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hyaluronic acid (HA), thereby linking binding to selectin, collagen, osteopontin, fibronectin and laminin
[60]
in the extracellular matrix . HA binding to CD44 also facilitates complex activation of receptor protein
[61]
tyrosine kinases of the epidermal growth factor receptor family in several types of cancer . Upon HA
binding to CD44 on the cell surface, increased levels of cell proliferation and survival occur through
[62]
downstream signal activation of the MAPK and PI3K/AKT pathways . Studies have also indicated that
CD44 plays a significant role in the invasive and tumorigenic stemness capacity of several tumor cell types,
[64]
[65]
including breast , prostate , pancreatic and mesothelioma [66,67] . CD44 surface levels on tumor cells were
[63]
[68]
shown to positively correlate with prostate cancer CTCs in the bloodstream of patients .
[58]
Genetic knockdown of CD44 prevented the formation of tumors by colorectal CSCs . CD44 either alone or
in combination with other cell surface markers can be used to enrich for CSCs from multiple tumor types
[75]
[65]
including breast [69,70] , prostate [71,72] , colon [73,74] , pancreas , and head and neck squamous cell carcinomas .
However, given the relatively high levels of CD44 expressed on the cancer cell population, it would appear
that CD44 is more a marker of the invasive, metastatic cell population and is not specific, per se for the
CSCs.

CD13
2+
The CD13 gene encodes the enzyme aminopeptidase N, a Zn dependent membrane-bound ectopeptidase
preferentially degrading proteins and peptides with an N-terminal neutral amino acid. CD13 is
overexpressed in multiple cancer types as well as on the surface of vasculature endothelial cells in tumors
[76]
+
undergoing angiogenesis, making it a promising target . CD13 cells form clusters or foci within the
tumors, are typically in the G phase of the cell cycle and also found present in the fractionated “SP”
0
+
of CSCs from hepatocarcinoma [77,78] . Haraguchi et al. showed that CD13 cancer cells surviving after
[79]
chemotherapy or radiation treatment in tumors removed from hepatocarcinoma patients were found present
within enriched fibrous capsular regions of the tumors and had decreased levels of DNA damage caused
by reactive oxygen species (ROS). Thus, they proposed that the cancer cells appeared to be protected from
treatment-induced apoptosis by expressing CD13. In their mouse xenograft models treated simultaneously
with a CD13 inhibitor (either using a neutralizing antibody or the peptidase inhibitor, Ubenimex) and
the genotoxic chemotherapeutic drug, 5-fluorouracil (5-FU), the combination diminished tumor levels
[79]
significantly and was far more effective than 5-FU used alone . Their studies have since been confirmed
in other studies where CD13 inhibitors were used as an adjuvant therapy together with ROS-inducing
chemotherapy or radiation treatment as a potential treatment method that could improve survival rates
of patients with hepatocarcinoma [79-81] . The tripeptide NGR that targets CD13, conjugated with anticancer
drugs has been similarly used to target human hepatocarcinoma growth by killing cancer stem cells and
suppressing angiogenesis . Although CD13 expression has been associated with human colon cancer and
[82]
[83]
poorer prognosis , targeting its functional role as a marker on CR-CSCs has not yet been tested such that
the usefulness of CD13 as a CR-CSC marker remains unclear.

LGR5
LGR5 is a member of the Leucine-rich-repeat-containing G-protein-coupled receptors (also known
as GPR49) which belong to the seven transmembraneous G-protein-coupled receptor superfamily. The
[84]
LGR1-5 family are regulatory receptors involved in WNT signalling and can bind to the furin-like repeat
FU2 domains of the R-spondin 1-4 stem cell growth factors to potentiate WNT signalling [85,86] . However,
R-spondin 1 (RSPO1)/LGR5 can also directly activate TGFb signalling in a cooperative interaction with
the TGFb type II receptor on colon cancer cells to enhance the TGFb-mediated growth inhibition and
[87]
stress-induced apoptosis . Knockdown of Lgr5 attenuated downstream TGFb signaling and increased
[87]
cell proliferation, survival, and metastasis in an orthotopic model of colon cancer in vivo . Upon RSPO1
[87]
stimulation, LGR5 formed complexes with TGFb receptors . Hence, the net effects of LGR5 expression will
also depend on its interplay with both the WNT and TGFb signalling systems as well.