Over a century of cancer research: Inconvenient truths and promising leads.

Despite over a century of intensive efforts, the great gains promised by the War on Cancer nearly 50 years ago have not materialized. Since 1999, we have analyzed the lack of progress in explaining and "curing" cancer by examining the merits of the premises that determine how cancer is understood and treated. Our ongoing critical analyses have aimed at clarifying the sources of misunderstandings at the root of the cancer puzzle while providing a plausible and comprehensive biomedical perspective as well as a new theory of carcinogenesis that is compatible with evolutionary theory. In this essay, we explain how this new theory, the tissue organization field theory (TOFT), can help chart a path to progress for cancer researchers by explaining features of cancer that remain unexplainable from the perspective of the still hegemonic somatic mutation theory (SMT) and its variants. Of equal significance, the premises underlying the TOFT offer new perspectives on basic biological phenomena.

Information, programme, signal: dead metaphors that negate the agency of organisms.

The metaphorical adoption of the concepts of information, program and signal introduced into biology the logic and implicit causal structure of the mathematical theories of information; this is inimical to biology. In turn, those metaphors have hindered the development of a theory of organisms by transferring the agency of organisms to natural selection and to DNA. Moreover, those metaphors introduced into biology the dualism software-hardware and a Laplacian causal structure. Instead, we propose to uphold the agency of the living by adopting three foundational principles for a theory of organisms: namely, 1) the principle of biological inertia (i.e., the default state of cells is proliferation and motility), 2) the principle of variation, and 3) the principle of organization.

Characterization of MCF-12A cell phenotype, response to estrogens, and growth in 3D.

BACKGROUND: Three-dimensional cultures of mammary epithelial cells allow for biologically-relevant studies of the development of the mammary gland in rodents and humans under normal and pathological conditions, like carcinogenesis. Under these conditions, mammotropic hormones play significant roles in tissue morphogenesis. Therefore, a system that recreates the normal, hormonally responsive epithelium would be a valuable tool to study the normal state and its transition to carcinogenesis. MCF-12A cells have been claimed to be non-tumorigenic mammary epithelial cells with reported sensitivity to estrogens. In this study, we aimed at characterizing MCF-12A cells for use in a hormone-responsive 3D culture system to determine their usefulness as a tool to identify normal and abnormal microenvironmental cues. METHODS: MCF-12A cells were single-cell cloned in order to investigate their heterogeneous makeup. The parental cells were then treated with estradiol to investigate proliferative and transcriptional responses through the estrogen receptor alpha. Finally, parental cells and epithelial-like cell-derived clones were seeded in rat-tail collagen I to profile the morphogenesis of multicellular 3D structures. The resultant structures were then analyzed using unsupervised morphometric analysis. RESULTS: MCF-12A cells consist of epithelial-like colonies which shed elongated, freely growing cells on the colony's edges. The cells express E-cadherin as well as mesenchymal vimentin but do not express markers associated with myoepithelial cells or fibroblasts. Treatment with estradiol does not affect either the proliferation rate or the induction of gene expression in MCF-12A cells. Parental MCF-12A cells form acini, solid spheres and elongated branching ducts when grown in rat-tail collagen type I matrix, the geometries and distribution of which are altered following the removal of fibroblast-like cells. CONCLUSIONS: MCF-12A cells are a heterogeneous pseudo-epithelial cell line capable of forming a variety of multicellular structures in 3D culture. We found no indication that the cells display estrogen-responsive characteristics, thus refuting previous studies which reported estrogen responsiveness. We report that MCF-12A cells are not suited for use in studies in which differential behaviors of "normal" and "cancerous" estrogen-responsive cells are to be compared.

Reductionism, Organicism, and Causality in the Biomedical Sciences: A Critique.

Biology is undergoing a crisis whereby technical innovations designed to overcome the difficulties encountered in explaining complex biological phenomena have not delivered the expected results. To overcome this problem, mainstream biomedical researchers favor adopting new technological wonders without considering that what hinders their research could be due to their philosophical stance, theoretical frame, or looking into the wrong level of biological organization. We address the conceptual problems underlying the scientific crisis by examining the philosophical stances that have illuminated biological thought for the last 200 years and their evolution into the conceptual frames now known as reductionism and organicism. We also analyze how these stances seek to establish causality. The reductionist fixation on bottom-up causation is based on physicalism, a stance that does not allow for emergent phenomena. Organicism, by contrast, allows for emergence and asserts that biological entities are defined by the relation between what they are and what they do; thus, they generate novel qualities and structures. Also, only organisms experience illness and health. Organicism recognizes the role of agency and normativity in determining biological phenomena. Based on these premises, we favor adopting organicism to resolve the current crisis in the biomedical sciences.

Evidence of Absence: Estrogenicity Assessment of a New Food-Contact Coating and the Bisphenol Used in Its Synthesis.

Consumer concerns about exposure to substances found in food contact materials with estrogenic activity (EA) have created substantial demand for alternatives. We assessed the potential EA of both a new bisphenol monomer used to synthesize polymeric coatings for metal food-contact applications and the nonintentionally added substances (NIAS) that may migrate into food. We evaluated tetramethyl bisphenol F (TMBPF) using in vitro and in vivo assays. We extracted the polymeric coating using food simulants ethanol (50% v/v) and acetic acid (3% w/v) and measured migration using tandem liquid chromatography (LC)/mass spectrometry (MS) and LC time-of-flight MS for TMBPF and NIAS, respectively. We also tested migrants for EA using the E-SCREEN assay. TMBPF did not show estrogenic activity in the uterotrophic assay and did not alter puberty in male and female rats or mammary gland development in female rats. Neither TMBPF nor the migrants from the final polymeric coating increased proliferation of estrogen-sensitive MCF7 cells. TMBPF did not show estrogen-agonist or antagonist activity in the estrogen receptor-transactivation assay. TMBPF migration was below the 0.2 parts per billion detection limit. Our findings provide compelling evidence for the absence of EA by TMBPF and the polymeric coating derived from it and that human exposure to TMBPF would be negligible.

Bisphenol A alters the development of the rhesus monkey mammary gland.

The xenoestrogen bisphenol A (BPA) used in the manufacturing of various plastics and resins for food packaging and consumer products has been shown to produce numerous endocrine and developmental effects in rodents. Exposure to low doses of BPA during fetal mammary gland development resulted in significant alterations in the gland's morphology that varied from subtle ones observed during the exposure period to precancerous and cancerous lesions manifested in adulthood. This study assessed the effects of BPA on fetal mammary gland development in nonhuman primates. Pregnant rhesus monkeys were fed 400 µg of BPA per kg of body weight daily from gestational day 100 to term, which resulted in 0.68 ± 0.312 ng of unconjugated BPA per mL of maternal serum, a level comparable to that found in humans. At birth, the mammary glands of female offspring were removed for morphological analysis. Morphological parameters similar to those shown to be affected in rodents exposed prenatally to BPA were measured in whole-mounted glands; estrogen receptor (ER) α and ß expression were assessed in paraffin sections. Student's t tests for equality of means were used to assess differences between exposed and unexposed groups. The density of mammary buds was significantly increased in BPA-exposed monkeys, and the overall development of their mammary gland was more advanced compared with unexposed monkeys. No significant differences were observed in ER expression. Altogether, gestational exposure to the estrogen-mimic BPA altered the developing mammary glands of female nonhuman primates in a comparable manner to that observed in rodents.

A novel pathogenic classification of cancers.

According to contemporary epidemiological and experimental evidence, we propose a novel classification of cancers based on pathogenesis instead of classifications based on histological appearance of cancer. This new scheme first defines cancers as either 1. inborn errors of development or 2. sporadic ones, and then sub-defines the former into 1A. inborn inherited errors of development, being those due to mutations contributed by one or both parents' gametes to the developing conceptus, and 1B. inborn induced errors of development when the malformations and/or cancers are due to environmental carcinogenic exposure during pregnancy. It is anticipated that the origin of an increasing number of so-called sporadic cancers will turn out to be linked to the inborn induced errors of development group.

[Cancer and the elusive cancer genes]. / Le cancer et ses gènes insaisissables.

For almost a century, the somatic mutation theory (SMT) has been the prevalent theory to explain carcinogenesis. The SMT posits that the accumulation of mutations in the genome of a single normal cell is responsible for the transformation of such cell into a neoplasm. Implicitly, this theory claims that the default state of cells in metazoan is quiescence and that cancer is a cell-based, genetic and molecular disease. From lessons learned while performing our own research on control of cell proliferation and while adopting an organicist perspective, in 1999, we proposed a competing theory, the tissue organization field theory (TOFT). In contraposition to the SMT, (1) the TOFT posits that cancer is a tissue-based disease whereby carcinogens (directly) and mutations in the germ-line (indirectly) may alter normal interactions between the stroma and their adjacent epithelium. And (2) the TOFT explicitly acknowledges that the default state of all cells is proliferation and motility, a premise that is relevant to and compatible with evolutionary theory. Theoretical arguments and experimental evidence are presented to compare the merits of the original SMT and its variants and those of the TOFT in organizing principles, construct objectivity, and ultimately explain carcinogenesis.

Does cancer start in the womb? altered mammary gland development and predisposition to breast cancer due to in utero exposure to endocrine disruptors.

We are now witnessing a resurgence of theories of development and carcinogenesis in which the environment is again being accepted as a major player in phenotype determination. Perturbations in the fetal environment predispose an individual to disease that only becomes apparent in adulthood. For example, gestational exposure to diethylstilbestrol resulted in clear cell carcinoma of the vagina and breast cancer. In this review the effects of the endocrine disruptor bisphenol-A (BPA) on mammary development and tumorigenesis in rodents is used as a paradigmatic example of how altered prenatal mammary development may lead to breast cancer in humans who are also widely exposed to it through plastic goods, food and drink packaging, and thermal paper receipts. Changes in the stroma and its extracellular matrix led to altered ductal morphogenesis. Additionally, gestational and lactational exposure to BPA increased the sensitivity of rats and mice to mammotropic hormones during puberty and beyond, thus suggesting a plausible explanation for the increased incidence of breast cancer.

Modeling Mammary Organogenesis from Biological First Principles: A Systems Biology Approach.

Stromal-epithelial interactions mediate mammary gland development and the formation and progression of breast cancer. To study these interactions in vitro, 3D models are essential. We have successfully developed novel 3D in vitro models that allow the formation of mammary gland structures closely resembling those found in vivo and that respond to the hormonal cues that regulate mammary gland morphogenesis and function. Due to their simplicity when compared to in vivo studies, and to their accessibility to visualization in real time, these models are well suited to conceptual and mathematical modeling.

The Conflict between Regulatory Agencies over the 20,000-Fold Lowering of the Tolerable Daily Intake (TDI) for Bisphenol A (BPA) by the European Food Safety Authority (EFSA).

BACKGROUND: The European Food Safety Authority (EFSA) recommended lowering their estimated tolerable daily intake (TDI) for bisphenol A (BPA) 20,000-fold to 0.2 ng/kg body weight (BW)/day. BPA is an extensively studied high production volume endocrine disrupting chemical (EDC) associated with a vast array of diseases. Prior risk assessments of BPA by EFSA as well as the US Food and Drug Administration (FDA) have relied on industry-funded studies conducted under good laboratory practice protocols (GLP) requiring guideline end points and detailed record keeping, while also claiming to examine (but rejecting) thousands of published findings by academic scientists. Guideline protocols initially formalized in the mid-twentieth century are still used by many regulatory agencies. EFSA used a 21st century approach in its reassessment of BPA and conducted a transparent, but time-limited, systematic review that included both guideline and academic research. The German Federal Institute for Risk Assessment (BfR) opposed EFSA's revision of the TDI for BPA. OBJECTIVES: We identify the flaws in the assumptions that the German BfR, as well as the FDA, have used to justify maintaining the TDI for BPA at levels above what a vast amount of academic research shows to cause harm. We argue that regulatory agencies need to incorporate 21st century science into chemical hazard identifications using the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) nonguideline academic studies in a collaborative government-academic program model. DISCUSSION: We strongly endorse EFSA's revised TDI for BPA and support the European Commission's (EC) apparent acceptance of this updated BPA risk assessment. We discuss challenges to current chemical risk assessment assumptions about EDCs that need to be addressed by regulatory agencies to, in our opinion, become truly protective of public health. Addressing these challenges will hopefully result in BPA, and eventually other structurally similar bisphenols (called regrettable substitutions) for which there are known adverse effects, being eliminated from all food-related and many other uses in the EU and elsewhere. https://doi.org/10.1289/EHP13812.

Science and policy on endocrine disrupters must not be mixed: a reply to a "common sense" intervention by toxicology journal editors.

The "common sense" intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

The tissue organization field theory of cancer: a testable replacement for the somatic mutation theory.

The somatic mutation theory (SMT) of cancer has been and remains the prevalent theory attempting to explain how neoplasms arise and progress. This theory proposes that cancer is a clonal, cell-based disease, and implicitly assumes that quiescence is the default state of cells in multicellular organisms. The SMT has not been rigorously tested, and several lines of evidence raise questions that are not addressed by this theory. Herein, we propose experimental strategies that may validate the SMT. We also call attention to an alternative theory of carcinogenesis, the tissue organization field theory (TOFT), which posits that cancer is a tissue-based disease and that proliferation is the default state of all cells. Based on epistemological and experimental evidence, we argue that the TOFT compellingly explains carcinogenesis, while placing it within an evolutionarily relevant context.

European Medicines Agency Conflicts With the European Food Safety Authority (EFSA) on Bisphenol A Regulation.

The European Food Safety Authority (EFSA) has revised their estimate of the toxicity of bisphenol A (BPA) and, as a result, have recommended reducing the tolerable daily intake (TDI) by 20 000-fold. This would essentially ban the use of BPA in food packaging such as can liners, plastic food containers, and in consumer products. To come to this conclusion, EFSA used a systematic approach according to a pre-established protocol and included all guideline and nonguideline studies in their analysis. They found that Th-17 immune cells increased with very low exposure to BPA and used this endpoint to revise the TDI to be human health protective. A number of regulatory agencies including the European Medicines Agency (EMA) have written formal disagreements with several elements of EFSA's proposal. The European Commission will now decide whether to accept EFSA's recommendation over the objections of EMA. If the Commission accepts EFSA's recommendation, it will be a landmark action using knowledge acquired through independent scientific studies focused on biomarkers of chronic disease to protect human health. The goal of this Perspective is to clearly articulate the monumental nature of this debate and decision and to explain what is at stake. Our perspective is that the weight of evidence clearly supports EFSA's proposal to reduce the TDI by 20 000-fold.

Is systems biology a promising approach to resolve controversies in cancer research?

At the beginning of the 21st century cancer research has reached an impasse similar to that experienced in developmental biology in the first decades of the 20th century when conflicting results and interpretations co-existed for a long time until these differences were resolved and contradictions were eliminated. In cancer research, instead of this healthy "weeding-out" process, there have been attempts to reach a premature synthesis, while no hypothesis is being rejected. Systems Biology could help cancer research to overcome this stalemate by resolving contradictions and identifying spurious data. First, in silico experiments should allow cancer researchers to be bold and a priori reject sets of data and hypotheses in order to gain a deeper understanding of how each dataset and each hypothesis contributes to the overall picture. In turn, this process should generate novel hypotheses and rules, which could be explored using these in silico approaches. These activities are significantly less costly and much faster than "wet-experiments". Consequently, Systems Biology could be advantageously used both as a heuristic tool to guide "wet-experiments" and to refine hypotheses and test predictions.

The cancer puzzle: Welcome to organicism.

During the fifty years since President Nixon declared the "War on Cancer", those inside and outside the cancer community have witnessed the systematic moving of the goalposts attitude to accommodate evidence into an inadequate theory, that is, the Somatic Mutation Theory (SMT). This sorry state promoted a renewable yearly promise that at the end of the next 10-year period the promises uttered in 1971 would become reality. Each failure triggered calls to do more of the same research under the same theory, routinely using more and more sophisticated technology. Meanwhile, in the last few years, an unambiguous general consensus has emerged acknowledging that this overall long, intensive effort has failed, and that it is likely that the solution to the cancer problem resides elsewhere, namely, in alternative theoretical principles of biology. In this essay we concentrate, first, on the big picture, from the philosophical stance (reductionism versus organicism) to the need to adopt rigorous theories. From this novel perspective we conceptualize cancer as a disease of tissue organization akin to development gone awry. Finally, having identified both a promising stance and a useful theory, i.e., the tissue organization field theory (TOFT), we call for abandoning the SMT and for adopting the more promising TOFT.

From Wingspread to CLARITY: a personal trajectory.

In the three decades since endocrine disruption was conceptualized at the Wingspread Conference, we have witnessed the growth of this multidisciplinary field and the accumulation of evidence showing the deleterious health effects of endocrine-disrupting chemicals. It is only within the past decade that, albeit slowly, some changes regarding regulatory measures have taken place. In this Perspective, we address some historical points regarding the advent of the endocrine disruption field and the conceptual changes that endocrine disruption brought about. We also provide our personal recollection of the events triggered by our serendipitous discovery of oestrogenic activity in plastic, a founder event in the field of endocrine disruption. This recollection ends with the CLARITY study as an example of a discordance between 'science for its own sake' and 'regulatory science' and leads us to offer a perspective that could be summarized by the motto attributed to Ludwig Boltzmann: "Nothing is more practical than a good theory".

Matrix Composition Modulates Vitamin D3's Effects on 3D Collagen Fiber Organization by MCF10A Cells.

Vitamin D3 (vitD3) has been implicated in various cellular functions affecting multiple tissue types. Epidemiological and laboratory studies suggest that vitD3 may be effective as a preventive or therapeutic option for breast cancer. However, randomized clinical trials have yet to confirm these suggestions. Breast neoplasias can arise from developmental alterations; based on this evidence, we seek to understand vitD3's role in normal breast development, particularly its role in epithelial morphogenetic processes such as ductal elongation, branching, and alveolar formation. These processes require extensive changes in the extracellular microenvironment, such as collagen fiber organization, and are largely influenced by hormones. Here, we build upon our past work to shed light on calcitriol's effects on collagen fiber organization by breast epithelial cells, and how such effects are modulated by extracellular matrix composition. We embedded MCF10A normal human breast epithelial cells in two different matrices-collagen type I and collagen type I + 10% Matrigel; treatment with calcitriol resulted in flatter epithelial structures. Next, using two-photon microscopy, we examined changes in collagen fiber organization and corresponding changes in epithelial structures. Applying a novel three-dimensional (3D) image analysis method, we show that increasing doses of calcitriol result in denser collagen fiber bundles in the localized area surrounding the epithelial structures, and that these bundles are aligned in a more parallel direction to epithelial structures when exposed to the highest vitD3 dose. Changed patterns in fiber organization may explain the flattening of epithelial structures; in turn, changes in biophysical forces in the matrix abutting these structures may be responsible for changes in the referred patterns. Addition of 10% Matrigel dampened the effects of calcitriol on both epithelial morphogenesis and fiber organization. Overall, we report novel functions of calcitriol in the breast epithelium and add to the growing body of evidence documenting how hormones affect biophysical processes. Impact statement In this study, we report novel functions of calcitriol in the breast epithelium and use a novel quantitative metric to parse the effects of calcitriol on collagen fiber organization that cannot be detected through conventional histological procedures. Despite the large body of literature on vitamin D3 (vitD3) and calcitriol's effects on cellular functions across tissue types, little is known about how they affect collagen fiber organization, an early critical step for breast epithelial development. This work provides further evidence that hormones affect morphogenesis by means of biophysical forces, with implications for a comprehensive view on vitD3's effects in breast development and neoplasia.

The microenvironment determines the breast cancer cells' phenotype: organization of MCF7 cells in 3D cultures.

BACKGROUND: Stromal-epithelial interactions mediate breast development, and the initiation and progression of breast cancer. In the present study, we developed 3-dimensional (3D) in vitro models to study breast cancer tissue organization and the role of the microenvironment in phenotypic determination. METHODS: The human breast cancer MCF7 cells were grown alone or co-cultured with primary human breast fibroblasts. Cells were embedded in matrices containing either type I collagen or a combination of reconstituted basement membrane proteins and type I collagen. The cultures were carried out for up to 6 weeks. For every time point (1-6 weeks), the gels were fixed and processed for histology, and whole-mounted for confocal microscopy evaluation. The epithelial structures were characterized utilizing immunohistochemical techniques; their area and proliferation index were measured using computerized morphometric analysis. Statistical differences between groups were analyzed by ANOVA, Dunnett's T3 post-hoc test and chi-square. RESULTS: Most of the MCF7 cells grown alone within a collagen matrix died during the first two weeks; those that survived organized into large, round and solid clusters. The presence of fibroblasts in collagen gels reduced MCF7 cell death, induced cell polarity, and the formation of round and elongated epithelial structures containing a lumen. The addition of reconstituted basement membrane to collagen gels by itself had also survival and organizational effects on the MCF7 cells. Regardless of the presence of fibroblasts, the MCF7 cells both polarized and formed a lumen. The addition of fibroblasts to the gel containing reconstituted basement membrane and collagen induced the formation of elongated structures. CONCLUSIONS: Our results indicate that a matrix containing both type I collagen and reconstituted basement membrane, and the presence of normal breast fibroblasts constitute the minimal permissive microenvironment to induce near-complete tumor phenotype reversion. These human breast 3D tissue morphogenesis models promise to become reliable tools for studying tissue interactions, therapeutic screening and drug target validation.