PRELIMINARY   VALIDATION   OF   THE   MULTIANTIBODY   EPEHSIA   DESIGN   WITH   CELL   LINES

CELL LINE MODELS FOR VALIDATION OF NEW DESIGN (D4) AND TARGET ANTIBODY

For testing the utility of this new Ephesia design, we used three different cell lines, one having low EpCAM and high CD44 expression (Hs578T), one having high EpCAM and low CD44 expression (SKBR3) and a cell line having moderate EpCAM and high CD44 expression, Table 3-4-1. All cell lines are originated from breast cancer patients and they were purchased from ATCC. The absence of mycoplasma was regularly tested for all cell lines. More detailed cell culture protocol can be found in the Annex I section.

TABLE 3-4-1 CELL LINES FOR INVESTIGATION OF DIFFERENT EPCAM AND CD44 EXPRESSION PROFILE ON THE NEW DESIGN TARGETING DOUBLE SUBPOPULATION

Cell Lines  EpCAM Level  CD44 Level  Phenotype 

Hs578T  low  high  Mesenchymal‐like, Basal B⁶⁷  MDA‐MB231  low/moderate  high  Mesenchymal‐like, basal B⁶⁷ 

SKBR‐3  high  low  Epithelial , luminal B⁶⁸ 

EpCAM and CD44 expressions were also confirmed by double staining flow cytometry (Figure 3-4-1). According to flow cytometry experiment, it is seen that for MDA-MB231 cell line, EpCAM expression is very low similar to Hs578T cell line. But according to the cell capture experiments with EpCAM beads, MDA-MB231 cell line is captured at a higher rate so either normally MDA-MB231 has higher EpCAM expression and flow cytometry experiment parameters is still need to be optimized or this cell line can differentiate at a higher rate which can increase its EpCAM expression depending on the conditions and passage number of the culture. So, more experiments are needed to conclude better on this observation.

104

FIGURE 3-4-1 EXPRESSIONS OF EPCAM AND CD44 BY FLOW CYTOMETRY, BLUE PEAK IS FLUORESCENCE OF IGG CONTROL CELLS, ORANGE PEAK IS FOR ANTIBODY STAINED CELLS.

105

INVESTIGATING CELL CAPTURE EFFICIENCY WITH CD44-CONJUGATED BEADS ON DOUBLE-ANTIBODY DESIGN

Following the establishment of the of the new design and fluidic protocol as well as the optimization of bead conjugation method, series of capture efficiency experiments were performed to validate the target antibody CD44 with cell lines mimicking possible scenarios of patients’ blood sample having different profiles of CTCs. Majority of the capture efficiency experiments were performed by Sophie Gontier, an internship student, under my supervision.

First, the capture profile of MDA-MB231 cell line was investigated comparing the capture efficiency obtained with only EpCAM or only CD44 antibody, cells lines being spiked into PBS solution. Since this cell line is classified as mesenchymal-like, lower capture efficiency is expected with EpCAM based capture compared to CD44. We obtained a capture efficiency of 41.6% with only EpCAM and 71.6% with only CD44 beads (Figure 3-4-2) suggesting that our EpCAM based method fails to capture efficiently CTCs with mesenchymal-like features confirming other studies discussed in previous sections.

Considering the relationship between EMT process and induction of stem-like cell properties and the fact that MIC subset in CTCs frequently found in stem-cell like population, this result shows that our system loses highly relevant CTC subpopulation.

FIGURE 3-4-2 COMPARISON OF CAPTURE EFFICIENCY FOR MDA-MB231 CELL LINE USING EPHESIA WITH SINGLE ANTIBODY, SPIKING INTO PBS SOLUTION, ERROR BARS ARE STANDARD DEVIATIONS.

106

Later, we have tested the capture profile on the double anybody system with all cell lines spiked into PBS with either 100 cells or 1000 cells. The samples were subjected to first EpCAM and then CD44 antibody capture (Figure 3-4-3).

SKBR3 cell lines are highly epithelial with very high expression of EpCAM, as expected they are mainly captured in the EpCAM chamber, however there is always a proportion of cells captured CD44 capture zone less than 10%. This may be due to the stem-cell-like/mesenchymal population in this cell line giving rise to more dedifferentiated stem-like cell population. By characterizing this population more in detail, it would give clue how this this behavior fits to CSC model.

Capture profile of Hs578T cell line shows how significantly EpCAM based methods can lose CTCs with only 0.3% capture efficiency on EpCAM beads. This cell line is classified as basal like breast cancer with highly expression of mesenchymal markers and it might be the reason why CTCs are detected less frequently compared to other subtypes of breast cancer^69,70. Compared to EpCAM antibody, CD44 may be considered as a good candidate for targeting mesenchymal-like CTCs, which has an 80.3% of capture efficiency for Hs578T cell line. As mentioned before, comparing the EpCAM expressions with flow cytometry, Hs578T and MDA-MB231 shows very similar expressions so it would be expected to obtain similar capture efficiency values, however obviously, MDA-MB231 has a higher EpCAM expression that is captured at rate of 67.6% with EpCAM and 12% with CD44. So probably, flow cytometry experiment should be repeated.

Another remark is higher capture efficiency for MDA-MB231 cell line on EpCAM zone (67.6%) obtained with D4 chip; however this is definitely not due to the design but probably due change in expression of EpCAM in time in the culture in which this cell line might be more prone to transformation/differentiation. Another reason could be the way of counting and spiking the cells. For spiking high number of cells, >500, the cells are counted by Scepter™ 2.0 Cell Counter after trypsinization and the required amount of cells is obtained by series of dilution of the solution in which for each dilution factor there is risk increasing the experimental error and not obtain the expected number of cells in the final solution. Also this protocol is more sensitive as cells sediments so pipetting is very important at low concentration of cells. To avoid these errors in dilution, for very low number of cells, <100, the successive dilutions are performed on 96 well plates and the required number of cells are collected from the wells by counting cells under the microscope before and after retrieval. This way exact number of cells can be calculated more precisely by checking how many cells have been collected and how many are left in the wells.

107

FIGURE 3-4-3 CAPTURE PROFILE OF CELL LINES HAVING VARIOUS EXPRESSION OF EPCAM AND CD44 PERFORMED WITH D4-EPHESIA, ERROR BARS ARE STANDARD DEVIATION.

The capture mode with first EpCAM and then CD44 could give us clue how much and what kinds of cells are missed by EpCAM based methods by further molecular characterization and finally they also demonstrate the feasibility of the two antibodies Ephesia device. We have also been interested in capture profile by reverse mode, first capture on CD44 and then EpCAM; see Figure 3-4-4. These experiments as said before should be repeated since the whole capture efficiency seems more than 100%. However, by only looking at the ratio of what is captured in EpCAM and CD44 could also give some insight about features of CTCs. The ratio of captured cells on EpCAM/CD44 for SKBR-3 is conserved which is around 10-fold, but for MDA-MB231 cell line is not (Figure 3-4-5). Majority of the MDA-MB-231 cells are captured in the first capture zone in both capture mode, either in EpCAM or CD44, this might suggest that this cell line reflect CTCs profile that is transient in EMT process possessing both epithelial and mesenchymal markers.

108

FIGURE 3-4-4 CAPTURE PROFILE OF CELL LINES WITH REVERSE MODE WITH FIRST APPLIED TO CD44 AND THEN EPCAM BASED CAPTURE PERFORMED WITH D4-EPHESIA, ERROR BARS ARE STANDARD DEVIATION.

FIGURE 3-4-5 COMPARISON OF CAPTURE PROFILE BETWEEN DIRECT MODE AND REVERSE

MODE FOR MDA-MB231 AND SKBR-3 CELL LINES 9.8

109