Tumor Cell Inoculation Research Articles

Abstract 637: Establishment of a realistic patient-derived xenograft (PDX) model for prostate cancer bone metastasis

Abstract Bone metastases are frequent and fatal outcome of advanced prostate cancer. Many of the currently used preclinical models lack typical characteristics of the heterogenic human disease. We improved existing methodology by using fresh patient-derived material in a xenograft model of bone metastasis. Clinical prostate tumor specimens were collected from robotic-assisted laparoscopic radical prostatectomy operations in Turku University Hospital (Turku, Finland). Patient-derived tumor tissue of Gleason grade 8-10 tumors (n = 5) were cut into small pieces, digested overnight, and then cell suspension was intratibially inoculated into the bone marrow cavity of nude mice (n = 8-10/patient). X-ray pictures were taken and blood samples were collected once a month. Mice were sacrificed 6 months after tumor cell inoculation, and hind limbs and lungs were collected for histological and immunohistochemical analysis. PSA was measured from serum using commercial kit. X-rays demonstrated osteosclerotic bone lesions. Immunohistochemical analysis showed that 80% of tumors in bone expressed similar characteristics compared with original tumor (androgen receptor AR, prostate specific antigen PSA, proliferation marker Ki67). However, no changes were seen in serum PSA. Lung metastases were detected in 50% of tumor-bearing mice. Interestingly, the most of lung metastases were negative for AR and PSA, which indicates that the cell population that forms metastases may be undifferentiated clone of heterogenic tumor and therefore the most aggressive. Our platform provides new tools for prostate cancer research. Citation Format: Yvonne Konkol, Maija Valta, Jenni Bernoulli, Pekka Taimen, Peter Boström, Pirkko Härkönen, Jussi Halleen, Johanna M. Tuomela. Establishment of a realistic patient-derived xenograft (PDX) model for prostate cancer bone metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 637.

Abstract 4084: Crosstalk between TNBC and stromal components via secreted factors enhances cell motility that can be attenuated by a CXCR1 inhibitor

Abstract Triple negative breast cancer (TNBC) as a metastatic disease is currently incurable. Unfortunately reliable and reproducible methods for testing drugs against metastasis are not available. We have previously developed a robust metastatic model in which mice are pretreated with tumor cell-conditioned media (TCM) from human TNBC cells (MDA-MB-231 and SUM149) for 2 weeks prior to tumor cell inoculation. In this model we found reproducible metastases in lymph nodes (LN) and lungs within 4-5 weeks after orthotropic tumor inoculation. We have discovered that the TNBC tumor cells secrete large amounts of interleukin-6 (IL-6) that “educates” lymphatic endothelial cells (LEC) in the LN and lungs. Stat3, a transcription factor, gets activated and induces the synthesis of CCL5 and VEGF among other factors. CCL5 recruits the tumor cells to the LN and lungs; VEGF helps build blood vessels in the LN to facilitate tumor cell survival; VEGF produced in the lung helps the tumor cells extravasate into the lung. We have confirmed the importance of these factors by showing that inhibitors of these factors significantly inhibit metastasis. In this report, using a human antibody array, we identified factors secreted by fibroblasts (HGF, IL-6, IL-8, CCL7, MIF, GDF-15, EMMPRIN and VEGF) and by macrophages (CXCL5, IL-8 and uPAR) upon induction by MDA-MB-231 TCM. We ranked the expression level of each factor by real time qRT-PCR and determined that interleukin 8 (IL-8) was the top candidate. We confirmed by ELISA that IL-8 secreted from either fibroblasts or macrophages treated with MDA-MB-231 TCM was upregulated compared to treatment with serum free media (SFM). Our data showed that the proliferation of MDA-MB-231 cells incubated with conditioned media (CM) from TCM-induced fibroblasts or TCM-induced macrophages was enhanced compared to SFM treatment. Furthermore, MDA-MB-231 cell migration, a key step in tumor metastasis, was promoted by CM from TCM-induced fibroblasts or macrophages. Significantly, inhibition of the IL-8 signaling pathway by Reparixin, an inhibitor of the IL-8 receptor CXCR1, abrogated the upregulation of MDA-MB-231 cell proliferation and migration induced by TCM-induced fibroblasts or macrophages. These findings implicate IL-8 signaling as a critical event in TNBC cell proliferation and migration via crosstalk with stromal components. Further, these studies suggest that IL-8 acts as a key regulator orchestrating TNBC metastatic breast cancer. Therefore, we have provided evidence that supports the hypothesis that functional antagonism of the IL-8 signaling pathway has the potential to circumvent TNBC breast cancer growth and metastasis. Citation Format: Kideok Jin, Niranjan B. Pandey, Aleksander S. Popel. Crosstalk between TNBC and stromal components via secreted factors enhances cell motility that can be attenuated by a CXCR1 inhibitor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4084.

Abstract 1319: A novel formulation of CX-5461, a small-molecule inhibitor of rRNA synthesis, and its use for treatment of acute myeloid leukemia models

Abstract CX-5461 is a RNA polymerase I inhibitor currently in Phase I clinical trial in Australia for patients with advanced hematologic malignancies. In the pre-clinical setting, CX-5461 is efficacious in a wide range of hematologic and solid tumor models when given orally or intraperitoneally. Currently, the compound is given intravenously (iv) in the first-in-human clinical trial. CX-5461 is solubilized in low pH (3.5) 50 mM sodium phosphate as it is sparingly soluble in water (<1 mg/mL) and at physiological pH. The use of such low pH phosphate buffers affects infusion tolerance. While CX-5461 has nanomolar range activity in vitro, in vivo studies are conducted with either high dosages or frequent dosing. The chemical structure of CX-5461 also suggests potential drug instability under physiological conditions. We have addressed these solubility and stability issues by developing a lipid-based nanoparticulate (LNP) formulation which relies on a previously unrecognized interaction between CX-5461 and copper. Copper (Cu) coordination with CX-5461 was demonstrated using 1H NMR and UV-Vis. Cu-LNPs were prepared with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol (CHOL) (55:45 molar ratio) using extrusion methods. CX-5461 was loaded into Cu-LNPs at 60°C for 30 minutes and the external buffer was exchanged to Hepes Buffered Saline (pH 7.4). Pharmacokinetic (PK) studies were performed in CD-1 mice given a dose of 30 mg/kg (iv) and plasma concentrations of CX-5461 were determined by HPLC. Efficacy studies were performed in RAG2M mice with established subcutaneous (sc) MV-4-11 tumors and in a MV-4-11 bone marrow engraftment model (30 mg/kg given iv, Q4Dx3). Our 1H NMR data suggests that CX-5461 binds to copper at a 1:1 molar ratio. Copper complexation does not affect the anticancer activity of CX-5461 as determined in several cancer cell lines in vitro. Using copper-complexation as a driving force, CX-5461 was efficiently loaded into LNPs (∼100 nm) prepared to contain copper (300 mM CuSO4). Resulting LNPs have a drug-to-lipid-ratio of 0.2. The LNP formulation has significantly improved the PK profile of CX-5461 and increased the total exposure to drug by an order of magnitude (AUC = 4156 μg-hr/mL for LNP and 319 μg-hr/mL for free CX-5461). The LNP formulation of CX-5461 is more active than the current low-pH formulation of CX-5461 when given iv in leukemia xenograft models. Specifically 37 days after sc tumor cell inoculation the tumor sizes were 720 mm3 and 240 mm3 in control and LNP-CX5461-treated animals respectively. The clinical formulation administered at the same dose exhibited tumor volumes that were comparable to the untreated controls. We have generated the first LNP formulation of CX-5461 using the copper complexation technology developed in our laboratory. LNP-CX-5461 is suitable for iv administration and is more efficacious than the current formulation in our pre-clinical studies. Citation Format: Ada W.Y. Leung, Malathi Anantha, Kathleen E. Prosser, Mohamed Wehbe, Charles J. Walsby, Marcel B. Bally. A novel formulation of CX-5461, a small-molecule inhibitor of rRNA synthesis, and its use for treatment of acute myeloid leukemia models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1319.

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Enhancing the Apoptotic Effect of a Low Dose of Paclitaxel on Tumor Cells in Mice by Arabinoxylan Rice Bran (MGN-3/Biobran)

ABSTRACTIn this study, we examine the ability of arabinoxylan rice bran (MGN-3/Biobran) to enhance the apoptotic effect of paclitaxel (Taxol) at low concentration [2 mg/kg body weight (BW)] in animals bearing Ehrlich ascites carcinoma (EAC) cells and elucidate its mechanisms of action. On Day 8 following tumor cells inoculation, mice bearing tumors were administered MGN-3 alone (40 mg/kg BW), paclitaxel alone, or MGN-3 plus paclitaxel. On Day 30 post-tumor inoculation, we observed significant suppression of tumor volume (TV) with paclitaxel alone (59%), MGN-3 alone (77%), and MGN-3 plus paclitaxel (88%). Inhibition of tumor growth post-treatment with both agents, as compared with either treatment alone, was associated with a decrease in cell proliferation, a marked increase in the sub-G0/G1 population, an increase in DNA damage and apoptosis of tumor cells, and a significant maximization of the apoptosis index (AI)/proliferation index (PrI) ratio. Histopathological and electron microscopy examination of the combined treatment group showed an increase in the degenerative regions of the solid tumor tissue and abundant apoptotic cells. These data suggest that MGN-3 supplementation enhances tumor cell demise in the presence of a low dose of chemotherapeutic agent via apoptotic mechanism.

Lung cancer inhibitory activity of dietary berries and berry polyphenolics

BACKGROUND: Blueberry (BB) and black raspberry (BRB) have been shown to be chemopreventive against estrogenmediated breast cancer in pre-clinical studies. However, therapeutic efficacy of these berries against lung cancer is not known. METHODS: In this study we investigated i) relative efficacy of individual anthocyanidins vs. respective anthocyanins, ii) relative antiproliferative activity of mixture of anthocyanidins compared to individual anthocyanidins, iii) antitumor activity of dietary BB, iv) Tumor inhibitory activity of diet supplemented with BB, alone and in combination with BRB, against lung tumor xenograft using nude mice, and finally, v) the efficacy of select polyphenolics present in BB and BRB against lung tumor xenograft. RESULTS: Our findings indicated that individual anthocyanidins (aglycones) were significantly more potent (2-3 fold lower IC50) in inhibiting the non-small-cell lung cancer (NSCLC) cell growth vs. respective anthocyanins (glycones). Further, anthocyanidins mixture at equimolar concentrations exhibited synergistic antiproliferative activity vs. individual anthocyanidins. When tested against NSCLC (A549 and H1299) cells in nude mice, dietary BB (7.5%, w/w) showed >40% reduction in tumor volume against H1299 xenografts. The maximal growth inhibition occurred with 5% BB dose, with no additional protection occurring at a higher dose (7.5%). However, somewhat lower protection was found when the BB diet initiated prior to tumor cell inoculation. The mixture of BB (5%, w/w) and BRB (2.5%) resulted in higher inhibition of tumor growth vs. BB alone (71% vs 42%). Likewise, a combination of delphinidin (bioactive of BB) and punicalagins (a bioactive of BRB, which gets converted to ellagic acid in vivo) showed higher tumor growth inhibition compared to delphinidin. CONCLUSIONS: The therapeutic effects of the berries and berry polyphenolics observed against lung cancer in this study are highly encouraging. Further investigation into the mechanism of action of the combinations of the berry bioactives will be valuable for clinical use of this potent natural product against lung cancer.

Cancer cachexia-when proteasomal inhibition is not enough.

Cachexia is a life threatening syndrome associated with several diseases, such as end‐stage heart failure, end‐stage renal disease, chronic obstructive pulmonary disease, chronic inflammation (i.e. rheumatoid arthritis), acquired immune deficiency syndrome, and cancer.1, 2 Cachexia is found in 31–87% of cancer patients especially in advanced disease stages.3 It is characterized by progressive weight loss, metabolic alterations, fatigue, and persistent reduction of body cell mass in response to a malignant tumour.2, 4, 5, 6 The incidence of cachexia in cancer patients is dependent on the type and site of the tumour. While patients with non‐Hodgkin's lymphoma, breast cancer, and sarcomas show low incidences, rates up to 83% in pancreatic cancer patients, and over 85% in patients with gastric cancer have been found. Additionally, around 60% of small‐cell and non‐small‐cell lung cancer patients develop cachexia.7, 8, 9 Cancer cachexia affects the function of several organs such as muscle, adipose tissue, liver, brain, immune system, and heart, collectively decreasing patients' quality of life and worsening their prognosis. Therefore, cachexia must be considered as a true multi‐organ syndrome.10 Because cancer cachexia leads to a decrease in physical performance and quality of life,11 and is associated with poor survival (accounting for more than 20% of cancer deaths,7, 12, 13, 14, 15) it is of major clinical relevance. Even more so since cachectic patients show lower response rates to chemotherapy7 and a reduced tolerance to anticancer treatment.16 Despite its importance, weight loss in cancer patients is rarely recognized, assessed,17 or treated actively.18, 19 Thus, cancer cachexia represents an important underappreciated clinical syndrome.

Co-option of pre-existing vascular beds in adipose tissue controls tumor growth rates and angiogenesis.

Many types of cancer develop in close association with highly vascularized adipose tissues. However, the role of adipose pre-existing vascular beds on tumor growth and angiogenesis is unknown. Here we report that pre-existing microvascular density in tissues where tumors originate is a crucial determinant for tumor growth and neovascularization. In three independent tumor types including breast cancer, melanoma, and fibrosarcoma, inoculation of tumor cells in the subcutaneous tissue, white adipose tissue (WAT), and brown adipose tissue (BAT) resulted in markedly differential tumor growth rates and angiogenesis, which were in concordance with the degree of pre-existing vascularization in these tissues. Relative to subcutaneous tumors, WAT and BAT tumors grew at accelerated rates along with improved neovascularization, blood perfusion, and decreased hypoxia. Tumor cells implanted in adipose tissues contained leaky microvessel with poor perivascular cell coverage. Thus, adipose vasculature predetermines the tumor microenvironment that eventually supports tumor growth.

Effects of different doses of compound Xuelian capsule on bone cancer pain in rats

Objective To evaluate the effects of different doses of compound Xuelian capsule on bone cancer pain (BCP) in rats. Methods Fifty pathogen-free adult female Sprague-Dawley rats, weighing 200-220 g, aged 7-8 weeks, were randomly divided into 5 groups (n=10 each) using a random number table: sham operation group (group S), group BCP, and compound Xuelian capsule 50, 100 and 200 mg·kg-1·d-1 groups (group CX50, group CX100 and group CX200). BCP was produced by injecting Walker256 mammary gland carcinoma cells into the intramedullary space of the right femur bone.At 11-21 days after inoculation of the tumor cells, normal saline containing compound Xuelian capsule 50, 100 and 200 mg·kg-1·d-1 was injected through a tube into stomach once a day in CX50, CX100 and CX200 groups, respectively.The mechanical paw withdrawal threshold (MWT) and limb use score were measured at 1 day before inoculation of the tumor cells (baseline) and 4, 7, 11, 14, 17, 19, and 21 days after inoculation of the tumor cells. Results Compared with group S, the MWT at 4-21 days after inoculation of the tumor cells was significantly decreased, and limb use score was significantly decreased at 11-21 days after inoculation of the tumor cells in BCP, CX50, CX100 and CX200 groups (P<0.05). Compared with group BCP, the MWT was significantly increased at 19-21 days after inoculation in group CX50, at 17-21 days after inoculation in group CX100 and at 14-21 days after inoculation in group CX200, and limb use score was significantly increased at 14-21 days after inoculation in group CX100 and at 17-21 days after inoculation in group CX200 (P<0.05). Conclusion Compound Xuelian capsule 50, 100 and 200 mg·kg-1·d-1 (for 11 consecutive days) can reduce BCP in a dose-dependent manner in rats. Key words: Asteraceae; Capsules; Bone neoplasms; Pain; Dose-response relationship, drug

TNF Neutralization Results in the Delay of Transplantable Tumor Growth and Reduced MDSC Accumulation.

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells (IMCs) that, under normal conditions, may differentiate into mature macrophages, granulocytes, and dendritic cells. However, under pathological conditions associated with inflammation, cancer, or infection, such differentiation is inhibited leading to IMC expansion. Under the influence of inflammatory cytokines, these cells become MDSCs, acquire immunosuppressive phenotype, and accumulate in the affected tissue, as well as in the periphery. Immune suppressive activity of MDSCs is partly due to upregulation of arginase 1, inducible nitric oxide synthase, and anti-inflammatory cytokines, such as IL-10 and TGF-β. These suppressive factors can enhance tumor growth by repressing T-cell-mediated anti-tumor responses. TNF is a critical factor for the induction, expansion, and suppressive activity of MDSCs. In this study, we evaluated the effects of systemic TNF ablation on tumor-induced expansion of MDSCs in vivo using TNF humanized (hTNF KI) mice. Both etanercept and infliximab treatments resulted in a delayed growth of MCA 205 fibrosarcoma in hTNF KI mice, significantly reduced tumor volume, and also resulted in less accumulated MDSCs in the blood 3 weeks after tumor cell inoculation. Thus, our study uncovers anti-tumor effects of systemic TNF ablation in vivo.

Effect of ALPPS procedure in an in vivo rat model of colorectal cancer metastases in the liver

Introduction: Associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) is a promising method to increase resectability rates of colorectal cancer (CRC) liver metastases. Residual latent micrometastases are considered a cause of tumour relapse. This work investigates the influence of ALPPS procedure on the growth of CRC liver metastases in a syngeneic rat model. Methods: Liver tumours were induced in 10 male WAG/Rij rats by subcapsular inoculation of one million CC531 rat CRC cells in the liver. ALPPS procedure was performed by ligation of the portal vein at the right lobe (RL), left lobe (LL) and right medium lobe (RML) with an “in situ” liver transection at the medium lobe, dividing RML and left medium lobe (LML). Rats were divided into three groups: sham-treated group with no surgical procedures performed, ALPPS procedure group with inoculation in the RML, and ALPPS procedure group with inoculation in the LML. USS was performed 4 weeks after the procedure and sacrifice. Size, location and histopathological features of the metastases were analysed. Results: Tumour growth was significantly greater in ALPPS than in sham treated group. In rats with ALPPS procedure and tumour cell inoculation in RML, tumour growth was inferior than that in the group of ALPPS procedure. Conclusion: ALPPS seems to be a promising method to increase resectability rates of liver tumours. However, its possible effects on latent tumour cells that might remain in the liver should be taken into account.

Resistance exercise prevents impaired homocysteine metabolism and hepatic redox capacity in Walker-256 tumor-bearing male Wistar rats

ObjectiveThe aim of this study was to investigate changes in homocysteine (Hcy) metabolism and redox balance in response to exercise treatment in a tumor-bearing rat model. MethodsMale Wistar rats were exposed, or not, to a resistance exercise program 6 wk before inoculation with Walker-256 tumor cells or vehicle. After application, rats maintained their routine for 12 d and were then sacrificed for plasma and liver analyses. ResultsImpaired Hcy metabolism was evident after 12 d of tumor cell inoculation as demonstrated by significantly increased (P < 0.05) plasma total homocysteine (tHcy) concentration (53%) and decreased plasma cysteine, methionine, and vitamin B12 concentrations. Decreased hepatic cystathionine β-synthase (CBS) and betaine-homocysteine S-methyltransferase mRNA levels were found in tumor-bearing rats but not in controls. Tumor inoculation also decreased levels of liver reduced glutathione (GSH) and increased hepatic oxidative stress compared with non-tumor controls. However, resistance exercise prevented the tumor-impaired transsulfuration pathway as demonstrated by the decreased plasma tHcy, hepatic CBS expression, and increased GSH in tumor-exercised versus tumor-sedentary rats. Remarkably, all measures of liver oxidative stress were suppressed by exercise training. Tumor weight was unchanged between groups. ConclusionResistance exercise prevented tHcy accumulation and liver oxidative damage caused by Walker-256 tumor cell inoculation; the modulatory effects of resistance exercise on Hcy metabolism appear to be at the level of transsulfuration pathway.

Human lung adenocarcinoma cell cultures derived from malignant pleural effusions as model system to predict patients chemosensitivity.

BackgroundLung cancer is the leading cause of cancer related deaths and Malignant Pleural Effusion (MPE) is a frequent complication. Current therapies suffer from lack of efficacy in a great percentage of cases, especially when cancer is diagnosed at a late stage. Moreover patients’ responses vary and the outcome is unpredictable. Therefore, the identification of patients who will benefit most of chemotherapy treatment is important for accurate prognostication and better outcome. In this study, using malignant pleural effusions (MPE) from non-small cell lung cancer (NSCLC) patients, we established a collection of patient-derived Adenocarcinoma cultures which were characterized for their sensitivity to chemotherapeutic drugs used in the clinical practice.MethodsTumor cells present in MPEs of patients with NSCLC were isolated by density gradient centrifugation, placed in culture and genotyped by next generation sequencing. In a subset of cases patient derived xenografts (PDX) were obtained upon tumor cell inoculation in rag2/IL2 knock-out mice. Isolated primary cultures were characterized and tested for drug sensitivity by in vitro proliferation assays. Additivity, antagonism or synergy for combinatorial treatments were determined by analysis with the Calcusyn software.ResultsWe have optimized isolation procedures and culture conditions to expand in vitro primary cultures from Malignant Pleural Effusions (MPEs) of patients affected by lung adenocarcinomas, the most frequent form of non small cell lung cancer. Using this approach we have been able to establish 16 primary cultures from MPEs. Cells were banked at low passages and were characterized for their mutational pattern by next generation sequencing for most common driver mutations in lung cancer. Moreover, amplified cultures were shown to engraft with high efficiency when injected in immunocompromised mice. Cancer cell sensitivity to drugs used in standard chemotherapy regimens was assessed either individually or in combination. Differential chemosensitivity and different mutation profiles were observed which suggests that this isolation method could provide a platform for predicting the efficacy of chemotherapy in the clinical setting. Most importantly for six patients it was possible to establish a correlation between drug response in vitro and response to therapy in the clinic.ConclusionsResults obtained using primary cultured cells from MPEs underscore the heterogeneity of NSCLC in advanced stage as indicated by drug response and mutation profile. Comparison of data obtained from in vitro assays with patients’ responses to therapy leads to the conclusion that this strategy may provide a potentially useful approach for evaluating individual chemosensitivity profile and tailor the therapy accordingly. Furthermore, combining MPE-derived primary cultures with their genomic testing allows to identify patients eligible to trials with novel targeted agents.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0816-x) contains supplementary material, which is available to authorized users.

JNK in spinal cord facilitates bone cancer pain in rats through modulation of CXCL1.

In patients with advanced cancer, cancer-induced bone pain (CIBP) is a severe and common problem that is difficult to manage and explain. As c-Jun N-terminal kinase (JNK) and chemokine (C-X-C motif) ligand 1 (CXCL1) have been shown to participate in several chronic pain processes, we investigated the role of JNK and CXCL1 in CIBP and the relationship between them. A rat bone cancer pain model was established by intramedullary injection of Walker 256 rat gland mammary carcinoma cells into the left tibia of Sprague-Dawley rats. As a result, intramedullary injection of Walker 256 carcinoma cells induced significant bone destruction and persistent pain. Both phosphorylated JNK1 (pJNK1) and pJNK2 showed time-dependent increases in the ipsilateral spinal cord from day 7 to day 18 after tumor injection. Inhibition of JNK activation by intrathecal administration of SP600125, a selective pJNK inhibitor, attenuated mechanical allodynia and heat hyperalgesia caused by tumor inoculation. Tumor cell inoculation also induced robust CXCL1 upregulation in the ipsilateral spinal cord on day 18 after tumor injection. Inhibition of CXCL1 by intrathecal administration of CXCL1 neutralizing antibody showed a stable analgesic effect. Intrathecal administration of SP600125 reduced CXCL1 increase in the spinal cord, whereas inhibition of CXCL1 in the spinal cord showed no influence on JNK activation. Taken together, these results suggested that JNK activation in spinal cord contributed to the maintenance of CIBP, which may act through modulation of CXCL1. Inhibition of the pJNK/CXCL1 pathway may provide a new choice for treatment of CIBP.

Intrahepatic Tissue Implantation Represents a Favorable Approach for Establishing Orthotopic Transplantation Hepatocellular Carcinoma Mouse Models

Mouse models are commonly used for studying hepatocellular carcinoma (HCC) biology and exploring new therapeutic interventions. Currently three main modalities of HCC mouse models have been extensively employed in pre-clinical studies including chemically induced, transgenic and transplantation models. Among them, transplantation models are preferred for evaluating in vivo drug efficacy in pre-clinical settings given the short latency, uniformity in size and close resemblance to tumors in patients. However methods used for establishing orthotopic HCC transplantation mouse models are diverse and fragmentized without a comprehensive comparison. Here, we systemically evaluate four different approaches commonly used to establish HCC mice in preclinical studies, including intravenous, intrasplenic, intrahepatic inoculation of tumor cells and intrahepatic tissue implantation. Four parameters—the latency period, take rates, pathological features and metastatic rates—were evaluated side-by-side. 100% take rates were achieved in liver with intrahepatic, intrasplenic inoculation of tumor cells and intrahepatic tissue implantation. In contrast, no tumor in liver was observed with intravenous injection of tumor cells. Intrahepatic tissue implantation resulted in the shortest latency with 0.5cm (longitudinal diameter) tumors found in liver two weeks after implantation, compared to 0.1cm for intrahepatic inoculation of tumor cells. Approximately 0.1cm tumors were only visible at 4 weeks after intrasplenic inoculation. Uniform, focal and solitary tumors were formed with intrahepatic tissue implantation whereas multinodular, dispersed and non-uniform tumors produced with intrahepatic and intrasplenic inoculation of tumor cells. Notably, metastasis became visible in liver, peritoneum and mesenterium at 3 weeks post-implantation, and lung metastasis was visible after 7 weeks. T cell infiltration was evident in tumors, resembling the situation in HCC patients. Our study demonstrated that orthotopic HCC mouse models established via intrahepatic tissue implantation authentically reflect clinical manifestations in HCC patients pathologically and immunologically, suggesting intrahepatic tissue implantation is a preferable approach for establishing orthotopic HCC mouse models.

The influence of Saw palmetto extract on immune function in GL261 glioma

Objective To investigate the effect of saw palmetto extract (SR) on GL261 glioma in rats and immune system. Methods The 40 rats were divided into 4 groups randomly, one was the control group without tumor (n=10), the other 30 rats were given subcutaneous inoculation of tumor cells and then divided into 3 groups: tumor-bearing group (n=10), low dose SR group (n=10), and high dose SR group (n=10). After 1 weeks feeded, the rats of SR groups were given the saw palmetto extract, low dose group 50 mg/kg once a day every other day and 300 mg/kg of high dose group every other day. The tumor-bearing groups received the same dose of distilled water. After 4 weeks feeding, we measured the tumor weight and the inhibition rate was calculated. The tumor cell apoptosis was detected by TdT mediated dUTP nick-end labeling (TUNEL) staining. The splenic T lymphocyte proliferation was tested by methyl thiazolyl tetrazolium (MTT). Results ⑴ The SR groups compared to tumor group, the tumor weight was significantly reduced (F=62.678, P=0.000). The tumor inhibition rate was significantly higher in high dose group. ⑵ The apoptosis of tumor cells in tumor-bearing group was significantly less than SR groups and the apoptosis was significantly increased after treatment with SR , especially in high dose SR group(F=1.287E3, P=0.000). ⑶ Compared to SR groups and control group, T lymphocyte proliferation of tumor-bearing group reduced obviously. After treated with SR, T lymphocyte proliferation significantly increased and was higher in high-dose group (F=103.565, P=0.000). Conclusions Saw palmetto extract can activate T lymphocytes and induce apoptosis to tumor cells. Its function was related to SR concentration. Key words: Plant extracts /PD; Glioma/IM/DT

Creatine supplementation prevents hyperhomocysteinemia, oxidative stress and cancer-induced cachexia progression in Walker-256 tumor-bearing rats.

The purpose of this study was to investigate (1) the impact of tumor growth on homocysteine (Hcy) metabolism, liver oxidative stress and cancer cachexia and, (2) the potential benefits of creatine supplementation in Walker-256 tumor-bearing rats. Three experiments were conducted. First, rats were killed on days 5 (D5), 10 (D10) and 14 (D14) after tumor implantation. In experiment 2, rats were randomly assigned to three groups designated as control (C), tumor-bearing (T) and tumor-bearing supplemented with creatine (TCr). A life span experiment was conducted as the third experiment. Creatine was supplied in drinking water for 21days (8g/L) in all cases. Tumor implantation consisted of a suspension of Walker-256 cells (8.0×10(7) cells in 0.5mL of PBS). The progressive increase (P<0.05) in tumor mass coincided with a progressively lower body weight and higher hepatic oxidative stress; plasma Hcy concentration was 80% higher (P<0.05) by 10days of tumor implantation. Impaired Hcy metabolism was evidenced by decreased hepatic betaine-homocysteine methyltransferase (Bhmt), glycine N-methyltransferase (Gnmt) and cystathionine beta synthase (CBS) gene expression. In contrast, creatine supplementation promoted a 28% reduction of tumor weight (P<0.05). Plasma Hcy (C 6.1±0.6, T 10.3±1.5, TCr 6.3±0.9, µmol/L) and hepatic oxidative stress were lower in the TCr group compared to T. Creatine supplementation was unable to decrease Hcy concentration and to increase SAM/SAH ratio in tumor tissue. These data suggest that creatine effects on hepatic impaired Hcy metabolism promoted by tumor cell inoculation are responsible to decrease plasma Hcy in tumor-bearing rats. In conclusion, Walker-256 tumor growth is associated with progressive hyperhomocysteinemia, body weight loss and liver oxidative stress in rats. Creatine supplementation, however, prevented these tumor-associated perturbations.

The protein kinase IKKepsilon contributes to tumour growth and tumour pain in a melanoma model

Inhibitor-kappaB kinase epsilon (IKKε) constitutes a non-canonical I-κB kinase, which amongst others modulates NF-κB activity. IKKε and NF-κB have both been described for their role in cell proliferation and their dysregulation has been associated with tumourigenesis and metastasis in multiple cancer types. Accordingly, overexpression and constitutive activation of NF-κB have also been shown in melanoma, however, the role of IKKε in this cancer type has not been investigated so far. Thus, we determined IKKε expression in malignant melanoma cells and we were able to show a significant overexpression of IKKε in tumour cells in comparison to melanocytes. Inhibition of IKKε either by shRNA or the pharmacological inhibitor amlexanox resulted in reduced cell proliferation associated with a cell cycle block in the G1-phase. Functional analysis indicated that NF-κB, Akt1 and MAPK pathways might be involved in the IKKε-mediated effects. In vivo, we applied a mouse melanoma skin cancer model to assess tumour growth and melanoma-associated pain in IKKε knockout mice as well as C57BL/6 mice after inoculation with IKKε-negative cells. In IKKε knockout mice, tumour growth was not altered as compared to IKKε wild type mice. However, melanoma associated pain was strongly suppressed accompanied by a reduced mRNA expression of a number of pain-relevant genes. In contrast, after inoculation of IKKε-depleted tumour cells, the development of melanoma was almost completely prevented.In conclusion, our data suggest that IKKε in the tumour plays an essential role in tumour initiation and progression while IKKε expression in tumour surrounding tissues contributes to melanoma-associated pain.

Metallothinein 1E Enhances Glioma Invasion through Modulation Matrix Metalloproteinases-2 and 9 in U87MG Mouse Brain Tumor Model.

Malignant glioma cells invading surrounding normal brain are inoperable and resistant to radio- and chemotherapy, and eventually lead to tumor regrowth. Identification of genes related to motility is important for understanding the molecular biological behavior of invasive gliomas. According to our previous studies, Metallothionein 1E (MT1E) was identified to enhance migration of human malignant glioma cells. The purpose of this study was to confirm that MT1E could modulate glioma invasion in vivo. Firstly we established 2 cell lines; MTS23, overexpressed by MT1E complementary DNA construct and pV12 as control. The expression of matrix metalloproteinases (MMP)-2, -9 and a disintegrin and metalloproteinase 17 were increased in MTS23 compared with pV12. Furthermore it was confirmed that MT1E could modulate MMPs secretion and translocation of NFkB p50 and B-cell lymphoma-3 through small interfering ribonucleic acid knocked U87MG cells. Then MTS23 and pV12 were injected into intracranial region of 5 week old male nude mouse. After 4 weeks, for brain tissues of these two groups, histological analysis, and immunohistochemical stain of MMP-2, 9 and Nestin were performed. As results, the group injected with MTS23 showed irregular margin and tumor cells infiltrating the surrounding normal brain, while that of pV12 (control) had round and clear margin. And regrowth of tumor cells in MTS23 group was observed in another site apart from tumor cell inoculation. MT1E could enhance tumor proliferation and invasion of malignant glioma through regulation of activation and expression of MMPs.