Metastatic Potential Prediction by a Visual Grading System of Cell Motility: Prospective Validation in the Dunning R-3327 Prostatic Adenocarcinoma Model1

Home , Grading (tumors)

(CANCER RESEARCH 48. 4312-4317, August 1, 1988] Metastatic Potential Prediction by a Visual Grading System of Cell Motility: Prospective Validation in the Dunning R-3327 Prostatic Adenocarcinoma Model1

James L. Mohler,2 Alan W. Partin,3 John T. Isaacs, and Donald S. Coffey

Department of Urology, The Johns Hopkins University School of Medicine and The Johns Hopkins Oncology Center, Baltimore, Maryland 21205

ABSTRACT At present many grading systems are used to assess the metastatic potential of prostatic carcinoma. Whether these A method for accurate prediction of prognosis in individual patients grading systems describe architectural (3), cytological (4), or with prostatic carcinoma does not exist. The limitations of pathological some combination of both features (5-8), they share the short grading systems may result from the failure of standard pathological examination of fixed dead tissue to accurately assess the biological and coming of relying upon the examination of dead tissue to predict metastatic behavior of live tumor cells. Many of the sublines of the the biological behavior of a living dynamic tumor system. At Dunning R-3327 rat prostatic adenocarcinoma are histologically similar present no grading system has utilized living cancer cells that yet differ in metastatic potential. Cells from the Dunning model were may possess biological properties that are related to the aggres grown in culture and filmed by time-lapse videomicroscopy. These cells siveness of neoplasms. Cancer cell motility is an obvious ex exhibited characteristic membrane ruffling, pseudopodal extension, and ample of a property that would be lost with fixation but may cellular translation that could be graded with 80% reproducibility. Indi prove important as a predictor of patient prognosis. vidual cells from sublines with high metastatic potential were separated Cancer cell motility was suggested in 1863 by Rudolph Vir- from cells from sublines of low metastatic potential in 96% of cases. We chow (9) but was first documented by George Gey with time- have applied our cell motility grading system to prospectively classify the metastatic potential of neoplastic cells. The mean motility grades of lapse cinematography in 1931 (10). Sumner Wood demon sublines of high and low metastatic potential differed significantly (Mann- strated that cancer cell motility occurred in vivo (11). Donald Whitney-Wilcoxon, I' < 0.0005). Among seven sublines in which the Merchant was the first to recognize the motility of human prostatic carcinoma in primary culture.4 Hosaka and coworkers grading system was developed, individual cells were correctly classified as high or low metastatic in 71% of cases by ruffling or pseudopodal demonstrated differences in cell motility between various rat extension, 73% of cases by translation, and 75% of cases by motility hepatoma cell lines that exhibited different propensities for index, an average of the three parameters of motility. Among four newly metastasis (12). Haemmerli and Strauli studied the motility of tested sublines, cells from a low metastatic and high metastatic subline human cells from six squamous carcinoma cell lines and sug were perfectly classified. Cells from two other low metastatic sublines gested that their motility in vitro reflected their invasive behav were misclassified. When all 88 cells from the 11 sublines were classified, ior in vivo (13, 14). Time-lapse videomicroscopy has replaced high metastatic cells were detected with 94% sensitivity and 50% speci the more cumbersome time-lapse cinematography and many ficity. The predictive value of a determinationof low metastatic was 93%, less precise methods for studying cell motility (15-19). Manger whereas the predictive value of an assignment of high metastatic was 52%. The ability to detect and accurately classify most highly metastatic and Heckman used time-lapse videomicroscopy to quantify cells while rarely erring in a classification of low metastatic potential translation by mean angular change of direction in addition to suggests that a grading system of cancer cell motility should be evaluated mean cellular velocity (20). Verschueren and Van Larebeke (21) in human prostatic carcinoma. The motility of live prostatic carcinoma developed a method to measure cellular translation as well as cells may predict patient prognosis better than standard pathological changes in cell contour. Fulton directed attention to three grading systems. subtypes of cell motion (22). She described swimming due to flagella and cilia, ameboid motion and fibroblastic movement INTRODUCTION by pseudopodal extension, and bleb formation and ruffling. These studies more precisely described cellular motility and In the United States, prostatic carcinoma is the second lead suggested that cancer cell motility may be related to metastatic ing cause of death from cancer in males. Although autopsy potential. However, no quantitative system has been used to studies have revealed prostatic carcinoma in over 30% of men grade cellular motility and predict metastatic potential, and over 50 yr of age (1), only 26,000 American men die of prostatic once developed, any grading system of cancer cell motility must carcinoma annually (2). The outcome of men with prostatic be validated prospectively. We developed a grading system and carcinoma is largely dependent upon the aggressiveness of their characterized the in vitro motility of living cancer cells from the tumor. Because death results rarely from failure to control local Dunning R3327 rat prostatic adenocarcinoma captured by disease, outcome is usually determined by the development of time-lapse videomicroscopy (23). Membrane ruffling, pseudo metastatic disease. We evaluated a grading system of cancer podal extension, and cellular translation (Fig. 1) described cell cell motility for assessment of the metastatic potential of pros motility with reproducibility superior to current histolÃ³gica! tatic cancer in an animal model that provided a series of tumors grading systems. Characteristic cell motility patterns permitted with a wide range of metastatic potential. identification of five histologically similar Dunning R-3327 Received 7/15/87; revised 2/29/88; accepted 5/4/88. prostatic adenocarcinoma sublines. The grading system of cell The costs of publication of this article were defrayed in part by the payment motility was then applied to the separation of 7 sublines of the of page charges. This article must therefore be hereby marked advertisement in Dunning system by metastatic potential (24). Although no accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by Grant CA 15416 from the National Cancer previous biological, biochemical, or morphological test accu Institute. rately described the metastatic potential of the Dunning sub- 2To whom requests for reprints should be addressed, at University of North Carolina at Chapel Hill, Division of Urology, Burnett-Womack Clinical Sciences lines (25), we distinguished the sublines and individual cells of Building CB#7050, Chapel Hill, NC 27599-7050. high and low metastatic potential. Mean motility grades of 3Supported by the Medical Scientist Training Program, Training Grant 5T32 GM07309. 4 Personal communication. 4312

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1988 American Association for Cancer Research. METASTATIC POTENTIAL AND CANCER CELL MOTILITY ruffling, pseudopodal extension, or translation distinguished 3 250 nM dexamethasone (Sigma Chemical, St. Louis, MO), 100 units/ high metastatic sublines from 4 low metastatic sublines. The ml of potassium penicillin G, and 100 units/ml of streptomycin sulfate combination of translation and pseudopodal extension identi under an atmosphere of 5% CO2:95% air. Twelve to 24 h later cells fied correctly 27 of 28 cells as high or low metastatic potential were viewed with an inverted microscope (Zeiss IM35, Thornwood, NY) fitted with Hoffman optics. A programmable control box (Red (24). Lion Controls, York, PA) coordinated illumination of the field for The motility grades which separated retrospectively cells of manual maintenance of focus and image capture. Images magnified 400 high and low metastatic potential have been used to predict times were recorded every 15 s by a high-resolution black and white metastatic potential. Cells from 7 sublines in which the grading video camera (Model MTI 66; Dage, Michigan City, IN) and time- system was developed were evaluated blindly and prospectively. lapse Videorecorder (TL AG-6050; Panasonic, Secaucus, NJ) on one- The motility grading system was tested in 4 sublines that have half in vertical helical scan. The 720 frames were played at 24 frames not been studied previously. per s which produced a 30-s film at 360 times normal speed. Eighty-six films of single cells were graded by two observers who had no knowledge of the identity of the specimens. Membrane ruffling, MATERIALS AND METHODS pseudopodal extension, and cellular translation (Fig. 1) were graded Eleven sublines with a wide range of metastatic potential from the from 0 (no motility observed) to 5 (excessive motility) by each observer. Dunning R-3327 tumor system of serially transplantable rat prostatic The grades of both observers were summed to yield final scores from 0 adenocarcinoma were studied. All sublines grew when inoculated into to 10 for each motility parameter. The three motility grades were inbred Copenhagen rats (HarÃanSprague Dawley, Indianapolis, IN). summed and divided by three to yield a motility index for each cell. In The characteristics of 3 high metastatic (AT-3, MAT-Lu, MAT-LyLu) addition, motility parameter grades were averaged to determine whether and 5 low metastatic (G, H, HI-F, AT-1, AT-2) sublines are well any two types of motility provided information superior to single grades established and were summarized in Table 1 (25-28). The anaplastic or the motility index. Chiba University B (CUB) subline was described in 1983 (29, 30) and We prospectively compared the motility grades of 32 cells from high was provided by Jun Shimazaki and colleagues of Chiba University, metastatic sublines and 56 cells from low metastatic sublines. For each Chiba, Japan. The poorly differentiated PIF-1 and anaplastic PAT-2 group, motility grades were described by mean Â±SE and compared by were developed at Johns Hopkins from tumors originally obtained from Mann-Whitney-Wilcoxon analysis for the probability that motility Norman H. Altman of the Papanicolaou Cancer Institute, Miami, FL. grade could distinguish metastatic potential. Next, we determined For these 2 sublines, the in vivo tumor volume-doubling time and prospectively whether the analysis of only 8 cells from each of 11 androgen sensitivity were determined as described previously (31). The sublines could predict the metastatic potential of the sublines. From metastatic ability of the various sublines was determined as described each subline the motility grades of 8 cells were averaged to yield subline previously (31). This entailed inoculation of at least 10 intact male motility grades. The grades from each subline were compared to pre Copenhagen rats/group s.c. with 1 x IO6viable cells from each of the viously reported motility grades which distinguished cells of high and Dunning sublines. All rats were followed until death. Complete autop low metastatic potential to classify the metastatic potential of these 11 sies were performed on each rat to determine the number and sites of sublines. Finally we prospectively tested the accuracy of this motility distant mÃ©tastasesineach rat. grading system to analyze a single cell from a subline to predict the Except for the H tumor, all sublines were maintained as continuous metastatic potential of that subline. The number of individual cells in vitro cell lines as described previously (25). H tumor was harvested correctly identified as high or low metastatic was tested for statistical from a tumor-bearing animal. The tissue was minced and then digested difference from chance assignment by x2 analysis. The sensitivity (true to single cell suspensions by serial treatment with 0.5% collagenase and positive divided by true positive + false negative), specificity (true 0.1% trypsin solutions. Eight time-lapse videomicroscopic films of negative divided by true negative + false positive), and predictive values isolated cells from each of the 11 sublines were made under similar of positive (true positive divided by true positive + false positive) and conditions with a standard technique. Approximately 10* cells were negative (true negative divided by true negative + false negative) tests plated in T 25 plastic tissue culture flasks (Falcon, Oxnard, CA) and were determined for prediction of metastatic potential. All analyses equilibrated at 37Â°Cin 3 ml of Roswell Park Memorial Institute 1640 were performed for 56 cells from the 7 sublines in which the grading medium with 2 mM L-glutamine (MA Bioproducts, Walkersville, MD) system was developed as well as 32 cells from 4 sublines which had not which contained 10% fetal calf serum (Hiclone Labs, Inc., Logan, UT), been previously evaluated.

Table 1 Biological characteristics of eleven sublines of the Dunning R3327 rat prostatic adenocarcinoma Eleven sublines of the Dunning R3327 rat prostatic adenocarcinoma differed in histological differentiation, growth rate, androgen sensitivity, and metastatic potential. The histological differentiation was interpreted from histological sections of in vivo tumors from the 11 sublines. The in vivo tumor doubling time, androgen sensitivity, and metastatic potential were determined as described previously (31).

rate survival typeLowCell (days)"4.0 sensitivityYesYesNoNoNoNoNoNoNoNoNoMetastaticpotential*Low mÃ©tastasesLungsof (days)75 potentialGmetastatic tumorH differentiatedWell Â±0.2C21 (50)"Low Â±6365 tumorHI-F differentiatedPoorly Â±6.04.8 (50)Low Â±21120Â± tumorPIF-1 differentiatedModerately Â±1.83.7 (25)Low 587 tumorCUB differentiatedAnaplasticAnaplasticAnaplasticAnaplasticAnaplasticAnaplasticAnaplasticGrowthÂ±1.13.4 (10)Low Â±867 tumorAT-1 Â±0.62.5 (10)Low Â±451 tumorAT-2 Â±0.22.5 (25)Low Â±763 tumorHigh Â±0.22.7 (50)High Â±845

potentialMAT-Lumetastatic tumorMAT-LyLu Â±0.31.7 (50)High [50-350]'Lymph Â±339 tumorAT-3 Â±0.31.8 (50)High (25-250)Lymphnodes and lung Â±540 tumorPAT-2 Â±0.21.8 (50)High Â¡20-200]Lymphnodes and lung Â±732 tumorHistologyPoorly Â±0.2Androgen (10)Site nodes and lungs (40-300]Host Â±5 " Tumor volume doubling time (days). * Low metastatic potential. <10% of s.c.-inoculated rats develop distant mÃ©tastases;high metastatic potential, >90% develop distant mÃ©tastases. c Mean Â±SD. rfNumbers in parentheses, the number of animals/group upon which these data are based. ' Numbers in brackets, range in the number of lung mÃ©tastasesper rat. 4313

PSEUOOPOOAL E MOTILITY INDEX

VECTORAL TRANSLATION

PSEUDOPODAL EXTENSION Fig. I. Cell motility grading system. Ruffling and pseudopodal extension were demonstrated by differences in cell membrane at Time 1 (left) and Time 2 (right). Ruffling represented rapid rhythmic movement of short segments of cell mem METASTATIC POTENTIAL brane. Pseudopodal extension was nonrhythmic extension and retraction of short Fig. 2. Cell motility profiles of 11 Dunning tumor sublines. Ruffling, pseu segments of cell contour over comparatively longer distances. Vectora! translation dopodal extension, translation grades, and motility index for 8 cells from each of graded displacement of cell centroid over time. (Reprinted by permission of the 11 Dunning sublines were plotted. The low metastatic G, H, and AT-2 sublines Journal of Urology from J. Urol., 138: 168-170, 1987.) had lower motility grades and indices than the high metastatic MAT-LyLu, Al 3, and PAT-2 sublines. The motility grades of low metastatic HIF, PIF-1, CUB, Table 2 Separation of cells of high and low metastatic potential by motility and AT-1 sublines and high metastatic MAT-Lu were similar. Columns, mean, parameters bars, SE. Thirty-two cells of high metastatic potential and 56 cells of low metastatic potential were filmed in vitro by time-lapse videomicroscopy. Ruffling, pseudo podal extension, and translation were graded from 0 to 10. The motility grades of high and low metastatic cells were compared. grade grade -1 , Lu coâ€¢z IO-,A86420 of 32 high of 56 low met *mÂ¡yC? PAT-2 IO-I8-A* MotilityparametersRuffling metastaticcells8.90 astaticcells6.46 â€¢IA Â±0.20* Â±0.36 1. Pseudopodal extension 7. 12 Â±0.38 4.22 Â±0.40 0.000015 Translation 7.04 Â±0.44 3.90 Â±0.44 0.00047 Ruffling + pseudopodal 8.02 Â±0.247.96 5.34 Â±0.365.18 0.000000810.0000028 extension 642-0-10-8-6-4â€¢ L_>co=!iij Ã¬.'~%1<%O Ruffling + translation Â±0.28 Â±0.38 Translation -I-pseudopo 7.08 Â±0.387.68 4.06 Â±0.424.86 0.0000110.0000024 20-Â» dal extension 6-!Â«â€¢XLe Motility index'Motility Â±0.30Motility Â±0.38P"0.00000029 * Probability of separation by Mann-Whitney-Wilcoxon analysis.

* Mean Â±SEM. bJ1Ã•'YI ' Motility index = (ruffling + pseudopodal extension + translation) divided by 3. O2OMAT-LUÂ«AT â€¢iB

RESULTS

Eleven sublines of the Dunning R-3327 rat prostatic adeno- RUFFLING PSEUDOPODAL TRANSLATION MOTILITY EXTENSION INDEX carcinoma models with low and high metastatic potential were Fig. 3. Classification of sublines of high metastatic potential by a grading described in Table 1. system of cancer cell motility. The motility grades which distinguished 3 Dunning The 88 individual cells of the 11 Dunning sublines were sublines of high metastatic potential from 4 Dunning sublines of low metastatic separated into two statistically distinct populations by single potential ( ) were used to predict prospectively the metastatic potential of each of 3 Dunning sublines in which the grading system was developed and the motility grades, paired motility grades, and motility indices newly developed PAT-2 subline which had not been studied previously. The (Table 2). The cells of high metastatic potential had higher metastatic potential of each highly metastatic subline was predicted correctly by mean motility grades than the cells of low metastatic potential. all motility grades except pseudopodal extension which misclassified MAT-Lu. Regardless of the motility descriptor(s) examined, the two groups of cells were clearly different (Mann-Whitney-Wil- established motility grades which distinguished sublines of high coxon, P = 0.00047 to 0.00000029). and low metastatic potential (24) to test this grading system A motility profile for each subline resulted when the motility prospectively. Whether single motility parameters, pairs of grades for 8 cells from each subline were averaged. Ruffling, parameters, or the motility index was used, classification errors pseudopodal extension translation, and motility index varied resulted (Figures 3 and 4). Among the 7 sublines in which the widely between Dunning tumor sublines (Fig. 2). Among sub- classification scheme was generated, ruffling misclassified AT- lines of low metastatic potential, G, H, and AT-2 had low 1 tumor as a high metastatic subline. Pseudopodal extension ruffling, pseudopodal extension, and translation grades which misclassified HI-F as high metastatic and MAT-Lu as low resulted in low motility indices. These three sublines were metastatic. Translation mislabeled 2 sublines of low metastatic separated clearly from the high metastatic MAT-LyLu, AT-3, potential, HI-F and AT-1, as high metastatic. Motility index and PAT-2 sublines. Considerable overlap existed between the also labeled HI-F and AT-1 as high metastatic. Pairs of motility low metastatic PIF-1, CUB, and AT-1 and high metastatic grades did not improve classification accuracy. Four sublines MAT-Lu sublines. Within sublines, variation of motility grades which had not contributed to the generation of classification averaged 33%. Intraassay reproducibility, (100 â€”% of coeffi motility grades also varied widely in their motility profiles. The cient variation) 67%, was similar to the 75% intraassay repro high metastatic PAT-2 subline and the low metastatic H subline ducibility determined previously for the 5 sublines G, AT-1, were properly classified by ruffling, pseudopodal extension, AT-2, AT-3, and MAT-LyLu (23). translation, and motility index. All 4 scores misclassified the Subline motility grades were compared to the previously CUB and PIF-1 sublines which had low metastatic potential. 4314

Table 4 Individual cell motility grades as a test for high metastatic potential of 0C^W Dunning sublines The metastatic potential predicted by a grading system of cell motility for 88 individual cells was compared to the actual metastatic potential of their subline 0 H IO-I Lu? of origin. The sensitivity, specificity, and predictive value of a positive and negative CUBPIF-t8-Â§ test were calculated for the motility parameters ruffling, pseudopodal extension, 0Ã›Q and translation as well as motility index. C/)^,-. "0â€¢gwO^ p8 dal '* i0^ RufflingPseudopo-indexActualextensionTranslationMotility 8"t<â€” 6-oo4-A20-0IO-I8-6-0o o2-0-o0lO-iog8-06-a4-2 potentialPredicted metastatic 'â€¢%o metastatic Low29 Low26 Low30 Low30 111 6"O^0 potential ^UC0 High 30 22 292 28 LowSensitivity"' 3 2691 6 3481 2794 2 2894 â€¢0-â€¢%Â¿Ã•Â¿Ã•u10 4-& * iitÂ£ Specificity*' ' 46 6154 48 5052 2-0-GHffAT-IAT-2 Predictive value + test*1' 48 5193High Predictive value - test*' 'High 90High 85High 93 " Sensitivity = (true positive) divided by (true positive + false negative). ' Percentage. RUFFLING PSEUOOPODAL TRANSLATION MOTILITY ' Specificity â€”(true negative) divided by (true negative + false positive). EXTENSION INDEX * Predictive value + test = (true positive) divided by (true positive -I- false positive). Fig. 4. Classification of sublines of low metastatic potential by a grading ' Predictive value â€”test = (true negative) divided by (true negative + false system of cancer cell motility. The motility grades which distinguished 3 Dunning sublines of high metastatic potential from 4 Dunning sublines of low metastatic negative). potential ( ) were used to predict prospectively the metastatic potential of 7 sublines of low metastatic potential. Among the G, HI-F, AT-1. and AT-2 sublines in which the grading system was developed, ruffling misclassified All. pseudo- were detected with 94% sensitivity and 50% specificity. The podal extension misclassified HI-F, and translation and motility index misclas predictive value of a positive test was 52%, and the predictive sified both sublines as high metastatic. In 3 sublines not studied previously, the value of a negative test was 93%. Thus, if these cells were low metastatic potential of H was predicted correctly by all motility scores. CUB and PIF-I were classified incorrectly as high metastatic by all motility scores. representative of primary tumors in Copenhagen rats that had not metastasized and were surgically excised, 94% of tumors of Table 3 Classification accuracy by motility parameters high metastatic potential would be identified and removed, The motility of single cells from 11 Dunning sublines was graded and compared whereas 48% of rats would have undergone unnecessary re to motility grades established previously in 7 Dunning sublines that distinguished moval of the primary tumor (100 â€”52% predictive value of a sublines of high and low metastatic potential. Accuracy of classification by ruffling, pseudopodal extension, translation, and motility index was determined positive test). An error would have been made in 7% of rats for 56 cells from 7 sublines in which the grading system was developed and 32 who were spared operation by this test. The high metastatic cells from 4 previously untested sublines. potential of their tumors would not have been identified. Cells from 7 sublines in Cells from 4 Motility which test previously un DISCUSSION parametersRuffling developed40 testedsublines15(47) 88cells55 (71)Â°'* (63)Â° A spontaneous prostatic adenocarcinoma was found in 1961 40(71)Â°41 60(68)Â°57 Pseudopodal 10(63)16(50) in the dorsal prostate of the 3327th rat examined by Dr. W. F. extension Translation (73)" (65)Â° Dunning (32). Since its discovery, the transplantable Dunning Motility index56 42 (75)Â°32 16(50)All 58 (66)Â° R-3327 rat prostatic adenocarcinoma line has spontaneously " Statistically different from random assignment by x2 analysis (/' < 0.05). given rise to several distinct variants with differing histology, *Numbers in parentheses, percentage. growth rates, androgen sensitivity, metastatic potential, and lethality (25-30). The usefulness of this model for human The 88 cells were individually classified by comparison of prostatic carcinoma has stimulated many investigators to search each parameter, pairs of parameters, and motility index with for a technique which distinguishes sublines of high and low previously established grades which distinguished cells of high metastatic potential. Failed biological discriminators include in and low metastatic potential (24) (Table 3). Fifty-six cells from vivo growth rate, androgen sensitivity, in vitro growth rate, 7 sublines in which the test was developed were correctly culture saturation density, and androgen-dependent and -inde classified with regard to metastatic potential with an accuracy pendent clonogenic potential (25). Lowe and Isaacs (28) derived of 75% by motility index, 73% by translation grade, and 71% an index from 3 of 9 measured hydrolytic enzymes which by ruffling or pseudopodal extension grade. When 32 cells from separated 3 sublines of high metastatic potential from 2 sublines 4 sublines which had not been previously studied were classified, of low metastatic potential. Brendler et al. (33, 34) applied this accuracy did not differ from metastatic potential assignment by concept to discrimination of normal, benign, and malignant random chance (x2, P > 0.5). When all 88 cells were considered, human prostatic tissue and prediction of hormonal responsive each parameter and motility index classified cells by metastatic ness of prostatic carcinoma with some success. Failed morpho potential with an accuracy statistically greater than by random logical discriminators can be classified into ultrastructural, assignment (x2, P < 0.05) (Table 3). Pseudopodal extension cytological, and architectural techniques. Electron microscopy was most accurate and classified 68% of 88 cells correctly. In of sublines in vivo and in vitro failed to distinguish the 2 groups each group of cells tested, when motility parameters were of sublines. Chromosomal number and DNA content per cell paired, no improvement in classification accuracy resulted. were similar (25). Nuclear size and shape measured by flow When the high metastatic potential of a primary tumor was cytometric forward and perpendicular laser light scatter iden predicted by examination of the motility of a single cell, motility tified characteristic indices for 4 Dunning sublines (35). Unfor index was the best classifier (Table 4). Highly metastatic cells tunately, the index was heavily influenced by growth rate and 4315

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1988 American Association for Cancer Research. METASTATIC POTENTIAL AND CANCER CELL MOT1LITY thus failed to recognize metastatic potential independently. administration of adjunctive therapy unnecessary. On the other Lastly, high and low metastatic sublines cannot be distinguished hand, appreciation of micrometastases may enable curative by their appearance in culture or by their histological appear adjunctive therapy. Isaacs (36) demonstrated in the Dunning ance in vivo (25). prostatic carcinoma model that chemo- and hormonal therapies We combined time-lapse videomicroscopy and a visual grad were much more effective at low tumor volumes. Unfortuntely, ing system to evaluate cell membrane ruffling, pseudopodal the tumor volumes used in that animal study were well below extension, and translation in normal and malignant rat prostate the tumor volumes required for detection of mÃ©tastasesin cells. The characteristic patterns of motility of normal and human patients by current methods. In humans, if hormonal neoplastic cells from the Dunning sublines enabled us to iden or chemotherapy is initiated after metastatic disease is detected tify these cells (23). Furthermore, the characteristic motility of by current methods, results are palliative at best without proven cells of the Dunning tumor sublines enabled us to distinguish improved survival. Therefore, if adjunctive therapy with cur cells of high and low metastatic potential. For each motility rently available agents is to have an effective role in prostatic parameter, a grade was established that most accurately sepa carcinoma, metastatic disease must be detected or its presence rated cells of high and low metastatic potential (24). We applied predicted at microscopic tumor burdens. this test for metastatic potential in a prospective and blinded If the absence of motility reliably predicts the inability of a fashion to cells from 11 sublines of the Dunning R-3327 pros tumor to metastasize, then removal of the primary is required tate adenocarcinoma model. Among the 7 sublines in which the only if death would occur as the result of primary tumor growth grading system test was developed, both HI-F and AT-1 were or later acquisition of metastatic capability. Unnecessary sur usually misclassified as high metastatic, whether subline grades gical intervention in patients with incidental prostatic carci or individual cell grades were considered. Four sublines not noma could be avoided. However, primary tumors of high previously studied by time-lapse videomicroscopy had met metastatic potential must be removed and hence should not be astatic potential predicted. The H tumor which metastasizes mislabeled. In the Dunning model, when 8 cells were sampled rarely and the recently developed and highly metastatic PAT-2 to provide a subline motility grade, highly metastatic sublines tumor were classified with near-perfect accuracy. CUB and PIF- were not misclassified. When subjected to a more rigorous 1 sublines have low metastatic potential and were misclassified analysis where metastatic potential was determined by the by the visual grading system of cancer cell motility as high examination of a single cell, 6% of cells of high metastatic metastatic sublines. Overall, the metastatic potential of 68% of potential had motility grades insufficient for recognition of 88 individual cells was correctly classified by motility index their high metastatic potential. Conversely, when a cell was which represented a significant improvement over assignment classified as low metastatic, only 7% were from high metastatic by chance alone. Although many cells of low metastatic poten sublines. The 24% variability in cancer cell motility grades tial were misclassified, this test for high metastatic potential within cells of the Dunning sublines suggested that assignment rarely failed to detect a cell of high metastatic potential. of metastatic potential by examination of a single cell from a Motility may be required for metastasis but not sufficient. subline was inadequate. When the Dunning tumor model is Although motility is required for movement from the primary subjected to motility analysis by in vivo harvest of single cells, tumor and through tissue and vascular walls, a cell or cluster the need for analysis of many cells may increase. In human of cells must also be able to exist independently of its neighbors, tumors which have not been subjected to the rigorous selection use hydrolytic enzymes to degrade ground substance and inter of maintenance for many years in vitro, the heterogeneity of rupt cell-cell contact, exist within the vascular space, and grow motility within the primary tumor may be even greater. A more at the metastatic site in order to establish a metastasis. In the reproducible quantitative grading system of cell motility with a Dunning model, cells from the highly metastatic sublines MAT- capacity to sample larger populations of cells in an automated Lu, MAT-LyLu, AT-3, and PAT-2 have extremely high motil fashion may be required to extend this grading system from the ity. Both individual cell and subline mean motility grades were in vitro to the in vivo Dunning model and eventually to the uniformly high, which permitted the recognition of 94% of human patient. However, we have developed a technique which highly metastatic cells by our grading system. Among sublines permitted separation of the Dunning sublines by metastatic of low metastatic potential, H tumor, G tumor, and AT-1 have potential and tested it successfully in a prospective blinded very little motility and are rarely misclassified as highly met fashion. Analysis of the motility of live cancer cells to predict astatic cells or tumors. In contrast, HI-F and AT-2 have inter the biological behavior of neoplasms is conceptually more at mediate motility and are misclassified as highly metastatic in tractive than examination of dead fixed histological sections. 50% of cases. CUB and PIF-1 are highly motile and are mis- Grading systems based upon cancer cell motility may offer classified as high metastatic in almost all cases. The failure of additional information beyond standard pathological grading correct classification could not be explained by heterogeneity systems. Cancer cell motility grading systems deserve further of motility within sublines. Homogeneously motile cells lacked development and testing for the prediction of prognoses of metastatic potential; most cells from the AT-2, HI-F, CUB, cancer patients. and PIF-1 tumors possess the capacity for metastasis presumed by the presence of motility. Because these cells metastasized in ACKNOWLEDGMENTS less than 10% of animals at the time of death from the primary tumor, these cells must have lacked other key characteristics Susan Dalrymple assisted with management of the Dunning sublines, furnished celis for time-lapse videomicroscopy, and assured their ano necessary for metastasis. nymity to the investigators. Leslie Stoll McKee assisted in preparation Recognition of highly motile cancer cells harvested from the of the manuscript. primary tumor would suggest presence of metastatic disease. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1988 American Association for Cancer Research. Metastatic Potential Prediction by a Visual Grading System of Cell Motility: Prospective Validation in the Dunning R-3327 Prostatic Adenocarcinoma Model

James L. Mohler, Alan W. Partin, John T. Isaacs, et al.

Cancer Res 1988;48:4312-4317.

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