Chaperonin Containing TCP1 As A Marker For Identification Of ... - PLOS

Optimization of CCT2 staining in the CSS

A limitation with standard CTC detection methods is the reliance on epithelial markers. Such methods could miss cancer cells with hybrid (mixed epithelial and mesenchymal) or mesenchymal features. To include cells in our study that also had mesenchymal markers, we used breast cancer cell lines in which we had previously evaluated CCT2 levels and then determined their expression of epithelial and mesenchymal markers. T47D cells, representative of early-stage luminal A breast cancer, have lower protein levels of CCT2 compared to TNBC cells, like MDA-MB-231 [12, 13, 19]. In our published study of the role of CCT2 in the proliferation of cancer cells, we generated T47D cells that expressed exogenous CCT2 tagged with FLAG (T47D-CCT2) [13]. We assessed these T472-CCT2 cells for SNAI1 (SNAIL), TWIST, EpCAM, vimentin, E-cadherin, and N-cadherin mRNA expression, Fig 3A. SNAIL and TWIST are transcription factors that are associated with the activation of EMT. EpCAM and E-cadherin are epithelial markers, while vimentin and N-cadherin are mesenchymal markers. Statistically significant increases in TWIST (p = 0.0023), N-cadherin (p = 0.0170), and E-cadherin (p = 0.0215) mRNA were present in the T47D-CCT2 cells compared to the lentiviral control cells, with a significant decrease in vimentin (p = 0.0194) mRNA. EpCAM levels decreased, and SNAIL was increased, but neither of these were statistically significant. Therefore, since T47D-CCT2 cells expressed some mesenchymal characteristics without complete loss of epithelial ones, we used these cells for subsequent experiments. According to the online database CCLE, RNAseq expression of EMT markers indicated that T47D cells are more epithelial-like, and TNBC MDA-MB-231 breast cancer cells are more mesenchymal-like (S3 Fig); hence, we also used MDA-MB-231 cells in our experiments. We examined the membrane protein levels of EpCAM, E-cadherin, and N-cadherin by flow cytometry for both cell lines. T47D-CCT2 cells had detectable levels of EpCAM and E-cadherin and lower N-cadherin, Fig 3B. MDA-MB-231 were N-cadherin positive with a slightly lower expression of EpCAM and E-cadherin than T47D-CCT2, Fig 3C. In total, these data demonstrate that these two cell lines represent a range of epithelial and mesenchymal features.

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Fig 3. EpCAM, vimentin, E-cadherin, and N-cadherin expression in MDA-MB-231 and T47D cells.

(A) RT-PCR data comparing lentiviral control (T47D-GFP) (black) with T47D-CCT2 (grey) for expression of EMT genetic markers: SNAIL and TWIST, epithelial markers: EpCAM and E-cadherin, and mesenchymal markers: vimentin and N-cadherin. p-values are shown on the graph. (B) Flow cytometry data detecting surface expression of EpCAM, E-cadherin, and N-cadherin protein in T47D-CCT2 cells (green) compared to isotype controls (grey). (C) Flow cytometry data detecting surface expression of EpCAM, E-cadherin, and N-cadherin proteins in MDA-MB-231 cells (red) compared to isotype controls (grey). All experiments were performed in duplicate.

https://doi.org/10.1371/journal.pone.0264651.g003

To validate the anti-CCT2 antibody for intracellular staining, various antibody concentrations were tested in MDA-MB-231 cells and T47D-CCT2 cells to differentiate specific and non-specific signals under the conditions similar to those occurring during automated CTC collection and staining with the CSS. Saturation of the response was achieved around 20 μg/mL of antibody, S4 Fig. To optimize the cell-based signal for CCT2 in the CSS Analyzer II, we manually performed the intracellular staining for CCT2 in cells [30] and adjusted the exposure time in the CSS Analyzer II and the anti-CCT2 antibody concentration. A 0.2 second exposure time showed the least background noise, while both 8 μg/mL and 12 μg/mL of anti-CCT2 antibody resulted in a detectable signal, S5 Fig. We chose 8 μg/mL as the anti-CCT2 antibody concentration moving forward.