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Morphological features of breast cancer circulating tumor cells in blood after physical and biological type of isolation

Tanja Jesenko
Tanja Jesenko
, 
Ziva Modic
Ziva Modic
, 
Cvetka Grasic Kuhar
Cvetka Grasic Kuhar
, 
Maja Cemazar
Maja Cemazar
, 
Urska Matkovic
Urska Matkovic
, 
Simona Miceska
Simona Miceska
, 
Jerneja Varl
Jerneja Varl
, 
Anamarija Kuhar
Anamarija Kuhar
 und 
Veronika Kloboves-Prevodnik
Veronika Kloboves-Prevodnik
   | 10. Aug. 2021
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Article Category: Research Article
Online veröffentlicht: 10. Aug. 2021
Seitenbereich: 292 - 304
Eingereicht: 05. Juli 2021
Akzeptiert: 09. Juli 2021
DOI: https://doi.org/10.2478/raon-2021-0033
© 2021 Tanja Jesenko, Ziva Modic, Cvetka Grasic Kuhar, Maja Cemazar, Urska Matkovic, Simona Miceska, Jerneja Varl, Anamarija Kuhar and Veronika Kloboves-Prevodnik, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1

Schematic presentation of both separation techniques. (A) Parsortix® separation cassette contain a stepped structure, gradually narrowing in diameter until reaching a final gap of 6.5 μm, therefore all of the cells that are larger than 6.5 μm are retained and isolated while all smaller cells continue to flow through the cassette into a waste container. The technique enables the isolation of CTCs with different phenotypes. After the whole sample has been processed, the liquid flow is reversed and CTCs can be harvested. (B) MACS® separation column uses magnetic beads covered with anti-epithelial cell adhesion molecule (EpCAM) antibodies for positive selection of CTCs with epithelial origin. When magnetic bead labeled CTCs are passed through a dense magnetic column, they are retained by a strong magnetic field. Other non-labeled cells are passed through the column into the waste tube. After the whole sample has been passed through the column, the column is removed from the magnet and retained CTCs can be eluted.
Schematic presentation of both separation techniques. (A) Parsortix® separation cassette contain a stepped structure, gradually narrowing in diameter until reaching a final gap of 6.5 μm, therefore all of the cells that are larger than 6.5 μm are retained and isolated while all smaller cells continue to flow through the cassette into a waste container. The technique enables the isolation of CTCs with different phenotypes. After the whole sample has been processed, the liquid flow is reversed and CTCs can be harvested. (B) MACS® separation column uses magnetic beads covered with anti-epithelial cell adhesion molecule (EpCAM) antibodies for positive selection of CTCs with epithelial origin. When magnetic bead labeled CTCs are passed through a dense magnetic column, they are retained by a strong magnetic field. Other non-labeled cells are passed through the column into the waste tube. After the whole sample has been passed through the column, the column is removed from the magnet and retained CTCs can be eluted.

Figure 2

Parsortix® and MACS® separation techniques induced hydropic degeneration of cells. (A) Giemsa stained, (B) AE1/ AE3 cytokeratin stained and (C) estrogen receptor stained non-separated MCF7 cells (No separation) and separated MCF7 cells by Parsortix® (Parsortix®) or MACS® (MACS®). Scale bar represents 50 μm. Note that estrogen receptor stain is nuclear therefore cells appear smaller due to the poor contrasting of the cytoplasm. (D) Presentation of measured and evaluated cell sizes. Non-separated and Parsortix® or MACS® separated (E) cell diameter of, n=100 cells, (F) nuclear diameter, n=100 cells and (G) thickness of cytoplasm, n=100 cells. (H) Cytoplasmic staining of non-separated MCF7 cells and Parsortix® or MACS® separated cells, n= 100 cells. Values in (E, F, G) represent median with interquartile range and (H) mean with 95% confidence interval. Statistical significance was determined by Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparisons test for (E, F, G) and by Fisher’s exact test for (H).** = p< 0.01; *** = p<0.001; **** = p< 0.0001; ns = not significant
Parsortix® and MACS® separation techniques induced hydropic degeneration of cells. (A) Giemsa stained, (B) AE1/ AE3 cytokeratin stained and (C) estrogen receptor stained non-separated MCF7 cells (No separation) and separated MCF7 cells by Parsortix® (Parsortix®) or MACS® (MACS®). Scale bar represents 50 μm. Note that estrogen receptor stain is nuclear therefore cells appear smaller due to the poor contrasting of the cytoplasm. (D) Presentation of measured and evaluated cell sizes. Non-separated and Parsortix® or MACS® separated (E) cell diameter of, n=100 cells, (F) nuclear diameter, n=100 cells and (G) thickness of cytoplasm, n=100 cells. (H) Cytoplasmic staining of non-separated MCF7 cells and Parsortix® or MACS® separated cells, n= 100 cells. Values in (E, F, G) represent median with interquartile range and (H) mean with 95% confidence interval. Statistical significance was determined by Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparisons test for (E, F, G) and by Fisher’s exact test for (H).** = p< 0.01; *** = p<0.001; **** = p< 0.0001; ns = not significant

Figure 3

Parsortix® and MACS® separation techniques induced degenerative cytoplasmic changes but retained the viability of MCF7 cells. (A) Morphologically non-degenerated cells presented with homogenous cytoplasm and intact smooth plasma membrane. Scale bar represents 10 μm. (B) Cytoplasmic villous projections as observed degenerative changes are indicated by black arrow. Scale represents 10 μm. (C) Membrane blebbing (indicated by black arrow) and cytoplasmic vacuolization (indicate by white arrow) as observed degenerative changes. Scale represents 10 μm. (D) Fraction of cells with villous projections, blebs or vacuolization. Values represent mean with 95% confidence interval, n = 100 cells. Statistical significance was determined by Fisher’s exact test. * = p<0.05, ** = p< 0.01, *** = p<0.001, **** = p< 0.0001, ns = not significant. (E) Representative dot plots of Annexin V and 7AAD staining in non-separated cells and Parsortix and MACS separated cells (F) Percent of live cells (Annexin V-, 7AAD-), n=3. The values represent mean ± (SE), n = number of biological replicates. Statistical significance was determined by one-way ANOVA followed by a Dunnett’s multiple comparisons test; ns- not significant.
Parsortix® and MACS® separation techniques induced degenerative cytoplasmic changes but retained the viability of MCF7 cells. (A) Morphologically non-degenerated cells presented with homogenous cytoplasm and intact smooth plasma membrane. Scale bar represents 10 μm. (B) Cytoplasmic villous projections as observed degenerative changes are indicated by black arrow. Scale represents 10 μm. (C) Membrane blebbing (indicated by black arrow) and cytoplasmic vacuolization (indicate by white arrow) as observed degenerative changes. Scale represents 10 μm. (D) Fraction of cells with villous projections, blebs or vacuolization. Values represent mean with 95% confidence interval, n = 100 cells. Statistical significance was determined by Fisher’s exact test. * = p<0.05, ** = p< 0.01, *** = p<0.001, **** = p< 0.0001, ns = not significant. (E) Representative dot plots of Annexin V and 7AAD staining in non-separated cells and Parsortix and MACS separated cells (F) Percent of live cells (Annexin V-, 7AAD-), n=3. The values represent mean ± (SE), n = number of biological replicates. Statistical significance was determined by one-way ANOVA followed by a Dunnett’s multiple comparisons test; ns- not significant.

Figure 4

Parsortix® separation enabled identification of more CTCs and their size was larger compared to MACS® separation. (A) Total number of identified CTCs in the patient cohort. (B) Number of identified CTCs for each individual patient after Parsortix® or MACS® separation. (C) Mean cell diameter of CTCs after Parsortix® or MACS® separation. (D) Histogram showing CTC size distribution after Parsortix® separation and (E) MACS® separation.**** = p< 0.0001; n = total number of cells
Parsortix® separation enabled identification of more CTCs and their size was larger compared to MACS® separation. (A) Total number of identified CTCs in the patient cohort. (B) Number of identified CTCs for each individual patient after Parsortix® or MACS® separation. (C) Mean cell diameter of CTCs after Parsortix® or MACS® separation. (D) Histogram showing CTC size distribution after Parsortix® separation and (E) MACS® separation.**** = p< 0.0001; n = total number of cells

Figure 5

Effect of separation techniques on morphology of breast cancer CTCs. (A) Number of morphologically preserved or unpreserved CTCs after Parsortix® separation. (B) Number of morphologically preserved or unpreserved CTCs after MACS® separation. (C) Images of CTCs with preserved or unpreserved morphology after both separation techniques. Scale bar represents 20 μm. Cells were stained with Giemsa (G) and cytokeratin AE1/AE3 (CK).
Effect of separation techniques on morphology of breast cancer CTCs. (A) Number of morphologically preserved or unpreserved CTCs after Parsortix® separation. (B) Number of morphologically preserved or unpreserved CTCs after MACS® separation. (C) Images of CTCs with preserved or unpreserved morphology after both separation techniques. Scale bar represents 20 μm. Cells were stained with Giemsa (G) and cytokeratin AE1/AE3 (CK).

Figure 6

Morphological polymorphism of CTCs. Images of identified CTCs on a single patient level in four patients (patient 18, 23, 30, 31) are presented following Parsortix® or MACS® separation from blood obtained in a single blood draw. CTCs varied in size and their morphological characteristics. Scale bar represents 10 μm.
Morphological polymorphism of CTCs. Images of identified CTCs on a single patient level in four patients (patient 18, 23, 30, 31) are presented following Parsortix® or MACS® separation from blood obtained in a single blood draw. CTCs varied in size and their morphological characteristics. Scale bar represents 10 μm.

Patient characteristics

Characteristics Frequency N (%)
Histology
    Invasive ductal carcinoma 28 (93.3)
    Invasive lobular carcinoma 2 (6.7)
Tumor stage
    T1 5 (16.7)
    T2 18 (60.0)
    T3 7 (23.3)
N stage
    N0 5 (16.7)
    N1 9 (30.0)
    N2 3 (10.0)
    N3 7 (23.3)
    Unknown 6 (20.0)
Grade
    Grade I 1 (3.3)
    Grade II 9 (30.0)
    Grade III 19 (63.3)
    Unknown 1 (3.3)
Hormone receptor
    Estrogene receptor positive 24 (80.0)
    Estrogene receptor negative 6 (20.0)
    Progesterone receptor positive 21 (70.0)
    Progesterone receptor negative 9 (30.0)
HER2 status
    Positive 4 (13.3)
        Negative 26 (86.7)
Molecular subtype
    Luminal A-like 8 (25.8)
    Luminal-B like HER2 negative 13 (43.3)
    Luminal-B like HER2 positive 3 (10.0)
    HER2 positive 1 (3.3)
    Triple negative 5 (16.7)
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