ARTICLE https://doi.org/10.1038/s41467-020-16496-y OPEN Blockade of VEGF-C signaling inhibits lymphatic malformations driven by oncogenic PIK3CA mutation Ines Martinez-Corral1,9, Yan Zhang1,9, Milena Petkova 1, Henrik Ortsäter1, Sofie Sjöberg1, Sandra D. Castillo 2, Pascal Brouillard 3, Louis Libbrecht4, Dieter Saur5, Mariona Graupera2, Kari Alitalo6, Laurence Boon3,7, Miikka Vikkula 3,8 & Taija Mäkinen1 ✉ 1234567890():,; Lymphatic malformations (LMs) are debilitating vascular anomalies presenting with large cysts (macrocystic) or lesions that infiltrate tissues (microcystic). Cellular mechanisms underlying LM pathology are poorly understood. Here we show that the somatic PIK3- CAH1047R mutation, resulting in constitutive activation of the p110α PI3K, underlies both macrocystic and microcystic LMs in human. Using a mouse model of PIK3CAH1047R-driven LM, we demonstrate that both types of malformations arise due to lymphatic endothelial cell (LEC)-autonomous defects, with the developmental timing of p110α activation determining the LM subtype. In the postnatal vasculature, PIK3CAH1047R promotes LEC migration and lymphatic hypersprouting, leading to microcystic LMs that grow progressively in a vascular endothelial growth factor C (VEGF-C)-dependent manner. Combined inhibition of VEGF-C and the PI3K downstream target mTOR using Rapamycin, but neither treatment alone, promotes regression of lesions. The best therapeutic outcome for LM is thus achieved by co- inhibition of the upstream VEGF-C/VEGFR3 and the downstream PI3K/mTOR pathways. 1 Uppsala University, Department of Immunology, Genetics and Pathology, Dag Hammarskjölds väg 20, 751 85 Uppsala, Sweden. 2 Vascular Signaling Laboratory, Institut d´Investigació Biomèdica de Bellvitge (IDIBELL), 08908L´Hospitalet de Llobregat, Barcelona, Spain. 3 Human Molecular Genetics, de Duve Institute, University of Louvain, Brussels, Belgium. 4 Center for Vascular Anomalies, Division of Pathology, Cliniques universitaires Saint Luc, University of Louvain, 10 avenue Hippocrate, B-1200 Brussels, Belgium. 5 Department of Internal Medicine 2, Klinikum rechts der Isar, Technische Universität München, Ismaningerstr. 22, 81675 München, Germany. 6 Wihuri Research Institute and Translational Cancer Biology Program, Biomedicum Helsinki, FIN-00014 University of Helsinki, Helsinki, Finland. 7 Center for Vascular Anomalies, Division of Plastic Surgery, Cliniques universitaires Saint Luc, University of Louvain, 10 avenue Hippocrate, B-1200 Brussels, Belgium. 8 Walloon Excellence in Lifesciences and Biotechnology (WELBIO), University of Louvain, Brussels, Belgium. 9 These authors contributed equally: Ines Martinez-Corral, Yan Zhang. ✉email: taija.makinen@igp.uu.se NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16496-y V ascular malformations are chronic, often congenital treatment of microcystic LM in human and suggest that therapies pathologies that can manifest in different types of blood targeting the key upstream pathway in combination with PI3K and lymphatic vessels. These diseases commonly arise pathway inhibition may be relevant for other PIK3CA-driven from abnormalities in the endothelial cells (ECs) of the affected pathologies. vessel type(s) that lead to structural and functional vascular defects causing deformation, pain, morbidity, and organ dys- Results function1. Genome sequencing efforts have identified causative PIK3CAH1047R mutation underlies micro- and macrocystic mutations for different types of malformations and opened up LMs. To address whether the two subtypes of LM require dif- possibilities for therapeutic intervention specifically targeting the ferent PIK3CA mutations, potentially driving different cellular aberrant signal transduction pathways2,3. responses, or if the same mutation can underlie both microcystic Somatic activating mutations in the PIK3CA gene, encoding and macrocystic LMs, we focused on patients with a somatic the p110α catalytic subunit of phosphatidylinositol 3-kinase PIK3CAH1047R mutation. Clinical features of five patients selected (PI3K), were identified as causative of ~20% of venous mal- for the study are summarized in Table 1. Histologic features of formations (VM)4–6, and the majority of lymphatic malforma- the lesions were investigated using tissue sections from the tions (LM)7,8. The most common VM/LM mutations affecting patients to confirm lymphatic identity of the lesions and the LM the helical domain (E542K, E545K) or the kinase domain subtype (Fig. 1a, b, Supplementary Fig. 1a). (H1047R, H1047L) of p110α are identical to those previously Lesions from two patients with macrocystic LM were found in cancer and other genetic syndromes characterized by characterized by convoluted structures with large lumens, which tissue overgrowth9. Both types of mutations result in basal acti- in some cases contained lymph, lymphocytes and/or red blood vation of the PI3K pathway by enhancing dynamic events in the cells (RBCs) (Fig. 1a). The endothelium lining the cystic lumens natural activation of p110α that lead to increased lipid binding10. was positive for PDPN and PROX1 (Supplementary Fig. 1a), The PI3K lipid kinases control a variety of cellular functions confirming lymphatic endothelial identity. LYVE1 (Supplemen- and developmental and homeostatic processes in response to tary Fig. 1a) and VEGFR3 (Fig. 1b, Supplementary Fig. 1b) extracellular signals by regulating the plasma membrane phor- staining was weak or lacking in the largest cysts, in particular in phatidylinositol (3,4,5)-triphosphate (PIP3) levels11. Of the four areas surrounded by αSMA+ smooth muscle cells. High VEGFR3 p110 isoforms, the ubiquitously expressed p110α has emerged as expression was instead observed in small-caliber lymphatic the key downstream effector of growth factor receptor signaling vessels in areas within the cyst wall containing infiltrated CD45 in most cell types and in particular in the endothelium. Genetic + cells (Fig. 1b). Microcystic LMs were characterized by smaller loss-of-function studies in mice demonstrated an important role of p110α in the development of both blood and lymphatic ves- cysts and vessels with irregular lumens and no smooth muscle cell sels12–14. Conversely, conditional expression of the PIK3CA- layer (Fig. 1b). The endothelium of microcystic LMs was positive activating mutation in endothelial cells led to vascular overgrowth for VEGFR3 (Fig. 1b), PDPN and PROX1 (Supplementary and malformations in mice4,5,15,16. Identification of PIK3CA Fig. 1a). LYVE1 staining was patchy, similarly as in macrocystic mutations as drivers of vascular malformations has opened up a LMs (Supplementary Fig. 1a). Similar to the macrocystic LM, and possibility for the therapeutic use of PI3K inhibitors in these in agreement with previous reports21, a large number of CD45+ diseases. Rapamycin and its analogues (sirolimus, everolimus) cells (Fig. 1b) and presence of lymphoid aggregates (Supplemen- that target the PI3K downstream effector mTOR can stop the tary Fig. 1c) were also observed in all three microcystic LMs. progression of vascular malformations in mice and human, and These data show that the two distinct LM subtypes showing improve the patients’ quality of life3,5,15–19. However, regression different morphological and molecular features result from the of lesions is observed only in a minority of patients3, which calls same somatic mutation in PIK3CA. for a need to develop new more effective therapies. Compared with the malformations affecting the blood vascu- Developmental timing of PI3K activation determines LM lature, LMs have received less attention despite often severe subtype. To investigate if both macrocystic and microcystic complications for patients. LMs are characterized by large fluid- PIK3CA-driven LMs are caused by lymphatic endothelial cell filled cysts (macrocystic LM), or diffuse, infiltrative lesions (LEC)-autonomous defects, we generated a mouse model that sometimes consisting of small vesicles containing lymph or blood allows inducible LEC-specific activation of the causative mutation (microcystic LM)19,20. Many patients show a mixed phenotype PIK3CAH1047R. Mice carrying a knock-in PIK3CAH1047R allele in with a combination of large and small cysts. Lesion growth may the Rosa26 locus, silenced by a lox-stop-lox cassette until Cre be progressive and, depending on the location, result in severe exposure22, were crossed with the Vegfr3-CreERT2 mice expres- complications such as infections and impairment of breathing or sing tamoxifen-inducible Cre recombinase specifically in lym- swallowing. Macrocystic LM can be usually effectively treated phatic endothelia23 (Fig. 2a). Since LMs are often present at birth, with sclerotherapy or surgical resection. By contrast, the treat- we first induced activation of PIK3CAH1047R expression at the ment of microcystic LM is challenging due to their infiltrative early stage of lymphatic development by administering 4- growth, and curative therapies are currently lacking. hydroxytamoxifen (4-OHT) to pregnant females at embryonic Here we studied the pathophysiological mechanisms of day (E)11 (Fig. 2a). Whole-mount immunofluorescence of the PIK3CA-driven LM. We show that both macrocystic LM and skin of embryos at E17 revealed two types of lesions in the microcystic LM are driven by the activating PIK3CAH1047R lymphatic vasculature. The majority of lesions appeared as large mutation, with the developmental timing of activation of the isolated cysts that were localized mainly to the cervical, and less p110α PI3K signaling in lymphatic endothelia determining the frequently to the sacral region of the skin (Fig. 2b, c, Supple- LM subtype. We further show that the growth of PIK3CAH1047R- mentary Fig. 2a, b). In addition, areas of lymphatic vessel driven microcystic LM in mice is dependent on the upstream hypersprouting were observed in the thoracic region of the skin lymphangiogenic vascular endothelial growth factor C (VEGF- (Fig. 2b). Lymphatic endothelium showed normal identity and C)/VEGFR3 signaling. Combined inhibition of VEGF-C signaling expression of LEC markers including PROX1 (Fig. 2b, Supple- and the PI3K downstream target mTOR using Rapamycin, but mentary Fig. 2b). Histological examination of the skin revealed neither treatment alone, promotes the regression of experimental that the cysts consisted of a single lumen (Fig. 2d) that frequently LM in mice. Our findings provide important implications for the contained RBCs (Supplementary Fig. 2b, c). The morphological 2 NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16496-y ARTICLE Table 1 Clinical features of patients with LM driven by H1047R mutation in PIK3CA. Case LM type Gender Onset of Symptoms Treatment disease 1 Microcystic M 10 y Localized soft tissue hypertrophy and dermal Surgical resection at 13 years of age lymphatic vesicles causing oozing on the right flank 2 Microcystic M Congenital Right hemifacial cheek hypertrophy; lesion invading Surgical resection at 2 years of age the facial nerve and the parotid gland; intrabuccal mucosal small vesicles causing daily oozing 3 Microcystic M Congenital Born with a small discoloration on his right Surgical resection at 6 years of age hypothenar area that slowly indurated. By the age of 2 years, an ill-defined 5 × 5 cm diameter mass appeared progressively with time; pain on palpation 4 Macrocystic M Congenital Lesion on the right neck and lower cheek; increased Sclerotherapy, intralesional injection of in size during chickenpox infection at picibanyl at 22 months of age; surgical removal 19 months of age at 27 months of age 5 Macrocystic F Congenital Lesion on the right neck englobing the parotid gland; Intralesional injection of picibanyl at 3 and 4 increased in size at 15 days of life and at 1.5 years of years; surgical removal at 7 years due to age with important intralesional bleeding frequent infections and increase in volume features of the lesions and their predominant localization to the PIK3CA mutant vasculature showed an increase in the number of neck region of the skin were thus reminiscent of human mac- vessel ends, the majority of which had the spiky morphology of rocystic LM. active sprouts (Fig. 3b). Staining for phosho-S6 as a readout of Next, we induced activation of PIK3CAH1047R expression in PI3K activity26 revealed signal selectively in active lymphatic LECs during late embryonic (Supplementary Fig. 2a) or early sprouts in the mutant vasculature, similar to lymphangiogenic postnatal development (Fig. 2a). Administration of 4-OHT to sprouts of embryonic vessels (Fig. 3c). LECs within the active pregnant females at E14 led to increased lymphatic vessel sprouts also displayed straight zipper-like cell-cell junctions, a branching in all regions of the dorsal skin of E17 PIK3- hallmark of lymphangiogenic vessels27, as opposed to oak-leaf CAH1047R;Vegfr3-CreERT2 embryos, but no macrocystic lesions shaped LECs of normal capillaries that possess button-like were observed (n = 6 embryos) (Supplementary Fig. 2d, e). junctions (Fig. 3d). Similarly, analysis of the vasculature in the ear skin of neonatally To study more advanced lesions, we utilized models permitting induced mice revealed well-defined malformations that were robust and reproducible induction of localized vs. generalized characterized by a dense network of hyperbranched lymphatic PIK3CAH1047R-driven response in the postnatal ear skin using the vessels (Fig. 2e). These lesions further developed into large sheet- Prox1-CreERT2 and Vegfr3-CreERT2 models. Topical application like vascular structures that frequently showed bleeding in the of a low dose of 4-OHT (0.5-2 µg) into one ear of a lesion area (Fig. 2e). A similar lymphatic hypersprouting PIK3CAH1047R;Prox1-CreERT2 mouse induced locally restricted phenotype was observed using another LEC-specific Cre line, Cre recombination and lesions in the treated ear (Supplementary the Prox1-CreERT224 (Supplementary Fig. 3a–c). Histological Fig. 3a, b). A similar sprouting response and progression of examination of the lesions in the PIK3CAH1047R;Prox1-CreERT2 lesions was observed as previously seen in the Vegfr3-CreERT2 ear showed multiple lumens (Fig. 2f) and presence of RBCs both model (Supplementary Fig. 3c, d). In contrast, topical adminis- inside and outside of the lymphatic vessels (Supplementary tration of a higher dose of 4-OHT (100 µg) into one ear of a Fig. 3d), reminiscent of microcystic LMs in human patients Vegfr3-CreERT2 mouse led to systemic Cre-mediated recombina- (Fig. 1b). tion (Supplementary Fig. 4a, b), resulting in a generalized In summary, these results demonstrate that developmental PIK3CAH1047R-driven sprouting response of the entire dermal timing of activation of the p110α signaling in LECs determines lymphatic vasculature (Supplementary Fig. 4c, d). Progressive the LM subtype. Early embryonic activation leads predominantly increase in lymphatic vessel hyperbranching (Supplementary to formation of lesions that recapitulate features of human Fig. 4e) was associated with increase in the number of active macrocystic LM, whereas activation during late embryonic or sprouts (Supplementary Fig. 4f), as well as in LEC proliferation neonatal development results in microcystic LM (Supplementary (Supplementary Fig. 4g) and numbers (Supplementary Fig. 4h). Table 1). Our data further suggest that the physical location of the To characterize the LEC phenotype associated with p110α lesion also impacts on their characteristics, with most macrocystic activation, we isolated primary dermal LECs from the PIK3- lesions developing in the cervical region of the skin. CAH1047R;Vegfr3-CreERT2 mice by sequential selection of PECAM1+ ECs and LYVE1+ LECs, as previously described23. PIK3CAH1047R expression confers migratory phenotype in Staining for VE-cadherin and PROX1 demonstrated high purity LECs. We focused our investigation on microcystic LMs that are of the isolated LEC population (Supplementary Fig. 5a). PIK3- difficult to treat due to their infiltrative pattern of growth. To CAH1047R -expressing LECs, treated in vitro with 4-OHT, showed allow analysis of the early steps of microcystic LM development, reorganization of cell-cell junctions and cytoskeleton character- we analyzed the acute response of postnatal ear vasculature of ized by increase in serrated junctions with VE-cadherin fingers, PIK3CAH1047R;Vegfr3-CreERT2 mice 10 days after neonatal 4- and increased actin stress fiber formation compared with OHT administration (Fig. 3a). We observed formation of new untreated controls (Fig. 3e). Such changes in junctional vessels showing a typical ‘spiky’ appearance of active vessel morphology have previously been linked to active EC migration sprouts (Fig. 3b). Quantification of vessel ends and their mor- and vessel sprouting28–30. To test if the observed morphological phology demonstrated blunted morphology of most lymphatic changes correlated with increased migratory behavior, we capillaries and only a few active sprouts in the ear skin of 3-week- exposed PIK3CAH1047R -expressing and control LECs to scratch old mice (Fig. 3b), in agreement with previous observations25. wound assay. Live imaging of scratch wounded cell monolayers NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16496-y a Microcystic LM Macrocystic LM Case 1 Case 4 lum * Case 2 Case 5 lum * lum Case 3 lum * b H&E VEGFR3 αSMA CD45 VEGFR3 lum Case 4 lum * * * * Macrocystic Case 2 lum Microcystic lum lum lum 0 μm 75 0 μm 75 Fig. 1 PIK3CA mutations underlie both micro- and macrocystic LM. a Hematoxylin and eosin stained sections of three microcystic (on the left) and two macrocystic (on the right) LMs. Note RBCs inside and outside of the malformations (asterisks), and lymphoid cell infiltration (yellow arrows). b On the left: hematoxylin and eosin stained sections of macrocystic and microcystic LM. On the right: immunofluorescence staining showing VEGFR3 expression in the endothelium lining both macrocystic and microcystic LM (arrows), except for areas associated with smooth muscle cells (arrowhead). Boxed area is shown in both hematoxylin and eosin staining and immunofluorescence staining. Note high expression of VEGFR3 in small-caliber lymphatic vessels in areas containing infiltrated CD45+ cells (asterisks). lum = vessel/cyst lumen. Images in (a, b) are representative of n = 3 microcystic LM and n = 2 macrocystic LM lesions. Scale bars: 200 µm (a, b). 4 NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16496-y ARTICLE a PIK3CAH1047R Early embryonic Early postnatal induction 4-OHT R26 STOP E11 E17 * P2-5 2-5 3-4 w Vegfr3 CreERT2 Analysis Analysis b LSL-PIK3CAH1047R ; Vegfr3-CreERT2 e LSL-PIK3CAH1047R ; Vegfr3-CreERT2 4-OHT: early embryonic macrocystic LM 4-OHT: early postnatal microcystic LM E17 back skin NRP2 P21 ear skin LYVE1 4-OHT (100 mg): P5 Cervical Analysis: P21 i Thoracic ii LYVE1 4-OHT (100 mg): P5 i PROX1 Analysis: P21 NRP2 PECAM1 PROX1 LYVE1 ii PROX1 4-OHT (5 mg): P2 Analysis: 4 w c d 4-OHT: early embryonic f 4-OHT: early postnatal Macrocystic lesions Ctrl Ctrl 6 Number of lesions 4 Vegfr3-CreERT2 PIK3CAH1047R ; Prox1-CreERT2 PIK3CAH1047R ; 2 0 E17 back skin 5 w ear skin Lu cic Sa r or l al Th ica ba cr a m v er VEGFR3 EMCN VEGFR3 EMCN C Fig. 2 Developmental timing of activation of PIK3CA determines LM subtype. a Genetic constructs and experimental plan for tamoxifen-inducible activation of PIK3CAH1047R expression in lymphatic endothelia at early embryonic (blue, 4-OHT 2 mg) or postnatal (gray, 4-OHT dose as indicated) development. Asterisk indicates the date of birth. b–d Characterization of PIK3CAH1047R-driven macrocystic LM. Whole mount immunofluorescence of back skin from an E17 PIK3CAH1047R;Vegfr3-CreERT2 embryo (b). Antibodies and regions of skin are indicated, and boxed areas (i, ii) are magnified below. Arrows point to cyst-like malformations in the neck (cervical) region of the skin that were quantified in (c). Data represent mean ± s.e.m. Immunofluorescence analysis of paraffin-embedded sections of lymphatic lesions showing overgrowth of lymphatic (VEGFR3+) but not blood vessels (EMCN+) (d). e, f Characterization of PIK3CAH1047R -driven microcystic LM. Immunofluorescence analysis of skin whole-mounts (e; Vegfr3-CreERT2 model) or paraffin sections (f; Prox1-CreERT2 model), and boxed areas (e) are magnified. 4-OHT dosage, timing of administration and analysis are indicated. Inset in (e) shows bleeding in the lesion area. Dotted lines in (d, f) indicate epidermis. Scale bars: 50 µm (d, f), 250 µm (b (i, ii), e), 2 mm (overview images in b, e). Source data are provided as a Source Data file. NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16496-y a b Vessel ends Acute microcystic LM 100 p=0.0003 4-OHT: short-term induction LYVE1 No of vessel ends 80 P10 3w 60 * LSL-PIK3CAH1047R; 40 Vegfr3-CreERT2 Analysis of initial steps 20 Blunt of microcystic LM formation Spiky 0 Ctrl PIK3CAH1047R (n=3) (n=6) c 3 w PIK3CAH1047R 3 w wild type E14 wild type d 3 w PIK3CAH1047R 3 w wild type pS6 VEGFR3 pS6 LYVE1 pS6 NRP2 LYVE1 VE-cad LYVE1 VE-cad pS6 pS6 pS6 VE-cad VE-cad High PI3K activity Straight zippers e Primary LEC: PIK3CAH1047R ; Vegfr3-CreERT2 f Primary LEC: PIK3CAH1047R ; Vegfr3-CreERT2 t=0h t = 48 h -4-OHT (Ctrl) Wound closure Relative wound density [%] +4-OHT (H1047R) -4-OHT (Ctrl) 100 80 60 +4-OHT (H1047R) 40 Ctrl 20 H1047R 0 0 10 20 30 40 50 60 70 Time (hours) Phalloidin PROX1 Phalloidin VE-cadherin VEGFR3 g h i Primary LEC: Primary LEC: PIK3CAH1047R ; Vegfr3-CreERT2 pAkt (Ser473) PIK3CAH1047R ; Vegfr3-CreERT2 Serum starvation 40 p=0.0025 Ctrl PIK3CAH1047R R R Relative pAkt levels VEGF-C - 15’ 30’ - 15’ 30’ p=0.0045 47 47 10 10 30 H H 4-OHT - - - + + + A A C C 70 kDa pAkt K3 K3 trl trl 20 PI PI C C 50 kDa Ser473 p=0.0005 4-OHT - + - + 70 kDa 70 kDa pAkt pAkt 50 kDa Thr308 10 50 kDa Ser473 70 kDa 70 kDa Akt Akt 0 50 kDa 50 kDa VEGF-C - 15’ 30’ Density High Low Ctrl PIK3CAH1047R showed enhanced wound closure of 4-OHT-treated LECs Together, these results demonstrate that p110α activation expressing the constitutive active p110α compared with vehicle- confers a migratory LEC phenotype and induces lymphatic vessel treated control LECs (Fig. 3f). No further increase in the speed of sprouting that leads to the hyperbranched network of vessels wound closure in the PIK3CAH1047R-expressing primary LECs defining LM lesions. was observed in the presence of the specific lymphangiogenic growth factor VEGF-C (Supplementary Fig. 5b), suggesting that the change in the cellular phenotype represents a cell- VEGF-C induces hyperactivation of Akt in PIK3CAH1047R- autonomous effect of oncogenic PI3K activity on the LEC. LECs. Next, we studied the effect of p110α activation on 6 NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16496-y ARTICLE Fig. 3 Migratory phenotype and VEGF-C-induced hyperactivation of Akt in PIK3CAH1047R -expressing LECs. a Experimental plan for short-term 4-OHT induction for the analysis of initiation of microcystic LM formation in PIK3CAH1047R;Vegfr3-CreERT2 mice. Asterisk indicates the date of birth. b Whole- mount immunofluorescence of ear skin of 4-OHT-treated (100 µg) P21 PIK3CAH1047R;Vegfr3-CreERT2 mice (on the left) and quantification of the morphology of vessel ends in control (n = 3) and PIK3CAH1047R (n = 6) mice (mean ± s.e.m., Two-tailed unpaired Student’s t-test) (on the right). Mutant shows increase in spiky vessel ends representing active sprouts (arrow) in comparison to blunt ends of normal capillaries (arrowhead). c Staining for phospho-S6 staining as a readout of PI3K activity, showing high signal in active lymphatic vessel sprouts (arrows) in 3-week-old mutant skin and embryonic back skin. No signal was detected in normal capillaries (arrowheads). d Staining for VE-cadherin showing straight zipper-like junctions, typical of lymphangiogenic vessels, in active lymphatic sprouts (arrows) in mutant skin as opposed to oakleaf shaped LECs with button junctions in normal capillaries (arrowhead). e Immunofluorescence of primary LECs isolated from PIK3CAH1047R;Vegfr3-CreERT2 mice, showing increase in stress fiber formation and disruption of cell-cell junctions in 4-OHT-treated (PIK3CAH1047R-expressing) compared with vehicle-treated (Ctrl) cells. Images are representative of three independent experiments. f IncuCyte scratch assay showing faster wound closure in 4-OHT-treated (PIK3CAH1047R-expressing) compared with vehicle- treated (Ctrl) primary LECs. Data is representative of two independent experiments showing mean relative wound density (n = 7 (Ctrl) or 5 (H1047R) wells ± s.d.). The difference between area under the curves: P < 0.0001 (Two-tailed unpaired Student’s t-test). Representative images of wells at t = 0 h and t = 48 h are shown on the right. Wound area is highlighted in light gray. g–i, Western blot analysis (g, h) and quantification of phospho-Akt (Ser473) (i) in primary dermal LECs from PIK3CAH1047R;Vegfr3-CreERT2 mice treated in vitro with 4-OHT and/or VEGF-C for indicated times. Data are representative of one experiment (g) or three independent experiments (h). Data in (i) represent mean (n = 3 independent experiments) ± s.e.m. Two-tailed one-sample t- test (untreated Ctrl vs. PIK3CA in (i)) or Two-tailed unpaired Student’s t-test (all others). Scale bars: 50 µm (b–e), 300 µm (f). Source data are provided as a Source Data file. downstream signaling in LECs. 4-OHT-induced expression of the in LM, which may critically contribute to driving pathological constitutive active p110α in primary LECs isolated from the vessel growth. PIK3CAH1047R;Vegfr3-CreERT2 mice led to increased Akt activa- Next, we analyzed the response of PIK3CA-driven vascular tion under basal growth conditions in both sparse and confluent malformations to inhibition of VEGF-C signaling in vivo. For this cells (Fig. 3g). To assess the response to growth factor activation, purpose, we used the model of progressive microcystic LM in serum starved LECs were stimulated with VEGF-C, the major postnatal mice (Supplementary Fig. 4e). To achieve continuous regulator of LEC migration31. As expected, serum starved 4-OHT inhibition of the VEGF-C pathway, we used adeno-associated treated PIK3CAH1047R -expressing LECs showed again increased vectors (AAVs) encoding the soluble VEGF-C-trap (AAV- Akt phosphorylation compared with vehicle-treated cells in the VEGFR3[1-4]-Ig36). Control mice were treated either with PBS absence of growth factor simulation (Fig. 3h, i). VEGF-C sti- or AAVs encoding the non-ligand binding region of the VEGFR3 mulation induced a comparable increase in Akt phosphorylation extracellular domain (AAV-VEGFR3[4-7]-Ig). Soluble VEGFR31- in control LECs. PIK3CAH1047R -expressing LECs however 4-Ig protein was detected in the serum 3 weeks after intraper- showed a further increase in Akt activity in response to VEGF-C itoneal AAV administration at a concentration of 121 ± 30 ng µl−1 (Fig. 3h, i). (n = 5) (Supplementary Fig. 7a). In agreement with previous Taken together, VEGF-C stimulation induces hyperactivation studies37, this was sufficient to block VEGF-C-induced lymphan- of Akt in PIK3CAH1047R -expressing LECs. This is in agreement giogenesis in the ear skin (Supplementary Fig. 7b). with previous studies in cancer cells showing that the H1047R Administration of AAVs to PIK3CAH1047R;Vegfr3-CreERT2 mutation residing in the kinase domain of p110α relies on mice one week after induction of lymphatic overgrowth upstream receptor tyrosine kinase (RTK) stimulation to reach a (Supplementary Fig. 4e, Fig. 4e) led to an efficient inhibition of critical threshold of membrane binding and activity needed for the growth of LM (Fig. 4f, g). As previously reported38, VEGF-C transformation32. inhibition did not affect mature vasculature in control mice (Fig. 4f). VEGF-C inhibition specifically affected the growth of PIK3CAH1047R-driven LM is dependent on VEGF-C signaling. lymphatic lesions, as it did not have an effect on the growth of To investigate the potential involvement of VEGF-C signaling in lesions within the blood vasculature in PIK3CAH1047R;Cdh5- LM pathology in vivo, we analyzed expression of the pathway CreERT2 mice that express the constitutive active p110α in all ECs components in mouse LM lesions in comparison to normal (Supplementary Fig. 8a–d). vasculature. The VEGF-C receptors VEGFR3 and NRP2, but not VEGFR2, were strongly upregulated in the abnormal sprouts in Co-inhibition of VEGF-C and mTOR promotes LM regression. the PIK3CAH1047R;Vegfr3-CreERT2 ears (Fig. 4a, b, Supplemen- Rapamycin (also known as sirolimus) is an allosteric inhibitor tary Fig. 6a). Increase in VEGFR3, which correlated with increase of the PI3K downstream target mTOR that has been used in the in lymphatic vessel branching, was also observed in a knock-in treatment of patients with vascular malformations including mouse model33, where Pik3caH1047R expression is driven by the both VM and LM3. To avoid body weight loss associated with endogenous promoter after Cre recombination (Supplementary treatment with a high dose of Rapamycin39, mice were admi- Fig. 6a–c), as well as in the localized lesions in the PIK3CAH1047R; nistered with daily intraperitoneal injections of 10 mg kg−1 of Prox1-CreERT2 mice (Supplementary Fig. 6d). We could not, Rapamycin during 5 consecutive days. As expected, Rapamycin however, detect a significant increase in VEGFR3 mRNA or inhibited the overgrowth of both blood and lymphatic vessels in protein levels in primary PIK3CAH1047R -expressing LECs (Sup- the PIK3CAH1047R;Cdh5-CreERT2 mice when administered at plementary Fig. 6e, f), suggesting that upregulation observed the time of 4-OHT-mediated induction of PIK3CAH1047R in vivo is not due to direct regulation by PI3K. As seen in human expression (Supplementary Fig. 9a–c). In addition, it sig- patients, we observed an accumulation of immune cells around nificantly reduced vessel growth and hyperbranching when advanced lesions in the PIK3CAH1047R;Prox1-CreERT2 mice administered one week after induction of vascular overgrowth (Fig. 4c). In agreement with this, analysis of ears in the model of (Supplementary Fig. 9d–f). progressive microcystic LM showed a modest increase in the To test the therapeutic effect on more advanced lesions, we number of macrophages (Fig. 4d), that are the major source of initiated Rapamycin treatment two weeks after induction of VEGF-C34,35. These results suggest increased VEGF-C signaling vascular overgrowth in the PIK3CAH1047R;Vegfr3-CreERT2 model NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16496-y a LSL-PIK3CAH1047R ; Vegfr3-CreERT2 Wild type b VEGFR3 staining intensity VEGFR3 VEGFR3 4 VEGFR3 LYVE1 p<0.0001 CTCF (fold change) 3 2 VEGFR2 VEGFR2 1 VEGFR2 LYVE1 0 Ctrl (n=4) LSL-PIK3CAH1047R (n=5) NRP2 NRP2 d Macrophages NRP2 LYVE1 CD11b+ F4/80+ [% of CD45+] p=0.0005 60 50 40 c PIK3CAH1047R ; Prox1-CreERT2 Ctrl 30 10 0 CD45 LYVE1 Ctrl (n=6) LSL-PIK3CAH1047R (n=7) e f LSL-PIK3CAH1047R ; Vegfr3-CreERT2 VEGFR3 Progressive microcystic LM Ctrl +4-OHT 4 weeks AAV-sR3 or 4-OHT vehicle 3w 4w 5w 6w 7w 8w H1047R LSL-PIK3CA ; Vegfr3-CreERT2 +AAV-sR3 Analysis +4-OHT +4-OHT + sR3 5 weeks g Lymphatic vessel branching p<0.0001 160 p<0.0001 140 p<0.0001 Branchpoints (mm–2) 120 +4-OHT +4-OHT + sR3 100 p=0.0089 6 weeks 80 60 40 20 n=6 n=10 n=6 n=8 n=12 n=12 n=13 n=4 n=10 0 +4-OHT +4-OHT + sR3 4OHT - + - + + + + + + 8 weeks AAV-sR3 - - + - + - + - + 4w 5w 6w 8w of progressive microcystic LM (Fig. 5a). Analysis of the c). However, combined treatment with the VEGF-C trap and vasculature after 1.5 weeks of Rapamycin treatment, administered Rapamycin showed an additive effect and promoted regression of every second day, showed only a modest inhibition of vascular the vasculature (Fig. 5b, c), and effectively blocked LEC growth during the treatment period, and no regression (Fig. 5b, proliferation (Fig. 5d). Remaining areas of hyperbranched c). Treatment with the VEGF-C trap also inhibited the growth but vasculature in mice treated with both the VEGF-C trap and did not promote regression of the lymphatic vasculature (Fig. 5b, Rapamycin frequently showed blunted morphology of vessel 8 NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16496-y ARTICLE Fig. 4 PIK3CAH1047R-driven LM is dependent on VEGF-C signaling. a Whole-mount immunofluorescence of ear skin of 5 weeks old PIK3CAH1047R;Vegfr3- CreERT2 (left panels) mice showing upregulation of VEGFR3 and NRP2 expression but unchanged VEGFR2 levels in abnormal sprouts (arrows) in comparison to capillaries with normal appearance (arrowheads), and those in wild-type mice (right panels). 4-OHT (100 µg) was administered at P21. b Quantification of VEGFR3 staining intensity in the dermal lymphatic vasculature in the ear skin of 4-OHT-treated PIK3CAH1047R;Vegfr3-CreERT2 mice (n = 5), compared with littermate controls (n = 4) ± s.d. c Immunofluorescence staining of paraffin sections of ear skin showing increase in CD45+ immune cells around LYVE1+ lymphatic lesions in a PIK3CAH1047R;Prox1-CreERT2 mouse compared with a control (Ctrl). d Percentage of CD45+CD11b+F4/80+ macrophages relative to all CD45+ cells in the ear skin of 5 weeks old PIK3CAH1047R;Vegfr3-CreERT2 mice treated with the vehicle (n = 6) or 4-OHT (100 µg, PIK3CAH1047R; n = 7) at P21 (mean ± s.d.). e Experimental plan for the induction of progressive microcystic LM and inhibition using the soluble VEGF-C trap (AAV-VEGFR3-Ig; AAV-sR3) or vehicle (PBS). f Whole-mount staining of ears from untreated PIK3CAH1047R;Vegfr3-CreERT2 mice (Ctrl), and mice treated with 4-OHT (100 µg) and AAV-sR3 or vehicle, and analyzed at different stages after induction. Images in red frame show immunofluorescence of ears from AAV-sR3 treated mice. g Quantification of lymphatic vessel branching in the progressive LM model. Red squares represent data from AAV-sR3 treated mice. Data represent mean (n = number of ears as indicated) ± s.d. Two-tailed unpaired Student’s t-test. Scale bars: 50 µm (a), 200 µm (c, f). Source data are provided as a Source Data file. sprouts, compared with spiky appearance characteristic of active generalized, rather than localized response characterized by sprouting process in vehicle-treated mice (Fig. 5e, f). progressive lymphatic vascular hyperplasia. This is in agreement with a previous study modeling generalized lymphatic anomaly Discussion (GLA) in mice upon LEC-specific induction of PIK3CAH1047R at LM are vascular anomalies presenting with large fluid-filled cysts 4 weeks of age16. The exquisite sensitivity of quiescent lymphatic (macrocystic) or lesions that infiltrate tissues (microcystic) and endothelium to p110α activation may be related to the require- cause morbidity, pain and organ dysfunction. Using a mouse ment of inhibition of the pathway for the maintenance of endo- model of LM driven by an activating PIK3CA mutation, we here thelial quiescence48. This finding raises the question of why characterize molecular and cellular mechanisms of lymphatic PIK3CA-driven lymphatic vascular overgrowth is not a frequent lesion formation and therapy response. Notably, we show that the manifestation in adult vasculature. It is possible that a critical progressive growth of PIK3CAH1047R-driven microcystic LM is threshold of mutant cells needed to cause vascular overgrowth is dependent on the upstream lymphangiogenic VEGF-C/ efficiently induced in our experimental model, but is not achieved VEGFR3 signaling, which permits a novel effective therapeutic in quiescent endothelium hit by a single somatic mutation. It is intervention to treat LM. also possible that endothelial cells that acquire PIK3CA mutation Here we focused on the common activating PIK3CA mutation require a permissive local environment to engage in signaling H1047R causative of both VM and LM5–7, but also cancer and with normal neighbors that allow them to collectively drive overgrowth syndromes9. We found that the H1047R mutation is pathological vascular growth. Clonal co-operation in vascular causative of both macrocystic and microcystic LM in a LEC- malformations, a concept that is well-established in cancer49, autonomous manner, with the developmental timing of PIK3CA may be supported by the observation that only a small proportion activation determining the LM subtype. In mouse skin, embryo- of endothelial cells forming CCM lesions in human carry a nic LEC-specific activation of PIK3CA led to macrocystic lesions mutation50,51. In addition, in a mouse model of CCM, initial characterized by large cysts localized predominantly to the neck clonal expansion of Ccm3 deficient cells was followed by incor- region of the skin, similarly as in human LM patients. In contrast, poration and phenotypic change of normal cells driving further late embryonic or early postnatal activation caused microcystic growth of the lesion52,53. Of interest, heterogeneity between malformations with hyperbranched lymphatic lesions. Why neighboring LECs in the level of VEGFR3, which is regulated by activation of the same signaling pathway can cause different LEC PI3K/mTOR signaling (our study and39,54), was shown to non- responses leading to morphologically different types of mal- cell-autonomously drive pathological vessel hyperplasia through formations remains to be studied. It is possible that the cellular cell contact-dependent regulation of Notch signaling55. Long- response may differ depending on the developmental origin and itudinal magnetic resonance imaging of the LM lesions could differentiation status40 (progenitor vs. lining a lumenized vessel) provide important insights into the mechanisms of LM growth. as well as the cellular state (proliferating vs. quiescent). Differ- Macrocystic LM can be generally effectively treated with ences in the tissue environment may also play a role. For example, sclerotherapy or surgical resection. The treatment of microcystic tissue stiffness and composition of the immune cells that are both LM is more challenging due to the infiltrative growth of the known to regulate LEC responses and lymphangiogenesis34,41,42 lesions. Ongoing clinical trials assessing the efficacy of Sirolimus differ between embryonic and postnatal skin. (mTOR inhibitor Rapamycin) for the treatment of PIK3CA-dri- The development of several types of vascular malformations, ven vascular malformations have shown promising results. Sir- including cerebral cavernous malformations (CCM) and arter- olimus efficiently stops the growth of vascular malformations in iovenous malformations (AVM) require active angiogenesis. The mice and human, and improves the patients’ quality of life3,5,15– 19. However, regression of the lesion is achieved only in rare formation of these vascular lesions can be efficiently induced experimentally in mice during developmental vascular growth, cases3. It is also currently unclear if patients need life-long but require reactivation of quiescent endothelium at adult treatment, which is associated with a risk of side effects of the stages43–47. The congenital nature of LM in human suggests that drug. Consistent with the clinical data and previous studies on a lymphatic endothelium is similarly sensitive to activation of mouse model of PIK3CA-driven generalized lymphatic anom- p110α signaling during the active growth of the vasculature, yet aly16, we found that Rapamycin inhibited lymphatic vascular lesions may also appear later in life and show progressive growth. growth in the experimental model of PIK3CAH1047R-driven Using the mouse model of PIK3CAH1047R-driven LM, we found microcystic LM. The treatment resulted in an efficient inhibition that, as expected, both embryonic and early postnatal lymphatic of the initial growth and expansion of early lesions, but only a vasculature is responsive to p110α activation, leading to forma- modest inhibition, and no regression, could be observed in more tion of localized vascular lesions. However, LEC-specific activa- advanced lesions. Interestingly, in a mouse model of VEGF-C tion of PIK3CAH1047R expression at 3 weeks of age led to a induced pulmonary lymphangiectasia, which persisted even after NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications 9 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16496-y a d LEC proliferation Progressive microcystic LM p<0.0001 20 4-OHT or AAV-sR3 or vehicle AAV-CTRL 15 p=0.0124 % Ki67+ 3w 4w 5w 6w 7w 10 LSL-PIK3CAH1047R ; Analysis Analysis Vegfr3-CreERT2 5 n=4 Ctrl Rapamycin(10 mg/kg) n=4 or vehicle (DMSO) n=5 n=4 n=5 0 i.p. every 2nd day 4-OHT - + + + + b LSL-PIK3CA H1047R ; Vegfr3-CreERT2 Rapamycin - - + - + LYVE1 sR3 - - - + + Ctrl +4-OHT 5w 6.5 w 5 weeks e DMSO + AAV-CTRL Rapa + AAV-sR3 AAV-CTRL + DMSO AAV-sR3 + DMSO LYVE1 LYVE1 6.5 weeks +4-OHT 6.5 w +4-OHT f Vessel ends AAV-CTRL + Rapa AAV-sR3 + Rapa 300 Spiky Blunt No of vessel ends 200 100 c Lymphatic vessel branching 0 p=0.0080 p=0.0169 Spiky ends 200 p<0.0001 p<0.0001 250 Branchpoints (mm-2) 150 200 No of vessel ends 150 100 100 50 50 n=7 n=8 n=14 n=13 n=14 n=17 n=17 n=14 n=14 n=17 n=17 0 0 4-OHT - + + + + + 4-OHT - + + + + Rapamycin - - - + - + Rapamycin - - + - + sR3 - - - - + + sR3 - - - + + 5w 6.5 w 5w 6.5 w normalization of VEGF-C levels, Rapamycin treatment effectively required for induction of lymphatic vessel regression in children promoted regression of abnormal lymphatic vessels without and young adolescents. Interestingly, Alpelisib (BYL719), a spe- affecting normal vessels39. However, a significant reversal of cific PIK3CA inhibitor, has shown a higher efficacy than Rapa- lymphangiectasia required a high dose (20 mg kg−1 daily) of mycin in the treatment of PIK3CA-related overgrowth syndromes Rapamycin that was associated with body weight loss39. Although (PROS) in mice and humans56. Alpelisib improved organ compatible with survival of adult mice39, high dose of Rapamycin abnormalities and dysfunction, also in the vasculature, with could not be tolerated during neonatal period of growth. It may minimal side effects. Future studies should address the efficacy of thus not be feasible to use a high enough dose of Rapamycin Alpelisib in patients with LM. 10 NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16496-y ARTICLE Fig. 5 Co-inhibition of the upstream VEGF-C/VEGFR3 and the downstream mTOR signaling pathways promotes LM regression. a Experimental plan for the induction and treatment of progressive microcystic LM using a combination therapy with VEGF-C trap and Rapamycin. b Whole-mount staining of ears from PIK3CAH1047R;Vegfr3-CreERT2 control (Ctrl) and 4-OHT-treated (100 µg) mice at 5 weeks of age, or following a 1.5-week treatment with Rapamycin, AAV-sR3 and/or vehicles at 6.5 weeks of age. c Quantification of lymphatic vessel branching in the PIK3CAH1047R;Vegfr3-CreERT2 mice. Stages of analysis and treatments are indicated. Data represent mean (n = number of ears as indicated) ± s.e.m. d Flow cytometry analysis of dermal LEC proliferation in the PIK3CAH1047R;Vegfr3-CreERT2 mice treated as indicated. Data represent mean (n = number of mice as indicated) ± s.d. e Lymphatic vessel morphology in areas of hypersprouting in 6.5-week-old 4-OHT-treated PIK3CAH1047R;Vegfr3-CreERT2 ears. Note blunt morphology of lymphatic sprouts in mice treated with Rapamycin and AAV-sR3 compared with vehicle-treated mice (yellow arrows). f Quantification of all vessel ends and their morphology (upper graph), with data shown separately for spiky ends representing active sprouts (lower graph), in the indicated groups. Data represent mean (n = number of mice as indicated ± s.e.m.) Two-tailed unpaired Student’s t-test. Scale bars: 200 µm (b), 100 µm (e). Source data are provided as a Source Data file. In addition to the cell-autonomous migration-promoting effect exons of PIK3CA. Genetic variants were analyzed using Highlander software of p110α on LECs observed in vitro, lymphangiogenic VEGF-C/ (http://sites.uclouvain.be/highlander/). Histology was examined by a pathologist expert in vascular anomalies. VEGFR3 signaling in the LM lesion was found to significantly contribute to the further vascular growth of the lesions in vivo. Strong upregulation of the VEGF-C receptors VEGFR3 and Mouse lines and treatments. R26-LSL-PIK3CAH1047R 22, knock-in LSL- Pik3caH1047R 33, Cdh5-CreERT2 59, Vegfr3-CreERT2 23, Prox1-CreERT2 24, and R26- NRP2 was observed in the lymphangiogenic sprouts, consistent mTmG60 mice were analyzed on a C57BL/6J background. Both female and male mice with previous observations in LM patients57,58. In addition, we were used for analyses and no differences in the phenotype between the genders were observed increased infiltration of immune cells, including mac- observed. The morning of vaginal plug detection was considered as embryonic day 0 rophages, which are an important source of pro-lymphangiogenic (E0). For inducing recombination during embryonic development 4-hydroxytamo xifen (4-OHT, H7904, Sigma) dissolved in peanut oil (10 mg ml−1) was administered growth factors such as VEGF-C34,35. Although LECs expressing intraperitoneally to pregnant females. For postnatal induction, 4-OHT dissolved in the oncogenic p110α showed increased Akt activity already under acetone (10 mg ml−1), was applied topically to the dorsal side of the right ear of each basal conditions, VEGF-C stimulation led to hyperactivation of mouse. The 4-OHT doses and days of administration are indicated in the figures and/ the pathway. These findings suggest a positive feedback reinfor- or figure legends and summarized in Supplementary Table 1. Additional information cing the upstream pathway that subsequently promotes abnor- on the routes of administration and the effect are provided in Supplementary Table 1. For inhibition of VEGF-C signaling, we used adeno-associated viral (AAV)-derived mally high activity of the downstream signaling, making the VEGF-C trap encoding the ligand binding domains 1–4 of VEGFR3, fused to the IgG pathway a potential target for therapeutic intervention. Indeed, Fc domain (AAV9-mVEGFR31–4-Ig) and control AAVs that encoded the inactive we found that inhibition of VEGF-C/VEGFR3 signaling using the domains four to seven of VEGFR3-Ig (AAV9-mVEGFR34-7-Ig)36. Mice received a soluble VEGF-C trap inhibited LM lesion growth in vivo. Nota- single intraperitoneal injection of 1 × 1010 virus particles diluted in PBS at a total volume of 100 µl at the indicated time point. PBS or AAV9-mVEGFR34-7-Ig were bly, treatment with the soluble VEGF-C trap was more effective used as control treatments. Rapamycin (R-5000 Rapamycin, >99% LC Labs) was than Rapamycin in inhibiting LM growth, and when adminis- dissolved in DMSO at 10 mg ml−1 and injected intraperitoneally either once per day tered in combination with Rapamycin it was able to promote (Supplementary Fig. 9) or every second day (Fig. 5) at the dose of 10 mg kg−1. DMSO lymphatic vessel regression. Our results suggest that, like in was used as a control. A single dose (2.5 × 1010 viral particles per ear in 10 µl) of AAVs encoding VEGF-C61,62 was injected intradermally into both ears of 5 weeks old cancer, optimal therapeutic benefit may require targeting both the adult mice. All experimental procedures were approved by the Uppsala Animal endothelial cell-autonomous PIK3CA-driven signaling and the Experiment Ethics Board and performed in compliance with all relevant Swedish microenvironment-derived paracrine signaling that contribute to regulations, or the Catalan Departament d’ Agricultura, Ramaderia i Pesca, following driving pathological vessel growth. The relative contributions of protocols approved by the local Ethics Committees of IDIBELL-CEEA. the two mechanisms may be different at different stages of lesion formation, which can affect response to therapy. It is possible that Antibodies. The details of primary antibodies used for immunofluorescence of the beneficial effect of Rapamycin is partly due to its immuno- whole mount tissues, cells and paraffin-embedded sections are provided in Sup- plementary Table 2. Secondary antibodies conjugated to Cy3, Alexa Fluor 405, 488 suppressive functions, although this should be addressed in future or 647 were obtained from Jackson ImmunoResearch. studies. In conclusion, we show that the progressive growth of PIK3- Immunofluorescence. Whole-mount tissue was fixed in 4% paraformaldehyde at CAH1047R-driven microcystic LM is dependent on the upstream room temperature for 2 h followed by permeabilization in 0.3% Triton X-100 in lymphangiogenic VEGF-C/VEGFR3 signaling. Combined inhi- PBS (PBST) for 10 min and blocking in PBST plus 3% milk for 2 h. Primary bition of VEGF-C signaling using the soluble VEGF-C trap and antibodies were incubated at 4 °C overnight in blocking buffer and washed in PBST the PI3K downstream target mTOR using Rapamycin, but neither before incubating them with fluorescence-conjugated secondary antibodies in blocking buffer for 2 h at room temperature. Stained samples were then washed treatment alone, promotes the regression of experimental LM in and mounted in Mowiol. Cells were fixed with 4% paraformaldehyde at room mice. These results suggest that the best therapeutic outcome for temperature for 20 min, washed twice with PBS and permeabilized in 0.5% Triton PIK3CAH1047R-driven microcystic LM is achieved by co- X-100 in PBS for 5 min, and blocked in PBST plus 2% BSA for 1 h. Cells were inhibition of the VEGF-C/VEGFR3 and the downstream incubated with primary antibodies diluted in the blocking buffer at room tem- perature for 1 h. After washing three times with PBS plus 0.2% BSA, the cells were (mTOR) pathways. Therapies targeting the key upstream path- incubated with fluorescence-conjugated secondary antibodies at room temperature way in combination with PI3K pathway inhibition may be rele- for 45 min, followed by staining with DAPI (D9542, Sigma, 1:1000) for 5 min, and vant also for other PIK3CA-driven pathologies. fixing with 4% paraformaldehyde for 4 min before further washing and mounting in Mowiol. Methods Immunostaining of paraffin sections. Tissue was fixed in 10% neutral buffered Patients. Residual tissue of macrocystic and microcystic LMs were collected from formalin (human) or 4% paraformaldehyde (mouse) and embedded in paraffin. patients undergoing therapeutical surgery. All participants gave their informed Deparaffinization and rehydration steps were performed manually on 4 μm consent, as approved by the ethical committee of the Medical Faculty of the (human) or 5–6 μm (mouse) paraffin sections. Heat-induced antigen retrieval was University of Louvain, Brussels, Belgium. All protocols were compliant with the performed using Sodium Citrate buffer (10 mM, pH 6) at 95 °C. Immunohis- Belgian laws governing research on human subjects. The phenotypes of the patients tochemistry with antibodies against PROX1 and PDPN (Supplementary Table 1) were evaluated as accurately as possible by the referring clinicians. DNAs were were performed on an automatic immunostainer, Ventana Benchmark Ultra. For extracted from crunched (powderized) snap-frozen tissues, using Wizard genomic immunofluorescence, paraffin sections were blocked with TNB (Tris-NaCl-block- DNA purification kit (Promega). DNAs were screened by Ion Torrent technology ing buffer, prepared according to the Tyramide Signal Amplification kit (TSATM, with a custom Ampliseq panel (www.ampliseq.com) designed to cover the coding NENTM Life Science Products) and washed with TNT buffer (0.1 M Tris pH 7.5, NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-16496-y 0.15 M NaCl, 0.05% Tween 20). The TSATM kit was used to enhance and detect the Flow cytometry. For FACS analysis of proliferating cells ear skin of adult mice signal of goat anti-mouse VEGFR3 (AF743, R&D Systems) and rat anti-mouse were dissected, cut into small pieces and digested in Collagenase IV (Life Tech- CD45 antibody (ab25386, Abcam) in the mouse paraffin sections and mouse anti- nologies) 10 mg ml−1, DNase I (Roche) 0.1 mg ml−1 and FBS 0.5 % (Life Tech- human VEGFR3 (clone 9D9F9, Millipore) antibody in the human paraffin sections. nologies) in PBS at 37 °C for about 30 min with vigorous shaking every 5 min. All other primary antibodies (see Supplementary Table 1) were used at 4 °C Collagenase activity was quenched by dilution with FACS buffer (PBS, 0.5% FBS, 2 overnight and visualized with fluorescence-conjugated secondary antibodies after mM EDTA) and digestion products were filtered twice through 70 µm nylon filters the TSA kit. The majority of the slides were mounted in Mowiol except the ones (BD Biosciences). Cells were again washed with FACS buffer and immediately with CD45 primary antibody, where Autofluorescence Quenching kit (Vector® processed for immunostaining first by blocking Fc receptor binding with rat anti- TrueVIEWTM) together with VECTASHIELD® HardSetTM Antifade Mounting mouse CD16/CD32 (eBiosciences) followed by incubation with antibodies target- Media was used to decrease autofluorescence. ing PDPN, CD31/PECAM1, CD45 and CD11b (all obtained from eBioscience, see Supplementary Table 2). Two different panels of antibodies were used depending on if samples were taken from mice with R26-mTmG reporter or not. After Primary LEC culture. Murine primary dermal LECs were isolated from the tail staining, cells were washed with PBS and then stained for dead cells using the blue skin of 6-13 weeks old LSL-PIK3CAH1047R;Vegfr3-CreERT2 mice by sequential LIVE/DEAD® fixable dead cell stain kit (Life Technologies), followed by fixation selection with PECAM1 (Mec13.3, Pharmingen) and LYVE1 (Aly7, Abnova) and permeabilization using the Foxp3/Transcription factor staining kit antibodies bound to Dynabeads, as described previously23. Mouse LECs were (eBioscience) according to the manufacturer’s instructions. Finally cells were cultured on 0.5% gelatin (G1393, Sigma)-coated dishes in complete DMEM incubated with rat serum and KI67 antibody (eBioscience). Cells were analyzed on medium. To induce Cre-mediated recombination, 4-OHT was dissolved in abso- a BD LSR Fortessa cell analyzer equipped with 5 lasers (355, 405, 488, 561, and 643 lute ethanol and added to the culture medium at the final concentration of 5 µM for nm). Compensation was performed using the anti-rat/hamster compensation bead 48 h. Cell were then reseeded in 12-wells at (low) density of 3 × 104 cells per kit and the ArC amine reactive compensation bead kit (Life Technologies). Single cm2 for western blot analysis and qRT-PCR analysis. For serum starvation, cells viable cells were gated from FSC-A/SSC-A, FSC-H/FSC-W and SSC-H/SSC-W were reseeded after 4-OHT treatment at the low density and starved overnight in plots followed by exclusion of dead cells in the UV dump channel. FMO controls 0.5% fetal bovine serum (FBS) containing medium and stimulated with VEGF-C were used to set up the subsequent gating scheme to obtain cell populations and (R&D Systems, 200 ng ml−1) for time periods indicated in the figures. To assess cell quantification of proliferating cells (Supplementary Fig. 10). migration in vitro, wound-healing assays were performed with IncuCyte ZOOMTM For FACS analysis of immune cells the ear skin was dissected into dorsal and according to the manufacturer’s instructions. Cells were reseeded in 96-well Ima- ventral parts, cut into pieces and digested in Liberase TL (Roche) 100 µg ml−1, geLock plate (Essen BioScience) at density of 6.25 × 104 cells per cm2 after 4-OHT DNase I (Roche) 0.5 mg ml−1 in PBS with 0.2% FBS at 37 °C for 1.5 h at 500 r.p. treatment. WoundMakerTM (Essen BioScience) was used to create homogeneous m. Liberase TL activity was quenched by adding 2 mM EDTA and the product scratch wounds, and the cells were then washed twice with fresh medium to was filtered through 50 µm filters (CellTricks). The cells were washed with FACS remove any debris, followed by adding 100 µl fresh complete DMEM medium buffer (PBS, 0.5% FBS, 2 mM EDTA) and incubated first with rat anti-mouse supplemented with or without VEGF-C (25 ng ml−1) to each well. The plate was CD16/32 antibody (eBioscience) for blocking of the Fc receptor binding and placed into the IncuCyte ZOOMTM apparatus and images of cells were acquired then with CD45, CD11b and F4/80 targeting antibodies (Supplementary automatically every 1 h for a total duration of 72 h. All cells were cultured at 37 °C Table 2). Cell death was analyzed by adding SYTOX Blue dead stain (Life in a humidified atmosphere with 5% CO2. Technologies). The cell suspension was filtered again immediately before analysis. The analysis was obtained on CytoFLEX Flow Cytometer (Beckman Coulter). Single cells were gated from SSC-H/FSC-H and FSC-Width/FSC-H Western blot analysis. Total protein extract was obtained by lysing cells in lysis plots followed by gating for CD45+ viable cells and CD11b+F4/80+ cells buffer [150 mM NaCl, 1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 50 (Supplementary Fig. 9). All flow data was processed using FlowJo software mM Tris-HCl (pH 7.6) supplemented with 25 mM NaF, 1 mM Na3VO4 and version 10.5.0 (TreeStar). protein inhibitor cocktail (11873580001, Roche)]. Cell lysates were spinned for 10 min at 13200 r.p.m. at 4 °C and supernatants were collected. Protein concentration was determined using a BCA™ Protein Assay Kit (23227, Thermo Fisher Scientific) according to manufacturer’s instructions and measured using Gen5 All-In-One Image acquisition and quantification. All confocal images represent maximum microplate reader. Equal amounts of proteins were loaded and separated in 4-20% intensity projections of Z-stacks of single tiles or multiple tile scan images. Images gradient polyacrylamide gels (Invitrogen), transferred to a polyvinylidene were acquired using Leica SP8 confocal microscope or Leica DMi8 fluorescence difluoride membrane (PVDF; 88520, Thermo Fisher Scientific) and blocked for 1 h microscope with Leica LAS X software. Quantification of lymphatic vessel at room temperature in 1× tris buffered saline tween (TBST) [150 mM NaCl, branching was done with Angiotool plugin of ImageJ (version 2.0.0-rc-65/1.5lu) 10 mM Tris-HCl (pH 7.4) and 0.1% Tween] containing 5% (w/v) bovine serum using tile scan images of adult ear skin (xy = 3260 μm × 2200 μm, tip region) or albumin (BSA). The membranes were subjected to overnight incubation at 4 °C dorsal skin of E17 embryos (xy = 2322 μm × 4644 μm, cervical/thoracic region). with primary antibodies diluted in 1× TBST plus 5% BSA. Following this incu- Quantification of VEGFR3 staining intensity was done by measuring corrected bation, membranes were rinsed three times with 1× TBST for 5 min each and total cell fluorescence (CTCF) = integrated density – (area of selected cell × mean incubated for 1 h at room temperature with HRP-conjugated secondary antibodies fluorescence of background readings) using ImageJ, and the vessel area was (diluted in 1× TBST plus 5% BSA). Membranes were rinsed three times with TBST determined by combined staining of LYVE1 and VEGFR3. Vessel ends with blunt for 5 min each and specific binding was detected using the enhanced chemilumi- or spiky morphology were marked with Photoshop CS6 software and counted nescence (ECL) system (WP20005, Invitrogen) and the ChemiDoc XRS + System manually using tile scan images of adult ear skin (xy = 3260 μm × 2200 μm at the (Biorad). All images were collected using Image Lab software. Protein molecular tip of the ear excluding the region 34 μm from the edge of the ear) and presented as masses were estimated relatively to the electrophoretic mobility of co-transferred number of ends per ear. All quantifications include littermate controls; for Cdh5- prestained protein marker (26634, Thermo Fisher Scientific). The following anti- CreERT2 model Cre- littermates treated with 4-OHT were used (Supplementary bodies were used: rabbit anti-mouse phospho-Akt (Ser473) (#4060), phospho-Akt Figs. 8, 9), Vegfr3-CreERT2 model Cre+ untreated (Figs. 4, 5) or vehicle-treated (Thr308) (#2965), Akt (#9272), VEGFR2 (#2479), GAPDH (#2118, all from CST (Figs. 4, 5) mice were used. At least two different litters of mice were analyzed. For and 1:2000), goat anti-mouse VEGFR3 (AF743, R&D Systems, 1:1000). validation of the purity of isolated primary LEC populations, % of VE-cadherin+ VEGFR31–4-Ig and VEGFR34–7-Ig proteins in serum were detected by western cells and % of PROX1+ cells were quantified using maximum intensity projection blotting of 0.5 µl serum samples collected at the indicated time points as images based on staining of DAPI, VE-cadherin and PROX1 (n = 1386 cells in described63. 50, 100, and 200 ng of recombinant mouse VEGFR3 chimera protein total from 15 experimental conditions and 2 batches of cells). The cells were (743-R3-100, R&D Systems) were used as a standard for calculating the marked using Photoshop CS6 software and counted manually. concentration of VEGFR31–4-Ig and VEGFR34–7-Ig proteins. Mouse VEGFR3 The images acquired by IncuCyte ZOOMTM were processed and analyzed using domains 1–4 and 4–7 were detected with polyclonal goat anti–mouse VEGFR3 IncuCyteTM scratch wound cell migration software module. Cell density in the antibody (AF743, R&D Systems, 1:1000) against the extracellular domain of wound area expressed relative to the cell density outside of the wound area over VEGFR3. The blots were quantified using ImageJ software. Full blots are shown in time (relative wound density%) was quantified. The quantification of images from source data. western blots was done using ImageJ software. qRT-PCR analysis. Total RNA was isolated from dermal mouse LECs using RNeasy Mini kit (Qiagen). Complementary DNA (cDNA) was synthesized using Statistics and reproducibility. Graphpad Prism was used for graphic repre- Superscript VILO Master Mix (Invitrogen). cDNA was analyzed by real-time sentation and statistical analysis of the data. Data between two groups were quantitative PCR (qRT-PCR) (StepOne Plus system, Applied Biosystems) using compared with One-sample t-test (Fig. 3i), or paired (Supplementary Fig. 4d) or TaqMan gene expression assays and TaqMan gene expression Master Mix (Applied unpaired (all others) two-tailed Student’s t-test, assuming equal variance. Differ- Biosystems). Relative quantifications of gene expression were performed using the ences were considered statistically significant when P < 0.05. The experiments were comparative cycle threshold method (ΔCT) with Gapdh as the reference gene. not randomized, and no blinding was done in the analysis and quantifications. No TaqMan Assays used for mouse LECs were as followed: Vegfr2 statistical methods were used to predetermine the sample size. All microscopy (Mm01222421_m1), Vegfr3 (Mm01292604_m1), Gapdh (Mm99999915_g1). 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Cancer Res. 73, 4212–4221 (2013). 62. Antila, S. et al. Development and plasticity of meningeal lymphatic vessels. J. Correspondence and requests for materials should be addressed to T.M. Exp. Med. 214, 3645–3667 (2017). 63. Karaman, S., Nurmi, H., Antila, S. & Alitalo, K. Stimulation and inhibition of Peer review information Nature Communications thanks the anonymous reviewer(s) for lymphangiogenesis via adeno-associated viral gene delivery. Methods Mol. their contribution to the peer review of this work. Peer reviewer reports are available. Biol. Clifton NJ 1846, 291–300 (2018). Reprints and permission information is available at http://www.nature.com/reprints Acknowledgements Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in We thank Sagrario Ortega (CNIO, Madrid) for the Vegfr3-CreERT2 mice and Ralf Adams published maps and institutional affiliations. (Max Planck Institute for Molecular Biomedicine, Münster) for the Cdh5-CreERT2 mice. We also thank the BioVis facility (Uppsala University, Sweden) for flow cytometer usage and support, and Simon Stritt for help with flow cytometry and Sofie Lunell-Sergerqvist Open Access This article is licensed under a Creative Commons for technical assistance. This work was supported by the Swedish Cancer Society (CAN Attribution 4.0 International License, which permits use, sharing, 2016/535), the European Research Council (ERC-2014-CoG-646849), Knut and Alice adaptation, distribution and reproduction in any medium or format, as long as you give Wallenberg Foundation (2015.0030) and the Swedish Research Council (542-2014-3535) appropriate credit to the original author(s) and the source, provide a link to the Creative to T.M.; the F.R.S.-FNRS (Fonds de la Recherche Scientifique, Belgium), grant Commons license, and indicate if changes were made. The images or other third party [T.0026.14] to M.V. and [T.0146.16] to L.B., and the Walloon Excellence in Lifesciences material in this article are included in the article’s Creative Commons license, unless & BIOtechnology (FNRS-WELBIO) grant [WELBIO-CR-2010-15R] and the Fund indicated otherwise in a credit line to the material. If material is not included in the Generet managed by the King Baudouin Foundation to M.V.; and Academy of Finland article’s Creative Commons license and your intended use is not permitted by statutory (grants 314498, 320249) to K.A. Laboratories of M.V. and T.M. are part of V.A.Cure, and regulation or exceeds the permitted use, you will need to obtain permission directly from thus received funding from the European Union’s Horizon 2020 research and innovation the copyright holder. To view a copy of this license, visit http://creativecommons.org/ programme under the Marie Skłodowska-Curie grant agreement No 814316. P.B. is a licenses/by/4.0/. Senior Platform Manager of the University of Louvain. MG lab is supported by Minis- terio de Ciencia, Innovación y Universidades, which is part of Agencia Estatal de © The Author(s) 2020 14 NATURE COMMUNICATIONS | (2020)11:2869 | https://doi.org/10.1038/s41467-020-16496-y | www.nature.com/naturecommunications