This slideshow covers the basic aspects of CTCs, their relevance in Oncology and the diagnostic / analytical methods being used to study them

1. CTCs
( Circulating Tumor Cells )
Review of Diagnostic
and
Analytical Methods
Sreepadmanabh M
Indian Institute of Science Education and Research - Bhopal

2. The Known
• Circulating tumor cells : reason for metastases
• Aggressive CTCs lead to secondary tumor formation
• strong prognostic factor for overall survival in patients with
metastatic breast, colorectal or prostate cancer

5. Seek , and ye MAY find
• Enumeration of CTCs -> extent of metastases , intensity of
cancer
• Characterization of CTCs -> molecular nature of cancer /
possible therapeutic cures
• Analogy : liquid biopsy

7. Why even bother
Tissue biopsies suffer from following fundamental limitations :
• Invasive
• Cannot be reused
• Ineffective to understand metastasis
• Cannot predict extent of spread of disease

8. Aims of current CTC research
• Facilitate culturing of CTCs -> personalized
medicine
• Phenotypic and molecular profiling of cancer
• Info beyond just simple enumeration
• Improving sensitivity of diagnostic tests : more
specificity and versatility

9. Glimpses
of
CTC Capture
methods and principles

10. Affinity Sorting
• Variation in types of antibodies as capture agents
• Broadening of applicability – micro level
• micropost method –> CD45 –ve , cytokeratin +ve immunostaining
• Major goal : to include low EpCAM CTCs and non-epithelial tumors
example : MagSweeper *

11. Analysis of Affinity Sorting Research
• Reduction to microscale level drastically increases
performance
• Reduction in #pre-processing steps will increase the
sensitivity
• FUTURE GOALS : To make isolation of cells after
enumeration process more efficient

12. Size based sorting
• Relative size difference in Tumor cells vs normal blood cells
• Microfabricated filters –ISET – very sensititve
• Bead separation method – tumor cell surface specific
markers

13. Analysis of Size based sorting
• MERITS :
label free method of cell isolation
cells can be used for post-capture analysis
• LIMITATION :
Inefficient separation
low sample purity
CTCs of small radii not isolatable

14. Dielectric Separation
• DEP ( dielectrophoresis) used
• Parameters like total capacitance etc. are used
• Refinements : Hydrodynamic Sorting combined with DEP
• LIMITATION : cytoplasmic and membrane conductivities vary
cell wall interactions hamper DEP

16. Marker-Free CTC Isolation
• NO Selection bias - allows uncharacterized phenotypes too to be
analyzed
• Usually employs a negative depletion strategy
• LIMITATION : purity of sample is lower than conventional positive
depletion methods ( Small size CTCs get through )

18. Common Barriers / Limitations
• Capture methods are destructive ; post-capture isolation
isn’t possible
• Limited precision wrt selectivity due to similarity btw CTC
and WBCs
• High susceptibility of CTCs released after enumeration to
get destroyed

19. Common Barriers / Limitations
• Stem cells have low magnetic susceptibility – weak
biomarkers , hence tougher to detect
• Integrating capture and analysis will limit feasible sampling
size , hence sensitivity must be high enough
• Profiling CTC heterogeneity is difficult

20. CTC Sub-Populations and Heterogeneity
• EMT ( Epithelial to Mesenchymal Transition) is the primary
cause for CTC heterogeneity
• CTCs are morphologically different from solid-tumor cells
• Morphological heterogeneity can indicate metastatic
potential due to changes in pro-metastatic cell signaling
pathways

22. A more mature approach ?

23. Signposts for CTC research
( what we’ve learnt )
• CTC heterogeneity exists and hence CTCs exhibit variety of
metastatic phenotypes
• Heterogeneity of CTCs implies 2 possible approaches :
1. Use a variety of bio-markers
2. Use marker-free isolation techniques ( as CTCs have variety of
phenotypes )

24. Signposts for CTC Research
( what we aim at )
• In-Vivo (inside organism/system ) CTC analysis , post-
capture – fluorescent ligands
• In-Situ ( onsite ) characterization of molecular properties of
CTCs
• Post capture recovery and isolation of CTCs

25. When too many cooks
DIDN’T
spoil the broth

26. Culture and Expansion of CTCs
• Essential to understand drug specificity , genomic makeup ,
phenotypic analysis of CTCs .
• Viable Devices : Graphene Oxide surface ; combination of magnetic
nanoparticles and microstructures
• LIMITATION : Specific factors influencing CTCs to form secondary
tumors are unidentified

27. CTC Heterogeneity - methods of analysis
• FACS – Fluorescence activated cell sorting ;
LIMITATION : needs large blood sample ; does not work for all blood
samples
• Surface antigen selectivity based on relative affinities shown by
different sub-pops of CTCs for particular antigens
PROs : Sensitivity is excellent ; Sorting is effective
CONs : Low output quantity ; low magnetic susceptibility of
nanoparticles

29. Integrated Molecular CTC Analysis
• Relies on understanding of invasomes (currently inadequate)
• Invasomes : bio markers that reveal whether the cells possess
invasive properties leading to metastases .
• Most technologies rely on bio-marker analysis using magnetic
nanoparticles and electro-chemical detectors
• Eg : NanoFlares , CellSearch , micro-Hall detectors (uHD)

30. CellSearch
• Predominant CTC analysis method
• Immunofluorescence followed by magnetic separation
• Immunofluorescence is done using a EpCAM* specific antibody
• Limitations : Low sensitivity , esp. in high EpCAM content cells
inability to access cellular material after enumeration