人源性肺癌组织移植瘤模型的研究进展

人源性肺癌组织移植瘤模型（patient-derived xenografts ,PDX ）是指将肺癌患者的肿瘤组织移植于免疫缺陷鼠进行增殖传代,传代的肺癌组织高度保存肿瘤生长所处的微环境,且保留原发肺癌组织的生物学、组织病理学特征以及肿瘤标志物等,是一种理想的模拟人体内环境的动物模型。该模型对肿瘤临床前期评估、抗肿瘤药物疗效评价、分析生物标志物等有重要意义,为肺癌的个体化治疗研究带来了新方向。     

Afatinib treatment for her-2 amplified metastatic colorectal cancer based on patient-derived xenograft models and next generation sequencing

Background: Substantial progress has been made in metastatic colorectal cancer (mCRC) treatment, but there is still a fraction of patients cannot find any effective therapeutic strategy after guideline-recommended standard chemotherapy and molecular targeted therapy.

Case presentation: Here we present a KRAS/NRAS/BRAF wild-type mCRC patient who has been previously treated with FOLFIRI (fluorouracil, leucovorin, and irinotecan), XELOX (capecitabine and oxaliplatin), cetuximab and bevacizumab, and then received the next generation sequencing (NGS) and whose metastatic subcutaneous nodule was resected to generate patient-derived xenograft (PDX) models. The NGS revealed HER-2 amplification as well as an activating mutation S310F and PDX models tested several drugs finding that afatinib was the optimal agent with notable efficacy and well tolerance among 6 regimens. Therefore, this patient started to take afatinib orally and achieved 3 months progression-free survival (PFS) and relief of clinical symptoms without severe adverse effects.

Conclusions: NGS and PDX models have great significance for precision and individualized medicine in the mCRC treatment, especially for patients whose diseases have been progressed after multiline standard therapies.     

Capturing colorectal cancer inter-tumor heterogeneity in patient-derived xenograft (PDX) models

Patient-derived xenograft (PDX) models have become an important asset in translational cancer research. However, to provide a robust preclinical platform, PDXs need to accommodate the tumor heterogeneity that is observed in patients. Colorectal cancer (CRC) can be stratified into four consensus molecular subtypes (CMS) with distinct biological and clinical features. Surprisingly, using a set of CRC patients, we revealed the partial representation of tumor heterogeneity in PDX models. The epithelial subtypes, the largest subgroups of CRC subtype, were very ineffective in establishing PDXs, indicating the need for further optimization to develop an effective personalized therapeutic approach to CRC. Moreover, we showed that tumor cell proliferation was associated with successful PDX establishment and able to distinguish patient with poor clinical outcomes within CMS2 group.     

Bitter melon juice intake with gemcitabine intervention circumvents resistance to gemcitabine in pancreatic patient-derived xenograft tumors

Chemoresistance to gemcitabine (GEM)—a frontline chemotherapeutic, resulting from its dysfunctional uptake and metabolism in cancer cells, is a major contributing factor for failed therapy in pancreatic cancer (PanC) patients. Therefore, there is an urgent need for agents that could reverse GEM resistance and allow continued chemosensitivity to the drug. We employed natural nontoxic agent (with anti-PanC potential) bitter melon juice (BMJ) and GEM to examine their combinatorial benefits against tumorigenesis of PanC patient-derived xenograft (PDX)—pancreatic ductal adenocarcinomas explants PDX272 (wild-type KRAS), PDX271 (mutant KRAS and SMAD4), and PDX266 (mutant KRAS). Anti-PanC efficacy of single agents vs combination in the three tumor explants, both at the end of active dosing regimen and following a drug-washout phase were compared. In animal studies, GEM alone treatment significantly inhibited PDX tumor growth, but effects were not sustained, as GEM-treated tumors exhibited regrowth posttreatment termination. However, combination-regimen displayed enhanced and sustained efficacy. Mechanistic assessments revealed that overcoming GEM resistance by coadministration with BMJ was possibly due to modulation of GEM transport/metabolism pathway molecules (ribonucleotide reductase regulatory subunit M1, human equilibrative nucleoside transporter 1, and deoxycytidine kinase). Study outcomes, highlighting significantly higher and sustained efficacy of GEM in combination with BMJ, make a compelling case for a clinical trial in PanC patients, wherein BMJ could be combined with GEM to target and overcome GEM resistance. In addition, given their specific effectiveness against KRAS-mutant tumors, this combination could be potentially beneficial to a broader PanC patient population.     

Ovarian cancer stem cells: Working towards the root of stemness

Despite medical advances made over the past decade, ovarian cancer remains one of the more lethal gynecologic cancers in the United States. While current therapeutic strategies are relatively effective, there is a high incidence of recurrent chemoresistant disease. This has been attributed, in part, to a regenerative tumor cell sub-population that has acquired stem cell properties which allows these cells to escape standard chemotherapeutics and drive recurrent disease. To date, a number of laboratories have identified these cancer stem cell (CSC) sub-populations in ovarian cancer cell lines, tumors or ascites and the collective findings suggest ovarian CSCs are likely to be as heterogeneous as the disease itself. Moreover, the multiple ovarian histophenotypes and possible sites of disease origin together with the potential for differential hierarchal contributions of multiple CSCs populations represent significant challenges to the identification, functional characterization and therapeutic targeting of ovarian CSC. This review will highlight the markers and methodology currently used to identify and isolate these cells. We will discuss some of the underlying ovarian CSC biology, the signaling pathways implicated in their survival, replication and differentiation and potential therapeutic targeting strategies.     

基于穿刺活检的复发转移性结直肠癌患者源性异种移植模型的建立

背景与目的:现行的结直肠癌患者源性异种移植(patient-derived xenografts,PDXs)模型建模样本多来源于外科手术,但复发转移性结直肠癌(metastatic colorectal cancer,mCRC)患者手术机会少,难以获取标本.该研究旨在利用穿刺活检术建立mCRC的PDXs模型.方法:入组12例结直肠癌根治术后患者,存在临床症状和(或)影像学提示术后复发和(或)转移,需行穿刺活检明确诊断、且不存在穿刺活检禁忌证者,保留用于常规病理诊断包括基因检测的组织量后,剩余的标本用于PDXs模型的建立.结果:共成功建立7例mCRC的PDXs模型,成功率为77.8%.结论:基于穿刺活检术建立mCRC的PDXs模型成功率高,可较完整的复制原始肿瘤特性,且操作安全、简便.     

Correlation between tumor engraftment in patient-derived xenograft models and clinical outcomes in colorectal cancer patients

Despite numerous studies involving patient-derived xenograft (PDX) models, few studies have investigated the relationship between the ability of the tumor to engraft (tumorigenicity) and the clinical features of colorectal cancer (CRC). The aim of this study was to determine whether tumorigenicity correlates with clinical outcomes of CRC patients. We included 241 CRC patients who underwent radical surgery from 2010 to 2013. PDX models were established by implanting tumor fragments obtained from these patients into the subcutaneous layer of immunodeficient mice. Xenografts were successfully established from 62.2%. Successful engraftment was associated with advanced stage (p < 0.001) and moderate/poor differentiation (p = 0.029). Three-year disease-free survival (DFS) rates were lower for patients with tumorigenicity (p = 0.011). In stage III patients, tumorigenicity was an independent predictor of poor DFS (p = 0.034). In addition, mutation of TP53 was most frequently detected in stage III patients with tumorigenicity. Two models of stage IV disease without KRAS mutations showed high sensitivity to EGFR-targeted agents, while none of the models with KRAS mutations showed high sensitivity. In conclusion, PDX models may provide an effective preclinical tool for predicting cancer progression and could be used to further genomic and pharmacologic research on personalized treatments.     

An increase in cancer stem cell population after primary systemic therapy is a poor prognostic factor in breast cancer

Background:

The cancer stem cell (CSC) hypothesis has important clinical implications for cancer therapeutics because of the proposed role of CSCs in chemoresistance. The aim of this study was to investigate changes in the CSC populations before and after primary systemic therapy (PST) and their prognostic role in human breast cancer.

Methods:

Paired samples (before and after PST) of breast cancer tissue were obtained from clinical stage II or III patients (n=92) undergoing PST with the regimen of doxorubicin plus docetaxel (AD) (n=50) or doxorubicin plus cyclophosphamide (AC) (n=42) and subsequent breast resection. The proportions of putative CSCs with CD44+/CD24− or aldehyde dehydrogenase 1+ (ALDH1+) phenotypes were determined by immunohistochemistry.

Results:

A higher proportion of CD44+/CD24− tumour cells and ALDH1 positivity in pre-chemotherapy tissue was correlated with higher histologic grade, oestrogen receptor (ER) negativity, high Ki-67 proliferation index and basal-like subtype of breast cancer. Aldehyde dehydrogenase 1 positivity in pre-chemotherapy biopsy was also associated with a higher rate of pathologic complete response following PST. In comparisons of putative CSC populations before and after PST, the proportions of CD44+/CD24− and ALDH1+ tumour cells were significantly increased after PST. The cases with increased CD44+/CD24− tumour cell populations after PST showed high Ki-67 proliferation index in post-chemotherapy specimens and those with increased ALDH1+ tumour cell population after PST were associated with ER negativity and p53 overexpression. Furthermore, cases showing such an increase had significantly shorter disease-free survival time than those with no change or a reduced number of CSCs, and the survival difference was most notable with regard to the changes of ALDH1+ tumour cell population in the patients who received AC regimen.

Conclusion:

The present study provides the clinical evidence that the putative CSCs in breast cancer are chemoresistant and are associated with tumour progression, emphasising the need for targeting of CSCs in the breast cancer therapeutics.     

Cabozantinib inhibits tumor growth and metastasis of a patient-derived xenograft model of papillary renal cell carcinoma with MET mutation

MET plays an important role in the development and progression of papillary renal cell carcinoma (pRCC). Evaluation of efficacy of MET inhibitors against pRCC has been hampered by limited preclinical models depicting MET abnormalities. We established a new patient-derived xenograft (PDX) model of pRCC carrying an activating mutation of MET and tested the ability of cabozantinib, an inhibitor of receptor tyrosine kinases including MET, to inhibit tumor growth and metastasis. Precision-cut, thin tissue slices from a pRCC specimen obtained by nephrectomy were implanted under the renal capsule of RAG2^?/?γC^?/? mice to establish first generation TSG-RCC-030. Histologic and genetic fidelity and metastatic potential of this model were characterized by immunohistochemistry, direct DNA sequencing and quantitative polymerase chain reaction (qPCR). The effect of cabozantinib on tumor growth and metastasis was evaluated. Whether measurements of circulating tumor DNA (ctDNA) by allele-specific qPCR could be used as a biomarker of tumor growth and response to therapy was determined. Subrenal and subcutaneous tumor grafts showed high take rates and metastasized to the lung. Both primary tumors and metastases expressed typical markers of pRCC and carried the same activating MET mutation as the parental tumor. Cabozantinib treatment caused striking tumor regression and inhibited lung metastasis in TSG-RCC-030. Plasma ctDNA levels correlated with tumor volume in control mice and changed in response to cabozantinib treatment. TSG-RCC-030 provides a realistic preclinical model to better understand the development and progression of pRCC with MET mutation and accelerate the development of new therapies for pRCC.     

Surgical efficacy and quality of wide resection of the pelvic peritoneum in patients with epithelial ovarian cancer

PurposeThe aim of the study was to evaluate the impact of adding an extensive pelvic peritoneal stripping procedure, termed “wide resection of the pelvic peritoneum,” (WRPP) to standard surgery for epithelial ovarian cancer on survival effectiveness and to investigate the role of ovarian cancer stem cells (CSCs) in the pelvic peritoneum.MethodsA total of 166 patients with ovarian cancer undergoing surgical treatment at Kumamoto University Hospital between 2002 and 2018 were retrospectively analyzed. Eligible patients were divided into three groups based on the surgical approach: standard surgery (SS) group (n = 36), WRPP group (standard surgery plus WRPP, n = 100), and rectosigmoidectomy (RS) group (standard surgery plus RS, n = 30). Survival outcomes were compared between the three groups. CD44 variant 6 (CD44v6) and EpCAM expression, as markers of ovarian CSCs, in peritoneal disseminated tumors were evaluated using immunofluorescence staining.ResultsWith respect to patients with stage IIIA–IVB ovarian cancer, there were significant differences in overall and progression-free survival between the WRPP and SS groups, as revealed by univariate (hazard ratio [HR], 0.35; 95% confidence interval [CI], 0.17–0.69; P = 0.003 and HR, 0.54; 95% CI, 0.31–0.95; P = 0.032, respectively) and multivariate Cox proportional hazards models (HR, 0.35; 95% CI, 0.17–0.70; P = 0.003 and HR, 0.54; 95% CI, 0.31–0.95; P = 0.032, respectively). Further, no significant differences were observed in survival outcomes between the RS group and the SS or WRPP group. Regarding the safety of WRPP, no significant differences in major intraoperative and postoperative complications were found between the three groups. Immunofluorescence analysis revealed a high percentage of CD44v6/EpCAM double-positive ovarian cancer cells in peritoneal disseminated tumors.ConclusionThe present study demonstrates that WRPP significantly contributes to improved survival in patients with stage IIIA–IVB ovarian cancer. WRPP could result in eradicating ovarian CSCs and disrupting the CSC niche microenvironment in the pelvic peritoneum.     

Outcomes from ovarian cancer screening in the PLCO trial: Histologic heterogeneity impacts detection,overdiagnosis and survival

AimA mortality benefit from screening for ovarian cancer has never been demonstrated. The aim of this study was to evaluate the screening outcomes for different histologic subtypes of ovarian cancers.MethodsWomen in the screening arm of the Prostate, Lung, Colorectal and Ovarian Screening Trial underwent CA-125 and transvaginal ultrasound annually for 3–5 years. We compared screening test characteristics (including overdiagnosis) and outcomes by tumour type (type II versus other) and study arm (screening versus usual care).ResultsOf 78,215 women randomised, 496 women were diagnosed with ovarian cancer. Of the tumours that were characterised (n = 413; 83%), 74% (n = 305) were type II versus 26% other (n = 108). Among screened patients, 70% of tumours were type II compared to 78% in usual care (p = 0.09). Within the screening arm, 29% of type II tumours were screen detected compared to 54% of the others (p < 0.01). The sensitivity of screening was 65% for type II tumours versus 86% for other types (p = 0.02). 15% of type II screen-detected tumours were stage I/II, compared to 81% of other tumours (p < 0.01). The overdiagnosis rate was lower for type II compared to other tumours (28.2% versus 72.2%; p < 0.01). Ovarian cancer–specific survival was worse for type II tumours compared to others (p < 0.01). Survival was similar for type II (p = 0.74) or other types (p = 0.32) regardless of study arm.ConclusionsTest characteristics of screening for ovarian cancer differed for type II tumours compared to other ovarian tumours. Type II tumours were less likely to be screen diagnosed, early stage at diagnosis or overdiagnosed.     

A large collection of integrated genomically characterized patient-derived xenografts highlighting the heterogeneity of triple-negative breast cancer

Triple-negative breast cancer (TNBC) represents 10% of all breast cancers and is a very heterogeneous disease. Globally, women with TNBC have a poor prognosis, and the development of effective targeted therapies remains a real challenge. Patient-derived xenografts (PDX) are clinically relevant models that have emerged as important tools for the analysis of drug activity and predictive biomarker discovery. The purpose of this work was to analyze the molecular heterogeneity of a large panel of TNBC PDX (n = 61) in order to test targeted therapies and identify biomarkers of response. At the gene expression level, TNBC PDX represent all of the various TNBC subtypes identified by the Lehmann classification except for immunomodulatory subtype, which is underrepresented in PDX. NGS and copy number data showed a similar diversity of significantly mutated gene and somatic copy number alteration in PDX and the Cancer Genome Atlas TNBC patients. The genes most commonly altered were TP53 and oncogenes and tumor suppressors of the PI3K/AKT/mTOR and MAPK pathways. PDX showed similar morphology and immunohistochemistry markers to those of the original tumors. Efficacy experiments with PI3K and MAPK inhibitor monotherapy or combination therapy showed an antitumor activity in PDX carrying genomic mutations of PIK3CA and NRAS genes. TNBC PDX reproduce the molecular heterogeneity of TNBC patients. This large collection of PDX is a clinically relevant platform for drug testing, biomarker discovery and translational research.     

Epigenetic regulation of RGS2 (Regulator of G-protein signaling 2) in chemoresistant ovarian cancer cells

Regulator of G-protein signaling 2 (RGS2) is a GTPase-activating protein functioning as an inhibitor of G-protein coupled receptors (GPCRs). RGS2 dysregulation was implicated in solid tumour development and RGS2 downregulation has been reported in prostate and ovarian cancer progression. However, the molecular mechanism by which RGS2 expression is suppressed in ovarian cancer remains unknown. The expression and epigenetic regulation of RGS2 in chemosensitive and chemoresistant ovarian cancer cells were determined by qRT-PCR and chromatin immunoprecipitation assays, respectively. In the present study, the molecular mechanisms contributing to the loss of RGS2 expression were determined in ovarian cancer. The data indicated that suppression of RGS2 gene in chemoresistant ovarian cancer cells, in part, due to accumulation of histone deacetylases (HDACs) and DNA methyltransferase I (DNMT1) at the promoter region of RGS2. Inhibition of HDACs or DNMTs significantly increases RGS2 expression. These results suggest that epigenetic changes in histone modifications and DNA methylation may contribute to the loss of RGS2 expression in chemoresistant ovarian cancer cells. The results further suggest that class I HDACs and DNMT1 contribute to the suppression of RGS2 during acquired chemoresistance and support growing evidence that inhibition of HDACs/DNMTs represents novel therapeutic approaches to overcome ovarian cancer chemoresistance.     

Downregulation of XIAP expression in ovarian cancer cells induces cell death in vitro and in vivo

A hallmark of cancer cells is an ability to evade apoptosis. Overexpression and/or activating mutations of prosurvival molecules such as the X-linked inhibitor of apoptosis (XIAP) contribute to this inappropriate cell survival. Our objectives were to investigate the effects of downregulation of XIAP in ovarian cancer cells in vitro and in vivo using the clinical candidate antisense oligonucleotide against XIAP, AEG35156 (AS XIAP). Three ovarian cancer cell lines were transfected with AS XIAP in vitro, and the effects on cell survival were assessed. Downregulation of XIAP resulted in significant apoptosis. To investigate the in vivo efficacy of AS XIAP, CD-1 nude mice were xenografted intraperitoneally with A2780-cp cells, treated with intraperitoneal AS XIAP and evaluated for survival time and tumor histology. Mice treated with 10 mg/kg/day AS XIAP showed a significant improvement in survival time compared to animals treated with control oligonucleotides. Histological analysis of the tumors showed significantly fewer viable cells in the AS XIAP-treated tumors. Downregulation of XIAP expression in ovarian cancer cells resulted in apoptosis in vitro and a prolonged survival time of ovarian cancer-bearing mice, which indicate that XIAP may be a valuable therapeutic target in ovarian cancers, and supports the ongoing clinical investigation of AEG35156.     

Emerging role of cancer stem cells in the biology and treatment of ovarian cancer: basic knowledge and therapeutic possibilities for an innovative approach

In 2013 there will be an estimated 22,240 new diagnoses and 14,030 deaths from ovarian cancer in the United States. Despite the improved surgical approach and the novel active drugs that are available today in clinical practice, about 80% of women presenting with late-stage disease have a 5-year survival rate of only 30%. In the last years a growing scientific knowledge about the molecular pathways involved in ovarian carcinogenesis has led to the discovery and evaluation of several novel molecular targeted agents, with the aim to test alternative models of treatment in order to overcome the clinical problem of resistance. Cancer stem cells tend to be more resistant to chemotherapeutic agents and radiation than more differentiated cellular subtypes from the same tissue. In this context the study of ovarian cancer stem cells is taking on an increasingly important strategic role, mostly for the potential therapeutic application in the next future. In our review, we focused our attention on the molecular characteristics of epithelial ovarian cancer stem cells, in particular on possible targets to hit with targeted therapies.     

A clinically relevant orthotopic xenograft model of ependymoma that maintains the genomic signature of the primary tumor and preserves cancer stem cells in vivo

Limited availability of in vitro and in vivo model systems has hampered efforts to understand tumor biology and test novel therapies for ependymoma, the third most common malignant brain tumor that occurs in children. To develop clinically relevant animal models of ependymoma, we directly injected a fresh surgical specimen from a 9-year-old patient into the right cerebrum of RAG2/severe complex immune deficiency (SCID) mice. All five mice receiving the initial transplantation of the patient tumor developed intracerebral xenografts, which have since been serially subtransplanted in vivo in mouse brains for 4 generations and can be cryopreserved for long-term maintenance of tumorigenicity. The xenograft tumors shared nearly identical histopathological features with the original tumors, harbored 8 structural chromosomal abnormalities as detected with spectral karyotyping, maintained gene expression profiles resembling that of the original patient tumor with the preservation of multiple key genetic abnormalities commonly found in human ependymomas, and contained a small population (<2.2%) of CD133⁺ stem cells that can form neurospheres and display multipotent capabilities in vitro. The permanent cell line (BXD-1425EPN), which was derived from a passage II xenograft tumor and has been passaged in vitro more than 70 times, expressed similar differentiation markers of the xenograft tumors, maintained identical chromosomal abnormalities, and formed tumors in the brains of SCID mice. In conclusion, direct injection of primary ependymoma tumor cells played an important role in the generation of a clinically relevant mouse model IC-1425EPN and a novel cell line, BXD-1425EPN. This cell line and model will facilitate the biological studies and preclinical drug screenings for pediatric ependymomas.     

Isolation and characterization of cancer stem-like side population cells in human oral cancer cells

Recent studies suggest that cancer stem cells may be responsible for tumorigenesis and contribute to some individuals’ resistance to cancer therapy. Some studies demonstrate that side population (SP) cells isolated from diverse cancer cell lines harbor stem cell-like properties; however, there are few reports examining the role of SP cells in human oral cancer. To determine whether human oral cancer cell lines contain a SP cell fraction, we first isolated SP cells by fluorescence activated cell sorting, followed by culturing in serum-free medium (SFM) using the SCC25 tongue cancer cell line, so that SP cells were able to be propagated to maintain the CSC property. Differential expression profile of stem cell markers (ABCG2, Oct-4 and EpCAM) was examined by RT-PCR in either SP cells or non-SP cells. Growth inhibition by 5-FU was determined by the MTT assay. Clonogenic ability was evaluated by colony formation assay. SCC25 cells contained 0.23% SP cells. The fraction of SP cells was available to grow in SFM cultures. SP cells showed higher mRNA expression of stem cell markers (ABCG2, Oct-4 and EpCAM) as compared with non-SP cells. Moreover, SP cells demonstrated more drug resistance to 5-FU, as compared with non-SP cells. The clone formation efficiency of SP cells was significantly higher than non-SP cells at an equal cell number (P < 0.01). We isolated cancer stem-like SP cells from an oral cancer cell line. SP cells possessed the characteristics of cancer stem cells, chemoresistance, and high proliferation ability. Further characterization of cancer stem-like SP cells may provide new insights for novel therapeutic targets.     

A single intravenous injection of oncolytic picornavirus SVV-001 eliminates medulloblastomas in primary tumor-based orthotopic xenograft mouse models

Difficulties of drug delivery across the blood-brain barrier (BBB) and failure to eliminate cancer stem cells (CSCs) are believed to be the major causes of tumor recurrences in children with medulloblastoma (MB). Seneca Valley virus-001 (SVV-001) is a naturally occurring oncolytic picornavirus that can be systemically administered. Here, we report its antitumor activities against MB cells in a panel of 10 primary tumor-based orthotopic xenograft mouse models. We found that SVV-001 killed the primary cultured xenograft cells, infected and replicated in tumor cells expressing CSC surface marker CD133, and eliminated tumor cells capable of forming neurospheres in vitro in 5 of the 10 xenograft models. We confirmed that SVV-001 could pass through BBB in vivo. A single i.v. injection of SVV-001 in 2 anaplastic MB models led to widespread infection of the preformed intracerebellar (ICb) xenografts, resulting in significant increase in survival (2.2-5.9-fold) in both models and complete elimination of ICb xenografts in 8 of the 10 long-term survivors. Mechanistically, we showed that the intracellular replication of SVV-001 is mediated through a subverted autophagy that is different from the bona fide autophagic process induced by rapamycin. Our data suggest that SVV-001 is well suited for MB treatment. This work expands the current views in the oncolytic therapy field regarding the utility of oncolytic viruses in simultaneous targeting of stem and nonstem tumor cells.     

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.