Tumor Cell Mass Research Articles

Intracellular reactive oxygen species determine cancer stem cell radiosensitivity related to predictive biomarker for radiotherapy

Cancer cells display a higher level of reactive oxygen species (ROS) mainly due to increased metabolic activities resulting in altered redox balance. Imbalance in redox arises when the generation of ROS exceeds antioxidants defense system. ROS are generated in cells from multiple pathways, but mitochondria contribute significantly to cellular ROS pool by oxidative phosphorylation. Elevated levels of ROS are implicated in cell transformation, proliferation, and tumorigenesis. ROS-mediated signaling pathways activate pro-oncogenes which regulate cancer progression, angiogenesis, and survival. Normal cells maintain intracellular homeostasis by developing an array of enzymatic antioxidant systems such as catalase, superoxide dismutase, and glutathione peroxidase. Chemotherapy and radiotherapy exert their cytotoxic effects on tumor cells by the generation of excessive ROS. The failure of therapies is attributable to a small fraction of core cells in tumor mass called cancer stem cells (CSCs) which have self-renewal property and exhibit proliferation, differentiation, and resistance to treatments. Both normal and CSCs maintain low-ROS level ascribed to stemness. This review describes role and relevance of ROS in CSC with particular emphasis on developing predictive biomarker for outcome of cancer radiotherapy. It is pointed out that CSCs maintain lower ROS homeostasis and evade cell death by increased level of endogenous antioxidants capacity in cancer cells. Search for regulators of ROS and surface markers in CSC may render them sensitive to radiation offering new and effective strategy for cancer treatment.

IMMU-32. NON-INFLAMMATORY EFFECT OF DIPG ON TUMOR-ASSOCIATED MICROGLIA

Diffuse intrinsic pontine glioma (DIPG) is a diffusely infiltrating high-grade glioma that has a peak incidence from ages 6-8 and only a 9-12 month median survival. Like adult gliomas, DIPG exhibits a significant expansion of myeloid cells, with microglia/macrophages accounting for ~30% of the tumor cell mass (Caretti & Monje, 2014). In adult glioma, these glioma-associated macrophages have been implicated as supporting angiogenesis and tumor cell invasion, proliferation, and survival (Glass & Synowitz, 2014). However, genomic and epigenomic characterizations of adult and pediatric high-grade gliomas revealed striking differences in the molecular nature of these tumors (Sturm et al., 2014), and traditional chemotherapeutic approaches to adult glioblastoma are not effective in DIPG, suggesting these are unique diseases with distinct pathophysiology. Our data demonstrates that tumor-associated macrophages from primary DIPG samples exhibit gene expression signatures that are associated with pro-tumorigenic functions, but are also significantly less inflammatory than tumor-associated macrophages isolated from adult glioblastoma samples. Moreover, the cytokine secretion profiles of patient-derived DIPG cell cultures are dramatically less inflammatory when compared to patient-derived adult high-grade glioma cell cultures, and are more similar to the profile of human neural precursor cell cultures. This suggests that DIPG is less immunogenic than adult GBM, which may allow the tumor cells to evade detection and infiltrate healthy tissue. We additionally show that DIPG cells express CD200, a ligand that inhibits myeloid cell activation. Knockdown of DIPG expression of CD200 increases primary microglial cell phagocytosis in direct co-culture experiments, and also inhibits DIPG progression in in vivo xenograft models. Thus, CD200 may represent a mechanism by which DIPG evades normal immune responses to tumor growth. By understanding the DIPG-microglial interaction in molecular detail, we will gain insight into the pathophysiology of DIPG that could direct future targeted therapies.

Intravenous leiomyomatosis of the uterus: A clinicopathological analysis of nine cases and literature review

ObjectiveIntravenous/intravascular leiomyomatosis is characterized by intravenous proliferation of a histologically benign smooth muscle cell tumor mass that is non-tissue-invasive. Although benign, intravenous leiomyomatosis may cause remarkable systematic complications, presents significant diagnostic difficulties, and also is characterized by a relatively increased possibility of recurrence. We determine patients’ characteristics, and recurrence and treatment of intravenous leiomyomatosis. Materials and methodsPrognostic factors are analyzed with univariate analysis. Differences in categorical data are evaluated by the X2 test. A P value below 0.05 is regarded as indicating a significant difference. ResultsThe data results accord with the widely held view that complete excision of intravenous leiomyomata achieves favorable prognoses regarding remission. The efficacy of using Gonadotropin releasing hormone agonists to prevent growth or recurrence of tumors in unresected or incompletely resected intravenous leiomyomatosis foci. ConclusionIf complete surgical resection is not possible, partial resection followed by hormone therapy using gonadotropin-releasing hormone agonists is recommended, which in this study achieved the same favorable prognosis with regard to remission.

Nanostructured lipid carriers employing polyphenols as promising anticancer agents: Quality by design (QbD) approach

Cancer is one of the leading causes of death worldwide. There are several hurdles in cancer therapy because of side-effects which limits its usage. Nanoparticulate drug delivery systems have been tested against cancer in a range of scientific studies. In the recent years, advanced research on Nanostructured Lipid Carriers (NLCs) has garnered considerable attention owing to the advantages over their first-generation counterparts, Solid Lipid Nanoparticles (SLN). NLCs facilitate efficient loading of poorly water soluble drugs with simple methods of drug loading. Recently, there is an increased interest in polyphenols because of the evidence of their promising role in prevention of cancer. Polyphenols are produced as secondary metabolites by plants. Their role in prevention of development of tumors through variety of mechanisms and reduction of tumor cell mass has been reported. This article aims to review the science behind development of NLCs and role of polyphenols as promising anticancer agents. Principles of Quality by Design (QbD) have also been explained which are used in formulation-development of many nanoparticles, including NLCs, as reported in literature.

Growth inhibitory effect of Scrophularia oxysepala extract on mouse mammary carcinoma 4T1 cells in vitro and in vivo systems

IntroductionMedical plants have been intensively studied as a source of antitumor compounds. In the present study, we determine the effect of Scrophularia oxysepala on triggering apoptosis and diminishing growth, size and weight of the tumor in the allograft model of Balb/c mice. Material & methodsThe cytotoxic effects of Scrophularia oxysepala extract on 4T1 cells were studied using MTT (3-[4,5-dimethyl-2-thiazolyl]-2, 5 diphenyl tetrazolium bromide) assay and Trypan blue staining. DNA fragmentation assay was done for apoptosis detection. After the establishment of tumor in Balb/c mice, two groups of mice were received the extract at two doses of 50 and 100mg/kg respectively using intraperitoneal injection once every two days for 28 days. In order to assess the induction of apoptosis in cancer cells, the TUNEL assay was carried out in tumoral tissues. Moreover, the Ki67 test was used to evaluate tumor proliferation. ResultsAccording to the findings, the Scrophularia oxysepala extract inhibited cell growth. In vivo results showed that tumor size in mice treated with the extract was significantly reduced. The weight of tumor mass in treated mice after resection was less than the control group. According to the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay results, the herbal extract induced apoptosis in tumoral cells. Ki67 test also demonstrated that administration of the extract suppressed the growth of tumor cells. ConclusionOur data well approved the anti-proliferative effect of Scrophularia oxysepala extract, and clearly showed that, the plant extract can decrease the growth of breast cancer cells in tumor mass. Thus it may represent an ideal therapeutic tool for breast cancer.

Cancer stem cell metabolism: a potential target for cancer therapy.

Cancer Stem cells (CSCs) are a unipotent cell population present within the tumour cell mass. CSCs are known to be highly chemo-resistant, and in recent years, they have gained intense interest as key tumour initiating cells that may also play an integral role in tumour recurrence following chemotherapy. Cancer cells have the ability to alter their metabolism in order to fulfil bio-energetic and biosynthetic requirements. They are largely dependent on aerobic glycolysis for their energy production and also are associated with increased fatty acid synthesis and increased rates of glutamine utilisation. Emerging evidence has shown that therapeutic resistance to cancer treatment may arise due to dysregulation in glucose metabolism, fatty acid synthesis, and glutaminolysis. To propagate their lethal effects and maintain survival, tumour cells alter their metabolic requirements to ensure optimal nutrient use for their survival, evasion from host immune attack, and proliferation. It is now evident that cancer cells metabolise glutamine to grow rapidly because it provides the metabolic stimulus for required energy and precursors for synthesis of proteins, lipids, and nucleic acids. It can also regulate the activities of some of the signalling pathways that control the proliferation of cancer cells.This review describes the key metabolic pathways required by CSCs to maintain a survival advantage and highlights how a combined approach of targeting cellular metabolism in conjunction with the use of chemotherapeutic drugs may provide a promising strategy to overcome therapeutic resistance and therefore aid in cancer therapy.

Chylothorax and Chylous Ascites - A Rare Manifestation of Signet Ring Cell Carcinoma

Introduction: Chylous ascites is the accumulation of milk-like peritoneal fluid rich in triglycerides in the abdominal cavity. Chylothorax is the accumulation of this lipid-rich fluid in the thoracic cavity, and has rarely been reported in the literature as a consequence of malignancy. We present a case of a patient with signet ring cell carcinoma presenting with chylothorax and chylous ascites. Case Presentation: A 68 year old man with severe COPD and atrial fibrillation presented with shortness of breath. A chest x-ray (Figure 1A) and CT Chest (Figure 1B) revealed a right pleural effusion. Multiple thoracenteses revealed cloudy, white fluid. Infectious and cytologic studies on the fluid, as well as a whole body PET-CT were negative for malignancy.Figure 1Three months later the patient was admitted for reaccumulating hydrothorax, as well as new-onset ascites. A diagnostic paracentesis revealed milky, white chylous fluid (Figure 1C). Ascitic fluid analysis revealed Triglycerides of 500, WBC 605 (76% lymph), Amylase 13, Total Protein 3.1, Albumin 1.6 and a SAAG of 2.1. Blood work was not consistent with cirrhosis. A triple phase CT revealed normal liver size and parenchyma, with no identifiable hepatic or intra-abdominal lesions. EGD revealed a small area of mucosal nodularity in the 2nd portion of the duodenum (Figure 2A). A colonoscopy revealed mucosal thickening in the rectosigmoid colon (Figure 2B). Biopsies of both lesions were positive for poorly differentiated adenocarcinoma with signet ring cells.Figure 2The patient initiated therapy with FOLFOX regimen, however ultimately opted for hospice and expired three months later. Discussion: Chylous ascites is rare, with an incidence of approximately 1 per 20,000 admissions over the last 20 years. In the setting of malignancy, chylous fluid extravasation is thought to result from obstruction of the lymphatic channels by either direct tumor cell invasion or mass effect. Chylous ascites and chylothorax are often with lymphoma. There are only a few reported cases of chylous ascites or chylothorax from signet ring cell carcinoma. All reported cases depict this finding as a late manifestation of metastatic disease. Conclusion: Chylous ascites and chylothorax are exceedingly rare manifesations of signet ring cell carcinoma. All reported cases have been diagnosed in the setting of late stage metastatic disease. These findings should prompt further evaluation for an underlying gastrointestinal malignancy with EGD and colonoscopy.

Novel epi-virotherapeutic treatment of pancreatic cancer combining the oral histone deacetylase inhibitor resminostat with oncolytic measles vaccine virus

Oncolytic viruses (OV) constitute highly promising innovative biological anticancer agents. However, like every other antitumoral compound, OV are also faced with both primary and secondary mechanisms of resistance. To overcome those barriers and moreover amplify the therapeutic potential of OV, we evaluated a novel combined approach composed of the oral histone deacetylase inhibitor resminostat and an oncolytic measles vaccine virus (MeV) for a future epi‑virotherapy of pancreatic ductal adenocarcinoma. Cytotoxicity assays revealed that combined epi-virotherapeutic treatment of four well-characterized human pancreatic cancer cell lines resulted in a beneficial tumor cell killing as compared to either monotherapeutic approach. Notably, epi-virotherapeutic treatment of MIA PaCa-2 and partly also of PANC‑1 pancreatic cancer cells resulted in a tumor cell mass reduction being significantly more pronounced than it would be expected in case of an additive effect only, indicating a synergistic mode of action when combining resminostat with MeV. We further found that the epigenetic compound resminostat neither impaired MeV growth kinetics nor prevented the activation of the interferon signaling pathway which plays an important role in mediating primary and secondary resistances to OV. Moreover, we yielded information that the pharma-codynamic function of resminostat was presumably not altered in the course of pancreatic cancer cell infections with MeV. Taken together, these promising results favor the onset of epi-viro-thera-peutic clinical trials in patients suffering from advanced pancreatic ductal adenocarcinoma.

Abstract 628: A method to examine spatial relationships between tumor cells and vasculature using a mouse orthotopic PDX glioblastoma model

Abstract Introduction: Glioblastoma progression requires angiogenesis and/or vascular co-option. A major pathway for its infiltration is the perivascular space. We present herein a method to visualize glioblastoma cells with respect to tumor-associated vasculature within and beyond the tumor mass. Experimental procedures: A neurosphere cell line, HF3253, developed from cultures of a human glioblastoma biopsy sample was labeled with PKH26, a red fluorescent dye, using a commercially available kit (Sigma-Aldrich). The labeled cells were implanted into the right striatum in immunocompromised nude mice (n = 5) and allowed to grow for 2-4 weeks. Terminally (one mouse at 2, two at 3 and the remaining two at 4 weeks), the mice were intravenously injected with fluorescent isothiocyanate (FITC) dextran of 70 kDa molecular weight. The brains were removed after 5 min of dextran circulation, immersion fixed in 10% neutral buffered formalin for 2 days and then sectioned into 100 μm thick coronal slices using a vibratome. Slices were stained with the nuclear counterstain 4’,6-diamidino-2-phenylindole (DAPI) to visualize native mouse brain cells at 405 nm in an Olympus FV1200 laser scanning confocal microscope. Relative distributions of the dextran and PKH26 fluorochromes were also examined using the same microscope. FITC-dextran (green fluorochrome)-labeled microvessels and PKH26-labeled tumor cells (red fluorochrome) were excited by a laser beam at 488 and 568 nm, respectively, and emissions were detected with a photomultiplier tube through 522 and 585 nm emission filters, respectively. Results: The normal vasculature on the contralateral side appeared well perfused, filled with the green dextran fluorescence. Intricate, fine networks of microvessels were present. The ipsilateral hemisphere showed bright red spots of tumor cells in the striatum in closely distributed groups among the blue DAPI-stained mouse cells. Compared to contralateral side, the vasculature associated with such groups was dilated and irregular, with fewer interconnections. Interestingly, these irregular networks were also observed away from the main tumor cell mass in 3- and 4-week-old tumors, suggesting probable future infiltrative pathways. Red-labeled single cells and groups of tumor cells could be seen along vascular branches penetrating brain tissue adjacent to the tumor. The strong fluorescence of the tumor cell tracer PKH26 persisted over the 4 week experimental period, suggesting its utility in imaging tumor infiltration in this model. Conclusions: This technique generated high resolution images of tumor cell distribution and associated microvasculature in an orthotopic glioblastoma model. With further quantification, it can help in establishing infiltrative patterns of different PDX tumor models, and may also be useful to measure discrete responses to therapies that affect tumor cells and/or vasculature. Citation Format: Tavarekere N. Nagaraja, Kelly Keenan, Susan Irtenkauf, Laura Hasselbach, Swayamprava Panda, Glauber Cabral, James R. Ewing, Tom Mikkelsen, Ana deCarvalho. A method to examine spatial relationships between tumor cells and vasculature using a mouse orthotopic PDX glioblastoma model. [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 628.

Endogenous GABAA receptor activity suppresses glioma growth.

Although genome alterations driving glioma by fueling cell malignancy have largely been resolved, less is known of the impact of tumor environment on disease progression. Here, we demonstrate functional GABAA receptor-activated currents in human glioblastoma cells and show the existence of a continuous GABA signaling within the tumor cell mass that significantly affects tumor growth and survival expectancy in mouse models. Endogenous GABA released by tumor cells, attenuates proliferation of the glioma cells with enriched expression of stem/progenitor markers and with competence to seed growth of new tumors. Our results suggest that GABA levels rapidly increase in tumors impeding further growth. Thus, shunting chloride ions by a maintained local GABAA receptor activity within glioma cells has a significant impact on tumor development by attenuating proliferation, reducing tumor growth and prolonging survival, a mechanism that may have important impact on therapy resistance and recurrence following tumor resection.

Identification of Glioblastoma Subpopulations by Flim

Glioblastoma multiforme (GBM) is one of the most aggressive forms of brain cancer. The tumor is known to be composed of heterogeneous subpopulations of cells, potentially playing a role in therapeutic resistance. Therefore, an understanding of the tumor heterogeneity is essential for the development of effective therapies. To further study the tumor heterogeneity, a culture method was developed to isolate distinct subpopulations from an established glioma cell line. These subpopulations include neural stem-like cells and tumor mass cells.

Abstract A095: New genes for enhancing T cell function in adoptive cell therapy of cancer

Abstract A growing number of clinical trials point to the exquisite ability of adoptively transferred T cells to eradicate vast masses of tumor cells, including aggressive metastatic tumors. In one therapeutic approach T cells are derived from tumor biopsies (tumor-infiltrating lymphocytes, or TILs) and express endogenous TCRs. In others T cells are expanded from the peripheral blood lymphocyte pool and redirected to recognize tumor antigens by virtue of exogenous TCR or chimeric antigen receptor (CAR) genes. Although reports on enduring clinical responses are encouraging, a significant portion of patients who do not respond, or respond only partially and transiently, underscore the critical challenge of improving T cell function and survival at the tumor site. In attempt to improve the clinical efficacy of adoptive cell therapy we created three classes of genetic adjuvants designed to operate autonomously in gene-modified T cells. One class encodes constitutively-active (ca) toll-like receptors devoid of their ligand-binding domain, the other produces membrane-anchored immunostimulatory cytokines and the third encodes a novel homo-oligomerizing configuration of tumor necrosis factor receptors, inducing constitutive ligand-free activation. For gene delivery into human TILs or peripheral blood T cells we have been using electroporation of in-vitro-transcribed mRNA. Here we present data obtained in preliminary ex-vivo experiments designed to assess the function of caTLR4, membrane IL-2, IL-12 (single chain) and IL-15 and trimeric caCD40 and their level of cooperation. The mere expression of caTLR4 mRNA in polyclonal human CD8 and CD4 T cells induced the production of IFN-γ, triggered the surface expression of CD25, CD69 and 4-1BB and upregulated a panel of cytokines and chemokines. In all samples of anti-melanoma TILs tested, caTLR4 induced a burst of IFN-γ secretion, which usually ceased within 24 hours post-transfection. Furthermore, caTLR4 enhanced the cytolytic activity of TILs and augmented the secretion of IFN-γ, TNF-α and GM-CSF in the presence of autologous, but not mismatched, melanoma for at least 4 days post-transfection. The membrane cytokines were found to act mainly in-cis and each could support the proliferation of human CD8 and CD4 T cells for at least 6 days post-transfection, at a magnitude comparable to a saturating dose of soluble IL-2. Following mRNA electroporation of anti-melanoma TILs, membrane cytokines cooperated with caTLR4 in inducing IFN-γ secretion and upregulating the activation and costimulatory molecules CD25, CD69, 4-1BB and OX40. Different combinations of caTLR4, membrane cytokines and caCD40 mRNA induced spontaneous cytokine secretion in mRNA-transfected TILs, typically acting in a synergistic manner. Even when the spontaneous, initial effect waned, a considerably higher fraction of mRNA-transfected, compared to control-transfected TILs could still respond robustly to autologous, but not allogeneic, melanoma. In the presence of the three classes of adjuvants, transfected short-term cultured ‘young’ TILs revealed exceptional upregulation of 4-1BB, OX40, CD25 and CD28 and an increase in IFN-γ production and no, or only minimal change in the inhibitory receptors PD-1 and CTLA-4. Here, too, the different adjuvants displayed marked synergy, with caCD40 often exerting a prominent effect even when delivered as a single gene. Enhanced killing of autologous melanoma cells by electroporated young TILs was observed up to ten days post-electroporation. Our findings suggest that this new panel of genetic adjuvants can substantially improve the functional properties of antitumor T cells for extended periods, offering a new tool in all approaches for cancer immunotherapy employing adoptive T cell transfer. Citation Format: Gideon Gross, Aviad Pato, Hadas Weinstein-Marom, Noam Levin, Alon Margalit, Orit Itzhaki, Michal Besser, Tamar Peretz, Arthur Machlenkin, Michal Lotem. New genes for enhancing T cell function in adoptive cell therapy of cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A095.

Cell-based polymerase chain reaction for canine transmissible venereal tumor (CTVT) diagnosis.

Canine transmissible venereal tumor (CTVT) is the only naturally contagious tumor that is transmitted during coitus or social behaviors. Based on the tumor’s location, the diagnosis of genital TVT (GTVT) is comparably easier than those in the extragenital area (ETVT) that are more easily incorrectly diagnosed. Fortunately, CTVT cells contain a specific long interspersed nuclear elements (LINE), inserted upstream of the myc gene, allowing a diagnostic polymerase chain reaction (PCR) based detection assay. The objectives of this study were aimed to improve the diagnostic accuracy by applying the diagnostic LINE1-c-myc PCR assay and fine needle aspiration (FNA) collection in direct comparison with standard cytological and histopathological analyses. Seventy-four dogs, comprised of 41 and 31 dogs with tumor masses at their external genitalia and extragenital areas (e.g. skin and nasal cavity), respectively, were included in this study. The signalment of these 65 dogs and clinical history of 20 client-owned dogs were collected. Samples were taken by biopsy for both histopathological examination and FNA for cytological examination and diagnostic PCR. The PCR products from 10 apparently CTVT samples were purified and sequenced. Sixty-one CTVT cases were diagnosed by cytological and histological analyses, but 65 were positive by the PCR assay. Overall, the PCR assay improved the accuracy of diagnostic CTVT results, especially for the more difficult ETVT tumors. Moreover, this PCR-based approach can facilitate the decision as to discontinue chemotherapy by discrimination between residual tumor cell masses and fibrotic tissue.

Upregulation of EGFR signaling is correlated with tumor stroma remodeling and tumor recurrence in FGFR1-driven breast cancer.

IntroductionDespite advances in early detection and adjuvant targeted therapies, breast cancer is still the second most common cause of cancer mortality among women. Tumor recurrence is one of the major contributors to breast cancer mortality. However, the mechanisms underlying this process are not completely understood. In this study, we investigated the mechanisms of tumor dormancy and recurrence in a preclinical mouse model of breast cancer.MethodsTo elucidate the mechanisms driving tumor recurrence, we employed a transplantable Wnt1/inducible fibroblast growth factor receptor (FGFR) 1 mouse mammary tumor model and utilized an FGFR specific inhibitor, BGJ398, to study the recurrence after treatment. Histological staining was performed to analyze the residual tumor cells and tumor stroma. Reverse phase protein array was performed to compare primary and recurrent tumors to investigate the molecular mechanisms leading to tumor recurrence.ResultsTreatment with BGJ398 resulted in rapid tumor regression, leaving a nonpalpable mass of dormant tumor cells organized into a luminal and basal epithelial layer similar to the normal mammary gland, but surrounded by dense stroma with markedly reduced levels of myeloid-derived tumor suppressor cells (MDSCs) and decreased tumor vasculature. Following cessation of treatment the tumors recurred over a period of 1 to 4 months. The recurrent tumors displayed dense stroma with increased collagen, tenascin-C expression, and MDSC infiltration. Activation of the epidermal growth factor receptor (EGFR) pathway was observed in recurrent tumors, and inhibition of EGFR with lapatinib in combination with BGJ398 resulted in a significant delay in tumor recurrence accompanied by reduced stroma, yet there was no difference observed in initial tumor regression between the groups treated with BGJ398 alone or in combination with lapatinib.ConclusionThese studies have revealed a correlation between tumor recurrence and changes of stromal microenvironment accompanied by altered EGFR signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0649-1) contains supplementary material, which is available to authorized users.

Persistent enhancement of bacterial motility increases tumor penetration.

Motile bacteria can overcome the transport limitations that hinder many cancer therapies. Active bacteria can penetrate through tissue to deliver treatment to resistant tumor regions. Bacterial therapy has had limited success, however, because this motility is heterogeneous, and within a population many individuals are non-motile. In human trials, heterogeneity led to poor dispersion and incomplete tumor colonization. To address these problems, a swarm-plate selection method was developed to increase swimming velocity. Video microscopy was used to measure the velocity distribution of selected bacteria and a microfluidic tumor-on-a-chip device was used to measure penetration through tumor cell masses. Selection on swarm plates increased average velocity fourfold, from 4.9 to 18.7 μm/s (P < 0.05) and decreased the number of non-motile individuals from 51% to 3% (P < 0.05). The selected phenotype was both robust and stable. Repeating the selection process consistently increased velocity and eliminated non-motile individuals. When selected strains were cryopreserved and subcultured for 30.1 doublings, the high-motility phenotype was preserved. In the microfluidic device, selected Salmonella penetrated deeper into cell masses than unselected controls. By 10 h after inoculation, control bacteria accumulated in the front 30% of cell masses, closest to the flow channel. In contrast, selected Salmonella accumulated in the back 30% of cell masses, farthest from the channel. Selection increased the average penetration distance from 150 to 400 μm (P < 0.05). This technique provides a simple and rapid method to generate high-motility Salmonella that has increased penetration and potential for greater tumor dispersion and clinical efficacy.

Circulating CD133+/ESA+ cells in colorectal cancer patients

BackgroundTumor initiating cells are a small subset of cancer cells responsible for tumor growth and recurrence. The status of tumor initiating cells was measured using the surface markers CD133 (prominin-1) and ESA (epithelial-specific antigen). The aims of this study were to investigate the significance of CD133+/ESA+ cells in mesenteric venous blood (MVB) and tumor mass (TM) for overall survival (OS) and disease-free survival (DFS) in colorectal cancer (CRC) patients undergoing curative resection. Materials and methodsA total of 229 CRC patients undergoing curative resection were prospectively enrolled in the study. Using CD133 and ESA as surface markers, CD133+/ESA+ cells were enumerated from MVB and TM using flow cytometry. ResultsWe analyzed the presence of CD133+/ESA+ cells in TM from 158 patients and found no correlation to patient DFS, OS, or clinical stage. In 135 patients, an analysis of CD133+/ESA+ cells in MVB showed an inverse correlation with both DFS and OS (P = 0.014 and P = 0.008, respectively). It exhibited an increase-then-decrease pattern with the peak in stage II patients. A multivariate Cox analysis demonstrated that the status of CD133+/ESA+ cells in MVB, but not the TM, was a significant prognostic factor for DFS and OS (P = 0.003 and P = 0.011, respectively). ConclusionsThe status of CD133+/ESA+ cells in MVB, but not in TM, could be a useful indicator for predicting tumor recurrence and a prognostic marker for CRC patients.

Establishment of a human multiple myeloma xenograft model in the chicken to study tumor growth, invasion and angiogenesis.

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA). In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Huge Intravascular Tumor Extending to the Heart: Leiomyomatosis.

Intravenous leiomyomatosis (IVL) is a rare neoplasm characterized by histologically benign-looking smooth muscle cell tumor mass, which is growing within the intrauterine and extrauterine venous system. In this report we aimed to present an unusual case of IVL, which is originating from iliac vein and extended throughout to right cardiac chambers. A 49-year-old female patient, who was treated with warfarin sodium due to right iliac vein thrombosis, was admitted to our department with intermittent dyspnea, palpitation, and dizziness. Physical examination was almost normal except bilateral pretibial edema. On magnetic resonance venography, there was an intravenous mass, which is originated from right internal iliac vein and extended into the inferior vena cava. Transthoracic echocardiography and transesophageal echocardiography revealed a huge mass extending from the inferior vena cava through the right atrium, with obvious venous occlusion. Thoracic, abdominal, and pelvic MR showed an intravascular mass, which is concordant with leiomyomatosis. Surgery was performed through median sternotomy. A huge mass with 25-cm length and 186-gr weight was excised through right atrial oblique incision, on beating heart with cardiopulmonary bypass. Histopathologic assessment was compatible with IVL. Exact strategy for the surgical treatment of IVL is still controversial. We used one-stage approach, with complete resection of a huge IVL extending from right atrium to right iliac vein. In such cases, high recurrence rate is a significant problem; therefore it should be kept in mind.

Sporadic hemangioblastomas are characterized by cryptic VHL inactivation.

Hemangioblastomas consist of 10-20% neoplastic “stromal” cells within a vascular tumor cell mass of reactive pericytes, endothelium and lymphocytes. Familial cases of central nervous system hemangioblastoma uniformly result from mutations in the Von Hippel-Lindau (VHL) gene. In contrast, inactivation of VHL has been previously observed in only a minority of sporadic hemangioblastomas, suggesting an alternative genetic etiology. We performed deep-coverage DNA sequencing on 32 sporadic hemangioblastomas (whole exome discovery cohort n = 10, validation n = 22), followed by analysis of clonality, copy number alteration, and somatic mutation. We identified somatic mutation, loss of heterozygosity and/or deletion of VHL in 8 of 10 discovery cohort tumors. VHL inactivating events were ultimately detected in 78% (25/32) of cases. No other gene was significantly mutated. Overall, deep-coverage sequence analysis techniques uncovered VHL alterations within the neoplastic fraction of these tumors at higher frequencies than previously reported. Our findings support the central role of VHL inactivation in the molecular pathogenesis of both familial and sporadic hemangioblastomas.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-014-0167-x) contains supplementary material, which is available to authorized users.

Circulating and tumor-based biomarkers predict clinical activity in cancer patients treated with the engineered anti-PD-L1 antibody MPDL3280A

PD-L1 expressed in the tumor microenvironment regulates Th1 immune responses and mediates cancer immune evasion through interactions with PD-1 or B7.1 receptors on activated T cells. MPDL3280A, an engineered human monoclonal antibody, targets PD-L1 and inhibits its function. To identify immunologic predictive and pharmacodynamic biomarkers of MPDL3280A treatment, we performed a comprehensive analysis of tumors and blood samples collected at baseline and/or on treatment from ≈280 patients with locally advanced or metastatic solid tumors, including NSCLC, RCC, melanoma and bladder cancer. Regardless of tumor type, clinical responses were characterized by PD-L1 expression, the presence of markers of T cell activation (Th1 gene signature and CTLA4), and the absence of fractalkine at baseline in the tumor microenvironment. Elevated baseline expression of IFN-γ and IFN-γ-inducible genes (e.g., IDO1 and CXCL9) was associated with MPDL3280A response in melanoma but not NSCLC or RCC. On treatment, responding tumors showed increased infiltration of Th1-dominant immune infiltrate and evidence of adaptive PD-L1 up-regulation. In contrast, progressing tumors displayed the following patterns of tumor-infiltrating lymphocytes (TILs) and PD-L1 expression: (1) few/no TILs and absent PD-L1 expression (immunologic ignorance), (2) TILs present with minimal/no PD-L1 expression (non-functional immune responses), or (3) TILs residing solely around the tumor cell mass outer edge (excluded infiltrate), suggesting that resistance to MPDL3280A may be associated with impaired T cell trafficking and/or function. Profiling of ≈180 circulating biomarkers revealed that plasma concentrations of IL-18 and interferon-inducible T cell alpha chemoattractant (ITAC) increased in all patients following MPDL3280A treatment, representing a pharmacodynamic measurement of PD-L1 inhibition. In addition, analysis of PBMC showed an increase in T cell activation, as measured by IFN-γ and granzymes A and B gene expression in responders following MPDL3280A treatment, consistent with the observations in responding tumors. Baseline soluble PD-L1 was not associated with response. Some indication-specific biomarkers, such as plasma VEGF, decreased in responders with RCC but not with other indications. In NSCLC, a decrease in tumor burden markers, CA-125 and CEA, was associated with response. Similarly, IL-6 and IL-8 were differentially expressed on treatment in responders vs non-responders. Additionally, responders exhibited a decrease in circulating tumor DNA (ctDNA) in plasma, suggesting that ctDNA may be used to monitor MPDL3280A clinical activity in NSCLC. In conclusion, these data provide general and indication-specific mechanistic insights into immune checkpoint inhibition, potential mechanisms of response and resistance, as well as identification of potential predictive and pharmacodynamic biomarkers of anti-PD-L1/PD-1 clinical activity across multiple tumor types.