Initiation and Characterization of Small Cell Lung Cancer Patient-Derived Xenografts from Ultrasound-Guided Transbronchial Needle Aspirates

Small cell lung cancer (SCLC) is a devastating disease with limited treatment options. Due to its early metastatic nature and rapid growth, surgical resection is rare. Standard of care treatment regimens remain largely unchanged since the 1980’s, and five-year survival lingers near 5%. Patient-derived xenograft (PDX) models have been established for other tumor types, amplifying material for research and serving as models for preclinical experimentation; however, limited availability of primary tissue has curtailed development of these models for SCLC. The objective of this study was to establish PDX models from commonly collected fine needle aspirate biopsies of primary SCLC tumors, and to assess their utility as research models of primary SCLC tumors. These transbronchial needle aspirates efficiently engrafted as xenografts, and tumor histomorphology was similar to primary tumors. Resulting tumors were further characterized by H&E and immunohistochemistry, cryopreserved, and used to propagate tumor-bearing mice for the evaluation of standard of care chemotherapy regimens, to assess their utility as models for tumors in SCLC patients. When treated with Cisplatin and Etoposide, tumor-bearing mice responded similarly to patients from whom the tumors originated. Here, we demonstrate that PDX tumor models can be efficiently established from primary SCLC transbronchial needle aspirates, even after overnight shipping, and that resulting xenograft tumors are similar to matched primary tumors in cancer patients by both histology and chemo-sensitivity. This method enables physicians at non-research institutions to collaboratively contribute to the rapid establishment of extensive PDX collections of SCLC, enabling experimentation with clinically relevant tissues and development of improved therapies for SCLC patients.     

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.     

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.     

Whole Exome Sequencing of Rapid Autopsy Tumors and Xenograft Models Reveals Possible Driver Mutations Underlying Tumor Progression

Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal malignancy due to its propensity to invade and rapidly metastasize and remains very difficult to manage clinically. One major hindrance towards a better understanding of PDAC is the lack of molecular data sets and models representative of end stage disease. Moreover, it remains unclear how molecularly similar patient-derived xenograft (PDX) models are to the primary tumor from which they were derived. To identify potential molecular drivers in metastatic pancreatic cancer progression, we obtained matched primary tumor, metastases and normal (peripheral blood) samples under a rapid autopsy program and performed whole exome sequencing (WES) on tumor as well as normal samples. PDX models were also generated, sequenced and compared to tumors. Across the matched data sets generated for three patients, there were on average approximately 160 single-nucleotide mutations in each sample. The majority of mutations in each patient were shared among the primary and metastatic samples and, importantly, were largely retained in the xenograft models. Based on the mutation prevalence in the primary and metastatic sites, we proposed possible clonal evolution patterns marked by functional mutations affecting cancer genes such as KRAS, TP53 and SMAD4 that may play an important role in tumor initiation, progression and metastasis. These results add to our understanding of pancreatic tumor biology, and demonstrate that PDX models derived from advanced or end-stage likely closely approximate the genetics of the disease in the clinic and thus represent a biologically and clinically relevant pre-clinical platform that may enable the development of effective targeted therapies for PDAC.     

An Advanced Preclinical Mouse Model for Acute Myeloid Leukemia Using Patients' Cells of Various Genetic Subgroups and In Vivo Bioluminescence Imaging

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups.     

Establishment and Characterization of 7 Novel Hepatocellular Carcinoma Cell Lines from Patient-Derived Tumor Xenografts

Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX) models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903) by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR) analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both in vitro and in vivo. STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3) was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening.     

Appropriateness of Using Patient-Derived Xenograft Models for Pharmacologic Evaluation of Novel Therapies for Esophageal/Gastro-Esophageal Junction Cancers

The high morbidity and mortality of patients with esophageal (E) and gastro-esophageal junction (GEJ) cancers, warrants new pre-clinical models for drug testing. The utility of primary tumor xenografts (PTXGs) as pre-clinical models was assessed. Clinicopathological, immunohistochemical markers (p53, p16, Ki-67, Her-2/neu and EGFR), and global mRNA abundance profiles were evaluated to determine selection biases of samples implanted or engrafted, compared with the underlying population. Nine primary E/GEJ adenocarcinoma xenograft lines were further characterized for the spectrum and stability of gene/protein expression over passages. Seven primary esophageal adenocarcinoma xenograft lines were treated with individual or combination chemotherapy. Tumors that were implanted (n=55) in NOD/SCID mice had features suggestive of more aggressive biology than tumors that were never implanted (n=32). Of those implanted, 21/55 engrafted; engraftment was associated with poorly differentiated tumors (p=0.04) and older patients (p=0.01). Expression of immunohistochemical markers were similar between patient sample and corresponding xenograft. mRNA differences observed between patient tumors and first passage xenografts were largely due to loss of human stroma in xenografts. mRNA patterns of early vs late passage xenografts and of small vs large tumors of the same passage were similar. Complete resistance was present in 2/7 xenografts while the remaining tumors showed varying degrees of sensitivity, that remained constant across passages. Because of their ability to recapitulate primary tumor characteristics during engraftment and across serial passaging, PTXGs can be useful clinical systems for assessment of drug sensitivity of human E/GEJ cancers.     

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.     

SPA: A Quantitation Strategy for MS Data in Patient-derived Xenograft Models

With the development of mass spectrometry (MS)-based proteomics technologies, patient-derived xenograft (PDX), which is generated from the primary tumor of a patient, is widely used for the proteome-wide analysis of cancer mechanism and biomarker identification of a drug. However, the proteomics data interpretation is still challenging due to complex data deconvolution from the PDX sample that is a cross-species mixture of human cancerous tissues and immunodeficient mouse tissues. In this study, by using the lab-assembled mixture of human and mouse cells with different mixing ratios as a benchmark, we developed and evaluated a new method, SPA (shared peptide allocation), for protein quantitation by considering the unique and shared peptides of both species. The results showed that SPA could provide more convenient and accurate protein quantitation in human–mouse mixed samples. Further validation on a pair of gastric PDX samples (one bearing FGFR2 amplification while the other one not) showed that our new method not only significantly improved the overall protein identification, but also detected the differential phosphorylation of FGFR2 and its downstream mediators (such as RAS and ERK) exclusively. The tool pdxSPA is freely available at https://github.com/Li-Lab-Proteomics/pdxSPA.     

Methods for production of monoclonal antibodies with specificity for human lung cancer cells

We have developed a screening strategy and technology to produce monoclonal antibodies with specificity for human lung cancer cells. Mice and rats were immunized with well-characterized tissue culture lines of human small cell lung cancer (SCLC), mouse myeloma x spleen hybrids formed by the technique of Kohler and Milstein, and the resulting culture fluids were screened for antibody binding phenotype using a radioimmunoassay. To facilitate testing large numbers of culture fluids, a 96-well, microtiter based, reusable, replicating device was designed. Using this, many hybridoma culture fluids were replica plated for antibody binding tests on a series of human target cell plates. Hybrids producing antibodies that reacted with the immunizing SCLC line and another independent SCLC line, but not with autologous B-lymphoblastoid cells derived from one of the patients, were identified, selected, and then repeatedly recloned using the same screening strategy. With this technology, hybridomas representing less than 0.5% of all hybrids generated could be isolated and stable antibody producing cultures derived. Such antibodies reacted with a panel of well-characterized SCLC lines and SCLC samples taken directly from patients but not with a variety of normal tissues. Using these antibodies we can demonstrate: tumor cell contamination of bone marrow specimens, marked heterogeneity of antigen expression on cells within individual SCLC lines and individual patients, and inhibition of clonal growth of SCLC lines in soft agarose assays. All of these findings have potential clinical and cell biologic application.     

Stemness and inducing differentiation of small cell lung cancer NCI-H446 cells

Small cell lung cancer (SCLC) accounts for nearly 15% of human lung cancers and is one of the most aggressive solid tumors. The SCLC cells are thought to derive from self-renewing pulmonary neuroendocrine cells by oncogenic transformation. However, whether the SCLC cells possess stemness and plasticity for differentiation as normal stem cells has not been well understood thus far. In this study, we investigated the expressions of multilineage stem cell markers in the cancer cells of SCLC cell line (NCI-H446) and analyzed their clonogenicity, tumorigenicity, and plasticity for inducing differentiation. It has been found that most cancer cells of the cell line expressed multilineage stem cell markers under the routine culture conditions and generated single-cell clones in anchorage-dependent or -independent conditions. These cancer cells could form subcutaneous xenograft tumors and orthotopic lung xenograft tumors in BALB/C-nude mice. Most cells in xenograft tumors expressed stem cell markers and proliferation cell nuclear antigen Ki67, suggesting that these cancer cells remained stemness and highly proliferative ability in vivo. Intriguingly, the cancer cells could be induced to differentiate into neurons, adipocytes, and osteocytes, respectively, in vitro. During the processes of cellular phenotype-conversions, autophagy and apoptosis were two main metabolic events. There is cross-talking between autophagy and apoptosis in the differentiated cancer cells. In addition, the effects of the inhibitor and agonist for Sirtuin1/2 on the inducing osteogenic differentiation indicated that Sirtuin1/2 had an important role in this process. Taken together, these results indicate that most cancer cells of NCI-H446 cell line possess stemness and plasticity for multilineage differentiation. These findings have potentially some translational applications in treatments of SCLC with inducing differentiation therapy.     

Two monoclonal antibodies against small-cell lung cancer show existence of synergism in binding

Summary Murine IgG1 monoclonal antibodies (mAbs), ITK-2 and ITK-3, were generated against a small-cell lung cancer (SCLC) cell line. Enzyme-linked immunosorbent assay using a variety of established cell lines as substrates, immunoperoxidase staining of freshly frozen tissue sections, and fluorescence-activated cell sorter analysis of peripheral blood leukocytes showed that these mAbs recognize a part of the SCLC-associated cluster 1 antigen. In immunoprecipitation studies, both ITK-2 and ITK-3 bound to a 145-kDa glycoprotein of SCLC cell membrane extracts, as did MOC-1 and NKH-1, which both recognize the cluster 1 antigen. However, because the binding of¹²⁵I-labeled ITK-2 to SCLC cells was not inhibited by MOC-1 or NKH-1, the binding site of ITK-2 on SCLC cells appeared to be different from that of either MOC-1 or NKH-1. Unexpectedly, binding of¹²⁵I-labeled ITK-2 to SCLC cells increased in the presence of ITK-3. This ITK-3-induced increase in ITK-2 binding was due partly to an increase in the number of binding sites for ITK-2 on SCLC cells. Addition of ITK-3 may, therefore, improve the effectiveness of ITK-2-based tumor detection or therapy.     

Methods for production of monoclonal antibodies with specificity for human lung cancer cells

Summary We have developed a screening strategy and technology to produce monoclonal antibodies with specificity for human lung cancer cells. Mice and rats were immunized with well-characterized tissue culture lines of human small cell lung cancer (SCLC), mouse myeloma x spleen hybrids formed by the technique of Kohler and Milstein, and the resulting culture fluids were screened for antibody binding phenotype using a radioimmunoassay. To facilitate testing large numbers of culture fluids, a 96-well, microtiter based, resuable, replicating device was designed. Using this, many hybridoma culture fluids were replica plated for antibody binding tests on a series of human target cell plates. Hybrids producing antibodies that reacted with the immunizing SCLC line and another independent SCLC line, but not with autologous B-lymphoblastoid cells derived from one of the patients, were identified, selected, and then repeatedly recloned using the same screening strategy. With this technology, hybridomas representing less than 0.5% of all hybrids generated could be isolated and stable antibody producing cultures derived. Such antibodies reacted with a panel of well-characterized SCLC lines and SCLC samples taken directly from patients but not with a variety of normal tissues. Using these antibodies we can demonstrate: tumor cell contamination of bone marrow specimens, marked heterogeneity of antigen expression on cells within individual SCLC lines and individual patients, and inhibition of clonal growth of SCLC lines in soft agarose assays. All of these findings have potential clinical and cell biologic application. Presented in the symposium on The Biology of Hybridomas at the 32nd Annual Meeting of the Tissue Culture Association, Washington, D.C., June 7–11, 1981. This symposium was supported in part by the following organizations: Bethesda Research Laboratories, Cetus Corporation, Hybritech Incorporated, MAB-Monoclonal Antibodies, Inc., National Capital Area Branch of the Tissue Culture Association, New England Nuclear Corporation, and Ortho Pharmaceutical Corporation.     

Development of Novel Patient-Derived Preclinical Models from Malignant Effusions in Patients with Tyrosine Kinase Inhibitor–Resistant Clear Cell Renal Cell Carcinoma

PURPOSE: Although targeting angiogenesis with tyrosine kinase inhibitors (TKIs) has become standard of care in the treatment of clear cell renal cell carcinoma (RCC), resistance mechanism are not fully understood, and there is a need to develop new therapeutic options overcoming them. METHODS AND MATERIALS: To develop a preclinical model that predicts clinical activity of novel agents in 19 RCC patients, we established patient-derived cell (PDC) and xenograft (PDX) models derived from malignant effusions or surgical specimen. RESULTS: Successful PDCs, defined as cells that maintained growth following two passages, were established in 5 of 15 malignant effusions and 1 of 4 surgical specimens. One PDC, clinically refractory to TKIs, was implanted and engrafted in mice, resulting in a comparable histology to the primary tumor. The PDC-PDX model also showed similar genomic features when tested using targeted sequencing of cancer-related genes. When we examined the drug effects of the PDX model, the tumor cells showed resistance to TKIs and everolimus in vitro. CONCLUSION: The results suggest that the PDC-PDX preclinical model we developed using malignant effusions can be a useful preclinical model to interrogate sensitivity to targeted agents based on genomic alterations.     

Chimeric antigen receptor T cells targeting CD147 for non-small cell lung cancer therapy

Non-small cell lung cancer (NSCLC) is a highly malignant tumor, with a significant mortality and morbidity. With the development of tumor immunotherapy, chimeric antigen receptor T cells (CART) gets increasingly attention and achieves prominent contributions in the treatment of hematologic malignancies. However, CART therapy for NSCLC proceeds slowly and further researches need to be investigated. In our study, we performed bioinformatics analysis to evaluate the significant role of CD147 in NSCLC. The expression level of CD147 was detected in human NSCLC cell lines and NSCLC tissues. Meanwhile, CD147-CART was constructed and identified. Cell cytotoxicity and cytokine secretion were performed to evaluate the efficacy of CD147-CART. We also constructed cell-derived xenograft (CDX) model and patient-derived xenograft (PDX) model, which was used to further investigate the safety and efficacy of CD147-CART in vivo. Our observations show that CD147 is a specific tumor antigen of NSCLC and plays an essential role in NSCLC progression, which can be used as a target for CART therapy in NSCLC. CD147-CART cells exhibit robust cytotoxicity and cytokine production in vitro, suggesting a strong anti-tumor activity against NSCLC tumor cells. Importantly, CD147-CART cells have strong anti-tumor activity against NSCLC cells in vivo in both CDX and PDX models and no adverse side effects. Our findings show that CD147-CART immunotherapy for NSCLC is safe and effective, which is an ideal and promising medical patch for treating NSCLC.     

Fc-engineered anti-CD33 monoclonal antibody potentiates cytotoxicity of membrane-bound interleukin-21 expanded natural killer cells in acute myeloid leukemia

BackgroundQualitative and quantitative defects in natural killer (NK) cells have been noted in patients with acute myeloid leukemia (AML), providing rationale for infusion of donor-derived NK cells. We previously showed that decitabine enhances expression of NKG2D ligands in AML with additive cytotoxicity when NK cells and Fc (fragment crystallizable region)-engineered CD33 monoclonal antibody (CD33mAb) was used. We conducted a phase 1 study evaluating decitabine and haploidentical NK cells in relapsed AML. Using patient samples from this study, we evaluated whether ex vivo donor-derived expanded NK cells with or without CD33mAb was effective in decitabine-treated AML.MethodsBone marrow aspirates were collected from patients at pre- and post-NK cell infusion. NK cells from healthy donors were expanded for 14 days using irradiated K562 feeder cells displaying membrane-bound IL-21 (mbIL-21). Patient samples were used to test in vitro activity of mbIL-21 NK cells ± CD33m Ab-dependent cellular cytotoxicity (ADCC) and AML patient derived xenograft (PDX) mice were developed to test in vivo activity.ResultsUpon incubation with primary AML blasts, mbIL-21 NK cells showed variable donor-dependent intra-cellular interferon-γ production, which increased with CD33mAb-coated AML. ADCC assays revealed mbIL-21 NK cells effectively lysed primary AML blasts with higher activity on CD33mAb-coated AML. Importantly, CD33mAb-dependent enhanced cytotoxicity by mbIL-21 NK cells was maintained in AML cells from patients even 24 days post-decitabine treatment. In vivo infusion of mbIL-21 NK cells in AML PDX mice, treated with CD33mAb, reduced the tumor burden.DiscussionThese data show the therapeutic utility of mbIL-21 NK cells that can be further potentiated by addition of CD33mAb in AML.