Establishment and characterization of a carcinoembryonic antigen (CEA)-producing cell line from a human carcinoma of the exocrine pancreas

A human carcinoembryonic antigen (CEA)-producing cell line, T3M-4, has been established from explant cultures of a primary human pancreatic exocrine adenocarcinoma transplanted into nude mice. The tumor had metastasized in the patient. The tumor obtained from metastatic lymph nodes was the initial source for implantation in athymic nude mice. In the primary culture, host fibroblasts were eliminated by the use of the antiserum raised against nude mouse cells. T3M-4 cells have been continuously propagated in vitro during the past 26 months. The cells grew in a monolayered sheet with about 31 hours of population doubling time. The cells exhibited epithelial morphologic features resembling the structure of the original tumor, and they showed tumor takes when inoculated into athymic nude mice. Xenografts established from the cell line have retained a similar histology to the original tumor on serial transplantation. Chromosomal analysis revealed the cell line to be a human aneuploid one with a hyperdiploid mode. T3M-4 cells possess the characteristic function of CEA secretion in vitro in culture and in vivo in nude mice bearing the tumors produced by inoculation with the cultured cells. In view of these characteristics, T3M-4 cell line represents a new human pancreatic exocrine adenocarcinoma cell line that produces CEA.     

Establishment and characterization of a human pancreatic cancer cell line (SUIT-2) producing carcinoembryonic antigen and carbohydrate antigen 19-9

A new tumor cell line (SUIT-2) derived from a metastatic liver tumor of human pancreatic carcinoma has been established in tissue culture and in nude mice, and maintained for over five years. In tissue culture, the cells grew in a monolayered sheet with a population doubling time of about 38.2 hr, and floated or piled up to form small buds above the monolayered surface in relatively confluent cultures. Chromosome counts ranged from 34 to 176 with a modal number of 45. Subcutaneous injection of cultured cells into nude mice resulted in tumor formation, histopathologically closely resembling the original neoplasm which had been classified as moderately differentiated tubular adenocarcinoma. Electron microscopic observation of the neoplastic cells revealed a characteristic pancreatic ductal epithelium. SUIT-2 cell line produces and releases at least two tumor markers, carcinoembryonic antigen and carbohydrate antigen 19-9, propagates even in serum-free medium, and metastasizes to the regional lymph nodes in nude mice xenografts.     

Growth of human digestive-tumour xenografts in athymic nude rats.

The athymic nude rat rnu/rnu has been established as an in vivo model for the acceptance of human digestive-tumour xenografts. We report the successful xenografting of 7/12 (58%) primary explants from patients with digestive cancer. Successful xenografting also occurred in 21/25 (84%) pancreatic tumours derived from a pancreatic exocrine adenocarcinoma (GER) maintained in cell culture; 2 of those have been successfully passaged in nude rats. The simultaneous implantation of these tumours into nude mice led to an almost identical take rate. Passage of one colonic and one pancreatic xenograft from nude rats into nude mice, and transplantation back into nude rats, increased the take rates. The critical period for the establishment of primary tumour growth was usually 28-42 days. The xenografts maintained histological and cytological characteristics of the primary explants or of the original tumour from which the cell line derived. The karyotype of the cell line was also maintained in the solid tumour. Three murine tumours were successfully grown as xenografts. Despite their immunoincompetence, the rats in this study showed no increased morbidity or mortality when kept in conventional conditions, compared with animals housed in isolators. The athymic nude rat will become a valuable complementary tool to the nude mouse for the establishment and maintenance of human digestive tumours and for surgical and serial serological studies.     

Intraductal Delivery of Adenoviruses Targets Pancreatic Tumors in Transgenic Ela-myc Mice and Orthotopic Xenografts

Gene-based anticancer therapies delivered by adenoviruses are limited by the poor viral distribution into the tumor. In the current work we have explored the feasibility of targeting pancreatic tumors through a loco-regional route.We have taken advantage of the ductal network in the pancreas to retrogradelly inject adenoviruses through the common bile duct in two different mouse models of pancreatic carcinogenesis: The transgenic Ela-myc mice that develop mixed neoplasms displaying both acinar-like and duct-like neoplastic cells affecting the whole pancreas; and mice bearing PANC-1 and BxPC-3 orthotopic xenografts that constitute a model of localized human neoplastic tumors. We studied tumor targeting and the anticancer effects of newly thymidine kinase-engineered adenoviruses both in vitro and in vivo, and conducted comparative studies between intraductal or intravenous administration. Our data indicate that the intraductal delivery of adenovirus efficiently targets pancreatic tumors in the two mouse models. The in vivo application of AduPARTK^T plus ganciclovir (GCV) treatment induced tumor regression in Ela-myc mice. Moreover, the intraductal injection of ICOVIR15-TK^T oncolytic adenoviruses significantly improved mean survival of mice bearing PANC-1 and BxPC-3 pancreatic xenografts from 30 to 52 days and from 20 to 68 days respectively (p<0.0001) when combined with GCV. Of notice, both AduPARTK^T and ICOVIR15-TK^T antitumoral responses were stronger by ductal viral application than intravenously, in line with the 38-fold increase in pancreas transduction observed upon ductal administration.In summary our data show that cytotoxic adenoviruses retrogradelly injected to the pancreas can be a feasible approach to treat localized pancreatic tumors.     

Anti-tumor efficacy of the nucleoside analog 1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl) cytosine (4'-thio-FAC) in human pancreatic and ovarian tumor xenograft models

1-(2-deoxy-2-fluoro-4-thio-beta-D-arabinofuranosyl) cytosine (4'-thio-FAC) is a deoxycytidine analog that has been shown previously to have impressive anti-proliferative and cytotoxic effects in vitro and in vivo toward colorectal and gastric tumors. In our present studies, the pharmacokinetic behavior in nude mice and the effectiveness of 4'-thio-FAC against human pancreatic and ovarian tumor growth were assessed in comparison with standard chemotherapeutic agents. Potent in vitro anti-proliferative effects were observed against pancreatic (Capan-1, MIA-PaCa-2, BxPC-3) and ovarian (SK-OV-3, OVCAR-3, ES-2) cancer cell lines with IC(50) of 0.01-0.2 microM. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously (s.c.) implanted human pancreatic tumor xenografts or intraperitoneally (i.p.) disseminated human ovarian xenografted tumors. Oral daily administration of 4'-thio-FAC for 8-10 days significantly inhibited the growth of gemcitabine-resistant BxPC-3 pancreatic tumors and induced regression of gemcitabine-refractory Capan-1 tumors. 4'-Thio-FAC was also a highly effective inhibitor of ovarian peritoneal carcinomatosis. In the SK-OV-3 and ES-2 ovarian cancer models, 4'-thio-FAC prolonged survival to a greater extent than that observed with gemcitabine. Furthermore, the superiority of 4'-thio-FAC to carboplatin and paclitaxel was demonstrated in the ES-2 clear cell ovarian carcinoma model. Studies provide evidence that 4'-thio-FAC is a promising new alternative to gemcitabine and other chemotherapeutic drugs in the treatment of a variety of tumor indications, including pancreatic and ovarian carcinoma.     

Establishment and characterization of human pancreatic cancer cell lines in tissue culture and in nude mice

Three human pancreatic cancer cell lines, designated as KP-1N, KP-2 and KP-3 have been established in both tissue cultures and in nude mice. The KP-1N and KP-3 tumors were obtained from liver metastases of pancreatic tumors and the KP-2 tumors was of primary pancreatic origin. The patients' tumors from which KP-1N and KP-2 were derived showed characteristics of adenocarcinoma, and the KP-3 tumor had adenosquamous carcinoma characteristics. Inoculations of samples from surgical specimens into athymic nude mice resulted in tumor formation, with the tumors histologically closely resembling the original neoplasms. Subcutaneous injections of the established cell lines also induced tumor formation and the tumors histologically resembled the original lesion in the cases of KP-2 and KP-3 tumors, but the KP-1N tumors in the mice were histologically different from the surgical specimen. The KP-1N, KP-2 and KP-3 cell lines have been cultured continuously in a medium supplemented with 10% fetal calf serum for more than 20, 21 and 17 months, respectively. The KP-2 and KP-3 cell lines produced and released carbohydrate antigen 19-9 into the spent medium but the KP-1N cell line did not. KP-1N and KP-3, produced liver metastases after intrasplenic injection into nude mice, whereas KP-2 produced few liver colonies. Cell lines highly metastatic to the liver, KP-1NLs and KP-3Ls, were isolated from the liver colonies of KP-1N and KP-3, respectively.     

In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption

We previously demonstrated the synergistic therapeutic effect of the cetuximab (anti-epidermal growth factor receptor [EGFR] monoclonal antibody, mAb)-trastuzumab (anti-HER2 mAb) combination (2mAbs therapy) in HER2^low human pancreatic carcinoma xenografts. Here, we compared the 2mAbs therapy, the erlotinib (EGFR tyrosine kinase inhibitor [TKI])-trastuzumab combination and lapatinib alone (dual HER2/EGFR TKI) and explored their possible mechanisms of action. The effects on tumor growth and animal survival of the three therapies were assessed in nude mice xenografted with the human pancreatic carcinoma cell lines Capan-1 and BxPC-3. After therapy, EGFR and HER2 expression and AKT phosphorylation in tumor cells were analyzed by Western blot analysis. EGFR/HER2 heterodimerization was quantified in BxPC-3 cells by time-resolved FRET. In K-ras-mutated Capan-1 xenografts, the 2mAbs therapy gave significantly higher inhibition of tumor growth than the erlotinib/trastuzumab combination, whereas in BxPC-3 (wild-type K-ras) xenografts, the erlotinib/trastuzumab combination showed similar growth inhibition but fewer tumor-free mice. Lapatinib showed no antitumor effect in both types of xenografts. The efficacy of the 2mAbs therapy was partly Fc-independent because F(ab′)₂ fragments of the two mAbs significantly inhibited BxPC-3 growth, although with a time-limited therapeutic effect. The 2mAbs therapy was associated with a reduction of EGFR and HER2 expression and AKT phosphorylation. BxPC-3 cells preincubated with the two mAbs showed 50% less EGFR/HER2 heterodimers than controls. In pancreatic carcinoma xenografts, the 2mAbs therapy is more effective than treatments involving dual EGFR/HER2 TKIs. The mechanism of action may involve decreased AKT phosphorylation and/or disruption of EGFR/HER2 heterodimerization.     

Differentiation features of human pancreatic tumor cells maintained in nude mice and in culture: Immunocytochemical and ultrastructural studies

A poorly differentiated human pancreatic adenocarcinoma was maintained in nude mice for more than 3 years. When tumor fragments from xenografts were cultivated in suspension, some became adherent, allowing cell culture. Cytochemical, immunocytological and ultrastructural methods were used to study cell differentiation in both solid tumors and cultures. Pancreatic differentiation features such as cell polarization, production of secretory granules, and M₁ and M₃ mucus-associated antigens were maintained in the tumor cells, in vivo and in vitro. Moreover, in long-term cell cultures, cells were able to organize themselves spontaneously into duct-like structures. Other differentiation features such as production of pancreatic enzymes and hormones were not expressed. However, differentiation patterns such as an intestinal-like brush border and the presence of the M₃ antigen associated with intestinal mucus were observed in both xenografts and cultures. This study shows the possible differentiation patterns which can be expressed by the hypothetical tumor pancreatic stem cell in nude mice as well as in culture.     

Biologic instability of pancreatic cancer xenografts in the nude mouse

Tumor transplants into nude mice (NM) may reveal abnormal biological behavior compared with the original tumor. Despite this, human tumor xenografts in NM have been widely used to study the biology of tumors and to establish diagnostic and therapeutic modalities. Clearly, precise differences in the biology of a given tumor in human and in NM cannot be assessed. We compared the growth kinetics, differentiation pattern and karyotype of an anaplastic Syrian hamster pancreatic cancer cell line in NM and in allogenic hamsters. As with the original tumor, transplants in hamsters grew fast, were anaplastic and expressed markers related to tumor malignancy like galectin 3, TGF-alpha and its receptor EGFR at high levels. However, tumors in the NM were well-differentiated adenocarcinomas, grew slower, had increased apoptotic rate and had a high expression of differentiation markers such as blood group A antigen, DU-PAN-2, carbonic anhydrase II, TGF-beta(2) and mucin. Karyotypically, the tumors in the NM acquired additional chromosomal damage. Our results demonstrate significant differences in the morphology and biology of tumors grown in NM and the allogenic host, and call for caution in extrapolating data obtained from xenografts to primary cancer.     

Establishment and characterization of primary human pancreatic carcinoma in continuous cell culture and in nude mice.

Primary human panceratic exocrine adenocarcinoma has been established in tissue culture and as xenografts in immune-deficient nu/nu mice. The cell line has a doubling time of 36 h and grows as a confluent monolayer together with a constant population of free-floating cells. Evidence of tumourigenicity was provided by growth on an early diploid fibroblast monolayer and in soft agar, and as solid tumours in immune-deficient nu/mu mice. Chromosome analysis of the cultured cells confirmed their tumour origin. Xenografts established from the cell line or directly from primary tumour tissue have retained a similar histology to the original tumour on serial transplantation. An electrophoretic study of exportable pancreatic digestive enzymes and a number of intracellular enzymes has shown that the cell line and xenografts maintain a human intracellular enzyme profile, but do not produce pancreatic digestive enzymes.     

Increased glyceraldehyde-3-phosphate dehydrogenase gene expression in human pancreatic adenocarcinoma

To identify and characterize genes, the products of which play a role in pancreatic adenocarcinoma, we constructed a complementary DNA (cDNA) library using mRNA from the pancreatic adenocarcinoma cell line HPAF, grown as a nude mouse tumor. Through differential screening, we identified a cDNA clone, pII5B, that is homologous to an mRNA expressed at significantly higher levels in HPAF cells than in normal human pancreas. The pII5B cDNA was homologous to the 3'-untranslated region of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12)mRNA. Partial sequencing of several HPAF tumor GAPDH cDNA clones revealed no significant differences from previously published GAPDH cDNA sequences. Increased levels of GAPDH mRNA, relative to actin mRNA levels, were found in six pancreatic adenocarcinoma cell lines and two nude mouse tumors, when compared to normal pancreas. Enolase and glucose transporter mRNA levels were also increased in HPAF cells and nude mouse tumor, suggesting a general increase in expression of genes associated with glycolysis in pancreatic adenocarcinoma. Levels of GAPDH protein were elevated in nude mouse tumors and fresh human pancreatic adenocarcinomas compared to normal pancreas. High GAPDH levels may be characteristic of human adenocarcinomas, since colon adenocarcinomas also exhibited high levels of GAPDH compared to normal colon.     

Androgen receptor in human normal and malignant pancreatic tissue and cell lines

Estrogen and progestogen receptors have been demonstrated in human pancreatic adenocarcinoma tissue. Tumor growth as xenografts in nude mice is promoted by testosterone and retarded by cyproterone acetate but is not influenced by estrogens, progestogens, or their antagonists, although estrogen receptors were demonstrated in xenograft cytosol. A new sensitive microassay technique for sex steroid receptors which relies on affinity chromatography was used in this study. With this assay, androgen receptors were detected in five fresh human pancreatic adenocarcinoma specimens (three male), two pancreatic cancer cell lines (Mia PaCa 2 and Ger), one xenograft tumor which responded to androgens, five specimens of normal adult pancreas (two male), and a pool of fetal pancreatic tissue. The similarity of the androgen receptor in pancreatic carcinoma to that of classical androgen target organs was demonstrated by sedimentation behavior and competitive binding studies. The improved sensitivity of the microassay allowed low levels of estrogen, androgen, and progesterone receptors to be detected in normal adult pancreatic tissue.     

The neurotrophin-trk receptor axes are critical for the growth and progression of human prostatic carcinoma and pancreatic ductal adenocarcinoma xenografts in nude mice.

PURPOSE: Aberrant expression of trk receptor kinases and enhanced expression of various neurotrophins (NTs) have been implicated in the development and progression of human prostatic carcinoma and pancreatic ductal adenocarcinoma. We examined the antitumor efficacy of administration of NT neutralizing antibodies on the growth of established human prostatic carcinoma and pancreatic ductal adenocarcinoma xenografts in nude mice. EXPERIMENTAL DESIGN: In initial studies, tumor-bearing nude mice were treated with a mixture of NT antibodies [100 microg each of anti-nerve growth factor (NGF), anti-brain-derived neurotrophic factor, anti-NT-3, and anti-NT-4/5] or normal rabbit IgG (400 microg) intratumorally and peritumorally three times/week over a 15-day dosing period. In subsequent studies, tumor-bearing nude mice were treated with individual NT antibodies (100 microg), affinity-purified anti-NGF (0.1, 1.0, or 10.0 microg), or normal rabbit IgG (100 microg) using the same dosing schedule. RESULTS: Treatment with the antibody mixture inhibited significantly the growth of TSU-Pr1 and AsPC-1 xenografts as compared with IgG-treated controls (maximal inhibition of 53 and 53%, respectively), whereas this treatment caused significant regression in PC-3 xenografts. Treatment of TSU-Pr1 xenografts with either anti-NGF or anti-NT-3 resulted in maximal tumor growth inhibition of 67 and 64%, respectively, whereas anti-brain-derived neurotrophic factor and anti-NT-4/5 did not inhibit tumor growth in this tumor model. Administration of various concentrations (0.1, 1.0, or 10.0 microg) of affinity-purified anti-NGF resulted in maximal TSU-Pr1 tumor growth inhibition of 49, 62, and 66%, respectively. CONCLUSIONS: These data add further support for the therapeutic potential of disrupting trk-signaling events in select types of nonneuronal human cancers, specifically prostatic and pancreatic carcinomas.     

ESTABLISHMENT OF A HUMAN PANCREATIC ADENOCARCINOMA CELL LINE （JF305） WITH p53 EXPRESSION

李昕，王宏，姜奕，王晓华，贾兰玲，张宝庚ＥＳＴＡＢＬＩＳＨＭＥＮＴＯＦＡＨＵＭＡＮＰＡＮＣＲＥＡＴＩＣＡＤＥＮＯＣＡＲＣＩＮＯＭＡＣＥＬＬＬＩＮＥ（ＪＦ３０５）ＷＩＴＨｐ５３ＥＸＰＲＥＳＳＩＯＮ￥ＬｉＸｉｎ；ＷａｎｇＨｏｎｇ；ＪｉａｎｇＹｉ；Ｗａｎｇ...     

Hormone independent murine carcinoma BX-1 spontaneously arising from nude mouse bearing Br-10, a hormone dependent human breast carcinoma strain

BX-1, an adenocarcinoma spontaneously arising from nude mouse bearing Br-10, a human breast carcinoma strain was characterized. And the purification of a hormone independent murine carcinoma strain, BX-1 was found in August 1986 in three institutes where a hormone dependent transplantable human breast carcinoma cell line Br-10 has been serially passaged. The histological features of BX-1 were different from any other strains which were maintained in these three institutes. Estrogen receptor of BX-1 was negative and no estrogen dependency was observed while Br-10 was the receptor positive and the growth of Br-10 was dependent on estrogen. Although the graft of BX-1 into the thymus intact littermates was rejected, the chromosomal analysis revealed only murine chromosomes for BX-1, while both of human and murine chromosomes were detected in Br-10 tumor. By incubating Br-10 tumor in untreated female nude mice for 2 months and stimulating the growth of the tumor by exogenous estradiol, the purification of Br-10 from BX-1 could be achieved. Whereas the stability of human tumor xenografts in nude mice is confirmed, the spontaneously arising murine tumor from nude mice bearing human tumor xenografts should be considered for the experiments.     

Inhibition of platelet-derived growth factor receptor phosphorylation by STI571 (Gleevec) reduces growth and metastasis of human pancreatic carcinoma in an orthotopic nude mouse model.

PURPOSE: We evaluated the expression of platelet-derived growth factor (PDGF) ligands and receptors in clinical specimens of human pancreatic adenocarcinomas and determined the therapeutic effect of STI571 (Gleevec), a protein tyrosine kinase inhibitor of PDGF receptor (PDGFR), on human pancreatic carcinoma cells growing in the pancreas and liver of nude mice. EXPERIMENTAL DESIGN: Immunohistochemical staining for PDGF-AA and -BB ligands, PDGFR-alpha and -beta, and phosphorylated PDGFR-alpha and -beta was performed on 31 specimens of human pancreatic cancer and L3.6pl human pancreatic adenocarcinoma cell line. To determine the in vivo effects of STI571, nude mice with L3.6pl cells injected into the pancreas were randomized 7 days later to receive one of the following treatments: sterile water p.o. (control), STI571, gemcitabine, or a combination of STI571 and gemcitabine. RESULTS: In 29 of 31 clinical specimens of human pancreatic adenocarcinoma, both tumor cells and tumor-associated endothelial cells expressed phosphorylated PDGFR-alpha and -beta. L3.6pl cells growing in culture expressed moderate amounts of PDGF-AA and little to no PDGFR-alpha or -beta, whereas L3.6pl cells growing in the pancreas of nude mice expressed a high level of PDGF and receptors. Colocalization immunohistochemical analysis demonstrated expression of activated PDGFR-beta by tumor-associated endothelial cells in both the pancreas and in liver metastases. Tumors of mice treated for 4 weeks with STI571 (50 mg/kg or 100 mg/kg p.o. daily) were slightly smaller than controls. Tumors treated with gemcitabine and STI571 (50 mg/kg) were >70% smaller than tumors in control mice and 36% smaller than those in mice treated with gemcitabine only (P < 0.0002 and P < 0.04, respectively). Combination therapy also inhibited spontaneous metastasis to the liver. Tumors from mice treated with both STI571 and gemcitabine had decreased expression of activated (phosphorylated) PDGFR-alpha and -beta, decreased mean vessel density, decreased cell proliferation, and increased apoptosis of tumor cells. CONCLUSIONS: Collectively, these data show that activated PDGFR on tumor cells and tumor-endothelial cells can be a novel target for therapy of pancreatic carcinoma.     

Antitumor activity of Virulizin,a novel biological response modifier (BRM) in a panel of human pancreatic cancer and melanoma xenografts

PURPOSE: To define the anticancer efficacy of Virulizin in vivo as a single agent or in combination with conventional drugs in human pancreatic tumor and melanoma xenografts. METHODS: The therapeutic effect of Virulizin was evaluated in a series of human tumor xenografts in athymic nude mice. RESULTS: Virulizin had a high level of antitumor activity against all the pancreatic tumors (BxPC-3, SU 86.86. and Mia-PaCa-2) and melanomas (C8161 and A2058), as indicated by suppression of tumor growth with an optimal T/C value of 相似文献   

Cyclooxygenase inhibitors decrease the growth and induce regression of human esophageal adenocarcinoma xenografts in nude mice

Cyclooxygenase (COX) inhibition has been shown to prevent the development of esophageal adenocarcinoma (EAC). However, the potential of this approach for treatment of established cancer has been poorly investigated. Our objective was to determine whether non-selective or selective inhibition of the COX pathway affects the growth of esophageal adenocarcinoma xenografts in nude mice. A human esophageal adenocarcinoma xenograft model was established by subcutaneous inoculation of OE33 cells in nude mice. Small tumor slices harvested from four OE33 xenografts were implanted in the flanks of new mice that were randomized to different treatments (6 animals per group): indomethacin (3 mg/kg/day), parecoxib (0.11 and 0.22 mg/kg/day) or a selective prostaglandin E? receptor antagonist (AH-23848B, 1 mg/kg/day). For each treatment, a control group of 6 animals (vehicle) carrying xenografts from the same OE33 tumor was included. Tumor growth was measured twice a week. After 8 weeks mice were euthanized. Tumors were assessed by histological analysis, mRNA expression of COX isoenzymes, PGE? receptors and PGE? content. All OE33 tumors were poorly differentiated esophageal adenocarcinomas. Tumors expressed COX-2, EP?, EP? and EP? receptor mRNA. Treatment with parecoxib, higher dose or indomethacin significantly inhibited tumor growth. Furthermore, indomethacin induced tumor regression (74 vs 582% in control animals; p<0.01). However, AH-23848B or parecoxib low dose failed to affect tumor growth significantly. PGE? content in tumors was significantly decreased by high-dose parecoxib and indomethacin. Indomethacin and parecoxib inhibit the growth of human esophageal adenocarcinoma xenografts in nude mice, which suggests a potential role for NSAIDs or selective COX-2 inhibitors for EAC chemotherapy.