Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a "liquid biopsy". In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as "gold standard" reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0-32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 μm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1-4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

The presence of high expressing epithelial cell adhesion molecule (EpCAMhigh) circulating tumor cells (CTC) enumerated by CellSearch® in blood of cancer patients is strongly associated with poor prognosis. This raises the question about the presence and relation with clinical outcome of low EpCAM expressing CTC (EpCAMlow CTC). In the EU-FP7 CTC-Trap program, we investigated the presence of EpCAMhigh and EpCAMlow CTC using CellSearch, followed by microfiltration of the EpCAMhigh CTC depleted blood. Blood samples of 108 castration-resistant prostate cancer patients and 22 metastatic breast cancer patients were processed at six participating sites, using protocols and tools developed in the CTC-Trap program. Of the prostate cancer patients, 53% had ≥5 EpCAMhigh CTC and 28% had ≥5 EpCAMlow CTC. For breast cancer patients, 32% had ≥5 EpCAMhigh CTC and 36% had ≥5 EpCAMlow CTC. 70% of prostate cancer patients and 64% of breast cancer patients had in total ≥5 EpCAMhigh and/or EpCAMlow CTC, increasing the number of patients in whom CTC are detected. Castration-resistant prostate cancer patients with ≥5 EpCAMhigh CTC had shorter overall survival versus those with <5 EpCAMhigh CTC (p = 0.000). However, presence of EpCAMlow CTC had no relation with overall survival. This emphasizes the importance to demonstrate the relation with clinical outcome when presence of CTC identified with different technologies are reported, as different CTC subpopulations can have different relations with clinical outcome.

Circulating tumor cells (CTCs), potential precursors of most epithelial solid tumors, are mainly enriched by epithelial cell adhesion molecule (EpCAM)-dependent technologies. Hence, these approaches may overlook mesenchymal CTCs, considered highly malignant. Our aim was to establish a workflow to enrich and isolate patient-matched EpCAMhigh and EpCAMlow/negative CTCs within the same blood samples, and to investigate the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutational status within single CTCs. We sequentially processed metastatic breast cancer (MBC) blood samples via CellSearch® (EpCAM-based) and via Parsortix™ (size-based) systems. After enrichment, cells captured in Parsortix™ cassettes were stained in situ for nuclei, cytokeratins, EpCAM and CD45. Afterwards, sorted cells were isolated via CellCelector™ micromanipulator and their genomes were amplified. Lastly, PIK3CA mutational status was analyzed by combining an amplicon-based approach with Sanger sequencing. In 54% of patients′ blood samples both EpCAMhigh and EpCAMlow/negative cells were identified and successfully isolated. High genomic integrity was observed in 8% of amplified genomes of EpCAMlow/negative cells vs. 28% of EpCAMhigh cells suggesting an increased apoptosis in the first CTC-subpopulation. Furthermore, PIK3CA hotspot mutations were detected in both EpCAMhigh and EpCAMlow/negative CTCs. Our workflow is suitable for single CTC analysis, permitting—for the first time—assessment of the heterogeneity of PIK3CA mutational status within patient-matched EpCAMhigh and EpCAMlow/negative CTCs.

BackgroundGrb2-associated binder 2 (Gab2), a scaffolding adaptor protein, has recently been implicated in cancer progression. However, the role of Gab2 in the progression and metastasis of colorectal cancer (CRC) remains unclear.MethodsGab2 expression was assessed in CRC patient specimens as well as in CRC cell lines. Recombinant lentivirus vector containing Gab2 gene and its small interfering RNAs were constructed and introduced into CRC cells. Cell migration and invasion ability were evaluated by transwell assays in vitro, and in vivo metastasis was performed on nude mice model. Moreover, the expression of Gab2 and epithelial-to-mesenchymal transition (EMT)-associated proteins (E-cadherin and vimentin) were assessed by western blot and qRT-PCR in CRC cells to evaluate the correlation between Gab2 and EMT. Finally, we evaluated the impact of Gab2 on the activation of its downstream signaling effectors, and furthermore the effects of these pathways on Gab2 induced-EMT were also detected.ResultsWe confirmed that increased Gab2 expression correlated with higher tumor node metastasis stage and highly invasive CRC cell lines. Ectopic expression of Gab2 promoted metastasis of CRC cells, whereas silencing of Gab2 resulted in inhibited metastasis both in vitro and in vivo. Overexpression of Gab2 in CRC cells induced EMT, whereas knockdown of Gab2 had the opposite effect. Furthermore, upregulation of Gab2 expression obviously stimulated the activation of extracellular signal-regulated kinase-1/2 (ERK1/2), and increased the expression of matrix metalloproteinase-7 (MMP7) and matrix metalloproteinase-9 (MMP9) in CRC cells. Conversely, downregulation of Gab2 expression significantly decreased the activation of ERK1/2, and inhibited MMP7 and MMP9 expression. U0126, an inhibitor of mitogen-activated protein kinase (MEK), can reverse the effects of Gab2 on EMT.ConclusionsOur work highlights that Gab2 induces EMT through the MEK/ERK/MMP pathway, which in turn promotes intestinal tumor metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0280-0) contains supplementary material, which is available to authorized users.

Background: Inositol polyphosphate 4-phosphatase type II (INPP4B) has been identified as a negative regulator of phosphatidyl inositol 3-kinase (PI3K)/Akt signaling in human several cancers. However, the expression, clinical significance and biological function of INPP4B in human hepatocellular carcinoma (HCC) clinical tissues and cell lines are little known. Materials and methods: We evaluated the expression of INPP4B in 86 cases of paired human HCC samples by immunohistochemistry, and the clinical significance of INPP4B expression was analyzed. The expression of INPP4B in five HCC cell lines was detected through using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses. The role of INPP4B gene on HCC cell proliferation, apoptosis, migration, invasion as well as epithelial-to-mesenchymal transition (EMT) and chemoresistance was examined via INPP4B mammalian expression vector and small interfering RNA (siRNA) transfection in vitro. Western blot analysis was used to explore the downstream molecules modulated by INPP4B. Results: Immunohistochemistry analysis revealed that INPP4B was significantly downregulated in HCC tissues compared with the corresponding normal tissues. The rate of INPP4B-positive staining was markedly lower in metastatic samples than in those of non-metastatic samples. Univariate analysis showed that INPP4B expression was indicated to have a marked association with histological grades, tumor size and tumor metastasis. Moreover, INPP4B overexpression suppressed cell proliferation, migration, invasion and EMT, but induced cell apoptosis and chemosensitivity in human HCC cell lines. In contrast, INPP4B knockdown had the opposite effects on the biological behaviors of HCC cells. Furthermore, INPP4B was found to inhibit the activation of PI3K/Akt signaling in HCC cells. Conclusion: Our findings suggest that INPP4B is a tumor suppressing gene in human HCC, and might act as a novel therapeutic target for HCC patients.