Solid tumor assay systems Flashcards
Which of the following assays would NOT be useful for the purpose of quantifying the response of a tumor to irradiation?
A. Lung colony assay
B. Number of tumors per animal
C. Time to reach a certain size
D. Growth delay
E. Colony forming ability of cells explanted from the tumor
B
An increase in the number of tumors per animal would be a reflection of metastatic spread of the tumor, and would not necessarily reflect the radiation response of the primary tumor per se. All of the other assays can be used to quantify the response of tumors to irradiation. The lung colony assay is a technique to measure the radiosensitivity of tumor cells that are irradiated in situ. Following irradiation, the tumor is removed, made into a preparation of single cells, and then injected into the mouse tail vein. Cells that retain clonogenic capacity form colonies in the lung that are countable. This allows comparison of clonogenic survival in various in situ experimental scenarios (Answer choice A). Similarly, colony forming ability of cells explanted from a tumor are radiated in situ; however, radiation response is tested by counting the surviving colonies grown in vitro, also known as the in vivo/in vitro assay (Answer choice E). Lastly, tumor growth assays (Answer choices C and D), quantifies radiation response through the measurement of tumor diameter at various time points following radiation compared to untreated tumors.
The TCD50 assay:
A. Measures radiation-induced tumor growth delay
B. Can be conducted using mouse tumors but not human tumor xenografts
C. Gives a measure of the number of cells required to produce a tumor in a mouse
D. Yields results independent of the immune competence of the host animal
E. Measures tumor cure, making it a relevant endpoint for extrapolation to the clinic
E
The TCD50 assay quantifies the dose required to cure 50% of a group of matched tumors and is therefore a highly relevant endpoint for extrapolation to the clinic. The assay can be conducted using mouse tumors or human tumor xenografts (Answer Choice B), although suppression of the host immune system when using xenografts is crucial in order to minimize misleading results due to rejection of implanted cells (Answer Choice D). The TD50 assay can be used to measure the number of cells required to cause a tumor in mice and has historically been used to determine tumor cell survival curves, to assess the number of clonogens in a tumor, and to study host factors that influence tumor development (Answer Choice C).
A local tumor recurrence after radiotherapy can be caused by:
A. Any surviving cancer cell
B. Any proliferating cancer cell
C. Only cancer cells with the ability to form colonies in vitro
D. Only cancer cells with unlimited proliferative potential
E. Only cancer cells that were well-oxygenated during irradiation
D
Results from tumor transplantation experiments indicate that only a small proportion of all cancer cells have an unlimited proliferative capacity and demonstrate the capacity of self-renewal. In analogy to in vitro assays, tumor cells that demonstrate the ability to achieve a local recurrence following radiotherapy have been termed “clonogenic cells” and correspond to putative “cancer stem cells,” or “tumor initiating cells.” The existence of cancer stem cells, defined by the ability for self-renewal and generation of the heterogeneous lineage of cells within a tumor, has been hypothesized.
Which assay or endpoint would provide the best estimate of the radiation response of putative cancer stem cells?
A. Time to first evidence of tumor shrinkage following irradiation
B. Tumor regrowth delay
C. Determining the fraction of proliferating tumor cells 2 weeks after irradiation
D. 50% tumor control dose
E. Quantifying the number of apoptotic tumor cells 6 hours after irradiation
D
It has been suggested that a small proportion (< 1%) of all cells in a tumor are cancer stem cells. If correct, this hypothesis suggests that all cancer stem cells must be killed or lose replicative potential in order to achieve permanent local tumor control. In theory, one surviving cancer stem cell would be sufficient to cause a local recurrence following irradiation. Thus, the rate of permanent local tumor control is a direct measure of radiation response of cancer stem cells. In contrast, tumor shrinkage and growth delay are dominated by the response of the bulk of cancer cells and not specific for the radiation response of cancer stem cells. Cancer cells with a limited proliferative capacity, as well as doomed cancer stem cells, might undergo a number of cell divisions before they permanently stop proliferating and ultimately die. Determination of proliferating cells will therefore not provide information regarding the radiation response of cancer stem cells. Cancer cells can die following exposure to radiation in different ways, including interphase death (i.e. apoptosis) and mitotic catastrophe (apoptosis, autophagy, or necrosis). None of these modes of cell death is likely to be specific for cancer stem cells. Given that many solid tumors exhibit resistance to undergoing apoptosis and the controversial data from studies comparing the rate of apoptosis with radiation response of tumors, it is unlikely that the rate of apoptosis after irradiation will be a proper parameter to determine the response of irradiated cancer stem cells.
In some experiments, tumors treated with radiation and concurrent molecularly-targeted drugs against EGFR and VEGFR displayed longer regrowth delays, but not higher tumor control probabilities, compared to tumors that were treated with radiation only. Which of the following statements provides the most likely explanation for this?
A. The treatment is effective for the bulk of tumor cells, but not for cancer stem cells.
B. The drug did not reach most of the cells due to poor vascular perfusion in the tumor.
C. Experimental error accounts for this, because growth delay and tumor control assays usually yield similar results.
D. Tumor cells generally do not express receptors that are targeted by these drugs.
E. The radiosensitivity of tumor cells does not depend on vascular supply or physiology.
A
There are indeed some examples in the literature showing a discrepancy between growth delay and tumor control probability. In these experiments, various molecular targeting approaches in combination with radiation were investigated. Though difficult to prove, the assumption of a differential effect on cancer stem cells and non-cancer stem cells is the most likely explanation for these results. It is likely that the drug reached the tumor since there was an effect on tumor growth. Cancer cells generally express EGFR and cell survival following irradiation is affected by vascular supply. The observed discrepancy between growth delay and local tumor control in some experimental settings suggests that the latter assay is the preferable endpoint to evaluate new therapeutic approaches with curative intent.
