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Human tumor clonogenic assays: Growth conditions and applications
Human Tumor Clonogenic Assays: Growth Conditions and Applications Sydney E. Salmon
Increasing investigative efforts have been focused on the application of in vitro clonogenic assays for human tumors. Several semisolid matrices have been used for purposes of immobilizing the clonogenic cells, and a variety of growth factors and environmental conditions have been studied to enhance tumor colony growth. Although there remain some technical problems with these assays, the methodology has proven to have broad applicability to preclinical and clinical studies of human cancer. This report reviews some features and areas of application of this assay methodolagy. The ability to combine studies of drug effect with cytogenetic studies on clonogenic cells suggests that such assays may prove useful for evaluation of the emergence of drug resistance by primary and metastatic cancers.
ABSTRACT:
INTRODUCTION In 1977, a bioassay system that facilitated in vitro colony formation by clonogenic tumor cells from biopsy samples of h u m a n solid tumors [1] was reported. Similar colony-forming assays also have been developed for studying the leukemias [2, 3]. A variety of morphologic, histochemical, immunologic, and cytogenetic techniques have been applied, which have established that the colonies that form in semisolid medium are representative of the malignant cells in the patient's tumor [4-6]. Such clonogenic cells are thought to be closely related to tumor stem cells in vivo [7]. It is the tumor stem cell p o p u l a t i o n that is capable of repeated cycles of proliferation characteristic of the self-renewing malignant cell population w i t h i n a tumor, and is responsible for local or metastatic recurrence after subcurative therapy. Clonogenic assay methodology has been applied to studies of cancer biology, cellular interaction and tumor immunology, cytogenetics, and preclinical drug development, as well as clinically in cancer diagnosis and the prediction of response to chemotherapy. Some of the features of clonogenic assays that have made them desirable for application in these areas are summarized in Table 1.
TECHNICAL C O N S I D E R A T I O N S
A wide variety of h u m a n tumor types can be grown in colony forming assays. However, not all specimens from all patients with any given type of cancer will grow colonies in vitro and in general, for most solid tumors, the cloning efficiencies are low (about 0.1%). Correcting for the percentage of non-neoplastic cells and dead From the Department of Internal Medicine,and the Arizona Cancer Center, Universityof ArizonaCollege of Medicine,Tucson,AZ. Address requests for reprints to Dr. Sydney E. Salmon, Arizona Cancer Center, University of Arizona College of Medicine, Tucson, A Z 85724. Received August 5, 1985; accepted August 19, 1985.
21 © 1986 Elsevier Science PublishingCo., Inc.
52VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet 19:21-28 (1986) 0165-4608/86/$03.50
22
S.E. Salmon Table 1
Features of clonogenic culture in semisolid medium
Applicable to direct study of growth by primary human tumors and metastases Only a small fraction of cells within a tumor give rise to tumor colony growth (<1%) Normal fibroblasts and bone marrow progenitors do not give rise to colonies (in the absence of conditioned medium) Clonal heterogeneity is preserved and can be quantitated Provides a quantitative approach to measure drug sensitivity of human tumors
cells in tumor s p e c i m e n s prior to culture initiation does yield a s o m e w h a t higher cloning efficiency, but it is still generally less than 1.0%. Whether the low cloning efficiencies are related to the presence of only a limited s u b p o p u l a t i o n of tumor cells with clonogenic or stem cell properties or to s u b o p t i m a l growth conditions, remains to be determined. Current information supports the s u b p o p u l a t i o n hypothesis. T u m o r specimens are u s u a l l y brought to the laboratory w i t h i n several hours of surgery, u n d e r aseptic conditions in a transport or complete growth medium. Enzymatic disaggregation techniques (generally e m p l o y i n g DNAse and collagenase and/or other enzymes) [8] have increased the yield of viable t u m o r cells from a number of solid tumor types, c o m p a r e d with m e c h a n i c a l disaggregation techniques. Technical details addressing m e d i u m selection and disaggregation are delineated elsewhere in this issue [9]. A variety of alterations in m e t h o d o l o g y have also been e x a m i n e d in order to increase the cloning efficiency for specific types of cancer. Thus far, these efforts have had only modest success. It is important to point out that a ntrmber of s p e c i m e n s received by the laboratory are inadequate in size (less than 1 g), some are cytology negative, while others are either c o n t a m i n a t e d or have not been adequately disaggregated u n d e r aseptic conditions. At the present time, only about 50% of t u m o r s p e c i m e n s y i e l d adequate in vitro growth (i.e., > 2 0 colonies/105 cells plated). A m o n g the various t u m o r types, excellent in vitro growth (>70% success) is observed in ovarian cancer, melanoma, and lung cancer. Such results have been obtained in our laboratory w i t h solid t u m o r specimens cultivated under the conditions of the standard Hamburger and Salmon assay, with only minor modifications in w h i c h DEAE-Dextran, calcium chloride, and L-asparagine have been deleted. Insulin is an i m p o r t a n t growth factor in this system; the addition of other growth factors i n c l u d i n g transferrin, hydrocortisone, thyroxine, and epidermal growth factor can m o d e s t l y potentiate colony formation in a majority of cases, but instances of modest inhibition of growth of some specimens have been observed with m a n y of these factors, as well. Use of a synthetic HITES m e d i u m has been reported to support growth of lung cancer cells with at least equal efficacy as serum-containing m e d i u m [10]. Breast cancer presents a n u m b e r of special problems, including inadequate s p e c i m e n size in T1 and m a n y T2 tumors, simultaneous need for estrogen receptor testing, and difficulties in obtaining adequate numbers of cells after disaggregation procedures [11]. Colorectal and head and neck cancers can also be grown in this system, but c o n t a m i n a t i o n is more frequently observed when the p r i m a r y t u m o r biopsy is submitted. L y m p h o m a s and m y e l o m a s generally do not grow well in such colony assays unless c o n d i t i o n e d m e d i u m factors (which are still undefined) are a d d e d [12]. Growth stimuli that have proved useful are summarized in Table 2. For specific tumor types, there are significant differences in growth as a function
Human Tumor Clonogenic Assays Table 2
23
Growth stimuli in semisolid culture
Mineral oil Balb-C conditioned medium (Myeloma) PHA-leukocyte conditioned medium (Leukemia) Defined growth factors and micronutrients (epidermal growth factor, hydrocortisone, transferrin, ascorbic acid, thiols, other hormones) (various tumors) Hypoxia (3%-5% environmental oxygen)
of the specimen source submitted (e.g., skin or lymph node). For some tumor types, progressive increases in cloning efficiency have been observed in vitro in association with tumor progression in vivo. Environmental oxygen concentration also has an effect on in vitro colony growth in semisolid medium. Studies with both normal hematopoietic colony-forming cells and human tumor cell lines have shown that relatively low oxygen concentrations (e.g., 3%-5% 02, normally found in tissues of the body) potentiate colony formation, compared with 20% 02. Although 20% 02 is present in the air (and in standard CO2 incubators), such high concentrations of 02 are not found in mixed venouscapillary blood within solid tumors. A soft agar colony assay developed by Courtenay and Mills [13] for human tumor xenograft culture in vitro utilizes such low 02 concentrations. In our own lab, we have found that 3% 02 does potentiate growth of some types of fresh human tumors. The specific semisolid medium selected for clonogenic assay varies depending on the specific needs of the investigator with respect to the required experimental versatility, as well as to optimize growth conditions for the specific tumor type tested. Frequently used semisolid culture supports are summarized in Table 3. Although agar has been most widely used for studies of solid tumors, agarose may be slightly preferable. It is more expensive but has a neutral charge, gels somewhat more slowly, and may permit slightly better growth of some type of solid tumors than agar. In some labs, however, no significant differences have been observed in growth support between these two semisolid supports. Two layer gel systems (prepared with agar or agarose) permit rigid physical separation between the softer plating layer (in which colony formation occurs) and the feeder layer within which nutrients or supporting cells can be placed. Feeder cell types, such as adherent macrophages, have also been adhered to the surface of the Petri dish, immobilized with the rigid feeder layer, and their influence on colony formation (via soluble mediators) examined [14]. Methylcellulose is not truly a gel; it is a highly viscous liquid, thus, permiting some cellular or colony movements within the matrix. Unfortunately, because of this property, it has not been used successfully with automated colony counters, as colony positions shift in methylcellulose with movements of a mechanical stage. Methylcellulose has been used largely for colony-forming studies of acute nonlymphocytic leukemias (ANLL) and by normal bone marrow progenitors, and has not been applied as successfully to solid tumors, (perhaps because endogenous fibroblasts can proliferate at the bottom of the dishes, and because of the aforementioned
Table 3 Semisolid culture systems Soft agar (0.3% top layer, 0.5% underlayer) Soft agarose (two layer) Methylcellulose Methylcellulose upper layer, agar underlayer
24
s.E. Salmon problem with automated colony counting). A two-layer system w h e r e i n t u m o r cells in methylcellulose are plated over agar has been e m p l o y e d for studies of bladder cancer [15]. This m e t h o d permits separation of a feeder cell layer w h i l e still facilitating simple recovery of intact colonies from the matrix for morphologic studies, as methylcellulose can be t h i n n e d by a d d i t i o n of water or buffered saline. We have observed that fibroblasts present in t u m o r cell suspensions can proliferate in cracks or irregularities at the a g a r - m e t h y l c e l l u l o s e interface. Agarose and agar are gels that cannot be dissolved by a d d i t i o n of water and that do not melt on slight increase in environmental temperature. However, i n d i v i d u a l colonies in agar can readily be p l u c k e d with a m i c r o p i p e t t e or m i c r o m a n i p u l a t o r and, additionally, simple techniques have been d e v e l o p e d for recovering intact colonies from agar for cytogenetic studies [16]. S i m p l e m e t h o d s also have been d e v e l o p e d for recovering the colonycontaining plating layer, preparing dried agar films on microscope slides and staining colonies for morphologic s t u d y [5]. These c o m m e n t s are not i n t e n d e d to imply that all technical problems with the clonogenic assay have been solved. However, these considerations plus the use of positive controls for drug assays and the use of an automated colony counter [17, 18] have i m p r o v e d quality control for in vitro clonogenic assays.
APPLICATIONS OF CLONOGENIC A S S A Y H u m a n t u m o r clonogenic assays are n o w a p p l i e d in a variety of preclinical and clinical areas of cancer research. Table 4 lists some of the current areas of application. Discussion of several areas w h e r e i n I have a p p l i e d this assay technique to studies of t u m o r heterogeneity and for clinical c h e m o s e n s i t i v i t y testing are prov i d e d b e l o w for illustrative purposes.
Tumor Heterogeneity A major area of current interest in t u m o r biology, one that m a y have clinical implications with respect to the metastatic process and the acquisition of drug resistance, is the p h e n o m e n o n of t u m o r heterogeneity. Heterogeneity in behavior of clonogenic tumor cells can readily be observed in the clouogenic assay w i t h respect to comparative differences of in vitro behavior between histologically similar tumors from different patients in drug sensitivity or resistance to the same agents. Additionally, differences have been observed among different metastases from the same patient,
Table 4
Some current areas of a p p l i c a t i o n of h u m a n t u m o r colony assays
Biology of growth of human neoplasms Regulation of tumor growth: Stromal cell-tumor cell interactions, growth factors Kinetics of clonogenic tumor cells and the self-renewal process Studies of heterogeneity of clonogenic tumor cells Purification of clonogenic tumor cells and antigenic analysis Cytogenetic analysis of clonogenic tumor cells New drug screening and preclinical drug development Cancer diagnosis Studies of radiation therapy and hyperthermia Studies of effects of biological response modifiers (interferons, tumor necrosis factors, interleukins, retinoids) Mechanisms of drug resistance Clinical chemosensitivity studies
Human Tumor Clonogenic Assays
25
and among colony-forming response of cells within a single biopsy specimen. Several laboratories have recently noted differences in sensitivity in vitro between primary tumors and their metastases [19]. These findings, as well as morphologic and cytogenetic observations, have been accumulated and indicate that the clonogenic assay systems can be applied in a variety of ways to assess tumor heterogeneity. Recently, we have applied the clonogenic assay system to determine if heterogeneity was seen with respect to estrogen receptor expression in breast cancer [20]. In these studies, the estrogen receptor (ER)-positive MCF-7 breast cancer cell line was grown in soft agar, and ER was evaluated immunohistochemically using specific monoclonal antibodies to ER. Our initial question was whether ER was expressed clonally by MCF-7 cells with some colonies staining positively and others negatively, or if it expressed as a differentiation antigen with varying expression in all colonies as a function of time and colony size. We observed significant and reproducible heterogeneity in ER staining of MCF-7 cells both within and between colonies. These differences were definitely related to colony size and the duration of culture, and truly ER-negative clones were only observed up through the small cluster stage of colony growth. Of interest, addition of the antiestrogen, tamoxifen, at doses that would cause significant reduction in the number of tumor colonies also reduced the degree of expression of ER (although a small number of truly ER-negative colonies was observed in the presence of tamoxifen). These findings are most consistent with a model of breast cancer growth wherein ER positivity is not expressed at the level of the tumor stem cell, but rather is expressed increasingly with increasing tumor colony growth and differentiation of progenitor cells. Tumor stem cells represent the key subpopulation in a tumor that has the ability to self-renew and maintain a tumor stem cell pool, which is responsible for tumor recurrence following subcurative therapy. Both tumor stem cells and some of their differentiating progeny (transit cells) share the property of clonogenicity, but the replicative capacity of the transit cells is limited with increasing differentiation. Based on our findings with respect to estrogen receptor expression, endocrine therapy such as the use of tamoxifen would appear capable of suppressing tumor growth, but would be incapable of eradicating tumor stem cells in breast cancer. In that setting, long-term therapy with this agent would be essential. Figure 1 depicts the apparent level of action of endocrine therapy in a stem cell model of breast cancer growth.
Figure 1 A proliferation-differentiation model for tumor growth based on the tumor stem cell concept. Based on the studies of Kodama et al. [20], tamoxifen and other endocrine active agents would appear to act on a differentiating transit population of tumor cells and beyond the level of the tumor stem cell.
Stem Cell (Proliferate) ll,,,iolnnNnm°lgln
Jnlq
Transi ,~,, Celltional ........................ ~ ~ ~ (Proliferate) ,,,H°llllnnm,o..mlllm.
EndCell
.............indocrine Therapy
~~~',
26
s.E. Salmon
Chemosensitivity Testing One major application of in vitro clonogenic assay has been for chemosensitivity testing. These assays have been applied preclinically in the area of new drug development [21] and for clinical testing for anticancer drug selection for individual patients. Important to such testing has been the development of a pharmacologically based testing system, wherein the in vitro drug concentrations tested are relevant to those that can be obtained in vivo. The initial clinical correlations of in vitro drug sensitivity testing were reported in 1978 for a limited number of patients with ovarian cancer or multiple myeloma [22], and included predominately retrospectively drawn correlations. The results indicated good agreement between in vitro sensitivity and clinical tumor regression, and excellent agreement between in vitro and in vivo resistance. More recently, prospective analyses have been carried out by a number of investigative groups and, in general, have indicated good correlation between in vitro sensitivity or resistance and the clinical success or failure of specific treatments for individual patients [23]. Technical factors including poor growth of some tumor types, lack of effective anticancer drugs for many tumor types, and other factors, however, have thus far limited the clinical applicability of such assays primarily to a few tumor types, such as ovarian cancer. For preclinical discovery of new anticancer agents, clonogenic assays appear to have significant utility [21].
Potential Applications in Cytogenetic Analysis Cytogenetic analysis can provide unique insights with respect to clonal heterogeneity in human tumors, and this problem is addressed extensively elsewhere in this issue. Application of cytogenetic analysis to cells cultivated in vitro in clonogenic assay has the potential to be quite useful in this regard, as shown by Trent (this issue) and others. Some of the advantages of clonogenic assay for cytogenetic analysis of human solid tumors are listed in Table 5. One of the major concepts that arose from the study of cytogenetics of human neoplasms is that of clonal progression [24]. Studies of multiple biopsies of different metastases within individual patients, as well as serial biopsy studies of such patients older than 1-2 years, are quite feasible with clonogenic assay techniques and could be quite interesting with respect to the clonal progression theory. An area of particular interest, which is potentially exploitable through use of such clonogenic assay systems, is the interrelation of results of in vitro drug sensitivity and chromosomal results. This approach could prove useful in the identification of chromosomal abnormalities associated with inherent or acquired drug resistance. Such analyses could provide the basis for more detailed studies of molecular mechanisms of drug resistance and provide new insights and new approaches to cancer treatment.
Table 5 Advantages of semisolid media for chromosome cultures of human solid tumors Growth of normal cells is suppressed Mitotic index of tumor cells within colonies is very high (>25%) Gel matrix immobilizes clonogenic cells and permits inter- and intraclonal chromosome analysis Assay permits chromosome analysis on surviving clonogenic cells after drug exposure Technique compatible with detailed banding analysis
Human Tumor C l o n o g e n i c Assays
27
SUMMARY In vitro c l o n o g e n i c assays for h u m a n s p e c i m e n s h a v e p r o v e n t h e i r b r o a d a p p l i c a tion to the s t u d y of h u m a n c a n c e r b i o l o g y a n d to c h e m o s e n s i t i v i t y testing. The combination of e x p e r i m e n t a l v e r s a t i l i t y a n d a p p l i c a b i l i t y to fresh t u m o r s p e c i m e n s makes it p a r t i c u l a r l y attractive. F u r t h e r i m p r o v e m e n t in cell disaggregation and tissue culture t e c h n i q u e s , as w e l l as a d d i t i o n a l k n o w l e d g e of d e f i n e d g r o w t h factor requirements for h u m a n t u m o r s , are h i g h p r i o r i t y areas for further d e v e l o p m e n t . It seems likely that s u c h assays w i l l h a v e e v e r - i n c r e a s i n g a p p l i c a t i o n s to f u n d a m e n t a l and clinical areas of c a n c e r research. Supported in part by Grants CA-17094, CA-21389, and CA-23074 from the National Institutes of Health, Department of Health and Human Services, Bethesda, MD.
REFERENCES 1. Hamburger AW, Salmon SE (1977): Primary bioassay of human tumor stem cells. Science 197:461-463. 2. Buick RN, Till JE, McCulloch EA (1977): Colony assay for proliferative blast cells circulating in myeloblastic leukaemia. Lancet i:862-863. 3. Park CH, Amare M, Wiernik PH, Dutcher JP, Morrison FS, Maloney TR (1984): In vitro drug sensitivity of leukemia colony-forming cells in acute nonlymphocytic leukemia: Clinical correlation update with various drug exposure methods. In: Human Tumor Cloning, SE Salmon and JM Trent, eds. Grune and Stratton, Orlando, FL, pp. 619-628. 4. Salmon SE (1984): Cloning of Human Tumor Stem Cells. Alan R. Liss, New York. 5. Salmon SE, Buick RN (1979): Preparation of permanent slides of intact soft agar colony cultures of hematopoietic and tumor stem cells. Cancer Res 39:1133-1136. 6. Salmon SE, Trent JM (1984): Human Tumor Cloning. Grune and Stratton, Orlando, FL. 7. Steel GG (1977): Growth and survival of tumour stem cells. In: Growth Kinetics of Tumors. Clarendon Press, Oxford, pp. 217-262. 8. Slocum HK, Pavelic ZP, Rustum YM, (1981): Characterization of cells obtained by mechanical and enzymatic means from human melanoma, sarcoma and lung tumors. Cancer Res 41:1428-1434. 9. Leibovitz A (1985): Development of tumor cell lines. Cancer Genet Cytogenet 19:11-49. 10. Carney DN, Nau MM, Minna JD (1984): Variability of cell lines from patients with small cell lung cancer. In: Human Tumor Cloning, SE Salmon and JM Trent, eds. Grune and Stratton, Orlando, FL, pp. 67-82. 11. Jones SE, Dean JC, Salmon SE (1985): The human tumor clonogenic assay (HTCA) in human breast cancer. J Clin Oncol 3:92-97. 12. Hamburger AW, Salmon SE (1977): Primary bioassay of human myeloma stem cells. J Clin Invest 60:846-854. 13. Courtenay FD, Mills J (1978): An in vitro colony assay for human tumors grown in immune-suppressed mice and treated in vivo with cytotoxic agents. Br J Cancer 37:261-268. 14. Buick RN, Fry SE, Salmon SE (1980): Effect of host cell interactions in clonogenic carcinoma cells in human malignant effusions. Br J Cancer 41:695-704. 15. Buick RN, Stanisic TH, Fry SE, Salmon SE, Trent JM, Krasovich P (1979): Development of an agar-methyl cellulose clonogenic assay for cells in transitional cell carcinoma of the human bladder. Cancer Res 39:5051-5056. 16. Trent JM, Salmon SE (1980): Human tumor karyology: marked analytic enhancement via short term agar culture. Br J Cancer 41:867-874. 17. Salmon SE, Liu R, Hayes C, Persaud J, Roberts R (1983): Usefulness of abrin as a positive control for the human tumar clonogenic assay. Inv New Drugs 1:277-281. 18. Salmon SE, Young L., Lebowitz J, Thomson S, Einsphar J, Tong T, Moon TE (1984): Evaluation of an automated image analysis system for counting human tumor colonies. Intl. J Cell Cloning 2:142-160.
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19. Schlag P, Schreml W (1982): Heterogeneity in growth pattern and drug sensitivity of primary tumor and metastases in the h u m a n tumor colony forming assay. Cancer Res 42:4086-4089. 20. Kodama F, Greene GL, Salmon SE (1985): Relation of estrogen receptor expression to clonal growth and antiestrogen effects on h u m a n breast cancer cells. Cancer Res 45:27202724. 21. Shoemaker RH, Wolpert-DeFilippes MK, Kern DH, Lieber MM, Makuch RW, Melnick NR, Miller WT, Salmon SE, Simon RM, Venditti M], Von Hoff DD (1985): Application of a h u m a n tumor colony forming assay to new drug screening. Cancer Res 45:2145-2153. 22. Salmon SE, Hamburger AW, Soehnlen B, Durie BGM, Alberts DS, Moon TE (1978): Quantitatian of differential sensitivity of h u m a n tumor stem cells to anticancer drugs. N Engl J Med 298:1321-1327. 23. Salmon SE (1984): Human tumor colony assay and chemosensitivity testing. Cancer Treat Rep 68:117-125. 24. Nowell PC (1976): The clonal evolution of tumor cell population. Science 194:23-32.
Increasing investigative efforts have been focused on the application of in vitro clonogenic assays for human tumors. Several semisolid matrices have been used for purposes of immobilizing the clonogenic cells, and a variety of growth factors and environmental conditions have been studied to enhance tumor colony growth. Although there remain some technical problems with these assays, the methodology has proven to have broad applicability to preclinical and clinical studies of human cancer. This report reviews some features and areas of application of this assay methodolagy. The ability to combine studies of drug effect with cytogenetic studies on clonogenic cells suggests that such assays may prove useful for evaluation of the emergence of drug resistance by primary and metastatic cancers.
ABSTRACT:
INTRODUCTION In 1977, a bioassay system that facilitated in vitro colony formation by clonogenic tumor cells from biopsy samples of h u m a n solid tumors [1] was reported. Similar colony-forming assays also have been developed for studying the leukemias [2, 3]. A variety of morphologic, histochemical, immunologic, and cytogenetic techniques have been applied, which have established that the colonies that form in semisolid medium are representative of the malignant cells in the patient's tumor [4-6]. Such clonogenic cells are thought to be closely related to tumor stem cells in vivo [7]. It is the tumor stem cell p o p u l a t i o n that is capable of repeated cycles of proliferation characteristic of the self-renewing malignant cell population w i t h i n a tumor, and is responsible for local or metastatic recurrence after subcurative therapy. Clonogenic assay methodology has been applied to studies of cancer biology, cellular interaction and tumor immunology, cytogenetics, and preclinical drug development, as well as clinically in cancer diagnosis and the prediction of response to chemotherapy. Some of the features of clonogenic assays that have made them desirable for application in these areas are summarized in Table 1.
TECHNICAL C O N S I D E R A T I O N S
A wide variety of h u m a n tumor types can be grown in colony forming assays. However, not all specimens from all patients with any given type of cancer will grow colonies in vitro and in general, for most solid tumors, the cloning efficiencies are low (about 0.1%). Correcting for the percentage of non-neoplastic cells and dead From the Department of Internal Medicine,and the Arizona Cancer Center, Universityof ArizonaCollege of Medicine,Tucson,AZ. Address requests for reprints to Dr. Sydney E. Salmon, Arizona Cancer Center, University of Arizona College of Medicine, Tucson, A Z 85724. Received August 5, 1985; accepted August 19, 1985.
21 © 1986 Elsevier Science PublishingCo., Inc.
52VanderbiltAve., New York, NY 10017
Cancer Genet Cytogenet 19:21-28 (1986) 0165-4608/86/$03.50
22
S.E. Salmon Table 1
Features of clonogenic culture in semisolid medium
Applicable to direct study of growth by primary human tumors and metastases Only a small fraction of cells within a tumor give rise to tumor colony growth (<1%) Normal fibroblasts and bone marrow progenitors do not give rise to colonies (in the absence of conditioned medium) Clonal heterogeneity is preserved and can be quantitated Provides a quantitative approach to measure drug sensitivity of human tumors
cells in tumor s p e c i m e n s prior to culture initiation does yield a s o m e w h a t higher cloning efficiency, but it is still generally less than 1.0%. Whether the low cloning efficiencies are related to the presence of only a limited s u b p o p u l a t i o n of tumor cells with clonogenic or stem cell properties or to s u b o p t i m a l growth conditions, remains to be determined. Current information supports the s u b p o p u l a t i o n hypothesis. T u m o r specimens are u s u a l l y brought to the laboratory w i t h i n several hours of surgery, u n d e r aseptic conditions in a transport or complete growth medium. Enzymatic disaggregation techniques (generally e m p l o y i n g DNAse and collagenase and/or other enzymes) [8] have increased the yield of viable t u m o r cells from a number of solid tumor types, c o m p a r e d with m e c h a n i c a l disaggregation techniques. Technical details addressing m e d i u m selection and disaggregation are delineated elsewhere in this issue [9]. A variety of alterations in m e t h o d o l o g y have also been e x a m i n e d in order to increase the cloning efficiency for specific types of cancer. Thus far, these efforts have had only modest success. It is important to point out that a ntrmber of s p e c i m e n s received by the laboratory are inadequate in size (less than 1 g), some are cytology negative, while others are either c o n t a m i n a t e d or have not been adequately disaggregated u n d e r aseptic conditions. At the present time, only about 50% of t u m o r s p e c i m e n s y i e l d adequate in vitro growth (i.e., > 2 0 colonies/105 cells plated). A m o n g the various t u m o r types, excellent in vitro growth (>70% success) is observed in ovarian cancer, melanoma, and lung cancer. Such results have been obtained in our laboratory w i t h solid t u m o r specimens cultivated under the conditions of the standard Hamburger and Salmon assay, with only minor modifications in w h i c h DEAE-Dextran, calcium chloride, and L-asparagine have been deleted. Insulin is an i m p o r t a n t growth factor in this system; the addition of other growth factors i n c l u d i n g transferrin, hydrocortisone, thyroxine, and epidermal growth factor can m o d e s t l y potentiate colony formation in a majority of cases, but instances of modest inhibition of growth of some specimens have been observed with m a n y of these factors, as well. Use of a synthetic HITES m e d i u m has been reported to support growth of lung cancer cells with at least equal efficacy as serum-containing m e d i u m [10]. Breast cancer presents a n u m b e r of special problems, including inadequate s p e c i m e n size in T1 and m a n y T2 tumors, simultaneous need for estrogen receptor testing, and difficulties in obtaining adequate numbers of cells after disaggregation procedures [11]. Colorectal and head and neck cancers can also be grown in this system, but c o n t a m i n a t i o n is more frequently observed when the p r i m a r y t u m o r biopsy is submitted. L y m p h o m a s and m y e l o m a s generally do not grow well in such colony assays unless c o n d i t i o n e d m e d i u m factors (which are still undefined) are a d d e d [12]. Growth stimuli that have proved useful are summarized in Table 2. For specific tumor types, there are significant differences in growth as a function
Human Tumor Clonogenic Assays Table 2
23
Growth stimuli in semisolid culture
Mineral oil Balb-C conditioned medium (Myeloma) PHA-leukocyte conditioned medium (Leukemia) Defined growth factors and micronutrients (epidermal growth factor, hydrocortisone, transferrin, ascorbic acid, thiols, other hormones) (various tumors) Hypoxia (3%-5% environmental oxygen)
of the specimen source submitted (e.g., skin or lymph node). For some tumor types, progressive increases in cloning efficiency have been observed in vitro in association with tumor progression in vivo. Environmental oxygen concentration also has an effect on in vitro colony growth in semisolid medium. Studies with both normal hematopoietic colony-forming cells and human tumor cell lines have shown that relatively low oxygen concentrations (e.g., 3%-5% 02, normally found in tissues of the body) potentiate colony formation, compared with 20% 02. Although 20% 02 is present in the air (and in standard CO2 incubators), such high concentrations of 02 are not found in mixed venouscapillary blood within solid tumors. A soft agar colony assay developed by Courtenay and Mills [13] for human tumor xenograft culture in vitro utilizes such low 02 concentrations. In our own lab, we have found that 3% 02 does potentiate growth of some types of fresh human tumors. The specific semisolid medium selected for clonogenic assay varies depending on the specific needs of the investigator with respect to the required experimental versatility, as well as to optimize growth conditions for the specific tumor type tested. Frequently used semisolid culture supports are summarized in Table 3. Although agar has been most widely used for studies of solid tumors, agarose may be slightly preferable. It is more expensive but has a neutral charge, gels somewhat more slowly, and may permit slightly better growth of some type of solid tumors than agar. In some labs, however, no significant differences have been observed in growth support between these two semisolid supports. Two layer gel systems (prepared with agar or agarose) permit rigid physical separation between the softer plating layer (in which colony formation occurs) and the feeder layer within which nutrients or supporting cells can be placed. Feeder cell types, such as adherent macrophages, have also been adhered to the surface of the Petri dish, immobilized with the rigid feeder layer, and their influence on colony formation (via soluble mediators) examined [14]. Methylcellulose is not truly a gel; it is a highly viscous liquid, thus, permiting some cellular or colony movements within the matrix. Unfortunately, because of this property, it has not been used successfully with automated colony counters, as colony positions shift in methylcellulose with movements of a mechanical stage. Methylcellulose has been used largely for colony-forming studies of acute nonlymphocytic leukemias (ANLL) and by normal bone marrow progenitors, and has not been applied as successfully to solid tumors, (perhaps because endogenous fibroblasts can proliferate at the bottom of the dishes, and because of the aforementioned
Table 3 Semisolid culture systems Soft agar (0.3% top layer, 0.5% underlayer) Soft agarose (two layer) Methylcellulose Methylcellulose upper layer, agar underlayer
24
s.E. Salmon problem with automated colony counting). A two-layer system w h e r e i n t u m o r cells in methylcellulose are plated over agar has been e m p l o y e d for studies of bladder cancer [15]. This m e t h o d permits separation of a feeder cell layer w h i l e still facilitating simple recovery of intact colonies from the matrix for morphologic studies, as methylcellulose can be t h i n n e d by a d d i t i o n of water or buffered saline. We have observed that fibroblasts present in t u m o r cell suspensions can proliferate in cracks or irregularities at the a g a r - m e t h y l c e l l u l o s e interface. Agarose and agar are gels that cannot be dissolved by a d d i t i o n of water and that do not melt on slight increase in environmental temperature. However, i n d i v i d u a l colonies in agar can readily be p l u c k e d with a m i c r o p i p e t t e or m i c r o m a n i p u l a t o r and, additionally, simple techniques have been d e v e l o p e d for recovering intact colonies from agar for cytogenetic studies [16]. S i m p l e m e t h o d s also have been d e v e l o p e d for recovering the colonycontaining plating layer, preparing dried agar films on microscope slides and staining colonies for morphologic s t u d y [5]. These c o m m e n t s are not i n t e n d e d to imply that all technical problems with the clonogenic assay have been solved. However, these considerations plus the use of positive controls for drug assays and the use of an automated colony counter [17, 18] have i m p r o v e d quality control for in vitro clonogenic assays.
APPLICATIONS OF CLONOGENIC A S S A Y H u m a n t u m o r clonogenic assays are n o w a p p l i e d in a variety of preclinical and clinical areas of cancer research. Table 4 lists some of the current areas of application. Discussion of several areas w h e r e i n I have a p p l i e d this assay technique to studies of t u m o r heterogeneity and for clinical c h e m o s e n s i t i v i t y testing are prov i d e d b e l o w for illustrative purposes.
Tumor Heterogeneity A major area of current interest in t u m o r biology, one that m a y have clinical implications with respect to the metastatic process and the acquisition of drug resistance, is the p h e n o m e n o n of t u m o r heterogeneity. Heterogeneity in behavior of clonogenic tumor cells can readily be observed in the clouogenic assay w i t h respect to comparative differences of in vitro behavior between histologically similar tumors from different patients in drug sensitivity or resistance to the same agents. Additionally, differences have been observed among different metastases from the same patient,
Table 4
Some current areas of a p p l i c a t i o n of h u m a n t u m o r colony assays
Biology of growth of human neoplasms Regulation of tumor growth: Stromal cell-tumor cell interactions, growth factors Kinetics of clonogenic tumor cells and the self-renewal process Studies of heterogeneity of clonogenic tumor cells Purification of clonogenic tumor cells and antigenic analysis Cytogenetic analysis of clonogenic tumor cells New drug screening and preclinical drug development Cancer diagnosis Studies of radiation therapy and hyperthermia Studies of effects of biological response modifiers (interferons, tumor necrosis factors, interleukins, retinoids) Mechanisms of drug resistance Clinical chemosensitivity studies
Human Tumor Clonogenic Assays
25
and among colony-forming response of cells within a single biopsy specimen. Several laboratories have recently noted differences in sensitivity in vitro between primary tumors and their metastases [19]. These findings, as well as morphologic and cytogenetic observations, have been accumulated and indicate that the clonogenic assay systems can be applied in a variety of ways to assess tumor heterogeneity. Recently, we have applied the clonogenic assay system to determine if heterogeneity was seen with respect to estrogen receptor expression in breast cancer [20]. In these studies, the estrogen receptor (ER)-positive MCF-7 breast cancer cell line was grown in soft agar, and ER was evaluated immunohistochemically using specific monoclonal antibodies to ER. Our initial question was whether ER was expressed clonally by MCF-7 cells with some colonies staining positively and others negatively, or if it expressed as a differentiation antigen with varying expression in all colonies as a function of time and colony size. We observed significant and reproducible heterogeneity in ER staining of MCF-7 cells both within and between colonies. These differences were definitely related to colony size and the duration of culture, and truly ER-negative clones were only observed up through the small cluster stage of colony growth. Of interest, addition of the antiestrogen, tamoxifen, at doses that would cause significant reduction in the number of tumor colonies also reduced the degree of expression of ER (although a small number of truly ER-negative colonies was observed in the presence of tamoxifen). These findings are most consistent with a model of breast cancer growth wherein ER positivity is not expressed at the level of the tumor stem cell, but rather is expressed increasingly with increasing tumor colony growth and differentiation of progenitor cells. Tumor stem cells represent the key subpopulation in a tumor that has the ability to self-renew and maintain a tumor stem cell pool, which is responsible for tumor recurrence following subcurative therapy. Both tumor stem cells and some of their differentiating progeny (transit cells) share the property of clonogenicity, but the replicative capacity of the transit cells is limited with increasing differentiation. Based on our findings with respect to estrogen receptor expression, endocrine therapy such as the use of tamoxifen would appear capable of suppressing tumor growth, but would be incapable of eradicating tumor stem cells in breast cancer. In that setting, long-term therapy with this agent would be essential. Figure 1 depicts the apparent level of action of endocrine therapy in a stem cell model of breast cancer growth.
Figure 1 A proliferation-differentiation model for tumor growth based on the tumor stem cell concept. Based on the studies of Kodama et al. [20], tamoxifen and other endocrine active agents would appear to act on a differentiating transit population of tumor cells and beyond the level of the tumor stem cell.
Stem Cell (Proliferate) ll,,,iolnnNnm°lgln
Jnlq
Transi ,~,, Celltional ........................ ~ ~ ~ (Proliferate) ,,,H°llllnnm,o..mlllm.
EndCell
.............indocrine Therapy
~~~',
26
s.E. Salmon
Chemosensitivity Testing One major application of in vitro clonogenic assay has been for chemosensitivity testing. These assays have been applied preclinically in the area of new drug development [21] and for clinical testing for anticancer drug selection for individual patients. Important to such testing has been the development of a pharmacologically based testing system, wherein the in vitro drug concentrations tested are relevant to those that can be obtained in vivo. The initial clinical correlations of in vitro drug sensitivity testing were reported in 1978 for a limited number of patients with ovarian cancer or multiple myeloma [22], and included predominately retrospectively drawn correlations. The results indicated good agreement between in vitro sensitivity and clinical tumor regression, and excellent agreement between in vitro and in vivo resistance. More recently, prospective analyses have been carried out by a number of investigative groups and, in general, have indicated good correlation between in vitro sensitivity or resistance and the clinical success or failure of specific treatments for individual patients [23]. Technical factors including poor growth of some tumor types, lack of effective anticancer drugs for many tumor types, and other factors, however, have thus far limited the clinical applicability of such assays primarily to a few tumor types, such as ovarian cancer. For preclinical discovery of new anticancer agents, clonogenic assays appear to have significant utility [21].
Potential Applications in Cytogenetic Analysis Cytogenetic analysis can provide unique insights with respect to clonal heterogeneity in human tumors, and this problem is addressed extensively elsewhere in this issue. Application of cytogenetic analysis to cells cultivated in vitro in clonogenic assay has the potential to be quite useful in this regard, as shown by Trent (this issue) and others. Some of the advantages of clonogenic assay for cytogenetic analysis of human solid tumors are listed in Table 5. One of the major concepts that arose from the study of cytogenetics of human neoplasms is that of clonal progression [24]. Studies of multiple biopsies of different metastases within individual patients, as well as serial biopsy studies of such patients older than 1-2 years, are quite feasible with clonogenic assay techniques and could be quite interesting with respect to the clonal progression theory. An area of particular interest, which is potentially exploitable through use of such clonogenic assay systems, is the interrelation of results of in vitro drug sensitivity and chromosomal results. This approach could prove useful in the identification of chromosomal abnormalities associated with inherent or acquired drug resistance. Such analyses could provide the basis for more detailed studies of molecular mechanisms of drug resistance and provide new insights and new approaches to cancer treatment.
Table 5 Advantages of semisolid media for chromosome cultures of human solid tumors Growth of normal cells is suppressed Mitotic index of tumor cells within colonies is very high (>25%) Gel matrix immobilizes clonogenic cells and permits inter- and intraclonal chromosome analysis Assay permits chromosome analysis on surviving clonogenic cells after drug exposure Technique compatible with detailed banding analysis
Human Tumor C l o n o g e n i c Assays
27
SUMMARY In vitro c l o n o g e n i c assays for h u m a n s p e c i m e n s h a v e p r o v e n t h e i r b r o a d a p p l i c a tion to the s t u d y of h u m a n c a n c e r b i o l o g y a n d to c h e m o s e n s i t i v i t y testing. The combination of e x p e r i m e n t a l v e r s a t i l i t y a n d a p p l i c a b i l i t y to fresh t u m o r s p e c i m e n s makes it p a r t i c u l a r l y attractive. F u r t h e r i m p r o v e m e n t in cell disaggregation and tissue culture t e c h n i q u e s , as w e l l as a d d i t i o n a l k n o w l e d g e of d e f i n e d g r o w t h factor requirements for h u m a n t u m o r s , are h i g h p r i o r i t y areas for further d e v e l o p m e n t . It seems likely that s u c h assays w i l l h a v e e v e r - i n c r e a s i n g a p p l i c a t i o n s to f u n d a m e n t a l and clinical areas of c a n c e r research. Supported in part by Grants CA-17094, CA-21389, and CA-23074 from the National Institutes of Health, Department of Health and Human Services, Bethesda, MD.
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19. Schlag P, Schreml W (1982): Heterogeneity in growth pattern and drug sensitivity of primary tumor and metastases in the h u m a n tumor colony forming assay. Cancer Res 42:4086-4089. 20. Kodama F, Greene GL, Salmon SE (1985): Relation of estrogen receptor expression to clonal growth and antiestrogen effects on h u m a n breast cancer cells. Cancer Res 45:27202724. 21. Shoemaker RH, Wolpert-DeFilippes MK, Kern DH, Lieber MM, Makuch RW, Melnick NR, Miller WT, Salmon SE, Simon RM, Venditti M], Von Hoff DD (1985): Application of a h u m a n tumor colony forming assay to new drug screening. Cancer Res 45:2145-2153. 22. Salmon SE, Hamburger AW, Soehnlen B, Durie BGM, Alberts DS, Moon TE (1978): Quantitatian of differential sensitivity of h u m a n tumor stem cells to anticancer drugs. N Engl J Med 298:1321-1327. 23. Salmon SE (1984): Human tumor colony assay and chemosensitivity testing. Cancer Treat Rep 68:117-125. 24. Nowell PC (1976): The clonal evolution of tumor cell population. Science 194:23-32.