BackgroundCirculating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited.MethodsWe quantified by RT-qPCR CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+, and mammaglobin gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer.ResultsRT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, CK-19 was detected in 42.4%, HER-2 in 13.6%, MAGE-A3 in 21.2%, hMAM in 13.6%, TWIST-1 in 42.4%, and hTERT α+β+ in 10.2%. In 26 patients with verified metastasis, CK-19 was detected in 53.8%, HER-2 in 19.2%, MAGE-A3 in 15.4%, hMAM in 30.8%, TWIST-1 in 38.5% and hTERT α+β+in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch.ConclusionsMolecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.
Type I collagen is the main component of the extra-cellular matrix (ECM). In vitro, under a narrow window of physico-chemical conditions, type I collagen self-assembles to form complex supramolecular architectures reminiscent of those found in native ECM. Presently, a major challenge in collagen-based biomaterials is to couple the delicate collagen fibrillogenesis events with a controlled shaping process in non-denaturating conditions. In this work an ice-templating approach promoting the structuration of collagen into macroporous monoliths is used. Instead of common solvent removal procedures, a new topotactic conversion approach yielding self-assembled ordered fibrous materials is implemented. These collagen-only, non-cross-linked scaffolds exhibit uncommon mechanical properties in the wet state. With the help of the ice-patterned micro-ridge features, Normal Human Dermal Fibroblasts and C2C12 murine myoblasts successfully migrate and form highly-aligned populations within the resulting 3D biomimetic collagen scaffolds. These results open a new pathway to the development of new tissue engineering scaffolds ordered across various organization levels from the molecule to the macropore, and are of particular interest for biomedical applications where large scale 3D cell alignment is needed such as for muscular or nerve reconstruction.
IntroductionComparison studies between different analytical methodologies for circulating tumor cells (CTC) detection and molecular characterization are urgently needed, since standardization of assays is essential before their use in clinical practice.MethodsWe compared three different CTC molecular assays. To avoid discrepancies due to pre-analytical errors we used the same cDNAs throughout our study. CTC were isolated using anti-EpCAM and anti-MUC1 coated magnetic beads from 2 × 5 ml of peripheral blood of 254 early and 51 metastatic breast cancer patients and 30 healthy individuals. The same cDNAs were analyzed by: a) singleplex RT-qPCR assay for CK-19; b) multiplex RT-qPCR for CK-19, HER-2, MAGE- A3, and PBGD; and c) a commercially available molecular assay (AdnaTest BreastCancer) for GA733-2, MUC-1, HER-2 and beta-actin.ResultsIn early breast cancer, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest, were positive for the presence of CTC in 14.2%, 22.8% and 16.5% subjects, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 72.4% while between the AdnaTest and multiplex RT-qPCR was 64.6%. In patients with overt metastasis, CK-19 RT-qPCR, multiplex RT-qPCR and the AdnaTest were positive in 41.2%, 39.2% and 54.9% patients, respectively. The concordance between the AdnaTest and CK-19 RT-qPCR was 70.6% while between the AdnaTest and multiplex RT-qPCR was 68.6%.ConclusionsAll CTC assays gave similar results in about 70% of cases. Better agreement was found in the metastatic setting, possibly explained by the higher tumor load in this group. Discordances could be attributed to the different gene transcripts used to evaluate CTC positivity. Our results indicate the importance of CTC heterogeneity for their detection by different analytical methodologies.
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