醛脱氢酶同工酶:人类黑色素瘤肿瘤干细胞的标志物。

Nicholas T. Nguyen, Yuchun Luo, M. Fujita
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In the 1990s, Dick described a subset of human leukemic cells that retained the capacity to self-renew and differentiate [3]. Following the identification of CSCs in acute myelogenous leukemia and other hematologic malignancies, numerous reports of CSCs in solid organ tumors surfaced. The gold standard assay to validate the presence of a candidate CSC subpopulation that fulfills the criteria of ‘self-renewal’ (serially transplantable) and ‘differentiation’ (generating heterogeneous lineages recapitulating an original tumor) is serial transplantation of tumor cells in an immunocompromised mouse model [1,2,4]. Thus far, these CSCs have been identified and characterized by specific cell-surface markers. In the context of human melanoma, cell-surface markers such as CD133 [5], ABCB5 [6], and CD271 [7] have been used to identify a phenotypically distinct CSC in the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. Despite a large body of evidence supporting the presence of CSCs in other cancers and the identification of the aforementioned melanoma CSC surface markers, the existence of CSCs in human melanoma has remained controversial. In two seminal studies, Quintana et al. [8,9] used a highly immunocompromised NOD/SCID IL-2Rγnull (NSG) mouse model to demonstrate an increased frequency of tumorigenic cells, such that one in four human melanoma cells were tumorigenic. The NSG model is unique in that residual natural killer cell activity which characterizes the NOD/SCID mouse has been eliminated. Some have used these data to reject the existence of CSCs in human melanoma, while others consider that multiple CSC populations can coexist within a tumor. The studies also failed to identify a cell surface marker which reliably correlated with tumorigenic capacity, including previously described markers for CSCs in human melanoma in the NOD/SCID model (ABCB5, CD271, CD133). It should be noted, however, that the aforementioned studies did not examine high aldehyde dehydrogenase (ALDH), a key intracellular CSC marker thought to be less vulnerable to CSC isolation procedures. Furthermore, Quintana et al. did not conduct in vivo serial transplantation experiments, the gold standard of assays to validate the presence of a CSC population [8,9]. Since Quintana et al.’s first report in 2008 [8], four groups including ours have compared tumorigenesis of human melanoma cells in NOD/SCID mice and NSG mice [7,10–12]. Although these four independent groups have also identified increased tumorigenicity in the NSG mouse model, the magnitude of the increase described by Quintana et al. has not been validated. These discrepancies may be explained in part by variations in methodologies. Nonetheless, the sum of the evidence certainly favors the existence of a CSC population in human melanoma. More recently, physiological and functional stem cell properties have been utilized to identify CSCs. Due to the conflicting data on the existence of CSCs in human melanoma, we decided to employ these properties to identify CSCs in human melanoma in lieu of cell-surface markers that could be susceptible to experimental conditions. Specifically, we studied ALDH, a detoxifying enzyme responsible for the oxidation of retinal to retinoic acid, which in turn regulates many genes associated with cell proliferation and differentiation. Indeed, increased ALDH activity has been reported in normal stem cells [13]. Moreover, the ALDH isozyme family has been identified as reliable intracellular markers for CSCs in other solid organ malignancies [14]. We investigated tumor initiating capacity of ALDH+ versus ALDH− human melanoma cells via intradermal injections into NOD/SCID and NSG mice [12]. We demonstrated a higher frequency of tumor initiating cells in ALDH+ cells in both mouse models. These findings validated our hypothesis that ALDH activity can distinguish tumorigenic and nontumorigenic melanoma cells in both NOD/SCID and NSG mice. While we and others [10] provide strong evidence that ALDH+ human melanoma cells were enriched with tumorigenic cells, Prasmickaite et al. [15] were unable to corroborate these findings. The discrepancy is multifactorial and probably secondary to variations in tumor cell samples, methodologies and data interpretation. For instance, cultured cells were used in Prasmickaite et al.’s report rather than patient tumors or xenografted patient tumors. Of note, neither Boonyaratanakornkit et al.’s report [10] nor Prasmickaite et al.’s report [15] analyzed the self-renewal capacity of ALDH+ cells by serially transplanting human melanoma cells into mice, an important criterion for CSCs. Therefore, to investigate whether ALDH+ cells and/or ALDH− cells possess the CSC properties of self-renewal and differentiation, tumor cells derived from ALDH+ and ALDH− tumors were serially transplanted in vivo [12]. Whereas secondary and tertiary tumors were observed from ALDH+ cells, ALDH− cells failed to form palpable tumors during the 24-week observation period. This observation confirms the self-renewal capacity of ALDH+ cells. The ability of ALDH+ cells to differentiate was examined in both the in vitro and in vivo setting. In vitro, ALDH+ cells demonstrated the capacity to differentiate into ALDH− cells, whereas ALDH− cells retained the ALDH− phenotype. In vivo studies validated these findings. Producing both ALDH+ and ALDH− cells, tumors generated from ALDH+ cells re-established tumor heterogeneity. Conversely, tumors generated from ALDH− cells were comprised entirely of ALDH− cells. Collectively, our findings confirm the presence of a phenotypically distinct subpopulation of tumorigenic melanoma cells and validate the existence of a CSC population in human melanoma. To date, we are among three groups [6,7,12] who have examined the self-renewal and differentiation properties of CSCs in human melanoma. Whether these melanoma CSCs (ABCB5+, CD271+ or ALDH+) are distinct or overlapping populations remains under investigation. The Aldefluor® assay was utilized in our study and others to identify CSCs measures ALDH enzymatic activity in living tissue specimens. By contrast, immunohistochemical staining quantifies ALDH expression in formalin-fixed tissue specimens and has been utilized to characterize the clinical significance of increased ALDH expression in solid-organ cancers. The availability of ALDH isozyme-specific antibodies has allowed for immunohistochemical analysis of tissues. Previously, it was thought that ALDH1A1 activity was responsible for Aldefluor assay positivity [16]. Therefore, initial studies investigating ALDH expression analyzed tumor specimens using ALDH1A1 specific antibodies for immunohistochemical staining. In many, but not all of these studies, ALDH1A1 expression correlated with prognosis [17]. Recently, however, Marcato et al. identified ALDH1A3 in Aldefluor-positive cells from human breast cancer, thereby providing evidence that the ALDH activity measured by the Aldefluor assay is not necessarily due to ALDH1A1 isozyme [18]. Furthermore, they demonstrated a positive correlation between prognostic factors and ALDH1A3, but not ALDH1A1, expression. These data underscore the need to understand the biological significance of each ALDH isoform in CSCs. We identified ALDH1A1 and ALDH1A3 as the predominant ALDH isozymes expressed in human melanoma tumors. Further biologic analysis of the ALDH+ melanoma cells revealed that these ALDH isozymes are vital to the function of these cells. Silencing ALDH1A3 by siRNA or shRNA led to cell cycle arrest, apoptosis, decreased cell viability in vitro and reduced tumorigenesis in vivo. We also noted decreased chemoresistance in ALDH+ cells following silencing of ALDH1A3 expression [12]. Collectively, these findings not only underscore the diagnostic, prognostic and therapeutic potential for ALDH isozymes in human melanoma, but they also open the door for future investigations. For example, the diagnostic utility of ALDH isozyme immunohistochemical staining may be assessed by examining the differential expression of ALDH isozymes in benign nevi versus melanoma. Immunohistochemical staining with ALDH1A1 and ALDH1A3 may also provide valuable prognostic information. Finally, development of isozyme-specific enzyme inhibitors may broaden our understanding of ALDH isozyme physiology and pathophysiology, potentially aiding in the creation of targeted therapeutics selective for CSCs [19].","PeriodicalId":12255,"journal":{"name":"Expert Review of Dermatology","volume":"23 1","pages":"111-113"},"PeriodicalIF":0.0000,"publicationDate":"2013-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"3","resultStr":"{\"title\":\"Aldehyde dehydrogenase isozymes: markers of cancer stem cells in human melanoma.\",\"authors\":\"Nicholas T. Nguyen, Yuchun Luo, M. Fujita\",\"doi\":\"10.1586/EDM.13.2\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"In recent years, the cancer stem cell (CSC) hypothesis has challenged conventional models of cancer initiation and growth. 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The gold standard assay to validate the presence of a candidate CSC subpopulation that fulfills the criteria of ‘self-renewal’ (serially transplantable) and ‘differentiation’ (generating heterogeneous lineages recapitulating an original tumor) is serial transplantation of tumor cells in an immunocompromised mouse model [1,2,4]. Thus far, these CSCs have been identified and characterized by specific cell-surface markers. In the context of human melanoma, cell-surface markers such as CD133 [5], ABCB5 [6], and CD271 [7] have been used to identify a phenotypically distinct CSC in the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. Despite a large body of evidence supporting the presence of CSCs in other cancers and the identification of the aforementioned melanoma CSC surface markers, the existence of CSCs in human melanoma has remained controversial. In two seminal studies, Quintana et al. [8,9] used a highly immunocompromised NOD/SCID IL-2Rγnull (NSG) mouse model to demonstrate an increased frequency of tumorigenic cells, such that one in four human melanoma cells were tumorigenic. The NSG model is unique in that residual natural killer cell activity which characterizes the NOD/SCID mouse has been eliminated. Some have used these data to reject the existence of CSCs in human melanoma, while others consider that multiple CSC populations can coexist within a tumor. The studies also failed to identify a cell surface marker which reliably correlated with tumorigenic capacity, including previously described markers for CSCs in human melanoma in the NOD/SCID model (ABCB5, CD271, CD133). It should be noted, however, that the aforementioned studies did not examine high aldehyde dehydrogenase (ALDH), a key intracellular CSC marker thought to be less vulnerable to CSC isolation procedures. Furthermore, Quintana et al. did not conduct in vivo serial transplantation experiments, the gold standard of assays to validate the presence of a CSC population [8,9]. Since Quintana et al.’s first report in 2008 [8], four groups including ours have compared tumorigenesis of human melanoma cells in NOD/SCID mice and NSG mice [7,10–12]. Although these four independent groups have also identified increased tumorigenicity in the NSG mouse model, the magnitude of the increase described by Quintana et al. has not been validated. These discrepancies may be explained in part by variations in methodologies. Nonetheless, the sum of the evidence certainly favors the existence of a CSC population in human melanoma. More recently, physiological and functional stem cell properties have been utilized to identify CSCs. Due to the conflicting data on the existence of CSCs in human melanoma, we decided to employ these properties to identify CSCs in human melanoma in lieu of cell-surface markers that could be susceptible to experimental conditions. Specifically, we studied ALDH, a detoxifying enzyme responsible for the oxidation of retinal to retinoic acid, which in turn regulates many genes associated with cell proliferation and differentiation. Indeed, increased ALDH activity has been reported in normal stem cells [13]. Moreover, the ALDH isozyme family has been identified as reliable intracellular markers for CSCs in other solid organ malignancies [14]. We investigated tumor initiating capacity of ALDH+ versus ALDH− human melanoma cells via intradermal injections into NOD/SCID and NSG mice [12]. We demonstrated a higher frequency of tumor initiating cells in ALDH+ cells in both mouse models. These findings validated our hypothesis that ALDH activity can distinguish tumorigenic and nontumorigenic melanoma cells in both NOD/SCID and NSG mice. While we and others [10] provide strong evidence that ALDH+ human melanoma cells were enriched with tumorigenic cells, Prasmickaite et al. [15] were unable to corroborate these findings. The discrepancy is multifactorial and probably secondary to variations in tumor cell samples, methodologies and data interpretation. For instance, cultured cells were used in Prasmickaite et al.’s report rather than patient tumors or xenografted patient tumors. Of note, neither Boonyaratanakornkit et al.’s report [10] nor Prasmickaite et al.’s report [15] analyzed the self-renewal capacity of ALDH+ cells by serially transplanting human melanoma cells into mice, an important criterion for CSCs. Therefore, to investigate whether ALDH+ cells and/or ALDH− cells possess the CSC properties of self-renewal and differentiation, tumor cells derived from ALDH+ and ALDH− tumors were serially transplanted in vivo [12]. Whereas secondary and tertiary tumors were observed from ALDH+ cells, ALDH− cells failed to form palpable tumors during the 24-week observation period. This observation confirms the self-renewal capacity of ALDH+ cells. The ability of ALDH+ cells to differentiate was examined in both the in vitro and in vivo setting. In vitro, ALDH+ cells demonstrated the capacity to differentiate into ALDH− cells, whereas ALDH− cells retained the ALDH− phenotype. In vivo studies validated these findings. Producing both ALDH+ and ALDH− cells, tumors generated from ALDH+ cells re-established tumor heterogeneity. Conversely, tumors generated from ALDH− cells were comprised entirely of ALDH− cells. Collectively, our findings confirm the presence of a phenotypically distinct subpopulation of tumorigenic melanoma cells and validate the existence of a CSC population in human melanoma. To date, we are among three groups [6,7,12] who have examined the self-renewal and differentiation properties of CSCs in human melanoma. Whether these melanoma CSCs (ABCB5+, CD271+ or ALDH+) are distinct or overlapping populations remains under investigation. The Aldefluor® assay was utilized in our study and others to identify CSCs measures ALDH enzymatic activity in living tissue specimens. By contrast, immunohistochemical staining quantifies ALDH expression in formalin-fixed tissue specimens and has been utilized to characterize the clinical significance of increased ALDH expression in solid-organ cancers. The availability of ALDH isozyme-specific antibodies has allowed for immunohistochemical analysis of tissues. Previously, it was thought that ALDH1A1 activity was responsible for Aldefluor assay positivity [16]. Therefore, initial studies investigating ALDH expression analyzed tumor specimens using ALDH1A1 specific antibodies for immunohistochemical staining. In many, but not all of these studies, ALDH1A1 expression correlated with prognosis [17]. Recently, however, Marcato et al. identified ALDH1A3 in Aldefluor-positive cells from human breast cancer, thereby providing evidence that the ALDH activity measured by the Aldefluor assay is not necessarily due to ALDH1A1 isozyme [18]. Furthermore, they demonstrated a positive correlation between prognostic factors and ALDH1A3, but not ALDH1A1, expression. These data underscore the need to understand the biological significance of each ALDH isoform in CSCs. We identified ALDH1A1 and ALDH1A3 as the predominant ALDH isozymes expressed in human melanoma tumors. Further biologic analysis of the ALDH+ melanoma cells revealed that these ALDH isozymes are vital to the function of these cells. Silencing ALDH1A3 by siRNA or shRNA led to cell cycle arrest, apoptosis, decreased cell viability in vitro and reduced tumorigenesis in vivo. We also noted decreased chemoresistance in ALDH+ cells following silencing of ALDH1A3 expression [12]. Collectively, these findings not only underscore the diagnostic, prognostic and therapeutic potential for ALDH isozymes in human melanoma, but they also open the door for future investigations. For example, the diagnostic utility of ALDH isozyme immunohistochemical staining may be assessed by examining the differential expression of ALDH isozymes in benign nevi versus melanoma. Immunohistochemical staining with ALDH1A1 and ALDH1A3 may also provide valuable prognostic information. 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引用次数: 3

摘要

近年来,癌症干细胞假说对传统的癌症发生和生长模型提出了挑战。这一假说不仅认为癌症是由异质细胞群组成的,而且还提出癌症拥有一种独特的细胞群,这些细胞群保持着增强的自我更新和分化能力。当移植到免疫功能低下的小鼠体内时,被命名的CSCs也可能具有肿瘤启动和繁殖的倾向,因此它们获得了两个额外的名称:癌症启动细胞和肿瘤启动细胞。有人提出,目前的癌症治疗方式针对非csc,其中包括大部分肿瘤。在肿瘤减体积后,由于内在的生存机制和化疗耐药,CSCs可能保持活力[1,2]。在20世纪90年代,Dick描述了人类白血病细胞的一个亚群,它们保留了自我更新和分化的能力[3]。随着在急性髓性白血病和其他血液系统恶性肿瘤中发现CSCs,大量关于CSCs在实体器官肿瘤中的报道浮出水面。验证满足“自我更新”(可连续移植)和“分化”(产生再现原始肿瘤的异质谱系)标准的候选CSC亚群存在的金标准试验是在免疫功能低下的小鼠模型中连续移植肿瘤细胞[1,2,4]。到目前为止,这些CSCs已经通过特定的细胞表面标记物进行了鉴定和表征。在人类黑色素瘤的背景下,细胞表面标记物如CD133[5]、ABCB5[6]和CD271[7]已被用于鉴定非肥胖糖尿病/严重联合免疫缺陷(NOD/SCID)小鼠模型中表型不同的CSC。尽管有大量证据支持CSC在其他癌症中的存在以及上述黑色素瘤CSC表面标记物的鉴定,但CSC在人类黑色素瘤中的存在仍然存在争议。Quintana等人[8,9]在两项开创性的研究中,使用免疫功能高度低下的NOD/SCID il - 2r - γ缺失(NSG)小鼠模型,证明致瘤细胞的频率增加,因此四分之一的人类黑色素瘤细胞具有致瘤性。NSG模型的独特之处在于消除了NOD/SCID小鼠特征的残留自然杀伤细胞活性。一些人利用这些数据来否定人类黑色素瘤中CSC的存在,而另一些人则认为多个CSC群体可以在肿瘤中共存。这些研究也未能确定与致瘤能力可靠相关的细胞表面标志物,包括先前描述的NOD/SCID模型中人类黑色素瘤中CSCs的标志物(ABCB5, CD271, CD133)。然而,值得注意的是,上述研究没有检测高醛脱氢酶(ALDH),这是一种关键的细胞内CSC标志物,被认为不太容易受到CSC分离程序的影响。此外,Quintana等人没有进行体内连续移植实验,这是验证CSC群体存在的金标准[8,9]。自2008年Quintana等人首次报道以来[8],包括我们在内的四个研究小组比较了NOD/SCID小鼠和NSG小鼠中人类黑色素瘤细胞的肿瘤发生[7,10 - 12]。尽管这四个独立的研究小组也发现了NSG小鼠模型的致瘤性增加,但Quintana等人描述的增加幅度尚未得到验证。这些差异部分可以用方法的不同来解释。尽管如此,总的证据肯定支持在人类黑色素瘤中存在CSC群体。最近,利用干细胞的生理和功能特性来鉴定csc。由于关于人类黑色素瘤中CSCs存在的相互矛盾的数据,我们决定利用这些特性来识别人类黑色素瘤中的CSCs,而不是可能易受实验条件影响的细胞表面标记。具体来说,我们研究了ALDH,一种负责视网膜氧化为视黄酸的解毒酶,它反过来调节许多与细胞增殖和分化相关的基因。事实上,在正常干细胞中,ALDH活性增加已被报道[13]。此外,ALDH同工酶家族已被确定为其他实体器官恶性肿瘤中CSCs的可靠细胞内标记物[14]。我们通过对NOD/SCID和NSG小鼠皮内注射,研究了ALDH+和ALDH -人黑色素瘤细胞的肿瘤启动能力[12]。我们发现在两种小鼠模型中,ALDH+细胞中肿瘤起始细胞的频率更高。这些发现证实了我们的假设,即ALDH活性可以在NOD/SCID和NSG小鼠中区分致瘤性和非致瘤性黑色素瘤细胞。虽然我们和其他人[10]提供了强有力的证据,证明ALDH+的人类黑色素瘤细胞富含致瘤细胞,但Prasmickaite等[15]无法证实这些发现。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Aldehyde dehydrogenase isozymes: markers of cancer stem cells in human melanoma.
In recent years, the cancer stem cell (CSC) hypothesis has challenged conventional models of cancer initiation and growth. The hypothesis not only maintains that cancers consist of heterogeneous cell populations, but it also proposes that cancers harbor a unique population of cells that retain an increased capacity for self-renewal and differentiation. Dubbed CSCs may also possess a propensity for tumor initiation and propagation when transplanted into immunocompromised mice, thereby earning them two additional designations: cancer initiating cells and tumor initiating cells. It has been proposed that current cancer treatment modalities target non-CSCs, which comprise the bulk of the tumor. Following tumor debulking, CSCs may remain viable due to intrinsic survival mechanisms and chemoresistance [1,2]. In the 1990s, Dick described a subset of human leukemic cells that retained the capacity to self-renew and differentiate [3]. Following the identification of CSCs in acute myelogenous leukemia and other hematologic malignancies, numerous reports of CSCs in solid organ tumors surfaced. The gold standard assay to validate the presence of a candidate CSC subpopulation that fulfills the criteria of ‘self-renewal’ (serially transplantable) and ‘differentiation’ (generating heterogeneous lineages recapitulating an original tumor) is serial transplantation of tumor cells in an immunocompromised mouse model [1,2,4]. Thus far, these CSCs have been identified and characterized by specific cell-surface markers. In the context of human melanoma, cell-surface markers such as CD133 [5], ABCB5 [6], and CD271 [7] have been used to identify a phenotypically distinct CSC in the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse model. Despite a large body of evidence supporting the presence of CSCs in other cancers and the identification of the aforementioned melanoma CSC surface markers, the existence of CSCs in human melanoma has remained controversial. In two seminal studies, Quintana et al. [8,9] used a highly immunocompromised NOD/SCID IL-2Rγnull (NSG) mouse model to demonstrate an increased frequency of tumorigenic cells, such that one in four human melanoma cells were tumorigenic. The NSG model is unique in that residual natural killer cell activity which characterizes the NOD/SCID mouse has been eliminated. Some have used these data to reject the existence of CSCs in human melanoma, while others consider that multiple CSC populations can coexist within a tumor. The studies also failed to identify a cell surface marker which reliably correlated with tumorigenic capacity, including previously described markers for CSCs in human melanoma in the NOD/SCID model (ABCB5, CD271, CD133). It should be noted, however, that the aforementioned studies did not examine high aldehyde dehydrogenase (ALDH), a key intracellular CSC marker thought to be less vulnerable to CSC isolation procedures. Furthermore, Quintana et al. did not conduct in vivo serial transplantation experiments, the gold standard of assays to validate the presence of a CSC population [8,9]. Since Quintana et al.’s first report in 2008 [8], four groups including ours have compared tumorigenesis of human melanoma cells in NOD/SCID mice and NSG mice [7,10–12]. Although these four independent groups have also identified increased tumorigenicity in the NSG mouse model, the magnitude of the increase described by Quintana et al. has not been validated. These discrepancies may be explained in part by variations in methodologies. Nonetheless, the sum of the evidence certainly favors the existence of a CSC population in human melanoma. More recently, physiological and functional stem cell properties have been utilized to identify CSCs. Due to the conflicting data on the existence of CSCs in human melanoma, we decided to employ these properties to identify CSCs in human melanoma in lieu of cell-surface markers that could be susceptible to experimental conditions. Specifically, we studied ALDH, a detoxifying enzyme responsible for the oxidation of retinal to retinoic acid, which in turn regulates many genes associated with cell proliferation and differentiation. Indeed, increased ALDH activity has been reported in normal stem cells [13]. Moreover, the ALDH isozyme family has been identified as reliable intracellular markers for CSCs in other solid organ malignancies [14]. We investigated tumor initiating capacity of ALDH+ versus ALDH− human melanoma cells via intradermal injections into NOD/SCID and NSG mice [12]. We demonstrated a higher frequency of tumor initiating cells in ALDH+ cells in both mouse models. These findings validated our hypothesis that ALDH activity can distinguish tumorigenic and nontumorigenic melanoma cells in both NOD/SCID and NSG mice. While we and others [10] provide strong evidence that ALDH+ human melanoma cells were enriched with tumorigenic cells, Prasmickaite et al. [15] were unable to corroborate these findings. The discrepancy is multifactorial and probably secondary to variations in tumor cell samples, methodologies and data interpretation. For instance, cultured cells were used in Prasmickaite et al.’s report rather than patient tumors or xenografted patient tumors. Of note, neither Boonyaratanakornkit et al.’s report [10] nor Prasmickaite et al.’s report [15] analyzed the self-renewal capacity of ALDH+ cells by serially transplanting human melanoma cells into mice, an important criterion for CSCs. Therefore, to investigate whether ALDH+ cells and/or ALDH− cells possess the CSC properties of self-renewal and differentiation, tumor cells derived from ALDH+ and ALDH− tumors were serially transplanted in vivo [12]. Whereas secondary and tertiary tumors were observed from ALDH+ cells, ALDH− cells failed to form palpable tumors during the 24-week observation period. This observation confirms the self-renewal capacity of ALDH+ cells. The ability of ALDH+ cells to differentiate was examined in both the in vitro and in vivo setting. In vitro, ALDH+ cells demonstrated the capacity to differentiate into ALDH− cells, whereas ALDH− cells retained the ALDH− phenotype. In vivo studies validated these findings. Producing both ALDH+ and ALDH− cells, tumors generated from ALDH+ cells re-established tumor heterogeneity. Conversely, tumors generated from ALDH− cells were comprised entirely of ALDH− cells. Collectively, our findings confirm the presence of a phenotypically distinct subpopulation of tumorigenic melanoma cells and validate the existence of a CSC population in human melanoma. To date, we are among three groups [6,7,12] who have examined the self-renewal and differentiation properties of CSCs in human melanoma. Whether these melanoma CSCs (ABCB5+, CD271+ or ALDH+) are distinct or overlapping populations remains under investigation. The Aldefluor® assay was utilized in our study and others to identify CSCs measures ALDH enzymatic activity in living tissue specimens. By contrast, immunohistochemical staining quantifies ALDH expression in formalin-fixed tissue specimens and has been utilized to characterize the clinical significance of increased ALDH expression in solid-organ cancers. The availability of ALDH isozyme-specific antibodies has allowed for immunohistochemical analysis of tissues. Previously, it was thought that ALDH1A1 activity was responsible for Aldefluor assay positivity [16]. Therefore, initial studies investigating ALDH expression analyzed tumor specimens using ALDH1A1 specific antibodies for immunohistochemical staining. In many, but not all of these studies, ALDH1A1 expression correlated with prognosis [17]. Recently, however, Marcato et al. identified ALDH1A3 in Aldefluor-positive cells from human breast cancer, thereby providing evidence that the ALDH activity measured by the Aldefluor assay is not necessarily due to ALDH1A1 isozyme [18]. Furthermore, they demonstrated a positive correlation between prognostic factors and ALDH1A3, but not ALDH1A1, expression. These data underscore the need to understand the biological significance of each ALDH isoform in CSCs. We identified ALDH1A1 and ALDH1A3 as the predominant ALDH isozymes expressed in human melanoma tumors. Further biologic analysis of the ALDH+ melanoma cells revealed that these ALDH isozymes are vital to the function of these cells. Silencing ALDH1A3 by siRNA or shRNA led to cell cycle arrest, apoptosis, decreased cell viability in vitro and reduced tumorigenesis in vivo. We also noted decreased chemoresistance in ALDH+ cells following silencing of ALDH1A3 expression [12]. Collectively, these findings not only underscore the diagnostic, prognostic and therapeutic potential for ALDH isozymes in human melanoma, but they also open the door for future investigations. For example, the diagnostic utility of ALDH isozyme immunohistochemical staining may be assessed by examining the differential expression of ALDH isozymes in benign nevi versus melanoma. Immunohistochemical staining with ALDH1A1 and ALDH1A3 may also provide valuable prognostic information. Finally, development of isozyme-specific enzyme inhibitors may broaden our understanding of ALDH isozyme physiology and pathophysiology, potentially aiding in the creation of targeted therapeutics selective for CSCs [19].
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