1263.Full.Pdf

[CANCER RESEARCH 46, 1263-1274, March 1986]

Drug and Radiation Sensitivity Measurements of Successful Primary Monolayer Culturing of Human Tumor Cells Using Cell-adhesive Matrix and Supplemented Medium1

Fraser L. Baker,2 Gary Spitzer,3 Jaffer A. Ajani, William A. Brock, John Lukeman, Sen Pathak, Barbara Tomasovic, Diva Thielvoldt, Marcia Williams, Charlotte Vines, and Philip Tofilon

Departments of Hematology [G. S., B. T., D. T.], Gastroenterology [J. A. A.], Pathology [J. L], Genetics [S. P.], Experimental Radiotherapy [W. A. B., M. W., C. V., P. T.], and the Division ol Medicine, The University of Texas at Houston, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030

ABSTRACT tumor cell culturing is the agar suspension culture method (2). The main feature of this method is its selection of transformed The limitations of the agar suspension culture method for cells for growth (3), although agar suspension cultures also primary culturing of human tumor cells prompted development support growth of normal hematopoietic cells (4), benign tumors of a monolayer system optimized for cell adhesion and growth. (5), and anchorage-dependent cells if the medium is supple This method grew 83% of fresh human tumor cell biopsy speci mented with high serum levels (6) or transforming growth factor mens, cultured and not contaminated, from a heterogeneous (7). group of 396 tumors including lung cancer (93 of 114, 82%); Culturing primary human tumor cells in soft agar has problems. melanoma (54 of 72, 75%); sarcoma (46 of 59, 78%); breast More than 50% of tumors cannot be cultured adequately to cancer (35 of 39, 90%); ovarian cancer (16 of 21, 76%); and a enable routine testing of drug sensitivity (8, 9). Those specimens miscellaneous group consisting of gastrointestinal, genitourinary, that can be tested for drug sensitivity routinely show low cloning mesothelioma, and unknown primaries (78 of 91, 86%). Cell efficiencies (less than 0.1 %) (10,11 ), unusual cisplatin resistance growth was characterized morphologically with Papanicolaou- in ovarian cancer (12), and unusual radiation survival curves (13, stained coverslip cultures and cytogenetically with Giemsa- 14). These findings may represent some of the true biology of stained metaphase spreads. Morphological features such as human tumors such as low frequency of clonogenic cells in nuclear pleomorphism, chromatin condensation, basophilic cy human tumors (15) and cell heterogeneity with tumors containing toplasm, and melanin pigmentation were routinely seen. Aneu- cells of varying drug sensitivities. Alternatively, the culture con ploid metaphases were seen in 90% of Ã©valuablecultures, with ditions may be so inadequate and subject to variability that the 15 of 28 showing 70% or more aneuploid metaphases. Colony- results are artifactual. Because of the technical difficulties asso forming efficiency ranged between 0.01 and 1% of viable tumor ciated with embedding the cells in agar, it is difficult to routinely cells, with a median efficiency of 0.2%. This culture system uses prepare cultures for morphologic, cytogenetic and histochemical a low inoculum of 25,000 viable cells per well which permitted analysis. The agar suspension culture method also limits the chemosensitivity testing of nine drugs at four doses in duplicate periods of drug exposure either to a 1-hr preincubation, which is from 2.2 x 106 viable tumor cells and radiation sensitivity testing frequently accompanied by severe cell aggregation, or to contin at five doses in quadruplicate from 0.6 x 106 cells. Cultures were uous exposure. analyzed for survival by computerized image analysis of crystal Recognizing that cell attachment plays a major role in growth violet-stained cells. Drug sensitivity studies showed variability in of normal and malignant epithelial and mesenchymal cell lines sensitivity and in survival curve shape with exponential cell killing (16) and that the relationship between growth in agar suspension for cisplatin, Adriamycin, and etoposide, and shouldered survival culture and cancer is not absolute (17,18,19), we developed a curves for 5-fluorouracil frequently seen. Radiation sensitivity liquid monolayer culture system optimized for cell adhesion and studies also showed variability in both sensitivity and survival cell growth. curve shape. Many cultures showed exponential cell killing, Cell adhesion was optimized through the use of culture sur although others had shouldered survival curves. This method for faces prepared from CAM,4 a preparation composed in part of growing cells from primary human biopsy specimens is more fibronectin and fibrinopeptides. Cell growth was optimized efficient than the agar culture method, enables easier and better through the use of hormone- and growth factor-supplemented biological analysis of the actual cells grown, and permits im culture medium. proved characterization of drug and radiation survival curves. We here report the successful growth of adhesive cells from biopsy specimens of human tumors. We document the malignant INTRODUCTION origin of cell growth, improved CFEs, and excellent morphologi cal and cytogenetic details. Survival curves determined for radia The method currently in use for quantitative primary human tion and several common chemotherapy drugs, quantitated by automated image analysis of crystal violet-stained cultures, showed sensitivities and shapes expected for human tumor cells Received 4/29/85; revised 9/16/85,11/22/85; accepted 11/26/8S. 'This work was supported by Contract JMV:bg 11783 from LifeTrac, Ltd., in vitro. Irvine, CA. This work was presented at the 76th Meeting of the American Associ ation for Cancer Research at Houston, TX, 1985 (1). "The abbreviations used are: CAM, cell-adhesive matrix; CFE, colony-forming 2 Present address: LifeTrac, 18300 Von Kamen Avenue, Suite 930, Irvine, CA efficiency; GM-CFC, granulocyte-macrophage colony-forming cell; EGF, epidermal 92715. growth factor; IA, integrated absorbance; ICÂ»,concentration that reduced survival 3 To whom requests for reprints should be addressed. to 50%; CFC, colony-forming cell.

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MATERIALS AND METHODS 95% ethanol after 24-h incubation to provide a record of the starting cell population; one well with culture medium to provide two control cultures Materials per plate; one well with 5 MCi of tritiated thymidine per ml to provide a record of the background cell population; and one well cultured without Cell-adhesive matrix was from Lifetrac, Irvine, CA. Culture medium was Ham's F-12 (K. C. Biological, Lenexa, KS) with: 4-(2-hydroxyethyl)- added EGF to determine EGF sensitivity. Drug Responses. Drug survival studies were carried out in the 1-piperazineethanesulfonic acid buffer (2.7 mg/ml) (Sigma, St. Louis, remaining four columns of four wells. Four drugs were tested each at MO); 10% swine serum (J. R. Scientific, Woodland, CA); and penicillin- four concentrations over a narrow concentration range which bracketed streptomycin (100 units/ml) (GIBCO, Grand Island, NY) and supple the ICso of GM-CFC (23). The drugs were added to duplicate cultures mented with hormones (20, 21): transferrin (10 Mg/ml); hydrocortisone after 24 h of incubation and removed after 6 days of incubation, resulting (0.5 Mg/ml); EGF (5 ng/ml); and insulin (5 Mg/ml) (Collaborative Research, in a 5-day exposure period. After an additional 7 days of incubation (13- Lexington, MA). Attachment medium was the same formulation as the day incubation period), the cultures were stained, and survival at each culture medium plus 0.6% Methylcellulose 4000 (Fisher Scientific, Hous dose was determined quantitatively by image analysis. ton, TX). Enzyme disaggregation medium was F-12 with 10% fetal calf Radiation Response. Each of the 24 wells of multiwell plates was serum, 0.075% collagenase type III (Cooper Biomedicai, Malvern, PA), and 0.005% DNase (Sigma). Phosphate-buffered saline was Dulbecco's inoculated with 25,000 cells. A 3-mm lead shield was made that permitted a 250-kV X-ray source to irradiate a single column of four wells, thus (GIBCO). Culture vessels used were 24-well and 6-well multiwell plates permitting quadruplicate cultures, consisting of a control set and five (Costar, Cambridge, MA). Twenty-two-mm-square polyester coverslips other sets each receiving doses of 1.0, 2.0, 3.0, 4.0, and 6.0 Gy. The were from Raylabcon, Shirley, NY. cultures were irradiated after they had been washed and refed after 24 h of incubation. After incubation for a total of 13 days, the cultures were Method for Primary Human Tumor Cell Culture

Culture Surface Preparation. CAM is a complex containing 50 to 70% fibronectin and fibrinopeptides supplied in solution at approximately 0.25 mg of protein per ml. Culture surfaces were prepared by coating the bottom of tissue culture dishes or wells by sequentially dispensing and aspirating the solution leaving a thin film of CAM. To produce an even coating in the small diameter wells of 24-well multiwell plates, the plates were cooled to the dew point (5 to 15Â°C,depending on the relative humidity) during coating. No precautions were necessary for coating larger 35- and 60-mm dishes. Plastic coverslips were coated 100 at a time in a small chamber and transferred to 6-well multiwell plates for use. After coating, the dishes/wells were covered, and the film of CAM was allowed to dry overnight before use. The culture surfaces were clear, transparent, showed no residue, and were stable at room temper ature for at least 3 mo. Cell Preparation. Biopsy specimens of human solid tumors were obtained from the Department of Pathology of the University of Texas, M. D. Anderson Hospital and Tumor Institute at Houston, during routine diagnostic procedures, transported to the laboratory, and placed in tissue culture medium. Effusions were anticoagulated with preservative-free heparin (10 units/ml) immediately after aspiration. Solid specimens were minced with scalpels to 1-mm pieces. The solid- and fluid-derived tissues were disaggregated to single cells by incubating in enzyme disaggrega tion medium for 16 h with constant stirring. Cell Culture Inoculation. The yield of trypan blue-viable cells was determined by a hemocytometer count of trypan blue-negative nucleated cells larger than 10 Mm (excluding lymphocytes, granulocytes, and mes- othelial cells) with a microscope fitted with a phase-gradient imaging apparatus (22) at a magnification of x 400. The cell suspension was diluted with attachment medium to 25,000 cells/ml, and 24-well plates were inoculated with: (a) a cell inoculum titration consisting of 25,000, 12,500, 6,250, and 3,125 cells per well in the first column of four wells; and (b) 25,000 cells in each of the 20 remaining wells of each plate. Adherent Cell Harvest. After 24 h of incubation, the attachment medium was aspirated, and the adherent cells were washed with phos phate-buffered saline and refed with culture medium, drugs, or experi mental conditions as described below. The number of adherent cells harvested from the inoculum was counted using an inverted microscope fitted with a phase-gradient apparatus (22) at magnifications of x 100 or x 200. The yield of adherent cells per well was calculated by multiplying the average field count (N = 5) by 82 or 330 for x 100 or x 200 Fig. 1. A and 8, ovarian and lung tumor cells, respectively, cultured over magnification, respectively. surfaces consisting of tissue culture plastic (i), agarose (2), and agarose plus 8% (3). 16% (4),32% (5),and 50% (6) CAM. These photographs show no growth over Controls. Columns 1 and 2 were reserved for control and EGF studies. Column 1 consisted of the inoculum titration described in "Cell Culture the agarose layer (2) and increased growth following addition of CAM to the agarose layer (3 to 6). Growth over CAM was denser than growth over tissue Inoculation." The second 4-well column consisted of: one well fixed with culture plastic (7).

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Fig. 2. A and B, Day 1 and Day 13 photographs, respectively. Cultures of melanoma. The Day 1 culture showed pleomorphic cells with dendritic-like cytoplasmic processes. Two of the three cells shown were broadly spread. Only one of the two spread cells was pigmented. The Day 13 culture showed pleomorphic fusiform cells with elongated cytoplasmic processes that contained melanin granules. Nuclei were oval and contained one to three small round nucleoli, x 400.

0525alUJ liter tubs of reagent to reduce intrates! staining variability. QuantitÃ¤ten of Cell Growth and Survival. A Nikon/Joyce-Loeble Magiscan2 image analysis system was used to determine the absor- 20 bance of crystal violet-stained cultures integrated over the surface of the 85_JI monolayer cultures. The resulting parameter, IA, reached maximum 15* values exceeding 3000 units relative to the Day 1 fixed cultures. Cultures with an IA of 300 units or less above background were not Ã©valuable. 10UJOPERCENl Growth in such cultures was approximately 15 to 20 colonies per control well or 0.06 to 0.08% colony-forming efficiency. Overall variability of the culture system appears to be 3 to 4%, as an analysis of a 24-well plate Oen---Â»â€”Median-||l in which all wells were treated as controls showed a mean IA of 2295 units and a SD of 84 units, resulting in a coefficient of variation of 3.7%. n0 -n n Survival was calculated as the fraction of IA of treated cultures relative to the IA of control cultures. We have reported that the surviving fraction 5 10 15 20 of an established cell line, measured by a standard clonogenic assay, ADHERENT CELLS was in close agreement to results obtained with this method over the x 103 CELLS/WELL radiation dose range of 0 to 6 Gy (24). It was also found that radiation- Fig. 3. Histogram of the yield of adherent cells per 16-mm well in 80 consecutive induced growth delay and growth kinetics were not significantly different assays. from control across the dose range used. Even though this assay is not a direct clonogenic assay, the results suggest that survival calculated stained, and survival at each dose was determined by quantitative image from IA measurements closely resembles the true clonogenic potential analysis. The data were then computer fitted to a linear quadratic model of the irradiated cells. for radiation survival, using least-squares regression analysis. Morphology. Cultures for morphological analysis were set up on two Incubation. Cultures were incubated in a humidified atmosphere of 22-mm plastic coverslips coated with CAM prior to use. The coverslips 5% CO2 in air for 13 days. All cultures were refed by 100% medium were placed in 35-mm wells which were inoculated with 100,000 cells in exchange after 6 days of incubation. 4 ml. After 20 to 24 h of incubation, a coverslip was removed from a Fixing and Staining. The cultures were washed with phosphate- well, washed, clipped to a microscope slide, and stored in 95% ethanol. buffered saline, fixed in 70% ethanol for 20 min, and stained with 0.5% After 2 wk of incubation, the second coverslip was removed, washed, crystal violet. The multiwell plates were processed four at a time in 2- and clipped to the same microscope slide. The Day 1 and Day 13

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Fig. 4. Photographs of typical cell dose titrations of 25,000 (A), 12,500 (B), 6,250 (C),and 3,125 (D) viable cells per well, showing merged colonies at the 25,000-cell inoculum level, decreasing cell density with decreasing inoculum, and individualcolonies at the 6,250- and 3,125-cell inocula levels.

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40 r Improvement in growth was also shown in 47 of 115 primary human tumor cell cultures by the addition of EGF to the culture O medium. This effect was primarily on colony size with increases 30 from 50 to 200 cells routinely seen with no change in colony CD OC number (25). This high response to EGF was frequently observed O

O 3.1 6.2 12.5 25 yield of adherent cells from an inoculum of 25,000 viable cells over 80 consecutive assays is shown in Fig. 3. The yield of INOCULUM CELLS/WELL X 103 adherent cells increased linearly with increased cell inoculum. Fig. 5. Relationship between cell growth and inoculum showing linearity be The yield of adherent cells was not correlated with the content tween growth and the inoculum titration over the range used in the assay for three assays (A, O, D). Cell growth was evaluated by measuringthe IA of crystal violet- of tumor cells in the specimen. stained cultures using a Nikon/Joyce-LoebleMagiscan2 image analysissystem. CFE. Inoculating 25,000 viable cells into a 16-mm well gener ally resulted in merged colonies so that individual colonies were not scorable. To evaluate CFE, the colony count was done at uj 20 the dilution of the cell inoculum titration (25,000, 12,500, 6,250, o IM and 3,125 cells per well) at which individual colonies were EL W 15 discernible. Fig. 4 shows a typical cell inoculum titration after 13 _ days of incubation, illustrating merged colonies with the highest Â»â€”Median i 10 inoculum, a decrease in cell density over the culture surface with decreasing inoculum, and very large individual colonies at the lower inocula. CFE (number of colonies per 25,000 viable cells) eÂ» was calculated by multiplying the actual colony count by a factor u depending on the dilution at which the count was made. The IE 0 2O 40 60 80 100 120 140 160 180 >200 number of colonies per 25,000 viable cells varied from 5 to over Â£ 200, reflecting a CFE of 0.01 to 1%. The distribution of CFE is COLONIES PER 25.000 VIABLE CELLS shown in Fig. 5. Fig. 6. Histogram of colonies (>50 cells) formed from an inoculum of 25,000 Colony size, an evaluation much more appreciated in mono- viable cells. layer culture, routinely exceeded 100 cells and often exceeded 250 cells. Occasionally, colony size was small, 8 to 32 cells per coverslip cultures were stained with Papanicolaou stain. Cytogenetic Analysis. Cultures for cytogenetic analysis were also colony. set up on CAM-coated 22-mm coverslips. After 11 days of incubation, Because we were unable to count colonies over the cell the medium was exchanged to increase the harvest of metaphase cells inoculum titration at the inoculum used in this monolayer assay, 2 days subsequent. After 13 days of incubation, cultures were harvested we analyzed the linearity of cell growth over the dose response in situ for cells in metaphase by incubating with 1% hypotonie citrate for using the Magiscan2 image analysis system. With this instrument 20 min, fixing with 1:3 acetic acid:methanol for 10 min, and staining with we quantitated the IA of crystal violet-stained cultures over the 4% Giemsa stain for 8 min. The stained coverslip cultures were washed cell-dose titration and found it to be linear as shown by three with water, air dried, and mounted with cells facing up on a microscope cases in Fig. 6. slide. The metaphase spreads were examined, and the chromosome Morphology. Cells spread very broadly over culture surfaces constitution was determined by counting the number of chromosomes at x 1000 magnification. Banding studies were not attempted. Chro of CAM and showed morphologies that varied from polygonal to mosome analyses were not done on patient specimens. fusiform, often with long cytoplasmic processes. Fig. 2 shows a melanoma culture, as evidenced by the pre- dominence of brown pigment granules in the cytoplasm of the RESULTS Day 1 culture and in the majority of cells in the Day 13 culture. Melanoma cells grew in a predominantly fusiform morphology, Effect of CAM and EGF. The ability of CAM to support cell with round to oval nuclei, multiple small irregular nucleoli, and adhesion and growth was demonstrated by comparing mono- pale-staining chromatin. The Day 1 cell and the cells that formed layer cell growth over culture surfaces prepared from agarose in culture after 13 days of incubation appeared similar in terms with and without CAM. Agarose culture surfaces are nonadhe- of overall morphology, nuclear structure, and pigmentation. sive. Cells did not attach to agarose and were lost from the A sarcoma is shown in Fig. 7. The Day 1 culture showed large cultures. Including CAM in the agarose culture surface resulted fusiform cells with intense cytoplasmic granularity, an irregular in cell adhesion and monolayer cell growth that routinely ex nucleus with prominent nucleoli, and coarse chromatin. The Day ceeded that over tissue culture plastic in over 20 primary human 13 culture grew colonies of similar cells. tumor cell cultures (Fig. 1). A lung adenocarcinoma culture is shown in Fig. 8. The Day 1

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Fig. 7. A and B, photographs of Day 1 and Day 13 cultures, respectively, of a sarcoma. The Day 1 culture showed a large fusiform cell with an irregular nucleus and many cytoplasmic organelles. The Day 13 culture showed growth of large fusiform cells with irregular nuclei and many prominent cytoplasmic organelles. x 400.

culture showed polygonal and broadly spread cells with large, individual chromatids. The renal cell metaphase showed 52 round nuclei; prominent round to irregular nucleoli; thick nuclear chromosomes, and the lung cancer culture showed 71 chromo rims; and clumped, densely stained chromatin. The Day 13 somes coated with amorphous substance, some of which had culture shows polygonal cells featuring nuclei similar to that of spiral structures. the Day 1 cells. Twenty-eight samples were processed for chromosome count Day 1 and Day 13 cultures of a lung squamous cell cancer are ing. Aneuploid metaphases were observed in 90% of the cultures shown in Fig. 9. The Day 1 culture shows a fusiform and a evaluated. Fifteen assays showed greater than 90% aneuploid polygonal cell, each with a similar elongated nucleus, two small metaphases, 11 assays showed 10 to 30% diploid metaphases, round nucleoli, thin nuclear rims, and pale chromatin. The culture and 2 assays showed a predominance of diploid normal-appear grew large pleomorphic cells with pleomorphic elongated nuclei; ing metaphases. Pathological analysis of the biopsy specimen multiple, often irregular nucleoli; and pale-staining, slightly gran for one of the diploid assays showed a mixed tumor composed ular chromatin. of an ovarian fibroma and adenocarcinoma. The other predomi Cell cultures from Days 1, 5, 9, and 13 from a fluid specimen nantly diploid culture was a melanoma that rapidly grew in the from a transitional cell cancer are shown in Fig. 10. The Day 1 assay (forming 85 cell colonies after 10 days of incubation) and culture showed the cell population to be composed predomi showed resistance to drugs. The patient experienced progres nantly of well-spread, round cells with a foamy cytoplasm and a sive disease and died 3 mo after the assay. small nucleus that were considered histiocytes. Elongated cells An additional 11 randomly selected cultures were processed with long cytoplasmic processes and large nuclearcytoplasmic for evaluation of sister chromatid exchange, and simultaneously ratio were seen and were considered as tumor cells. The time chromosome number was also determined. Of 200 metaphases sequence of photographs showed rounding up and diminished examined in this group, extreme variation in chromosome num content of histiocytes, increased content of elongated cells, and ber was seen. Only two diploid metaphases were seen in this their development of epithelial morphology by the Day 13 culture. study (1%). Cytogenetics. Thirty-eight % of cultures yielded Ã©valuable Radiation Survival Curves. Radiation survival curves were metaphases. Photographs of Giemsa-stained metaphase constructed by plotting the log of the survival versus the radiation spreads harvested from cultures of colon, ovarian, renal, and dose. Survival curves from a melanoma and a sarcoma are lung tumor cells are shown in Fig. 11. The colon metaphase shown in Fig. 12. Variation in sensitivity and in the degree of showed 69 chromosomes. The ovarian metaphase had a very shouldering in the survival curves between cultures was ob high number of chromosomes, with chromosome fragments and served. We have analyzed over 40 irradiated cultures thus far,

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Fig. 8. A and B, photographs of Day 1 and Day 13 cultures, respectively, of lung adenocarcinoma. The Day 1 culture showed oval cells with thin, occasionally vacuolated cytoplasm and round nuclei with one or two prominent nucleoli. The Day 13 culture showed numerous similarly appearing cells with prominent single nucleoli. Occasional giant multinucleated cells were seen, x 400. and the range of sensitivities varies but falls within the range from 0.03 to 0.7 ^g/ml for cisplatin, 0.0014 to 0.028 /ug/ml for expected for cell cultures. The calculated range of survival after Adriamycin, 0.025 to 0.3 yug/ml for etoposide, and 0.11 to 0.6 2 Gy was from 20 to almost 90%. ing/ml for 5-fluorouracil. Although a constant inoculum of 25,000 cells was used, the Performance. The performance rate of a drug assay based yield of adherent cells varied between assays as did the final cell on this culture system was determined. Of 475 specimens re density in the control cultures. High-density cultures may be ceived by the laboratory, 328 drug sensitivity reports were made subject to inhibition of cell growth by density-dependent inhibi reflecting an overall performance of 69% (Table 1). The perform tion, to depleted medium, or both. To study whether variation in ance rate according to the number of assays actually set up and the final cell density affected the measurement of radiation not contaminated reached 83% (Table 2). This performance was sensitivity, we determined radiation survival curves at four cell fairly consistent over a spectrum of tumor types (Table 2). inocula: 50,000; 25,000; 12,500; and 6,250 cells per well. The Hematopoietic neoplasms are nonadherent and are lost from the survival curves obtained at the lower inocula showed similar culture system. radiation sensitivities; however, cultures with the highest inocu lum showed decreased sensitivity and an increased shoulder DISCUSSION (Fig. 13). These results show that it is necessary to use lower cell inocula numbers so that the cells in the unirradiated controls We have described a monolayer cell culture system for adher are still growing at the end of the culture period. ent cells that is 70 to 80% efficient in growing colonies from Drug Survival Curves. Drug survival curves were constructed tumors of diverse histology and origin. The CFE (0.01 to 1%; by plotting the log of the survival versus the drug dose. Examples median, 0.2%) of this system suggests improvement over the from melanoma and squamous cell lung cancer biopsy speci Hamburger and Salmon (2) agar suspension culture method, mens with different sensitivities are shown for each drug (cispla- although agar suspension culture shows improved efficiency tin, Adriamycin, etoposide, and 5-fluorouracil) in Fig. 14. Expo when cultured at low densities (26) or with the modifications nential responses were observed for cisplatin, Adriamycin, and described by Courtney (27). Colony size routinely exceeded 100 etoposide, and a shouldered response was observed for 5- cells and frequently exceeded 250 cells after 13 days of incu fluorouracil in the sensitive assay. A shouldered response was bation. observed for all the other drugs in the resistant assay. In over The morphology of the cells grown in this culture system 110 to 170 responsive drug sensitivity assays, the IC50 varied varies from polygonal to fusiform. The expression of fusiform

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Fig. 9. A and B. photographs of Day 1 and Day 13 cultures, respectively, of lung squamous carcinoma. The Day 1 culture showed well-spread cells with elongation of the cytoplasm that resembled fibrous cells. Nuclei were oval with multiple small nucleoli. The Day 13 culture showed growth of the elongated fibrous-like cells, which had delicate thin cytoplasm and pleomorphic oval nuclei with up to five irregular nucleoli, x 400. morphology in these primary human tumor cell cultures raises grated over the culture surface. This end point, IA, has been the concern that growth of fibroblasts may occur. However, the correlated with the traditional colony assay by us (24) and others finding of morphological features of cancer, such as pigment in (29). The plating of nonviable aggregates and debris does not melanoma cells, large nucleus:cytoplasm ratio, nuclear pleo- contribute error in IA as they do not attach and are removed morphism, prominent nucleoli, basophilic cytoplasm, and cording from the culture during medium exchange. Error in this end point (28) in many cultures showing fusiform cell morphology, suggests due to the plating of viable aggregates of CFCs appears minimal, that any potential contamination of the cultures with fibroblast as an aggregate of CFCs grows into a larger colony that contrib growth is small. utes proportionally more to the IA of the culture. This contrasts To ascertain the cancer of the cells grown, ploidy was deter with the agar suspension clonogenic assay, in which a colony mined by counting the number of Giemsa-stained chromosomes derived from an aggregate of CFCs is counted as one regardless in expanded metaphases. The distribution of chromosome num of its size. This error, inherent in the agar suspension clonogenic ber was generally broad, with aneuploid metaphases seen in assay, places a stringent need for the inoculum into the agar 95% of Ã©valuablecultures. Frequently, both hypodiploid and culture system to be a pure single cell suspension, a criterion hyperdiploid populations were seen in the same culture, sug that may not be attainable due to the propensity for tumor cells gesting a high incidence of aneuploidy and heterogeneous DMA to aggregate. Aggregates of CFCs would be expected to pro populations in the cultured cells. In a study determining the duce plateaus in survival curves and limit the dose response of frequency of sister chromatid exchange in primary human tumor the agar suspension clonogenic assay, particularly in the mea cells cultured in this system, 11 randomly selected cultures were surement of radiation sensitivity. evaluated for chromosome number, and in over 200 metaphases The adhesive tumor cell culture system produced a wide range examined, diploid metaphases were rarely seen. In a total of 39 of radiation sensitivities, all in the range expected for in vitro cultures examined for chromosome number, only 2 (5%) were cultures. The ability of the adhesive tumor cell culture system to predominantly diploid. These results suggest that, when cells produce classical radiation survival curves suggests that the from biopsies of malignant tumors are cultured in this culture system may have potential usefulness in determining the sensi system, predominantly malignant cells grow and that normal cell tivity of human tumors to radiation. Reviews of published radia growth, if it occurs, is a minority proportion of the culture. tion survival curves of established human tumor cell lines showed The end point for the measurement of growth in our method that there are significant differences in survival curves at 2.0 Gy, of analysis is the absorbance of crystal violet-stained cells inte but not in D0 (30, 31). Small differences in survival at 2.0 Gy

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Fig. 10. A to D, photographs of Day 1, 5, 9, and 13 cultures, respectively, of a fluid specimen from a transitional cell cancer. The Day 1 culture showed two cell populations. The predominant population was round, was very well spread, had a foamy cytoplasm, and had a small bean-shaped nucleus. This population resembled histiocytes. The minor population was very elongated, having a prominent nucleus and nucleolus. The Day 5 culture showed decreased numbers and degree of spreading by the histiocytes and increased numbers of the elongated cells. The Day 9 culture showed a dramatic increase in the number of elongated cells and further loss of histiocytes. The level of cell growth in the Day 13 culture was similar to the Day 9 culture; however, the Day 13 culture showed marked development of epithelial morphology and giant cells with multiple heterogeneous nuclei, x 100.

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Fig. 11. Metaphase spreads from colon (A), ovarian (H), renal (C), and lung (D) cancer. The colon cancer metaphase was hyperdiploid with 72 chromosomes. The ovarian cancer metaphase also was hyperdiploid with numerous chromosomes and chromosomefragments. The renal cancer metaphase contained 52 chromosomes, and the lung cancer culture showed 71 chromosomes covered with amorphous material and having spiral morphology.

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a Cisplatin b Adriamycin Melanoma 1.0

.05

O Ã¼ OC .01 2 4 u. .1 .2 .4 .6 .0045.009 .018 .027 0 c Etoposide d 5-Fluorouracil DOSE (Gy) z Fig. 12. Radiation-survival curves of cells from a melanoma (â€¢)anda sarcoma 1.0 (â€¢)biopsy specimen showing differences in sensitivity. Bars, SE. (E .5

1.0 CO

.05

o .01 .045 .09 .18 .27 .13.27 .54 .8 0.1 o Fig. 14. Drug survival curves of cells from melanoma (A) and a squamous cell lung cancer biopsy (â€¢)specimenfor Cisplatin (a), Adriamycin (b), etoposide (c). and 5-fluorouracil (d) for two assays showing different sensitivities. Exponential re sponses were observed for Cisplatin, Adriamycin, and etoposide in the sensitive assay. A shoulder in the response was observed for 5-fluorouracil in the sensitive assay and for all the drugs in the resistant assay.

Table 1 Performance of 475 primary cell cultures from biopsy specimens of human tumors in the adhesive tumor cell culture system Specimens/assaysSpecimens

123456 assessed RADIATION DOSE (Gy) Specimens with inadequate yield 44(9)436 of cells Fig. 13. Radiation survival curves at inocula of 50,000 (â€¢),25,000 (â€¢),12,500 Assays cultured (92) (O), and 6,250 P) cells per well show similar sensitivities for the lower three inocula Assays contaminated 33(7) and decreased sensitivity at the highest inoculum. (Standard errors were omitted Assays with low growth 58(12) for clarity). Assays with technical error 12(3) Completed assaysNo.475(100)Â° 328 (69) a Numbers in parentheses, percentage. would result in great differences in survival after several fractions, assuming equal effect per fraction. Because this measurement could have important diagnostic value, we plan to test for a marrow GM-CFC, confirming the findings of Hug ef al. (34). possible correlation between survival at 2.0 Gy and clinical The adhesive tumor cell culture system offers advantages over outcome in radiotherapy patients with this assay. the agar culture system for primary cultures of human tumor Cisplatin, Adriamycin, and etoposide showed simple exponen cells. It is efficient and grows most tumor types. Morphological tial cell killing, typical of in vitro responses for these drugs (32), and cytogenetic analyses suggest that the system grows malig and shouldered responses in more resistant tumors. The sensi nant cells, and the CFE rate suggests that the majority of tivity of different tumors to these drugs varied. The IC5o rates proliferating cells in tumors are grown. The drug and radiation reported herein are similar to the dose range reported for these survival curves are classical and show sensitivities expected of drugs by Hill ef al. (33), using cell lines from human tumors. For cells in vitro. This method is relatively easy to perform, and the myelosuppressive drugs, the drug dose range showing toxicity cultures can be read by image analysis systems. It appears to human tumor cells falls within the range of toxicity to normal capable of assaying human tumors for drug and radiation sen-

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Table2 Salmon (ed.), Cloning of Human Tumor Stem Cells, pp. 85-99 New York- Performance of adhesive tumor cell culture system by tumor type relative to Alan R. Liss, Inc., 1980. assays not contaminated 15. Tepper, J. Clonogenic potential of human tumors. Acta Radiol. Oncol., 20: 283-288, 1981. of assays of assays 16. Ben-Ze'ev, A., and Raz, A. Multinucleation and inhibition of cytokinesis in TumortypeLung setup114 completed93 suspended cells: reversal upon reattachment to a substrate. Cell, 26' 107- (82)a 115,1981. Breast 39 35 (89) 17. Danielson, K. G., Anderson, L. W., and Hosick, H. L. Selection and character Sarcoma 59 46 (78) ization in culture of mammary tumor cells with distinctive growth properties in vivo. Cancer Res., 40: 1812-1819,1980. Melanoma 72 59 (82) 18. Shields, R. Transformation and tumorigenicity. Nature (Lond ) 262- 348-349 Ovarian 21 16(76) Genitourinary 23 20 (87) 1978. 19. Yates, J., and King, R. J. B. Lack of correlation between transformed charac Gastrointestinal 21 18(86) teristics in culture and tumorigenicity of mouse mammary tumour cells. Eur. J. MiscellaneousNo. 47No. 40 (85) Cancer Clin. Oncol., 18: 399-403,1982. 20. Hug, V., Haynes, M., Rashid, R., Spitzer, G., Blumenshein, G., and Hortabagyi, Totals 396 327 (83) G. Improved culture conditions for clonogenic growth of primary human breast a Numbers in parentheses, percentage. tumors. Br. J. Cancer, 50: 207-213, 1984. 21. Singletary, S. E., Tucker, S. L., Spitzer, G., Tomasovic, B., Hug, V., and Drewinko, B. Effects and interactions of epidermal growth factor, insulin, sitivity, which is potentially useful in predicting clinical response hydrocortisone, and estradiol in the human tumor stem cell assay. Int. J. Cell and in screening new chemotherapy agents. Cloning, 3:407-414, 1985. 22. Axelrod, D. Zero-cost modification of brightfield microscopes for imaging phase gradients on cells: Schlieren optics. Cell Biophys., 3: 167-173, 1981. 23. Umbach, G., Singletary, S. E., Tomasovic, B., Spitzer, G., and Drewinko, B. REFERENCES Dose-survival curves for cisplatin, melphalan, and velban in human granulocyte- macrophage progenitor cells. Int. J. Cell Cloning, 2: 335-340,1984. 24. Brock, W. A., Williams, M., Bahdkamkar, V. A., Spitzer, G., and Baker, F. L. 1. Baker, F. B., Spitzer, G., Ajani, J., Lukeman, J., Pathak, S., Brock, W., Radiosensitivity testing of primary cultures from human tumor biopsy speci Tomasovic, B.. and Thielvoldt, D. Adhesive tumor cell culture system: suc mens. In: Proceedings of the Third International Meeting of Progress in cessful primary culture of human tumor cells on an adhesive substrate. Proc. Radiation Oncology, Vienna, Austria, in press, 1985. Am. Assoc. Cancer Res., 26: 367, 1985. 25. Singletary, E., Romsdahl, M., Spitzer, G., Baker, F., Ajani, J., Tomasovic, B., 2. Hamburger, A. W., and Salmon, S. E. Primary bioassy of human myeloma and Thielvoldt, D. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1986 American Association for Cancer Research. Drug and Radiation Sensitivity Measurements of Successful Primary Monolayer Culturing of Human Tumor Cells Using Cell-adhesive Matrix and Supplemented Medium

Fraser L. Baker, Gary Spitzer, Jaffer A. Ajani, et al.

Cancer Res 1986;46:1263-1274.

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