Slow Growing Cell Lines Research Articles

Abstract 2168: A growth rate corrected metric for cytotoxic co-culture systems

Abstract Tumor/T-cell co-cultures in combination with large-scale genome-editing tools emerged as powerful tools for discovery of drivers of resistance to immunotherapies. Traditionally, chromium or lactate-dehydrogenase release and/or target cell viability counts before and after co-culture treatment are used as surrogate endpoint assays to estimate the selection pressure applied. These assays cannot dissect growth arrest, cell death, cell viability and target cell outgrowth, all of which occur as a consequence of released cytokines and direct tumor/T-cell interactions, hence, significantly impact the interpretation of small- and large-scale screens in co-culture models. These biases are particularly relevant when comparing cell lines or genotypes with different growth kinetics. Here, we use a dual reporter system and adopt pharmaco-mathematical principles to dissect these biases to create a growth-rate corrected co-culture metric (GRC), which normalizes T-cell mediated antiproliferative and cytotoxic effects in tumor target cells. We illustrate the value of this metric by comparing fast and slow-growing tumor cells in autologous patient-derived melanoma/T-cell co-culture pairs. At tumor-T-cell ratios resulting in a half maximal relative reduction of target cell counts compared to controls, the fast-proliferating cell line had not changed in absolute cell counts since baseline while the slow-growing cell line showed a 50% reduction. This indicates that in one instance (fast growing cell line) growth-retardation occurred due to a balance of immune editing and target cell proliferation, while in the other (slow-growing cell line) T-cell mediated killing resulted in net loss of tumor cells. Using GRC, these differences can be readily distinguished, and comparable selection conditions can be selected. This has important implications for large-scale screens with perturbations within cancer cells: drop-outs from a screen in the fast-growing cell line would not only contain perturbations that increase specific sensitivity to T-cell mediated cytotoxicity, but also perturbations causing a general loss in proliferation/survival fitness. In addition, some of the discrepancy in recently published screens may be explained by differences in models with varying proliferation rates and therefore selection conditions. In summary, the GRC metric enhances the interpretation of co-culture experiments and could contribute to improved selection of targets from large-scale screens investigating resistance to T-cell mediated toxicity for drug development. Citation Format: Johannes Christian Melms, Clarence Yapp, Caitlin Elizabeth Mills, Peter Sorger, Benjamin Izar. A growth rate corrected metric for cytotoxic co-culture systems [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2168.

Immunosurveillance and immunoediting in MMTV-PyMT-induced mammary oncogenesis

ABSTRACTEvidence of cancer immunosurveillance and immunoediting processes has been primarily demonstrated in mouse models of chemically induced oncogenesis. Although these models are very tractable, they are characterized by high mutational loads that represent a minority of human cancers. In this study, we sought to determine whether cancer immunosurveillance and immunoediting could be demonstrated in a more clinically relevant oncogene-induced model of carcinogenesis, the MMTV-PyMT (PyMT) mammary carcinoma model. This model system in the FVB/NJ strain background was previously used to demonstrate that adaptive immunity had no role in limiting primary cancer formation and in fact promoted metastasis, thus calling into question whether cancer immunosurveillance operated in preventing the development of breast cancer. Our current study in the C57BL/6 strain backgrounds provides a different conclusion, as we report here the existence of an adaptive immunosurveillance of PyMT mammary carcinomas using two independent models of immune deficiency. PyMT mice bred onto a Rag1−/− background or immune suppressed by chronic tacrolimus therapy both demonstrated accelerated development of mammary carcinomas. By generating a bank of cell lines from these animals, we further show that a subset of PyMT cell lines had delayed growth after transplantation into wild-type (WT) syngeneic, but not immune-deficient hosts. This reduced growth rate in immunocompetent animals was characterized by an increase in immune cell infiltration and tissue differentiation. Furthermore, loss of the immune cell infiltration that characterized immunoediting of slow growing cell lines, changed them into fast growing variants capable of progressing in the immunocompetent model. In conclusion, our study provides evidence that immunosurveillance and immunoediting of PyMT-derived cell lines modulate tumor progression in this oncogene-induced model of cancer.

Effects of Abiotic Factors on Cell Biomass and Rosmarinic Acid Production in Cell Suspension Cultures of Orthosiphon stamineus Benth

Orthosiphon stamineus Benth. is a medicinal plant belongs to Lamiaceae family. Due to the high variations in rosmarinic acid produced from the field-derived plants, cell suspension culture techniques was previously suggested and practiced as the alternative for cell biomass and rosmarinic acid (RA) production. In the present study, the effects of medium pH, photoperiod, temperature and sucrose were evaluated on cell biomass and rosmarinic acid production using three different cell lines of O. stamineus. This study revealed that the slow and intermediate growing cell lines had similarities in terms of growth pattern. However, cells cultured in medium supplemented with lower sucrose concentration (30 g/L) were more productive in terms of cell biomass and RA in the intermediate and fast growing cell lines however slow growing cells cultured in proliferation medium with 45 g/l sucrose produced higher amount of RA. Medium pH in the range of 5.65-5.8 was optimal for both cell biomass and RA production. Photoperiod did not have a consistent effect on the three cell line’s biomass production but the alternation of darkness and illumination led to high RA production in the slow growing cell line. This study proved that abiotic factors including medium pH, photoperiod, temperature and carbohydrate source had impacts on cell biomass and rosmarinic acid production although in a diverse range among different growing cell lines

Inhibiting Translation as a Novel Strategy to Target Multiple Myeloma

AbstractAbstract 2995 Background:Multiple myeloma (MM) cell survival is dependent on the ability to produce and secrete immunoglobulins, requiring precise coordination of protein synthesis and degradation. Inhibition of these processes may be responsible for the exquisite sensitivity of MM to drugs such as bortezomib and histone deacetylase inhibitors, which respectively block proteosomal and aggresomal-mediated protein degradation. We hypothesized that MM cells would also be sensitive to inhibition of mRNA translation and have tested silvestrol, a plant-derived natural product that has previously been shown to inhibit ribosome recruitment and to be cytotoxic to B lymphocytes. Mehods:Silvestrol was resuspended in DMSO and diluted for use in RPMI medium. A panel of human MM cell lines, representing a range of chromosomal abnormalities observed in this disease, were cultured in RPMI with 10% FCS and appropriate supplementation. Primary mouse embryonic fibroblasts (MEFs) were prepared and passaged 7–10 times until senescence was observed by viability studies. Cytotoxicity was determined using MTT viability assay and apoptosis by flow cytometry using annexin V-PE, CD138-PECy5 conjugated antibodies and 7-AAD. Primary patient samples obtained by bone marrow aspiration were prepared by ficoll isolation and peripheral blood mononuclear cells were cultured in IMDM with 20% FCS with or without silvestrol followed by flow cytometric analysis for apoptosis. Polysome profiling and immunoblotting were performed on extracts of cells treated with silvestrol at various time points in order to determine a mechanism of action as well as to observe cell signaling pathways. Results:All MM cell lines tested with silvestrol showed profound inhibition of cell growth and exhibited cell death within 48h, with an average IC50 of 10nM. Furthermore, the slow growing cell line, MM1.S and its dexamethasone resistance counterpart, MM1.R, were equally sensitive to silvestrol and at the same IC50 range as more rapidly growing lines. Apoptotis was confirmed by AnnexinV flow cytometric assay in MM cell lines following exposure to 10–20nM silvestrol for 24h. As MM is not usually characterized by a high proliferative index, we tested silvestrol in non-dividing, senescent MEFs and observed cytotoxicity and apoptosis at similar concentrations. Silvestrol was observed to be cytotoxic to the CD138 fraction of primary MM patient bone marrow samples at 10 and 50nM concentrations after 24 and 48h of incubation. Silvestrol was shown to inhibit translation in MM cell lines as evidenced by inhibition of ribosome binding and a decrease in polysome content with a concomitant increase in 80S ribosomes. Western blot analyses suggest that silvestrol significantly decreases NFKB activity in cell lines that constitutively process NFKB2. MCL1, a BCL2 family inhibitor of apoptosis, is also rapidly downregulated in silvestrol treated MM cells, suggesting a possible mechanism for silvestrol-induced apoptosis. In vivo, we have determined that no toxicity was evident at daily 0.2mg/kg injections in the transgenic and immunocompetent vk*MYC mouse model of MM that has shown biologic and therapeutic fidelity to human MM. Silvestrol has already shown tumoricidal activity in xenographic models of other malignancies and its efficacy in vk*MYC MM mice is currently being explored. Conclusions:These preclinical studies suggest that, because of its unique biology, MM is particularily susceptible to the cytotoxic effects of translation inhibition by silvestrol. These observations warrant clinical evaluation of silvestrol in MM. Disclosures:No relevant conflicts of interest to declare.

Involvement of arabinogalactan proteins in the control of cell proliferation of Cucurbita pepo suspension cultures

Arabinogalactan proteins (AGPs) secreted by zucchini squash (Cucurbita pepo L.) cell cultures into the medium are implicated in cell proliferation. Conditioned medium derived from cell suspensions of squash cultivar Dundoo could enhance multiplication rate of slow-growing cell line Cx3005. To examine the role of AGPs, a precipitation assay was performed using Yariv reagent which binds selectively to AGPs. This AGP precipitation as well as proteinase application arrested cell division. However, chitinase treatment successfully increased embryogenic callus mass. A growth promotion was also obtained by arabinogalactan addition to the culture medium. Immunoblotting analysis using the MAC 207 anti-AGP monoclonal antibody showed high AGP expression in Dundoo cell cultures.

The human epithelial cell cytotoxicity assay for determining tissue specific toxicity: method modifications.

The Human Epithelial Cell Cytotoxicity (HECC) Assay (Meth Cell Sci, 22: 17-24, 2000) has been modified to include three additional cell lines and to allow protocol adjustments for slow growing cell lines. This manuscript presents methods using human epithelial cells from ten different normal human tissues including: skin, mammary, prostate, renal, bronchial, lung, oral, ecto-cervix, colon, and liver. The HECC Assay can also be used to evaluate other types of drugs, personal care products, environmental chemicals, and potential toxicants. Human epithelial cells at an early passage are seeded into multi-well dishes. The cells are exposed to multiple concentrations of each test agent. A preliminary assay using an exposure of five days at 1 mM (if soluble) and four log dilutions is used to determine the highest concentration for the HECC Assay. In the HECC Assay, cultures are exposed for three to four days. Following the exposure period, endpoint measurements for inhibition of growth, mitochondrial function, and PCNA (proliferating cell nuclear antigen) expression or albumin synthesis (hepatocytes) are made. Data are analyzed to determine the concentration that inhibited an endpoint by 50 percent (TC(50)) for each agent in each target epithelial cell line or culture and the data are compared to determine the relative sensitivity of each epithelial cell line to the test agent.

Pharmacokinetics of the thymidine analog 2′-Fluoro-5-[ 14C]-methyl-1-β-D-arabinofuranosyluracil ([ 14C]FMAU) in rat prostate tumor cells

2′-Fluoro-5-[ 14C]-methyl-1-β-D-arabinofuranosyluracil (FMAU) is an analog of thymidine (TdR) that is resistant to catabolism, is incorporated into DNA, and has been labeled with 11C for use with positron emission tomography. We compared the uptake and metabolism of [ 14C]FMAU with that of [ 3H]TdR in fast- and slow-growing cell lines of a rat prostate tumor. Although FMAU was incorporated much less rapidly than TdR, FMAU behaved very similarly to TdR with respect to correlation between uptake velocity and cell growth rate, saturability of cellular incorporation, and intracellular metabolite pools. Thus, FMAU warrants further evaluation as an in vivo indicator of tumor cell division.

Comparison of mathematical formulas used for estimation of DNA synthesis time of bromodeoxyuridine-labelled cell populations with different proliferative characteristics.

Growth kinetic data of human tumours, obtained by flow cytometric analysis of cells labelled with bromodeoxyuridine (BrdUrd) might provide prognostic information and allow prediction of response to radio- and chemotherapy. However, the theoretical models applied for calculation of growth kinetic data are not fully evaluated. The purpose of this study was to investigate the dependence of the estimation of DNA synthesis time (Ts) on sampling time after BrdUrd labelling, using four different mathematical formulas (Begg et al., White & Meistrich, White et al. and Johansson et al.) which have been developed for the evaluation of flow cytometry-derived data of BrdUrd-labelled cells. In addition, we have investigated the influence of the growth kinetic properties of the cell populations using two cultured cell lines (one slow and one fast growing), and two hetero-transplanted human tumours. The dependence of the estimation of Ts on sampling time was more or less pronounced, depending on the cell population examined and on the formula used. In the fast growing cell line, the estimates of Ts did not vary significantly with sampling time when using the formulas by White et al., whereas in the slow growing cell line, the estimates of Ts did not show any significant dependence on sampling time when using the formula by Johansson et al. In the tumours, the estimation of Ts depended on sampling time with all formulas used, although to different degrees. In one of the tumours, this was mainly caused by the influence of mouse cells, as we demonstrate. Our results indicate that the proliferative characteristics of a cell population should be taken into consideration when choosing a mathematical formula in order to attain Ts values that are independent of sampling time.

Relation between Nucleolar Size and Growth Characteristics in Small Cell Lung Cancer Cell Lines

Relationships among cytological features, doubling time, S‐phase percentage, expression of myc‐family oncogenes, DNA ploidy and biochemical properties were studied in thirteen small cell lung cancer cell lines. Six cell lines that grew slowly (average doubling time 99 h) and had lower S‐phase percentages (average 32%) showed inconspicuous nncleoli (average area of 1.5 μm2), and the remaining seven cell lines that grew quickly (average doubling time 45 h) and had higher S‐phase percentages (average 44%) showed large and prominent nncleoli (average area of 6.1 μm2). DNA index value obtained from flow cytometric DNA histograms showed that all cell lines except for H‐69 cell line displayed aneuploidy. Ribbon‐like cell arrangements were observed in the 7 cell lines that grew quickly, and in 1 cell line that grew slowly. Biochemically, six slow‐growing cell lines and four fast‐growing cell lines showed high levels of aromatic L‐amino acid decarboxylase activity, while in the remaining three fast‐growing cell lines its level was low. A high level of c‐myc or N‐myc oncogene expression was observed in all 7 cell lines that grew quickly, but not in any of the 6 cell lines that grew slowly. It appears that small cell lung cancer cell lines that grow quickly can be expected to have large nucleoli and ribbon‐like cell arrangements and to express high levels of mycfamily oncogenes, and that nucleolar size is a good indicator for growth characteristics.

Markers and characteristics of human SCLC cell lines

Permanent human small cell lung cancer (SCLC) cell lines established in our laboratory were investigated for their expression of the enzymatic neuroendocrine markers L-DOPA decarboxylase (DDC), neuron-specific enolase (NSE), and creatine kinase (CK), including its BB isoenzyme (CK-BB), the classical tumor markers carcinoembryonic antigen (CEA), the alpha and beta subunits of human chorionic gonadotropin (alpha-HCG, beta-HCG), and alpha-fetoprotein (alpha-FP), and their chromosomal characteristics. DDC activities were detectable in 5/6 SCLC cell lines and absent in non-SCLC. NSE levels ranged from 160 to 1422 ng/mg soluble protein and were less than 290 ng/mg soluble protein in non-SCLC. Activities of CK and levels of CK-BB clearly distinguished SCLC from non-SCLC with CK activities greater than 1000 munits/mg soluble protein and CK-BB levels greater than 3000 ng/mg soluble protein in SCLC and less than 300 munits/mg soluble protein and less than 2000 ng/mg soluble protein in non-SCLC. CEA was detectable in 5/6 SCLC cell lines but absent in non-SCLC, and its level seemed to correlate with those of DDC, NSE, and CK. One cell line, SCLC-16H, lost some of its neuroendocrine properties and CEA after 1 year of in vitro cultivation. Generally, marker levels were low in fast growing cell lines and high in slow growing cell lines. HCG alpha and beta subunit and alpha-FP were not detectable in SCLC cell lines. All SCLC cell lines examined had near diploid DNA indices and modal chromosome numbers. Double minute chromosomes and homogeneously staining regions were found in 2/5 and 4/5 SCLC cell lines respectively. With respect to chromosomal aberrations, we found a deletion of the short arm of at least one chromosome 3 in all SCLC cell lines (5/5). These data show that SCLC expresses neuroendocrine markers and CEA; CK is the most sensitive marker, and DDC and CEA are the most specific markers for SCLC in vitro; individual marker levels correlate with each other and the in vitro malignancy of SCLC; and SCLC cell lines have relatively uniform chromosomal characteristics. Our results suggest that patients whose tumors have high levels of DDC, NSE, CK-BB, and CEA have a better prognosis than those with low marker levels. This hypothesis could be proved by comparing pairs of patients that are matched for all known prognostic parameters, in particular tumor spread, for their serum and tumor marker levels with respect to the patients' outcome and prognosis.