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Collagen gel droplet embedded culture-drug sensitivity test (CD-DST) is the newly developed in vitro chemosensitivity test that has several advantages over the conventional ones. The aim of the present study is to examine the clinical usefulness of this test in the prediction of response to chemotherapy in breast cancer patients. Seventy patients with primary (n ‫؍‬ 45) or locally recurrent (n ‫؍‬ 25) breast cancers were recruited, and each patient underwent tumor biopsy before chemotherapy. The biopsy specimens were used for CD-DST and immunohistological examination of 6 biological markers (P-gp, erbB2, p53, BCL2, MIB1 and ER-␣). As chemotherapy, cyclophosphamide 600 mg/m 2 plus epirubicin 60 mg/m 2 q3w (CE, n ‫؍‬ 28) or docetaxel 60 mg/m 2 q3w (DOC, n ‫؍‬ 42) was given. Interpretable results using the CD-DST assay were obtained from 84.3% (59/70) of tumor specimens studied. Of the 18 tumors diagnosed as CE sensitive by CD-DST, 15 (83.3%) exhibited a response to CE therapy and none of the 5 tumors diagnosed as CE resistant by CD-DST exhibited a response to CE therapy. Of the 14 tumors diagnosed as DOC sensitive by CD-DST, 13 (92.9%) exhibited a response to DOC therapy and only one of the 22 tumors diagnosed as DOC resistant by CD-DST exhibited a response to DOC therapy. P-gp expression was found to exhibit a significant (p < 0.05) association with the resistance to CE therapy but not to DOC therapy. Diagnostic accuracy (72.7%) achieved by P-gp was lower than that (87.0%) achieved by CD-DST in CE therapy. Expressions of other biological markers (erbB2, p53, BCL2, MIB1 and ER-␣) were not significantly associated with response to CE or DOC therapy. These results demonstrate that CD-DST can predict the response to CE and DOC therapy with a high accuracy in breast cancer patients and seems to be superior to the conventional predictors.

To develop a simpler method of performing the collagen gel droplet-embedded culture drug sensitivity test (CD-DST), we examined the introduction of colorimetric quantitative determination of images for evaluation of anticancer effect against cancer cells alone in the presence of fibroblasts, based on differences in proliferative morphology and stainability with neutral red of cells within collagen gel drops determined using a video-microscope and NIH Image software. In examinations using a human cancer cell line and a fibroblast cell line, a high degree of linearity between number of cancer cells and image-optical density was found within the range of 10 2 -10 6 cells/droplet (r 2 = = = =0.933). Using NIH Image, fibroblast cells could be eliminated at a cut-off value of 128, and an immunocytochemical method demonstrated that the cells eliminated from the image were indeed fibroblasts, and those remaining were cancer cells. CD-DST was carried out with mixtures of cancer cells with fibroblasts at various ratios, and the feasibility of evaluating anticancer activity in cancer cells alone with no effect of fibroblasts at any mixing ratio was confirmed. In addition, for CD-DST of primary cell cultures of human lung cancers collected at the time of surgery, a high correlation between results obtained with the volume supplementation method, a current cell quantification method, and those with the imaging colorimetric quantification method was obtained (r = = = =0.933). These results indicate that introduction of imaging colorimetric quantification utilizing NIH Image makes CD-DST a quick and simple method that should be highly useful for clinical chemosensitivity testing using primary cell cultures of human cancers.

The results show that CD-DST is capable of selecting the responders and the respective optimal regimens, and also delineating the patients less likely benefit from treatment.

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