An exosome‑based liquid biopsy signature for pre‑operative identification of lymph node metastasis in patients with pathological high‑risk T1 colorectal cancer

Background: According to current guidelines, more than 70% of patients with invasive submucosal colorectal cancer (T1 CRC) undergo a radical operation with lymph node dissection, even though only ~ 10% have lymph node metastasis (LNM). Hence, there is imperative to develop biomarkers that can help robustly identify LNM-positive patients to prevent such overtreatments. Given the emerging interest in exosomal cargo as a source for biomarker development in cancer, we examined the potential of exosomal miRNAs as LNM prediction biomarkers in T1 CRC. Methods: We analyzed 200 patients with high-risk T1 CRC from two independent cohorts, including a training (n = 58) and a validation cohort (n = 142). Cell-free and exosomal RNAs from pre-operative serum were extracted, followed by quantitative reverse-transcription polymerase chain reactions for a panel of miRNAs. Results: A panel of four miRNAs (miR-181b, miR-193b, miR-195, and miR-411) exhibited robust ability for detecting LNM in the exosomal vs. cell-free component. We subsequently established a cell-free and exosomal combination signature, successfully validated in two independent clinical cohorts (AUC, 0.84; 95% CI 0.70–0.98). Finally, we developed a risk-stratification model by including key pathological features, which reduced the false positive rates for LNM by 76% without missing any true LNM-positive patients. Conclusions: Our novel exosomal miRNA-based liquid biopsy signature robustly identifies T1 CRC patients at risk of LNM in a preoperative setting. This could be clinically transformative in reducing the significant overtreatment burden of this malignancy

Family with Sequence Similarity 5, Member C (FAM5C) Increases Leukocyte Adhesion Molecules in Vascular Endothelial Cells: Implication in Vascular Inflammation

Identification of the regulators of vascular inflammation is important if we are to understand the molecular mechanisms leading to atherosclerosis and consequent ischemic heart disease, including acute myocardial infarction. Gene polymorphisms in family with sequence similarity 5, member C (FAM5C) are associated with an increased risk of acute myocardial infarction, but little is known about the function of this gene product in blood vessels. Here, we report that the regulation of the expression and function of FAM5C in endothelial cells. We show here that FAM5C is expressed in endothelial cells in vitro and in vivo. Immunofluorescence microscopy showed localization of FAM5C in the Golgi in cultured human endothelial cells. Immunohistochemistry on serial sections of human coronary artery showed that FAM5C-positive endothelium expressed intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1). In cultured human endothelial cells, the overexpression of FAM5C increased the reactive oxygen species (ROS) production, nuclear factor-κB (NF-κB) activity and the expression of ICAM-1, VCAM-1 and E-selectin mRNAs, resulting in enhanced monocyte adhesion. FAM5C was upregulated in response to inflammatory stimuli, such as TNF-α, in an NF-κB- and JNK-dependent manner. Knockdown of FAM5C by small interfering RNA inhibited the increase in the TNF-α-induced production of ROS, NF-κB activity and expression of these leukocyte adhesion molecule mRNAs, resulting in reduced monocyte adhesion. These results suggest that in endothelial cells, when FAM5C is upregulated in response to inflammatory stimuli, it increases the expression of leukocyte adhesion molecules by increasing ROS production and NF-κB activity

Family with Sequence Similarity 5, Member C (FAM5C) Increases Leukocyte Adhesion Molecules in Vascular Endothelial Cells: Implication in Vascular Inflammation

Identification of the regulators of vascular inflammation is important if we are to understand the molecular mechanisms leading to atherosclerosis and consequent ischemic heart disease, including acute myocardial infarction. Gene polymorphisms in family with sequence similarity 5, member C (FAM5C) are associated with an increased risk of acute myocardial infarction, but little is known about the function of this gene product in blood vessels. Here, we report that the regulation of the expression and function of FAM5C in endothelial cells. We show here that FAM5C is expressed in endothelial cells in vitro and in vivo. Immunofluorescence microscopy showed localization of FAM5C in the Golgi in cultured human endothelial cells. Immunohistochemistry on serial sections of human coronary artery showed that FAM5C-positive endothelium expressed intercellular adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1). In cultured human endothelial cells, the overexpression of FAM5C increased the reactive oxygen species (ROS) production, nuclear factor-κB (NF-κB) activity and the expression of ICAM-1, VCAM-1 and E-selectin mRNAs, resulting in enhanced monocyte adhesion. FAM5C was upregulated in response to inflammatory stimuli, such as TNF-α, in an NF-κB- and JNK-dependent manner. Knockdown of FAM5C by small interfering RNA inhibited the increase in the TNF-α-induced production of ROS, NF-κB activity and expression of these leukocyte adhesion molecule mRNAs, resulting in reduced monocyte adhesion. These results suggest that in endothelial cells, when FAM5C is upregulated in response to inflammatory stimuli, it increases the expression of leukocyte adhesion molecules by increasing ROS production and NF-κB activity

Expression and intracellular localization of FAM5C in cultured human endothelial cells.

<p><b>A</b>, Expression of FAM5C mRNA. The expression of FAM5A, FAM5B, FAM5C, and GAPDH mRNAs was determined with conventional RT–PCR. <b>B–E</b>, Intracellular localization of FAM5C. HUVECs were stained with the indicated antibodies. The nuclei were stained with DAPI. The results shown are representative of three to four independent experiments, with identical results obtained. ER, endoplasmic reticulum.</p

Colocalization of FAM5C with ICAM-1 and VCAM-1 in the endothelium of human coronary arteries.

<p>Serial sections of coronary arteries obtained from a 68-year-old man with chronic renal failure (<b>A</b>) and a 75-year-old man with hepatocellular carcinoma (<b>B</b>) were subjected to immunofluorescence staining for FAM5C and ICAM-1 (<b>A</b>) or VCAM-1 (<b>B</b>).</p

Involvement of FAM5C in the increases in ROS production and NF-κB activity.

<p>HUVECs transfected with control or FAM5C siRNAs were cultured for 48 h, and were then cultured in the presence of 10 ng/ml TNF-α for 6 h. The increase in TNF-α-induced ROS production (<b>A</b>) and NF-κB activity (<b>B</b>) by knockdown of FAM5C. The results shown are representative of three independent experiments (<b>A</b>). The luciferase activity was normalized by the β-galactosidase activity (<b>B</b>) (n = 4).</p

FAM5C increases ROS production and NF-κB activity.

<p><b>A and B</b>, HUVECs were transfected with 0.1 µg of FLAG (Mock) or FLAG–FAM5C (FAM5C) cDNAs and then incubated with 10 mM NAC or 10 µM TTFA for 16 h. ROS production assessed by dihydroethidium staining (<b>A</b>) and NF-κB activity (<b>B</b>) (n = 3–5) were analysed. <b>C and D</b>, HUVECs were transfected with 0.1 µg of FLAG (Mock) or FLAG–FAM5C (FAM5C) cDNAs and then incubated with 10 mM NAC, 10 µM TTFA, or 10 µM BAY 11-7085 (BAY) for 16 h. Involvement of ROS and NF-κB in the increase in FAM5C-induced ICAM-1 mRNA expression (<b>C</b>) (n = 4–5) and monocyte adhesion to HUVECs (<b>D</b>) (n = 6) were analyzed. ^†P<0.01 vs Mock, ^‡P<0.05, ^§P<0.01 vs FAM5C.</p

Upregulation of leukocyte adhesion molecules by FAM5C.

<p><b>A and B</b>, Total RNA was extracted from HUVECs 48 h after transfection with 0.1 µg of FLAG (Mock) or FLAG–FAM5C (FAM5C) cDNAs. An increase in FAM5C mRNA levels (<b>A</b>) (n = 3). and upregulation of ICAM-1, VCAM-1 and E-selectin mRNA levels (<b>B</b>) (n = 3) were analyzed by qPCR. <b>C</b>, 48 h after transfection, monocyte adhesion assays were performed and the numbers of monocytes that adhered to the HUVEC monolayers were analyzed (n = 5). HUVECs incubated with 10 ng/ml TNF-α for the last 16 h were used as the controls. *P<0.05, ^†P<0.01 vs Mock.</p

Upregulation of FAM5C mRNA by inflammatory stimuli in an NF-κB- and JNK-dependent manner.

<p><b>A</b>, HUVECs were cultured in the presence of 10 ng/ml TNF-α, 50 ng/ml IL-6, 10 ng/ml IL-1β, or 10 ng/ml LPS for 6 or 24 h (n = 11). <b>B and C</b>, HUVECs were incubated with 10 µM BAY 11-7085 (BAY), SP600125 (SP), SB203580 (SB), or DMSO (D) for 30 min and then cultured in the presence of 10 ng/ml TNF-α (<b>B</b>) (n = 8) or 10 ng/ml IL-1β (<b>C</b>) (n = 5) for 6 h. Values are expressed as the fold increase over the unstimulated control. ^*P<0.05, ^†P<0.01 vs control (<b>A</b>); *P<0.05, ^†P<0.01 vs DMSO (<b>B, C</b>). <b>D</b>, Upregulation of FAM5C mRNA expression in aortas of TNF-α-injected mice. RNA was extracted from mouse aortas and brains of the control (n = 5) and TNF-α-injected (n = 5) mice.</p