CTCs( Circulating Tumor Cells )

Review of Diagnostic and

Analytical Methods

Sreepadmanabh MIndian Institute of Science Education and Research - Bhopal

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

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

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

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

Glimpses of

CTC Capture methods and principles

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 *

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

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

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

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

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 )

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

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

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

A more mature approach ?

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 )

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

When too many cooksDIDN’T

spoil the broth

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

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

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)

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