Establishment and preclinical application of a patient-derived xenograft model for uterine cancer.

BACKGROUND: The patient-derived xenograft (PDX) model is a promising translational platform for duplicating the characteristics of primary tumors. Here, we established and characterized PDX models of uterine cancer to demonstrate their utility for preclinical drug testing. MATERIALS AND METHODS: We generated PDX tumors surgically derived from 58 cases of uterine cancer. Subrenal capsule xenografts and primary tumors were compared using microscopic examination, short tandem repeat analyses, and targeted sequencing analyses. A phosphatidylinositol 3-kinase (PI3K) inhibitor was administered to mice whose PDX tumors harbored a PTEN deletion or PIK3CA mutation. We also generated an orthotopic PDX model using uterine horn implantation. RESULTS: Thirty-three (56.9%) PDXs were successfully generated and passaged to maintain tumors. The histological features of the PDX tumors were stable over subsequent passages. By contrast, the proportions of epithelial and mesenchymal components of carcinosarcoma PDX models varied by generation. Targeted sequencing analyses revealed that all mutated cancer-related genes were stable during establishment and subgrafting. Treatment with a PI3K inhibitor cased a significant decrease in tumor weight in the clear cell carcinoma PDX harboring a frameshift PTEN deletion (p = 0.049) and in the serous carcinoma PDX harboring a missense PI3KCA mutation (p = 0.003) compared with matched controls. We also successfully established orthotopic PDX models (3/3; 100.0%). CONCLUSIONS: The histological and genetic features of PDXs were similar to those of primary tumors. This model is a promising translational platform for preclinical testing of new anticancer drugs and will enable the personalized development of therapeutic options for uterine cancer.

Patient-derived xenografts of gastrointestinal cancers are susceptible to rapid and delayed B-lymphoproliferation.

Patient-derived cancer xenografts (PDX) are widely used to identify and evaluate novel therapeutic targets, and to test therapeutic approaches in preclinical mouse avatar trials. Despite their widespread use, potential caveats of PDX models remain considerably underappreciated. Here, we demonstrate that EBV-associated B-lymphoproliferations frequently develop following xenotransplantation of human colorectal and pancreatic carcinomas in highly immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice (18/47 and 4/37 mice, respectively), and in derived cell cultures in vitro. Strikingly, even PDX with carcinoma histology can host scarce EBV-infected B-lymphocytes that can fully overgrow carcinoma cells during serial passaging in vitro and in vivo. As serial xenografting is crucial to expand primary tumor tissue for biobanks and cohorts for preclinical mouse avatar trials, the emerging dominance of B-lymphoproliferations in serial PDX represents a serious confounding factor in these models. Consequently, repeated phenotypic assessments of serial PDX are mandatory at each expansion step to verify "bona fide" carcinoma xenografts.

A patient-derived subrenal capsule xenograft model can predict response to adjuvant therapy for cancers in the head of the pancreas.

BACKGROUND: Although gemcitabine is commonly used as adjuvant therapy for pancreatic adenocarcinoma and pancreaticobiliary-type periampullary cancers, not all patients appear to benefit. This translational study evaluates the potential of a patient-derived subrenal capsule pancreatic cancer xenograft (SRCPCX) model to identify within eight weeks after surgery those tumours which will respond to gemcitabine. METHODS: SRCPCXs from 32 pancreatectomy patients were established in six to ten NOD/SCID mice per patient. After four weeks the mice were randomly assigned to receive gemcitabine or saline for four more weeks. After eight weeks, gemcitabine response in the grafts was evaluated by the percentage of tumour growth inhibition (%TGI), histological morphology and immunohistochemical markers (Ki-67, CK7 and cleaved caspase-3). These were collated into an Overall Response. Survival was assessed by Kaplan-Meier and Cox multivariate analyses. RESULTS: 375 of 450 pieces of tissue from 27 of 31 patients were evaluable. In 90% of patients, histopathological and immunostaining features of saline-treated control grafts were concordant with their original tumours. At follow up, six of 15 patients whose tumours had an Overall Response to gemcitabine died, compared with ten of 12 whose tumours did not respond (P = 0.025, Fisher's exact test). This was associated with improved survival on Kaplan-Meier analysis (P = 0.013). Cox multivariate analysis indicated that Overall Response, stage and grade were independent predictors of survival. CONCLUSION: This SRCPCX model retains major histopathological and immunohistochemical characteristics of the original tumour and when a combination of measures is used, enables early assessment of tumour sensitivity to gemcitabine in pancreatic cancers.

[Establishment of a xenograft model of human prostate cancer in mouse].

OBJECTIVE: To establish the murine xenograft model of human prostate cancer by grafting tumor tissues beneath the renal capsule of intact male athymic mouse. METHODS: Fifteen SCID mice were randomly divided into 3 groups (n = 5 each). Tissue recombinants were prepared in vitro with newborn BALB/c murine seminal vesicle mesenchyme (SVM) and surgical isolated human prostate cancer tissues by using recombination technique and then grafted beneath the renal capsule of intact male athymic mouse. At Week 4 after initial implantation, grafts were harvested and tumor sizes calculated. The expressions of human specific markers CK8/18 and vimentin were evaluated by immunohistochemistry to identify the human prostatic origin in grafts. P63 protein, a basal cell marker, was detected in prostate basal membrane to identify whether it was benign or malignant tissue. And the study control was prepared by implanting prostate cancer tissues alone under the renal capsule in SCID mouse. RESULTS: Of all 78 implantation cases in 15 mice, the tumor-forming rates were 100% (39/39) and 94.1% (37/39) respectively in the recombination and prostate cancer alone grafting groups. The recombination group was shown to be more efficient in terms of tumor size and weight in comparison with the prostate cancer alone group [(9.7 ± 3.1) vs (6.8 ± 2.0) mm(3), (12.1 ± 3.6) vs (8.2 ± 2.2) µg, P < 0.01]. There was no difference in serum PSA level between two groups. Grafts were confirmed as human prostate cancer tissues with the expressions of CK8/18 and vimentin. No expression of P63 was detected. CONCLUSION: The xenograft murine model of human prostate cancer is successfully established. It contains stroma components and is particularly suitable for studying the interaction of stroma and epithelia in the in vivo progression of prostate cancer.

Ex vivo model of cranial suture morphogenesis and fate.

BACKGROUND/AIMS: Craniosynostosis, the premature fusion of cranial sutures, is a common congenital defect. In vivo models for studying cranial suture biology impose inherent restrictions on tissue accessibility and manipulation. The present study was performed to investigate the utility of the renal capsule assay in overcoming these limitations and providing a reproducible model system for studying cranial suture morphogenesis and fate. MATERIALS AND METHODS: The posterior frontal suture, which fuses physiologically, and the coronal and sagittal sutures, which remain patent, were dissected from postnatal and embryonic mouse calvaria and placed under the renal capsule of syngeneic recipient mice (n = 72 in total). Sutures were harvested from 1-14 days after transplantation for histological and morphometric analysis. Suture transplants were compared with nonmanipulated sutures at equivalent developmental stages. The derivation of cells associated with the growing transplants was analyzed using beta-actin-GFP (green fluorescent protein) transgenic mice. RESULTS: Sutures transplanted under the renal capsule maintained normal suture morphology and fate with the posterior frontal suture fusing and the coronal and sagittal sutures remaining patent. In posterior frontal suture transplants, the fusion process mimicked in vivo suture fusion with a delay of 1-2 days. In comparison to in vivo suture complexes, transplant thickness and trabeculation were significantly increased. In addition, we found that osteoblasts within the growing transplant were derived from the transplant itself rather than the host. CONCLUSION: The renal capsule supports the growth of cranial sutures. In this system transplanted sutures recapitulate the anatomical development and fate (fusion or patency) of cranial sutures in vivo. This model system will facilitate controlled ex vivo manipulations of both embryonic and postnatal sutures.

Tissue Recombination and Kidney Capsule Transplantation Assays for the Study of Epithelial-Mesenchymal Interactions.

Tissue interactions are crucial during the development of organs. Among the most studied tissue interactions are those that take place between the epithelial cells and the underlying mesenchymal cells, known as epithelial-mesenchymal interactions. Tissue recombination assay is a valuable model to study the mechanisms involved in the regulation of such interactions. Here, we describe how to dissociate and recombine the epithelial and mesenchymal components of the embryonic tooth. In addition, we explain how to transplant the recombined tissues under the kidney capsule of immunocompromised mice in order to allow their further development into a mature tooth.

Occurrence of autoimmunity after xenothymus transplantation in T-cell-deficient mice depends on the thymus transplant technique.

Xenothymus transplantation under the kidney capsule in athymic rodents frequently leads to multiorgan autoimmunity. Herein, we explore whether this is an intrinsic risk of xenothymus grafting or whether it depends on the transplant technique. We developed a new technique of "venous pouch" thymus grafting (heart-xenothymus) and compared this with the conventional kidney subcapsular technique (kidney-xenothymus) in a rat-into-nude-mouse model. Whereas lethal autoimmunity developed in 90% of kidney-xenothymus recipients, all heart-xenothymus grafted mice remained completely healthy. Autoimmunity in heart-xenothymus recipients was absent despite a significantly improved T-cell generation and was associated with significantly higher CD4+CD25+ T-cell frequencies and CD4+CD25+ cell Foxp3 mRNA levels than those observed in kidney-xenothymus recipients. In conclusion, we describe a novel vascular pouch technique of xenothymus transplantation that prevents the development of autoimmunity in nude mice. Our data further suggest that prevention of autoimmunity is related to a superior development of regulatory T-cells.

The tumor microenvironment controls primary effusion lymphoma growth in vivo.

Certain lymphomas in AIDS patients, such as primary effusion lymphoma (PEL), are closely associated with the lymphotropic gamma herpes virus Kaposi's sarcoma-associated herpes virus (KSHV), also called human herpesvirus 8. The virus is thought to be essential for tumorigenesis, yet systems to investigate PEL in vivo are rare. Here we describe PEL tumorigenesis in a new xenograft model. Embedded in Matrigel, PEL cells formed rapid, well-organized, and angiogenic tumors after s.c. implantation of C.B.17 SCID mice. Without Matrigel we did not observe comparable tumors, which implies that extracellular support and/or signaling aids PEL. All of the tumors maintained the KSHV genome, and the KSHV latent protein LANA/orf73 was uniformly expressed. However, the expression profile for key lytic mRNAs, as well as LANA-2/vIRF3, differed between tissue culture and sites of implantation. We did not observe a net effect of ganciclovir on PEL growth in culture or as xenograft. These findings underscore the importance of the microenvironment for PEL tumorigenesis and simplify the preclinical evaluation of potential anticancer agents.

Stability and repeat regeneration potential of the engineered liver tissues under the kidney capsule in mice.

Liver tissue engineering using hepatocyte transplantation has been proposed as a therapeutic alternative to liver transplantation toward several liver diseases. We have previously reported that stable liver tissue with the potential for liver regeneration can be engineered at extrahepatic sites by transplanting mature hepatocytes into an extracellular matrix. The present study was aimed at assessing the liver tissue persistence after induced regeneration by hepatectomy and repeat regeneration potential induced by repeat hepatectomy. Mouse isolated hepatocytes mixed in EHS extracellular matrix gel were transplanted under both kidney capsules of isogenic mice. The hepatocyte survival persisted for over 25 weeks. In some of the mice, we confirmed that the grafted hepatocytes developed a thin layer of liver tissues under the kidney capsule, determined by specific characteristics of differentiated hepatocytes in cord structures between the capillaries. We then assessed the regenerative potential and persistence of the exogenous liver tissue. To induce liver regeneration, we performed a two-thirds hepatectomy at 70 days after hepatocyte transplantation. Three weeks after this procedure, the engineered liver tissues showed active regeneration, reaching serum marker protein levels of 261 +/- 42% of the prehepatectomy level. We found that the regenerated liver tissue was stably maintained for 100 days (length of the experiment). Repeat regeneration potential was established by performing a repeat hepatectomy (that had been two-thirds hepatectomized at day 70) 60 days after the initial hepatectomy. Again, the regenerated engineered liver tissues showed active regeneration as there was an approximately twofold increase in the serum marker protein levels. The present studies demonstrate that liver tissue, which was recognized as a part of the host naive liver in terms of the regeneration profile, could be engineered at a heterologous site that does not have access to the portal circulation.

Development of a high metastatic orthotopic model of human renal cell carcinoma in nude mice: benefits of fragment implantation compared to cell-suspension injection.

In this study we compared the metastatic rate of human renal cell carcinoma SN12C in two orthotopic nude mouse models: surgical orthotopic implantation (SOI) of histologically intact tumor tissue and cellular orthotopic injection (COI) of cell suspensions in the kidney. The primary tumors resulting from SOI were larger and much more locally invasive than primary tumors resulting from COI. SOI generated higher metastatic expression than COI. The differences in metastatic rates in the involved organs (lung, liver, and mediastinal lymph nodes) were 2-3 fold higher in SOI compared to COI (P < 0.05). Median survival time in the SOI model was 40 days, which was significantly shorter than that of COI (68 days) (P < 0.001). Histological observation of the primary tumors from the SOI model demonstrated a much richer vascular network than the COI model. Lymph node and lung metastases were larger and more cellular in the SOI model compared to COI. We conclude that the tissue architecture of the implanted tumor tissue in the SOI model plays an important role in the initiation of primary tumor growth, invasion, and distant metastasis. This study directly demonstrates that the implantation of histologically intact tumor tissue orthotopically allows accurate expression of the clinical features of human renal cancer in nude mice. This model should be of value in cancer research and antimetastatic drug discovery for renal cancer, a currently very poorly responding malignancy.

Drug-sensitivity testing in patients with human oesophageal squamous cell carcinoma.

To evaluate the response to chemotherapeutic agents against human oesophageal cancer, 19 samples were tested by the human tumour clonogenic assay (HTCA), 21 samples by subrenal capsule assay (SRCA) and 33 samples by SRCA with immunosuppressant (IS-SRCA). The evaluability rate of was 21% for HTCA, 95% for SRCA and 91% for IS-SRCA. No active agent was detected by HTCA, however, 29% of the drugs tested by SRCA and 22% by IS-SRCA were considered to be active. Histological analysis revealed substantial inflammatory infiltrates and poor tumour cell preservation with SRCA; however, infiltrates were minimal and there was a high degree of tumour cell preservation with IS-SRCA. The response rates of IS-SRCA were comparable with those of prior clinical tests for each drug. These results suggested that IS-SRCA is the most useful drug sensitivity test for human oesophageal cancer.

Engraftment of splenic tissue as a method to investigate repopulation by hematopoietic cells from host and donor marrow.

The lymphohematopoietic function of the spleen in mice varies dependent on age and hematopoietic requirements. A method was developed to study splenic repopulation of mature and progenitor cell populations by grafting neonatal or adult spleen tissue under the renal capsule of splenectomized mice. Two weeks following implant of irradiated syngeneic neonatal spleens into B6-Ly 5.1 or B6-gfp recipients, host lymphoid (B220(+), CD4/8(+)) and myeloid cells (CD11b(+)) had repopulated the splenic grafts and constituted the majority of cells contained in these heterotopic implants. Notably, the percentage of lymphoid and myeloid cells approximated adult levels in contrast to preimplant neonatal spleen levels. This observation indicated relatively rapid repopulation of the grafted tissue by adult host cells and suggests that the repopulation patterns were regulated by the host. Three months post-implantation, the cell composition in the graft remained comparable to adult levels. Microscopic examination demonstrated normal splenic architecture including follicles and red pulp. Lymphocytes within the graft were functional as indicated by their proliferation in response to lipopolysaccharide (LPS) and concanavalin A (ConA) stimulation. Progenitor cell activity determined by colony-forming unit interleukin-3 (CFU-IL-3) levels was also present in these grafts. Splenic implants were then assessed in transplant models following lethal irradiation and syngeneic or allogeneic bone marrow transplantation (BMT). Two weeks post-BMT, adult splenic tissue implants contained donor-derived B cells, T cells, and myeloid cell populations. As typically detected in the host spleen post-BMT, the grafted tissue also contained elevated levels of donor progenitor cells. By 3 months post-BMT, CFU-IL3 levels in the graft reflected the decreased levels characteristic of adult levels. The functional integrity of post-transplant splenocytes in the implants was also demonstrated by mitogenic responsiveness. In summary, this method should provide a useful model for the transfer of the splenic microenvironment to study the biology of the spleen in non-transplant and BMT settings.

Nondepleting anti-CD4 and soluble interleukin-1 receptor prevent autoimmune destruction of syngeneic islet grafts in diabetic NOD mice.

BACKGROUND: Successful islet transplantation in type 1 diabetes requires tolerance induction of both allo- and autoreactive T-cell responses. Monoclonal antibodies targeting the CD4 coreceptor on T-helper cells have been shown to be effective in this regard. In type 1 diabetes, there is some evidence to suggest that cytokines such as interleukin (IL)-1 may be involved in beta-cell destruction. The high glucose levels associated with type 1 diabetes are also known to be toxic to beta cells. METHOD: The tempo of T-cell and macrophage infiltration into syngeneic islets transplanted into diabetic nonobese diabetic (NOD) mice was examined by immunohistochemistry. We investigated the ability of a nondepleting anti-CD4 monoclonal antibody (YTS177) to induce tolerance to syngeneic islet grafts in female spontaneous diabetic NOD mice and in an adoptive transfer model of diabetes in NOD mice. The spontaneous model was used to test the effect on graft function of perioperative insulin therapy in mice treated with YTS177. The ability of soluble interleukin (sIL)-1 receptor (R) type II (sIL-1RII) to inhibit IL-1 effects in syngeneic islet transplants was also assessed. RESULTS: Cellular infiltration of CD3 cells and macrophages into the islet graft coincided with loss of graft function in untreated mice. Self-tolerance to beta cells was restored with YTS177, allowing long-term graft survival in a proportion of animals. The use of perioperative insulin therapy increased the number of successful grafts in spontaneously diabetic NOD mice treated with YTS177. The combination of YTS177 with sIL-1RII significantly improved the rates of graft survival compared with graft survival in YTS177-treated spontaneously diabetic NOD mice. CONCLUSIONS: Nondepleting anti-CD4 antibodies restore self tolerance to syngeneic islet transplants in diabetic NOD mice. Insulin therapy improves graft survival in mice treated with YTS177. Preventing the action of IL-1 greatly improves graft survival induced with YTS177.

Improved islet yield and function with ductal injection of University of Wisconsin solution before pancreas preservation.

BACKGROUND: Ensuring sufficient islet yield from preserved pancreases is a critical step in clinical islet transplantation. The aim of this study was to investigate whether pancreatic ductal injection, performed at procurement, using a small volume of preservation solution before cold storage (ductal preservation method) would improve islet yield and function from rat pancreases preserved for 6 and 24 hr. MATERIALS AND METHODS: Islets were isolated from Lewis rats. Pancreases were classified into five groups: fresh (group 1); preserved for 6 hr in University of Wisconsin solution without and with ductal preservation (groups 2 and 3); and preserved for 24 hr in University of Wisconsin solution without and with ductal preservation (groups 4 and 5). We assessed islet yield, function, and viability of pancreatic ductal cells. RESULTS: Islet yields per pancreas in groups 1 to 5 were 2010+/-774, 674+/-450, 1418+/-528, 527+/-263, and 1655+/-618 (islet equivalent) (+/-SD), respectively. Stimulation indices in groups 1 to 5 were 11.97+/-3.17, 6.48+/-4.04, 12.44+/-5.65, 2.56+/-2.03, and 5.55+/-2.71. Functional success rates in groups 1 to 5 were 100%, 0%, 100%, 0%, and 66.7%. Percentages of nonviable pancreatic duct cells in groups 1 to 5 were 3.8+/-2.7%, 59.7+/-4.4%, 19.5+/-7.3%, 64.7+/-4.5%, and 17.2+/-2.6%. In all experiments, the differences were significant between the groups without versus the groups with ductal preservation (P<0.05, group 2 vs. group 3 and group 4 vs. group 5). CONCLUSIONS: Ductal preservation improved islet yield and function after 6 and 24 hr of preservation. Well-preserved pancreatic ducts maintained good distribution of collagenase solution.

Development of an immunoprivileged site to prolong islet allograft survival.

Sertoli cells (SC) play a critical role in the maintenance of the immunoprivileged environment of the testis. We hypothesized that preengrafting SC would allow one to develop a vascularized immunoprivileged ectopic site that provides protection for mouse islet allografts. SC, prepared from 9-day Balb/c mice, were transplanted under the kidney capsule in adult Balb/c mice. After SC engraftment (approximately 30 days), mice were rendered diabetic and subsequently implanted with Balb/c or CBA/J islets directly adjacent to the established SC grafts. Preengrafted SC (5.7 +/- 0.2 x 106) had no adverse effect on syngeneic islet graft function. When allogeneic islets were transplanted into the immunoprivileged ectopic site created by preengrafting 6.4 +/- 0.3 x 10(6) SC, mean graft survival was slightly prolonged (32.4 +/- 6.0 days) compared with control mice that received allogeneic islets alone (16.3 +/- 1.5 days; p = 0.329). In contrast, when 4.8 +/- 0.4 x 10(6) SC were preengrafted, islet allograft survival was significantly prolonged (66.1 +/- 9.8 days; p = 0.001). Four of eight mice, preimplanted with 4.8 +/- 0.4 x 10(6) SC, remained normoglycemic throughout the follow-up period (83.8 +/- 8.6 days) and returned to a diabetic state only when the kidneys bearing the composite grafts were removed. Transplantation of islets into an immunoprivileged ectopic site created by preengrafting SC did not affect islet function and, moreover, provided a means of developing an immunopriveliged ectopic site that permits prolonged islet allograft survival without systemic immunosuppression.

Beta-cell sparing in transplanted islets by vascular endothelial growth factor.

We have reported that vascular endothelial growth factor (VEGF) promotes the revascularization of transplanted islets, thereby reducing the initial number required to prevent diabetes. The present study was undertaken to assess other mechanisms of beta-cell sparing by VEGF. For in vitro studies, islets were cultured for 14 days with versus without 20 ng/mL VEGF. Viability, necrosis, and apoptosis were examined by specific staining (Alcein AM, propidium iodide, and annexin/phosphatidylserine). The effects of VEGF on islets were also examined in a proteomic study. In vivo streptozotocin-treated diabetic Lewis rats received 1000 Lewis or Sprague-Dawley islets beneath the renal capsule. Oxygen levels at the transplant site were monitored by a Clark-type oxygen electrode. Fasting blood glucose served as an indicator of islet survival and function. VEGF enhanced oxygen levels at the transplant site. Syngeneic recipients were euglycemic for over 6 months, whereas control islets failed within 30 to 60 days. VEGF prevented allograft rejection for over 14 days, whereas controls were rejected within 6 to 7 days. Immunostaining suggested that VEGF inhibited the presentation of MHC II antigen and promoted islet survival by the inhibition of necrosis and apoptosis. Our proteomic study suggested VEGF preserved systems required for cellular preservation (heat shock proteins) and insulin secretion. VEGF promotes the preservation of isolated and transplanted islets by a variety of mechanisms, including enhanced oxygenation and inhibition of immune rejection, necrosis, and apoptosis. The provision of exogenous VEGF may be a useful adjunct to islet transplantation.

[Methodological aspects on the histological evaluation of subrenal capsule assay (SRCA)].

For the purpose to establish the reliability of SRCA using clinical materials, a histological assessment under the suppression of host reaction was introduced; 1) for the immune suppression cyclosporin A (CsA) was used, 2) to overcome the heterogeneity of fragments, the numbers of implants were increased to ten per group (two pieces/kidney), 3) time flame was 6 days as original, 4) the amounts of drugs were essentially similar to those of Bogden's original method. Drugs solely enough to induce immunosuppression such as 5-FU, CPA and MTX were administered by themselves and those not enough such as MMC, ADM and CDDP were done with least enough usage of CsA, 5) histological changes were analysed from three factors such as grade of degeneration (5 grades), tumor cell amounts (3 grades) and numbers of mitosis (3 grades). The histological analysis form representing the numbers of implants of each group on the indicated places by the individual grades of those 3 factors was devised. With this form, chemosensitivity results were expressed as ++, +, +/- and -. In a panel of more than one +/- results a sensitivity ranking was put on. 5) Macroscopic assessment was made by the measurement of height of the implants at day 6 for simplicity only for future relevance. This method of histological SRCA was applied on one parotid, one pancreas and ten colon cancers where viable tumor cells with frequent mitosis were seen in the CsA treated control groups and, on the other hand, occasional apparent degenerations were observed in some of drug treated groups. These facts verified that this histological assessment was practical and rational and covers the disadvantage of macroscopic findings.

[Subrenal capsule assay using nude mice].

Six-day subrenal capsule assay (SRCA) using normal immunocompetent mice developed by Bogden et al. is a promising in vivo chemosensitivity test. This method, however, has a problem of influence of the host reaction. We compared the tumor growth kinetics and host reaction between normal immunocompetent and nude mice. The tumor diameter increased until day 6 in normal and day 16 in nude mice, respectively. However the histological finding revealed many host reactive cells and few viable tumor cells on day 6 in normal mice, and well preserved tumour cells on day 16 in nude mice. These results were supported by flow cytometrical analysis. Then, we examined two immunosuppressive drugs; cyclophosphamide (EX) and cyclosporin A (CSA) in SRCA. The tumors increased in the diameter until day 16 in both EX and CSA-treated groups, but the results of the histological examination showed that tumor cells were preserved in tissue on day 14 in CSA-treated and day 6 in EX-treated group. These results were also supported by flow cytometrical analysis. From the investigation of antitumour activities of adriamycin (ADR) and mitomycin C, it was suggested that the 12-day assay was suitable if nude mice were used in SRCA, and six-day assay also, if EX-treated normal mice were used. In CSA-treated group, more toxicity of anticancer drugs was manifested than usual. We studied whether or not CSA had a usefulness in SRCA with normal immunocompetent mice. Sixty mg/kg of CSA was given to BDF1 mice daily SC, and various dosage of ADR was given i.v. on day 2. The body weight of BDF1 mice decreased over 20% within 10 days when ADR was given at more than 5 mg/kg. MX-1, a human breast carcinoma cell line, is known to be sensitive to ADR. This tumor was implanted SC in the back of BALB/c nu/nu mice and chemosensitivity was tested against ADR. ADR resulted to be positive at the dose of 8 mg/kg. On the other hand, the dose of 5 mg/kg proved to be negative, and hence the result of SRCA would be false negative, if the dose of ADR is reduced to avoid the toxicity of CSA. The tumor grew slowly when only 60 mg/kg of CSA was given daily for three weeks, and the inhibition rate was 56.2%. The toxicity of CSA was neglected because the body weight loss was approximately 13%. CSA may have the antitumor effect by itself, and EX did not suppress the host reaction sufficiently.(ABSTRACT TRUNCATED AT 400 WORDS)