3 TARGETING SUBPOPULATIONS OF CIRCULATING TUMOR CELLS WITH EPHESIA
3.5 CONCLUSIONS
This part of my PhD is still on going so there are still many aspects to confirm, especially for the validation of the new device with the patients’ samples. Up to now, an advanced structure was designed for Ephesia and a new fluidic protocol has been established with no cross-contamination of beads for column formation or cells retrieval. Preliminary validation of the design with cell lines showed that distinct CTCs profile can be easily obtained with this design which targets MICs/CSC/ mesenchymal population. Main advantage of the design is the ability to observe distinct two subpopulations of CTCs directly on the chip without requiring a further separation rather than increasing yield of captured CTCs in one mixed subpopulation with more than one antibody. Moreover, the possibility to retrieve each cell population with minimal contamination is another plus which would allow further molecular characterization of CTCs such as mutational analysis by qPCR.
In order to be able to confirm the utility of the device for clinical samples, the priority is now to confirm the specificity of the of target antibody, CD44 for CTCs. The concern about this antibody is that CD44 has also role in lymphocytes homing and T-lymphocytes activation71, therefore expression of CD44 on lymphocytes may impede the device specificity significantly. However, this might be minimal since we are using WBC depletion with RosetteSep which can achieve 3.4 to 4.7 log depletion of targeted CD45+. Thus, capture efficiency of spiked blood sample experiments are urged to determine sensitivity and specificity. If the capture is highly limited by the contaminant WBC, then other isoforms of CD44, such as CD44v6 found to be expressed in tumor cells46,21 can be also tested.
Otherwise other markers that were discussed before such as EGFR, cell surface vimentin and N-cadherin could be used, that we could test their applicability on a wide range of patients as CTCs capture marker.
Moreover mixture of cell lines with variant epithelial and mesenchymal properties could be captured with various percentages to map CTC subpopulation profile of patients from different cancer types.
Another critical aspect is to verify the possible loss of captured cells while retrieving them from the two separate tubing in the chip and confirm whether the sample is amenable to apply further molecular characterization such as qPCR which could be used for scanning specific MIC gene signatures.
110 REFERENCES
1. Krebs, M. G. et al. Molecular analysis of circulating tumour cells-biology and biomarkers. Nat. Rev. Clin. Oncol. 11, 129–144 (2014).
2. Thiery, J. P., Acloque, H., Huang, R. Y. J. & Nieto, M. A. Epithelial-Mesenchymal Transitions in Development and Disease. Cell 139, 871–890 (2009).
3. Armstrong, A. J. et al. Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers. Mol. Cancer Res. 9, 997–1007 (2011).
4. Yu, M. et al. Circulating Breast Tumor Cells Exhibit Dynamic Changes in Epithelial and Mesenchymal Composition. Science (80-. ). 339, 580–584 (2013).
5. Aktas, B. et al. Stem cell and epithelial-mesenchymal transition markers are frequently overexpressed in circulating tumor cells of metastatic breast cancer patients.
Breast Cancer Res. 11, R46 (2009).
6. Barrière, G., Riouallon, A., Renaudie, J., Tartary, M. & Rigaud, M. Mesenchymal and stemness circulating tumor cells in early breast cancer diagnosis. BMC Cancer 12, 114 (2012).
7. Rao, C. et al. Expression of epithelial cell adhesion molecule in carcinoma cells present in blood and primary and metastatic tumors. Int. J. Oncol. 27, 49–57 (2005).
8. Rhim, A. D. et al. EMT and dissemination precede pancreatic tumor formation. Cell 148, 349–361 (2012).
9. Willipinski-Stapelfeldt, B. et al. Changes in cytoskeletal protein composition indicative of an epithelial-mesenchymal transition in human micrometastatic and primary breast carcinoma cells. Clin. Cancer Res. 11, 8006–8014 (2005).
10. Gorges, T. M. & Pantel, K. Circulating tumor cells as therapy-related biomarkers in cancer patients. Cancer Immunol. Immunother. 62, 931–939 (2013).
11. Soysal, S. D. et al. EpCAM expression varies significantly and is differentially associated with prognosis in the luminal B HER2+, basal-like, and HER2 intrinsic subtypes of breast cancer. Br. J. Cancer 108, 1480–1487 (2013).
12. Pecot, C. V. et al. A novel platform for detection of CK + and CK - CTCs.
Cancer Discov. 1, 580–586 (2011).
111
13. Gorges, T. M. et al. Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition. BMC Cancer 12, 178 (2012).
14. Farace, F. et al. A direct comparison of CellSearch and ISET for circulating tumour-cell detection in patients with metastatic carcinomas. Br. J. Cancer 105, 847–53 (2011).
15. Barrière, G., Tartary, M. & Rigaud, M. Epithelial mesenchymal transition: a new insight into the detection of circulating tumor cells. ISRN Oncol. 2012, 382010 (2012).
16. Fina, E. et al. Did circulating tumor cells tell us all they could? The missed circulating tumor cell message in breast cancer. Int. J. Biol. Markers 30, e429–e433 (2015).
17. Wu, S. et al. Classification of circulating tumor cells by epithelial-mesenchymal transition markers. PLoS One 10, 1–14 (2015).
18. Wit, S. de et al. The detection of EpCAM+ and EpCAM– circulating tumor cells.
Sci. Rep. 5, 12270 (2015).
19. Adams, D. L. et al. Cytometric characterization of Circulating Tumor Cells Captured by microfiltration and their correlation to the cellsearch ® CTC test. Cytom. Part A 87, 137–144 (2015).
20. Baccelli, I. & Trumpp, A. The evolving concept of cancer and metastasis stem cells.
J. Cell Biol. 198, 281–293 (2012).
21. Baccelli, I. et al. Identification of a population of blood circulating tumor cells from breast cancer patients that initiates metastasis in a xenograft assay. Nat. Biotechnol.
31, 539–44 (2013).
22. Al-Hajj, M., Wicha, M. S., Benito-Hernandez, A., Morrison, S. J. & Clarke, M. F.
Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. U.
S. A. 100, 3983–8 (2003).
23. Chao, M. P. et al. Therapeutic antibody targeting of CD47 eliminates human acute lymphoblastic leukemia. Cancer Res. 71, 1374–1384 (2011).
24. Trusolino, L., Bertotti, A. & Comoglio, P. M. MET signalling: principles and functions in development, organ regeneration and cancer. Nat. Rev. Mol. Cell Biol. 11, 834–
48 (2010).
112
25. Kallergi, G. et al. Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients. Breast Cancer Res. 13, R59 (2011).
26. Mitra, A., Mishra, L. & Li, S. EMT, CTCs and CSCs in tumor relapse and drug-resistance. Oncotarget 6, 10697–711 (2015).
27. Li, Y.-M. et al. Epithelial-mesenchymal transition markers expressed in circulating tumor cells in hepatocellular carcinoma patients with different stages of disease. Cell Death Dis. 4, e831 (2013).
28. Singh, A. & Settleman, J. EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29, 4741–4751 (2010).
29. Mani, S. A. et al. The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells. Cell 133, 704–715 (2008).
30. Polyak, K. & Weinberg, R. A. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer 9, 265–273 (2009).
31. Kasimir-Bauer, S., Hoffmann, O., Wallwiener, D., Kimmig, R. & Fehm, T.
Expression of stem cell and epithelial-mesenchymal transition markers in primary breast cancer patients with circulating tumor cells. Breast Cancer Res. 14, R15 (2012).
32. Iinuma, H. et al. Clinical significance of circulating tumor cells, including cancer stem-like cells, in peripheral blood for recurrence and prognosis in patients with dukes’ stage B and C colorectal cancer. J. Clin. Oncol. 29, 1547–1555 (2011).
33. Kang, Y. & Pantel, K. Tumor Cell Dissemination: Emerging Biological Insights from Animal Models and Cancer Patients. Cancer Cell 23, 573–581 (2013).
34. Giancotti, F. G. Mechanisms governing metastatic dormancy and reactivation. Cell 155, 750–764 (2013).
35. Brabletz, T. et al. Variable beta-catenin expression in colorectal cancers indicates tumor progression driven by the tumor environment. Proc. Natl. Acad. Sci. U. S. A.
98, 10356–10361 (2001).
36. Hou, J. M. et al. Clinical significance and molecular characteristics of circulating tumor cells and circulating tumor microemboli in patients with small-cell lung cancer.
J. Clin. Oncol. 30, 525–532 (2012).
113
37. Muller, V. et al. Circulating Tumor Cells in Breast Cancer: Correlation to Bone Marrow Micrometastases, Heterogeneous Response to Systemic Therapy and Low Proliferative Activity. Clin. Cancer Res. 11, 3678–3685 (2005).
38. Wu, Y. et al. Isolation and analysis of rare cells in the blood of cancer patients using a negative depletion methodology. Methods 64, 169–82 (2013).
39. Balasubramanian, P. et al. Multiparameter analysis, including EMT markers, on negatively enriched blood samples from patients with squamous cell carcinoma of the head and neck. PLoS One 7, (2012).
40. Königsberg, R. et al. Detection of EpCAM positive and negative circulating tumor cells in metastatic breast cancer patients. Acta Oncol. 50, 700–710 (2011).
41. Karabacak, N. M. et al. Microfluidic, marker-free isolation of circulating tumor cells from blood samples. Nat. Protoc. 9, 694–710 (2014).
42. Labib, M. et al. Aptamer and Antisense-Mediated Two-Dimensional Isolation of Specific Cancer Cell Subpopulations. J. Am. Chem. Soc. 138, 2476–2479 (2016).
43. Yan, Y., Zuo, X. & Wei, D. Concise Review: Emerging Role of CD44 in Cancer Stem Cells: A Promising Biomarker and Therapeutic Target. Stem Cells Transl. Med.
4, 1033–1043 (2015).
44. Zhang, Y., Toy, K. A. & Kleer, C. G. Metaplastic breast carcinomas are enriched in markers of tumor-initiating cells and epithelial to mesenchymal transition. Mod.
Pathol. 25, 178–184 (2011).
45. Morel, A.-P. et al. Generation of Breast Cancer Stem Cells through Epithelial-Mesenchymal Transition. PLoS One 3, e2888 (2008).
46. Todaro, M. et al. CD44v6 is a marker of constitutive and reprogrammed cancer stem cells driving colon cancer metastasis. Cell Stem Cell 14, 342–356 (2014).
47. Satelli, A., Brownlee, Z., Mitra, A., Meng, Q. H. & Li, S. Circulating Tumor Cell Enumeration with a Combination of Epithelial Cell Adhesion Molecule – and Cell-Surface Vimentin – Based Methods for Monitoring Breast Cancer Therapeutic Response. Clin. Chem. 266, 259–266 (2015).
48. Ithimakin, S. et al. HER2 Drives Luminal Breast Cancer Stem Cells in the Absence of HER2 Amplification: Implications for Efficacy of Adjuvant Trastuzumab. Cancer Res.
73, 1635–1646 (2013).
114
49. Korkaya, H., Paulson, A., Iovino, F. & Wicha, M. S. HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion. Oncogene 27, 6120–6130 (2008).
50. Korkaya, H. & Wicha, M. S. HER2 and breast cancer stem cells: More than meets the eye. Cancer Res. 73, 3489–3493 (2013).
51. Nicholson, R. I., Gee, J. M. & Harper, M. E. EGFR and cancer prognosis. Eur.
J. Cancer 37 Suppl 4, S9–S15 (2001).
52. Vuoriluoto, K. et al. Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer. Oncogene 30, 1436–48 (2011).
53. Ivaska, J., Pallari, H. M., Nevo, J. & Eriksson, J. E. Novel functions of vimentin in cell adhesion, migration, and signaling. Exp. Cell Res. 313, 2050–2062 (2007).
54. Satelli, A. & Li, S. Vimentin as a potential molecular target in cancer therapy Or Vimentin, an overview and its potential as a molecular target for cancer therapy. Cell Mol Life Sci. 68, 3033–3046 (2011).
55. Armstrong, A. J. et al. Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers. Mol. Cancer Res. 9, 997–1007 (2011).
56. Satelli, A. et al. Universal Marker and Detection Tool for Human Sarcoma Circulating Tumor Cells. Cancer Res. 74, 1645–1650 (2014).
57. Mitra, A. et al. Cell-surface Vimentin: A mislocalized protein for isolating csVimentin(+) CD133(-) novel stem-like hepatocellular carcinoma cells expressing EMT markers. Int. J. Cancer 137, 491–6 (2015).
58. Bitting, R. L. et al. Development of a method to isolate circulating tumor cells using mesenchymal-based capture. Methods 64, 129–36 (2013).
59. Thiery, J. P. & Sleeman, J. P. Complex networks orchestrate epithelial–mesenchymal transitions. Nat. Rev. Mol. Cell Biol. 7, 131–142 (2006).
60. Lustberg, M. B. et al. Heterogeneous atypical cell populations are present in blood of metastatic breast cancer patients. Breast Cancer Res. 16, R23 (2014).
61. Barriere, G. et al. Circulating tumor cells and epithelial, mesenchymal and stemness markers: characterization of cell subpopulations. Ann. Transl. Med. 2, 109 (2014).
115
62. Yokobori, T. et al. Plastin3 Is a Novel Marker for Circulating Tumor Cells Undergoing the Epithelial-Mesenchymal Transition and Is Associated with Colorectal Cancer Prognosis. Cancer Res. 73, 2059–2069 (2013).
63. Albarracin, C. & Dhamne, S. Ki67 as a Biomarker of Prognosis and Prediction: Is it Ready for Use in Routine Pathology Practice? Curr. Breast Cancer Rep. 6, 260–
266 (2014).
64. Yerushalmi, R., Woods, R., Ravdin, P. M., Hayes, M. M. & Gelmon, K. A. Ki67 in breast cancer: prognostic and predictive potential. Lancet Oncol. 11, 174–183 (2010).
65. Kallergi, G. et al. Apoptotic Circulating Tumor Cells (CTCs) in early and metastatic breast cancer patients. Mol. Cancer Ther. 12, 1886–1896 (2013).
66. Vishnoi, M. et al. The isolation and characterization of CTC subsets related to breast cancer dormancy. Sci. Rep. 5, 17533 (2015).
67. May, C. D. et al. Epithelial-mesenchymal transition and cancer stem cells: a dangerously dynamic duo in breast cancer progression. Breast Cancer Res. 13, 202 (2011).
68. Xing, L., Hung, M., Bonfiglio, T., Hicks, D. G. & Tang, P. Breast Cancer : Basic and Clinical Research The Expression Patterns of ER , PR , HER2 , CK5 / 6 , EGFR , Ki-67 and AR by Immunohistochemical Analysis in Breast Cancer Cell Lines. 35–41
69. Banys-Paluchowski, M. et al. Prognostic Relevance of Circulating Tumor Cells in Molecular Subtypes of Breast Cancer. Geburtshilfe Frauenheilkd. 75, 232–237 (2015).
70. Punnoose, E. a et al. Molecular biomarker analyses using circulating tumor cells.
PLoS One 5, e12517 (2010).
71. Cagle, P. T. & Allen, T. C. Basic Concepts of Molecular Pathology. (Springer US, 2009). at <https://books.google.fr/books?id=uwGRTL2UqdIC>
116 ANNEX I
CONJUGATION OF ANTIBODY WITH STREPTAVIDIN BEADS
Resuspend the vial and wash the desired amount of beads with 1 ml of PBS/BSA-0.01% three times by placing the tube into magnetic holder and removing the supernatant.
Resuspend the beads with initial volume of PBS/BSA-0.01%
Incubate the beads with biotinylated CD44 antibody in PBS for 30 mins. at room temperature
Wash the conjugated beads with PBS/BSA-1%
Resuspend the beads at a desired concentration.
CONJUGATION OF ANTIBODY WITH CARBOXYLIC ACID BEADS
MES= 2-[N-morpholino] ethane sulfonic acid, coupling buffer: 1M MES in water, pH=5 Sulfo-NHS= N-hydroxysulfosuccinimide,
MES= 2-(N-morpholino)ethanesulfonic acid
Resuspend the vial and wash 1 mg particle with 1ml of 0.1 MES four times using magnetic holder.
Dilute particles in 100 μl of MES
Dissolve 7.5 mg of EDC in 200μl of MES and dissolve 1.25 mg of sulfo-NHS in MES.
Quickly mix the solution of EDC and sulfo-NHS and mix it with the particles.
Incubate the beads for 10 min on rotating wheel (activation of particles)
Wash the beads with 1 ml of MES buffer and remove the supernatant.
Add 25μg of antibodies with particles in 1 ml of MES buffer.
Incubate overnight at 4°C with rotating wheel.
Wash particles four times with 1 ml of MES buffer, one time with solution of 0.1 M MES in 1 M NaCl solution and then three times with 0.1 MES buffer.
CONJUGATION OF ANTIBODY WITH SHEEP ANTI-MOUSE IGG
Resuspend the vial and wash 25μl of beads with PBS-BSA-1% using magnetic holder.
Add 1.5μg of antibody and incubate for at least 30 min at 4 °C on rotating wheel.
Wash the particles with 2 ml of PBS/BSA-1% three-four times using magnetic holder.
Remove supernatant and resuspend the beads in 1 ml of PBS/BSA-1%.
Use desired amount of beads.
117 CELL CULTURE
Cells are culture with the appropriate medium containing 10% FBS (fetal bovine serum), 0.1% penicillin-streptomycin (1X) and if the medium is not enriched with L-glutamine, then, 0.1% is added at 37°C in a humidified atmosphere with 5% CO2. Cells are passed two times per week at around 70-90% confluency.
TABLE 3-I-1 CELL LINES USED IN THE VALIDATION OF NEW EPHESIA DESIGN
Cell Lines Medium Passage ratio
Hs578T DMEM 1/3
MDA‐MB231 DMEM 1/5
SKBR‐3 McCoy's 5A (Modified) Medium 1/2
CELL LABELING FOR FLOW CYTOMETRY
The experiments were done in cytometry: cell sorting and analysis platform in Institut Curie with BD LSR II Flow cytometry instrument with FITC and APC filter. The different control tubes and the details of the primary and secondary are shown in the table below.
TABLE 3-I-2 ANTIBODIES, CONTROL ISTOYPES AND CONJUGATION PAIRS USED IN FLOW CYTOMETRY
Expand the cell with a T75 flask at 90% of confluency
Disassociate the cells with Trypsin
Resuspend the pellet in 3mL of PBS-BSA 1% (keep them on ice to preserve cell integrity)
Prepare a dilution from previous solution at 1/10 (in 200μL) to count with the Scepter (Millipore), usually 3-15.10^6 cells.
Centrifuge the cells for 4 min at 1000rpm and aspirate the supernatant
118
Fix them with PFA 4% in 1000μL for 15 min and wash them three times with 1000μL of PBS-BSA 1% at 1500rpm for 3 min.
Permeabilize with PBS-BSA 1%-Triton 0.1% for 15 min
Wash them three times with 1000μL of PBS-BSA 1% at 1500rpm for 3 min.
Distribute the cells to tubes having 10^6 cells in 400 μl PBS-BSA 1%
Primary antibodies are labeled with Zenon antibody labeling kit.
Primary antibodies are incubated at 100 times dilution for 30 min in 400 μL on ice.
Wash them three times with 1000μL of PBS-BSA 1% at 1500rpm for 3 min.
Wash them three times with 1000μL of PBS-BSA 1% at 1500rpm for 3 min.
Resuspend the cell in 400 μL.
TABLE 3-I- 3 CONTROL SAMPLES TO ANALYASE THE DOUBLE STAINING OF HER2 AND HER3 WITH FLOW CYTOMETRY
119
120