Ahmed GroupLecture 11 SOLID TUMOR ASSAY SYSTEMS LECTURE 11.

Ahmed GroupLecture 11

SOLID TUMOR ASSAY SYSTEMS

LECTURE 11


• TD50 limiting dilution assay

• Tumor re-growth assay

• TCD50 tumor control assay

• Lung colony assay

• In vitro/ in vivo assay

• Monolayers vs. 3-D spheroid cultures


Transplant Tumors

Isologous (syngenic) tumorsXenografts

Approaches to measure tumor response

1. Conventional tumor growth measurements2. Tumor cure (TCD50)3. Dilution assay4. Lung colony assay5. Tumor cell survival


Transplantable solid tumor systems in experimental animals

• Experimental tumors of various histologic types are used for radiobiologic studies.

• To produce identical tumors, transplantation from one generation to the next is used, which makes it mandatory that the animals be isologous.

• In practice, pure inbred strains of rats or mice are used and the brother-sister mating serves the function of reducing the variability among the animals to a minimum.


Tumor transplantation

Tumor removed from the animal and prepared intosingle-cell suspension;

To produce a transplant, 104 to 106 cells are inoculatedsubcutaneously into recipient animal of the same strain;

The site of transplantation varies widely; the flank or backare commonly used, but sometimes a special tumor requiresa special site;

Within days or weeks, depending on the type of tumor andthe strain of animals, palpable tumors appear that are uniformin size, histologic type, and so on.


Hundreds to thousands of animals can be used, making it possible to design quantitative studies of tumor response to different radiations, fractionation regimens, sensitizers, and combinations of radiation and chemotherapeutic studies.


There are five commonly used techniques to assaythe response of solid tumors to a treatment regimen:

1. Tumor cell survival determined in vivo by the dilution assay technique

2. Tumor growth measurements

3. Tumor cure (TCD50)

4. Tumor cell survival assayed by the lung colony system

5. Tumor cell survival – in vivo treatment followed by in vitro assay


Dilution Assay

The dilution assay technique was devised by Hewitt andWilson, who used it to produce the first in vivo survivalcurve in 1959.The dilution assay technique has become the basis for obtaining an in vivo cell survival curve


Dilution Assay


Dilution assay: dose-response curve


Tumor Growth Measurements

Tumor Growth Measurements is possibly the simplestendpoint to use and involves the daily measurementof each tumor to arrive at a mean diameter.For tumor-growth experiments a large number oftransplanted tumors are prepared as previouslydescribed. When they have grown to a specified sizethey are treated according to the plan of theparticular experiment.


Tumor Growth Measurements


Growth delay


TCD50 tumor control assay

Tumor control provides data of most obvious relevance toradiotherapy. In experiments of this kind a large numberof animals with tumors of uniform size is divided intoseparate groups, and the tumors are irradiated locallywith graded doses. The tumors subsequently are observedregularly for recurrence or local control. The proportion of tumors that are locally controlled can be plotted as afunction of dose, and data of this kind are amenable to astatistical analysis to determine the TCD50, the dose atwhich 50% of the tumors are locally controlled.


TCD50 tumor control assay


Lung colony assay

Hill and Bush have devised a technique to assaythe clonogenicity of the cells of a solid tumorirradiated in situ by injecting them into recipientanimals and counting the number of lung coloniesproduced.The general principles of the method are illustratedon the next slide.


Lung colony assay


In-vivo / in-vitro assay

A limited number of cell lines have been adapted so thatthey grow either as a transplantable tumor in ananimal or as clones in a Petri dish. These cells can bereadily transferred from in vivo to in vitro and back.

The three most commonly used systems are:- a rhabdomyosarcoma in the rat;- a fibrosarcoma in the mouse;- the EMT6 mammary tumor in the mouse


In-vivo / in-vitro assay


Monolayers vs. 3-D spheroid cultures

Mammalian cells in culture may be grown either as amonolayer attached to a glass or plastic surface or insuspension, in which case they are prevented fromsettling out and attaching to the surface of the culturevessel by continuous gentle stirring. Most cells insuspension remain as a single cells.Some cells however, notably several rodent tumor cell lines, do not behave in this way but instead grow as spheroids.


Monolayers vs. 3-D spheroid cultures

The spheroid system is simpler, more reproducible,less expensive, and easier to manipulate than animal tumors, and yet the cells can be studied in anenvironment that includes the complexities of cell-to-cell contact and nutritional stress from diffusionlimitations that are characteristic of a growing tumor.


Comparison of the various model tumor systems

Xenografts of human tumors are used on a much more limited scale. They are somewhat an artificial system;

Spheroids represent an intermediate model betweenmonolayers of cells in culture and tumors in vivo.Spheroids are much less expensive than xenografts inimmunosuppressed animals and perform much the samefunction


Spheroids

• Cells grow in large spheric clump of cells are stuck together, with a diameter of 200-800 m.

• The center of spheroid are deficient of oxygen and nutrients, with a buildup of waste products.

• Mature spheroids consist of three population of cells with varied radiation sensitivity


Photo-micrograph of spheroid


Types of cells in a spheroid

Asynchronous, aerobic cyclic cells

Non-cycling G1-like cells

Necrosis

Non-cycling G1-like hypoxic cells


Xenograft Tumors

• Tumors are induced in immuno-deficient mice• There is greater tendency for the tumors to get

rejected• Human tumor cells undergo kinetic changes

and cell selection• Histological characteristics are well

maintained, however, the stroma is of mouse origin.

• Growth delay and cell survival are not affected


Response of Human tumor xenografts and clinical complete remission rates



In all five transplantable systems described the tumor is treated in situ, with all of the realism and complexities of the in vivo situation, such as cell-to-cell contact and the presence of hypoxic cells, factors that cannot be fully simulated in a Petri dish.



- The TCD50 and growth delay – are both leftin situ undisturbed after treatment. In the other threetechniques the tumor must be removed, minced,and prepared into a single-cell suspension beforesurvival is assessed.- However, the TCD and growth delay systems are very expensive because they require a large number ofanimals to produce the information. The same information can be obtained in 10 days with one ortwo mice and six Petri dishes using the in vivo/in vitrotechniques

Ahmed GroupLecture 11 SOLID TUMOR ASSAY SYSTEMS LECTURE 11.

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