Third Harmonic Generation (THG) microscopy as a non-invasive, label free imaging methodology, allows linkage of lipid profiles with various breast cancer cells. The collected THG signal arise mostly from the lipid droplets and the membrane lipid bilayer. Quantification of THG signal can accurately distinguish HER2-positive cells. Further analysis using Fourier transform infrared (FTIR) spectra reveals cancer-specific profiles, correlating lipid raft-corresponding spectra to THG signal, associating thus THG to chemical information. THG imaging of a cancer cell.

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Nonlinear optical imaging techniques have created new opportunities of research in the biomedical field.Specifically, Third Harmonic Generation (THG) seems to be a suitable noninvasive imaging tool for the delineation and quantification of biological structures at the microscopic level. The aim of this study was to extract information as to the activation state of different cell types by using the THG imaging microscopy as a diagnostic tool.BV-2 microglia cell line was used as a representative biological model enabling the study of resting and activated state of the cells linked to various pathological conditions. Third Harmonic Generation (THG) andTwo Photon Excitation Fluorescence (TPEF) measurements were simultaneously collected from stained breast cancer cells, by employing a single homemade experimental apparatus and it was shown that high THG signals mostly arise from lipid bodies. Continuously, BV-2 microglia cells were examined with or without activation by lipopolysaccharide (LPS) in order to discriminate between control and activated cells based on the quantification of THG signals. Statistically quantification was accomplished in both mean area and mean intensity values of THG. The values for mean total area and mean THG intensity values have been increased in activated versus the non-activated cells. Similar studies of quantification are underway in breast cancer cells for the exact discrimination on different cell lines. Furthermore, laser polarization dependence of SHG and THG signal in unstained biological samples is investigated.

Triple negative (ER-negative, PR-negative, HER2-negative) considered as poor prognosis breast cancer patients without many therapeutic options. Circulating Tumor Cells (CTCs) have been proposed as a “real time liquid biopsy” in breast cancer patients. CTCs are associated with disease relapse and could serve as a target for molecular cancer therapies. The aim of the present study was the phenotypic characterization of CTCs in triple negative patients to explore new therapeutic strategies for these patients. We evaluated peripheral blood mononuclear cells (PBMC) cytospins from 29 triple negative patients, before the initiation of adjuvant (19 early) and front-line (10 metastatic) treatment. The expression of Cytokeratins (CK) (marker for CTCs), Estrogen Receptor (ER), Progesterone Receptor (PR), EGFR and HER2 in CTCs was assessed using double immunofluorescent experiments. Our results revealed that median expression per patient for ER, PR, EGFR and HER2 was 16.7%, 0%, 42.9% and 91.4%. The respective numbers for early versus (vs) metastatic patients were 29.7% vs 0% for ER, 27.1% vs 0% for PR, 70.8% vs 52.3% for EGFR and 50% vs 48.7% for HER2. In addition ER, PR, EGFR and HER2 were expressed in 33.1%, 28%, 67.7% and 45.1%, respectively, of the total examined CTCs. Triple staining experiments with the Ariol system could not revealed any no co-expression of CK/EGFR/ER in CTCs; however, there was only one patient with CTCs co-expressing CK/HER2/ER. Consequently in order to identify whether chemotherapy could induce phenotypic changes of CTCs, 18 patients were analyzed after the completion of adjuvant treatment. ER, PR, EGFR and HER2 median expression per patients was identified in 0.2%, 15.2%, 66.7%, and 50% suggesting that adjuvant chemotherapy does not significantly influence the phenotype of CTCs in triple negative patients.The majority of CTCs in triple negative patients revealed expression of HER2 and EGFR implying that these receptors could be a potential target for the limitation of metastasis in these patients. Citation Format: Galatea Kallergi, Haris Markomanolaki, Dimitrios Mavroudis, Vassilis Georgoulias, Sofia Agelaki. Phenotypic characterization of circulating tumor cells (CTCs) in triple negative breast cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5107. doi:10.1158/1538-7445.AM2013-5107

e22101 Background: Clinical dormancy is frequently observed in breast cancer (BC). In the present study, we aimed to characterize CTCs in dormancy candidates (DC) with BC in terms of proliferation and apoptosis. Methods: Cytospins of peripheral blood mononuclear cells (PBMCs) were obtained from DC (n=122) disease-free for ≥5 yrs and from metastatic pts (n=37) on relapse that occurred ≥5 yrs after surgery. Sequential samples (n=27) of 8 DC with late relapse and from 8 relapse-free (n=38), were also analyzed. 106 PBMCs were stained with pancytokeratin antibody along with ki-67 and M30 as proliferation and apoptosis markers, respectively. Results: CTCs were identified in 40 (32%) of 122 DC. In 25 (61.5%), all CTCs were ki-67(-)/M30(-), 7 (17.5%) had ki-67(+), 4 (10%) M30(+) CTCs and 4 had both phenotypes. Among 243 CTCs detected, 201 (82.5%) were ki-67(-)/M30(-), 14 (5.7%) ki-67(+) and 29 (11.9%) M30(+). Of 13 (35%) CTC(+) metastatic pts, 6 (46%) had ki-67(+) CTCs (p=0.037) whereas 54 (40%) of total CTCs were ki-67(+) (p<0.001). No M30(+) CTCs were detected. When sequential samples of the 8 DC who relapsed were analyzed, 2 (25%), had only ki-67(-)/M30(-) CTCs, 6 (75%) had ki-67(+) and 4 (50%) M30(+) CTCs. The respective numbers in the non-relapsed group were 4 (50%), 3 (37.5%) and 3 (37.5%). Moreover, ki-67(+) CTCs were more frequently observed in samples of the relapsed pts (29.6% vs 13.1%). Among 382 CTCs detected in all sequential samples from the relapsed group, 337 (88.14%) were ki-67(-)/M30(-), 26 (6.7%) ki-67(+) and 20 (5.1%) M30(+), whereas the respective percentages among 78 CTCs in non-relapsed pts were 77.6%, 6.5% and 15.8% (p=0.001). Conclusions: The great majority of CTCs detected in DC with BC express neither proliferation, nor apoptosis markers. However, the apoptotic index in CTCs is increased during clinical dormancy, whereas the proliferation index is increased on relapse. In addition, apoptotic CTCs are more frequently encountered during follow-up in DC free of relapse. The above observations suggest that monitoring proliferation and apoptosis in CTCs during clinical dormancy could be used to predict subsequent late relapse.