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Culture of Peripheral Monocytes in Vitro in Patients with Renal Cell Carcinoma: A Possible Prognostic Indicator
GU'.J,.2-5347/83/1308-0597$02.G'O/G V cL 1301 c:,e;,cemoe1
TuE JOURNAL OF UROLC.GY
Printed
Copyright© 1983 by The Wiiiian1s & "vViikins Co.
PERIPHERAL
VITHO IN PATIENTS POSSIBLE PROGNOSTIC
CELL INDICATOR
ENGIKOLAI C. KRISHNAN,* WINSTON K MEBUST, JOHN W. WEIGEL
AND
WILLIAM R. JEWELL
From the Department of Surgery, University of Kansas Medical Center, Kansas City, Kansas
ABSTRACT
An :in vitro assay has been applied in 23 patients with :renal cell carcinoma. A mononuclear cellrich fraction was cultured from peripheral blood of patients with .renal cell carcinoma. The number of monocytes maturing into macrophages was quantitated over a period of 7 days. Monocyte maturation was significantly lower in patients with :renal cell carcinoma than in normal individuals. Of 10 patients tested both pre- and post-nephrectomy, 8 patients showed clinical improvement after surgery. In all 8, the postt:reatment macrophage yield improved significantly from the pretreatment values to approach normal values. In 2 patients found to have metastasis postoperatively, the macrophage yield continued below normal levels. Our results suggest that the in vitro maturation of macrophages from mononudea:r cells may paxallel clinical events. Patients with renal cell carcinoma often present with metastasis at the time of initial (m1gi101,1s, 1 Nearly 20 per cent show clinical metastasis within a year of diagnosis, in·espective of the treatment initiated, 2 Surgical :resection has been the treatment of choice where the neoplasm is confined to the kidney or the fascia, However, even after definitive surgery, the cure rate has not exceeded 50 per cent of the cases. Advanced adenocarcinoma of the kidney has in general been refractory to cytotoxic drugs3 • 4 and to hormones. Mean survival of patients with single regional metastasis was 5 to 11 months, and of those with multiple metastasis it was around 20 months. 3 Recently, infarction of the involved kidney by emboli.zation of the renal artery was demonstrated to result in spontaneous regTession of metastasis. 5- 7 The mechanism for this spontaneous regression is not well understood. It has been suggested8 that infarction imbalances the antigen ar1t1il)o,cty ratio in circulation resulting in the alteration of complexes, which leads to improved immune response in patient. We have investigated the tumor-host relationship in 23 patients with primary operable renal cell carcinoma using an in vitro assay ,Nhich quantitates the conversion of peripheral blood monocytes to --·--··--~·"Ten patients without metastasis have been pre- and the treatment period. Previous reports demonstrated a decrease rn conversion in ,...-,o·······M vvith breast 111,,cit;;Han,c vu'"-'·°'"''u"'""'• and cell carcinoma, s- ll suggest that this conversion rate clinical events and may therefore be useful as a progno,st,Lc indicator and in following patients po,stJ,eo,ec:tH)R Patient u1__,,uiiw,iu;; A r-.y·,-,"n"''"-r'"'" was undertaken on cell renal carcinoma and patients with renal cell carcinoma and meta.stasis. The patients were evaluated preoperatively by intravenous pyelog-.rams, comrmt;ea axial tomography (CAT) scans of the abdomen, renal arteriogrnphy, vena cavography as indicated, lung tomograms, and serum chemistry profiles. Renal infarction was done by a gel foam and Gian Turcott coil technique. One week later the patients underwent a radical nephrectomy. The patients were then randomized into immune RNA as adjuvant therapy or Provera. Observations were made on the patients' clinical and immunologic status, preinfarction, post radical nephrectomy and at 3Accepted for publication May 3, 1983. * Requests for reprints: Department of Surgery, University of Kansas Medical Center, Rainbow Boulevard at 39th St., Kansas City, Kansas 66103.
month intervals. The details of treatment and clinical followup will be reported separately. MATERIALS AND METHODS
Fifteen ml. of hepari.nized blood was obtained by venipuncture from patients with renal cell carcinoma and from normal donors. The blood was mixed with an equal volume of RPMI 1640 medium, layered on an equal volume of sterile lymphocyte separation medium (density of l.077-1.080 gm./ml.), and centrifuged for 45 minutes at 400 X g. After the red blood cells sedimented, the mononuclear cell band was removed and washed twice with RPMI 1640 centrifuging for 10 minutes at 500 X g. The final pellet was suspended in 3.0 ml. of 50 per cent autologous plasma and the cell concentration was determined. The cell concentration was adjusted to 2 X 106 mononuclear cells/ml. with 50 pe:r cent autologous plasma. The cell preparation consisted largely of mononuclear cells, including both lymphocytes and monocytes. Poly:morphonuclear cell contamination was often observed, especially in patients with malignant disease. After assurii.,g 95 per cent cell viability trypan blue exclusion, 0.2 ml. (4 X 105 cells) of this suspension on at least 10 wells of Costar flat bottom tissue The were incubated in 5 per cent CO2 at 37C in nuLu1_ttuueu for 7 days. At this point the wells were washed 3 times with normal saline. To each well, 0.05. ml of a 0.1 M citric acid solution covac,u,.uu 14 cent naphthol blue black was added. The were dil'-'"''"'-' to stand fo:r 30 minutes at room temperature. The contents in each well were vigorously agitated by eu,em,eu aspirations with a micropipet, and the number of cells was determined by taking the mean of the number of nuclei counted in 10 wells. The results were expressed as both the percentage of macrophages and the number of macrophages per roJ. of blood. The procedure is schematically shown in figure L Twenty-five µI. of the original cell suspension was diluted to 10 times the volume using 50 per cent autologous plasma, and used for cytocentrifugation. All slides for differential and nonspecific esterase staining were prepared in a Shandon Cytospin II at 1,500 rpm for 10 minutes. Nonspecific esterase staining. The cytocentrifuged slides were air dried and the cells were fixed in cold (4C) buffered formalin acetone (pH 6.6) for 30 seconds and washed for 10 seconds in running tap water. While still wet the cells were stained with freshly prepared substrate mixture containing diazonum salt (fast blue BB), napthoic acetate (L-napthal ace-
597
598
KRISHNAN AND ASSOCIATES
ASSAY FOR PERIPHERAi.. MONOCYTES/
DILUTE AND LA YER OVER FICOLL HYPAQUE CENTRIFUGE 400G x 40 MIN
REMOVE MONONUCLEAR CELL BAND
PERIPHERAL BLOOD
---------~~~---------WASH OFF NONADHERENT CELLS
INCUBATE 16 HRS
CULTURE 7 DAYS
WASH OFF NONVIABLE CELLS
COUNT NUMBER OF ADHERENT CELLS
FIG. 1. Schematic representation of peripheral monocyte culture.
tate) in trismaleate buffer, pH 7.8. After 20 minutes the slides were washed and stained with Wright stain. The cytocentrifuge preparations were further counterstained with dilute Giemsa stain for 20 minutes. Standardization of culture conditions. Preliminary experiments to ascertain the number of cells that could be cultured without being affected by cell crowding were conducted. No crowding effects in these experiments up to a concentration of 4 X 106 cells/ml. were found. The results were reproducible up to 2 X 106 cells/ml. or 4 X 105 cells/well. Several different plasma sources were tested. Autologous plasma gave more reproducible results than allogeneic or xenogeneic plasma. Reproducibility of the assay. The mononuclear cell preparation from each patient was plated in at least 10 independent wells. At the end of the culture period the number of macrophages was quantitated by averaging the counts of 4 separate 16-block fields of a hemacytometer. This was done on all 10 independently plated wells. The final number of monocytes per ml. of blood was calculated by taking the average of the counts of the 10 wells. The results of the experiment were subjected to statistical analyses by using Student's t test for significance. P values less than 0.05 are considered significant.
yield in the case of the patients with renal cell carcinoma was 1.47 ± 1.16 X 104 as compared with 8.29 ± 3.14 X 104 in the normal volunteers. These values are significant at p <0.005. Each well is plated with 400 X 103 cells. If we analyze the actual number ofmonocytes plated (table 2) and number ofmonocytes that remained adherent to the plates and matured into macrophages at the end of the culture period it was significantly different (5.89 ± 4.29 X 103 cells in patients against 33.34 ± 10.67 X 103 cells in the case of normal volunteers). Controls tested repeatedly for monocyte maturation over a period of 2 years had results which fell within the statistical variation of mean value of normal populations. To evaluate the change in mononuclear cell culture pre- and posttreatment we analyzed 23 patients during the pretreatment period and 10 of these patients in the postsurgery and treatment period. The analysis of Ficoll-Hypaque gradient separated leukocytes is shown in table 3. The number of leukocytes in preand posttreatment periods was 103.25 ± 45.48 X 103 and 115.08 ± 55.61 X 103 ml. of blood respectively. There was no difference in the number of monocytes plated among the groups. There was a decrease in the polymorphonuclear cell counts but the decrease was not statistically significant (p = 0.05). But the number of lymphocytes in the Ficoll-Hypaque was improved from a mean of 34.33 ± 12.39 X 104 to 49.21 ± 16.26 during the pretreatment period to the posttreatment period. These values are significant at p <0.05. The analysis of monocytes and the macrophage yield in the in vitro cultures is shown in table 4. TABLE 1. Number of macrophages recovered by culturing mononuclear cells in autologous serum over a period of 7 days
No. Patients with renal cell carcinoma Normal individuals
No. Macrophages No. Leukocytes per Recovered per ml. of ml. of Blood x 104 ' 1 Blood x 104 ' t
23
103.25 ± 45.48:f:
1.47 ± 1.16§
17
113.0 ± 39.70
8.29 ± 3.14§
* Values are expressed as the mean ± the standard deviation.
t Cells isolated from Ficoll-Hypaque interface.
+Statistically not significant.
§ Statistically significant at p < 0.005. TABLE 2.
Proportion ofmonocytes that remain in culture and develop into macrophages
No.
No. Cells Plated/ Well X 10't
In renal cell carcinoma pa-
23
400
tients' In normal volunteers
17
400
RESULTS
AH the experiments were conducted within 60 to 90 minutes from the time the blood was drawn. Most of the adherent monocytes were attached to the plastic dishes within 2 hours. However, the number of monocytes attached to the plates was slightly higher after culturing 16 hours in 50 per cent autologous plasma. N onmonocyte attachment to the culture plates was 25 per cent at the end of 16 hours. After 16 hours in culture, the nonadherent lymphocytes and other cells were washed off with RPMI 1640 culture media, replaced with fresh 50 per cent autologous plasma and the culture continued for a total of 7 days. Our initial experiments showed better consistency and optimum yield of macrophages when culturing with media containing more than 30 per cent autologous plasma or serum. After removal of the nonadherent cell population, 90-95 per cent of the remaining cells were nonspecific esterase reactive. In this study we had a total of 23 patients with renal cell carcinoma. Table 1 shows the results of 23 patients and 17 normal volunteers. The number ofleukocytes that were isolated in Ficoll-Hypaque gradient separation in both normal volunteers and patients with renal cell carcinoma was not statistically different (103.23 ± 47.7 for renal cell carcinoma and 113.00 ± 39.70 in the case of normal volunteers). However, when comparing the final yield of macrophages at the end of 7 days, the
103.25 ± 45.48 116.00 ± 36.93
5.89 ± 4.29 33.34 ± 9.05
6.32 ± 4.60 30.60 ± 10.67
* Peripheral monocytes from a total of 23 patients before treatment were cultured and processed identically to that of normal volunteers as described in MATERIALS AND METHODS.
t Values are expressed as the mean ±
the standard deviation.
+Statistically no difference between normal volunteers and patients with renal cell carcinoma. § Statistically significant at p <0.005. TABLE 3. Analysis of mononuclear cells obtained using FicollHypaque density gradient from patients with renal cell carcinoma
No. Polymorphonuclear Cells Recovered per ml. of Blood X 104 t
No. Lymphocytes:f: Recovered per ml. of Blood X lO't
No. Monocytes Recovered per ml. of Blood X lO't
23
103.25 ± 45.48 40.00 ± 15.85
34.33 ± 12.39
32.36 ± 19.74
10
115.08 ± 55.61 32.98 ± 18.34
49.21 ± 16.26 35.92 ± 14.99
No.
Pretreatment Posttreatment
No. Leukocytes* Recovered per ml. of Blood X l0 4 t
* No statistical difference between pre- and posttreatment values.
t Values are expressed as the mean ± the standard deviation.
+Statistically significant at p <0.05.
MONOCYTE MATURATION AND CANCER TABLE 4.
Morphological and functional analysis of mononuclear cell preparation
No. mononuclear cells recovered per ml. of blood X 104 No. macrophages per ml. of blood X 104 % monocytes recovered after 7 day culture X 104 No. monocytes per ml. of blood X 104
Pretreatment (No.= 23)*
Posttreatment (No.= 10)*
Statistical Significance
103.25 ± 47.70
115.08 ± 55.61
NSt
1.47 ± 1.16
3.45 ± 2.52
XXt
6.32 ± 4.60
13.00 ± 5.00
XXX§
31.36 ± 19.12
31.22 ± 13.03
NS
* Values are expressed as the mean ± the standard deviation. t NS = Not significant. Significant at p <0.05. § XXX = Significant at p <0.01.
t XX =
The number of monocytes in the pre- and posttreatment period were similar. However, the macrophage yield in the case of patients with renal cell carcinoma was 1.47 ± 1.16 and 3.45 ± 2.52 during the pre- and posttreatment period respectively. This difference is statistically significant at p <0.05. The difference is more significant (p <0.01) if we look at the number of monocyte cultures which actually become macrophages. To check the number of monocytes actually cultured, the cells isolated from Ficoll-Hypaque were analyzed using both histological staining and nonspecific esterase staining. Using these techniques, there was no difference found in circulating monocytes in these patients in the pre- and posttreatment periods (table 4). Maturation of mononuclear cells. Macrophages cultured using our assay technique were nondividing cells. Cultures were examined over a period of 7 days to observe changes in morphology. On the 1st and 2nd day most of the lymphocytes and other end cells began to degenerate. Within 2 to 4 hours after culturing, most of the monocytes were adhering to the plates. These adherent cells differentiated into macrophages in 4 to 7 days. By the 7th day, most of the adherent cells showed distinct maturational changes, such as increase in size and spreading of cytoplasm. The size of the monocytes was originally 12 to 18 pm., and it enlarged to as much as 40 to 50 µm. in diameter. The cells went through other modifications, such as ruffled membranes and filopodia. Some of the membranes spread their long, thin cytoplasmic extensions over the culture plate, adhering closely to the surface. After maturation, cells seemed larger with a rounded bulge at the center, and numerous uniformly sized microvilli could be seen at high magnification. DISCUSSION
The possibility that immune surveillance plays a part in the development and spread of neoplastic disease has been well accepted. However, there have been disappointingly few laboratory evaluations that can be used to recognize the recurrence of malignant disease. In a recent article 11 we showed a decreased macrophage yield in renal cell carcinoma patients compared to normal individuals. The technique described in this paper is used primarily to recognize the possible changes in in vitro monocyte cultures in renal cell carcinoma patients during preand posttreatment periods and to explore possible use of this assay for following patients during the course of treatment. Various cells have been carefully analyzed in the pursuit of understanding the mechanism of immunological changes. Among these cells monocytes seem to be involved in various phases of immune modulation both as an efficient effector cell12 and as an enhancer of immune response of both T and B cells. 13 It has been shown that the induction of antibody production and cell mediated immunity require the interaction of macrophages. 14, 15 Numerous reports give evidence of the capability of macrophages to elaborate factors that augment16• 17 or suppress 18• 19
hemopoietic cell proliferation. The principal cell capable of elaborating the colony-stimulating factor has been identified as monocyte; 16• 20 also, macrophages have been identified as a primary source of prostaglandins. 21 • 22 Recently, homogeneates of a variety of mouse tumor, but not normal tissue, have been shown to alter in vitro properties of macrophages. 23 ' 24 These studies clearly document the capability of the monocyte-macrophage to stimulate, enhance, or control the hemopoietic system lending to overall systemic changes in the tumor host. Studies on the quantitative changes in monocyte function and maturation are limited. In this study we were able to analyze Ficoll-Hypaque separated mononuclear cell rich fractions in an in vitro system. There are 23 patients on whom we have an analysis of the macrophage recovery in a 7-day in vitro culture system. The reports of Schrek and Rabinowitz25 have shown that the precursors of macrophages are more than likely peripheral monocytes. The number of mononuclear cells per ml. that can be isolated from Ficoll-Hypaque gradient centrifugation in normal individuals and patients with renal cell carcinoma is similar. However, the macrophage yield is considerably lower in the patients. This low macrophage yield may be affected by the low number of monocytes to start with. To eliminate the error due to this possibility we analyzed monocytes that were in our Ficoll-Hypaque gradient separated cells and calculated the number of monocytes actually plated in the study group and normal individuals (see table 2). The number of monocytes plated was comparable in both populations, but the number of monocytes maturing into macrophages was significantly lower in the patients (p <0.005). We have seen similar results in patients with malignant melanoma. In patients with melanoma10• 26 we found that the number of monocytes recovered from Ficoll-Hypaque gradient centrifugation was slightly elevated. However, when cultured in vitro in patients the large number of monocytes fail to stay alive and mature into macrophages, whereas in normal individuals more of these monocytes remain attached and mature into macrophages. We have done experiments 10 to find possible serum factors that might interfere with the macrophage yield in patients with malignant disease. Results of these experiments show that serum factors have no significant effect on the assay. The most interesting observation in this study is the values obtained in pre- and posttreatment periods of patients with renal cell carcinoma. Earlier we showed in a murine model27 that macrophage yield decreased as tumor burden increased. When the tumor was surgically excised the monocyte to macrophage yield improved and reached near-normal pretumor induction values. To evaluate the possible use of this assay in a clinical situation we evaluated mononuclear cell preparation and development of macrophages in an in vitro system in these patients. The results are shown in tables 3 and 4. They indicate that although there is no significant change in the number of cells recovered per ml. of blood from the Ficoll-Hypaque gradient centrifugation, there were significantly (p <0.05) fewer lymphocytes found in the gradient in the pretreatment period. This difference was in both the number oflymphocytes obtained per ml. of blood as well as in the percentage of lymphocytes in the Ficoll-Hypaque separated cells during the posttreatment period. The value increased toward normal during the posttreatment period. On the other hand, the number of polymorphonuclear cells found in the Ficoll-Hypaque gradient was higher in the pretreatment period, but reduced in the posttreatment period. The difference was not statistically significant (p = 0.05). This may become significant as more patients are evaluated. The reason for the excess polymorphonuclear cells in the Ficoll-Hypaque gradient is not known. In the case of patients with lung carcinomas, it has been shown that lymphocytes from the Ficoll-Hypaque gradient interface correlate with the state of the disease. 28 Analysis of the Ficoll-Hypaque gradient separated leukocytes has shown that patients who had a low percentage of lymphocytes had poor prognosis compared to pa-
600
KRISHNAN AND ASSOCIATES MONOCYTE MATURATION PER ML OF BLOOD PRE THERAPY AND POST THERAPY
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may reflect the extent of metabolic disturbance in the patient, or it may be that a subset of monocytes that matures into macrophages is deficient. Recently monocyte adherence has been known to improve in cancer patients after immunotherapy. 33 Since monocytes appear to be multi-functional cells controlling their own function as well as the function of other peripheral cells, we hope that the study of these important cells will provide clinically useful information. Though the series is small, the results are encouraging, especially in the absence of other quantitative laboratory evaluations in patients with renal cell carcinoma. The technique is simple and reproducibility is good, and appears to be amenable to regular laboratory use.
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REFERENCES
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PRE THERAPY
POST
THERAPY
Fm. 2. Number of macrophages recovered per ml. of blood pre- and posttreatment period. These values are obtained by calculating the number of macrophages obtained per culture, X the number of cells per ml. of blood. Nonresponders to the treatment are indicated with a star beside their values.
tients who had a higher percentage of lymphocytes in head and neck cancer. 29 In our study, the most statistically significant change found was in the percentage of plated monocytes giving rise to macrophages in in vitro culture (fig. 2). The percentage of macrophages and also the number of monocytes that matured per ml. of blood increased during the posttreatment period. In these patients the percentage of monocytes yielding macrophages changed from 6.32 ± 4.60 per cent pretreatment to 13.00 ± 5.00 per cent during the posttreatment period. In terms of the number of monocytes which matured into macrophages per ml. of blood, the mean pretreatment value of 1.47 ± 1.16 X 104 cells/ml. improved to 3.45 ± 2.52 cells/ml. In spite of the limited numbers, the values are statistically significant. These values continue to improve as patients improve clinically. Two patients who did not show improvement in in vitro monocyte maturation during the posttreatment evaluation period were found to have metastasis (fig. 2). We have been following 27 patients throughout their clinical course. Future communications would include the relevance of this kind of in vitro assay in detecting recurrence of the disease in nonresponders. In the 10 patients we studied in both the pre- and posttreatment periods, we had 2 patients who had recurrences by the time they finished their treatment regimen. In these 2 patients, the macrophage yield did not improve. The results are encouraging. We are in the process of correlating clinical outcome and in vitro evaluation of these results in a separate communication. It was shown that large numbers of mature macrophages accumulate in draining lymph nodes of nonmetastasizing tumors, while low numbers of macrophages were found in nodes of metastatic tumors. 30 It has been shown recently that maturation of monocytes is significantly depressed in patients with squamous cell carcinoma. 31 Earlier, in a group of patients with melanoma, 10 and in breast carcinoma, 11 we found similar depressed monocyte maturation. The decrease in monocyte maturation has been related to prognosis in primary breast carcinoma.32 The mechanism leading to the lack of maturability of monocytes in vitro into macrophages in patients with malignant disease is not known. This may indicate an intrinsic defect in the maturation processes of the monocytes in patients with malignant disease or
1. Skinner, D. G., Colvin, R. B., Vermillion, C. D., Pfister, R. C. and Leadbetter, W. F.: Diagnosis and management of renal cell carcinoma. Cancer, 28: 1165, 1971. 2. Lokich, J. J. and Harrison, J. H.: Renal cell carcinoma: natural history and chemotherapeutic experience. J. Urol., 114: 371, 1975. 3. Johnson, D. E. and Samuels, M. C.: Chemotherapy for metastatic renal cell carcinoma. In: Cancer Chemotherapy: Fundamental Concepts and Recent Advances. Chicago: Yearbook Medical Publishers, Inc., 1971. 4. Talley, R. W.: Proceedings: chemotherapy of adenocarcinoma of the kidney. Cancer, 32: 1062, 1973. 5. Mohr, S. J. and Whitesel, J. A.: Spontaneous regression of renal cell carcinoma metastases after preoperative embolization of primary tumor and subsequent nephrectomy. Urology, 14: 5, 1979. 6. Bracken, R. B., Johnson, D. E., Goldstein, H. M., Wallace, S. and Ayala, A. G.: Percutaneous transfemoral renal artery occlusion in patients with renal carcinoma: preliminary report. Urology, 6: 6, 1975. 7. Paster, S. B., Bergreen, P. and Schwarz, H.: Percutaneous catheteraided infarction of renal tumors: a preliminary report. J. Urol., 114: 351, 1975. 8. Johnson, D. E., Bracken, R. B., Swanson, D. A., et al.: Arterial occlusive techniques in the management of renal diseases. (in press) 9. Krishnan, E. C., Krishnan, L., Stephens, R. and Jewell, W.R.: In vitro culture of monocytes in normal individuals and patients with breast carcinoma. Breast Cancer Research and Treatment 1: 164, 1981. 10. Krishnan, E. C., Menon, C. D., Krishnan, L. and Jewell, W. R.: Deficiency in maturation process of macrophages in human cancer. JNCI, 65: 273, 1980. 11. Krishnan, E. C., Mebust, W. K., Weigel, J. W. and Jewell, W. R.: Maturation of monocytes in patients with renal cell carcinoma. Invest. Urol., 19: 4, 1981. 12. Tagliabue, A., Mantovani, A., Kilgallen, M., Herberman, R. B. and McCoy, J. L.: Natural cytotoxicity of mouse monocytes and macrophages. J. Immunol., 122: 2363, 1979. 13. Basten, A. and Mitchell, J.: Role of macrophages in T cell-B cell collaboration in antibody production. In: lmmunobiology of the Macrophage. Edited by D.S. Nelson. New York: Academic Press. p. 45, 1976. 14. Erb, P. and Feldmann, M.: Role of macrophages in in vitro induction of T-helper cells. Nature, 254: 352, 1975. 15. Bergholtz, B. 0. and Thorsby, E.: HLA-D restriction of the macrophage-dependent response of immune human T lymphocytes to PPD in vitro: inhibition by anti-HLA-DR antisera. Scand. J. lmmunol., 8: 63, 1978. 16. Chervenick, P.A. and LoBuglio, A. F.: Human blood monocytes: stimulators granulocyte and mononuclear colony formation in vitro. Science, 178: 164, 1972. 17. Gery, I. and Waksman, B. H.: Potentiation of the T-lymphocyte response to mitogens IL The cellular source of the potentiating mediator (s). J. Exp. Med., 136: 143, 1972. 18. Nelson, D. S.: Production by stimulated macrophages of factors depressing lymphocyte transformation. Nature, 246: 306, 1973. 19. Ichikawa, Y., Plunznic, D. H. and Sachs, L.: Feedback inhibition of the development of macrophage and granulocyte colonies. I. Inhibition by macrophage. Proc. Natl. Acad. Sci. USA, 58: 1480, 1967. 20. Moore, M. A. S. and Williams, N.: Physical separation of colony stimulating cells from in vitro colony forming cells in hemopoietic
MONOCYTE MATURATION AND CANCER
tissue. J. Cell. Physiol., 80: 195, 1972. 21. Brune, K., Glatt, M., Kiilin, H. and Peskar, B. A.: Pharmacological 22.
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control of prostaglandin and thromboxane release from macrophages. Nature, 274: 261, 1978. Humes, J. L., Bonney, R. J., Pelus, L., Dahlgren, M. E., Sadowski, S. J., Kuehl, F. A. and Davies, P.: Macrophages synthesize and release prostaglandins in response to inflammatory stimuli. Nature, 269: 149, 1977. Rhodes, J. M., Bennedsen, J., Larsen, S. 0., Riisgaard, S. and Spiirck, J. V.: Correlation between in vivo and in vitro functional tests for activated macrophages. Infect. Immun., 23: 34, 1979. Schultz, R. M., Pavlidis, N. A., Stylos, W. A. and Chirigos, M.A.: Regulation of macrophage tumoricidal function: a role for prostaglandins of the E series. Science, 202: 320, 1978. Schrek, R. and Rabinowitz, Y.: Effects of phytohemagglutinin on rat and normal and leukemic human blood cells. Proc. Soc. Exp. Biol. Med., 113: 191, 1963. Krishnan, E. C. and Jewell, W. R.: Deficiency of macrophage precursors in malignancy. Fed. Proc., 38: 1220, 1979. Krishnan, E. C., Krishnan, L. and Jewell, W.R.: Interrelationship
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31. 32. 33.
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between monocyte maturation in vitro and tumor growth in murine tumor host. J. Surg. Res., 31: 240, 1981. Check, I. J. and Hunter, R. L.: Changes in Ficoll-Hypaque gradient with advancing stage of lung cancer. Fed. Proc., 38: 1224, 1979. Check, I. J., Hunter, R. L., Lounsbury, B., Rosenberg, K. and Matz, G.: Prediction of survival in head and neck cancer based on leukocyte sedimentation in Ficoll-Hypaque gradients. Laryngoscope, 90: 1281, 1980. Carr, I., Underwood, J. C., McGinty, F. and Wood, P.: The ultrastructure of the local lymphoreticular response to an experimental neoplasm. J. Pathol., 113: 175, 1974. Dent, R. G. and Cole, P.: In vitro monocyte maturation in squamous carcinoma of the lung. Brit. J. Cancer., 43: 486, 1981. Taylor, S. A. and Currie, G. A.: Monocyte maturation and prognosis in primary breast cancer. Brit. Med. J., I: 1050, 1979. Hersh, E. M., Murphy, S. G., Quesada, J. R., Gutterman, J. U., Gschwind, C. R. and Morgan, J.: Effect of immunotherapy with Coryne bacterium parvum and methanol extraction residue of BCG administered intravenously on host defense function in cancer patients. JNCI, 66: 993, 1981.
TuE JOURNAL OF UROLC.GY
Printed
Copyright© 1983 by The Wiiiian1s & "vViikins Co.
PERIPHERAL
VITHO IN PATIENTS POSSIBLE PROGNOSTIC
CELL INDICATOR
ENGIKOLAI C. KRISHNAN,* WINSTON K MEBUST, JOHN W. WEIGEL
AND
WILLIAM R. JEWELL
From the Department of Surgery, University of Kansas Medical Center, Kansas City, Kansas
ABSTRACT
An :in vitro assay has been applied in 23 patients with :renal cell carcinoma. A mononuclear cellrich fraction was cultured from peripheral blood of patients with .renal cell carcinoma. The number of monocytes maturing into macrophages was quantitated over a period of 7 days. Monocyte maturation was significantly lower in patients with :renal cell carcinoma than in normal individuals. Of 10 patients tested both pre- and post-nephrectomy, 8 patients showed clinical improvement after surgery. In all 8, the postt:reatment macrophage yield improved significantly from the pretreatment values to approach normal values. In 2 patients found to have metastasis postoperatively, the macrophage yield continued below normal levels. Our results suggest that the in vitro maturation of macrophages from mononudea:r cells may paxallel clinical events. Patients with renal cell carcinoma often present with metastasis at the time of initial (m1gi101,1s, 1 Nearly 20 per cent show clinical metastasis within a year of diagnosis, in·espective of the treatment initiated, 2 Surgical :resection has been the treatment of choice where the neoplasm is confined to the kidney or the fascia, However, even after definitive surgery, the cure rate has not exceeded 50 per cent of the cases. Advanced adenocarcinoma of the kidney has in general been refractory to cytotoxic drugs3 • 4 and to hormones. Mean survival of patients with single regional metastasis was 5 to 11 months, and of those with multiple metastasis it was around 20 months. 3 Recently, infarction of the involved kidney by emboli.zation of the renal artery was demonstrated to result in spontaneous regTession of metastasis. 5- 7 The mechanism for this spontaneous regression is not well understood. It has been suggested8 that infarction imbalances the antigen ar1t1il)o,cty ratio in circulation resulting in the alteration of complexes, which leads to improved immune response in patient. We have investigated the tumor-host relationship in 23 patients with primary operable renal cell carcinoma using an in vitro assay ,Nhich quantitates the conversion of peripheral blood monocytes to --·--··--~·"Ten patients without metastasis have been pre- and the treatment period. Previous reports demonstrated a decrease rn conversion in ,...-,o·······M vvith breast 111,,cit;;Han,c vu'"-'·°'"''u"'""'• and cell carcinoma, s- ll suggest that this conversion rate clinical events and may therefore be useful as a progno,st,Lc indicator and in following patients po,stJ,eo,ec:tH)R Patient u1__,,uiiw,iu;; A r-.y·,-,"n"''"-r'"'" was undertaken on cell renal carcinoma and patients with renal cell carcinoma and meta.stasis. The patients were evaluated preoperatively by intravenous pyelog-.rams, comrmt;ea axial tomography (CAT) scans of the abdomen, renal arteriogrnphy, vena cavography as indicated, lung tomograms, and serum chemistry profiles. Renal infarction was done by a gel foam and Gian Turcott coil technique. One week later the patients underwent a radical nephrectomy. The patients were then randomized into immune RNA as adjuvant therapy or Provera. Observations were made on the patients' clinical and immunologic status, preinfarction, post radical nephrectomy and at 3Accepted for publication May 3, 1983. * Requests for reprints: Department of Surgery, University of Kansas Medical Center, Rainbow Boulevard at 39th St., Kansas City, Kansas 66103.
month intervals. The details of treatment and clinical followup will be reported separately. MATERIALS AND METHODS
Fifteen ml. of hepari.nized blood was obtained by venipuncture from patients with renal cell carcinoma and from normal donors. The blood was mixed with an equal volume of RPMI 1640 medium, layered on an equal volume of sterile lymphocyte separation medium (density of l.077-1.080 gm./ml.), and centrifuged for 45 minutes at 400 X g. After the red blood cells sedimented, the mononuclear cell band was removed and washed twice with RPMI 1640 centrifuging for 10 minutes at 500 X g. The final pellet was suspended in 3.0 ml. of 50 per cent autologous plasma and the cell concentration was determined. The cell concentration was adjusted to 2 X 106 mononuclear cells/ml. with 50 pe:r cent autologous plasma. The cell preparation consisted largely of mononuclear cells, including both lymphocytes and monocytes. Poly:morphonuclear cell contamination was often observed, especially in patients with malignant disease. After assurii.,g 95 per cent cell viability trypan blue exclusion, 0.2 ml. (4 X 105 cells) of this suspension on at least 10 wells of Costar flat bottom tissue The were incubated in 5 per cent CO2 at 37C in nuLu1_ttuueu for 7 days. At this point the wells were washed 3 times with normal saline. To each well, 0.05. ml of a 0.1 M citric acid solution covac,u,.uu 14 cent naphthol blue black was added. The were dil'-'"''"'-' to stand fo:r 30 minutes at room temperature. The contents in each well were vigorously agitated by eu,em,eu aspirations with a micropipet, and the number of cells was determined by taking the mean of the number of nuclei counted in 10 wells. The results were expressed as both the percentage of macrophages and the number of macrophages per roJ. of blood. The procedure is schematically shown in figure L Twenty-five µI. of the original cell suspension was diluted to 10 times the volume using 50 per cent autologous plasma, and used for cytocentrifugation. All slides for differential and nonspecific esterase staining were prepared in a Shandon Cytospin II at 1,500 rpm for 10 minutes. Nonspecific esterase staining. The cytocentrifuged slides were air dried and the cells were fixed in cold (4C) buffered formalin acetone (pH 6.6) for 30 seconds and washed for 10 seconds in running tap water. While still wet the cells were stained with freshly prepared substrate mixture containing diazonum salt (fast blue BB), napthoic acetate (L-napthal ace-
597
598
KRISHNAN AND ASSOCIATES
ASSAY FOR PERIPHERAi.. MONOCYTES/
DILUTE AND LA YER OVER FICOLL HYPAQUE CENTRIFUGE 400G x 40 MIN
REMOVE MONONUCLEAR CELL BAND
PERIPHERAL BLOOD
---------~~~---------WASH OFF NONADHERENT CELLS
INCUBATE 16 HRS
CULTURE 7 DAYS
WASH OFF NONVIABLE CELLS
COUNT NUMBER OF ADHERENT CELLS
FIG. 1. Schematic representation of peripheral monocyte culture.
tate) in trismaleate buffer, pH 7.8. After 20 minutes the slides were washed and stained with Wright stain. The cytocentrifuge preparations were further counterstained with dilute Giemsa stain for 20 minutes. Standardization of culture conditions. Preliminary experiments to ascertain the number of cells that could be cultured without being affected by cell crowding were conducted. No crowding effects in these experiments up to a concentration of 4 X 106 cells/ml. were found. The results were reproducible up to 2 X 106 cells/ml. or 4 X 105 cells/well. Several different plasma sources were tested. Autologous plasma gave more reproducible results than allogeneic or xenogeneic plasma. Reproducibility of the assay. The mononuclear cell preparation from each patient was plated in at least 10 independent wells. At the end of the culture period the number of macrophages was quantitated by averaging the counts of 4 separate 16-block fields of a hemacytometer. This was done on all 10 independently plated wells. The final number of monocytes per ml. of blood was calculated by taking the average of the counts of the 10 wells. The results of the experiment were subjected to statistical analyses by using Student's t test for significance. P values less than 0.05 are considered significant.
yield in the case of the patients with renal cell carcinoma was 1.47 ± 1.16 X 104 as compared with 8.29 ± 3.14 X 104 in the normal volunteers. These values are significant at p <0.005. Each well is plated with 400 X 103 cells. If we analyze the actual number ofmonocytes plated (table 2) and number ofmonocytes that remained adherent to the plates and matured into macrophages at the end of the culture period it was significantly different (5.89 ± 4.29 X 103 cells in patients against 33.34 ± 10.67 X 103 cells in the case of normal volunteers). Controls tested repeatedly for monocyte maturation over a period of 2 years had results which fell within the statistical variation of mean value of normal populations. To evaluate the change in mononuclear cell culture pre- and posttreatment we analyzed 23 patients during the pretreatment period and 10 of these patients in the postsurgery and treatment period. The analysis of Ficoll-Hypaque gradient separated leukocytes is shown in table 3. The number of leukocytes in preand posttreatment periods was 103.25 ± 45.48 X 103 and 115.08 ± 55.61 X 103 ml. of blood respectively. There was no difference in the number of monocytes plated among the groups. There was a decrease in the polymorphonuclear cell counts but the decrease was not statistically significant (p = 0.05). But the number of lymphocytes in the Ficoll-Hypaque was improved from a mean of 34.33 ± 12.39 X 104 to 49.21 ± 16.26 during the pretreatment period to the posttreatment period. These values are significant at p <0.05. The analysis of monocytes and the macrophage yield in the in vitro cultures is shown in table 4. TABLE 1. Number of macrophages recovered by culturing mononuclear cells in autologous serum over a period of 7 days
No. Patients with renal cell carcinoma Normal individuals
No. Macrophages No. Leukocytes per Recovered per ml. of ml. of Blood x 104 ' 1 Blood x 104 ' t
23
103.25 ± 45.48:f:
1.47 ± 1.16§
17
113.0 ± 39.70
8.29 ± 3.14§
* Values are expressed as the mean ± the standard deviation.
t Cells isolated from Ficoll-Hypaque interface.
+Statistically not significant.
§ Statistically significant at p < 0.005. TABLE 2.
Proportion ofmonocytes that remain in culture and develop into macrophages
No.
No. Cells Plated/ Well X 10't
In renal cell carcinoma pa-
23
400
tients' In normal volunteers
17
400
RESULTS
AH the experiments were conducted within 60 to 90 minutes from the time the blood was drawn. Most of the adherent monocytes were attached to the plastic dishes within 2 hours. However, the number of monocytes attached to the plates was slightly higher after culturing 16 hours in 50 per cent autologous plasma. N onmonocyte attachment to the culture plates was 25 per cent at the end of 16 hours. After 16 hours in culture, the nonadherent lymphocytes and other cells were washed off with RPMI 1640 culture media, replaced with fresh 50 per cent autologous plasma and the culture continued for a total of 7 days. Our initial experiments showed better consistency and optimum yield of macrophages when culturing with media containing more than 30 per cent autologous plasma or serum. After removal of the nonadherent cell population, 90-95 per cent of the remaining cells were nonspecific esterase reactive. In this study we had a total of 23 patients with renal cell carcinoma. Table 1 shows the results of 23 patients and 17 normal volunteers. The number ofleukocytes that were isolated in Ficoll-Hypaque gradient separation in both normal volunteers and patients with renal cell carcinoma was not statistically different (103.23 ± 47.7 for renal cell carcinoma and 113.00 ± 39.70 in the case of normal volunteers). However, when comparing the final yield of macrophages at the end of 7 days, the
103.25 ± 45.48 116.00 ± 36.93
5.89 ± 4.29 33.34 ± 9.05
6.32 ± 4.60 30.60 ± 10.67
* Peripheral monocytes from a total of 23 patients before treatment were cultured and processed identically to that of normal volunteers as described in MATERIALS AND METHODS.
t Values are expressed as the mean ±
the standard deviation.
+Statistically no difference between normal volunteers and patients with renal cell carcinoma. § Statistically significant at p <0.005. TABLE 3. Analysis of mononuclear cells obtained using FicollHypaque density gradient from patients with renal cell carcinoma
No. Polymorphonuclear Cells Recovered per ml. of Blood X 104 t
No. Lymphocytes:f: Recovered per ml. of Blood X lO't
No. Monocytes Recovered per ml. of Blood X lO't
23
103.25 ± 45.48 40.00 ± 15.85
34.33 ± 12.39
32.36 ± 19.74
10
115.08 ± 55.61 32.98 ± 18.34
49.21 ± 16.26 35.92 ± 14.99
No.
Pretreatment Posttreatment
No. Leukocytes* Recovered per ml. of Blood X l0 4 t
* No statistical difference between pre- and posttreatment values.
t Values are expressed as the mean ± the standard deviation.
+Statistically significant at p <0.05.
MONOCYTE MATURATION AND CANCER TABLE 4.
Morphological and functional analysis of mononuclear cell preparation
No. mononuclear cells recovered per ml. of blood X 104 No. macrophages per ml. of blood X 104 % monocytes recovered after 7 day culture X 104 No. monocytes per ml. of blood X 104
Pretreatment (No.= 23)*
Posttreatment (No.= 10)*
Statistical Significance
103.25 ± 47.70
115.08 ± 55.61
NSt
1.47 ± 1.16
3.45 ± 2.52
XXt
6.32 ± 4.60
13.00 ± 5.00
XXX§
31.36 ± 19.12
31.22 ± 13.03
NS
* Values are expressed as the mean ± the standard deviation. t NS = Not significant. Significant at p <0.05. § XXX = Significant at p <0.01.
t XX =
The number of monocytes in the pre- and posttreatment period were similar. However, the macrophage yield in the case of patients with renal cell carcinoma was 1.47 ± 1.16 and 3.45 ± 2.52 during the pre- and posttreatment period respectively. This difference is statistically significant at p <0.05. The difference is more significant (p <0.01) if we look at the number of monocyte cultures which actually become macrophages. To check the number of monocytes actually cultured, the cells isolated from Ficoll-Hypaque were analyzed using both histological staining and nonspecific esterase staining. Using these techniques, there was no difference found in circulating monocytes in these patients in the pre- and posttreatment periods (table 4). Maturation of mononuclear cells. Macrophages cultured using our assay technique were nondividing cells. Cultures were examined over a period of 7 days to observe changes in morphology. On the 1st and 2nd day most of the lymphocytes and other end cells began to degenerate. Within 2 to 4 hours after culturing, most of the monocytes were adhering to the plates. These adherent cells differentiated into macrophages in 4 to 7 days. By the 7th day, most of the adherent cells showed distinct maturational changes, such as increase in size and spreading of cytoplasm. The size of the monocytes was originally 12 to 18 pm., and it enlarged to as much as 40 to 50 µm. in diameter. The cells went through other modifications, such as ruffled membranes and filopodia. Some of the membranes spread their long, thin cytoplasmic extensions over the culture plate, adhering closely to the surface. After maturation, cells seemed larger with a rounded bulge at the center, and numerous uniformly sized microvilli could be seen at high magnification. DISCUSSION
The possibility that immune surveillance plays a part in the development and spread of neoplastic disease has been well accepted. However, there have been disappointingly few laboratory evaluations that can be used to recognize the recurrence of malignant disease. In a recent article 11 we showed a decreased macrophage yield in renal cell carcinoma patients compared to normal individuals. The technique described in this paper is used primarily to recognize the possible changes in in vitro monocyte cultures in renal cell carcinoma patients during preand posttreatment periods and to explore possible use of this assay for following patients during the course of treatment. Various cells have been carefully analyzed in the pursuit of understanding the mechanism of immunological changes. Among these cells monocytes seem to be involved in various phases of immune modulation both as an efficient effector cell12 and as an enhancer of immune response of both T and B cells. 13 It has been shown that the induction of antibody production and cell mediated immunity require the interaction of macrophages. 14, 15 Numerous reports give evidence of the capability of macrophages to elaborate factors that augment16• 17 or suppress 18• 19
hemopoietic cell proliferation. The principal cell capable of elaborating the colony-stimulating factor has been identified as monocyte; 16• 20 also, macrophages have been identified as a primary source of prostaglandins. 21 • 22 Recently, homogeneates of a variety of mouse tumor, but not normal tissue, have been shown to alter in vitro properties of macrophages. 23 ' 24 These studies clearly document the capability of the monocyte-macrophage to stimulate, enhance, or control the hemopoietic system lending to overall systemic changes in the tumor host. Studies on the quantitative changes in monocyte function and maturation are limited. In this study we were able to analyze Ficoll-Hypaque separated mononuclear cell rich fractions in an in vitro system. There are 23 patients on whom we have an analysis of the macrophage recovery in a 7-day in vitro culture system. The reports of Schrek and Rabinowitz25 have shown that the precursors of macrophages are more than likely peripheral monocytes. The number of mononuclear cells per ml. that can be isolated from Ficoll-Hypaque gradient centrifugation in normal individuals and patients with renal cell carcinoma is similar. However, the macrophage yield is considerably lower in the patients. This low macrophage yield may be affected by the low number of monocytes to start with. To eliminate the error due to this possibility we analyzed monocytes that were in our Ficoll-Hypaque gradient separated cells and calculated the number of monocytes actually plated in the study group and normal individuals (see table 2). The number of monocytes plated was comparable in both populations, but the number of monocytes maturing into macrophages was significantly lower in the patients (p <0.005). We have seen similar results in patients with malignant melanoma. In patients with melanoma10• 26 we found that the number of monocytes recovered from Ficoll-Hypaque gradient centrifugation was slightly elevated. However, when cultured in vitro in patients the large number of monocytes fail to stay alive and mature into macrophages, whereas in normal individuals more of these monocytes remain attached and mature into macrophages. We have done experiments 10 to find possible serum factors that might interfere with the macrophage yield in patients with malignant disease. Results of these experiments show that serum factors have no significant effect on the assay. The most interesting observation in this study is the values obtained in pre- and posttreatment periods of patients with renal cell carcinoma. Earlier we showed in a murine model27 that macrophage yield decreased as tumor burden increased. When the tumor was surgically excised the monocyte to macrophage yield improved and reached near-normal pretumor induction values. To evaluate the possible use of this assay in a clinical situation we evaluated mononuclear cell preparation and development of macrophages in an in vitro system in these patients. The results are shown in tables 3 and 4. They indicate that although there is no significant change in the number of cells recovered per ml. of blood from the Ficoll-Hypaque gradient centrifugation, there were significantly (p <0.05) fewer lymphocytes found in the gradient in the pretreatment period. This difference was in both the number oflymphocytes obtained per ml. of blood as well as in the percentage of lymphocytes in the Ficoll-Hypaque separated cells during the posttreatment period. The value increased toward normal during the posttreatment period. On the other hand, the number of polymorphonuclear cells found in the Ficoll-Hypaque gradient was higher in the pretreatment period, but reduced in the posttreatment period. The difference was not statistically significant (p = 0.05). This may become significant as more patients are evaluated. The reason for the excess polymorphonuclear cells in the Ficoll-Hypaque gradient is not known. In the case of patients with lung carcinomas, it has been shown that lymphocytes from the Ficoll-Hypaque gradient interface correlate with the state of the disease. 28 Analysis of the Ficoll-Hypaque gradient separated leukocytes has shown that patients who had a low percentage of lymphocytes had poor prognosis compared to pa-
600
KRISHNAN AND ASSOCIATES MONOCYTE MATURATION PER ML OF BLOOD PRE THERAPY AND POST THERAPY
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may reflect the extent of metabolic disturbance in the patient, or it may be that a subset of monocytes that matures into macrophages is deficient. Recently monocyte adherence has been known to improve in cancer patients after immunotherapy. 33 Since monocytes appear to be multi-functional cells controlling their own function as well as the function of other peripheral cells, we hope that the study of these important cells will provide clinically useful information. Though the series is small, the results are encouraging, especially in the absence of other quantitative laboratory evaluations in patients with renal cell carcinoma. The technique is simple and reproducibility is good, and appears to be amenable to regular laboratory use.
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REFERENCES
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PRE THERAPY
POST
THERAPY
Fm. 2. Number of macrophages recovered per ml. of blood pre- and posttreatment period. These values are obtained by calculating the number of macrophages obtained per culture, X the number of cells per ml. of blood. Nonresponders to the treatment are indicated with a star beside their values.
tients who had a higher percentage of lymphocytes in head and neck cancer. 29 In our study, the most statistically significant change found was in the percentage of plated monocytes giving rise to macrophages in in vitro culture (fig. 2). The percentage of macrophages and also the number of monocytes that matured per ml. of blood increased during the posttreatment period. In these patients the percentage of monocytes yielding macrophages changed from 6.32 ± 4.60 per cent pretreatment to 13.00 ± 5.00 per cent during the posttreatment period. In terms of the number of monocytes which matured into macrophages per ml. of blood, the mean pretreatment value of 1.47 ± 1.16 X 104 cells/ml. improved to 3.45 ± 2.52 cells/ml. In spite of the limited numbers, the values are statistically significant. These values continue to improve as patients improve clinically. Two patients who did not show improvement in in vitro monocyte maturation during the posttreatment evaluation period were found to have metastasis (fig. 2). We have been following 27 patients throughout their clinical course. Future communications would include the relevance of this kind of in vitro assay in detecting recurrence of the disease in nonresponders. In the 10 patients we studied in both the pre- and posttreatment periods, we had 2 patients who had recurrences by the time they finished their treatment regimen. In these 2 patients, the macrophage yield did not improve. The results are encouraging. We are in the process of correlating clinical outcome and in vitro evaluation of these results in a separate communication. It was shown that large numbers of mature macrophages accumulate in draining lymph nodes of nonmetastasizing tumors, while low numbers of macrophages were found in nodes of metastatic tumors. 30 It has been shown recently that maturation of monocytes is significantly depressed in patients with squamous cell carcinoma. 31 Earlier, in a group of patients with melanoma, 10 and in breast carcinoma, 11 we found similar depressed monocyte maturation. The decrease in monocyte maturation has been related to prognosis in primary breast carcinoma.32 The mechanism leading to the lack of maturability of monocytes in vitro into macrophages in patients with malignant disease is not known. This may indicate an intrinsic defect in the maturation processes of the monocytes in patients with malignant disease or
1. Skinner, D. G., Colvin, R. B., Vermillion, C. D., Pfister, R. C. and Leadbetter, W. F.: Diagnosis and management of renal cell carcinoma. Cancer, 28: 1165, 1971. 2. Lokich, J. J. and Harrison, J. H.: Renal cell carcinoma: natural history and chemotherapeutic experience. J. Urol., 114: 371, 1975. 3. Johnson, D. E. and Samuels, M. C.: Chemotherapy for metastatic renal cell carcinoma. In: Cancer Chemotherapy: Fundamental Concepts and Recent Advances. Chicago: Yearbook Medical Publishers, Inc., 1971. 4. Talley, R. W.: Proceedings: chemotherapy of adenocarcinoma of the kidney. Cancer, 32: 1062, 1973. 5. Mohr, S. J. and Whitesel, J. A.: Spontaneous regression of renal cell carcinoma metastases after preoperative embolization of primary tumor and subsequent nephrectomy. Urology, 14: 5, 1979. 6. Bracken, R. B., Johnson, D. E., Goldstein, H. M., Wallace, S. and Ayala, A. G.: Percutaneous transfemoral renal artery occlusion in patients with renal carcinoma: preliminary report. Urology, 6: 6, 1975. 7. Paster, S. B., Bergreen, P. and Schwarz, H.: Percutaneous catheteraided infarction of renal tumors: a preliminary report. J. Urol., 114: 351, 1975. 8. Johnson, D. E., Bracken, R. B., Swanson, D. A., et al.: Arterial occlusive techniques in the management of renal diseases. (in press) 9. Krishnan, E. C., Krishnan, L., Stephens, R. and Jewell, W.R.: In vitro culture of monocytes in normal individuals and patients with breast carcinoma. Breast Cancer Research and Treatment 1: 164, 1981. 10. Krishnan, E. C., Menon, C. D., Krishnan, L. and Jewell, W. R.: Deficiency in maturation process of macrophages in human cancer. JNCI, 65: 273, 1980. 11. Krishnan, E. C., Mebust, W. K., Weigel, J. W. and Jewell, W. R.: Maturation of monocytes in patients with renal cell carcinoma. Invest. Urol., 19: 4, 1981. 12. Tagliabue, A., Mantovani, A., Kilgallen, M., Herberman, R. B. and McCoy, J. L.: Natural cytotoxicity of mouse monocytes and macrophages. J. Immunol., 122: 2363, 1979. 13. Basten, A. and Mitchell, J.: Role of macrophages in T cell-B cell collaboration in antibody production. In: lmmunobiology of the Macrophage. Edited by D.S. Nelson. New York: Academic Press. p. 45, 1976. 14. Erb, P. and Feldmann, M.: Role of macrophages in in vitro induction of T-helper cells. Nature, 254: 352, 1975. 15. Bergholtz, B. 0. and Thorsby, E.: HLA-D restriction of the macrophage-dependent response of immune human T lymphocytes to PPD in vitro: inhibition by anti-HLA-DR antisera. Scand. J. lmmunol., 8: 63, 1978. 16. Chervenick, P.A. and LoBuglio, A. F.: Human blood monocytes: stimulators granulocyte and mononuclear colony formation in vitro. Science, 178: 164, 1972. 17. Gery, I. and Waksman, B. H.: Potentiation of the T-lymphocyte response to mitogens IL The cellular source of the potentiating mediator (s). J. Exp. Med., 136: 143, 1972. 18. Nelson, D. S.: Production by stimulated macrophages of factors depressing lymphocyte transformation. Nature, 246: 306, 1973. 19. Ichikawa, Y., Plunznic, D. H. and Sachs, L.: Feedback inhibition of the development of macrophage and granulocyte colonies. I. Inhibition by macrophage. Proc. Natl. Acad. Sci. USA, 58: 1480, 1967. 20. Moore, M. A. S. and Williams, N.: Physical separation of colony stimulating cells from in vitro colony forming cells in hemopoietic
MONOCYTE MATURATION AND CANCER
tissue. J. Cell. Physiol., 80: 195, 1972. 21. Brune, K., Glatt, M., Kiilin, H. and Peskar, B. A.: Pharmacological 22.
23.
24.
25.
26. 27.
control of prostaglandin and thromboxane release from macrophages. Nature, 274: 261, 1978. Humes, J. L., Bonney, R. J., Pelus, L., Dahlgren, M. E., Sadowski, S. J., Kuehl, F. A. and Davies, P.: Macrophages synthesize and release prostaglandins in response to inflammatory stimuli. Nature, 269: 149, 1977. Rhodes, J. M., Bennedsen, J., Larsen, S. 0., Riisgaard, S. and Spiirck, J. V.: Correlation between in vivo and in vitro functional tests for activated macrophages. Infect. Immun., 23: 34, 1979. Schultz, R. M., Pavlidis, N. A., Stylos, W. A. and Chirigos, M.A.: Regulation of macrophage tumoricidal function: a role for prostaglandins of the E series. Science, 202: 320, 1978. Schrek, R. and Rabinowitz, Y.: Effects of phytohemagglutinin on rat and normal and leukemic human blood cells. Proc. Soc. Exp. Biol. Med., 113: 191, 1963. Krishnan, E. C. and Jewell, W. R.: Deficiency of macrophage precursors in malignancy. Fed. Proc., 38: 1220, 1979. Krishnan, E. C., Krishnan, L. and Jewell, W.R.: Interrelationship
28. 29.
30.
31. 32. 33.
601
between monocyte maturation in vitro and tumor growth in murine tumor host. J. Surg. Res., 31: 240, 1981. Check, I. J. and Hunter, R. L.: Changes in Ficoll-Hypaque gradient with advancing stage of lung cancer. Fed. Proc., 38: 1224, 1979. Check, I. J., Hunter, R. L., Lounsbury, B., Rosenberg, K. and Matz, G.: Prediction of survival in head and neck cancer based on leukocyte sedimentation in Ficoll-Hypaque gradients. Laryngoscope, 90: 1281, 1980. Carr, I., Underwood, J. C., McGinty, F. and Wood, P.: The ultrastructure of the local lymphoreticular response to an experimental neoplasm. J. Pathol., 113: 175, 1974. Dent, R. G. and Cole, P.: In vitro monocyte maturation in squamous carcinoma of the lung. Brit. J. Cancer., 43: 486, 1981. Taylor, S. A. and Currie, G. A.: Monocyte maturation and prognosis in primary breast cancer. Brit. Med. J., I: 1050, 1979. Hersh, E. M., Murphy, S. G., Quesada, J. R., Gutterman, J. U., Gschwind, C. R. and Morgan, J.: Effect of immunotherapy with Coryne bacterium parvum and methanol extraction residue of BCG administered intravenously on host defense function in cancer patients. JNCI, 66: 993, 1981.