Information

Is a large tumor is more likely to develop hypoxic regions?


It is known that cancerous tumors in humans can develop hypoxic regions where no blood nor oxygen arrive to some volume of its cells, creating a dead lump inside or around the tumor. See Wikipedia - Tumor hypoxia.

  • Are hypoxic regions and regions without blood supply are more common in large tumors than in small tumors?
  • What is the likelyhood or frequency of hypoxic regions in small (< 2 cm) and large (> 4 cm) tumors?
  • What is the typical size of an hypoxic region within a tumor?

The article in Wikipedia is too techincal and very hard to be read and understood, and doesn't have an explicit answer to my question.


The prescence of hypoxia is independent of size, grade, or histology. It occurs due to a cut off in blood supply to the tumour. Or insufficient vasculature systems, meaning the oxygen cannot diffuse all the way into the tumour. Aberrant growth of tumours can increase the likelyhood of tumours developing hypoxic regions.


Physical and Biologic Basis of Radiation Therapy

Eric J. Hall , James D. Cox , in Radiation Oncology (Ninth Edition) , 2010

Nicotinamide and Carbogen Breathing

Hypoxic cell radiosensitizers such as the nitroimidazoles were designed primarily to overcome chronic hypoxia, which is diffusion-limited hypoxia resulting from the inability of oxygen to diffuse further than about 100 μm through respiring tissue. However, hypoxia also arises through acute mechanisms (i.e., the intermittent blockage of blood vessels). Nicotinamide, a vitamin B3 analogue, has been shown in mouse tumors to prevent the transient fluctuations in tumor blood flow that lead to the development of acute hypoxia.

The combination of nicotinamide to overcome acute hypoxia with carbogen breathing to overcome chronic hypoxia is the basis of the ARCON trials being conducted in several European centers. These trials also involve accelerated and hyperfractionated radiation therapy to avoid tumor proliferation and damage to late-responding normal tissues.


Introduction

The evolution of our multicellular ancestors from their single-celled predecessors required the development of an ability to sense changes in oxygen tension and respond with both an acute change in cell phenotype to preserve survival, but also a more long-term rearrangement of the surrounding architecture to allow better oxygen perfusion. The diffusion limit for oxygen is

100–200 μm, which means that for adequate oxygenation, cells must be within this radius. 1, 2 However, hypoxia is not a binary stimulus, and gradients between one functional blood vessel and the next often allow for appropriate cell and tissue development and function. During fetal development, hypoxia represents a positive, necessary stimulus, required for the appropriate patterning and function of most organs. 3 Indeed, in some organs, a gradient in the oxygen tension across the tissue is required throughout life for their function—an example being in liver zonation. 4 However, it is becoming increasingly clear that the cellular effects of exposure to low-oxygen tensions represent a pernicious facet of many diseases, such as cancer, cardiovascular disease, dementia and diabetes.


The biology of hypoxia

Tumor oxygen levels

Earlier studies assessing oxygen tension (pO2) in tumors by use of the Eppendorf electrodes have clearly demonstrated that hypoxia is a common feature of cervical cancer. Cohort-based median pO2 levels ranging from 2 mmHg (0.3% O2) to 14 mmHg (1.8% O2), as measured before the start of treatment, have been reported [1, 3, 11,12,13]. There are, however, considerable differences between individual tumors, and hypoxic fraction, in terms of pO2 readings below 5 mmHg (0.7% O2), can differ from 0 and up to 100%. A large pre-treatment hypoxic volume has been associated with the presence of lymph node metastases at diagnosis and poor overall or disease free survival [1,2,3,4]. Similar associations have been reported for locoregional control, but the data are less consistent probably due to few relapses in small patient cohorts. The significance is generally retained in multivariate analysis with clinical markers such as tumor stage and size [1, 2, 4]. Assessment of hypoxia status would, therefore, add valuable information to the traditional diagnostics in treatment planning.

Although some tumors are more hypoxic than others, a large intratumor heterogeneity in the oxygen levels exists, and sampling from several regions is required to achieve reliable hypoxia estimates that can be compared across patients [14]. Imaging is particularly appealing in this respect by providing information from the entire tumor. A challenge is, however, the small size of the hypoxic regions, which can be below the spatial resolution of medical images. Hence, pO2 levels ranging from 1 to 20 mmHg (0.1–2.6% O2) over a distance of less than 1 mm have been measured [15], and even smaller hypoxic patches of a couple of cell diameters have been detected with immunohistochemistry, using the hypoxia marker pimonidazole [16].

Tumor hypoxia is not static, but evolves and diminishes in a dynamic process depending on tumor growth, neo-angiogenesis, and treatment [17]. Thus, temporal fluctuation in pO2 over short time periods of less than 1 h has been detected in cervical cancer xenografts [18]. In an orthotopic cervical cancer mouse model, exposing the mice to varying O2 concentrations in cycles during tumor growth was shown to enhance the capability of tumor cells to metastasize to local lymph nodes [19]. This suggests that O2 fluctuations could be of clinical relevance. There is a lack of information on cycling hypoxia in human tumors, and imaging could facilitate such investigations by repetitive non-invasive measurements.

Changes in tumor hypoxia have been measured during fractionated radiotherapy [13, 20,21,22]. In a study comparing pO2 data before treatment and after 10 Gy of radiation, increased oxygenation was primarily found in the most oxygenated tumors, while the hypoxic tumors showed no change or a decrease [22]. This observation was attributed to more treatment-induced cell death and thereby probably a larger decrease in oxygen consumption in oxygenated tumors. The benefit of assessing hypoxia during therapy for outcome prediction is, however, not clear. Suzuki et al. [13] found a stronger association with locoregional control for pO2 data measured after 2 weeks of external radiation, whereas pre-treatment data performed better regardless of end-point in the study by Lyng et al. [4]. Tumor pO2 measured after 26–52 Gy of external radiotherapy seems to be less useful [21].

Physiological and molecular hypoxia markers

Hypoxic tumors show specific physiological and molecular characteristics that can be visualized in medical images. Hypoxia occurs in poorly vascularized tumors, where the impaired oxygen supply is not capable of meeting the oxygen demand of the cells. Necrosis may develop under severe, long-lasting hypoxia, depending on the cells’ ability to adapt to the oxygen and nutrient deprived conditions. Hence, a negative correlation has been found between fraction of necrosis and pO2 in cervix tumors [23]. Several studies have addressed relationships between hypoxia and immunohistochemistry markers of oxygen supply and demand. Poorly vascularized tumors or tumor regions have shown low pO2 [24, 25], and long intercapillary distance has been associated with locoregional relapse [25]. Vascular parameters may, therefore, serve as surrogate markers of hypoxia in cervix tumors before the start of treatment. However, associations with poor outcome have also been found in cases of high vascular density in hot spots, which may reflect high angiogenic activity not necessarily related to hypoxia [25, 26]. Treatment-induced cell death has been shown to be more important than vascular changes for reoxygenation during the early phase of radiotherapy, probably because only small changes in vascular density have occurred at this stage [22]. Tumor cellularity, therefore, seems to also influence the hypoxia status, most likely reflecting the oxygen demand.

Tumor cells adapt to the hypoxic environment partly through stabilization of hypoxia inducible factors HIF1A and EPAS1 (HIF2A), enhancing glycolytic activity to maintain ATP levels when mitochondrial activity has slowed down [27]. Proteins involved in glucose metabolism have been investigated as possible endogenous hypoxia markers in immunohistochemical studies. High expression of HIF1A and its target proteins glucose transporter SLC2A1 (GLUT1) and pH regulator CA9 has been found in cervix tumors that were identified as hypoxic by pimonidazole staining or electrode measurements [28,29,30,31,32]. However, the spatial overlap between protein expression and pimonidazole staining has been found to be poor in many tumors [28, 31]. Moreover, although significant association between clinical outcome and expression of HIF1A, SLC2A1, CA9, or the glycolytic enzymes HK2 and PFKM2 has been found both for early [33,34,35] and late stage disease [32, 36,37,38,39,40,41,42,43], conflicting results have been reported [29, 30, 41]. This can probably be explained, because HIF1A and its target genes may be regulated by other factors than hypoxia, including reactive oxygen species (ROS), oncogenes, and metabolic stressors like lactate [27]. In addition, tumor cells rely in general on glycolysis rather than oxidative phosphorylation as energy source even in the presence of oxygen, a phenomenon termed the Warburg effect [27]. Rapidly proliferating cells can, therefore, have high glycolytic activity and express proteins involved in glucose metabolism regardless of hypoxia.

In addition to activation of the HIF pathway, hypoxia tolerance in tumors is also mediated by activation of the unfolded protein response (UPR) and inhibition of MTOR signaling [44]. In global gene expression studies, we found that both HIF targets and UPR genes were required to construct a robust hypoxia gene classifier that could be validated across two independent cervical cancer cohorts [45, 46]. The importance of UPR has been further emphasized by the observations that high expression of the UPR regulated protein LAMP3 promoted hypoxia-driven metastasis in an orthotopic cervical cancer model and was associated with hypoxia in patient tumors [47]. Increased understanding of the mechanisms underlying hypoxia-related aggressiveness will be important for development of new molecular imaging approaches.


Oxygen sensing and the hypoxia-inducible factor (HIF) system

About 2.5 billion years ago, the evolution of photosynthesis in cyanobacteria liberated oxygen into Earth's atmosphere 1 . Under standard temperature and pressure, two atoms of oxygen combine to form molecular oxygen (i.e. O2 or ‘dioxygen’), a colourless and odourless gas. Increases in atmospheric oxygen coincided with the development of multicellular organisms (Metazoa) 2 , which depend upon oxygen for a range of physiological processes, most notably energy production. Although oxygen is essential to generate cellular energy, it is toxic at high concentrations 3 . Thus, oxygen homeostasis must match supply and demand across distinct cell types that inhabit environments with varying oxygen levels 4 . In higher animals, the respiratory, cardiovascular, and haematopoietic systems are responsible for oxygen transport. Physiological control occurs through acute regulation of ventilation by peripheral and central chemoreceptors, interweaved with slower adaptive responses, including transcriptional effects mediated through the HIF system 5, 6 . Pivotal discoveries pertaining to this pathway have recently been recognised by the award of the Nobel Prize in Physiology or Medicine 2019 7 .

HIF functions as a heterodimer of an oxygen-regulated α subunit (either HIF-1α, HIF-2α, or HIF-3α) and a β subunit (HIF-β), which is widely expressed in an oxygen-independent manner. HIF-α subunits are hydroxylated in an oxygen-dependent manner by the prolyl hydroxylase domain-containing enzymes (PHD1, 2, and 3), facilitating recognition by the von Hippel–Lindau ubiquitin E3 ligase (pVHL) and subsequent proteasomal degradation (Figure 1). The PHDs belong to the 2-oxoglutarate (2-OG)–dependent dioxygenase family, which in humans contains about 70 enzymes that target different substrates. These enzymes require dioxygen, the tricarboxylic acid (TCA) cycle intermediate 2-OG, and ferrous iron (Fe 2+ ) as reaction co-factors. Other members of the 2-OG-dependent dioxygenase family include collagen prolyl hydroxylases and epigenetic modifiers such as the Jumonji domain-containing family of histone demethylases and the ten-eleven translocation (TET) DNA 5-methylcytosine hydroxylases. In hypoxia, reduced oxygen availability inhibits the enzymatic activity of PHDs, leading to HIF-α stabilisation, heterodimerisation with HIF-β, co-activator recruitment (itself regulated in an oxygen-dependent manner by the asparaginyl hydroxylase, factor inhibiting HIF [FIH], another member of the dioxygenase family) and transcription of many hundreds of genes. Overall, this entrains a gene-expression profile that facilitates cellular adaptation to hypoxia, for example, by switching cellular metabolism from oxidative phosphorylation to glycolysis (to reduce oxygen demand) and by promoting erythropoiesis and angiogenesis (to restore tissue oxygenation), but also causes responses less intuitively related to oxygenation 8 . Like most physiological systems, activation of the HIF pathway is regulated by a number of feedback loops that shape its overall output 9 . In humans, the HIF-1α-PHD2-VHL axis is ubiquitously expressed and most closely represents ‘archaic’ components of the pathway, whereas HIF-2α, HIF-3α, PHD1, and PHD3 are ‘modern’ genes, derived via gene duplication events, that demonstrate tissue-restricted expression and have evolved to perform specific functions. Reflecting this paradigm, HIF-1α and HIF-2α modulate expression of both overlapping and distinct target genes 10 .

The effects of HIF pathway activation are dependent upon changes to transcriptional output and, consequently, represent medium- to long-term adaptations to hypoxia occurring over hours, days, and weeks. Other less well-defined, or still to be discovered, oxygen-sensing systems must exist that mediate acute responses over seconds to minutes. Intriguingly, a mammalian homologue of a plant oxygen sensor has recently been identified as cysteamine (2-aminoethanethiol) dioxygenase (ADO) 11 . This enzyme alters target protein stability directly through post-translational modification and, therefore, is likely to transduce more rapid responses to hypoxia than those mediated by the HIF system (Figure 2).


Role of ferroptosis in disease and therapeutic opportunities

Although the physiological function of ferroptosis remains obscure, its role in a plethora of human diseases has been extensively documented. Importantly, pharmacological modulation of ferroptosis has been demonstrated to be a promising therapeutic venue for the treatment of cancer and IRI in diverse preclinical animal models. While in this section we focus on the role and therapeutic implication of ferroptosis in cancer and IRI, ferroptosis has been implicated in the pathogenesis of many other diseases, as discussed in Box-1.

Box 1 –

Diseases possibly related to ferroptosis

In addition to cancer and ischemic organ injuries, ferroptosis has been implicated in the pathogenesis of a growing list of other diseases, such as neurodegeneration 19,165�,182 , liver and lung fibrosis 80,183 , autoimmune diseases 184,185 , Mycobacterium-tuberculosis-induced tissue necrosis 186 , cigarette-smoking-associated chronic obstructive pulmonary disease 187,188 , and a rare genetic neurological disorder called Pelizaeus-Merzbacher Disease 189 . While this long list speaks to the clinical relevance and therapeutic potential of ferroptosis-modulating approaches, further investigation is required to determine if there is indeed a causative role of ferroptosis in these diseases. For example, in most cases the general observation is that non-apoptotic cell death was observed in the disease tissue, and that a ferroptosis inhibitor, often a lipophilic RTA, could mitigate the observed cell death and in some cases, alter the severity of the symptom. However, lipid peroxides can regulate immunity and inflammation, processes that play important roles in all the listed diseases. Therefore, caution is needed to distinguish whether the observed effect of lipophilic RTA is via modulation of inflammation or ferroptosis, or both. A detailed mechanistic interrogation of tissue cell death associated with the disease, including examining specific in vivo ferroptosis biomarkers (which the field sorely miss), will be crucial for this purpose.

Ferroptosis in cancer.

Ferroptosis has been linked to cancer since the very beginning of the field: the initial discovery of chemical inducers of ferroptosis is the result of a hunting for novel cancer therapeutic compounds 20,21 . Subsequent mechanistic studies have revealed that numerous cancer-relevant genes and signaling pathways regulate ferroptosis. The observations that mesenchymal and dedifferentiated cancer cells, which are often resistant to apoptosis and common therapeutics, as well as so called “therapy-persister” cancer cells, are highly susceptible to ferroptosis inducers 39,133,134 , further underscores the promise of ferroptosis induction as a novel cancer therapy.

Conceptually, since ferroptosis is an oxidative stress-induced, metabolic form of cell death, it seems logical to propose that cancer cells may have higher tendency to undergo ferroptosis, due to overall more active metabolism and higher ROS load. Adding to this notion, it has been shown that cancer cells often demand high iron contents 135,136 , which may further sensitize them to ferroptosis. However, cancer cells may also harness additional genetic or epigenetic alterations to counter these metabolic and oxidative burdens, such as increased expression of SLC7A11 or upregulation of the anti-oxidative transcription factor NRF2 137 . Therefore, whether a given cancer is more sensitive or resistant to ferroptosis induction is dictated by its specific genetic background. The genomics of cancer, as well as various other parameters as discussed below, should be considered for the development of ferroptosis induction-based cancer therapy.


Figure 1. Tumour hypoxia. When a small, localised tumour outgrows its vascular supply (distances >100 ?m) tumour hypoxia arises in regions with impaired oxygen delivery. Consequently, hypoxic cells switch on target genes involved in angiogenesis [vascular endothelial growth factor (VEGF)], glucose transport [glucose transporter 1 (GLUT-1)] and cell migration [urokinase-type plasminogen activator receptor (u-PAR) and plasminogen activator inhibitor 1 (PAI-1)]. Increased vascular supply to the tumour via the induction of new blood vessel formation (angiogenesis) encourages tumour growth and facilitates metastasis to distant sites. One of the functions of the mammalian hypoxic response in development and cancer is the generation of nascent vascular networks through angiogenesis. Through transcriptional regulation of the vascular endothelial growth factor A (VEGF-A) and other angiogenic factors, the Hypoxia-Inducible Factor 1 and 2 alpha (HIF-1alpha and HIF-2alpha) can increase angiogenesis in an oxygen dependent fashion, giving a survival and growth advantage to HIF wild type tumours (UCSD n.d.). Angiogenesis is an important mediator of tumor progression. As tumors expand, diffusion distances from the existing vascular supply increases resulting in hypoxia. Sustained expansion of a tumor mass requires new blood vessel formation to provide rapidly proliferating tumor cells with an adequate supply of oxygen and metabolites. The key regulator of hypoxia-induced angiogenesis is the transcription factor hypoxia inducible factor (HIF)-1 (Liao & Johnson, 2007). Deficiencies in oxygenation are widespread in solid tumors. The transcription factor HIF-1a is an important mediator of the hypoxic response of tumor cells and controls the up-regulation of a number of factors important for solid tumor expansion, including the angiogenic factor vascular endothelial growth factor (VEGF). The evidence from these experiments by Ryan et al, (2000) indicates that hypoxic response via HIF-1a is an important positive factor in solid tumor growth and that HIF-1a affects tumor expansion in ways unrelated to its regulation of VEGF expression. Cells undergo a variety of biological responses when placed in hypoxic conditions, including activation of signalling pathways that regulate proliferation, angiogenesis and death. Cancer cells have adapted these pathways, allowing tumours to survive and even grow under hypoxic conditions, and tumour hypoxia is associated with poor prognosis and resistance to radiation therapy. Many elements of the hypoxia-response pathway are therefore good candidates for therapeutic targeting (Harris, 2002). Nobel Laureate, Dr. Otto Warburg, in his article “The Prime Cause and Prevention of Cancer” (1966) wrote, “Cancer, above all other diseases, has countless secondary causes. Almost anything can cause cancer. But, even for cancer, there is only one prime cause. The prime cause of cancer is the replacement of the respiration of oxygen (oxidation of sugar) in normal body cells by fermentation of sugar… In every case, during the cancer development, the oxygen respiration always falls, fermentation appears, and the highly differentiated cells are transformed into fermenting anaerobes, which have lost all their body functions and retain only the now useless property of growth and replication.“ Dr. Otto Warburg investigated the metabolism of tumors and the respiration of cells, particularly cancer cells, and in 1931 was awarded the Nobel Prize in Physiology or Medicine for his “discovery of the nature and mode of action of the respiratory enzyme.” These conclusions are important since we have already proved the following key findings: 1. Sick patients (various chronic diseases) breathe much more than the norm. 2. Overbreathing or hyperventilation reduces CO2 content in the lungs and arterial blood. 3. Due to numerous uses of CO2 in the human body, hypocapnia (lowered CO2) leads to reduced oxygenation of all vital organs and tissues due to chest breathing, vasoconstriction, and suppressed Bohr effect. The Bohr effect explains oxygen release in capillaries or why red blood cells unload oxygen in tissues. The Bohr effect was first described in 1904 by the Danish physiologist Christian Bohr (father of famous physicist Niels Bohr). Christian Bohr stated that at lower pH (more acidic environment, e.g., in tissues), hemoglobin will bind to oxygen with less affinity. Since carbon dioxide is in direct equilibrium with the concentration of protons in the blood, increasing blood carbon dioxide content causes a decrease in pH, which leads to a decrease in affinity for oxygen by hemoglobin. Do modern scientists have a different opinion about the prime cause of cancer on cell level? It has been known for decades that malignant cells normally and constantly appear and exist in any human organism due to the billions of cell divisions and mutations. These abnormal cells, under normal conditions, are quickly detected by the immune system and destroyed. However, the work of macrophages, enzymes and other agents of the immune system is severely hampered when the conditions of hypoxia exists. That was the conclusion of various studies. For example, Dr. Rockwell from Yale University School of Medicine (USA) studied malignant changes on the cellular level and wrote, “The physiological effects of hypoxia and the associated micro environmental inadequacies increase mutation rates, select for cells deficient in normal pathways of programmed cell death, and contribute to the development of an increasingly invasive, metastatic phenotype” (Rockwell, 1997). The title of this publication is ” Oxygen delivery: implications for the biology and therapy of solid tumors”. Summarizing the results of numerous studies, a group of biological scientists from University of California (San Diego) chose the following title for their article, “The hypoxia inducible factor-1 gene is required for embryogenesis and solid tumor formation” (Ryan et al, 1998). Under normal conditions, even a group of hypoxic cells dies (or is easily destroyed). What about cells in malignant tumors? Researchers from the Gray Laboratory Cancer Research Trust (Mount Vernon Hospital, Northwood, Middlesex, UK) concluded, “Cells undergo a variety of biological responses when placed in hypoxic conditions, including activation of signalling pathways that regulate proliferation, angiogenesis and death. Cancer cells have adapted these pathways, allowing tumors to survive and even grow under hypoxic conditions…” (Chaplin et al, 1986). There is so much professional evidence about the fast growth of tumors when the condition of hypoxia is present that a large group of Californian researchers recently wrote a paper “Hypoxia – inducible factor-1 is a positive factor in solid tumor growth” (Ryan et al, 2000). Echoing their paper, a British oncologist Dr. Harris from the Weatherhill Institute of Molecular Medicine (Oxford) went further with the manuscript “Hypoxia – a key regulatory factor in tumor growth” (Harris, 2002). When the solid tumor is large enough and the disease progresses, cancer starts to invade other tissues. This process is called metastasis. Does poor oxygenation influence it? “…Therefore, tissue hypoxia has been regarded as a central factor for tumor aggressiveness and metastasis” (Kunz & Ibrahim, 2003). That was the conclusion of a group of German researchers from the University of Rostock and the University of Leipzig. Since dozens of medical and physiological studies yield the same result, what about the following title? “Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma” (Brizel et al, 1996). This title claims that tumor oxygenation predicts chances of cancer invasion. The reader can probably guess about the effect of cancer treatment and the chances of survival for those who suffer from severe chronic hyperventilation. Indeed, “… tumor hypoxia is associated with poor prognosis and resistance to radiation therapy” (Chaplin et al, 1986). American scientists from Harvard Medical School noted “… Hypoxia may thus produce both treatment resistance and a growth advantage” (Schmaltz et al, 1998). “Low tissue oxygen concentration has been shown to be important in the response of human tumors to radiation therapy, chemotherapy and other treatment modalities. Hypoxia is also known to be a prognostic indicator, as hypoxic human tumors are more biologically aggressive and are more likely to recur locally and metastasize” (Evans & Koch, 2003). “Clinical evidence shows that tumor hypoxia is an independent prognostic indicator of poor patient outcome. Hypoxic tumors have altered physiologic processes, including increased regions of angiogenesis, increased local invasion, increased distant metastasis and altered apoptotic programs” (Denko et al, 2003). Another factor is iron metabolism. Iron is an essential element in all living organisms and is required as a cofactor for oxygen-binding proteins. Iron metabolism, oxygen homeostasis and erythropoiesis are consequently strongly interconnected. Iron needs to be tightly regulated, as iron insufficiency induces a hypoferric anemia in mammals, coupled to hypoxia in tissues, whereas excess iron is toxic, and causes generation of free radicals. Given the links between oxygen transport and iron metabolism, associations between the physiology of hypoxic response, and the control of iron availability are important. Numerous lines of investigation have proven that the HIF transcription factors function as central mediators of cellular adaptation to critically low oxygen levels in both normal and compromised tissues. Several of these target genes are involved in iron homeostasis, reflecting the molecular links between oxygen homeostasis and iron metabolism (Peyssonnaux et al, 2008). Copper is another factor in hypoxia. Cellular oxygen partial pressure is sensed by a family of prolyl-4-hydroxylase domain (PHD) enzymes that modify hypoxia-inducible factor (HIF)alpha subunits. Ceruloplasmin, the main copper transport protein in the plasma and a known HIF-1 target in vitro, was also induced in vivo in the liver of hypoxic mice. Both hypoxia and CuCl(2) increased ceruloplasmin (as well as vascular endothelial growth factor [VEGF] and glucose transporter 1 [Glut-1]) mRNA levels in hepatoma cells, which was due to transcriptional induction of the ceruloplasmin gene (CP) promoter (Martin et al, 2005). The authors of one of the studies cited above mused about the origins of all these problems, “Surprisingly little is known, however, about the natural history of such hypoxic cells” (Chaplin et al, 1986). Why do they appear? What is the source of tissue hypoxia? The answer is unknown and likely systemic but the conclusion is therefore, appearance, development and metastasis of tumors are based on cell hypoxia. References Brizel DM, Scully SP, Harrelson JM, Layfield LJ, Bean JM, Prosnitz LR, Dewhirst MW, Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma, Cancer Research 1996, 56: p. 941-943. Chaplin DJ, Durand RE, Olive PL, Acute hypoxia in tumors: implications for modifiers of radiation effects, International Journal of Radiation, Oncology, Biology, Physics 1986 August 12(8): p. 1279-1282. Denko NC, Fontana LA, Hudson KM, Sutphin PD, Raychaudhuri S, Altman R, Giaccia AJ, Investigating hypoxic tumor physiology through gene expression patterns, Oncogene 2003 September 1 22(37): p. 5907-5914. Evans SM & Koch CJ, Prognostic significance of tumor oxygenation in humans, Cancer Letters 2003 May 30 195(1): p. 1-16. Harris AL, Hypoxia: a key regulatory factor in tumor growth, National Review in Cancer 2002 January 2(1): p. 38-47. Kunz M & Ibrahim SM, Molecular responses to hypoxia in tumor cells, Molecular Cancer 2003 2: p. 23-31. Liao D, Johnson RS. Hypoxia: A key regulator of angiogenesis in cancer. Cancer and Metastasis Reviews. 2007 Volume 26, Number 2, 281-290, DOI: 10.1007/s10555-007-9066-y Martin F, Linden T, Katschinski DM, et al. Copper-dependent activation of hypoxia-inducible factor (HIF)-1: implications for ceruloplasmin regulation. Blood. 2005 Jun 15105(12):4613-9. Peyssonnaux C, Nizet V, Johnson RS. Role of the hypoxia inducible factors HIF in iron metabolism. Cell Cycle. 2008 Jan 17(1):28-32. Rockwell S, Oxygen delivery: implications for the biology and therapy of solid tumors, Oncology Research 1997 9(6-7): p. 383-390. Ryan H, Lo J, Johnson RS, The hypoxia inducible factor-1 gene is required for embryogenesis and solid tumor formation, EMBO Journal 1998, 17: p. 3005-3015. Ryan HE, Poloni M, McNulty W, Elson D, Gassmann M, Arbeit JM, Johnson RS, Hypoxia-inducible factor-1 is a positive factor in solid tumor growth, Cancer Res, August 1, 2000 60(15): p. 4010 – 4015. Schmaltz C, Hardenbergh PH, Wells A, Fisher DE, Regulation of proliferation-survival decisions during tumor cell hypoxia, Molecular and Cellular Biology 1998 May, 18(5): p. 2845-2854. UCSD. (n.d.) http://biology.ucsd.edu/labs/johnson/cancer.html

[…] Dr. Rockwell from Yale University School of Medicine (USA) studied malignant changes on the cellular level and wrote, “The physiological effects of hypoxia and the associated micro environmental inadequacies increase mutation rates, select for cells deficient in normal pathways of programmed cell death, and contribute to the development of an increasingly invasive, metastatic phenotype”[3] […]

VINDICATED AT LAST’
The first Cancer conference I attended 16 years ago was a joint Lorne (Vic Aust) AACR (American Association of Cancer Research) conference. A lecture on the second day was about a bowel cancer in which the number one tumor repressor p53 was mutated, presumably the reason for the tumor, and it secreted a prostaglandin PGF2. Now I knew that PGF2 constricted or narrowed blood vessels supplying the gastrointestinal tract and suddenly I had a different spin on the sequence of events. At question time I suggested that hypoxia or oxygen starvation was the cause of the bowel cancer, not the mutations in the p53 tumor repressor. I suggested that the mutations in the p53 were a RESULT of the hypoxia. As I said this tumor secreted a prostaglandin PGF2 which is a vasoconstrictor. Adrenalin activates PGF2 in the gastro-intestinal tract in response to ‘fight or flight’, stress in other words. My theory went like this. Stress>adrenalin>PGF2>vasoconstriction>HYPOXIA>p53 mutation>CANCER.

At the end of the symposium a chap approached me and suggested that this was a very interesting idea as he was working on the same cells. It turned out that he was the AACR convener. I saw him later and explained to him what is called the RAS pathway by which adrenalin works. He said, “You must be a very good biochemist” I was nonplussed by this as I didn’t thing my suggestion was anything extraordinary and I replied that “I’m not a biochemist, I’m an electronic technician, this is just a hobby for me. He looked dumbfounded as if I was taking the Mickey out of him and didn’t say anything, then took off. I didn’t run into him again for the rest of the Conference.

The following year when I went to pay the Conference secretary for everything, registration, all meals and accommodation she whispered as she handed me my bag, “Don’t worry Noddy, it all been taken care off” ($750>. I was gobsmacked as there was no explanation as to why. The same thing happened for the following two years the gradually tapered off. I know now. You see my answer that day involved endocrinology, biochemistry, hematology, oncology, molecular biology etc. all of which I had studied BECAUSE I DIDN’T KNOW THAT I WASN’T SUPPOSED TO

HYPOXIA 2.
All of the delegates are molecular biologist looking for GENES to blame, then design the appropriate drug. It is pure commerce and that blinds totally. This, plus Dawkinist dogma, makes it IMPOSSIBLE to understand cancer, let alone find a cure. How could the delegates POSSIBLY admit that an amateur scientist had worked something out that the entire global community of cancer researchers couldn’t. Yet taking the multidisciplinary approach made it easy, hence my amazement at the response to my suggestion. These conferences are run by private enterprise and they are not gonna give away thousands of dollars to some amateur out the goodness of their hearts. Now I know this sounds incredibly mean spirited, but I really am grateful.

At the last conference I attended there were two lectures on the discovery of the HIF (hypoxia inducable factor) proteins and their associated genes. No kudos for me, however. If I had said “I suggested that years ago” I would have been howled down.

OXYGEN TENSION AS PRIMARY DIFFERENTIATION FACTOR.
There would appear to be overwhelming evidence that OXYGEN Tension is the PRIMARY differentiating factor, ie that which determines at what stage of development or differentiation (going forward) that cells are at or what stage of DEDIFFERENTIATION (going backward) that cancer cells are at. What if, after transformation of epithelial cells, angiogenesis (the growth of blood vessels) is PROGRAMMED to lag behind tumor growth, so that as oxygen tension goes down in the tumor, it’s cells increasingly DEDIFFERENTIATE, those furthest from the blood vessels and thus exposed to the LEAST oxygen tension. undergoing EMT (epithelial to mesenchymal transfer) to become cancer stem cells.

Then as these cells with a mesenchymal phenotype crawl toward the blood vessels to metastasise they are exposed to increased oxygen tension and revert to an epithelial phenotype? The question is why all this order? HIF (hypoxia inducable factor) proteins affect 800 genes, 1/13 of all coding genes. That an AWFUL lot of genes for something that is supposed to be a stuff up, eh

HIFs IN CANCER PROGRESSION. (Semenza. PDF)
This is from an excellent paper, with several exceptions, by Jeff Semenza MD, on hypoxia. The text in brackets is mine.

HIFs play key roles in many crucial aspects of cancer biology including 1. angiogenesis, (the growth of blood vessels to accommodate the growth of a tumour) 2. stem cell maintenance, (preventing the cell from fully differentiating to a mature cell going onto terminal differentiation, ie apoptosis or cell suicide) 3. metabolic reprogramming (from oxidative metabolism to non oxidative fermentation of glucose), 4. autocrine growth factor signalling (self signalling rather than paracrine , juxtacrine , or endocrine signalling, 5. epithelial-mesenchymal transition or EMT (not limited to a single or monolayer of cells, eg those lining a milk duct and conversion of resultant tumor cells from an epithelial phenotype to a mesenchymal phenotype), 6. invasion (of surrounding tissue), 7. metastasis (cells moving into nearby bloodvessels then circulating to set up a secondary tumor at a a distant site), and 8. resistance to radiation therapy and chemotherapy.

AMAZING.
ALL attributes of CANCER, built into almost every cell in response to HYPOXIA. WHY? Why don’t cancer researchers ask this question? This and a lot of other factors suggest ‘wild type’ cancer. There is one very important word glaringly missing in this paper. DEDIFFERENTIATION of the cancer cell, going BACKWARDS, retracing the steps that it took during embryogenesis. Older books eg my ‘Ocology 1972’ are full of the word. It is not questioned. It is a word that has fallen into almost total misuse, maybe for the very reason that is might suggest Larmarkism, ie back all the way to the germ line and THAT is heresy. (This blindness seems to coincide with the rise of Richard Dawkins). Thus the stem cells referred to might have arisen from fully differentiated cells that have dedifferentiated in response to hypoxia.

Instead of the paper suggesting addressing the CAUSES of the hypoxia, ie stress and arteriosclerosis it instead suggests different sites whereby the action of HIF’s might be blocked by a drug or two or three. Still, getting rid of the stress in the US, might make these drugs, and their myriad side effects, the only option. (I live in Australia.)

ADRENALIN.
In ‘fight or flight’ adrenalin, mediated by prostaglandin PGF2, CONSTRICS all blood vessels not essential to same, the gastrointestinal tract and associated organs and presumably to breast as an animal cannot breast feed and run simultaneously. Enter HYPOXIA, not a problem short term, but long term due to persistent stress, malabsorption of IRON (and cobalt) thus adding ANEMIA, making the hypoxia global, but obviously worse in tissue in which vasocontriction prevails. (Then there is malabsorption of nutrients, such as anti-oxidants, vitamins, minerals etc.)

NORADRENALIN.
A mood enhancer at low levels, as evidenced by the SNRI’s blocking noradrenalin’s reuptake in the synapse. However at higher levels associated with panic attacks, paranoid schizophrenia etc.

At high concentrations noradrenalin constricts ALL ARTERIES AND VEINS. Global HYPOXIA.
However not everybody gets panic attacks, so what about something that is almost universal and can profoundly effect oxygen transport to the tissues.

REFINED SUGAR and ARTERIOSCLEROSIS.
Almost the entire world pigs out on HUGE amounts of sugar. In high blood concentrations albeit transitory with reactive hypoglycaemia, glucose binds to blood vessel walls via NON-ENZYMATIC GLYCOSATION of PROTEIN, also trapping cholesterol. The use of Statins to control blood cholesterol can lead to diabetes 2 because they block the pathway between Acetyl CoA and the Melovanate pathway. Thus excess glucose is converted to cholesterol, maybe to limit OSMOTIC HYPERTENSION caused by excess blood glucose.

Thus sugar also contributes cholesterol to the plaques gunking up blood vessels and possibly smothering receptors on epithelial cells, so inhibiting the action of endocrine hormones on hemostasis.

BLOOD PRESSURE and BETA BLOCKERS.
Blood vessels have carbon dioxide detector nodes along them, so that if CO2 builds up in the bloodstream, meaning reduced oxygen, then the heart increases its output, the result being higher blood pressure. Now gunked up blood vessels inhibit the flow of blood through them so the heart has to increase it’s output to compensate, meaning higher blood pressure and risk of stroke. So you take blood pressure controls e.g. beta blockers and you lower your blood pressure but increase your RISK OF CANCER by definition, the Warburg effect.

INDUCER AND PROMOTER.
If HYPOXIA can be the prime INDUCER of cancer, albeit slow growing, then what about a toxic blood concentration of estradiol (due to impaired deactivation resulting from liver pathology, as the liver is the primary site of estradiol deactivation), acting as the PROMOTER of breast cancer.
Early ductal cell carcinoma cells have up to 1,000 times more ER than a normal cell. This would suggest 1,000 times more deactivation of estradiol than a normal ductal cell. Hundreds of times more cells in the tumor and you have a MASSIVE increase in the ability of the tumor to deactivate the hormone, thus protecting the body from estradiol mediated kidney and liver damage AND osteoporosis, as a high concentration estradiol stimulates the secretion of PROLACTIN which demineralises bone.

It is thus difficult not to reason that THIS IS WHY THE CANCER GROWS IN THE FIRST PLACE, ie. to compensate for LIVER pathology. Thus to use chemo which is hepatoxic does seem a little irrational I think one just might agree, unless one is drowning in Dawkinism.


Diffusion Tensor Imaging and White Matter Tractography

Diffusion tensor imaging (DTI) is a rapidly growing area of research in technical optimization and image processing, as well as clinical application in a variety of intracranial disorders. DTI is distinguished from DWI by its sensitivity to anisotropic or directionally dependent diffusion and provides unique information on 3D diffusivity, which is characterized by 3 eigenvectors (direction) and 3 eigenvalues (magnitude). In humans, brain DTI provides a 3D depiction of white matter connectivity that allows unprecedented opportunity to study brain cytoarchitecture at a microscopic level. The term “tensor” is a mathematic construct adopted from physics and engineering to describe tension forces in solid bodies with an array of 3D vectors. In DTI, tensor is made up of a matrix of numbers derived from diffusion measurements in at least 6 independent diffusion-encoding directions to calculate orientation-dependent diffusion in all spatial directions for each image voxel. The anisotropic diffusion in the brain is largely attributed to 2 unique cytoarchitectural compositions of the brain: myelin and axons. The lipid bilayer of myelin sheath creates a unique microsopic diffusion barrier that results in different degrees of diffusion along different planes. 17 Myelin alone, however, cannot be responsible for anisotropic diffusion, because nonmyelinated nerves have been shown to demonstrate striking diffusion anisotropy. 18 The attenuation and packing of axons and the subcomponents of axons such as the micro- and neurofilaments, microtubules, and membranes also contribute significantly to the anisotropic diffusion. Although the biologic basis of diffusion anisotropy is not yet completely understood, the differential degree of water diffusivity that is maximal parallel to and minimal perpendicular to the long axes of collimated axonal bundles and myelin sheath create a unique situation to measure and tract fiber orientation, especially in large white matter tracts such as the corticospinal tract.

In brain tumors, DTI tractography has had a tremendous impact on intraoperative guidance in tumor resection. 19 DTI tractography maps of desired white matter tracts can be overlaid onto high-resolution anatomic images and provide information on alterations in fiber tract directionality and integrity due to neighboring brain tumor (Fig 3). DTI tractography of the corticospinal tract has gained popularity among neurosurgical colleagues as a noninvasive guide to avoid injuring the corticospinal tract during tumor resection.

A 37-year-old man with right frontal low-grade astrocytoma. A series of axial postcontrast T1-weighted images of the brain demonstrate a nonenhancing right frontal insular mass. The diffusion tensor tractogram, which has been coregistered and overlaid onto the contrast-enhanced T1-weighted images, demonstrates corticospinal tract (white marks) that is displaced but not invaded by the tumor.


Oxygen-starved tumor cells have survival advantage that promotes cancer spread

Using cells from human breast cancers and mouse breast cancer models, researchers at the Johns Hopkins Kimmel Cancer Center say they have significant new evidence that tumor cells exposed to low-oxygen conditions have an advantage when it comes to invading and surviving in the bloodstream.

The experiments mapping the "fate" of the cells in two- and three-dimensional lab-created tissue systems and in live animals specifically showed that cells from a primary cancer exposed to low oxygen levels, or hypoxia, have a four times greater probability of becoming viable circulating tumor cells -- and likely spreading to distant tissues -- than those under normal oxygen conditions.

The results were described Oct. 24 in the journal Nature Communications.

"Our findings also show that these post-hypoxic cells have six times the probability of forming lung metastases, suggesting that oxygen starvation enhances their metastatic capabilities," says study leader Daniele Gilkes, Ph.D., assistant professor of oncology and researcher in the breast and ovarian cancer program of the Johns Hopkins Kimmel Cancer Center.

Gilkes and her team also identified a pattern of genetic expression in post-hypoxic cells that appears to help the cells survive oxidative stress when they enter the bloodstream. Some tumor cells retain parts of this genetic signature as a "hypoxic memory" even after they have been reoxygenated, the researchers found.

"Cancer cells tend to become more aggressive as they adapt to low oxygen levels," says Gilkes, "but we were surprised to find that cells that were exposed to hypoxia in the primary tumor maintained their aggressive features even when they were reoxygenated in the blood."

In the future, the unique features of the hypoxic cells might be used as biomarkers to identify patients at risk for metastasis, or might be targeted directly by therapies to prevent or limit metastasis, the research team suggested.

Hypoxia occurs in 90% of solid tumors and is known to have an adverse impact on a patient's prognosis. However, little is known about how tumor cells change in response to low oxygen. Gilkes says most research teams -- including her own -- grow and experiment with tumor cells using the same oxygen concentrations as normal air.

"This is actually a much higher level of oxygen than what is found in our bodies," Gilkes says. "For example, the average concentration of oxygen in breast tissue is on the order of 6% to 8%, whereas solid breast tumors have a gradient of oxygen concentrations that reach much less than 1% oxygen in some regions."

For their new experiments, designed to capture the changes that occur as normal breast cells become malignant, Gilkes and colleagues developed an experimental system that uses oxygen as a switch to make tumor cells "light up" with a fluorescent marker after they are exposed to low oxygen conditions of 0.5% or less, comparable to the levels measured in human tumors.

The study's first author and member of Gilkes' lab, Inês Godet, used this marker to follow the fate of these cells as they multiplied and moved around within 2D and 3D tissue "spheres " and "mini-organs" created in the laboratory, as well as in live mouse models of breast cancer.

Using fluorescence activated cell sorting to capture red or green (oxygen deprived) breast cancer cells, followed by RNA sequencing, the team found that the expression of many gene products, including integrin alpha 10 (ITGA10) and ceruloplasmin (CP) are induced in cells that experienced hypoxia within tumors, but not in cells exposed to hypoxia in the lab. The tumor-based hypoxia pattern was also better at predicting the survival of patients free of distant metastases, they concluded after studying similar genetic expression data from primary tumors from more than 1000 patients with breast cancer.

Among the next questions to answer, say the researchers, are whether post-hypoxic tumor cells at metastatic sites are more resistant to chemotherapy than other cells and whether targeting these post-hypoxic cells will be beneficial for treating patients with metastatic cancers.


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