Solid Tumor Origin Research Articles

Selective Vulnerability to Pyrimidine Starvation in Hematologic Malignancies Revealed by AG-636, a Novel Clinical-Stage Inhibitor of Dihydroorotate Dehydrogenase.

Agents targeting metabolic pathways form the backbone of standard oncology treatments, though a better understanding of differential metabolic dependencies could instruct more rationale-based therapeutic approaches. We performed a chemical biology screen that revealed a strong enrichment in sensitivity to a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic versus solid tumor origin. Differential AG-636 activity translated to the in vivo setting, with complete tumor regression observed in a lymphoma model. Dissection of the relationship between uridine availability and response to AG-636 revealed a divergent ability of lymphoma and solid tumor cell lines to survive and grow in the setting of depleted extracellular uridine and DHODH inhibition. Metabolic characterization paired with unbiased functional genomic and proteomic screens pointed to adaptive mechanisms to cope with nucleotide stress as contributing to response to AG-636. These findings support targeting of DHODH in lymphoma and other hematologic malignancies and suggest combination strategies aimed at interfering with DNA-damage response pathways.

Clinical and analytical validation of FoundationOne Liquid CDx, a novel 324-Gene cfDNA-based comprehensive genomic profiling assay for cancers of solid tumor origin.

As availability of precision therapies expands, a well-validated circulating cell-free DNA (cfDNA)-based comprehensive genomic profiling assay has the potential to provide considerable value as a complement to tissue-based testing to ensure potentially life-extending therapies are administered to patients most likely to benefit. Additional data supporting the clinical validity of cfDNA-based testing is necessary to inform optimal use of these assays in the clinic. The FoundationOne®Liquid CDx assay is a pan-cancer cfDNA-based comprehensive genomic profiling assay that was recently approved by FDA. Validation studies included >7,500 tests and >30,000 unique variants across >300 genes and >30 cancer types. Clinical validity results across multiple tumor types are presented. Additionally, results demonstrated a 95% limit of detection of 0.40% variant allele fraction for select substitutions and insertions/deletions, 0.37% variant allele fraction for select rearrangements, 21.7% tumor fraction for copy number amplifications, and 30.4% TF for copy number losses. The limit of detection for microsatellite instability and blood tumor mutational burden were also determined. The false positive variant rate was 0.013% (approximately 1 in 8,000). Reproducibility of variant calling was 99.59%. In comparison with an orthogonal method, an overall positive percent agreement of 96.3% and negative percent agreement of >99.9% was observed. These study results demonstrate that FoundationOne Liquid CDx accurately and reproducibly detects the major types of genomic alterations in addition to complex biomarkers such as microsatellite instability, blood tumor mutational burden, and tumor fraction. Critically, clinical validity data is presented across multiple cancer types.

Abstract B43: Genetic analysis of circulating tumor cells of colorectal cancer patients captured by multiantibodies technique

Abstract Background: Accurate genetic analysis is essential for molecular therapy. Conventionally, DNA derived from tumor tissue has been the source of the gold standard of DNA information in solid tumors including colorectal cancer (CRC). However, it is well known that molecular therapy induces emerging mutations that are not found in primary tumor; thus, real-time monitoring of gene information is ideal. Circulating tumor cells (CTCs) are circulating malignant cells of solid tumor origin that are found in the bloodstream and can be a powerful candidate to play an important role. Capturing a large amount of CTCs improves the accuracy of genetic analysis of DNA derived from CTC (ctcDNA). We used 3 (EpCAM, Her2, Trop2) or 4 (EpCAM, Her2, Trop2, EGFR) antibodies to capture CTCs, and analyzed ctcDNA using next-generation sequencing (NGS). Methods: Cohort 1: Untreated CRC patients were enrolled. Ten mL of whole blood was collected from each patient. The blood was processed using 3 antibodies (EpCAM, Her2, Trop2) and CTCs were collected. Tumor tissue was also collected from each patient. Tumor tissue DNA and ctcDNA were extracted and analyzed using NGS. Cohort 2: CRC patients, both treated and untreated, were enrolled. Twenty mL of whole blood was collected from each patient. Ten mL of the blood was processed using 3 antibodies (EpCAM, Her2, Trop2), and the remaining 10 mL was processed using 4 antibodies (EpCAM, Her2, Trop2, EGFR). The numbers of collected CTCs were counted and compared. Results: Cohort 1: We enrolled 34 patients (stage II: n=4, stage III: n=7, stage IV: n=23). Median number of extracted CTC was 34 cells. From tumor tissue DNA, 53 mutations were detected. The most frequent mutation was within TP53 (n=18), followed by mutations in APC (n=13) and KRAS (n=12). From ctcDNA, 16 mutations, including 5 mutations which were not found in tissue DNA, were detected. The most frequent mutation was within TP53 (n=5), followed by mutations in KRAS and APC (n=4 each). Cohort 2: We enrolled 10 patients (stage II: n=1, stage III: n=1, stage IV: n=8). Using 3 antibodies, the median number of collected CTCs was 27 cells (range, 2–112). Using 4 antibodies, the median number of collected CTCs was 33 cells (range, 7–260). There were no statistically significant differences between the 2 groups (p=0.40). Conclusions: Mutations not detected in primary tumors can be identified in ctcDNA, indicating the potential of CTCs in complementing gene analysis. The technique to capture CTCs using 3 antibodies appears to increase the detection rate and yield of CTCs. However, the present study did not show advantages of the 4-antibodies method, and future studies should investigate the best combination of antibodies to extract more CTCs with higher specificity. Citation Format: Kohki Takeda, Takeshi Yamada, Michihiro Koizumi, Seiichi Shinji, Akihisa Matsuda, Ryo Ohta, Yasuyuki Yokoyama, Goro Takahashi, Masahiro Hotta, Takuma Iwai, Keisuke Hara, Koji Ueda, Sho Kuriyama, Hiroshi Yoshida. Genetic analysis of circulating tumor cells of colorectal cancer patients captured by multiantibodies technique [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B43.

Correlation of gene expression profile to identify tumors with extreme sensitivity to the alkylating agent LP184.

e21021 Background: Lantern Pharma is advancing LP184, a next generation alkylating acylfulvene class prodrug. LP184 damages DNA that is subject to preferential repair by Transcription-Coupled Nucleotide Excision Repair. LP184 efficacy in 35 cell lines from solid tumor origin with known transcriptomic profiles identifies gene expression patterns that strongly correlate with response in cell lines. This gene expression profile allows the interrogation of clinical transcriptomics datasets to identify tumors that would potentially be highly responsive to LP184. Methods: LP184 cytotoxicity in cell lines was tested in a concentration range from 14 nM to 10 uM. Cell viability readout using CellTiter Fluor reagent was measured 72h post drug addition, and IC50 value was generated using GraphPad Prism. Bioinformatic analyses were run using the Morpheus tool from the Broad Institute. CCLE microarray expression data were used for gene signature findings and TCGA patient data were used for gene signature validation. Results: LP184 showed broad anti-tumor cytotoxicity in solid tumor cell lines including liver, NSCLC, prostate, ovarian, kidney and thyroid cancers (median IC50: 358.9 nM). Across this spectrum, LP184 sensitivity was found to be most highly correlated with high transcript levels of PTGR1, and with low transcript levels of KDM4A. Analysis of top 10 sensitive cell lines showed that NSCLC cell lines had the highest representation (5 of 10). Differential sensitivity and gene expression pattern analyses on NSCLC cell lines correlated with high relative expression of PTGR1, CASP6, NAMPT, SMARCD3 and GLRX genes along with low relative expression of KMD4A, HPGD, PTPRF and KRT14 genes in the highly sensitive cell lines. The oxidoreductase activity of PTGR1 is likely involved in bioactivation of LP184 and CASP6 mediates drug-induced apoptosis. HPGD acts upstream of PTGR1 in the prostaglandin metabolism pathway in turn modulating the turnover of endogenous PTGR1 substrate. Assessing relative expression of these genes in selected clinical NSCLC data indicates that around 37% of such tumors would potentially respond with high sensitivity to LP184. Conclusions: Overall, LP184 is a novel alkylating agent with nanomolar potency in solid tumor cell lines, especially NSCLC, and can serve as a molecularly guided therapy option in multiple tumors. The correlated genes KDM4A/ HPGD that impart high sensitivity at low expression levels, suggest potential synergistic combinations of their respective inhibitors in development PKF118-310/ ML147 with LP184.

Nano Meets Micro-Translational Nanotechnology in Medicine: Nano-Based Applications for Early Tumor Detection and Therapy.

Nanomaterials have great potential for the prevention and treatment of cancer. Circulating tumor cells (CTCs) are cancer cells of solid tumor origin entering the peripheral blood after detachment from a primary tumor. The occurrence and circulation of CTCs are accepted as a prerequisite for the formation of metastases, which is the major cause of cancer-associated deaths. Due to their clinical significance CTCs are intensively discussed to be used as liquid biopsy for early diagnosis and prognosis of cancer. However, there are substantial challenges for the clinical use of CTCs based on their extreme rarity and heterogeneous biology. Therefore, methods for effective isolation and detection of CTCs are urgently needed. With the rapid development of nanotechnology and its wide applications in the biomedical field, researchers have designed various nano-sized systems with the capability of CTCs detection, isolation, and CTCs-targeted cancer therapy. In the present review, we summarize the underlying mechanisms of CTC-associated tumor metastasis, and give detailed information about the unique properties of CTCs that can be harnessed for their effective analytical detection and enrichment. Furthermore, we want to give an overview of representative nano-systems for CTC isolation, and highlight recent achievements in microfluidics and lab-on-a-chip technologies. We also emphasize the recent advances in nano-based CTCs-targeted cancer therapy. We conclude by critically discussing recent CTC-based nano-systems with high therapeutic and diagnostic potential as well as their biocompatibility as a practical example of applied nanotechnology.

Imaging intercellular interaction and extracellular vesicle exchange in a co-culture model of chronic lymphocytic leukemia and stromal cells by lattice light-sheet fluorescence microscopy.

Recent advances in live cell imaging allow investigating processes that take place over the entire cell volume with unprecedented time and spatial resolution. Here we describe a protocol to study intercellular communication, including extracellular vesicle exchange, between cancer cells and their microenvironment, using lattice light sheet fluorescence microscopy. While the described protocol is intended to study the interactions between chronic lymphocytic leukemia cells and bone marrow stromal cells, many components of it can be applied to study other cancers of hematopoietic or solid tumor origin, as well as to characterize other modalities of intercellular communication.

Hematologic Malignancies Exhibit Selective Vulnerability to Inhibition of De Novo Pyrimidine Biosynthesis By AG-636, a Novel Inhibitor of Dihydroorotate Dehydrogenase in Phase 1 Clinical Trials

Rapidly proliferating cells reprogram metabolism to support increased biosynthetic demands, a feature that can expose targetable vulnerabilities for therapeutic intervention. A chemical biology screen was performed in an effort to identify metabolic vulnerabilities in particular tumor subtypes, and revealed potent and selective activity of a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic origin. In contrast, cancer cell lines of solid tumor origin exhibited comparatively poor sensitivity. Evaluation of a lymphoma cell line panel demonstrated broad responsiveness to DHODH inhibition, independent of clinical subtype (e.g. ABC, GCB, double-hit). The on-target cellular activity of AG-636 was evaluated by examining the metabolic effects of AG-636 on cells and by evaluating the ability of extracellular uridine to rescue the effects of AG-636 on proliferation and viability. The metabolic changes incurred upon treatment of cells with AG-636 were consistent with a mechanism of action driven by inhibition of DHODH and de novo pyrimidine biosynthesis. Supraphysiologic concentrations of extracellular uridine rescued the effects of AG-636 on growth and viability as well as the effects on metabolism, further confirming on-target activity. The mechanistic basis for differential sensitivity to AG-636 was assessed by comparing the activity of the de novo pyrimidine biosynthesis and uridine salvage pathways in cancer cell lines of hematologic or solid tumor origin with similar proliferative rates. Differential response to AG-636 could not be attributed to varying abilities to utilize the de novo pyrimidine biosynthesis pathway or to salvage extracellular uridine. Real-time imaging of cells treated with AG-636, along with monitoring of extracellular uridine concentrations, demonstrated immediate effects on the viability of lymphoma cell lines in the setting of depleted extracellular uridine. In contrast, solid tumor cell lines were able to maintain growth for an additional period of time, suggestive of adaptive mechanisms to supply pyrimidine pools and/or to cope with nucleotide stress. The high in vitro activity of AG-636 in cancer cells of hematologic origin translated to xenograft models, including an aggressive, patient-derived xenograft model of triple-hit lymphoma and an ibrutinib-resistant model of mantle cell lymphoma in which complete tumor regression occurred. These studies support the development of AG-636 for the treatment of hematologic malignancies. A phase 1 study has been initiated in patients with relapsed/refractory lymphoma (NCT03834584). Disclosures Ulanet: Agios: Employment, Equity Ownership. Chubukov:Agios: Employment, Equity Ownership. Coco:Agios: Employment, Equity Ownership. McDonald:Agios: Employment, Equity Ownership. Steadman:Agios: Employment, Equity Ownership. Narayanaswamy:Agios: Employment, Equity Ownership. Ronseaux:Agios: Employment, Equity Ownership. Choe:Agios: Employment, Equity Ownership. Truskowski:Agios: Employment, Equity Ownership. Nellore:Aurigene Discovery Technologies: Employment. Rao:Firmus Laboratories: Employment, Equity Ownership. Lenz:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Agios: Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy; AstraZeneca: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Employment, Honoraria, Research Funding, Speakers Bureau. Cooper:Agios: Employment, Equity Ownership. Murtie:Agios: Employment. Marks:Agios: Employment, Equity Ownership.

A Detection System for Circulating Tumor Cells (Ctcs) Using Gfp Expressing Telomerase-Specific Replication-Competent Adenovirus in Bone and Soft Tissue Sarcoma

ABSTRACT Aim: The concept of circulating tumor cell (CTC) is of great interest recently in the field of cancer treatment. CTCs defined as cancer cells of solid tumor origin found in the peripheral blood, and have been detected in the patients with advanced cancer. However, CTCs have also detected in patients with localized cancers, indicating the risk of progression to metastatic disease. Although many papers on CTCs have been published, there is no report on CTCs detected in patients with bone and soft tissue tumors. We analyzed CTCs in the peripheral blood of patients with sarcoma. Methods: 7.5ml of blood was collected from the patients, and CTCs were assessed using TelomeScan (OBP-401), which GFP-expressing attenuated adenovirus-5 vector, in which the hTERT promoter regulates viral replication. A total of 22 patients with bone (6 osteosarcoma) and soft tissue sarcoma (4 myxofibrosarcoma, 4 liposarcoma, 3 DFSP, 2 synovial sarcoma, 3 others) patients were enrolled. GFP-expressing cells were labeled with anti-CD45 (a marker for blood cell), anti-vimentin antibody (a marker for mesenchymal cell) and DAPI stain (a marker for cell nucleus) 24 hours after the virus infection to ensure that cells labeled with GFP were indeed sarcoma cells. Results: CTCs were detected in 11 of 22 sarcoma patients (4 osteosarcoma, 3 myxofibrosarcoma, 2 DFSP, 2 others). Average number of CTCs was 3.5 cells per 7.5ml. 3 patients with positive CTC showed negative CTC after sarcoma resection, and 4 patients with positive CTC showed negative CTC after chemotherapy. 5 of 22 patients developed lung metastasis, 4 of those showed positive CTC at diagnosis. Conclusions: CTCs were detected in 50% of patients with bone and soft tissue sarcoma in our series. CTC detection using TelomeScan has been reported only in patients with cancer. We identified that the detection system using TelomeScan can be a new approach to visualize live human CTCs from bone and soft tissue sarcoma, and that CTC can be also a biomarker for bone and soft tissue sarcoma. Disclosure: All authors have declared no conflicts of interest.

Abstract 2160: Development of selective MELK kinase inhibitors for cancer treatments.

Abstract Maternal embryonic leucine zipper kinase (MELK) is an atypical member of the AMPK family of serine-threonine kinases that has been implicated in stem cell renewal, cell cycle progression, cytokinesis, mRNA splicing and apoptosis. MELK activity is correlated with its phosphorylation level, is cell cycle dependent and maximal during mitosis although direct upstream regulators of MELK kinase activity are unknown. This kinase is highly expressed in several types of solid cancers: colon, breast, ovary, lung, and brain and shows relatively low expression levels in normal tissues. MELK overexpression in patient-derived tumors strongly correlates with poor prognosis in glioblastoma and breast cancer. Moreover, siRNA mediated knockdown of MELK kinase significantly inhibits growth of tumor cell lines both in vitro and in vivo. Therefore, MELK kinase is an emerging and interesting target of significant potential for therapeutic intervention in cancer. In this study, we are reporting results for a series of new MELK kinase inhibitors that were developed at Selvita. Newly synthesized derivatives exert good selectivity and potency in MELK inhibitions with the low sub-micromolar range. Anticancer effects of these compounds were investigated in several cancer cell lines of solid tumor origin where the compounds were shown to induce cell death with low micromolar ED50 values. Compounds were also analyzed for their effects on cell death, proliferation, apoptosis, cell cycle parameters and ADME properties. Taken altogether, the presented data supports our rationale of using inhibitors of MELK kinases as a novel approach for the cancer therapy, especially for the treatment of glioblastoma and breast cancer. Citation Format: Piotr Kowalczyk, Paulina Wegrzyn, Przemyslaw Zawadzki, Edyta Palacz, Ewa Trebacz, Katarzyna Wiklik, Mariusz Milik, Adrian Zarebski, Karolina Krawczynska, Krzysztof Brzózka. Development of selective MELK kinase inhibitors for cancer treatments. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2160. doi:10.1158/1538-7445.AM2013-2160

Abstract 4499: In vitro and in vivo evaluation of novel Pim kinase inhibitors with potent anticancer activity

Abstract The Pim serine-threonine kinase family is composed of three members that play an important role in intracellular signaling and contribute to pathways involved in cell survival, proliferation, stress response and cellular motility. Out the three family members, Pim-1 has been studied most extensively and was shown to be a crucial downstream effector of several oncogenes such as Jak2 and FLT3 kinases. Pim-1 overexpression has been also reported in a variety of cancers such as diffuse B cell lymphoma, chronic lymphocytic leukemia, Flt3-mediated acute myelogenous leukemia and solid tumors including prostate and pancreatic cancers. For this reason, Pim-1 kinase emerged as a novel and interesting target of significant potential for therapeutic intervention. In the current study, the optimization of the initial lead compound - which presented moderate potency in Pim kinase inhibition, low solubility and bioavailability - led to the identification of a group of new compounds. Among the newly synthesized compounds several derivatives exerted increased potency in Pim kinase inhibition with IC50 values below 5 nM. Profiling of best inhibitors on a KINOMEscanTM Max kinase panel revealed superior selectivity towards Pim kinases. Anticancer activity of these new derivatives was investigated in various neoplastic cell lines of hematological and solid tumor origin where the compounds were shown to induce apoptosis both as a single agent, but also synergistically with standard therapies, such as docetaxel and cytarabine. FACS analyses revealed accumulation of subG0 phase, indicating apoptosis in time and concentration dependant manner. In the living cells, two phenotypes could be observed, namely a cell cycle arrest of the cells either in the G0/G1 or G2/M phases, depending on the cell line. In order to confirm that the observed cytotoxic and cell cycle effects were due to inhibition of the Pim kinases, we performed analyses of direct downstream substrates of these kinases. Pim kinases were shown to phosphorylate p27KIP1, 4E-BP1 and Bad proteins, and treatment of cells in vitro with our new compounds caused a dramatic reduction in the phosphorylation levels of these targets. Best performing compounds were then chosen for an efficacy screen in a mouse xenograft model, proving their anticancer activity. Treatment of the animals also led to in vivo biomarker inhibition after single administration of the compounds. Results of Selvita's Pim kinase inhibitor optimization efforts are shown and discussed, supporting further development of this class of inhibitors in oncology indications. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4499.

Abstract A242: Identification of novel and specific Pim-1 kinase inhibitors with potent anticancer activity in hematological and solid tumor malignancies

Abstract The Pim serine-threonine kinase family is composed of three members which play an important role in intracellular signaling and contribute to pathways involved in cell survival, proliferation, stress response and cellular motility. Out the three family members, Pim-1 has been studied most extensively and was shown to be a crucial downstream effector of several oncogenes like Jak2 and FLT3 kinases. Pim-1 overexpression has been also reported in a variety of cancers such as diffuse B cell lymphoma, chronic lymphocytic leukemia, Flt3-mediated acute myelogenous leukemia and solid tumors such as prostate and pancreatic cancers. For this reason, Pim-1 kinase emerged as a novel and interesting target of significant potential for therapeutic intervention. In the current study we have performed lead optimization of a new small molecule Pim-1 kinase inhibitor. Initial compound exerted moderate potency in Pim-1 kinase inhibition accompanied by high selectivity for the Pim kinase family. In parallel to biochemical characterization, the original compound had been extensively tested in vitro and in vivo for anticancer activity. In order to improve potency and selectivity of the original compound a series of new derivatives was synthesized and screened for Pim-1 inhibition. Among the newly synthesized compounds several exerted increased potency in Pim-1 inhibition with IC50 values as low as 4,8 nM. Further biochemical profiling of best inhibitors on a 440 KINOMEscanTM Max kinase panel revealed superior selectivity in binding and inhibition of the Pim-1 kinase with over 20-fold off-target selectivity. Anticancer effect of new derivatives was investigated in several cancer cell lines of hematological and solid tumor origin where the compounds was shown to induce of apoptosis. As Pim-1 kinase was shown to regulate cell cycle progression by phosphorylation of several key proteins such as p27 and cdc25A in the G1/S checkpoint and cdc25C and c-TAK-1 in the G2/M checkpoint, we investigated cell cycle effects after compounds administration. We could observe two phenotypes in cell that did not undergo apoptosis, namely a time and dose dependent arrest of the cells either in the G0/G1 or G2/M phase, depending on the cell line. To confirm that the observed cytotoxic and cell cycle effects were due to inhibition of the Pim-1 kinase by the new derivatives, a biomarker analysis was performed. Pim-1 kinase was shown to phosphorylate p27KIP1 at threonine 198 both in vitro and in the cells. As soon as 1h after treatment with the compounds, a dramatic inhibition of phospho-p27KIP1 was observed on K562 cells proving the expected mechanism of action of Pim-1 kinase inhibiting compounds in the cells. Results of the Selvita's Pim-1 inhibitor lead optimization efforts are shown and discussed, supporting further development of this class of inhibitors in oncology indications. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A242.

The role of soluble and cell-surface expressed 4-1BB ligand in patients with malignant hemopoietic disorders

The TNFR family member 4-1BB and its ligand 4-1BBL are involved in the costimulation of T-cells and tumor-derived soluble (s)4-1BBL may influence the interaction of malignant cells with the immune system. Here, we report that cell-surface-expressed (c)4-1BBL can be expressed on mononuclear blood cells from patients with acute myeloid leukemia (AML) (n = 35), myelodysplasia (n = 5) or non-Hodgkin lymphoma (n = 11) and can be coexpressed on varying proportions of lymphoid or myeloid malignant cells and on dendritic cells differentiated from AML-blasts. Direct correlations between c- and s4-1BBL were not found in the investigated cases. Up to now expression of 4-1BBL has not been described on primary myeloid malignant cells, but only on malignant cells of lymphoid or solid tumor origin or on tumor cell lines. With our work we further contribute to the understanding of the potential role of c/s4-1BBL in immune reactions and its influence on the interaction of tumor and immunoreactive cells.

Establishment and characterization of a highly tumorigenic human diploid endometrial cancer cell line

A new cell line designated CUME-1 has been established from a poorly differentiated endometrial adenocarcinoma of the uterus. This cell line grew well without interruption for more than 88 months and 110 serial passages were successively carried out. The cells were highly tumorigenic in nude mice (85%). Repeated karyotype analyses from early (4th) to late (55th) passages of this cell line revealed a diploid stable clone in each passages without any noticeable structural or numerical aberrations. But from the 80th passage, a subpopulation with reciprocal translocation between chromosomes 1q and 9q consistently appeared and was observed in about 30% of the cells. This cell line is one of the rare examples of experimentally proved tumorigenic cells of human solid tumor origin that retains the diploid karyotype in vitro. HLA typing indicated the presence of DR4, DR13, DQ3, and DQ6. Cytosol estrogen and progesterone receptors were found both in fresh primary tumor and in this cell line. Gonadotropin-releasing hormone (Gn-RH) receptor mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) in cultured cells. Using the single-strand conformation polymorphism (SSCP) technique, we have screened CUME-1 cells for p53 mutation in exons 4 to 9. No mobility shift was observed. This cell line may be useful in studying the in vitro chromosomal evolution of the cell line and the in vivo properties of human endometrial adenocarcinoma.

A wide range of human cancers express interleukin 4 (IL4) receptors that can be targeted with chimeric toxin composed of IL4 and Pseudomonas exotoxin

A chimeric toxin has been constructed by fusion of a gene encoding human interleukin 4 (hIL4) to a gene encoding a mutant form of Pseudomonas exotoxin A (PE) which cannot bind to its receptors (PE4E). The chimeric gene was expressed in Escherichia coli where large amounts of the chimeric toxin, hIL4-PE4E, was produced. Purified hIL4-PE4E was very cytotoxic to cancer cell lines of both hematopoietic and solid tumor origin. In the HUT 102 T cell leukemia and Daudi B cell lymphoma cell lines, protein synthesis was inhibited by 50% (ID50) at a hIL4-PE4E concentration of 2 and 7 ng/ml (25 and 86 pM, respectively). hIL4-PE4E was also very cytotoxic to cell lines derived from carcinomas of the colon, breast, stomach, liver, adrenals, and prostate, as well as melanoma and epidermoid carcinoma, indicating that hIL4 receptors are widely expressed on human malignancies. We also found that human phytohemagglutinin-activated peripheral blood lymphocytes were extremely sensitive to hIL4-PE4E with an ID50 of 0.2 ng/ml (2.5 pM). The cytotoxic action of hIL4-PE4E was specific because it was blocked by an excess of hIL4 and not of human interleukin 2. In addition, hIL4-PE4ED553, an enzymatically inactive form of the chimeric toxin, was not cytotoxic. These results suggest that the hIL4 receptor may be a target for therapy in malignant and immunologic disorders using hIL4 chimeric toxin.

SK-UT-1B, a human tumorigenic diploid cell line

SK-UT-1B (ATCC HTB 115) produced tumors 2 weeks after subcutaneous injection of 10 million cells into athymic nude mice. Karyotypic analysis at the 500–600 subband level, however, did not reveal any noticeable aberrations. This cell line therefore may represent the first example of experimentally proven human tumorigenic cells of solid tumor origin that retains the diploid karyotype in vitro indefinitely.

Influence of cultivation, trypsinization and aggregation on the transplantability of melanoma B16 cells.

AbstractTo elucidate the usefulness of tumour cells grown in vitro for transplantation studies, Melanoma B16 was grown in monolayer culture. Three types of suspensions were prepared from the cell cultures: two virtually monocellular suspensions, one produced by mechanical procedures and Versene (CV), the other with the addition of trypsinization (CTV); a third suspension containing cell aggregates (CVA) was produced by centrifuging a CV suspension. For comparison, a well‐dissociated suspension of solid tumor origin was prepared by a trypsin‐Versene procedure (STV). All suspensions were characterized with regard to dissociation grade, cell volumes and cellular electrophoretic mobility and were transplanted in graded cell doses subcutaneously, intraperitoneally and intravenously. While inoculum‐doses needed for progressive growth were lower IP than SC, the transplantabilities were similar in these sites for all suspensions, indicating similar viabilities and absence of major dilution errors. The growth patterns for IV infused cells exhibited significant differences between the suspensions. CV cells gave rise to lung tumours almost exclusively, while CVA animals, in addition to a large total lung tumour volume, had some extrapulmonary takes. CTV had the greatest number of extrapulmonary tumours, but in combination with small lung tumours, STV cell recipients were intermediate with respect to pulmonary as well as extrapulmonary tumours. These observations were collated with data regarding aggregation, cell volumes and electrophoretic mobility. It was concluded that (1) cultivated tumour cells can have similar transplantation characteristics to the tumour of origin; (2) such cells may be useful in tumour dissemination studies; (3) trypsinization as well as the presence of cell aggregates affects the growth pattern of IV infused tumour cells by yielding an increased extrapulmonary tumour crop.