The somatic mutation theory of cancer: growing problems with the paradigm?

The somatic mutation theory has been the prevailing paradigm in cancer research for the last 50 years. Its premises are: (1) cancer is derived from a single somatic cell that has accumulated multiple DNA mutations, (2) the default state of cell proliferation in metazoa is quiescence, and (3) cancer is a disease of cell proliferation caused by mutations in genes that control proliferation and the cell cycle. From this compelling simplicity, an increasingly complicated picture has emerged as more than 100 oncogenes and 30 tumor suppressor genes have been identified. To accommodate this complexity, additional ad hoc explanations have been postulated. After a critical review of the data gathered from this perspective, an alternative research program has been proposed. It is based on the tissue organization field theory, the premises of which are that carcinogenesis represents a problem of tissue organization, comparable to organogenesis, and that proliferation is the default state of all cells. The merits of these competing theories are evaluated herein.     

The tumor suppressor Apc controls planar cell polarities central to gut homeostasis

The stem cells (SCs) at the bottom of intestinal crypts tightly contact niche-supporting cells and fuel the extraordinary tissue renewal of intestinal epithelia. Their fate is regulated stochastically by populational asymmetry, yet whether asymmetrical fate as a mode of SC division is relevant and whether the SC niche contains committed progenitors of the specialized cell types are under debate. We demonstrate spindle alignments and planar cell polarities, which form a novel functional unit that, in SCs, can yield daughter cell anisotropic movement away from niche-supporting cells. We propose that this contributes to SC homeostasis. Importantly, we demonstrate that some SC divisions are asymmetric with respect to cell fate and provide data suggesting that, in some SCs, mNumb displays asymmetric segregation. Some of these processes were altered in apparently normal crypts and microadenomas of mice carrying germline Apc mutations, shedding new light on the first stages of progression toward colorectal cancer.     

Emergentism as a default: Cancer as a problem of tissue organization

During the last fifty years the dominant stance in experimental biology has been reductionism. For the most part, research programs were based on the notion that genes were in ’the driver’s seat’ controlling the developmental program and determining normalcy and disease (genetic reductionism and genetic determinism). Philosophers were the first to realize that the belief that the Mendelian genes were reduced to DNA molecules was questionable. Soon after these pronouncements, experimental data confirmed their misgivings. The optimism of molecular biologists, fueled by early success in tackling relatively simple problems, has now been tempered by the difficulties found when attempting to understand complex biological problems. Here, we analyse experimental data that illustrate the shortcomings of this sort of reductionism. We also examine the prevailing paradigm in cancer research, the somatic mutation theory (SMT), the premises of which are: (i) cancer is derived from a single somatic cell that has accumulated multiple DNA mutations; (ii) the default state of cell proliferation in metazoa is quiescence; and (iii) cancer is a disease of cell proliferation caused by mutations in genes that control proliferation and the cell cycle. We challenge the notion that cancer is a cellular problem caused by mutated genes by assessing data gathered both from within the reductionist paradigm and from an alternative view that regards carcinogenesis as a developmental process gone awry. This alternative view, explored under the name of the tissue organization field theory (TOFT), is based on premises that place cancer in a different hierarchical level of complexity from that proposed by the SMT, namely: (i) carcinogenesis represents a problem of tissue organization comparable to organogenesis, and (ii) proliferation is the default state of all cells. We propose that the organicist view, in which the TOFT is based, is a good starting point from which to explore emergent phenomena. However, new theoretical concepts are needed in order to grapple with the apparent circular causality of complex biological phenomena in development and carcinogenesis.     

Clustering of apoptotic cells via bystander killing by peroxides.

Clustering of apoptotic cells is a characteristic of many developing or renewing systems, suggesting that apoptotic cells kill bystanders. Bystander killing can be triggered experimentally by inducing apoptosis in single cells and may be based on the exchange of as yet unidentified chemical cell death signals between nearby cells without the need for cell-to-cell communication via gap junctions. Here we demonstrate that apoptotic cell clusters occurred spontaneously, after serum deprivation or p53 transfection in cell monolayers in vitro. Clustering was apparently induced through bystander killing by primary apoptotic cells. Catalase, a peroxide scavenger, suppressed bystander killing, suggesting that hydrogen peroxide generated by apoptotic cells is the death signal. Although p53 expression increased the number of apoptoses, clustering was found to be similar around apoptotic cells whether or not p53 was expressed, indicating that there is no specific p53 contribution to bystander killing. Bystander killing through peroxides emitted by apoptotic cells may propagate tissue injury in different pathological situations and be relevant in chemo-, gamma-ray, and gene therapy of cancer.     

The aging of the 2000 and 2011 Hallmarks of Cancer reviews: A critique

Two review articles published in 2000 and 2011 by Hanahan and Weinberg have dominated the discourse about carcinogenesis among researchers in the recent past. The basic tenets of their arguments favour considering cancer as a cell-based, genetic disease whereby DNA mutations cause uncontrolled cell proliferation. Their explanation of cancer phenotypes is based on the premises adopted by the somatic mutation theory (SMT) and its cell-centered variants. From their perspective, eight broad features have been identified as so-called ‘Hallmarks of Cancer’. Here, we criticize the value of these features based on the numerous intrinsic inconsistencies in the data and in the rationale behind SMT. An alternative interpretation of the same data plus data mostly ignored by Hanahan and Weinberg is proposed, based instead on evolutionarily relevant premises. From such a perspective, cancer is viewed as a tissue-based disease. This alternative, called the tissue organization field theory, incorporates the premise that proliferation and motility are the default state of all cells, and that carcinogenesis is due to alterations on the reciprocal interactions among cells and between cells and their extracellular matrix. In this view, cancer is development gone awry.     

Ultra High Content Image Analysis and Phenotype Profiling of 3D Cultured Micro-Tissues

In many situations, 3D cell cultures mimic the natural organization of tissues more closely than 2D cultures. Conventional methods for phenotyping such 3D cultures use either single or multiple simple parameters based on morphology and fluorescence staining intensity. However, due to their simplicity many details are not taken into account which limits system-level study of phenotype characteristics. Here, we have developed a new image analysis platform to automatically profile 3D cell phenotypes with 598 parameters including morphology, topology, and texture parameters such as wavelet and image moments. As proof of concept, we analyzed mouse breast cancer cells (4T1 cells) in a 384-well plate format following exposure to a diverse set of compounds at different concentrations. The result showed concentration dependent phenotypic trajectories for different biologically active compounds that could be used to classify compounds based on their biological target. To demonstrate the wider applicability of our method, we analyzed the phenotypes of a collection of 44 human breast cancer cell lines cultured in 3D and showed that our method correctly distinguished basal-A, basal-B, luminal and ERBB2+ cell lines in a supervised nearest neighbor classification method.     

Of lice and men: The return of the ’comparative parasitology‘ debate

The question of whether or not parasite phylogeny provides information about host relationships (‘comparative parasitology’) reached a peak in 1957 in a vigorous debate between Gunther Timmermann and Ernst Mayr. Timmermann argued that parasites were associated with their hosts by descent and that this produced congruent host and parasite phylogenies. In contrast, Mayr argued that parasites were often associated by colonization and that this led to incongruence between host and parasite phylogenies. To test these differing views. Adrian Paterson, Russell Gray and Graham Wallis derived a procellaniform phylogeny. This tree is here compared with Timmermann's tree based on the relationships of feather lice. Timmermann's tree is more similar to the seabird phylogeny than would be expected by chance. Thus, support is found for the ‘comparative parasitology’ approach.     

Visual pattern discrimination by population retinal ganglion cells’ activities during natural movie stimulation

In the visual system, neurons often fire in synchrony, and it is believed that synchronous activities of group neurons are more efficient than single cell response in transmitting neural signals to down-stream neurons. However, whether dynamic natural stimuli are encoded by dynamic spatiotemporal firing patterns of synchronous group neurons still needs to be investigated. In this paper we recorded the activities of population ganglion cells in bullfrog retina in response to time-varying natural images (natural scene movie) using multi-electrode arrays. In response to some different brief section pairs of the movie, synchronous groups of retinal ganglion cells (RGCs) fired with similar but different spike events. We attempted to discriminate the movie sections based on temporal firing patterns of single cells and spatiotemporal firing patterns of the synchronous groups of RGCs characterized by a measurement of subsequence distribution discrepancy. The discrimination performance was assessed by a classification method based on Support Vector Machines. Our results show that different movie sections of the natural movie elicited reliable dynamic spatiotemporal activity patterns of the synchronous RGCs, which are more efficient in discriminating different movie sections than the temporal patterns of the single cells’ spike events. These results suggest that, during natural vision, the down-stream neurons may decode the visual information from the dynamic spatiotemporal patterns of the synchronous group of RGCs’ activities.     

Inverse Correlation between an Organ’s Cancer Rate and Its Evolutionary Antiquity

Human cancer rates vary dramatically across the range of internal organs in the body, but there is no single model to explain the variation and there is also no obvious overall pattern to it. Theories have been proposed to account for high rates in particularly cancer-prone organs, and they usually concentrate on the peculiar vulnerability of certain cells to mutation (eg lung cells’ direct exposure to airborne carcinogens). These explanations are valuable for understanding mechanisms of disease and also for cancer prevention, but they neither address the overall distribution of cancers nor the possibility that some states of differentiation may be intrinsically less stable than others to the effects of random mutation, a possibility predicted on purely theoretical grounds many years ago. This brief report describes an overall pattern to human organ-specific cancer incidence data and shows that organ-specific cancer rates correlate negatively with an organ’s evolutionary antiquity. Although the relationship may just be coincidental, it suggests the possibility that recently-evolved differentiation states may be intrinsically more vulnerable to neoplastic change. Extrapolation of the regression line to a cancer incidence of zero equates to a level of tissue organization typical of 660Myr ago; the inferred beginning of neoplasia therefore coincides with the rise of complex multicellular animals.     

Heterogeneity of anticancer drug sensitivity in squamous cell carcinoma of the tongue

Heterogeneity is known to be present to varying degrees in cancer cell groups. There have been no reports, however, of studies in which a single cell clone was prepared from a cancer cell group to examine heterogeneity with respect to anticancer drug sensitivity. Thus, the authors herein report an investigation into the heterogeneity of cancer cells within the same tumor with respect to anticancer drug sensitivity. Anticancer drug sensitivity was investigated in primary tumors, metastatic lymph node tumors, recurrent tumors and established cell lines obtained from four cases of tongue cancer using an oxygen electrode apparatus. As differences were observed in anticancer drug sensitivity from one case to another, even though all four were of the same pathological tissue type, the individual differences were apparently significant. Moreover, primary tumors and recurrent tumors demonstrated different sensitivities to the anticancer drugs even in the same patient. When single cell clones were prepared from primary tumors and anticancer drug sensitivity testing was carried out, sensitivity to anticancer drugs that was not seen in the primary tumors was observed. We performed RT-PCR on cell groups derived from this single cell using MDR1, MRP1, MRP2 and ERCC1, which are primary genes that are resistant to anticancer drugs. Expression of MDR and ERCC1 was not observed in single cell clones nos. 1-10. MRP1 and MRP2, on the other hand, were expressed in all of these single cell clones. Because cells with different sensitivity levels were initially present in the cancer cell groups, even when large numbers of cancer cells died in response to anticancer drug therapy, the results suggest the possibility that recurrence and metastasis occur based on cells with differing sensitivities. After examining anticancer drug sensitivity at the single cell level, we believe that anticancer drug-resistant genes may be involved in the heterogeneity of anticancer drug sensitivity with respect to cancer cell groups.     

Identification of cancer stem cells: from leukemia to solid cancers

Cancer stem cells (CSCs) are widely considered to be a small cell population in leukemia and many solid cancers with the properties including self-renewal and differentiation to non-tumorigenic cancer cells. Identification and isolation of CSCs significantly depend on the special surface markers of CSCs. Aberrant gene expression and signal transduction contribute to malignancies of CSCs, which result in cancer initiation, progression and recurrence. The inefficient therapy of cancers is mainly attributed to the failure of elimination of the malignant CSCs. However, CSCs have not been detected in all cancers and hierarchical organization of tumors might challenge cancer stem cell models. Additionally, opinions about the validity of the CSC hypothesis, the biological properties of CSCs, and the relevance of CSCs to cancer therapy differ widely. In this review, we discuss the debate of cancer stem cell model, the parameters by which CSCs can or cannot be defined, and the advances in the therapy of CSCs.     

Viruses in Biology

During the first half of the twentieth century, many scientists considered viruses the smallest living entities and primitive life forms somehow placed between the inert world and highly evolved cells. The development of molecular biology in the second half of the century showed that viruses are strict molecular parasites of cells, putting an end to previous virocentric debates that gave viruses a primeval role in the origin of life. Recent advances in comparative genomics and metagenomics have uncovered a vast viral diversity and have shown that viruses are active regulators of cell populations and that they can influence cell evolution by acting as vectors for gene transfer among cells. They have also fostered a revival of old virocentric ideas. These ideas are heterogeneous, extending from proposals that consider viruses functionally as living beings and/or as descendants of viral lineages that preceded cell evolution to other claims that consider viruses and/or some viral families a fourth domain of life. In this article, we revisit these virocentric ideas and analyze the place of viruses in biology in light of the long-standing dichotomic debate between metabolist and geneticist views which hold, respectively, that self-maintenance (metabolism) or self-replication and evolution are the primeval features of life. We argue that whereas the epistemological discussion about whether viruses are alive or not and whether some virus-like replicators precede the first cells is a matter of debate that can be understood within the metabolism-versus-genes dialectic; the claim that viruses form a fourth domain in the tree of life can be solidly refuted by proper molecular phylogenetic analyses and needs to be removed from this debate.     

The heterogeneity of epithelial ovarian cancers: reconciling old and new paradigms

Epithelial ovarian cancer comprises several subtypes of tumours that exhibit diverse histopathological features. The intriguing assumption by many epithelial ovarian cancers of specialised features of nonovarian tissue lineages has promoted considerable debate as to whether these tumours arise from the deceptively simple surface epithelium of the ovary. This review focuses on recent molecular and pathological studies of epithelial ovarian cancers that support and challenge their surface-epithelial derivation, and discusses the findings in the context of current views of the 'cell-of-origin' of solid tumours.     

Multimodal microscale mechanical mapping of cancer cells in complex microenvironments

The mechanical phenotype of the cell is critical for survival following deformations due to confinement and fluid flow. One idea is that cancer cells are plastic and adopt different mechanical phenotypes under different geometries that aid in their survival. Thus, an attractive goal is to disrupt cancer cells’ ability to adopt multiple mechanical states. To begin to address this question, we aimed to quantify the diversity of these mechanical states using in vitro biomimetics to mimic in vivo two-dimensional (2D) and 3D extracellular matrix environments. Here, we used two modalities Brillouin microscopy (～GHz) and broadband frequency (7–15 kHz) optical tweezer microrheology to measure microscale cell mechanics. We measured the response of intracellular mechanics of cancer cells cultured in 2D and 3D environments where we modified substrate stiffness, dimensionality (2D versus 3D), and presence of fibrillar topography. We determined that there was good agreement between two modalities despite the difference in timescale of the two measurements. These findings on cell mechanical phenotype in different environments confirm a correlation between modalities that employ different mechanisms at different temporal scales (Hz-kHz versus GHz). We also determined that observed heterogeneity in cell shape is more closely linked to the cells’ mechanical state. Moreover, individual cells in multicellular spheroids exhibit a lower degree of mechanical heterogeneity when compared with single cells cultured in monodisperse 3D cultures. The observed decreased heterogeneity among cells in spheroids suggested that there is mechanical cooperativity between cells that make up a single spheroid.     

Imaging protein-protein interactions in living cells

The complex organization of plant cells makes it likely that the molecular behaviour of proteins in the test tube and the cell is different. For this reason, it is essential though a challenge to study proteins in their natural environment. Several innovative microspectroscopic approaches provide such possibilities, combining the high spatial resolution of microscopy with spectroscopic techniques to obtain information about the dynamical behaviour of molecules. Methods to visualize interaction can be based on FRET (fluorescence detected resonance energy transfer), for example in fluorescence lifetime imaging microscopy (FLIM). Another method is based on fluorescence correlation spectroscopy (FCS) by which the diffusion rate of single molecules can be determined, giving insight into whether a protein is part of a larger complex or not. Here, both FRET- and FCS-based approaches to study protein-protein interactions in vivo are reviewed.     

Raman microspectroscopic discrimination of TCam-2 cultures reveals the presence of two sub-populations of cells

TCam-2 cells are the main in vitro model for investigations into seminomatous tumors. However, despite their widespread use, questions remain regarding the cells’ homogeneity and consequently how representative they are of seminomas. We assess the TCam-2 cell line using routine and novel authentication methods to determine its homogeneity, identify any cellular sub-populations and resolve whether any changes could be due to generational differentiation. TCam-2, embryonal carcinoma cells (2102EP) and breast cancer cell (MCF7) lines were assessed using qRT-PCR, immunocytochemistry, flow cytometry and short tandem repeat analyses. Raman maps of individual cells (minimum of 10) and single scan spectra from 200 cells per culture were obtained. TCam-2s displayed the characteristic marker gene expression pattern for seminoma, were uniform in size and granularity and short tandem repeat analysis showed no contamination. However, based only on physical parameters, flowcytometry was unable to differentiate between TCam-2 and 2102EPs. Raman maps of TCam-2s comprised three equally distributed, distinct spectral patterns displaying large intercellular single spectral variation. All other cells showed little variation. Principal component, cluster and local spectral angle analyses indicated that the TCam-2s contained two different types of cells, one of which comprised two subgroups and was similar to some 2102EP cells. Protein expression corroborated the presence of different cells and generational differences. The detailed characterization provided by the Raman spectra, augmented by the routine methods, provide substantiation to the long-held suspicion that TCam-2 are not homogeneous but comprise differing cell populations, one of which may be embryonal carcinoma in origin.     

Identification of single chain antibodies to breast cancer stem cells using phage display

Recent evidence suggests that most malignancies are driven by “cancer stem cells” sharing the signature characteristics of adult stem cells: the ability to self renew and to differentiate. Furthermore these cells are thought to be quiescent, infrequently dividing cells with a natural resistance to chemotherapeutic agents. These studies theorize that therapies, which effectively treat the majority of tumor cells but ‘miss’ the stem cell population, will fail, while therapies directed at stern cells can potentially eradicate tumors. In breast cancer, researchers have isolated ‘breast cancer stem cells’ capable of recreating the tumor in vivo and in vitro. Generated new tumors contained both additional numbers of cancer stem cells and diverse mixed populations of cells present in the initial tumor, supporting the intriguing self‐renewal and differentiation characteristics. In the present study, an antibody phage library has been used to search for phage displayed‐single chain antibodies (scFv) with selective affinity to specific targets on breast cancer stem cells. We demonstrate evidence of two clones binding specifically to a cancer stem cell population isolated from the SUMl59 breast cancer cell line. These clones had selective affinity for cancer stem cells and they were able to select cancer stem cells among a large population of non‐stem cancer cells in paraffin‐embedded sections. The applicability of these clones to paraffin sections and frozen tissue specimens made them good candidates to be used as diagnostic and prognostic markers in breast cancer patient samples taking into consideration the cancer stern cell concept in tumor biology. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A micro-anatomical model of the distribution of myocardial endomysial collagen

Myocardial connective tissue probably provides passive support for regulating heart tensile strength and stiffness and ultimately for controlling heart mechanics through its endomysial part. However, endomysial collagen micro-arrangement is still a matter of debate. In order to define the fine distribution of left ventricle endomysial collagen, we applied the NaOH-scanning electron microscopy (SEM) maceration method (one of the techniques of choice for studying collagen micro-arrangement) to rabbit heart. Gomori-reticulum staining was used for correlated light microscopy (LM) observations. The SEM-NaOH method allowed isolation of collagen by removing other extracellular matrix components and cells and preserved collagen structure and position. Endomysial collagen appeared arranged in laminae that delimited the lacunae that were left empty by macerated myocytes and small vessels (mostly capillaries). These laminae were formed by reticular fibers, as confirmed by LM observations of Gomorireticulum-stained samples, and were organized in irregularly meshed networks made of thin (single) and thick (composed) filaments. In longitudinal views, collagen laminae extended the entire length of lacunae. In transversal views, the cut surface of the laminae appeared to be made of collagen bundles. These observations provide an updated microanatomical view of endomysial collagen distribution, which integrates previous studies. This model is based on the evidence that collagen laminae enveloped the surface of small vessels and myocytes. Thus, a type of myocyte-myocyte or capillary-myocyte "laminar connection" anchored to the entire cell length here is emphasized, rather than a type of "strut connection" anchored to defined loci, as usually described. This structure explains better how endomysium may provide the necessary support for heart compliance and protection against overstretch.