Journal o(

J Cancer Res Clin Oncol 97, 15-21 (1980) Cancer Research and

Clinical Oncology Original Papers ~ Springer-Verlag 1980

Adjuvant Chemotherapy of Human Coloreetal Adenocarcinoma After Growth on Mice with Congenital Thymic Dysplasia ("Nude")*

2. Tumor Remission Following Treatment with 5-FU and VCR of Xenutransplanted Primary Human Culorectal Adenucarcinumas

R. Schmitz, C.A. Schmitz, J. Hiiper, and R. Pichlmayr

Clinics of Abdominal Surgery and Transplantation, Department of Surgery (Chairman: Prof. Dr. R. Pichlmayr), Medical School Hospitals, Hannover, Karl-Wiechert-Allee 9, D-3000 Hannover, Federal Republic of Oe~Tnany

Summary. The poor 5-year survival rate of patients with malignant colorectal tumors calls for clarification of the question as to whether it is advisable to car- ry out postoperative adjuvant chemotherapy. In the present study a simple "in vivo" test is described with which the sensitivity of individual human adenocar- cinomas of the colon, primary xenotransplanted onto syngeneic "nude"-mice suffering from congenital thymic dysplasia, to cytostatic therapy with 5-FU and VCR could be determined. Five of six primary xenotransplanted tumors showed a significant reduction in size following combined therapy with these drugs in doses comparable to those administered clinically, whereas one of the transplanted tumors remained unresponsive to therapy. On the basis of these results it is absolutely necessary that a comparison be carried out between the rate of tumor remission in groups of patients and animals undergoing the same chemotherapy.

Key words: Nude mice - Combined chemotherapy - VCR - 5-FU - Adjuvant chemotherapy - Xenotransplantation - Human colorectal adenocarcinomas - Tumor remission

As the prognosis for patients with primary carcinomas is relatively poor once the reginal lymph nodes have been invaded - the 5-year survival rate being approxi- mately 20% (Groteliischen et al. 1975) - the question arises whether directed ad- juvant chemotherapy, i.e., chemotherapy with the prospect of success, should be administered postoperatively. Recently, many reports of the successful transplan- tation of primary human cancers onto "nude"-mice have been published. The fact that these experimental animals which are very susceptible to infection can also be used for experiments with the cytostatic drugs 5-Fluorouracil (5-FU) and Vincris- tine (VCR) (Schmitz 1979) will be reported in the present study. Following the suc- cessful xenotransplantation of primary human colorectal adenocarcinomas, the

* Supported by the Deutsche Forschungsgemeinschaft Offprint requests to: Dr. R. Schmitz (address see above)

0171-5216/80/0097/0015/$1.40

16 R. Schmitz et al.

treatment of these animals with 5-FU and VCR resulted in most cases in the re- mission of tumor growth.

Materials and Methods

The breeding of syngeneic Balb/c-"nude"-mice and the animal experiments were performed in the lab- oratories of the Zentrales Tierlabor of the Medical School Hospitals, Hannover (Director: Prof. Dr. K. G/irtner) under spf-conditions at a temperature of 25 + 2 ~ and with a relative atmospheric humid- ity of 50-60%. The animals were kept in Macrolon cages. The experiments were carried out on over 5-week-old female mice, the tumor material from the patients - except for adenocarcinomas - being transplanted within 60 rain. The tissue was first freed from necrotic material and then washed with cold medium TC 199, followed by a second washing with medium containing neomycin sulfate and baci- tracin. The ratio of medium to antibiotica was 2:1 (500 mg neomycin sulfate, 500 mg bacitracin dis- solved in 100 mt physiological saline to give a 1~ solution). The tissue was cut into 1 m m pieces which were placed with the help of a trocar in the lateral flank o f a mouse under ether narcosis. As a rule, t0 animals were transplanted from one tumor patient and the size of the tumor was measured regularly with calipers. The tumor volume was calculated according to the formula:

n V = ~ (d 1 x d z x d3) with the empirical correction factor 1.4

d 1 = greatest diameter d a = smallest diameter d 3 = height tangential to the body surface

The cytostatic drugs 5-FU and VCR were injected i.p. (volume = 0.2 ml; concentration: VCR = 0.05 mg/kg BW; 5-FU = 10 mg/kg BW).

At the end of the experiment a complete autopsy was performed on all animals and macroscopic and microscopic changes were noted. The heart, lungs, kidneys, spleen, small intestine, bladder, and lymph nodes were embedded in paraffin and stained with HE and either azan or van Gieson according to the question being investigated. The tumor which had grown on the "nude ' -mice and undergone che-

Table 1. Growth of primary human rectal tumors after xenotransplantat ion onto syngeneic Balb/c-"nude"-mice with congenital dysplasia of the thymus

No. Sex Tumor Original Tumor Resting" Histology localization histology take (%) phase after

(days) transplant.

26.05.39 f Sigm/Rect. Mod. diff. 80 36 Adeno-Ca 10.02.29 m Rect. Well diff. 63 48 Adeno-Ca 01.03.22 f Sigm/Rect. Mod. diff. 100 19 Adeno-Ca 30.10.03 b f Sigm/Rect. Mod. diff., 90 46 Adeno-Ca

Mucilagn. 24.10.01 * f Rect. Mod. diff. 100 26 Adeno-Ca 04.07.27 m Rect. Mod. diff. 52 30 Adeno-Ca 30.10.03 b f Sigm/Rect. Mod. diff. 57 44 Adeno-Ca 24.10.01 ~ f Sigm/Rect. Mod. diff. 100 22 Adeno-Ca 23.02.08 m Sigm. Mod. diff. 75 28 Adeno-Ca 17.10.99 m Rect. Mod. diff. 100 36 Adeno-Ca 28.05.06 f Rect. Mod. diff. 85 39 Adeno-Ca

a Time in days until au tonomous tumor growth after transplantation b The tumor material was sufficient for 2 experimental groups

The tumor material was sufficient for 2 experinaental groups

Chemotherapy of Human Colorectal Adenocarciuoma 17

Fig. 1. Infiltration of an adenocarcinoma in the sigma/rectum transitional area (patient female, 26.05.39) H&E x 125

Fig. 2. Successful primary xenotransplanted adenocarcinoma following combined chemotherapy with 5-FU and VCR Human adenocarcinoma growing on a nude mouse. H&E 125 x Stain

18

vol(%)

R. Schmitz et al.

/ / / /

/ J 1OO

so ] ++l'

I I I I I I �9

1 0 3 0 5 0 t ( d ) p . t r a n s p l .

Fig. 3. Decrease and increase in the tumor volumes of groups of syngeneic Balb/c-"nude"-mice receiving combined cytostatic therapy with 5-FU and VCR

motherapy was compared histologically with the original tumor from the respective patient. At specific points of time differential blood counts were performed.

The course of the growth or remission in tumor size was determined for each animal. The median course for each experimental group was then calculated whereby the tumor votumes were expressed as percentages. A volume of 100% was said to be attained if the tumor had shown a clearly measurable increase in growth for at least 14 days after reaching a volume minimum. In most cases it was approxi- mately 1 cm 3.

Results From Table 1 it can be seen that in an average of 80-90% of the animals in each group of syngeneic Balb/c-"nude"-mice (8-9 animals), the rate of tumor growth after primary xenotransplantation was so high that enough tumor carriers were available for subsequent experiments. Following a pause in which the post-trans- plantation tumor volume at first decreases before showing a constant increase, the same growth pattern could be found histologically as in the primary tumor of the patient. The growing tissue met all criteria of malignancy discernible on the light microscope level (Fig. 1 and 2).

Attempts to obtain a significant remission in tumor growth in a group of tumor carrying "nude"-mice were started on the 50 th day after transplantation. At this time an average of 8 animals were available. The chemotherapy was carried out ac- cording to the following schedule:

day 1 2 3

5 -FU (10 mg/kg BW) x x x i.p. VCR (0.05 mg/kg BW) x i.p.

Chemotherapy of Human Colorectal Adenocarcinoma 19

Voi(%)

125

100. .

. . . . . " "

75

5 0

/ / 1 o .1"

/ � 9 / O

.i.I'I[ !

. s O , ~ LI 4.

~,,..

\ \ l "�9149149 o

�9 �9 .�9 L ' , .. 1 "%

".."> l .-T ?' - -o -" L " . I o" t

O~ 1 s I t l ' - o " [

L

n:. 5, Pat.~ 78J.

n : 15, Pat. O 4 5 2 J

FU x x x

VCR x

I 1 I I

1 3 9 18 t (d )

x~SEM

Fig. 4. Different development in the tumor volumes of primary xenotransplanted adenocarcinomas on- to Balb/c-"nude"-mice under the same therapy with 5-FU and VCR

As shown in Fig. 3, under this therapy tumor growth had stopped by the fol- lowing day, the remission lasting between 1-9 days. On the 10 th day the tumor vol- ume started to increase slowly�9

As demonstrated in previous experiments on the response of animals to chemo- therapy (Schmitz 1979), the organs of the tumor-carrying "nude"-mice treated here showed corresponding pathohistological changes. Thus, the transitional epitheli- um of the bladder and the jejunal epithelium underwent regressive changes to 100~o and 50~, respectively, which could mainly be attributed to the chemotherapy�9 The very high number of animals with hepatitis (89~) and pneumonia (93~) lay far over the number expected so that a laboratory infection could not be ruled out.

Figure 4 shows the course of tumor growth in the groups of "nude"-mice treated with 5-FU and VCR. The dotted curve represents the remission in tumor growth shown by the animals from five experimental groups under chemotherapy. The transplanted tumors of one of these groups (lower curve) were particularly re- sponsive to therapy�9 The growth of one xenotransplanted tumor in another ex-

20

Table 2

R. Schmi tz et al.

No. Tumor Original Tumor Respon- Therapy Localisation Histology Take % siveness n animals

Star t End

24.10.01 Rect. Mod. diff. 100 ~ 5 5 26.05.39 Sigm/Rect . Mod. d i f f . 80 b 8 4 10.02.29 Rect. Wel l diff. 63 b 6 4 01.03.22 Sigm/Rect . Mod. diff. 100 b 4 2 30.10.03 Sigm/Rect . Mod. diff. 90 b 9 4

Muci lagn. 04.07.27 Rect. Mod. diff. 52 ~ 15 10

a = very respons ive = respons ive

~ = unrespons ive

perimental group (upper curve) - not belonging to the dotted durve - was not sig- nificantly affected under the same therapy. The tumors depicted in Fig. 4 are shown in detail in Table 2.

Discussion

In recent years a large number of tumors were transplanted onto "nude"-mice showing thymic dysplasia once it became clear that the murine mutant "nude" - a known T cell defect - was immune tolerant (Povlsen and Rygaard 1972). Never- theless, the rate of tumor growth after primary transplantation remained low so that results from different tumors and from different experiments were combined (Povlsen 1971 ; Bastert 1977). Our results show that it is possible to transplant pri- mary human adenocarcinomas with a high tumor take provided sufficient tumor tissue is available. The rate of local infection after transplantation is insignifiant if the tissue is washed in medium supplemented with neomycin.

The tumors grown in mice are histologically identical to the original tumor apart from the lack of connective tissue. It could be shown earlier that the growing tumors retain the human genome (Lindenberger 1978) so that we dispensed with this test in these experiments.

We were interested in determining the possible individual responses of primary transplanted human tumors undergoing standardised chemotherapy in the initial passage as up till now, clinical data about adjuvant palliative chemotherapy has been lacking. With the help of this experimental animal model we were able to dem- onstrate, with one exception, tumors responsive to chemotherapy so that for these patients it could be recommended that chemotherapy with 5-FU and VCR from part of the tumor follow-up, with the reservation that results obtained in an animal model may not be completely valid for human beings. It is well known, however, that chemotherapy represents a considerable physiological burden for the patient and that it can only be carried out as part of restricted surgical program for tumor follow-up. Thus, the selection of patients suitable for chemotherapy with the help of this relatively simple test system appears justified.

Chemotherapy of Human Colorectal Adenocarcinoma 21

References

Bastert G, Schmidt-Matthiesen H, Michel RT, Fortmeyer HP, Sturm R, Nord D, Gerner R (1977) Hu- man Mammary Cancers in nu/nu-Mice. A Model for Testing in Research and Clinic. Klin Wo- chenschr 55:83 84

Groteliischen B, Reilmann H, Pichlmayr R (1975) Entfernung mehrerer Organgebiete als erweiterter Carcinomeingriff. Langenbecks Arch Klin Chir 339:714-715

Lindenberger J, Ganzer U, Fortmeyer HP (1978) Die Heterotransplantation b6sartiger Kopf- und Halsgeschwfilste auf thymusaplastische nackte M/iuse. Arch Oto-Rhino-Laryng 220:117-128

Povlsen CO, Rygaard J (1971) Heterotransplantation of human adenocarcinomas of the colon and rec- tum to the mouse mutant nude. A study of nine consecutive transplantations. Acta Pathol Micro- biol Scand [A] 79:159-169

Povlsen CO, Rygaard J (1972) Heterotransplantation of human epidermoid carcinomas to the mouse mutant nude. Acta Pathol Microbiol Scand [A] 80:713--717

Schmitz CA, Schmitz R, Echtermeyer V, Pichlmayr R (1980) Adjuvante Chemotherapie humaner ko- lorektaler Adeno-Carcinome nach Wachstum auf kongenital thymusdysplatischen Mfiusen ,,Nu- de". 1. Untersuchungen zur Durchffihrbarkeit einer zytostatischen Kombinationstherapie mit Vin- cristin (VCR) und 5-Fluorouracil (5-FU). J Cancer Res Clin Oncol 96:269 279

Received August 21, 1979/Accepted January 14, 1980