Primary Cancer Cell Cultures Research Articles

Thyroid Hormone Promotes β-Catenin Activation and Cell Proliferation in Colorectal Cancer.

Thyroid hormone status has long been implicated in cancer development. Here we investigated the role of thyroxine (T4) in colorectal cancer cell lines HCT 116 (APC wild type) and HT-29 (APC mutant), as well as the primary cultures of cancer cells derived from patients. Cell proliferation was evaluated with standard assay and proliferation marker expression. β-Catenin activation was examined according to nuclear β-catenin accumulation and β-catenin target gene expression. The results showed that T4 increased colorectal cancer cell proliferation while cell number and viability were elevated by T4 in both established cell lines and primary cells. Moreover, the transcriptions of proliferative genes PCNA, CCND1, and c-Myc were enhanced by T4 in the primary cells. T4 induced nuclear β-catenin accumulation, as well as high cyclin D1 and c-Myc levels compared to the untreated cells. In addition, the β-catenin-directed transactivation of CCND1 and c-Myc promoters was also upregulated by T4. CTNNB1 transcription was raised by T4 in HCT 116, but not in HT-29, while the boosted β-catenin levels were observed in both. Lastly, the T4-mediated gene expression could be averted by the knockdown of β-catenin. These results suggested that T4 promotes β-catenin activation and cell proliferation in colorectal cancer, indicating that an applicable therapeutic strategy should be considered.

Spheroid Cultures of Primary Urothelial Cancer Cells: Cancer Tissue-Originated Spheroid (CTOS) Method.

Increasingly, it has been recognized that studying cancer samples from individual patients is important for the development of effective therapeutic strategies and in endeavors to overcome therapy resistance. Primary cultures of cancer cells acutely dissected from individual patients can provide a platform that enables the study and characterization of individual tumors. To that end, we have developed a method for preparing cancer cells in the form of multi-cellular spheroids. The cells can be derived from patient tumors (primary cells), from patient-derived xenografts, or from genetically- or chemically induced animal tumors. This method of culturing spheroids composed of cells derived from cancer tissues can be applied to various types of cancer, including urothelial cancer. The method is based on the principle of retaining cell-cell contact throughout cancer cell preparation and culturing. The first step is a partial digestion of the tumor specimen into small fragments; these fragments spontaneously form spheroidal shapes within several hours. The spheroid is referred to as a cancer tissue-originated spheroid (CTOS). The advantage of the CTOS method is that it allows one to prepare pure cancer cells at high yield. CTOSs can be stably cultured in serum-free conditions. The CTOS method can be applied to drug sensitivity assays, drug screening, and analyses of intracellular signaling. Moreover, the CTOS method provides a platform for studying the nature of cancer cell clusters.

Abstract 5516: Regulation of TFPI-2 in the progression of ovarian cancer

Abstract Ovarian cancer metastasis occurs when malignant cells migrate from the primary tumor into the ascitic fluid and form a secondary tumor in the peritoneal cavity. The cells responsible for this process, Metastases Initiating Cells (MICs), suffer a series of changes in protein expression, with increased Epithelial Mesenquimal Transition and stem cell markers having been reported. Tissue Factor Pathway Inibitor-2 (TFPI-2) is a serine proteinase inhibitor and potential tumor suppressor gene, whose reduction is correlated with more metastases and poor prognosis. As the exact stage of TFPI-2 loss is still unknown, the purpose of our study was to determine TFPI-2 during ovarian cancer progression. Protein and RNA levels were assessed in primary tumor and metastatic tissue from papillary serous carcinoma by immunohistochemistry and qPCR, respectively. TFPI-2 RNA and protein levels were analyzed in cancer cell lines and primary cultured cancer cells isolated from ascitic fluid of advanced ovarian cancer patients and in cancer spheres derived from these cultures. Epigenetic changes of 3 CpG were analized by bisulfite and pirosequencing technique in ovarian cancer cell lines and in cancer spheres derived from these cultures. We report that significantly decreased TFPI-2 protein levels in metastatic tissue compared to the primary tumor, a result confirmed in matched primary and metastatic lesions from the same ovarian cancer patients. Furthermore, cancer spheres (representing potential MICs) displayed significantly lower levels of TFPI-2 than corresponding cell line and some primary cultured cancer cells. The cancer spheres showed a modest increase in methylation percentage in three specific CpG sites within the TFPI-2 promoter. Our results suggest that TFPI-2 loss plays a role in the cancer cell escape from the primary tumor. Future studies will determine the mechanism and the biological consequences of the progressive loss of TFPI-2 in cancer. Funding: FONDECYT 11140657, 1160800, 1140970, 3140335, 3150028, CORFO L2 13IDL2-18608, IMII-P09/016-F, CONICYT-FONDAP 15130011, BMRC 13CTI21526-P6 Citation Format: Jacqueline Fry, Sumie Kato, Lorena Abarzua, Pamela Gonzalez, Carolina Ramirez, Elisa Cumsille, Erasmo Bravo, Raimundo Correa, Juan E. Leiva, Cesar Paredes, Juan C. Roa, Carolina Ibañez, Mauricio Cuello, Gareth I. Owen, Maria L. Bravo. Regulation of TFPI-2 in the progression of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5516. doi:10.1158/1538-7445.AM2017-5516

Abstract 410: Identifying ovarian cancer specific targeted drugs using high-throughput drug sensitivity profiles of primary cancer cells

Abstract Ovarian cancer (OvCa) is the sixth most common cancer in women and leading cause of death from gynecologic diseases. Poor prognosis in OvCa is due to late diagnosis and acquired resistance to the commonly used platinum-based regimens. A significant setback for OvCa treatment is the lack of reliable biomarkers and effective targeted therapies. In order to discover novel therapeutic opportunities with approved and emerging drugs for OvCa, we have established primary cultures using ascites or tumor tissue samples from chemo-refractory ovarian cancer patients for ex vivo Drug Sensitivity and Resistance Testing (DSRT) and genomic profiling. In addition, we have performed DSRT with 31 established OvCa cell lines. Comparison of the drug sensitivity profiles of ten primary cancer cell cultures and 31 OvCa cell lines revealed previously unanticipated cancer selective drug vulnerabilities. Several drug groups were identified suggesting that the sensitive samples were addicted to the corresponding signaling networks. For example, in a 53-year old chemorefractory low grade serous OvCa patient, genomic and transcriptomic analyses revealed a fusion gene of NRG-1, a target that was recently reported to involve the NRG1/ERBB3 activation loop in OvCa. We found high expression of ERBB2 and ERBB3 by RNA-seq as well as high levels of phospho-ERBB3, phospho-ERBB2 and phospho-EGFR by immunohistochemistry. In agreement with the molecular mechanism, DSRT analysis identified significant sensitivity of primary cancer cells to EGFR inhibitors, such as erlotinib and to dual EGFR and Her2 inhibitor afatinib. The patient has received combination therapy of gemcitabine and erlotinib during nine months, followed by afatinib monotheraphy that has lead to complete remission after six months treatment. We have detected activated NRG1/ERBB3 activation loop also in several additional ovarian cancer patient cases, highlighting the importance of this signaling pathway in ovarian cancer pathogenesis. Our study reveals that a subset of serous ovarian cancer patients with activating NRG1/ERBB3 signaling loop have clinical benefits from repurposing dual EGFR/Her2 inhibitor afatinib, providing a basis for the clinical use of EGFR and dual EGFR/Her2 tyrosine kinase inhibitors in clinical treatment. In conclusion, DSRT technology together with molecular profiling provides a powerful strategy to identify tumor driver signals and select clinically actionable inhibitors. Hence, this type of systems medicine approach can significantly improve the power of mainly genomics-oriented personalized medicine approaches. Citation Format: Astrid Murumägi, Akira Hirasawa, Suleiman Khan, Daniela Ungureanu, Mariliina Arjama, Teijo Pellinen, Samuli Eldfors, Riitta Koivisto-Korander, Arto Leminen, Ralf Bützow, Tero Aittokallio, Olli Kallioniemi. Identifying ovarian cancer specific targeted drugs using high-throughput drug sensitivity profiles of primary cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 410. doi:10.1158/1538-7445.AM2017-410

Abstract 789: Establishment of an in vitro model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer cells

Abstract Introduction: A key step in cancer progression is tumor irrigation. The process of angiogenesis can be complemented in sub-sets of highly aggressive cancers by the process of vasculogenic mimicry, which is the formation of tubular structures by tumor cells. Herein, we sought to establish an in vitro assay of vasculogenic mimicry and determine the percentage of ovarian and gastrointestinal primary cultures capable of undergoing this process. Materials & Methods: Gastric cancer cell lines, AGS and Hs746T, and the ovarian cancer cell lines SKOV-3 and HEY were used in conjunction with gastrointestinal and ovarian primary cultured cancer cells extracted from peritoneal fluid. Cells were seeded on matrigel and monitored for a week. Hollow channels formation was evaluated by fluorescent dye microinyection, periodic acid Schiff staining, confocal microscopy and X-Ray microtomography (Micro-CT). Results: SKOV-3, HEY and AGS cell lines underwent the process of vasculogenic mimicry. Both confocal microscopy and Micro-CT reconstruction were able to show the presence of a lumen of the structures in 3D. In 22 primary cancer cultures (Ovarian, Colon and Gastric) only 38% could underwent vasculogenic mimicry. Discussion: We have standardized an in vitro assay to assay and quantify vasculogenic mimicry. Regardless of origin, only a low percentage of cultures have the ability to undergo vasculogenic mimicry. An understanding of this process could shed light on new therapies for this sub-set of highly aggressive cancers. Funding: FONDECYT 1120292, 3150028 & 1140970. CORFO L2 13IDL2-18608, BMRC 13CTI 21526-P6, IMII P09/016-F, CONICYT-FONDAP #1513001 Citation Format: Andres Valdivia, Dusan Racordon, Raul Aravena, Gabriel Mingo, Alejandra Sandoval, Maria Loreto Bravo, Mauricio A. Cuello, Sumie Kato, Rafaela Erices, Carolina Ramirez, Pamela Gonzalez, Beatriz Sanchez, Alejandro H. Corvalan, Gareth I. Owen. Establishment of an in vitro model for the study of vasculogenic mimicry in ovarian and gastrointestinal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 789. doi:10.1158/1538-7445.AM2017-789

Diabetic concentrations of metformin inhibit platelet-mediated ovarian cancer cell progression.

Clinical studies have suggested a survival benefit in ovarian cancer patients with type 2 diabetes mellitus taking metformin, however the mechanism by which diabetic concentrations of metformin could deliver this effect is still poorly understood. Platelets not only represent an important reservoir of growth factors and angiogenic regulators, they are also known to participate in the tumor microenvironment implicated in tumor growth and dissemination. Herein, we investigated if diabetic concentrations of metformin could impinge upon the previously reported observation that platelet induces an increase in the tube forming capacity of endothelial cells (angiogenesis) and upon ovarian cancer cell aggressiveness. We demonstrate that metformin inhibits the increase in angiogenesis brought about by platelets in a mechanism that did not alter endothelial cell migration. In ovarian cancer cell lines and primary cultured cancer cells isolated from the ascitic fluid of ovarian cancer patients, we assessed the effect of combinations of platelets and metformin upon angiogenesis, migration, invasion and cancer sphere formation. The enhancement of each of these parameters by platelets was abrogated by the present of metformin in the vast majority of cancer cell cultures tested. Neither metformin nor platelets altered proliferation; however, metformin inhibited the increase in phosphorylation of focal adhesion kinase induced by platelets. We present the first evidence suggesting that concentrations of metformin present in diabetic patients may reduce the actions of platelets upon both endothelial cells and cancer cell survival and dissemination.

Primary cancer cell culture: mammary-optimized vs conditional reprogramming

The impact of different culture conditions on biology of primary cancer cells is not always addressed. Here, conditional reprogramming (CRC) was compared with mammary-optimized EpiCult-B (EpiC) for primary mammary epithelial cell isolation and propagation, allograft generation, and genome-wide transcriptional consequences using cancer and non-cancer mammary tissue from mice with different dosages of Brca1 and p53 Selective comparison to DMEM was included. Primary cultures were established with all three media, but CRC was most efficient for initial isolation (P<0.05). Allograft development was faster using cells grown in EpiC compared with CRC (P<0.05). Transcriptome comparison of paired CRC and EpiC cultures revealed 1700 differentially expressed genes by passage 20. CRC promoted Trp53 gene family upregulation and increased expression of epithelial differentiation genes, whereas EpiC elevated expression of epithelial-mesenchymal transition genes. Differences did not persist in allografts where both methods yielded allografts with relatively similar transcriptomes. Restricting passage (<7) reduced numbers of differentially expressed genes below 50. In conclusion, CRC was most efficient for initial cell isolation but EpiC was quicker for allograft generation. The extensive culture-specific gene expression patterns that emerged with longer passage could be limited by reducing passage number when both culture transcriptomes were equally similar to that of the primary tissue. Defining impact of culture condition and passage on the transcriptome of primary cells could assist experimental design and interpretation. For example, differences that appear with passage and culture condition are potentially exploitable for comparative studies targeting specific biological networks in different transcriptional environments.

Abstract A104: Development of pathophysiologically relevant in vitro tumor models: Cytotoxicity of anticancer drugs in two dimensional and three dimensional cell line cultures versus primary colorectal tumor cells

Abstract Chemotherapy is an effective modality for cancer therapy. However, treatment of solid tumors is hampered by several factors, such as development of acquired resistance, nonspecific drug effects, etc. Moreover, high attrition rate of anticancer drugs is an additional clinical hurdle often encountered in cancer patients. One of the potential ways to overcome these issues is to use in vitro tumor models that reliably capture in vivo tumor conditions for effective drug selection in the preclinical stages. Although two-dimensional (2D) cultures of cancer cells are widely used for drug screening and target validation; they are less predictive in vitro tumor models than three-dimensional (3D) cultures and primary cultures of patient-derived tumor cells. Cytotoxic drugs, nanoparticles, and antitumor antibodies display altered activity in 2D, 3D, and primary cancer cell cultures, potentially due to cellular and molecular differences in these culture types. Recently, the use of multicellular 3D spheroid cultures of cancer cells have gained considerable attention among researchers as more predictive in vitro tumor models to study the effects of tumor microenvironment on cancer cell response to drugs. In addition to 3D cultures, patient-derived tumor cells are also becoming increasingly popular in drug research for their relative closeness to tumors. It is expected that drugs characterized using these pathophysiologically relevant in vitro tumor models will improve their translation to higher disease models, and significantly reduce the cost associated with failure of drugs in the clinical trials. In this study, we compare the cytotoxic effects of 12 standard anticancer drugs in 2D and 3D cultures of colorectal carcinoma (CRC) cells to &amp;gt;500 primary CRC cultures. Furthermore, using primary cultures of patient-derived ovarian, breast, and lung cancer cells, we present the potential use of primary tumor cells as in vitro predictive model for chemotherapy. Our data indicates that compared to 2D cultures, 3D cultures of CRC cells better reflect the drug effects seen in primary CRC cultures. Our cytotoxicity data from primary cultures also show how the drug sensitivity profiles differ across histologically different solid tumors types. Acknowledgements This work was supported by grants from the Czech Ministry of Education, Youth and Sports (CZ.1.07/2.3.00/30.0041 to V.D.; LO1304 to M.H.), the Czech Ministry of Health (NT14282 to M.H.), and the Technological Agency of the Czech Republic (TE01020028 to M.H.). Citation Format: Viswanath Das, Marta Zbozinkova, Martin Holoubek, Petr Dzubak, Marian Hajduch. Development of pathophysiologically relevant in vitro tumor models: Cytotoxicity of anticancer drugs in two dimensional and three dimensional cell line cultures versus primary colorectal tumor cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A104.

Abstract 329: Establishment of three-dimensional primary tumor cell culture method and novel drug sensitivity test

Abstract Background Predicting sensitivity to anticancer drug and selecting the most effective drug may help to enhance the therapeutic value, leading to improve patients’ QOL. For these purposes, primary cancer cell from patient specimen is being studied on anticancer drug sensitivity test. In general cell culture method, primary cancer cells are difficult to culture because of fibroblast cells are increasing predominantly than cancer cells. For resolving the problem, collagen-gel-embedding culture method has been developed. However this culture method remain having some problems, for example, complexity of the cell culture preparation from small cancer tissue like a biopsy sample, low efficiency of primary cancer cells and difficulty in multi-drug sensitivity test. Therefore we developed a three dimensional (3D) cancer cell culture kit that could conduct a primary tumor cell culture and an evaluation of the anticancer drug sensitivity in one-step process after dissociated small cancer tissue. For 3D cell culture we have used NanoCulture Plate (NCP) which is scaffold based 3D culture system and easily form spheroids than other culture systems. Spheroids microenvironment are closely resemble to actual tumor biology, and are therefore more relevant for drug sensitivity. In this report, we carried out a sensitivity test in a lung adenocarcinoma specimen harboring EGFR gene exon19 deletion mutation indicating susceptibility to gefitinib to confirm the efficacy of the system. Method Dissociation of cancer tissue and primary cancer cell culture were performed by using 3D cancer cell culture kit and NCP. Partially digested cancer fragments from surgical specimens were seeded and cultured for 3 days. The spheroids were composed of both CK-18 expressing cancer cells and α-SMA expressing fibroblast cells, then spheroids were treated with gefitinib. After 4 days of drug treatment, the susceptibility of the cells to gefitinib was investigated an index of viability by ATP assay. Detection of EGFR mutations was performed by SRL Inc., using real-time PCR that combines scorpion technology and Amplification Refractory Mutation System (ARMS). Result and conclusion We found that primary cells with EGFR exon19 deletion were highly susceptible to gefitinib, whereas the cells with intact EGFR were not. These finding indicated the feasibility of 3D cancer cell culture kit and NanoCulture Plate for primary cell culture and screening of effective anticancer drugs. The cell lines generated through this procedure may help to advance our knowledge of certain forms of lung cancer and may also be useful for developing patient-specific anti-cancer drug screening procedures. To clarify the causal relationship between the mutation type and susceptibility, additional experiment and optimization of this kit are currently underway. Citation Format: Hiroshi Goji, Manami Shimomura, Yasushi Uemura, Tetsuya Nakatsura, M.Mamunur Rahman, Manabu Itoh. Establishment of three-dimensional primary tumor cell culture method and novel drug sensitivity test. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 329. doi:10.1158/1538-7445.AM2015-329

Colorectal cancer-derived tumor spheroids retain the characteristics of original tumors

Primary cultures of cancer cells are useful for developing personalized medicine. In this study, we characterized three lines of three-dimensional (3D) tumor spheroids established directly from tumor tissues of patients with colorectal cancers (CRCs). Each line mainly included EpCAM-positive cells and cells expressing putative cancer stem cell markers such as CD133, CD44, CD24, ALDH1, and LGR5. These characteristic stem cell markers remained identically for months in vitro. Short tandem repeat genotyping suggested that genetic fingerprints of these tumor spheroids were similar to those of the original tumor tissues from which they were derived. Mutational analysis showed that each line had the same mutation profile for APC, KRAS, MLH1, serine–threonine kinase 11, and TP53 as its parental tumor tissue. One line harboring an activating KRAS mutation was resistant to cetuximab while the remaining two lines harboring wild-type KRAS showed different responses to cetuximab. Immunohistochemical analysis showed that xenograft tumors derived from these lines retained the histopathological and mutational patterns of their parental tumors. Collectively, these results clearly showed that 3D tumor spheroids directly generated from tumor tissues of patients with CRCs preserved the characteristics of their parental tumor tissues and could be used for developing personalized medicines for CRCs.

Analysis of ERBB ligand-induced resistance mechanism to crizotinib by primary culture of lung adenocarcinoma with EML4-ALK fusion gene.

Using cell line-based assays, the secretion of erythroblastic leukemia viral oncogene homologue (ERBB) ligands has been reported to contribute to resistance against crizotinib in lung cancer with the echinoderm microtubule-associated protein-like 4 and anaplastic lymphoma kinase fusion gene. However, it is difficult to predict the role of the ligands in each patient. Here, we report an analysis of the mechanism of resistance behind crizotinib resistance using a primary culture of cancer cells from pleural effusion of an anaplastic lymphoma kinase-positive lung cancer patient who was clinically resistant to crizotinib. Primary cancer cells were prepared as cancer tissue-originated spheroids (CTOSs) according to previously described methods. CTOSs were maintained in StemPro medium, and a sensitivity assay was performed under growth factor-free conditions, or under stimulation with epidermal growth factor (EGF) or neuregulin-1/heregulin. The effect of treatment with crizotinib alone or a combination of crizotinib and erlotinib was examined. Cancer cells (LB53) were established to be CTOSs from a patient who was clinically resistant to crizotinib. The CTOSs were sensitive to crizotinib under growth factor-free conditions in vitro, whereas resistant under stimulation with EGF or neuregulin-1. These ligands rescued the inhibition of intracellular signaling by crizotinib. Pleural effusion from the patient also activated EGF receptor signaling to the similar extent of EGF stimulation. The resistance to crizotinib by EGF was reversed by blocking EGF receptor signaling by erlotinib in vitro. Stimulation by ERBB ligands is suggested to be responsible for resistance to crizotinib in this patient. The CTOS method may enable analysis of resistance mechanism for targeted therapy in individual patients.

The effect of primary cancer cell culture models on the results of drug chemosensitivity assays: the application of perfusion microbioreactor system as cell culture vessel.

To precisely and faithfully perform cell-based drug chemosensitivity assays, a well-defined and biologically relevant culture condition is required. For the former, a perfusion microbioreactor system capable of providing a stable culture condition was adopted. For the latter, however, little is known about the impact of culture models on the physiology and chemosensitivity assay results of primary oral cavity cancer cells. To address the issues, experiments were performed. Results showed that minor environmental pH change could significantly affect the metabolic activity of cells, demonstrating the importance of stable culture condition for such assays. Moreover, the culture models could also significantly influence the metabolic activity and proliferation of cells. Furthermore, the choice of culture models might lead to different outcomes of chemosensitivity assays. Compared with the similar test based on tumor-level assays, the spheroid model could overestimate the drug resistance of cells to cisplatin, whereas the 2D and 3D culture models might overestimate the chemosensitivity of cells to such anticancer drug. In this study, the 3D culture models with same cell density as that in tumor samples showed comparable chemosensitivity assay results as the tumor-level assays. Overall, this study has provided some fundamental information for establishing a precise and faithful drug chemosensitivity assay.

Abstract 1341: Rapid decrease of ATP followed by necrosis-like cell death in bladder cancer cells after exposure to high-dose chemotherapeutics used in intravesical therapy

Abstract Objectives) Non-muscle invasive bladder cancer is often treated by intravesical chemotherapy following transurethral resection. Mechanism of the effect of intravesical chemotherapy remains elusive. In this experiment, we studied cell death mechanism induced by high-dose chemotherapeutics using a novel culture method for primary cancer cells. Materials and methods) We have developed a new culture method which enables preparation and culture of primary cancer cells as multicellular spheroids which are termed cancer tissue-originated spheroids (CTOSs) (Okuyama 2013 J Urol). First we generated a bladder cancer patient-derived xenograft. We prepared CTOS from the xenograft, and exposed to high-dose (1mg/ml) or low-dose (0.01mg/ml) of epirubicin (e-ADM) or mitomycin C (MMC) for 2 hours. We investigated absorption of chemotherapeutics into CTOSs and assessed the mode of cell death. Next, we prepared CTOSs directly from surgical specimens of bladder cancer and tested the individual responses after exposure to high-dose e-ADM and MMC. Results) At high dose, e-ADM was promptly and homogenously delivered into cancer cells in the CTOSs. High-dose e-ADM and MMC decreased ATP levels in CTOSs within an hour accompanied with mitochondrial swelling and reduction of mitochondrial membrane potential, while plasma membrane integrity was mainteined. Some of the molecules in mitochondrial apoptotic pathway were subsequently activated, while at later time point the cells lost integrity of cell membrane without DNA laddering. In contrast, CTOSs exposed to low-dose of e-ADM or MMC exhibited typical DNA laddering. Intriguingly, the decrease of ATP levels by e-ADM and MMC was varied among CTOSs derived from 20 surgical specimens. Conclusions) High-dose chemotherapeutics used in intravesical therapy may induce necrosis-like cell death, different from typical apoptosis caused by chemotherapeutics at the dose of systemic chemotherapy. Each bladder cancer may have a different response to high-dose chemotherapeutics used in intravesical therapy, indicating necessity of chemosensitivity test aiming for personalized medicine. Citation Format: Takahiro Yoshida, Hiroaki Okuyama, Masashi Nakayama, Kazuo Nishimura, Norio Nonomura, Masahiro Inoue. Rapid decrease of ATP followed by necrosis-like cell death in bladder cancer cells after exposure to high-dose chemotherapeutics used in intravesical therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1341. doi:10.1158/1538-7445.AM2014-1341

Abstract 495: Inactive status of cancer cells with suppression of AKT activity contributes to survival in chronic hypoxia

Abstract A hypoxic microenvironment in tumors has been recognized as a cause of malignancy or resistance to various cancer therapies. In contrast to the recent progress in understanding the acute response of cancer cells to hypoxia, the characteristics of tumor cells in chronic hypoxia remain elusive. We have identified a pancreatic cancer cell line, AsPC-1, which is exceptionally able to survive for weeks under hypoxic conditions (1%O2) without changing medium at all, while most of the tested cancer cell lines died after several days under these conditions. Under hypoxic conditions, the AsPC-1 cells entered an “inactive status” characterized by no proliferation or death and by metabolic suppression. They recovered to active status after being placed again in optimal culture conditions. Under chronic hypoxia, ATP turnover, an indicator of energy demand, was markedly decreased, as was AKT phosphorylation. Forced activation of AKT resulted in increased ATP turnover and massive cell death in vitro. Xenograft tumors generated with AKT over-expressing cells showed remarkable decrease of inactive zone, where BrdU uptake and S6 phosphorylation were eliminated without any sign of cell death. We recently developed a novel culture system for primary cancer cells. In contrast to most cancer cell lines, primary colorectal cancer cells entered inactive status with AKT suppression under hypoxia combined with growth factor-depleted conditions. The ability of cancer cells to survive in a deteriorated microenvironment by entering into an inactive status might be a common property among cancer cells. The cancer cells in inactive status of both primary cultured cancer cells and AsPC-1 cells were resistant to chemotherapy. Targeting the regulatory mechanism inducing this inactive status might provide a new strategy for overcoming resistance to various cancer therapies. Citation Format: Masahiro Inoue, Hiroaki Okuyama, Hiroko Endo, Masayuki Ohue. Inactive status of cancer cells with suppression of AKT activity contributes to survival in chronic hypoxia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 495. doi:10.1158/1538-7445.AM2014-495

Abstract 3918: Characterizing growth features, allograft generation and transcriptomes of cultured conditionally reprogrammed cells (CRC) prepared from primary triple negative cancer from Brca1-mutant mice

Abstract Background: Efficient acquisition of primary cancer cells for genetic and pharmacological testing is a means towards individualized cancer treatment. Genetically engineered mouse models of triple negative mammary cancer were used to compare culture conditions conditionally reprogrammed cell culture CRC with a rho kinase inhibitor (Y-27632) and irradiated J2 feeder cells, EpiCult™, DMEM for establishment efficiency, differentiation, fibroblast contamination, and allograft generation. Goals: 1) Evaluate mammary cancer progenitor cells by allograft and secondary culture 2) Compare RNA-seq transcriptomes in cells and allografts with different Brca1 gene dosages (100%, 50%, 0%) Methods: Triple negative invasive adenocarcinomas from 4 mouse genotypes, all carrying a p53-null allele, were analyzed: A) 1 mutant Brca1 allele; B) 2 mutant Brca1 alleles; C) 1 mutant Brca1 allele and 1 human aromatase transgene; D) human aromatase transgene. Cancers were divided and growth assessed in CRC, EpiCult™ and DMEM cultures. Illumina (V2scriptseq) RNA-seq libraries were prepared from cell pellets and allografts. One million cells injected into mammary gland fat pads of nude mice generated allografts divided for histology/DNA analyses, secondary culture and RNAseq. Transcript abundance and differentially expressed genes were determined after assembling transcriptomes with Cufflinks. Results: CRC was the most efficient methodology based on rapidity and percentage cancer specimens successfully cultured and paucity of fibroblasts. Brca1 gene dosage made no difference in culture efficiency. Differentiation markers were expressed at significantly higher levels in CRC whereas markers of epithelial mesenchymal transition were expressed higher in EpiCult™. Allografts were derived from CRC and EpiCult™ but not DMEM. Genetic testing confirmed allografts were derived from original endogenous cancer cells. Some individual allografts derived from the same culture exhibited different growth curves and histology, suggesting tumor cell heterogeneity within cultures. Overall, palpable EpiCult™ allografts appeared significantly earlier than CRC allografts (p&amp;lt;0.05 Chi Square). RNAseq analyses distinguished gene expression patterns dependent upon culture condition as distinct from those determined by underlying genetics. Summary: CRC technology was an efficient means of generating primary cancer cell cultures, maintained epithelial cell differentiation better than other methods, had no fibroblast contamination, and preserved cancer progenitor cells. Citation Format: Ahmad M. Alamri, Svenja Groeneveld, Keunsoo Kang, Sarah Dabydeen, Weisheng Wang, Lothar Hennighausen, Bhaskar Kallakury, Xuefeng Liu, Priscilla A. Furth. Characterizing growth features, allograft generation and transcriptomes of cultured conditionally reprogrammed cells (CRC) prepared from primary triple negative cancer from Brca1-mutant mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3918. doi:10.1158/1538-7445.AM2014-3918

243 Glycan modification of podoplanin enhances growth factor production through stabilization of platelet aggregation in bladder cancer cell

INTRODUCTION AND OBJECTIVES: Non-muscle invasive bladder cancer is often treated by intravesical chemotherapy following transurethral resection. Mechanism of the effect of intravesical chemotherapy has yet to be elucidated. In this study, we investigated cell death mechanism induced by high-dose chemotherapeutics using a newly-developed culture method for primary cancer cells. METHODS: We have developed a novel culture method which enables preparation and culture of primary cancer cells as multicellular spheroids which are termed cancer tissue-originated spheroids (CTOSs) (Okuyama 2013 J Urol). First we generated a bladder cancer patient-derived xenograft. We prepared CTOSs from the xenograft, and exposed to high-dose (1mg/ml) of epirubicin (e-ADM) or mitomycin C (MMC) for 2 hours, or low-dose (0.01mg/ml) of those chemotherapeutics. We investigated absorption of chemotherapeutics into CTOSs and assessed the mode of cell death. Next, we prepared CTOSs directly from surgical specimens of bladder cancer and tested the individual responses after exposure to high-dose e-ADM and MMC. RESULTS: At high dose, e-ADM was promptly and homogenously delivered into cancer cells in the CTOSs. High-dose e-ADM and MMC decreased ATP levels in CTOSs within an hour accompanied with mitochondrial swelling and reduction of mitochondrial membrane potential, while plasma membrane integrity was maintained. Some of the molecules in mitochondrial apoptotic pathway were subsequently activated, while at later time point the cells lost integrity of cell membrane without DNA laddering. In contrast, CTOSs exposed to low-dose of eADM or MMC exhibited typical DNA laddering. Intriguingly, the decrease of ATP levels was varied among CTOSs derived from 20 surgical specimens. CONCLUSIONS: High-dose chemotherapeutics used in intravesical therapy may induce necrosis-like cell death, different from typical apoptosis caused by chemotherapeutics at the dose of systemic chemotherapy. Each bladder cancer may have a different response to high-dose chemotherapeutics used in intravesical therapy, indicating necessity of chemosensitivity test aiming for personalized medicine.

MP21-07 HIGH-DOSE CHEMOTHERAPEUTICS USED IN INTRAVESICAL THERAPY INDUCE MITOCHONDRIAL DYSFUNCTION FOLLOWED BY NECROSIS-LIKE CELL DEATH IN BLADDER CANCER CELLS

INTRODUCTION AND OBJECTIVES: Non-muscle invasive bladder cancer is often treated by intravesical chemotherapy following transurethral resection. Mechanism of the effect of intravesical chemotherapy has yet to be elucidated. In this study, we investigated cell death mechanism induced by high-dose chemotherapeutics using a newly-developed culture method for primary cancer cells. METHODS: We have developed a novel culture method which enables preparation and culture of primary cancer cells as multicellular spheroids which are termed cancer tissue-originated spheroids (CTOSs) (Okuyama 2013 J Urol). First we generated a bladder cancer patient-derived xenograft. We prepared CTOSs from the xenograft, and exposed to high-dose (1mg/ml) of epirubicin (e-ADM) or mitomycin C (MMC) for 2 hours, or low-dose (0.01mg/ml) of those chemotherapeutics. We investigated absorption of chemotherapeutics into CTOSs and assessed the mode of cell death. Next, we prepared CTOSs directly from surgical specimens of bladder cancer and tested the individual responses after exposure to high-dose e-ADM and MMC. RESULTS: At high dose, e-ADM was promptly and homogenously delivered into cancer cells in the CTOSs. High-dose e-ADM and MMC decreased ATP levels in CTOSs within an hour accompanied with mitochondrial swelling and reduction of mitochondrial membrane potential, while plasma membrane integrity was maintained. Some of the molecules in mitochondrial apoptotic pathway were subsequently activated, while at later time point the cells lost integrity of cell membrane without DNA laddering. In contrast, CTOSs exposed to low-dose of eADM or MMC exhibited typical DNA laddering. Intriguingly, the decrease of ATP levels was varied among CTOSs derived from 20 surgical specimens. CONCLUSIONS: High-dose chemotherapeutics used in intravesical therapy may induce necrosis-like cell death, different from typical apoptosis caused by chemotherapeutics at the dose of systemic chemotherapy. Each bladder cancer may have a different response to high-dose chemotherapeutics used in intravesical therapy, indicating necessity of chemosensitivity test aiming for personalized medicine.

High antitumor activity of pladienolide B and its derivative in gastric cancer

The antitumor activity of pladienolide B, a novel splicing inhibitor, against gastric cancer is totally unknown and no predictive biomarker of pladienolide B efficacy has been reported. We investigated the antitumor activity of pladienolide B and its derivative on gastric cancer cell lines and primary cultured cancer cells from carcinomatous ascites of gastric cancer patients. The effect of pladienolide B and its derivative on six gastric cancer cell lines was investigated using a MTT assay and the mean IC50 values determined to be 1.6 ± 1.2 (range, 0.6–4.0) and 1.2 ± 1.1 (range, 0.4–3.4) nM, respectively, suggesting strong antitumor activity against gastric cancer. The mean IC50 value of pladienolide B derivative against primary cultured cells from 12 gastric cancer patients was 4.9 ± 4.7 nM, indicative of high antitumor activity. When 18 SCID mice xenografted with primary cultured cells from three patients were administered the pladienolide B derivative intraperitoneally, all tumors completely disappeared within 2 weeks after treatment. Histological examination revealed a pathological complete response for all tumors. In the xenograft tumors after treatment with pladienolide B derivative, immature mRNA were detected and apoptotic cells were observed. When the expressions of cell-cycle proteins p16 and cyclin E in biopsied gastric cancer specimens were examined using immunohisctochemistry, positivities for p16 and cyclin E were significantly and marginally higher, respectively, in the low-IC50 group compared with the high-IC50 group, suggesting the possibility that they might be useful as predictive biomarkers for pladienolide B. In conclusion, pladienolide B was very active against gastric cancer via a mechanism involving splicing impairment and apoptosis induction.

Metformin, at Concentrations Corresponding to the Treatment of Diabetes, Potentiates the Cytotoxic Effects of Carboplatin in Cultures of Ovarian Cancer Cells

The use of the type 2 diabetics drug metformin has been correlated with enhanced progression-free survival in ovarian cancer. The literature has speculated that this enhancement is due to the high concentration of metformin directly causing cancer cell death. However, this explanation does not fit with clinical data reporting that the women exposed to constant micromolar concentrations of metformin, as present in the treatment of diabetes, respond better to chemotherapy. Herein, our aim was to examine whether micromolar concentrations of metformin alone could bring about cancer cell death and whether micromolar metformin could increase the cytotoxic effect of commonly used chemotherapies in A2780 and SKOV3 cell lines and primary cultured cancer cells isolated from the peritoneal fluid of patients with advanced ovarian cancer. Our results in cell lines demonstrate that no significant loss of viability or change in cell cycle was observed with micromolar metformin alone; however, we observed cytotoxicity with micromolar metformin in combination with chemotherapy at concentrations where the chemotherapy alone produced no loss in viability. We demonstrate that previous exposure and maintenance of metformin in conjunction with carboplatin produces a synergistic enhancement in cytotoxicity of A2780 and SKOV3 cells (55% and 43%, respectively). Furthermore, in 5 (44%) of the 11 ovarian cancer primary cultures, micromolar metformin improved the cytotoxic response to carboplatin but not paclitaxel or doxorubicin. In conclusion, we present data that support the need for a clinical study to evaluate the adjuvant maintenance or prescription of currently approved doses of metformin during the chemotherapeutic treatment of ovarian cancer.

Abstract 2787: PrimePanel provides a high throughput in vitro drug screening platform that intimately links to in vivo pharmacological analysis in PDX models.

Abstract Patient derived xenografts (PDX) are in vivo animal tumor models established directly from patient tumor samples without any in vitro manipulation. It has been shown that these tumor xenografts maintain essential histopathological features and genetic profiles of the original tumors, and thus are the most clinically relevant animal models for cancer drug discovery. Comparing to the traditional cell line derived xenograft (CDX) tumor models, however, one challenge encountered by the PDX in vivo studies is the lack of corresponding in vitro cell culture system for cost-effective and high throughput drug screening and model selection. Here we set out to establish such an in vitro platform, so-called PrimePanelTM, by deriving homogeneous primary cancer cell cultures from the PDX tumors in several major tumor types. These cells are early passage cultures (usually p&amp;lt;10), which maintain similar cellular characteristics as the corresponding tumor xenografts, including the genomic mutational status, biochemical signaling, and responses to tumor cell autonomously targeted therapeutics. Thus, PrimePanelTM offers a higher throughput, faster turnaround, more versatile and lower cost platform to assess drug efficacy prior to in vivo study, which could fulfill a critical need in early stage drug discovery. Citation Format: Yanxia Zhang, Yuqiang Ge, Yanan Liu, Jing Zhao, Lanhua Li, Meng Qiao, Jinying Ning, Jiping Zha, Guizhong Liu. PrimePanel provides a high throughput in vitro drug screening platform that intimately links to in vivo pharmacological analysis in PDX models. [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 2787. doi:10.1158/1538-7445.AM2013-2787 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.