[citation report] CCN2-induced lymphangiogenesis is mediated by the integrin αvβ5–ERK pathway and regulated by DUSP6 (2024)

“…The cells were used up to six passages in the experiments. C57BL/6 mouse-derived primary cultured LECs (Cell Biologics, Chicago, IL) were cultured in 0.1% gelatin-coated culture dishes at 37 °C with 5% CO 2 in EGM2-endothelial cell growth medium-2 (Lonza, Basel, Switzerland), and used in in vitro experiments up to six passages 54 . For transwell culture, 5 × 10 4 LECs were seeded into 24-well plates and incubated overnight.…”

confidence: 99%

Section: Methodsmentioning

et al. 2022

Cancer cells often metastasize to the lymph nodes (LNs) before disseminating throughout the body. Clinically, LN metastasis correlates with poor prognosis and influences treatment options. Many studies have shown that cancer cells communicate with immune and stromal cells to prepare a suitable niche for metastasis. In this study, mice were injected with B16–F10 murine melanoma cells to generate a tongue submandibular lymph node (SLN) metastasis model in which genes of interest could be investigated. Microarray analyses were performed on SLNs, identifying 162 upregulated genes, some of which are known metastasis genes. Among these upregulated genes, Kcne4, Slc7a11, Fscn1, and Gadd45b were not associated with metastasis, and increased expression of Kcne4 and Slc7a11 was confirmed by real-time PCR and immunohistochemistry. The roles of KCNE4 in chemokine production and cell adhesion were examined using primary lymphatic endothelial cells, and demonstrated that Ccl17 and Ccl19, which are involved in melanoma metastasis, were upregulated by KCNE4, as well as Mmp3 matrix metalloproteinase. Expression of KCNE4 was detected in human LNs with metastatic melanoma. In conclusion, we found that LN metastatic melanoma induces KCNE4 expression in the endothelium of LNs.

Metachronous liver metastases (MLM) are characterized by high incidence and high mortality in clinical colorectal cancer treatment. Currently traditional clinical methods cannot effectively predict and prevent the occurrence of metachronous liver metastasis in colorectal cancer. Based on 5hmC-Seal analysis of blood and tissue samples, this study found that portal venous blood was more relevant to tumor gDNA than peripheral blood. We performed a novel epigenetic liquid biopsy strategy using the 10 5hmC epigenetic alterations, to accurately distinguish MLM patients from patients without metastases. Among these epigenetic alterations, phosphodiesterase 4 (PDE4D) was highly increased in MLM patients and correlated with poor survival. Moreover, our studies demonstrated that PDE4D was a key metastasis-driven target for drug development. Interfering with the function of PDE4D significantly repressed liver metastases. Similarly, roflumilast, a PDE4 inhibitor approved for Chronic obstructive pulmonary disease (COPD) also inhibits liver metastases. Roflumilast treatment significantly inhibited the protein levels of HIF-1α, CCN2, p-AKT and p-ERK, thus suppressing metastases. To develop a more efficient PDE4D inhibitor and reduce the occurrence of adverse events, we also designed several new compounds based on 2-arylbenzofurans and discovered lead L11 with potent affinity for PDE4D and significant suppression of liver metastases. Interestingly, we further found that compound L11 was better tolerated in terms of emetic activity on beagle dogs than roflumilast. Summarily, our study provides a promising strategy for predicting metachronous liver metastasis and discovers roflumilast as a potential repurposed drug for inhibiting liver metastasis, which have the potential to benefit patients with CRC in the future.

No abstract