Characterization of a slow-growing, transplantable rat mammary tumor (MCR-83): a model for endocrine-related cell kinetic studies.

Out of 24 primary mammary tumors, arising in rats of the WAG/Rij Wistar strain after low dose irradiation, with or without prolonged treatment with estrogen, a slow-growing, well differentiated adenocarcinoma (MCR-83) was selected. This tumor, induced by radiation alone, is independent of estrogen pellets for growth after transplantation into adult female rats, but nontransplantable into males or ovariectomized females. Measurements of tumor growth and contents of both estrogen and progesterone receptors on three successive passages are not indicative of a rapid progression in growth rate or to hormone independency. Ovariectomy and treatment with tamoxifen give a pronounced inhibition of tumor growth, whereas neither methotrexate nor cyclophosphamide is effective. Growth rate is significantly increased when rats are given 17 beta-estradiol. Flow cytometric DNA analysis as well as in situ S-phase cell detection with anti-bromodeoxyuridine antibodies show a 3-fold increase in S-phase fraction cells within 4 days after the onset of estrogen treatment. No spontaneous metastases have been found so far, but lung nodules develop after i.v. inoculation of tumor cells. From one of these nodules a fast-growing, hormone independent subline (MCR-86) has been derived, showing both lymphatic and hematogenous dissemination upon s.c. transplantation. By showing several features of hormone responsive human disease in its early stage of progression the MCR-83 tumor system may be a clinically relevant model for studies on endocrine regulation of tumor growth and its therapeutic manipulation.

Two types of tumour sensitivity test compared for platinum derivatives.

For early passages of 15 human tumours grown in nude mice two types of test for prediction of sensitivity to cytostatic drugs were carried out for four platinum compounds. The subrenal capsule assay of Bogden was not found to be useful for these early passages, since the response of duplicate tests was not very different from randomly distributed results. The clonogenic assay as described by Hamburger and Salmon gave reproducible results on the drug sensitivity of those tumour cells that grew colonies in vitro. However, in a limited number of cases irregular colony growth occurred. Lower drug concentrations were apparently more effective in killing cells than higher concentrations. From the replicate test results it became clear that such results are not a reliable indicator of drug sensitivity. Furthermore, the critical drug concentrations for optimal testing of drug effectiveness is probably not well represented by a uniform relation to peak plasma level.

Tumor dependent growth kinetics of human tumor xenografts using the subrenal capsule assay.

Using the subrenal capsule assay modified by us in order to decrease the ingrowth of host cells, evaluation of the growth kinetics of three human ovarian tumors and one human lung tumor were made by multiple measurements of the tumor implantation site over the 6-day growth period. For all tumors, a lag period of 2-3 days was noticed before growth occurred in the subrenal location. In general, estimation of the composition of the fragments growing under the renal capsule did not change greatly in terms of percentage tumor of which they consisted but by day 6 most had shown a significant degree of host cell infiltration despite the effects of pre-implantation immunosuppression. We would suggest that only certain human tumors are suitable for implantation using this technique. Further, it appears that within even this group of selected tumors only some are suitable for drug studies, those having established exponential growth early enough so that a measurable endpoint can be reached within the 6-day time limit of the assay.

Optimal timing of adjuvant chemotherapy in mouse models.

In three mouse tumor models the effect of timing of adjuvant chemotherapy with a single dose of cyclophosphamide was studied. In one tumor, preoperative adjuvant therapy was better than postoperative therapy. In a second tumor, the reverse was the case, and in the third model there was no difference resulting from variation in the timing of cytostatic drug therapy. Obviously, the concept that a standard drug dose always gives a constant relative cell kill is not always a valid simplification of dose-effect relationships in chemotherapy.

Effectiveness of P-aminobenzoyl-O-phenylenediamine (Goe 1734) against mouse, rat, and human tumour cells.

The new N-phenylbenzamide derivative Goe 1734 was tested for its antitumour effects against mouse, rat, and human tumours. The preparation showed marginal activity against leukaemia L1210, moderate activity against Lewis lung carcinoma, and high activity against osteosarcoma C22LR and Brown Norway myeloid leukaemia. In the subrenal capsule assay the drug was active against four (cisplatin: 2) of nine human tumours. An in vitro clonogenic assay did not reveal any activity of Goe 1734 when mouse osteosarcoma or human tumour cells were exposed for only 1 h. However, continuous exposure led to 70% or greater inhibition of colony formation at concentrations of 0.1-1 microgram/ml (osteosarcoma) or 0.2-2 micrograms/ml (human tumours).

Improvements and limitations of the subrenal capsule assay for determining tumour sensitivity to cytostatic drugs.

Cyclophosphamide (CY) prevented the host response from occurring in treated animals, and we therefore evaluated CY and other immunosuppressive forms of pretreatment in normal mice using the subrenal capsule assay initially for transplanted and later also for primary tumours. CY pretreatment, 4 or 4.5 Gy whole-body irradiation and cortisone were superior to silica in reducing host cell infiltration, and irradiation as pretreatment has become our routine technique. The addition of cortisone acetate to irradiation was of minor benefit in only 1/3 transplanted lines. When primary tumours were tested, the irradiation was only rarely able to completely prevent cellular infiltration. Only 7/11 ovarian tumours and 3/9 lung tumours were evaluable as tumour specimens (greater than 50% tumour) in preirradiated mice. The degree of infiltration and fibrosis was similar in transplants in irradiated normal mice or in athymic nude mice, suggesting that these phenomena are largely due to properties of the tumours rather than of the host. The limitations of the technique to some cell lines and occasional primary tumours is obvious.

Preclinical studies on toxicity, antitumour activity and pharmacokinetics of cisplatin and three recently developed derivatives.

Preclinical studies were performed in mice, rats and dogs of cis-diamminedichloroplatinum(II) (CDDP) and its derivatives cis-1,1-di(aminomethyl) cyclohexane platinum(II) sulphate (TNO-6), cis-diammine-1,1-cyclobutanedicarboxylate platinum(II) (CBDCA) and cis-dichloro, trans-dihydroxybis-isopropylamine platinum(IV) (CHIP). In mice toxicity and antitumour activity were determined. All three derivatives were at least as toxic as CDDP for haemopoietic stem cells and were less active than CDDP against the mouse tumours leukaemia L1210 and osteosarcoma C22LR. Toxicology studies in rats revealed no renal toxicity after a single dose of TNO-6. Fractionated doses of TNO-6 and CBDCA did cause renal toxicity but less than CDDP. CHIP produced little or no kidney damage. In dogs, TNO-6 (1.5 mg/kg) produced more severe kidney damage--although this was reversible--than CDDP (2 mg/kg). Half-lives of distribution were 4.0-5.1 min for TNO-6 and 9.7 min for CDDP, while half-lives of elimination were 3.6-6.6 days and 5.9 days respectively. Plasma levels, normalized for the dose, were at least two times higher after TNO-6 than after CDDP. Twelve weeks after drug administration, plasma levels were undetectable, while tissue concentrations could still be measured. The platinum concentration in kidney cortex was higher after CDDP than after TNO-6.

Comparison between macroscopic and microscopic evaluation of tumour responsiveness using the subrenal capsule assay.

Using the subrenal capsule assay in normal mice, a histologic evaluation was made of 8 human primary ovarian tumours and 3 human colon, 2 lung and 5 ovarian carcinomas growing in serial passage in nude mice. The results of the evaluation indicated that there is a tumour- and drug-dependent correlation between the macroscopically and microscopically evaluated effects, with cyclophosphamide demonstrating excellent concordance but adriamycin and cisplatin both demonstrating consistently more tumour cell killing on histologic analysis than could be appreciated macroscopically. Leukocyte infiltration and fibrosis were greatly increased by the latter 2 drugs, leading to unrepresentative macroscopic measurements. Variable amounts of host cell infiltration can also be demonstrated in the untreated control when normal mice are used. The use of nude mice decreases the discrepancy between macroscopic and microscopic evaluation.

Model studies in adjuvant chemotherapy.

We have focused on three aspects of adjuvant chemotherapy applied to mice with one of the metastasizing tumors: Lewis lung carcinoma (LL) or mammary carcinoma 2661 (M2661). The first aspect concerned the timing of adjuvant chemotherapy. To investigate this, tumor-bearing mice were treated postoperatively with cyclophosphamide using a standard regimen. In M2661, adjuvant therapy was marginally effective in contrast to the clearly significant results obtained in LL. Any delay in the initiation of adjuvant therapy decreased the efficacy of the treatment. The effect of administering chemotherapy before surgery was also studied; normally, marginally effective adjuvant therapy was found to become effective when started preoperatively in M2661. In LL, effective adjuvant therapy was found to become less effective when started preoperatively. The second aspect considered was the comparability between the increase in relapse-free survival time and the increase in cure rate as alternate goals of adjuvant therapy. To study this, mice with small, medium, or large residual tumor loads were subjected to surgery and subsequently treated with cyclophosphamide. While the effect of adjuvant therapy on the cure rate increased proportionally with decreasing tumor load, the increase in lifespan in nonsurvivors was not related to tumor load. The final aspect of study was the selection procedure for drugs to be applied in adjuvant treatment in our models. Taking the volume response of large tumors as being predictive for the successful use of the same agent in adjuvant therapy, we obtained both false-negative and false-positive results in our tumor lines.

Comparison of metastatic disease after local tumour treatment with radiotherapy or surgery in various tumour models.

Spontaneous metastases in lymph nodes and/or the lung were obtained after tumour cell inoculation of four mouse tumours and one rat tumour into the foot-pads of syngeneic animals or their F1 hybrids. Following local radiotherapy with doses of 45-80 Gy, significantly more mice died with metastases than following local amputation of the tumour-bearing foot when the 2661 carcinoma was involved. No significant difference was observed after these treatments for the other tumours. The enhancement of metastastic growth after local radiotherapy in the 2661 carcinoma seems not to be due to incomplete killing of tumour cells in the foot. The presence of irradiated normal structures and tumour tissue after radiotherapy promoted the outgrowth of 261 carcinoma cells which were outside the radiation field at the time of treatment. Evidently, even under similar experimental conditions, radiotherapy may enhance the growth of metastases from some tumours and not from others.

Differences and similarities in adjuvant chemotherapy in mouse and man.

The effectiveness of adjuvant chemotherapy is easily demonstrated in many mouse models, whereas in man the response is good but far less spectacular. With the aid of a mathematical model the following variables in which differences may exist between mouse and man were tested: 1. Uniformity of tumour load in population 2. Cell kinetic factors in drug sensitivity 3. Variation of drug sensitivity among tumours 4. Drug penetration into tumour 5. Tumour antigenicity 6. Variation in growth rate among tumours It is concluded that the factors 1 and 3 are responsible for the difference. Mouse models are more sensitive by selection of an effective drug and an optimal tumour load. Other factors seem not to affect the results.