The Effect of UFI"M Therapy on Primary and Metastatic Colon Cancer froni the Same Human Xenotransplanted into Nude Mice Yutaka TAKAHASHI,Takahito OrrrA, Akishi Ooi, Tomomi OGINO and Masayoshi MAI ABSTRACT: Successful simultaneous transplants of a cancer of the ascending colon from a 60 year old woman, taken from 3 sites: the primary focus, a lymph node metastasis, and a hepatic metastasis, into nude mice yielded KHC (-P, -N, -H) strains. These three strains were compared under uniform conditions of nude mouse transplantation from the standpoints of morphological variation, growth rate, and sensitivity to chemotherapy. The results showed no major differences in morphology or growth rate. However, an effect on chemotherapeutic sensitivity was observed in KHC-P and KHC-N, with reduction rates of 25.8 per cent and 31.4 per cent, respectively, in the MMC only treatment group with large doses, and in KHC-N and KHC-H, with reduction rates of 46.5 per cent and 34.9 per cent, respectively, in the U F r M group. Chemotherapy sensitivity not only exhibited heterogeneity by site, but also differed according to the chemotherapeutic agent used. These results indicate that this method of nude mouse transplantation is a good experimental system for comparing primary foci and metastases under uniform conditions, and also strongly suggest the presence of heterogeneity in sensitivity to chemotherapy. KEY WORDS: cancer chemotherapy, colon cancer, nude mouse, metastatic colon cancer

INTRODUCTION A

_/"Mthough various aspects of hetrogeneity among primary loci and metastases of cancer have been studied, 1-3 there have been few if any studies using h u m a n tumors. This is presumably because there has been no good experimental system for making comParisons under identical conditions. In the preThe Department of Surgery, Cancer Research In -~ stitute, Kanazawa University, Kanazawa, Japan Reprint requests to: Yutaka Takahashi, MD, The Department of Surgery, Cancer Research Institute, Kanazawa University, 4-86 Yoneizumi, Kanazawa 921, Japan

sent study, the authors used a case of cancer of the colon, in which the same patient had metastases in both the lymph nodes and the liver, to attempt simultaneous transplantation into nude mice from three sites: the primary focus, a lymph node metastasis, and a hepatic metastasis. Transplantation and passage were successful for all three sites and the following report describes a comparative study by site of morphological variation, growth rate, and chemotherapeutic efficacy. MATERIALSAND METHODS TUTgtOTS

The tumors used in this study were ex.

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tracted u n d e r aseptic surgical conditions from three sites of a cancer of the ascending colon in a 60 year woman: the primary focus, a lymph node metastasis, and a hepatic metastasis. These were then divided into 5 ram-diameter sections which were separately implanted subcutaneously in the nude mice. Successive transplantations were performed by the same technique and the strains thus obtained n a m e d KHC-P (primary focus), KHC-N (lymph node metastasis), and KHCH (hepatic metastasis). Test animals BALB/c. n u / n u mice aged 6 to 8 weeks were maintained with Isolac in a specific pathogen free state and 5 mice were used in each group. Histological examination The transplanted tumors were extracted from each strain, fixed in formalin, embedded, thinly sliced, and subjected to HE staining. They were then examined by an optical microscope and compared with the corresponding tumors of the patient. Growth rate The major axes (mm) and minor axes (ram) of the transplanted tumors were measured every three days, and approximate weight values (rag) calculated using the following formula: major axis X minor axis2/2 Doubling time in days was obtained from the exponential proliferation period as a measure of the growth rate for each strain and compared. Sensitivity to chemotherapy Two medication groups were established: one which received massive doses of mitomydn C (MMC) only (the MMC-only group), and another, which received MMC combined with UFF as previously reported (the UFTM group). 4 The MMC-only group received 3.0 mg/kg of MMC intra-abdominally twiceon the first and fourth day of treatment respectively, and the UFTM group received 20rag/kg of UFT orally on a daily basis, with 0.5rag/kg ofMMC intra-abdominally once a week (Fig. 1).

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Fig. 1. Schedule of drug administration. Efficacy was determined by finding approximate weight values every three days as described above and comparing changes in the ratio of this value to the value at the start of drug administration, that is, the relative weight, for each group. Treatment was defined as being effective when the ratio of this relative weight to that of the non-therapeutic group was 42 per cem or less, in accordance with the Battelle Columbus Laboratories protocol? Body weights of the nude mice were measured on a regular basis to provide an indicator of side effects. R~SULTS Morphological variation Histological examination revealed mucinous carcinoma with virtually no difference observed between the primary focus and the metastases o f the patient. Moreover, no variation was seen in the t r a n s p l a n t e d tumors of the nude mice {Figs. 2 and 3). Growth rate The doubling time of the three strains showed little disparity at 8 days for KHC-P, 11 days for KHC-N, and 8 days for KHC-H. These results remained virtually the same with successive transplants (Table 1). Sensitivity to chemotherapy On the 24th day, the ratios of relative weight for the MMC-only large dose group

Takahashi et al.

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Fig. 2. a. Primary lesion in the patient, b. Primary lesion transplanted into a nude mouse.

Fig. 3. a. Lymphnode metastasis in the patient, b. Lymphnode metastasis transplanted into a nude mouse.

Table 1. Comparison of Doubling Times among the Three Lines

the lymph node and hepatic metastases. These results iiadiCated differences between sites not only in chemotherapeutic sensitivity, but also according to the chemotherapeutic agent used. Neither o f the treated groups exhibited any marked loss o f weight compared to the non-therapeutic group.

Line

Doubling Time

KHC-P KHC-H KHC-N

8 days 11 days 8 days

vis-gz-vis the non-therapeutic group were 25.8 per cent for KHC-P, 31.4 per cent for KHC-N, and 63.6 per cent for KHC-H. T h e ratios for the UF]?M group were 86.9 per cent, 41.5 per cent and 34.9 per cent respectively (Fig. 4 and Table 2). Thus, in the MMC-only group, treatment was effective against the primary focus and lymph node metastasis, but not against the hepatic metastasis, whereas in the UFTM group, treatment was effective against

DISCUSSION A single malignant tumor will be composed of non-homogeneous cancer cells, and a metastasis originating in one part of the primary focus of a cancer can easily be expected to show marked biological differences from the primary focus. Indeed, such differences are frequently manifested mot-

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UFTM therapy for primary and metastatic colon cancer MMC-only Group

UFTM Group

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Fig. 4. Comparison of drug sensitivity among the three lines. I~, KHC-P (primary focus); A KHC-N (lymph node metastasis); O, KI-IC-H (hepatic metastasis); II, KHC-P (primary focus); A, KHC-N (lymph node metastasis); 0, KHC-H (hepatic metastasis) Table 2. Rations of Relative Weight for Each Group vis-a-vis the Control on the 24th Day KHC-P (primary focus) KHC-N (lymph node metastasis) KHC-H (hepatic metastasis) phologically, as well as clinically by the efficacy o f chemotherapy. In clinical comparisons o f primary foci and their metastases, however, a major problem is posed by conditions related to differences in environment such as blood flow and accessibility to medication. We therefore devised a method 0fconducting a comparative study o f primary and metastatic tumors from a variety o f aspects u n d e r the uniform conditions provided by nude mouse implantation. 6 We succeeded in creating h u m a n colonic cancer strains by successful simultaneous transplantation into nude mice from three sites: KHCP from the primary focus, KHC-N from a lymph node metastasis, and KHC-H from a hepatic metastasis. T h e r e was virtually no morphological variation between the primary focus and the metastases o f this tumor, with histological e x a m i n a t i o n indicating

MMC-only Group

UFI'-M Group

25.8% (effective) 31.4% (effective) 63.6%

86.9% 41.5% (effective) 34.9% (effective)

mucinous a d e n o c a r c i n o m a . Moreover, no differences were observed after nude mouse implantation. As regards growth rate, the doubling time was 8 days for the KHC-P and KHC-H strains, and 11 days for the KHC-N strain, indicating no substantial difference among the three. Although the doubling time for each site in the patient could not be ascertained, the hepatic metastasis was markedly larger than the primary focus at the time o f surgery, suggesting that the rate o f growth o f the hepatic metastasis was c o n s i d e r a b l y faster than that of the primary tumor. However, the fact that virtually no difference was observed among the nude mouse transplants suggests that environmental differences exe r t e d a m u c h g r e a t e r i n f l u e n c e o n the growth rate than did c a n c e r cell heterogeneity.

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Takahashi et al.

T h e d i f f e r e n c e s in sensitivity to c h e m o t h e r a p y w e r e as follows: i n t h e M M C - o n l y l a r g e d o s e group, a n d effect was s e e n o n the K H C - P a n d K H C - N strains, w h i l e in t h e U F T M group, c o m b i n i n g M M C a n d UFT, a n effect was s e e n o n t h e K H C - N a n d K H C - H strains. T h u s , c h e m o t h e r a p e u t i c sensitivity r e f l e c t e d n o t o n l y h e t e r o g e n e i t y a m o n g sites, b u t also v a r i a t i o n a c c o r d i n g to t h e c h e m o t h e r a p e u t i c a g e n t used. Clinically s p e a k i n g , t h e s e results a p p e a r to i n d i c a t e t h a t w h e r e M M C a l o n e is n o t effective, a n effect c a n b e a n t i c i p a t e d i f U F T is c o m b i n e d in t h e regimen. T h e p r e s e n t study was b a s e d o n o n l y o n e case, h o w e v e r , u n d e r t h e u n i f o r m c o n d i t i o n s o f n u d e m o u s e t r a n s p l a n t a t i o n , with t h e lack o f s u b s t a n t i a l v a r i a t i o n in m o r p h o l o g y a n d g r o w t h r a t e n o t w i t h s t a n d i n g , t h e results o f this study s t r o n g l y suggest t h e p r e s e n c e o f h e t e r o g e n e i t y i n c h e m o t h e r a p e u t i c sensitivity.

(Received f o r p u b l i c a t i o n o n J u n . 22, 1989) RE~RENCES 1. Fidler IJ. Selection of successive tumor lines for metastases. Nature 1973; 242: 148-149. 2. Tsnruo T, Eidler IJ. Differences in drug sensitivity among tumor cells from parent tumors, selected variants, and spontaneous metastases. Cancer Res 1981; 41: 3058-3064. 3. Schnipper LE. Clinical implications of tumor cell heterogeneity. N EngJ Med 1986; 314: !423-1431. 4. Takahashi Y, Kikuchi R, Ueno M, Mai M. Effect of combination of UFI" and MMC (UFFM Therapy) of human colonic cancer xenotransplanted in nude mice. Jpn J Cancer Chemother 1987; 14: 1345-1347. (in Japanese) 5. Ovejera AA. Sensitivity of human xenograft in nude mice to various clinicallyactive drugs. In Proc. 2nd Int. Workshop on Nude Mice. Tokyo: Univ Tokyo Press 1977; 451-461. 6. Regaard J, Povlsen CD. Heterotransplantation of a human malignant tumour to "nude" mice. Acta Pathol Microbiol Scand 1969; 77: 758-760.