Establishment and evaluation of four different types of patient-derived xenograft models

Background

Patient-derived xenografts (PDX) have a biologically stable in tumor architecture, drug responsiveness, mutational status and global gene-expression patterns. Numerous PDX models have been established to date, however their thorough characterization regarding the tumor formation and rates of tumor growth in the established models remains a challenging task. Our study aimed to provide more detailed information for establishing the PDX models successfully and effectively.

Methods

We transplanted four different types of solid tumors from 108 Chinese patients, including 21 glioblastoma (GBM), 11 lung cancers (LC), 54 gastric cancers (GC) and 21 colorectal cancers (CRC), and took tumor tissues passaged for three successive generations. Here we report the rate of tumor formation, tumor-forming times, tumor growth curves and mortality of mice in PDX model. We also report H&E staining and immunohistochemistry for HLA-A, CD45, Ki67, GFAP, and CEA protein expression between patient cancer tissues and PDX models.

Results

Tumor formation rate increased significantly in subsequent tumor generations. Also, the survival rates of GC and CRC were remarkably higher than GBM and LC. As for the time required for the formation of tumors, which reflects the tumor growth rate, indicated that tumor growth rate always increased as the generation number increased. The tumor growth curves also illustrate this law. Similarly, the survival rate of PDX mice gradually improved with the increased generation number in GC and CRC. And generally, there was more proliferation (Ki67+) in the PDX models than in the patient tumors, which was in accordance with the results of tumor growth rate. The histological findings confirm similar histological architecture and degrees of differentiation between patient cancer tissues and PDX models with statistical analysis by GraphPad Prism 5.0.

Conclusion

We established four different types of PDX models successfully, and our results add to the current understanding of the establishment of PDX models and may contribute to the extension of application of different types of PDX models.

     

Lymphoma immunotherapy with CpG oligodeoxynucleotides requires TLR9 either in the host or in the tumor itself

Established widely metastatic tumor was cured in a transplanted mouse B cell lymphoma model, by the combination of chemotherapy plus intratumoral injection of oligodeoxynucleotides containing unmethylated C-G motifs (CpG). This therapeutic effect required that the CpG be injected directly into the tumor and was dependent on CD8 T cells. Although the efficacy of CpG oligodeoxynucleotides has been thought to depend on the expression of TLR9, we unexpectedly found that tumor rejection did not require host expression of TLR9. By using a TLR9-deficient tumor and a TLR9KO host, we demonstrate that TLR9 expression either by the host or the tumor is required. These results indicate that activation of Ag presentation by cells within the tumor via TLR9 stimulation can be an effective form of immunotherapy. This study forms the basis of an ongoing clinical trial in patients with lymphoma.     

人源性食管癌异种移植模型的建立及应用进展*

食管癌是全球第十大常见癌症,其发病率和病死率较高,主要包括食管鳞状细胞癌(ESCC)和食管腺癌(EAC),通常发展到晚期才被诊断发现。食管癌的标准治疗方法包括放化疗、内窥镜疗法和外科手术,但其预后效果不甚理想。良好的动物模型可用于研究食管癌的发生发展和生物学机制。患者来源的异种移植(PDX)模型最大程度上保留了原始肿瘤的细胞形态、组织结构特征和与患者相似的遗传特征。PDX模型为研究食管癌患者对放化疗的反应性,寻求新的治疗靶点,改善预后效果提供了新的平台,使个性化精准治疗研究迈入新阶段。就食管癌PDX模型的特点、构建时常用的实验动物、优化模型建立的方法以及PDX模型在食管癌研究中的应用进行综述,并讨论了食管癌PDX模型的局限性和未来发展前景,以期为食管癌的个性化精准治疗、改善患者预后提供新的研究方向。     

Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry

To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers for metastatic progression, we have developed a highly metastatic variant of human MDA-MB-435 breast cancer cell line through in vivo stepwise selection of pulmonary metastatic cells caused by parental MDA-MB-435 cells in the athymic mice. Comparative proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein spots were reproducibly altered more than three-fold between the selected variant and its parental counterpart. Eleven differentially expressed protein spots were identified with high confidence using SEQUEST with uninterpreted tandem mass raw data. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (TPM1), TPM2, TPM3, TPM4, 14-3-3 protein epsilon, and tumor protein D54 were up-regulated in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parental counterpart. Differential expression was confirmed for four proteins including PDX6, CRAB, TPM4, and HSP60 by real-time quantitative PCR and Western blotting analysis in our model. Immunohistochemical analysis in 80 breast cancer donors demonstrated a significant association of TPM4 (p = 0.002), HSP60 (p = 0.001), PDX6 (p = 0.002) but not CRAB (p = 0.113) staining with the presence of lymph node metastasis. In addition, TPM4 staining was also associated with clinical stage (p = 0.000), but no significant association was found between TPM4, PDX6, CRAB, and HSP60 expression and tumor size, hormone receptor, and HER-2 status (p > 0.05). The functional implication of these identified proteins was also discussed. These proteomic data are valuable and informative for understanding breast cancer metastasis and searching for potential markers for metastatic progression.     

Establishment and characterization of patient-derived xenograft and its cell line of primary leiomyosarcoma of bone

Primary leiomyosarcoma (LMS) of bone is a rare and aggressive mesenchymal malignancy that differentiates toward smooth muscle. Complete resection is the only curable treatment, and novel therapeutic approaches for primary LMS of bone have long been desired. Patient-derived xenografts (PDXs) and cell lines are invaluable tools for preclinical studies. Here, we established PDXs from a patient with primary LMS of bone and a cell line from an established PDX. Bone primary LMS tissue was subcutaneously implanted into highly immune-deficient mice. After two passages, a piece of the tumor was subjected to tissue culturing, and a morphological evaluation and proteomic analysis were performed on the PDX and the established cell line. Moreover, the responses of the established cell line to anti-cancer drugs were examined. Microscopic observations revealed that the PDX tumors retained their original histology. The cell line was established from the third-generation PDX and named NCC-LMS1-X3-C1. The cells were maintained for over 18 mo and 40 passages. The cells exhibited a spindle shape and aggressive growth. Mass spectrometric protein identification revealed that the original tumor tissue, PDX tumor tissue, and NCC-LMS1-X3-C1 cells had similar but distinct protein expression profiles. We previously established the cell line, NCC-LMS1-C1, from the tumor tissue of same patient. We found that the response to drug treatments was different between NCC-LMS1-X3-C1 and NCC-LMS1-C1, suggesting the heterogeneous traits of tumor cells in the identical tumor tissue. This set of PDXs and stable cell line will be a useful resource for bone LMS research.     

A new method of cryopreserving colorectal carcinoma cells for patient derived xenograft model generation

Patient derived xenograft (PDX) models provide an efficient way to study anti-tumor drug efficacy. In this respect, it is essential to study the optimal method needed to cryopreserve the starting cells obtained from tumor samples for PDX model generation. Cryopreservation of cells prior to xenografting is necessary for cross-verification of results obtained by xenografting and also for practical planning of experiments. In the present work, we studied the cryopreservation of colorectal carcinoma (CRC) cells isolated from patient tumor samples for generating their patient derived xenograft models. CRC therapeutics study is essential for early stage intervention and treatment of the disease. CRC cell lines do not ideally depict the molecular characteristics of patient CRC tumor samples. This necessitates the generation of CRC PDX models for drug discovery. We show that CRC cells isolated from patient tumor samples have comparable recovery, viability and growth with both conventional cryopreservation methods as well as Fibulas BioFlash Drive™. However, xenograft tumor formation was much more effective with Fibulas BioFlash Drive™ cryopreserved cells than with cells cryopreserved with conventional methods. Therefore, we put forward an effective way to cryopreserve primary cells obtained from patient tumor samples for PDX model generation in this study.     

Machine-learning aided in situ drug sensitivity screening predicts treatment outcomes in ovarian PDX tumors

Long-term treatment outcomes for patients with high grade ovarian cancers have not changed despite innovations in therapies. There is no recommended assay for predicting patient response to second-line therapy, thus clinicians must make treatment decisions based on each individual patient. Patient-derived xenograft (PDX) tumors have been shown to predict drug sensitivity in ovarian cancer patients, but the time frame for intraperitoneal (IP) tumor generation, expansion, and drug screening is beyond that for tumor recurrence and platinum resistance to occur, thus results do not have clinical utility. We describe a drug sensitivity screening assay using a drug delivery microdevice implanted for 24 h in subcutaneous (SQ) ovarian PDX tumors to predict treatment outcomes in matched IP PDX tumors in a clinically relevant time frame. The SQ tumor response to local microdose drug exposure was found to be predictive of the growth of matched IP tumors after multi-week systemic therapy using significantly fewer animals (10 SQ vs 206 IP). Multiplexed immunofluorescence image analysis of phenotypic tumor response combined with a machine learning classifier could predict IP treatment outcomes against three second-line cytotoxic therapies with an average AUC of 0.91.     

Functional analysis of PDX2 from Arabidopsis, a glutaminase involved in vitamin B6 biosynthesis

Vitamin B6 is an essential metabolite in all organisms, being required as a cofactor for a wide variety of biochemical reactions. De novo biosynthesis of the vitamin occurs in microorganisms and plants, but animals must obtain it from their diet. Two distinct and mutually exclusive de novo pathways have been identified to date, namely deoxyxylulose 5-phosphate dependent, which is restricted to a subset of eubacteria, and deoxyxylulose 5-phosphate independent, present in archaea, fungi, plants, protista, and most eubacteria. In these organisms, pyridoxal 5'-phosphate (PLP) formation is catalyzed by a single glutamine amidotransferase (PLP synthase) composed of a glutaminase domain, PDX2, and a synthase domain, PDX1. Despite plants being an important source of vitamin B6, very little is known about its biosynthesis. Here, we provide information for Arabidopsis thaliana. The functionality of PDX2 is demonstrated, using both in vitro and in vivo analyses. The expression pattern of PDX2 is assessed at both the RNA and protein level, providing insight into the spatial and temporal pattern of vitamin B6 biosynthesis. We then provide a detailed biochemical analysis of the plant PLP synthase complex. While the active sites of PDX1 and PDX2 are remote from each other, coordination of catalysis is much more pronounced with the plant proteins than its bacterial counterpart, Bacillus subtilis. Based on a model of the PDX1/PDX2 complex, mutation of a single residue uncouples enzyme coordination and in turn provides tangible evidence for the existence of the recently proposed ammonia tunnel through the core of PDX1.     

Burkitt's lymphoma - a human tumor model system for immunological studies.

Burkitt's lymphoma occurs mainly in parts of tropical Africa and has attracted the attention of experimental workers due to its epidemiological and clinical features, which indicate a viral etiology and a host immune response to the tumor. As a result of virological studies, Epstein-Barr virus (EBV) DNA has been demonstrated in almost all tested biopsies of African BL. This contrasts to the absence of EBV in all, or almost all, of the non-African Burkitt's lymphoma-like tumors, even though the number of tested tumors in this group is small, and to the lack of EBV in all other types of lymphoma or leukemia. Immunological studies have revealed the presence of antibodies to different EBV-associated antigens in all African patients with Burkitt's lymphoma. However the antibodies are not specific for Burkitt's lymphoma but are found in most adults all over the world, although at lower levels. They cannot therefore serve diagnostic purposes, but they can give prognostic information and occasionally give clues to the mechanisms behind late tumor recurrences, and possibly guide so-called immunotherapy. Burkitt's lymphoma patients contrast to appropriate control groups where some of the persons are anti-EBV seronegative, and this, together with the presence of EBV in Burkitt's lymphoma biopsies and the absence of EBV in other lymphomas, even though the cell type involved may be infectable by EBV in vitro and the tumor may arise in an EBV-carrying person, favors an etiological role in EBV in Burkitt's lymphoma and speaks against the "passenger" hypothesis, according to which EBV is picked up by the Burkitt's lymphoma cell which happens to be particularly suitable for EBV persistence. To explain the geographical distribution, a cofactor, such as certain forms of malaria, has been implied.     

The conjugation of RGDS peptide with CM-chitin augments the peptide-mediated inhibition of tumor metastasis

A water soluble 6-O-carboxymethyl chitin (CM-chitin) containing cell adhesive Arg-Gly-Asp-Ser (RGDS) sequence, i.e. CM-chitin-RGDS conjugate was synthesized, and the inhibitory effects of this compound on lung or liver metastasis of lung-metastatic B16-BL6 melanoma or liver-metastatic L5178Y-ML25 lymphoma cells in mice was examined. CM-chitin-RGDS showed the inhibitory effects on lung metastasis of melanoma cells in a dose-dependent manner (ranging from 100 to 1000 μg) and on liver metastasis of lymphoma cells. A mixture of CM-chitin and RGDS peptide or CM-chitin alone did not show any inhibitory effect on experimental lung metastasis as compared with the conjugate CM-chitin-RGDS on a molar basis. GRGDS peptide, however, required a higher dose (3000 μg) to obtain a sufficiently antimetastatic effect. The in-vitro tumor invasion study showed that CM-chitin-RGDS was apparently more effective for the inhibition of tumor cell penetration into reconstituted basement membrane Matrigel than RGDS or the mixture of RGDS and CM-chitin on a molar basis. Intermittent i.v. administration of CM-chitin-RGDS after the inoculation of B16-BL6 cells caused significant inhibition of spontaneous lung metastasis produced by intrafootpad injection of tumor cells as compared with the multiple administration of RGDS, CM-chitin or untreated control. These results demonstrate the importance of the conjugation of RGDS peptide with CM-chitin as a polymeric carrier for the increased therapeutic potential to cancer metastasis, thus implying a possibility that RGDS-polymer conjugation may lead to the prolongation of antimetastatic action of RGDS peptide in vivo.     

HOXC8通过调控PDX1的表达促进非小细胞肺癌的生长与EMT过程

摘要 目的：探索HOXC8与PDX1在非小细胞肺癌（non-small lung cancer, NSCLC）细胞生长及上皮间质转化（Epithelial-mesenchymal transition, EMT）的作用机制。方法：通过转录组测序、荧光定量PCR及染色质免疫沉淀等方法筛选并鉴定HOXC8调控的靶基因;通过Western blot、CCK-8、克隆集落生成及生物信息学等手段分析靶基因PDX1在非小细胞肺癌中的作用。结果：实验证明HOXC8可结合到PDX1基因的启动子上,并作为转录因子激活PDX1的表达。PDX1的表达促进NSCLC细胞的生长与EMT过程,而沉默PDX1能显著地抑制NSCLC细胞的生长与EMT过程,并诱导细胞的凋亡。通过分析已知的肿瘤数据库, 我们发现在NSCLC中PDX1的表达显著高于正常组织,且PDX1的高表达与肺癌患者的预后不良呈显著的相关性。结论：本研究发现HOXC8-PDX1轴在非小细胞肺癌中起着重要的调节作用, 可有望成为非小细胞肺癌治疗的新靶点。     

Opposing roles for complement component c5a in tumor progression and the tumor microenvironment

Promoting complement (C) activation may enhance immunological mechanisms of anti-tumor Abs for tumor destruction. However, C activation components, such as C5a, trigger inflammation, which can promote tumor growth. We addressed the role of C5a on tumor growth by transfecting both human carcinoma and murine lymphoma with mouse C5a. In vitro growth kinetics of C5a, control vector, or parental cells revealed no significant differences. Tumor-bearing mice with C5a-transfected xenografted tumor cells had significantly less tumor burden as compared with control vector tumors. NK cells and macrophages infiltrated C5a-expressing tumors with significantly greater frequency, whereas vascular endothelial growth factor, arginase, and TNF-α production were significantly less. Tumor-bearing mice with high C5a-producing syngeneic lymphoma cells had significantly accelerated tumor progression with more Gr-1(+)CD11b(+) myeloid cells in the spleen and overall decreased CD4(+) and CD8(+) T cells in the tumor, tumor-draining lymph nodes, and the spleen. In contrast, tumor-bearing mice with low C5a-producing lymphoma cells had a significantly reduced tumor burden with increased IFN-γ-producing CD4(+) and CD8(+) T cells in the spleen and tumor-draining lymph nodes. These studies suggest concentration of local C5a within the tumor microenvironment is critical in determining its role in tumor progression.     

Downregulation of PDGFRß Signaling Overcomes Crizotinib Resistance in a TYRO3 and ALK Mutated Neuroendocrine-Like Tumor

Patient-derived xenografts provide significant advantages over long-term passage cell lines when investigating efficacy of treatments for solid tumors. Our laboratory encountered a high-grade, metastatic, neuroendocrine-like tumor from a pediatric patient that presented with a unique genetic profile. In particular, mutations in TYRO3 and ALK were identified. We established a human patient-derived xenoline (PDX) of this tumor for use in the current study. We investigated the effect of crizotinib, a chemotherapeutic known to effectively target both TYRO3 and ALK mutations. Crizotinib effectively decreased viability, proliferation, growth, and the metastatic properties of the PDX tumor through downregulation of STAT3 signaling, but expression of PDGFRß was increased. Sunitinib is a small molecule inhibitor of PDGFRß and was studied in this PDX independently and in combination with crizotinib. Sunitinib alone decreased viability, proliferation, and growth in vitro and decreased tumor growth in vivo. In combination, sunitinib was able to overcome potential crizotinib-induced resistance through downregulation of ERK 1/2 activity and PDGFRß receptor expression; consequently, tumor growth was significantly decreased both in vitro and in vivo. Through the use of the PDX, it was possible to identify crizotinib as a less effective therapeutic for this tumor and suggest that targeting PDGFRß would be more effective. These findings may translate to other solid tumors that present with the same genetic mutations.     

Metformin Treatment Does Not Inhibit Growth of Pancreatic Cancer Patient-Derived Xenografts

There is currently tremendous interest in developing anti-cancer therapeutics targeting cell signaling pathways important for both cancer cell metabolism and growth. Several epidemiological studies have shown that diabetic patients taking metformin have a decreased incidence of pancreatic cancer. This has prompted efforts to evaluate metformin, a drug with negligible toxicity, as a therapeutic modality in pancreatic cancer. Preclinical studies in cell line xenografts and one study in patient-derived xenograft (PDX) models were promising, while recently published clinical trials showed no benefit to adding metformin to combination therapy regimens for locally advanced and metastatic pancreatic cancer. PDX models in which patient tumors are directly engrafted into immunocompromised mice have been shown to be excellent preclinical models for biomarker discovery and therapeutic development. We evaluated the response of four PDX tumor lines to metformin treatment and found that all four of our PDX lines were resistant to metformin. We found that the mechanisms of resistance may occur through lack of sustained activation of adenosine monophosphate-activated protein kinase (AMPK) or downstream reactivation of the mammalian target of rapamycin (mTOR). Moreover, combined treatment with metformin and mTOR inhibitors failed to improve responses in cell lines, which further indicates that metformin alone or in combination with mTOR inhibitors will be ineffective in patients, and that resistance to metformin may occur through multiple pathways. Further studies are required to better understand these mechanisms of resistance and inform potential combination therapies with metformin and existing or novel therapeutics.     

Circulating tumor cell-derived organoids: Current challenges and promises in medical research and precision medicine

Traditional 2D cell cultures do not accurately recapitulate tumor heterogeneity, and insufficient human cell lines are available. Patient-derived xenograft (PDX) models more closely mimic clinical tumor heterogeneity, but are not useful for high-throughput drug screening. Recently, patient-derived organoid cultures have emerged as a novel technique to fill this critical need. Organoids maintain tumor tissue heterogeneity and drug-resistance responses, and thus are useful for high-throughput drug screening. Among various biological tissues used to produce organoid cultures, circulating tumor cells (CTCs) are promising, due to relative ease of ascertainment. CTC-derived organoids could help to acquire relevant genetic and epigenetic information about tumors in real time, and screen and test promising drugs. This could reduce the need for tissue biopsies, which are painful and may be difficult depending on the tumor location. In this review, we have focused on advances in CTC isolation and organoid culture methods, and their potential applications in disease modeling and precision medicine.     

Immunological enhancement of a murine allogeneic tumor in absence of the spleen.

An AKR lymphoma, conditioned to grow in BALB mice by inoculating it within a subcutaneously implanted glass cylinder, led to the development of an allogeneic tumor, lymphoma P, in 65% of the animals killing them in an average of 39 days. Splenectomy performed 12 days before tumor challenge significantly decreased the incidence of lymphoma P to 40%. However, the condition of maximal tumor enhancement obtained by pretreatment of the host with soluble tumor antigen 10 days before tumor challenge remained unaltered by splenectomy, 91% of the animals dying of tumor as compared to 92% of the controls.