Rapid Method for Permanent Slide Preparation of Colonies in Soft Agar Cultures

Transcription

1 International Journal of Cell Cloning 4: (1986) Rapid Method for Permanent Slide Preparation of Colonies in Soft Agar Cultures Jazbieh Moeui, Francis Ali-Osman, Martin J. Murphy, Jr. Hipple Caqcer Research Center and Wright State University School of Medicine, Dayton, OH, USA Key Words. Slide preparation - Human tumor clonogenic assay Colony characterization Abstract. A method is presented for preparing permanent microscopic slides from colony-bearing agar layers in soft agar cultures. The main advantages of this technique are its simplicity, rapidity and accurate colony preservation. This method could have broad applications in the human tumor clonogenic assay (HTCA), particularly in the quantitative morphological, cytochemical and immunocytochemical assessment of colonies that form in both control and drug-treated cultures. Thus, this method opens up possibilities for using cytopathological criteria as a quantitative endpoint of the HTCA. Introduction Since the development of the soft agar double-layer assay [l] for clonogenic tumor cells, several laboratories have used it in various modifications to evaluate in vitro drug sensitivities of many human tumor biopsies. Generally, tumor colonies in the native state are enumerated visually by inverted phase microscopy. More recently, colonies have been counted using computerized image analysis [2, 31. The main limitation of these methods has been the presence of pre-existing cellular aggregates and non-viable tissue debris, which often are misinterpreted as newly formed colonies. When a metabolizable tetrazolium stain is applied directly to the Petri dishes [4], it is possible to discriminate between viable versus non-viable Correspondence: Dr. Martin J. Murphy, Jr., Hipple Cancer Research Center, 4100 South Kettering Boulevard, Dayton, OH (USA). Received September 4, 1985; Accepted March 24, Press, Inc.

2 Moezzil Ali-Osman/Murphy 369 cell groups and allows artifactual viable aggregates to be scored as day 1 counts. However, none of these developments provides a means for the quantitative morphological assessment of tumor versus non-tumor colony growth. Although a method has been reported for preparing permanent slides of colonies [5], it is time-consuming and colony loss may be a significant problem because of the repeated washings involved. We present in this report an optimal method for preparing slides that allows the upper agar layer to be preserved without cell or colony loss; furthermore, it is rapid and easy to perform. Materials and Methods Single cell suspensions from human solid tumors were prepared using an improved enzymatic procedure employing a mixture of deoxyribonuclease, collagenase and neutral protease (Ah-Osman F, Moezzi J, Murphy MJ Jr. manuscript in preparation). Soft agar cultures of these solid tumor cells and/or those obtained from malignant effusions were prepared using a modification of the Hamburger Md Salmon technique [l]. Cells were plated in 1.0 ml of 0.3% agarose containing enriched CMRL 1066 medium (GIBCO Laboratories, Grand Island, NY) over a 1 ml feeder layer containing 0.5% agarose in enriched McCoy's 5A medium (GIBCO). Cultures were incubated at 3PC in an atmosphere of 5% C02 in humidified air for two to three weeks. After adequate growth was attained, the colonies were enumerated first with an inverted microscope and then with an Omnicon FAS 111 Image Analyzer (Bausch & bmb, Rochester, NY). The slide preparation of colony-bearing (upper) agar layers was performed as follows. Each Petri dish (Flow Laboratories, McLean, VA) was filled with fixative (preferably 95% alcohol for Papanicolaou stain or 3% glutaraldehyde for hematoxylin-eosin stain) for min. Using a Pasteur pipette and tilting the dish at a 30"angle, we aspirated the supernatant fixative without disturbing the agar. Using a surgical scalpel, we cut the agar into pieces approximately the same size as a microscope field of the Omnicon FAS III Image Analyzer (e.g., 3 X 4 mm), or any other desired size, as suggested in Figure 1. We carefully placed a dry strip of filter paper (Whatman #2, Whatman Inc,, Clifton, NJ) about twice the size of the cut agar frame on the agar surface, covering the entire cut frame. Immediately after the filter paper was moistened evenly with water, it was removed gently from the agar surface. We attached the cut section of the agar's top layer, which contains cells and colonies, to the filter paper with the bottom layer remaining in the dish. Occasionally, e.g., when the bottom layer is loose on the plastic, both layers will be removed together from the dish. In this case, another piece of dry filter paper should be put on the other surface of the agar botttm layer. It can then be removed easily from the upper layer by sliding off the filter pap& We placed the filter paper containing the top agar layer on a clean glass slide and added a few drops of water or fixative to the paper. The filter strip then is detached easily, leaving the agar with the colonies on the slide. A thinner agar layer can be achieved by leaving the filter paper on the agar for min at room temperature; it can be removed easily by re-moistening the paper with water. The slide may be dried with or without the

3 Fig. 1. Preparation of permanent slides from colony-bearing agar layers. The upper agar layer is cut into rectangular frames of desirable size (1). A dry strip of filter paper is placed on the surface of the cut agar. After the paper is moistened, it is removed (2). The cut section of the agar top layer attached to the filter paper is placed on the glass slide (3). After min, the filter paper strip is detached and a thin layer of colony-bearing agar remains on the slide (4). After drying, the slide can be stained with any desirable stain (5).

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5 Slide Preparation in Soft Agar Cultures 372 $ 300 U Fig. 3. Correlation between the number of colonies formed in agar plates prior to and after removal of the colony-bearing upper agar layer, enumerated with an inverted microscope. aid of a fan. Slides may be stained immediately after drying or stored for later staining. We find that a modified Fapanicolaou or hematoxylin-eosin staining technique [6] is satisfactory for routine evaluation of tumor colonies. Results Figure 1 illustrates the method for removing the agar top layer and depositing it onto a microscope slide. Figure 2A shows intact colonies and clusters of an 18-day-old cultured colon cancer specimen in a small (3 x 3 mm) cut frame of an upper agar layer. These were later transferred onto a slide and stained using the Papanicolaou technique (Fig. 2B). Figure 3 shows the close correlation between colony counts in agar plates before and after the upper agar layer has been removed and permanently stained

6 Moezzi/Ali-Osman/Murphy 373 with the Papanicolaou technique. The correlation coefficient of 0.94 obtained with 21 different tumor specimens shows that there was a quantitative transfer of colonies onto the slides for this group of tumors. Discussion Preparation of colonies grown in the soft agar culture for purposes of quantitative characterization by morphological, cytochemical and immunocytochemical techniques has been difficult. Therefore, we have developed an improved method for colony preparation. Besides being easy and quick to perform, the most important advantage of this technique is that almost all cells and colonies in the cut agar frames remain intact and spatially in place. Consequently, colonies can be enumerated accurately by directly evaluating malignant colonies according to the size and number of cells within a given colony. Pre-existing cell aggregates (viable and non-viable) can be identified by microscopic inspection of slides prepared on day 1. Using this technique, one can evaluate the colony size-range, and approximate the number of cells per colony for different tumor types, without having to pluck individual colonies. Other applications of this slide preparation technique include the morphological evaluation of cytological changes in colonies and clusters of drug-treated culture plates and the quantitative characterization of colonies with special or variant morphology. This uncomplicated and useful method promises broad application in various aspects of the soft agar culture. Acknowledgment Supported by the Hipple Cancer Research Center Foundation. References 1 Hamburger AW, Salmon SE. Primary bioassay of human tumor stem cells. Science 1977;197: Kressner BE, Morton RRA, Martens AE, Salmon SE, Von Hoff DD, Soehnlen B. Use of an image analysis system to count colonies in stem cell assays of human tumors. In: Salmon SE, ed. Cloning of human tumor stem cells. New York: Alan R. Liss, Inc. 1980: Salmon SE, Young L, Lebowitz J, et al. Evaluation of an automated image analysis system for counting human tumor colonies. Int J Cell Cloning 1984;2: Alley MC, Uhl CB, Lieber MM. Improved detection of drug cytotoxicity in the soft agar colony formation assay through the use of a metabolizable tetrazolium salt. Life Sciences 1982;31:

7 Slide Preparation in Soft Agar Cultures Salmon SE, Buick RN. Preparation of permanent slides of intact soft-agar colony cultures of hematopoietic and tumor stem cells. Cancer Res 1979;39: Luna LG. Manual of histologic staining methods of the Armed Forces Institute of Pathology. New York: McGraw Hill Book Co., 1968:70-71.