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

methods in heamatopoietic stem cell assays - part -2

Heamatopoietic stem cell assays


I Surrogate short-term in vivo and in vitro assays for detecting HSCs and their progeny:

I) Immunophenotypical Analysis of HSC / Progenitors
- This method relay on fluorescence –activated cell sorting (FACS)-based methods. The most commonly used FACS-purified populations of HSC/progenitors cells include the following:
ia)- Thy1.1(10), Lin- Sca1+Cells:– is for isolation of short term repopulating HSCs and based on expression of their stem-cell antigen (scs-1-, low expression of Thy1.1 and lack of expression of lineage markers
ib) Lin-c-Kit+ Sca-1+ Cells (LKS+)
- in recent advance, expression of CD34 and Flt3(CD135) has been used to further purify long-term repopulating HSC ( LKS+ CD34- Flt-3-) from short-term repopulating HSCs( LKS+CD34+ Flt-3-) and multipotent progenitors ( LKS+ CD34+Flt-3+)

ii) Fluorescent Dyes on High Drug Efflux Properties of HSCs: Rhodamine 123, Hoescht 33342, and the side population:

here two different dyes mitochondrial binding dye Rhodamine 123 (Rh123) and DNA-binding dye Hoescht 33342 (Ho 33342) used either alone or combination for isolation of HSC; and isolation of Hoescht 33342 (Ho 33342) side population (SP), which emits Ho 33342 at two wavelengths simultaneously, resulting in a distinct ‘tail ‘profile, which disappears with the drug Verapamil (the drug used for blocking the activity of ATP-binding cassettes (ABC) transporters superfamily.

iii) SLAM Family Members: SLAM proteins are a family of cell surface glycoproteins in the immunoglobulin superfamily with specific SLAM antigen (CD150+ CD244-CD48-), which is also applying to purify a population of which approximately 50% of single cells reconstituted lethally irradiated animals.
- this SLAM method more useful to detect HSCs in older, mobilised, or transplanted mice,


II
In vitro and Short-term in Vivo Assays for detecting Functional Potential of HSCs and Progenitors:

This method often sued for measuring the mature progenitors,
i) Colony –Forming Cell or CFU mixed Assays:
- the Colony –Forming Cell(CFC) assay measures the progenitor cells in a give n population using semisolid agar- or more commonly, well defined methylcellulose –based culture media,
- the majority of CFCs consists of lineage restricted colonises such as BFU-E, CFU-G, CFU-GM, etc,
- the most immature (multipotent) CFC measurable contains Granulocytes, Erythrocytes, Macropages, and often Megakaryocytes (CFU-GEMM),
- unfortunately ,t his CFC method do not measure the HSCs cells

ii) Cobblestone Area-Forming Cells (CAFC) /Long-term Culture Initiating Cells(LTC-IC):
- It’s a coculture system, used to measure the HSCs frequencies,

iii) Short-term in vivo Assays:
- the cells which is accountability is Colony-forming unit- spleen (CFU-S),
- this cells once injected into an irradiated mice recipient, hoe to spleen and form macroscopic colonies that provide very short term (usually 1-3 weeks ) in vivo repopulation of the mouse,

III) In vivo assays to measure HSC numbers and their Functional potential:

-this assays, in general, is focusing on the long-term repopulating assays, and the most common one is Competitive Repopulating Assay (CRA). This CRA measures the functional potential of an unknown source of HSCs against a set known numbers of HSCs (usually whole bone marrow cells from the congenic wilt-type mice),
- this method also provide qualitative or semi quantitative information about the HSCs within a given population but it can’t distinguish between the numbers of HSCs o their quality (progeny produced peer HSC),


- while competitive repopulating units (CRU), which uses minimal number of HSCS, measure the quantity of HSCs, repopulating units (RU) measures the functional quality of HSCs.

Molecular methods /assays for study of (tumour) microenvironment / niche - part - 1

- illustrations of isolation and characterization of each cell type comprising normal and cancerous breast tissue: ->Please refer the above picture<-

- stroma specific genes, stromal component of breast tumours, Gene expression profiling, determination of stromal signatures, confirm, localize, observations of the expression of solitary fibrous tumour (SFT) and desmoid- type fibromatosis (DTF) specific genes, etc: gene microarray / tissue microarray (TMA)

-key genes associated with a specific disease (Roche???*!!!): cantilever-array (label free and amplification free)

-study of signalling in microenvironments, for manipulating the
behaviour of embryonic stem cells: synthesized biomaterial microarrays (Anderson et al, 2004) and ECM microarrays (Flaim et al, 2005) Additional platforms or small molecules (Bailey et al, 2004) in cell-based assays.

-proteomic profiling of the cancer microenvironment: antibody arrays

-study of micrometastasis: q-RT-PCR, as well as cellular imaging techniques, such as brighter and dual-fluorescence cell markers, inorganic labels that do not photo-bleach and longitudinal single cell imaging by MRI,

-circulating tumour cells (CTS): laser scanning cytometry with a multimarker real-time RT-PCR assay, immunomagnetic enrichment and fluorescent immunocytochemical characterization (an automated FDA approved system, CellSearch™, etc)


-study the cell fusion (HSCs and local stem-cell niche): Staining: staining cells expressing both the donor’s double marker (EGFP and beta-gal) and the recipients Y chromosome.

-reveal and analyse the cellular complexity: Living –cell micro array (by R&D Molecular cytomics),

-for stem-cell behaviour: labelling in animal studies: retroviral transduction with a market gene or labelling with thymidine or bromodeoxyuridine (BrdU)

-clinical detection of stem-cells: magnetic labelling and in vivo tracking of bone marrow cells by the use of magnetodentrimers or radioactive detection methods

-for assessment of time course of proliferation of stem-cells: generally using reporter gene: LecZ, by identification of galactosidase positive cells in tissue sections and the chromosome analysis by FISH (fluorescent insitu hybridisation),

-for separate different cell populations in cancer stroma expression: flow cytometry,

-to generate gene expression profiles of different components of the tumour microenvironment, including neoplastic and stromal cell types: SAGE (serial analysis of gene expression),


-epithelial–stromal interactions during mammary gland development: mouse model (paper to read: Review: Mouse models of breast cancer metastasis
By Anna Fantozzi and Gerhard Christofor). any other techniques?

-study of tumour microenvironment and drug resistance in hematologic malignancies (or study of environment mediated-drug resistance (EM-DR)): Stromal model using transwell (for reference: Tumour microenvironment and drug resistance in hematologic malignancies by Zhi-Wei Li etal. There is an illustration too)

- illustrations of isolation and characterization of each cell type comprising normal and cancerous breast tissue: ( here i will up-date the picture soon from library)

->Please refer the picture<-

curiosity question ....

one curiosity thought is, though there are number of drugs which is modulating or targeting the cellular components within the microenvironment, however, there are also reports about drug resistance to a particular therapy. Is that due to not finding and validating the right molecular target or any development of isoform within the molecular target or development of any cross cell - signalling followed by drug resistance. ….???? Lots of curiosity thoughts and questions....!!!!

drugs towards cancer microenvironment,...

Clinical experience with targeting cancer and its microenvironment

Drug Target Trial Phase

Soluble factors:

Gefitinib EGFR II
Trastuzumab Her-2-neu II
Imatinib mesylate PDGFR-b I


Bone targeted:

Zolendronic acid Osteoclast III
Atrasentan Osteoblast II
Strontium Bone interface II
Gene therapy Osteoblast I

Angiogenesis:

Bevacizumab VEGF II
Thalidomide bFGF IL-8 II
GVAX immunotherapy Unknown II

+


Teriparatide (brand name Forteo) Eli Lilly - alter the bone: bone marrow interface.

Natalizumab (brand name Tysabri,_ Biogen Idec) - affects VLA-4 adhesion interactions (one of regulatory components for pre-metastatic niche), but the drug is meant for multiple sclerosis,
Cinacalcet (brand name Sensipar, Amgen) - renal failure-associated hypercalcemia – for modulates calcium receptors,
Ostabolin-C (Zelos Therapeutics) also modulates osteoblasts,

Anything else?

A sensitive but motivating facts for cancer research communities…

Cancer deaths to hit 17 million in 2030:

Cancer deaths will more than double to 17 million people each year in 2030 with poor countries shouldering the heaviest burden from the disease, the head of the United Nation's cancer agency said on Monday.

An ageing population will bump up cancer rates worldwide in the coming years, especially in developing countries where the number of people who smoke and drink is on the rise, said Peter Boyle, director of the International Agency for Research on Cancer.

And the disease will hit poorer countries harder because of limited health budgets and a lack of treatments such as radiotherapy that can extend people's lives, he told the European Cancer Conference.

"If we put population growth and ageing to one side the exportation of cancer risk factors, primarily tobacco smoking, from developed countries will continue to be a major determinant of cancer risk and cancer burden in less developed countries," he said.

For many years, many thought cancer was mainly a problem in rich nations in part because health officials assumed people in poorer countries did not live long enough to develop cancer.
This trend is changing, however, as residents of these nations live longer and continue cancer-causing activities like smoking that are declining in Western countries, Boyle said.

This will fuel a dramatic increase in worldwide cancer with the disease likely killing 17 million people each year by 2030, up from the current 7 million. The number of people diagnosed and living with cancer will treble to 75 million, he said.

"The big issue is ageing," he said. "The speed of the ageing of the population is something which is dramatically increasing, especially in the low and medium resource countries."

But he said Europe offers an example that something can be done because even as cancer cases rise, the disease is killing fewer people these days than expected.

This shows that programs such as increased screening and education aimed at preventing tobacco use helped whittle EU cancer deaths to 935,219 in 2000, nearly 10 percent below expectations.

"This approach has clearly paid off," he said. "In Europe good quality care exists for the great majority of people."

This is not the case in many poor countries, however, he said, noting that at least 30 African and Asian countries do not have radiotherapy machines.