LINC00460 Enhances Bladder Carcinoma Cell Proliferation and Migration by Modulating miR-612/FOXK1 Axis.

INTRODUCTION: LincRNA (long intergenic noncoding RNA) has been indicated as a mediator in tumorigenesis of bladder carcinoma. This study was performed to evaluate the role of LINC00460 in bladder carcinoma progression. METHODS: Expression levels of LINC00460 in bladder carcinoma tissues and cell lines were analyzed via qRT-PCR. MTT, EdU (5-ethynyl-2'-deoxyuridine) staining, and colony formation assays were utilized to evaluate cell viability and proliferation. The wound healing assay was performed to evaluate bladder cancer cell migration, and the transwell assay was used to evaluate cell invasion. The microRNA (miRNA) target of LINC00460 and the corresponding target gene were validated via the dual luciferase activity assay. The tumorigenic function of LINC00460 was determined via establishment of a xenotransplanted tumor model. RESULTS: LINC00460 was elevated in bladder carcinoma tissues and cell lines. Elevated LINC00460 was associated with shorter overall survival of bladder carcinoma patients. Overexpression of LINC00460 promoted cell viability, proliferation, invasion, and migration, while silencing of LINC00460 indicated the opposite effect on bladder carcinoma progression. LINC00460 could directly bind to miR-612 and inhibit miR-612 expression. Moreover, LINC00460 expression was negatively correlated with miR-612 in patients with bladder carcinoma. FOXK1 (Forkhead Box K1) was identified as the target of miR-612 and upregulated in patients with bladder carcinoma. Overexpression of FOXK1 attenuated interference of LINC00460-inhibited bladder carcinoma progression. Knockdown of LINC00460 suppressed in vivo bladder carcinoma growth. CONCLUSIONS: LINC00460 promoted bladder carcinoma progression via sponging miR-612 to facilitate FOXK1 expression, suggesting that LINC00460 might have the potential of being explored as a therapeutic target for treatment of bladder carcinoma.

Modeling rectal cancer to advance neoadjuvant precision therapy.

Progress in rectal cancer therapy has been hindered by the lack of effective disease-specific preclinical models that account for the unique molecular profile and biology of rectal cancer. Thus, we developed complementary patient-derived xenograft (PDX) and subsequent in vitro tumor organoid (PDTO) platforms established from preneoadjuvant therapy rectal cancer specimens to advance personalized care for rectal cancer patients. Multiple endoscopic samples were obtained from 26 Stages 2 and 3 rectal cancer patients prior to receiving 5FU/RT and implanted subcutaneously into NSG mice to generate 15 subcutaneous PDXs. Second passaged xenografts demonstrated 100% correlation with the corresponding human cancer histology with maintained mutational profiles. Individual rectal cancer PDXs reproduced the 5FU/RT response observed in the corresponding human cancers. Similarly, rectal cancer PDTOs reproduced significant heterogeneity in cellular morphology and architecture. PDTO in vitro 5FU/RT treatment response replicated the clinical 5FU/RT neoadjuvant therapy pathologic response observed in the corresponding patient tumors (p < 0.05). The addition of cetuximab to the 5FU/RT regiment was significantly more sensitive in the rectal cancer PDX and PDTOs with wild-type KRAS compared to mutated KRAS (p < 0.05). Considering the close relationship between the patient's cancer and the corresponding PDX/PDTO, rectal cancer patient-derived research platforms represent powerful translational research resources as population-based tools for biomarker discovery and experimental therapy testing. In addition, our findings suggest that cetuximab may enhance RT effectiveness by improved patient selection based on mutational profile in addition to KRAS or by developing a protocol using PDTOs to identify sensitive patients.

Genomic DNA PCR analysis to assess xenograft development in mouse mammary gland.

The mouse transplantation model remains the most relevant methodology to assess the functional capacities of mammary cells and is particularly appropriate for investigations regarding mammary stem cells, whatever the species studied. Following xenotransplantation in mice mammary fat pad, the development of the xenograft is commonly evaluated by immunohistology. Here, we present a simple and rapid method to control the species specificity of a xenograft based on genomic DNA PCR amplification. DNA is extracted from the fixed samples intended for histology, thus allowing the reuse of precious samples. Standard and digital droplet PCR (requiring low DNA quantities) methods have been used to make the present method suitable for the analysis of xenotransplanted samples.

SPZ1 promotes deregulation of Bim to boost apoptosis resistance in colorectal cancer.

Colorectal cancer (CRC) is the third most common malignancies in adults. Similar to other solid tumors, CRC cells show increased proliferation and suppressed apoptosis during the development and progression of the disease. Previous studies have shown that a novel tumor oncogene, spermatogenic basic helix-loop-helix transcription factor zip 1 (SPZ1), can promote proliferation. However, it is unclear whether SPZ1 plays a role in suppressing apoptosis, and the molecular mechanism behind SPZ1's suppression of apoptosis in CRC remains unclear. Here, we found that silencing endogenous SPZ1 inhibits cell growth and induces apoptosis, and overexpression of SPZ1 promotes cell growth. These findings were corroborated by in vitro and in vivo studies. Interestingly, SPZ1 overexpressing cells were resistant to 5-fluorouracil, a drug commonly used to treat cancer. Moreover, knocking down SPZ1 led to the activation of caspase through the deregulation of Bim by ERK1/2, we found that CRC tissues had significantly higher SPZ1 and lower Bim expression, and SPZ1HBimL were associated with advanced clinical stage of CRC. Collectively, our findings demonstrate that SPZ1 contributes to tumor progression by limiting apoptosis. SPZ1 reduces apoptosis by altering the stability of Bim, suggesting SPZ1 may serve as a biomarker and therapeutic target for CRC.

Calcium-sensing stromal interaction molecule 2 upregulates nuclear factor of activated T cells 1 and transforming growth factor-ß signaling to promote breast cancer metastasis.

BACKGROUND: Stromal interaction molecule (STIM) 2 is a key calcium-sensing molecule that regulates the stabilization of calcium ions (Ca2+) and therefore regulates downstream Ca2+-associated signaling and cellular events. We hypothesized that STIM2 regulates epithelial-mesenchymal transition (EMT) to promote breast cancer metastasis. METHODS: We determined the effects of gain, loss, and rescue of STIM2 on cellular motility, levels of EMT-related proteins, and secretion of transforming growth factor-ß (TGF-ß). We also conducted bioinformatics analyses and in vivo assessments of breast cancer growth and metastasis using xenograft models. RESULTS: We found a significant association between STIM2 overexpression and metastatic breast cancer. STIM2 overexpression activated the nuclear factor of activated T cells 1 (NFAT1) and TGF-ß signaling. Knockdown of STIM2 inhibited the motility of breast cancer cells by inhibiting EMT via specific suppression of NFAT1 and inhibited mammary tumor metastasis in mice. In contrast, STIM2 overexpression promoted metastasis. These findings were validated in human tissue arrays of 340 breast cancer samples for STIM2. CONCLUSION: Taken together, our results demonstrated that STIM2 specifically regulates NFAT1, which in turn regulates the expression and secretion of TGF-ß1 to promote EMT in vitro and in vivo, leading to metastasis of breast cancer.

Randomized study to prove the quality of human ovarian tissue cryopreservation by xenotransplantation into mice.

PURPOSE: To study the quality of our human ovarian tissue cryopreservation technique as performed in the first official "International Fertility Protection Centre" in China in patients with certain cancer types using a mouse model, and to find the best site for tissue transplantation in the mouse. METHODS: Thirty-six BALB/C female nude mice were randomly divided into 3 groups, group 1: control group; group 2: ovariectomized group; group 3: ovarian tissue transplantation group. Seventy-two pieces obtained from six ovarian tissue samples from each of three cancer patients were transplanted into the ovarian bursa cavity (OBC), the subcutaneous thigh (TS) and the subcutaneous neck (NS) and removed after 1.5 and 2.5 months, respectively. Follicular growth rate (FGR), total follicle surviving rate (TFSR), tissue recovery rate (TRR), antral follicles (AF), follicle stimulating hormone (FSH), estradiol (E2) and anti-Mullerian hormone (AMH) levels were measured. RESULTS: No significant differences in FGR, OBC, NS (p > 0.05); TFSR was 100% in OBC, NS and TS. No significant differences in TRR (p > 0.05); AF were found only in OBC; TFSR was 100% after transplantation; significantly higher FGR in the 2.5 months compared to the 1.5 months-group (p < 0.05). AMH- and E2-level in group 1 and 3 were significantly higher than in group 2 (p < 0.05); in contrast, FSH was significantly lower. CONCLUSIONS: After transplantation in the mice, the thawed ovarian tissue survived and follicles developed. The ovarian fossa site was the best site for transplantation. Our animal experiments can verify that our human ovarian tissue cryopreservation technique can preserve the quality of ovarian tissue. This is the essential precondition for successful re-transplantation into the patients after performing chemo/radiotherapy to protect ovarian function and fertility.

Xenotransplantation of pre-pubertal ovarian cortex and prevention of follicle depletion with anti-Müllerian hormone (AMH).

OBJECTIVE: To determine whether recombinant AMH (rAMH) could prevent post-transplant follicular depletion by acting on the stemness markers Oct-4, Sox2, and NANOG. MATERIALS AND METHODS: This was an experimental study where 12 ovariectomized nude mice were xenotransplanted with vitrified/warmed ovarian cortex obtained from a pre-pubertal girl and Alzet pumps delivering rAMH, or placebo (control), were inserted intra-abdominally. Previously vitrified/warmed ovarian cortex fragments were transplanted after 7 days and then harvested after 14 days from pump placement. We performed real-time RT-PCR analyses, ELISA for AMH, FSH, and estradiol, histologic measurement of ovarian follicles, and immunohistochemistry for Ki67 and TUNEL. The main outcome measures were serum levels and tissue expression of the parameters under investigation and follicle count. RESULTS: Serum AMH, FSH, and estradiol reflected post-ovariectomy profiles and were mildly influenced by rAMH administration. Ovarian cortex expression of AMH, AMH-R2, VEGF, GDF9, Oct-4, and Sox2 was lower in rAMH mice than in controls, while NANOG was upregulated. There was a non-significant decrease in primordial follicles after vitrification-warming, and xenotransplantation further decreased this number. There were lower cell replication and depressed apoptosis in the rAMH group. CONCLUSIONS: Administration of recombinant AMH in the peri-transplant period did not protect the initial follicular depletion but decreased apoptosis and cellular activation and regulated stem cell markers' tissue expression. These results aid our understanding of the inhibitory effects of AMH on follicular development and show the benefit of administering exogenous AMH at the time of pre-pubertal ovarian cortex transplant to protect the follicles from pre-activation and premature depletion.

Saponins isolated from Schizocapsa plantaginea inhibit human hepatocellular carcinoma cell growth in vivo and in vitro via mitogen-activated protein kinase signaling.

The underground cane of Schizocapsa plantaginea (Hance) has long been used by Chinese ethnic minority as a constituent of anti-cancer formulae. Saponins are abundant secondary metabolic products located in the underground cane of this plant. The potential therapeutic effects of total saponins isolated from Schizocapsa plantaginea (Hance) (SSPH) on human hepatocellular carcinoma (HCC) were tested in vitro in human liver cancer cell lines, SMMC-7721 and Bel-7404. Apoptosis and cell cycle arrest were determined using flow cytometry, caspase activation was determined by ELISA, and PARP, cleaved PARP, mitogen-activated protein kinase (MAPK) expression and phosphorylation were measured using Western blotting analysis. In vivo anti-HCC effects of SSPH were verified in nude mouse xenograft model. SSPH exerted markedly inhibitory effect on HCC cell proliferation in time- and concentration-dependent manner. Moreover, SSPH significantly induced apoptosis through caspase-dependent signaling and arrested cell cycle at G2/M phase. These anti-proliferation effects of SSPH were associated with up-regulated phosphorylation of extracellular signal-regulated kinase-1/2 (Erk1/2) and c-jun-NH2-kinase-1/2 (JNK1/2) and reduced phosphorylation of p38MAPK. Furthermore, inhibitors of ERK, UO126, and JNK, SP600125 inhibited the anti-proliferation effects by SSPH, suggesting that Erk and JNK were the effector molecules in SSPH induced anti-proliferative action. During in vivo experiments, SSPH was found to inhibit xenograft tumor growth in nude mice, with a similar mechanism in vitro. Our study confirmed that SSPH exerted antagonistic effects on human liver cancer cells both in vitro and in vivo. Molecular mechanisms underlying SSPH action might be closely associated with MAPK signaling pathways. These results indicated that SSPH has potential therapeutic effects on HCC.

The effects of type I interferon on glioblastoma cancer stem cells.

Glioblastomas (GBMs) are highly invasive brain tumors that are extremely deadly. The highly aggressive nature of GBM as well as its heterogeneity at the molecular and cellular levels has been attributed to a rare subpopulation of GBM stem-like cells (GSCs). Interferons (IFNs) are a family of endogenous antiviral proteins that have anticancer activity in vitro, and have been used clinically to treat GBM. IFN inhibits the proliferation of various established GBM cell lines, but the effects of IFNs on GSCs remain relatively unknown. The present study explored the effects of IFN on the proliferation and the differentiation capacity of GSCs isolated from GBM patient-derived xenolines (PDXs) grown as xenografts in immunocompromised mice. We show that IFN inhibits the proliferation of GSCs, inhibits the sphere forming capacity of GSCs that is a hallmark of cancer stem cells, and inhibits the ability of GSCs to differentiate into astrocytic cells. In addition, we show that IFN induces transient STAT3 activation in GSCs, while induction of astrocytic differentiation in GSCs results in sustained STAT3 activation.

A zebrafish xenograft model for studying human cancer stem cells in distant metastasis and therapy response.

Lethal and incurable bone metastasis is one of the main causes of death in multiple types of cancer. A small subpopulation of cancer stem/progenitor-like cells (CSCs), also known as tumor-initiating cells from heterogenetic cancer is considered to mediate bone metastasis. Although over the past decades numerous studies have been performed in different types of cancer, it is still difficult to track small numbers of CSCs during the onset of metastasis. With use of noninvasive high-resolution imaging, transparent zebrafish embryos can be employed to dynamically visualize cancer progression and reciprocal interaction with stroma in a living organism. Recently we established a zebrafish CSC-xenograft model to visually and functionally analyze the role of CSCs and their interactions with the microenvironment at the onset of metastasis. Given the highly conserved human and zebrafish genome, transplanted human cancer cells are able to respond to zebrafish cytokines, modulate the zebrafish microenvironment, and take advantage of the zebrafish stroma during cancer progression. This chapter delineates the zebrafish CSC-xenograft model as a useful tool for both CSC biological study and anticancer drug screening.

High-fat Western diet-induced obesity contributes to increased tumor growth in mouse models of human colon cancer.

Strong epidemiologic evidence links colon cancer to obesity. The increasing worldwide incidence of colon cancer has been linked to the spread of the Western lifestyle, and in particular consumption of a high-fat Western diet. In this study, our objectives were to establish mouse models to examine the effects of high-fat Western diet-induced obesity on the growth of human colon cancer tumor xenografts, and to examine potential mechanisms driving obesity-linked human colon cancer tumor growth. We hypothesize that mice rendered insulin resistant due to consumption of a high-fat Western diet will show increased and accelerated tumor growth. Homozygous Rag1tm1Mom mice were fed either a low-fat Western diet or a high-fat Western diet (HFWD), then human colon cancer xenografts were implanted subcutaneously or orthotopically. Tumors were analyzed to detect changes in receptor tyrosine kinase-mediated signaling and expression of inflammatory-associated genes in epididymal white adipose tissue. In both models, mice fed an HFWD weighed more and had increased intra-abdominal fat, and tumor weight was greater compared with in the low-fat Western diet-fed mice. They also displayed significantly higher levels of leptin; however, there was a negative correlation between leptin levels and tumor size. In the orthotopic model, tumors and adipose tissue from the HFWD group displayed significant increases in both c-Jun N-terminal kinase activation and monocyte chemoattractant protein 1 expression, respectively. In conclusion, this study suggests that human colon cancer growth is accelerated in animals that are obese and insulin resistant due to the consumption of an HFWD.

Lamp2a is required for tumor growth and promotes tumor recurrence of hepatocellular carcinoma.

Exploring the function of chaperone-mediated autophagy (CMA) in cancer has promoted progress in cancer treatment through the regulation of CMA pathways. However, CMA status and function in hepatocellular carcinoma (HCC) by focusing on the regulatory role of lyso-some-associated membrane protein type 2a (Lamp2a) remain to be clarified. We examined Lamp2a in a normal human liver cell line, 6 HCC cell lines, 10 normal liver samples as well as 42 HCC tissue and para-tumor tissues samples, and then validated it in 228 HCC patients to assess the relationship between Lamp2a and clinical prognosis. Gain and loss of Lamp2a function were also explored in HCC cell lines and xenograft models. Significantly lower level of Lamp2a expression was found in HCC cells and tissues compared with normal hepatic cells, para-tumor tissues and normal livers. Although no differences in HCC cell morphology or function were observed in relation to Lamp2a expression under normal culture or short-term starvation conditions, Lamp2a blockage significantly inhibited HCC cell viability under prolonged starvation. Critically, Lamp2a is required for HCC xenograft growth in vivo by helping cells to avoid apoptosis and promoting cell proliferation. Furthermore, a significant correlation between Lamp2a expression and tumor size or cumulative recurrence was uncovered in HCC patients. Collectively, the present study shows that impaired Lamp2a expression in HCC contributes to tumor cell viability and promotes tumor growth and recurrence. Targeting chaperone-mediated autophagy through Lamp2a may also imply a potentially novel treatment strategy for HCC.

ABT737 reverses cisplatin resistance by regulating ER-mitochondria Ca2+ signal transduction in human ovarian cancer cells.

Bcl-2, which belongs to the Bcl-2 family, is frequently overexpressed in various types of cancer cells and contributes to drug resistance. However, the function of Bcl-2 in cisplatin resistance in human ovarian cancer cells is not fully understood. In this study, we found that the pharmacological inhibitor ABT737 or genetic knockdown of Bcl-2 increased cisplatin cytotoxicity in cisplatin-resistant ovarian cancer cells. Additionally, treatment with ABT737 or Bcl-2 siRNA increased cisplatin-induced free Ca2+ levels in the cytosol and mitochondria, which increased endoplasmic reticulum (ER)-associated and mitochondria-mediated apoptosis. In addition, ABT737 or Bcl-2 siRNA increased the ER-mitochondria contact sites induced by cisplatin in cisplatin-resistant SKOV3/DDP ovarian cancer cells. Consistently with the in vitro results, ABT737 potently synergized with cisplatin in inhibiting the growth of human ovarian cancer xenografts in nude mice. Collectively, these results indicate that pharmacological inhibitors or genetic knockdown of Bcl-2 may be a potential strategy for improving cisplatin treatment of ovarian cancer.

Establishment and Characterization of a Human Neuroendocrine Tumor Xenograft.

Neuroendocrine tumors (NETs) are increasing in incidence yet the cause of these tumors remains unknown. Familial associations have shed light on the genetic basis of some of these tumors, but sporadic tumors seem to have primarily epigenetic dysregulation. The rarity of cell lines and animal models has been a barrier to studies of treatment modalities. We set out to develop a xenograft model of gastrointestinal NETs. Primary human NETs were collected at the time of surgery under sterile conditions and xenografted into the flanks of immunodeficient mice. Tumor growth was measured and when tumors reached 1500 mm(3), they were excised and half was re-xenografted through multiple generations. The other half was bisected; a part was frozen and a part was fixed for morphologic and immunohistochemical characterization as well as molecular validation of fidelity of a successful xenograft. Of 106 human NETs, seven were successfully engrafted of which only one tumor was successfully propagated for eight passages. Two years later, the tumor retains its neuroendocrine features and similarity to the original primary human tumor. It has retained expression of keratin as well as chromogranin A reactivity. The establishment of a NET xenograft provides a model for further study of the biological behavior of these tumors and can be used to examine the in vivo effects of various medical and targeted radiotherapeutic agents on tumor growth.

Successful xenogeneic germ cell transplantation from Jundia catfish (Rhamdia quelen) into adult Nile tilapia (Oreochromis niloticus) testes.

Fish germ cell transplantation presents several important potential applications for aquaculture, including the preservation of germplasm from endangered fish species with high genetic and commercial values. Using this technique in studies developed in our laboratory with adult male Nile tilapias (Oreochromis niloticus), all the necessary procedures were successfully established, allowing the production of functional sperm and healthy progeny approximately 2months after allogeneic transplantation. In the present study, we evaluated the viability of the adult Nile tilapia testis to generate sperm after xenogeneic transplant of germ cells from sexually mature Jundia catfish (Rhamdia quelen) that belong to a different taxonomic order. Therefore, in order to investigate at different time-periods post-transplantation, the presence and development of donor PKH26 labeled catfish germ cells were followed in the tilapia seminiferous tubules. From 7 to 20days post-transplantation, only PKH26 labeled spermatogonia were observed, whereas spermatocytes at different stages of development were found at 70days. Germ cell transplantation success and progression of spermatogenesis were indicated by the presence of labeled PKH26 spermatids and sperm on days 90 and 120 post-transplantation, respectively. Confirming the presence of the catfish genetic material in the tilapia testis, all recipient tilapias evaluated (n=8) showed the genetic markers evaluated. Therefore, we demonstrated for the first time that the adult Nile tilapia testis offers the functional conditions for development of spermatogenesis with sperm production from a fish species belonging to a different order, which provides an important new venue for aquaculture advancement.

[Lentiviral vector-mediated short hairpin RNA targeting survivin inhibits abdominal growth of human endometrium xenograft in nude mice].

OBJEVTIVE: To investigate the inhibitory effect of lentiviral vector-mediated short hairpin RNA targeting survivin (LV-survivin shRNA) on the growth of human endometrium xenograft in the abdominal cavity of nude mice. METHODS: The endometrium xenografts from 8 women with endometriosis were injected into the peritoneal cavities of 45 nude mice. The mice were then randomly assigned to receive intraperitoneal injection of LV-survivin shRNA, pGCL-NC-GFP (negative control) or PBS (blank control). Two weeks later, the number and morphometry of endometriotic lesions were quantified and the expression of survivin protein were detected by immunohistochemistry. RESULTS: The formation of endometriotic lesions was significantly suppressed in mice receiving LV-survivin shRNA injection as compared with those in the two control groups (P/0.001). The mice in LV-survivin-shRNA group showed significantly down-regulated expression levels of survivin protein compared with those in the negative and blank control groups, presenting also necrosis in the endometriosis-like lesions in microscopic observation. CONCLUSION: Lentiviral vector-mediated shRNA can effectively inhibit the expression of survivin in human endometrium xengrafts and suppress the formation and growth of endometriotic lesions in the abdominal cavities of nude mice.

Generation of human acute lymphoblastic leukemia xenografts for use in oncology drug discovery.

The establishment of reproducible mouse models of acute lymphoblastic leukemia (ALL) is necessary to provide in vivo therapeutic test systems that recapitulate human ALL, and for amplification of limited amounts of primary tumor material. A popular assay is the primary xenograft model that utilizes immunocompromised mice. The protocol includes injection of primary patient tumor specimens into mice with subsequent serial passaging of the tumors by retransplants of cells harvested from the mouse bone marrow and spleen. The tumors generated are then used for genomic profiling, ex vivo compound testing, mechanistic studies and retransplantation. Detailed in this unit are procedures for the establishment and maintenance of primary ALL xenograft panels for use in basic research and translational studies.

Local and systemic protumorigenic effects of cancer-associated fibroblast-derived GDF15.

The tumor stroma is vital to tumor development, progression, and metastasis. Cancer-associated fibroblasts (CAF) are among the abundant cell types in the tumor stroma, but the range of their contributions to cancer pathogenicity has yet to be fully understood. Here, we report a critical role for upregulation of the TGFß/BMP family member GDF15 (MIC-1) in tumor stroma. GDF15 was found upregulated in situ and in primary cultures of CAF from prostate cancer. Ectopic expression of GDF15 in fibroblasts produced prominent paracrine effects on prostate cancer cell migration, invasion, and tumor growth. Notably, GDF15-expressing fibroblasts exerted systemic in vivo effects on the outgrowth of distant and otherwise indolent prostate cancer cells. Our findings identify tumor stromal cells as a novel source of GDF15 in human prostate cancer and illustrate a systemic mechanism of cancer progression driven by the tumor microenvironment. Further, they provide a functional basis to understand GDF15 as a biomarker of poor prognosis and a candidate therapeutic target in prostate cancer.

Temporal characterization of lymphatic metastasis in an orthotopic mouse model of oral cancer.

BACKGROUND: The overall mortality rate in cases of head and neck squamous cell carcinoma (HNSCC) has not improved over the past 30 years, mostly because of the high treatment failure rate among patients with regionally metastatic disease. To better understand the pathobiologic processes leading to lymphatic metastasis development, there is an urgent need for relevant animal models. METHODS: HNSCC cell lines were implanted into the tongues of athymic nude mice. Histology, immunohistochemistry, and ex vivo 2-photon microscopy were used to evaluate tumor progress and spread. RESULTS: Orthotopic xenografts of different HNSCC cell lines produced distinct patterns of survival, tumor histology, disease progression rate, and lymph node metastasis development. Remarkably, all injected cell types reached the lymph nodes within 24 hours after injection, but not all developed metastasis. CONCLUSION: This orthotopic xenograft model closely mimics several characteristics of human cancer and could be extremely valuable for translational studies focusing on lymphatic metastasis development and pathobiology.

Generation of a patient-derived chordoma xenograft and characterization of the phosphoproteome in a recurrent chordoma.

OBJECT: The management of patients with locally recurrent or metastatic chordoma is a challenge. Preclinical disease models would greatly accelerate the development of novel therapeutic options for chordoma. The authors sought to establish and characterize a primary xenograft model for chordoma that faithfully recapitulates the molecular features of human chordoma. METHODS: Chordoma tissue from a recurrent clival tumor was obtained at the time of surgery and implanted subcutaneously into NOD-SCID interleukin-2 receptor gamma (IL-2Rγ) null (NSG) mouse hosts. Successful xenografts were established and passaged in the NSG mice. The recurrent chordoma and the derived human chordoma xenograft were compared by histology, immunohistochemistry, and phospho-specific immunohistochemistry. Based on these results, mice harboring subcutaneous chordoma xenografts were treated with the mTOR inhibitor MLN0128, and tumors were subjected to phosphoproteome profiling using Luminex technology and immunohistochemistry. RESULTS: SF8894 is a novel chordoma xenograft established from a recurrent clival chordoma that faithfully recapitulates the histopathological, immunohistological, and phosphoproteomic features of the human tumor. The PI3K/Akt/mTOR pathway was activated, as evidenced by diffuse immunopositivity for phospho-epitopes, in the recurrent chordoma and in the established xenograft. Treatment of mice harboring chordoma xenografts with MLN0128 resulted in decreased activity of the PI3K/Akt/mTOR signaling pathway as indicated by decreased phospho-mTOR levels (p = 0.019, n = 3 tumors per group). CONCLUSIONS: The authors report the establishment of SF8894, a recurrent clival chordoma xenograft that mimics many of the features of the original tumor and that should be a useful preclinical model for recurrent chordoma.