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Application of human tumor colonyforming assay to new drug screening
Abstract
The applicability of a human tumor colony-forming assay to drug screening was investigated in terms of feasibility, validity, and potential for discovering new antitumor drugs. Feasibility was addressed in a pilot study during which basic methods, appropriate assay quality controls, and a standardized protocol for screening were developed. Considerable variability was noted in the availability and colony growth of different tumor types. The majority of the evaluable experiments utilized breast, colorectal, kidney, lung, melanoma, or ovarian tumors. For many tumor types, little evidence of growth was observed, or only rare specimens formed colonies. Colony-forming efficiencies ranged from 0.05 to 0.11% for the six most useful tumors listed above. A set of quality control measures was developed to address technical problems inherent in the assay. Testing of standard agents in the pilot study established that most of these agents could be detected as active. However, it also identified three assay limitations: compounds requiring systemic metabolic activation are inactive; medium constituents may block the activity of certain antimetabolites; and compounds without therapeutic efficacy may be positive in the assay. The assay categorized nontoxic clinically ineffective agents as true negatives with 97% accuracy. Of 79 compounds which were negative in the current National Cancer Institute prescreen (leukemia P388), 14 were active in the assay. Several demonstrated outstanding in vitro activity and are structurally unrelated to compounds already in development or in clinical trials. A subset of these active compounds were found to lack activity in a P388 in vitro colony-forming assay. This indication of differential cytotoxicity to human tumor cells makes this subset of compounds particularly interesting as antitumor drug leads. The demonstrated sensitivity to most standard agents, discrimination of nontoxic compounds, reproducibility of survival values within assays and between laboratories, and evidence of ability to identify active compounds which were negative in the in vivo prescreen suggest that the human tumor colony-forming assay may be a valuable tool for antitumor drug screening. However, because of technical limitations inherent in the current assay methodology, this must be confined to selected tumor types and limited to screening on a moderate scale.
... The colony-forming property is a sign of undifferentiated cancer stem cells [24,25]. Therefore, we assessed the colony-forming ability in TNBC cell lines upon Disarib treatment. ...
Targeted cancer therapy aims to disrupt the functions of proteins that regulate cancer progression, mainly by using small molecule inhibitors (SMIs). SMIs exert their effect by modulating signalling pathways, organelle integrity, chromatin components, and several biosynthetic processes essential for cell division and survival. Antiapoptotic protein BCL2 is highly upregulated in many cancers compared with normal cells, making it an ideal target for cancer therapy. Around 75% of primary breast cancers overexpress BCL2, providing an opportunity to explore BCL2 inhibitors as a therapeutic option. Disarib is an SMI that has been developed as a selective BCL2 inhibitor. Disarib works by disrupting BCL2-BAK interaction and activating intrinsic apoptotic pathways in leukemic cells while sparing normal cells. We investigated the effects of Disarib, a BCL2 specific inhibitor, on breast cancer cells and xenografts. Cytotoxicity and fluorometric assays revealed that Disarib induced cell death by increasing reactive oxygen species and activating intrinsic apoptotic pathways in Triple-Negative Breast Cancer cells (MDA-MB-231 and MDA-MB-468). Disarib also affected the colony-forming properties of these cells. MDA-MB-231- and MDA-MB-468-derived xenografts showed a significant reduction in tumours upon Disarib treatment. Through the transcriptomics approach, we also explored the influence of BCL2 inhibitors on energy metabolism, mitochondrial dynamics, and epithelial-to-mesenchymal transition (EMT). Mitochondrial dynamics and glucose metabolism mainly regulate energy metabolism. The change in energetics regulates tumour growth through epithelial–mesenchymal transition, and angiogenesis. RNA sequencing (RNAseq) analysis revealed that BCL2 inhibitors ABT-199 and Disarib maintain Oxphos levels in MDA-MB-231. However, key glycolytic genes were significantly downregulated. Mitochondrial fission genes were seen to be downregulated both in RNAseq data and semi quantitative real time polymerase chain reaction (qRTPCR) in Disarib-treated TNBC cells and xenografts. Lastly, Disarib inhibited wound healing and epithelial-to-mesenchymal transition. This study showed that Disarib disrupts mitochondrial function, activates the intrinsic apoptotic pathway in breast cancer, and inhibits epithelial-to-mesenchymal transition both in vitro and in vivo. These findings highlight Disarib’s potential as a multifaceted therapeutic strategy for patients with Triple-Negative Breast Cancer.
... To remove EAD, the cells were washed, and the colony formation was observed for 10 days. After 10 days, the colonies were stained with 0.25% crystal violet; the colonies were manually counted (Kumar et al. 2022;Shoemaker, Wolpert-DeFilippes et al. 1985). ...
Neuroblastoma, the most common childhood solid tumor, originates from primitive sympathetic nervous system cells. Epoxyazadiradione (EAD) is a limonoid derived from Azadirachta indica, belonging to the family Meliaceae. In this study, we isolated the EAD from Azadirachta indica seed and studied the anti-cancer potential against neuroblastoma. Herein, EAD demonstrated significant efficacy against neuroblastoma by suppressing cell proliferation, enhancing the rate of apoptosis and cycle arrest at the SubG0 and G2/M phases. EAD enhanced the pro-apoptotic Caspase 3 and Caspase 9 and inhibited the NF-kβ translocation in a dose-dependent manner. In order to identify the specific EAD target, a gel-free quantitative proteomics study on SH-SY5Y cells using Liquid Chromatography with tandem mass spectrometry was done in a dose-dependent manner, followed by detailed bioinformatics analysis to identify effects on protein. Proteomics data identified that Enolase1 and HSP90 were up-regulated in neuroblastoma. EAD inhibited the expression of Enolase1 and HSP90, validated by mRNA expression, immunoblotting, Enolase1 and HSP90 kit and flow-cytometry based bioassay. Molecular docking study, Molecular dynamic simulation, and along with molecular mechanics/Poisson-Boltzmann surface area analysis also suggested that EAD binds at the active site of the proteins and were stable throughout the 100 ns Molecular dynamic simulation study. Overall, this study suggested EAD exhibited anti-cancer activity against neuroblastoma by targeting Enolase1 and HSP90 pathways.
... Functional precision medicine (see Glossary) is a rapidly advancing strategy to inform personalized treatment decisions for cancer patients based on functional readouts such as direct drug sensitivity testing of patient cancer cells (Box 1) [1]. This approach was introduced more than four decades ago [2,3], but initial reports found that functional assays were too premature for clinical implementation, in part because of the low fraction of tumor samples that could successfully be cultivated and tested for drug sensitivity in the laboratory [4,5]. For solid tumors, studies have so far mostly been conducted retrospectively, and prospective evidence that is crucial for adopting such technologies in clinical decision making, is still lacking. ...
Functional precision medicine is a new, emerging area that can guide cancer treatment by capturing information from direct perturbations of tumor-derived, living cells, such as by drug sensitivity screening. Precision cancer medicine as currently implemented in clinical practice has been driven by genomics, and current molecular tumor boards rely extensively on genomic characterization to advise on therapeutic interventions. However, genomic biomarkers can only guide treatment decisions for a fraction of the patients. In this review we provide an overview of the current state of functional precision medicine, highlight advances for drug-sensitivity screening enabled by cell culture models, and discuss how artificial intelligence (AI) can be coupled to functional precision medicine to guide patient stratification.
... In our previous in vitro study [23], it was demonstrated that the measured radiation survival by metabolic cell assay is comparable to that of clonogenic assays. Additionally, the metabolic assay measures all viable cells thus representing cells from a true tumour population rather than just clonogenic cells [34,35]. The FDG scan uses a glucose analogue and is the most commonly used PET tracer to assess tumour metabolism. ...
Background
The prediction of response is one of the major challenges in radiation-based therapies. Although the selection of accurate linear–quadratic model parameters is essential for the estimation of radiation response and treatment outcome, there is a limited knowledge about these radiobiological parameters for liver tumours using radionuclide treatments.
Methods
The “clinical radiobiological” parameters ( $$T_{{\text{p}}}$$ T p , $$T_{{\text{k}}}$$ T k , $$\alpha$$ α , $$\alpha {/}\beta$$ α / β ) for twenty-five patients were derived using the generalised linear–quadratic model, the diagnostic ([ ¹⁸ F] FDG PET/CT) and therapeutic ([ ⁹⁰ Y]-SIR-Spheres PET/CT) images to compute the biological effective dose and tumour control probability (TCP) for each patient.
Results
It was estimated that the values for $$\alpha$$ α and $$\alpha {/}\beta$$ α / β parameters range in ≈ 0.001–1 Gy ⁻¹ and ≈ 1–49 Gy, respectively. We have demonstrated that the time factors, $$T_{{\text{p}}}$$ T p , $$T_{{\text{k}}}$$ T k and $$T_{{{\text{critic}}}}$$ T critic are the key parameters when evaluating liver malignancy lesional response to [ ⁹⁰ Y]SIR-Spheres treatment. Patients with cholangiocarcinoma have been shown to have the longest average $$T_{{\text{p}}}$$ T p (≈ 236 ± 67 d), highest TCP (≈ 53 ± 17%) and total liver lesion glycolysis response ( $$\Delta {\text{TLG}}_{{{\text{liver}}}}$$ Δ TLG liver ≈ 64%), while patients with metastatic colorectal cancer tumours have the shortest average $$T_{{\text{p}}}$$ T p (≈ 129 ± 19 d), lowest TCP (≈ 28 ± 13%) and $$\Delta {\text{TLG}}_{{{\text{liver}}}}$$ Δ TLG liver ≈ 8%, respectively.
Conclusions
Tumours with shorter $$T_{{\text{k}}}$$ T k have shown a shorter $$T_{{{\text{critic}}}}$$ T critic and thus poorer TCP and $$\Delta {\text{TLG}}_{{{\text{liver}}}}$$ Δ TLG liver . Therefore, these results suggest for such tumours the [ ⁹⁰ Y]SIR-Spheres will be only effective at higher initial dose rate (e.g. > 50 Gy/day).
... In vitro reproductive cell survival assays are the gold standard tool to evaluate the sensitivity of cancer cells to therapeutic agents (Shoemaker et al. 1985) as they evaluate all mechanisms of cell death and impairment of proliferative capacity simultaneously by measuring the ability of single cells to divide and generate colonies. Colony-forming assays were conducted on SKOV3 cells after exposure to AuNP treatments to determine the health of cells and their ability to survive and reproduce ( Figure 3). ...
Gold nanoparticles (AuNP) are promising anti-cancer agents because of their modifiable properties and high biocompatibility. This study used multiple parallel analyses to investigate the cytotoxic properties of 5 nm AuNP conjugated to four different ligands with distinct surface chemistry: polyethylene glycol (PEG), trimethylammonium bromide (TMAB), 4-dimethylaminopyridine (DMAP), and carboxyl (COOH). We used a range of biochemical and high-content microscopy methods to evaluate the metabolic function, oxidative stress, cell health, cell viability, and cell morphology in SKOV3 ovarian cancer cells. Each AuNP displayed a distinct cytotoxicity profile. All AuNP species assessed exhibited signs of dose-dependent cytotoxicity when morphology, clonogenic survival, lysosomal uptake, or cell number were measured as the marker of toxicity. All particles except for AuNP-COOH increased SKOV3 apoptosis. In contrast, AuNP-TMAB was the only particle that did not alter the metabolic function or induce significant signs of oxidative stress. These results demonstrate that AuNP surface chemistry impacts the magnitude and mechanism of SKOV3 cell death. Together, these findings reinforce the important role for multiparametric cytotoxicity characterization when considering the utility of novel particles and surface chemistries.
... The cells were re-suspended in a mixture of binding buffer, 5 µL Annexin-V and 5 µL of propidium iodide, and incubated in the dark for 15 min at room temperature. Thereafter, fluorescence was measured using Accuri C6 Plus flow cytometer [6]. ...
Purpose: To Investigate the antiproliferative effect of n-hexane-diethyl ether fraction of Costus speciosus (NP) on triple negative breast cancer (MDA-MB-231) cells, and the mechanism involved. Methods: Maceration with methanol (CH3OH) was used for extraction of Costus speciosus rhizomes. Chromatographic separation was used to obtain the non-polar fraction (NP) via elution with n-hexane:(C2H5)2O at a volume ratio of 9:1. The cytotoxic effect of NP was evaluated against two breast cancer cell lines i.e., triple negative (MDA-MB-231) and positive ER (MCF-7) employing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT) assay, and the IC50 values were estimated. Cell cycle was determined with flow cytometry, while the likely mechanism involved in the cytotoxic effect was investigated using comet assay, immunofluorescence, clonogenic and scratch assays, zymography and detection of the antioxidant markers. Results: NP produced potent cytotoxicity against MDA-MB-231, with IC50 value of 4 ± 0.03 µg/mL, whereas its IC50 for MCF-7 was 27 ± 1.3 µg/mL. It induced apoptosis via cell cycle arrest at G1 phase. Moreover, NP markedly decreased levels of superoxide dismutase (SOD), reduced glutathione (GSH), and matrix metalloprotease-2 (MMP-2), in MDA-MB-231 cells. Moreover, it inhibited cancer cell migration and colony formation. Conclusion: Non-polar fraction of Costus speciosus (NP) exerted cytotoxic effect on triple negative breast cancer cells (MDA-MB-231) and positive ER (MCF-7). It inhibited cancer cell migration and colony formation. Interestingly, NP arrested the breast cancer cell cycles at sub-G1 phase, inhibited SOD and MMP-2, and decreased GSH levels. It induced apoptosis via DNA damage, downregulation of mutant p53, and over-expressions of the cell cycle inhibitors p21 and p27.
... An alternative assay is the colony forming clonogenic assay, in which cancer cells are incubated with the compound for a short period, then seeded into flasks, and eventual colonies formed by single cells are monitored (Hamburger & Salmon, 1977;Shoemaker et al., 1985). The inability of cells to form colonies after termination of treatment with the compounds indicates growth inhibition and senescence, in particular of stem cells. ...
G-quadruplexes are specific nucleotide structures of stacked planes of guanines, and can form in G-rich telomeres and promoter regions of oncogenes. Their stabilisation by small molecules has a profound anti-cancer effect, as it leads to telomerase inhibition and telomere uncapping followed by DNA damage response and senescence, as well as chromosomal instability, and down-regulation of oncogene expression. This thesis reports on the design and synthesis of small molecule G-quadruplex ligands and their biophysical and biological evaluation. A series of highly water soluble naphthalene diimides (NDs) with N-methylpiperazine end groups was synthesised, which have high affinity and selectivity for human telomeric G-quadruplexes over duplex DNA, as shown in FRET melting temperature assays. The compounds were conceived by molecular modelling as derivatives of previously described potent NDs, indicating enhanced selectivity through groove interactions between protonated N-methylpiperazine groups and phosphates. Circular dichroism studies indicate that the ligands induce a parallel G-quadruplex topology in solution. In 96 h SRB assays, the compounds show high toxicity to a panel of cancer cell lines including pancreatic cancer cells, with IC50 values in the order of 0.1 μM and up to 100 fold selectivity over a normal fibroblast cell line. Treatment with the compounds at sub-cytotoxic concentrations over several weeks decreases growth of pancreatic cancer cells, resulting in senescence. The fluorescent lead compound was visualised inside the nuclei and cytoplasm of cancer cells by confocal microscopy. It causes DNA damage in cancer cells, which was indicated by detection of the phosphorylated histone H2AX. Cell cycle analysis was undertaken with propidium iodide in relation to further cellular responses to the compounds. The thesis examines the likely mode of action of these compounds in the light of this and further data, and suggests that they are promising candidates for the treatment of pancreatic cancers. The thesis also reports on the design and synthesis of other novel G-quadruplex ligands.
... Then the cells were washed with PBS (phosphate buffered saline) preheated to 37 °C, trypsinized and plated in 6-well plates (100 cells/wells). After 12 days of incubation in complete culture medium, the colonies were stained with Giemsa's stain after fixation with 2% para-formaldehyde [29]. ...
Cisplatin (CDDP) is a potent chemotherapeutic drug, but its severe side-effects often prohibit its use. Combined treatment with CDDP plus Farnesol (FAR) and their co-encapsulated nano form were investigated in in vitro to examine if synergistic cytotoxicity of this combination could reduce unwanted side-effects of CDDP chemotherapy and potentiate CDDP anticancer activity against hepatocellular carcinoma (HCC) cells. After finding combination therapy of CDDP and FAR successfully combat HCC we formulated co-encapsulation of CDDP and FAR within poly(lactic-co-glycolic acid) copolymer (NCDDPFAR) by following the standardized solvent displacement method. NCDDPFAR treatment caused faster drug mobility, sustained particle release, site-specific action and higher percentage of apoptotic death compared with single drug treatment even at relatively low concentrations. Co-encapsulation of two drugs exhibited additive effects against HCC; FAR reduced CDDP-induced glutathione level by increasing expression of CYP2E1 while CDDP directly interacted with DNA; FAR up-regulated the expression of TopII, thereby promoting DNA breaks and escaping DNA repair machinery. Expression pattern of apoptotic genes like p53, Bax, cytochrome c and caspase-3 suggested that NCDDPFAR induced HCC cell death through mitochondrial intrinsic pathway. Administration of NCDDPFAR had better ability of drug carriage and enhanced anticancer potentials against HCC cells.
... A recent study highlighted the emerging use of 3D cell culture, given its potential impact on early drug discovery and therapeutics for the treatment of diseases, such as cancer 19 because it can more closely mimic in vivo cell environments. 20 Therefore, we conducted the soft-agar colony-forming assay, which has certain advantages, including simplicity, validity, and potential for discovering new antitumor drugs, 21,22 using in cancer stem cells (CSCs). 23,24 Both HT29 and SW620 cell lines were treated in a layer of soft agar media containing various concentrations of ginsenoside Rg3R. ...
Background
Cancer stem cells (CSCs) have been proposed as central drivers of cancer relapse in many cancers. In the present study, we investigated the inhibitory effect of 20(R)-Ginsenoside Rg3 (Rg3R), a major active component of ginseng saponin, on CSC-like cells and the Epithelial-Mesenchymal Transition (EMT) in colorectal cancer (CRC).
Methods
The effects of ginsenoside Rg3R on the colony-forming, migration, invasion, and wound-healing abilities of CRC cells were determined in HT29 and SW620 cell lines in vitro. Further, ginsenoside Rg3R was given intraperitoneally at 5mg/kg of mouse body weight to check its effect on the metastasis of CRC cells in vivo.
Results
Ginsenoside Rg3R significantly inhibited CSC properties, but did not affect cell proliferation. Moreover, ginsenoside Rg3R treatment significantly inhibited the motility of CRC cells based on migration, invasion, and wound-healing assays. The inhibitory effects of ginsenoside Rg3R on CRC are potentially mediated by significant down-regulation of the expression of stemness genes and EMT markers in CRC cells in a SNAIL-dependent manner. Furthermore, ginsenoside Rg3R treatment decreased both the number and size of tumor nodules in the liver, lung, and kidney tissues in a metastasis mouse model.
Conclusion
These findings highlighted the potential use of ginsenoside Rg3R in clinical applications for colorectal cancer treatment.
... In comparison to the established clonogenic assay, the MTS (or MTT) assays are sensitive and accurate methods with several advantages such as requiring a short time to assess the viability, acquiring the results easily and accurately [25,[27][28][29][30][31][32]. It measures all viable cells thus representing cells from a true tumour population rather than just clonogenic cells (i.e. cells that can form colonies) [33,34]. ...
Background:
Dose rate variation is a critical factor affecting radionuclide therapy (RNT) efficacy. Relatively few studies to date have investigated the dose rate effect in RNT. Therefore, the aim of this study was to benchmark 90Y RNT (at different dose rates) against external beam radiotherapy (EBRT) in vitro and compare cell kill responses between the two irradiation processes.
Results:
Three human colorectal carcinoma (CRC) cell lines (HT29, HCT116, SW48) were exposed to 90Y doses in the ranges 1-10.4 and 6.2-62.3 Gy with initial dose rates of 0.013-0.13 Gy/hr (low dose rate, LDR) and 0.077-0.77 Gy/hr (high dose rate, HDR), respectively. Results were compared to a 6-MV photon beam doses in the range from 1-9 Gy with constant dose rate of 277 Gy/hr. The cell survival parameters from the linear quadratic (LQ) model were determined. Additionally, Monte Carlo simulations were performed to calculate the average dose, dose rate and the number of hits in the cell nucleus. For the HT29 cell line, which was the most radioresistant, the α/β ratio was found to be ≈ 31 for HDR-90Y and ≈ 3.5 for EBRT. LDR-90Y resulting in insignificant cell death compared to HDR-90Y and EBRT. Simulation results also showed for LDR-90Y, for doses ≲ 3 Gy, the average number of hits per cell nucleus is ≲ 2 indicating insufficiently delivered lethal dose. For 90Y doses [Formula: see text] 3 Gy the number of hits per nucleus decreases rapidly and falls below ≈ 2 after ≈ 5 days of incubation time. Therefore, our results demonstrate that LDR-90Y is radiobiologically less effective than EBRT. However, HDR-90Y at ≈ 56 Gy was found to be radiobiologically as effective as acute ≈ 8 Gy EBRT.
Conclusion:
These results demonstrate that the efficacy of RNT is dependent on the initial dose rate at which radiation is delivered. Therefore, for a relatively long half-life radionuclide such as 90Y, a higher initial activity is required to achieve an outcome as effective as EBRT.
... The applicability of the HTSC assay for drug screening purposes in terms of feasibility, validity, and potential to identify new antitumor agents was investigated [3]. The testing of established standard chemotherapeutic agents in this pilot study revealed that most agents were found to be active with the exception of those requiring systemic activation. ...
Cancer is a disease characterized by uncontrolled proliferation of cells that have transformed from the normal cells of the body. The widely used cancer drugs suffers from the drawback of high toxicity not within the reach of a common man. This urgently necessitating the screening of these compounds. This review focuses on the major contributions of preclinical screening models to anticancer drug development over the years till recent times, from the empirical drug screening of cytotoxic agents against uncharacterized tumor models to the target-orientated drug screening of agents with defined mechanisms of action,, a general transition has been observed. The newer approaches to anticancer drug development involve the molecular characterization of models along with an appreciation of the pharmacodynamics and pharmacokinetic properties of compounds [e. g., the US National Cancer Institute (NCI) in vitro 60-cell line panel, hollow fibre assay, and s. c. xenograft]. In vivo tumor models including orthotopic, metastatic, and genetically engineered mouse models are also reviewed. The preclinical screening efforts of the European are also included. In 2015 with the rapid development of cancer modeling in zebrafish, great opportunities exist for chemical screens to find anticancer drug since 1970 the European Organisation for Research and Treatment of Cancer and Cancer Research UK, have been collaborating with the NCI in the acquisition and screening of compounds.
... The CFA monitors directly the cell's ability to produce a viable colony after treatment (Franken et al., 2006). This test is unbiased to the mode of cell growth inhibition or death and is able to detect the cytotoxic effect of test material, regardless of mechanism, as long as the agent affects the cell's reproductive ability (Franken et al., 2006;Rafehi et al., 2011;Rosenblum et al., 1975;Shoemaker et al., 1985). The disadvantage is that this test requires multiple cell replication cycles in order to detect any adverse effects, versus biochemical methods which detect earlier changes more rapidly. ...
... However, 2 does not induce cell death in both lines tested (l " Fig. 4) even though mexicanolide-type compounds like erythrocarpine E (CEB4) induced apoptosis in the oral tumour cell line HSC-4 [49]. The applicability of the tumour colony-forming assay for drug screening has long been accepted to investigate new anti-tumour drugs [50]. We observed that 1 at 10 µM completely inhibited clonogenic capacity in both cell lines, since no surviving cells could be detected. ...
The seeds of Carapa procera are exploited extensively in West African ethnopharmacy for the treatment of several pathologies, including inflammation. They also are effective as insect antifeedants and as a mosquito repellent. With the aim of identifying bioactive principles, an ethyl acetate extract of the defatted seeds was made and fractionated. Two principle compounds were isolated. One of these, 5,6-dehydro-7-deacetoxy-7-oxogedunin (1), while known from another genus of the Meliaceae, is newly identified from the genus Carapa and its X-ray structure is described for the first time. In addition, 1 displayed strong anti-clonogenic activity at 10 µM. The other compound, mexicanolide (2), is known from this species and showed neither cytotoxicity nor anti-clonogenicity. These differences in efficacy are discussed in relation to known structure-activity relationships of limonoids.
Georg Thieme Verlag KG Stuttgart · New York.
... After 12 days of incubation in complete culture medium, the colonies were stained with Giemsa's stain after fixation with 2% para-formaldehyde. [16] Proliferation assay ...
Objective: Conium maculatum extract is used as a traditional medicine for cervix carcinoma including homeopathy. However, no systematic work has so far been carried out to test its anticancer potential against cervix cancer cells in vitro. Thus, in this study, we investigated whether ethanolic extract of conium is capable of inducing cytotoxicity in different normal and cancer cell lines including an elaborate study in HeLa cells. Materials and Methods: Conium's effects on cell cycle, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential (MMP) and apoptosis, if any, were analyzed through flow cytometry. Whether Conium could damage DNA and induce morphological changes were also determined microscopically. Expression of different proteins related to cell death and survival was critically studied by western blotting and ELISA methods. If Conium could interact directly with DNA was also determined by circular dichroism (CD) spectroscopy. Results: Conium treatment reduced cell viability and colony formation at 48 h and inhibited cell proliferation, arresting cell cycle at subG stage. Conium treatment lead to increased generation of reactive oxygen species (ROS) at 24 h, increase in MMP depolarization, morphological changes and DNA damage in HeLa cells along with externalization of phosphatidyl serine at 48 hours. While cytochrome c release and caspase3 activation led HeLa cells toward apoptosis, downregulation of Akt and NFkB inhibited cellular proliferation, indicating the signaling pathway to be mediated via the mitochondriamediated caspase3 dependent pathway. CDspectroscopy revealed that Conium interacted with DNA molecule.
... The ability of lung cancer cells to form colonies is another relevant endpoint of cytotoxicity and genotoxicity (Shoemaker et al., 1985). A very small amount of treated cells were seeded on plates in order to perform single cell forming colonies. ...
The pulmonary route has attracted great attention for the delivery of nanomedicines due to the non invasiveness, a weak enzymatic activity and potential alveolar retention and/or absorption. However, the potential pulmonary toxicity of nanoparticles raises a lot of concern, especially for manufactured, non-biodegradable nanoparticles. In this study, we design and characterize an in vitro model of lung epithelium based on a co-culture of alveolar epithelial-like cells (A549) and macrophages (differentiated from THP-1 monocytes), and use it to assess the potential toxicity of various biodegradable nanoparticles in the form of nanocarriers, as compared to non-biodegradable nanoparticles used in manufactured products. The ultimate goal is to provide a safety pattern of nanoparticles relevant for drug delivery to the lung.The co-culture was characterized by flow cytometry (analysis of three cell membrane receptors: CD14, CD11b and CD54) and confocal laser scanning microscopy. The presence of two different cell types was evidenced, as well as several cellular interactions. For instance, after exposure to a pro-inflammatory compound, synergistic effects were observed, in terms of cytokine secretions ( IL-6, IL-8, TNF-α and MCP-1). Such co-cultures are thus a valuable tool to investigate the inflammatory response following exposure to nanoparticles. On the other hand, the cell membrane receptor CD14 (expressed only by macrophages) was used as an identification tool to distinguish each cell population in co-culture.Biodegradable nanoparticles having size around 230 nm, were prepared according to an emulsion-evaporation process using poly(lactide-co-glycolide) (PLGA). The use of polyvinylalcohol (PVA), chitosan (CS) or poloxamer (PF68) as stabilizers allows the formation of, respectively, neutral, positively- or negatively-charged nanoparticles. In addition, stabilizer-free nanoparticles (negatively charged) were prepared. Commercial titanium dioxide and polystyrene nanoparticles were used as non-biodegradable nanoparticles.After exposure to nanoparticles, uptake kinetics (flow cytometry and confocal microscopy) were performed in cells in mono and co-culture. Negatively-charged nanoparticles (stabilizer-free PLGA and PLGA/PF68 nanoparticles) were found in higher quantity in each cell population. Several cytotoxicity tests (MTT, trypan blue, selective membrane permeability, lactate dehydrogenase (LDH) release) have shown a low to medium cytotoxicity of PLGA nanoparticles. The cytotoxicity of PLGA/CS nanoparticles was attributed to the cytotoxicity of the chitosan in solution, whereas the cytotoxicity of PLGA/PF68 nanoparticles was attributed to their higher uptake. After 24 h exposure to a low dose of PLGA nanoparticles, a low inflammatory response was detected. Non-biodegradable nanoparticles have shown a slightly higher toxicity.Differences observed among PLGA nanoparticles in terms of cytotoxicity, cell uptake quantity and inflammatory response highlight the importance of the coating of nanocarriers for drug delivery application.
... Tumor colony assay-To quantitatively compare peritoneal tumor burden, an ex vivo tumor colony assay was adapted from Shoemaker and colleagues (27). Briefly, peritoneal cells were plated in 10-fold serial dilutions starting at 1×10 6 cells/mL in 48-well plates (Costar). ...
Immune checkpoint blockade has shown significant therapeutic efficacy in melanoma and other solid tumors, but results in ovarian cancer have been limited. With evidence that tumor immunogenicity modulates the response to checkpoint blockade, and data indicating that BRCA-deficient ovarian cancers express higher levels of immune response genes, we hypothesized that BRCA(-) ovarian tumors would be vulnerable to checkpoint blockade. To test this, we used an immunocompetent BRCA1-deficient murine ovarian cancer model to compare treatment with CTLA-4 or PD-1/PD-L1 antibodies alone or combined with targeted cytotoxic therapy using a PARP-inhibitor. Correlative studies were performed in vitro using human BRCA1(-) cells. We found that CTLA-4 antibody, but not PD-1/PD-L1 blockade, synergized therapeutically with the PARP-inhibitor, resulting in immune-mediated tumor clearance and long-term survival in a majority of animals (p<0.0001). The survival benefit of this combination was T cell-mediated and dependent on increases in local IFN-gamma production in the peritoneal tumor environment. Evidence of protective immune memory was observed greater than 60 days after completion of therapy. Similar increases in the cytotoxic effect of PARP-inhibition in the presence of elevated levels of IFN-γ in human BRCA1(-) cancer cells supports the translational potential of this treatment protocol. These results demonstrate that CTLA-4 blockade combined with PARP-inhibition induces protective anti-tumor immunity and significant survival benefit in the BRCA1(-) tumor model, and support clinical testing of this regimen to improve outcomes for women with hereditary ovarian cancer.
Copyright © 2015, American Association for Cancer Research.
... After 12 days of incubation in complete culture medium, the colonies were stained with Giemsa's stain after fixation with 2% para-formaldehyde. [16] ...
Objective:
Conium maculatum extract is used as a traditional medicine for cervix carcinoma including homeopathy. However, no systematic work has so far been carried out to test its anti-cancer potential against cervix cancer cells in vitro. Thus, in this study, we investigated whether ethanolic extract of conium is capable of inducing cytotoxicity in different normal and cancer cell lines including an elaborate study in HeLa cells.
Materials and Methods:
Conium's effects on cell cycle, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential (MMP) and apoptosis, if any, were analyzed through flow cytometry. Whether Conium could damage DNA and induce morphological changes were also determined microscopically. Expression of different proteins related to cell death and survival was critically studied by western blotting and ELISA methods. If Conium could interact directly with DNA was also determined by circular dichroism (CD) spectroscopy.
Results:
Conium treatment reduced cell viability and colony formation at 48 h and inhibited cell proliferation, arresting cell cycle at sub-G stage. Conium treatment lead to increased generation of reactive oxygen species (ROS) at 24 h, increase in MMP depolarization, morphological changes and DNA damage in HeLa cells along with externalization of phosphatidyl serine at 48 hours. While cytochrome c release and caspase-3 activation led HeLa cells toward apoptosis, down-regulation of Akt and NFkB inhibited cellular proliferation, indicating the signaling pathway to be mediated via the mitochondria-mediated caspase-3-dependent pathway. CD-spectroscopy revealed that Conium interacted with DNA molecule.
Conclusion:
Overall results validate anti-cancer potential of Conium and provide support for its use in traditional systems of medicine.
... The ( We next tested the effects of NGEN and NGENCuB for their ability to inhibit colony formation in MDA-MB-231 tumor cells ( figure 3AD). The applicability of the tumor colony-forming assay for drug screening has long been accepted to investigate new antitumor drugs [40]. NGENCuB (1, 10 and 100 mM) was significantly more effective inhibiting both colony number Figure 5B). ...
Cancer is the second leading cause of death worldwide and there is epidemiological evidence that demonstrates this tendency is emerging. Naringenin (NGEN) is a trihydroxyflavanone that shows various biological effects such as antioxidant, anticancer, anti-inflammatory, and antiviral activities. It belongs to flavanone class, which represents flavonoids with a C6-C3-C6 skeleton. Flavonoids do not exhibit sufficient activity to be used for chemotherapy, however they can be chemically modified by complexation with metals such as copper (Cu) (II) for instance, in order to be applied for adjuvant therapy. This study investigated the effects of Cu(II) and 2,2'-bipyridine complexation with naringenin on MDA-MB-231 cells. We demonstrated that naringenin complexed with Cu(II) and 2,2'-bipyridine (NGENCuB) was more efficient inhibiting colony formation, proliferation and migration of MDA-MB-231 tumor cells, than naringenin (NGEN) itself. Furthermore, we verified that NGENCuB was more effective than NGEN inhibiting pro-MMP9 activity by zymography assays. Finally, through flow cytometry, we showed that NGENCuB is more efficient than NGEN inducing apoptosis in MDA-MB-231 cells. These results were confirmed by gene expression analysis in real time PCR. We observed that NGENCuB upregulated the expression of pro-apoptotic gene caspase-9, but did not change the expression of caspase-8 or anti-apoptotic gene Bcl-2. There are only few works investigating the effects of Cu(II) complexation with naringenin on tumor cells. To the best of our knowledge, this is the first work describing the effects of Cu(II) complexation of a flavonoid on MDA-MB-231 breast tumor cells.
... In an NCI-funded study, four laboratories evaluated the technology and its use in drug testing. Six tumor entities, breast, colorectal, kidney, lung, melanoma, and ovarian yielded cultures that passed the quality controls established by the laboratories, for 20-38% of patients tested at samples sizes of >160 patients each [15]. The protocol was then used to re-test 79 compounds, which had been inactive in the in vivo screening assay, on 15 tumors each. ...
Cancers are complex and heterogeneous pathological “organs” in a dynamic interplay with their
host. Models of human cancer in vitro, used in cancer biology and drug discovery, are generally
highly reductionist. These cancer models do not incorporate complexity or heterogeneity. This
raises the question as to whether the cancer models’ biochemical circuitry (not their genome) represents,
with sufficient fidelity, a tumor in situ. Around 95% of new anticancer drugs eventually fail
in clinical trial, despite robust indications of activity in existing in vitro pre-clinical models. Innovative
models are required that better capture tumor biology. An important feature of all tissues,
and tumors, is that cells grow in three dimensions. Advances in generating and characterizing simple
and complex (with added stromal components) three-dimensional in vitro models (3D models)
are reviewed in this article. The application of stirred bioreactors to permit both scale-up/scaledown
of these cancer models and, importantly, methods to permit controlled changes in environment
(pH, nutrients, and oxygen) are also described. The challenges of generating thin tumor
slices, their utility, and potential advantages and disadvantages are discussed. These in vitro/ex
vivo models represent a distinct move to capture the realities of tumor biology in situ, but significant
characterization work still remains to be done in order to show that their biochemical circuitry
accurately reflects that of a tumor.
... Anchorage-independent growth was assayed as previously described (29). A total of 5,000 cells were plated and colonies were counted after 14 days. ...
The discovery of somatic mutations in epidermal growth factor receptor (EGFR) and development of EGFR tyrosine kinase inhibitors (TKIs) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all patients and currently no effective treatment is available. Here we show that β-catenin is essential for development of EGFR mutated lung cancers. β-catenin was upregulated and activated in EGFR mutated cells. Mutant EGFR preferentially bound to and tyrosine-phosphorylated β-catenin, leading to increase in β-catenin-mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacological inhibition of β-catenin suppressed EGFR-L858R-T790M mutated lung tumor growth and genetic deletion of the β-catenin gene dramatically reduced lung tumor formation in EGFR-L858R-T790M transgenic mice. These data suggest that β-catenin plays an essential role in lung tumorigenesis and that targeting the β-catenin pathway may provide novel strategies to prevent lung cancer development or overcome resistance to EGFR TKIs.
... Soft agar colony formation assays are commonly used as a monitoring system for anchorage-independent (three-dimensional) growth, measuring proliferation by the counting of colonies subsequent to 3-4 weeks in a semi-solid culture medium. This traditional method has been widely published (10)(11)(12). As the technique delivers results that are comparable to those obtained when injecting tumorigenic cells into nude mice, the soft agar colony formation assay is regarded as the gold standard for testing the tumorigenicity of cells in vitro (13). ...
Our previous study reported that an extract of an Indonesian marine sponge, Haliclona sp., showed potent cytotoxicity and induced apoptosis. The major cytotoxic chemical compound was identified as papuamine, which caused reduction of cell survival through activation of cJun Nterminal kinase (JNK) in human breast cancer MCF7 cells. Doxorubicin (DOX), a Streptomyces metabolite, is used in chemotherapy against a wide range of cancers, including breast cancer. The present study examined the combined effect of papuamine and DOX on MCF7 cells. The effect of these reagents on cell growth was assessed by a colony formation assay. Incubation with either of the reagents alone resulted in concentrationdependent decreases in the colony formation of the MCF7 cells. Incubation with the reagents together at subcytotoxic concentrations resulted in significant decreases in colony formation. The phosphorylation of JNK, the activated form of the protein, was elevated in a concentrationdependent manner upon coincubation with papuamine and DOX. Fluorescence intensity analysis demonstrated that papuamine caused a small, but nonsignificant, decrease in cellular accumulation of DOX. These results indicate that the combinatory effect of papuamine and DOX is not associated with changes in the cellular accumulation of DOX, and may instead reflect additive effects on JNK activation. This study indicates that papuamine may represent a novel type of modulator for DOX chemotherapy.
... The ( We next tested the effects of NGEN and NGENCuB for their ability to inhibit colony formation in MDA-MB-231 tumor cells ( figure 3AD). The applicability of the tumor colony-forming assay for drug screening has long been accepted to investigate new antitumor drugs [40]. NGENCuB (1, 10 and 100 mM) was significantly more effective inhibiting both colony number Figure 5B). ...
For many years, ginger or ginger root, the rhizome of the plant Zingiber officinale, hasbeen consumed as a delicacy, medicine, or spice. Several studies have been conducted on the medicinal properties of ginger against various disorders, including cancer. Cancer is the second leading cause of death, and chemoprevention is defined as the use of natural or synthetic substances to prevent cancer initiation or progression. Evidence that ginger-derived compounds have inhibitory effects on various cancer cell types is increasingly being reported in the scientific literature. In this review we focused on the cancer chemopreventive effects of [6]-gingerol, the major pungent component of ginger, and its impact on different steps of the metastatic process.
... The applicability of the tumor colony-forming assay for drug screening has long been accepted to investigate new anti-tumor drugs [18]. Cedrelone was able to inhibit the colony formation in MDA-MB-231 in concentration as low as 1 M in almost 50%. ...
Introduction: Cancer is the second leading cause of death, preceded only by cardiovascular diseases, and epidemiological evidence demonstrates that this tendency is emerging worldwide. The number of cancer-related deaths is expected to increase 45% between 2007 and 2030. Excluding skin cancer, breast cancer is the most common malignancy among women, accounting for nearly 1 in 3 cancers diagnosed in the United States. Brazilian data confirm that breast cancer is the leading type of cancer in women and that, over the past 30 years, mortality has increased. Brazil has an extensive vegetal biodiversity with more than 55,000 species listed. Such biodiversity collaborates with the finding of compounds which could be the basis for the design of new anti-tumor drugs, with fewer side effects than the conventional chemotherapy used currently. Cedrelone is a limonoid isolated from Trichilia catigua (Meliaceae), a native Brazilian plant popularly known as “catuaba” or “catigua”.
Material and Methods: Cedrelone was isolated from the fruits of Trichilia catigua collected at Escola Superior de Agricultura Luiz de Queiroz (ESALQ), Piracicaba, Brazil. The aryls were reduced to powder and extracted with dichloromethane and methanol at room temperature. Dichloromethane fraction was chromatographed on a silica gel, similar fractions were grouped and fraction eight was recristalized and produced 323.4 mg of pure cedrelone. MDA-MB-231 human breast tumor and HF cell lines, obtained from ATCC, were maintained at 37°C in 5% CO2 in DMEM medium (Vitrocell) containing FBS 10%, penicillin (100 UI/ml), streptomycin (100 mg/ml) and L-glutamine (2 mM). For proliferation assays, the MTT method was used (Mosmann et al., 1983). Wound-healing migration assay was based on the repopulation of wounded cultures. Adhesion assays were performed in 96 well plates. Different concentrations of cedrelone were incubated with MDA-MB-231 cells before being plated on the wells, which were previously coated with a solution of collagen type I. Unbound cells were washed and adhered cells were stained with crystal violet solution. Absorbance reading was performed at 595nm. The effect of cedrelone on MDA-MB-231 tumor cell invasiveness was determined by the ability to transmigrate through a layer of matrigel in a transwell chamber (BD Biosciences). Zymography experiments were performed with supernatants of the wound healing assay which were collected and tested for MMP activity as previously described (Leber and Negus, 2001). Finally, the apoptotic activity of cedrelone on MDA-MB-231 cells was analyzed by flow cytometry with the FITC Annexin V Apoptosis Detection Kit (BD Biosciences).
Results: This study demonstrates that cedrelone inhibits proliferation, adhesion, migration and invasion of breast tumor cells from the line MDA-MB-231. The effects of cell migration and invasion on MDA-MB-231 cell may be explained, at least in part, by the ability of cedrelone to inhibit MMP activity. We also demonstrate that cedrelone is able to induce apoptosis in MDA-MB-231 cells. There are only a few works investigating the effect of limonoids in cellular processes closely related to tumor progression such as adhesion, migration and invasion. To the best of our knowledge, this is the first work describing the effects of a limonoid on tumor and non-tumor cell adhesion process. Furthermore, after nimbolide, this is the second work to demonstrate the effects of a limonoid on cell migration and invasion, in addition to the effect on MMP activity, and the first work performed with MDA-MB-231 breast tumor cell line.
Conclusion: Our results show that cedrelone could be the basis for the design of a new anti-tumor drug to be used in chemotherapy.
Citation Format: Amanda Blanque Becceneri, Angelina Maria Fuzer, Julio Cesar Conceição Filho, Paulo Cezar Vieira, João Batista Fernandes, Cristiane Cazal, Márcia Regina Cominetti. Effects of limonoid cedrelone on MDA-MB-231 breast tumor cells in vitro. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B10.
Guided by the molecular hybridization principle, a novel series of 4-chloropyridazinoxyphenyl conjugates (3a-h, 4a-e, and 5) was designed and synthesized as proposed apoptotic inducers and PARP-1 inhibitors. The growth inhibition % of the designed hybrids was investigated in eleven cancer cell lines, where the anticancer activities were found to be in the following order: 4-chloropyridazinoxyphenyl-aromatic ketones hybrids (3a-h) > 4-chloropyridazinoxyphenyl-benzyloxyphenylethan-1-one hybrids (4a-e) > 4-chloropyridazinoxyphenyl-thiazolidine-2,4-dione hybrid (5). Further, the most sensitive three cancer cell lines (HNO97, FaDu, and MDA-MB-468) were selected to measure the IC50 values of the new hybrids. Moreover, the frontier three members (3c, 3e, and 4b) were selected for the measurements of apoptotic protein markers (p53, BAX, caspase 3, caspase 6, BCL-2, and CK 18). Besides, the impact of compounds 3a-e and 4b on the activity of PARP-1 was investigated, where 3c, 3d, and 3e demonstrated comparable efficiencies to Olaparib. Furthermore, γ-H2Ax, a well-established marker for double-strand DNA breaks, was examined and the occurrence of DNA damage was observed. In addition, a significant inhibition of cell proliferation and a remarkable 15 to 50-fold reduction in the number of colonies compared to the control group was recorded. Finally, the PARP-1 inhibitory potential of the novel hybrids was compared to the co-crystal of the target receptor (PDB ID: 6NTU) using molecular docking.
The concept that continuous cell lines derived from one histological type of tumour provide an in vitro model system for that disease was examined. Biological characteristics and in vitro drug sensitivities of lines derived from transitional cell carcinomas (TCC) of the human bladder are described. The isozyme phenotype, cytological appearance, cell kinetics, colony-forming efficiency, tumorigenicity and monoclonal antibody reactivities of twenty-two urothelial lines were assessed. Three types were identified; 1) fourteen distinct lines derived from different patients, 2) five cross-contaminated with one of the distinct lines (T24) and 3) three non-tumorigenic lines. Xenograft morphology and isozyme pattern were used to select representative TCC cell lines. Ten clonogenic assay procedures for measuring cytotoxicity were compared using one cell line (RT112) exposed to adriamycin and methotrexate. Significant differences between dose-response curves were obtained for the same drug indicating methods should be standardised. The relative cytotoxicities of a 24h exposure to twelve chemotherapeutic drugs against RT112 cells were compared using a clonogenic assay. Drug concentrations reducing clonogenic cell survival by 10% ranged from 2.9 ng/ml for mitoxantrone to 27.0 pg/ml for hydroxyanisole. The range and reproducibility of in vitro sensitivities to adriamycin and methotrexate were compared using eight distinct lines and four lines cross-contaminated with T24. The cross-contaminated lines had similar sensitivities, demonstrating stability during long-term culture. However there was a broads pectrum of sensitivities (>50 fold) amongst the distinct lines to methotrexate and a narrow range (<3 fold) to adriamycin. In summary, these data show that cell lines derived from human bladder tumours can retain certain biological characteristics of the tumours of origin. Taken in conjunction with published data, these results indicate that cell lines also retain the pattern of drug sensitivities of the tissue of origin, and if the differences in pharmacokinetics in vivo can be taken into account, might be used for screening new agents.
Verapamil was evaluated as a chemosensitizer for reversing multidrug resistance in multiple myeloma both in vitro and in clinical trials. Bone marrows from 59 myeloma patients in relapse were evaluated for several resistance parameters: expression of p-glycoprotein (MDR1), doxorubicin (Adriamycin) and vincristine sensitivity, and the ability of added verapamil to reduce resistance to the cytotoxic agents. We found that verapamil was capable of sensitizing myeloma cells that exhibited resistance to doxorubicin and vincristine in vitro, but did not enhance sensitivity of cells that were drug sensitive (P less than .001). Myeloma cells expressing MDR1 immunohistochemically tended to be more doxorubicin resistant in vitro than MDR1-negative cells. In the clinical trials, 22 patients with myeloma refractory to vincristine- Adriamycin-dexamethasone (VAD) were treated with VAD plus high-dose intravenous verapamil (Ve). Among the 22 patients treated with VAD/Ve, five achieved a partial remission (23%). The median relapse-free survival for the VAD/Ve responders was 5.4 months and their overall survival from the start of VAD/Ve was better than that of the nonresponders. Among the subset of 10 patients whose myeloma cells were MDR1 positive, four responded clinically (40%), whereas none of five patients with MDR1-negative myeloma cells achieved remission with VAD/Ve. We also observed that myeloma cells from three of four VAD/Ve clinical responders exhibited in vitro chemosensitization with verapamil, whereas in vitro verapamil chemosensitization was seen in only one of six clinical nonresponders. Our observations demonstrate that clinical reversal of multidrug resistance can be achieved in some patients with VAD-refractory myeloma with the use of verapamil. In addition to their value in drug development, in vitro tests of MDR1 expression and of chemosensitizers plus cytotoxic drugs on the patients' bone marrow myeloma cells may identify patients who will respond clinically to chemosensitizer-containing regimens. We anticipate that chemosensitizer regimens capable of inhibiting multidrug resistance will play an increasing role in the treatment of hematologic malignancies, including B-cell neoplasms such as multiple myeloma and the non-Hodgkin's lymphomas.
Overview
Over the past 10 years, the approach to drug development has changed considerably. Owing to the enormous increase in insights into the molecular biology of cancer cells, a largely empirical approach has been mostly abandoned and replaced with a biology‐driven, hypothesis‐based, and translational approach. Notwithstanding this dramatic development, the hierarchical sequence of clinical drug‐development trials basically remained unchanged and is divided into three phases. This chapter provides an overview of the various steps of early clinical development with milestones of the drug‐development process, the role of the National Cancer Institute, and the switch to mechanism‐based drug development. It outlines challenges in the clinical development of these new agents and provides a fundamental understanding of the complexity involved in early clinical drug development.
This chapter is not intended to provide a comprehensive review of applications of the National Cancer Institute (NCI) 60-cell screen to anticancer drug discovery and development. The literature is now replete with such examples, given the NCI operation and provision of the screen to researchers worldwide for well over a decade. Selected examples are used here to illuminate the kind of output that has been routinely available from the screen and to show how some of the simplest applications of this output have been and perhaps remain of substantial utility to researchers engaged in the challenging and uncertain field of anticancer drug discovery and development. Readers may also wish to examine and consider the current operational details, as well as the wealth of related information and research tools based on the 60-cell screen, now provided by the NCI Developmental Therapeutics Program (DTP) at its internet website (http:// dtp. nci. nih. gov). What follows here is intended as an historical and personal perspective on how the 60-cell screen came to be and the value and legitimacy of the screen as a research tool. I attempt to convey a sense of the breadth and depth of the diverse participants and their contributions to the screen’ s conceptual development, implementation, and oversight, and I offer one participant’ s view of obstacles encountered, choices and compromises made, and other issues that may have contributed to the utility as well as limitations of the current screen.
Early clinical investigations in oncology are distinct from therapeutics evaluations in other medical disciplines. First, the risks associated with medicines for oncologic diseases dictate that they be evaluated in cancer patients only, and not in healthy volunteers. Second, dose-response relationships are essential to the understanding of potential therapeutic benefit and toxic side effects. Third, determining the specificity of therapeutic value for subsets of patients is essential to the development of effective therapies. For these reasons, a rational system of evaluation has been constructed to optimize the selection of tolerable and effective therapies for comparison with standard treatments in subsequent clinical trials.
Further advances in cancer biology and therapeutics depend, among other things, on the development of knowledge of the organization and biological potential of cells within tumors. Two widely supported but extreme views of tumor cell organization have been expressed: 1) the view of tumors as consisting of undifferentiated cells, each with potential for tumor development but with phenotypic heterogeneity imposed by genetic and epigenetic events. Also, often superimposed on this model is the notion of “de-differentiation” which implies that carcinogenesis can occur in differentiated cells which can subsequently re-express the phenotype of more primitive cells. 2) The view of tumors as representing a form of the normal differentiating tissue from which the tumor derived. This implies heterogeneity of cells within tumors with respect to differentiation, and also implies that the likely target for carcinogenesis is the stem cell population. Much of the support for the former view is based on study of transplantable tumors in animals while the importance of cell differentiation has been reinforced mostly by study of spontaneous animal or human tumors.
Therapeutic clinical trials are essential components of clinical research in oncology. They are superior to nonexperimental or observational or survey studies in their ability to answer important medical questions. Clinical trials are carried out under predetermined conditions, as defined in the treatment protocol. By specifying in this document the target group of patients, the treatment conditions, and the desired criteria of success or failure, potential sources of bias are minimized. The treatment evaluation is thus based on objective results rather than deductive reasoning (1).
Since the first description of the Dunning rat prostate tumor (Dunning 1963) several sublines of this tumor, originating from the dorsal lobe of rat prostatic tissue, have been described (Claflin and McKinney 1977; Isaacs et al. 1978; Isaacs et al. 1981; Smolev et al. 1977). One of these tumor lines, the MATLyLu tumor line which shows metastatic potential to lymph nodes and lung tissue (Isaacs et al. 1981), has been investigated over several years in our laboratory.
As an immunologist, my research interests had been focused on immunotherapy of cancer involving monoclonal antibodies as a mode of drug delivery and cytokines as therapeutic agents. I had no real interest or in-depth understanding of photobiology until September 1986, when I joined the Baylor Research Foundation (now Baylor Research Institute) where several photobiology related projects were already in progress. In one of these projects lead by Dr. Lester Matthews, it was demonstrated that in vivo chloroquine resistant parasitemia caused by P. berghei (resistant to at least three daily doses of 0.4 mg/kg of chloroquine) could be significantly reduced when treated with hematoporphyrin derivative (HPD) prior to chloroquine injection.1 Therefore, the following question was asked; if HPD plus chloroquine = death of the parasite, then what other compound (say X) plus HPD will induce death in tumor cells? Finding the answer to this question was one of my research projects, and it involved the determination of the dark toxicity of HPD in the presence of compound X against cultured tumor cells. This was the beginning of my inculcation into the field of photo-dynamic therapy.
Once a cancer has metastasized widely, chemotherapy would be expected to offer the best hope for control. However, there are few agents with a high degree of efficacy against the common cancers, and continued drug discovery remains a priority in cancer research. The development of new methods for testing anticancer drugs constitutes a rapidly evolving and, at times, controversial field. The frustrations experienced in identifying useful new compounds arise in part from the subtlety of the biochemical differences between normal and neoplastic cells, and the difficulties encountered in developing model systems for screening and testing experimental compounds1—4. In vivo screens, using rapidly growing transplantable murine tumours such as the P388 and L1210 leukaemias, in general have proved disappointing in identifying agents effective in the treatment of carcinomas. Whereas these classical murine tumours have identified agents active against rapidly proliferating tumours, there has been little impact on long-term survival rates for patients with the common malignancies which are characterized by low growth fractions.
Numerous methods exist to measure drug and radiation resistance in cell culture. Each method has committed proponents, but no method has been widely accepted for clinical application. Virtually all of the methods are based on sound biologic principles and are capable, in my view, of providing useful clinical information. However, I have also observed that even experienced investigators (and grant reviewers) tend to have unrealistic expectations and an incomplete understanding of the biologic and statistical principles relating to these assays. My message to investigators and reviewers alike will be to (1) understand the principles, (2) have realistic expectations (i.e. look to use the information as a tool for a clinical improvement which may fall short of a revolutionary breakthrough), (3) realize that calibration of these assays for clinical application is a very slow process involving considerable trial and error, and (4) demand convincing validation, but do not erect insurmountable validation barriers which prevent utilization of the assays as a tool for clinical improvement. While I am not a radiobiologist, I will also attempt to address the issue of cell culture assays for radiation resistance. Other reviews relating to assays for both radiation (Peters et al., 1984; Peters et al., 1988; Elkind, 1988) and drug resistance (Weisenthal and Lippman, 1985; Weisenthal, 1981; Von Hoff and Weisenthal, 1980; Taetle and Koziol, 1985; Von Hoff, 1987; McGuire et al., 1988; Weisenthal et al., 1988; Von Hoff, 1989; Ichihashi, 1989; Von Hoff, 1988; Von Hoff, 1990; Dendy and Hill, 1983; Hofmann et al., 1984) are available elsewhere. In vivo assays, such as the subrenal capsule assay (Bogden et al., 1988) and nude mouse xenograft assays (Guichard, 1989; Leonetti et al., 1989;Mattern et al., 1988;Bamberg et al., 1988; Houghton and Houghton, 1983) are beyond the scope of this discussion.
The problem of curing cancer is steadily coming into sharper focus and the picture emerging is the challenge of dealing with metastatic cancer in new ways. Of the 870,000 new cases of cancer each year, approximately half will be localized at presentation. The majority of these, about 400,000 patients per year, will be cured with either surgery or radiotherapy. The remainder will require some form of systemic therapy, either to prevent recurrence (adjuvant therapy) or to treat actual recurrent disease. Of this number about 50,000 will be cured. The remainder, constituting 50% of patient with cancer, will die. The primary cause of their deaths will be metastases.
Since 1955, the US National Cancer Institute (NCI) has provided screening support to cancer researchers worldwide. Until 1985, the NCI screening program and the selection of compounds for further preclinical and clinical development under NCI auspices had relied predominantly on the in vivo L1210 and P388 murine leukemias and certain other transplantable tumor models (1). From 1975–1985, the in vivo P388 mouse leukemia model was used almost exclusively as the initial or primary screen. With few exceptions, agents that showed minimal or no activity in the P388 system were not selected by the NCI for further evaluation in other tumor models or alternative screens. Most of the available clinical anticancer agents are active in the P388 system; however, most were discovered prior to 1975 or by observations initially in test systems other than the NCI-operated P388 primary screen.
This chapter discusses the use of mechanism-based assays to detect the potential antitumor drugs that interfere with processes relevant to the neoplastic growth and cites examples of compounds identified by such approaches. Agents in preclinical development, discovered by more traditional approaches, are also included, with examples limited to those drugs that demonstrate a degree of preclinical efficacy that would appear to warrant the development to clinical trial. Compounds that have recently entered Phase I clinical trial are also included. Many of these clinical candidates have demonstrated only the modest activity in preclinical tumor models as has been the case for majority of the drugs developed for cancer in the past. Several clinical candidates, however, have shown a spectrum and degree of preclinical efficacy comparable or superior to that of the most effective established antitumor agents. Significant progress has been made in adopting new strategies for the discovery of novel antitumor drugs using mechanism-based assays. Such approaches apply to the targets of the cyiotoxic drugs with established selectivity for tumors as well as to the targets evolving from the study of oncogenes and tumor cell biology. A number of other targets beside those mentioned in the chapter can be exploited. Examples are: recombination pathways and deoxyribonucleic acid (DNA) damage repair, proteases and adhesion molecules involved in invasion and metastasis, endothelial receptors for tumor-derived angiogenesis factors, and protein kinases and phosphatases involved in the control of cell division. Major advances in the understanding of each of these areas are likely to provide further opportunities for drug discovery. It should be noted, however, that the less selective biological screens for new antitumor agents discussed in this chapter continue to identify the novel compounds with interesting activity and selectivity in animal tumor models and, hopefully, in cancer patients. These novel compounds could lead to the identification of new targets relevant to cancer therapy.
The development of methods for growing human cancer cells in culture has raised the question of whether it might be possible to use culture methods to model all the features important for clinical antitumor activity. Cell culture techniques play a key role in the development of new anticancer drugs by imposing additional constraints on those of receptor interaction alone, such as drug uptake and efflux, interaction with other cellular receptors, and cellular metabolism. Clonogenic assays, integrating multiple cell death pathways, are particularly useful in measurement of cell survival following exposure to cytotoxic drugs. On the other hand, microcultures, combined with colorimetric and other methods for measuring antiproliferative effects, have provided the basis for large-scale screening of cytotoxic and cytostatic drugs. The U.S. National Cancer Institute program has made it possible to use 60 or more cell lines to compare the growth inhibition profiles of potential new drugs with those of tens of thousands of previously tested compounds, providing information on mechanism of action as well as antiproliferative activity. Some of the most promising new approaches include the use of three-dimensional cell cultures to allow the measurement of rates of drug diffusion, as well as the assessment of the effects of cell contact and tumor microenvironment on drug sensitivity.
Cancer stem cells (CSC) are recently proposed to be the cancer initiating cells responsible for tumorigenesis and contribute to cancer resistance. Like normal stem cells, cancer stem cells should be rare, quiescent and capable of self renewing & maintaining tumor growth and heterogeneity. Although the concept of cancer stem cells originates from that of normal stem cells, cancer stem cells are not necessarily aberrant counterparts of normal stem cells. In fact, they may arise from stem cells or committed progenitors of corresponding tissues & even cells from other tissues. At the molecular level, the alteration of stem cell self-renewal pathways has been recognized as an essential step for cancer stem cells transformation. Better understanding of cancer stem cell will no doubt lead to new era of both basic & clinical research, reclassification of human tumors & development of novel therapeutic strategies specifically targeting cancer stem cells.
Biodegradable nanoparticles exhibit high potentialities for local or systemic drug delivery through lung administration making them attractive as nanomedicine carriers. However, since particulate matter or some inorganic manufactured nanoparticles exposed to lung cells have provoked
cytotoxic effects, inflammatory and oxidative stress responses, it becomes important to investigate nanomedicine toxicity towards the lungs. This is the reason why, in the present review, the behavior of biodegradable nanoparticles towards the different parts of the respiratory tract as well
as the toxicological consequences, measured on several models in vitro, ex vivo or in vivo, are described. Taken all together, the different studies carried out so far conclude on no or slight toxicity of biodegradable nanoparticles.
Image-processing programs are used to identify and classify eukaryotic cell colonies as spots following seeding at low density on dishes or in multiwell plates. The output from such approaches, however, is generally limited to 1-2 parameters, and there is no ability to extract phenotypic information at the single colony level. Furthermore, there is a lack of user-friendly pipelines for analysis of clonogenicity in the context of high-content analysis. This article describes an experimental and multiparametric image analysis workflow for clonogenic assays in multiwell format, named the Colony Assay Toolbox (CAT). CAT incorporates a cellular-level resolution of individual colonies and facilitates the extraction of phenotypic information, including the number and size of colonies and nuclei, as well as morphological parameters associated with each structure. Furthermore, the pipeline is capable of discriminating between colonies composed of senescent and nonsenescent cells. We demonstrate the accuracy and flexibility of CAT by interrogating the effects of 2 preclinical compounds, Nutlin-3a and ABT-737, on the growth of human osteosarcoma cells. CAT is accessible to virtually all laboratories because it uses common wide-field fluorescent microscopes, the open-source CellProfiler program for colony image analysis, and a single fluorescent dye for all the segmentation steps.
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Pluripotent cells can be grown in clonogenic assays. The tumour stem-cell fraction, which accounts for <0.4% of the total cells, and which is considered the most relevant cell type in the development of metastases and recurrences, is able to divide and to form colonies in a semisolid matrix (agar or methylcellulose). Major applications of the tumour clonogenic assay (TCA) are chemosensitivity testing of tumours and xenografts, and for assessments within drug discovery programmes. Of critical relevance for the usefulness of the TCA is whether it can predict sensitivity or resistance towards clinically used agents. When we compared the response of human tumours established as xenografts in nude mice in the TCA in vitro to that of the clinical response, 62% of the comparisons for drug sensitivity, and 92% of the comparisons for drug resistance were correct. The same percentage of true/false observations was found when tumours were tested after serial passage in nude mice in the TCA in vitro and their response compared to in vivo activity in corresponding xenografts (60% and 90%, respectively). The highest correct predictive values were, however, found when the clinical response of tumours was compared to their explants established in the nude mouse and treated in vivo. Of 80 comparisons performed, we observed a correct prediction for tumour resistance in 97% and for tumour sensitivity in 90%. In our opinion, the TCA with established human tumour xenografts has an important role in current drug discovery strategies. We therefore included the TCA as secondary assay in our approach to anticancer drug discovery and found that a number of novel agents were active; these are now in advanced preclinical development or clinical trials. Thus, the tumour clonogenic assay has proven predictive value in the chemosensitivity testing of standard and experimental anticancer drugs.
Primary cultures of cells from patients with chronic lymphocytic leukemia (CLL) and ovarian carcinoma (Ovca) were compared with the renal carcinoma ACHN and the lymphocytic CCRF-CEM human tumor cell lines in response to 63 toxic or nontoxic compounds. The experiments were conducted at 1, 10, and 100 &mgr;/ml in 96-well microtiter plates for an assay time of 72 h. The plates were analyzed by the fully automated Dynatech Immuno Assay System (DIAS) using Alamar blue as a fluorometric/colorimetric indicator of metabolic activity. Drug response data were reported as the area under the tumor cell survival-concentration curve (AUC). Noncytotoxic compounds were classified as inactive by all cell systems. According to the AUC, CCRF-CEM and CLL cells were the most sensitive, followed by ACHN, and then Ovca. Many of the clinically active drugs were detected by all cell systems. However, the sensitivity pattern differed considerably between the cell types as judged from correlation analysis, and a higher proportion of clinically inactive drugs were scored as active by the cell lines compared with the primary cultures. Ovca showed the highest ratio of clinically solid tumor active/clinically inactive agents followed by CLL. The results indicate that primary cultures of human tumor cells may be a useful and valuable model for anticancer drug screening.
Syntheses based on podophyllotoxin have led to the inclusion among antitumoral agents valuable in practice of the preparations
etoposide(I) and teniposide(II). These compounds are, respectively, the 4,6-ethylidene and 4,6-thenylidene derivatives of
4′-dimethylepipodophyllotoxin β-D-glucopyranoside. A review has been made of methods of synthesizing (I) and (II) and for
their experimental investigation. Some conclusions are given from the results of clinical trials. Although (I) and (II) have
been accepted for practical use, their clinical activity is limited. The principles of the experimental selection of antitumoral
agents are considered particularly. Considerations are expressed concerning the possibilities of improving the results of
the search for antitumoral agents of plant origin.
Attempts to develop in vitro assays to predict in vivo response to antineoplastic therapy in patients date back more than 40 years for radiotherapy and more than 25 years for chemotherapy (Von Hoff and Weisenthal 1980; Weisenthal 1981; Weisenthal and Lippman, to be published). More than 2,500 individual clinical correlations between assay results and results of therapy have been published. The combined average of the accuracy of “true positive” results of these tests is 66%, while the “true negative” accuracy average is 90%. The published data do not indicate that any one type of approach to in vitro chemosensitivity testing has superior validity over all other approaches.
A prospective clinical trial was performed to evaluate the usefulness of a human tumor cloning system for selecting single- agent chemotherapy for patients with advanced cancers. Six hundred four single-agent trials were performed in the 470 patients whose tumors were submitted for drug sensitivity test ing. Only 246 of these 604 trials (41%) could be directed by the cloning system results because of inadequate tumor growth and other difficulties. In these 246 prospective trials, there was a 60% true positive and an 85% true negative rate for predicting for response or lack of response of an individual patient's tumor to the single agent. There was also a relationship between the percentage of decrease in survival of tumor colony-forming units and the probability of a clinical response of the patient's tumor to the same drug used in vivo. Despite these encouraging findings, work to improve tumor growth and additional prospec tive clinical trials of the system are needed before the system can be recommended for routine clinical use.
Two techniques for growing colonies of human tumour cells in soft agar have been applied to cell suspensions derived from fresh tumour tissue from 48 patients. Colonies were obtained in 31 cases, with plating efficiencies between 0.01 and 15%. In 11 cases the plating efficiencies were 1% or above. There was evidence that some categories of tumour grew more readily than others under these conditions. The potential applications of the methods to clinical and experimental oncology are discussed.
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A potential application of the human tumor stem cell colony assay is to guide Phase II clinical investigations by identifying classes of tumors (or individual patients) which are sensitive in vitro to a new antitumor compound. We have tested human tumor stem cells from 140 tumor biopsies representing 20 different tumor types for chemosensitivity to the Phase II drug 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was defined as a reduction in the number of tumor colony-forming cells to 30% of the control or less after a 1-hr exposure to one-tenth of the pharmacologically achievable plasma concentration of 4'-(9-acridinylamino)methanesulfon-m-anisidide. In vitro sensitivity was found in 29 cases: non-Hodgkin's lymphoma (2 of 2); cervical carcinoma (1 of 1); sarcoma (3 of 6); neuroblastoma (1 of 2); acute myelogenous leukemia (6 of 16); chronic myelogenous leukemia (1 of 3); melanoma (8 of 34); uterine carcinoma (1 of 5); lung carcinoma (1 of 9); ovarian carcinoma (4 of 36); and breast carcinoma (1 of 11). Prospective in vitro-in vivo correlations in eight patients with various tumor types showed that three of three patients sensitive in vitro to 4'-(9-acridinylamino)methanesulfon-m-anisidide responded in vivo, while five of five patients resistant in vitro had no clinical response. The results provide support for further evaluation of the utility of the human tumor stem cell colony assay for targeting Phase II clinical trials.
We have examined the in vitro sensitivity to Adriamycin of human ovarian cancer colonies cloned in soft agar. In the 26 patients tested, 3 different patterns of sensitivity to Adriamycin were observed: (a) in 75% of the previously untreated patients, there was greater than 70% reduction in colony-forming cells after exposure to Adriamycin (1.0 micrograms/ml), a level which approximates the peak plasma level after i.v. therapy; (b) in all the patients who had progressive disease while on a chemotherapy regimen without Adriamycin, a greater than 70% reduction in colony-forming cells was observed only at a concentration of 10 micrograms/ml, a level not achievable by i.v. administration; (c) in 80% of patients with progressive disease after treatment with Adriamycin as part of the primary chemotherapy regimen, a 70% reduction in tumor colony-forming cells could not be achieved even at 10 micrograms/ml. These in vitro results are in agreement with clinical observations regarding the effectiveness of Adriamycin in previously untreated patients (42% response rate) with ovarian cancer as well as its ineffectiveness (0 to 6% response rate) as a second-line therapy in relapsed patients. The results also have provided a rationale for an ongoing Phase I trial of i.p. Adriamycin in patients with ovarian cancer from Group b above since cytotoxic levels can be produced i.p. using large-volume dialysis via a Tenckhoff dialysis catheter. The relative cytotoxicity of Adriamycin to its two major metabolites, adriamycinol and adriamycin aglycone, was also determined in the clonogenic assay. Both derivatives produced suppression of ovarian cancer colony formation; however, Adriamycin was more cytotoxic that was either metabolite.
To study the usefulness of an in vitro colony-forming assay in predicting individual clinical responses to chemotherapy, tumor cells obtained from 150 melanoma metastases (119 patients) were grown in soft agar according to the method of Courtenay and Mills (1978), and tested for sensitivity to DTIC, CCNU, vinblastine, procarbazine, abrin and ricin. In 83% of the cases colony formation was observed (plating efficiency, PE, greater than 0.01%). Twenty-seven per cent of the tumors gave PEs ⪖1%, 45% gave PEs in the range 0.1-0.9%, whereas 11% of the tumors gave 0.01-0.09%. The PEs were not correlated with the degree of pigmentation or with the clinical course. Evaluable chemosensitivity data were obtained on 104 metastases from 83 patients. Large differences in sensitivity were seen. In cases which were evaluable both in vivo and in vitro a clear correlation was found between the in vitro chemosensitivity, expressed as the expected growth delay, and the clinical response to chemotherapy. Tumors from patients with partial response, mixed response or stable disease after prior progression, all had rather high in vitro sensitivity to the drug used (expected growth delay ⪖2.0), whereas patients with progression had lower sensitivity. The results confirm that the soft agar method used here provides good culture conditions for human melanoma cells and show that chemosensitivity data can be obtained in a high percentage of melanoma patients. The approach used seems promising in aiding clinicians to adjust chemotherapy to individual patients.
With a direct in vitro tumor-colony assay developed to measure sensitity of human-tumor stem cells to anticancer drugs, we performed 32 retrospective or prospective clinical studies in nine patients with myeloma and nine with ovarian cancer treated with standard agents that were tested in vitro. The results were clearly correlated (P is less than 0.00001). Unique patterns of sensitivity and resistance to the six drugs tested were observed for individual patients. In eight cases of myeloma and three of obarian carcinoma in vitro sensitivity corresponded with in vivo sensitivity whereas in one case of myeloma it did not. In vitro resistance correlated with clinical resistance in all five comparisons in myeloma and all 15 in ovarian cancer. We conclude that this assay shows sufficient promise to warrant larger-scale testing to determine its efficacy for selection of new agents and individualized cancer chemotherapy regimens.
A simple method has been developed to support human tumor stem cell colony growth in soft agar. The technique appears suitable
for culture of a variety of neoplasms of differing histopathology. Tumor stem cell colonies arising from different types of
cancer have differing growth characteristics and colony morphology. This bioassay should be suitable for clinical studies
of effects of anticancer drugs or irradiation on human tumor stem cells.
The effect of mechanical and enzymatic disaggregation on human malignant melanoma, soft-tissue sarcoma and lung carcinoma colony growth in soft agar was studied. The enzymatic disaggregation was advantageous in most cases of melanoma and sarcoma, giving a larger number of colonies and increasing the probability of achieving growth in soft agar. Enzymatically treated pulmonary carcinoma cell populations had lower clonogeneic potential, especially in the case of anaplastic carcinomas. Morphological studies showed that the cells growing in soft-agar colonies had the same characteristics as those of the original tumor. A linear relationship was obtained between the number of enzymatically and mechanically treated tumor cells plated and the number of colonies. Delayed plating decreased the number of colonies.
Data are presented from two cell culture systems which support the notion that a multi-drug-resistant phenotype occurs in human tumor cell populations. Human small cell lung cancer cell lines derived from patients in relapse following intensive combination chemotherapy demonstrate broad cross-resistance to nine standard drugs in vitro. However, analysis of [14C]glucosamine-labeled glycoproteins in the small cell lung cancer cell lines failed to identify any consistent association between a specific glycoprotein marker and the drug-resistant phenotype. Evaluation of drug sensitivity of human tumor cells in primary culture (colony-forming assay) has indicated that multidrug-resistant cells may be present in tumor cell populations even in the absence of prior drug therapy. Several features of the multidrug-resistant phenotype, as observed in these human tumor cell populations, differ from those observed in Chinese hamster cell systems. In particular, the variability in patterns of resistance to various agents and in expression of glycoprotein markers suggests that a substantial amount of genetic heterogeneity underlies this phenotype in human tumors.
Multidrug resistance is a complex pleiotropic phenotype of cross-resistance and collateral sensitivity to unrelated compounds observed in many mammalian cell mutants selected for resistance to single agents. In Chinese hamster ovary cells, colchicine-resistant mutants expressing this phenotype have been characterized extensively. Such mutants arise apparently from a single genetic event, and the basis of this phenotype appears to be localized at the membrane level, resulting in altered drug permeability. Expression of a 170,000-dalton surface glycoprotein (P-glycoprotein) has been identified to correlate with the multidrug-resistance phenotype. Selection of a second mutation in colchicine-resistant mutants, for resistance to phytohemagglutinin, results in an alteration of the carbohydrate moiety in P-glycoprotein and other surface components. This mutation does not noticeably affect the multi-drug-resistance phenotype. The altered permeability of mutant cells to drugs, however, can be modulated by nonionic detergents or metabolic inhibitors. These findings are consistent with a molecular mechanism of multidrug resistance whereby the pleiotropic response of the cell is mediated by an overexpression of a cell-surface protein, the P-glycoprotein.
Studies of Chinese hamster and mouse cells with high levels of acquired resistance to dactinomycin, daunorubicin, or vincristine have shown that these different permeability mutants all display similar phenotypic alterations: dramatic changes toward normal cell morphology and growth behavior and substantially reduced oncogenic potential. All three resistant cell types show increased expression of a high molecular weight plasma membrane glycoprotein species, gp150. Uniquely, vincristine-resistant cells contain gene amplification-associated chromosome abnormalities (homogeneously staining regions or double minute chromosomes), and they oversynthesize a low molecular weight cytosolic protein (V19). Cells grown in the absence of drug are phenotypically unstable. Revertant cells decline in resistance, length or number of homogeneously staining regions or double minute chromosomes, and expression of gp150 and V19. These proteins are thus candidate products of amplified genes which may or may not be manifested cytogenetically. The phenomena of drug resistance and reverse transformation are currently being addressed in protein phosphorylation studies.
The human tumor stem cell assay (HTSCA) provides a means of performing drug sensitivity measurements on human tumor cells in primary culture. Results from such assays offer potential for improving cancer chemotherapy by identifying drugs useful for treatment of individual patients' tumors and through application to screening new compounds for antitumor activity. While existing data supports the potential of the assay in both areas, the assay also poses significant drawbacks. Many of these drawbacks relate to technical aspects of the assay and can be eliminated or reduced by further assay development. In this paper, we describe some of the technical drawbacks in detail and some approaches which have been successful in minimizing them. Continued advances in this area should make it possible to more fully realize the potential of the human tumor stem cell assay.
An in vitro clonogenic assay was used to test the chemosensitivity of the human medulloblastoma cell line TE-671. Dose-response relationships for reduction in colony formation were generated for cyclophosphamide, vincristine, Adriamycin, 1,3-bis(2-chloroethyl)-1-nitrosourea (NSC 409962), and 1,4-cyclohexadiene-1,4-dicarbamic acid, 2,5-bis(1-aziridinyl)-3,6-dioxo-diethylester (NSC 182986); and the in vitro drug dose at which there is a 75% reduction in the number of colonies in comparison to controls (ID75S) were derived from these data. Methotrexate produced no colony reduction at any dose tested up to 1000 micrograms/ml. The in vitro results were compared to growth delays in s.c. TE-671 xenografts in athymic mice treated with the same agents. Agents with an ID75 less than assumed in vivo plasma drug concentrations were all active in vivo, whereas two of the three agents with an ID75 greater than assumed in vivo plasma drug concentrations demonstrated no in vivo activity. These results suggest that for these agents, the relationship between the ID75 of the drug and its in vivo concentration allows in vitro clonogenic assay results to agree with in vivo growth delay responses.
To improve clinical interpretation and use of in vitro clonogenic assay results, the authors reviewed their experience to date with chemosensitivity testing of over 1500 solid tumors. All clonogenic assays were performed using a double-layer-soft-agar system with continuous exposure of cells to one concentration of standard anticancer drugs. Significant growth was defined as greater than or equal to 30 colonies/control plate. Clinical responses were determined according to standard criteria. Data were analyzed using two different criteria of in vitro sensitivity (greater than or equal to 50% and greater than or equal to 75% inhibition of colony formation) and independently for each histologic type of tumor. Overall, 68% of specimens plated produced significant growth in vitro. Cloning ability varied from 57% to 82% depending on tumor histology. The assay was 57% reliable for predicting in vivo sensitivity, and 92% reliable for in vivo resistance. Predictive accuracy for sensitivity varied from 30% to 86%, depending on the tumor histology. Use of greater than or equal to 50% ICF (inhibition of colony formation) as criteria for differentiating sensitivity from resistance proved most reliable, although criteria should be individualized for each tumor type to maximize predictive accuracy.
In vitro studies of the schedule dependency and cytotoxicity of didemnin B, a novel depsipeptide isolated from a Carribean tunicate (Didemnidae), were carried out in fresh tumor cells obtained from biopsies from 39 cancer patients using the human tumor stem cell assay. Two schedules of drug exposure were examined (1 h and continuous exposure in the agar). Tumor cells from nine of 26 patients (34.6%) showed reduced survival of tumor-colony forming units to 30% of control or less at the 0.01 microgram level in the continuous exposure studies. Cells from eight of 17 patients (47%) showed a similar degree of sensitivity to didemnin after a 1-h exposure to 0.1 microgram prior to plating. The median ID50 values were 4.2 X 10(-3) micrograms/ml and 46 X 10(-3) micrograms/ml for continuous and 1-h exposures, respectively. A clear dose-response relationship was observed with both dosage schedules. Comparison of the slopes of the continuous and 1-h exposures and of the ID50 of the drug schedules suggests that didemnin is a cell-cycle-non-specific cytotoxic agent. Significant in vitro antitumor activity was observed at low concentrations against carcinomas of the breast, ovary, and kidney, and also mesothelioma and sarcoma. These results can provide pharmacologic goals to be achieved in phase I clinical trial. Further in vitro testing should help select tumor types for study in phase II trials of this very promising new anticancer drug.
Ten anthracyclines, including doxorubicin (DX) and daunorubicin (DNR), and eight analogs with modifications in structure or stereochemistry of the aglycone and/or the aminosugar moiety were simultaneously tested in serial vitro titration studies against human adenocarcinomas in the human tumor stem cell assay. More than a two-log range in cytotoxicity of the various anthracyclines was observed with the tumors tested. Marked individual differences in sensitivity of specific tumors (breast, lung, peritoneal) were observed for the various analogs. By assessing average effects on survival of tumor colony-forming units (TCFU) in the tumors tested, the three compounds lacking the methoxyl group in position 4 of the aglycone (4-demethoxyDX, 4-demethoxy-4'-epiDX, 4-demethoxyDNR) all proved to be more cytotoxic than their parent compounds. Compounds modified in position 4' of the aminosugar were on average either as toxic (4' epiDX) or more toxic (4'-deoxyDX and 4'-0-methylDX) to TCFU than the parent compound DX. On average, 11-deoxyDX was less toxic than DX or the other eight anthracyclines tested. The results obtained are also in good general agreement with those previously reported for anthracyclines with human tumors in xenografts or cancer patients. These antitumor results viewed in concert with toxicology studies in normal mice (including evidence of a lack of cardiac toxicity) suggest that 4'deoxyDX may prove to be a clinically useful anthracycline analog. We also conclude that use of this clinically predictive in vitro soft agar assay provides a rapid and relatively inexpensive means of simultaneously testing a large number of analogs of a parent compound against a spectrum of human tumors.
The National Cancer Chemotherapy Program was initiated in 1955. It is administered and integrated by the Division of Cancer Treatment at the National Cancer Institute. The program involves the discovery and development of potential new antitumor agents; their screening in preclinical experimental systems for antitumor effect, and, if active, for toxicology; and, for selected agents, preliminary, and more definitive clinical trials. While serendipity and empiricism played a substantial role in the early years of the program, the program has increasingly emphasized and been influenced by advances in tumor biology, drug development, clinical pharmacology, and the science of clinical trials. There has been effective interaction between investigator-initiated research on the one hand and developmental research at preclinical and clinical levels on the other. Over 30 chemotherapeutic agents with substantial clinical antitumor activity have been discovered, and their proper use, often in combination and often integrated with surgery or radiotherapy, has resulted in significant progress in the effective treatment of many forms of cancer.
Of 1,014 human solid tumors of various histologic types, 690 (68%) showed evidence of colony formation within 2 to 4 weeks. Tumors which grew particularly well were colon carcinoma (104/175), melanoma (134/155), lung carcinoma (62/85), breast cancer (100/140), ovarian carcinoma (50/67), and sarcoma (72/122). Histologic examination indicated that the colony-forming cells retained functional and morphologic features similar to those of the original tumor. Plating efficiencies varied between 0.01% and 0.2%, and the numbers of colonies observed formed a direct linear correlation with the number of cells plated. Recovery of viable tumor cells was increased when enzymatic tumor dissociation was used rather than a mechanical method. A simplified, supplemented medium resulted in improved cloning efficiencies when compared to previously reported methods (Hamburger and Salmon, 1977 b).
We have utilized a recently developed human tumor cloning system to screen for antitumor effects in vitro of a new anthracenedione derivative, Mitoxantrone. The object was to determine if the system is useful for pinpointing the types of tumors in patients which should be studied in early Phase II clinical trials. Tumors from 267 patients were placed in culture (20 different histological tumor types). One hundred seventy tumors both grew and formed enough colonies for drug sensitivity assays. Excellent in vitro antitumor activity was noted for Mitoxantrone against human adenocarcinoma of the lung, small cell lung cancer, melanoma, and biliary tree cancer. Good antitumor activity was noted against breast cancer, ovarian cancer, non-Hodgkin's lymphoma, head and neck cancer, squamous cell lung cancer, soft tissue sarcoma, gastric cancer, and hepatomas. The drug showed no in vitro activity against colon cancer. These data indicate that Mitoxantrone has a wide spectrum of in vitro antitumor activity. A comparison of these in vitro results with the results of Phase II clinical trials with the drug should allow an evaluation of the utility of the human tumor cloning system for predicting clinical antitumor activity of a new compound.
The successful development of a soft agar clonogenic assay for human tumor stem cells provides an in vitro technique with a high degree of accuracy for predicting in vivo clinical response to standard anticancer drugs. We used this system to conduct an "in vitro phase II trial" in human ovarian cancer and melanoma. This approach can potentially identify active phase I--II drugs suitable for treatment of given tumor types for specific patients and eliminates the need to subject patients (who would be predicted not to respond) to toxic side effects. In vitro sensitivity for new agents was operationally defined as at least a 70% reduction of tumor colony-forming units (TCFU) at concentrations which are readily achievable pharmacologically. The new agents AMSA and vindesine (as well as vinblastine) appeared to have activity in melanoma, while PALA and thymidine were inactive. Pentamethylmelamine, mitomycin C, methyl-GAG, and AMSA were relatively ineffective in ovarian cancer. Vinblastine and vindesine had definite activity. The human tumor stem cell assay may thus provide the basis for a useful alternative to the current clinical phase II testing approach for identifying antitumor activity of new agents. Validation of this concept with correlative in vitro and in vivo phase II trials of new agents in patients with tumor types predicted to be sensitive is clearly warranted.
An in vitro tumor colony-forming assay was utilized to measure the sensitivity of 800 individual patients' tumors to a variety of antineoplastic agents. Thirty-six separate histologic types of cancer were represented. Only 199 of the 800 patients' tumors (25 percent) both formed colonies in vitro and had enough cells in the biopsy or fluid specimen to perform drug sensitivity assays. In 123 instances the drug tested in vitro against the tumor was also used clinically to treat the patient. The clinician caring for the patient did not know the results of the in vitro test. When analyzed in a retrospective manner, the probability of a positive prediction from the assay, given the patient responded clinically, was 0.88. The probability for a negative prediction of the assay given the patient did not respond, was 0.94. Associations of in vitro and in vivo results in the 123 correlations were highly significant (p less than 0.001). We conclude that, as now constituted, the human tumor colony-forming assay can provide useful sensitivity information for only about 25 percent of the general oncology patients. Secondly, a prospective clinical trial of the assay is needed to insure that the assay is indeed predictive of which drug will produce a patient response and that it is not merely an indicator that a particular patient's tumor is highly responsive in vivo.
We have utilized a recently developed human tumor cloning system to screen for antitumor effects in vitro of a new anthracene derivative, CL216,942. The object was to determine whether the system is useful for pinpointing the types of tumors in patients which should be studied in early phase II clinical trials. Tumors from 684 patients were placed in culture (27 different histologic tumor types). Two hundred seventy-three tumors both grew and formed enough colonies for drug sensitivity assays. In vitro antitumor activity was noted for CL216,942 against human breast cancer, ovarian cancer, renal cancer, squamous cell, small cell and large cell lung cancer, lymphoma, acute myelogenous leukemia, melanoma, adenocarcinoma of unknown origin, adrenal cancer, gastric cancer, pancreatic cancer, and head and neck cancer. The drug definitely showed no in vitro activity against colon cancer. These data indicate that CL216,942 has a wide spectrum of in vitro antitumor activity. A comparison of these in vitro results with the results of phase II clinical trials with the drug should allow an evaluation of the utility of the human cloning system for predicting clinical activity of a new compound.
Objective methods have been developed to quantitate results of the in vitro human tumor stem cell assay, the degree of the association between the in vitro assay and clinical response as well as the likelihood of response. Methods considered to quantitate in vitro assay data included first-order kinetics of percent survival with drug concentration, minimal percent of tumor colony-forming unit survival at low drug concentrations, and area under the in vitro percent survival-drug concentration curve. Based upon experimental data, the percent tumor colony survival and the area under the curve (i.e., in vitro sensitivity indices) were concluded to better account than other methods for the commonly observed nonlinear shape of the in vitro curves. The two methods also yielded equivalent quantitative descriptions of the in vitro data. A logistic regression model was used for explicit quantitation of the relationship between the in vitro sensitivity index and predicted probability of clinical response. Very high association was observed between the predicted in vivo and actual clinical response for the cytotoxic drugs considered. Incorporation of other pharmacologic and patient prognostic factors into the quantitative methods is discussed and shown to improve their effectiveness.
Letter to the editor
- M S Aapro
- J R Eliason
Aapro, M. S., and Eliason, J. R. Letter to the editor. N. Engl. J. Med., 308: 1478-1479,1983.
In vitro Phase II evaluation of cisplatm and three analogs against various human carcinomas
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- G D Wilbanks
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- J E Graham
- C G Kittle
- S G Economou
Rossof, A. H., Johnson, P. A., Kimmell, B. D., Wilbanks, G. D., Yordan, E. L., Graham, J. E., Kittle, C. G., and Economou, S. G. In vitro Phase II evaluation of cisplatm and three analogs against various human carcinomas. Proc. Am. Soc. Clin. Oncol., 2: 37,1983.
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- L M D E Weisenthal
- C P Page
- A B Crux
- J F Sandbach
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- P Selby
- R N Buick
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Potentials to rationale and methods.Semin. colony formingassay results Prospective clinical trial of a human tumor cloning system
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American Association for Cancer Research Copyright © 1985 WITH A HUMAN TUMOR COLONY-FORMING ASSAY Growth of human tumour cell colonies from biopsies using two soft-agar techniques
American Association for Cancer Research
Copyright © 1985
WITH A HUMAN
TUMOR
COLONY-FORMING
ASSAY
Growth of human tumour cell colonies from biopsies using two soft-agar
techniques. Br. J. Cancer, 38: 77-81,1978.