Individual human tumors in short-term micro-organ cultures: Chemosensitivity testing by fluorescent cytoprinting

Abstract

Using innovative approaches, we addressed several problems often associated with in vitro chemosensitivity testing of individual human tumors: 1) obtaining a high rate of evaluability; 2) excluding participation of nonmalignant stromal and vascular components usually present in tumor specimens; 3) preserving cell-to-cell interactions present in the original tumor; 4) assessing drug-induced cytotoxicity without sacrificing the tumor culture. To circumvent these problems, tumor specimens were processed as follows: i) tissue (fresh or cryopreserved) was mechanically or enzymatically dissociated under mild conditions into cellular clusters (termed micro-organs); ii) large micro-organs were separated by a brief decantation, resuspended, and then exposed to fluorescein acetate to visualize (under naked eye) viable micro-organs; iii) fluorescent (i.e., viable) micro-organs were collected using a Pasteur pipette, and then planted on a solid support made of cellulose fibers impregnated with collagen. Since tumor micro-organs have been previously shown to consist solely of malignant cells, the procedure described here not only preserves a critical portion of the tumor architecture but eliminates at the onset necrotic tissue and nonmalignant cellular components that could interfere with the chemosensitivity testing. Drug-induced cytotoxicity was measured by "fluorescent cytoprinting", a novel, nondestructive procedure for assessing micro-organ viability in situ. The key feature of fluorescent cytoprinting is that cytotoxic effects are not measured against control cultures but against a baseline provided by a cytoprint of the same culture before drug addition. Using three experimental designs, we tested the potential of the method for clinical applications. The results using 469 human malignant tumors showed that the micro-organ culture assay can distinguish individual tumor chemosensitivity profiles with an overall success rate of 96%. For three commonly used chemotherapeutic drugs, the observed frequency of responding tumors was found to be comparable to previously reported clinical results using single agents.

Chemotherapy of non-small cell lung carcinoma guided by an in vitro drug resistance assay measuring total tumour cell kill

“…These theoretical considerations also apply to other assay systems measuring cell damage in the total (largely non-dividing) tumour cell population (e.g. (Rotman et al, 1988;Campling et al, 1988)). In our present study, it was striking that none of the eight patients assigned to receive chemotherapy which included 5FU responded.…”

In vitro assay-assisted treatment selection for women with breast or ovarian cancer.

“…The FCPA measures the enzyme activity of membrane cytosolic esterases (Fig. 6) (Rotman et al 1988, Leone et al 1991. Esterase activity is related to cell viability, and is assayed by monitoring hydrolysis of fluorescein-monoacetate to fluorescein.…”

In vitro chemosensitivity testing and mechanisms of drug resistance

“…The fluorescent cytoprint assay was developed by Rotman et al [33] as a means to overcome some of the limitations of the HTCA. To improve plating efficiency, this method uses small tumor tissue fragments of approximately 50 or more cells, so-called "micro-organs" [22••].…”