Modifying the tumour microenvironment and reverting tumour cells: New strategies for treating malignant tumours

The tumour microenvironment (TME) plays a pivotal role in tumour fate determination. The TME acts together with the genetic material of tumour cells to determine their initiation, metastasis and drug resistance. Stromal cells in the TME promote the growth and metastasis of tumour cells by secreting soluble molecules or exosomes. The abnormal microenvironment reduces immune surveillance and tumour killing. The TME causes low anti‐tumour drug penetration and reactivity and high drug resistance. Tumour angiogenesis and microenvironmental hypoxia limit the drug concentration within the TME and enhance the stemness of tumour cells. Therefore, modifying the TME to effectively attack tumour cells could represent a comprehensive and effective anti‐tumour strategy. Normal cells, such as stem cells and immune cells, can penetrate and disrupt the abnormal TME. Reconstruction of the TME with healthy cells is an exciting new direction for tumour treatment. We will elaborate on the mechanism of the TME to support tumours and the current cell therapies for targeting tumours and the TME—such as immune cell therapies, haematopoietic stem cell (HSC) transplantation therapies, mesenchymal stem cell (MSC) transfer and embryonic stem cell‐based microenvironment therapies—to provide novel ideas for producing breakthroughs in tumour therapy strategies.     

Effects of mitomycin C and porfiromycin on exponentially growing and plateau phase cultures

Abstract. Laboratory studies and clinical trials are exploring the use of hypoxia-directed cytotoxic agents as adjuncts to radiotherapy. Because hypoxia and the microenviron-mental inadequacies associated with hypoxia in solid tumours inhibit cell proliferation, an essential requirement for the successful use of hypoxia-directed drugs in cancer therapy is that these drugs be toxic to quiescent tumour cells, as well as tumour cells progressing rapidly through the cell cycle. The experiments reported here compared the cytotoxicities of mitomycin C and porfiromycin to exponentially growing and plateau phase cultures of EMT6 mouse mammary tumour cells. The proliferative status of the cultures did not influence the cytotoxicity of mitomycin C under either aerobic or hypoxic conditions, or the cytotoxicity of porfiromycin in air. Exponentially growing cultures were slightly more sensitive than plateau phase cultures to porfiromycin in hypoxia, but the difference between the sensitivities of proliferating and quiescent cells was much smaller than the difference between aerobic and hypoxic cells. No evidence for repair of potentially lethal damage was found after treatment with porfiromycin in air or in hypoxia; this is in agreement with previous findings for mitomycin C. Mitomycin C and porfiromycin therefore exhibit the toxicity to quiescent cells needed for effective use as hypoxia-directed drugs for the treatment of solid tumours.     

Multiple Drug Resistance Mechanisms in Cancer

Multiple drug resistance (multidrug resistance; MDR), a phenomenon whereby human tumours that acquire resistance to one type of therapy are found to be resistant to several other drugs that are often quite different in both structure and mode of action, has been recognised clinically for several decades. An important advance in our understanding of MDR came with the identification of P-glycoprotein and other related transporters that were expressed in some cancer cells and could recognise and catalyse the efflux of diverse anticancer drugs from cells. A second advance came from an understanding of the mechanism of programmed cell death or apoptosis, leading to MDR mediated by increased to resistance to anticancer drug-induced apoptosis. A third advance came with the finding that the proliferation of human tumours was driven by a small population of self-renewing tumour cells, focussing attention on the MDR properties of these so-called tumour stem cells rather than on the cells that comprised the majority of the tumour population. A fourth advance was the delineation of features of the tumour microenvironment, including immunosuppression, which essentially provided tumour stem cells with an MDR phenotype. Most published work on the overcoming of MDR has concentrated on inhibition of drug transporters but the complexity of mechanisms contributing demands a broad strategy for the development of methods to overcome MDR in a clinical setting.     

CELL KINETIC ALTERATIONS IN NORMAL AND NEOPLASTIC CELL POPULATIONS IN VITRO AND IN VIVO FOLLOWING VINCRISTINE*. A REPLY TO DR CAMPLEJOHN'S REVIEW ARTICLE†

It is shown that the lethal action of vincristine (VCR) is dose-dependent and may occur at interphase and mitosis. In general, the VCR dose used to destroy cells must be approximately ten times higher than that used to arrest cells in mitosis at metaphase. There is strong evidence that cells can survive metaphase arrest by a sublethal dose of VCR either completing cytokinesis normally after metabolism of the drug or becoming polyploid because of an impaired mitotic spindle apparatus. These cells are not doomed to die, at least in some cell systems. Furthermore, there is strong evidence in three animal tumour systems (transplantable and autochthonous tumours) that VCR is able to induce in vivo partial synchronization of proliferating tumour cells and/or recruitment of resting cells into the proliferating compartment. Failures to induce partial synchrony in cell populations by VCR may be attributed to resistance to VCR or cytolysis or slow proliferation of cells in badly vascularized tumours. Chemotherapy after synchronization seems to be effective as shown by non-randomized trials in bad-risk patients with solid tumours and acute leukaemias. In a randomized co-operative trial results of the two-drug synchronization protocol in patients with non-Hodgkin's lymphoma of high grade malignancy were statistically better than those of a four-drug protocol (COPP) established empirically. The two-drug protocol was equally effective as the four-drug protocol in Hodgkin's disease. Side-effects were less pronounced with the so-called synchronization scheme.     

Cell resensitization after delivery of a cycle-specific anticancer drug and effect of dose splitting: learning from tumour cords

After a single dose of an anticancer agent, changes due to cell death are expected to occur in the distribution of cells between proliferating and quiescent compartment as well as in the oxygenation and nutritional state of surviving cells. These changes are transient because tumour regrowth tends to restore the pretreatment status. The reoxygenation due to the decrease of oxygen consumption is expected to induce cell recruitment from quiescence into proliferation, and consequently to increase the sensitivity of the cell population to a successive treatment by a cycle-specific drug. In previous papers we proposed a model of the response of tumour cords (cylindrical arrangements of tumour cells growing around a blood vessel of the tumour) to single-dose treatments. The model included the motion of cells and oxygen diffusion and consumption. On the basis of that model suitably extended to better account for the action of anticancer drugs, we study the time course of the oxygenation and of the redistribution of cells between the proliferating and quiescent compartments. By means of simulations of the response to a dose delivered as two spaced equal fractions, we investigate the dependence of tumour response on the spacing between the fractions and on the main parameters of the system. A time window may be found in which the delivery of two fractions is more effective than the delivery of the undivided dose.     

Cancer stem cells – the current status of an old concept: literature review and clinical approaches

As regards their morphology and biology, tumours consist of heterogeneous cell populations. The cancer stem cell (CSC) hypothesis assumes that a tumour is hierarchically organized and not all of the cells are equally capable of generating descendants, similarly to normal tissue. The only cells being able to self-renew and produce a heterogeneous tumour cell population are cancer stem cells. CSCs probably derive from normal stem cells, although progenitor cells may be taken into consideration as the source of cancer stem cells. CSCs reside in the niche defined as the microenvironment formed by stromal cells, vasculature and extracellular matrix. The CSC assays include FACS sorting, xenotransplantation to immunodeficient mice (SCID), incubation with Hoechst 33342 dye, cell culture in non-adherent conditions, cell culture with bromodeoxyuridine. CSCs have certain properties that make them resistant to anticancer therapy, which suggests they may be the target for potential therapeutic strategies.     

Tumour-initiating cells vs. cancer 'stem' cells and CD133: what's in the name?

Recent evidence suggests that a subset of cells within a tumour have 'stem-like' characteristics. These tumour-initiating cells, distinct from non-malignant stem cells, show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumour cells, resistance to chemotherapy or radiation, and they are often characterised by elevated expression of the stem cell surface marker CD133. Understanding the molecular biology of the CD133(+) cancer cells is now essential for developing more effective cancer treatments. These may include drugs targeting organelles, such as mitochondria or lysosomes, using highly efficient and selective inducers of apoptosis. Alternatively, agents or treatment regimens that enhance sensitivity of these therapy-resistant "tumour stem cells" to the current or emerging anti-tumour drugs would be of interest as well.     

Brain tumour stem cells: the undercurrents of human brain cancer and their relationship to neural stem cells

Conceptual and technical advances in neural stem cell biology are being applied to the study of human brain tumours. These studies suggest that human brain tumours are organized as a hierarchy and are maintained by a small number of tumour cells that have stem cell properties. Most of the bulk population of human brain tumours comprise cells that have lost the ability to initiate and maintain tumour growth. Although the cell of origin for human brain tumours is uncertain, recent evidence points towards the brain's known proliferative zones. The identification of brain tumour stem cells has important implications for understanding brain tumour biology and these cells may be critical cellular targets for curative therapy.     

CD133: holy of grail of neuro‐oncology or promiscuous red‐herring?

The CD133 glycoprotein is a controversial cancer stem cell marker in the field of neuro‐oncology, based largely on the now considerable experimental evidence for the existence of both CD133+ve and CD133?ve populations as tumour‐initiating cells. It is thought that decreasing oxygen tension enhances the complex regulation and phenotype of CD133 in glioma. In light of these ideologies, establishing the precise functional role of CD133 is becoming increasingly critical. In this article, we review the complex regulation of CD133 and its extracellular epitope AC133, and associated alterations, to tumour cell behaviour by hypoxia. Furthermore, its role in functional modulation of tumours, rather than determination of a specific stem cell type is therefore alluded to, while evidence for and against its ability as a cancer stem cell marker in primary brain tumours, is critically evaluated. Thus, the suggestion that CD133 may be a central ‘holy grail’ in identifying core cells for propagation of malignant glial neoplasms seems increasingly less convincing. It remains to be seen, however, whether CD133 is randomly expressed on such brain tumour cell populations or whether it is of major significance to brain biological behaviour.     

Human pancreatic cancer cells under nutrient deprivation are vulnerable to redox system inhibition

Large regions in tumor tissues, particularly pancreatic cancer, are hypoxic and nutrient-deprived because of unregulated cell growth and insufficient vascular supply. Certain cancer cells, such as those inside a tumor, can tolerate these severe conditions and survive for prolonged periods. We hypothesized that small molecular agents, which can preferentially reduce cancer cell survival under nutrient-deprived conditions, could function as anticancer drugs. In this study, we constructed a high-throughput screening system to identify such small molecules and screened chemical libraries and microbial culture extracts. We were able to determine that some small molecular compounds, such as penicillic acid, papyracillic acid, and auranofin, exhibit preferential cytotoxicity to human pancreatic cancer cells under nutrient-deprived compared with nutrient-sufficient conditions. Further analysis revealed that these compounds target to redox systems such as GSH and thioredoxin and induce accumulation of reactive oxygen species in nutrient-deprived cancer cells, potentially contributing to apoptosis under nutrient-deprived conditions. Nutrient-deficient cancer cells are often deficient in GSH; thus, they are susceptible to redox system inhibitors. Targeting redox systems might be an attractive therapeutic strategy under nutrient-deprived conditions of the tumor microenvironment.     

Macrophage-Based Anti-Cancer Therapy: Modelling Different Modes of Tumour Targeting

Tumour hypoxia is associated with poor drug delivery and low rates of cell proliferation, factors that limit the efficacy of therapies that target proliferating cells. Since macrophages localise within hypoxic regions, a promising way to target hypoxic tumour cells involves engineering macrophages to express therapeutic genes under hypoxia. In this paper we develop mathematical models to compare the responses of avascular tumour spheroids to two modes of action: either the macrophages deliver an enzyme that activates an externally applied prodrug (bystander model), or they deliver cytotoxic factors directly (local model). The models we develop comprise partial differential equations for a multiphase mixture of tumour cells, macrophages and extracellular fluid, coupled to a moving boundary representing the spheroid surface. Chemical constituents, such as oxygen and drugs, diffuse within the multiphase mixture. Simulations of both models show the spheroid evolving to an equilibrium or to a travelling wave (multiple stable solutions are also possible). We uncover the parameter dependence of the wave speed and steady-state tumour size, and bifurcations between these solution forms. For some parameter sets, adding extra macrophages has a counterintuitive deleterious effect, triggering a bifurcation from bounded to unbounded tumour growth. While these features are common to the bystander and local models, the crucial difference is where cell death occurs. The bystander model is comparable to traditional chemotherapy, with poor targeting of hypoxic tumour cells; however, the local mode of action is more selective for hypoxic regions. We conclude that effective targeting of hypoxic tumour cells may require the use of drugs with limited mobility or whose action does not depend on cell proliferation.     

Intermittent hypoxia is a key regulator of cancer cell and endothelial cell interplay in tumours

Solid tumours are complex structures in which the interdependent relationship between tumour and endothelial cells modulates tumour development and metastasis dissemination. The tumour microenvironment plays an important role in this cell interplay, and changes in its features have a major impact on tumour growth as well as on anticancer therapy responsiveness. Different studies have shown irregular blood flow in tumours, which is responsible for hypoxia and reoxygenation phases, also called intermittent hypoxia. Intermittent hypoxia induces transient changes, the impact of which has been underestimated for a long time. Recent in vitro and in vivo studies have shown that intermittent hypoxia could positively modulate tumour development, inducing tumour growth, angiogenic processes, chemoresistance, and radioresistance. In this article, we review the effects of intermittent hypoxia on tumour and endothelial cells as well as its impacts on tumour development.     

Using a stem cell and progeny model to illustrate the relationship between cell cycle times of in vivo human tumour cell tissue populations, in vitro primary cultures and the cell lines derived from them

There is increasing evidence that the growth of human tumours is driven by a small proportion of tumour stem cells with self-renewal properties. Multiplication of these cells leads to loss of self-renewal and after division for a finite number of times the cells undergo programmed cell death. Cell cycle times of human cancers have been measured in vivo and shown to vary in the range from two days to several weeks, depending on the individual. Cells cultured directly from tumours removed at surgery initially grow at a rate comparable to the in vivo rate but continued culture leads to the generation of cell lines that have shorter cycle times (1–3 days). It has been postulated that the more rapidly growing sub-population exhibits some of the properties of tumour stem cells and are the precursors of a slower growing sub-population that comprise the bulk of the tumour. We have previously developed a mathematical model to describe the behaviour of cell lines and we extend this model here to describe the behaviour of a system with two cell populations with different kinetic characteristics and a precursor–product relationship. The aim is to provide a framework for understanding the behaviour of cancer tissue that is sustained by a minor population of proliferating stem cells.     

Multiplexing Spheroid Volume,Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.     

Mathematical model for chemotherapeutic drug efficacy in arresting tumour growth based on the cancer stem cell hypothesis

OBJECTIVE: Cancer stem cells have been identified as the growth root for various malignant tumours and are thought to be responsible for cancer recurrence following treatment. MATERIALS AND METHODS: Here, a predictive mathematical model for the cancer stem cell hypothesis is used to understand tumour responses to chemotherapeutic drugs and judge the efficacy of treatments in arresting tumour growth. The impact of varying drug efficacies on different abnormal cell populations is investigated through the kinetics associated with their decline in response to therapy. RESULTS AND CONCLUSIONS: The model predicts the clinically established 'dandelion phenomenon' and suggests that the best response to chemotherapy occurs when a drug targets abnormal stem cells. We compare continuous and periodic drug infusion. For the latter, we examine the relative importance of the drug cell-kill rate and the mean time between successive therapies, to identify the key attributes for successful treatment.     

Stem cells and cancer: a deadly mix

Stem cells and cancer are inextricably linked; the process of carcinogenesis initially affects normal stem cells or their closely related progenitors and then, at some point, neoplastic stem cells are generated that propagate and ultimately maintain the process. Many, if not all, cancers contain a minority population of self-renewing stem cells, “cancer stem cells”, that are entirely responsible for sustaining the tumour and for giving rise to proliferating but progressively differentiating cells that contribute to the cellular heterogeneity typical of many solid tumours. Thus, the bulk of the tumour is often not the clinical problem, and so the identification of cancer stem cells and the factors that regulate their behaviour are likely to have an enormous bearing on the way that we treat neoplastic disease in the future. This review summarises (1) our knowledge of the origins of some cancers from normal stem cells and (2) the evidence for the existence of cancer stem cells; it also illustrates some of the stem cell renewal pathways that are frequently aberrant in cancer and that may represent druggable targets.     

Polyether ionophores—promising bioactive molecules for cancer therapy

The natural polyether ionophore antibiotics might be important chemotherapeutic agents for the treatment of cancer. In this article, the pharmacology and anticancer activity of the polyether ionophores undergoing pre-clinical evaluation are reviewed. Most of polyether ionophores have shown potent activity against the proliferation of various cancer cells, including those that display multidrug resistance (MDR) and cancer stem cells (CSC). The mechanism underlying the anticancer activity of ionophore agents can be related to their ability to form complexes with metal cations and transport them across cellular and subcellular membranes. Increasing evidence shows that the anticancer activity of polyether ionophores may be a consequence of the induction of apoptosis leading to apoptotic cell death, arresting cell cycle progression, induction of the cell oxidative stress, loss of mitochondrial membrane potential, reversion of MDR, synergistic anticancer effect with other anticancer drugs, etc. Continued investigation of the mechanisms of action and development of new polyether ionophores and their derivatives may provide more effective therapeutic drugs for cancer treatments.     

Murine Mammary Tumour Cells In Vitro. Ii. Recruitment of Quiescent Cells

Abstract The development of a pure quiescent (Q) tumour cell population can be induced in three mouse mammary tumour lines (66, 67 and 68H) by nutrient deprivation. When these Q cells were removed from nutrient-deprived cultures and replated in fresh medium at a lower cell concentration within 72 hr of entering quiescence virtually all of the Q cells could re-enter the proliferating (P) state. This recruitment was characterized by an increase in cell volume, an increase in total cellular RNA, and a resumption of cell division. the length of the Q to P transition varied among the three cell lines and the depth of the quiescent state depended on the amount of time the cells had been quiescent. Once re-entry into the P compartment was completed, cell-cycle times, as estimated by the culture doubling time, were the same as the cells that had not entered the Q state. however, after 72 hr in quiescence, not all of the 66 cells could reattach after trypsinization and of those that could reattach 50% were incapable of either increasing their RNA levels to that of proliferating G₁ cells or entering S. Clonogenicity of the nutrient-deprived Q cells in these lines decreases exponentially from time the cells enter quiescence with approximate half-times of 32, 34, and 96 hr for the 66, 68H and 67 cells, respectively. Slnce clonogenicity was already declining at a time when all the Q cells could re-enter the P compartment, the ability of a Q cell to form a colony is not determined solely by its capacity to re-enter the proliferating compartment.     

Plasma membrane electron transport: a new target for cancer drug development

The view that mitochondrial electron transport is the only site of aerobic respiration and the primary bioenergetic pathway in mammalian cells is well established in the literature. Although this paradigm is widely accepted for most tissues, the situation is less clear for proliferating cells. Increasing evidence indicates that glycolytic ATP production contributes substantially to fulfilling the energy requirements of rapidly dividing somatic cells, many tumour cells, and self-renewing stem cells in hypoxic environments. Glycolytic cells have been shown to consume oxygen at the cell surface via plasma membrane electron transport (PMET), a process that oxidises intracellular NADH, supports glycolytic ATP production and may contribute to aerobic energy production. PMET, as determined by reduction of a cell-impermeable tetrazolium dye, is highly active in rapidly-dividing tumour cell lines, where it ameliorates intracellular reductive stress, originating from the mitochondrial TCA cycle. Thus, mitochondrial NADH production is linked to dye reduction outside the cell via the malate-aspartate shuttle. PMET activity increases several-fold under hypoxic conditions, consistent with the view that oxygen competes for electrons from this PMET system. In addition, rho(o) cells that lack mitochondrial electron transport are characterised by elevated PMET presumably to recycle NADH, a role traditionally assumed by lactate dehydrogenase. PMET presents an excellent target for developing novel anticancer drugs that exploit its unique plasma membrane localisation. We propose that PMET is a ubiquitous, high-capacity acute NADH redox-regulatory system responsible for maintaining the mitochondrial NADH/NAD+ ratio. Blocking this pathway compromises the viability of rapidly proliferating cells that rely on PMET.