Tumor-derived Cell Cultures Research Articles

Functional proteomics of patient derived head and neck squamous cell carcinoma cells reveal novel applications of trametinib

ABSTRACT In this study, we report a differential response of mitogen-activated protein kinase–kinase (MEK) inhibitor trametinib in 20 head and neck squamous cell carcinoma (HNSCC) patients’ tumor-derived cell cultures. Relatively sensitive and resistant cases to trametinib were identified using high throughput metabolic assays and validated in extended dose response studies in vitro. High throughput metabolic assays exploring combination therapies with trametinib were subjected to synergy models and maximal synergistic dose analyses. These yielded several candidates, including axtinib, GDC-0032, GSK-690693, and SGX-523. The combination regimen of trametinib and AXL/MET/VEGFR inhibitor glesatinib showed initial efficacy both in vitro and in vivo (92% reduction in tumor volume). Sensitivity was validated in vivo in a patient-derived xenograft (PDX) model in which trametinib as a single agent effected reduction in tumor volume up to 72%. Reverse Phase Protein Arrays (RPPA) demonstrated differentially expressed proteins and phosphoproteins upon trametinib treatment. Furthermore, resistant cell lines showed a compensatory mechanism via increases in MAPK and non-MAPK pathway proteins that may represent targets for future combination regimens. Intrinsic-targeted options have potential to address paucity of medical treatment options for HNSCC cancer patients, enhance response to extrinsic targeted agents, and/or reduce morbidity as neoadjuvant to surgical treatments.

If Artificial In Vitro Microenvironment Can Influence Tumor Drug Resistance Network via Modulation of lncRNA Expression?\u2014Comparative Analysis of Glioblastoma-Derived Cell Culture Models and Initial Tumors In Vivo

The tumor resistance of glioblastoma cells in vivo is thought to be enhanced by their heterogeneity and plasticity, which are extremely difficult to curb in vitro. The external microenvironment shapes the molecular profile of tumor culture models, thus influencing potential therapy response. Our study examines the expression profile of selected lncRNAs involved in tumor resistance network in three different glioblastoma-derived models commonly utilized for testing drug response in vitro. Differential expression analysis revealed significant divergence in lncRNA profile between parental tumors and tumor-derived cell cultures in vitro, including the following particles: MALAT1, CASC2, H19, TUSC7, XIST, RP11-838N2.4, DLX6-AS1, GLIDR, MIR210HG, SOX2-OT. The examined lncRNAs influence the phenomenon of tumor resistance via their downstream target genes through a variety of processes: multi-drug resistance, epithelial–mesenchymal transition, autophagy, cell proliferation and viability, and DNA repair. A comparison of in vivo and in vitro expression identified differences in the levels of potential lncRNA targets, with the highest discrepancies detected for the MDR1, LRP1, BCRP and MRP1 genes. Co-expression analyses confirmed the following interrelations: MALAT1–TYMS, MALAT1–MRP5, H19–ZEB1, CASC2–VIM, CASC2–N-CAD; they additionally suggest the possibility of MALAT1–BCRP, MALAT1–mTOR and TUSC7–PTEN interconnections in glioblastoma. Although our results clearly demonstrate that the artificial ex vivo microenvironment changes the profile of lncRNAs related to tumor resistance, it is difficult to anticipate the final phenotypic effect, since this phenomenon is a complex one that involves a network of molecular interactions underlying a variety of cellular processes.

Tumor-infiltrating B cells affect the progression of oropharyngeal squamous cell carcinoma via cell-to-cell interactions with CD8+ T cells

BackgroundStandard treatment of oropharyngeal squamous cell carcinoma (OPSCC) is associated with high morbidity, whereas immunotherapeutic approaches using PD-1:PD-L1 checkpoint blockade only show moderate response rates in OPSCC patients. Therefore, a better stratification of patients and the development of novel therapeutic protocols are crucially needed. The importance of tumor-infiltrating B cells (TIL-Bs) in shaping antitumor immunity remains unclear; therefore, we analyzed frequency, phenotype, prognostic value and possible roles of TIL-Bs in OPSCC.MethodsWe utilized transcriptomic analysis of immune response-related genes in 18 OPSCC samples with respect to human papillomavirus (HPV) status. The density and localization of CD20+, CD8+ and DC-LAMP+ cells were subsequently analyzed in 72 tissue sections of primary OPSCC samples in relation to patients’ prognosis. The immunohistochemical approach was supplemented by flow cytometry-based analysis of phenotype and functionality of TIL-Bs in freshly resected primary OPSCC tissues.ResultsWe observed significantly higher expression of B cell-related genes and higher densities of CD20+ B cells in HPV-associated OPSCC samples. Interestingly, CD20+ TIL-Bs and CD8+ T cells formed non-organized aggregates with interacting cells within the tumor tissue. The densities of both intraepithelial CD20+ B cells and B cell/CD8+ T cell interactions showed prognostic significance, which surpassed HPV positivity and CD8+ TIL density in stratification of OPSCC patients. High density of TIL-Bs was associated with an activated B cell phenotype, high CXCL9 production and high levels of tumor-infiltrating CD8+ T cells. Importantly, the abundance of direct B cell/CD8+ T cell interactions positively correlated with the frequency of HPV16-specific CD8+ T cells, whereas the absence of B cells in tumor-derived cell cultures markedly reduced CD8+ T cell survival.ConclusionsOur results indicate that high abundance of TIL-Bs and high density of direct B cell/CD8+ T cell interactions can predict patients with excellent prognosis, who would benefit from less invasive treatment. We propose that in extensively infiltrated tumors, TIL-Bs might recruit CD8+ T cells via CXCL9 and due to a highly activated phenotype contribute by secondary costimulation to the maintenance of CD8+ T cells in the tumor microenvironment.

Unraveling the Heterogeneous Mutational Signature of Spontaneously Developing Tumors in MLH1-/- Mice.

Mismatch repair deficient (MMR-D) tumors exemplify the prototypic hypermutator phenotype. Owing to the high mutation rates, plenty of neo-antigens are present on the tumor cells’ surface, ideally shared among different cancer types. The MLH1 knock out mouse represents a preclinical model that resembles features of the human MMR-D counterpart. While these mice develop neoplasias in a sequential twin-peaked manner (lymphomas > gastrointestinal tumors (GIT)) we aimed at identification of underlying molecular mechanisms. Using whole-genome sequencing, we focused on (I) shared and (II) mutually exclusive mutations and describe the process of ongoing mutational events in tumor-derived cell cultures. The landscape of MLH1−/− tumors is heterogeneous with only a few shared mutations being detectable among different tumor entities (ARID1A and IDH2). With respect to coding microsatellite analysis of MMR-D-related target genes, partial overlap was detectable, yet recognizing shared antigens. The present study is the first reporting results of a comparison between spontaneously developing tumors in MMR-D driven tumorigenesis. Additionally to identifying ARID1A as potential causative mutation hotspot, this comprehensive characterization of the mutational landscape may be a good starting point to refine therapeutic concepts.

Tumor-induced peripheral immunosuppression promotes brain metastasis in patients with non-small cell lung cancer.

Brain metastases are a significant source of morbidity and mortality for patients with lung cancer. Lung cancer can induce local and systemic immunosuppression, promoting tumor growth and dissemination. One mechanism of immunosuppression is tumor-induced expansion of programmed death-ligand 1 (PD-L1) expressing myeloid cells. Here, we investigate peripheral blood immune phenotype in NSCLC patients with or without brain metastasis. Peripheral blood was collected from patients with lung metastatic brain tumors and pre-metastatic lung cancer. Immunosuppressive monocytes, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs) were quantified through flow cytometry. T cell reactivity was analyzed via ELISpot. Brain metastasis conditioned media was collected from tumor-derived cell cultures and analyzed for cytokines by ELISA. Naïve monocytes were stimulated with brain metastasis conditioned media to evaluate PD-L1 stimulation. Patients with brain metastatic lung carcinoma demonstrated increased peripheral monocyte PD-L1, MDSC abundance, and Treg percentage compared to early stage pre-metastatic patients and healthy controls. Patients with elevated peripheral monocyte PD-L1 had less reactive T cells and worse survival. Brain metastasis conditioned media stimulation increased monocyte PD-L1, and conditioned media IL-6 levels correlated with PD-L1 induction. Treatment with anti-IL-6 or anti-IL-6 receptor antibodies reduced PD-L1 expression. In summary, patients with lung cancer and brain metastases exhibit multiple markers of peripheral immunosuppression. The frequency of PD-L1+ myeloid cells correlated with the presence of brain metastases.Tumor-derived IL-6 was capable of inducing PD-L1+ myeloid cellsin vitro, suggesting thatmonitoring of immunosuppressive factors in peripheral blood may identify new targets for therapeutic intervention in selected patients.

A new mouse model to study the role of ectopic Nanos3 expression in cancer

BackgroundNANOS3 is a gene conserved throughout evolution. Despite the quite low conservation of Nanos sequences between different organisms and even between Nanos paralogs, their role in germ cell development is remarkably universal. Human Nanos3 expression is normally restricted to the gonads and the brain. However, ectopic activation of this gene has been detected in various human cancers. Until now, Nanos3 and other Nanos proteins have been studied almost exclusively in germ cell development.MethodsTransgenic mice were generated by targeted insertion of a human Nanos3 cDNA into the ROSA26 locus. The transgene could be spatiotemporally induced by Cre recombinase activity removing an upstream floxed STOP cassette. A lung tumor model with ectopic Nanos3 expression was based on the lung-specific activation of the reverse tetracycline transactivator gene, in combination with a tetO-CMV promoter controlling Cre expression. When doxycycline was provided to the mice, Cre was activated leading to deletion of TP53 alleles and activation of both oncogenic KRasG12D and Nanos3. Appropriate controls were foreseen. Tumors and tumor-derived cell cultures were analyzed in various ways.ResultsWe describe the successful generation of Nanos3LSL/− and Nanos3LSL/LSL mice in which an exogenous human NANOS3 gene can be activated in vivo upon Cre expression. These mice, in combination with different conditional and doxycycline-inducible Cre lines, allow the study of the role of ectopic Nanos3 expression in several cancer types. The Nanos3LSL mice were crossed with a non-small cell lung cancer (NSCLC) mouse model based on conditional expression of oncogenic KRas and homozygous loss of p53. This experiment demonstrated that ectopic expression of Nanos3 in the lungs has a significant negative effect on survival. Enhanced bronchiolar dysplasia was observed when Nanos3-expressing NSCLC mice were compared with control NSCLC mice. An allograft experiment, performed with cell cultures derived from primary lung tumors of control and Nanos3-expressing NSCLC mice, revealed lymph node metastasis in mice injected with Nanos3-expressing NSCLC cells.ConclusionsA new mouse model was generated allowing examination of Nanos3-associated pathways and investigation of the influence of ectopic Nanos3 expression in various cancer types. This model might identify Nanos3 as an interesting target in cancer therapeutics.

Inhibitor of DNA binding-4 promotes angiogenesis and growth of glioblastoma multiforme by elevating matrix GLA levels

Inhibitor of differentiation-4 is highly expressed in glioblastoma multiforme (GBM). We report a novel pro-angiogenic function for inhibitor of differentiation-4 in the growth of glioblastoma xenografts. Tumor-derived cell cultures expressing elevated levels of ID4 produced enlarged xenografts in immunosuppressed mice that were better vascularized than corresponding control tumors and expressed elevated matrix GLA protein (MGP) that mediated enhanced tumor angiogenesis. Inhibition of MGP resulted in smaller and less vascularized xenografts. Our finding shows a novel function for ID4 in tumor angiogenesis, and identifies ID4 and MGP as possible therapeutic targets for GBM.

OCT4 expression in human non-small cell lung cancer: implications for therapeutic intervention,,

Here we investigate the expression of OCT4 human lung adenocarcinoma and bronchioloalveolar carcinoma (BAC) tumor biopsies and tumor-derived primary cell cultures. OCT4 has been detected in several human tumors suggesting a potentially critical role in tumorigenesis. We assessed the presence of OCT4 in clinical tumor samples of both adenocarcinoma and BAC at the cellular and transcriptional levels, respectively. Furthermore, we evaluated tumor-derived cell cultures for potential differences in OCT4 expression. Immunohistochemical analysis depicted OCT4 in 2 of 8 adenocarcinoma tumor samples and 3 of 5 BAC tumor samples, with no apparent difference in the degree of expression among the sections examined. These results were validated by transcript analysis. Flow cytometric assessment of 11 adenocarcinoma-derived cell cultures and 3 BAC-derived cell cultures revealed significantly higher OCT4 expression in adenocarcinoma tumors compared to their normal counterparts. This, however, was not observed in the BAC cultures. Comparative studies of OCT4 in adenocarcinoma and BAC tumor cell cultures demonstrated a dramatically higher expression in the former. The expression of OCT4 may represent a specific and effective target for therapeutic intervention in adenocarcinoma and BAC. In addition, the aberrant expression and distribution of OCT4 may indicate important parameters concerning the differences between adenocarcinoma and BAC.

Metastatic lung disease to the central nervous system: in vitro response to chemotherapeutic agents.

Metastatic lung disease to the central nervous system (CNS) comprises a significant percentage of cranial metastases. For those cases where chemotherapy may be of palliative or therapeutic benefit, in vitro chemoresponse testing may identify the agent(s) most likely to be effective clinically. Tumor-derived cell cultures were established from 14 surgically excised lung lesions metastatic to the CNS. In vitro chemoresponse testing was performed with a variety of anticancer agents on the tumor cells that grew out of the cultured tissue specimens. Drug concentrations and exposure times were adjusted to bracket approximate average peak plasma levels that are observed typically in vivo. For each tumor-derived cell culture, a complete dose-response curve was established for each chemotherapeutic agent tested. Approximately 80% of the 14 tumor cell cultures had a definitive response to one or more chemotherapeutic agents in vitro, with approximately one-third of these cultures displaying a response to at least three of the drugs tested. There was considerable heterogeneity in the response of individual tumor cell cultures to the chemotherapeutic drugs. The agents that showed the highest cytotoxic response rate against the individual tumor cell cultures included lomustine, carboplatin, cisplatin and etoposide. The tumor cells isolated from individuals with lung lesions metastatic to the brain demonstrated differential chemoresponses to the agents tested. No single agent was effective against every tumor cell culture. These data suggest that in vitro chemoresponse testing of cultured tumor cells may be useful to identify biologically effective chemotherapeutic agents for individual patients, thereby addressing at least one factor in this complex therapeutic challenge.

Different simian virus 40 genomic regions and sequences homologous with SV40 large T antigen in DNA of human brain and bone tumors and of leukocytes from blood donors

BACKGROUND Many studies found only a small fragment of the large T-antigen coding sequences in human tumors, raising doubts on authenticity of SV40 sequences detected in these samples. METHODS Five different regions of SV40 DNA were investigated in 106 fresh human tumor biopsies (25 brain, 69 bone, 12 Wilms' tumors), 71 tumor-derived cell cultures (38 from brain and 33 from bone tumors) and normal tissues (5 fresh bone biopsies and 38 buffy coats) by polymerase chain reaction (PCR) techniques and filter hybridization with specific oligoprobes. Expression of SV40 Tag sequences was analyzed in human tumor specimens by RT-PCR. RESULTS SV40 large T-antigen sequences were detected at high prevalence, in human biopsies of primary brain (37–44%) and bone (21–37%) tumors, in cell cultures derived from brain (30–54%) and bone (53–80%) tumors. SV40 Tag sequences were detected in 29% of buffy coats of blood donors. However, only four brain tumor cell lines showed all the five regions of the SV40 genome investigated. Expression of SV40 Tag sequences was found in 11 of 27 (41%) human tumor samples. DNA sequence analysis indicated that the PCR-amplified products belong to the SV40 wild type. Polymerase chain reaction products of Tag middle portion from 20 of 78 (26%) samples showed a 97% homology with telomeric sequences of human chromosomes 10 and 11. CONCLUSIONS Authentic SV40 sequences were detected in human samples. The expression of SV40 Tag sequences indicates that SV40 could play a role, as a cofactor, in the onset/progression of specific human cancers. The inability to detect some regions of the virus genome may suggest that those regions are not required for tumor persistence or growth and have been lost or, alternatively, may be the result of assay conditions that were unable to PCR-amplify those regions in the tumors. Cancer 2002;94:1037–48. © 2002 American Cancer Society. DOI 10.1002/cncr.10272

Different simian virus 40 genomic regions and sequences homologous with SV40 large T antigen in DNA of human brain and bone tumors and of leukocytes from blood donors

Many studies found only a small fragment of the large T-antigen coding sequences in human tumors, raising doubts on authenticity of SV40 sequences detected in these samples. Five different regions of SV40 DNA were investigated in 106 fresh human tumor biopsies (25 brain, 69 bone, 12 Wilms' tumors), 71 tumor-derived cell cultures (38 from brain and 33 from bone tumors) and normal tissues (5 fresh bone biopsies and 38 buffy coats) by polymerase chain reaction (PCR) techniques and filter hybridization with specific oligoprobes. Expression of SV40 Tag sequences was analyzed in human tumor specimens by RT-PCR. SV40 large T-antigen sequences were detected at high prevalence, in human biopsies of primary brain (37-44%) and bone (21-37%) tumors, in cell cultures derived from brain (30-54%) and bone (53-80%) tumors. SV40 Tag sequences were detected in 29% of buffy coats of blood donors. However, only four brain tumor cell lines showed all the five regions of the SV40 genome investigated. Expression of SV40 Tag sequences was found in 11 of 27 (41%) human tumor samples. DNA sequence analysis indicated that the PCR-amplified products belong to the SV40 wild type. Polymerase chain reaction products of Tag middle portion from 20 of 78 (26%) samples showed a 97% homology with telomeric sequences of human chromosomes 10 and 11. Authentic SV40 sequences were detected in human samples. The expression of SV40 Tag sequences indicates that SV40 could play a role, as a cofactor, in the onset/progression of specific human cancers. The inability to detect some regions of the virus genome may suggest that those regions are not required for tumor persistence or growth and have been lost or, alternatively, may be the result of assay conditions that were unable to PCR-amplify those regions in the tumors.

Expression and modulation by cytokines of the intercellular adhesion molecule‐1 (ICAM‐1) in human central nervous system tumor cell cultures

The intercellular adhesion molecule (ICAM-1) has been shown to be important in interactions involving cells of the immune system and to be upregulated in a number of cell culture systems by cytokines, including immune interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). In the present study, we have determined by fluorescence-activated cell sorter (FACS) analysis and the anti-ICAM-1 monoclonal antibody (MAb) CL203.4 the base-line expression of ICAM-1 and its modulation by recombinant IFN-beta, IFN-gamma and TNF-alpha in early passage (less than 15) human central nervous system (CNS) tumor-derived cell cultures. These cultures were established from various malignancies, including glioblastoma multiforme (GBM), astrocytoma (AST), ganglioglioma, medulloblastoma, meningioma and a pineal tumor. ICAM-1 expression was highest in the GBM- and AST-derived cell cultures and was lowest in the ganglioglioma and normal pineal cell cultures. Variable ICAM-1 expression was found, however, in tumors of the same histological group. In several cell cultures the variable expression observed by FACS was substantiated by the intensity of the molecular species immunoprecipitated by the anti-ICAM-1 MAb CL203.4 from these cells. All the cell cultures displayed variable but consistent increases in ICAM-1 expression following treatment with IFN-gamma or TNF-alpha. In general, the degree of increase in ICAM-1 expression was greatest in cultures exposed to TNF-alpha. Upregulation of ICAM-1 expression in an established glioblastoma multiforme cell line was of greater magnitude and more rapid following TNF-alpha treatment (within 2 to 3 hr) than exposure to IFN-gamma (by 24 hr). In several cultures, IFN-beta also increased ICAM-1 expression and enhanced the increase induced by TNF-alpha. The results of the present study indicate that variable expression of ICAM-1 is a common property of early passage cultures of CNS tumors and recombinant interferons and TNF-alpha can differentially upregulate ICAM-1 expression in these CNS tumor cell cultures.

Longterm survival of normal, diploid Syrian hamster cells in tumors induced by transfection with v-Ha- ras and v- myc oncogenes

Tumors were induced following transfection of normal, diploid hamster embryo cells with plasmids containing the viral Harvey ras and viral myc oncogenes. Direct cytogenetic studies of the tumors performed at 3–7 weeks after injection of the transfected hamster cells into nude mice revealed that 100% of the hamster cells were aneuploid and no detectable diploid cells in mitosis were observed. However, when tumor explants were cultured in vitro, diploid Syrian hamster cells were frequently detected at early passages. The percentage of diploid hamster cells in the cultures varied from 2 to 94% at the first passage. After several passages in vitro, only aneuploid hamster cells were observed. The diploid hamster cells in culture had a flat morphology and senesced. The aneuploid cells in the cultures were readily cloned and formed tumors after reinjection in nude mice. Reconstruction experiments consisting of injecting cloned, aneuploid tumor cells mixed with 6 × 10 6 normal Syrian hamster embryo cells were performed. Cell cultures derived from these mixed tumors contained both normal and aneuploid hamster cells indicating that normal cells survived in vivo during the growth of the tumor cells for up to 4 weeks. During this period, some normal cells transplanted in vivo did not die or terminally differentiate. Since normal cells can persist in vivo, tumor-derived cell cultures are mixed populations and caution should be exercised in interpreting quantitative molecular or cellular studies of these uncloned populations.

Human non-malignant and malignant brain tumor derived cell cultures: proliferation and sensitivity to natural human fibroblast (beta) interferon

Twenty-one human brain tumor biopsies were processed by mechanical and enzymatic methods to produce mixed cell suspensions. Cultures were prepared in small plastic flasks, and primary outgrowth occurred in 16/21 cultures. The period required for primary outgrowth ranged from 3 days to 14 days. We established serial propagation with 15/16 of the primary cultures. Sensitivity to HuIFN-beta was determined between passages 3 to 12, using a microassay based on cell viability (uptake of a supravital stain, neutral red). Extracted dye was quantified in acidic-methanol using the MR580 Microelisa Autoreader (Dynatech). We observed a broad range of responsiveness to the drug among the 12 cell-strains tested. Thus, 4 cell strains were relatively sensitive; 4 were resistant to 10(4) IRU/ml of purified HuIFN-beta. Four cell strains exhibited a level of responsiveness that was intermediate to that of these two groups. During propagation of these biopsies, cytopathology suggestive of paramyxovirus-infection appeared in 4 of the cell-strains. This characteristic was not uniformly associated with high sensitivity to human beta interferon which is a very potent, naturally occurring antiviral substance. Our results support the concept that information concerning sensitivity to HuIFN-beta and other cytostatic agents may be rapidly obtained using microcultures of brain tumor cultures in conjunction with supravital stain uptake studies. Additionally, these results suggest that further clinical studies with beta interferon should be undertaken to define the parameters which determine successful in vivo application.