High mitochondrial mass identifies a sub-population of stem-like cancer cells that are chemo-resistant

Chemo-resistance is a clinical barrier to more effective anti-cancer therapy. In this context, cancer stem-like cells (CSCs) are thought to be chemo-resistant, resulting in tumor recurrence and distant metastasis. Our hypothesis is that chemo-resistance in CSCs is driven, in part, by enhanced mitochondrial function. Here, we used breast cell lines and metastatic breast cancer patient samples to begin to dissect the role of mitochondrial metabolism in conferring the CSC phenotype. More specifically, we employed fluorescent staining with MitoTracker (MT) to metabolically fractionate these cell lines into mito-high and mito-low sub-populations, by flow-cytometry. Interestingly, cells with high mitochondrial mass (mito-high) were specifically enriched in a number of known CSC markers, such as aldehyde dehydrogenase (ALDH) activity, and they were ESA+/CD24-/low and formed mammospheres with higher efficiency. Large cell size is another independent characteristic of the stem cell phenotype; here, we observed a >2-fold increase in mitochondrial mass in large cells (>12-μm), relative to the smaller cell population (4–8-μm). Moreover, the mito-high cell population showed a 2.4-fold enrichment in tumor-initiating cell activity, based on limiting dilution assays in murine xenografts. Importantly, primary human breast CSCs isolated from patients with metastatic breast cancer or a patient derived xenograft (PDX) also showed the co-enrichment of ALDH activity and mitochondrial mass. Most significantly, our investigations demonstrated that mito-high cells were resistant to paclitaxel, resulting in little or no DNA damage, as measured using the comet assay. In summary, increased mitochondrial mass in a sub-population of breast cancer cells confers a stem-like phenotype and chemo-resistance. As such, our current findings have important clinical implications for over-coming drug resistance, by therapeutically targeting the mito-high CSC population.     

Antibiotics that target mitochondria effectively eradicate cancer stem cells,across multiple tumor types: Treating cancer like an infectious disease

Here, we propose a new strategy for the treatment of early cancerous lesions and advanced metastatic disease, via the selective targeting of cancer stem cells (CSCs), a.k.a., tumor-initiating cells (TICs). We searched for a global phenotypic characteristic that was highly conserved among cancer stem cells, across multiple tumor types, to provide a mutation-independent approach to cancer therapy. This would allow us to target cancer stem cells, effectively treating cancer as a single disease of “stemness”, independently of the tumor tissue type. Using this approach, we identified a conserved phenotypic weak point – a strict dependence on mitochondrial biogenesis for the clonal expansion and survival of cancer stem cells. Interestingly, several classes of FDA-approved antibiotics inhibit mitochondrial biogenesis as a known “side-effect”, which could be harnessed instead as a “therapeutic effect”. Based on this analysis, we now show that 4-to-5 different classes of FDA-approved drugs can be used to eradicate cancer stem cells, in 12 different cancer cell lines, across 8 different tumor types (breast, DCIS, ovarian, prostate, lung, pancreatic, melanoma, and glioblastoma (brain)). These five classes of mitochondrially-targeted antibiotics include: the erythromycins, the tetracyclines, the glycylcyclines, an anti-parasitic drug, and chloramphenicol. Functional data are presented for one antibiotic in each drug class: azithromycin, doxycycline, tigecycline, pyrvinium pamoate, as well as chloramphenicol, as proof-of-concept. Importantly, many of these drugs are non-toxic for normal cells, likely reducing the side effects of anti-cancer therapy. Thus, we now propose to treat cancer like an infectious disease, by repurposing FDA-approved antibiotics for anti-cancer therapy, across multiple tumor types. These drug classes should also be considered for prevention studies, specifically focused on the prevention of tumor recurrence and distant metastasis. Finally, recent clinical trials with doxycycline and azithromycin (intended to target cancer-associated infections, but not cancer cells) have already shown positive therapeutic effects in cancer patients, although their ability to eradicate cancer stem cells was not yet appreciated.     

Brain tumor stem cells: The cancer stem cell hypothesis writ large

Brain tumors, which are typically very heterogeneous at the cellular level, appear to have a stem cell foundation. Recently, investigations from multiple groups have found that human as well as experimental mouse brain tumors contain subpopulations of cells that functionally behave as tumor stem cells, driving tumor growth and generating tumor cell progeny that form the tumor bulk, but which then lose tumorigenic ability. In human glioblastomas, these tumor stem cells express neural precursor markers and are capable of differentiating into tumor cells that express more mature neural lineage markers. In addition, modeling brain tumors in mice suggests that neural precursor cells more readily give rise to full blown tumors, narrowing potential cells of origin to those rarer brain cells that have a proliferative potential. Applying stem cell concepts and methodologies is giving fresh insight into brain tumor biology, cell of origin and mechanisms of growth, and is offering new opportunities for development of more effective treatments. The field of brain tumor stem cells remains very young and there is much to be learned before these new insights are translated into new patient treatments.     

Defining the dimensions of circulating tumor cells in a large series of breast,prostate, colon,and bladder cancer patients

Circulating tumor cells (CTCs) in the blood of cancer patients are of high clinical relevance. Since detection and isolation of CTCs often rely on cell dimensions, knowledge of their size is key. We analyzed the median CTC size in a large cohort of breast (BC), prostate (PC), colorectal (CRC), and bladder (BLC) cancer patients. Images of patient‐derived CTCs acquired on cartridges of the FDA‐cleared CellSearch^® method were retrospectively collected and automatically re‐analyzed using the accept software package. The median CTC diameter (μm) was computed per tumor type. The size differences between the different tumor types and references (tumor cell lines and leukocytes) were nonparametrically tested. A total of 1962 CellSearch^® cartridges containing 71 612 CTCs were included. In BC, the median computed diameter (CD) of patient‐derived CTCs was 12.4 μm vs 18.4 μm for cultured cell line cells. For PC, CDs were 10.3 μm for CTCs vs 20.7 μm for cultured cell line cells. CDs for CTCs of CRC and BLC were 7.5 μm and 8.6 μm, respectively. Finally, leukocytes were 9.4 μm. CTC size differed statistically significantly between the four tumor types and between CTCs and the reference data. CTC size differences between tumor types are striking and CTCs are smaller than cell line tumor cells, whose size is often used as reference when developing CTC analysis methods. Based on our data, we suggest that the size of CTCs matters and should be kept in mind when designing and optimizing size‐based isolation methods.

Abbreviations

ACCEPT

Automated CTC Classification, Enumeration, and PhenoTyping software

BC

breast cancer

BLC

bladder cancer

CD

computed diameter

CEL

cultured tumor cell (cell line)

CK

cytokeratin

CRC

colorectal cancer

CTC‐L

circulating tumor cells derived from cerebrospinal fluid (liquor)

CTCs

circulating tumor cells

DAPI

4′6‐diamidino‐2‐phenylindole

EMT

epithelial–mesenchymal transition

EpCAM

epithelial cell adhesion molecule

IQR

interquartile range

KW test

Kruskal–Wallis test

MWU test

Mann–Whitney U test

NCR

nucleus/cytoplasm ratio

P2A

perimeter to area

PC

prostate cancer

TIF

tagged Image Format files

TXT

text file

μm

micrometer

µm²

square micrometers

     

Identification of a cancer stem-like population in the Lewis lung cancer cell line

Objective: Although various human cancer stem cells (CSCs) have been defined, their applications are restricted to immunocompromised models. Developing a novel CSC model which could be used in immunocompetent or transgenic mice is essential for further understanding of the biomolecular characteristics of tumor stem cells. Therefore, in this study, we analyzed murine lung cancer cells for the presence of CSCs. Methods: Side population (SP) cells were isolated by fluorescence activated cell sorting, followed by serum-free medium (SFM) culture, using Lewis lung carcinoma cell (LLC) line. The self-renewal, differentiated progeny, chemosensitivity, and tumorigenic properties in SP and non-SP cells were investigated through in vitro culture and in vivo serial transplantation. Differential expression profiles of stem cell markers were examined by RT-PCR. Results: The SP cell fraction comprised 1.1% of the total LLC population. SP cells were available to grow in SFM, and had significantly enhanced capacity for cell proliferation and colony formation. They were also more resistant to cisplatin in comparison to non-SP cells, and displayed increased tumorigenic ability. Moreover, SP cells showed higher mRNA expression of Oct-4, ABCG2, and CD44. Conclusion: We identified SP cells from a murine lung carcinoma, which possess well-known characteristics of CSCs. Our study established a useful model that should allow investigation of the biological features and pharmacosensitivity of lung CSCs, both in vitro and in syngeneic immunocompetent or transgenic/knockout mice.     

Tumor heterogeneity and cancer stem cell paradigm: Updates in concept,controversies and clinical relevance

Although tumor heterogeneity is widely accepted, the existence of cancer stem cells (CSCs) and their proposed role in tumor maintenance has always been challenged and remains a matter of debate. Recently, a path‐breaking chapter was added to this saga when three independent groups reported the in vivo existence of CSCs in brain, skin and intestinal tumors using lineage‐tracing and thus strengthens the CSC concept; even though certain fundamental caveats are always associated with lineage‐tracing approach. In principle, the CSC hypothesis proposes that similar to normal stem cells, CSCs maintain self renewal and multilineage differentiation property and are found at the central echelon of cellular hierarchy present within tumors. However, these cells differ from their normal counterpart by maintaining their malignant potential, alteration of genomic integrity, epigenetic identity and the expression of specific surface protein profiles. As CSCs are highly resistant to chemotherapeutics, they are thought to be a crucial factor involved in tumor relapse and superficially appear as the ultimate therapeutic target. However, even that is not the end; further complication is attributed by reports of bidirectional regeneration mechanism for CSCs, one from their self‐renewal capability and another from the recently proposed concept of dynamic equilibrium between CSCs and non‐CSCs via their interconversion. This phenomenon has currently added a new layer of complexity in understanding the biology of tumor heterogeneity. In‐spite of its associated controversies, this area has rapidly emerged as the center of attention for researchers and clinicians, because of the conceptual framework it provides towards devising new therapies.     

LoVo细胞系中结肠癌干细胞样细胞的分离、培养及鉴定

摘 要　目的：从结肠癌LoVo细胞系中分离、鉴定具有CD44+/EPCAM high特异表型的结肠癌干细胞样细胞,观察其生物学行为,证实该细胞系中结肠癌干细胞样细胞的存在。方法：从普通血清培养的LoVo细胞系中以流式细胞仪分选具有CD44+/EPCAM high表型的细胞,接种于添加生长因子的无血清培养基中,观察其增殖过程,继而诱导分化。MTT法、流式细胞术检测CD44+/EPCAM high、EPCAM low和未分选LoVo细胞的增殖能力及细胞周期分布。3种细胞接种裸鼠,比较不同细胞的成瘤率;免疫荧光技术检测小鼠次代CD44+/EPCAM high细胞中 CD44/EPCAM的表达。结果：LoVo细胞中有17.4%的CD44+/EPCAM high细胞,并能在添加生长因子的无血清培养基中呈细胞球样生长,且可连续传代;在血清的诱导下,呈贴壁分化生长,其形态与未分选LoVo细胞无差别。CD44+/EPCAM high细胞增殖能力高于EPCAM low细胞及未分选LoVo细胞,且细胞周期多集中在G0/G1期。以500个CD44+/EPCAM high细胞接种裸鼠成瘤率为90%（9/10）,而1×104个EPCAM low细胞成瘤率为0（0/10)。小鼠移植瘤中次代CD44+/EPCAM high细胞仍能少量表达CD44和EPCAM。结论：LoVo细胞中存在CD44+/EPCAM high结肠癌干细胞样细胞,CD44+/EPCAM high可用于结肠癌肿瘤干细胞的深入研究。     

Isolation of cancer stem-like cells from a side population of a human glioblastoma cell line,SK-MG-1

Accumulating evidence suggests that in several types of brain tumors, including glioma, only a phenotypic subset of tumor cells called brain cancer stem cells (BCSCs) may be capable of initiating tumor growth. Recently, the isolation of side population (SP) cells using Hoechst dye has become a useful method for obtaining cancer stem cells in various tumors. In this study, we isolated cancer stem-like cells from human glioma cell lines using the SP technique. Flow cytometry analysis revealed that SK-MG-1, a human glioblastoma cell line, contained the largest number of SP cells among the five glioma cell lines that were analyzed. The SP cells had a self-renewal ability and were capable of forming spheres in a neurosphere culture medium containing EGF and FGF2. Spheres derived from the SP cells differentiated into three different lineage cells: neurons, astrocytes and oligodendrocytes. RT-PCR analysis revealed that the SP cells expressed a neural stem cell marker, Nestin. The SP cells generated tumors in the brains of NOD/SCID mice at 8 weeks after implantation, whereas the non-SP cells did not generate any tumors in the brain. These results indicate that SP cells isolated from SK-MG-1 possess the properties of cancer stem cells, including their self-renewal ability, multi-lineage differentiation, and tumorigenicity. Therefore, the SP cells from SK-MG-1 may be useful for analyzing BCSCs because of the ease with which they can be handled and their yield.     

Characterization of sphere-forming cells with stem-like properties from the small cell lung cancer cell line H446

A relatively novel paradigm in tumor biology hypothesizes that cancer growth is driven by tumor cells with stem-like properties. However, direct proof of a population of stem cells in small cell lung cancer (SCLC) remains elusive. In this study, we enriched for stem-like cells from the SCLC cell line H446 by growing them as spheres in a defined serum-free medium. Sphere-derived cells have increased in vitro clonogenic and in vivo tumorigenic potentials as well as drug-resistant properties. After enrichment for stem-like cells, we used multiple candidate stem cell markers to examine the expression profile and found that the sphere-derived cells contained a higher proportion of cells expressing the stem cell surface markers uPAR and CD133 when compared with parental cells. To identify a selectable marker for the sphere-forming cells, we evaluated the sphere-forming abilities of uPAR(+) and uPAR(-) cells as well as the sphere-forming abilities of CD133(+) and CD133(-) cells. Both CD133(+) and CD133(-) cell fractions were capable of forming spheres, and no statistically significant difference was observed in the sphere-forming efficiency between these two populations. In contrast, cells derived from the uPAR(+) fraction were capable of forming spheres, whereas cells derived from the uPAR(-) fraction remained as single cells. Moreover, uPAR(+) cells efficiently formed transplantable tumors, whereas uPAR(-) cells were unable to initiate tumors when transplanted at equivalent cell numbers. In addition, uPAR(+) cells could differentiate into CD56(+)cells, CK(+) cells, and uPAR(-) cells. These data support the existence of a population of tumor sphere-forming cells with stem cell properties in the H446 SCLC cell line. Furthermore, the stem cell population may be enriched in cells expressing the uPAR cell surface marker.     

Systematic dissection of phenotypic,functional, and tumorigenic heterogeneity of human prostate cancer cells

Human cancers are heterogeneous containing stem-like cancer cells operationally defined as cancer stem cells (CSCs) that possess great tumor-initiating and long-term tumor-propagating properties. In this study, we systematically dissect the phenotypic, functional and tumorigenic heterogeneity in human prostate cancer (PCa) using xenograft models and >70 patient tumor samples. In the first part, we further investigate the PSA^−/lo PCa cell population, which we have recently shown to harbor self-renewing long-term tumor-propagating cells and present several novel findings. We show that discordant AR and PSA expression in both untreated and castration-resistant PCa (CRPC) results in AR⁺PSA⁺, AR⁺PSA⁻, AR⁻PSA⁻, and AR⁻PSA⁺ subtypes of PCa cells that manifest differential sensitivities to therapeutics. We further demonstrate that castration leads to a great enrichment of PSA^−/lo PCa cells in both xenograft tumors and CRPC samples and systemic androgen levels dynamically regulate the relative abundance of PSA⁺ versus PSA^−/lo PCa cells that impacts the kinetics of tumor growth. We also present evidence that the PSA^−/lo PCa cells possess distinct epigenetic profiles. As the PSA^−/lo PCa cell population is heterogeneous, in the second part, we employ two PSA⁻ (Du145 and PC3) and two PSA⁺ (LAPC9 and LAPC4) PCa models as well as patient tumor cells to further dissect the clonogenic and tumorigenic subsets. We report that different PCa models possess distinct tumorigenic subpopulations that both commonly and uniquely express important signaling pathways that could represent therapeutic targets. Our results have important implications in understanding PCa cell heterogeneity, response to clinical therapeutics, and cellular mechanisms underlying CRPC.     

人结肠癌CCL187细胞肿瘤球细胞干细胞特性研究

目的：研究体外培养条件下人结肠癌CCL187细胞系肿瘤球细胞的干细胞特性。方法：用无血清培养液和低吸附6孔培养板培养CCL187细胞,显微镜观察肿瘤球形成,荧光染料Hoechst33342染色观察干样细胞,用24孔培养板接种绘制CCL187细胞和肿瘤球细胞的生长曲线,顺铂和依托泊苷处理检测两种细胞抵抗化疗的能力。结果：CCL187细胞在无血清培养条件下可形成肿瘤球,肿瘤球细胞中存在浅染的干样细胞。两种细胞的生长曲线显示肿瘤球细胞的自我繁殖能力大于CCL187细胞。化疗药物处理显示肿瘤球细胞比一般肿瘤细胞具有更强的化疗抵抗性。结论：CCL187细胞的肿瘤球细胞具有肿瘤干细胞特性。     

Live single cell mass spectrometry reveals cancer‐specific metabolic profiles of circulating tumor cells

Recently, there has been increased attention on the analysis of circulating tumor cells (CTCs), also known as liquid biopsy, owing to its potential benefits in cancer diagnosis and treatment. Circulating tumor cells are released from primary tumor lesions into the blood stream and eventually metastasize to distant body organs. However, a major hurdle with CTC analysis is their natural scarcity. Existing methods lack sensitivity, specificity, or reproducibility required in CTC characterization and detection. Here, we report untargeted molecular profiling of single CTCs obtained from gastric cancer and colorectal cancer patients, using live single cell mass spectrometry integrated with microfluidics‐based cell enrichment techniques. Using this approach, we showed the difference in the metabolomic profile between CTCs originating from different cancer groups. Moreover, potential biomarkers were putatively annotated to be specific to each cancer type.     

非小细胞肺癌新辅助化疗后肿瘤体积变化规律及其影响因素

目的:探讨非小细胞肺癌（NSCLC）新辅助化疗后肿瘤体积变化规律及其影响因素。方法:回顾性分析2010年1月至2013年12月在我院肿瘤科确诊并治疗的80例NSCLC患者,使用治疗计划系统（TPS）自带体积测量软件及ImageJ图像处理软件对肿瘤体积进行测量,分析比较不同新辅助化疗周期后肿瘤体积变化情况及其可能的影响因素。结果:化疗1周期后,NSCLC患者平均原发灶肿瘤体积由（89.33±29.24）cm3降至（58.36±23.62）cm3,差异具有统计学意义（t=7.369,P<0.01）;化疗2、3、4周期后, 肿瘤体积变化不显著（P>0.05）;化疗第5周期后,肿瘤体积较第4周期增大（P<0.01）;但第6周期较第5周期无明显变化（P>0.05）。对可能影响化疗1周期后肿瘤体积变化的因素进行单因素分析显示,原发灶肿瘤体积及T分期对化疗1周期后肿瘤体积变化的影响有统计学意义（P<0.05）,而患者的性别、年龄、肿瘤分类、肿瘤部位以及病理类型与其无关。Logistic回归分析显示,原发灶肿瘤体积及T分期均是影响化疗1周期后肿瘤退缩率的独立影响因素(P均<0.05）。结论:NSCLC新辅助化疗1周期肿瘤体积回缩最显著,原发灶肿瘤体积和T分期是影响化疗1周期后疗效的独立因素,建议新辅助化疗1周期后开始介入放疗或同步放化疗。     

Some chemotherapeutics-treated colon cancer cells display a specific phenotype being a combination of stem-like and senescent cell features

Colorectal cancer (CRC) is the second leading cause of death among cancer patients in the Northern countries. CRC can reappear a long time after treatment. Recent clinical studies demonstrated that, in response to chemotherapy, cancer cells may undergo stress-induced premature senescence (SIPS), which typically results in growth arrest. Nonetheless, these senescent cells were reported to divide in an atypical manner and thus contribute to cancer re-growth. Therefore, we examined if SIPS escape may follow treatment with chemotherapeutics used clinically: 5-fluorouracil (5-FU), oxaliplatin (OXA) and irinotecan (IRINO).

To mimic the therapeutic regimes we exposed human colon cancer HCT116 and SW480 cells to repeated cycles of drug treatment. The cells treated with 5-FU or IRINO exhibited several hallmarks of SIPS: growth arrest, increased size and granularity, polyploidization, augmented activity of the SA-β-galactosidase, accumulation of P21 and CYCLIN D1 proteins, and the senescence-associated secretory phenotype. Moreover, re-population of the cancer cell cultures was delayed upon treatment with the senescence-inducing agents. At the same time, we detected a subpopulation of senescent colon cancer cells with features of stemness: elevated NANOG expression, exclusion of Hoechst 33342 (typical for side population) and increased CD24 expression. Additionally, rare, polyploid cells exhibited blastocyst-like morphology and produced progeny. In parallel, majority of chemotherapeutics-treated cells underwent mesenchymal to epithelial transition, as the percentage of CD44-positve cells was reduced, and levels of E-cadherin (epithelial marker) were elevated.

Our study demonstrates that a subpopulation of chemotherapeutics-treated colon cancer cells display a specific phenotype being a combination of stem-like and senescent cell features. This may contribute to their resistance to chemotherapy and their ability to re-grow cancer after completion of therapeutic intervention.     

人肝癌组织中肿瘤干细胞样细胞的分离培养及鉴定

目的：从人肝癌组织中分离培养肝癌干细胞样细胞,为进一步研究肝癌干细胞靶向治疗奠定基础。方法：采用酶消化法和以原代培养结合短暂传代培养的方法从人肝癌组织中分离出含人肝癌干细胞样细胞（human liver cancer stemlike cells, hLCSLCs) 的连续传代细胞,分别加入肝素、白蛋白、氢化可的松以筛选适用于hLCSLCs的无血清悬浮成球培养基。以流式细胞术检测hLCSLCs和由无血清悬浮成球培养所获得的成球细胞中旁群（side population, SP）细胞、CD133及CD90等的表达,裸鼠成瘤实验检测这两种细胞的致瘤能力。结果：成功地从人肝癌组织分离获得hLCSLCs,其SP、CD133+和CD90+细胞含量分别为0.9%、0.8%和12.7%,裸鼠皮下接种2 000个SP细胞的成瘤率为100%。含有肝素的无血清培养基更适于hLCSLCs的成球培养,所获成球细胞中SP、CD133+和CD90+细胞含量分别为4.6%、9.7%和48%,接种10 000个成球细胞即可全部成瘤。结论：成功建立了从人肝癌组织中分离肿瘤干细胞样细胞的方法,添加肝素的无血清悬浮成球培养法可有效富集肝癌干细胞样细胞。