Therefore, we decided EGFR expression on different epithelial tumor cell lines used in our assays. metastasis development. Importantly, preoperative treatment with specific tumor-targeting monoclonal antibodies (mAb) prevented surgery-induced liver metastasis development in rats. This study investigated whether the epidermal growth factor receptor (EGFR) represents a suitable target for preoperative antibody treatment of colorectal Cabergoline malignancy patients undergoing surgery. The majority of patients with resectable colorectal liver metastases were shown to have EGFR?+?CTCs. Three different anti-EGFR mAbs (cetuximab, zalutumumab, and panitumumab) were equally efficient in the opsonization of tumor Cabergoline cell lines. Additionally, all three mAbs induced antibody-dependent cellular phagocytosis (ADCP) of tumor cells by macrophages at low antibody concentrations and induced phagocytosis. Furthermore, neither proliferation nor migration of epithelial cells was affected migration/wound healing experiments (Cat no: 80209, Ibidi, Martinsried, Germany). Culture inserts were placed in 24 well values <0.05 were considered statistically significant. 3. Results 3.1. Colorectal Malignancy Patients Have Increased Numbers of EpCAM?+?EGFR?+?Cells in Their Blood circulation We optimized the protocol based on the detection of CTCs in blood by circulation cytometry to be Cabergoline able to detect EGFR?+?CTCs [39]. Healthy donor blood samples were spiked with no, 50, 100, or 500 HT29 cells. After isolation of the PBMC layer and enrichment by EpCAM beads, samples were stained for the presence of EpCAM?+?EGFR?+?cells. Tumor cell recovery was more than 85% in samples spiked with as low as 100 HT29 cells (Supplementary Physique 1(a)). Additionally, we investigated whether cetuximab binding interferes with the detection of CTCs, as patients will be treated with cetuximab prior to medical procedures in a clinical establishing. We confirmed that tumor cells that had been preincubated with cetuximab showed similar binding to the anti-EGFR detection antibody (Supplementary Physique 1(b)), indicating that both anti-EGFR antibodies bind to different epitopes and therefore do not interfere with each other. Next, the number of EpCAM?+?EGFR?+?cells in blood samples of patients with metastatic colorectal malignancy Cabergoline was determined. Increased numbers of EpCAM?+?EGFR?+?cells were detected in the majority of the patients compared to healthy donors (Physique 1). Open in a separate window Physique 1 Detection of EpCAM?+?EGFR?+?cells in peripheral blood samples. Absolute numbers of EpCAM?+?EGFR?+?cells in blood samples of healthy donors (open diamonds) and colorectal malignancy patients (closed diamonds). < 0.05. 3.2. Comparable Opsonization of EGFR-Expressing Tumor Cells by Cetuximab, Panitumumab, and Zalutumumab Although approximately Rabbit Polyclonal to RHOBTB3 80% of colorectal malignancy patients have EGFR-expressing tumors, the level of EGFR expression can differ between tumors. Therefore, we decided EGFR expression on different epithelial tumor cell lines used in our assays. The epidermoid carcinoma cell collection A431 experienced high EGFR overexpression (>300,000 molecules per cell) (Table 1). The colorectal carcinoma cell lines Caco2, HCT116, HT29, and SW948 experienced similar EGFR expression, ranging from 20,000 to 40,000 molecules per cell. The colorectal carcinoma cell collection RKO experienced about 10 occasions less EGFR expression, whereas the colorectal carcinoma cell collection SW620 hardly expressed EGFR. Tumor cells were opsonized by different concentrations of the anti-EGFR mAbs in a dose-dependent manner. In most cases, 0.05?< 0.05, < 0.01. Table 1 EGFR expression. < 0.05, < 0.01 compared to the least expensive concentration. Next, culture inserts were used to mimic wound healing of a gap (Physique 4(a)). Untreated HCT116 cells were able to completely close the space in approximately 27?h (Physique 4(b)). No significant differences were observed in the presence of the different anti-EGFR mAbs at concentrations up to 30?< 0.01. 4. Conversation The presence of CTCs correlates with poor survival in patients with colorectal malignancy, even after resection of the primary tumor/liver metastases with curative intention [11, 12]. Perioperative treatment that eliminates CTCs may significantly improve individual outcomes, as perioperative chemotherapy has shown limited clinical efficacy [23]. In this study, we exhibited that EGFR.