SummaryEnlargement of the lymphatic vascular network in tumor-draining lymph nodes (LNs) often precedes LN metastasis, likely providing a lymphovascular niche for tumor cells. We investigated morphological and molecular changes associated with the lymphatic remodeling process, using the 4T1 breast cancer and B16F10 melanoma models. Lymphatic expansion in tumor-draining LNs is mediated by sprouting and proliferation of lymphatic endothelial cells (LECs) as early as 4 days after tumor implantation. RNA sequencing revealed an altered transcriptional profile of LECs from tumor-draining compared to naive LNs with similar changes in both tumor models. Integrin αIIb is upregulated in LECs of tumor-draining LNs and mediates LEC adhesion to fibrinogen in vitro. LEC-associated fibrinogen was also detected in LNs in vivo, suggesting a role of integrin αIIb in lymphatic remodeling. Together, our results identify specific responses of LN LECs to tumor stimuli and provide insights into the mechanisms of lymphovascular niche formation in tumor-draining LNs.
The incidence of melanoma, the most fatal dermatological cancer, has dramatically increased over the last few decades. Modern targeted therapy with kinase inhibitors induces potent clinical responses, but drug resistance quickly develops. Combination therapy improves treatment outcomes. Therefore, novel inhibitors targeting aberrant proliferative signaling in melanoma via the MAPK/ ERK and PI3K/AKT pathways are urgently needed. Biosensors were combined that report on ERK/AKT activity with image-based highcontent screening and HPLC-based activity profiling. An in-house library of 2576 plant extracts was screened on two melanoma cell lines with different oncogenic mutations leading to pathological ERK/AKT activity. Out of 140 plant extract hits, 44 were selected for HPLC activity profiling. Active thymol derivatives and piperamides from Arnica montana and Piper nigrum were identified that inhibited pathological ERK and/or AKT activity. The pipeline used enabled an efficient identification of natural products targeting oncogenic signaling in melanoma.
The discovery of bioactive natural
products remains a time-consuming
and challenging task. The ability to link high-confidence metabolite
annotations in crude extracts with activity would be highly beneficial
to the drug discovery process. To address this challenge, HPLC-based
activity profiling and advanced UHPLC-HRMS/MS metabolite profiling
for annotation were combined to leverage the information obtained
from both approaches on a crude extract scaled down to the submilligram
level. This strategy was applied to a subset of an extract library
screening aiming to identify natural products inhibiting oncogenic
signaling in melanoma. Advanced annotation and data organization enabled
the identification of compounds that were likely responsible for the
activity in the extracts. These compounds belonged to two different
natural product scaffolds, namely, brevipolides from a Hyptis
brevipes extract and methoxylated flavonoids identified in
three different extracts of Hyptis and Artemisia spp. Targeted isolation of these prioritized compounds led to five
brevipolides and seven methoxylated flavonoids. Brevipolide A (1) and 6-methoxytricin (9) were the most potent
compounds from each chemical class and displayed AKT activity inhibition
with an IC50 of 17.6 ± 1.6 and 4.9 ± 0.2 μM,
respectively.
A library of more than 2500 plant
extracts was screened
for activity
on oncogenic signaling in melanoma cells. The ethyl acetate extract
from the aerial parts of Artemisia argyi displayed
pronounced inhibition of the PI3K/AKT pathway. Active compounds were
tracked with the aid of HPLC-based activity profiling, and altogether
21 active compounds were isolated, including one novel dimerosequiterpenoid
(1), one new disesquiterpenoid (2), three
new guaianolides (3–5), 12 known
sesquiterpenoids (6–17), and four
known flavonoids (19–22). A new eudesmanolide
derivative (13b) was isolated as an artifact formed by
methanolysis. Compound 1 is the first adduct comprising
a sesquiterpene lactone and a methyl jasmonate moiety. The absolute
configurations of compounds 1 and 3–18 were established by comparison of their experimental and
calculated ECD spectra. The absolute configuration for 2 was determined by X-ray diffraction analysis. Guaianolide 8 was the most potent sesquiterpene lactone, inhibiting the
PI3K/AKT pathway with an IC50 value of 8.9 ± 0.9 μM.
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