Serine/Threonine Protein Kinases and Tyrosine Protein Kinases in stroma- cancer microenvironment, with some (basic) reference pictures

Microecology of the tumour–host invasion field (picture above):
(Invasive carcinoma is viewed as a pathology of multiple cell societies inhabiting the epithelial/mesenchymal stromal unit. Transition to invasive carcinoma is preceded by the activation of host fibroblasts, immune cells and endothelial cells. Invasion occurs in a localized zone of cross-talk and cooperation between the stromal cells and the premalignant epithelium (depicted as zones demarked by dashed lines). Cytokine and enzyme exchange between the participating cells stimulates migration of both cell types towards each other and modifies the adjacent extracellular matrix/basement membrane. The result is a breakdown of normal tissue boundaries)









Molecular cross-talk at the invasion front (picture above):
(Example mediators are shown. Motility and invasion is a bi-directional process. Fibroblasts produce chemoattractants such as SF/HGF, which stimulates motility of tumour cells by binding to the Met receptor (c-Met). Tumour cells produce angiogenesis factors such as VEGF and bFGF, which bind to receptors on stromal vascular cells and cause increased vascular permeability, endothelial proliferation, migration and invasion. Fibroblasts and endothelial stromal cells elaborate latent enzymes, including MMPs and uPA, which dock on the surface of the carcinoma invadopodia and become activated, thereby degrading the ECM, and clearing a pathway. ECM degradation releases bound growth factors such as TGF- and EGF, which bind to cognate receptors (TGF- R and uPAR) on the carcinoma cell. ECM proteolysis also exposes cryptic RGD sites, which are recognized by integrins. Cross-talk between signal pathways within the carcinoma cells links motility, proliferation and pro-survival signals. For example, phosphorylation of FAK through Met and integrin signalling transduces signals through Ras, PI(3)K, -catenin and MLCK, causing cytoskeletal remodelling, ERK activation of mitogenesis, and sustainment of survival through phosphorylation of Akt)

Reference:
Lance A. Liotta etal Progress: The microenvironment of the tumour–host interface Nature 411, 375-379 (17 May 2001)








Epithelia can be reactive to a changing stromal environment (picture above):
(a, Homeostatic interactions between the epithelia and fibroblasts are maintained by positive and negative signals that influence the proliferation and differentiation of both the stroma and epithelia. b, When signalling by a suppressive growth factor (TGF- ) to the stromal fibroblasts is lost (red starburst), it leads to elevated fibroblast proliferation. Resulting paracrine factors (for example, HGF) and potential modifications in the ECM can stimulate the proliferation and transformation of epithelial cells in vivo in some tissues)







Regulation of epithelial growth, differentiation and apoptosis (table)

Reference:

Neil A. Bhowmick etal Stromal fibroblasts in cancer initiation and progression Nature 432, 332-337 (18 November 2004)





While studying the stromal cancer (stem) microenvironment, one area are really fascinating me lot especially their high level of involvement towards identify novel therapeutic targets followed by develop therapeutics, find solutions for various cancers associated problems as well as helping to study the basic of stem-cell biology, etc etc. Such one field is also known as Serine/Threonine Protein Kinases and Tyrosine Protein Kinases.

The surprise thing is that though there are more than 70 numbers of Kinase families playing role within the human system, but just few of them only have been studied (ing), on the field of stroma- cancer cells microenvironment.

Having interest to gain some knowledge on the role of this cross-talk, complex system within the stromal – cancer –microenvironment I would like to begin from the cell fibroblasts (as one of the components of stroma cancer microenvironment ) and its association with kinases.

Since new to this field I would also likes to add the basic, key informative abstracts for further (quickest) reference purpose.


Cross-talk or associated role of fibroblast with stroma components

:- >(pictures above)<-:

stroma cancer microenvironment -basics

An increasing body of research indicates that stroma surrounding cancer cells plays an important role in the development and subsequent behavior of the tumor. Studies using a wide range of techniques, including stromal cell isolation, modification of stromal-specific gene expression, and recreation of specific microenvironment conditions in culture, have demonstrated that stroma can promote cancer and that the expression patterns within the stroma can influence clinical outcome. Major hurdles in the study of the cancer stroma revolve around the cellular complexity of the tumor microenvironment,
both in modeling the microenvironment and discovering/isolating pure populations of stromal cell types.

An alternative, emerging, avenue of therapy focuses on targeting various non-neoplastic cells that are associated with the tumor microenvironment, such as endothelial cells. Since stromal cells within the tumor are thought to be ‘normal’ and less genetically labile than the neoplastic cells, development of acquired resistance to therapy may be less likely. As such, the tumor stroma may be an excellent target for directed therapy.

CELLS OF THE STROMA:
The stroma consists of a compilation of cells, including fibroblasts/myofibroblasts, glial, epithelial, fat, immune, vascular, smooth muscle, and immune cells along with the extra cellular matrix (ECM) and extra cellular molecules. Among this, the most widely studied to date are the fibroblasts, immune cells, and the vascular cells. Rrecently, research groups are also trying to identify new subtypes of fibroblasts and their role in cancer, develop markers, etc by using the studies of profiles from two different STTs (soft tissue tumors (STTs), desmoid-type fibromatosis (DTF) and solitary fibrous tumor (SFT).


References:

1 Li H etal Tumor microenvironment: the role of the tumor stroma in cancer. J Cell Biochem. 2007 Jul 1;101(4):805-15.

2) Robert B West etal Experimental approaches to the study of cancer–stroma interactions: recent findings suggest a pivotal role for stroma in carcinogenesis Laboratory Investigation (2007), 1–4

Some interesting facts....

- 60-80 tumour cells can initiate angiogenesis,
- 100 tumour cells may initiate neovascularisation,
- In vivo, cancer cell move at high speed of up to 15 μm in a minute,
- The minimum diameter of capillaries where cancer cells are able to migrate is
approximately 8 μm/h,
- Non-dividing cancer –cells in capillaries over 8 um in diameter could migrate up to 48.3
μm/h,
- Micrometastasis occur either with 1 cells or fewer than 10 tumour cells,
- The GFP+ cells migrated dorsoventrally at an average speed of 9.8 ± 2.1 µm/h,
- Mouse epidermal stem cells have been shown to migrate 1 mm in 24 hours,
- Embryonic cortical neural stem cells migrate at an average speed of 1.5 ± 1.8 μm/h,
- The migration of transplanted cells labeled with magnetic nanoparticles migrated towards
the ischemic parenchyma at a mean speed of 65 μm/h,
- In pre-metastatic microenvironment, the mean speed of migration of EGF-treated cells
on LN5 was 48 ± 14 μm/h,
- The average rate of migration of the mutant cells (example, myosin) was 1.8 μm/h,
- invasive glioma cells migrate at an average speed on tissue-culture plastic surfaces was
12.5 μm/h,
- colorectal cancer tumor cell migration varied from 5.6 plus/min 2.5 μm/h (demonstrated
in Panc-1, Human pancreatic carcinoma, epithelial-like cell line),

Emerging technologies in the biomolecular imaging..part-2

• Fluorescence mediated tomography: A development of fluorescence reflectance imaging that captures images from multiple angles. Subsequent computational analysis calculates the point of origin of the fluorescent light in three dimensions.
• Fluorescence lifetime imaging: The length of time that a fluorophore remains in an excited state can provide additional information. For example, fluorescence resonance energy transfer usually leads to a decrease in the length of time the donor fluorophore is excited. Measuring the lifetime of intrinsic fluorophores can provide more information either about the exact fluorophore or its local environment.
• Protease-activated probes: Fluorophores are less effective at high local concentrations. Many fluorophores fused to a cleavable substrate generate a high local concentration, and therefore weak fluorescence. Upon substrate cleavage the fluorophore concentration drops and fluorescence increases.
• Diffuse optical tomography: Diffuse light is propagated through tissue at multiple angles and captured, subsequent computational analysis determines the optical properties of the tissue in three dimensions. Provides good tissue penetration, haemoglobin is easily imaged, but limited resolution and contrast agents.
• Photoacoustic microscopy: Absorption of light by tissue can lead to the emission of sound waves that can be recorded and analysed. Provides good penetration and ability to detect intrinsic signals. Still being developed, difficult to detect exogenous contrast agents, resolution limited to the cellular level.
• Optical coherence tomography: Coherence between light reflected from the sample and a reference signal is measured. Greater than mm penetration, ability to detect intrinsic signals, good resolution and three-dimensional information. Problems detecting exogenous contrast agents.
• Photoacoustic flow cytometry: Similar to flow cytometry except that circulating cells are analysed in vivo using blood flow in small vessels to generate a flow of cells through the imaging path. Many anatomical sites can be imaged. Either conventional reflectance, fluorescence or photoacoustic measurements can be made. Can analyse cells in circulation with sub-micron resolution. Very complicated to set up.
• Laser scanning endoscopes: Modified objectives with a narrow 1–2 mm diameter enable a laser scanning microscope to be used endoscopically. High resolution images of internal organs can be obtained.

Reference:
Illuminating the metastatic process by Erik Sahai , Nature Reviews cancer, volume 7, October 2007

Imaging probes (for references)

• Conventional organic fluorophores: emit light of a longer wavelength than they are
illuminated with. Near-infrared light is most efficiently transmitted through tissue,
and fluorophores in this range are best suited to intravital imaging (for example
Cy5.5 and Cy7).
• Fluorescent proteins: are genetically encoded and have intrinsic fluorescent
properties. Green fluorescent protein (GFP) was the first to be widely used, and
various mutants with enhanced or altered spectral properties remain popular. Red
fluorescent proteins have recently been developed, and may supercede GFP
owing to the more efficient transmission of red light through tissue.
• Luminescent proteins: are genetically encoded, and in the presence of an
appropriate injected substrate catalyse a light-generating reaction. Both firefly
and Renilla luciferase have been used, but simultaneous use in vivo is
problematic.
• Intrinsic signals: Numerous endogenous molecules have fluorescent properties, and
reflectance imaging can be used to determine information about the structure of
tissues. Second harmonic generation by fibrous collagen can also be imaged.
• Quantum dots: are highly efficient inorganic fluorophores that do not bleach, and a
large difference in their excitation and emission reduces problems with tissue
autofluorescence. Quantum dots can also be detected in electron microscopy,
thereby facilitating correlative analysis of light and electron microscopy. Quantum
dots linked to luminescent proteins will fluoresce in the absence of an external
light source.
• Affinity probes: Fluorescently-labelled antibodies can be used to probe the
localization of tumour antigens. Alternatively, molecules that bind to biomolecules
of interest can be labelled with fluorophores and used for imaging.
Examples include hydroxyapatite for bone imaging and RGD peptides for
integrin distribution.
• Protein–protein interaction probes: Fluorescence resonance energy transfer can be
used to investigate the distance between two fluorophores, and hence protein–
protein interactions and/or protein conformation. GFP genetically split into two
can be fused to two different proteins, so that when the proteins interact the two
parts of GFP are brought together and the fluorescent properties of GFP are
recreated. A related strategy uses light generated by bioluminescence to excite a
local fluorophore.

+
There is a special molecule: bimodal contrast agent Gadolinium-RhodamIne Dextran [GRID] which could be detectable by both magnetic resonance imaging (MRI) and fluorescent microscopy and it’s utilizing in the field of pre-labelling neural stem cells

General imaging methods..

-> Whole body/tissue fluorescence (fluorescence reflectance imaging):
• Principle: animal or tissue is illuminated and the emitted fluorescence is captured
using a camera.
• Advantages: non-invasive, multiple colours or probes and spectral analysis can
provide additional information, can detect a few hundred cells, can image day after
day. It can provide information about populations of cells.
• Disadvantages: limited resolution, not all anatomical sites image equally well, often
requires the introduction of exogenous fluorophores, autofluorescence.

-> Whole body/tissue bioluminescence:
• Principle: tumour is engineered to express a protein that catalyses a luminescence
reaction, substrate is injected and the emitted light is captured using a camera.
• Advantages: non-invasive, can detect a few hundred cells, can image day after day.
• Disadvantages: limited resolution, not all anatomical sites image equally well, threedimensional
reconstruction is difficult, requires the introduction of exogenous
luminescent enzymes and substrates.


-> Invasive confocal/epifluorescence:
• Principle: similar to fluorescence reflectance imaging except that a much smaller
region is imaged, usually after some surgical manipulation or implantation of a
‘window chamber’.
• Advantages: high spatial and temporal resolution — subcellular structures can be
visualized, multiple colours or probes, confocal techniques provide threedimensional
information.
• Disadvantages: imaging depth currently limited to ~500 ìm even with multiphoton
microscopy, usually requires some surgical manipulation.

-> Raman spectroscopy:
• Principle: light shifted in wavelength from the illuminating source is analysed, the
shifting is influenced by the chemical composition of the tissue being analysed
(changes in the type of C–C bond and amide and CH2 groups can be
identified).
• Advantages: no need for imaging probes, can provide useful chemical information,
can be combined with confocal techniques to improve spatial resolution. Coherence
methods offer much greater sensitivity.
• Disadvantages: sensitivity, still difficult to obtain high resolution images in a useful
time frame.

Emerging technologies in the biomolecular imaging..part-1

Basics of fluorescent proteins:
-Behaviour of highly metastatic cancer cells: label with green fluorescent protein (GFP)
-low metastatic cancer cells: labelling with red fluorescent protein (RFP)
-both can be done in-vivo

Or alternatively, the host and the tumour calls can be differentially labelled with fluorescent proteins –a transgenic mouse expressing GFP in all of its cells (or in specific cells such as endothelial cells) transplanted with tumour cells expressing RFP enables the interaction between the tumour cell and the hoist cells to be visualised in the real time.

Ex-vivo imaging, using the fluorescent proteins:
- examples include micro metastasis (including dormant cells ) which visualised in unfixed or unprocessed tissues
Intra vital imaging using the fluorescent proteins:
- the technique, High resolution Intravital video microscopy of GFP-expressing tumour cells gives directly observing steps in the metastatic process, individual, non-dividing cells, as well as micro- and macro metastasis, cellular details such as pseudopodial projections, tumour cell s motility (including moving in, and out of the blood vessels ). Along with the confocal microscopy, the polarity of tumour cells, response to the chemotatic cytokines etc can be visualised by the intra vital imaging technique.

Multiphoton laser-scanning microscopy (MPLSM): provides high resolution three –dimensional images of angiogenesis –associated gene expression and this technique also useful to investigate deeper regions of GFP-expressing tumours. Example include, monitored the” activity of VEGF promoter in transgenic mice and their subsequent blood vessel formation,


- Real time fluorescent imaging technique: also helping to discover new drugs and genes that mitigate cancer growth and progression.

-GFP-fluorescent tumour nodules can be detected by Fluorescence stereomicroscopy

-imaging the metastatic cells can be detected by confocal laser scanning microscope



-for tumour pathophysiology studies such as differentiating tumour vessels from both perivascular cells and matrix, assaying the ability of microparticles to access the tumour, and monitoring the trafficking of precursor cells: quantum dots and multiphoton intravital microscopy,


Colour-coding metastatic cells:
- in addition to the DNA micro array, this technique useful for visualisation of tumour cell types that have different properties in the live animal, visualising the action of specific genes in tumour growth and metastasis. Examples, include, Nucleoside diphosphate kinase A (NDPKA) a metastatic suppressor in certain tumours including breast cancer.

- This technique also useful for studies of different subpopulation of cancer cells to investigate the metastatic capacity of different cancer cells and also to understand how the expression of particular proteins drives or inhibits the metastatic process

Imaging dormant cells:

Dormancy means the tumour cells may lodge in some organs for instance lung but it won’t grow. This situation i.e. whether a cell that reaches a distant organ and proliferate or die or any factors that influence this process can b visualise by seeing the fluorescent proteins as well as fluorescent microscopy. Examples include studies of isogenic pair of metastatic and non-metastatic, GFP-labelled human breast cancer

(The studies of dormant tumour or stem-cells in in- vivo not only understanding of the dormant cells but also address why cancer patients can relapse many years after the eradication of the primary tumours, especially true for breast cancer patients. So there are no surprises that there are some biotech companies working on this field at USA!)

ps: Vey interestingly, the basics and the principles behind this fluorescent methods, proteins etc are also utilising in the field of cancer stem cells..

Reference: The multiple uses of fluorescent proteins to visualize cancer in vivo by Robert M. Hoffman, Nature Reviews Cancer 5, 796-806 2005