Challenges associated with the detection of the CTCs

ChallengeExplanationWhat requiresRef.
Rarity

  • As few as 1 CTC per billion blood cells

  • Fewer than 1 CTC per 10 mL in early-stage cancer

  • Efficient enrichment

  • Depletion of unwanted cells

  • High detection accuracy

  • High sensitivity

  • Processing a large amount of blood

[23–26]
Heterogeneity

  • Phenotypic and genotypic variation across tumor types

  • Marker-dependence (EpCAM)

  • CTC size differs across cancer types; cell line-based filters may overestimate CTC size.

  • Can miss CTCs with other characteristics, resulting in false negative results

  • Multiple biomarkers

  • Combining more than one parameter: size- and label-based approaches

  • Deformability-based microfluidics

  • Combining with other components of liquid biopsy (cfDNA, exosomes)

  • Label-free detection

  • Real-time detection

[27–32]
Complex blood environment

  • Non-specific binding from blood cells and plasma proteins creates high background noise.

  • Antifouling surfaces

  • Improving surface-cell interaction

  • Dual-selective ligands

[33–35]
Low viability of
captured CTCs

  • Delay in processing affects downstream analysis.

  • High shear stress during isolation causes apoptosis/necrosis, damages CTCs, and reduces recovery.

  • Real-time detection

  • Gentle capture via microfluidics or flexible biosensors

  • Integrated culture systems

  • Low-shear microfluidic designs to mimic physiological conditions

[33, 36–38]
Clinical implementation

  • Rapid processing needed

  • Short tube storage times, logistical hurdles for analysis

  • Freezing and thawing may cause CTC loss, aggregation, and reduced integrity.

  • High cost of devices

  • Performance of devices is limited.

  • Use preservative tubes

  • Automate capture and analysis

  • Miniaturized real-time biosensors

  • Standardized cryopreservation

  • Use of cryoprotectants

  • Integrated biobanking protocols

[1, 37, 39, 40]

CTC: circulating tumor cell; cfDNA: cell-free DNA; EpCAM: epithelial cell adhesion molecule