Putative roles of EMT and mesenchymal-to-epithelial transition (MET) in tumour cell dissemination. In the primary tumour, a subpopulation of tumour cells (red) acquires a mesenchymal-like migratory phenotype during tumour progression. They lose their epithelial properties (ie, downregulate EpCAM and CK) through the EMT process and enter the bloodstream. These specific CTCs are thought to have stem cell properties. After extravasation into distant organs, these tumour cells (DTCs) have to re-express their epithelial properties through MET to form tumour cell clusters (micrometastases). Another subpopulation of CTCs, which is not able to undergo EMT (blue), can also disseminate through the bloodstream into distant organs but lacks cancer stem cell properties and, therefore, does not form (micro)metastases. These cells are detected by current CTC technologies, whereas the EMT-induced CTCs (red) are missed. This figure is reprinted from the article by Pantel K, et al. (42).

Epithelial (E-cadherin) and mesenchymal (vimentin) markers in circulating tumor microemboli (CTM) and circulating tumor cells (CTCs) profiled by dual-color IHC. Blood samples from lung cancer patients were subjected to ISET filtration and CTM/CTCs were profiled for both epithelial (E-cadherin) and mesenchymal (vimentin) markers by dual-color IHC. (A) Vimentin-positive CTM (cytoplasm stained in red) with negative staining for E-cadherin (brown). (B) E-cadherin positive CTM (brown) without vimentin expression. (C) Displays CTC (black arrow), which is positively stained for E-cadherin in cytosol and negative for vimentin. White blood cells (white arrow in C and cells in D) serve as control and demonstrate positive staining for vimentin, negative staining for E-cadherin, and typical neutrophil nuclear morphology. This picture is reprinted from the article by Hou JM, et al. (17).

Illustrative cartoon demonstrating a patient with non-small cell lung cancer (NSCLC) donating a tube of peripheral blood which is then processed in the circulating tumor cell (CTC)-chip immediately, without any required preprocessing. CTCs are captured against the sides of the anti-Ep-CAM-coated posts (epithelial cell adhesion molecule), and then can be stained with fluore-scently-labeled markers for enumeration or undergo genomic DNA extraction for epidermal growth factor receptor mutation or other molecular analysis. This picture is reprinted from the article by Sequist LV, et al. (28).

Deposition of a single CTC on the AmpliGrid chip. (A) AmpliGrid chip with 48 reaction sites for single cell deposition and PCR analysis. (B) A single CTC is deposited with a capillary on the reaction site of the Ampligrid chip. CTCs are normally resuspended in physiological buffer and the volume of deposition is approxi-mately 20 nl. (C) A single CTC can be easily visualised on the reaction site when the DNA is stained with Hoechst dye. This pricture is reprinted from the article by Alunni-Fabbroni M, et al. (43).

(A) Distribution of CTC count in primary lung cancer patients without distant metastasis and in patients with distant metastasis. (B) ROC curves for CTC count and serum CEA level to predict the absence or presence of distant metastasis among primary lung cancer patients. CTC: Circulating tumor cell, CEA: Carcinoembryonic Antigen. This picture is reprinted from the article by Tanaka F, et al. (36).

Disease progression and progression-free survival in surgically resected NSCLC patients according to paired presurgery and postsurgery TTF-1 (＋) CTC status. When pairing presurgery and postsurgery TTF-1 (＋) CTC status and allocating patients to four groups, the Pre (−) Post (＋) group (TTF-1 (＋) CTCs were not present at presurgery, but detected at postsurgery time) most frequently developed disease progression (A), and showed shortest progression-free survival (B) as compared to other three groups. This picture is reprinted from the article by Yoon SO, et al. (37).

Serial Analyses of Circulating Tumor Cells during Therapy. (A) Shows serial analyses of the numbers of circulating tumor cells (CTCs) per milliliter and the radiographic tumor burden in centimeters in four patients with non-mall-cell lung cancer with EGFR mutations, as measured at multiple time points during the course of treatment with gefitinib, another chemotherapy agent (chemo), or an experimental agent (exp). The duration of each therapy is indicated by the gray bars. The genotypes of circulating tumor cells are shown for various time points. Mutations in brackets are those that were present at low allele frequencies. (B) SARMS analysis of EGFR genotypes in Patient 9 shows an increased abundance of the T790M drug-resistance allele during disease progression. Arrows denote the cycle of threshold for amplification cycles (Ct) required for detection of the primary mutation (Del or Deletion, referring to the grouped exon 19 deletions) and the T790M mutation, as compared with the exon 2 control. Δ Ct reflects the difference in allele frequency between the primary mutation and T790M in the tumor-biopsy specimen, the circulating tumor cells that were isolated at the time of response to gefitinib therapy, and the circulating tumor cells that were isolated at the time of disease progression. This picture is reprinted from the article by Maheswaran S, et al. (38).

Correlation of circulating tumor cell (CTC) counting and computed tomography (CT) scanning with a follow-up study. Correlation of CT scanning and CTC counting was evaluated on a small scale of follow-up study. CTC was enumerated from each patient before chemotherapy started. After two courses of first line chemotherapy, patient CTC was measured, followed by CT examination. Eight of 12 patients (patient 1-) including 5 adenocarcinoma (ADC), 2 squamous cell carcinoma (SCC) and 1 small cell lung cancer (SCLC) were classified by CT as stable disease. Among those 8 patients, 5 patients dropped their CTC to 0, 1 patient dropped from 21 to 3, 2 patients remained 0, and in 1 patient the count increased from 2 to 3. Three patients had a partial radiologic response: For partial response (PR) patients (patients 9-1), one was noted to have a CTC drop from 52 to 0, while the CTC in the other 2 patients remained below 2. Only one patient was noted to have an increasing CTC, from 2 to 12 after treatment, and this patient was confirmed to have progressive disease (PD). Clinical responses are classified by CT scanning according to the Response Evaluation Criteria in Solid Tumors (RECIST). This pricture is reprinted from the article by Wu C, et al. (39).