**2. Expression of special surface markers in LSCs**

Bonnet et al. revealed that the CD34+ CD38<sup>−</sup> subpopulation is similar to normal HSCs with surface markers and can be used to identify cells with unlimited proliferation and differentiation in AML [4]. Subsequent studies have shown that the surface markers of LSCs are extremely complex and vary from person to person. Previous experiments have demonstrated that in some cases, subpopulations of cells with different phenotypes have LSC activity [13–15]. CD34 and CD38 are no longer specific markers that define LSCs. Recent studies have identified various new markers such as CLL-1, CD96, TIM3, CD47, CD32, and CD25. The current study summarizes some of the specific markers expressed by LSCs (**Table 1**) [16] and has been utilized to successfully validate LSCs in recent clinical trials [17].

### **2.1 CLL-1**

*Advances in Hematologic Malignancies*

transplanted sublethal doses of CD34<sup>+</sup>

LSC in NOD/SCID mice [4]. Inoculation of 106

logical malignancies but also in some solid tumors.

*Comparison of the normal and AML human hematopoietic systems.*

row expression both of CD34+

CD34+

also found CD34+

identified and confirmed to play an important role in the occurrence and development of leukemia. In 1994, Lapidot et al. reported that AML contains LSC. It is believed that only 0.1–1% cells have the ability to produce AML [3]. The researchers

CD38<sup>+</sup>

CD38<sup>−</sup> cells, could survive and pass to the next generation. The researchers

isolated from the bone marrow of a patient with AML into non-obese diabetic mice with severe combined immunodeficiency disease (NOD/SCID mice). After 4–8 weeks, human AML cells isolated from the engrafted murine bone mar-

M3, thus indicating that this subpopulation of cells has stem cell-like strong selfrenewal and reproductive ability. In 1997, Bonnet et al. confirmed the presence of

human AML in animals; this finding suggested that the "source of all evils" is LSC (**Figure 1**) [5]. Since then, the existence of LSC has been recognized, which is a significant milestone. The presence of LSC has been confirmed not only in hemato-

Although LCSs were identified and thought to be the main cause of leukemia origin, recurrence, and drug resistance, there is still controversy regarding the origin of this distinct population [6]. Several hypotheses have been proposed with regard to the origin of LCS: (1) from hematopoietic stem cells (HSCs) [7]; (2) from partially differentiated hematopoietic progenitor cells [8]; (3) from blood vascular stem cells and granulocyte macrophage precursors (GMPs) [9–11]; and (4) from relatively mature leukemia cells [12]. Although the number of LSCs is small, LSCs have the same potential for self-renewal, multidirectional differentiation, and unlimited proliferation, resistance to cell death, multidrug resistance, metastasis, and recurrence. Because they can escape inhibition by most chemotherapeutic

and CD34+

CD38<sup>−</sup> cells can induce various subtypes of leukemia other than

CD38<sup>−</sup>. The recipient mouse, re-implanted with

CD38<sup>−</sup> subpopulations

LSCs resulted in the formation of

**12**

**Figure 1.**

C-type lectin domain family 12 member A (CLL1, also known as CLEC12A) positive cells show high tumorigenicity in immunodeficient mice, indicating that this cell subpopulation has the characteristics of LSCs. Moreover, the side population cells enriched in LSCs isolated by flow cytometry from patients with AML also express CLL-1 [18]. Jiang et al. have reported that CLL1-antibody-drug conjugate (CLL1-ADC) could become an attractive target therapy for AML [19]. The use of a


**Table 1.** *Summary of cell surface marker expression on AML LSCs.* DNA-binding payload in CLL1-ADC is critical because such a payload affords the ADC the ability to kill both proliferative and quiescent cells, thus making CLL1- ADC a very compelling candidate for the treatment of patients with AML.
