Dermal Lymphatic Vessels Research Articles

A neuro-lymphatic communication guides lymphatic development by CXCL12/CXCR4 signaling.

Lymphatic vessels grow through active sprouting and mature into a vascular complex including lymphatic capillaries and collecting vessels that ensure fluid transport. However, the signaling cues that direct lymphatic sprouting and patterning remains unclear. In this study, we demonstrated the chemokine signaling, specifically through CXCL12/CXCR4 plays critical roles in regulating lymphatic development. We showed that LEC specific CXCR4 deficient embryos and CXCL12 mutant embryos exhibited server defects in lymphatic sprouting, migration, and lymphatic valve formation. We also discovered that CXCL12, originating from peripheral nerves, directs the migration of dermal lymphatic vessels to align with nerves in developing skin. Deletion CXCR4 or blockage of CXCL12/CXCR4 activity results in reduced VEGFR3 levels on the LEC surface. This, in turn, impairs VEGFC mediated VEGFR3 signaling and downstream PI3K/AKT activities. Taken together, these data identify previously unknown chemokine signaling originating from peripheral nerves that guides dermal lymphatic sprouting and patterning. Our work identifies for the first time a neuro-lymphatics communications during mouse development and reveals a novel mechanism by which CXCR4 modulates VEGFC/VEGFR3/AKT signaling.

Abstract PO5-27-02: A Tumor Emboli Platform for Morphometric Analysis, Drug screening and for Intravital Optical Imaging in a Murine Model to Simulate Clinicopathological Features of Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is distinguished by the presence of tumor emboli, clusters of tumor cells, found within breast parenchyma and dermal lymphatic vessels. These emboli serve as a crucial clinicopathological hallmark, believed to underlie the characteristic diffuse growth pattern and aggressive progression observed in IBC patients, with nearly 30% of patients presenting with metastatic disease at diagnosis. However, the rarity of this cancer and the absence of a solid tumor mass pose challenges in obtaining biospecimens, particularly those with evidence of tumor emboli. To address this pressing need for preclinical models that replicate this distinctive tumor growth pattern, we describe a tumor emboli culture platform that facilitates live imaging, enables compound screening by assessing various phenotypes of the emboli, and permits evaluation of emboli in an in vivo murine model. Methods: The tumor emboli culture system was evaluated for physiological measurements corresponding to dermal lymphatic vessels and subsequently employed to generate tumor emboli from diverse patient-derived cell lines and PDX. Confocal microscopy was employed to characterize cellular information across multiple slices of the tumor emboli. This provided a comprehensive, multi-parametric analysis of single-cell and spheroid phenotypes, including measurements of number, size, shape, and viability of the organoids. Furthermore, we developed imaging algorithms to evaluate phenotypic changes in tumor emboli in response to anti-cancer drug treatment. We also optimized a technique that allows for in vivo tumor emboli implantation including in a transgenic mice with red fluorescent lymphatic vasculature that were surgically implanted with a dorsal skin window chamber. This allowed for intravital optical imaging of the local tumor microenvironment and adjacent skin. Results: Our culture platform enables live imaging of the tumor emboli generated over a span of 7-10 days, maintaining key parameters including kinematic and dynamic viscosity, density along with sheer wall stress within the reported physiological range for lymphatics. Through higher resolution confocal image acquisition and multi-parametric analysis, we conducted particle counting and classification, providing statistically significant insights into the integrity of tumor emboli over time. Additionally, the developed algorithms allowed for quantitative analysis of images from tumor emboli cultures subjected to anti-cancer drugs, revealing disparities in drug efficacy on tumor emboli integrity and dispersion including inter-cluster space within the emboli. Intravital optical imaging, coupled with quantitative analysis of dispersion characteristics of the tumor emboli implanted in the murine model over 5-6 days, demonstrated a diffuse spread reminiscent of observations in IBC patients. Furthermore, immunohistochemistry staining showed marked Ki67, CD45, F4-80 revealing a proliferative tumor microenvironment with high levels of macrophage infiltration. Conclusions: As of now specific targeted therapies for IBC patients remain elusive. Our culture platform for tumor emboli, when combined with advanced imaging techniques both in vitro and in vivo and traditional approaches like immunohistochemistry and genomic evaluation, forms a powerful toolkit for assessing the phenotype and dispersion characteristics of this unique pathological feature in IBC. This preclinical model offers crucial insights, potentially paving the way for precision medicine strategies in the treatment of this understudied cancer. Funding in part from American Cancer Society Mission Boost MBG-20-141-01-MBG grant (GRD), Department of Defense W81XWH-20-1-0153 (GRD), Duke Consortium for Inflammatory Breast Cancer Education (AB, CW, RE). Citation Format: Dorababu Sannareddy, Theresa Charity, Alexandra Bennion, Caroline Way, Edwin Yu, Ralph Erdmann, Gregory M Palmer, Gayathri R Devi. A Tumor Emboli Platform for Morphometric Analysis, Drug screening and for Intravital Optical Imaging in a Murine Model to Simulate Clinicopathological Features of Inflammatory Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-02.

β-arrestin1/2 are essential for embryonic lymphatic vessel development

Lymphatic dysfunction can lead to fetal loss due to interstitial edema known as hydrops fetalis during development and lymphedema in adulthood. Lymphedema affects an estimated 250 million individuals worldwide, yet there is no effective treatment available. Numerous studies have established G protein-coupled receptors (GPCRs) as key lymphangiogenic signaling pathways and ideal druggable therapeutical targets. β-arrestins (β-arrestin 1 and 2) are ubiquitously expressed cytosolic adaptor proteins which perform a wide range of functions in GPCR pathways, such as promoting agonist-induced internalization and forming signaling scaffolds with internalized GPCRs in endosomes. However, the roles of β-arrestins in lymphatic vessel development and function remain undefined. We hypothesize that β-arrestin1/2 are essential in embryonic lymphatic vessel development. To test this hypothesis, I used an inducible β-arrestin1fl/fl (Arrb1 fl/fl), β-arrestin2fl/fl (Arrb2 fl/fl);Prox1 CreERT2 mouse line. β-arrestin1 and 2 were deleted by oral gavage of tamoxifen at embryonic day 10.5 (E10.5) and E12.5, critical points for developmental lymphangiogenesis. Compared to Arrb1/2fl/flembryos, the Arrb1/2fl/fl;Prox1CreERT2 embryos exhibit profound hydrops fetalis, subcutaneous hemorrhages, and increased embryonic lethality between E14.5 and E16.5. At E15.5, the Arrb1/2fl/fl;Prox1CreERT2 embryos with edema show decreased weight indicating growth restriction compared to controls. Jugular lymphatic sacs are enlarged and filled with blood in Arrb1/2fl/fl;Prox1CreERT2 embryos compared to controls. Dermal lymphatic vessel length in the Arrb1/2fl/fl;Prox1CreERT2 embryos is shorter than controls. Collectively, our results demonstrate that loss of β-arrestin1/2 expression in lymphatics causes mid-gestational arrest in lymphatic development and fetal growth restriction, indicating that β-arrestins are required for developmental lymphangiogenesis in mammals. American Physiological Society postdoctoral Fellowship (to YT) and NIH R01 HL129086 (to KMC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

VE-cadherin junction dynamics in initial lymphatic vessels promotes lymph node metastasis.

The endothelial junction component vascular endothelial (VE)-cadherin governs junctional dynamics in the blood and lymphatic vasculature. Here, we explored how lymphatic junction stability is modulated by elevated VEGFA signaling to facilitate metastasis to sentinel lymph nodes. Zippering of VE-cadherin junctions was established in dermal initial lymphatic vessels after VEGFA injection and in tumor-proximal lymphatics in mice. Shape analysis of pan-cellular VE-cadherin fragments revealed that junctional zippering was accompanied by accumulation of small round-shaped VE-cadherin fragments in the lymphatic endothelium. In mice expressing a mutant VEGFR2 lacking the Y949 phosphosite (Vegfr2 Y949F/Y949F ) required for activation of Src family kinases, zippering of lymphatic junctions persisted, whereas accumulation of small VE-cadherin fragments was suppressed. Moreover, tumor cell entry into initial lymphatic vessels and subsequent metastatic spread to lymph nodes was reduced in mutant mice compared with WT, after challenge with B16F10 melanoma or EO771 breast cancer. We conclude that VEGFA mediates zippering of VE-cadherin junctions in initial lymphatics. Zippering is accompanied by increased VE-cadherin fragmentation through VEGFA-induced Src kinase activation, correlating with tumor dissemination to sentinel lymph nodes.

Lymphangiogenesis and Lymphatic Zippering in Skin Associated with the Progression of Lymphedema

Secondary lymphedema often develops after lymph node dissection or radiation therapy for cancer treatment, resulting in marked skin fibrosis and increased stiffness owing to insufficiency of the lymphatic system caused by abnormal structure and compromised function. However, little is known about the associated changes of the dermal lymphatic vessels. In this study, using the lower limb skin samples of patients with secondary lymphedema, classified as types 1-4 by lymphoscintigraphy, we first confirmed the presence of epidermal thickening and collagen accumulation in the dermis, closely associated with the progression of lymphedema. Three-dimensional characterization of lymphatic capillaries in skin revealed prominent lymphangiogenesis in types 1 and 2 lymphedema. In contrast, increased recruitment of smooth muscle cells accompanied by development of the basement membrane in lymphatic capillaries was observed in types 3 and 4 lymphedema. Remarkably, the junctions of dermal lymphatic capillaries were dramatically remodeled from a discontinuous button-like structure to a continuous zipper-like structure. This finding is consistent with previous findings in an infection-induced mouse model. Such junction tightening (zippering) could reduce fluid transport and cutaneous viral sequestration during the progression of lymphedema and might explain the aggravation of secondary lymphedema. These findings may be helpful in developing stage-dependent treatment of patients with lymphedema.

Resident memory T cells in the lymph node balance localized and systemic protection

Abstract The endogenous mechanisms that protect against pathogen infection in the lymph node (LN) and LN metastasis during early tumor development remain poorly understood. Resident memory T cells (Trm), which do not circulate, provide highly localized protection to both pathogens and cancer, and correlate with better clinical outcomes. Here we track LN Trm development using models of cutaneous viral infection to determine the kinetics and requirements for their differentiation. We show that after viral skin infection, Trm form in the draining LN concurrent with their establishment at the initial site of infection in skin. LN Trm formed specifically in LNs draining infected skin expressing cognate antigen and did not form in contralateral controls. These data supported a hypothesis that T cell egress out of infected skin through lymphatic vessels was necessary for LN Trm formation. Consistent with this hypothesis, LN Trm were absent from mice lacking dermal lymphatic vessels indicating that either fluid transport or cellular transport from the skin to the LN was necessary for the establishment of LN Trm. To decouple LN priming from T cell egress we resected infected skin days 5 and 20 post infection. Ear resection at 5 days post infection impaired Trm formation but circulating memory T cell responses were unaffected. We demonstrated that LN Trm are poised for cell killing as evidenced by constitutive expression of granzymes, and were able to protect against viral rechallenge. These findings indicate that LN Trm are poised to provide rapid, localized protection specific to the draining LN. These data may suggest that LN’s harbor populations capable of controlling regional cancer metastasis and pathogenic infections. Supported by grants from NIH (R01CA238163)

Primary Melanoma miRNA Trafficking Induces Lymphangiogenesis

Melanoma, the deadliest cutaneous tumor, initiates within the epidermis; during progression, cells invade into the dermis and become metastatic through the lymphatic and blood system. Prior to melanoma cells invasion into the dermis, an increased density of dermal lymphatic vessel is observed, generated by a not fully understood mechanism. Here, we show that, while at the primary epidermal stage (in-situ), melanoma cells secret extra cellular vesicles termed melanosomes, which are up taken by dermal lymphatic cells, leading to transcriptional and phenotypic pro-lymphangiogenic changes. Mechanistically, melanoma-derived melanosomes traffic mature let-7i to lymphatic endothelial cells, which mediate pro-lymphangiogenic phenotypic changes by the induction of type I interferon signaling. Further, transcriptome analysis upon treatments with melanosomes or let-7i reveal the enhancement of IFI6 expression in lymphatic cells. Since melanoma cells metastasize primarily via lymphatic vessels, our data suggest that blocking this lymphangiogenesis process by repressing either melanosome release or type I interferon signaling will prevent melanoma progression to the deadly metastatic stage.

Genetic Engineering of Lymphangiogenesis in Skin Does Not Affect Blood Pressure in Mouse Models of Salt-Sensitive Hypertension.

Recent studies have indicated that sodium storage is influenced by macrophages that secrete VEGF-C (vascular endothelial growth factor) during salt stress thus stimulating lymphangiogenesis, thereby acting as a buffer against increased blood pressure (BP). We aimed to explore the role of dermal lymphatics in BP and sodium homeostasis. Our hypothesis was that mice with reduced dermal lymphatic vessels were more prone to develop salt-sensitive hypertension, and that mice with hyperplastic vessels were protected. Mice with either hypoplastic (Chy), absent (K14-VEGFR3 [vascular endothelial growth factor receptor 3]-Ig), or hyperplastic (K14-VEGF-C) dermal lymphatic vessels and littermate controls were given high-salt diet (4% NaCl in the chow), deoxycorticosterone acetate (DOCA)-salt diet and 1% saline to drink or nitric oxide blocker diet L-NG-nitro arginine methyl ester (followed by high salt diet). BP was measured by telemetric recording, and tissue sodium content by ion chromatography. In contrast to previous studies, high salt diet did not induce an increase in BP or sodium storage in any of the mouse strains investigated. DOCA-salt, on the other hand, gave an increase in BP in Chy and K14-VEGFR3-Ig not different from their corresponding WT controls. DOCA induced salt storage in skin and muscle, but to the same extent in mice with dysfunctional lymphatic vessels and WT controls. Lymph flow as assessed by tracer washout was not affected by the diet in any of the mouse strains. Our results suggest that dermal lymphatic vessels are not involved in salt storage or blood pressure regulation in these mouse models of salt-sensitive hypertension.

Abstract P1-24-06: Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression

Abstract Background. Lymphovascular invasion (LVI) and breast tumor emboli within dermal and breast lymphatic vessels are prognostic for metastatic spread and poor outcomes, and are abundant in Inflammatory breast cancer (IBC). IBC is an aggressive breast cancer that presents suddenly with breast swelling and redness due to tumor emboli in lymphatics. Lack of breast-feeding and obesity are IBC risk factors. We sought to demonstrate the combinatorial effects of a high-fat diet and nursing on lymphatic function and compare these to IBC tumor induced changes in lymphatic function. We hypothesize that risk factors for aggressive breast cancer may alter lymphatic function in the normal gland prior to tumor initiation. Methods. Following two rounds of pregnancy in 20 multiparous SCID Beige immunocompromised mice, half of the mice were force weaned while half nursed pups. Prior to forced weaning, half of each of these groups were fed a high fat diet (HFD: 60 Kcal %, N = 10) while the other half received a low-fat diet (LFD: 10 Kcal %, N = 10). Consecutive dynamic near-infrared fluorescence (NIRF) lymphatic imaging was performed at 6-7 months (covid interruption) and 14 months after initiating the diet by injecting a near-IR fluorophore into the mammary fatpad and recording lymphatic pulsing over 8 minutes using V++. Matlab and ImageJ were used to quantify pulsing rates on the ventral lymphatics in each animal. Fatpads were subsequently inoculated with SUM149 IBC cells and imaging was repeated 16 months post diet initiation. Lymphatic imaging over time by HFD vs LFD was further studied in nulliparous animals. Tissues were collected for further analyses. ResultsData analysis prior to tumor injection, demonstrated lymphatic pulsing (pulses/4 minutes) increased over time in HFD force weaned (HFFW) and HFD nursing (HFN) animals only (65.5 vs 72.6, P=0.059; 60.1 vs 76.6, P=0.0099, respectively). Comparing HFFW and HFN to matched LFD groups (LFFW and LFN), at 14 weeks HFD was associated with increased pumping after forced weaning (62.3 vs. 72.6, P=0.074), and nursing (62.5 vs 76.6, P=0.0023). There was an increase in pulsing after tumor initiation (16 months after initiation of diet) in all groups (80.1, 84.1, 83.2, 82.4, P > 0.05 all comparisons to initial timepoint). In a separate experiment examining HFD (N=5) vs LFD (N=5) in nulliparous mice, lymphatic contractile activity increased in all animals over. time, average ventral lymphatic contractile frequency for LFD and HFD at week 8,11 and 14 weeks after diet initiation were 5, 8.64, 15.9 pumps/4 mins vs 11.8, 18.5, 28.2 pumps/4 mins, (P = 0.01, 0.05, and 0.0005 respectively). ConclusionsHFD increased lymphatic pulsing rate over time to a significantly greater extent than LFD continuing over 14 months independent of reproductive and nursing status. Tumor initiation prompted further increased pulsing rates beyond that observed after HFD across all groups. The magnitude of the effect of HFD on lymphatic pulsing approached the rate after tumor initiation, while reproductive variables did not impact lymphatic pulsing. Further studies are warranted to demonstrate the relationship if any between lymphatic pumping pre-initiation and LVI after tumor initiation and examine the role of intervention on reducing LVI. Citation Format: Wintana Balema, Janelle Morton, Richard Larson, Fred C Velasquez, Eva Sevick-Muraca, Wendy A Woodward. Risk factor modeled microenvironment effects lymphatics activity and IBC invasiveness and progression [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-24-06.

No Effect of Skin Lymphangiogenesis on Blood Pressure in Mouse Models of Salt‐Sensitive Hypertension

The pathogenesis of hypertension is not well understood, but high sodium intake has been associated with blood pressure (BP) rise. Studies have indicated that sodium can be stored in skin without commensurate water accumulation, and macrophages may secrete VEGF-C that can stimulate lymphangiogenesis, thereby acting as a buffer against increased BP. Here we aimed to explore the role of dermal lymphatics in BP and sodium homeostasis. Our hypothesis was that mice with reduced dermal lymphatic vessels were more prone to develop salt-sensitive hypertension, and that mice with hyperplastic vessels were protected against this outcome. Mice with either absent (K14-VEGFR3-Ig), hypoplastic (Chy) or hyperplastic (K14-VEGF-C) dermal lymphatic vessels and littermate controls were given high salt diet (HSD) (4% NaCl in the chow and 1% saline to drink), DOCA-salt diet (16mg/week 11-deoxycorticosterone acetate - 50mg/21 days slow release tablet subcutaneously and 1% saline to drink) or nitric oxide (NO) blocker diet (L-NAME 0.5mg/ml in drinking water for 3 weeks, followed by one week washout and thereafter 2 weeks HSD). BP was measured by telemetric recording, and tissue sodium content by ion chromatography. In contrast to previous studies, HSD did not induce an increase in BP or sodium storage in any of the mouse strains investigated. DOCA-salt, on the other hand, induced an increase in mean arterial pressure (MAP) in Chy of 13.4 ± 7.1 (SD) mm Hg (n=4), not different from a corresponding pressure in WT control of 14.7 ± 6.4 mm Hg, n=5. In K14-VEGFR3-Ig mice, DOCA raised the MAP 24 ± 12.1 mm Hg (n=6), not different from the corresponding WT control (22.8 ± 8.3 mm Hg, n=7). DOCA induced salt storage in skin and muscle, but to the same extent in mice with dysfunctional lymphatic vessels and WT controls. L-NAME diet tended to give a higher diastolic pressure in mice lacking dermal lymphatic vessels compared with WT control as suggested by the rise in diastolic BP of 11.6 ± 10.8 (SD) mm Hg (n=9) and 1.9 ± 2.4 (SD) mm Hg (n=6) (p<0.05, One-way ANOVA), respectively, without a concomitant increase in sodium content in skin or muscle in any of the strains. Our results indicate that there is no association between dermal lymphangiogenesis, sodium accumulation and blood pressure response. The response seen in NO-blocker diet in mice lacking dermal lymphatic vessels was salt storage independent. This suggests that dermal lymphatic vessels are not involved in salt storage or blood pressure regulation in these mouse models of salt-sensitive hypertension.

Whole-mount Immunohistochemistry to Visualize Mouse Embryonic Dermal Lymphatic Vasculature.

Lymphatic vessels are abundant in the skin where they regulate interstitial fluid uptake and immune surveillance. Defects in dermal lymphatic vessels, such as fewer vessels and abnormal lymphatic vessel coverage with mural cells, are frequently associated with lymphedema and other lymphatic disorders. Whole-mount immunohistochemistry allows the visualization of dermal lymphatic vessels and identifies morphogenetic defects. Most dermal lymphatic vessels start growing during embryogenesis from lymph sacs that are located close to the axilla towards the dorsal and ventral midlines. Here, we present an approach that we have developed to permeabilize, immunolabel, clear, and visualize the lymphatic vessels. These simple and inexpensive techniques reproducibly generate images of dermal lymphatic vessels with great clarity.

Light sheet microscopy-based 3-dimensional histopathology of the lymphatic vasculature in Emberger syndrome

Abstract Introduction Lymphovascular diseases represent a heterogenous group of inherited and sporadic disorders and refer to a range of possible underlying pathologies and pathogenesis.Emberger Syndrome, an inherited form of lymphedema, is characterized by bilateral lower limb lymphedema, however, upper limbs do not show any signs of swelling.To identify disease-associated histopathological alterations in patients with Emberger Syndrome and to elucidate potential histological differences between the lymphatic vasculature of upper and lower limbs, a detailed knowledge on the 3-dimensional tissue and vessel architecture is essential. However, the current gold standard in 2-dimensional histology provides only very limited spatial information. Material and methods To elucidate the underlying vascular pathology in Emberger Syndrome on the cellular level, we applied the 3-dimensional visualization and analysis approach VIPAR (volume information-based histopathological analysis by 3D reconstruction and data extraction) to entire wholemount immunofluorescence-stained human tissue samples. VIPAR is a light sheet microscopy-based imaging technique, which allows 3-dimensional reconstruction of entire tissue biopsies followed by automated and semi-automated analysis of vascular parameters in 3-dimensional space. Results Using VIPAR we could show that in Emberger Syndrome the dermal lymphatic vasculature is intact and non-disrupted.However, lower limbs showed an hypoplastic lymphatic vasculature with absence of lymphatic valves in pre-collecting and collecting vessels. In contrast to the lower limbs, the lymphatic vasculature of the upper limbs showed no morphological alterations of lymphatic vessels and lymphatic valves compared to healthy controls. Discussion Based on the 3-dimensional histopathological analysis we were able to perform a detailed phenotyping of lymphatic vessels in the upper and lower limb in Emberger Syndrome and to identify the underlying vascular pathology. In addition, we could show vascular alteration between the upper and lower limbs indicating a vascular heterogeneity of dermal lymph vessels causing the lower limb lymphedema.

TGF-beta and TNF-alpha cooperatively induce mesenchymal transition of lymphatic endothelial cells via activation of Activin signals.

Lymphatic systems play important roles in the maintenance of fluid homeostasis and undergo anatomical and physiological changes during inflammation and aging. While lymphatic endothelial cells (LECs) undergo mesenchymal transition in response to transforming growth factor-β (TGF-β), the molecular mechanisms underlying endothelial-to-mesenchymal transition (EndMT) of LECs remain largely unknown. In this study, we examined the effect of TGF-β2 and tumor necrosis factor-α (TNF-α), an inflammatory cytokine, on EndMT using human skin-derived lymphatic endothelial cells (HDLECs). TGF-β2-treated HDLECs showed increased expression of SM22α, a mesenchymal cell marker accompanied by increased cell motility and vascular permeability, suggesting HDLECs to undergo EndMT. Our data also revealed that TNF-α could enhance TGF-β2-induced EndMT of HDLECs. Furthermore, both cytokines induced the production of Activin A while decreasing the expression of its inhibitory molecule Follistatin, and thus enhancing EndMT. Finally, we demonstrated that human dermal lymphatic vessels underwent EndMT during aging, characterized by double immunostaining for LYVE1 and SM22α. These results suggest that both TGF-β and TNF-α signals play a central role in EndMT of LECs and could be potential targets for senile edema.

P38 MAPK Isoforms as Modulators of Oncogenic Phenotypes in Inflammatory Breast Cancer

Inflammatory Breast Cancer (IBC) is a rare, aggressive, and highly metastatic subtype of breast cancer; accounting for approximately 1–6% of all breast cancer diagnoses in the United States and 7–10% of breast cancer‐associated deaths in Western countries. The aggressiveness of this disease is generally attributed to the formation of diffuse tumor cell clusters, known as tumor emboli, that block the dermal lymphatic vessels near the chest wall, a key hallmark of IBC. Clinically, it has been documented that most of IBC cases are either HER2 amplified or Triple Negative (TN‐IBC), the latter being the most difficult to treat. As a result, there is a dire need to understand the molecular profile of IBC to further develop more efficient targeted therapies. It has been reported that 17β‐Estradiol (E2) exerts non‐genomic signaling in ER‐negative IBC and non‐IBC cells using estrogen receptors GPR30 and estrogen receptor alpha variant 36 (ERα‐36), which activate the PI3K/AKT/ERK pathways involved in oncogenic phenotypes. p38 isoforms alpha, beta, gamma, and delta have been demonstrated to modulate proliferation, migration, apoptosis, and cell senescence via stress‐induced pathways or mitogen‐activated pathways. Also, it has been shown that these isoforms have context‐dependent functions depending on tissue type, expression and activation level, and the pathways involved. Nevertheless, the effects of these isoforms have yet to be elucidated in IBC. Preliminary data suggest that estrogen mediates p38 activation when IBC cells are treated with E2. For this reason, we hypothesize that estrogenmediated activation is involved in p38‐induced oncogenesis via non‐genomic signaling in an isoform‐dependent manner in IBC cell lines. A comparison of p38 isoforms expression was done on both IBC and non‐IBC cell lines via Western blot and qRT‐PCR, presenting differential expression profiles. This data was then used to design a knockdown model to study the effects of the most predominant isoforms on oncogenic phenotypes. Treatment using SB202190, a potent p38 inhibitor, caused a significant decrease in cell viability of several breast cancer cell lines when performing a dose response curve. Ongoing studies include assessing the effects of p38 inhibition and knockdown on proliferation, invasion, migration, and tumor emboli formation under basal and endocrine conditions. To recapitulate, understanding E2 responsiveness by ER‐negative cell lines may provide great insight into novel targeted therapies using the non‐genomic signaling pathway; in particular, p38 signaling appears to be a key effector of prooncogenesis in IBC.

Abstract P3-01-08: Infiltrating immune cells in the tumor emboli of inflammatory breast cancer

Abstract INTRODUCTION: Inflammatory breast cancer (IBC) is a rare and aggressive type of locally advanced breast cancer. The hallmark of IBC is the presence of E-Cadherin positive tumor emboli within the dermal and parenchymal lymphatic vessels. These tumor emboli are thought to be responsible for the clinical presentation by the physical obstruction of draining lymph vessels. In addition, the intravascular growth of tumor emboli may account for the typical diffuse growth pattern and the fast local spread associated with IBC. Tumor emboli can result from encircling lymphovasculogenesis or lymphovascular invasion and little is known about the infiltrating immune cells in these emboli. This is the first study to investigate the immune infiltrate in the tumor emboli of patients with IBC. METHODOLOGY: To analyze the immune composition of the emboli, four slides of 24 IBC patients were stained according to a validated protocol and scored using the VISIOPHARM® software that makes virtual multiplexing possible after the alignment of different digital slides. We used four validated antibodies: CD79α (B-cell lineage), CD8 (cytotoxic T-cells), FOXP3 (Tregs) and CD163 (Tumor associated macrophages; TAMs), combined with a standard H&amp;E stained section to mark the tumor area and tumor emboli on pretreatment biopsy sections. RESULTS: Half of the IBC patients presented with a hormone receptor positive carcinoma (54.2% - n= 13/24) and 8 out of 21 patients with initially localized disease achieved complete pathological response (pCR) after neo-adjuvant chemotherapy (NACT). Our results are summarized in table 1. Table 1: For every staining we report the number of patients that have infiltrated emboli, the median density in the invasive carcinoma (IC), the median density in the emboli (E) and the ratio between the invasive tumor and emboli in table 1.# patientsDensity (IC) #/μm2Density (E) #/μm2Ratio Density E/ICP-valueCD812/24 (50 %)19.62.40.11&amp;lt; .001CD16319/23 (83 %)35.412.80.43.02CD79α11/24 (46 %)18.23.50.31.01FOXP34/23 (17 %)5.10.30.04.003 Patients with more immune cells in the invasive carcinoma (IC) also had more immune cells in the emboli (E). This correlation was significant for all stainings (Table 1). CD163+ macrophages are the most abundant immune cells in emboli, especially when compared to the invasive carcinoma, whereas CD8+ cells were uncommon in the emboli. The density of CD8+ cells in the invasive carcinoma or emboli did not predict pCR. Interestingly, patients with a higher IC/E CD8 density ratio achieved more often pCR (Kruskal-Wallis χ = 3.6, P = 0.05). Only 20.8% (n= 5/24) had invasive carcinoma directly surrounding the intravascular emboli, however, median overall survival in these patients was significantly shorter (P= 0.003). CONCLUSIONS: This is the first study looking at infiltrating immune cells in IBC. Although based on a small discovery cohort of only 24 patients, we showed that there is a strong correlation between the density of immune cells in the emboli and the density in the invasive carcinoma. Furthermore, we demonstrate that TAMs are the predominant immune cell type in IBC emboli. Cytotoxic T cells are less common and having more CD8+ cells in the invasive carcinoma compared to the emboli was associated with pCR. Patients with invasive carcinoma directly surrounding the emboli had a worse prognosis. Citation Format: Christophe Van Berckelaer, Charlotte Rypens, Peter Vermeulen, Steven Van Laere, Peter Van Dam, Cecile Colpaert. Infiltrating immune cells in the tumor emboli of inflammatory breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-08.

IFNγ-activated dermal lymphatic vessels inhibit cytotoxic T cells in melanoma and inflamed skin.

Mechanisms of immune suppression in peripheral tissues counteract protective immunity to prevent immunopathology and are coopted by tumors for immune evasion. While lymphatic vessels facilitate T cell priming, they also exert immune suppressive effects in lymph nodes at steady-state. Therefore, we hypothesized that peripheral lymphatic vessels acquire suppressive mechanisms to limit local effector CD8+ T cell accumulation in murine skin. We demonstrate that nonhematopoietic PD-L1 is largely expressed by lymphatic and blood endothelial cells and limits CD8+ T cell accumulation in tumor microenvironments. IFNγ produced by tissue-infiltrating, antigen-specific CD8+ T cells, which are in close proximity to tumor-associated lymphatic vessels, is sufficient to induce lymphatic vessel PD-L1 expression. Disruption of IFNγ-dependent crosstalk through lymphatic-specific loss of IFNγR boosts T cell accumulation in infected and malignant skin leading to increased viral pathology and tumor control, respectively. Consequently, we identify IFNγR as an immunological switch in lymphatic vessels that balances protective immunity and immunopathology leading to adaptive immune resistance in melanoma.

Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds.

Endothelial cells line blood and lymphatic vessels and form intercellular junctions, which preserve vessel structure and integrity. The vascular endothelial cadherin, VE-cadherin, mediates endothelial adhesion and is indispensible for blood vessel development and permeability regulation. However, its requirement for lymphatic vessels has not been addressed. During development, VE-cadherin deletion in lymphatic endothelial cells resulted in abortive lymphangiogenesis, edema, and prenatal death. Unexpectedly, inducible postnatal or adult deletion elicited vessel bed-specific responses. Mature dermal lymph vessels resisted VE-cadherin loss and maintained button junctions, which was associated with an upregulation of junctional molecules. Very different, mesenteric lymphatic collectors deteriorated and formed a strongly hyperplastic layer of lymphatic endothelial cells on the mesothelium. This massive hyperproliferation may have been favored by high mesenteric VEGF-C expression and was associated with VEGFR-3 phosphorylation and upregulation of the transcriptional activator TAZ Finally, intestinal lacteals fragmented into cysts or became highly distended possibly as a consequence of the mesenteric defects. Taken together, we demonstrate here the importance of VE-cadherin for lymphatic vessel development and maintenance, which is however remarkably vessel bed-specific.

PROX1 is a transcriptional regulator of MMP14

The transcription factor PROX1 is essential for development and cell fate specification. Its function in cancer is context-dependent since PROX1 has been shown to play both oncogenic and tumour suppressive roles. Here, we show that PROX1 suppresses the transcription of MMP14, a metalloprotease involved in angiogenesis and cancer invasion, by binding and suppressing the activity of MMP14 promoter. Prox1 deletion in murine dermal lymphatic vessels in vivo and in human LECs increased MMP14 expression. In a hepatocellular carcinoma cell line expressing high endogenous levels of PROX1, its silencing increased both MMP14 expression and MMP14-dependent invasion in 3D. Moreover, PROX1 ectopic expression reduced the MMP14-dependent 3D invasiveness of breast cancer cells and angiogenic sprouting of blood endothelial cells in conjunction with MMP14 suppression. Our study uncovers a new transcriptional regulatory mechanism of cancer cell invasion and endothelial cell specification.

Histological and Morphological Characterization of Developing Dermal Lymphatic Vessels.

The capacity to visualize the lymphatic vasculature in three-dimensions has revolutionized our understanding of the morphogenetic mechanisms important for constructing the lymphatic vascular network during development. Two complementary approaches are commonly employed to assess the function of genes and signaling pathways important for development of the dermal lymphatic vasculature in the mouse embryo. The first of these is whole-mount immunostaining of embryonic skin to analyze dermal lymphatic vessel network patterning and morphology in two and three dimensions. The second is immunostaining of thin tissue sections to examine lymphatic vessel identity, lumen formation and protein localization within discrete lymphatic endothelial cells in a two-dimensional setting. Here we present detailed protocols for multicolor immunofluorescent immunostaining of embryonic dorsal skin and thin tissue cryosections. Each of these methods generates high-resolution images of the dermal lymphatic vasculature, yielding information integral to in-depth characterization of lymphatic vessel phenotypes in the developing mouse embryo.

A Case of Primary Pleural Melanoma

SESSION TITLE: Rare Thoracic Neoplasms SESSION TYPE: Global Case Report Poster PRESENTED ON: Tuesday, October 31, 2017 at 01:30 PM - 02:30 PM INTRODUCTION: This case report describes a rare presentation of melanoma and emphasizes the importance of the cytological and immunocytochemical analyses of pleural fluid. CASE PRESENTATION: An 83-year-old Japanese male presented with left-sided chest pain and dyspnea lasting a few hours. He had a history of old myocardial infarction and paroxysmal atrial fibrillation. He drank alcoholic beverages occasionally and was non-smoker. Initial vital signs were unremarkable. There was no lymphadenopathy or hepatosplenomegaly. Breath sounds were decreased and dullness was present at the base of the left lung. Cardiovascular examination was normal. Laboratory test values were as follows: white blood count cell, 4770/mm3; hemoglobin, 14.8 g/dL; platelet count, 84,000/mm3; random serum glucose, 147 mg/dl; serum lactate dehydrogenase, 387 U/l; serum albumin, 3.3 g/dl; serum C-reactive protein, 0.34 mg/dl. Chest radiograph showed a large amount of pleural effusion in the left hemi-thorax, and the chest computed tomography scan showed multiple nodular soft tissue thickenings of the left hemi-pleura and mediastinum with pleural effusion. Thoracentesis was performed and macroscopically the pleural effusion was bloody. The cytological examination of the plural fluid revealed mono or multinucleate large malignant cells in loose aggregates. There were large eccentric nuclei with prominent nucleoli and granular cytoplasmic melanin pigment. On the immunehistochemical examination, the malignant cells were positive for the expression of human melanoma black-45 and S-100, and negative for calretinin, D2-40, desmin, EMA, TTF-1 and Napsin A, which further supported the diagnosis of melanoma. An ultrasonography-guided transthoracic biopsy of the nodular soft tissue thickening of the left hemi-pleura was performed, and the histological/immunohistochemical examinations confirmed the diagnosis. Detailed physical examinations of the patient failed to reveal any site for the primary melanoma in the skin, eyes, oral cavity, genital or anal regions.The patient received chemical pleurodesis using OK-432 for the relief of dyspnea due to the malignant plural effusion, and thereafter, he declined any further treatment. He died of respiratory failure 1 month after the diagnosis. The autopsy disclosed muddy black-colored tumor widely disseminated in the left thoracic cavity. The tumor mainly located in the left visceral pleura and invaded into the left lung tissue. Moreover, the tumor also disseminated to the left parietal pleura, diaphragm, peritoneum and mesenterium. No primary site was found anywhere, including skin and other mucous membranes. From these findings, this patient can be designated as primary pleural melanoma. DISCUSSION: Melanoma predominantly arises in the skin or mucosa close to the skin, and primary pleural melanoma is extremely rare. To our best knowledge, only four cases have been reported in the English literature. Not for pleural but for primary pulmonary melanoma, Jensen and Egedorf proposed the following six clinical criteria for diagnosis: (1) no previously removed pigmented skin tumors, (2) no ocular tumors removed, (3) a solitary tumor in the surgical specimen, (4) tumor morphology compatible with a primary tumor, (5) no demonstrable melanoma in other organs at time of operation, and (6) autopsy without primary melanomas being demonstrated elsewhere. These criteria should be also applied well to primary pulmonary melanoma, and our patient satisfied all of them. It has been considered that melanoblasts arise from the neural crest and migrate to all parts of the body. Moreover, it is also considered that cutaneous nerve cells can enter dermal lymphatic vessels and travel through the lymphatic system. These mechanisms may explain the pathogenesis of primary pleural melanoma. CONCLUSIONS: In conclusion, our patient can be diagnosed as primary pleural melanoma which is extremely rare condition. This case highlights the importance of cytomorphologic and immunocytochemical analyses in the differential diagnosis of pleural effusion. Reference #1: Nigogosyan G, de la Pava S, Pickren JW. Melanoblasts in vaginal mucosa. Origin for primary malignant melanoma. Cancer 1964;17: 912-913. Reference #2: Biddle DA, Evans H, Kemp BL, et al. Intraparenchymal nevus cell aggregates in lymph nodes: A possible diagnostic pitfall with malignant melanoma and carcinoma. Am J Surg Pathol 2003;27: 673-681. Reference #3: Jensen OA, Egedorf J. Primary malignant melanoma of the lung. Scand J Respir Dis. 1967;48:127-35. DISCLOSURE: The following authors have nothing to disclose: Kojin Murakami, Kazunori Tobino No Product/Research Disclosure Information