyrinthine vasculature and attenuate SL formation. CONCLUSIONS:Our findings provide evidence that downregulating uPARAP expression can induce a beneficial remodeling of lymphatic vasculature that attenuates lymphedema through a cell junction-based mechanism, offering a novel therapeutic pathway for lymphedema.

Boxplot of the expression of genes encoding proteins involved in capecitabine metabolism (TYMP, TYMS, TK1, DPYD) and cell-cycle control (RB1, CDKN2A, CCND1).