Clonogenic assay with established human tumour xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery

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Abstract

Pluripotent cells can be grown in clonogenic assays. The tumour stem-cell fraction, which accounts for <0.4% of the total cells, and which is considered the most relevant cell type in the development of metastases and recurrences, is able to divide and to form colonies in a semisolid matrix (agar or methylcellulose). Major applications of the tumour clonogenic assay (TCA) are chemosensitivity testing of tumours and xenografts, and for assessments within drug discovery programmes. Of critical relevance for the usefulness of the TCA is whether it can predict sensitivity or resistance towards clinically used agents. When we compared the response of human tumours established as xenografts in nude mice in the TCA in vitro to that of the clinical response, 62% of the comparisons for drug sensitivity, and 92% of the comparisons for drug resistance were correct. The same percentage of true/false observations was found when tumours were tested after serial passage in nude mice in the TCA in vitro and their response compared to in vivo activity in corresponding xenografts (60% and 90%, respectively). The highest correct predictive values were, however, found when the clinical response of tumours was compared to their explants established in the nude mouse and treated in vivo. Of 80 comparisons performed, we observed a correct prediction for tumour resistance in 97% and for tumour sensitivity in 90%. In our opinion, the TCA with established human tumour xenografts has an important role in current drug discovery strategies. We therefore included the TCA as secondary assay in our approach to anticancer drug discovery and found that a number of novel agents were active; these are now in advanced preclinical development or clinical trials. Thus, the tumour clonogenic assay has proven predictive value in the chemosensitivity testing of standard and experimental anticancer drugs.

Introduction

Many normal cells show the phenomenon of adherence, i.e. they grow and divide only if attached to a solid inert support, as is provided for example by the glass or plastic surfaces of tissue-culture dishes. The clonogenic assay is a classical way of evaluating colony formation of pluripotent cells with the potential for anchorage-independent growth in semisolid media, e.g. transformed cells or haematopoietic stem cells. Semisolid media reduce cell movement and allow individual cells to develop into clones that are identified as single colonies. The assay is widespread in oncological research where it is used to test the proliferative capacity of cancer cells after radiation and/or treatment with anticancer agents 1, 2, 3.

Patients’ tumours can be studied directly in the clonogenic assay, or after being established as a permanent xenograft in serial passages in nude mice. The xenograft should be characterised for chemosensitivity and for molecular markers relevant to the pathogenesis of a tumour. Clonogenicity is a hallmark of transformed and malignant cell types; thus, permanent human tumour cell lines can also be used, but many of them have changed during long-term serial passaging, with the selection of subclones 4, 5, 6. In addition, murine tumours such as the leukaemias P388 and L1210, as well as the solid models B16, Lewis-Lung, Colon 36, Colon 28, and others, grow very well in the clonogenic assay [7].

Haematopoietic stem cells (the normal tissue being clinically dose limiting for about half of all compounds) are obtained from bone marrow, peripheral blood or umbilical cord blood. The effect of novel compounds can be tested against human tumours and human haematopoietic stem cells, allowing evaluation, based on in vitro studies only, of whether a new agent is tumour specific and will have a therapeutic index. As a result, large and expensive up-scaling of compound synthesis or refermentation can be avoided at an early stage.

Most investigators use a three-layer technique with a base layer consisting of 0.5–0.8% agar, a second layer containing cells with 0.4% agar and a third layer containing medium or test drugs 2, 3, 8. Human haematopoietic stem cells can be grown to form colonies in semisolid media after the addition of placenta-conditioned medium 7, 9, or in methylcellulose media supplemented with defined growth factors (e.g. granulocyte-macrophage-colony-stimulating factor, interleukin 3, erythropoietin) 10, 11, 12. Up to 1990, most studies were done in Petri dishes of 35 mm dia. Since the 1990s the use of 24-well cell-culture microplates of 16 mm dia. has been made possible, allowing for miniaturisation and easier handling [13]. Another aspect of miniaturisation was accomplished by using capillaries of 1–1.5 mm dia. into which agar containing stem cells was introduced 14, 15. The capillaries are 1.5 cm long and the number of colonies is usually small, ranging between 3–10 per capillary however with great variability. In our experience, the 24-well microplate is clearly the most reliable format [13].

To individualise chemotherapy regimens by preclinically assessing the chemosensitivity of tumours to registered anticancer agents in vitro has been a goal of oncological research for many years. The tumour clonogenic assay (TCA), as described by Hamburger and Salmon 1, 16, is one of the most intensively studied in vitro methods for chemosensitivity testing. Its role in patient sensitivity testing in addition to in vitro methods such as the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay 17, 18, the histoculture drug-response assay 19, 20, 21, the collagen gel droplet-embedded culture drug-sensitivity test 22, 23, or the ATP-based tumour chemosensitivity assay 24, 25, 26 is well documented 2, 27, 28, 29, 30. However, there are no phase III studies demonstrating a significant increase in survival compared to empirically determined standard chemotherapy. Therefore, the TCA has not found a practical established role in the individualisation of patient therapy.

In another major application, clonogenic assays have been widely used for assessing the efficacy of novel compounds in anticancer drug discovery programmes, such as that of the Institute for Experimental Oncology in Freiburg [7]. Since the assay is labour intensive and automation not as easy to achieve as in experimental set-ups using adherent or suspended cells, the TCA is not useful as a primary screening method but has its credentials as a secondary screen, e.g. for prioritised compounds after cell-based assays with tumour cell lines 31, 32, 33, 34. We test novel lead compounds from primary screenings in the TCA in 24 models. The IC70 and IC50 in such a tumour panel are then compared with the sensitivity of human haematopoietic stem cells obtained from cord blood or peripheral blood to define a ‘therapeutic window’. In addition, the in vitro profile is compared to the fingerprint of standard agents in these tumour models and to 35 known, validated molecular targets in our database. The latter comparison will help to define novelty or similarity to known drugs. Once in vivo activity is observed, TCA testing is extended to 48 tumours and the resulting in vitro IC70 profile can be correlated with our cDNA-expression database (based on the Affymetrix HU133A gene chip; 22000 genes/tumour) in order to identify gene clusters that might be essential for drug activity. With this approach, genes important for the activity of novel compounds with novel mechanisms might be discovered. Large studies demonstrating high correlations between the results of the in vitro TCA and the patient's response or resistance to established agents have been published 8, 35, 36, 37, 38. Secondary screening of experimental agents for anticancer efficacy has also been described as feasible 7, 39.

Established tumour xenografts provide a rich source of regrowable human tumour tissue, which can be broadly characterised. In target-directed drug development, we first determine the expression of a target at the RNA and protein level by using our cDNA gene-expression database and tissue microarrays. Between 12 and 24 tumour models that over-express or are deficient for a particular target are then selected, and potential inhibitors tested in the TCA. This procedure allows us to determine rationally the most sensitive tumours, which can subsequently be evaluated for in vivo activity.

The application of the TCA in large-scale anticancer drug development has been hampered by the following factors:

  • 1.

    Tumours resected for diagnostic or therapeutic purposes provide highly relevant material, but tumour specimens originating from patients have growth rates that range between 40–60% only. Tests are not reproducible and further characterisation of the tumours is mostly impossible 40, 41, 42.

  • 2.

    Cell lines are frequently used as a tumour source for drug screening, but such lines show considerable alterations in biological properties and chemosensitivity pattern as compared to the original tumours 4, 5, 6.

  • 3.

    Interpretation of data is sometimes difficult because of a lack of standardisation of experiments and inadequate quality-control measures 41, 42, 43, 44.

By introducing quality-control criteria for the minimum colony number per well, positive controls, background control plates and a coefficient of variation in the control groups of <50%, a substantial increase in assay reliability with a very good reproducibility has been achieved [42].

In this paper, we report our experience with the growth and predictivity of the TCA by performing the following in vitro/in vivo correlations comparing the response to standard agents in the same tumour, relating these findings to our earlier work and to published material:

  • 1.

    Patients’ tumours established subcutaneously in nude mice studied in the TCA in vitro compared with the same tumour treated in the patient.

  • 2.

    Patients’ tumours grown in nude mice studied in the TCA compared with those treated in vivo in the nude mouse.

  • 3.

    A summary of our earlier experiences in comparing the drug response of a tumour treated in vivo in the nude mouse with that in the patient.

  • 4.

    A literature survey of work in which patients’ tumours were studied directly in the TCA and compared with the patients’ responses.

We also describe here our concept of integrating the TCA into a combined in vitro/in vivo drug discovery programme and the advanced preclinical development of experimental anticancer drugs.

Section snippets

Tumours

For direct testing on patients, living tumour tissue from primary tumours or metastatic lesions, resected for diagnostic or therapeutic purposes, was placed in a sterile tube with RPMI 1640 medium supplemented with 20% fetal bovine serum and 0.05% gentamicin. The tissue was processed within 0.5–2 h of resection. For xenograft testing, fresh human tumour specimens were first cut into slices (5×5×0.5–1 mm dia.) and implanted subcutaneously into nude mice of NMRI genetic background. The animals

Biological properties of human tumours grown in the TCA

The properties of both normal haematopoietic and neoplastic cell populations are consistent with a model in which cells with proliferative potential can carry out a limited number of potential divisions or have the capacity to renew the entire cell population, including themselves. These self-renewing and population-renewing cells, which may constitute only a small proportion of the total population, are known as stem cells. Tumour stem cells are the relevant cell population responsible for the

Sensitivity testing on patients—future perspectives

Although there is evidence that clinical response rates may be superior for in vitro assay-directed chemotherapy rather than chemotherapy selected by an oncologist 37, 74, 75, there has been no prospective randomised controlled trial comparing survival between patients given an in vitro-tested drug, patients treated by surgery alone, and patients treated by standard chemotherapy. Many different laboratories have demonstrated the value of the TCA's correct predictivity. In all published studies,

Acknowledgements

We are grateful to our coworkers Anke Masch, Sibyll Driever, Sandra Kissel, Elke Simon, Verena Haberstroh, Cathy Scholz and Ute Winterhalter for their important contributions to this project.

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