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[CANCER RESEARCH 39, 1133-1136, March 1979]
0008-5472/79/0039-0000$02.0O
Communication
Preparationof PermanentSlidesof Intact Soft-AgarColonyCultures
of HematopoieticandTumorStemCells1
Sydney E. Salmon and Ronald N. Buick
Sectionof Hematologyand Oncology,Departmentof InternalMedicine,and TheCancerCenter-Universityof Arizona,ArizonaHealthSciencesCenter,
Tucson, Arizona 85724
ABSTRACT used for permanent fixation of protein electrophonesis on
immunodiffusion in agamon glass slides or plastic films (13)
Asimple technique is described for fixing colony-contain might be adaptable to intact agamlayers from in vitro colony Downloaded from http://aacrjournals.org/cancerres/article-pdf/39/3/1133/2404145/cr0390031133.pdf by guest on 14 September 2022
ing layers of soft agar and drying them onto microscopic assays. This perspective provided the basis for a simple
slides. The method is extrapolated from techniques used in method for fixation and preservation of agamcultures for a
immunology for permanent preservation of immunodiffu wide variety of studies. The purpose of this paper is to
sion or immunoalectrophonasis plates. Slides prepared in describe the method which we standardized and to provide
this fashion are eminently suitable for subsequent analysis suitable illustrations of the morphological quality and var
with a variety of techniques including conventional Papani satility of this technique.
colaou onother staining methods as well as histochemistry,
immunofluonascenca, and autonadiognaphy. In addition to METHODSAND RESULTS
research applications, the technique may have diagnostic
applications and should greatly enhance both qualitative Soft-agan cultures of normal human granulocyte precum
and quantitative analysis of the biology of hamatopoietic sons(colony-forming units in culture) were prepared by the
and tumor colony formation. method of Pike and Robinson (10), and cultures of human
INTRODUCTION tumor biopsies waneprepared by the method of Hamburger
and Salmon (5-7). Calls wane plated in 1.0 ml of 0.3% agar
The development of clonogenic assays in soft agar has containing the appropriate medium and additives oven a
greatly increased our understanding of the biology of self feeder layer of 1.0 ml of 0.5% agar containing plain or
renewal compartments in the bone marrow (8). The recent conditioned medium or feeder cells. Cultures were incu
development of soft-agar clonogenic assays for human bated at 37°for 7 to 21 days in a humidified incubator
tumor stem calls may provide this same potential (5-7), as supplied with 5% CO2 in air. At appropriate times, plates
well as practical application to measurement of drug sensi wane examined with an inverted microscope and selected
tivity (11). Until the present time, morphological study of for permanent preservation with the following method.
such colonies by conventional histological and histocham 1. With a Pasteur pipet, each Petni dish is carefully filled
ical techniques has bean an arduous task, inasmuch as with Hanks' balanced salt solution and incubated for 15 mm
most staining techniques have required careful picking of atroom temperaturetoelutethemajorityofextracellulam
individual colonies from the agar under an inverted micro proteins from the plating layer. The supernatant solu
scope followed by deposition on microscope slides. With tion is then aspirated or decanted from the dish, taking cane
that approach, careful selection of relatively nondismuptad not to disrupt on pour out the colony-containing plating
stained colonies for review is usually required, further layer. This initial wash step is optional but reduces back
limiting morphological study. Additionally, the relationship ground staining somewhat.
of the small fraction of calls which form colonies in vitro 2. Each dish is than filled with a fixative solution consist
from normal marrow or spontaneous human tumors to ing of 3% glutanaldehyda in Hanks' balanced salt solution
background nonproliferativa and “stromal―calls has not and is allowed to fix for 10 mm at room temperature. The
been studied, inasmuch as only colonies or clusters have supennatant fixative is then removed as above.
been picked for fixation and staining. Several techniques 3. Each dish is than filled with distilled water, agitated,
have been described in an attempt to simplify onstandardize submerged in a tray (e.g., a disposable plastic weigh boat)
the picking procedure (2, 4, 12); however, these are, in our containing at least 50 ml of distilled water, and allowed to
opinion, suboptimal. incubate for 10 mm to alute the salts and fixative from the
In the course of our reviewing various techniques which plating layer. During this tmme,the plates can be agitated
might be suitable for preservation and morphological study and decanted gently by hand in order to displace the plating
of whole Patmidishes of marrow and tumor colonies, we layer from the Petni dish so that it may float freely in the
considered that the general approach which has long bean water. Frequently, the 0.3% agan plating layer will slip free
from the 0.5% aganfeeder layer which remains attached to
the plate. When this occurs, the dish containing the feeder
1 The authors' tumor colony work is supported in part by Grant CA-21839 layer can be discarded. Should the plating layer and feeder
from the National Cancer Institute, NIH, Bethesda, Md.
Received November 17, 1978; accepted January 3, 1979. layer decant attached to one another, gentle spurts of water
MARCH1979 1133
S. E. Salmon and R. N. Buick
from a Pasteur pipat can be used to tease the plating layer ization of fluorescent antiimmunoglobulin in dried slides of
free from the thicken feeder layer. Sometimes, a small rant human myaloma colonies. Autoradiographic slides pra
must bemadein the plating layerso that the pipat can be pared with the high-speed scintillation technique (3)
introduced between the 2 layers. They may then be disso showed specific localization of DNA-synthesizing calls
ciatad from each other with additional gentle spurts of which had been pulsed with high-specific-activity
water. The separated plating layer is thin and filmy and can [3H]thymidina shortly before fixation; however, prominent
usually be identified easily even when both the plating and background grains remain unless a longer period of wash
feeder layers are floating in the water. After the feeder layer ing is used.
is discarded, the tray is carefully drained, leaving the
plating layer in a few drops of water. The thin plating layer DISCUSSION
can than be poured gently onto a microscope slide. Altar
natively, a cleaned microscope slide can be introduced into The development of this simple technique for fixation and
an undrained tray by hand, and the plating layer (or a piece drying of agancultures onto microscope slides will probably
of it) is allowed to spread on the slide which is than picked have wide application in experimental studies of hamato
up gently out of the water. If the entire area of the colony poietic and tumor colony formation. Such films can also be Downloaded from http://aacrjournals.org/cancerres/article-pdf/39/3/1133/2404145/cr0390031133.pdf by guest on 14 September 2022
containing layer is not needed, standard microscope slides dried onto hydmophilic plastic supports or directly onto Patni
(2.5 x 7.5 cm) can be utilized, and the few mm of excess dishes if there is not a separate feeder layer. We have
agan can be pinched off with a finger where it hangs oven illustrated only straightforward morphological applications,
the edges of the slide. Wider slides can be utilized when the and many mona sophisticated analyses should also be
entire 35-mm diameter layer must be recovered. possible. One such example would be the identification of
4. A prawatted cellulose acetate membrane (as is used “variant―myaloma colonies with respect to immunoglobu
for alactrophonesis) which has bean cut to the dimension of lin synthesis (1). While the majority of applications of this
the slide is carefully placed on top of the still-wet agar layer, method will probably be directed towards research quas
and any bubbles are gently expressed. The cellulose acetate tions, we recognize that the ability to prepare permanent
strip provided for uniform evaporation of the water from the stained slides of such colonies may also have practical
agamwhich otherwise dries unevenly onto the slide. Altar application in clinical diagnosis. Additionally, the ability to
natively, low-fiber filter paper can be substituted for callu provide uniform staining of such preparations may greatly
lose acetate strips; however, filter paper frequently leaves facilitate colony counting and potentially permit automation
undesired fibers adherent to the agar which may obscure of the process.
morphology. In the low-humidity atmosphere of our labo
ratony, such agan layers dry firmly onto the slide within 4 hr; REFERENCES
however, in a higher-humidity environment, overnight
drying might be required. Once the slide has dried, the 1. Coffino, P., Baumal, R., Laskov, R., and Scharff, M. D. Cloning of
cellulose acetate strip either comas loose spontaneously or mouse myeloma cells and detection of rare variants. J. Cell. Physiol.,
79: 429-440, 1972.
can be moistened and gently pulled off the slide. 2. Dicke, K.A., and Platenburg,M. G. C. Technical manualofthethin agar
Slides prepared as described above can either be stored layer technique. In: D. van Bekkum and K. Dicke (ads.), in Vitro Culture
in the unstained state or be stained immediately with any of of Hemopoietic Cells, pp. 466-470. Rijswijk, The Netherlands: Radio
biological Institute TNO, 1972.
avariety of stains. 3. Dune, B. G. M., and Salmon, S. E. High speed scintillation autoradiog
The most useful general stain which we have applied is raphy. Science, 190: 1093—1095,1975.
4. Goube De Laforest, P., Riou-Lasma-Vons, N., and Boizard, G. A micro
the standard Papanicolaou technique (9). While standard cytocentrifugation technique for cytological studies on hemopoietic
staining times are usually satisfactory, longer staining times colonies grown in agar. Exp. Hematol., 6: 361-364, 1978.
for the nuclear hamatoxylin are sometimes required. Ro 5. Hamburger, A., and Salmon, S. E. Primary bioassay of human myeloma
stemcells.J.Clin.Invest.,60:846—854,1977.
manovsky stains such as Wnight-Giemsa often stain the 6. Hamburger,A.W.,andSalmon,S.E.Primarybioassayofhumantumor
background agan a somewhat bluish-pink but are usable stem cells. Science, 197: 461-463, 1977.
when theplatinglayeristhin.Aganplatesofcolony-forming 7. Hamburger, A.W., Salmon, S. E., Kim, M. B., Trent, J. M., Soehnlen, B.
J,, Alberts, D. S., and Schmidt, H. J. Direct cloning of human ovarian
units in culture which have bean stained histochemically carcinoma cells in agar. Cancer Res.,38: 3438—3443,1978.
for penoxidasa (14) prior to fixation maintain good histo 8. Metcalf,D.HemopoieticColonies.Berlin:SpringerVerlag,1977.
9. Papanicolaou,G. N. Atlas of ExfoliativeCytology,p. 6. Cambridge,
chemical localization through the fixation and drying steps. Mass.:Harvard University Press, 1954.
When 2 colonies are above one another in the wet film (a 10. Pike, B. L., and Robinson, W. A. Human bone-marrow colony growth in
maneevent), they may be partially superimposed upon one agar-gel. J. Cell. Physiol., 76: 77—84,1970.
11.Salmon,S.E.,Hamburger,A.W.,Soehnlen,B.J.,Dune,B.G. M.,
another. Discrimination between such colonies can be Alberts, D. S., and Moon, T. C. Quantitation of differential sensitivities
achieved with fine focusing. Figs. 1 to 5 illustrate typical of human tumor stem cells to anticancer drugs. N. Engi. J. Med., 298:
stained, dried films of human bone marrow granulocyte 1321-1327,1978.
12.Testa,N.G.,andLord,B.I.Atechniqueforthemorphologicalexami
colonies and tumor colonies from a variety of human nation of hemopoietic cells grown in agar. Blood, 36: 586-589, 1970.
cancers which we have cloned directly in soft agar from 13. Weime, R. J. Studies on Agar Gel Electrophoresis. Brussels: Arscia
UitgavenN.V.,1959.
biopsy samples. Of particular note are the excellent mom 14. Zucker-Franklin, D., and Grusky, G. The identification of eosinophil
phology and staining qualities of hamatopoietic and tumor colonies in soft agar cultures by differential staining for peroxidase. J.
colonies. We have also observed specific cytoplasmic local Histochem. Cytochem., 24: 1270-1272, 1976.
1134 CANCER RESEARCH VOL.39
Slides of Intact Agar cultures of Tumor Stem cells
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Fig. 1.a, morphological appearance of normal human bone marrow colony-forming units in culture grown in soft agar. The stain produces a stippled
background staining of the agar which does not interfere with morphology in thin films. Lower left, nonproliferative megakaryocyte. Wright-Giemsa, x 100.
b, high-power photomicrograph of human neutrophils in a soft-agar colony grown from the bone marrow. Wright-Giemsa, x 1000.
Fig.2.a, morphologicalappearanceofhumanmelanomacoloniesgrowndirectlyfrombiopsyinsoftagar.Differentamountsofmelaninareexpressedin
various cells. Papanicolaou stain, x 100. b, high-power photomicrograph of melanoma cells shows excellent preservation of cellular morphology in dried
films from soft-agar culture. Cytoplasmic stippling is due to diffuse distribution of fine melanin granules. Papanicolaou stain, x 1000.
MARCH 1979 1135
S. E. Salmon and A. N. Buick
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Fig. 3. a, morphological appearance of human squamous cell carcinoma colonies grown in soft agar from a needle biopsy of the liver from a patient with
knownlivermetastases.The dark centralareasandcrackingnearthe centerofthe right handcolonyareduetoflatteningofthe largesphericalcolonyonto
a microscope slide in the agar film. Papanicolaou stain, x 100. b, high-power photomicrograph of squamous cells in a colony depicted in a. The wide
intercellular spacing and bridging were characteristic morphological features manifest in squamous carcinoma colonies. x 1000.
Fig. 4. Typical malignant adenocarcinoma colony at 14 days grown in soft agar from a patient with metastatic colonic carcinoma and malignant ascites.
Excellentmorphologyof the cancer cells inthe colonyaswell asa backgroundnonproliferativeplasmacell at the leftsideof the field are preservedinthe
dried agar film. Papanicolaou stain, x 400.
Fig. 5. Human ovarian cancer colony from a patient with a metastatic papillary cystadenocarcinoma. The colony size was typical for 14-day culture.
Papanicolaou stain with hematoxylin exposure prolonged to 12mm to enhance nuclear detail, x 400.
1136 CANCER RESEARCH VOL.39
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