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.

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.

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.

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.

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.

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.

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.

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.

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.

Testing prospective anticancer drugs against human tumors using simple in vivo models.

The activities of established and experimental antitumor drugs were tested against the BRO and MeWo human melanomas transplanted in the subrenal capsule of phenotypically immunocompetent (CBA x C57BL/6)F1 mice. Immunosuppression was achieved with preliminary whole-body radiation, and cytological examination showed that the tumors of untreated mice consisted largely of viable tumor cells. Several drugs tested showed moderate activity against one or both tumors. The system described provides a basis for testing anticancer agents against a panel of human melanomas implanted in immunocompetent mice.

Implanted solid human tumours grow under the renal capsule of cyclosporin-immunosuppressed rats.

Cyclosporin (CsA) is a potent immunosuppressive drug widely used in organ transplantation. We transplanted fresh surgical samples from human solid malignant tumours into 45 CsA-immunosuppressed rats. Eight out of nine tumour types grew and remained viable for 5 weeks or more in at least two of the transplanted rats. In 29 rats (64%) a distinct growth of primary human tumours was recorded. Five malignancies (intestinal-type gastric carcinoma, adenocarcinoma of the lung, lymph node metastasis of a testicular teratocarcinoma, soft tissue malignant fibrous histiocytoma (MFH), and small-cell sarcoma) showed invasive and progressive growth. In all five cases the largest tumours were 0.9 cm or over in diameter when the rats were killed 5-9 weeks after transplantation. In three cases (adenocarcinoma of the colon, hypernephroma, and a second MFH) the growth of the implants under the kidney capsule was slow, but small living tumour transplants were still found 3-6 weeks later. In every case the microscopic morphology of the xenograft tumour was identical with the original tumour. In two cases the primary xenografts (teratocarcinoma and small-cell sarcoma) were retransplanted into 11 CsA-immunosuppressed rats. In both types the second passage tumours grew, and the take-off and growth rates were comparable to the primary xenografts. Cyclosporin-treated laboratory rats are an alternative to immunodeficient nude and SCID mice for growing fresh human tumour transplants in vivo. Although a few infections were encountered, most of the rats survived the CsA treatment well for up to 2 months.

Mouse subrenal capsule assay chemosensitivity and DNA flow cytometry parameters of human melanoma metastases.

Chemosensitivity was analyzed by mouse subrenal capsule assay (SRCA) in 103 human melanoma metastases 79 of which were also analyzed by DNA flow cytometry. The most effective combination was dacarbazine, vincristine, and carmustine (DOB), followed by cisplatin plus etoposide (Plat-VP16). By the original criteria of the assay, 35% of tumors were sensitive to DOB treatment while 15% responded to Plat-VP16. Chemosensitivity of DNA diploid and aneuploid tumors showed no significant difference; 35% of the aneuploid tumors were sensitive to DOB and 19% to Plat-VP16, whereas 41% and 13% respectively of the diploid tumors were sensitive. An association between DNA index and chemosensitivity was observed. Growth of the implant was observed in 44% of tumors with a DNA index above 1.5 receiving DOB treatment, whereas only 12% of tumors with an aneuploid DNA index < or = 1.5 grew. No significant difference was observed in the SPF of chemotherapy sensitive and insensitive tumors, though a tendency to lower SPF was observed in sensitive tumors.

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.

Subrenal capsule assay using nude mice as a predictor of the response of the gastric cancer to chemotherapy.

Feasibility of utilizing human gastric cancers as first transplant generation xenografts in nude mice for determining tumor sensitivity to chemotherapeutic agents was demonstrated by applying subrenal capsule (SRC) assay. A total of 55 human gastric tumors from patients were tested in this assay. Mitomycin-C (MMC) and hexycarbamyl-5-FU (HCFU, 5-FU derivative) were selected for the treatment of these patients after surgery and also for this assay as first transplant. Evaluable rate of MMC in this assay was 92.7% and that of HCFU was 90.9%. Sensitivity of tumors to MMC was 25% and to HCFU was 32%. Correlation between response to chemotherapy of human tumors in patients and in nude mice was 78.6%. These results indicate that this assay could predict effective drugs for patients with gastric cancer.

The usefulness of cyclophosphamide pretreatment for subrenal capsule assay against human esophageal cancer.

In order to suppress the host immune reaction in subrenal capsule assay (SRCA) using normal immunocompetent mice, the effects of cyclophosphamide (CPM) pretreatment were compared to those obtained after cyclosporine A (CSA) treatment. CPM and CSA suppressed the host reaction until day 6 and day 12, respectively, however, the histological evaluation of the tumors on day 6 revealed no differences between the two groups. The cytotoxicity of CPM in mouse serum against P388 cells was measured to evaluate the residual activity of CPM against the implanted tumor. No cytotoxicity was observed in the serum 36 hours after the CPM injection. In the histological analysis of clinical samples, inflammatory infiltration was observed in only 4 of 25 samples and tumor cell preservation recognized in 27 of 28 samples. A persistence of tumor cells was recognized in the samples rich in tumor cells. Adequate growth of the tumor in the control groups was obtained from 30 of 33 tumors (91 per cent), and the response rates of this assay were comparable to those of prior clinical experiences on each of these drugs. These results indicated the feasibility of SRCA with CPM pretreatment as a predictive test of chemotherapeutic agents for esophageal cancer.

Subrenal capsule assay in selection of chemotherapy after operation for recurrent ovarian cancer.

Forty-six patients with recurrent ovarian cancer were reoperated, and cancer samples for the subrenal capsule assay (SRCA) were collected from 23 of them, whereas this test was not done in the remaining 23 control patients. The SRCA was evaluable in 22 cases (96%). Taken together, no significant difference appeared in the 3 years' survival figures between the groups: seven of 22 patients (32%) with the evaluable SRCA and six of 23 control patients (26%) were alive. However, a further analysis of the data revealed that the SRCA guided the selection of chemotherapy only in 15 patients, whereas tumour samples were resistant to all cytostatics tested in six cases and toxic side-effects limited the clinical application of the test results in the remaining one case. Four of the 11 patients (36%) whose further chemotherapy was strictly chosen based on the SRCA and seven of the 24 patients (29%) whose treatment was based on physician's choice survived at least 3 years. Our conclusion is that the SRCA is of limited value in the selection of second-line chemotherapy in recurrent ovarian cancer.