Mesenchymal Phenotype of CTC-Enriched Blood Fraction and Lymph Node Metastasis Formation Potential

Abstract

IntroductionCirculating tumor cells (CTCs) that present mesenchymal phenotypes can escape standard methods of isolation, thus limiting possibilities for their characterization. Whereas mesenchymal CTCs are considered to be more malignant than epithelial CTCs, factors responsible for this aggressiveness have not been thoroughly defined. This study analyzed the molecular profile related to metastasis formation potential of CTC-enriched blood fractions obtained by marker unbiased isolation from breast cancer patients without (N−) and with lymph nodes metastases (N+).Materials and MethodsBlood samples drawn from 117 patients with early-stage breast cancer were enriched for CTCs using density gradient centrifugation and negative selection with anti-CD45 covered magnetic particles. In the resulting CTC-enriched blood fractions, expression of CK19, MGB1, VIM, TWIST1, SNAIL, SLUG, HER2, CXCR4 and uPAR was analyzed with qPCR. Results were correlated with patients' clinicopathological data.ResultsCTCs (defined as expression of either CK19, MGB1 or HER2) were detected in 41% (20/49) of N− and 69% (34/49) of N+ patients (P = 0.004). CTC-enriched blood fractions of N+ patients were more frequently VIM (P = 0.02), SNAIL (P = 0.059) and uPAR-positive (P = 0.03). Positive VIM, CXCR4 and uPAR status correlated with >3 lymph nodes involved (P = 0.003, P = 0.01 and P = 0.045, respectively). In the multivariate logistic regression MGB1 and VIM-positivity were independently related to lymph node involvement with corresponding overall risk of 3.2 and 4.2. Moreover, mesenchymal CTC-enriched blood fractions (CK19−/VIM+ and MGB1+ or HER2+) had 4.88 and 7.85-times elevated expression of CXCR4 and uPAR, respectively, compared with epithelial CTC-enriched blood fractions (CK19+/VIM− and MGB1+ or HER2+).ConclusionsTumors of N+ patients have superior CTC-seeding and metastatic potential compared with N- patients. These differences can be attributed to VIM, uPAR and CXCR4 expression, which endow tumor cells with particularly malignant phenotypes.

The Landscape of Circulating Tumor Cell Research in the Context of Epithelial-Mesenchymal Transition

“…Presence of mesenchymal markers was higher in MBC patients than in early stage BC patients [129,130] and was associated with lymph node involvement [20] , suggesting that EMT phenotype is directly related to the metastatic potential of CTCs. However, plastin-3 expressed in CTCs undergoing EMT was found with a similar frequency in all stages of BC, from I to IV [42] .…”

“…Further studies showed that mesenchymal CTCs are detected in 41% of MBC patients, especially in patients with triple negative or HER2+ tumors, where mesenchymal CTCs are the major subpopulation of CTCs [19] . Patients whose CTCs expressed EMT markers had a worse prognosis than patients with fully epithelial CTCs or no CTCs [4,20] . Therefore, clinical data point to asymmetry in frequency and malignancy of epithelial/mesenchymal phenotypes of CTCs, which has meaningful clinical implications.…”

Immune System and the Efficacy of Cancer Treatment

“…Оптимальные условия для индукции ЭМП возникают при развитии в строме опухоли варианта воспаления, основным эффектором которого являются макрофаги с М2-фенотипом. Именно они секретируют ростовой фактор TGFβ, индуцирующий ЭМП (Giordano A. et al, 2012;Mego M. et al, 2012;Markiewicz A. et al, 2014). В результате ЭМП клетки опухоли приобретают свойства стволовости и выходят в кровоток, что является необходимым условием диссеминации.…”

Circulating Tumor Cells in Breast Cancer: A Potential Liquid Biopsy

“…Likewise, Papadaki et al found that nuclear Twist localization was detected in the CTCs of 70.3% of MBC patients, whereas it was detected in only 32.3% of CTCs from early breast cancer patients[89]. Moreover, Markiewicz et al found that CTCs isolated from lymph node-positive breast cancer patients are more frequently Vimentin and Snail mRNA expression positive compared to those from lymph node-negative patients[95]. Polioudaki et al reported that 1-year OS of patients with high cytokeratin + CTCs was 73.3%, whereas 1-year OS declined by 46.2% in patients with low cytokeratin + CTCs (p = 0.038)[91].…”