Examination of in vitro chemosensitivity test using collagen gel droplet culture method with colorimetric endpoint quantification.

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.

Immunohistochemical p53 protein status in nonsmall cell lung cancer is a promising indicator in determining in vitro chemosensitivity to some anticancer drugs.

BACKGROUND AND OBJECTIVES: The tumor suppressor oncogene p53 abnormalities have been closely associated with resistance or sensitivity of cancer cells to some chemotherapeutic agents. We examined the association between p53 protein status in nonsmall cell lung cancer (NSCLC) and in vitro chemosensitivity to several chemotherapeutic agents. METHODS: Using 146 surgically resected specimens of NSCLC, p53 status was immunohistochemically evaluated, and in vitro chemosensitivity to 5-fluorouracil (5-Fu), cisplatin (CDDP), mitomycin C (MMC), etoposide (VP-16), doxorubicin hydrochloride (ADM), and vindesine sulfate (VDS) was examined by a collagen gel-droplet embedded culture drug sensitivity test (CD-DST, Int J Oncol, 1997;11:449). RESULTS: Sixty-five of 146 materials (45%) showed immunohistochemically abnormal p53 protein accumulation in >10% of cancer cells within the tumor tissue, being regarded as p53+, whereas 81 (55%) were to p53-, in which no or less than 10% positive immunostaining cancer cells were detected. By CD-DST, the incidence of chemosensitive, borderline, and resistant p53- materials (N=81) to 5-Fu was 37%(N=30), 14%(N=11), and 49%(N=40), whereas that of p53+ materials (N=65) was 20%(N=13), 6%(N=4), and 74%(N=48), respectively, showing that p53- materials were significantly more sensitive to 5-Fu than p53+ materials (P=0.011), especially in the adenocarcinoma type. As similar borderline association between p53 protein status and in vitro chemosensitivity was also shown in ADM (P=0.078), but not in other chemoagents. CONCLUSIONS: Immunohistochemically detected p53 protein status in NSCLC patients may be a promising indicator in determining in vitro chemosensitivity to some anticancer drugs, especially 5-Fu and ADM.

An in vitro chemosensitivity test for solid human tumors using collagen gel droplet embedded cultures.

In vitro chemosensitivity testing using a collagen gel droplet embedded culture drug sensitivity test (CD-DST), was conducted with several types of solid cancer. The overall evaluable rate was 80% (443/554), including 76% for lung (n=243), 78% for breast (n=110), 87% for gastric (n=62), 83% for colorectal (n=107) cancers and 88% for 32 metastatic brain tumors. The in vitro sensitivity of breast, gastric and colorectal cancers to mitomycin C (MMC), cisplatin (CDDP), 5-fluorouracil (5-FU) and doxorubicin (DXR) was similar to the efficacy rates reported for each drug. This was also observed with lung cancer, the sensitivity of which to MMC, CDDP, vindesine (VDS) and etoposide (VP-16) was similar to the clinical efficacy. The clinical response to chemotherapy was compared with the results of in vitro chemosensitivity testing in Il patients: the clinical correlation was 91%, with a 80% true positive and 100% true negative rate. These results suggest that the CD-DST may be clinically useful by allowing the prediction of clinical response in various solid cancers.

In vitro chemosensitivity testing of human tumours by collagen gel droplet culture and image analysis.

It is necessary to develop an in vitro test to overcome the problems often associated with in vitro chemosensitivity tests on individual human tumours. We have developed a collagen gel droplet culture technique that allows for a three-dimensional in vitro growth and drug response assay for human solid tumour cells. Important features of chemosensitivity testing by the collagen gel droplet culture technique include the maintenance of high cloning efficiencies resulting in the need for fewer tumour cells, sufficient suppression of the in vitro proliferation of contaminating non-malignant cells by serum-free medium, and the application of the image analysis system which automatically discriminates between cancer cell colonies and fibroblasts. We described in vitro-in vivo correlations for drug response using 7 human lung cancer xenografts grown in the collagen gel droplet culture and as xenografts in nude mice. Results demonstrated significant correlations with the in vitro drug concentration at 1/10 of the peak plasma concentrations (1/10 Cmax) with the correlation coefficient 0.84 for all four drugs tested. We have cultured 206 tumours thus far obtaining 86% of evaluability for drug response. The drug response data of the fresh lung cancers were similar to not only to data for lung cancer lines but the reported clinical pattern. These results suggested that the collagen gel droplet culture at 1/10 Cmax may have potential in predicting clinical drug responses.

In vitro-in vivo correlation in anticancer drug sensitivity test using AUC-based concentrations and collagen gel droplet-embedded culture.

To improve the ability of an in vitro drug sensitivity test to predict in vivo effects, we applied a drug concentration that was pharmacokinetically equivalent to plasma levels and collagen gel droplet-embedded culture with a high cloning efficiency. We reported that the cell-killing effect of cell cycle phase-nonspecific drugs such as mitomycin C, cisplatin and Adriamycin depends on the area under the drug concentration-time curve (AUC). The plasma AUC values of these drugs were estimated after an injection into nude mice at the maximal tolerated doses (MTD). Tumor cells isolated from human tumor xenografts implanted into nude mice and cultured in collagen gel droplets were exposed to drugs under conditions that can reproduce the plasma AUC in vitro. The in vitro sensitivity to a drug was compared with the in vivo response of the same tumor treated with the MTD of the drug. When the criterion of sensitivity was taken as 50% or less of the growth inhibition (growth rate of treated group/that of control group, T/C), the correlation between the in vitro and in vivo growth inhibition of all 3 drugs tested was relatively high (86% of the true-positive rate, 82% of the true-negative rate and 83% of the correlation rate).

[Development of new in vitro chemosensitivity test using collagen gel droplet embedded culture and its clinical usefulness].

We developed a new in vitro assay for chemosensitivity test using collagen gel droplet embedded culture and image analysis. In this in vitro assay, we successfully minimized the cancer cell number required for culture to approximately 3-10 x 10(3) cells for each 30 microliters collagen gel droplet, obtained the sufficient growth of cancer cells using serum-free medium while suppressing the growth of fibroblastic cells, and measured the volume of cancer cells by eliminating the contaminating fibroblastic cells by an image processing technique. Anticancer effects of the in vitro assay showed a very good correlation with those of in vivo nude mouse assay using human cancer cell lines. The success rates of the in vitro assay for 141 surgical specimens of primary lung cancers and for 65 of primary breast cancers were 89 and 80%, respectively. The accumulated in vitro assay response rates of MMC, CDDP, VDS and VP-16 for primary lung cancers and of MMC, 5-FU and ADR for primary breast cancers were similar to the respective clinical response rates. These results suggest that this in vitro chemosensitivity test may be practically useful for clinical applications.