??Original Contribution
BCG AS AN ADJUNCT
TO RADIOTHERAPY:
JAMES C. KENNEDY, M.D., Division of Cancer Research,
Department
Ph.D.
A MOUSE
and THOMAS NOVAK,
of Pathology, Queen’s University Kingston, Ontario, Canada
MODEL? B.Sc.
and the Kingston
General Hospital,
A mouse model was used to mimic conditions in tumors in which radiotherapy had failed to inactivate a small residue of malignant stem cells. This model permitted separate assessment of both the masking effect of a large number of radiation-damaged malignant cells and the damaging effect of the irradiation itself on the capacity of the host defenses to destroy those few malignant stem cells which had survived irradiation with their ability to proliferate indefinitely still intact. It was found that the local administration of Bacillus Calmette Gukin (BCG) stimulated host defenses against CaD2 mammary carcinoma cells enough to overcome both the harmful effects of 400 rad total-body irradiation and the decreased effectiveness of host defense mechanisms in the presence of large numbers of heavily-irradiated CaD2 cells. We suggest therefore that local administration of BCG following radiotherapy might reduce the incidence of local recurrence by sthnulating radiation-damaged and overloaded host defense mechanisms to the extent that they become capable of eliminating those malignant stem cells which survive irradiation.
BCG (Bacillus Calmette Guerin), Radiotherapy,
Irradiation,
INTRODUCTION
Host defenses, Malignant
stem cells.
host defense mechanisms are still functional. It would appear that procedures capable of either increasing the maximum capacity of the host defenses or decreasing the rate of proliferation of the surviving malignant stem cells below that maximum capacity also might result in reduction of the local recurrence rate following radiotherapy. We suspected that the capacity of host defense mechanisms to destroy residual malignant stem cells in an irradiated tumor might be reduced both by the damaging effects of the radiotherapy itself 2~3~5~‘o~18~2’~22~3’ and by ~34the presence of a relatively large number of irradiated stem cells which had lost their ability for unlimited proliferation but which were otherwise quite indistinguishable from the small number of fully functional survivors hidden among them ‘03’6*23226227*28V32*33. These suspicions were verified by the use of a mouse model system. However, the presence of BCG in close proximity to the malignant cells largely restored the capacity of either radiationdamaged or overloaded host defenses to normal levels. We suggest therefore that the injection of BCG or other stimulators of host defense mechanisms into accessible malignant tumors im-
Local recurrence of a malignant tumor following radiotherapy is inevitable whenever two conditions are met: at least one stem cell survives the irradiation with its capacity for unlimited proliferation intact; and the number of malignant daughter cells produced per unit period of time by the surviving stem cells is greater than the maximum capacity of the various host defense mechanisms to destroy such cells within that same period of time. The number of stem cells that are inactivated by irradiation is a function of the dose of radiation used, the quality of that radiation, the total number of stem cells originally present in the tumor, and the in situ radiosensitivity of those cells; therefore, it is quite reasonable to attempt to minimize recurrence by modifying dose schedules, trying various types of radiation, reducing the total number of stem cells in the target area by surgery and/or chemotherapy prior to radiotherapy, or increasing the radiosensitivity of those cells by the use of chemical sensitizers or metabolic manipulation. However, the failure of radiotherapy to remove the capacity for unlimited proliferation from a few residual stem cells in a tumor will not necessarily lead to recurrence if tSupported by the Medical Research Council and the National Cancer Institute of Canada. Acknowledgements-We wish to acknowledge the excellent
technical assistance Frances Paul.
685
of Mrs. Margaret
Legault
and Mrs.
686
Radiation Oncology 0 Biology 0 Physics
mediately following radiotherapy incidence of local recurrence. METHODS
might reduce
the
AND MATERIALS
Mice
DBA/2 males 6-8 weeks old were obtained from the Jackson Laboratories, Bar Harbor, Maine, and maintained undisturbed in our own facilities until they were 10 to 14 weeks old. The various experimental and control groups in each experiment were always matched for both the age of the mice and the date of their arrival. CaD2
mammary
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1977, Volume 2, No. 7 and No. 8
absorbed dose measurements utilizing the ferrous ammonium sulphate dosimete? with an assumed G value of 15.5 ferric ions/lOOeV, and by air ionization measurements made with a Victoreen Condenser RMeter. Variation in dose of radiation from center to edge of the irradiation chamber was + 4%. CaD2 cell suspensions in well-aerated ice-cold medium were placed in polystyrene tubes (Falcon, Oxnard, Ca.) in the centre of the irradiation field, surrounded by crushed ice, and given ‘37Cs irradiation at a dose rate of 107 rad/min. Cell suspensions not being irradiated were kept on ice until used.
gland adenocarcinoma
This transplantable tumor line originated spontaneously in a DBA/2 mouse in 1960, and since that time has gone through many animal passages at the Jackson Laboratories, Bar Harbor, Maine. We obtained our line directly from them and adapted it to tissue culture. For the past 3 years we have carried it in continuous culture in vitro as a pure line free of all host cells, growing it as a monolayer on glass in our standard medium (a modified Eagle’s minimum essential medium plus 10% fetal calf serum) whose composition has been described in detail elsewhere.14 Single cell suspensions of CaD2 cells were prepared by incubating the monolayers at 37°C for up to 10 min in a few ml of 0.85% NaCl containing 0.05% (w/v) trypsin (Sigma Chemical Company, St. Louis, MO.). Further digestion of the suspended cells was blocked by the addition of an equal volume of medium containing 10% fetal calf serum, and the cells then were washed free of trypsin by centrifugation. Complete medium (containing 10% fetal calf serum) was used for resuspension if the cells were to be used for in vitro experiments, and serum-free medium if they were to be injected into mice. Total cell counts and Trypan Blue excluding cell counts were made in 0.04% Trypan Blue in saline by means of Neubauer hemocytometers. BCG vaccine
This was a freeze-dried preparation from Connaught Laboratories, Willowdale, Ontario, Canada, obtained originally from the Pasteur Institute, Paris, France. Exposure to strong light was avoided during preparation, handling, and injection of all suspensions containing BCG. Viability (assessed by Connaught Laboratories) was 12.9 x lo6 colony-forming organisms per mg. Irradiation
Mice were given various doses of total-body irradiation by means of a Gammacell 20 13’Cs irradiator (Atomic Energy of Canada) at a dose rate of 107 rad/min. The irradiator was calibrated both by
In vitro
assay for
proliferative
capacity
of CaD2
cells
1 ml aliquots of each CaD2 cell suspension to be assayed were added to each of 5 polystyrene tissue culture Petri dishes (60 x 15 mm, Falcon, Oxnard, Calif.) containing an additional 4 ml of complete medium. The cells were allowed to settle and proliferate without vibration or other disturbance for 7 days, after which the medium was poured off and the colonies fixed and stained by the addition of 1% (w/v) methylene blue in 50% ethanol and water. The stained colonies are macroscopic, and ordinarily there is no problem counting them. However, heavily-irradiated CaD2 cell suspensions contain a large number of cells which, although badly damaged by the radiation, are still capable of going through a few cell divisions and forming micro-colonies before that damage is fully expressed. Such micro-colonies (less than 50 cells per colony) were never counted, since their cells were too badly damaged to permit continuous proliferation. As the dose of radiation increased, the proportion of microcolonies to normal colonies also increased. Since the total number of cells plated per petri was adjusted for each dose of radiation so that there would always be approximately 100 normal colonies per Petri, the background of micro-colonies became troublesome and eventually obscured the normal colonies as the dose of radiation was increased. Evaluation sions
of host resistance
to CaD2
cell suspen-
0.1 ml of medium (without fetal calf serum) containing the required number of CaD2 cells (it 0.1 mg BCG, _t various numbers of heavily-irradiated CaD2 cells) was injected subcutaneously into the flank of each mouse. The mice were examined for tumors by palpation every Monday, Wednesday and Friday for a minimum of 60 days after the CaD2 cell injections, and the dimensions of each individual tumor were recorded (maximum length, and maximum diameter at right angles to the maximum length) until the product of these measurements was greater than
BCG
as
100 mm’. A “take” was product was 25 mm*.
an adjunct to radiotherapy:
a mouse model 0
first
the
recorded
when
RESULTS Radiation
sensitivity
of
in
colony-forming
vitro
capacity of CaD2 cells These experiments were undertaken to determine how much 13’Cs radiation would be sufficient to completely destroy the proliferative capacity of lo6 CaD2 cells. Aliquots of a single cell suspension of CaD2 cells were prepared, irradiated, and assayed for in vitro colony-forming ability as described in Methods and Materials. Figure 1 combines the data from two similar experiments. The average number of colonyforming cells surviving per aliquot of irradiated cell suspension and the 95% confidence limits at each dose of radiation have been expressed as the decimal fraction of the average number of colony-forming cells per aliquot of non-irradiated cell suspension. The D,, of the resulting curve is 136 rad, and its extrapolation number is 14. Both of these parameters were calculated by the technique of least squares regression analysis, using only those points which lie below 10% survival in order to avoid influence from the non-linear portion of the curve. The non-irradiated CaD2 cells had an absolute plating efficiency of 90%.
J. C. KENNEDY
and
Radiation sensitivity CaD2 cells
T.
NOVAK
of tumor-forming
687
capacity
of
We wanted to find out how much irradiation must be given to a CaD2 cell suspension in order to ensure that the injection of lo6 cells subcutaneously would not result in the formation of a tumor. The radiation sensitivity of the in vitro color,y-forming ability of CaD2 cells is not necessarily the same as the radiation sensitivity of their ability to produce tumors following injection into syngeneic recipients. The repair of radiation-damaged cells might be expected to in vivo ; on the other hand, the be more efficient various host defense mechanisms which can destroy malignant cells in viva are not normally present in Petri dishes. Aliquots of CaD2 cells suspensions were given various doses of radiation (0, 1000, 2000 or 5000rad), following which lo6 cells from each were injected subcutaneously into syngeneic DBA/2 recipients. The lo6 non-irradiated cells produced detectable tumors in 8 out of 8 recipients within less than 6 days. The lo6 cells given 1000 rad produced tumors in 8 of 8 recipients also, but the average time interval between cell injection and first detection of a tumor was increased to 14 days. The lo6 cells given 2000rad or 5000 rad produced no tumors whatever during the 60 days the mice remained under observation. It is possible to calculate from the Do and extrapolation number of the radiation survival curve in Fig. 1 that approximately lo4 of the original lo6 CaD2 cells should survive 1000 rad with their proliferative potential intact; approximately 6 cells should survive 2000rad, and no cells should survive 5000rad. It appears then that survival of the proliferative potential of irradiated CaD2 cell suspensions is no better in viva than in vitro. Consequently, the radiation survival curve for in uitro colony-forming ability can be used to obtain a safe estimate of the minimum dose of radiation required to completely eliminate the tumor-forming capacity of lo6 CaD2 cells.
Effect of BCG on the increased tumor incidence associated with the presence of heavily-irradiated CaD2 cells
,
0
200
,
, 400
,
, 600
I
800
I
1000
I
,
,
1200
Rod r Radiation
Fig. 1. In vitro “‘Cs radiation sensitivity of proliferative function of CaD2 mammary carcinoma cells, assessed by colony formation in vitro. Each point represents the mean normalized colony count and 95% confidence limits of 10 replicate cultures.
Figure 2 summarizes the results of an experiment in which the tumor-forming capacity of various numbers of CaD2 cells was determined in the presence and in the absence of lo6 heavily-irradiated CaD2 cells, with and without the addition of 0.1 mg of BCG. The number of fully-functional CaD2 cells injected per mouse ranged from 10’ to 106; these sometimes were injected alone, sometimes along with lo6 CaD2 cells which had previously been given 10,000 rad, sometimes with 0.1 mg BCG, and sometimes with both lo6 irradiated cells and 0.1 mg BCG. The incidence of tumor formation in each group of 8 mice
688
Radiation Oncology 0 Biology
0
Physics
July-August
1977, Volume 2, No. 7 and No. 8
tive in the presence of lo6 heavily-irradiated CaD2 cells, but that even under such adverse conditions, their effectiveness can be greatly improved by the presence of BCG. Effect of BCG on host resistance to CaD2 cells in irradiated mice Figure 3 summarizes the results of two experiments of similar design in which the incidence of tumor formation at various times following the injection of 4 x lo3 CaD2 cells 2 0.1 mg BCG was determined in normal mice and in mice which had been given 400 rad of total-body irradiation 24 hr prior to injection. Since the results of the two experiments were similar, the data have been combined. Each group contained 60 mice. Figure 3 shows the change in tumor incidence in each group with time following injection of the CaD2 cells + BCG.
IO’
102
Number
103
Id
103
106
of CODZ cells injected
Fig. 2. Effect of BCG on increased incidence of tumor formation associated with the presence of heavily-irradiated CaD2 cells. Each point represents the incidence of tumor formation within 60 days following injection of the indicated numbers of non-irradiated CaD2 cells into 8 mice. 0, normal control; 0, plus 0.1 mg BCG; Cl, plus lo6 heavilyirradiated CaD2 cells; ?? , plus both 0.1 mg BCG and heavily-irradiated CaD2 cells. 60 days later is recorded
in Fig. 2. As expected, none of the irradiation control group (which had been injected with lo6 of the heavily-irradiated cells alone) developed any tumors during the 60 day period of observation. However, the injection of lo6 such cells along with small numbers of non-irradiated CaD2 cells led to an incidence of tumor formation much greater than that observed when the same number of non-irradiated CaD2 cells were injected by themselves. The addition of 0.1 mg BCG to injected CaD2 cell suspensions led to a reduction in tumor incidence, and effectively nullified the enhancing effect of lo6 heavily-irradiated CaD2 cells. These differences can be expressed quantitatively in terms of the number of non-irradiated CaD2 cells which must be injected to produce a tumor incidence of 50%. The 50% end-point for CaD2 cells by themselves was approximately 1 x 104, the presence of 0.1 mg BCG increased this by a factor of 4.4. When lo6 heavilyirradiated CaD2 cells were added, the 50% end-point dropped to 56, but the addition of 0.1 mg BCG increased it by a factor of approximately 178 and returned it to the normal range. It may be concluded that the host’s defense mechanisms against CaD2 cells with tumor-forming potential are not very effec-
z 2 E 2 \, E 2 E 3 0, .u E z 2
70
60 50 40 30 20 )-L-s--*--l
-L-c--
&~.___.
IO
E t
0 0
IO
20
30 Days
after
40 injection
50
60
Fig. 3. Effect of BCG on host resistance to 4 x 10’ CaD2 cells injected into irradiated mice. 0, non-irradiated recipients; 0, non-irradiated recipients given 0.1 mg BCG also; 0, recipients given 400 rad 24 hr prior to injection; H, irradiated recipients given 0.1 mg BCG also. It is apparent that the host defenses against CaD2 cells were somewhat damaged by the 400 rad of totalbody irradiation given 24 hr prior to injection of the cells, but that the effect of this damage could be nullified to a large extent by the presence of BCG. The irradiation treatment increased the tumor incidence by a factor of 2.0 above the non-treated value; BCG reduced the incidence in these irradiated mice by a factor of 2.7 below this same value. Three tumors in the group which received both irradiation and BCG grew slightly larger than 0.5 cm in diameter and then regressed. This shows as a slight fluctuation in the corresponding curve. There were no regressions in any other group.
BCG as an adjunct to radiotherapy:
a mouse modelOJ.
Assessment
of direct toxicity of BCG for CaD2 cells Is the decrease in the in viuo plating efficiency of
CaD2 cells which is observed when BCG is mixed with them immediately prior to injection the result of direct toxicity of BCG for CaD2 cells? CaD2 cells were mixed with BCG in various proportions so that each 5 ml aliquot contained 100 CaD2 cells and from 0.0001 mg to 1.0 mg BCG. Five aliquots of each mixture were put into 60 x 1.5 mm Petri dishes and incubated for 7 days, after which the colonies were stained and counted. The plating efficiency of the CaD2 cells in the absence of BCG was 94%. Those petris which had been given 1.0 mg of BCG along with lOOCaD cells showed an 18% reduction in colony count, but none of the other ratios tested produced any significant reduction. In contrast, the addition of 0.1 mg BCG to lo5 CaD2 cells, a ratio equivalent to 0.0001 mg BCG per 100 CaD2 cells, caused a substantial reduction in CaD2 cell plating efficiency in uivo (Fig. 2). It is apparent then that direct toxicity of BCG for CaD2 cells is not sufficient to account for its effects on tumor incidence observed in the experiments described above. Effect of immunization against CaD2
It is possible to induce some degree of immunity to CaD2 cells in syngeneic recipients. Table 1 summarizes data from an experiment in which agematched DBA/2 mice were injected subcutaneously with either 1 x lo6 heavily-irradiated (10,000 rad) CaD2 cells, 4 x lo3 non-irradiated CaD2 cells, or an equivalent volume of the suspending medium (Eagles’s MEM), and then challenged 53 days later by the subcutaneous injection of a threshold dose of 4 x lo3 CaD2 cells into each flank. The number of tumors which appeared in these mice during the subsequent 58 days are recorded in Table 1 as a fraction of the total number of injected sites. It is Table 1.
Original subcutaneous injection None 0.1 ml. Eagle’s MEM 4 x 10’non-irradiated CaD2 cells4 1 x lo6heavily-irradiated CaD2 cells§
Number of tumors/Number of injection sites? (95% confidence limits) 24/60 = 0.40 (0.27-0.54) 21/60 = 0.35 (0.23-0.49) 53/144 = 0.37 (0.29-0.46) 4/60 = 0.07 (0.01-o. 17)
t4 X lo3 CaD2 cells were injected subcutaneously into one site on each flank. SOnly those mice which did not develop palpable tumors within 53 days of the original injection were given the second injection. §No tumors developed within 53 days of the original injection.
C.
KENNEDY
and T.
NOVAK
689
apparent that the prior injection of 1 X lo6 irradiated CaD2 cells can induce some degree of resistance to tumor formation by a subsequent threshold dose of cells. Stronger resistance to a threshold dose was demonstrable following the surgical removal of CaD2 tumors which had been allowed to grow in the flank until their cross-sectional area was between 50 and 100 mm*. Of the 20 mice so treated and then injected in the contralateral flank with 4 x lo3 CaD2 cells, none developed tumors during the subsequent 63 days of observation. The control mice of Table 1 were controls for this experiment also, since the mice were age-matched and the same CaD2 cell suspension was used in both experiments. However, the ability of some host defense mechanism to destroy a threshold dose of malignant cells is largely irrelevant to the question of whether or not it will be able to destroy a similar or even smaller number of fully functional malignant cells hidden in an irradiated tumor among a very much larger number of cells which lost their ability to proliferate as a result of radiation damage. The results of the experiment described below indicate that DBA/2 mice which had been immunized by the amputation of a CaD2 tumor were not able to destroy as few as 1 x lo6 CaD2 cells even though these cells were not clustered together in a tumor but were injected as a single cell suspension, thus permitting free access by the host defenses to each malignant cell. The 8 mice in each group were injected in the foot-pad of the left hind leg with either 1 x lo5 live CaD2 cells or 0.1 mg BCG, or were left without treatment. Fifteen days later, when obvious tumor growth had taken place in the first group, the left hind legs of all mice were amputated at the knee. Four days later, 1 x lo6 CaD2 cells were injected subcutaneously into the flank. Tumors developed at the site of the injection in all mice. The average time interval from injection of the CaD2 cells to the first evidence of tumor formation was the same in all groups, an indication that relatively few of the injected CaD2 cells had been destroyed as a result of the previous immunization. Moreover, the slopes of the growth curves of the tumors in each group were indistinguishable. It appears then that the immune responses induced by growing CaD2 tumors were not strong enough either to prevent tumor formation by 1 X lo6 CaD2 cells or to perceptibly slow the growth of CaD2 tumors large enough to be detected by palpation. It should be noted that the smallest palpable CaD2 tumor contains more than 1 x lo6 cells. DISCUSSION The local recurrence rate following radiotherapy of various types of strongly immunogenic cancers can
690
Radiation
Oncology
0
Biology
0
Physics
be reduced (in experimental animals) either by prior immunization against the specific cancer in question4S9*25V3’ or by stimulation of both immunologically specific and non-specific host defense mechanisms via systemic administration of such “adjuvants” as methanol-extracted residue of BCG36 or various anaerobic Corynebacteria.” Unfortunately, the spontaneous cancers which arise in humans are only rarely so immunogenic as the carcinogen or virus-induced cancers which generally are used in experimental animal models; consequently, therapeutic techniques which are demonstrably very effective against strongly immunogenic cancers in animals will not necessarily be of great value clinically. Animal experiments support the view that the curability of weakly immunogenic tumors will not be increased by specific immunization prior to radiotherapy.30 Under such conditions, the only useful host defenses will be those which make use of immunologically non-specific mechanisms, and the only useful type of “immunotherapy” will be that which results in the stimulation of such mechanisms. The CaD2 mammary carcinoma in syngeneic mice provides a useful model system for evaluating various techniques for stimulating immunologically non-specific host defense mechanisms, since any tumor-specific responses which might also be stimulated should have little effect on subsequent growth of the tumor. The mouse model system described above was designed to mimic conditions in and about heavilyirradiated tumors in which there are still a few stem cells capable of unlimited proliferation. The model permits distinction between the effect on host defense mechanisms of the irradiation as such, and the effect of a relatively large number of irradiated cancer cells that are physically and chemically indistinguishable from the small number of stem cells which survived the irradiation with their full proliferative potential still intact. We asked the following question: can the administration of BCG significantly improve the effectiveness of host defenses which have been damaged by irradiation or overwhelmed by the presence of a large number of irradiated cancer cells? The data presented above indicate that the improvement to be expected is indeed significant. We suggest therefore that the injection of BCG or other stimulators of host defenses into accessible malignant tumors following radiotherapy might compensate for both the local damage done to the host defenses by the irradiation and the confusion of the host defense mechanisms resulting from the presence of a large number of radiation-damaged decoys among which are
July-August
1977, Volume
2, No.
7 and No.
8
hidden the few all-important cells still capable of forming a tumor. The question of whether or not localized irradiation can result in clinically significant damage to a patient’s immunological reactivity against his cancer is still controversial.37 Consequently, in order to ensure that the mice in our experiments received significant radiation damage to their immune system, we used 400 rad total-body irradiation. This dose is close to the maximum tolerated by DBA/2 males of this age, is strongly immunosuppressive, and is severely damaging to any mechanism requiring cell proliferation.lS.19.20.29 The relatively small decrease in host resistance to injected CaD2 cells which resulted (see Fig. 3) does not permit distinction between a host defense mechanism which requires cell proliferation but recovers quickly by rapid replacement of the radiation-damaged cells, and one which involves cells such as macrophages, which can remain functional for days after a dose of radiation sufficient to severely inhibit cell proliferation.7.‘3.2o The precise mechanism by which BCG stimulates host defenses against malignant cells had not yet been identified with certainty. It is widely accepted that the cells ultimately responsible for the increased effectiveness of host defenses in the presence of BCG are macrophages.‘.8.“.‘2.24 However, a recent report implicates a cell with quite different characteristics.3’ There also is controversy about the mechanism by which heavily-irradiated cells interfere with host defenses against malignant cells. This phenomenon was first reported in 1956,2” when the suggestion was made that it might be the result of either specific stimulation by certain cell products, a non-specific feeder effect, or a response to inflammation. Another early reportI suggested that the irradiated cells interfere with local immune mechanisms. More reimmune cently,” evidence that classical cell-mediated mechanisms are not involved has been presented, along with indirect evidence that fibrin deposition at the site of injection may be an important factor.23 However, all of the data available at present, and our finding that the administration of BCG nullifies the protective effect of heavily-irradiated cells, are compatible with the concept that immunologically nonspecific mechanisms involving the activation of macrophages are primarily responsible for eliminating the last few surviving stem cells in an irradiated tumor. Consequently, any technique leading to the activation of macrophages within and in the immediate vicinity of an irradiated tumor might be expected to reduce the incidence of local recurrence.
REFERENCES 1. Alexander, P.: Activated macrophages and the antitumor action of BCG. Nat1 Cancer Instit. Monograph
39: 127-133,
1973. H.A.S.,
2. van den Brenk,
Burch,
W.M.,
Orton,
C., Shar-
BCG
as an adjunct
to radiotherapy:
a mouse
pington, C.: Stimulation of clonogenic growth of tumor cells and metastases in the lungs by local X-irradiation. Br. J. Cancer 27: 291-306, 1973. 3. Brown, J.M.: The effect of lung
irradiation on the of pulmonary metastases in mice. Br. J. Radial. 46: 613-618, 1973. Cohen, A., Cohen, L.: Radiobiology of the C3H mouse mammary carcinoma: Increased radiosensitivity of the radiation-attenuated isografts. Br. J. Cancer 10: 312317, 1956. Cohen, A., Cohen, L.: Estimation of the cellular lethal dose and the critical cell number for the C3H mouse mammary carcinoma from radiosensitivity studies in vivo. Nature 185: 262-263, 1960. Fricke, H., Hart, E.J.: Chemical dosimetry. In Radiation Dosimetry. 2nd Edn, ed. by Attix, F. H., Roesch, W.C. New York, Academic Press, 1966, Vol. 2, pp. 181-197. Geiger, B., Gallily, R.: Effect of X-irradiation on various functions of murine macrophages. Clin. Exp.
incidence 4.
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lmmunol. 16: 643-655, 1974. 8. Germain, R.N., Williams, R.M., Benacerraf,
B.: Specific and nonspecific antitumor immunity-II. Macrophagemediated non-specific affector activity induced by BCG and similar agents. J. Natl Cancer Znstit. 54: 709-718,
1975. 9. Haddow,
10.
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A., Alexander, P.: An immunological method of increasing the sensitivity of primary sarcomas to local irradiation with X-rays. Lancet (i): 452-457, 1964. Hewitt, H.B., Blake, E., Porter, E.H.: The effect of lethally irradiated cells on the transplantability of murine tumors. Br. J. Cancer 28: 123-135, 1973. Hibbs, Jr., J.B.: Macrophage non-immunologic recognition: target cell factors related to contact inhibition. Science 180: 868-870, 1973. Hopper, D.G., Pimm, M.V., Baldwin, R.W.: Methanol extraction residue of BCG in the treatment of transplanted rat tumors. Br. J. Cancer 31: 176-181, 1975. Jones, T.L., Kennedy, J.C.: Inhibition of parental tumor colony formation by cells from a semi-allogeneic mixed spleen cell reaction. Transplantation 22: 176-183, 1976. Kennedy, J.C., Ekpaha-Mensah, J.A.: Genetics of stimulation of in vitro hemolytic plaque-forming cell responses by irradiated allogeneic spleen cells. J. Zmmunol. 110: 1108-1117, 1973. Kennedy, J.C., Till, J.E., Siminovitch, L., McCulloch, E.A.: Radiosensitivity of the immune response to sheep red cells in the mouse, as measured by the hemolytic plaque method. J. Zmmunol. 94: 715-722, 1965. Mazurek, C., Duplan, J.F.: Stimulation et inhibition de la croissance du carcinome ascitique d’Ehr1ich par des celles tumorales irradites. Bull. Cancer 46: 119-131, 1959. Milas, L., Hunter, N., Withers, H.R.: Combination of local irradiation with systemic application of anaerobic Corynebacteria in therapy of a murine fibrosarcoma.
Cancer Res. 35: 1274-1277, 1975. 18. Moore, M., Lawrence, N., Nisbet,
hibition
mediated by BCG Znt. J. Cancer 15: 897-911, 19. Nichols, W.S., Troup, G.M., sitivity of sensitized and
N.W.: Tumour inin immunosuppressed rats. 1975. Anderson, R.E.: Radiosennonsensitized human lym-
modelOJ.
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NOVAK
691
phocytes evaluated in vitro. Am. J. Pathol. 79: 499-507. 1975. 20. Osoba, D.: Some physical and radiobiological properties of immunologically reactive mouse spleen cells. J. Exp. Med. 132: 368-383, 1970. 21. van Peperzeel, H.A.: Effects of single doses of radiation on lung metastases in man and experimental animals. Europ. J. Cancer 8: 665-675, 1972. of syngeneic murine tumour 22. Peters, L,.J.: Enhancement transplantability by whole body irradiation-a non-immunological phenomenon. Br. J. Cancer 31: 293-300. 1975. 23. Peters,
L.J., Hewitt, H.B.: The influence of fibrin formation on the transplantability of murine tumour cells: Implications for the mechanism of the RCvCsz effect. Br. J. Cancer 29: 279-291, 1974. 24. Pimm, M.V., Baldwin, R.W.: BCG immunotherapy of rat tumours in athymic nude mice. Nature (Land.) 254: 77-78, 1975. 25. Powers, W.E., Palmer, L.A., Tolmach, L.J.: Cellular radiosensitivity and tumor curability. Natl Cancer Inst. Monograph 24: 169-184, 1967. 26. Rev&z, L.: Effect of tumour
cells killed by X-rays upon the growth of admixed viable cells. Nature (Land.) 178: 1391-1392, 1956. 27. Rev&z, L.: Effect of lethally damaged tumour cells upon the development of admixed viable cells. J. Nat/ Cancer Znstit. 20: 1157-l 186, 1958. 28. Scott, O.C.A.: A model system for examining the radiosensitivity of metabolizing layers of cells. Br. J. Cancer 11: 130-136, 1957. 29. Sprent, J., Anderson, R.E., Miller, J.F.A.P.: Radiosensitivity of T and B lymphocytes-II. Effect of irradiation on response of T cells to alloantigens. Europ. J. Zmmunol. 4: 204-210, 1974. 30. Suit, H., Kastelan, A.: Tumor control
by irradiation: a C3H/He mouse mammary carcinoma in mammary-tumor-agent-positive and mammary-tumor-agent-free mice. J. Nat1 Cancer Inst. 40: 945-950, 1968. 31. Suit, H.D., Kastelan, A.: Immunologic status of host and response of a methylcholanthrene-induced sarcoma to local X-irradiation. Cancer 26: 232-238, 1970. 32. Toda, J.K., Yatvin, M.B., Clifton, K.H.: Source of stimulation of tumour inocula by lethally-irradiated cells. Proc. Sot. Exp. Biol. Med. 125: 241-245, 1967. 33. Wallace, A.C.: Effect of delayed addition of irradiated cells to small viable tumor inocula. Cancer Res. 25: 355-357, 34. Withers,
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H.R., Milas, L.: Influence of pre-irradiation of lung on development of artificial pulmonary metastases of fibrosarcoma in mice. CancerRes. 33: 1931-1936,1973. 35. Wolfe, S.A., Tracey, D.E., Henney, C.S.: Induction of “natural killer” cells by BCG. Nature (Lond.) 262: 584-586, 1976. 36. Yron, I., Weiss, D.W., Robinson,
E., Cohen, D., Adelberg, M.G., Mekory, T., Haber, M.: Immunotherapeutic studies in mice with the methanol-extraction residue (MER) fraction. Nat1 Cancer Znstit Monograph 39:
33-54, 1973. 37. Alexander, P.: The bogey of immune suppressive action of local radiotherapy. Znt. J. Radiat. Oncol. Biol. Phip. 1: 369-371, 1976.
BCG AS AN ADJUNCT
TO RADIOTHERAPY:
JAMES C. KENNEDY, M.D., Division of Cancer Research,
Department
Ph.D.
A MOUSE
and THOMAS NOVAK,
of Pathology, Queen’s University Kingston, Ontario, Canada
MODEL? B.Sc.
and the Kingston
General Hospital,
A mouse model was used to mimic conditions in tumors in which radiotherapy had failed to inactivate a small residue of malignant stem cells. This model permitted separate assessment of both the masking effect of a large number of radiation-damaged malignant cells and the damaging effect of the irradiation itself on the capacity of the host defenses to destroy those few malignant stem cells which had survived irradiation with their ability to proliferate indefinitely still intact. It was found that the local administration of Bacillus Calmette Gukin (BCG) stimulated host defenses against CaD2 mammary carcinoma cells enough to overcome both the harmful effects of 400 rad total-body irradiation and the decreased effectiveness of host defense mechanisms in the presence of large numbers of heavily-irradiated CaD2 cells. We suggest therefore that local administration of BCG following radiotherapy might reduce the incidence of local recurrence by sthnulating radiation-damaged and overloaded host defense mechanisms to the extent that they become capable of eliminating those malignant stem cells which survive irradiation.
BCG (Bacillus Calmette Guerin), Radiotherapy,
Irradiation,
INTRODUCTION
Host defenses, Malignant
stem cells.
host defense mechanisms are still functional. It would appear that procedures capable of either increasing the maximum capacity of the host defenses or decreasing the rate of proliferation of the surviving malignant stem cells below that maximum capacity also might result in reduction of the local recurrence rate following radiotherapy. We suspected that the capacity of host defense mechanisms to destroy residual malignant stem cells in an irradiated tumor might be reduced both by the damaging effects of the radiotherapy itself 2~3~5~‘o~18~2’~22~3’ and by ~34the presence of a relatively large number of irradiated stem cells which had lost their ability for unlimited proliferation but which were otherwise quite indistinguishable from the small number of fully functional survivors hidden among them ‘03’6*23226227*28V32*33. These suspicions were verified by the use of a mouse model system. However, the presence of BCG in close proximity to the malignant cells largely restored the capacity of either radiationdamaged or overloaded host defenses to normal levels. We suggest therefore that the injection of BCG or other stimulators of host defense mechanisms into accessible malignant tumors im-
Local recurrence of a malignant tumor following radiotherapy is inevitable whenever two conditions are met: at least one stem cell survives the irradiation with its capacity for unlimited proliferation intact; and the number of malignant daughter cells produced per unit period of time by the surviving stem cells is greater than the maximum capacity of the various host defense mechanisms to destroy such cells within that same period of time. The number of stem cells that are inactivated by irradiation is a function of the dose of radiation used, the quality of that radiation, the total number of stem cells originally present in the tumor, and the in situ radiosensitivity of those cells; therefore, it is quite reasonable to attempt to minimize recurrence by modifying dose schedules, trying various types of radiation, reducing the total number of stem cells in the target area by surgery and/or chemotherapy prior to radiotherapy, or increasing the radiosensitivity of those cells by the use of chemical sensitizers or metabolic manipulation. However, the failure of radiotherapy to remove the capacity for unlimited proliferation from a few residual stem cells in a tumor will not necessarily lead to recurrence if tSupported by the Medical Research Council and the National Cancer Institute of Canada. Acknowledgements-We wish to acknowledge the excellent
technical assistance Frances Paul.
685
of Mrs. Margaret
Legault
and Mrs.
686
Radiation Oncology 0 Biology 0 Physics
mediately following radiotherapy incidence of local recurrence. METHODS
might reduce
the
AND MATERIALS
Mice
DBA/2 males 6-8 weeks old were obtained from the Jackson Laboratories, Bar Harbor, Maine, and maintained undisturbed in our own facilities until they were 10 to 14 weeks old. The various experimental and control groups in each experiment were always matched for both the age of the mice and the date of their arrival. CaD2
mammary
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1977, Volume 2, No. 7 and No. 8
absorbed dose measurements utilizing the ferrous ammonium sulphate dosimete? with an assumed G value of 15.5 ferric ions/lOOeV, and by air ionization measurements made with a Victoreen Condenser RMeter. Variation in dose of radiation from center to edge of the irradiation chamber was + 4%. CaD2 cell suspensions in well-aerated ice-cold medium were placed in polystyrene tubes (Falcon, Oxnard, Ca.) in the centre of the irradiation field, surrounded by crushed ice, and given ‘37Cs irradiation at a dose rate of 107 rad/min. Cell suspensions not being irradiated were kept on ice until used.
gland adenocarcinoma
This transplantable tumor line originated spontaneously in a DBA/2 mouse in 1960, and since that time has gone through many animal passages at the Jackson Laboratories, Bar Harbor, Maine. We obtained our line directly from them and adapted it to tissue culture. For the past 3 years we have carried it in continuous culture in vitro as a pure line free of all host cells, growing it as a monolayer on glass in our standard medium (a modified Eagle’s minimum essential medium plus 10% fetal calf serum) whose composition has been described in detail elsewhere.14 Single cell suspensions of CaD2 cells were prepared by incubating the monolayers at 37°C for up to 10 min in a few ml of 0.85% NaCl containing 0.05% (w/v) trypsin (Sigma Chemical Company, St. Louis, MO.). Further digestion of the suspended cells was blocked by the addition of an equal volume of medium containing 10% fetal calf serum, and the cells then were washed free of trypsin by centrifugation. Complete medium (containing 10% fetal calf serum) was used for resuspension if the cells were to be used for in vitro experiments, and serum-free medium if they were to be injected into mice. Total cell counts and Trypan Blue excluding cell counts were made in 0.04% Trypan Blue in saline by means of Neubauer hemocytometers. BCG vaccine
This was a freeze-dried preparation from Connaught Laboratories, Willowdale, Ontario, Canada, obtained originally from the Pasteur Institute, Paris, France. Exposure to strong light was avoided during preparation, handling, and injection of all suspensions containing BCG. Viability (assessed by Connaught Laboratories) was 12.9 x lo6 colony-forming organisms per mg. Irradiation
Mice were given various doses of total-body irradiation by means of a Gammacell 20 13’Cs irradiator (Atomic Energy of Canada) at a dose rate of 107 rad/min. The irradiator was calibrated both by
In vitro
assay for
proliferative
capacity
of CaD2
cells
1 ml aliquots of each CaD2 cell suspension to be assayed were added to each of 5 polystyrene tissue culture Petri dishes (60 x 15 mm, Falcon, Oxnard, Calif.) containing an additional 4 ml of complete medium. The cells were allowed to settle and proliferate without vibration or other disturbance for 7 days, after which the medium was poured off and the colonies fixed and stained by the addition of 1% (w/v) methylene blue in 50% ethanol and water. The stained colonies are macroscopic, and ordinarily there is no problem counting them. However, heavily-irradiated CaD2 cell suspensions contain a large number of cells which, although badly damaged by the radiation, are still capable of going through a few cell divisions and forming micro-colonies before that damage is fully expressed. Such micro-colonies (less than 50 cells per colony) were never counted, since their cells were too badly damaged to permit continuous proliferation. As the dose of radiation increased, the proportion of microcolonies to normal colonies also increased. Since the total number of cells plated per petri was adjusted for each dose of radiation so that there would always be approximately 100 normal colonies per Petri, the background of micro-colonies became troublesome and eventually obscured the normal colonies as the dose of radiation was increased. Evaluation sions
of host resistance
to CaD2
cell suspen-
0.1 ml of medium (without fetal calf serum) containing the required number of CaD2 cells (it 0.1 mg BCG, _t various numbers of heavily-irradiated CaD2 cells) was injected subcutaneously into the flank of each mouse. The mice were examined for tumors by palpation every Monday, Wednesday and Friday for a minimum of 60 days after the CaD2 cell injections, and the dimensions of each individual tumor were recorded (maximum length, and maximum diameter at right angles to the maximum length) until the product of these measurements was greater than
BCG
as
100 mm’. A “take” was product was 25 mm*.
an adjunct to radiotherapy:
a mouse model 0
first
the
recorded
when
RESULTS Radiation
sensitivity
of
in
colony-forming
vitro
capacity of CaD2 cells These experiments were undertaken to determine how much 13’Cs radiation would be sufficient to completely destroy the proliferative capacity of lo6 CaD2 cells. Aliquots of a single cell suspension of CaD2 cells were prepared, irradiated, and assayed for in vitro colony-forming ability as described in Methods and Materials. Figure 1 combines the data from two similar experiments. The average number of colonyforming cells surviving per aliquot of irradiated cell suspension and the 95% confidence limits at each dose of radiation have been expressed as the decimal fraction of the average number of colony-forming cells per aliquot of non-irradiated cell suspension. The D,, of the resulting curve is 136 rad, and its extrapolation number is 14. Both of these parameters were calculated by the technique of least squares regression analysis, using only those points which lie below 10% survival in order to avoid influence from the non-linear portion of the curve. The non-irradiated CaD2 cells had an absolute plating efficiency of 90%.
J. C. KENNEDY
and
Radiation sensitivity CaD2 cells
T.
NOVAK
of tumor-forming
687
capacity
of
We wanted to find out how much irradiation must be given to a CaD2 cell suspension in order to ensure that the injection of lo6 cells subcutaneously would not result in the formation of a tumor. The radiation sensitivity of the in vitro color,y-forming ability of CaD2 cells is not necessarily the same as the radiation sensitivity of their ability to produce tumors following injection into syngeneic recipients. The repair of radiation-damaged cells might be expected to in vivo ; on the other hand, the be more efficient various host defense mechanisms which can destroy malignant cells in viva are not normally present in Petri dishes. Aliquots of CaD2 cells suspensions were given various doses of radiation (0, 1000, 2000 or 5000rad), following which lo6 cells from each were injected subcutaneously into syngeneic DBA/2 recipients. The lo6 non-irradiated cells produced detectable tumors in 8 out of 8 recipients within less than 6 days. The lo6 cells given 1000 rad produced tumors in 8 of 8 recipients also, but the average time interval between cell injection and first detection of a tumor was increased to 14 days. The lo6 cells given 2000rad or 5000 rad produced no tumors whatever during the 60 days the mice remained under observation. It is possible to calculate from the Do and extrapolation number of the radiation survival curve in Fig. 1 that approximately lo4 of the original lo6 CaD2 cells should survive 1000 rad with their proliferative potential intact; approximately 6 cells should survive 2000rad, and no cells should survive 5000rad. It appears then that survival of the proliferative potential of irradiated CaD2 cell suspensions is no better in viva than in vitro. Consequently, the radiation survival curve for in uitro colony-forming ability can be used to obtain a safe estimate of the minimum dose of radiation required to completely eliminate the tumor-forming capacity of lo6 CaD2 cells.
Effect of BCG on the increased tumor incidence associated with the presence of heavily-irradiated CaD2 cells
,
0
200
,
, 400
,
, 600
I
800
I
1000
I
,
,
1200
Rod r Radiation
Fig. 1. In vitro “‘Cs radiation sensitivity of proliferative function of CaD2 mammary carcinoma cells, assessed by colony formation in vitro. Each point represents the mean normalized colony count and 95% confidence limits of 10 replicate cultures.
Figure 2 summarizes the results of an experiment in which the tumor-forming capacity of various numbers of CaD2 cells was determined in the presence and in the absence of lo6 heavily-irradiated CaD2 cells, with and without the addition of 0.1 mg of BCG. The number of fully-functional CaD2 cells injected per mouse ranged from 10’ to 106; these sometimes were injected alone, sometimes along with lo6 CaD2 cells which had previously been given 10,000 rad, sometimes with 0.1 mg BCG, and sometimes with both lo6 irradiated cells and 0.1 mg BCG. The incidence of tumor formation in each group of 8 mice
688
Radiation Oncology 0 Biology
0
Physics
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1977, Volume 2, No. 7 and No. 8
tive in the presence of lo6 heavily-irradiated CaD2 cells, but that even under such adverse conditions, their effectiveness can be greatly improved by the presence of BCG. Effect of BCG on host resistance to CaD2 cells in irradiated mice Figure 3 summarizes the results of two experiments of similar design in which the incidence of tumor formation at various times following the injection of 4 x lo3 CaD2 cells 2 0.1 mg BCG was determined in normal mice and in mice which had been given 400 rad of total-body irradiation 24 hr prior to injection. Since the results of the two experiments were similar, the data have been combined. Each group contained 60 mice. Figure 3 shows the change in tumor incidence in each group with time following injection of the CaD2 cells + BCG.
IO’
102
Number
103
Id
103
106
of CODZ cells injected
Fig. 2. Effect of BCG on increased incidence of tumor formation associated with the presence of heavily-irradiated CaD2 cells. Each point represents the incidence of tumor formation within 60 days following injection of the indicated numbers of non-irradiated CaD2 cells into 8 mice. 0, normal control; 0, plus 0.1 mg BCG; Cl, plus lo6 heavilyirradiated CaD2 cells; ?? , plus both 0.1 mg BCG and heavily-irradiated CaD2 cells. 60 days later is recorded
in Fig. 2. As expected, none of the irradiation control group (which had been injected with lo6 of the heavily-irradiated cells alone) developed any tumors during the 60 day period of observation. However, the injection of lo6 such cells along with small numbers of non-irradiated CaD2 cells led to an incidence of tumor formation much greater than that observed when the same number of non-irradiated CaD2 cells were injected by themselves. The addition of 0.1 mg BCG to injected CaD2 cell suspensions led to a reduction in tumor incidence, and effectively nullified the enhancing effect of lo6 heavily-irradiated CaD2 cells. These differences can be expressed quantitatively in terms of the number of non-irradiated CaD2 cells which must be injected to produce a tumor incidence of 50%. The 50% end-point for CaD2 cells by themselves was approximately 1 x 104, the presence of 0.1 mg BCG increased this by a factor of 4.4. When lo6 heavilyirradiated CaD2 cells were added, the 50% end-point dropped to 56, but the addition of 0.1 mg BCG increased it by a factor of approximately 178 and returned it to the normal range. It may be concluded that the host’s defense mechanisms against CaD2 cells with tumor-forming potential are not very effec-
z 2 E 2 \, E 2 E 3 0, .u E z 2
70
60 50 40 30 20 )-L-s--*--l
-L-c--
&~.___.
IO
E t
0 0
IO
20
30 Days
after
40 injection
50
60
Fig. 3. Effect of BCG on host resistance to 4 x 10’ CaD2 cells injected into irradiated mice. 0, non-irradiated recipients; 0, non-irradiated recipients given 0.1 mg BCG also; 0, recipients given 400 rad 24 hr prior to injection; H, irradiated recipients given 0.1 mg BCG also. It is apparent that the host defenses against CaD2 cells were somewhat damaged by the 400 rad of totalbody irradiation given 24 hr prior to injection of the cells, but that the effect of this damage could be nullified to a large extent by the presence of BCG. The irradiation treatment increased the tumor incidence by a factor of 2.0 above the non-treated value; BCG reduced the incidence in these irradiated mice by a factor of 2.7 below this same value. Three tumors in the group which received both irradiation and BCG grew slightly larger than 0.5 cm in diameter and then regressed. This shows as a slight fluctuation in the corresponding curve. There were no regressions in any other group.
BCG as an adjunct to radiotherapy:
a mouse modelOJ.
Assessment
of direct toxicity of BCG for CaD2 cells Is the decrease in the in viuo plating efficiency of
CaD2 cells which is observed when BCG is mixed with them immediately prior to injection the result of direct toxicity of BCG for CaD2 cells? CaD2 cells were mixed with BCG in various proportions so that each 5 ml aliquot contained 100 CaD2 cells and from 0.0001 mg to 1.0 mg BCG. Five aliquots of each mixture were put into 60 x 1.5 mm Petri dishes and incubated for 7 days, after which the colonies were stained and counted. The plating efficiency of the CaD2 cells in the absence of BCG was 94%. Those petris which had been given 1.0 mg of BCG along with lOOCaD cells showed an 18% reduction in colony count, but none of the other ratios tested produced any significant reduction. In contrast, the addition of 0.1 mg BCG to lo5 CaD2 cells, a ratio equivalent to 0.0001 mg BCG per 100 CaD2 cells, caused a substantial reduction in CaD2 cell plating efficiency in uivo (Fig. 2). It is apparent then that direct toxicity of BCG for CaD2 cells is not sufficient to account for its effects on tumor incidence observed in the experiments described above. Effect of immunization against CaD2
It is possible to induce some degree of immunity to CaD2 cells in syngeneic recipients. Table 1 summarizes data from an experiment in which agematched DBA/2 mice were injected subcutaneously with either 1 x lo6 heavily-irradiated (10,000 rad) CaD2 cells, 4 x lo3 non-irradiated CaD2 cells, or an equivalent volume of the suspending medium (Eagles’s MEM), and then challenged 53 days later by the subcutaneous injection of a threshold dose of 4 x lo3 CaD2 cells into each flank. The number of tumors which appeared in these mice during the subsequent 58 days are recorded in Table 1 as a fraction of the total number of injected sites. It is Table 1.
Original subcutaneous injection None 0.1 ml. Eagle’s MEM 4 x 10’non-irradiated CaD2 cells4 1 x lo6heavily-irradiated CaD2 cells§
Number of tumors/Number of injection sites? (95% confidence limits) 24/60 = 0.40 (0.27-0.54) 21/60 = 0.35 (0.23-0.49) 53/144 = 0.37 (0.29-0.46) 4/60 = 0.07 (0.01-o. 17)
t4 X lo3 CaD2 cells were injected subcutaneously into one site on each flank. SOnly those mice which did not develop palpable tumors within 53 days of the original injection were given the second injection. §No tumors developed within 53 days of the original injection.
C.
KENNEDY
and T.
NOVAK
689
apparent that the prior injection of 1 X lo6 irradiated CaD2 cells can induce some degree of resistance to tumor formation by a subsequent threshold dose of cells. Stronger resistance to a threshold dose was demonstrable following the surgical removal of CaD2 tumors which had been allowed to grow in the flank until their cross-sectional area was between 50 and 100 mm*. Of the 20 mice so treated and then injected in the contralateral flank with 4 x lo3 CaD2 cells, none developed tumors during the subsequent 63 days of observation. The control mice of Table 1 were controls for this experiment also, since the mice were age-matched and the same CaD2 cell suspension was used in both experiments. However, the ability of some host defense mechanism to destroy a threshold dose of malignant cells is largely irrelevant to the question of whether or not it will be able to destroy a similar or even smaller number of fully functional malignant cells hidden in an irradiated tumor among a very much larger number of cells which lost their ability to proliferate as a result of radiation damage. The results of the experiment described below indicate that DBA/2 mice which had been immunized by the amputation of a CaD2 tumor were not able to destroy as few as 1 x lo6 CaD2 cells even though these cells were not clustered together in a tumor but were injected as a single cell suspension, thus permitting free access by the host defenses to each malignant cell. The 8 mice in each group were injected in the foot-pad of the left hind leg with either 1 x lo5 live CaD2 cells or 0.1 mg BCG, or were left without treatment. Fifteen days later, when obvious tumor growth had taken place in the first group, the left hind legs of all mice were amputated at the knee. Four days later, 1 x lo6 CaD2 cells were injected subcutaneously into the flank. Tumors developed at the site of the injection in all mice. The average time interval from injection of the CaD2 cells to the first evidence of tumor formation was the same in all groups, an indication that relatively few of the injected CaD2 cells had been destroyed as a result of the previous immunization. Moreover, the slopes of the growth curves of the tumors in each group were indistinguishable. It appears then that the immune responses induced by growing CaD2 tumors were not strong enough either to prevent tumor formation by 1 X lo6 CaD2 cells or to perceptibly slow the growth of CaD2 tumors large enough to be detected by palpation. It should be noted that the smallest palpable CaD2 tumor contains more than 1 x lo6 cells. DISCUSSION The local recurrence rate following radiotherapy of various types of strongly immunogenic cancers can
690
Radiation
Oncology
0
Biology
0
Physics
be reduced (in experimental animals) either by prior immunization against the specific cancer in question4S9*25V3’ or by stimulation of both immunologically specific and non-specific host defense mechanisms via systemic administration of such “adjuvants” as methanol-extracted residue of BCG36 or various anaerobic Corynebacteria.” Unfortunately, the spontaneous cancers which arise in humans are only rarely so immunogenic as the carcinogen or virus-induced cancers which generally are used in experimental animal models; consequently, therapeutic techniques which are demonstrably very effective against strongly immunogenic cancers in animals will not necessarily be of great value clinically. Animal experiments support the view that the curability of weakly immunogenic tumors will not be increased by specific immunization prior to radiotherapy.30 Under such conditions, the only useful host defenses will be those which make use of immunologically non-specific mechanisms, and the only useful type of “immunotherapy” will be that which results in the stimulation of such mechanisms. The CaD2 mammary carcinoma in syngeneic mice provides a useful model system for evaluating various techniques for stimulating immunologically non-specific host defense mechanisms, since any tumor-specific responses which might also be stimulated should have little effect on subsequent growth of the tumor. The mouse model system described above was designed to mimic conditions in and about heavilyirradiated tumors in which there are still a few stem cells capable of unlimited proliferation. The model permits distinction between the effect on host defense mechanisms of the irradiation as such, and the effect of a relatively large number of irradiated cancer cells that are physically and chemically indistinguishable from the small number of stem cells which survived the irradiation with their full proliferative potential still intact. We asked the following question: can the administration of BCG significantly improve the effectiveness of host defenses which have been damaged by irradiation or overwhelmed by the presence of a large number of irradiated cancer cells? The data presented above indicate that the improvement to be expected is indeed significant. We suggest therefore that the injection of BCG or other stimulators of host defenses into accessible malignant tumors following radiotherapy might compensate for both the local damage done to the host defenses by the irradiation and the confusion of the host defense mechanisms resulting from the presence of a large number of radiation-damaged decoys among which are
July-August
1977, Volume
2, No.
7 and No.
8
hidden the few all-important cells still capable of forming a tumor. The question of whether or not localized irradiation can result in clinically significant damage to a patient’s immunological reactivity against his cancer is still controversial.37 Consequently, in order to ensure that the mice in our experiments received significant radiation damage to their immune system, we used 400 rad total-body irradiation. This dose is close to the maximum tolerated by DBA/2 males of this age, is strongly immunosuppressive, and is severely damaging to any mechanism requiring cell proliferation.lS.19.20.29 The relatively small decrease in host resistance to injected CaD2 cells which resulted (see Fig. 3) does not permit distinction between a host defense mechanism which requires cell proliferation but recovers quickly by rapid replacement of the radiation-damaged cells, and one which involves cells such as macrophages, which can remain functional for days after a dose of radiation sufficient to severely inhibit cell proliferation.7.‘3.2o The precise mechanism by which BCG stimulates host defenses against malignant cells had not yet been identified with certainty. It is widely accepted that the cells ultimately responsible for the increased effectiveness of host defenses in the presence of BCG are macrophages.‘.8.“.‘2.24 However, a recent report implicates a cell with quite different characteristics.3’ There also is controversy about the mechanism by which heavily-irradiated cells interfere with host defenses against malignant cells. This phenomenon was first reported in 1956,2” when the suggestion was made that it might be the result of either specific stimulation by certain cell products, a non-specific feeder effect, or a response to inflammation. Another early reportI suggested that the irradiated cells interfere with local immune mechanisms. More reimmune cently,” evidence that classical cell-mediated mechanisms are not involved has been presented, along with indirect evidence that fibrin deposition at the site of injection may be an important factor.23 However, all of the data available at present, and our finding that the administration of BCG nullifies the protective effect of heavily-irradiated cells, are compatible with the concept that immunologically nonspecific mechanisms involving the activation of macrophages are primarily responsible for eliminating the last few surviving stem cells in an irradiated tumor. Consequently, any technique leading to the activation of macrophages within and in the immediate vicinity of an irradiated tumor might be expected to reduce the incidence of local recurrence.
REFERENCES 1. Alexander, P.: Activated macrophages and the antitumor action of BCG. Nat1 Cancer Instit. Monograph
39: 127-133,
1973. H.A.S.,
2. van den Brenk,
Burch,
W.M.,
Orton,
C., Shar-
BCG
as an adjunct
to radiotherapy:
a mouse
pington, C.: Stimulation of clonogenic growth of tumor cells and metastases in the lungs by local X-irradiation. Br. J. Cancer 27: 291-306, 1973. 3. Brown, J.M.: The effect of lung
irradiation on the of pulmonary metastases in mice. Br. J. Radial. 46: 613-618, 1973. Cohen, A., Cohen, L.: Radiobiology of the C3H mouse mammary carcinoma: Increased radiosensitivity of the radiation-attenuated isografts. Br. J. Cancer 10: 312317, 1956. Cohen, A., Cohen, L.: Estimation of the cellular lethal dose and the critical cell number for the C3H mouse mammary carcinoma from radiosensitivity studies in vivo. Nature 185: 262-263, 1960. Fricke, H., Hart, E.J.: Chemical dosimetry. In Radiation Dosimetry. 2nd Edn, ed. by Attix, F. H., Roesch, W.C. New York, Academic Press, 1966, Vol. 2, pp. 181-197. Geiger, B., Gallily, R.: Effect of X-irradiation on various functions of murine macrophages. Clin. Exp.
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lmmunol. 16: 643-655, 1974. 8. Germain, R.N., Williams, R.M., Benacerraf,
B.: Specific and nonspecific antitumor immunity-II. Macrophagemediated non-specific affector activity induced by BCG and similar agents. J. Natl Cancer Znstit. 54: 709-718,
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A., Alexander, P.: An immunological method of increasing the sensitivity of primary sarcomas to local irradiation with X-rays. Lancet (i): 452-457, 1964. Hewitt, H.B., Blake, E., Porter, E.H.: The effect of lethally irradiated cells on the transplantability of murine tumors. Br. J. Cancer 28: 123-135, 1973. Hibbs, Jr., J.B.: Macrophage non-immunologic recognition: target cell factors related to contact inhibition. Science 180: 868-870, 1973. Hopper, D.G., Pimm, M.V., Baldwin, R.W.: Methanol extraction residue of BCG in the treatment of transplanted rat tumors. Br. J. Cancer 31: 176-181, 1975. Jones, T.L., Kennedy, J.C.: Inhibition of parental tumor colony formation by cells from a semi-allogeneic mixed spleen cell reaction. Transplantation 22: 176-183, 1976. Kennedy, J.C., Ekpaha-Mensah, J.A.: Genetics of stimulation of in vitro hemolytic plaque-forming cell responses by irradiated allogeneic spleen cells. J. Zmmunol. 110: 1108-1117, 1973. Kennedy, J.C., Till, J.E., Siminovitch, L., McCulloch, E.A.: Radiosensitivity of the immune response to sheep red cells in the mouse, as measured by the hemolytic plaque method. J. Zmmunol. 94: 715-722, 1965. Mazurek, C., Duplan, J.F.: Stimulation et inhibition de la croissance du carcinome ascitique d’Ehr1ich par des celles tumorales irradites. Bull. Cancer 46: 119-131, 1959. Milas, L., Hunter, N., Withers, H.R.: Combination of local irradiation with systemic application of anaerobic Corynebacteria in therapy of a murine fibrosarcoma.
Cancer Res. 35: 1274-1277, 1975. 18. Moore, M., Lawrence, N., Nisbet,
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mediated by BCG Znt. J. Cancer 15: 897-911, 19. Nichols, W.S., Troup, G.M., sitivity of sensitized and
N.W.: Tumour inin immunosuppressed rats. 1975. Anderson, R.E.: Radiosennonsensitized human lym-
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phocytes evaluated in vitro. Am. J. Pathol. 79: 499-507. 1975. 20. Osoba, D.: Some physical and radiobiological properties of immunologically reactive mouse spleen cells. J. Exp. Med. 132: 368-383, 1970. 21. van Peperzeel, H.A.: Effects of single doses of radiation on lung metastases in man and experimental animals. Europ. J. Cancer 8: 665-675, 1972. of syngeneic murine tumour 22. Peters, L,.J.: Enhancement transplantability by whole body irradiation-a non-immunological phenomenon. Br. J. Cancer 31: 293-300. 1975. 23. Peters,
L.J., Hewitt, H.B.: The influence of fibrin formation on the transplantability of murine tumour cells: Implications for the mechanism of the RCvCsz effect. Br. J. Cancer 29: 279-291, 1974. 24. Pimm, M.V., Baldwin, R.W.: BCG immunotherapy of rat tumours in athymic nude mice. Nature (Land.) 254: 77-78, 1975. 25. Powers, W.E., Palmer, L.A., Tolmach, L.J.: Cellular radiosensitivity and tumor curability. Natl Cancer Inst. Monograph 24: 169-184, 1967. 26. Rev&z, L.: Effect of tumour
cells killed by X-rays upon the growth of admixed viable cells. Nature (Land.) 178: 1391-1392, 1956. 27. Rev&z, L.: Effect of lethally damaged tumour cells upon the development of admixed viable cells. J. Nat/ Cancer Znstit. 20: 1157-l 186, 1958. 28. Scott, O.C.A.: A model system for examining the radiosensitivity of metabolizing layers of cells. Br. J. Cancer 11: 130-136, 1957. 29. Sprent, J., Anderson, R.E., Miller, J.F.A.P.: Radiosensitivity of T and B lymphocytes-II. Effect of irradiation on response of T cells to alloantigens. Europ. J. Zmmunol. 4: 204-210, 1974. 30. Suit, H., Kastelan, A.: Tumor control
by irradiation: a C3H/He mouse mammary carcinoma in mammary-tumor-agent-positive and mammary-tumor-agent-free mice. J. Nat1 Cancer Inst. 40: 945-950, 1968. 31. Suit, H.D., Kastelan, A.: Immunologic status of host and response of a methylcholanthrene-induced sarcoma to local X-irradiation. Cancer 26: 232-238, 1970. 32. Toda, J.K., Yatvin, M.B., Clifton, K.H.: Source of stimulation of tumour inocula by lethally-irradiated cells. Proc. Sot. Exp. Biol. Med. 125: 241-245, 1967. 33. Wallace, A.C.: Effect of delayed addition of irradiated cells to small viable tumor inocula. Cancer Res. 25: 355-357, 34. Withers,
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H.R., Milas, L.: Influence of pre-irradiation of lung on development of artificial pulmonary metastases of fibrosarcoma in mice. CancerRes. 33: 1931-1936,1973. 35. Wolfe, S.A., Tracey, D.E., Henney, C.S.: Induction of “natural killer” cells by BCG. Nature (Lond.) 262: 584-586, 1976. 36. Yron, I., Weiss, D.W., Robinson,
E., Cohen, D., Adelberg, M.G., Mekory, T., Haber, M.: Immunotherapeutic studies in mice with the methanol-extraction residue (MER) fraction. Nat1 Cancer Znstit Monograph 39:
33-54, 1973. 37. Alexander, P.: The bogey of immune suppressive action of local radiotherapy. Znt. J. Radiat. Oncol. Biol. Phip. 1: 369-371, 1976.
