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Tumor Clonogenic Assay (TCA)

Your Benefit

In contrast to normal cells, pluripotent cancer cells including stem cells possess the potential for anchorage-independent growth and colony formation in semi-solid medium. In the clonogenic assay, inhibition of in vitro colony formation of tumor cells is examined. The assay is frequently used in oncological research since the original publication by Salmon et al. in 1977. The TCA has been found to be a highly predictive test for further in vivo evaluation of anti-cancer drugs. When we compared the response of patient-derived tumor models in the TCA in vitro to clinical response rates, 62% of comparisons for
 drug sensitivity, and 92% for drug resistance were correct. By preselecting the most sensitive tumor models, the TCA improves the probability of in vivo activity, reduces the costs of random in vivo testing and the use of animals from an ethical point of view. The growth-inhibiting effect of test compounds on tumor cells can be compared to that on hematopoietic stem cells (representative of normal tissue) and to the antitumor activity of standard chemotherapeutic agents.

Methodology

Tumor cell suspensions containing tumor stem cells, which are responsible for tumor growth, metastatic and infiltrative potential, are prepared by mechanical disaggregation of freshly explanted xenografts growing in nude mice followed by incubation with an enzyme cocktail. The cell suspensions are cultured in a 3-layered assay using 24-well microplates, and treated with test compounds (drug overlay). Each plate contains 6 untreated control wells and 18 drug-treated wells (6 concentrations in triplicate). Cultures are incubated for up to 20 days and monitored closely for colony growth. Colony formation is finally quantified using an automatic image analysis system.

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TCA Handling

Our Services

Almost every in vivo suitable tumor of the ONCOTEST collection has an in vitro TCA counterpart. Therefore the potency and selectivity profile of test compounds can be determined in vitro in a large proportion of the ONCOTEST patient-derived tumor collection. We also offer customized panels of 48 and 24 tumor models representing different histotypes. Profiling a compound by using one of these panels guarantees a fast turnaround at reduced costs. The anti-tumor activity of compounds can be compared to activity towards human hematopoietic stem cells from umbilical cord blood. We also offer a comparison to historic ONCOTEST clonogenic assay data for standard drugs.
Based upon your needs, the assay can be performed by adding selected growth factors or done as a combination study to analyze additive or synergistic activities. The assay is suitable for small molecule inhibitors as well as therapeutic antibodies. For antibodies we offer a feasibility study to exclude any effect of the enzyme treatment on the target protein.

Selected Example

Anti-tumor activity profile of the HSP90 inhibitor 17-DMAG in the clonogenic assay and in vivo.

The activity profile of the geldanamycin analog 17-DMAG was assessed in vitro in 104 human tumor xenograft models (A). In the next step, 11 tumor models were selected based on this clonogenic assay data, and retested with 17-DMAG in vivo in xenograft-bearing nude mice.
Examples of melanoma tumors MEXF 276 and MEXF 989 tested in nude mice are shown in detail in B and all data are summarized in C. The Box-Whisker plot in D shows that in vivo results grouped into sensitive (optimal T/C 50%) correlate very well with sensitivity in the clonogenic assay, given as IC70 values.

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