Qian Liu Department of histology and embryology Cancer Stem Cells Qian Liu Department of histology and embryology

Origin of the Theory of Cancer Stem Cells Only a small subset of cancer cells is capable of extensive proliferation Liquid Tumors In vitro colony forming assays: - 1 in 10,000 to 1 in 100 mouse myeloma cells obtained from ascites away from normal hematopoietic cells were able to form colonies In vivo transplantation assays: - Only 1-4% of transplanted leukaemic cells could form spleen colonies Solid Tumors - A large number of cells are required to grow tumors in xenograft models - 1 in 1,000 to 1 in 5,000 lung cancer, neuroblastoma cells, ovarian cancer cells, or breast cancer cells can form colonies in soft agar or in vivo

Normal stem cells Cancer stem cells Rare cells within organs with the ability to self-renew and give rise to all types of cells within the organ to drive organogenesis (differentiation) Cancer stem cells Rare cells within tumors with the ability to self-renew and give rise to the phenotypically diverse tumor cell population to drive tumorigenesis

Properties shared by normal stem cells and cancer stem cells Basic Characterizations: Self renewal Tissue-specific normal stem cells must self-renew throughout the lifetime of the animal to maintain specific organs Cancer stem cells undergo self-renewal to maintain tumor growth Differentiation into phenotypically diverse cell types Give rise to a heterogeneous population of cells that compose the organ or the tumor but lack the ability for unlimited proliferation (hierarchical arrangement of cells) Regulated by similar pathways Pathways that regulate self-renewal in normal stem cells are dys-regulated in cancer stem cells

Pathways involved in self-renewal that are deregulated in cancer cells Wnt, Shh, and Notch pathways have been shown to contribute to the self-renewal of stem cells and/or progenitors in a variety of organs, including the haematopoietic and nervous systems. When dysregulated, these pathways can contribute to oncogenesis. Mutations of these pathways have been associated with a number of human tumours, including colon carcinoma and epidermal tumours (Wnt), medulloblastoma and basal cell carcinoma (Shh), and T-cell leukaemias (Notch).

Properties shared by normal stem cells and cancer stem cells 干细胞和肿瘤干细胞均通过两种分裂方式, 一种是对称分裂: 即形成两个相同的干细胞, 另一种是非对称分裂:即由于细胞质中调节分化蛋白不均匀地分配, 使得一个子细胞不可逆地走向分化的终端成为功能专一的分化细胞; 另一个保持亲代的特征, 仍作为干细胞保留下来。 干细胞与肿瘤干细胞都具有端粒酶活性以及扩增的端粒重复序列, 而人类终末分化体细胞不具或具有极低的端粒酶活性。在人类干细胞的恶性转化实验中发现, 端粒酶活性和附加的基因突变是正常干细胞恶性转化的两个重要条件。 干细胞具有迁移的特性, 肿瘤干细胞具有转移的能力。

Different properties in normal stem cells and cancer stem cells 肿瘤干细胞没有分化为完全成熟细胞的能力, 其分化程序异常, 这与有正常分化程序的干细胞有着本质的不同。最后, 肿瘤细胞倾向于积累复制错误, 而正常干细胞的发育机制要防止这种现象的出现。 干细胞可以在某一时间内连续分裂, 也可在较长时间内处于相对静止状态, 干细胞的自我更新具有反馈机制调节, 增殖和分化处于平衡状态, 有序的; 而在肿瘤干细胞中, 这一调节机制并不存在, 它的增殖分化是无序的, 失控的。 干细胞的增殖具有自稳定性, 当组织处于稳定状态时, 平均一个干细胞产生一个子代干细胞和一个特定分化细胞, 其干细胞总数保持恒定。而肿瘤细胞无自稳定性的特点。

Mutual conversion 在一定的条件下可以两者可以相互转化, 如:生殖嵴或胚胎植入体内可以诱导成畸胎瘤，而畸胎瘤细胞注入鼠囊胚内细胞团可以形成正常胚胎。

The origin of cancer stem cells (d)

Two General Models for Cancer Heterogeneity 1. All cancer cells are potential cancer stem cells but have a low probability of proliferation in clonogenic assays 2. Only a small definable subset of cancer cells are cancer stem cells that have the ability to proliferate indefinitely.

Self renewal and differentiation are random. All cells have equal but low probability of extensive proliferation. Only cells with self renewal capacity can sustain tumor growth. Distinct classes of cells exist within a tumor. Only a small definable subset, the cancer stem cells can initiate tumor growth.

Isolation of cancer stem cells ①CSC筛选最常用的手段是利用CSC细胞表面特异标志物，通过流式细胞术或磁珠分选法分离得到CSC。确定更多的CSC表面标志物及组合使用这些标志物将有助于提高CSC筛选的正确率。 ②根据肿瘤细胞中一群称为SP细胞(side population cell)能将核酸染料Hoechst33342排出胞外的特性，通过流式细胞术来分选．虽然一些SP细胞也具备自我更新和分化能力，但究竟是不是CSC还需进一步研究。 ③根据CSC的克隆形成能力来筛选．将肿瘤细胞放在软琼脂培养基上培养，CSC在无血清添加生长因子条件仍具有很强的自我更新和保持非分化状态的能力，进而能形成克隆，而其他肿瘤细胞则在传代过程中消亡。

Isolation of cancer stem cells ④鉴定CSC细胞的金标准是看分选得到的CSC能否在移植动物体内形成新肿瘤。将分离出来的各细胞亚群按不同细胞浓度梯度接种NOD／SCID小鼠比较各细胞群成瘤能力，从而筛选出优势细胞表面标志，或者将几种优势细胞表面标志进行组合，筛出各种组合的细胞群，这一方法又称系列原位移植法。 ⑤非粘附球囊分析法相比于系列原位移植法更简便，逐渐被用来分选鉴定CSC，CSC细胞能在无血清添加生长因子的培养基中保持未分化状态并形成致密球体，非CSC则贴壁缓慢生长，CSC得以富集。非粘附肿瘤细胞具备裸鼠体内致瘤能力和分化能力。但非粘附肿瘤球囊细胞是否是由于渗人的正常干细胞引起的这效应还有待进一步证实。

Self-renewal Assay in NOD/SCID Mice (Non-obese diabetic/severe combined immunodeficiency) FACS Cell Sorter Cancer Cells (ex: Leukaemia cells) Sublethally irradiated NOD/SCID Mice

Detection of cancer stem cells 特殊标记物 1. nestin 2. CD133 特定基因的表达 Oct-4，Sox-2，bmi-1，HIF-2 具有自我更新的能力 形成神经球 分化潜能 1. 神经元 2. 胶质细胞 肿瘤干细胞的致瘤性分析（金标准）

Brain Tumor Stem Cells: CD133+ CD133 – neuronal stem cell marker 细胞膜上的抗原（跨膜蛋白） 选择性表达于造血干细胞、内皮前体细胞、神经干细胞和神经前体细胞，在成熟血细胞、内皮细胞及神经细胞则没有表达 Singh等最新的研究证明在脑肿瘤中也存在小部分表达CD133的肿瘤干细胞

CD133 is only expressed on NSC/BTSC.

The CD133+ fraction were incubated with BrdU for 12h and stained with antibody against BrdU (×400)

The isolated CD133+ cells generated neurospheres The isolated CD133+ cells generated neurospheres. Phase contrast images of growing spheres at day 7 (×200)

Neurospheres derived from CD133+ cells were plated on an adhesive coverslip in presence of mitotic factors (20ng/ml EGF, 20ng/ml bFGF). The cells were stained with antibody against nestin (green), and nuclei were counterstained with PI (red).

脑肿瘤干细胞球贴壁分化

脑肿瘤干细胞球分化后的MAP 染色阳性神经元

脑肿瘤干细胞球分化后的G F A P 染色阳性的星形胶质细胞

Breast Cancer Stem Cells: CD44+ CD24low Lin- B38.1+ ESA+ CD44 and CD24 – adhesion molecules B38.1 – breast/ovarian cancer-specific marker ESA – epithelial specific antigen Al-Hajj, Muhammad et al. (2003) Proc. Natl. Acad. Sci. USA 100, 3983-3988

The isolated CD44+CD24－ cells generated neurospheres The isolated CD44+CD24－ cells generated neurospheres. Phase contrast images of growing spheres at day 7 (×200)

Hypoxia-Inducible Factors Regulate Tumorigenic Capacity of Glioma Stem Cells Cancer Cell 15, 501-513, June 2, 2009

Glioma Stem Cells (GSCs) Glial cells that self-renew and propagate glioma phenotypically similar to the parental tumor The same as normal neural stem cells: the expression of neural stem cell markers, the capacity for self-renewal and long-term proliferation, the formation of neurospheres, and the ability to differentiate into multiple nervous system lineage. Different from normal neural stem cells: frequency proliferation aberrant expression of differentiation markers chromosomal abnormalities tumor formation

Molecular Regulators of GSCs GSCs contribute to glioma progression, thus, the elucidation of specific molecular inhibitors of GSCs might translate into improved antineoplastic therapies. Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in tumorigenesis has been receiving increased attention. HIF is one of the master regulators that orchestrate the cellular responses to hypoxia. The authors therefore hypothesized that unique HIF responses might contribute to glioma initiation and maintenance of GSCs.

Schematic Representation of HIF Family Member Protein Domains bHLH: basic helix-loop-helix PAS: PER-ARNT-SIM ODD: oxygen-dependent degradation domain TAD: transactivation domains

Mechanisms of HIF Activation

List of HIF-regulated Genes Promoting Key Aspects of Tumorigenesis.

Purpose of the Study Previous data have linked HIFs to tumorigenesis in the aspects of angiogenesis, metabolism, proliferation and metastasis. The authors sought to determine HIF expression and its biological consequences in the context of the GSCs and non-stem glioma cell subpopulations.

Figure 1. Glioblastoma Stem and Non-Stem Cells Differentially Expressed Hypoxia Response Genes

Figure 1. Glioblastoma Stem and Non-Stem Cells Differentially Expressed Hypoxia Response Genes

Figure 2. GSCs and Normal Neural Progenitors Differentially Expressed Hypoxia Response Genes

Figure 2. GSCs and Normal Neural Progenitors Differentially Expressed Hypoxia Response Genes

Figure 3. Hypoxia Potently Induced HIF2a Protein Expression in GSCs

Figure 3. Hypoxia Potently Induced HIF2a Protein Expression in GSCs

Figure 4. HIF2a Coexpressed with Cancer Stem Cell (CSC) Markers in Human Glioblastoma Biopsy Specimens

Figure 4. HIF2a Coexpressed with CSC Markers in Human Glioblastoma Biopsy Specimens

Figure 5. HIF Knockdown Altered GSCs Neurosphere Formation

Figure 5. HIF Knockdown Altered GSCs Neurosphere Formation

Figure 6. HIF Knockdown Reduced GSC Growth due to Elevated Apoptosis

Figure 6. HIF Knockdown Reduced GSC Growth due to Elevated Apoptosis

Figure 7. HIF Knockdown Decreased GSC Mediated Angiogenesis

Figure 7. HIF Knockdown Decreased GSC Mediated Angiogenesis

Figure 8. HIF Knockdown Suppressed CSC Mediated Tumor Growth

Figure 8. HIF Knockdown Suppressed CSC Mediated Tumor Growth

Conclusions GSCs preferentially express HIF2α on both mRNA and protein levels under normoxic and hypoxic conditions. Acute hypoxic conditions can induce the expression of HIF1α protein in GSCs, non-stem cancer cells and neural progenitor cells. HIF2α is required for the survival and VEGF expression in GSCs, but not non-stem cells, whereas HIF1α is required in both tumor subpopulations. Targeting HIF2α in GSCs decreases tumorigenic capacity and increases the survival of mice bearing intracranial xenografts as well as or significantly more than HIF1α. Elevated HIF2α expression is associated with poor surval of glioma patients.

Significance Considering the specific expression in GSCs, HIF2α appears to be an attractive target for antineoplastic agents.