1. Ahmed GroupLecture 11 SOLID TUMOR ASSAY SYSTEMS LECTURE 11

2. Ahmed GroupLecture 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

3. Ahmed GroupLecture 11 Transplant Tumors Isologous (syngenic) tumors Xenografts Approaches to measure tumor response 1.Conventional tumor growth measurements 2.Tumor cure (TCD 50 ) 3.Dilution assay 4.Lung colony assay 5.Tumor cell survival

4. Ahmed GroupLecture 11 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.

5. Ahmed GroupLecture 11 Tumor transplantation Tumor removed from the animal and prepared into single-cell suspension; To produce a transplant, 10 4 to 10 6 cells are inoculated subcutaneously into recipient animal of the same strain; The site of transplantation varies widely; the flank or back are commonly used, but sometimes a special tumor requires a special site; Within days or weeks, depending on the type of tumor and the strain of animals, palpable tumors appear that are uniform in size, histologic type, and so on.

6. Ahmed GroupLecture 11 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.

7. Ahmed GroupLecture 11 There are five commonly used techniques to assay the 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 (TCD 50 ) 4.Tumor cell survival assayed by the lung colony system 5.Tumor cell survival – in vivo treatment followed by in vitro assay

8. Ahmed GroupLecture 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

9. Ahmed GroupLecture 11 Dilution Assay The dilution assay technique was devised by Hewitt and Wilson, who used it to produce the first in vivo survival curve in 1959. The dilution assay technique has become the basis for obtaining an in vivo cell survival curve

10. Ahmed GroupLecture 11 Dilution Assay

11. Ahmed GroupLecture 11 Dilution assay: dose-response curve

12. Ahmed GroupLecture 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

13. Ahmed GroupLecture 11 Tumor Growth Measurements Tumor Growth Measurements is possibly the simplest endpoint to use and involves the daily measurement of each tumor to arrive at a mean diameter. For tumor-growth experiments a large number of transplanted tumors are prepared as previously described. When they have grown to a specified size they are treated according to the plan of the particular experiment.

14. Ahmed GroupLecture 11 Tumor Growth Measurements

15. Ahmed GroupLecture 11 Growth delay

16. Ahmed GroupLecture 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

17. Ahmed GroupLecture 11 TCD 50 tumor control assay Tumor control provides data of most obvious relevance to radiotherapy. In experiments of this kind a large number of animals with tumors of uniform size is divided into separate groups, and the tumors are irradiated locally with graded doses. The tumors subsequently are observed regularly for recurrence or local control. The proportion of tumors that are locally controlled can be plotted as a function of dose, and data of this kind are amenable to a statistical analysis to determine the TCD 50, the dose at which 50% of the tumors are locally controlled.

18. Ahmed GroupLecture 11 TCD 50 tumor control assay

19. Ahmed GroupLecture 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

20. Ahmed GroupLecture 11 Lung colony assay Hill and Bush have devised a technique to assay the clonogenicity of the cells of a solid tumor irradiated in situ by injecting them into recipient animals and counting the number of lung colonies produced. The general principles of the method are illustrated on the next slide.

21. Ahmed GroupLecture 11 Lung colony assay

22. Ahmed GroupLecture 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

23. Ahmed GroupLecture 11 In-vivo / in-vitro assay A limited number of cell lines have been adapted so that they grow either as a transplantable tumor in an animal or as clones in a Petri dish. These cells can be readily 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

24. Ahmed GroupLecture 11 In-vivo / in-vitro assay

25. Ahmed GroupLecture 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

26. Ahmed GroupLecture 11 Monolayers vs. 3-D spheroid cultures Mammalian cells in culture may be grown either as a monolayer attached to a glass or plastic surface or in suspension, in which case they are prevented from settling out and attaching to the surface of the culture vessel by continuous gentle stirring. Most cells in suspension 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.

27. Ahmed GroupLecture 11 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 an environment that includes the complexities of cell-to- cell contact and nutritional stress from diffusion limitations that are characteristic of a growing tumor.

28. Ahmed GroupLecture 11 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 between monolayers of cells in culture and tumors in vivo. Spheroids are much less expensive than xenografts in immunosuppressed animals and perform much the same function

29. Ahmed GroupLecture 11 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

30. Ahmed GroupLecture 11 Photo-micrograph of spheroid

31. Ahmed GroupLecture 11 Types of cells in a spheroid Asynchronous, aerobic cyclic cells Non-cycling G 1 -like cells Necrosis Non-cycling G 1 -like hypoxic cells

32. Ahmed GroupLecture 11 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

33. Ahmed GroupLecture 11 Response of Human tumor xenografts and clinical complete remission rates

34. Ahmed GroupLecture 11 Comparison of the various model tumor systems 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.

35. Ahmed GroupLecture 11 Comparison of the various model tumor systems - The TCD 50 and growth delay – are both left in situ undisturbed after treatment. In the other three techniques the tumor must be removed, minced, and prepared into a single-cell suspension before survival is assessed. - However, the TCD and growth delay systems are very expensive because they require a large number of animals to produce the information. The same information can be obtained in 10 days with one or two mice and six Petri dishes using the in vivo/in vitro techniques

36. Ahmed GroupLecture 11 Comparison of the various model tumor systems