Intro Cancer progresses in several stages - cancers of epithelial cells (ones which line the surfaces which require the smooth flow of fluids, like blood vessels), also known as carcinomas, will initially grow locally before infecting the neighbouring tissue (stroma, made of an extracellular matrix, fibroblast cells, immune cells and capillary vessels). The primary tumor's rapid growth is controlled by 3 key elements: (i) Accumulation of gene mutations (ii) Biochemical environment (iii) Mechanical environment These factors are difficult to accurately isolate and therefore determine the importance of these factors individually. Mechanical Stress Recent studies have suggested that mechanical stress plays a role in the progression of tumor - when applied to genetically predisposed tissues or tumor spheroids grown artificially it induces signalling pathways which are characteristic of cancer invasion, while it has also been shown that an increase of mechanical stress leads to a reduction in cancer cell growth (artificially) and "drives apoptosis though the mitochondrial pathway" (i.e. a mechanism is activated by stress on the cells, which generate intracellular signals, which is a process of programmed cell death which occurs in multicellular organisms). Also - Mitochondria are known as the powerhouses of the cell. They are organelles that act like a digestive system which takes in nutrients, breaks them down, and creates energy rich molecules for the cell. The biochemical processes of the cell are known as cellular respiration. The Theory of This Experiment A theoretical framework was developed to describe the balance between cell division and apoptosis on tumor growth under stress, in order to try to better understand the relationship between the tumor and its microenvironment.  This theory is based on the existence of a homeostatic state of tissue (the body attempting to maintain an equilibrium within its internal environment), so when the rate of tissue cell division and cell death are equal. Homeostatic Stress The homeostatic stress is a function of the biochemical state of the tissue and depends on the local concentrations of nutrients, oxygen and growth factors, as well as on the environment of the tissue. For example, signalling induced by the stroma can modify the homeostatic state.  In the simple case, (where biochemical stress can be kept constant) the homeostatic stress is that which the tissue can exert steadily against the walls of a confining chamber. Hence, to grow against surrounding tissue, cells have to exert mechanical stress on the neighbouring cells. The Aim of This Expriment The aim here was to test the relevance of the concept of the effects of homeostatic stress.This was done by measuring the effect of known external stress on the growth of a mock-tumor (with time scales longer than the typical time scales of cell division or apoptosis. Well defined mechanical stress is applied to multicellular tumor spheroids for a period longer than 20 days.

Method CT26 cell lines (from a mouse) were used to derive a colon carcinoma cell spheroid Well plate sides were covered in gel Cell suspensions seeded on gels Cells self assemble into spheroids (in less than 24hrs) Cells were cultured Confocal microscopy was used to check the spheroid shape Constant stress applied to tissue over long timescales using neutral polymer (which is not metabolized by mammalian cells) Also it was confirmed that it is neither a growth nor death factor by plating cells for 3 days with dextran and measuring cell concentration and viability   First, indirect stress measurements were performed - this is done by positioning a growing spheroid inside a dialysis bag which is then placed in an external medium with added dextran. Oxmotic stress induces a force on the dialysis membrane, which is transmitted in the quasistatic equilibrium to the spheroid and calibrated  Stress exerted on this system can be seen as network stress that tends to reduce the volume occupied by the spheroid, and acts directly on the cells and not the interstitial fluid. Volume of spheroid is measured In absence of stress, spheroid reaches steady state of typical diameter 900 When dextran is added to the medium, growth rate decreases as does the steady state volume Interestingly, after a release of stress, the growth resumes until it reaches the same steady state volume of ~900   Also, direct experiments were conducted where the osmotic stress (the minimum pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane) is applied to the spheroid in the absence of the dialysis membrane (a type of semi-permeable membrane tubing used in separation techniques, that facilitates the removal or exchange of small molecules from macromolecules in solution based on differential diffusion). It was also verified that the dextran cannot diffuse inside the spheroid It was observed that the dependence of growth rate and the steady state size on stress is very similar to that observes in the indirect experiment, validating the approach.   Finally, we investigate the spatial dependence of cell division and apoptosis using cyrosections and immunofluorescence.  Spheroids of comparable diameters are embedded in a freezing medium, frozen, cut into slices at the level of their equatorial line Recently divided cells were labelled, as were apoptotic cells

Results and Discussion The approach was validated by the observed dependence of growth on the applied mechanical stress. Interestingly, when the stress is above 10kPa, the effect of stress saturates and the growth curves become indistinguishable from one another. The direct experiment is based on the application of a mechanical stress on the surface of the spheroid through an osmotic shock. Osmotic stress is known to have direct effects on cell growth and apoptosis, in particular, through the mitogen activated protein kinase pathway.  Drawbacks- in all these studies, the effect of an osmotic shock is only measured for an osmotic stress 2 orders of magnitude larger than the one applied in our experiments. Also apoptosis was not observed at the surface of the spheroid (where osmotic stress is exerted). The balance of chemical potentials inside and outside the spheroid shows that the concentration gradient of small solutes induced by the presence of dextran is negligible. The chemical potential of water in the cell is dominated by the small ions and it is only slightly modified by the presence of dextran. In the last part of this experiment it was found that in the absence of external stress, cell division is distributed over all the spheroid with an increase in periphery, whereas for an external stress of 1 kPa, it was greatly reduced in the center of the sections. As in previous sudies we observe the accumulation of apoptotic cells in the center of the spheroid but with no measurable effects of stress on this localistation. In order to better understand this stress dependence of cell division and to interpret the genetic trends of the experimental findings, we performed numerical simulations similar to those of another experiment [see references]. These simulations were adapted to the suitable geometry and setup and we see a steady state that depends on applied stress.

Assignment: Topic Reading Topic Question Answer AnnoBib References: Search tools - Web of Science, Google Scholar, ERIC More than 1 type of source   Additional:  Assoc. papers   Question: The paper mentions cell death via two routes: apoptosis, and a necrotic core which generates death at the inner surface of the viable rim of the cancer cell. What is the difference between cell death through apoptosis and necrosis and what causes each of these processes to occur, and how do these relate to tumor growth? Elaboration: As the paper focuses on the effects of mechanical stress on tumor growth, it is important to understand the balance between cell death and cell division which this involves. Hence, it is vital to understand the difference between cell death caused by apoptosis and cell death which is caused by the formation of a necrotic core, such as is mentioned in pg 3 of the report.  Answer/References:  [1] - Explanation of apoptosis as programmed cell death (Ch10) - explains how a disruption in the balance between cell death and division can lead to disease. Explanation of necrosis as an accidental unprogrammed cell death (Ch 20) Apoptotic Molecular Advances in Breast Cancer Management, By Pontsho Moela and Lesetja R. Motadi, DOI: 10.5772/61654, 2236-4, Published: December 16, 2015 under CC BY 3.0 license. © The Author(s). Pontsho Moela and Lesetja R. Motadi (2015). Apoptotic Molecular Advances in Breast Cancer Management, Cell Death - Autophagy, Apoptosis and Necrosis, Dr. Tobias Ntuli (Ed.), InTech, DOI: 10.5772/61654. Available from: https://www.intechopen.com/books/cell-death-autophagy-apoptosis-and-necrosis/apoptotic-molecular-advances-in-breast-cancer-management Explains how cancers form and how disrupting the balance between cell death and cell division can lead to disease, since cancer growth leads to the interruption of several important processes in the cells, one of which is apoptosis, the programmed death of cells. Also explains what exactly happens in the biochemistry of the body for the cancer cells to prevent the apoptosis occurring.  [2] - Necrosis - types and further definition of necrosis (and a little on apoptosis) https://www.ncbi.nlm.nih.gov/books/NBK430935/ [3] - How apoptosis is related to cancer  https://www.biooncology.com/pathways/cancer-tumor-targets/bcl-2/evading-apoptosis.html [4] Cell death can switch between the two. The section 'Switches From Necrosis to Apoptosis After Ischemia/Reperfusion' explains how the 2 processes con conflict and interact with one another. Also it explains how ATP depletion can effect the cells and what processes this starts, and how this can cause necrosis.  http://academicdepartments.musc.edu/ccdir/Malhi.pdf   [5] Explanation of apoptosis and further explanation of the difference between that and necrosis https://www.ncbi.nlm.nih.gov/books/NBK26873/   [7] Explanation of how apoptosis is different from necrosis in how it doesn't damage surrounding cells. Explains how the cells round up before programmed death to prevent themselves from damaging other cells Cite this chapter as: Thangaraj P. (2016) Induction of Apoptosis. In: Pharmacological Assays of Plant-Based Natural Products. Progress in Drug Research, vol 71. Springer, Cham DOIhttps://doi-org.ezproxy.is.ed.ac.uk/10.1007/978-3-319-26811-8_27 Publisher NameSpringer, Cham Print ISBN978-3-319-26810-1 Online ISBN978-3-319-26811-8 [8] What is ATP? https://www.nature.com/scitable/definition/atp-318 https://www.humpath.com/spip.php?article8790

Cancer Research, Microenvironment Info: http://scienceblog.cancerresearchuk.org/2013/02/11/i-want-to-break-free-the-microenvironment-and-metastasis/