Capillary cloning of primary human tumor cells: assay miniaturization for drug efficacy testing

The conventional double-layer agar method of cloning human tumor cells requires a substantial number of viable tumor cells and 14-21 days of culture. These prerequisites frequently limit its utility as an assay. In an attempt to circumvent these limitations and to reduce the amount of drug that is needed in the assay, we have further developed and miniaturized the assay in which human tumor cells are cloned in glass microcapillary tubes. Cultures consisted of 50 microliters containing 15,000 nucleated cells in 975 mm capillary tubes which were incubated for seven days. The results from 50 consecutive tumor biopsies resulted in cloning efficiencies, ranging from 0.007% to 1.0% with an overall successful cloning of 88% of all tumors tested and a good linear growth relationship and chemotherapy sensitivity. This miniaturized assay offers distinct advantages for drug efficacy testing including high cloning efficiencies, small tumor sample and drug requirements, quicker assay turnaround time and a general conservancy of reagents and incubator space.     

Methods and goals for the use of in vitro and in vivo chemosensitivity testing

Sensitive, specific, and accurate methods to assay chemosensitivity are needed to (1) screen new therapeutic agents, (2) identify patterns of chemosensitivity for different tumor types, (3) establish patterns of cross-resistance and sensitivity in treatment of naïve and relapsing tumors, (4) identify genomic and proteomic profiles associated with sensivity, (5) correlate in vitro response with preclinical in vivo effects and clinical outcomes for a particular therapeutic agent, and (6) tailor chemotherapy regimens to individual patients. Various methods are available to achieve these end points, including several in vitro clonogenic and proliferation assays, cell metabolic activity assays, molecular assay to monitor expression of markers for responsiveness, drug resistance, and for induction of apoptosis, in vivo tumor growth and survival assays in metastatic and orthotopic models, and in vivo imaging assays. The advantages and disadvantages of the specific assays are discussed. A summary of research questions related to chemosensitivity testing is also included.     

Dynamic BH3 profiling identifies active BH3 mimetic combinations in non-small cell lung cancer

Conventional chemotherapy is still of great utility in oncology and rationally constructing combinations with it remains a top priority. Drug-induced mitochondrial apoptotic priming, measured by dynamic BH3 profiling (DBP), has been shown in multiple cancers to identify drugs that promote apoptosis in vivo. We therefore hypothesized that we could use DBP to identify drugs that would render cancers more sensitive to conventional chemotherapy. We found that targeted agents that increased priming of non-small cell lung cancer (NSCLC) tumor cells resulted in increased sensitivity to chemotherapy in vitro. To assess whether targeted agents that increase priming might enhance the efficacy of cytotoxic agents in vivo as well, we carried out an efficacy study in a PC9 xenograft mouse model. The BH3 mimetic navitoclax, which antagonizes BCL-xL, BCL-w, and BCL-2, consistently primed NSCLC tumors in vitro and in vivo. The BH3 mimetic venetoclax, which electively antagonizes BCL-2, did not. Combining navitoclax with etoposide significantly reduced tumor burden compared to either single agent, while adding venetoclax to etoposide had no effect on tumor burden. Next, we assessed priming of primary patient NSCLC tumor cells on drugs from a clinically relevant oncology combination screen (CROCS). Results confirmed for the first time the utility of BCL-xL inhibition by navitoclax in priming primary NSCLC tumor cells and identified combinations that primed further. This is a demonstration of the principle that DBP can be used as a functional precision medicine tool to rationally construct combination drug regimens that include BH3 mimetics in solid tumors like NSCLC.Subject terms: Non-small-cell lung cancer, Apoptosis, Predictive markers     

The human tumor stem cell assay revisited

The human tumor stem cell assay (HTSCA) is a bilayer soft agar system for growing fresh human tumor specimens in vitro to determine drug sensitivity and improve our understanding of tumor biology. Recent clinical correlations of 60% accuracy for predicting a positive clinical response and a 90% accuracy for predicting a lack of response to therapeutic agents suggest promising clinical usefulness. However, the clinician should be aware of the assay's inherent pitfalls, such as heterogeneity of the tumor specimen, inability to obtain pure single-cell suspensions, low cloning efficiency, unusual drug dose-dependent survival curves, uncertain validity of in vitro pharmacology, non-standardized criteria for in vitro sensitivity, and the variability of in vitro results. A brief summary of the concepts, potential, and limitations of this assay are discussed.     

Detection of substrates of keratinocyte transglutaminase in vitro and in vivo using a monoclonal antibody to dansylcadaverine.

A method providing more sensitive detection of transglutaminase substrates was developed to localize transglutaminase activity in tissue and to identify in vivo substrates in epidermal extracts. The enhanced sensitivity of this method was achieved via the generation of a monoclonal antibody (designated E7) made to dansylcadaverine. Transglutaminase substrates were visualized by western blot after a 1-min incubation with dansylcadaverine in contrast to the 2 h required when [14C]putrescine incorporation was measured by autoradiography of SDS-polyacrylamide gels. In addition, putative substrates not apparent using conventional methods were readily detected by western analysis. An ELISA assay to measure transglutaminase activity showed similar sensitivity to the traditional radiometric assay (Lorand et al., 1972). The correlation between the ELISA procedure and the radiometric assay was high (r2 = 0.924). Strips of neonatal human and mouse skin incubated in dansylcadaverine-supplemented culture medium were used to localize enzyme activity and to detect substrates in vivo. Transglutaminase activity was demonstrated at the cellular periphery in the upper spinous and granular cell layers of the epidermis. Substrates detected in epidermal extracts were similar to those detected using the in vitro assay. This technique allows for highly sensitive and nonradiometric analysis of both enzymatic activity and the substrates involved. The extension of this methodology to an in vivo system is the first demonstration of a system in which the dynamics of cornified envelope assembly may be further studied.     

Correlation of intralesional in vivo chemotherapy of line 10 hepatoma with in vitro drug sensitivity

The effects of intralesional chemotherapy with 7 different drugs on line 10 hepatoma grown in strain 2 guinea pigs were compared with the sensitivity of line 10 tumor cells in vitro, using a micro modification of the tumor stem assay with capillary tubes. A modified method was used to evaluate the in vitro dose-response curves. The correlation for in vivo/in vitro resistance was found to be 100% and for in vivo/in sensitivity it was 80%.     

Monitoring of tumor response to chemotherapy in vivo by a novel small-molecule detector of apoptosis

Utilization of molecular imaging of apoptosis for clinical monitoring of tumor response to anti-cancer treatments in vivo is highly desirable. To address this need, we now present ML-9 (butyl-2-methyl-malonic acid; MW = 173), a rationally designed small-molecule detector of apoptosis, based on a novel alkyl-malonate motif. In proof-of-concept studies, induction of apoptosis in tumor cells by various triggers both in vitro and in vivo was associated with marked uptake of ³H-ML-9 administered in vivo, in correlation with the apoptotic hallmarks of DNA fragmentation, caspase-3 activation and membrane phospholipid scrambling, and with correlative tumor regression. ML-9 uptake following chemotherapy was tumor-specific, with rapid clearance of the tracer from the blood and other non-target organs. Excess of non-labeled “cold” compound competitively blocked ML-9 tumor uptake, thus demonstrating the specificity of ML-9 binding. ML-9 may therefore serve as a platform for a novel class of small-molecule imaging agents for apoptosis, useful for assessment of tumor responsiveness to treatment. H. Grimberg, G. Levin and A. Shirvan contributed equally to this article.     

Tumor-cell resistance to death receptor--induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax

The importance of Bax for induction of tumor apoptosis through death receptors remains unclear. Here we show that Bax can be essential for death receptor--mediated apoptosis in cancer cells. Bax-deficient human colon carcinoma cells were resistant to death-receptor ligands, whereas Bax-expressing sister clones were sensitive. Bax was dispensable for apical death-receptor signaling events including caspase-8 activation, but crucial for mitochondrial changes and downstream caspase activation. Treatment of colon tumor cells deficient in DNA mismatch repair with the death-receptor ligand apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selected in vitro or in vivo for refractory subclones with Bax frameshift mutations including deletions at a novel site. Chemotherapeutic agents upregulated expression of the Apo2L/TRAIL receptor DR5 and the Bax homolog Bak in Baxminus sign/minus sign cells, and restored Apo2L/TRAIL sensitivity in vitro and in vivo. Thus, Bax mutation in mismatch repair--deficient tumors can cause resistance to death receptor--targeted therapy, but pre-exposure to chemotherapy rescues tumor sensitivity.     

Systemic Activation of Macrophages by Liposomes Containing Muramyltripeptide Phosphatidylethanolamine for therapy of Cancer Metastasis

Abstract

The heterogeneous response of metastases to conventional therapy is a major cause of failure in cancer treatment. Evidence that activated macrophages can recognize and destroy neoplastic cells in vitro without regard to their phenotypic diversity has stimulated efforts to develop effective approaches to the activation of macrophages in situ. Systemic administration of liposomes containing immunomodulators activates macrophages in situ and augments host destruction of spontaneous metastases.

Liposomes are a useful carrier system for the transport of agents to phagocytic cells in vivo. Once in the circulation, liposomes are cleared by phagocytic cells, and this passive localization provides an effective mechanism for targeting liposome-entrapped materials, such as muramyltripeptide phosphatidylethanolamine (MTP-PE), to macrophages.

Macrophage destruction of metastases in vivo is significant, provided that the total tumor burden at start of treatment is minimal. For this reason, we advocate using chemotherapy or radiotherapy first to reduce the tumor burden in patients with metastases. Tumoricidal macrophages that can differentiate neoplastic from bystander nonneoplastic cells are then used to destroy the few tumor cells that escape destruction by conventional therapeutic methods.     

Development of an in vitro colony formation assay for the evaluation of in vivo chemotherapy of a rat brain tumor.

An in vitro colony formation assay for the evaluation of in vivo brain tumor therapy has been developed. When plated, disaggregated cells derived from solid tumors proliferated to form relatively homogeneous colonies after a latency period of 2 to 6 days. Increasing concentrations of fetal calf serum enhanced colony-forming efficiency (CFE) with a plateau between 7 and 16%. Supplementation with either irradiated feeder cells (10(3) to 10(5) cells per dish), or medium conditioned by 1 to 3 days of in vitro incubation with the same cell line, doubled the CFE. The density of tumor cells (untreated or previously treated with chemotherapeutic agents) did not affect the CFE when a minimum of 10(4) total cells (tumor plus feeder) were plated. Therefore, in this system the optimal experimental conditions for evaluating chemotherapy and radiotherapy require incubation of disaggregated tumor cells for 12 days in medium containing 10% of fetal calf serum and enough feeder cells to provide a minimum of 10(4) cells per dish. The CFE for untreated tumors was 18 +/- 10% (+/-S.D.), demonstrating that there is significant biological variation. The assay appeared sensitive, with reproducible results, when applied to individual chemically treated tumors. An estimate of the percentage of clonogenic cells affected by in vivo chemotherapy may be obtained by comparing the CFE of cells from treated and untreated tumors. This assay can measure up to a 5 log(10) cell kill, and it should prove to be valuable in developing more effective regimens for the treatment of solid tumors in animals and man.     

Cholinergic responsiveness of respiratory and vascular tissues in two different rat strains

The airway and systemic arterial smooth muscle responsiveness to cholinergic agents of two strains of rats, Rat Albino (RA) and Brown Norway (BN), was compared in vivo and in vitro. In vivo, we measured the doses of carbachol that induced a 100% increase in lung resistance (PD100 RL), a 50% decrease in dynamic lung compliance (PD50 Cdyn), and the value of systolic blood pressure at the carbachol dose of 10 micrograms (Pa 10 micrograms). In vitro airway smooth muscle and systemic arterial smooth muscle responsiveness was assessed by measuring the maximal response to acetylcholine, the slope of the linear portion of the dose-response curve, and the negative logarithm of the molar concentration of acetylcholine producing 50% of the maximal response (pD2). PD100 and PD50 were about four times greater in BN rats than in RA rats. In contrast, Pa 10 micrograms was 1.5 lower in the BN rats. These differences persisted after bivagotomy. Tracheal pD2 was 25% greater in the RA than in the BN strain. The mean dose-response curve of parenchymal strips of RA rats was situated upward and to the left of the BN curve, but the reverse was observed for aortic smooth muscle dose-response curves. Thus 1) airway smooth muscle responsiveness to cholinergic agents is greater in RA strain than in BN, but the reverse is true for systemic arterial smooth muscle responsiveness; and 2) these differences are not due to factors extrinsic to the smooth muscle, since they occurred in vitro and may depend on different densities of muscarinic receptors.     

Deorphanizing vertebrate olfactory receptors: recent advances in odorant-response assays

Olfactory receptors (ORs) comprise the largest multigene G protein-coupled receptor families in organisms from fish to primates, and play a critical role in recognizing thousands of odorant molecules. Recent achievement of functional OR expression in heterologous cells led to identification of ligands for some ORs, revealing a combinatorial receptor coding scheme in the olfactory sensory system. Using the functional assay, the odorant-binding site in ORs has been elucidated, showing that a binding pocket constructed by transmembrane helices provides the molecular basis for agonist and antagonist specificity. To retrospectively identify ORs that recognize a particular odorant of interest, two functional cloning strategies have been developed: one is a strategy wherein OR genes are amplified from single olfactory neurons that show odorant responsiveness in Ca(2+) imaging, and another is an approach based on glomerular activity by combining in vivo bulbar Ca(2+) imaging and retrograde dye labeling of innervating olfactory neurons. The conventional ligand-screening approach and the functional cloning strategies in an odorant-directed manner have allowed us to match ORs to the cognate odorants both in vitro and in vivo.     

Relationship between in vitro tumor stem cell assay and in vivo antitumor activity using the P388 leukemia

The relationship between in vitro tumor stem cell sensitivity and in vivo antitumor efficacy using 13 active antineoplastic agents was examined by means of the intraperitoneal P388 mouse leukemia system. The doses, which produced a 50% increase in life span after one, five and nine days of treatment, were all significantly correlated with the in vitro concentration producing a 70% reduction in colony formation during a seven-day continuous drug exposure. The five-day correlation was the best, followed by nine days and one day. Correlations were not improved by corrections for either in vitro drug stability or toxicity (LD50). The highly significant correlations observed in this simple retrospective analysis provide a basis for the development of more sophisticated models for the prediction of in vivo results from in vitro data.     

Nutritional intervention with omega-3 fatty acids enhances tumor response to anti-neoplastic agents

Nutritional intervention with specific fatty acids depresses tumor growth and enhances tumor responsiveness to chemotherapy. Supplementation of tumors with long chained omega-3 polyunsaturated fatty acids results in enrichment of tumor phospholipid fractions with omega-3 fatty acids resulting in an altered membrane composition and function. Tumors enriched with long chained omega-3 polyunsaturated fatty acids possess membranes with increased fluidity, an elevated unsaturation index, enhanced transport capabilities that results in accumulation of selective anti-cancer agents, increased activity of selected drug activating enzymes, and alteration of signaling pathways important for cancer progression. These nutritionally induced changes in tumor fatty acid composition result in increased sensitivity to chemotherapy, especially in tumor lines that are resistant to chemotherapy and cause specific enhancement of cytotoxicity to tumor cells and protection of normal cells. Pre-disposing tumors to increased chemo-sensitivity through nutritional intervention with specific fatty acids has the potential to improve patient response to chemotherapy with fewer untoward side effects if these pre-clinical findings carry over into a clinical setting.     

Multi-gene expression predictors of single drug responses to adjuvant chemotherapy in ovarian carcinoma: predicting platinum resistance

Despite advances in radical surgery and chemotherapy delivery, ovarian cancer is the most lethal gynecologic malignancy. Standard therapy includes treatment with platinum-based combination chemotherapies yet there is no biomarker model to predict their responses to these agents. We here have developed and independently tested our multi-gene molecular predictors for forecasting patients' responses to individual drugs on a cohort of 55 ovarian cancer patients. To independently validate these molecular predictors, we performed microarray profiling on FFPE tumor samples of 55 ovarian cancer patients (UVA-55) treated with platinum-based adjuvant chemotherapy. Genome-wide chemosensitivity biomarkers were initially discovered from the in vitro drug activities and genomic expression data for carboplatin and paclitaxel, respectively. Multivariate predictors were trained with the cell line data and then evaluated with a historical patient cohort. For the UVA-55 cohort, the carboplatin, taxol, and combination predictors significantly stratified responder patients and non-responder patients (p = 0.019, 0.04, 0.014) with sensitivity = 91%, 96%, 93 and NPV = 57%, 67%, 67% in pathologic clinical response. The combination predictor also demonstrated a significant survival difference between predicted responders and non-responders with a median survival of 55.4 months vs. 32.1 months. Thus, COXEN single- and combination-drug predictors successfully stratified platinum resistance and taxane response in an independent cohort of ovarian cancer patients based on their FFPE tumor samples.     

Studies of the proliferation and differentiation of immature myeloid cells in vitro: 4: Preculture proto-oncogene expression and the behaviour of myeloid leukemia cells in vitro

Studies were conducted to determine the relationship between the pretherapy characteristics of leukemia cells and their behaviour during culture in vitro. Leukemia cells which proliferated well in vitro also proliferated well in vivo. Cells which manifested myeloid or monocytic differentiation in vivo tended to manifest differentiation along these lines in vitro. Cells which manifested high levels of expression of c-fms, c-fes, or triose phosphate isomerase prior to culture were likely to differentiate in vitro, with high levels of c-fes expression being related to myeloid maturation. These observations suggest that differentiation at the molecular level prior to culture is a requisite for leukemia cell differentiation in vitro. The same may be true for differentiation in vivo under the influence of exogenously administered agents such as cytotoxic chemotherapy or recombinant growth factors.     

Automated Radiometric Detection of Bacteria in 2,967 Blood Cultures

A new radiometric method for the automatic detection of bacterial growth in blood cultures has been compared with conventional methods. A total of 2,967 cultures from 1,280 patients suspected of having bacteremia were studied. A 2-ml amount of blood was inoculated into culture media in which the glucose was labeled with carbon-14. The release of (14)CO(2) by bacterial metabolism was checked hourly for 18 to 24 hr, daily for the next 2 days, and, on the 12th day, with an automated instrument. A 10-ml amount of blood was studied by conventional bacteriological techniques. In 125 cultures from 50 patients, there was bacterial growth in at least one of the routine media. Of these, the radiometric method detected 102 cultures from 40 patients. In 111 cultures from 48 patients, there was radiometric detection of bacterial growth. In all of these cultures, there was detection of bacterial growth in subcultures from the radioactive medium. Of these, the routine laboratory detected 98 cultures from 40 patients. Neither method detected all patients with bacteremia. Among the 57 patients positive by one or both methods, routine techniques detected bacteria in 87% and the radiometric method detected bacteria in 85%. Seventy per cent of the cultures were detected first by the radiometric method, 65% on the day of inoculation. Our results suggest that the radiometric method is faster than conventional techniques and comparable in accuracy. Its great advantage is that it is simple, automatic, and can be extended to automatic detection of bacterial sensitivity to antibiotics.     

Epigenetic Regulation of RIP3 Suppresses Necroptosis and Increases Resistance to Chemotherapy in NonSmall Cell Lung Cancer

INTRODUCTION: The efficacy of chemotherapeutic agents in killing cancer cells is mainly attributed to the induction of apoptosis. However, the tremendous efforts on enhancing apoptosis-related mechanisms have only moderately improved lung cancer chemotherapy, suggesting that other cell death mechanisms such as necroptosis could be involved. In this study, we investigated the role of the necroptosis pathway in the responsiveness of nonsmall cell lung cancer (NSCLC) to chemotherapy. METHODS: In vitro cell culture and in vivo xenograft tumor therapy models and clinical sample studies are combined in studying the role of necroptosis in chemotherapy and mechanism of necroptosis suppression involving RIP3 expression regulation. RESULTS: While chemotherapeutic drugs were able to induce necroptotic cell death, this pathway was suppressed in lung cancer cells at least partly through downregulation of RIP3 expression. Ectopic RIP3 expression significantly sensitized lung cancer cells to the cytotoxicity of anticancer drugs such as cisplatin, etoposide, vincristine, and adriamycin. In addition, RIP3 suppression was associated with RIP3 promoter methylation, and demethylation partly restored RIP3 expression and increased chemotherapeutic-induced necroptotic cell death. In a xenograft tumor therapy model, ectopic RIP3 expression significantly sensitized anticancer activity of cisplatin in vivo. Furthermore, lower RIP3 expression was associated with worse chemotherapy response in NSCLC patients. CONCLUSION: Our results indicate that the necroptosis pathway is suppressed in lung cancer through RIP3 promoter methylation, and reactivating this pathway should be exploited for improving lung cancer chemotherapy.     

The human tumor clonogenic assay as a model system in cell biology

The human tumor stem cell (clonogenic) assay (HTCA) is a soft agar system designed for growing fresh human tumor specimens in vitro. The assay has been extensively used in studies both of individual patients' response to chemotherapy and for screening new agents. The technical limitations of this assay have been extensively discussed. The use of this test as a model system to study fundamentals of tumor cell growth has not been stressed. The potentials and limitations of this assay for the study of the regulation of tumor growth are presented.     

Loss of putative tumor suppressor EI24/PIG8 confers resistance to etoposide

Expression of p53-target gene EI24/PIG8 is lost in invasive breast cancers, suggesting that EI24/PIG8 is a tumor suppressor that prevents tumor spreading, and partially mediates p53-attributed tumor suppressor activity. EI24/PIG8 also has pro-apoptotic activity indicating that loss of EI24/PIG8 may modulate sensitivity to chemotherapy. Here it is demonstrated that suppression of EI24/PIG8 in fibroblasts and breast cancer cells significantly inhibits the apoptotic response to etoposide treatment. These findings suggest that loss of EI24/PIG8 contributes significantly to resistance of cells to chemotherapeutic agents that function through p53, and identify the EI24/PIG8 status as a potentially new prognostic marker of chemotherapy responsiveness.