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.
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.
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.
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.